GLYCOLIPIDS
New Comprehensive Biochemistry
Volume 10
General Editors
A. NEUBERGER London
L.L.M. van DEENEN Utrecht
ELSEVIER AMSTERDAM. NEW YORK . OXFORD
G1ycolipids
Editor
H. WIEGANDT Marburg/ Lahn
1985
ELSEVIER AMSTERDAM. NEW YORK.OXFORD
0
1985 Elsevier Science Publishers B.V. (Biomedical Division)
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher, Elsevier Science Publishers B.V. (Biomedical Division), P.O. Box 1527, lo00 BM Amsterdam, The Netherlands. Special regulations for readers in the USA: This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which the photocopying of parts of this publication may be made in the U.S.A. All other copyright questions, including photocopying outside of the U.S.A., should be referred to the publisher. ISBN for the series: 0-444-80303-3 ISBN for the volume: 0-444-80595-8
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Library of Congress Cataloging in Publication Data Main entry under title: GIycolipids. (New comprehensive biochemistry; v. 10) Bibliography: p. Includes index. 1. Glycolipids. 1. Wiegandt, H. 11. Series. [DNLM: 1. Glycolipids. W1 NE372F v. 10/ QU 85 G56861 QD415.N48 vol. 10 574.19’2 s [574.19’247] 84-21266 [QP752.G56] ISBN 0-444-80595-8 Printed in The Netherlands
Preface
Fundamental to all living cells is the utilization of membranes, that basically comprize of lipid as the main barrier to an aqueous environment. Therefore, a multiplicity of regulated modulations of the interphase between lipid and water is necessary to enable the membrane to perform its specialized functions; amongst many others these include provisions for cell communications and membrane rearrangements. The glycolipids, as judged by the ubiquity of their occurrence in all cells, and their special physicochemical properties as well as their strategic positioning (frequently at the outer cell surface membranes), appear to be molecules particularly well suited to serve as links at the lipid-water membrane interphase. Indeed, glycolipids are enabled to mediate between the hydrophilic and the lipophilic environments because of their unique constitution, the molecular combination of a hydrophdic carbohydrate and a lipophilic aliphatic hydrocarbon chain residue. Positioned in the lipid bilayer, the glycolipids can, with their lipid ‘tails’, dramatically influence the properties of biological membranes, as exemplified in the haloand thermophilic organisms. In addition, many glycolipids carry very complex carbohydrates that may enable highly specialized interactions towards the aqueous environment. The obvious multitude of modulatory requirements at the membrane interphase is possibly reflected by the diversity and variability of the structural constitutions of the glycolipids. Still, most glycolipids can be classified into three major groups, and are distinguished by the molecular entity to which the carbohydrate moiety is directly linked. These groups are: the sphingo-lipids, including their sialic acid-containing components, the gangliosides; and furthermore, the glycero- and isoprenol-glycolpids. Whereas the functional significance of the isoprenol-glycolipids may reside in their ability to mediate the transport of carbohydrates through lipid membranes as part of the biosynthesis of glycoproteins, the sphmgo- and glyceroglycolipids appear to serve more directly as fundamental membrane constituents.
VI
Glycolipids have received special attention in several areas of medical interest. Besides their participation in the immunological expression of cells (they may be involved in storage diseases), they have been implicated to play an important role in the regulation of the social behavior of cells, including cancer, and some of them have been even found to be therapeutically useful agents in the treatment of neurological disorders, such as peripheral nerve injury and other peripheral as well as central neuropathies. The advances in biochemical methodology in recent years has also considerably increased knowledge of the glycolipids; in fact, to such an extent that it is becoming difficult to find an easy access to all available information. With the present volume, we have attempted to describe, the main groups, and to collate the present knowledge of the glycolipids. This has been done, however, not only with the intention of reviewing the more recent advances, but also to allow for the interested nonspecialist reader to become introduced to the respective fields. In addition, in accordance with the title of the New Comprehensive Biochemistry series, some attempt was made to make the present volume as comprehensive as seemed reasonable, in order to be useful as a reference source of the most relevant hitherto published data on glycolipids.
H. Wiegandt Department of Biochemistry School of Medicine Philipps University Marburg an der Luhn F.R. G.
Contents Preface by H . Wiegandt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V
Chapter 1. Glycosphingolipids, by A . Makita and N . Taniguchi . . . . . . . . . . . . .
I
........................................
..........................
1 2 4
3.4. High performance liquid chromatography . . . . . . . . . . . . . . . ............................ 3.5 Determination of GSL constituents
6
1. Introduction
.....
3.2. Fractionation.
3.7.
............................
3.5.2. Sphingoid bases
.....................
6
Mass spectrometry of whole GSLs .
.....................
9
3.11. Radiolabelling of GSLs ........................ pports and macromolecules . . . . 3.12. Covalent attachment of 3.13. Immunological procedu ................ 4. The lipophilic moiety of GSLs ........................................ 4.1. Long-chain bases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
........................ 5.1. Gala series . . . . . . . . . . . . . . . . . . . . . . . . . .
15 15 16
...............
16
.................
19
5.2.1. Glucosylceramide . . . . . . . . . . . . . . . . . 5.2.2. Glucocerebroside-ester . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3. Lactosylceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Globo and isoglobo series . . . . . . . . . . . ........................... 5.3.1. Globotriaosylceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20 20
VlII 5.3.2. Globoisotriaosylceramide. . . . . 5.3.3. Isoglobotetraosylceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.
Ganglio series . . . . 5.4.1. Gangliotriaosy
5.5.1. 5.5.2. 5.5.3. 5.5.4. 5.5.5. 5.5.6.
...............................
Lactotriaosylceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neolactotetraosylceramide. . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lactotetraosylceramide . . . .. ... 1V3-a-Galactosyl-neolactotet ........................... IV3-~-Galactosyl-neolactotetraosylceramide . .. . ...... . . . .. . . . . . . . . . . . . IV4-a-Galactosyl-neolactotetraosylceramide. . ... . .
...............................
5.5.9. Neolactodecaglycosylceramide
........ .........
5.6.2. Lactosylceramide sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.3. Sulfated tri- and tetraglycosylceramides . . . 5.7.
Fucolipids . . . . . . . . . . . . . . . . .
........ ...........
5.9.1. Phosphorus-freeglycosphingolipids
...................................... 6.3. 6.4. 6.5.
........................ ...........
Biosynthesis of glucosylceramide . . . . . Biosynthesis of di- and trihexosylceramides . . . . . . . . . . a and 8-N-Acetylgalactosaminyltransferasesinvolved in
...........
....................................
substance . . . . . . . . . . . . . .
6.9.
Galactosylceramide sulfotransferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 .lCeramidase .
... . , .. ..... . ... . . . .. .
7.1.7. a-Fucosidase
................... ................................
.
....................................
7.2.2. Activator protein for the hydrolysis of 8-glucosides . .
. . . . .... . . . . .
21 21 21 22 22 23 23 23 24 24 24 25 25 25 26 26 26 26 26 27 27 27 28 36 38 41 42 43 43 44 45 46 46 47 48 48 50 50 51 51 51 52 53 53 54 54 55 55 56 56
IX 7.2.3. Activator protein for the hydrolysis of ganglioside II'NeuAc-Gg,Cer ....................... 7.2.4. Activator for arylsulfatase A 7.2.5. Transfer proteins ........................................ 7.3. Metabolic disorders of glycosphingol ................. ........................... 8. Glycosphingolipids in immunology . . . . . 8.1. Human blood group systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................ 8.1.1. ABO system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.2. Lewis system .......................................... 8.1.3. lisystem . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . ................................ 8.1.4. P system . . . . . . . 8.2. Heterophile antigen lycosphingolipids . . . . . 8.3. Stage-specific embryonic antigens . . ............................ .... , and effects on the antigens of lectins and 8.4. GSL antigen marke ...................... differentiation inducers . . . . . . . , . . . . . . . . . . . . . . 8.5. Antigenicity of simple glycosphingolipids and possible olvement of neutral and acidic ... GSLs in autoimmunization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Glycosphingolipid changes in transformation and malignancy . . . . . . . . . . . . . . . . . . . . . . . . 9.1. Glycosphingolipid pattern and metabolism in transformed cells and their possible relationship to cell behaviors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Glycosphingolipid changes in tumor tissues and GSLs as possible tumor markers . . . . . . ................................ References . . . . . . . . . . . . . . , . .
56 57 57 51 59 60 60 62 63 65 67 68 69 12 73 13 77 82
Chapter 2. G[ycoglycerolipids,by I. Ishizuka and T. Yamakawa . , . . . . . . . . . . 101 1. Introduction . . . . . . . . . . . .
2. Structure . . . . . . . . . . .
................... ...............
. . . . .... ....................... .......................
..................... ....................... 3.1. Plant . . . . . . . . . . . . . . 3.1.1. Tissue distribution 3.1.3. Differentiation . . . . . .
................. ....................
..............
........................ ......................
3.2.5. Growth stage . . . . . . . . . . .
.......... ........................... ......................... ............ 3.3.2. Regional distribution . . . . . . . . . . . . . . . . . . . . . . .
101 101
104 104 105 105 106 106 112 121 122 125 127 129 132 132 132 133 134 135 135 137 138 139 140 141 141 141
X 3.3.3. Developmental variations. . . ...................... .. 3.3.4. Turnover of lipophilic domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... ..... ... 3.3.5. Location in myelin 3.3.6. Hormonal regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Germcell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. Distribution 3.4.2. Location in g ..............................
142 142 143 144 144 144 145 145 146 3.5. Secretion of animal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 3.6. Molecular evolution of glycoglycerolipids . . . . . . . . . . . 147 3.6.1. The concept of molecular evolution ........... 147 3.6.2. Evolutionary convergence and adap ....................... 148 3.6.3. Phylogenetic divergence of domains in glycoglycerolipids . . . . . . . . . . . . . . . . . . 149 4. Metabolism 149 ................................ 150 4.1.1. Synthesis of lipophlic domain . . ..................... 150 4.1.2. Transfer of reducing carbohydrates . . . . . . . . . . . . . . . . . . . . . . 153 4.1.3. Transfer of sulfate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 4.1.4. Specificity of transferase to lipophilic domain . . . . . . . . . . 156 4.1.5. Transfer of sn-glycerol-1-phosphate and ribitol e. . . . . . . . . . . . . . . . . 158 4.1.6. Location of enzyme activity . . . . . . . . . . . . . . . . . . . . , , . . . . . . . . . . . . . . . . . 160 162 164 4.2. Biodegradation 166 166 166 . . . . . . . . . 168 5. Biological property . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 170 5.1.1. Lipophilic domain . . . . . . . . . . . . . . . 170 5.1.2. Micelles . . . . . . . . . . . . . . . . . . . . . 170 5.1.3. Unsaturation of lipophlic domain . . . . . . . . . . . . _ _ _ _ . . . 171 . 5.1.4. Electric charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 5.1.5. Seminolipid . . . . . . . . . . .................................. 172 5.1.6. Macroglycolipid . . . . . . . . . . . . . . . . . 172 173 5.2.1. Integration of membrane . . . . . ............................... 173 5.2.2. Ion trap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 5.3. Interaction with protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 176 177 178 179 ....................... 179 180 180 5.5.1. Interaction with cations . . . . . . . . . . . . . . . . . 180 181 182 Acknowledgement . . . . . . . . . . . . . . . . . . 183 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
XI
Chapter 3. Gangliosides, by H. Wiegandt . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Chemistry, physics and methods of preparation and analysis . . . . . . . . . . . . . . . .
2.4.1. Alteration of the ceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6. Physicochemical characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.2. Gangliosides in solution . . ....................
.........................................
es . . . . . . . . . . .
3.2.3. Peripheral nerves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Cellular localisation . . 4. Metabolism . . . . . ...................... 4.1. Biosynthesis 4.2. Biodegradatio 4.3.1. Developmental changes . . . . . . ... . .. ......... 4.3.2. Changes after nerve stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3. Temperature-adaptive changes in the brain . . . . . . . . . . 5. Immuno-properties of gangliosides . . . . . ............. 5.1. Gener. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Involvement in disease .............................. 5.2.1. Animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Anti-ganglioside immune activities in human pathology . . . . . . . . . . . . 6. Ligand-binding properties of gangliosides . . . . . . . . . . 6.1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Ganglioside complexing with ligand protein 6.3. Interaction with lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Interaction with toxins, hormones, interferon and cell growth and differentiation factors . 6.5. Interactions with .......................... 7. Concludingremarks . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
199 200 200 202 205 205 205 208 210 210 211 219 219 220 221 224 224 224 226 226 226 221 228 229 229 230 232 232 233 234 234 234 238 238 239 239 239 240 240 240 241 241 244 245 245
Chapter 4. Glycosyl phosphopolyprenols, by F. W. Hemming . . . . . . . , . . . . . . . 261 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Polycis-isoprenoid alcohols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
261 262
XI1
s. nomenclature and methods
.......................... ........
2.1.3. Eukaryotic polycis-prenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolism of polycis-prenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Formation and hydrolysis of phosphoryl derivatives . . . . 2.3. Biosynthesis of polycis-prenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2
2.3.2. Eukaryotic cells
...............................................
3.1. Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. General principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Phosphopolycis-prenols in prokaryotic glycosyl transfer . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. General principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Formation of peptidoglycan . . . . . . . . ...... 3.3.3. Formation of 0-antigen determinants and capsular ex 3.3.4. Formation of teichoic acids and related compounds . . . . . . . . . . . . . . . . . . . . . . 3.3.5. The formation of other bacterial polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Phosphopolycis-prenolsin eukaryotic glycosyl transfer . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. General . . . . . . . . . . . . . . . . . . . . . . . . . ......... 3.4.2. N-Glycosylation of proteins in animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3. N-Glycosylation of proteins in plants . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.4. 0-Glycosylation in plants .................................... 3.4.5. Miscellaneous glycosyl phosphodoli 3.5. Phosphoretinol in glycosyl transfer . . . . . . . .......................... 4 . The control of phosphopolyprenol-mediatedglyc 4.1. The significance of controlling the process 4.2. Manipulation by administration of antibioti ................ 4.2.1. Bacitracin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Tunicamycin ....................................... 4.2.3. 2-Deoxyglucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.4. Other antibiotics . . . . . . . . . . . . . . . . . . . . ........ 4.3, Variation in the concentration of phosphopolypre 4.3.1. General . . . . . . . . . . . . . . . . . . . . . . . ....................... 4.3.2. Control of the biosynthesis of phosphopol 4.3.3. The association of phosphopolyprenols wi 4.3.4. Changes in concentration of phosphodolichol during development . . . . . . . . . . . . 4.4. Changes in phosphopolyprenol-mediatedglycosylation in mutant cell lines . . . . . . . . . . . 4.5. The effect of analogues of natural phosphopolyprenols on glycosylation 5 . Summary . . . . . ............................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SubjectIndex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
262 262 263 263 265 265 268 268 268 271 271 271 273 213 273 275 277 278 279 279 280 286 287 288 289 290 290 291 291 291 294 294 295 295 295 296 297 297 298 298 299
307
Wiegundt (ed.) Glvcolrprd~~ I985 Elseoier Science Publisher.\ B. V. (Biomedical Diursron)
3:
CHAPTER 1
Glycosphingolipids * AKIRA MAKITA and NAOYUKI TANIGUCHI Hokkuido University School of Medicine, Sapporo 060, Japan
I . Introduction Glycosphingolipids (GSLs) are composed of a long-chain base (sphingoid), a fatty acid, and a carbohydrate. The hydrophobic moiety, which is a ceramide, consists of the long-chain base substituted at the amino group by a fatty acid. The carbohydrate moiety is linked at the primary hydroxy group of the sphingoid base, e.g., sphingosine (sphing-4-ene): N - Acyl-sphingosine (ceramide)
Glycosphingolipid
CH,(CH,),,-CH=CH-CH-CH-CH, CH,(CH,),,-CH=CH-CH-CH-CH, I l l I l l OH NH OH
I
R
=
Fatty acid
OH NH 0
I
I
co
co sugar
I R
I R
The lipophilic moiety of GSLs shows microheterogeneity, and the GSLs of particular animal species and organs have characteristic lipid distribution patterns. Although the GSLs of mammalian tissues and cells have been most extensively studied, GSLs are also known to be present in organisms other than vertebrates, such as molluscs, plants and even microorganisms, in which their constituents and structure differ in varying degrees from those of mammals (see Section 5.9). GSLs are both species and tissue specific with regard to their qualitative and quantitative distribution patterns. In cells GSLs exist mainly as components of cellular membranes, especially of cell surface membranes. Their hydrophobic moiety embeds in the lipid bilayer, while the carbohydrate moiety extends to the outside. Essentially all of the GSLs are antigenically active, and one of their biological
* Sialic acid-containing glycosphingolipids (the gangliosides) are discussed in a separate chapter (see Chapter 3).
2
properties is that they act as immunogens (see Section 10). Some of the GSLs play roles as cell receptors for bacterial toxins and possibly also for bacteria and virus (see the chapter on gangliosides). Although all the biological properties of GSLs are intimately associated with their carbohydrate moiety, their lipid moiety, which makes the GSL molecule amphipathic, is essential to strengthen such biological properties as antigenicity [1,2], selective glycosylation [3,4], and possibly the organization and orientation of the carbohydrate chains [ 5 ] .
2. Classification and nomenclature GSLs usually are classified with respect to the chemical structures found in their carbohydrate moiety. This includes the number and species of the constituent monosaccharides, their sequence, positional and anomeric linkages, and other components such as sulfate (“sulfatides”) or sialic acid (“gangliosides”). The latter group will be dealt with in a separate chapter. Although numerous GSLs have been assigned trivial names derived from their history, the nomenclature and abbreviations recommended by the IUPAC-IUB Nomenclature Commission (1977) [6] cover semi-systematically the structures of most GSLs and are used in this chapter as much as possible. The GSL series with two novel core carbohydrate sequences have been demonstrated recently in nonvertebrates (Section 5.9). These GSLs are classified newly into arthro (a name derived from arthropod) series and mollu (a name derived from mollusc) series in this chapter. In this nomenclature system, the principal classifications based on the skeletal structure of the carbohydrate moiety are indicated by prefixes as follows: Prefix
Abbreviation
Structure
globo
Gb
Gal( /?1 + 3)GalNAc( j3l 3)Gal(a1 4)Gal(b l + 4)Glc -+
-+
isoglobo
iGb
GalNAc( bl + 3)Gal(a1 + 3)Gal(/3l
lacto
Lc
(Gal( /3l 3)GlcNAc),,(bl + 3)Gal(/3l+ 4)Glc
neolacto
nLc
(Gal(b1- 4)GlcNAc)”(/3l-+3)Gal( pl-+ 4)Glc
-+
4)Glc
-+
GalNAc( /3l + 4)Gal( pl 3)GalNAc(81 .-, 4)Gal( /31--$4)Glc
ganglio
-+
gala
Ga
Gal( a1
arthro
Ar
GalNAc( a1 + 4)GalNAc( 81 -+ 4)GlcNAc( p l + 3)Man( P l - 4)Glc
mollu
MI
Fuc( a1 + 4)GlcNAc( bl-+ 2)Man(a1 + 3)Man( bl 4)Glc
-+
4)Gal
-+
3 The number of monosaccharide units in an oligosaccharide is indicated by the suffixes “-biaose”, “-triaose”, “-tetraose” etc. [6]. For example, globoside is designated as globotetraosylceramide, and the corresponding GSL with one less monosaccharide is globotriaosylceramide (refer to Table 1.3). Differences in linkage position (e.g., 1 -,4 uersus 1 -, 3) in an otherwise identical sequence are indicated by the prefixes “iso-” or “neo-”, as in isoglobotetraosylceramide (refer to Tables 1.1 and 1.3). The prefixes tabulated above imply the entire structure of the root oligosaccharide (family) of the GSLs, including the order of the sugars and the position and anomeric configuration of the glycosidic linkages. With regard to shorthand notations for GSLs, the symbols Cer for ceramide, Sph for sphingoid base [6], and the recommended symbols for the hexoses (Gal, Glc, etc.) [7] have been adopted. Galactosylceramide therefore is abbreviated GalCer, and lactosylceramide LacCer. For complex GSLs, oligosaccharides are represented by specific symbols in which the number of monosaccharide units (-oses) is indicated by Ose,, preceded by two or three letters of the family name of the oligosaccharide (Gg, nLc, etc.); for example, gangliotriaosylceramide is GgOse,Cer which may also be abbreviated as Gg,Cer. Examples of GSLs representing the structure and abbreviation in this way are shown in Table 1.1. For GSLs with five or more glycose units of either straight or branched sugar chains, the nomenclature and abbreviations [6] recommended for fucolipids and gangliosides are employed in this chapter. The location of a glycose residue is indicated by a Roman numeral (counting from the ceramide end) designating the position at which the residue is attached to parent oligosaccharide, and by an Arabic numeral superscript indicating the position within that parent sugar residue to which the glycose is attached. The anomeric symbol follows the Roman numeral and precedes the (specified) “glycosyl-”. Therefore, 4N-acetylglucosaminylfll -, 3galactosylpl -, galactosylal + 3galactosylfll 4glucosylceramide (refer to Table 1.3) is written as IV3-a-galactosylneolactotetraosylceramide and abbreviated as IV3aGal-nLc,Cer, while galactosylal -, 3galactosyl
-
TABLE 1 . 1 Examples of names and abbreviations of di-, tetra- and pentaglycosylceramides Structure
Name of GSL
Abbreviation
GalPl + 3GalNAcPl+ 3Galal + 4Gal/31+ 4GlcCer G a l N A c P l - + 3Galal- 3GalPI + 4GlcCer GalPl + 3GlcNAcPl+ 3 G a l P l - + 4GlcCer G a l P l + 4GlcNAcPl+ 3Gal/31+ 4GlcCer GalNAcPl + 4GalPl + 3GalNAcPl+ 4GalPl+ 4GlcCer Gala1 + 4GalCer Fucal + 4GlcNAcPl 2Manal + 3ManPl + 4GlcCer GalNAcal + 4GalNAcPl -+ 4GlcNAcPI 3Manpl -,4GlcCer
Globopentaosylceramide lsoglobotetraosylceramide Lactotetraosylceramide Neolactotetraosylceramide Gangliopen taosylceramide Galabiaosylceramide Mollupentaosylceramide
Gb, Cer iGb,Cer Lc,Cer n Lc, Cer Gg,Cer Ga,Cer MI Cer
Arthropentaosylceramide
Ar,Cer
-
-+
A A sole mollupentaosylcerarnide has not been isolated, but the lower and higher homologues are shown in Table 1.5.
4 ( 2 + alfucosyl)/31 + 3N -acetylglucosaminyl/31 3galactosyl/3l 4glucosylceramide becomes IV2-a-fucosyl-IV3-~-galactosyllactotetraosylceramide which is abbreviated IV2aFuc-IV3aGal-Lc4Cer. -+
-+
3. Preparation and analysis Although carbohydrates compose nearly half the molecular weight of a trihexosylceramide, and GSLs having three or more glycose units are soluble in water, GSLs are prepared according to the methods used for the isolation of such lipids as phospholipids. The procedure for the preparation of GSLs consists of lipid extraction from the tissue, removal of lipids other than GSLs, particularly phospholipids, and separation of the individual GSLs. Recent methods for the isolation and characterization of of GSLs [8], including other useful procedures [9,10], have been summarized. 3.1. EXTRACTION
Tissues or cells are homogenized directly with 19 volumes of chloroform-methanol (2 : 1, v/v) [ll]. The residue is often extracted further with chloroform-methanol (1 : 1) and (I : 2) to ensure complete extraction, and the extracts are combined. For a large scale preparation of GSLs, the tissue or erythrocyte ghost is homogenized with acetone, and essentially all the simple lipids are removed by filtration. The acetone powder is extracted for 10-30 min [12] with pure or 90% ethanol near its boiling point. Polyglycosylceramides, which contain very long carbohydrate chains, were extracted with phosphate buffer-butanol from the ethanol-extracted erythrocyte ghosts ~31. 3.2. FRACTIONATION
The chloroform-methanol extract is adjusted to a solvent ratio of 2 : 1 by the addition of chloroform, and 0.2 vol. of water are added [ll]. After partition, most gangliosides and neutral GSLs with long carbohydrate chains are recovered in the upper phase. Mono- to pentaglycosylceramides, most of the cerebroside sulfate and lipids other than GSLs, and a portion of the less polar gangliosides are recovered in the lower phase. Folch’s partition [ll] is best suited for GSL fractionation from adult brain tissue which contains little neutral GSL that has two or more glycose units but contains the more complex and freely water-soluble gangliosides. The lipid extract, which contains the bulk of the phospholipids, is subjected to mild alkaline hydrolysis [ 141, or peracetylation [ 151 followed by Florisil (activated magnesium silicate) column chromatography, after which the native GSLs are recovered by deacetylation with sodium methoxide [16]. Most of the phospholipids can also be removed by chromatography on silicic acid from which neutral GSLs, cerebroside sulfate and considerable amounts of the less polar gangliosides are eluted with
5
acetone-methanol (9 : 1, v/v) while phospholipids remain on the column [17]. Separation of neutral GSLs from acidic GSLs is achieved by chromatography on a diethylaminoethyl (DEAE) Sephadex column. Neutral GSLs are eluted with chloroform-methanol-water (30 : 60 : 8 by volume) and acidic GSLs with chloroformmethanol-0.8 M sodium acetate (30 : 60 : 8 by volume) [18]. It is probably not wise to directly apply GSL mixtures containing large amounts of phospholipids to a DEAE-Sephadex column. A DEAE-silica gel column may also be useful [19]. Sulfoglycosphingolipids in the acidic GSL fraction can be fractionated by chromatography on silicic acid. Non-lipid low-molecular weight substances are removed by dialysis against water or by gel filtration on a Sephadex G-25 column using chloroform-methanol-water (60 : 30 : 4.5 by volume) [20] at any step in the preparation. 3.3. ISOLATION OF INDIVIDUAL GLYCOSPHINGOLIPIDS
Isolation of individual GSLs is almost exclusively achieved by chromatography on silicic acid. The chromatography is performed by either stepwise elution with increasing proportions of methanol in a chloroform-methanol solution, or gradient elution with increasing concentrations of methanol and water in a chloroformmethanol-water system [21]. By the use of porous silica gel spheres (Iatrobeads), clear separations of mono- to tetraglycosylceramides were achieved with linear gradient elutions using a chloroform-methanol-water system [21]. To monitor the chromatographic separation of GSLs on a column, the color reaction of hexose and/or thin layer chromatography are used to examine an aliquot of the eluate. A GSL often appears as a double (or multiple) spot on thin layer chromatography; the fast-moving species include predominantly longer-chain fatty acids with 22-26 carbon atoms, and the slow-moving species with 16-18 carbon atoms and, if present, a-hydroxy acids. Separation of a GSL mixture which migrates closely during chromatography, such as isomers with the same number of glycose residues, can be achieved by repeated chromatography, or by chromatography on a column or a preparative thin layer plate of peracetylated GSLs using a less polar solvent mixture. Galactosylceramide and glucosylceramide are separated on a borate-impregnated thin layer plate [22], while there is no way at present to separate galabiosylceramide from lactosylceramide (for their structure see Table 1.3). For thin layer chromatography of a complex mixture of GSLs, two-dimensional chromatography is useful [23]. A GSL preparation obtained by repeated silica gel chromatography is often contaminated with “soluble” silica gel. Non-lipid contaminants can be effectively removed by passing an aqueous solution of the preparation through a hydrophobic column (Sep-Pak C18) [756]. GSL can be obtained in an amorphous, white solid form by dissolving at warm temperature in a minimum volume of methanol followed by precipitation with acetone. 3.4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Due to its rapid and fine separation, high performance liquid chromatography has proven to be of considerable use in the isolation and quantification of GSLs. Most
6
of the presently available apparatus is equipped with a UV detector and a refractometer. Benzoylated GSLs [24] can be used for detection of minute amounts of GSLs (on the order of nmol) using a UV monitor. With the use of high performance liquid chromatography, neutral GSLs containing one to four glycose residues were quantitatively separated as their perbenzoylated [25] and 0-acetyl-N-p-nitro-benzoylated [26,27] derivatives. Without derivatization, both five globo series GSLs with monoto pentaglycosyl residues and five blood group H-active GSLs with penta- to tetradecaglycosyl residues were separated by elution with a 2-propanol-hexane-water system in a high performance liquid chromatography apparatus. However, detection was made manually [28]. For widespread application to the separation and quantification of GSLs, improvement of detectors such as the moving wire flame ionization detector [29] or development of new detection devices seems necessary. 3.5. DETERMINATION OF GSL CONSTITUENTS
3.5.I . Fatty acids The purified GSL is methanolyzed and fatty acid methyl esters are extracted with hexane [576]. The methyl esters are analyzed by gas chromatography [576]. a-Hydroxy acid esters, if present in the GSL, can be separated from the straight-chain acid esters using a preparative thin layer plate * developed with hexane-diethyl ether (85 : 15, v/v) followed by extraction with diethyl ether and acetylation or trimethylsialylation to convert the hydroxy acid esters to volatile derivatives [8].
3.5.2. Sphingoid bases After removal of the fatty acid esters by hexane extraction, the methanolysate of GSL is made alkaline, and sphingoid bases are extracted with diethylether. Following periodate oxidation, the sphingoid bases are analyzed in the form of long-chain aldehydes [30] or trimethylsilyl derivatives [31] by gas chromatography. It should be noted that periodate oxidation of a dihydroxy sphingoid base with n carbon atoms yields the same aldehyde as does a trihydroxy base with n + 1 carbon atoms. The concentration of sphingoid bases can be estimated by colorimetry of a complex with methyl orange [32], or more sensitively either by a fluorometric procedure using fluorescamine after differentiation of the hexosamine amide [33,34] or by a radioisotopic method after N-acetylation with I4C-labeled acetic anhydride [86]. 3.5.3. Carbohydrates The composition of hexoses, including fucose and hexosamines. can be estimated by gas chromatography of their trimethylsilyl methyl glycoside derivatives [35]. In this
* The fatty acid methyl ester fraction is often contaminated with phthalic acid esters, such as plasticizer, which may come from the organic solvents used and which interfere heavily with fatty acid analysis by gas chromatography. Introduction of thin layer chromatography before gas chromatography can remove completely the contaminants, which are left at the solvent front.
7
case, the methanolysate of the GSL, after extraction of the fatty acid esters, is neutralized by an anion exchanger [36], and hexosamines (and sialic acids, if necessary) are re-N-acetylated with acetic anhydride and trimethylsilylated. To estimate the content of individual monosaccharides, the methanolysate is supplemented with a known amount of mannitol as an internal standard, and then processed as above. Another valuable technique for carbohydrate determination is gas chromatographic estimation of alditol acetates [37], whch are prepared by a procedure involving acid hydrolysis of the GSLs, removal of fatty acids, reduction of monosaccharides with NaBH,, and peracetylation. 3.6. DETERMINATION OF CARBOHYDRATE STRUCTURE
The structural determination of complex carbohydrates involves three principles; sequence, linkage position and anomeric configuration. 3.6.1. Analysis of sequence and anomeric configuration
Specific exoglycohydrolases are employed to determine simultaneously the sequence and the anomeric configurations of the carbohydrate moiety in a GSL [38,39]. Many of the exoglycosidases so far characterized from invertebrate sources are specific for the anomeric configurations in the respective glycosidic linkages, but do not differentiate positional isomers. By sequential treatment of a GSL with the respective exoglycosidases in the presence of a detergent, and identification of the products (mostly the ceramide-linked products are subjected to thin layer chromatography) with or without inactivation of the glycosidase at each hydrolysis step, the sequence and the anomeric configuration of the GSL are determined simultaneously [38,39]. The enzymes, such as a- and P-galactosidases, P-N-acetylhexosaminidase, a-Nacetylgalactosaminidase, P-glucosidase. and a-L-fucosidase, can be prepared from a variety of sources according to published procedures [9] and are commercially available. An endo-P-galactosidase from Escherichia freundii catalyzes the hydrolysis of the inner P-galactosidic linkages of GSLs of the lacto series [40], and this enzymatic hydrolysis was adopted for characterization of GSL sugar chains containing a repeating N-acetyllactosamine (GalP1 + 4GlcNAc) unit [41]. Treatment of acetylated hexopyranosides with CrO, easily oxidizes the P-glycosidic glycose units but only very slowly oxidizes the a-glycosidic ones, providing a valuable procedure for determination of anomeric configurations [42]. Gas chromatographic analysis of the composition of hexoses and hexosamines in the peracetylated GSLs, before and after CrO, oxidation, reveals that a-linked monosaccharides in the GSLs remain almost intact, whereas P-linked ones are considerably reduced [43]. However, when this oxidation method was applied to the GSLs containing 0-methyl monosaccharides which are found in shellfish GSLs, some demethylation occurred (probably enough to give ambiguous results) [343].
8
173
43
e
61 116 158 129
205 7
100
I
f
61
200
116
147 233
43 I
Fig. 1.l. Mass spectra of partially methylated alditol acetates. Blood group A-active hexaglycosylceraisolated from human lung [SO] was permethylated and mide, IV2Fuccr,lV3GalNAccr-nLcOse4Cer. hydrolyzed. The partially methylated monosaccharides were reduced, peracetylated and subjected to a combined gaschromatography-mass spectrometry. a, 2,3,4,-tri-O-methyl-l,5-di-O-acetyl-fucitol (unsubstituted Fuc); b, 3-substituted Gal: c, 4-substituted Glc: d, 2,3-substituted Gal; e, unsubstituted GalNAc; f, esubstituted GlcNAc.
9 3.6.2. Determination of glycoside position Methylation analysis is the most valuable technique for determination of the positions of glycosidic linkages in complex carbohydrates. A particularly useful application of methylation analysis is based on identification by combined gas chromatography and mass spectrometry of the partially methylated alditol acetates derived from the permethylated glycoconjugates (reviewed in Refs. 44 and 45). The GSL in dimethylsulfoxide is permethylated with methyl sulfinyl carbanion and methyl iodide [46,47]. The permethylated derivative of the GSL is then hydrolyzed, and partially methylated monosaccharides are converted to their alditol acetates and identified by gas chromatography or combined gas chromatography and mass spectrometry [48,49]. Relative retention times of partially methylated alditol acetates on a gas chromatogram and characteristic mass fragments are given in Refs. 44,48 and 49. The mass spectra of the partially methylated alditol acetates derived from III*aFuc, II13aGalNAc-neolactotetraosylceramideof blood group A-glycosphingolipid [50] are shown in Fig. 1.1 as examples. Partially methylated hexosaminitol acetates, present at lower than a certain level, usually give a considerably weaker response in gas chromatography than neutral monosaccharides. However, application of a sample containing more than 0.5 nmol of the hexosamine to a column 1.5 m in length or less can largely prevent the preferential loss of hexosamine derivatives [51] during gas chromatography. Periodate oxidation, a classical method for determining glycosidic positions, is still useful, especially in combination with other analytical methods, for the attainment of such information as the glycosidic position, number and species of periodate-sensitive glycoses in GSLs with very complex carbohydrate chains [52]. 3.7. MASS SPECTROMETRY OF WHOLE GSLS
Direct inlet mass spectrometry of GSLs in the form of their volatile derivatives yields considerable information about the number of glycose residues, the approximate sequence of the glycose units (for example, -hexose-hexosamine-hexose-),the nature of the terminal glycose, and the approximate composition of the fatty acid (chain length, hydroxy or nonhydroxy, and double bonds) and sphingoid base. Although the pertrimethylsilylated [53,54] and peracetylated GSLs [55] can be analyzed, the permethylated GSLs which give more stable mass fragments are the particularly useful derivatives [57-591. A direct inlet mass spectrum of permethylated IV 3GalNAca-globotetraosylceramide (Forssman antigen) is shown in Fig. 1.2. In electron impact mass spectrometry, the molecular ion and the fragment ions in the high mass range are scarcely obtained, unless the amide group in the ceramide moiety, and N-acetylhexosamine if present, is reduced with LiAIH, to a substituted amine [60].On the other hand, mass spectrometry of permethylated GSLs [61-631 by chemical ionization, a soft ionization, provides molecular ions which consist of a number of ions due to the heterogeneous composition of fatty acid and sphingoid base, and rarely gives fragments due to ring opening which are often observed in electron impact mass spectrometry.
10
A GSL mixture of permethylated and LiAlH,-reduced derivatives was subjected to temperature programming of the direct inlet probe, which led to successive evaporation of GSL species mainly according to the number of glycoses [64,65]. The mass spectra and the ion curves for selected mass ions of the mixture could, in most cases, be assigned to specific GSLs which were revealed by thm layer chromatography [64,651. Field desorption mass spectrometry [66-681, fast atom bombardment mass spectrometry [69], and secondary (or sputtered) ion mass spectrometry do not require chemical derivatization of the samples. In these techniques, GSLs yield the fragment ions derived from almost successive cleavages of the glycose units at their glycosidic
HexNAc - 0 - H C x N A c
260
i
-
*,
..
’ -0-Hex- O-Hex-O-Hex-, O+Cer
505;
-449
709:
408-
M’1793
913;
1117;
j660
408
%
0
0
200
160
360
400
I%
’ O O P
0
0 1000
1100
1200
1300
Fig. 1.2. Direct inlet mass spectra of permethylated IV3GalNAccr-Gb,Cer(Forssman antigen). (Performed by Dr. S. Gasa at Hokkaido University, School of Medicine.)
11
linkages. Fast atom bombardment mass spectrometry cleaved the amide linkage of the ceramide moiety in the case of lactosylceramide [69]. 3.8. NUCLEAR MAGNETlC RESONANCE (NMR) SPECTROSCOPY
Proton NMR spectroscopy is a valuable means of determining the stereochemical configurations of the anomeric linkages and of H-1 to H-5 of glycopyranoses, the geometrical positions of CH=CH bonds and amides, and other proton signals or GSLs. Anomeric proton signals are clearly separated from other proton signals, and those of the different monosaccharide residues of GSLs were assigned by their chemical shifts using native GSLs [70,71], a triglycosylsphingosine [72] and oligosaccharides [73-751 derived from GSLs, pertrimethylsilylated GSLs [39] and permethylated GSLs [77-791. The use of a high resolution N M R apparatus equipped with a 200-500 MHz resonance frequency magnet facilitated the analysis of protons other than anomeric protons, such as protons H-1 to H-6 in monosaccharide residues of peracetylated [80], permethylated [77-791 and native GSLs [84,85]. By proton N M R spectroscopy of native GSLs in deuterated dimethylsulfoxide solution, amide protons can also be measured, giving structural information about the lipid moiety as well [85,87]. As shown in Fig. 1.3, chemical shifts of anomeric protons of globotetraosylceramide are resonated in a narrow range between 4 and 5 ppm, amide protons between 7 and 7.5 ppm, and methyl protons of an acetamide group at
Gioboslde 1100
9
8
7
6
5
4
I
I
I
3
2
1
1
0
PPM
Fig. 1.3. Proton NMR spectrum of globotetraosylceramide in dimethyl-d, sulfoxide at 110 O C . N-Ac. methyl proton signal of N-acetyl group; GalNAc-NH and Cer-NH. amide proton signals at N-acetylgalactosamine and ceramide. respectively; Olefinic. olefinic proton signals in ceramide moiety. Others are anomeric protons. (Taken by Dr. S. Gasa at Hokkaido University. School of Medicine.)
L
h,
TABLE 1.2 Molar composition of glycosphingolipids by measurement of intensities of amide and anomeric protons a GSL
Required Cer
Glc
GlcCer GalCer LacCer Ga,Cer Gb,Cer Gg3Cer
1
1
Lc,Cer Gb,Cer
GalNAc
GlcNAc
1
1
1 1
1
1
1
1 1
1
1
1
Analyzed Gal
1
1 2 2
1
1
1 2
1 1
Cer
BGlc
1.0’ 1.o 1.0 1.o 1.0 1.0
1.1
1.2 1.2
0.8 1.1 1.1 1.1 1.3
1.0
1.1
1.o
1.0
1.2
1.3
B-Gal
1.1
a-Gal
p-GalNAc
a-GalNAc
8-GlcNAc
1.1
0.9 0.7’ (1.3)
0.9 1.o
1.1
(1.0)
’
(WhC Gg,Cer
1
1
2
1
1.0
1.1
2.3
1.1
(0.9)
nLc,Cer IV ’GalNAcaGb,Cer I ’SO,-GalCer
1 1 1
1 1
2 2 1
1
2
1.0 1.0 1.01
1.4 1.2
2.2
.o
1
1.1 0.9
’
0.9 (0.9)
’
(1.3)
0.8 (0.9)
1.1
The peak intensities of each GSL in a dimethyl-d, sulfoxide solution were integrated in the spectra taken at 110 OC (data taken from Ref. 87). The value was from amide proton. The value was from anomeric proton. For abbreviations of GSLs, see Table 1.3.
a
13
1.85 ppm. The molar composition [87] of ceramide and carbohydrates of GSLs which were measured by integrating the intensities of signals of the amide and anomeric protons, respectively, agrees reasonably with their known compositions (Table 1.2). Spin decoupling in NMR analysis [85] facilitates measurement of particular groups such as the acetyl ester group in a GSL molecule [loll. Carbon 13 NMR spectroscopy, though it requires a fairly large amount of sample, also yields some structural information about GSLs. 13C-NMR spectroscopy of glucosylceramide (300 mg in a CDCl,/CD,OD solution) confirmed the existence of a pyranose ring with /%configuration in the glucosyl moiety, and measured the contents of unsaturated hydrocarbon chains and of a-hydroxy acids in the lipid [89]. The presence of the latter was deduced from the spectra of the geometric orientation. 3.9. INFRARED SPECTROSCOPY
Infrared spectra of GSL commonly show a broad absorption maximum near 3500 cm- due to OH group, whch can be employed to confirm permethylation of GSLs, peaks at 2900 and 2850 cm-' (CH, and CH, respectively), and a peak at 1550 cm-' (NHCO) (Fig. 1.4). Sulfoglycosphingolipids show broad absorption maximum near 1240 (SO,) and 810 cm-' (C-0-S) (Fig. 1.4, lower). Monoglycosylceramides that contain an ester absorb at 1740 cm-' due to the aliphatic ester (Fig. 1.4, middle),
'
s w oI 3000 ,
I
I
V I
I
1
I
I
I
6
I
I
,
I
I
I
10
0
I
700 cm-1
1000 900 800
I
I
I
I
4
2
4000
1400 2000 1600 1200
I
I
I 12
I
I
I
14 B
I
I
I
3000 2000 1000 1600 1400 1200 1000 800 600 (cm-1)
Fig. 1.4. Infrared spectra of GSLs. Upper, glucosylceramide; middle, 6-fatty acyl-glucosylceramide; lower, I 'SO,-GalCer. For absorptions characteristic of functional groups in GSLs, see text.
14
and this peak can be a semi-quantitativeindicator as to whether or not a non-esterified GSL preparation is contaminated with some glycerophospholipids. 3.10. PREPARATION OF OLIGOSACCHARIDES AND OF GLYCOSYLSPHINGOSINE
Whole oligosaccharides are released from intact GSLs in reasonably high yields by oxidative ozonolysis of the olefinic double bond of the sphingenine residue [81,90]. The oligosaccharides can also be prepared by an osmium tetroxide-periodate oxidation procedure [83,91]. Partial acid hydrolysis [52] or alkaline hydrolysis [92] of the intact GSLs in an aqueous medium yields oligosaccharide fragments, and provides a useful technique for characterization of GSLs with very complex carbohydrates. In alkaline hydrolysis in a butanol solution, amide bonds of GSLs are cleaved as well, and glycosylsphingosines(lyso GSLs) are obtained in low yield [93]. 3.11. RADIOLABELING OF GSLr
GSLs can be radiolabeled either on the lipid moiety or on some of the carbohydrate chains. By reduction of GSLs in tetrahydrofuran solution with tritiated KBH,, using PdCl, or Pd on BaSO, as catalysts, GSLs which are labeled at their sphingenine residue with tritium are obtained [94]. The procedure for tritium labeling of GSLs which have galactose or N-acetylgalactosamine at their non-reducing termini involves oxidation with galactose oxidase [95] in the presence of horseradish peroxidase [96] followed by reduction with tritiated NaBH,. In our experience, the use of a solution containing more than 50% tetrahydrofuran in the oxidation reaction does not give labeled GSLs of high specific radioactivity, possibly due to some inactivation of galactose oxidase. The oxidation reaction in a phosphate buffer resulted in the high labeling [97]. 35 S-labeled sulfoglycosphingolipidscan be prepared from the brain and kidney of an animal which was administered inorganic [ 35S]sulfate,either intracerebrally [98] or intraperitoneally. 3.12. COVALENT ATTACHMENT OF GSLr TO SOLID SUPPORTS AND MACROMOLECULES
Peracetylated GSL is oxidized with KMnO, in the presence of a crown ether, dicyclohexyl-18-crown-6, yielding a carboxy-bearing product (‘glycolipid acid’) derived by cleavage of the olefinic double bond of the sphingenine residue [99]. ‘Glycolipid acid’ is also prepared by performic acid oxidation of glycolipid aldehyde [83]. The amide conjugate of the glycolipid acid and an amino-bearing solid support is successfully formed in an approximate yield of 50% or more through coupling of the glycolipid acid with alkylamine glass beads [99] or aminopropyl silica gel [19] in the presence of both N-hydroxy succinimide and dicyclohexyl carbodiimide followed by deacetylation with mild alkali. Deacetylated glycolipid acids can be coupled with aminoethyl-Sepharose, polyacrylic hydrazide or protein in the presence of N-hydroxy succinimide and l-ethyl-3(3-dimethylaminopropyl)-carbodiimideto give the corresponding GSL-polymers [991.
15 3.13. IMMUNOLOGICAL PROCEDURES
GSLs possess an antigenic reactivity whch is specific for the respective carbohydrate structure. To detect the antigenic reactivity of GSLs, one or more immunological methods are employed in the presence or absence of auxiliary lipids such as phosphatidylcholine and cholesterol. These include double immunodiffusion in agar, hemagglutination, immune hemolysis, complement fixation, quantitative precipitin reaction, immunoadherence, and an enzyme-linked immunosorbent assay. The procedures are summarized in Ref. 100. The murine hybridoma technique for producing monoclonal antibodies of Koehler and Milstein [82] is useful to obtain highly specific monoclonal antibodies. Multiple monoclonal antibodies directed against a GSL will each recognize a distinct portion of the carbohydrate moiety. The antibodies can be detected by a binding assay using GSL-antibody complex conjugated with I-labeled protein A of Stuphylococcus aweus in wells of a plastic micro plate [lo21 (solid phase radioimmunoassay). Another recent sensitive, simple method of detecting small amounts of GSL antigens is a solid phase immunoautoradiography assay [lo31 which is a modification of a method developed for the detection of gangliosides that bind to cholera toxin [104]; GSLs separated on a thin-layer plate are reacted with mouse antibody specific for a GSL and then with 1251-labeledF(+<)zfragments of rabbit immunoglobulin G directed against mouse immunoglobulins, followed by autoradiography.
4. The lipophilic moiety of GSLs The composition of fatty acid and long-chain base generally reflects the cells and tissues from which the GSLs are derived. 4.1.
LONG-CHAINBASES
Although sphingosine (sphing-4-ene) is a predominant long-chain base in many GSLs, other sphmgoids such as dihydrosphingosine or phytosphingosine may be more or less prevalent : CH i(CH 2 ) 1 4 -CH-CH-CH
I
l
Sphinganine
2
l
(Dihydrosphingosine)
OH N H 2 0 H CH l(CH2)ll-CH-CH-CH-CH
I
I
I
2
I
OH OH NH,OH
CH3(CH2)14-CH=CH-CH-CH-CH2
I
l
l
OH NH,OH
4-~-hydroxysphinganine (Phytosphingosine) Eicosasphingenine (~-eryfhro-2-amino-4trans-eicosene-1,3-diol)
16 Phytosphingosine wluch was found as a principal long-chain base of fungus [lo51 and plant [lo61 sphingolipids was identified in human kidney (2-15% of the total bases) [107]. GSLs with short carbohydrate chains of mono- to trihexosylceramides from extraneural tissues of herbivorous animals such as cattle [lo71 and horses [lo81 contain a large proportion of phytosphingosine. Karlsson [ 109,1101 describes about 60 naturally occurring sphingoid bases (for review, see also Refs. 111-113). 4.2. FATTY ACIDS
Regarding the constituent fatty acid, it is characteristic of most GSLs that they contain long-chain fatty acids with more than 20 carbon atoms and fewer unsaturated acids than glycerolipids, which makes them relatively stable. Moreover, lower molecular weight GSLs from certain tissues, such as brain, kidney and small intestine, contain a considerable quantity of a-hydroxy fatty acids (for review, see Ref. 114). It can be considered that the fatty acid composition of GSL in a tissue reflects the substrate (including the sphingoid moiety) specificity of glycosyltransferases which catalyze synthesis of the GSL. However, the brain P-galactosyltransferase which synthesizes galactosylceramide from UDP-galactose and ceramide acts in vitro almost exclusively on ceramides containing a-hydroxy acids but not on ceramides containing straight chain acids (4,1151. The reason is not clear at present, since brain galactosylceramides contain a substantial proportion of straight chain acids.
5. Glycosphingolipid components according to carbohydrate series GSLs are classified according to their carbohydrate structure. Sulfated GSLs and fucose-containing GSLs (fucolipids) can be separately grouped. Chemical structures of fucose-free GSLs as well as sulfated GSLs are shown in Table 1.3. Surveys [36,113,117] on GSL chemistry and biology have recently appeared. 5.1. GALA SERIES
GSLs which belong to the gala series are galactosylceramide, galabiosylceramide and I13~(GalNAccul-3GalNAc)-galabiosylceramide(1-3 in Table 3). No other compounds which may be extended into GSLs belonging to this series have been demonstrated in mammals. 5.1.1. Galactosylceramide (galactocerebroside, structure 1-1 in Table 1.3) Ths lipid was discovered in human brain by Thudichum (1884)[118] (for a review, see Ref. 111). Galactosylceramide has also been isolated from organisms other than mammals, such as Aspergillus niger [119], shellfish [120,121] fish testis [122], frog skin [123], and the salt gland of spiny dogfish [124]. Shapiro and Flowers [125] chemically synthesized galactosylceramide.
17 TABLE 1.3 Structure of neutral glycosphingolipids and sulfated glycosphingolipids No.
Gala series 1-1 GalPl
+
1-2
Galal
+
1-3
GalNAcal 3GalNAcPl 3Galal -+ 4GalB1 + lCer
lCer 4GalP1
+
lCer
-+
GalPl
-+
4GlcPl
G l o b and isoglobo series Galal + 4GalPl 1-6
GalCer
Galactosylceramide
Ga,Cer
Galabiosylceramide
II ' 4 N-acetylgalactosaminylal-3N-acetylgalactosaminyl)P-galabiosylceramide
3GalPI
GlcCer
Glucosylceramide
LacCer
Lactosylceramide
Gb,Cer
Globotriaosylceramide
iGb,Cer
Isoglobotriaosylceramide
+
Gb,Cer
Globotetraosylceramide
-+
iGb,Cer
Isoglobotetraosylceramide
IV'GalNAcaGb,Cer
IV '-~-acety~ga~actosaminy~aGb,Cer
II13(Galal 3Gal)a-Gb,Cer
III '-(ga~actosy~al 3galactosyl)aglobotriaosylceramide
Gb, Cer
Globopentaosylceramide
+
lCer
-+
4GlcPl
-+
4GlcB1 + lCer
+
1-7
Galal
1-8
GalNAcPl + 3Galal 4GlcP1 + lCer
1-9
GalNAcPl 3Galal 4GlcPI + lCer
1-10
GalNAcal -+ 3GalNAcPI 3Galal + 4GalP1 + GlcPl
+
Systematic name
-+
Glucosylceramide and lactosylceramide 1-4 GlcPl .+ lCer 1-5
Abbreviation
-+
lCer
-+
4GalPl
+
3GalP1
-+
-+
1-11
Galal + 3Galal + 3Galal + 4GalPI + 4GlcPl + lCer
1-12
GalPl 3GalNAcPl + 3Galal 4GalPI + 4GlcS1 lCer -+
lCer
-+
+
-+
-+
Ganglio series GalNAcPl 1-13 1Cer 1-14
+
4GalPl
GalPl 3GalNAcPl 4GlcBI lCer -+
-+
4GlcPl
+
Gg,Cer
Gangliotriaosylceramide
+
4GalP1-+
Gg,Cer
Gangliotetraosylceramide
Lc,Cer
Lactotriaosylceramide
+
nLc,Cer
Neolactotetraosylceramide
+
Lc,Cer
Lactotetraosylceramide
-+
Lacto and neolacto series GIcNAcPl -+ 3GalP1 + 4GlcPI 1-15 1 Cer 1-16
GalPl + 4GlcNAcPl 4GlcP1 + lCer
1-17
GalPl + 3GlcNAc/3I + 3GalPl 4GlcPI + lCer
-+
3GalPl
-
18 TABLE 1.3 (continued) No. a
Abbreviation
Systematic name IV 3-gaIactosy~a-neo~actote traosylceramide
1-18
Galal + 3GalB1 + 4GlcNAcb1+ 3GalP1+ 4Glcb1+ lCer
IV3Gala-nLc,Cer
1-19
GalPl + 3GalS1 + 4GlcNAcPl 3Galb1+ 4GlcPI + lCer
IV~G~I/I-~LC,C ~ I V~3-ga~actosy~fi-neo~actotetraosylceramide
1-20
Galal + 4GalP1 -B 4GlcNAc/31 + 3Gal/31+ 4GlcBl+ lCer
IV4Gala-nLc4Cer
~ v ~ - g a ~ a c ta-neo~actoosy~ tetraosylceramide
1-21
GalNAcPl + 3Gal/31-+ 4GlcNAc/31 + 4Gal/3l+ 4Glcb1 + lCer
IV3GalNAc/3nLc,Cer
IV '-~-acety~ga~actosaminy~~neolactotetraosylceramide
1-22
Galal + 3GalB1 + 4GlcNAcB1 +
IV4(Gal al-3Gal/3l4GlcNAc)BnLc4Cer
+
3GalP + 4GlcNAcPl- 3GalPl 4GlcP1+ lCer 1-23
-
Galal
-+
3GalB1 -B 4GlcNAcPlh 6
1V 3*6-bis(Gal a1-3GalPl4GlcNAc)P-nLc4Cer
GalPl-
Galal + 3GalP1 + 4GlcNAcal f 4GlcNAcPl + 3GalPl- 4Glc/3l + Cer
Sulfated glycosphingolipids HSO, + 3GalPI + lCer 1-24 1-25
HSO,
-
3Gal/3l + 4Glc/31 + lCer
13S0,-GalCer
Galactosylceramide-1,sulfate
1190,-~ac~er
Lactosylceramide-I13sulfate
11'SO,-Gg ,Cer
Gangliotriaosylceramide-
HSO,
1
1-26
3 GalNAc/?l+ 4GalP1 + 4Glcbl
+
~~~-su~fate
lCer
HSO, 1-27
HSO,
f
f
GalNAcPl+ 4GalPI
+
4GlcPI
+
lCer
1-28
Gg3Cer
HSO, 1 3 Gal/3l+ GalNAcPl
-
Gangliotriaosylceramide
I1 '.I11 '-disulfate
HSO,
+
-1 3 4GalPl
4Glc/31+ lCer 1-29
I1 'SO, ,111'SO,-
HS03 + 6GlcNAc/.31 + 3GalP1+ 4Glcb1 lCer
+
II~so,,Iv~so3Gg4Cer
III6S0,Lc,Cer
Gangliotetraosylceramide-
I1 ',IV '-disulfate LactotriaosylceramideW-sulfate
19 TABLE 1.3 (continued)
No.“
1-30
HSO, 1 6 GalPl + 4GlcNAcPl 4GlcPl
a
+
-
3GalP1
lCer
+
Abbreviation
Systematic name
I I I “SO,-
Neolactotetraosylceramide-
nLc,Cer
11 I “-sulfate
Numbers listed in the table are used for simple assignment of individual glycosphingolipids in this Chapter.
5.1.2. Galactocerebroside-esters
Glycosphingolipids which migrate much faster than cerebrosides during silica gel chromatography have been isolated from bovine brain and proved to be three specimens of galactosylceramides containing fatty acid esters [126]. The compounds were also isolated from human brain [116]. In a permethylation reaction, ester-linked material undergoes varying degrees of de-esterification owing to an alkaline condition, particularly if an incompletely dehydrated solvent is used, making it difficult to determine the position of the ester linkage. The fast-migrating GSL from human brain [128] and from pig brain [129] was found by methylation analysis as well as periodate oxidation [129] to contain an ester group linked at C-6 of the galactose moiety of the galactosylceramide. In addition to 6-acylgalactosylceramide, 3-acylgalactosylceramide from human [ 1281 and bovine [ 1301 brains, and 2-acylgalactosylceramide from whale brain [131], were characterized. Ester-linked fatty acids in these acylgalactosylceramides were C,, and C , , straight-chain acids but not a-hydroxy acids, in contrast to the pattern of amide-bound acids. The isolation of 6-acylgalactosylceramide along with lesser amounts of 3- and 4-acylgalactosylceramidesfrom hog stomach mucosa was described [132]. 5.1.3. Galabiaosylceramide (digalactosylceramide,structure I-2 in Table 1.3)
This lipid, along with globotriaosylceramide (1-6 in Table 1.3), was found by Sweeley and Klionsky [133] to be one of the lipids stored in the kidneys of a patient with Fabry’s disease, a genetic a-galactosidase deficiency [ 134,1351. The normal human kidney [ 136,1371 was demonstrated to contain both this lipid and lactosylceramide, and the Gall 4Gal structure was determined by a methylation method [137,138]. The anomeric configuration of the carbohydrate moiety was assigned by NMR spectrometry [73] of the disaccharide derived from this glycolipid, and by susceptibility of the lipid to a-galactosidase treatment [139]. By methylation analysis it has been shown that the ratios of digalactosylceramide to lactosylceramide in kidney dihexosylceramide fractions from different mouse strains differ considerably [ 1401. 5.1.4. II~(GalNAcal-3GalNAc)-galabiaosylceramide (structure I-3 in Table 3)
A tetraglycosylceramide(1-3 in Table 1.3) in which GalNAcd-3GalNAc is bound to galabiaosylceramide and which cross-reacts immunologically with anti-
20 IV3GalNAca-Gb,Cer antibody (Forssman antibody) was described by Gahmberg and Hakomori [141] in cultured hamster NIL cells. 5.2. GLUCOSYLCERAMlDE AND LACTOSYLCERAMIDE
Glucosylceramide and lactosylceramide are the common biogenic precursors of globo, ganglio and lacto series GSLs.
5.2.I. Glucosylceramide (glucocerebroside,structure I-4 in Table 1.3) Glucosylceramide was first demonstrated in the spleens of patients with Gaucher disease [142-1441 (galactosylceramide was earlier found in the central nervous system), and the structure (1-4 in Table 3) was established [145-1481. Later this GSL was isolated from normal human spleen [149,150] and many visceral organs. A trace amount of glucosylceramide was also isolated from bovine [lsl], mouse [152] and human [153] brains. Glucosylceramide has also been found in yeast [154], a variety of fungi [155-1571, plants (leaves [158,159], ginkgo nuts [160] and mushroom [161]), and invertebrates (sea star [162] and sea anemone [163]). A product resulting from the transfer of UDP-glucose to an endogenous acceptor present in a cellular slime mold (Dictyostelium discoideum) was shown to be glucosylceramide [164]. A synthetic analogue of glucosylceramide, L-glucosyl ceramide [ 1651, may be of value in metabolic study. 5.2.2. Glucocerebroside-ester 6-Acylglucosylceramidefrom Gaucher spleen [166] and 3-acylglucosylceramide from human and pig epidermis [167] were characterized. The latter compound of epidermis contained a-hydroxy acids with more than 30 carbon atoms [757]. The esterified fatty acids of the both acylglucosylceramides [166,167] were the straight-chain C,, and C,, acids. 5.2.3. Lactosylceramide (structure I-5 in Table 1.3) Klenk et al. first isolated this glycosphingolipid from bovine spleen [168] and from equine erythrocytes [169]. The compound, named cytolipin H [170], was later isolated from human cancer tissue as a lipid hapten. The structure was determined by means of methylation analysis [138,171] and a haptenic inhibition test using lactose [172]. Lactosylceramide was chemically synthesized by Shapiro and Rachaman [173]. 5.3. GLOB0 AND ISOGLOBO SERlES
In addition to GlcCer and LacCer, GSLs of the globo series are the major constituent glycolipids of most extraneural tissues of mammals. 5.3.1. Glohotriaosylceramide(structure I-6 in Table 1.3) This lipid was first isolated by Makita and Yamakawa [149] from bovine spleen. It was clearly distinguished from lactosylceramide on the basis of its chemical composi-
21 tion, dextrorotatory nature ( + 22.3" in pyridine) in contrast to the levorotatory power ( - 10 to - 12') of lactosylceramide, as well as its behavior in thin-layer chromatography. Patients with Fabry's disease were found to contain large quantities of globotriaosylceramide in their kidney [133] and other tissues [174,175] including nervous tissues [176].Normal human kidney [136,177],spleen [150],serum [178],and most other extraneural tissues and fluids have also been shown to contain substantial amounts of this lipid. The linkage position of the hexoses in globotriaosylceramide isolated from normal human kidney was determined by methylation analysis of the intact lipid and di- and mono-hexosylceramides derived by partial hydrolysis [137,138].An a-configuration between the two galactoses was proposed by Kawanami [72]based on NMR spectrometry of a triglycosylsphingosine which was derived by an alkaline hydrolysis procedure [93]from a mouse tumor triglycoTotal chemical sylceramide, and confirmed by several laboratories [39,70,73,74,179]. synthesis of globotriaosylceramide was achieved by Shapiro and Acher [180],and globotriaose was synthesized by Cox et al. [181].Globotriaosylceramide is the Pk antigen of the human P blood group system (see Section 10.1.4). 5.3.2. Globoisotriaosylceramide(structure I - 7 in Table 3) Stoffyn et al. [182]biosynthesized a triglycosylceramide from lactosylceramide and UDP-galactose using a particulate enzyme preparation from rat spleen or bone marrow (see Section 6.4). The digalactosyl linkage of the product had an al-3 bond, in contrast to the al-4 bond of globotriaosylceramide. Globoisotriaosylceramide isolated from rat spleen tissue [183]and rat tumor [184]was characterized. The triglycosylceramides of dog intestine were reported to consist largely of globoisotriaosylceramide with a minor amount of globotriaosylceramide [ 1851 (see also Section 5.3.5). 5.3.3. Isoglobotetraosylceramide (cytolipin R, structure I-9 in Table 3) Rapport et al. [186]isolated from rat lymphosarcoma a glycolipid, named cytolipin R, which had a carbohydrate composition similar to that of cytolipin K (globoside) but was less soluble in chloroform-methanol. Anti-rat lymphosarcoma serum reacted with the lipids from a variety of rat tumors but not with cytolipin K. Cytolipin R isolated from rat tumors [184,187]and normal rat tissues [188,189]had the same carbohydrate structure (1-9in Table 1.3), indicating that this glycolipid is species specific but not characteristic of rat tumors. 5.3.4. Globotetraosylceramide(globoside, globoside I, cytolipin K, structure 1-8 in Table 3) This lipid was isolated as a major glycosphingolipid of human erythrocyte membranes [190,191]and named globoside [191].Rapport et al. [192]called the compound cytolipin K and demonstrated its haptenic reactivity. The sequence and positions of the monosaccharides in this glycolipid from human erythrocytes [193,194] and kidney [ 1951 were established by partial acid hydrolysis and methylation procedures. The identical partial structure was found in globoside from pig lung
22 [196] and spleen [197], equine spleen and kidney [198], and dog kidney [199]. The anomeric j3-GalNAc linkage of globoside was determined by infrared spectroscopy of a terminal disaccharide obtained by partial acid hydrolysis of globoside [1951, and by susceptibility to a /3-N-acetylhexosaminidase [200,201]. The anomeric structures of the remaining glycosidic bonds were assigned by glycosidase experiments [ 39,2021 and NMR studies [74]. This established the whole carbohydrate structure of globoThis carbohydrate appears at present side: GalNAc~l-3Galal-4Gal~l-Glcfil-Cer. to exist not solely as a glycolipid, but to occur also as protein, since a human erythrocyte globoprotein [203] which had a structure that reacted with anti-globoside antibodies was described. Globoside was assigned as the P antigen in the human blood group P system (see Section 10.1.4.). 5.3.5. I V~-a-N-acetylgalactosaminyl-globotetraosylceramide(Forssman antigen, structure 1-10 in Table 1.3) The chemical structure of IV’GalNAca-Gb,Cer was determined using samples of this haptenic lipid from equine [48,204] and sheep [206] erythrocytes. However, the structure determined for the Forssman antigen [205] from dog intestine seems to be inconsistent with the observation [185] that the major triaosylceramide of the same tissue which could serve as a biogenic precursor of Forssman antigen was noted to be isoglobotriaosylceramide instead of globotriaosylceramide (see Section 5.3.2), and this will need further study. Total chemical synthesis of IV3GalNAca-GbOse, was recently achieved by Paulsen and Biinsch [207]. Some microorganisms, such as pneumococcus [208], possess Forssman-antigenic substances. A disaccharide GalNAc + GalNAc isolated from streptococcal polysaccharides cross-reacted with the antiserum against IV3GalNAca-Gb4Cer, and the glycosidic linkage in it was hence assumed to be a1 -,3 [209]. Recently, Ando et al. [210] isolated from human erythrocytes a pentaglycosylceramide which had the same sugar composition as IV3GalNAca-Gb4Cer(1-10 in Table 3) but did not show Forssman antigenicity. They proposed the structure, GalNAcPl -, 3 G a l N A c P l j 3Galal + 4Galfi1 + 4GlcBl + Cer (named para-Forssman glycolipid) for this GSL. 5.3.6. GIobo series - such as poly-a-galactosylated GSLs Angstrom et al. [211] recently found penta- to heptaglycosylceramides with globoside-like termini in the nonepithelial portion (mainly connective tissue and muscle) of rat small intestine. A permethylated GSL fraction containing penta- to nonaglycosylceramides was examined by direct inlet mass spectrometry by evaporation at increasing temperature, by proton nuclear magnetic resonance spectrometry and characterization of the partially methylated monosaccharides therefrom. On the basis of these results, they deduced the structures to be GalNAcPl (3Galal -+),-,4Galpl + 4Glcpl + 1Cer. The presence of octa- and nonaglycosylceramides containing one to two additional + 3Galal-3 units was also suggested. Breimer et a]. [65] proposed a tetrahexosylceramide (1-11 in Table 1.3) from nonepithelial tissue of rat small intestine which is composed only of hexoses. Homologues with one or
-
23 two additional internal + 3Gala + units were also described. In contrast to these poly-a-galactosylglycolipids in rat intestine, P-galactosylated GSLs (muco series) which can be classified into fucolipids and bear blood group A or H antigenicity were demonstrated in hog gastric mucosa by Slomiany et al. (see Section 5.7). One of their core structures is (GalPl + 3) f GalPl + 4GlcP1 + 1Cer. Kannagi et al. [212] isolated and characterized globopentaosylceramide, Gb, Cer or IV3GalP-Gb4Cer(I-12 in Table 1.3), its fucosylated derivative, and the sialylated derivative (see Section 8.1.3) from a human teratocarcinoma cell line. Schwarting et al. [758] demonstrated two GSLs of globo series, whose tentative structures are 1V"Fuca-Gb4Cer and IV3NeuAca-Gb4Cer, in a human teratocarcinoma cell line. 5.4. GANGLIO SERIES
GSLs of the ganglio series of which are devoid of sialic acid occur typically as GSL components together with the major accumulated gangliosides in the brains of patients with gangliosidoses (see Section 7.3 and Chapter 3). Although they are not distributed ubiquitously in tissues and cells, they have frequently been seen in particular cell types of extraneural origin. 5.4.1. Gungliotriuosylcerumide(structure I-I 3 in Table I .3)
This lipid was isolated from the brains of patients with Tay-Sachs disease, a genetic P-N-acetylhexosaminidase-Adeficiency (see Section 9), as an accumulated glycosphngolipid [213-2171 along with the principal ganglioside, I13NeuAca-Gg3Cer. Similarly, patients with Sandhoff-Jatzkewitz disease, a P-N-acetylhexosaminidase-A and -B deficiency (see Section 9), accumulate this lipid, along with globoside, in the brain [218] and in visceral organs [219]. Guinea pig erythrocytes contain this lipid as the predominant glycosphingolipid [75]. The carbohydrate structure of gangliotriaosylceramide was determined by a methylation procedure [75,214] and by NMR spectrometry [75] of the trisaccharide derived from this lipid. Tumors produced in mice by implantation of Kirsten virus-transformed 3T3 cells [220] and a rat ascites hepatoma cell [221] line contained substantial quantities of gangliotriaosylceramide (see also Section 11.2). Total chemical synthesis of t h s lipid was carried out by Shapiro et al. [222]. S.4.2. Gungliotetruosylcerumide (structure 1-14 in Table 1.3) An abnormal quantity of gangliotetraosylceramidewas demonstrated to be in the brain of patients with I13NeuAca-Gg4Cer(GM1) gangliosidosis [215], an inherited P-galactosidase deficiency (see Section 9), as well as in Tay-Sachs brain [223] for whch the accumulation cannot be explained by P-hexosaminidase deficiency. This lipid was also detected in normal human brain [153] in trace amounts. As for extraneural tissues and cells, gangliotetraosylceramide was isolated from rat hepatoma cells (AH794F) of the free-cell type [221]. The intestinal mucosa of germ-free mice contained substantial amounts of this lipid [224]. A glycosphingolipid hapten called cytolipin S from rat spleen was described [225]. This lipid had a
24 carbohydrate composition, chromatographic mobility and complement-fixation reaction similar to gangliotetraosylceramide, but differed in its hemagglutination inhibition reaction. 5.5. LACTO A N D NEOLACTO SERIES GLYCOSPHINGOLIPIDS
The glycolipids of the neolacto series have been isolated and characterized mainly from erythrocytes. Erythrocytes of the humans and animals examined so far contain neolacto (but not lacto) series GSLs and their fucosylated derivatives (see Section 5.7). The type 1 saccharide chain (-Galpl-3GlcNAc-) of the lacto series and type 2 chain (-Galpl4GlcNAc-) of the neolacto series can be differentiated by the mass spectra of the permethylated intact GSLs; the mass fragments characteristic of type 1 are a very intensive m/e 228 peak [226] and peaks at m/e 862 to 974 [299], while the type 2 chain has a small but significant peak at m/e 182 [227]. Human neutrophils were demonstrated to contain lactotriaosylceramide and neolactotetrae sylceramide in comparable quantities to those of mono- and diglycosylceramides and globotriaosylceramide [229]. The concentration of neolacto series glycolipids in tissues and cells is in general lower, and they overlap frequently with other major glycolipid classes such as the globo series in chromatography. It is therefore difficult to isolate the lacto series GSLs. In some cases, acetylation of the closely migrating glycolipid mixture followed by separation by thin layer chromatography affords satisfactory results. 5.5.1. Lactotriaosylceramide (amino CTH, structure I-! 5 in Table 1.3)
Lactotriaosylceramide is a common biosynthetic precursor of lacto- and neolactoseries GSLs and proceeds to a number of fucolipids through the action of specific glycosyltransferases. Ando et al. [71,230] isolated this lipid (designated amino CTH) from a triglycosylceramide fraction of human erythrocytes using preparative highspeed liquid chromatography. The purified lipid had a slightly greater mobility than globotriaosylceramide during thin layer chromatography in an alkaline solvent mixture, and its concentration (47 pg per 100 ml of packed type-B erythrocytes) was much lower than that (9.4 mg) of globoside, a major GSL of human erythrocytes. The chemical structure (1-15 in Table 1.3) was determined by means of proton NMR of the intact lipid and other degradative procedures [71]. The erythrocytes of patients with dyserythropoietic anemia (type 11) accumulate t h s lactotriaosylceramide along with a compound presumed to be paragloboside [231].
5.5.2. Neolactotetraosylceramide (paragloboside, structure I-1 6 in Table 1.3) Neolactotetraosylceramide, or paragloboside, is a precursor glycolipid of the blood group ABH-antigenic GSLs of human erythrocytes. An N-glycolylneuraminyl derivative of this glycolipid was earlier isolated by Kuhn and Wiegandt [232] from bovine erythrocytes and spleen, and the structure of the carbohydrate moiety liberated from the lipid by an ozonolysis-alkaline cleavage procedure [90] was determined [233]. The N-acetylneuraminyl derivative of neolactotetraosylceramide
25
was shown to be the major ganglioside of human erythrocytes [228]. The structure of neolactotetraosylceramide from human erythrocytes was established by means of methylation analysis [234,235], sequential hydrolysis with specific glycosidases [235], proton nuclear magnetic resonance spectrometry of a tetrasaccharide which was released from this lipid by an ozonolysis procedure [234], and by its reactivity [235] with antiserum against type XIV pneumococcal polysaccharide *. This glycolipid was found to have the same structure in human neutrophils [228,229], by similar procedures. Some human cancer tissues, in this there was marked infiltration of neutrophls, contained a relatively large amount of neolactotetraosylceramide as well as some lactotriaosylceramide, whereas the cancer tissues without neutrophil infiltration did not [237]. 5.5.3. Lactotetraosylceramide (structure I-17 in Table 1.3) GSLs having a type 1 disaccharide unit (i.e., -Galb1 + 3GlcNAcP1-) which can be precursors of the blood group Le system were isolated by Karlsson and Larson [238] from human meconium (1.9 mg of purified GSL from 12.5 g of dry meconium). The glycolipid moved just below paragloboside in thin layer chromatography, but the acetylated derivative of this GSL as well as acetylated globoside moved much faster than acetylated paragloboside. The chemical structure of lactotetraosylceramide was determined by methylation analysis and proton NMR spectrometry of the permethylated GSL [238]. 5.5.4. I V3-a-Galactosyl-neolactotetraosylceramide (rabbit B-active glycolipid, structure 1-18 in Table 1.3) Eto et al. [239] isolated and characterized a pentaglycosylceramide, a major glycolipid from rabbit erythrocyte membranes (73 mg of the purified lipid from 100 g of the acetone powder). The chemical structure of this GSL was established as IV3aGal-nLc,Cer by methylation analysis and sequential hydrolysis with specific glycosidases [48]. Bovine erythrocyte membranes contain this GSL as one of their major glycolipids [240]. 5.5.5. I V3-~-Galactosyl-neolactotetraosylceramide (structure I-19 in Table 1.3)
Stellner and Hakomori [241] isolated from human erythrocytes a pentaglycosylceramide for which the structure IV 3PGal-nLc,Cer was proposed. This GSL could not be separated from blood group H-active fucopentaosylceramide (H,, see Table 1.4) on thin layer chromatography, but the acetylated lipid migrated slower than acetylated H, lipid. Regarding the lipophilic moiety, the long-chain base was almost exclusively sphinganine, and normal and unsaturated C16- and C,,-acids composed two-thirds the total acids, although the inclusion of phospholipids in this glycolipid preparation was not noted.
* Type XIV pneumococcal polysaccharide was shown to be composed of a branched repeating structure of
-, 6GlcNAc(4 + 1PGal)PI + 3GalP1
-t
4Glc- (Ref. 236).
26 5.5.6. I V4-a-Galactosyl-neolactotetraosylceramide (P,gbcolipid, structure 1-20 in Table
1.3) P, antigen is one of three antigens involved in the human blood group P system (see Section 10.2). Hydatid cyst fluid in sheep liver contained a PI-active glycoprotein [242-2441. The P,-active trisaccharide from the glycoprotein, Galal-4GalBl4GlcNAc [245], was characterized as the immunodominant carbohydrate. On the other hand, the P, antigen on human erythrocytes was suggested to be a GSL [246]. Naiki and Marcus [247] isolated P,-antigenic pentaglycosylceramide from human erythrocyte membranes. The yield of PI glycolipid from 350 g of the acetone powder of the membranes was approximately 150-250 pg. The carbohydrate structure was determined by methylation analysis of the glycolipid before and after a-galactosidase treatment and by total mass spectrometry of the permethylated lipid [247]. 5.5.7. I V3-~-N-Acetylgalactosaminyl-neolactotetraosylceramide (structure I-21 in Table
1.3) Kannagi et al. [248] isolated a blood group P-antigenic glycosphingolipid from human erythrocytes by high performance liquid chromatography on a silicic acid column. This GSL was found by methylation analysis and glycosidase treatment to be a pentaglycosylceramide (1-21 in Table 1.3) with a neolactotetraosyl backbone and a GalNAcbl-3 linkage at its nonreducing terminus. 5.5.8. Neolactoheptoglycosylceramide (structure 1-22 in Table 1.3) and related longerchain glycosphingolipidF Chien et al. [249] isolated a heptaglycosylceramide (1.1 mg) along with a hexaglycosylceramide (0.4 mg) from a 0.1 M KC1-tetrahydrofuran extract of bovine erythrocyte acetone powder (100 g). The carbohydrate moiety of the heptaglycosylceramide 3GalP1 + 4GlcNAcB + [249]. (1-22 in Table 1.3) has two repeating units of In addition to neolactotetraosylceramide, three minor glycolipids containing longer sugar chains were fractionated from sheep erythrocytes in which IV 3GalNAccu-Gb,Cer (Forssman glycolipid) was a predonlinant lipid using h g h performance liquid chromatography of acetylated glycolipids [250]. Their molar compositions of Gal/Glc/GlcNAc/GalNAc were (4 : 1 : 3 : l), (5 : 1 :4 : 0.5) and (5 : 1 : 5 : 0.6), and they were suggested to possess repeating units of + 3Gal+ 4GlcNAc + based on methylation analysis.
-
5.5.9. Neolactodecaglycosylceramide (structure I-23 in Table 1.3) A branched decaglycosylceramide was found to be one of the major GSLs of rabbit erythrocytes by Hanfland et al. [251]. This GSL has both human blood group B and I antigenicities (see Section 10). The predominant constituents of the ceramide moiety are tetracosenoic acid (nervonic acid) and sphingosine. 5.6. SULFATED GL YCOSPHINGOLIPIDS (SULFATIDES)
A sulfur-containing lipid was found in human brain by Thudichum [118] and was identified as sulfated galactosylceramide (named cerebroside sulfuric ester) by Blix
27 [252]. The presence of the sulfated GSL as a major GSL component is primarily limited to the brain, kidney and small intestine. Sulfated GSLs other than cerebroside sulfate have recently been identified as minor GSL components. 5.6.I . Galactosylceramide sulfate (cerebroside sulfate, structure 1-24 in Table 1.3) Sulfate was established to be esterified at the 3-hydroxy group of galactose in cerebroside sulfate on the basis of methylation studies [138,171,253] and resistance of the galactose moiety to periodate oxidation, and the P-configuration of the galactosidic linkage was determined by, infrared spectrometry [138,171]. The occurrence of this compound as a major glycosphingolipid was demonstrated in human brain [118] and kidney [136,254], and pig 12551 and dog [256] small intestine, although the lipid is distributed as il minot glycolipid in many tissues [257]. Jatzkewitz [258] and Austin [259] found considerable accumulation of cerebroside sulfate in brain and kidney [259] from patients with metachromatic leukodystrophy which is caused by a genetic defect in a cerebroside sulfatase, namely arylsulfatase A [260,261]. A very small amount of galactosylceramide sulfate (3.3 mg from 6.7 kg wet cells) was isolated from human erythrocyte membranes [262]. The sulfate ester at galactose-3 in cerebroside sulfate is stable under alkaline conditions (for example, 0.2 M NaOH at 37OC for 1 h), while under mild acid conditions (0.1 M HC1 at 100°C for 1 h) approximately one-third of the sulfate was released [138], concurrent with some glycosidic cleavage [138]. Treatment with methanolic hydrogen chloride [263] did not result in complete desulfation of the sulfatides [264]. Complete desulfation bf galactose-3-sulfate at the non-reducing termini (13S0,-GalCer and I13S0,-LacCer) and in the inner sugar chain (I13S0,Gg3Cer) (Table 1.3) was attained by solvolysis in anhydrous dioxane at 100°C for 1-3 h [264]. 5.6.2. Lactosylceramide sulfate (structure I-25 in Table 1.3) This compound was first isolated from human kidney by M%rtensson[254]. It was also found in porcine gastric mucosa [265] and rat sublingual and submaxillary glands [266]. The chemical structure was determined by methylation studies [265,267], partial hydrolysis and periodate oxidation [267]. 5.6.3. Sulfated tri- and tetraglycosylceramides A sulfated trihexosylceramide isolated from hog gastric mucosa was reported to have the partial structure HSO, + 3Gall- 4Gall- 4Glc + Cer [265]. Slomiany et al. described two sulfated glycosphingolipids with the basic structure of the lacto-series. These lipids were extracted, with 0.4 M sodium acetate in chloroform/methanol/water, from hog gastric mucosa residue that had been extracted with chloroform/methanol (2 : l), and separated by several procedures. Based on the results of partial acid hydrolysis, sequential exoglycosidase hydrolysis and methylation methods, the chemical structures were proposed to be lactotriaosylceramide-HI6-sulfate (1-29 in Table 1.3) [268] and neolactotetraosylceramide1116-sulfate(1-30 in Table 1.3) (2691. Recently, Tadano and Ishizuka isolated three
.
,
28 sulfoglycosphingolipidsof the ganglio-series from rat kidneys. The first one, for the isolation of which an in vivo [ 35S]sulfatelabeling technique was adopted [270], was presented at about 23 nmol/g of tissue and composed 13%of the galactosylceramide sulfate. Solvolysis of this lipid with dioxane resulted in complete desulfation, and a glycolipid corresponding to gangliotriaosylceramide appeared on a thin layer plate. (1-26 in Table 1.3) For this lipid, the structure gangliotriaosylceramide-113-sulfate was proposed based on the results of several methods of structural characterization [264]. The second sulfated GSL (11.2 nmol/g tissue) of rat kidney was found to be a bis-sulfoglycosphingolipid [264] in which one mole of sulfate is esterified at Nacetylgalactosamine, and the structure was determined to be gangliotriaosylceramide-113,1113-disulfate(1-27 in Table 1.2) [271]. The third sulfated GSL (5.5 nmol/g tissue) isolated from rat kidney migrated slower than gangliotriaosylceramideI13,1113-disulfate.The chemical structure [272] was identified as gangliotetraosylceramide-I13,1V3-disulfate (11-28 in Table 1.3). These sulfatides (11-27 and 11-28 in Table 1.3) represent the first demonstration of bis-sulfated GSLs. Treatment of these bis-sulfated GSLs with dimethylsulfoxide/methanol(9 : 1) containing 8 mM H,SO, at 8OoC for 30 min [88]resulted in desulfation only at the non-reducing termini, leaving sulfate ester in the inner galactose [271,272]. 5.7. FUCOLIPIDS
Complex glycosphingolipids containing L-fucose (fucolipids) have attracted interest because they possess antigenic activity of the human ABH and Lewis blood group systems (see Section 8). Since demonstration of the existence of fucose as well as glucosamine in glycolipid fractions from human erythrocytes with blood group ABH antigenicities [273,274], human adenocarcinoma [275], the liver of a patient with fucosidosis [276], and from dog [277] and pig [255] small intestines, much effort has been devoted to the isolation and characterization of fucolipids, particularly those responsible for blood group specificities. The first pure fucolipid was isolated from human cancer tissue by Hakomori and Jeanloz [275], and as of now a number of fucolipids (Table 1.4) have been chemically defined by the laboratories of Hakomori, McKibbin, Karlsson and others (for reviews, see Ref. 280). Although many of the fucolipids have core saccharides with lacto and neolacto structures (refer to Section 5.5), fucolipids having other core structures have recently been elucidated. Fucolipids serve not only as cell surface antigens of human blood groups (Section 8), but also may play a role as tumor markers, as some tumors produce ectopically peculiar fucolipids which are not present, or present only in small quantities, in the normal tissue counterparts (Section 9.2). In general, epithelial tissues of glandular organs, such as intestine, stomach and pancreas, are rich sources of the fucolipids, whereas parenchymatous organs and red and white blood cells contain very small amounts of these lipids. As shown in Table 1.4, all the fucolipids characterized so far, except for fucosylceramide,possess the human blood group specificities of ABH, Lewis and/or Ii systems. Their antigenic properties are described in Section 8. In gastrointestinal
29 epithelia, fucose attachment in two tetraglycosylceramides with an isoglobo core differs between hog stomach (11-4 in Table 1.4) and rat intestine (11-5 in Table 1.4), and there is a corresponding difference in blood group antigenicity. A fucolipid (11-9 in Table 1.4) isolated from a rat benign tumor, a granuloma which consisted of large amounts of macrophages, has been reported to contain a tetrasaccharide sequence similar to that of gangliotetraose but differing from the latter in having a 3-linked galactose at the lactose residue [289]. Interestingly, although a fucosyl gangliotetraosylceramide (11-8 in Table 1.4) was found in rat hepatoma cells [286] and in the intestinal mucosa of conventional mice [287], both of which were enriched in a fucosyltransferase activity responsible for the synthesis of this lipid [224,280], the intestinal mucosa of germ-free mice did not contain this lipid, and instead contained gangliotetraosylceramide [287]. Therefore, it is probable that the fucosyltransferase can be induced in microvillus cells by the intestinal microflora or, though it seems less probable, the microflora may produce the enzyme. In rats, the fucosyl gangliotetraosylceramide appears to occur only in the freed ascites cell line and macrophages, but not in the island-forming subline (whch grows in a cell-to-cell adhesive state [684]), when examined by staining with fluorescein-conjugated antibodies directed to this GSL [686]. An a-fucosyltransferase activity which forms blood group H-active IV2Fuca-Gg,Cer from Gg,Cer was demonstrated in a rat ascites cell line of freed cell type but not in the island-forming cell line [288]. The presence of a difucosylated heptaglycosylceramide(s)in pig stomach [308] (11-29 in Table 1.4) and dog small intestine [312] has been reported. The compounds have similar carbohydrate compositions. Although all of the fucolipids from human erythrocytes so far known possess exclusively neolacto structures, including a type 2 chain (N-acetyllactosamine, GalPl 4GlcNAc), the fucolipids from glandular tissues of various other species possess both neolacto core and lacto core, including a type 1 chain (GalP1 3GlcNac) (Table 1.4). The complex fucolipids with longer and branched chains contain two or more repeating N-acetyllactosamine units in which the galactose is bound through Pl 3 linkage or pl 6 linkage (at a branching point). In a branched fucolipid (11-20 in Table 1.4) of rat intestine, the 3-linked branch was homogenous with the type 1 chain, while the 6-linked branch had both type 1 and type 2 chains [299]. The presence of fucolipids, 11-16 to -19 (Table 1.4), in human meconium was deduced and their structures determined from the analytical results of mass spectrometry, proton NMR spectrometry, gas chromatography of the permethylated compounds, and a hemagglutination test of the intact lipids, without isolation of the individual fucolipids [298]. Fucolipids (11-32 and -33 in Table 1.4) which possess one or more structural units of GalPl -,4GlcNAc(3 + 1aFuc) were isolated from human 0-erythrocytes [310]. Because the antigenicity (Lex antigen) of these fucolipids is expressed on blastomeres at a certain stage, as well as on some embryonic carcinoma cells, they are called stage-specific embryonal antigens (SSEA1) (Section 8.3). Slomiany and his collaborators reported the presence in hog gastric mucosa of a variety of fucolipids, including some that could be classified among the globo-series: -+
-+
-+
-+
w
TABLE 1.4 Fucolipids classified according to core carbohydrate structures No.
Structure
-
0
Systematic abbreviation (symbol used by author)
Series other than globo, ganglio dlacto series 11-1 Fucal lCer
2GalBl- 4GlcBl-lCer
11-2
Fucal
11-3
GalNAcal
-
2Galbl- 4GIcBl- lCer 2 f laFuc
Source
Ref.
Human colon cancer
281
Blood group H
Rat small intestine
282
Blood group A
Rat small intestine
283
Antigenicity
FucaCer
I1 Fuca-LacCer II’GalNAca,II (A-4)
Fuca-LacCer
Glob0 and isoglobo series
-
11-4
Fucal + 3Galal --t 3GalB1- 4GlcBl* lCer
1112Fuca-iGb,Cer
H
Hog stomach
284
11-5
Galal --t 3GalB1- 4GlcB1 2
112 Fuca-iGb,Cer
B
Rat large intestine
285
65
-
lCer
f
laFuc
11-6
Fucal 2Galal- 3Galal- 4GalB1+ 4GlcBl- lCer
I I I (Fuc a1-2Gal)a-Gb,Cer
H
Rat small intestine
11-7
Fucal 2GalBl- 3GalNAcBl- 3Galal4GalB1- 4GlcBl- lCer
IV 3( Fuc a1-ZGal)P-Gb,Cer
SSEA-3
Human teratocarcinoma cells
212
IV Fuca-Gg,Cer
H
Rat ascites hepatoma cells
286
H
Mouse small intestine
224
B
Rat granuloma
289
-
Canglio and isoganglio series 11-8 Fucal 2GalBl- 3GalNAcBl4GalP1- 4Glcfl1- lCer
11-9
Galal
+ 3GalB1
2
t
3GalB1 -P
-
3GalNAcB1
laFuc 4Glcj31+ lCer
-
IV ,Gal a.IV2 Fuc a-iGg,Cer (glycolipid RM)
Lacto series 11-10 Fucal + 2Gal/31+ 3GlcNAcj313Galfi1- 4Glc/31- lCer
IV2Fucu-Lc,Cer
H
Human pancreas 290 Small intestine 748
11-11
II14Fuca-Lc4Cer
Lea
Human small intestine
292
Human plasma
291
GalPl- 3GlcNAc/31+ 3Gal/31- 4Glc/314 4
t
IaFuc lCer 11-12
Gala1 + 3Gal/31 2
-
3GlcNAcpl
-
Lewis d H 3Gal/31-
IV3Galn,IV2Fuca-Lc,Cer
B
Pancreas of patient with Fahry’s disease
293
1V3GalNAcn,IV2Fuca-Lc4Cer
A
Human small intestine
283. 294
A
Pig stomach
295
Le”
Human plasma
291, 296
Le (B)
Human small intestine
297
t
laFuc 4Glc/31- Cer 11-13 GalNAcal + 3Gal@1+ 3GlcNAc/31+ 2
t
laFuc 3GalP1- 4Glc/31+ lCer 11-14
Fucal + 2GalS1- 3GlcNAc/31+ 3Gal/31+ 4
’
II14Fuca,IV2Fuca-Lc4Cer
f
laFuc 4Glc/31+ lCer 11-15
Gaia1
-
3 or 2Gal/3l+ 3GlcNAc/31+ 2(or3)
”
I
1aFuc 1aFuc 3Gal/31+ 4GlcB1+ lCer
-
11-16
Fucal 2Gal/31+ 4GlcNAc/31+ ?Gal/31 -t 3GlcNAcfiI- 3Gal/31+ 4Glc/31+ lCer
H
Human meconium
298
11-17
G a l b l - 4GlcNAc/31+ 3Gal/31+ 3GlcNAcfil3
Lea
Human
298
t
laFuc 3Galfi1- 4Glcfil- lCer
meconium
TABLE 1.4 (continued) No.
Structure
11-18
Fucal
-
W h,
Systematic abbreviation (symbol used by author) 2Galp1- 4GlcNAcpl- 3Galp13
f laFuc 3GlcNAcBl- 3Galp1- 4Glcp1- lCer 11-19
-
2Galfll- 4GlcNAcpl- 3GalB1 3 f laFuc 3GlcNAcp1* 3GalB1- 4GlcB1- lCer 4 Fucal
-
Antigenicity
Source
Le
Human meconium 298
Leb. H
Human meconium 298
H
Rat small intestine
299
A
Rat small intestine
373
A
Rat small intestine
313
Ref.
T
laFuc 11-20
-
2Galpl- 3 and 4GlcNAcp1\ 6 Galpl F u c a l d G a l ~ l - 3 G l c N A c 3~ l ~ 3GlcNAcBl- 3Galp1- 4Glcb1- lCer
Fucal
11-21
-
laFuc J
-
L
GalNAcal 3Galp1- 3GlcNAcpl I 6 Galpl GalNAc a1-3G~1~1-3GlcNAc/31 f 3
-
L
t
laFuc 3GlcNAc/31+ 3GalB1- 4Glcp1+ lCer 11-22
laFuc J 2 GalNAcal + 3Galp1- 4GlcNAcplL 6 GalNAcal
-
3Galb1- 3GlcNAcpl/* 3 2
3GlcNAcaI
-
Gala1 +
laFuc 3Galfi1+ 4Glc/31- lCer
Neolacto series 11-23 Fucal + 2GalBl- 4GlcNAc/31+ 3GalBl- 4GlcB1+ lCer
I V 2 Fuca-nLc,Cer ( H I , Ref. 301; H-I, Ref. 300)
H H
111 Fuca-nLc,Cer
11-24 GalBl- 4GlcNAcB1 + 3GalBl3
(X hapten. Ref. 37)
t
laFuc 4GlcB1 --t lCer
11-25
Galal
11-26
GalNAcal
11-27
3GalB1- 4GlcNAcj31- 3GalPl+ 2 t laFuc 4GlcB1- lCer +
3GalB1- 4GlcNAcBl+ 2 t laFuc 3GalBl- 4GlcBl- lCer
GalBl- 4GlcNAcB 2 3
t
-
laFuc 11-28
-
-
3GaIB1- 4GlcB1+1Cer
Human erythrocytes Dog small intestine
300,301
Human adenocarcinoma Dog stomach
37
302
295
(Lea isomer, Ref. 304)
Lea
Dog small intestine
304
IV3Gala.lV2Fuca-nLc,Cer (B-I. Ref. 300)
B
Human erythrocytes
300, 305
IV 3GalNAca,IV2Fuca-nLc,Cer (A")
A
Dog small intestine Humanlung
294 50
II13Fuca,IV2Fuca-nLc4Cer
Le
Dog small intestine
304
(H2. Ref. 307; H-11, Ref. 300)
H and I
Human erythrocytes
300,307
A
Pig stomach
308
B
Human erythrocytes
305
t
laFuc
Fucal 2GalS1- 4GlcNAcfil- 3GalP14GlcNAcSl- 3GalS1- 4GlcB1- lCer
11-29 GalNAcal + 3GalB1- 4GlcNAcBl+ 2
t
-
3
t
laFuc laFuc 3Galp1- 4GlcB1 lCer 11-30 Galal
- 3Gall 2
4GlcNAcl
-
3Gall-
t
laFuc 4GlcNAcl- 3Gall+ 4Glcl + lCer
'
(B-11, Ref. 305)
w w
W
P
TABLE 1.4 (continued) No.
Structure
Systematic abbreviation (symbol used by author)
Antigenicity
11-31 GalBl+ 4GlcNAc/31+ 3Gal/31+ 4GlcNAc/31 + 3 3 t
1aFuc 3Gal/?1+ 4Glc/31+ lCer
Source
Ref.
Human hepatoma 309
t
iaFuc
11-32 Gal/3l+ 4GlcNAc/31+ 3Gal/31+ 4GlcNAc/31+ 3 f laFuc 3Galgl- 4Glc/?1+ lCer
SSEA-1
Human erythrocytes
310
11-33 GalBl+ 4GlcNAcBI+ 3Gal/31+ 4GlcNAcB1+ 3 3 T t IaFuc IaFuc 3Gal/31+ 4GlcNAc/31+ 3Gal/31+ 4Glcp1- lCer
SSEA-I
Human erythrocytes
310
11-34 Fucal --t 2Gal/3l+ 4GlcNAc/31 L 3 Fucnl + 2Gal/3l+ 4GlcNAc/31~6
H and I
Human erythrocytes
307
-+
4GIcNArRl + W a l R 1 + 4GlcR1 + lCer
11-35
laFuc
I
2 GalNAcnl -+ 3Gal/31+ 4GlcNAcP1\
3
GalNAcnl
6
-+
3GalPl- 4GlcNAcBl? 2
A
Human erythrocytes
31 1
A
Human erythrocytes
31 1
GalPl-+
t
laFuc 4GlcNAcP1 + 3GalPI -t 4GlcPI + lCer 11-36
IaFuc
1
2 GalNAcnl + 3GalP1+ 4GlcNAcPl- 3GalP1-+ 4GlcNAcPl \ GalNAcnl + 3GalPl- 4GlcNAcPI? 2 ?
IaFuc ;GalPl-+ 4GlcNAcfll-+ 3GalPl+ 4GlcP1+ lCer
' For Lewis d H (Led") antigenicity, see Section 8.1.2. The preparation examined contained two compounds with type 1 and type 2 structures in a ratio of 4 : 1, based on the analysis of partially methylated glucosamine derivatives. The pig preparation contained both type 1 and type 2 chains, and no anomeric configurations of the inner saccharides were given. After selective removal of fucose bound to glucosamine, blood group B antigenicity occurred concomitant with loss of Leb antigenicity. The anomeric configurations of the saccharides were not given.
'
W
u l
36 GalPl -, 3GalP1 -,4GalP1 -,4Glc. GSLs of the globo-series have not yet been detected with internal -GalP1 + 4Gal-, however, in sources other than hog stomach. They include blood group A-active fucolipids such as: GalNAcal -,3(Fucal -, 2)GalPl -, 3GalP1 + 4(GalNAccul 3GalP1 + 6)GalPl + 4GlcP1 + lCer (3131, and GalNAcal + 3(Fucal -, 2)GalPl .+ 3GlcNAcPl -, 3GalP1 -, 4GalPl + 4GlcP1 -, lCer [314]. --.)
5.8. POLYGL YCOSYLCERAMIDES (MACROGLYCOLIPIDS, MEGALOGL YCOLIPIDS)
The presence of highly complex glycosphingolipids (megaloglycolipids) having large numbers of glycose residues was demonstrated in a phosphate buffer extract of lipid-extracted human erythrocyte membranes by Gardas and Koscielak [ 131. Polyglycosylceramides from human erythrocyte ghost bear blood group ABH and Ii activities but do not have Lewis and MN activities [13,315]. The number of glycoses has been reported to be more than 20 in polyglycosylceramide preparations of erythrocytes. Polyglycosylceramides can be delineated from the low-molecular weight GSLs, so far known, and glycoproteins based on the following points: they are a group of GSLs with unusually long sugar chains; they do not contain appreciable amounts of amino acids and mannose, and they migrate only moderately after acetylation in silica gel chromatography using a chloroform-methanolwater system in which the acetates of low-molecular weight glycosphingolipids run to the solvent front. However, the delineation with respect to the polymerization of glycoses in polyglycosylceramidesfrom glycosphingolipidsof “ low-molecular weight” is not necessarily clear at the present time, since fucosyl GSLs with 12 [311,373]and 14 glycoses [311] were found in human erythrocytes and rat intestine (Table 1.4). Separation procedures for polyglycosylceramideswere elaborated by Koscielak et al. [316]as follows: human erythrocyte membranes after extraction with 83% ethanol were extracted twice with a mixture of equal volumes of phosphate buffer and n-butanol. The pooled aqueous phase was concentrated, dialyzed and chromatographed successively on CM-cellulose and DEAE-Sephadex columns. The eluate from DEAE-chromatography was dialyzed, freeze-dried, acetylated and chromatographed on a silicic acid column. After washing the column with chloroform, the acetylated polyglycosylceramides were eluted with a mixture of chloroformmethanol-water. The acetylated materials were separated into subfractions by preparative thin layer chromatography. Deacetylated polyglycosylceramides were soluble in water but insoluble in organic solvents, and did not migrate during thin layer chromatography in a solvent system in which mono- to decaglycosylceramides migrated. The polyglycosylceramidesdid not contain amino acids (less than 0.4% by weight), and their carbohydrate moiety was demonstrated to consist of 22-59 glycosyl residues depending on the polyglycosylceramide subfraction [ 3161. A simple method for preparation of polyglycosylceramides described by Dejter-Juszynski et al. [317] started with direct extraction of human erythrocyte ghosts with chloroform-methanol-water.From the aqueous phase of the chloroform-methanolwater extract, n-butanol-soluble materials (containing residual low-molecular weight
37 glycosphingolipids) were removed by partition, and the n-butanol-insoluble, aqueous phase was chromatographed on a DEAE-cellulose column in the presence of a detergent to separate the glycoproteins. The flow-through fraction from the DEAEcolumn contained polyglycosylceramides (termed macroglycolipids), and the yield was about 5 mg per 200 ml of erythrocytes. This preparation of polyglycosylceramides [317], which was essentially free of proteins (less than 0.5% amino acid), gave a broad band in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and a streak on a thin layer plate after acetylation, suggesting the presence of a variety of homologues. In these preparative procedures, introduction of an acetylation-silica gel chromatography step reduced the content of amino acids from 2-78 to less then 1%,while the molar ratio of carbohydrates to sphingoid base remained unchanged before and after the acetylation step [315]. The complete isolation of individual polyglycosylceramide classes, and determinations of the exact number of glycose residues and of the chemical structure, appear to be difficult at present. The constituent monosaccharide species of human erythrocyte polyglycosylceramides are glucose (one mole), galactose, glucosamine and fucose. The polyglycosylceramides from type-A erythrocytes also contained galactosamine [13,317]. In some polyglycosylceramide preparations, a small percentage of sialic acid was detected [13,317]. The number of glycose residues has been represented by the ratio relative to sphingoid content (1-3.5% by weight) [13,315] or glucose (2-4%) [317]. The molar composition [318] of the constituents in two polyglycosylceramide fractions (I and 11) obtained from human type-0 erythrocytes was estimated as follows. In fraction I, fucose : galactose : glucose : glucosamine : sphingoid 2.7 : 8.7 : 1.0 : 9.9 : 0.6; and in fraction 11, 3.4 : 12.6 : 1.0 : 13.4 :0.7, respectively. The major fatty acids detected in human polyglycosylceramide preparations were C22- and C,,-acids, which comprise more than two-thirds of the total acids [316] similar to those in low-molecular weight glycosphingolipids. Methylation studies of the polyglycosylceramides, irrespective of blood group, revealed the presence of unsubstituted galactose, C-3-substituted galactose and C-3, 6-disubstituted galactose, C-4-substituted glucosamine, C-4-substituted glucose, and unsubstituted fucose [52,319]. On the basis of the results of Smith degradation, methylation analysis and partial acid hydrolysis after hydrazinolysis of human erythrocyte polyglycosylceramides, Zdebska and Koscielak [52] deduced two carbohydrate sequences: GalPl -+ 4GlcNAcPl + 6GalP3 + R and GalPl -,4GlcNAcPl + 4GlcNAc, which represent the first N-di-acetyl chitobiose unit in a GSL. The structure N-acetyllactosamine or GlcNAcPl + 3Gal has been suggested for the repeating unit of polyglycosylceramides, and branching has been proposed to occur at C-3 and C-6 of galactose in the inner core [ 5236,3201. The polyglycosylceramide fraction from adult human erythrocytes contained an average of about five branching points per 30 glycosyl residues, whle a hexaglycosylceramide obtained from cord blood by the same procedure had only 0.7 of a branching point [321]. These results suggest that polyglycosylceramide is not synthesized at the embryonic stage (see Section 8.3). Polyglycosylceramides [322] were also fractionated from a rabbit erythrocyte glycophorin fraction which was extracted with lithium diiodosalicylate [323]. The
38
chloroform/methanol-insoluble materials were gel-filtrated after acetylation, and the materials gave a single band on a thin layer plate. Assuming one ceramide residue per molecule, the average number of glycoses in the rabbit polyglycosylceramides was estimated to be 30. The partial structure of the rabbit polyglycosylceramides examined by several methods revealed a highly branched structure consisting of Gala1 + GalPl + 4GlcNAc at the non-reducing termini, and -3GalPl + 4GlcNAc repeating units in the inner core of the carbohydrate chain. Some of the inner galactose residues branched at the C-6 position [322]. Slomiany et al. [324] reported the isolation of six GSLs with 8-36 glycose residues, which displayed both blood group A and H activities, from an extract obtained from delipidized hog gastric mucosa using 0.4 M acetate buffer in chloroform-methanol-water, and proposed a highly branched structure for an eicosaglycosylceramide which contains an N-diacetylchitobiose unit. 5.9. GLYCOSPHINGOLIPIDS I N NON-VERTEBRATES
GSLs have been identified in invertebrates, plants and microorganisms. Pioneering work on plant GSLs was done by Carter and his collaborators [127,325]. Hori, Hayashi and their associates have found glycosphingolipids with unique structures in shellfish. GSLs obtained from these origins are markedly different from those of mammals with respect to their constituent carbohydrates * and lipophilic moiety. The constituent monosaccharides peculiar to GSLs from plants and water mollusc include mannose, glucuronic acid and 0-methyl-glycoses. Many linkage types found in the sugar chains of the non-vertebrate GSLs have not been detected in those of mammalian GSLs (see Table 1.5). The sphingoids in some glycolipids from freshwater shellfish [329] and plants [328] are composed of a variety of branched-chain bases including positional isomers of the carbon-to-carbon double bond. The sphingoids from fresh-water shellfish GSLs [330] are composed predominantly of sphing-monoenes, while those from sea-water shellfish [331] and other mollusc (sea anemone) [332] GSLs contain a relatively large amount of sphing-dienes. Lactosylceramide with a variety of long chain bases was isolated from a shellfish [332]. Some of these GSLs contain, in addition, phosphate (phosphoglycosphingolipids), phosphonate (phosphonoglycosphingolipids),inositol and alkyl amines. In the major type of complex GSLs in plants (phytoglycolipids), the carbohydrate moiety and ceramide are linked through a phosphodiester (111-26 in Table 1.5). All mannose-containing GSLs so far known have been found in fresh-water shellfish and in plants, but not in sea-water shellfish. On the other hand, phosphonoglycosphingolipids have been identified in sea-water shellfish. The structures of glycosphingolipids isolated from the non-vertebrates are listed in Table 1.5.
* Karlsson et al. 13261 reported the presence of a xylosylcerarnide in salt gland of herring gull.
39 TABLE 1.5 Glycosphingolipids in non-vertebrates No. a
Glycosphingolipid
Source
111-1
GlcUA
Cer
Flavobacterium devorans 333
111-2
Manbl
Cer
Hyriopsis schlegelii (fresh-water bivalve)
120
111-3
Manbl + ZManfil+ Cer
Hepatopancreas of H. schlegelii
335
111-4
Manal
Hepatopancreas of H. schlegelii
335
111-5
Manfil- 4Glc@l Cer (rnalabiaosylceramide)
Corbicula sanhi (fresh-water bivalve) H. schlegelii Wheat flour
327.336-338
4Manbl+ 4Glcbl+ Cer
Wheat flour
339
4Manbl-+ 4GlcfiI
C. sandai Spermatozoa of H. schlegelii
327,337.388 340
Haliotis japonica
347
-+
+
3Man/?1+ 2Manbl-+ Cer
-+
-+
111-6
Manbl
+
111-7
Manal
+
-+
Cer
Ref.
335 339
a1Fuc 1 111-8
Fucal
-+
3GalNAcal+ 3Ghbl
-+
4Glcbl- Cer 111-9
(abalone)
GlcNAcl -+ 3[(3-O-Me-Fuc)l+ 2]Gall-4GalNAcl\ (3-O-Me-GalNAc)l+ 3[Fucal+ 2]Gall/* 3Gall 4Glc + Cer :Gall -+
-+
Osreria gigas
274,318
(oyster)
Mollu series
111-10 Manal
-+
3Manbl + 4Glcb1+ Cer
Hepatopancreas of
335
H. schlegelii
111-11 Manal
+
PlXYl -1 2 3 M a n p l j 4Glcbl-+ Cer
Spermatozoa of
340
H. schlegelii
111-12 GlcNAcfll- 2Manal- 3 M a n f i l j 4GlcfiI + Cer
Spermatozoaof H. schlegelii
344
Spermatozoa of
345
PlXYI 111-13 GlcNAc/3l-+ 2Manal
-.
L
L
3Manpl-. 4Glcbl-+ Cer
H. schlegelii
40 TABLE 1.5 (continued) No. a
Glycosphingolipid
Source
Ref.
111-14 (4-0-Me-Gal)Bl --t 3GalNAcB1 + 3Fucal+ 4GlcNAc/31+ 2Manal3Manbl- 4Glc/31+ Cer 2 f a1 Xyl
C.sandai
341
111-15
Spermatozoa of H. schlegelii
342
Spermatozoa of H. schlegelii
343
Larvae of Lucilia caesar (green-bottle fly)
738
111-18 GalNAc/3l+ 4GlcNAc/3I + 3Manpl4Glc/3I + lCer
Larvae of L. caesar
738
111-19 GalNAcal + 4GalNAc/31+ 4GlcNAc/31+
Larvae of L. caesar
739
Larvae of L. caesar
739
Larvae of L. caesar
739
Isogala and neogala series 111-22 Galbl- 4GalbI + Cer (isogalabiaosylceramide)
Hepatopancreas of H. schlegelii
335
111-23 Gal/3l+ 6Gal/31 + Cer (neogalabiaosylceramide)
Turbo cornutus (marine snail)
346
111-24 Gala1 + 6Gal/3I + 6Gal/31 + Cer (neogalatriaosylceramide)
T. cornutus
346
Tobacco leaves
348
al(3-0-Me-GalNAc) 1 3 (4-O-Me-GlcUA)Bl- 4Fucal+ 4GlcNAc/31+ 2Manal- 3Man/31+ 4Glc/31+ Cer 2 T PXYl
111-16 (3-O-Me-Fuc)al+ 2(3-0-Me-Xyl)/31+ a1(3-0-Me-GalNAc)
1
3 4Fucal+ 4GlcNac/31+ 2Manalh 3Man/3l+ 4Glc/31+ Cer 2 T PlXYl Arthro series 111-17 GlcNAc/3l+ 3Man/31+ 4Glc/31+ lCer
3Man/3l+ 4Glc/?l+ lCer 111-20 Gala1 + 4GalNAcal- 4GalNAc/31+
4GlcNAc/31+ 3Manbl- 4Glc/3l+ lCer 111-21 GlcNAc/3l+ 4Galal- 4GalNAcal- 4GalNAc/31 4GlcNAcal + 3Man/3l + 4GlcPl+ lCer +
Phosphoglycosphingolipids 111-25 GlcN( Ac)al + 4GlcUAal + 2myoinotol-1-0-phosphate + Cer
*
41 TABLE 1.5 (continued) No. a
Glycosphingolipid
111-26 (4-O-Me-Gal)/31+ 3GalNAcPl + 3FucalalXyl
Source
Ref.
C. sandai
349
T. cornutus
350-352
Monodona labio (marine gastropod)
352
T. cornutus
350, 351, 353
.1 2 4GlcNAc/31- 2Manal+ 3Man/31-, 4Glc/31+ Cer 6 0
T I1
0- P-O-CH2CHzNH2
AH Phosphonoglycosphingolipids
fH
111-27 H2N-CH2CH2- P-0
+ 6Gal-
Cer
‘0 OH
111-28 HN-CH2CH,-
I P-0
A
+
6Gal+ Cer-
a Numbers listed in the Table are used for simple assignment of individual glycosphingolipids in text. GlcUA, glucuronic acid.
5.9.1. Phosphorus-free glycosphingolipids
Galactosylceramide and glucosylceramide, which are the only monoglycosylceramides (with the exception of fucosylceramide (Section 5.7)) found in vertebrates, are distributed widely in non-vertebrates (see Section 5.1.1 and 5.2.1). However, monoglycosylceramides containing less common monosaccharides have been identified in non-vertebrates. Examples include a glucuronylceramide (111-1 in Table 5) identified in a strain of Flauobacterium, and a mannosylceramide (111-2 in Table 5) found in a fresh-water shellfish which is extended into a dimannosylceramide (111-3 in Table 5) and a trimannosylceramide (111-4 in Table 1.5). Mannose was detected in mono- and diglycosylceramides from cultured mosquit cells [354]. MannosylPl-4glucosylceramide (malabiosylceramide * ) which is found in plants [339] and fresh-water shellfish [335,336] is presumed to be a direct, common biogenic precursor of compounds containing Manal + 4 (111-7 in Table 1.5), Manal + 3 (111-10 in Table lS), Manal + 3 and XylPl .+ 2 (111-11 in Table 1.5) at the non-reducing terminus of malabiosylceramide. The non-reducing terminus of a dimannosylglucosylceramide from wheat flour was assigned as a 0-anomer [339] (111-6 in Table l.5), in contrast to the compound from shellfish with the same carbohydrate sequence [337,338](111-7 in Table 1S ) . Two tetraglycosylceramides belonging to mollu series, Manal + 3(GalP1 *
In analogyto lactose, the name “malabiose” was suggested for the disaccharide ManPl
-, 4Glc.
42 + 2)Manpl + 4Glcp1- lCer and Manal + 2(?)Manal + 3Manpl- 4GlcPl + ICer, were recently characterized in ova of H . schlegelii [755]. Highly complex octaglycosylceramides (111-14 and 111-15 in Table 1.5) and nonaglycosylceramide (111-16 in Table 1.5) which may be derived from 113-a-mannosyl-malabiosylceramide or M1,Cer (111-10 in Table 1.5) include a variety of 0-methyl monosaccharides. These GSLs (111-14, -15 and -16) belonging to mollu series, and a phosphoglycosphingolipid (111-26) shown in Table 1.5, are the first demonstration of glycolipids (possibly including other glycoconjugates) containing internal fucose. The internal location of the L-fucose was determined by partial acid hydrolysis, methylation analysis, and proton NMR spectrometry [342,343]. All P-configurated digalactosylceramides (111-22 and 111-23 in Table 1.5) and trigalactosylceramide (111-24 in Table l S ) , which can be classified into isogala and neogala series, were found in shellfish [335,346]. A tetraglycosylceramide, (Gal),-Glc-Cer, was isolated from a microorganism (Neurosporu crussu) [355]. Sugita et al. [738,739] recently characterized, from larvae of green-bottle fly, five GSLs (111-17-21 in Table 1.5) belonging to arthro series (see Section 2). The long-chain bases of the larva GSLs comprised tetradeca-4-sphingenine (70-85%) and hexadeca-4-sphingenine (15-30%).
5.9.2. Phosphoglycosphingolipids The major complex GSLs (phytoglycolipids [356]) in plants (corn and soy bean [357]) contain phosphate and inositol. A compound having constituents similar to phytoglycolipids was identified in two species of yeast [358]. The predominant long-chain base obtained from corn lipids was named phytosphingosine [ 3571. Structural studies of phytoglycolipid carbohydrate moieties were done on sugar fractions isolated from partial hydrolysates of the lipids. As suggested earlier [356], phytoglycolipid was proposed to have the following basic structure with all a-anomeric configurations [92], based on the analysis of a tetrasaccharide isolated from the alkaline hydrolysate [359] of corn phytoglycolipid: 0
II
GlcNal + 4GlcUAal+ 6(Manal+ 3)myoinositol-1-0- P-0-Cer
AH
Lester et al. [360,361] have reported the presence of inositolphosphoceramides containing mannose in yeast. They also [362] fractionated from tobacco leaves several phosphoglycosphingolipids which possessed both arabinose (2-4 mol) and galactose (1-2 mol), and had the c p x n o n structure of GlcN( fAc)al -,4GlcUAal + 2-myoinositol-l+ phosphate + Cer. Of these, the chemical structure of a trisaccharide derivative, GlcNal + 4GlcUAd + 2(Manal + 3) myoinositol was found to be substituted by glucuronic acid in a manner different from that in corn phytoglycolipid [92] (see above). Two tetrasaccharides containing inositol were isolated from oligosaccharide fractions which were prepared from carboxy-reduced lipids of tobacco leaves by alkaline hydrolysis and phosphatase treatment, and the following
43 structures were determined for them [363]: Galbl
-
4GlcNAcal + 4GlcUAal+ 2-rnyoinositol
GlcNAcal + 4GlcUAal
+
?(Manal
+
?)myoinositol
The presence of phosphoinositoglycosphingolipidswas also demonstrated in microorganisms, such as Neurospora crassa, baker’s yeast [364] and Aspergillus niger [365]. Cellular slime mold ( Dyctyosteliurndiscoideum) was reported to have a sphingolipid(s) possibly serving as a cell aggregation-competent factor which is composed of GlcNAc : Fuc : Man : Cer (12 :5 :2 : I), phosphate and ethanolamine [366]. The carbohydrate composition of non-aggregating mutant cells was different from that of the aggregating cells. Itasaka and Hori [349] identified a complex phosphorylated glycosphingolipid (111-26 in Table 1.5) in which phosphate links ethanolamine to C-6 of mannose through a phosphodiester. 5.9.3. Phosphonoglycosphingolipids Aminoalkylphosphonic acids, H,N-CH,-CH,-PO,H, (2-aminoethylphosphonic acid) and CH ,-NH-CH ,-CH PO, H ( N-methyl-2-aminoethylphosphonicacid), are widely distributed in many organisms in free form and as constituents of lipids (reviewed in Refs. 367 and 368) and proteins [367]. The C-P bonding in these compounds is extremely resistant to usual acid hydrolysis conditions. Lipid-bound aminoalkylphosphonates (named phosphonolipids [368]) were found independently by Rouser et al. [369] and Hori et al. [370] in aquatic lower animals, in which phosphonolipids in the two compound groups, glycerolipids and sphingolipids, are abundant. Hayashi and Matsuura [350] isolated and characterized for the first time two galactosylceramides containing aminoalkylphosphonates (111-27 and 111-28 in Table 1.5). A phosphonoglycosphingolipid isolated from the skin of a marine gastropod ( Aplysia kurodai) was described as being composed of Glc/Gal/ 3-0MeGal/GalNAc/2-aminoethylphosphonate/Cer (1 : 2 : 1 : 1: 2 : 1) [371]. “Lipophosphonoglycan” containing ceramide, aminophosphonates, sugars, and inositol was found in the plasma membrane of a soil amoeba (Acanrhamoeba castellani) [372].
,-
,
6. Biosynthesis of glycosphingolipid The biosyntheses of GSLs, except the biosynthesis of the ceramide moiety, are essentially accomplished through the action of glycosyltransferases whch transfer sugar residues from specific nucleotides to ceramide or an oligosaccharide chain bound to ceramide [377]. The nucleotide moiety is usually a uridine diphospho-sugar which serves as the donor substrate in the formation of the glycosides of galactose, glucose, N-acetylgalactosamine and N-acetylglucosamine. Glycosides of the sialic acids and L-fucose are formed by transfer from cytidine monophospho-sialic acid
44 acids and L-fucose are formed by transfer from cytidine monophospho-sialic acid and guanosine diphospho-fucose, respectively. Most of the glycosyltransferases are presumed to exist in the Golgi membranes and require a detergent for solubilization. The enzymes are quite unstable and their activities towards GSL substrate are very low as compared to the enzymes involved in glycoprotein biosynthesis. Until recently very few glycosyltransferases involved in GSL metabolism had been purified [334,374,375]. Therefore, most of the kinetic and physicochemical properties were obtained from data on crude or partially purified enzyme preparations. Recently several specific ligands for affinity chromatography have been successfully employed for the purification of glycosyltransferases [ 334,375,3761. This technique, it is hoped, will make it possible to purify the enzymes involved in GSL metabolism and characterize further their enzymatic properties. The scope of this chapter is restricted to a description of the glycosyltransferases involved in the biosynthesis of GSLs, excluding gangliosides. Survey on GSL metabolism has recently appeared [455]. 6.1. BIOSYNTHESIS OF SPHINGOID BASE A N D CERAMIDE
Biosynthesis of the ceramide moiety is one of the key steps in GSL metabolism [377]. Radioactive serine and palmitoyl-CoA were incorporated into ceramide, in the presence of pyridoxal phosphate and NADP, in rat brain homogenate [378] and mouse brain microsomal fractions [379,380]. Palmitoyl-CoA and serine first combine to form sphingosine (sphing-4-enine). A second palmitoyl-CoA reacts with sphingosine to form ceramide [379,381]. The sphingosine is derived from dihydrosphingosine via a ketosphingosine intermediate [382]. Dihydrosphingosine is created by stereospecific reduction of 3-ketodihydrosphingosineby an NADPH-dependent enzyme [383]. Palmitoyl CoA + serine + (pyridoxal phosphate)
-P
3-Ketodihydrosphingosine(3-dehydrosphingonine) 3-Ketodihydrosphingosine+ NADPH -P dihydrosphingosine Dihydrosphingosine + D-sphingosine
The synthesis of ceramide (acylated sphingosine) from sphingosine in vitro was first reported by Sribney [381] in a rat brain microsome preparation. The reaction, Long-chain base + fatty acyl-CoA
+
ceramide + CoA
is catalyzed by the enzyme acyl-CoA :sphingosine N-acyltransferase. The pH optimum of the reaction is 7.5, The enzyme does not require a metal or other cofactors and is localized in microsomes. There are several acyl-CoA :sphngosine Nacyltransferases, which differ in substrate specificity with respect to the fatty acid-CoA moiety. Free fatty acids are not reactive. Stearoyl- and lignoceroyl-CoAs were better substrates than palmitoyl and oleoyl-CoAs. The rate and extent of
45 conversion of these CoAs to ceramide were in a ratio of 60 : 12 : 3 : 1 [380]. This ratio strongly resembles the relative distributions of these fatty acids in the rat brain. With respect to the long-chain base there is less specificity. The C,, and C,, sphingosines, as well as dihydrosphingosines, are good acceptors (3791. In mouse brain, there are two distinct transferases which react with stearoyl-CoA and lignoceroyl-CoA, respectively [384,385].The transferases for 2-hydroxystearoyl-CoA and for cerebronyl-CoA are also different enzymes [384]. The reverse reaction of the catabolically active ceramidase, also was considered to contribute to the biosynthesis of ceramides [386-3881. However, the nature of this reverse reaction remains to be elucidated because the product in some buffer systems [389] was not sphingosine but a condensation product between long-chain fatty acids and ethanolamine. Recently a third pathway has been proposed for the biosynthesis of ceramide in the rat brain [390,391]. This reaction is catalyzed by mitochondria1 fractions and requires a pyridine nucleotide in the presence of two water-soluble factors (one heat stable and the other heat labile) which are also required for fatty acid a-hydroxylation in the brain. The pH optimum is 7.0, and the activity is stimulated by KCI and Mg2+ but strongly inhibited by EDTA. The apparent K , value for lignoceric acid is 12 pM. 6.2. BIOSYNTHESIS OF GALACTOSYLCERA MIDE
The biosynthesis of galactosylceramide was demonstrated with particulate preparations of mammalian brains [3,4,115,392-3981. Two pathways for the biosynthesis of galactosylceramide from sphingosine have been proposed. Sphingosine + UDP-Gal
+
psychosine+ UDP
Psychosine + fatty acyl-CoA
+
galactosylceramide
Ceramide + UDP-Gal + galactosylceramide
+ CoA
+ UDP
(1) (2)
Because in the studies which led to the proposal of reaction (1) the acylation of psychosine occurred by a non-enzymatic reaction, it appears that the major pathway for the biosynthesis of galactosylceramide is by way of a ceramide intermediate [3,115]. The activity of a UDP-ga1actose:ceramide galactosyltransferase, which catalyzes reaction (2), increases in the rat brain during myelination and reaches a plateau 16-17 days after birth [399]. The enzyme is concentrated in the brain white matter rather than the gray matter. The enzyme was purified 105-fold from rat brain by a procedure involving detergent extraction, ion-exchange chromatography, and proteolytic digestion [397,400]. The enzyme appeared to exist as a lipoprotein complex of approximately 400 000-500 000 molecular weight containing tightly bound phospholipid [400]. The purified UDP-ga1actose:ceramide galactosyltransferase displayed no activity with ceramide containing a nonhydroxy fatty acid, and also showed no transfer activity towards glucosylceramide, lactosylceramide or a ganglioside, I13NeuAc-Gg,Cer [401]. Phospholipase treatment of the enzyme resulted in a loss of
46 activity [398], suggesting that amphiphiles can interact with the enzyme in addition to the normal substrate. 6.3. BIOSYNTHESIS OF GLUCOSYLCERAMIDE
Basu et al. [403] characterized a glucosyltransferase which transferred glucose from UDP-glucose to a ceramide containing normal fatty acids and yielded gluco-, in a particulate enzyme preparation from 13- to 14-day old embryonic chick brain. The activity profile of the enzyme coincided with the onset of ganglioside deposition and myelination in the brain. The enzyme was also found in the developing rat brain [3,404-4061 and glia cell lines [398]. The pH optimum was 7.8, and Mn2+ was required. It has been suggested that glucosylceramide is a precursor for the next step in the de novo synthesis of gangliosides. 6.4. BIOSYNTHESIS OF D I - A N D TRIHEXOSYLCERAMIDES
An enzyme activity incorporating galactose from UDP-Gal into glucosylceramide to form lactosylceramide was first reported by Hauser [407,408] in the rat spleen. The enzyme was subsequently found in the embryonic chick [403] and rat brains [408], and in rat retina [409]. The enzyme was distinct from galactosyltransferases that use lactosylceramide and ganglioside I13NeuAc-Gg,Cer as acceptors [182,410]. The enzyme has been detected also in hamster BHK and Nil cells [411], and adrenal cloned Y-I-K cells [412]. Bushway and Keenan [413] suggested that the enzyme may be the same as the galactosyltransferase involved in lactose synthesis based on observations of the modification of activity by a-lactalbumin, copurification of the two enzymes, and similar enzymatic properties. The substrate specificity of the enzyme is modulated by its interaction with a-lactalbumin. In the absence of a-lactalbumin, the transferase is specific for acceptors with a nonreducing terminal N-acetylglucosamine residue. In the presence of a-lactalbumin, glucose, maltose and a-methylglucoside are excellent acceptors. Yamamoto and Yoshida [414] isolated lactose synthase (named A protein) from human plasma and found that, in the presence of a-lactalbumin, the enzyme catalyzed the synthesis of lactose, lactosylceramide and neolactotetraosylceramide (paragloboside, GalPl 4GlcNAcPl -, 3GalP1 + 4Glcj31 -, 1Cer). The enzyme therefore is a GalPl -,4 :GlcNAc -, R (or Glc + Cer)galactosyltransferase. In a homogenate of mouse kidney, enzymes occur that catalyze the synthesis of both digalactosyl ceramide and globotriaosylceramide [415]. In rat tissue the latter enzyme is widely distributed, whereas the former was shown to be present only in the kidney [415-4181. The trihexosylceramide produced by rat spleen homogenates was found not to be globotriaosylceramide but globoisotriglycosylceramide[182], presumably a precursor of globoisotetraosylceramide(cytolipin R). Further studies showed that the rat kidney enzyme synthesized both isomers [410].
-
47 6.5. a A N D B-N-ACETYLGALACTOSAMINYLTRANSFERASES (GalNAc TRANSFERASES) I N V O L V E D IN T H E BIOSYNTHESIS O F G L O B O - S E R I E S G L Y C O L I P I D S
Steigerwald et a1 [419] described, in an extract of embryonic chick brain, an enzyme activity that catalyzed the reaction: UDP-GalNAc+ X-GaI/31-4Glcbl-lCer-
X(GalNAcPl+ 4)GalPl- 4Glc/31+ lCer + UDP
where X = a 2 + 3 sialic acid or H. Studies on the substrate specificity of this enzyme indicated that sialic acid is transferred to lactosylceramide before the attachment of N-acetylgalactosamine in the synthesis of ganglioside I13NeuAc-Gg3Cer. In embryonic chcken brain, GalNAcal + 4Galactoside P-GalNAc transferase, catalyzing the conversion of globotriaosylceramide to a globoside-like product, was found [420]. The enzyme required Mn2' and taurocholate for maximum activity. The product reacted with antigloboside serum and was destroyed by treatment with P-N-acetylhexosaminidase.Competition experiments suggested that this transferase was the same as the P1-4GalNAc transferase described by Steigerwald et al. [419]. However, the product differed from globotetraosylceramide in thin layer chromatographic behavior, which suggested it to be an isomer differing at the N-acetylgalactosaminidic linkage [420]. Embryonic chick brain [420], guinea pig kidney [421,422], mouse adrenal tumor cell lines [412], hamster cells [423,424], and canine spleen [375,402] contain both a GalNAcal -+ 4Galactoside Pl + 3GalNAc transferase and a GalNAcPl + 3GalNAcal + 3GalNAc transferase that synthesize globoside and Forssman glycolipid, respectively. These enzymes catalyze the following respective reactions: UDP-GalNAc+Galal + 4GalPl+ 4GlcPl
+ 1Cer-
GalNAcBl+ 3Galal + 4
GalPl+ 4Glc/31+ lCer + UDP UDP-GalNAc + GalNAcPl
+
3Galal- 4GalB1+ 4GlcPl- 1Cer-
+
GalNAcal + 3GalNAcPl-+3Galal+ 4Galbl-+ 4GlcPl+ lCer UDP
Recently Taniguchi et al. [375] purified the latter enzyme to apparent homogeneity by the use of affinity chromatography on globoside acid-Sepharose. The enzyme is composed of two non-identical subunits of molecular weights 66 000 and 56 000. The enzyme required Mn2+ for its activity and had a pH optimum of 6.7-6.9. It catalyzes the transfer of N-acetylgalactosamine in al-3 linkage to globoside. Fucosyl lactose(Fucal-2Gal~l-4Glc), blood group H substance and deglycosylated mucin were not acceptors. Studies on substrate specificity indicated that the preferred substrates have the general structure GalNAcfll-3Gal-O-R, whereby the nature of the R moiety has relatively little effect on activity. Kinetic analysis revealed that UDP acts as a competitive inhibitor with respect to UDP-GalNAc and a noncompetitive inhibitor with respect to globoside. Therefore, the enzyme was distinct from
48 both the N-acetylgalactosaminyl transferases that convert the blood group H substance to the A substance [425-4281 and those which catalyze the synthesis of the GalNAcal-0-Ser(Thr)- linkage occurring in mucin type glycoproteins [429,430]. The enzyme is also distinct from GalNAcd -,4Galactosidepl + 3GalNAc transferase which converts globotriaosylceramide to globotetraosylceramide. The Pl-3 GalN Ac transferase involved in the biosynthesis of globoside was purified over 18 000 fold from canine spleen, with the use of UDP-Sepharose and globotetraosylceramide acid-Sepharose and globotriaosylceramide acid-Sepharose [ 3341. The enzyme is composed of two non-identical subunits of molecular weight 64000 and 57000. Mn” is required for activity. The preferred substrates have the general structure Gala1 + 4Gal-0-R, in which the nature of the R moiety has relatively little effect on activity. 6.6. a-N-ACETYLGALACTOSAMINYLTRANSFERASE INVOLVED I N THE BIOS YNTHESIS OF BLOOD GROUP A SUBSTANCE
GalNAc:(Fucal-2)Galactosideal + 3GalNAc transferase that catalyzes the reaction UDP-GalNAc+Fucal
+
2Gal-R-
GalNAcal
+
3(Fucal+ 2)Gal-R+ UDP
has been purified to homogeneity from both porcine submaxillary gland [425,426] and human serum [427,428]. The enzyme from porcine submaxillary gland was enriched 38 000-fold with the use of UDP-hexanolamine Sepharose, whereas the enzyme from human serum was purified over 1000-fold by Whitehead et al. [427], and then 135 000-fold by Nagai et al. [428], using agarose. The acceptor specificity of the purified N-acetylgalactosaminyltransferasefrom both porcine and human sources appears to be identical in that the primary structural requirement for an acceptor is the nonreducing terminal sequence, F u c d + 2Gal. The linkage of galactose to the remainder of the oligosaccharide chain, and the size and hydrophobic character of the acceptor have little effect on the activity of the enzyme [426]. The enzyme can convert type-0 erythrocytes into type A erythrocytes [426]. Studies on substrate specificity indicated that the enzyme formed the A-blood group determinant from the Fucal -+ 2Gal structure irrespective of whether they occurred in isolated oligosaccharides, glycolipids or glycoproteins. The relative activity rates of the enzyme using a GSL as acceptor substrate were 29% for a Fucal + 2Galp1 + 4GlcNAcp1 + 3GalPl + 4Glcfi1 + lCer and 137 and 1008, respectively. for Fucal + 2Galpl + 4Glc and asialo porcine submaxillary mucin [426]. The GalNAc transferase is described as being composed of two identical subunits of molecular weight 46000. The enzyme is a glycoprotein with 16% hexose, 11% hexosamine and 2% sialic acid as carbohydrate constituents [425]. 6.7. a-FUCOSYLTRANSFERASES INVOLVED I N THE BIOSYNTHESIS OF FUCOLIPIDS
The synthesis of an H-active fucolipid from GDP-L-[‘‘C]fucose and lactoneotetraosylceramide, Galpl + 4GlcNAcPl -+3Galb1 + 4Glcb1 + 1Cer. was as demonstrated in a Golgi-rich membrane preparation of beef spleen homogenate [431]. The
49 reaction required Mn2+ and Mg2+ ions and a cationic detergent. Glucosylceramide served as a specific acceptor, and GDP-fucose as a specific donor. The product was an H-active glycolipid, as judged by immunological and chromatographic criteria, which showed the enzyme to be a fucose:galactoside ail + 2Fuc transferase. An enzyme which leads to the formation of products with H blood group activity has been purified 124000-fold to homogeneity from porcine submaxillary glands [432,433]. The enzyme was purified by repeated affinity chromatography on GDPhexanolamine agarose, employing both specific elution with GMP and nonspecific elution with NaC1. As estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis two forms, of molecular weights 60 000 and 55 000, composing the enzyme were isolated. The pH optimum was broad, ranging from pH 6-7. MnCl, stimulated the fucosyltransferase activity about six fold, but the enzyme was active in the absence of added metal ion. The enzyme can utilize glycoprotein or oligosaccharide acceptors that contain a nonreducing terminal 8-galactoside residue. The acceptor substrate specificity was unusual as compared to most other glycosyltransferases. The fucosyltransferase did not show an absolute requirement either for a specific sugar residue penultimate to the nonreducing terminal galactose or for a particular glycosidic linkage between this galactose and the penultimate residue. GSLs containing a fairly large oligosaccharide moiety, such as neolactotetraosylceramide and ganglioside I1 3NeuAc-Gg,Cer, are poor acceptors, whereas fucosetransfer to smaller GSLs such as galactosylceramide, lactosylceramide and lactotriaosylceramide could not be detected. The best acceptors were oligosaccharides that contained the disaccharide sequence GalPl-3GalNAc or GalP13GlcNAc at the nonreducing termini [433]. Fuc:GlcNAcal -+ 3- and a1 + 4-fucosyltransferases were purified from human milk [434]. The enzymes catalyze the following three reactions [434,435,436]: GDP-Fuc+Gal/3
3GlcNAc-R-
Fucal
GDP-Fuc+Galal+ 4GlcNAc-R-
Fucal
+
GDP-Fuc+GalPl+ 4Glc-
-
-
4(Galbl+ 3)GlcNAc-R+ GDP
(3)
3(Galp1+ 4)GlcNAc-R+GDP
(4)
Fucal- 3(Galb1-+4)Glc+GDP
(5)
The product of reaction (3) is the antigenic determinant of the Lewis a (Lea) blood group substance [437]. Lewis-negative individuals lack the enzyme catalyzing the reaction [435] (see discussion in Section 8.1.2). A single enzyme, independent of the Lewis system, catalyzes reactions (4) and ( 5 ) [438]. a1 + 3- and a1 + 4-fucosyltransferases were purified from human milk [434] by repeated affinity chromatography on GDP-hexanolamine agarose to over a 500 000-fold enrichment. The purified enzyme has nonidentical subunits of M , 51 000 and 53 000, as identified by sodium dodecyl sulfate gel electrophoresis. The study of acceptor specificity indicates that oligosaccharides, glycoprotein, and possibly GSLs with the non-reducing terminal sequences of GalPl + 4GlcNAc, GalPl + 3GlcNAc, or GalPl -,4Glc, can all act as acceptors [434,439].An a1 -+ 3 flucosyltransferase activity converting blood group
50
A-active hexaglycosylceramide (11-26 in Table 1.4) to the corresponding difucosyl GSL (11-29 in Table 1.4) was demonstrated in human serum [749]. 6.8. a-GALACTOSYLTRANSFERASEINVOLVED IN THE BIOSYNTHESIS OF BLOOD GROUP B SUBSTANCE
The blood group B-specific galactosyltransferase, which catalyzes the reaction: UDP-Gal+ Fucal-2Gal-R-
Galal
+
3(Fucal+ 2)Gal-R+ UDP,
has been purified to homogeneity from human serum by a 400000-fold enrichment, and its molecular properties have been extensively studied [440]. The enzyme consists of two identical subunits of molecular weight 80 000. Interestingly, antibodies raised against the purified blood group A-specific N-acetylgalactosaminyltransferase of human serum cross-reacted with the blood group B-specific a-galactosyltransferase and vice versa [440]. This indicates that the two enzymes are structurally very similar. The a-galactosyltransferase has an absolute specificity for acceptors with the nonreducing terminal sequence of Fucal-2Gal. Assays have been described utilizing 2’-fucosyllactose [440,441], lacto-N-fucopentaose I [441], and H-active glycoproteins [442]. A blood group B-specific galactosyltransferase from rabbit bone marrow has been reported by Basu and Basu [443]. The enzyme catalyzes the react ion : Gala1 + 3GlcNAcSl+ 3Galpl
-
+
+
4Glc/l1+ lCer UDP-Gal
Galal + 3Gala1+ 3GlcNAc/31+ 3Gal/31+ 4Glcj?1+ 1Cer+ UDP
This pentaglycosylceramide product carries serological blood group B activity. Acceptors substituted with fucose at the 2-position of the terminal galactose were less actively galactosylated by this enzyme than the nonfucosylated analogues. However, no other blood group B-specific galactosyltransferases that act on acceptors without a terminal sequence of Fucal-2Gal have been reported. 6.9. GALACTOS YLCERAMIDE SULFOTRANSFERASE
A galactosylceramide sulfotransferase (3’-phosphoadenylsulfate:galactosylceramide 3’-sulfotransferase) catalyzes the transfer of a sulfate group from 3‘-phosphoadenosine 5’-phosphosulfate (PAPS) to the 3-position of the galactose moiety to form 3-sulfo-galactosylceramide: PAPS + galactosylceramide-
3-sulfo-galactosylceramide+ PAP
The enzyme activity was detected in inicrosomes prepared from various mammalian tissues [444-4461. The main locus of the enzyme in kidney cells is the Golgi apparatus [447]. The enzyme was solubilized, and some physicochemical properties were reported [448].The enzyme was activated by Triton X-100 and Mn2+. The M, whilst that for PAPS apparent K , value for galactosylceramide was 1.3 X
51
was 2 x M. The enzyme catalyzed sulfate transfer to galactosylcerarnides containing hydroxy fatty acid, as well as those containing normal fatty acids [447]. Earlier, crude preparations of the enzyme had been considered to catalyze sulfation of a variety of galactolipids including galactosylceramide, psychosine (galactosylsphngosine) and lactosylceramide. However, Tennekoon and McKhann [449] demonstrated that a brain preparation of the sulfotransferase can utilize only galactosyland lactosylceramide as substrate, whereas other lipids such as monogalactosyl sphingosine and cholesterol have their own corresponding specific transferases. Tadano and Ishizuka [450] reported that monkey and canine kidney cell lines contained an enzyme(s) that catalyzed the transfer of sulfate to galactosylceramide and lactosylceramide. The enzyme required Ca2+ for its activity.
7. Biodegradation of glycosphingolipids The degradation of GSLs occurs through the stepwise hydrolytic cleavage of the non-reducing terminal glycoses at the carbohydrate moiety of the molecule. For example, the catabolism of galactosylceramide is initiated through the action of a P-galactosidase that cleaves the galactose of the molecule to yield ceramide. Ceramide is in turn hydrolyzed to sphingosine and fatty acid. Intensive efforts to elucidate the mechanism of sphingolipidoses, a group of biochemical mutations in man whch had been defined clinically and pathologically, also have provided fruitful results for the chemistry and catabolism of GSLs. 7.1. H Y D R A S E S
Some of the glycolipid hydrolases show typical differences between their activities towards natural and synthetic substrates. Generally, for the diagnosis of sphingolipidoses, synthetic substrates are employed. However, the natural substrate provides a valuable tool for more detailed biochemical studies of the corresponding hydrolases. Although it is generally believed that a glycohydrolase will be specific only for the anomeric configuration of the glycoside, irrespective of linkage position and glycoconjugate class, some glycohydrolases from mammalian sources exhibit an aglycon specificity. One interesting aspect of lysosomal hydrolases is the presence of activator proteins for several lysosomal hydrolytic enzymes. 7.1.I. Ceramidase Ceramidase ( N-acylsphingosine amidohydrolase) catalyzes the hydrolysis of ceramide to sphngosine and fatty acid, and possibly also catalyzes the reverse reaction [386,388]: ceramide( acylsphingosine) + H,O
+
long-chain base + fatty acid
Human tissues have three distinct ceramidases with pH optima in the acidic, neutral
52 and alkaline range, respectively. The acid ceramidase has been purified from rat brain by an approximate 200-fold enrichment [386, 3871. The enzyme has a pH optimum of 4.8 and appears to be lysosome-associated. The enzymatic hydrolysis is stimulated by the addition of cholate and taurocholate, and inhibited by sphingosine and fatty acid. In the tissue of patients with Farber’s disease, a genetic ceramidase deficiency or ceramidosis, less than 1%of the normal ceramidase activity was found at pH 4.0, which explained the accumulation of ceramide in the tissues. For the purposes of diagnosis both leukocytes and cultured fibroblasts are employed as enzyme sources [456]. Since no artificial chromogenic or fluorogenic substrates are commercially available, N-[l-’4C]oleylsphingosinewas synthesized from [l-’4C]oleic acid and sphingosine, and used as a substrate. After incubation of the substrate with an enzyme preparation, the liberated [1-14C]oleic acid is separated by solvent partitioning followed by thin layer chromatography, and its radioactivity is determined. Small intestine [457], cerebellum [388], and cultured skin fibroblasts [458] contain ceramidase activities with neutral to alkaline pH optima from 7.6 to 9.0. These reactions seem to be reversible. 7.1.2. 6-Galactosidase At least two, and possibly three, distinct P-galactosidases ( P-D-galactoside galactohydrolase) are involved in the catabolism of GSLs. One is galactosylceramide P-galactosidase which catalyzes hydrolysis of galactosylceramide to ceramide and galactose. The enzyme was first studied in human brain by Radin and h s co-workers [459-4621. The enzyme is lysosome-associated. A 300-fold enriched P-galactosidase was obtained by a procedure including extraction with detergent and DEAE-Sephadex chromatography [460]. The pH optimum was 4.5. Several inhibitors of this enzyme such as ceramide, sphingosine, lactosylceramide, galactose and galactonolactone, were found. The enzyme has been partially purified from human and other tissue [761-7631. The enzyme from humans has a molecular weight of 125000 and exists as mono-, di- and tetramers, as well as hexamers [762]. It is also active, to some extent, towards galactosyl-sphingosine (psychosine), monogalactosyldiglyceride and lactosyl ceramide. The enzyme activity is deficient in globoid cell leukodystrophy (Krabbe’s disease) [466]. The other is ganglioside I1 NeuAc-Gg4Cer (ganglioside GM1) P-galactosidase [467,468]. The enzyme hydrolyzes the terminal galactose from lactosylceramide, gangliotetraosylceramide and asialofetuin. This enzyme has been shown to be deficient in generalized gangliosidosis or “ G M l gangliosidosis” [467-4701. In human tissues three isozymes, designated as A,, A, (called acid P-galactosidase A) and A , (called acid P-galactosidase B), exist [463]. All three isozymes are deficient in “GM, gangliosidosis”. Major A, and A, forms have been purified to homogeneity from human liver [464,468]. A, is monomeric, A, is dimeric, and A, is a multimeric form of A, [464]. The molecular weight of A, is 660000, and that of A, is about 65 000. A monospecific antibody against A, precipitates both A, and A, forms, indicating that the two enzymes have a common antigenic site. The enzyme was also purified from
53
porcine spleen [764].Two forms of apparent molecular weights were 400 000-600000 and 70000-74000. The latter was a monomer at neutral pH and formed a dimer at acidic pH [765]. However, liver contains another neutral P-galactosidase whch possesses a neutral pH optimum and a hgher heat stability than the A form [573]. Monospecific anti-P-galactosidase A sera do not precipitate neutral P-galactosidase [468]. Therefore the neutral P-galactosidase is thought to be coded by a separate gene locus from the A and B types. 7.1.3. P-N-Acetylhexosaminidase
0-N-Acetylglucosaminidaseand P-N-acet ylgalactosaminidase (2-acetamido-2-deoxyP-D-hexoside acetamidodeoxyhexohydrolase)are considered to be the same enzyme even though their activities towards synthetic substrates containing P-N-acetylglucosaminide and N-acetyl-P-galactosaminideare somewhat different. The enzyme has two major isozymes designated as P-hexosaminidase A (Hex A) and P-hexosaminidase B (Hex B) [472,473]. The former is thermolabile with an acidic pl, and the latter is thermostable with a basic pl. The substrate specificities of these isozymes towards GSLs are different [473]. Kinetic studies indicated that ganglioside I13NeuAc-Gg,Cer is cleaved by Hex A but not by Hex B. Both enzymes cleave gangliotriaosylceramide and globotetraosylceramide as well as p-ni trophenyl and 4-methylumbelliferyl P-N-acetylhexosaminides.Hex A and Hex B from human placenta [473-4751 have molecular weights of 100000 and 108000, respectively. Hex A cross-reacts immunologically with anti-Hex B antibodies, because the two isozymes have a common subunit P. Hex A exists as an aP2 trimer, and Hex B is a &P2 tetramer [475]. Hex A was found to be profoundly deficient in the tissues and fluids of Tay-Sach’s disease patients, because the a-chain is lacking [476]. The tissues of the patients are unable to hydrolyze ganglioside I13NeuAc-Gg,Cer. Therefore, Hex A deficiency brings about juvenile and adult types of I13NeuAc-Gg,Cer gangliosidosis (reviewed in ref. 477). In the case of Sandhoff‘s disease, a variant form of Phexosaminidase deficiency, both Hex A and B were absent [478], because the P-chain is lacking. 7.I . 4. Glucosylceramide P-glucosidase The enzyme catalyzes the hydrolytic cleavage of glucose from glucosylceramide according to the reaction: glucosylceramide + H,O-
ceramide + glucose
The enzyme has been purified to an apparently homogeneous state from human placenta [479]. The apparent molecular weight is approximately 87 000-92 000 on Sephadex G-200 and 67 000 by sodium dodecyl sulfate gel electrophoresis. The enzyme seems to be a glycoprotein. The apparent K, value for the glucosylceramide is 87 pM, and the pH optimum is 4.5-7.5. Genetic deficiency of the enzyme is the cause of Gaucher’s disease [452]. Two groups [452,453] independently reported subnormal activity of gluco-
54 cerebrosidase in Gaucher’s disease. The enzyme is only one of a yet undetermined number of P-glucosidases. For the diagnosis of Gaucher’s disease, 14C- or H-labeled glucosylceramide is used as a substrate to distinguish between the several p-glucosidases existing in various mammalian tissues [480], including several isozymes with glucosylceramide P-glucosidase activity [481,482].
’
7.1.5.Arylsulfatase A Arylsulfatases (aryl sulfate sulfohydrolases) occur in various tissues and fluids and are classified into A, B and C enzymes [483,484]. Of these, the B and C enzymes act to desulfate sulfated glycosaminoglycans and steroid sulfates, respectively. Arylsulfatase A or cerebroside sulfatase is a lysosomal acid hydrolase, and its physiological substrate, 3-sulfo-galactosylceramide, was discovered during elucidation of the enzyme deficiency responsible for sulfatidosis, metachromatic leukodystrophy [260,455,485]. Cleavage of both p-nitrocatechol sulfate and 3-sulfo-galactosylceramide is catalyzed by the same enzyme. This enzyme has been purified from human liver [486,487] and from urine [488,489]. Multiple molecular forms of the enzyme have been found, and neuraminidase treatment eliminates the anodically migrating bands in certain preparations [490]. Sheep brain arylsulfatase A has been shown to have enzyme forms with high uptake rate which are phosphorylated at their carbohydrate moieties [491]. Isoelectric fractionation resolved three major peaks of activity towards p-nitrocatechol sulfate [490]. The arylsulfatase A hydrolyzes other galactosyl-3-sulfates such as lactosylsulfatide [492], seminolipids and psychosine sulfates [493]. The enzyme exists as a monomer with an approximate molecular weight of 100000 above pH 6, but below pH 6 the enzyme forms a tetramer. The enzyme is a glycoprotein and contains 10% carbohydrates [494]. 7.I . 6. a-Galactosidase and a-N-ucetylgalactosaminidase In an assay using an artificial substrate, two types (A and B) of a-galactosidase activity have been reported to occur in extracts of normal human tissues [495]. 7.1.6.1. a-Galactosidase A The thermolabile form, called a-galactosidase A ( a - ~ galactoside galactohydrolase A), has been highly purified from human placenta [495] and liver [496]. a-Galactosidase A catalyzes the hydrolysis of substrates possessing terminal a-galactosidic residues, including synthesis substrates and naturally occurring GSLs and glycoproteins. The molecular weight of the native enzyme from human tissue is approximately 103 000 with a homo- or hetero-dimeric structure [495]. Assay of globotriaosylceramide hydrolysis by a-galactosidase A in vitro requires the presence of detergent for optimal activity [496]. Sodium cholate, sodium taurocholate and Triton X-100 all have stimulatory effect on the enzyme. The primary defect of a-galactosidase A activity brings about Fabry’s disease [134]. This disease is the only known X-linked disorder among the sphingolipidoses. The accumulation of GSLs with terminal a-galactosyl moieties such as globotriaosylceramide, galabiosylceramide [133] and blood group B active GSL [293] was observed in the affected hemizygotes.
55 7.1.6.2. a-Galactosidase B a-Galactosidase B also catalyzes the hydrolysis of terminal a-N-acetylgalactosaminidic linkages of glycoconjugates and several water soluble synthetic substrates in addition to a-galactosidic linkages [455,457,458]. This applies to GSLs, such as globotriaosylceramide with an a-galactose residue or Forssman glycolipid with terminal a-N-acetylgalactosamine, from which the enzyme is able to catalyze the hydrolysis of these sugar residues. a-Galactosidase B from human liver [496] and placenta [495] has been purified to apparent homogeneity. The native enzyme is composed of two identical subunits and has a molecular weight of approximately 117 000 [495]. In vitro, a-galactosidase B probably functions as a-N-acetylgalactosaminidase [495,498]. 7. I . 7. a-Fucosidase The enzyme (a-L-fucoside fucohydrolase) is active towards natural oligosaccharide and glycosphingolipid substrates, and hydrolyzes both fucosyl d - 2 and al-6 linkages. A typical deficiency of a-L-fucosidase activity can be demonstrated in serum, leukocytes, fibroblasts and tissues from patients with the infantile, late infantile and juvenile forms of fucosidosis [499-5021. The accumulated materials have been identified as blood group H glycolipid [503] Gal a1 + 3GalPI 2
+
3(and 1 + 4)GlcNAc/Il+ 3GalPl- 4Glc -+ Cer
t
a1 Fuc
a mixture of oligosaccharides having an a-fucosyl linkage at their nonreducing termini [503,504], as well as Lea and Leb glycolipids [505]. In the urine of a patient with fucosidosis, 22 glycopeptides have been identified [506]. Fucosidase which is located in lysosomes has been purified from human liver [507]. The native molecular weight of the enzyme is 230000 and, it is composed of four identical subunits of 50000 molecular weight. The pH of optimal activity is around 4-5. Multiple isozymic forms of fucosidose enzyme have been reported [508-5111. These heterogeneities seem to be due to variable sialylation of the enzyme. 7.2. ACT1 VATOR PROTEINS FOR HYDROLASES AND TRANSFER PROTEINS
The degradation of GSLs by water-soluble lysosomal glycosidase requires the presence of either detergents or specific water-soluble proteins that facilitate the interaction between the enzymes and their lipid substrates [512]. Activators that stimulate the enzymatic hydrolysis of 3-sulfo-galactosylceramide[512-5141, glucosylceramide [471,5151, ganglioside I1 NeuAc-Gg3Cer [516-5211 and I1 NeuAcGg,Cer [522,523] have been highly purified. However, whether or not one given activator can stimulate the hydrolysis of only a GSL substrate or also many others is still unknown. The activator forms a complex with the lipid molecule, but again, whether or not it forms a complex with the enzyme remains to be elucidated. Several cases of storage diseases due to activator protein deficiency have been reported, and these will be discussed in this section. Recently Conzelman and h s group [524]
56 found that the activator protein for the hydrolysis of ganglioside 113 NeuAc-Gg,Cer acts in vitro as a GSL transfer protein in the absence of enzyme. This result will also be described in this section. 7.2.1. Activator protein for the hydrolysis of ganglioside I13Newic-Gg,Cer A low molecular weight glycoprotein obtained from human liver [516] stimulated Hex A but not Hex B to cleave ganglioside I13NeuAc-Gg,Cer. The molecular weight of the activator protein was determined to be 36000 by Hechtman and LeBlanc [516] and 22000-25 000 by Conzelmann and Sandhoff [517] and Li et al. [521]. The isoelectric point of this activator is 4.75. The activator has strict specificities towards both the enzyme and the substrate. Hechtman and LeBlanc [516] reported that the activator could not stimulate the hydrolysis of gangliotriaosylceramide by either Hex A or B. They suggested the presence of an enzyme-activator complex. On the contrary, Conzelman and Sandhoff [518] reported that ganglioside I13NeuAcGg,Cer, and its asialo form, and globotetraosylceramide were degraded by Hex A in the presence of the activator. They suggested that the activator formed complexes with lipid molecules but not with the enzyme. In several patients affected with the variant AB of infantile “G M2 gangliosidosis”. normal activities of P-hexosaminidases were found towards synthetic substrates, but no activity was noted towards the ganglioside II3NeuAc-Gg,Cer [517]. Cerebral accumulation of ganglioside Il’NeuAc-Gg,Cer was noted in these patients. Conzelman and Sandhoff ascribed this accumulation to severe deficiency of P-hexosaminidase activator proteins [517]. A similar case has been reported by Hechtman et al. [519]. The activator protein could extract glycolipid monomers from micelles and liposomes and yielded watersoluble complexes consisting of one mole of GSL per mole of activator protein [518,5241.
7.2.2. Activator protein for the hydrohsis of P-glucosides An activator protein (coglucosidase) which is effective in facilitating glucosylceramide P-glucosidase activity was discovered in the spleen of a patient with Gaucher’s disease [515,573]. The activator is a glycoprotein and has a molecular weight of about 6000 and a p l of about 4.3-4.4 [525]. The protein stimulates the glucosidase activity towards the unnatural substrate 4-methylumbelliferyl P-glucoside [525] as well as the natural lipid glucosylceramide. In this respect it is distinguished from the P-hexosaminidase activators described in Section 7.2.1. 7.2.3. Activator protein for the hydrolysis of ganglioside I13NeuAc-Gg,Cer The activator stimulates the enzymatic hydrolysis of ganglioside I13NeuAc-Gg,Cer catalyzed by human P-galactosidase. The activator is a heat stable glycoprotein and withstands heating in a bath of boiling water for 30 min at pH 7.0. The molecular weight is about 22000 and the p14.1 [522]. P-Galactosidase activator for ganglioside II3NeuAc-Gg,Cer forms complexes with the ganglioside but not with P-galactosidase [523]. It also enhances the hydrolysis of globotriaosylceramide catalyzed by human a-galactosidase and, to a smaller extent, the hydrolysis of ganglioside I13NeuAc-Gg,Cer catalyzed by human Hex A [747].
57 7.2.4. Activator for atylsulfatase A A heat stable protein “activator” for arylsulfatase A is present in tissues of normal individuals and patients with metachromatic leukodystrophy [514]. This is a nonenzymatic glycoprotein with a molecular weight of 21 500 and p l of 4.3. The activation effect for the arylsulfatase A is more prominent with this activator than with bile salts such as taurocholate [513,514]. Several cases [492,493] of sulfatidosis without arylsulfatase A deficiency have been reported. In all those cases the arylsulfatase activity in leukocytes, fibroblasts and urine was above the control range or reduced to only one half of the normal controls. The properties of the arylsulfatase A from the patients’ fibroblasts were identical to those of normal fibroblasts. However, the hydrolysis of 3-sulfo-galactosylceramide by the cultured skin fibroblasts from the patients was markedly reduced. Supplementation of the fibroblasts from one case with activator proteins corrected the defect in 3-sulfo-galactosylceramidehydrolysis [526]. This indicates that the cause of the disease is the deficiency of the activator protein.
7.2.5. Transfer proteins A nonspecific transfer protein from bovine liver has the ability to accelerate the transfer of glycosphingolipids, especially globotetraosylceramide [527]. The transfer proteins may exist in the same compartments in which the synthesis of complex oligosaccharides takes place. Senyal and Jungalwala [528] also reported the transfer protein for galactocerebroside, sulfatide and ganglioside I13NeuAc-Gg,Cer. Metz and Radin [529] purified a cerebroside transfer protein from a cytosolic extract of bovine spleen. The molecular weight of the active protein was about 20 300, and the protein facilitated the transfer of glucosylceramide, galactosylceramide and lactosylceramide from liposomal vesicles to red cell ghosts. However, the protein did not facilitate the transfer of lecithin, cholesterol or ceramide. These data suggested this protein might stimulate the enzymes which act on cerebrosides and lactosylceramide to form sialosylgalactosylceramide, ganglioside I13NeuAc-Gg,Cer and trihexosylceramide. Abe et al. also reported a transfer protein which facilitated the transfer of galactosylceramide, lactosylceramide and glucosylceramide [530]. 7.3. METABOLIC DISORDERS OF GLYCOSPHINGOLIPIDS
Lieb [451] reported for the first time a case in which spleen of a patient with Gaucher’s disease accumulated the lipid glucosylceramide instead of the then known galactosylceramide. The concept that the particular glycolipids which accumulate in the tissues of patients with glycosphmgolipidoses are normally existing ones, even if only in very small amounts, has been firmly established by extensive studies on both pathological and normal tissues. As to the metabolic dysfunction of Gaucher’s disease, Brady et al. [452] as well as Patrick [453] demonstrated the defect to be in a P-glucosidase, whch normally hydrolyzes glucosylceramide in the spleen and liver. On the other hand, a lack of arylsulfatase A activity was verified in tissues from patients with an inherited sulfatidosis by Austin et al. [454] and Mehl and Jatzkewitz
58 TABLE 1.6 Characteristics of the sphingolipidosis Deficient enzyme or activator
Name of disease
Principle glycolipid accumulated
Genetic trait
Acid ceramidase
Farber’s disease Ceramidosis
Ceramide
Autosomal recessive
Galactosylceramide 8-galactosidase
Krabbe’s disease, globoid cell leukodystrophy, Galactosylceramidosis
Galactosylceramide
Autosornal recessive
Ganglioside I13NeuAcGg,Cer galactosidase
GM, gangliosidosis, Type I (infantile type) Type 11 (juvenile type) Type 111 (adult type)
II’NeuAc-Gg,Cer
Autosomal recessive
8-Hexosaminidase A
Tay-Sachs disease, GM gangliosidosis, Type I
I13NeuAc-Gg,Cer
Autosomal recessive
/3-Hexosaminidases A and B
GM ganghosidosis, Sandhoff disease, Type I1 (infantile type)
11’NeuAc-Gg,Cer
8-Hexosaminidase A
GM, gangliosidosis, Type Ill (juvenile type) Type IV (adult type)
1I3NeuAc-Gg3Cer
8-Hexosaminidase activator protein
A( + )B( + ) variant of infantile GM gangliosidosis
Glucosylceramide 8-glucosidase
Gaucher’s disease, glucocerebrosidosis, Type I (adult, chronic nonneuropathic form) Type 11 (infantile, acute neuropathic form) Type Ill (subacute, neuropathic form)
Glucosylceramide
Autosomal recessive
Arylsulfatase A
Metachromatic leukodystrophy, sulfatidosis Adult type Late infantile type Juvenile type Congenital type
Sulfogalactosj ceramide
Au tosomal recessive
3-Sul fogalactosylceramide
Activator for arylsulfatase A Arylsulfatase A, B and C
Multiple sulfatase deficiency
3-Sulfogalactosylceramide, sulfated glycosaminoglycans
a-Galactosidase A
Fabry’s disease
Globotriaosylceramide, X-linked galabiosylceramide
59
[260]. Following these investigations a number of sphingolipidoses were proved to be due to defective specific lysosomal hydrolases. Most of the studied glycosphingolipidoses including ceramidosis are listed in Table 1.6, and the biochemical properties of each enzyme involved was discussed in the previous sections. Studies on GSL storage diseases brought about important progress in the understanding of the catabolism of GSLs as well as other glycoconjugates. Several disorders were shown to be due to the deficiencies of the glycosidases and sulfatases, whch resulted in the accumulation of GSLs. An excellent review of these metabolic disorders has been published [531]. Some biochemical characteristics of sphingolipidoses, of which hydrolases and activators were described in this section, are reviewed in Table 1.6. The stored GSLs in these diseases are described in Section 5.
8. Glycosphingolipids in immunologV Essentially all of the GSLs are immunologically active both in haptenic reactivity in vitro and in antibody-producing potency. Much of the current interest in GSL research has been directed to characterization of antigenic GSLs, such as those belonging to human blood group systems, and to the study of immunobiological properties of GSLs of known chemical structure. In many cases, the carbohydrate antigens are distributed both in glycoproteins and in glycolipids. Studies on the antigenicity of GSLs from tumors were initiated by Rapport et al. [170]. Many antigenic saccharide determinants, except for Ii antigens (Section 8.1.3), reside in nonreducing termini of the carbohydrate chains. It is thus known that multiple GSLs may display similar antigenic behavior if they possess a common oligosaccharide residue reactive to a specific antibody but differ from each other in the structures of their residual chains. Examples of such structural polymorphism of GSL antigens are seen in Table 1.4. The addition of inert lipids called as auxiliary lipids, such as phosphatidylcholine and cholesterol, to some haptenic GSLs in an antigen-antibody reaction often strengthens to some extent the haptenic reactivities (for a review, see Ref. 690). For manifestation of antigenic reactivity of haptenic oligosaccharides they must be used in fairly large quantities, either on a weight or a molar basis, relative to the intact whole antigens (for a principle of assay of antigenic potency, see Section 8.1.1.). For instance, blood group A glycoprotein showed antigenicity at only 0.01 yg in a hemagglutination inhibition test, while 30 yg of an A determinant trisaccharide, GalNAca -+ 3Ga1(2 + laFuc), was required to attain the same reactivity [532]. About lo3 times more IV3GalNAca-globotetrao~ethan IV3GalNAccu-Gb,Cer (Forssman antigen) is necessary for display of the same haptenic reactivity [2]. N-Deacylated GSLs (“lysoglycolipids”), which have marked hemolytic properties, gave rise to nonimmune precipitation with low density lipoproteins of nonimmune serum [l].On the other hand, a triglycosylceramide (11-2 in Table 1.4) [282], an H-active substance with the lowest molecular weight in the intact ABH antigens, exhibits a potency almost comparable to that of pentaglycosylcera-
60
mide (11-10 in Table 1.4) [290], and with branched decaglycosylceramide (11-20 in Table 1.4) [299] in the inhibition of hemagglutination of group 0 red cells by Bombay (Oh) serum. These observations suggest that, irrespective of the carbohydrate chain length, the lipid moiety in GSL antigens, as well as the peptide moiety in glycoprotein antigens, markedly strengthens the antigenic potency in vitro and probably in vivo. Antigenic expression of glycoconjugate antigens is determined by the presence or absence of genetically defined glycosyltransferases which are responsible for synthesis of the specific saccharide structures (see Section 6). The genetics of those glycosyltransferases related to blood group antigens was reviewed by Watkins [533] and others [534,544]. 8.1. H U M A N BLOOD GROUP SYSTEMS
In addition to those of blood group ABH and Lewis systems, antigens of P and Ii systems were recently described as GSLs. In an immunologically defined system, two or more related carbohydrate antigens often bear a precursor-product relationship; for example the H antigen is a precursor of A and B antigens. In other cases, an antigen may share a precursor-product relationship in two different immunological systems; for example P antigen (globotetraosylceramide) is a product of the precursor Pkantigen (globotriaosylceramide) in the P blood group, and also a precursor of Forssman antigen (IV3GalNAca-Gb,Cer), a heterophile antigen which does not belong to human blood group. Some antigenic GSLs possess two antigenicities belonging to two different blood group systems (refer to Table 1.4). An octadecaglycosylceramide from hog gastric mucosa was noted to have both A and H determinants [535], although the presence of both A and H determinants in a molecule has not been detected in the type-A GSL variants from human erythrocytes [311]. 8.1.1. ABO system
Recent investigations [536,537] have assigned the human ABH locus to chromosome 9. The H gene gives rise to a saccharide structure which is the immediate precursor of the A and B determinants; hence in group 0 persons the H-determinant does not undergo further change. ABH antigens occur in human erythrocytes, and possibly also in other cell types having ABO antigenicity in the forms of low-molecular weight GSLs [538], macroglycolipids (Section 5.8), mucin-type glycoprotein [539] present in glycophorin [323], and asparagine-N-linked glycoproteins [540] having a polylactosamine structure (erythroglyan [541]). There appears to be no consistent view about which category of the antigens is the major component in the cells and plays an essential role in the manifestation of antigenicity on the cell surface. For routine assay of the blood group activity of a GSL, hemagglutination inhibition of erythrocytes by the corresponding isoantibodies has been adopted, and the potency of the haptenic GSL is expressed in terms of the minimum quantity required to inhbit the reaction. Ulex europaeus lectin, eel serum or serum of an individual with Bombay (Oh) type is used as anti-H hemagglutinin. However, U.
61
europaeus lectin reacts exclusively with H antigens having type 2 chains [542]. The potency of the H-active GSLs assayed by the use of these “hemagglutinins” is rather weak as compared to those of A and B antigenic GSLs, and is abolished by the addition of a large excess (e.g., 20 times that of globotetraosylceramide [301]) of inert GSLs. Because the concentrations of blood group ABH-antigenic fucolipids in erythrocytes are very low, for example 128 pg of B-active GSLs compared to 9430 pg of globoside from 100 ml of packed B-erythrocytes [71], the early studies on saccharide structures responsible for the antigenic reactivities of blood group antigens were performed on glycoproteins and oligosaccharides obtained from secretions (for reviews, see Refs. 533, 534). Yamakawa and his collaborators found ABH antigenic specificities in a crude globoside fraction [545] which proved later, by column chromatographic fractionation on silicic acid [546], to contain more polar, antigenic fucose-containing GSLs in addition to globoside; the glycoprotein antigens and oligosaccharides were retained on the column, which suggested that the antigenic nature of the crude fraction was due to GSLs but not to other glycoconjugates [547]. Through extensive fractionation studies of blood group-active fucolipids from human erythrocytes [273,549,749]and tumor tissues [549,550], the ABH isoantigens were established to be indeed GSLs. The groups of Hakomori [550,551], McKibbin [552], Koscielak [300] and Karlsson and others purified fucolipids with ABH specificity and Lewis specificity, and contributed further examples of the structural polymorphism of the blood group GSL antigens. It is now recognized that fucolipids containing a GalNAcal --* 3Ga1(2 + laFuc) residue at the nonreducing terminus possess blood group A antigenicity; those that contain Gala1 3Ga1(2 + laFuc) possess the B antigenicity, and those that contain Fucal + 2Gal possess the H specificity. In addition, a saccharide structure lacking an a-fucosyl residue but possessing a galactosyl residue linked through a1 -+ 3 at the external N-acetyllactosamine unit expresses the B specificity, although the B-haptenic potency is 103-times lower than that of the fucosylated saccharides. GSLs with such a structure (1-18 and 1-23 in Table 1.3) were isolated from rabbit erythrocytes (Section 5.5). The chemically defined ABH-antigenic GSLs are tabulated in Table 1.4. Although sole saccharide chains of the antigenic GSLs from human red blood cells belong solely to the neolacto series, those from other sources include, in addition, core chains of the lacto, globo and ganglio series. A B-specific fucohexaosylceramide with an isoganglio core (11-9 in Table 1.4) in which the N-acetylglucosamine typically found in B-active GSLs of the lacto (11-12 in Table 1.4) and neolacto series was replaced by N-acetylgalactosamine had the same B activity level as B-active GSLs belonging to the neolacto series [289]. There exist two sub-groups, A, and A,, in blood group A [533]. Certain antisera (anti-A,) and Dolichos biflorus lectin [553] react only with A, erythrocytes, and A, erythrocytes have higher H activity than A, cells. The question of whether the difference between A, and A , is qualitative or quantitative has generated much discussion. Since all A-active GSLs present in erythrocytes are based on type 2 chain, the two sub-groups do not represent a difference in the type of chain to which
-
62 the A determinant is linked. Complex A-glycolipids (11-35 and -36 in Table 1.4) possessing two A determinants are more reactive than a less complex A-glycolipid (11-26 in Table 1.4) with one A determinant [311]. Fukuda and Hakomori [311] observed that, although the structures of A-antigenic GSL variants (11-24, -35, and -36 in Table 1.4) did not differ between A, and A, erythrocytes, the GSL pattern on a thin layer chromatogram showed that A, cells contain larger amounts of complex A variants (11-35 and -36 in Table 1.4) and smaller amounts of complex H variants (11-28 and -34 in Table 1.4) as compared to A, cells. A study of the binding of ferritin-labeled lectin from D. biflorus to red blood cells revealed that the A, cells contain 5-times more A-determinant than do the A, cells [554]. Alternately, activity level and qualitative differences between a-N-acetylgalactosaminyl transferases (A enzymes) acting on the H-determinant may result in the formation of different A antigens, A, and A,. The A enzyme from milk of A, and A, individuals exhibited the identical substrate specificity when either H-active oligosaccharides [555] or glycophorin [556] were used as acceptors, and the maximum incorporation of N-acetylgalactosamine into A-specific mucin-type oligosaccharide products derived therefrom, and their distribution patterns, were the same [556]. However, using H-active oligosaccharides, A, and A enzymes partially purified from human sera [557,558] and ovarian cyst [558] of the respective sub-groups were shown to be different from each other in K,, value, pH optimum and cation requirement [557] and in isoelectnc point [558]. Fuji and Yoshda [559] demonstrated that the A , enzyme highly purified from sera of the A , subjects converted the 0 and A, erythrocyte membranes, which were prepared freshly, to A,, and the A, enzyme converted the 0 membranes to A, which contained lower amounts of incorporated N-acetylgalactosamine than did the products of A, enzyme. The membrane products formed with A, enzyme were separated into two or three major components by isoelectric focusing, 80% of whch were assumed to be GSLs based on the results of Folch's partition, whereas only one component was formed with A, enzyme. H-active GSL variants however have not been examined for acceptor specificity of A, and A, enzymes. The observations described above imply that the disparity between A, and A, sub-groups may be ascribed to one or more differences in the properties of A , and A, enzymes, the quantitative distribution of A and H antigen GSLs, and the organization of GSLs in membranes. More comprehensive studies taking into consideration also macroglycolipids and erythrocyglycan, which were noted to exist in higher levels in red cells than do low-molecular weight GSL antigens, appear to be necessary to establish a definite conclusion.
,
8.1.2. Lewis system
The classical Lewis system consists of Lewis a (Lea) and Lewis b (Leb) antigens. This isoantigen system is related to the genetically determined secretion status of the ABH antigens in body fluids [560]; individuals who have Lea antigen on their red cells are non-secretors of ABH antigens, whereas those who have Leb antigen are secretors of ABH. In about 10%of individuals, the erythrocytes have neither Lea nor
63 Leb (Le(a-b-)). The Lea and Leh antigens on the erythrocyte surface are not original constituents of the cell membrane but are acquired from blood plasma [561]. Marcus and Cass [562] demonstrated that these antigens are GSLs in nature, and are acquired from low and high density lipoproteins in the plasma. Gastrointestinal mucosae are rich in Lewis fucolipids (Table 1.4), and hence these tissues are postulated to be the site of synthesis of Lewis GSLs. Macroglycolipids do not have Lewis specificity [13]. Hakomori and Strycharz [550], Smith et al. [292], and Hanfland [563] isolated Lewis-antigenic GSLs from human erythrocytes, small intestine and plasma, respectively. A variety of Lewis-related GSLs have recently been detected in human cancer tissues as tumor-associated antigens (refer to Section 9.2). As was established first [ 3031 for Lewis-antigenic human milk oligosaccharides [744,745], the Lea antigens possess a determinant structure of GalPl -, 3GlcNAc(4 + laFuc) and the Leb antigens that of Fucal 2GalP1 -, 3GlcNAc(4 + 1aFuc) at their nonreducing termini. Although both the Lea- and Leb-antigenic saccharides were originally identified to be derivatives of type 1 chains, a fucopentaosylceramide (11-24 in Table 1.4) having a type 2 chain, in which fucose linkage to N-acetylglucosamine is by al-3 bonding instead of al-4, was recently described as Le"-like [280], or the Lea isomer fucolipid [304]. However, the compound does not cross-react with anti-Lea, and the a-3-fucosyltransferase is not a product of a gene at the Lewis locus. The nomenclature such as Lea isomer or Leb isomer fucolipid (11-27 in Table 1.4) is confusing and incorrect. The same compound (11-24 in Table 4) isolated from human adenocarcinoma [37,564] was noted to have neither Lea nor Leh reactivity and was called X hapten [37] or Le' [565] or Le". Serologically, the antigenicity of Le(a-b-) antigens on red cells may be classified into LeC and Led [560], and their carbohydrate structures have been predicted to have type 2 chains: GalPl 4GlcNAc(3 + 1aFuc)- for Le' and Fucal 2GalPl + 4GlcNAc(3 + 1aFuc)- for Led [565]. Led antisera against putative Led antigens are subdivided into those which commonly react with red cells from all Le(a-b-) secretors, and those whose Lewis specificity depends additionally on the ABH secretion status [565]. The latter antisera, designated anti-LedH,agglutinated strongly red cells from OLe(a-b-) secretors, and agglutinated weakly cells from A 2Le(a-b-) secretors. Recently, Hanfland and Graham [291] demonstrated LedH antigen to be 1114-a-fucosyl-lactotetraosylceramide(11-11 in Table 1.4), which was purified from plasma of OLe(a-b-) secretors, based on the results of serological experiments with this lipid. This fucopentaosylceramide failed to react with Ulex europaeus lectin which has specificity only for H antigens with type 2 chains. The occurrence of LedH antigenicity was demonstrated in human lung squamous cell carcinoma irrespective of blood group status of the hosts [732] (Section 9.2). The structure-antigenicity relationship of another Led antigen, which might have a determinant structure containing two fucosyl residues described above, is not known. -+
-
-+
8.1.3. li System
Most human sera contain low titers of a cold hemagglutinin designated anti-I. This
64 agglutinin reacts best at 4 ° C and much more poorly at 37°C. Rat sera on the other hand do not contain anti-I, but have another hemagglutinin that is called anti-i. High titers of anti-I hemagglutinin are present in patients suffering from acquired hemolytic anemia (chronic cold agglutinin disease), which has many features of Waldenstroem macroglobulinemia. Such serum frequently is utilized for I-specific hemagglutination assays. These patients usually produce monoclonal hemagglutinins which are specific in terms of the Ii system, although each may react with one or several, but not all, of the variants of I antigen. Erythrocytes of most adults possess I antigen reactive to anti-I, while those of rare adults, of embryos, and of the newborns display only i antigenicity. The Ii blood group system, therefore, is of an autoimmune type in its immunological nature (for reviews, see Refs. 534, 567). Marcus et al. [568] demonstrated that treatment of I-erythrocytes with a Clostridium perfringens filtrate rich in P-galactosidase and P-N-acetylglucosaminidase destroyed the I antigenicity. Feizi, Kabat and associates found that ovarian cyst glycoproteins which did not have ABO and Lewis antigenicities reacted with certain anti-I sera [569], and the cyst glycoproteins having blood group A 0 specificities revealed I antigenicity after treatment with periodate [570]. This led to the speculation that the I-antigenic structure, therefore, presented a possible precursor of the ABH antigens. These and other observations established that the determinants of Ii antigens were carbohydrate in nature. Through investigations of several oligosaccharides which were derived from glycoproteins [570] or chemically synthesized [571], it could be demonstrated that the type 2 chain sequence of the neolacto series was recognized by certain anti-I antisera. More detailed chemical structures of Ii-antigenic determinants were furnished by antigenic analyses of the Ii-GSLs of erythrocytes by Hakomori et al., Feizi et al. and others. Human macroglycolipids [52,316] and H-active decaglycosylceramide [307] (11-28 in Table 1.4) and rabbit B-active decaglycosylceramide (1-23 in Table 1.3) [251] carry I antigenicity in addition to ABH antigenicity. The minimum structural requirement for the i antigen is a straight carbohydrate chain consisting of two Pl + 3 linked N-acetyllactosamine units in neolactohexaosylceramide, such as GalPl 4GlcNAcP1+ 3GalPl -+ 4GlcNAcPl- 3GalPl 4GlcP1 lCer [572]. Neither neolactotetraosylceramide nor IV'-(GalPl 4GlcNAc)P-nLc4Cer expresses i antigenicity. It is noteworthy that an H-active VI'Fuca-nLc,Cer structure (11-28 in Table 1.4) does not display i antigenicity, whereas a terminal sialyl a2 3 substitution of the same structure results in low but definite expression of i antigenicity [41,572]. On the other hand, the I antigenic determinant resides in structures having a branched N-acetyllactosamine. The B- and I-active branched decaglycosyl ganglioside of human red cells has the following structure [41] -+
-+
-+
-+
-+
NeuAcal2 Gala1
-
--f
3Gal/31-+ 4 G l c N A c b l ~ ,Gal/?l-+ 4GlcNAcb1+ 3GalPl 3Gal/31+ 4GlcNAcblp
--t
4GlcDl-+lCer
This ganglioside (structure 1) was modified by treatment with various exoglycosi-
65 dases. and the obtained derivatives were examined for this reactivity with eleven different anti-I sera [41,574]. The intact ganglioside (structure l), its desialylated derivative (structure 2), and the derivative (structure 3) freed from both a-galactosyl and sialyl residues at the termini (the unsubstituted N-acetyllactosamine) reacted with the majority of the anti-I sera. Therefore, this structure (3) represented the most reactive one with regard to both the range of different anti-I sera (10 out of 11 sera) as well as the potency [574]. No two anti-I sera were strictly identical in their reaction patterns. However, specificities could be classified into three main types. The first type recognized the N-acetyllactosamine (LacNAc) at the fll-6 branch, as in IV'LacNAcfl-, IV'GlcNAcfl-nLc,Cer (reactive with two of 11 anti-I sera) and IV' LacNAcfl-nLc,Cer (two of 11 antisera). The second reacted with N-acetyllactosamine at the fll-3 branch, as in IV3LacNAcfl-, IV'GlcNAcfl-nLc,Cer (four of 11 antisera) but not with IV 'LacNAcfl-nLc,Cer. The third required both N-acetyllactosamine branches, as in structures 1, 2 and 3 quoted above. None of eleven anti-I sera reacted with either IV3~6-bis-GlcNAcfl-nLc4Cer or IV'-GlcNAcfl-nLc,Cer. Structural differences between I and i antigens were also evidenced in macroglycolipids. Koscielak et al. [321] observed that macroglycolipids fractionated from human I-erythrocytes contained the average 30 glycoses and about five branching points, whereas the complex GSL fractions which were prepared by the same method from erythrocytes of cord blood and i-individuals contained six and 15 glycoses, respectively, and only 0.6 unit of branching point. The fact that the anti-I antibodies available are usually monoclonal, and every anti-I therefore recognizes a different, narrow region of the integral I determinant structure, will presumably explain the observed microheterogeneity in the I specificity of respective I antigens. 8.I.4. P system
The P system consists of three antigens, P k , P and PI. Five phenotypes, PI, P2, P:, Pl and p are now recognized, based upon the presence or absence of the three distinct serological specificities (Table 1.7) (reviewed, in Refs. 533, 575). Subjects with phenotypes Pz and Pt have no PI specificity on their red cells, the P,k and P i phenotypes have no P, and p does not have any P specificity. Naturally, individuals having the antigens on their red cells do not contain the corresponding isoantibodies in their sera, and hence only P subjects contain all three isoantibodies. Hydatid cysts from livers of sheep infested with Echinococcus grunulosus contain glycoproteins specific for PI as well as Pk antigenicities [533]. Partial acid hydrolysis of the glycoproteins yielded a P,-active trisaccharide, Gala1 + 4Galfl1 + 4GlcNAc, which was identified as the PI determinant by Cory et al. [245]. The antigens of human red cells were identified by Naiki, Marcus and their colleagues as globotriaosylceramide for P antigen, globotetraosylceramide (globoside) [577] for P, for P, [578] (Sections 5.3 and 5.5). and IV4-galactosyl-a-lactoneotetraosylceramide and the immunological properties of the antigens and the isoantibodies were characterized [579,580]. The antigenic expression of the P blood group system in individuals was substantiated by biochemical studies. The rare Pk erythrocytes [581,582], as well as the
66
fibroblasts [583], of P,k and P l phenotypes lack globoside, which is the most abundant GSL in P, and P2 red cells. In addition, the Pk cells possess a comparably hgher amount of globotriaosylceramide, while p cells contain virtually no globoside or globotriaosylceramide. “Small p” cells instead accumulate lactosylceramide and gangliosides, II’NeuAca-LacCer and IV’NeuAca-nLc,Cer. Erythrocytes from P, individuals were shown to contain more globotriaosylceramide and less lactosylceramide than did erythrocytes of Pz individuals [584]. As for the biosynthesis of the antigens in the P system (see also Sections 6.4 and 6.5), lactosylceramide is converted to globotriaosylceramide by an a-4-galactosyl transferase I. The P product is subsequently elongated to the P-substance, e.g., globotetraosylceramide by a ~-3-N-acetylgalactosaminyltransferase, while PI antigen can be synthesized from lactoneotetraosylceramide by an a-4-galactosyltransferase 11, of which activity has not been examined for the expression of the PI antigenicity. Since the PI and Pk antigens possess the same nonreducing terminal structure, Gala1 + 4GalP1 -,4R,it is possible that both the antigens are synthesized by the same a-galactosyltransferase. However, the existence of the Pz and P! phenotypes (Table 1.7) seems to suggest that the two antigens are synthesized by two different 1 and 11 enzymes from their respective immediate precursors. Enzymatic evidence exists for the manifestation of P k and p phenotypes [583]: Human P; fibroblasts lack the /3-N-acetylgalactosaminyltransferase, and those of p are deficient in both the a-galactosyltransferase I and the P-N-acetylgalactosaminyltransferase activities (Table 1.7). On the other hand, Fellous et al. [585]
TABLE 1.7 GSL antigens, related enzymes and antibodies in blood group P system Phenotype (frequency)
Antigens on erythrocytes
p (very rare) None P: (very rare) P k (Gb,Cer) P: (very rare) P k , P, (IV4GalanLc,Cer) Pk,P(Gb,Cer) Pz (251%) Pk. P. PI PI (75%)
Final GSL products LacCer Gb,Cer Gb,Cer IV4Gal a-nLc,Cer
Gb4Cer Gb,Cer I V ‘ G ~ a-nLc4Ccr I
Synthetic enzymes Gal(a1-4)transferase I -
a
~
+ +
-
+
+
+
Antibodies i n
GalNAc(B1-3)- Serum each phenotype transferase h
~
+
Anti-P, P Anti-P Anti-P
%‘
-P,
Anti-PI None
Gal( al-4)-transferase I acts on synthesis of Gb,Cer from LacCer. The activity of a putative cwyme, Gal(al-4)-transferase I1 which will catalyze synthesis of IV4Gala-nLc4Cer from nLc4Cer has not heen examined. GalNAc( /31-3)-transferase catalyzes synthesis of Gb4Cer from Gb,Cer. Anti-PIPkis cross-reactive with both PI and P k antigens, but separated into high affinity antibody which reacts with all phenotype cells except for p cells, and low affinity antibody which reacts only with P k cells (see text). The amount of Gb,Cer in Pk phenotype cells is 10-times more abundant than that in P, or P2 cells. I n sera of P:-persons. anti-PI has not been reported.
67 demonstrated that 30-40% of the hybrid cells created by fusion of p and Pk fibroblasts expressed the P antigenicity 3-4 days after hybridization, while about 60% of the hybrids of P2 and p cells expressed P antigenicity immediately after the fusion. Since the possibility of an inhibitor of the P-N-acetylgalactosaminyltransferase was eliminated by experiments with mixed preparations of p and P2 cells [583], a plausible explanation for these observations is that the gene coding for this enzyme is present in the p cells, and the enzyme is induced in P k p hybrids through the supply of globotriaosylceramide, after a time lag. Another P-like antigen of human erythrocytes, IV3-N-acetylgalactosaminyl-~-neolactotetraosylceramide (I21 in Table 1.3), was recently found [248], but a similar GSL exists in p-erythrocytes. The antigenicity of globotetraosylceramide has been established by the raising of antibodies against this lipid [192,587]. Although intact human fetal erythrocytes are agglutinated by anti-globoside antiserum, the hemagglutination of adult erythrocytes takes place only after the cells are treated with protease, which suggests that the adult cells are covered with proteins [587]. Sera containing monoclonal IgM paraprotein from individuals with Waldenstroem’s macroglobulinemia reacted with GSLs having N-acetylgalactosamine at their nonreducing termini, such as in globotetraosylceramide, gangliotriaosylceramide [586] and IV3GalNAca-Gb,Cer [586,588]. Similarly, sera from persons with paroxysmal cold hernoglobinuria, an autoimmune hemolytic anemia which occurs sometimes in certain viral and syphilis infections, contained antibodies against these GSLs [589]. Rabbit IgG antibodies against GSLs containing terminal N-acetylgalactosamine were specific for the respective GSLs although anti-globoside cross-reacted partially with gangliotetraosylceramide [590]. Anti-globotriaosylceramide antibodies raised in rabbits were prepared, characterized [591,592], and partially purified as IgM [592]. Of the isoantibodies present in the sera of the p-type human individuals (Table 1.7), all anti-Pk antibodies are cross-reactive with P, GSL, whereas the anti-P, antibodies present in the sera of the P,-type individuals are not cross-reactive with Gb3Cer [593]. In addition, most of anti-Pk, which have high affinity for a Pk oligosaccharide, are absorbed with P, erythrocytes. The antibodies which have been absorbed with P, erythrocytes react specifically with P k erythrocytes, though the titer of the antibodies is lowered [593]. 8.2. HETEROPHILE ANTIGEN AND ANTIBODY RELATED TO GLYCOSPHINGOWPIDS
Antibodies raised against an antigen derived from one particular species also react with the same antigen present in other species. Such an antigen was designated initially a heterogenetic antigen which is now called a heterophile antigen, since at an early time most antigens had been regarded as species-specific. Sera of some patients contain heterophile antibodies produced against antigens derived from other species, as will be described below. Forssman antigen was found to be a common antigen in guinea pig tissues and sheep red cells, as injection of a saline extract of guinea pig kidneys into rabbits led to the formation of an antiserum which lysed sheep red cells in the presence of complement [ 5941. According to early immunological work, mammals could be classified into Forssman-positive and negative species with respect to the presence or
68 absence of this antigen in their tissues or red cells, whereby man belonged to the negative species (reviewed in Ref. 595). However, recent studies have demonstrated that normal and cancer tissues of humans contain small amounts of Forssman antigen (see Section 9). After early studies the chemical identity of Forssman antigen, was suggested to be that of a complex lipid [596] or complex carbohydrate [597]. The antigen of sheep erythrocytes was established to be a GSL by Papirmeister and Mallete [598] and others [599,600]. The immunochemical properties of the antigen purified from equine organs were characterized [601]. Molecular polymorphism of Forssman antigen (1-3 in Table 1.3) was noted; the antigenic determinant, GalNAcal-3GalNAc, can be linked to a core chain different from that of IV’GalNAca-globotetraosylceramide. Since Forssman antigen (1-10 in Table 1.3) and blood group A antigen (Table 1.4) both possess a GalNAcal-3 pyranosyl linkage in their antigenic determinants, it seems plausible that these antigens would cross-react. In fact, Forssman antigen partially reacted with anti-A antibodies [601-6031, whereas Forssman antibodies, either raised in rabbits against Forssman antigen, or present in human sera at low titer [602-6041, scarcely reacted with human A red cells. Forssman antigen could be one of the antigens specific for a developmental stage: a monoclonal antibody recognizing this antigen reacted with early murine blastocysts of 3-4-day-old embryos as well as murine embryonal carcinoma cells [605]. A certain class of heterophile antibodies is present in patients who received therapeutic injections of foreign serum [606,607]. These antibodies called serum sickness antibodies or Hanganutziu-Deicher (H-D) antibodies, react with erythrocytes from various animal species such as horse, sheep, cow and rabbit. Using a specific inhibition test of equine hemagglutination with H-D antisera, Higashi et al. [608] found that gangliosides containing N-glycolylneuraminic acid at their non-reducing termini, such as 113NeuGca-LacCer (hematoside) and IV’NeuGca-nLc,Cer, as well as a mucin-type glycopeptide containing NeuGca2 + 3GalDl -+ 3(NeuGca2 6)GalNAc -,Thr (or Ser) [609], inhibited the hemagglutination and formed precipitating lines with H-D antisera in immunodiffusion, whereas gangliosides containing N-acetylneuraminic acid were totally inactive. An equine erythrocyte hematoside containing 4-0-acetyl-N-glycosylneuraminicacid did not exhibit H-D antigenicity [ 1011. Paul-Bunell antibodies [610], another well known class of heterophile antibodies, are clearly distinguishable from the H-D antibodies because they are unable to react with the H-D active gangliosides [608]. Since various species excluding man and chicken have N-glycolylneuraminic acid in glycoconjugates in their erythrocytes, sera and secretions [712], introduction into humans of the glycoconjugates containing H-D determinant, NeuGca2 -+ 3Ga1, by injection or other means, presumably results in the production of H-D antibodies. 8.1STAGE-SPECIFIC EMBRYONIC ANTIGENS
Embryonic stage-specific substances have been postulated to regulate cell interactions and cell sorting during embryogenesis and differentiation [611]. These subs-
69 tances, which indicate marked changes on the cell surface during development, can be detected by immunological techniques and include carbohydrate antigens belonging to human blood group ABH and Ii systems and Forssman antigen, a heterophile type as was noted above. Using purified rabbit antibodies, the antigenicity of globotetraosylceramide was found to be expressed first on two- to four-cell mouse embryos and maximally on morulae, whereas that of IV 3GalNAca-Gb4Cer was expressed first on late morulae and most intensely on early blastocysts [612]. However, antigenic expression may differ from the chemical presence of the antigens. In mouse embryonal carcinoma cells, the antigenic expression of these compounds was not proportional to the actual concentration in the cells [612]. A monoclonal antibody directed to a murine teratocarcinoma cell line reacts with embryonal carcinoma cells of mouse and human origin, as well as mouse embryo cells beginning at the eight-cell stage [613]. The corresponding antigen, named stage-specific embryonic antigen (SSEA-I), was suggested to be a GSL based on its lipid-extractability and fast migration during sodium dodecyl sulfate electrophoresis [613].The antigen was also detected in a GSL fraction of human type-0 erythrocytes [614] and in sheep gastric mucin glycoproteins [615,616]. Gooi et al. [615] found that a terminal GalPl -,4GlcNAc(3 + laFuc) structure in an oligosaccharide(s) from human ovarian cysts is essential for SSEA-1 antigenicity. Kannagi et al. [310] characterized three SSEA-1 active fucolipids from human type-0 erythrocytes, which all possess one (11-31, -32 in Table 1.4) or two (11-33 in Table 1.4) Fucal -,3GlcNAc, fucosylated repeating N-acetyllactosamine units. The reactivity of these fucolipids to monoclonal SSEA-1 antibody varied in proportion to their saccharide chain length. They did not exhibit Ii antigenicity [310]. On the other hand, normal murine blastomeres at the four- to eight-cell stage expressed another stage-specific embryonic antigen, termed SSEA-3, when surveyed with a monoclonal antibody directed to the embryonic cells [617]. This antigen was also expressed in human, but not murine, teratocarcinoma cells and was suggested to be present on cell surface GSLs as well as glycoproteins. Based on chemical and antigenic characterizations of GSLs fractionated from a human teratocarcinoma cell line, Kannagi et al. [212] demonstrated that SSEA-3 antibody recognized structures belonging to the globo series, whde globotriaosylceramide, which is the richest in the teratocarcinoma cells, was completely inactive. The antigenic GSLs from the teratocarcinoma cells included IV3Gal/?-Gb4Cer(Gb,Cer),V3NeuAca-Gb,Cer, V3FucaGb,Cer and Gb4Cer. Among these GSLs, the SSEA-3 antigenicity of Gb,Cer was strongest, and that of V3Fuca-Gb,Cer was weakest and comparable to that of IV3GalNAca-Gb,Cer (Forssman GSL) which was not detected in the teratocarcinoma cells. 8.4. G S L ANTIGEN MARKERS OF LYMPHOCYTE SUBSETS, A N D EFFECTS ON T H E ANTI-
GENS OF LECTINS A N D DIFFERENTIATION INDUCERS
Humoral and cellular immunities in organisms are displayed by the co-operative regulation of two sets of lymphocytes, thymus-derived immunoglobulin (1g)-negative T-cells and bursa-equivalent (or bone marrow-derived) Ig-positive B-cells, and
70
macrophages. These immuno-competent cells are distinguished according to their functions and antigenic markers on cell surface. Among the GSLs on cells of the immune system of mice and rats, the gangliotetraosylceramide was one of the components that perhaps has attracted more attention than others. Gangliotetraosylceramide appears to be present on Ig-negative lymphocytes of spleen [618] and lymph nodes, as well as peritoneal macrophages [629,631]. In splenic cells of normal mice, the quantity of Gg,Cer increases with age, reaching a peak at 5-10 weeks [626]. Gg,Cer was therefore suggested to represent a differentiation antigen [625]. Similarly, Gg,Cer appears on a majority of mouse thymus T-cells at an early embryonic stage when mature murine T-cell markers of thymocytes are not yet present, and decreases strikingly thereafter [628]. Shimamura et al. [627] showed that more than 90% of nylon wool-passed splenocytes, consisting of 90% lymphocytes and 10% monocytes, reacted with an anti-Gg,Cer antiserum. The Gg,Cer-positive lymphocytes, but not the monocytes, could be characterized as natural killer (NK) cells by binding to, and lysis of, target tumor cells. NK-cells whch are T-cell marker-negative and Ig-negative are capable of damaging a variety of tumor cells without previous sensitization. They are still ill-defined immune cells, because they lack most such detectable surface markers that otherwise are characteristic for mature T- and B-lymphocytes. The cytotoxic activity of murine NK-cells is abolished by their preincubation with anti-Gg,Cer antibodies and complement [619,620]. Therefore, it was suggested that Gg,Cer might represent a surface marker of NK-cells. Indeed, an intravenous injection of antibodies directed against Gg,Cer into T-cell-deficient athymic mice enhanced the growth of some tumors, and increased the tumor-take that was interpreted to be a consequence of a diminished NK-cell activity [621.622]. In addition, Gg,Cer, Gg,Cer and other antigens of non-lipid nature could be used as markers of cloned cells with NK activity [623]. There are indications that GSLs of the target cells also play a role in their being targeted and lyzed by NK-cells. Thus, the sensitivity for NK-cell-mediated lysis of several murine lymphoma YAC-1 cell lines were correlated with the quantities of Gg,Cer, as well as some gangliosides on these cells, suggesting that this GSL and gangliosides would be involved in NK function [624]. A glycosphingolipid hapten, called cytolipin S, from rat spleen was described [634]. This lipid had similar carbohydrate composition, chromatographic mobility and complement-fixation reaction to gangliotetraosylceramide, but differed in its hemagglutination inhibition reaction. T- and B-lymphocytes are blastogenized and proliferated in response to mitogens specific for the respective cells, i.e., concanavalin A for T-cells and lipopolysaccharide (LPS) for B-cells, or to their alloantigens. It is of interest to know whether mitogen- or alloantigen-stimulated cells generate a common specific GSL pattern different from that of control cells, or if the activated cells will exhibit disparate patterns in response to the respective stimulants. The GSL patterns of murine T-cells differed greatly from those of murine B-cells in isotope labeling experiments [632]. Gruner et al. [633] demonstrated, by metabolic labeling experiments, that there were prominent increases of two GSLs in murine T-cells activated by the alloantigen, but
71 much less in LPS-activated B-cells, and almost no change in concanavalin A-stimulated T-cells. These GSLs were partially characterized as Gal-Gal-Glc-Cer and GalNAc-Gal-Gal-Glc-Cer. The latter lipid, the partial structure of which resembles globotetraosylceramide and isoglobotetraosylceramide, appears to serve as a marker for alloantigen-activated T-cell subpopulations [633,760]. An interesting system for the study of immune cell differentiation is provided by a mouse myeloid leukemia cell line, named M1-cells [637]. This multipotential cell line can be induced to differentiate into multiple, other cell types, such as macrophage-type cells (termed M1 +-cells), by the action of various inducers. M1 +-cells acquire phagocytic activity with the expression of Fc receptor, but lose leukemogenicity in vivo and proliferative activity in vitro. Taki et al. [638] demonstrated recently that differentiation of M1 into M1+ brings about dramatic changes of the cellular GSLs: whereas the M1-cells contained GSLs of the ganglio series, Gg,Cer and Gg,Cer, as the major components, the M1 +-cells, which were generated by the addition of a culture medium of murine embryonic cells, contained GSLs of the globo and lacto series, with predominant Gb,Cer and Lc,Cer due to induction of a-galactosyltransferase and P-Nacetylglucosaminyltransferase, respectively. Concomitantly, marked decrease of ganglio series GSLs was observed. On the other hand, the treatment of M1-cells with other inducers, e.g., lymphokines, brought about different effects, i.e., a marked increase of Gg,Cer [639]. In general, it seems difficult at present to establish a consistent GSL pattern characteristic of lymphocyte responses to the specific stimulants. Polyclonal antibodies against a GSL can be produced by immunizing animals, usually rabbits, with the GSL. Since most pure GSLs are rather poor immunogens, it is necessary to mix them with Freund’s complete adjuvant or other immunogens such as bovine serum albumin, to obtain antisera with high titer (for a review, see Ref. 76). On the other hand, the recently developed procedure [82] using hybridoma cells provides a means of production of monoclonal antibodies against GSLs. The monoclonal antibodies prepared by this method are highly specific for the immunogens, as was demonstrated in monoclonal anti-Gg,Cer antibodies that recognize a narrow range of the molecular structure of the antigen (see below). Furthermore, monoclonal Ig can be produced at considerably higher concentrations, i.e., 106-times more than polyclonal antisera. If a permanently growing cell line producing an appropriate monoclonal Ig is established once, the monoclonal Ig is available whenever needed. For production of monoclonal Ig [548], mice, e.g., Balb/c strain, are immunized with cells or bacterial cells coated with a GSL. Spleen cells obtained from the immunized mice are fused with a syngeneic mutant murine myeloma cell line which is resistant to 8-azaguanine and does not grow in a selective medium (HAT medium [543]), using polyethylene glycol. The treated cells are incubated in HAT medium which permits growth only of hybrids (hybridoma cells) derived by fusion between the spleen cells and the myeloma cells. The established hybridoma cell line can be grown in vitro or in vivo, or stored in a frozen state in the presence of dimethylsulfoxide. Hybridoma cells secreting Ig are screened and cloned in microtiter plates. For screening Ig directed to a GSL from a large number of colonies, a
72 solid phase radioimmunoassay (Section 3.13) can be applied. This assay utilizes the finding [740] that GSLs are adsorbed from an aqueous medium onto plastic surfaces. The procedure [102,644] involves ( I ) incubation of GSL adsorbed on plastic surfaces with culture fluid after blocking nonspecific binding of Ig with bovine serum albumin, ( 2 ) labeling of antigen-antibody complex with '2SI-labeled protein A from Sruphylococcus aureus, and ( 3 ) detection of immune reaction by autoradiography of the microtiter plates on X-ray film. At each step, the plates are thoroughly washed with phosphate-buffered saline. Solid phase immunoautoradiography (Section 3.13) allows detection of a particular GSL antigen from a GSL mixture, and this method has been frequently utilized for detection of GSLs specific for tumor-associated monoclonal antibodies (Section 9.2). Rabbit polyclonal IgG, but not IgM, antibodies directed to Gg,Cer were hghly specific for this lipid [640,641], while antibodies against I13NeuAca-Gg,Cer were less specific and cross-reacted with Gg,Cer and 113 NeuAca, IV3NeuAca-Gg,Cer [642]. Rabbit polyclonal antibodies against Gg,Cer are specific for this lipid, and do not react with other GSLs having terminal GalNAc. As expected they agglutinate guinea pig erythrocytes [643]. Young et al. [lo21 prepared two monoclonal antibodies recognizing two distinct steric portions of the nonreducing terminal N-acetylgalactosamine of Gg3Cer; one, an IgM antibody, recognized the C-6 primary hydroxy group but not the C-2 acetamide group, whereas the other, an lg G3 antibody, recognized C-2 but not C-6. 8.5. ANTIGENICITY OF SIMPLE GLYCOSPHINGOLIPIDS AND POSSIBLE INVOLVEMENT OF NEUTRAL AND ACIDIC GSLs IN AUTOIMMUNIZATION
The antigenic activity of GSLs was first demonstrated in cytolipin H (latosylceramide) by Rapport and his coworkers in connection with the serological reaction of lipids extracted from a variety of human tumors [645]. Anti-lactosylceramide antibodies were detected in the sera of cancer patients and of pregnant women [646]. As for monohexosylceramides, rabbit anti-glucosylceramide antiserum was specific for the p-glucosyl moiety and cross-reacted little with galactosylceramide and 1'-sulfo-galactosylceramide (6471. Rabbit antiserum raised against 13-sulfo-galactosylceramide was specific for this lipid, except that it cross-reacted with a synthetic 1'-sulfo-galactosylceramide in a dissimilar pattern of complement fixation [648], and the antigen-antibody reaction was completely inhibited by a high concentration of p-nitrocatechol sulfate, a synthetic substrate for cerebroside sulfatase (arylsulfatase A) [649]. The antibodies existed exclusively in the IgM fraction [650]. However, mouse antibodies against 13S03-GalCerwere present in IgG fraction although the antibodies cross-reacted with GalCer [759]. Early studies have established the antigenicity of galactosylceramide (reviewed in Ref. 651). Of the experimental models of human autoimmune diseases in nervous tissues, experimental allergic encephalitis in the central nervous system, of which encephalitogen is a myelin basic protein, appears to be augmented by galactosylceramide [652]. On the other hand, experimental allergic neuritis, which is evoked by
73 two basic proteins [653] and bears a close relationship to Guillain-Barre syndrome in humans, could also be induced in rabbits by repeated immunization with galactosylceramide [654]. In central nervous tissue culture, antibodies directed to natural [655] and synthetic [656] galactosylceramide caused demyelination, and inhibited myelination and sulfatide synthesis. In addition to the basic proteins, this GSL can thus serve as a neuritogenic antigen. Anti-galactosylceramide antiserum cross-reacts with galactosyldiglyceride, and vice versa [657]. These antibodies which were purified by liposomes containing galactosylceramide [658] or by affinity chromatography [659] did not cross-react with GSLs having galactose at their nonreducing termini, other than lactosylceramide which cross-reacted weakly [659]. Anti-gangliotetraosylceramide antibody was detected at a high incidence in sera from patients with some autoimmune diseases such as systemic lupus erythematosus [635] and Grave’s disease [636]. Rabbits immunized with total brain gangliosides or purified gangliosides such as I13NeuAc-Gg,Cer and I13NeuAc, IV3NeuAc-Gg,Cer developed a variety of neurologic symptoms which closely resembled experimental allergic encephalomyelitis [746]. This neurologic disorder, named ganglioside syndrome, was suggested to be induced by a lymphocyte-mediated autoimmune response to gangliosides based on the immunological and histological examinations.
9. Glycosphingolipid changes in transformation and malignancy One of the major interests in the biological role of GSLs in cells has been nourished by the observations of their cancer-associated alterations. Certainly one of the most effective stimuli to the study of GSLs of the cell was the original observation made by Hakamori and Murakami in 1968 [663], that upon oncogenic transformation, a dramatic change in the pattern of GSL components takes place. These include alterations of GSL composition and metabolism in tumor cells derived either spontaneously or by intentional oncogenesis. The pioneering early work on the characterization of GSLs from tumor cells was done by Rapport and his co-workers (e.g., Ref. 645). The principal changes in GSLs that accompany oncogenic transformation and malignancy can be summarized as follows. ( I ) More complex GSLs of cells undergo shortening of their carbohydrate chains due to a deficiency of specific glycosyltransferases. Such observations were made frequently with cells in vitro. ( 2 ) Particular GSLs which are not present or are barely detectable in normal controls appear or increasz in quantity during malignancy, as demonstrated in malignant tissues and cells in vivo. 9.1. GLYCOSPHINGOLIPID PATTERN A N D METABOLISM IN TRANSFORMED CELLS, A N D
THEIR POSSIBLE RELATIONSHIP TO CELL BEHAVIORS
Normal differentiated cells in a tissue which is under regulation by homeostasis of the host do not proliferate, while tumor cells have more or less lost control and grow
74 autonomously, thereby exhibiting the behavior characteristic of malignant cells, such as invasive growth and metastasis. In order to obtain information about the characteristics of tumor cells, particularly of their cell surface, it is of considerable advantage to investigate cultured cells in vitro which are of a single defined type. Treatment of normal cells (largely fibroblasts) with oncogenic DNA or RNA viruses, which can produce tumors in vivo in animals, may transform the cells. Cells transformed in vitro are considered to be analogous to tumor cells derived in vivo either spontaneously or by treatment with carcinogenic agents (chemical carcinogens, viruses and radiation), since injection of such transformed cells into animals frequently results in tumor formation. The transformants then behave differently from their normal counterparts; among others, transformed cells exhibit a loss of contact inhibition of growth and movement, enhanced transport of sugars, increased cell agglutinability by lectins, and a lowered requirement for serum factors (for a review, see Ref. 660). Intensive investigations have been made to obtain biochemical insight into transformation-associated alterations of GSL patterns and metabolism in oncogenic transformed cells, with particular emphasis on those of cellular gangliosides (reviewed in Refs. 661, 662). Hakomori and Murakami [663] found that, whereas a baby hamster kidney cell line (BHK-21) contained predominantly a ganglioside, I13NeuAcar-LacCer, the polyoma virus-transformed derivative showed a reduced content of this ganglioside and an increased level of its precursor, LacCer (Table 1.8). In mouse cell lines [664] of Swiss 3T3 fibroblasts and epithelial-like embryo AL/N cells, viral transformation resulted in a striking decrease of gangliosides larger than II’NeuAccu-LacCer (Table 1.8). GSL components which are reduced upon transformation due to a block in glycolipid chain elongation largely reflect the type of cell, but not the nature of the carcinogenic agents employed. For instance, chemically, X-ray-transformed and virally transformed mouse 3T3 cells exhibited, equally, loss of mono- and disialogangliotetraosylceramides (I13NeuAcar-Gg4Cer and I13NeuAca,IV’NeuAcarGg4Cer, respectively) [665]. Some examples of GSL changes in transformed cells in vitro are summarized in Table 1.8. A generalization of those observations are however complicated by the following findings. Two clones of SV 40-transformed 3T3 fibroblasts showed elevated levels of the more complex gangliosides, whereas other clones transformed by SV 40 or polyoma virus showed a simplification in ganglioside pattern [666]. The chemical content of ganglioside II’NeuAcar-LacCer of mouse STU fibroblasts at third culture passage was considerably decreased upon transformation by SV 40 virus (DNA virus), in contrast to a marked increase of this ganglioside when the cells were transformed by Friend leukemic virus (RNA virus) [667]. Furthermore appearance of a Forssman-active GSL II’(GalNAccu1 + 3GalNAc)P-Ga2Cer [141] in transformed hamster NIL cells cannot be explained by a reduction in the activity of a glycosyltransferase. Nevertheless, most results of comparisons between transformed cell lines and their corresponding nontransformed cell lines in vitro are in general agreement with the proposal that transformation causes incomplete GSLs.
TABLE 1.8 Changes of GSL content and enzymatic block in transformed cells Species
Cell transformed
Gangliosides Neutral GSL LacCer Gb,Cer Gb,Cer GalNAc- SiaSiaGb,Cer
Hamster D N A virus BHK-Py a N I L-Fy
Mouse
Rat
(Sia),-
1
t
I 1
1
1
I
1
Ref. Enzymic block in transformant
Ref.
663 LacCer + Sia-LacCer (Sia-TF h , 675 LacCer + Gb,Cer (Gal-TF)
703
1
675
704 (Sia-LacCer + Sia-Gg,Cer (GalNAc-TF) 704 705
.I
D N A virus Swiss 3T3
1
1 1
I 1
664 Sia-LacCer + Sia667 Gg,Cer (GalNAc-TF) 664 Sia-LacCer + Sia-Gg,Cer (GalNAc-TF)
1
J
1
671
-sv, Py I
1
AL,”
g-SV
Embryocell -LV or -MC ‘
(Sia),-
LacCer Gg,Cer Gg,Cer Gg,Cer Gg,Cer
Embryo cell ‘-Py or -DMN * R N A virus NIL-HSV
Sia-
t
670 670
T or 1, increased or decreased contents as compared to untransformed counterpart, respectively. a Transformed by Abbreviations: Sia, sialic acid; (Sia),-Gg,Cer, disialosyl-gangliotetraosylceramides;(Sia),-Gg,Cer, trisialosylgangliotetraosylceramides. polyoma virus (Py). Sialyl transferase. ‘Secondary culture of hamster embryonic cells. * Transformed by a chemical carcinogen. dimethyl nitrosamine (DMN). Hamster sarcoma virus. Doubly transformed by SV40 (SV) and polyoma viruses. g Epithelial-like embryo cell. Rauscher leukemia virus (RNA virus). ’ 3-Methylcholanthrene.
76 It should be noted that one of the established cell lines which has been used in cancer research as a nontransformed normal control is tumorigenic. The Balb/3T3 mouse embryo cell line produced malignant, host-killing sarcomas when mice were subcutaneously inoculated with the cells attached to glass beads [668] or polycarbonate platelets [669]. In light of the possibility of similar tumorigenicity in other established, highly passaged cell lines which have never been transformed by deliberate oncogenesis [669], care must be taken in the choice of control cells. The shortening in sugar chains of GSLs in transformed cells is due to a decreased activity of specific glycosyltransferases. Cumar et al. [670] demonstrated that DNA virus-transformed mouse cell lines lacking the more complex gangliosides than NeuAc-LacCer have markedly reduced levels of an N-acetylgalactosaminyltransferase which catalyzes conversion of NeuAc-LacCer to 11'-sialyl-Gg,Cer as compared to those of the nontransformed counterparts (Table 1.8), whereas other glycosyltransferases leading to synthesis of higher ganglioside homologues (NeuAcGg,Cer and NeuAc-NeuAc-Gg,Cer) were present at comparable levels in the transformants and their counterparts [671]. The kinetic properties of these residual transferases are similar to those of the normal enzymes [665]. Reduction in the activity of specific glycosyltransferases of other transformed cell lines is thought to be responsible for the low levels of GSL products of the deficient transferases, and some of the results are cited in Table 1.8 (for reviews, see Refs. 662, 672). The steady-state level of a given GSL results from its formation by biosynthesis, degradation of hgher homologues, as well as its own degradation and its being used as a precursor for the synthesis of more complex components. The glycosidase activities examined with the GSLs present in the cells were at similar levels in transformed and non transformed cells. This implies that the catabolism of gangliosides is not responsible for shortening of the carbohydrate chains [664]. although this may warrant re-examination based on observations of increased activity levels of a number of glycosidases, using synthetic substrates [660,673] and of sialidase toward added extracellular di- and trisialogangliosides [674], are significantly elevated in transformed cells. Although the role of GSLs in mediating the altered behavior of malignant cells is speculative at the present time, many reports have attempted to correlate changes in cell surface GSL content or organization with the appearance of a transformed phenotype. Synthesis of certain GSLs is influenced by the cell density in vitro. The amounts of globotri- to pentaglycosylceramides were markedly increased when normal hamster cell lines became contact inhibited at high cell density, as compared to the levels in growing cells at sparse density, while no density effect was seen in transformed cells [675-6781. However, it is not clear at present, whether or not the density-dependent change of certain GSLs' components and the loss of this effect after transformation really is a general phenomenon. Murine nontransformed cell lines which exhibit strong density dependency of growth did not show a parallel change in neutral GSL [666,671]. However, in ganglioside the murine cells exhibit the chemical change in response to cell density, and the effect was lost upon transformation [685].
77
The possibility of the density dependence of GSL synthesis being related to whether or not cells are dividing was examined. NIL cells whose division was blocked with excess thymidine or by glutamine deprivation at sparse density had the GSL pattern of cells of sparse density, whereas cells blocked by serum deprivation showed increased incorporation of [I4C]palmitateinto Gg,Cer although density-dependent Gb4Cer and IV3GalNAca-Gb4Cer were little affected [679]. Relationship between the synthesis or chemical amounts of GSLs and cell division does not appear to be clear at present. In connection with cell division, the synthesis and chemical amount of cellular GSLs were examined. Globotriaosylceramide of NIL cells was synthesized preferentially in the G I phase of cell cycle, whereas the GSLs that show a density-dependent increase, i.e., Gg4Cer, IV3GalNAca-Gb4Cerand the ganglioside I13NeuAca-LacCer were not markedly affected by cell cycle when assayed either by [I4C]palmitic acid incorporation into GSLs [662,680] or by chemical means [141]. A marked increase in synthesis of GSLs was noted in human KB cells in M and G I phases [681]. To investigate cell density-dependent synthesis of GSLs, glycosyltransferases which might be present on cell surface were assumed to be responsible for the synthesis [682]. In this hypothesis, nucleotide sugars must exist extracellularly; however these compounds are easily hydrolyzed by a strong nucleotide pyrophosphatase present on the cell surface [683]. Thus, there is little evidence for the hypothesis at the present time. The possibility of a correlation between the GSL pattern and metastatic and tumorigenic properties of transformed cells was examined. Various metastatic sublines of transformed murine 3T3 fibroblasts contain gangliosids I13NeuAca-LacCer and Gg,Cer as predominant GSLs. The higher metastatic level into mouse lung was correlated with an increased chemical content of Gg,Cer as well as with an enhanced external labeling of this GSL by galactose oxidase-NaB3H4method [687]. This increased external labeling was also observed in a metastatic mouse B16 melanoma cell subline as compared to a nonmetastatic subline [688]. Comparing murine L-cell sublines with various tumorigenicities, the more highly tumorigenic sublines contained much diminished amounts of the more complex neutral and sialylated GSLs [689]. Intermediately tumorigenic L-cells derived by cell fusion of low- and high-tumorigenic sublines regained the pattern including the more complex GSLs. 9.2. GLYCOSPHINGOLIPID CHANGES IN TUMOR TISSUES AND GSLs A S POSSIBLE TUMOR
MARKERS
It is generally difficult to establish definitively whether the GSL pattern and metabolism of a tumor in vivo are different from those of the parent tissue, because of problems of the cellular heterogeneity that exists within the tumor tissue. That is, for example, the GSL pattern of human lung cancer tissues was demonstrated to be markedly influenced by GSLs stemming from leukocytes which had heavily infiltrated into the interstitial tissue of the tumors [237]. However, assuming that a tumor tissue contains a majority of a single type of tumor cell occurring either by
78 intended oncogenesis or spontaneously, comparisons with uninvolved tissue could be of some value. The case is different with blood cells, e.g., in leukemia, cells of myelogenous or lymphocytic origins constitute a major cell population. Furthermore, if a GSL which is not detectable in the normal tissue is found to occur in the tumor derived therefrom, such a GSL may potentially be useful as a tumor marker. Recent investigations of tumors, in particular using monoclonal antibodies raised against tumor-associated antigens, have demonstrated the immuno-exposure of GSLs associated with immunologically defined tumors. Recent reviews discuss also GSL alterations in tumor tissues [279,691]. The general observation, made with culture cell systems, that oncogenic transformation usually causes a reduction in the complexity of higher GSL components also holds true for many of the tumor tissues. They include rat hepatoma [692.694], human renal carcinoma [695], avian lymphoid tumor [696], rat mammary carcinoma [693] and human leukemic cells [697,698]. Human leukocytes bear different GSL compositions reflecting myeloid or lymphoid origins. Well-differentiated or chronic leukemia cells possess almost the same types of neutral GSLs as their normal counterparts; that is, chronic myeloid leukemia cells contain GSLs of the neolacto series [699], and cells of chronic lymphoid leukemia [700] contain compounds of the globo series. In contrast to this, acute leukemia cells either of myeloid or lymphoid origins contain mainly mono- and dihexosylceramides and, to some extent, Gb,Cer and nLc,Cer, but only negligible amounts of the more complex neutral GSLs [698]. No appearance of particular GSLs associated with leukemia cells could be shown in any type of leukemia. In lymphoid tissues of leukemic animals, it was demonstrated that leukemic mouse thymus [701] incorporated much more labeled monosaccharide precursors into gangliosides, while leukemia cattle thymus and lymph nodes had nearly equal levels of gangliosides [702], as compared to respective normal counterparts. Biochemical and immunological studies of malignant tissues have led to the detection of some particular GSLs which are absent or present only in small quantity in the parent normal tissues. Such cancer-related GSLs are shown in Table 1.9. Some cases of the ectopic occurrence of such GSLs appear to be oncofetal alterations. Thus, the sialylated Lea lipid isolated from human gastrointestinal cancers was also detected in human meconium [717], and the stage-specific embryonic antigen (SSEA-1) was also shown to be present in rectal cancer [720] (Table 1.9). The increase or appearance of the particular GSLs are also related to the histology of the tumors, such as adenocarcinomas growing in various organs (e.g., adenocarcinoma-related cerebroside sulfate, in Table 1.9). or to developmental origins such as neuroectodermal [728] (e.g., neuroectodermal cancer-related I13NeuAca-Gg,Cer in melanoma, glioma and neuroblastoma in Table 1.9). There are, however, inconsistent observations, and re-examination will be required for them. The observation [566] that human gastrointestinal cancers derived from Forssman-negative ( F - ) normal tissue contained IV3GalNAca-Gb,Cer, while tumors from F+-normal tissue were F - , was not confirmed in human lung cancers of F + individuals in which there was significantly enhanced expression of the antigen
TABLE 1.9 Increment or appearance of particular glycosphingolipids in tumors Glycosphingolipid
Tumor type
Human tumor Colonic adenocarcinoma Gastrointestinal adenocarcinomas Lung and stomach adenocarcinomas Biliary adenocarcinoma
Fucosylceramide I '-SO,-GalCer
IV 'GalNAca-Cer
Lung adenocarcinoma Blood group A-GSLs in 0- or B-type patients Gb,Cer Ill Fuca-nLc,Cer (LeXGSL) I I NeuAc a-Gg,Cer I1 3(NeuAca2-8NeuAc) aGg3Cer I13(NeuAca2-8NeuAc)aLacCer II14Fuca,IV3NeuAcaLc,Cer (sialylated Lea antigen) 111 Fuc a,IV Fuc a-Lc,Cer and possibly other Leb-active GSLs GSLs with terminal Gala1 + 3Ga1(2 + laFuc) residue (blood group B-determinant)
'
'
Hepatocarcinoma Gastric adenocarcinoma Burkitt lymphoma Gastric and colonic adenocarcinomas Carcinomas of ectodermal origin Melanoma and other carcinomas
Procedure for detection
Ref.
Chemical analysis Chemical analysis
281 706.707
Chemical and biosynthetic assays Chemical and antigenic analysis Chemical and biosynthetic assays Chemical and antigenic analyses Same as above
708,709, 71 1
(Chemical analysis [37])
710 711.97 713 707.714 715 564 306 741,742
Melanoma
716
Gastrointestinal and pancreatic adenocarcinomas Colonic and gastric adenocarcinomas
717. 718
Pancreatic adenocarcinoma
(Chemical and/or antigenic analysis [706,707])
719
Establishment of monoclonal antibodies to tumor cells followed by antigenic detection on solid phase immunoautoradiography and chemical analysis
720
GSLs with ~erminaland internal [309] Galbl 4GlcNAa3 + laFuc) residue (SSEA-1 determinant) GSLs with terminal NeuAca2 + 6Gal residue GSLs and glycoproteins with lacto series blood group H determinant (Fucal + 2GalPl + ~GIcNAc-)
-
Gg,Cer
nLc,Cer
I1 3NeuAca,IV FucuGg,Cer I1 NeuAca,IV* Fucu. IV3Galu-Gg,Cer
Colonic adenocarcinoma
720
Hepatocarcinoma Colonic and hepatic carcinomas
309 721
Squamous cell carcinomas
743
Experimental tumor Kirsten virus-transformed mouse 3T3 cells and tumors Methylcholanthrene-induced mouse lymphoma Hamster tumor implanted with polyoma virus-transformed NIL cells N-2,7-Fluorenyl-bis-trifluoroacet amide-induced rat hepat oma N-2-Acetylaminofluorene-induced rat hepatoma
Chemical and antigenic analyses Same as above
687.722 123
Chemical analysis
724
Chemical analysis
725
Chemical and antigenic analyses Induction of fucosyltransferase
726 727
' This GSL was detected by human monoclonal antibody, whereas other GSLs were detected using murine monoclonal antibodies.
CQ
0
81 [97,711]. Two different cancer alterations of blood group ABH GSLs, i.e., ectopic appearance of GSLs and shortening of GSL chains, have been reported. Recent observations on the ectopic occurrence of blood group A-active GSLs which are incompletely characterised, in blood group B- and 0-individuals (Table 1.9) seem to be contrary to the previously demonstrated reduced levels of GSLs with blood group A or B activity in cancerous tissues [729]. In these cancers much reduced levels of glycosyltransferases responsible for synthesis of A- and B-active GSLs, and accumulation of less complex precursor fucose-containing GSLs, were shown [730]. Tumor cells cultured in vitro often release the apparent tumor-associated antigens into the culture medium. Such antigens can be detected in the sera of patents with advanced cancer, such as colon cancer [731]. Monoclonal antibodies, produced by hybridomas obtained from mice immunized with human tumor cell lines which were cultured from tumor tissues, have an apparent tumor specificity when tested against a number of human cell lines derived from normal and tumor tissues. Although the monoclonal antibody-producing system which has been frequently used is that of mouse [82] (see also Section 8.4), systems of rat [715] and human cells [728] were recently introduced, and the latter will be valuable for clinical application of human antibodies. Ginsburg's, Koprowski's and Hakomori's groups and others have identified a number of human cancer-related GSL antigens of the monoclonal antibodies (Table 1.9) through a sensitive and simple method using solid phase immunoautoradiography coupled with thin layer chromatography (see Section 3.13 and 8.4). Conversely, a monoclonal antibody (antibodies) directed to a GSL of known structure can be first produced, and then particular tumor cells which react specifically with this reagent can be detected [721]. The apparent specificity of certain monoclonal antibodies for human digestive organs can be ascribed to the high levels of fucolipids with or without sialic acid having blood group antigenicities. Particularly, Lewis antigens have been detected as tumor-associated antigens regardless of the Lewis status of the donors (Table 1.9, and Ref. 732 for a blood group A- and Led-active antigen (A,Led) of human pulmonary squamous cell carcinoma). In some cases, a given tumor may express more than one type of tumor-associated GSL antigen (Table 1.9). A number of possible explanations exist for the mechanism of the increase or appearance of the GSLs which are barely detectable in normal parent tissues and cells. ( I ) Tumor cells have certain glycosyltransferases which are increased or induced upon cancerization, as demonstrated by elevated activity of an a-N-acetylgalacte saminyltransferase to form IV3GalNAca-Gb4Cer from Gb4Cer in human pulmonary cancer [97], and by induction of an a-fucosyltransferase activity in rat hepatoma [727]. Burkitt lymphoma (Table 1.9) consisting of malignant B lymphocytes may also represent one case of this sort, as normal human B lymphocytes contain a minute amount of Gb,Cer and a major one of LacCer [733]. ( 2) Malignant cells are deficient in glycosyltransferases responsible for synthesis of higher homologues than those GSLs that one finds accumulated in malignancy; that is, synthesis is incomplete (see Section 9.1 as well as review of Ref. 691).
( 3 ) There may be an organizational change of cell surface membrane constituents involving GSLs on malignant cells, as was evidenced in mouse lymphoma cells where the antigenic expression of Gg,Cer is masked largely by sialylated glycoconjugates [734]. In spite of the common, specific antigen on the surface of human melanoma cells that reacts with monoclonal antibody to disialyllactosylceramide (Table 1.9), the content of I13(NeuAc),-LacCer varies significantly between a variety of human melanoma cell lines [716] as well as between a number of individual melanoma tissues [735]. It was observed that the antigenic reactivity of the ganglioside does not necessarily correlate with its chemical quantity present in the cells [716]. Therefore, the presence of a surface GSL antigen which is more exposed in tumor cells than in normal counterparts could be responsible for the expression of a tumor-associated GSL antigenicity irrespective of the concentration of the GSL. Using antibodies against GSLs for therapy of experimental tumors, Hakomori and his collaborators demonstrated that the growth of murine lymphoma tissue which accumulates Gg,Cer was suppressed by passive immunization with murine monoclonal immunoglobulin IgG, directed to this GSL [736]. In another experiment, they derivatized polyclonal antibodies to Gg,Cer, and anti-cancer drugs (e.g., neocarzinostatin) were coupled with biotin separately [737]. Administration of the biotinyl antibodies to lursten virus-transformed mouse 3T3 cells in culture (Table 1.9) followed by addition of avidin and biotinyl drug resulted in successful targeting and killing of the transformed cells [737]. Hurlyn and Koprowski [750] demonstrated that an IgG isotype (IgG,,) of mouse monoclonal antibodies directed to various human carcinomas suppressed the growth of the respective carcinomas transplanted in nude mice. The suppressive effect was most probably by a mechanism of IgG,,-dependent macrophage-mediated cytotoxicity. Alterations in carbohydrate structure in GSLs can be ascribed to the changed levels of glycosyltransferases upon transformation and malignancy. However, the general glycoconjugate pattern through activities of glycosyltransferases can vary extensively depending on a number of factors - the presence of drugs such as sodium butyrate [751] and cyclic AMP [752], vitamin deficiency (e.g. retinoic acid [753]) and environmental conditions. The numerous GSL alterations could be in turn responsible for a variety of small nonlethal functional changes from which the malignant cell arises. Although the GSL carbohydrates could be epigenetic in nature, because they are not template-determined, the changes in carbohydrates observed in transformation and malignancy could be ultimately the result of a response to an activation or mutation of oncogenes [754] or transforming genes.
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101
CHAPTER 2
G1ycoglycerolipids INEO ISHIZUKA a and TAM10 YAMAKAWA * Department of Biochemisiry, Teikyo University School of Medicine, Kaga 2-11-1, Itabashi-ku, Tokyo 173, and The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18, Bunkyo-ku, Tokyo 113, Japan
I . Introduction This short account describes the present state of the studies on the glycoglycerolipids, laying stress on those of eubacterial, archaebacterial and animal kingdoms. Plant lipids will be referred to only marginally because the topic has already been extensively reviewed. 1.1. HISTORY
The first glycerol-containing glycolipid to be isolated was mannosylphosphatidylinositol (for the structure see Table 2.2.3(6)) from mycobacteria studied by the Anderson school in the 1930s. Mycobacterium tuberculosa received considerable attention at that time as the pathogen of tuberculosis [3]. Although this group of glycolipids have been customarily regarded as a special type of phospholipid, it is also possible to classify these lipids in a group of glycolipids, “glycoglycerophospholipids” 141. Kates [ 5 ] pointed out that the 1920s was “the early growth phase” of the studies on bacterial lipids, with the following three decades being “the exponential phase”. But even in his review written in 1964, only a few references concerning glycoglycerolipids were cited as “added in proof ’. About 80 years ago, the presence of high amounts of galactose in the lipid fraction of wheat flour was reported [6]. In 1956, Carter isolated glycolipids containing acyl esters, galactose and glycerol from this source. Subsequently the structures were identified as galactosyldiacylglycerol [7] and digalactosyldiacylglycerol [8] in 1961 (Table 2.1.1(5) and 2.1.2(1)), These represent the first complete structures of glycoglycerolipids including the stereochemical configurations of glycerol [13,142]. On the other hand, Benson and his colleagues discovered, in 1959, a sulfonoglycog1ycerolipid, a-sulfoquinovosyldiacylglycerol (Table 2.2.1(7)), from chlorella [9,10,195]. Various physicochemical techniques including nuclear magnetic resonance, infrared spectroscopy and X-ray crystallography were applied to characterize
102 this compound. In 1965, Carter reviewed plant glycoglycerolipids in detail along with the representative glycosphingolipids of animals in the Annual Review of Biochemistry [ll]. At about the same time, the pioneering works on erythrocyte glycolipids such as globoside and hematoside [116,352], as well as brain gangliosides were completed (see also Chapters 1 and 3). The establishment of the localization of “galactolipids” in chloroplasts [12] and the suggestion for their possible roles in photosynthesis stimulated the developments in the studies of plant glycolipids to a great extent. The literature on plant glycolipids until 1974 has been extensively reviewed [13,14]. Sastry [13] compared glycoglycerolipids from plants, microorganisms and animals for the first time. Shortly after the characterization of plant galactosyldiacylglycerols, Macfarlane demonstrated the presence of mannosyldiacylglycerol [ 151 and glucosyldiacylglycerol [ 161 in Micrococcus luteus (lysodeikticus) and Staphylococcus aureus, respectively. These discoveries, together with the necessity of characterizing the protoplast membrane of M. luteus [17], might trigger an enthusiasm in the search for new glycolipids in various microorganisms. The initial surveys indicated that gram-positive bacteria were the treasure box of glycoglycerolipids [18]. The varieties of the constituent carbohydrates, mode of linkages, configurations of the oligosaccharide, and the molecular species of lipophilic moieties have been listed. The first complete structure of glycoglycerolipids of gram-positive bacteria, Gal pcul-2Glcpal-3-sn-l,2diacylglycerol, was established by Brundish et al. [19] in 1965. Then the number of publications concerning bacterial glycolipids increased in a logarithmic mode. In the 1960s, the use of thin-layer chromatography for the analysis of lipids became very popular. Typing of gram-positive bacteria according to the structure of the disaccharide residue in dihexosyldiacylglycerol agreed well with the conventional classification and has been regarded as one of the valid parameters for chemical taxonomy [20,21]. From then on, many reviews on bacterial glycoglycerolipids appeared: Brundish and Baddiley [22]; Asselineau [23]; O’Leary [24,25]; Op den Kamp et al. [26]; Shaw [27,28]; Hakomori and Ishizuka [29]; Fischer [30,31]; Gigg [32]; and Ward [33]. The second wave of enthusiasm was aroused in 1965 by the isolation of a new class of glycoglycerophospholipids, glucosaminylphosphatidylglycerols,from Bacillus subtilis by van Deenen’s group [34], and from B. megaterium by Phizackerley and MacDougall [35] (Table 2.2.3(1)-(3)). Strominger’s proposal, that the antigen-carrier lipid” of Salmonellu typhimurium is a cardiolipin-type glycolipid, was also stimulating, although this “glycophospholipid’ turned out later to be a glycoside of isoprenolpyrophosphate by analysis using mass-spectrometry. Then two other new classes of glycoglycerolipids: glycerophosphoglycoglycerolipid (Table 2.2.4(7)) from Streptococcus hemolyticus by Yamakawa’s group [ 361; and phosphatidylglycoglycerolipids from S. fuecium [37] (Table 2.2.4(2)) and Pseudomonus diminufa [38] (Table 2.2.4(1)) by Fischer’s and Wilkinson’s groups, respectively, were isolated. Finally, the membrane teichoic acid of gram-positive bacteria was rediscovered as lipoteichoic acid [39], a macroglycoglycerolipid (poly(g1ycerophospho)-glycoglycerolipid) [31,40,41] (Fig. 2.3). More recently, the studies of membrane glyco“
103 glycerolipids of archaebacteria (a new kingdom located possibly between prokaryotes and eukaryotes) were accepted with excitement and gave an insight into the evolution of glycolipids in biosphere [112] (Table 2.1(5)). Glycoglycerolipids now include several classes of glycoconjugates ranging from simple monogalactosyldiacylglycerols to highly complex polymers. In parallel, the complex amphiphiles in mycobacteria and lipopolysaccharides of gram-negative bacteria [3,23,33,41,66]have also been extensively studied. Such tides must have also accelerated the establishment of the present concepts on mammalian sphingoglycolipids as a diverse entity of amphiphiles [116,250], ranging from simple oligoglycosylceramides to water-soluble “macroglycolipids”. About half of the glycoglycerolipids contain one or two of the following acidic groups: sialic acids, sulfate, phosphate or phosphono groups, and carboxyl or uronic acids. On the other hand, the animal galactosyl glycerolipids, which has been considered as the minor component of the central nervous system since 1963 [43,44], emerged as the major glycolipid of mammalian sperm in 1972 [45,46]. The structure of testicular seminolipid (1-O-alkyl-2-O-acyl-3(/3-3’-sulfogalactopyranosyl)-sn-glycerol)was the first complete one including the stereochemical configuration of glycerol moiety established for animal glycoglycerolipids (Fig. 2.la) [47]. Gastric or pulmonary secretions [48-501 and brain of cod fish, as well, were shown to contain glycoglycerolipids as the major glycolipid class [51,52]. Glucosyl glycerolipids from secretions are unique in that they contain isomaltotri- to octaose, and some of them have a sulfate ester on the primary hydroxyl (C-6)of the terminal glucose (Table 2.2.1(2)). Animal glycoglycerolipids were reviewed by Sweeley and Siddiqui [53] for the first time together with glycosphingolipids and have subsequently received attention of pathologists and biochemists [32,54-581. Metabolic studies have also been conducted using chloroplasts [59,60], and particulate fractions of bacteria [61-641. In all systems, hexoses were transferred from their nucleoside diphosphate derivatives to lipid acceptors. The reviews on the lipid metabolism of bacteria by Lennarz [65,66], O’Leary [25], Goldfine [67] and Ambron and Pieringer [68] contained substantial information on the metabolism of glycoglycerolipids in bacteria. Metabolism of lipoteichoic acids has been studied by the groups of Baddiley, Glaser, Pieringer and Chiu (for a review see Ref. 31). The studies on the biosynthesis and biodegradation of rat brain galactosyldiacylglycerolsin vitro were conducted by Pieringer’s group in the period following 1968 [69]. Several years later, the groups of Yamakawa [70] and Murray [71] examined the metabolism of testicular or cerebral seminolipid using both in vitro and in vivo systems. The “galactolipids” (galactosyldiacylglycerolsand their sulfates) localize in chloroplast membrane [12,72-741, cytoplasmic membrane of bacteria [17], myelin of central nervous system [69] and plasma membrane of spermatozoa [55,56]. Since it was established that glycoglycerolipids were the components of surface membrane of animal and unicellular organisms, or chloroplasts, various functions have been proposed. The more important of them are: maintainance of membrane fluidity (Section 5.1.3); regulation of the conformations of transporting proteins (Section
104
5.2.2); fixing of water, ions and other solutes on the external surface of the cells (Section 5.5.1); and the roles of the receptors to transduce information from the outside of the cell to the interior of the cell, or organelle (Section 5.3.3). 1.2. CLASSIFICATION AND NOMENCLATURE
I . 2. I. Classificntion Glycolipids in biosphere have been classified into four major categories: the glycosphingolipid, glycoglycerolipid, polyprenol phosphate glycoside and steryl glycoside, according to the structure of hydrophobic domains. The lipids containing erythritol, ethyleneglycol or D-( - )-glyceric acid, acylated carbohydrates [27] including rhamnolipid [75], lipopolysaccharides of gram-negative bacteria [33,41] and wax esters of mycobacteria [3] also have often been included in the glycolipids. Alternatively, we can classify all of the above in “glycoconjugates”, although we have been accustomed to call them “glycolipids” implying that they are one of the lipid classes. Glycoglycerolipids (GGroLs) can be further divided into two subgroups: neutral and acidic GGroLs. Such a classification reflects the frequent use of ion-exchange chromatographies for the separation of polar lipids and also the proposed functions of acidic compounds as ion-binding matrices of cell surface and organelles [361]. The neutral GGroL includes glycosylated diacylglycerol (DAG), or alkylacylglycerol (AAG) and acylated glycosylDAG (Table 2.1). The acidic GGroL contains one or more of acidic group(s), such as acidic carbohydrate(s). ester(s) of sulfate or HOCH,
EH~OR,
HO
T I ‘
R4CO-C-H
z o
(1)
(2)
OH
Fig. 2.1, Stereospecific structures of glycerol moiety. a, Seminolipid of mammalian germ cell: b. sn-Glycerol-l-phosphoryl-6glucopyranosylcrl-2(3-phosphatidyl-6)-glucopyranosylal-3. 1.2-diacyl-snglycerol from Streptococcusjaecalis.
105
phosphate, in the molecule (Table 2.2). The sulfo(su1fato)GGroL (451, glycerophosphoglycoglycerolipid (GroP-GGroL) or phosphatidylglycoglycerolipid (Ptd-GGroL) [31,76] contain sulfate, sn-glycerol-1-phosphates(GroP) [77] and/or a 3-sn-phosphatidyl (Ptd) group [78], respectively, on the carbohydrate moiety. On the other hand, the term “glycoglycerophospholipid” should be used to designate phosphoglycerolipids, which are glycosylated on their glycerol (Gro) or inositol moieties. Sulfono group (with C-S bond) has been found in only one GGroL from plant, 6-sulfoquinovosyldiacylglycerol(SQ-DAG). I . 2.2. Nomenclature Glycolipids, which contain diacylglycerol (DAG) have been designated as “monogalactosyl-” and “digalactosylglycerol lipids” (81, “galactolipids” [79-81,1421 or “glycerogalactolipids” [82], when the sugar moiety is galactose, and more generally as “glycosyl glycerides” [13,65,83], “glyceride glycolipids” [64] or “glyceroglycolipids” [84]. According to the IUPAC-IUB nomenclature [1,2], “glycolipid” is the generic term for compounds, in which the carbohydrate domains are linked via the reducing end through glycosidic linkage to lipophilic domains. The reverse, acylated carbohydrates (271 have been traditionally classified as carbohydrates. However, acylated GGroLs should be added to the category of the neutral GGroL because they have been recently shown to occur frequently in gram-positive bacteria [85]. Lipopolysaccharides of gram-negative bacteria also contain an acylated disaccharide as the lipid anchor [33]. When the lipophilic moiety contains monoacylglycerol (MAG) molecules, the lipid is called “glycoglycerolipid” (GGroL). The stereospecific numbering system of the Gro portion may require special explanations. If a Gro molecule is drawn in the Fischer projection, with the secondary hydroxyl group to the left of the central carbon (Fig. 2.1), the carbons are stereospecifically numbered 1, 2 and 3 from top to bottom. The prefix “sn” (for s tereospecifically n umbered), used immediately preceding the term “glycerol” and separated from it by a hyphen, differentiates this numbering from the conventional D-, L-system. The prefix, “rac” precedes the full name if the product is an equal mixture of both antipodes, and the prefix “ X ” is used if the configuration of the compound is either unknown or unspecified. To show an example, the compound shown in Fig. 2.la reads 1-O-alkyl-2-O-acyl-3-/3(3’-sulfo-~-galactopyranosyl)-snglycerol (seminolipid) [45]. The more complex sn-1-glycerophosphoglucosyl(phosphatidy1)-glucosyldiacylglycerol [77] (Fig, 2.lb) can be described as follows, 6’-( 1sn-g1ycerophospho)-a-D-glucopyranosyl(1-2)(6”-sn-phosphatidyl)-c~-~-glucopyranosyl-1-3, 1,2-diacyl-sn-glycerol: or shorter and more conveniently, using the symbols recommended by IUPAC-IUB [1,2], sn-Gro-l-P-6Glcal-2(3-sn-Ptd-6)Glcal-3-snGro-1,2-acy12.
2. Structure I t should be stressed at the beginning that, in addition to the major glycolipid class (GGroLs), plant tissues contain also glycosphingolipids, such as monohexosylcera-
106 mide (cerebrosides), mannosylglucosylceramide [ 861 or phytosphingolipids as the relatively minor components of cell membrane [ 1061. Phytoglycolipids are comprised from glucuronic acid (GlcU), arabinose, inositol etc. [87] (see also Chapter 1, Section 9). Glycolipids of gram-positive eubacteria and archaebacteria belong almost exclusively to the glycerol (Gro) type with various carbohydrate chains, ranging from simple oligoglycosylDAG to polyglycosyl or polyglycerophosphorylDAGs. Sphingoglycolipids have so far been detected in only two microorganisms: ceramide phosphorylglycerol in Bacteroides melaninogenicus [ 881 and GlcUp-ceramide from Flauobacterium deuorans [89]. In contrast to plants and bacteria, the plasma membranes of animals, with the notable exception of spermatozoa, always contain glycosphingolipids as the major glycolipid components [ 116,2501. 2.1. CARBOHYDRATE DOMAIN
Tables 2.1 and 2.2 show that the carbohydrate domain of plant GGroLs is comprised exclusively from galactose (Gal), quinovose (Q), rhamnose or Glc, and that of animal GGroLs from Gal or Glc. They are sometimes modified by a sulfono group [9,10] or sulfate group [10,57]. The carbohydrate composition of microbial GGroLs is more diverse. The neutral and basic components include Gal, Glc, Man, Q, mannoheptose, fucosamine, glucosamine (GlcN), N-acetylglucosamine (GlcNAc) and long-chain acylated GlcN in pyranose ( p ) form. Gal [90.94] and xylose [526] in furanose ( f) form are also reported. The acidic components include: galacturonic acid (GalU) and GlcU, sialic acid, sulfate, sulfonate and glycerophosphate (GroP) in the sn-Gro-1-P or 3-Ptd groups (Table 2.2). 2.1.1. Neutral glycoglycerolipid
The major monohexosylDAG (Hex, DAG) in plant is Galp/31-3-sn-DAG. Glcppll(3)DAG has been detected in rice bran [91] (Table 2.1.1(10)), and recently, evidence was presented for the presence of a GlcDAG in a cyanobacterium [498]. Hex, DAGs, whch are the biosynthetic precursors of the Hex, compounds, have been isolated in substantial amount in only a few microorganisms, e.g., Mycoplasma mycoides [96], Acholeplasma laidlawii strain B [97], and a group B streptococcus (20 molS) [76] (Table 2.1.1). DihexosylDAG (Hex,DAG) of plant is exclusively Galpal-6Galp/313-sn-DAG. Similarly, Ga1,DAG or Gal, AAG were suggested to occur as trace components of rat brain [92,93] and human testis [56] (Table 2.1.2(1), cf., 5.3.2.). The major glycolipids of gram-positive bacteria is usually Hex ,DAG, although Lactobacillus casei [ 8 5 ] and Thermus thermophilus [94,95] contained trihexosylDAG (Hex,DAG) and tetraglycosylDAG (Hex,DAG), respectively, as the major glycolipid (Table 2.1.3, 4). Since the first complete structure for the GGroL, Galal2Glcal-3DAG (Table 2.1.2(5)) of pneumococcus, was established [19], some 90 bacterial GGroLs have been characterized (cf., Section 3.2.). The typical thin-layer chromatographic profile of the glycolipids from a gram-positive bacterium ( Srreptococcus faecium (faecalis)) is shown in Fig. 2.2. Thermoplasma acidophilum was shown to contain a glycolipid consisting of as
107 TABLE 2.1 Neutral glycoglycerolipids and related glycosides The contents are weight% of the total lipid (w/w). unless otherwise specified. In some cases, the contents in tissue are also listed. 1. Monoglycosyl glycoglycerolipid
( I ) Galpal-3(1)DAG: Treponema pallidum Kazan 5 (9.5% of dry cell) [165,166]; Treponema hyodysenteriae (29.9%). I-alk-1-enyl form (88.3%). contain also an additional fatty acyl ester (1671. gal pal-3-rac-Gro: Porphyra umbilicalis (Rhodophyta) [168]. (2) Galpal-3-sn-Gro: Chemical synthesis [169]. (3) Galpal-1-sn-Gro: Chemical synthesis [169]. (4) Galpal-2Gro: Marine alga (lridaeu laminarioides) [170]; red alga ( Furcellaria /astigiata) [171]; chemical synthesis [172]. (5) Galpbl-3-sn-DAG: Plant: wheat flour, isolation and structure [11,142]; runner bean leaves, 15.6% = 2.02 pmol/g 1801; thylakoid membrane of chloroplast, about 50% of thylakoid membrane [74] or 22% of thylakoid from spinach [173], 0ryza satiua (rice plant) thylakoid, 85% [174]; envelope of spinach, 32.8% [173] or 13.2 mo1% [175]; potato tubers, 5.7% [176]; rice bran, 8.7% [91]. Unicellular organism: green alga (chlorella), 4.2% = 18.1 pmol/g of dry weight (1781; cyanobacterium 175,1791; halotolerant algae. 21-22 mol% of total lipid [515]. 37% of polar lipids [520]; nonphotosynthetic diatom, 0.98, 10.5 mg/g [180]; Chloropseudomonas [ISl]; Bifidobacrerium [182], presence of an additional acyl ester(s) on galactose [183]; Arthrobacter [184]; Treponema reiten' (1851. antigenicity [186]; Saccharomyces cereuisiae 13221. Galp/31-3-sn-DAG and AAG: Animal: central and peripheral nervous system: bovine spinal cord (DAG form) [43, 1871. 0.6% or 1.8 pmol/g=1.37 mg/g tissue, 15% [loll; bovine brain white matter, 0.4% (80% was AAG form) [44]; sheep brain, 0.4% or 2.4 mg = 0.4 pmol/g (8-128 was AAG form) [82]; rat brain, 1.46 pmol/g (DAG form) [ISS]; rat brain (DAG form) (69,1891; rat brain (DAG/AAG = ca. 1 : 1 ) [189],change in amounts with age of rat [l02]; porcine spinal cord (DAG form) (190); DAG form in 330-day-old rat brain =I46 p g / g =1.62 pmol/g or 0.19% of total lipid, 660 and 464 nmol/g in spinal cord and sciatic nerve, respectively [93]; 18-day-old mouse brain, 0.44 pmol/g 11911; cultured mouse brain cell [192]; 14-day-old rat brain stem, 413 nmol/g [193]; cod brain (DAG and AAG forms). 1.15 pmol/g [52]; cod spinal cord, 1.89 pmol/g (194); rabbit sciatic nerve (also AAG form) (1961; bull frog (Rana catesbeiana) brain. 0.72 mg/g and sciatic nerve, 0.27 mg/g [539]. Galpbl-3-sn-AAG: Mammalian spermatozoa and testis (germ cell) (exclusively AAG form): boar spermatozoa [45,47]; guinea pig testis [122]; rat testis [71] and spermatozoa (55.56.1971; human testis [47,199]; absence in seminoma (199); calf brain [187]; whale brain [304]. Acyl-0-6Galpj31-3(1)DAG:Wheat flour 12011; spinach homogenates, in vitro at 4OC for 3 h (pH 4.6). ca. 20%; Isolation: [202]; Structure (203); spinach envelope isolated at pH 8.5, 0.8 mol% [81]; Chemical synthesis: an ether analogue (2041. Gal pbl-diglyceryltetraether: Sulfolobus sp. [510,526]. Galppl-3(1)MAG: Rice bran [91]. Acyl-0-6Galp/31-3(1)MAG: Spinach homogenates, 0.6% [205]. Chemical synthesis of galactosylglycerol and galactosyldiacylglycerol:Gal ppl-3-sn-Gro [105,169]. Galpl-3-sn-DAG with 16 :0 and 18 :0 fatty acids. as well as its 6-0-acyl derivative [206]; 16: 0/16 :0, and 16: O/Lin species [207]; Ole/01e species [549] with desired fatty acids (partial synthesis) (2081, and their 6-0-acyl derivatives [203]. Gal pbl-3-sn-AAG (desulfoseminolipid): (2091. Galp~l-3-di-O-alkylglycerol (di-0-18 : 1 and di-0-16 :0 alkylglyceryl ether): [22]. Galp/31-3-sn-Gro: Chemical synthesis (1691. Galpbl-2Gro: Chemical synthesis [172].
108 TABLE 2.1 (continued)
(7)Galp/ll-2(acyl-O-l)ethylene glycol: Ripening (immature) corn seed, 1% of total lipid [210]; chemical synthesis by the same authors. (8) Galj@l-3(1)-sn-DAG:Bacteroides symbiosus [211]; Mycoplasma mycoides, 1.3%1961; Bifidohacterium biJidum [212], also 2’,3’-di-O-acyl and 3’-O-acyl derivatives [183]; Acholeplasma axanthum [213]. galactofuranoside is linked through a-configuration and also ring 2’.3’-di-O-acyl derivative; Buryriuibrio, in alk-I-enyl form and its n-valeryl or n-butyryl esters 12141. Gal//31-3-sn-Gro: Chemical synthesis 1215). (9) Glcpal-3-sn-DAG: fneumococcus (63,2161; Diplococcus pneumoniae, 11%= 11 mg/g [217]; fseudomonas diminura, 7.2-10.7% (ca. 31 mg/g) [38]; f. uesiculuris [108]; Streprococcus lactis and S. hemolyticus ( pyogenes. type 4). 4.7 and 5.1 mol%, respectively [30]; a group B streptococcus. 20 mol% [76]; Acholeplasma laidlawii (971; A. modicum [218]; S. faecium [64]; Lmtobacillus casei, 2.9 mol% [85]. Glcpal-l(3)Gro: Chemical synthesis [219]. Glcpal-3-sn-Gro: Chemical synthesis [32.220]. Glcpal-3(1)DAG and AAG: Human saliva, 0.58 mg/100 ml [221]; pulmonary lavage [222]. (10) ClcpP1-3-sn-DAG: Isolation: Staphylococcuq aureus 116); Structure: Staphylococcus lactis 13 [200]; Alteromonas (Pseudomonus) rubescens [169]; Mycoplasma neurolyticum [223]; rice bran (911; Arthrobacrer [ 1841. Glcp/31-3-sn-Gro: Chemical synthesis (2201. GlcpPl-3-DAG. 16 :0/16 : O species: [IOS]. GlcpPl-?-0-carditoltetraether, Sulfolobus [ 5261. (11) Glcp/?l-l-sn-Gro: Chemical synthesis (2201. (12) Glcp/31-2Gro: Chemical synthesis [172]. (13) Manpal-3-sn-DAG: Isolation: Micrococcus luteus (bsodeikricus) [IS]; Structure: M . luteus [61]. Manpal-3(1)-sn-Gro, Chemical synthesis: (61). Manpal-3DAG. 16 :0/16 :0 species, chemical synthesis: [IOS]. (14) GlcNAcpal-3(1)DAG: Streprococcus hemolyticus. minor component [224]. (15) GlcNAcpPl-3DAG: Bacillus megarerium. 5% [225]. (16) ~-glycero-~-glucoheptosepa1-3(1)DAG: Pseudomonus uesicularrs ( 1081.
2. Diglycosyl glycoglycerolipid (1) Galpa1-6Galp/31-3DAG: Plant: wheat flour [ll]; runner bean leaves, 3.5%. 0.4 pmol/g [go]; potato
tuber, 14.2% 1177); rice bran, 12.3% [Sl]; chlorella, 7.2% = 25.1 pmol/g dry weight 1178); halotolerant algae, 11-21 mol% (nonphotosynthetic marine diatom, Nirzschia alba, 0.9%, 10.0 mg/g [180]; spinach envelope, 33.6% [173]. 21.6 mol% (1751; spinach thylakoid, 15% [173]: a fungus ( Blastocladiella emersonii), structure not conclusive (3223. Animal: rat brain, not detectable by chemical method and the structure tentative [93,226,227]; human brain (921: a lipid tentatively ascribed to GalzAAG was also reported to occur in human testis and sperm 156.2281. Acyl-O-6Galpal-6Galp/3I-3DAG: Synthesis in vitro in the homogenates of spinach leaves, 1.61,40 p g / g leaves at pH 5.3 and 4’C for 4 h (2291; spinach envelope isolated at pH 7.2, 7.8 mol% [175]. Chemical synthesis: Gent and Gigg [230], Gigg [32]; partial synthesis [205]. Galpal-6Galp/31-3Gro: Wheat flour, stereochemical configuration of Gro [169]. (2) Galpal-6Galpal-3(1)-sn-Gro:Obtained by the degradation of lipoteichoic acid by a-galactosidase and hydrogen fluoride [158]. (3) GalPI-2GalPl-3-sn-DAG: Eifidoharterium bifidum [183]. (4) Galf/3l-2(3-O-acyl)Galf~1-3DAG: Bifidohacterium bifidum (1831. (5) Galpal-2Glcpal-3-sn-DAG: Pneumococcus 1-192, 18.8 mg/g dry weight. 34% [19]; Pneumococcus [63]; Diplococcus pneumoniae IR/44. 33%. 33 mg/g dry cell [217]; Loctobacillus casei (2311. 19.9 mol% (85.1451.
109 TABLE 2.1 (continued)
Galpnl-2(acyl-O-6)GIcpal-3DAG: L. case;, 0.7 mol% [85]. Galpal-2Glcpal-3-sn-Gro: Chemical synthesis (22,2201. ( 6 ) Galpl-6Gal~l-3(1)DAG: Arrhrobacrer sp. (1841. (7) Galf~1-6Galf~l-3-dibiphytanylglycerylether:Methanospirillum hungatei [271], also a di-0phytanylglycerol ether derivative, 2%. (8) Glcpal-2Glcpal-3DAG (a-kojibiosylDAG): Sireprococcus lacris (serogroup N) [18,37], 7.9 mol% [30]; S. faecium [64.135]; S. hemolyticus, 44.3% = 56.7 mol%, 66 mg/g dry cell (30,361; Acholeplasma laidlawri (971. Glcal-2(acyl-O-6)Glcal-3DAG: Streprococcus lacris [138,233]. (9) Glcpal-6Glcpal-3(1)-sn-DAG: Human saliva, 1.56 mg/100 ml, the alkylacyl form predominates [221]. (10) Glcp~1-6Glcp~l-3DAG ( 8-gentiobiosylDAG):Isolation and characterization: Sraphylococcus aureus [16,145,234]; S. lactis 13, 0.7 mg/g dry cell = 3% [18,22.200]; S. lacris 7944, I-2% [26]; S. epidermidis (1391; Bacillus suhtilis 118,221; B. cereus [235]; B. licheniformis 12361; Mycoplasma neurolyricurn (2231. Chemical synthesis: Also 1-2, 1-3 and 1-4 analogues [18,22.26]. (11) Manpal-3Manpal-3DAG: Isolation: Micrococcus lufeus (lysodeikticus)(151; Structure and metabolism: M. luteus, 7-15%. 0.75-1.5 mg/g [61]; Structure: arthrobacter [184]; Microbacterium lacricum. 46% [237]. (12) Glcpal-4GlcNp( N-acyl)pl-3(1)DAG: Bacillus acidocaldarius, 14.9%. monoacyl derivative, 26.5% (238.2391. (13) Glcyal-2GaI//31-3-dibiphytanyl glycerylether: Merhanospirillum hungarei GP 1. also a di-0-phytanyl glycerylether derivative, 17% [232]; Sulfolobus solfararicus (Caldariella acidophila) (a thermoacidophile) [240]; a thermoacidophile strain TA 1 (a Sulfolobus sp.) [518]. (14) Manpal-3Galpal-3Gro: Furcellariafastigiara. a marine red alga [171.241]. (IS) man pal-2Glcpal-1-, 2.3-di-0-phytanyl-sn-Gro(DGD): halophilic archaebacteria [500]. (16) Glcppl-4Glcp~l-3(1)DAG( /3-cellobiosylDAG): Chemical synthesis [105,207]. (17) Glcp~l-3Galp/3l-l-diglyceryltetraether: Sulfolobus sp. 1518.5261. (18) Xyl-Gal-diglyceryl-dialkyl-tetraether: Sulfolobus sp. [526]. (19) Quinovose-Gal-diglyceryl-dialkyl-tetraether: Sulfolobus sp. [526].
3. Triglycosyl glycoglycerolipid (1) (Galpal-6),Galp/31-3DAG: isolation and structure: Potato tuber, 1%. 10 pg/g tuber [177]; apple
pulp [176]; rice bran, 3.7% [91]; spinach envelope, 1.5% [173], 5.2 mol% [175]. Chemical synthesis [ 32,242).
( 2 ) (GalPl-2),GalPl-3(1)DAG: Bijidobacrerium bifidum (1831. (3) (Glcpal-2),Glcpal-3DAG (a-kojitriosylDAG): Srreptococcus hemolyticus, 0.98, 6.6 mol% 130,361; S. faecrum [78]. (Glcp a1 -2) (acyl-6)Glcpa1-3DAG: Lactobacillus case;. 3% [MI. (4) Glcp~l-6Galpal-2Glcpal-3(1)DAG: Isolation and partial structure: Lacrobacillus casei, less than 17% [231]; final structure: L. casei 26.1 mol% (851. Glcp~l-6Galpal-2(acyl-6)Glcpal-3(1)DAG: L. casei, 3.2 mol% [85], also occurs as the component of LTA. ( 5 ) Glcp~l-3Glcpal-ZGlcyal-3(1)Gro: A diacyl derivative, although the positions of the fatty acids not specified, Acholeplasma granularum [147]. ( 6 ) (Glcp~l-6),Glcp~l-3(1)DAG ( P-gentiotriosylDAG): Bacillus subrilis, B. lichenijormis (2431. structure unpublished. (7) Glcp~1-6Manpal-2Glcpal-l-sn-2.3-di-O-phytanylGro (TGD-2): Halobacterium marismortui, 11 molS (245).
,
110 TABLE 2.1 (continued) (8) Galp/31-6Manpal-2Glcpal-l -sn-2,3-di-O-phytanyl-Gro (TGD-I): H . curirubrurn [114]. (9) (Glcpal-6),Glcpal-3(1)DAG(a-isomaltotriosylDAG): Human saliva, 0.9 mg/100 ml. alkylacyl form predominates (2211. (10) Galpal-6Galpal-3(Galpal-2)Gro: The major glycoside obtained on hydrogen fluoride degradation of LTA from L. casei [158].
4. Tetraglymsyl glycoglycerolipid (1) (Glc/31-6),Galpal-2Glcpal-3(1)DAG: Lactobacillus casei, 5.5 moI%(27,853. ( 2 ) Galj/31-2Galpal-6GlcNp( N-acyl)/3l-2Glcpal-3(1)DAG:Thermw thermophifus (//auus). 70%.
amide-linked acyl mainly iso-pentadecanoyl [94,244]. (3) (Galpal-6),Galp/3l-3(1)DAG:Spinach chloroplasts, about 1%of GalGGroLs [246]; rice bran. 2.3% [91]; cassava tubers (tapioca), 7.3% [247]; spinach envelope, 2.1 mol%[175], but lower in envelope isolated at pH 8.5 [81]. (4) Rhampal-4Galp/31-4Galp/3l-4Galp/31-2AAG: 7-day mung bean ( Phaseolus mungo) sprout. 0.2% [248]. ( 5 ) Galp/31-6(Galfal-3)Manpal-2Glcpal-1-sn-,2,3-di-O-phytanyl-Gro (TeGD): Halobacterium cutirubrum [114].
5. Polyglycosyl glymglycerolipid
( I ) Gal p a1-2Gal p al-3-D-gIycero-D-mannoheptosep~l-3Glcp al-2Glcp al-(1)3DAG: Acholeplasma modicum [249]. ( 2 ) (Glcpal-6),Glcpal-3(1)DAG (a-isomaltopentaosylDAG) and AAG: Pulmonary lavage [222]. (3) (Gl~pal-6)~GIcpal-3(1)DAG (a-isomaltohexaosylDAG) and AAG: Human gastric contents, 5.2 mg/100 ml [48.50]; human saliva, 2.8 mg/100 ml [221], pulmonary lavage [222], in all fluids the alkylacyl form predominated. (4) (Glcpal-6),Glcpal-3(1)DAG (a-isomaltooctaosylDAG) and AAG: Human gastric contents, 4.7 mg/100 ml [48,50]; human saliva, 4.7 mg/100 ml [221].
6. Macroglycoglycerolipidor lipopolysaccharide
( I ) (Man p al-2Man p al-2Man p al-3),Glcpl-1(3)-di-O-phytanyl glyceryldiether: Thermoplasmu acidophilum, 3% of cell dry weight [98]. ( 2 ) (Man),DAG (non-succinylated lipomannan): Micrococcus luteus 12871. (3) Glcp~l-2Glcpal-4Glcpal-3,4FucNAc/3l-3(Gal p al-3),Galp al-3.4GIcNAc/31-3,4-GIcNAc~l4’Glcpb1-2Glcp al-4Glcp a1-3.4FucNAcbI -3(Gal p a1 -3),Gal p a1-3.4-GlcNAc/31 -3,4GlcNAc/314~,-Glcp/3l-2Glcpal-4Glc~al-3.4FucNAc/31-3(Galpal-3) *Gal pal-3,4GlcNAc/3l-3,4GIcNAcDAG (a lipoglycan): Achofepfasmagranularurn [SOS].
many as 24 Man and one Glc [98] (Table 2.1.6(1)). Lipomannan of Micrococcus luteus is a polymannosylDAG, a part (2.5%) of which is succinylated [252,287] (Table 2.1.6(2)). Lipomannan of mycoplasma share some properties with lipopolysaccharides of gram-negative bacteria, and lipomannan of M. luteus, e.g., the
111
extractability with phenol (991, although they are very different in chemical composition [41,100]. “Lipopolysaccharides” or lipoglycan of this type have been found in four acholeplasma species (about 1% of cell dry weight), and in two anaeroplasma species (about 2%) [99,100]. In addition to hexoses, most of them contained aminohexoses and fucosamine [99,505] (Table 2.1.6(3)). The structure of GalDAG from bovine spinal cord [loll and frog brain [539] was similar to that of plant GalDAG, except for the composition of fatty acids, which were mainly 16 : 0 and 18 : 1 (see Section 2.2.3). However, the content of GalAAG and GalDAG in spinal cord or brain of rat [44] was significantly lower than that of cerebrosides. Recently, the brain of some fishes was shown to contain higher concentrations of GGroLs [51,52,102]. Also the secretions of mammals, such as gastric juice [48], sublingual gland secretions, saliva and pulmonary lavages, have
Fig. 2.2. Carbohydrate-containing membrane lipids of Streptococcusfaecalis. The crude lipid extract was separated two dimensionally on a plate of silica gel (Merck). The first direction (upwards) was developed in chloroforni/methanol/water. 65 :25 : 4 (v/v), and the second direction was developed with chloroform/ace~one/methanol/acetic acid/water, 60 : 20 : 10 : 10 : 5 (v/v). Carbohydrate-containing lipids were visualized with a-naphthol/H,SO,,. (1) Glcal-3DAG; (2) Glcal-2Glcal-3DAG; (3) Ptd-6Glcal3DAG; (4) Glcal-2(Ptd-6)Glcal-3DAG;(5) sn-Gro-l-P-6Glcal-2(Ptd-6)Glcal-3DAG (for structure, see Fig. 2.lb); (6) sn-Gro-l-P-6Glcal-2Glcal-3DAG; (7-1 1 ) unidentified components; (7) stained also with ninhydrin and phosphate reagents; (8) is phosphate-negative. but is stained with ninhydrin; ( X ) is Glcal-2Glcal-2Glcal-3DAG. By the courtesy of Dr. W. Fischer (Erlangen) from Ref. 77.
112 been reported to contain isomaltose (Glcal-6Glca-) series of oligoglucosides on the position l(3) of AAG and DAG [50,103](Table 2.1.1(9), 2.1.2(9), 2.1.3(9). 2.1.5(2-4)). a-Isomaltose has not been detected in mammals, although recently a-isomaltotriose was characterized in a nephritogenic peptide of murine renal glomerulus [104]. Chemical synthesis of GGroLs has been extensively reviewed by Shvets [lo51 and Gigg [32]. The representatives of synthetic GGroL are included in Tables 2.1 and 2.
2.1.2. Acidic glycoglycerolipid 2.1.2.1. G G r o h containing uronic and sialic acid Glucuronic acid-containing glycolipids (GlcU-DAGs) (Table 2.2.2) are produced in small amounts in various typical pseudomonads under appropriate growth conditions [ 107,108], and can be a major [ 1091 but expendable lipid in the atypical Pseudomonas (now AIteromonas) rubescens. Moderately halotolerant bacteria also contain GalU-DAG [ 1101 or GlcUceramide [89]. A capsular polysaccharide from a gram-negative bacterium contained polymers of sialic acid linked to Ptd residue (Table 2.2.2.(5)). 2.1.2.2. Sulfoglycolipids Sulfate ester has been regarded as an analog of phosphate esters, since the time when F. Egami was engaged in studies to see if Glc 6-sulfate can serve as an analog of Glc 6-phosphate. It appears also, that when Tannhauser concluded that the sulfate ester of “sulfatide” was linked to the position 6 of Gal, it may have been because of the fact that Glc 3-phosphate was not known. Thus far, sulfate had been the servant of phosphate. The mycobacterial “sulfolipids” [lll] have been elucidated in great detail. However, they do not belong to the class of glycerol-type glycolipids. Sulfato-GGroL occur frequently in archaebacteria, while in eubacteria it has seldom been reported. Sulfate esters of di-, tri- and tetraglycosyl-di-0-phytanylglycerolethersin halophilic archaebacteria have been described [10,112-114,2501 (Table 2.2.1(4-6); c.f., Section 5.4). A sulfolobus contained 10 and 7% respectively, of sulfo- and sulfophosphoGGroLs [526]. The discovery of sulfoglycolipids appears to be delayed mainly because there are no specific detection methods which are sufficiently sensitive, as the resorcinol spray is for gangliosides. Most of the sulfur-containing glycolipids have been discovered by 35S-labeling and thin-layer autoradiography [9,115,127,131,195]. Sensitive and specific assay of sulfoglycolipids based on Azure A complexing of peracetylated derivatives, is also a relatively novel device [364]. This method is applicable even for highly polar structures. It is also very important to he aware of the highly polar nature of sulfate group [364]. Since GGroLs in the animal kingdom had been neglected as a minor component of central nervous system, the major glycolipid of rat testis and boar sperm was at first assigned to glycosphingolipid [117-1191. In 1972, Ishizuka et al. [45,120] and Kornblatt et al. [46,121] independently isolated a sulfogalactoglycerolipid (seminolipid) as the major glycolipid component of mammalian testis. The lipid was characterized as the 3’-sulfate ester of GalPl-3AAG [45,47,122] (Fig. 2.la). The content in boar testis and sperm was 0.3 and 1.0 pmol/g of tissue, respectively,
113 TABLE 2.2 Anionic glycoglycerolipids and related glycosides 1. Sulfo- and sulfonoglycoglycerolipid (1) HS03-O-3Galp/31-3-sn-DAG and AAG (seminolipid or sulfogalactosyldiacylglycerol):Mammalian germ cell: boar testis and spermatozoa, 0.3 and 1.0 pmol/g = 38 and 64 nmol/mg protein.
respectively, exclusively alkylacyl form 1451; guinea pig testis [122]; rat testis 146.1211; mouse testis [70]; human testis and sperm [47,71,199]; bovine epididymal spermatozoa [253]; comparative biochemistry [56]. Nervous system: AAG form, rat bain [71]; DAG form [124]; both AAG and DAG forms in rat brain [125.254], 2.1-7.1% of total sulfoglycolipids, change in content depending on age [102]; cod brain, 0.84 pmol/g [52]; cod spinal cord, mainly DAG form. 1.3 pmol/g [194]; guinea pig spinal cord [255]; chicken retina [256]; sheep brain (1931; mouse cerebellum, 950 nmol/g [540]; cultured mouse nerve cell (1921. Chemical synthesis I32.58.2091. HS03-O-3Galp~l-3-sn-l-monoalkylGro (lysoseminolipid): Boar testis 1451; human testis, first reported as sulfo-lactosylceramide because of very similar R, on TLC 1471; produced by mild alkaline deacylation of seminolipid [46,122.295,365]. (2) H S O , - O - ~ ( G ~ C ~ ~ - ~ ) ~ G(a-isomaltotriosylDAG IC~~~-~(~)DA sulfate) G and AAG: Human gastric contents, 42 mg/100 ml, AAG form predominating, AAG is a mixture of I-0-alkyl and 2-0-alkyl type [49]; human saliva, 1.2 mg/100 ml, AAG form predominated [221]; rabbit pulmonary lavage 12223. (3) HSO,-O-~(GIC~~-~)~~I-~(~)AAG (a-isomaltotetraosylDAG sulfate): Human gastric contents and mucous (50,103). (4) HSO3-O-6Manpal-2Glcpal-1,2,3-di-O-phytanyl-sn-Gro (S-DGD): A halophilic bacterium (R-4). 23% [500]. (5) HS03-O-3Galppl-6Man p a1 -2Glcp a1 -1 ,-2.3-di-O-phytanyl-sn-Gro(S-TGD-1 ): Halobacterium cuiirubrum, 24% of total acetone insoluble lipids 11121; H. salinarium [113]. ( 6 ) HS03-O-3Galppl-6(Galfal-3)Man p a1-2Glcpal-1,-2.3-di-O-phytanyl-sn-Gro (S-TeGD): Hulobucferium curirubrum [114]. (7) HS03-6Quinovosepal-3-sn-DAG (6-deoxy-6-suIfo-~-Glcp-al-3-sn-Gro 2,3-acyl 2 ) : Isolation: chloreIla [9.195.208.305], 1.9% [178]; halotolerant algae, 7-10 mol% [515]; spinach envelope, 0.8% 11731. 7.5 mol% [175], 7% of thylakoid membrane of spinach [173]; Bacillus acidocoldarius, 8.8%. 35 mg/g 12381; marine diatom (non-photosynthetic) Nifrschia ulba, 1.6%. 19.5 mg/g, 17% of "S incorporated into lipid, probably contain also sulfate ester 12571; distribution in algae [535]. Chemical synthesis [258]. HS0,-6Quinovosepal-3-sn-MAG (lyso derivative), N. alba, 0.3%. 3 mg/g [257]. (8) Dimethylarsinoyl~l-5(5-deoxyribose)3(HSO3-O-l)Gro: a green alga [527].
2. Glycoglycerolipids containing uronic acid or sialic acid
( I ) GlcUpal-3-sn-DAG: Pseudomonas diminura, 3.5% 15 mg/g [38]; Isolation and metabolism [259]; P. uesiculuris [ 1081. ( 2 ) GlcUpfil-3-sn-DAG: Alteromonas (Pseudomonas)rubescens, 3.0% of total lipid 11091; Bacillus cereus T [306]. (3) Glcp~I-4GlcUpal-3-sn-DAG: Pseudomonas diminura, 4.1 % 1381; P. uesiculuris [log]. (4) Glcpal-4GalUpal-3(1)DAG: Srrepromyces LA 7071, 6% acylated at position-2 or -3 of GalUp [260]. ( 5 ) (NeuAca2-9NeuAc)nNeuAca2-O-Ptd-DAG: Meningococcus [251]. (6) NeuAca2-9NeuAca2-3DAG: chemical synthesis 15471. (7) NeuAc~2-9NeuAca2-3DAG:chemical synthesis [547].
114 TABLE 2.2 (continued)
3. Glycosyl glycerophospholipid (1) 3-Ptd-l’-P-GlcNp-3’-sn-Gro (GlcNbl-3’-PtdGro): Structure: Bacillus megaterium. 251 nmol/g. 7.8 pgP/g cell when cultured at pH 5 [132], no obvious effect of pH on the content [133]. Chemical synthesis (2611. (2) 3-Ptd-l’-P-GlcNp-2’-sn-Gro (GlcNpbl-2’-PtdGro): Isolation: Bacillus rneguterium, at p H 5 , 5.6% of total lipid = ca. 11 mg/g cell [34,132], not detectable at p H 7.0. Structure: B. megaterium, 30-35% of P, no obvious effects of p H on the content [132,133]; Pseudornonas ovulis (2621. Chemical synthesis: a and p mixture [263]. (3) 3-Ptd-2’-a-GlcNp-l’-sn-Gro (GlcNpal-2’-PtdGro): Isolation: P. ovalis, 1.O pmol/g. 31 pgP/g cell [35]; Structure: Pseudomonas ovulis (133,2621. (4) 3-Ptd-2’-a-Glcp-l’-sn-Gro (Glcpcrl-2’-PtdGro): Isolation: a moderately halophilic bacterium, gramnegative 11341. (5) 2-O-aGlcp-I’,3’-bis( sn-3-Ptd)Gro (Glcpal-2‘-diPtdGro): Isolation and characterization: Group B streptococcus, 7-8 m o l l , 18% of lipid P [4]. (6) I-Ptd-2-a-Manp-~-myoinositol (mannosyl phosphatidylinositol) mycobacterium [265,266]: propionibacterium (267); nocardia [268,269]; streptomyces [269]. (7) l-Ptd-2,6-di-aManp-~-myoinositol(dimannosyl phosphatidylinositol) mycobacterium [265,266,270]. (8) 1-Ptd-2-aMan p-6(Man p al-2Man p al-6Man p al-6Man p al-6)-1.-myoinositol (phosphatidylinosi to1 pentamannoside): mycobacteria [265,266,270]. (9) Glcpal-2Gal/~1-l’-dibiphytanylglycerylphosphorylglycerol: Methanospirillurn hungutei. 50% [232]. (10) Gal/fil-6Gal/~1-3’-sn-Gro-l-P-3-sn-Gro: Methonospirillurn hungutei, 14% [271]. (11) GlcpSl-3-Ptd (phosphatidylglucose): 1-Myristoyl-2-oleoyl. chemical synthesis [272,530]. (12) 3-Ptd-6Glc (phosphatidyl-6-glucose): 1,2-dipalmitoyl, chemical synthesis [530]. (13) 0(CH3),As-5(5-deoxyribose~1-3’)PtdGro:a symbiotic unicellular green alga living in clam kidney [527].
4. Phosphatidyl- or glycerophosphoryl-glycoglycerolipids,glycerophospho-glycerophospho-glycoglycerolk pids and the combined forms
“Fischer number” indicates the number of glycolipid designated by W. Fischer (University of Nurnberg. Erlangen). Usually they are in the order of higher R , values on thin-layer chromatogram, i.e.. highly glycosylated compounds have usually higher numbers. (1) 3-sn-Ptd-6Glcpal-3(1)DAG (phosphatidyl glucosyldiacylglycerol. Fischer number I): Pseudomonas diminutu. 0.9%,isolation (381; structure [273]: P. vesiculuris, distribution [lOS]; metabolism [259];
Streptococcusfaeciurn, up to 1.1 mol% [243]; S. hemolyticus, 1.5 mo1%[243]. (2) Glcpcrl-2(3-Ptd-6)Glcpal-3(1)-sn-DAG (phosphatidyl a-kojibiosylDAG. Fischer number 111). Isolation: Srreprococcus /uecium, 8-20 mo18 [37,137] and S. luctis, 21.0 mol% [30]; old culture of Acholeplasma laidlawii, position of Ptd group not known [274]: Structure: S. /uecium, 12% [78]; partial structure (641, in group D streptococci, up to 28% of polar lipids (2431. (3)3-sn-PtddGlcpal-2Glcpal-3(1)DAG (glycerophosphoryl-a-kojibiosylDAG, Fischer number, 11): Streptococcus hemolyticus, 4.5 mol% [30]. (4) l-sn-GroP-6Gal//31-3DAG (glycerophosphoryl n~onogalactosyl DAG): Blfrdohucrerium hrfidum. 19%) is in pyranose form (2121; Isolation: butyrivibrio. a or p. mainly in plasmalogen type [214]; Biosynthesis 1275,2761. (5) 3-Ptd-6(Glcpal-2),Glcpal-3(1)DAG(phosphatidyl-a-kojltriosylDAG. Fischer nuniher I V ) : Streptococcus hemolyticus [155].
115 TABLE 2.2 (continued) (6) 1(3)-GroP-6Glcpl-3Gro: Isolation: Acholeplasma laidlawii 12771. (7) I-sn-GroP-6Glcp-al-2Glcpal-3(1)DAG (glycerophosphoryl-a-kojibiosylDAG, Fischer number V): Isolation and partial structure. S. hemolyticus, 1.8% [217]; Structure: 2.8 mol% [30]; partial structure: Acholeplasma laidlawii [97,278], S. Jaecalis [279]; Structure: S. Jaecalis and S. hemolyticus [78]; S. lactis NCDO 712, 8.6 mol% [145,243]. 34% substituted by D-Ala at the GroP moiety [31,507]; group B streptococcus 0.7% (76,1451; the initial segment of the LTA from S. lactis MCD 712 and S. Jaecalis
[311. l-sn-GroP-6Glcpal-2(acyl-0-6)GIcal-3(1)DAG(Fischer number VII), group N streptococci [ 138.1451. (8) Galpal-2-sn-Gro-l-P-6Glcal-2(acyl-0-6)Glcpal-3( 1)DAG (Fischer number XV): Streptococcus lactis Kiel 42172 (1381. ( 9 ) Galpal-3-sn-Gro-l-P-6Glcpal-2(acyl-0-6)Glcpal-3(1)DAG (Fischer number XVI): Streptococcus lactis Kiel 42172. (10) Galpal-2(Galpal-3)-sn-Gro-l-P-6Glcpal-2(acyl-O-6)Glcal-3(1)-DAG (Fischer number XVII): Streptococcus lacris Kiel 42172 [ 1381. (11) Gal p al-6Gal p al-3-sn-Gro-l-P-6Glcpal-2(acyl-0-6)Glc al-3(1)-DAG (Fischer number XVIII): Streptococcus lactis k e l 42172 [138]. (12) Gal pal-6Gal p al-3(Gal p a1 -2)-sn-Gro-l-P-6GlcaI-2(acyl-0-6)Glcpal-3( I)-DAG (Fischer number XIV). The initial segment of the LTA from Streptococcus 1a:tis Kiel 42172 [138,158]; S. lacris L e l 42172 (1381. (13) Gal p a l - 6 G a l p a1 -6Gal p al-3(Gal p al-2)-sn-Gro-l-P-6GIcp a1 -2(acyl-O-6)Glcp al-3( 1 )-DAG (Fischer number XX): S. lactis [31]. (14) I-sn-GroP-6Glcp~1-6Glcp~l-3(1)-DAG (glycerophosphoryl-8-gentiobiosylDAG) (Fischer number VIII): Structure: Staphylococcus epidermidis grown in 25% NaCI, 10%by weight of polar lipids [139]; S. aureus 0.5 mol% [145,243]; Bacillus licheniformis. 10.5 mol% [145,243]; the initial segment of the LTA from S. uureus, E. licheniformis and B. subtilis 1311 and is substituted with D-alanine at glycerol. when extracted with mild acid solvent (5071. (15) GroP-Galpal-2Glcpal-3(1)-DAG (Fischer number IX): Lacrobacillusplantarum [145]; Leuconostoc mesenteroides and Lisreria monocytogenes [ 2801. (16) l-sn-GroP-6Glcp~l-6Galpal-2Glcpal-3(1)-DAG (Fischer number X) and I-sn-GroP-6Glcp~l6Galpal-2(acyl-0-6)Glcpal-3(1)-DAG (Fischer number XI): Lacrobacillus casei 1.2 and 0.8 mol%, respectively [145.243], both lipids are the initial segments of the LTA from L. casei [141,154] L. helveticus [l50]. (17) l-sn-GroP-3-sn-Gro-l-P-6Glcpal-2Glcpa1-3(1)-DAG (Fischer number XIVa) and 1-sn-GroP-3-snGro-1 -P-6Glcp a1 -2(acyl-0-6)Glcpa1 -3(1)-DAG (Fischer number XI I): Streptococcus lacris NCDO 712, 1.6 mol% each [146,243], 37% substituted with D - a h i n e at GroP [31,507]. (18) I-.sn-GroP-3(Galpul-2)-sn-Gro-l-P-6Glcpal-2(acyl-O-6)Glcpal-3(1)-DAG (Fischer number XIVb): Streptococcus lactis NCDO 712 [146]. (19) l-sn-GroP-6Glcpal-2(3-sn-Ptd)Glcpal-3(1)-DAG (Fischer number VI): Streptococcus Jaecalis strains, 1.4-2.7 mol% [77,243], the initial segment of the LTA from S. Jaecalis strains [31]. (20) G l c p p l - 1-X-Gro-3-(~-glyceraldehyde-3-O)P-6Glcp a l - 2 G l c p al-3(1 ) - D A G : Acholeplasma granulurum. tentative structure containing a phosphotriester, 0.6% of cell weight = 25% of total lipid P (1471. (21) sn-Gro-3-P-6(Galpal-R) and sn-Gro-3-P-S(GalJal-R) (R indicates a carbohydrate chain): E. coli K2 antigen [503]. (22) D-Ala-2 or 3-sn-Gro-l-P-6GIcppl-6Glcppl-3( 1)DAG: Streptococcus lactis, Bacillus IicheniJormis. 7.4 mol% [507].
116 TABLE 2.2 (continued)
5. Lipoteichoic acid (1) (GroP),,. 2,-Glcpal-2(Ptd-6)GIcpal-3(1)-DAG:Isolation and structure ((GroP),,) from group D
streptococci as “intracellular teichoic acid” by Wicken and Baddiley [283] (without lipid anchor, but with D-alanine): S. faecalis (faecium) [155,161,282], 43% of the innermost Glc not substituted with Ptd. Chain substitution, given as mole alanine ester or glycosyl groups per mole LTA phosphorus or glycerol: Glca, Glcal-2Glca, (Glcal-2),Glca (82 to more than 90%), D-Ala inconstant [42.283]. About 10%of membrane GGroLs in the form of LTA [282]. Biosynthesis [284.360]. ( 2 ) (GroP),,-Glcpal-2(Ptd-6)Glcpal-3(1)-DAG: Streptococcus /uecalis subsp. zymogenes [ I 551, 62% of the innermost Glc not substituted with Ptd. Chain substitution: D-Ala (0.29). Glcal-2Glca (29W), and ~-Ala-Glcal-2Glca(19%). ( 3 ) ( G ~ o P ) ~ , - ~ G I c ~ ~ ~ - ~ GStreptococcus Ic~~~-~ hemolyticus ( ~ ) - DD-58 A G :[141]. Chain substitution of group A streptococci: D-Ma, 47-56% [285]. (4) (GroP) ,,-6Glcp a1 -2Glcpal-3(1)-DAG: Leuconostoc mesenteroides [ 141,1551. Chain substitution: D-Ala, 54-59%). ( 5 ) sn-Gro-l-(P-3-sn-Gr0-1)~~-P-6Glcpcrl-2Glcpal-3(1)-DAG: Streprococcus lacris NCDO 712 [42,150]. Acylation at the innermost Glc is 51%. Length of the hydrophilic chain (extended conformation) is 17 nm, 1 unit = 0.75 nm [l58]. Chain substitution: D-Ala, 21%; Gala, 39%; D-Ala-Gala (9%). LTA constitutes 10 mol% of the polar membrane lipids [31]. ( 6 ) G a l p al-6Gal p al-3-(Galp al-2)-sn-Gro-l-P-(6Gal p al-6Gal p al-3-(Gal p al-2)-sn-Gro-l )h.lO-P6Glcpal-2(acyl-0-6)Glcal-3(1)DAG: Sireprococcus luctis Kiel 42172 [158]. 5 % of the innermost Glc not acylated: length of the hydrophilic chain (extended conformation)= 12-19 nm, 1 unit = 1.63 nm. Chain not substituted with D-Ala [l55]. (7) (G~oP),,-~GIc~/~~-~GIc~/~~-~(~)DAG: Micrococcus uariuns (1551. Chain not substituted. (8) (GroP),,.,,-6Glcp/31-6Glcp/31-3( I)-DAG: Staphylococcus aureus [42,150,160,510], unbranched chain structure [31]. Chain substitution: GlcNAc, trace; D-Ma, 23-73s; decreasing with increasing NaCl concentration in the growth medium [l59]. LTA accounts for 8-15 mol% of the polar membrane lipids [31,545]. ( 9 ) (Gr0P)~,-Glcp/31-6Glcp/31-3( 1)-DAG: Bacillus licheni/ormis [236], G I c / ~ I - ~ G I c / ~ ~ - ~replaced DAG by DAG or PtdGro when the formation of UDP-Glc is blocked [281]. Chain substitution: no glycosyl residues, D-Ala not studied. (10) (GroP),,-6Glcp/31-6Glcp/3l-3( 1)-DAG: Bacillus subtilis (31,421. chain substitution: D-Ala, 38-42%; Glca, 17-20%; GlcNAc. 18-21%. (1 1) (Gr0P)~~-Glcp/31-6Galpal-2Glcpal-3(1)-DAG: Lactobacillus plantarum [141], 45% carries 6-0-acyl on the innermost Glc. Chain substitution: no glycosyl residues, D-Ala not studied. (12) (GroP),,,,-Glcp/31-6Galpa1-2Glcpal-3(1)-DAG: Lactobacillus casei [MI, 40% carries 6-0-acyl on the innermost Glc. Chain substitution: D-Ala 93% [412], no glycosyl residues. (13) (GroP),-Glcp/31-6Galpal-2Glcpal-3(1)-DAG: Lactobacillus helueticus [141], 45% carries 6-0-acyl on the innermost Glc. n = 38 instead of n = 24 (Fischer, personal communication). Chain substitution: D-Ala, 57%:.no glycosyl residues.
comprizing about 3% of the total lipid of testis (Table 2.2.1(1)). The level of seminolipid in boar sperm on protein basis [123] was calculated to be about 15-times higher than that of GalP1-3DAG in rat brain [93].
117
In analogy to the occurrence of both cerebrosides and their sulfate esters in the central nervous system, it was anticipated that a sulfate ester of GalDAG may also be present in brain. Actually, Flynn et al. [124] isolated a sulfated GalDAG and Levine et al. [71] characterized the alkyl-acyl (AAG) form, both from rat brain. It was shown later that both AAG and DAG forms coexist in the brain of rat [71,102,125] and mouse [540] as the minor components (c.f., Section 3.3.2). The molar ratio of GGroLs (including seminolipid) to glycosphingolipids in the cerebral hemisphere of Pacific cod, however, reached 0.38 [52]. The polyglucosylDAGs (isomaltose-series) and AAGs of secretory fluids also contain a sulfate ester at C6 of the terminal Glc [50](Table 2.2.1(2,3)). The primary sulfate ester of the sulfoGGroLs from gastric juice could be easily removed by acid solvolysis or mild acid hydrolysis [48]. However, neither in vivo biosynthetic studies nor confirmatory reports from other groups appeared [56,417,522], except for the preliminary accounts by Pritchard [129] (see Section 4.1.3). The recent developments in biochemistry of sulfoglycolipids have been the subject of detailed reviews by Farooqui et al. [57,126,419], Dulaney and Moser [54a], Karlsson [250], Ishizuka [131], Kates [544], Radin [533] and Kolodney and Moser [54b]. Also the conditions for solvolysis of sulfate esters from sphingoglycolipids was reviewed [10,127,512,546].Seminolipid could be successfully cleaved by heating in moist dioxan or acidic dimethylsulfoxide [102,127,128]. 2.1.2.3. Micro GGroLs with phosphate ester It is not necessary to stress the importance of phosphate in nature. Phosphate in biological membranes is usually present as monoester (R’O)P(O)(OH),, or diester (R’O(R’O)P(O)(OH), where R’ and R2 designate hydrophilic and/or hydrophobic alcohol. The classical “glycoglycerophospholipids” of mycobacteria, phosphatidylinostiol di-, tri-, tetra- and pentamannosides (Table 2.2.3(6-8)), possess the core carbohydrate structure (Manal-4myo-inositol-2-phosphate) in common with a class of complex plant glycophosphosphingolipids (phytoglycolipid). These acidic and amphiphilic glycoconjugates in microorganisms may be analogous in chemical properties to the acidic amphiphiles, such as gangliosides and sulfoglycolipids in animal cell membrane [106,127,131,198] (see also Section 2.7.1). These phosphoglyco-amphiphiles have been traditionally classified in the phospholipid, because usually phospholipids are the more abundant constituents of tissues and cells. Some representatives of these “phospholipids” are included in Table 2.2.3 for comparison with “phosphoglycolipids”. The glycosides of phosphatidylglycerol (PtdGro) and diphosphatidylglycerol (diPtdGro) in microorganisms are relatively new members of glycoglycerophospholipids. The first of this type, glucosamine derivatives of PtdGro, 3’-GlcN-PtdGro (Table 2.2.3(1)) [132,133] and 2’-GlcN-PtdGro (Table 2.2.3(2)) [26,34,35,132,133], were isolated independently by two groups from Bacillus megaterium and Pseudomonas ouafis, respectively, shortly after the discovery of 0-aminoacyl esters of PtdGro by Macfarlane. GlcaPtdGro (Table 2.2.3(4)) was also reported to occur in a halobacterium [134]. Glucosylated cardiolipin (GlcadiPtdGro, Table 2.2.3(5)) was isolated from a group B streptococcus [4]. The contents accounted for more than the
118 sum of lipid phosphate contributed by PtdGro and lysyl-PtdGro. The names given to the other two classes of phosphate-containing GGroLs, phosphatidyl-GGroLs” [135] and “glycerophospho-GGroLs” [78] (Table 2.2.4), imply that they are derived from glycolipids by the transfer of Ptd- (Section 4.1.1) and glycerophospho- (GroP) (Section 4.1.5) groups, respectively [78]. In 1965, Smith and Henrikson [288] described a novel class of lipid phosphatidyl-glucose” from Acholeplasma laidlawii. The quantitative composition of Glc, Gro and fatty acids might indicate that this preparation was a mixture of equimolar amounts of PtdGro and GroP-Glc,DAG, which are difficult to separate from each other [36]. The first pure GroP- and Ptd-GGroLs were obtained from Streptococcus hemolyticus by Ishizuka and Yamakawa [36] and from Streptococcus faecalis by Fischer and Seyferth [37], respectively. Thus, the neutral GGroL. Glca-2Glcal-3DAG (a-kojibiosylDAG), occurs in streptococci not only in a free form but also as a covalently bound component of “phospholipids” [30]. Then it was established that Glcal2(Ptd-6)Glcal-3DAG (Table 2.2.4(2)) [136.137] and Ptd-6Glcal-3DAG (Table 2.2.4(1)) [38] have their phosphate group attached to C6 of the internal Glc (Fig. 2.2, Lanes 3 and 4). GroP-Glc,DAG (Fig. 2.1, Lane 6) was shown to be a compound different from Glc(Ptd)GlcDAG, in that it contains the sn-l-GroP group at C6 of the external Glc of Glcal-2Glcal-3DAG (Table 2.2.4(7)) [135]. The stereoconfigurations of Gro-P and DAG-P were established as l-sn-GroP and 3-Ptd, respectively, by chemical methods [77,135,138,139]. GroP-GGroLs were later detected as the lipid anchor of the lipoteichoic acid (LTA), the accessory membrane polymer of most of gram-positive bacteria [31,33,41,140,141,510] (Fig. 2.3). Earlier, in vitro biosynthetic studies suggested the presence of unidentified polar lipids in some gram-positive bacteria. By incubation of UDP-[14C]Glc and the membrane vesicle fraction of Streptococcus faecium, an anionic lipid was formed in addition to GlcDAG and Glc,DAG [64]. A polar lipid, which at first appeared to be a Man,DAG, was synthesized from GDP-Man also in the system of Micrococcus luteus [61]. Later, the former was characterized as G l c d -2(Ptd-6)GlcaDAG (Table 2.2.4(2)) [68,137]. The latter was, however, identified as Man-l-P-undecaprenol [66,143], the precursor of lipomannan [41,144]. A combined (hybrid) form that had both sn-Gro-l-P and 3-Ptd groups attached to Glc (Fig. 2.lb; Fig. 2.2, spot 5; Table 2.2.4(19)) [77] and GroP-GGroLs with an O-acyl group on position 6 of the internal hexose (Table 2.2.4(7)) were also isolated [145]. These are key compounds for the biosynthesis of LTA by successive addition of GroP units [31] (Section 4.1.5). In 1978, the unique 1-sn-GroP-3’-sn-Gro-l’-P (Table 2.2.4(17)) residue, which indicated the next step in the scheme of the GroP chain elongation of GroP-GGroLs, was discovered in Streptococcus lactis [146]. Furthermore, an a-Gal (Table 2.2.4(18)) or D-Ala linked to positions 2 of GroP [31,146,507] reflected the chain substitution of LTA in this organism. To date, about 20 GroP-GGroLs have been characterized (Table 2.2.4). Recently a new phosphotriester structure (Table 2.2.4(20)) was proposed for a phosphoglycolipid of Acholeplasma granularum [ 1471. “
“
119 2.1.2.4. Mucro phospho-GGroLs The presence of GroP and an unidentified phosphate (presumably ribitol phosphate) in bound form in wall and membrane preparations of staphylococcus was reported by Mitchell and Moyle [17], but the techniques for preparing pure cell walls and membranes were insufficiently developed at that time [40], and the true nature of the phosphate-containing compounds was not established. A Forssman-antigenic F-polysaccharide of pneumococcus is also a kind of polysaccharide, which is now included in LTA, containing galactosamine, ribitol phosphate and fatty acids [148,149](see also Section 2.5.5). Later on, two classes of phosphate-containing accessory polymers of cell wall became discernible. “Wall teichoic acids” are linked to the peptidoglycan, which is the primary polymeric component of cell wall, through a phosphodiester bond. On the other hand, “membrane teichoic acid” could be extracted from protoplasts with trichloroacetic acid after digestion by proteolytic enzymes and removal of peptidoglycan [33]. In 1970, by the introduction of phenol for the extraction by Wicken and Knox [39], the amphiphilic nature of membrane teichoic acids was discovered. It was shown that the poly(g1ycerophosphate) chain is covalently linked to a glycolipid, which is anchored in the cytoplasmic membrane [39,41]. Since then, membrane teichoic acid has been called lipoteichoic acid (LTA) to emphasize its amphiphilic nature [140]. It was only recently that, using low p H and low temperature during the isolation procedure, “native” LTA which retains the original substitution with D-alanine ester and free from contaminants, was isolated [150,534]. The putative “lipoteichoic acid carrier” (LTC) which, in vitro, serves as the assembly point for teichoic acid polymerase (Section 4.1.5) [151]. remains structurally undefined [152,153] until its identity with lower or ubsubstituted LTA has been demonstrated [141]. Recently, an interesting observation was made: that Triton X-100 extracts of the membrane of a group A streptococci (S. pyogenes) contained a greater amount of deacyl-LTA than LTA [130]. The former appeared to be derived from LTA by the activity of a membrane-bound lipase, which was most likely activated by Triton treatment (see Section 4.2.3). Usually LTAs have a 1-3 linked poly(g1ycerophosphate) chain of 20-40 units in length (Table 2.2.5; Fig. 2.3) [33,40,41,140].For a given LTA the chain length varies within a narrow range [31] but from species to species [141,154,155]. The sn-1 configuration of the GroP units, which had been suggested from biosynthetic studies [156,157],was proved chemically [42,158]. Depending on the species of organism, the GroP units are substituted at position 2 by various mono-, di- and trisaccharides and/or by D-alanine ester [42]. The LTA from Streptococcus luctis Kiel42172, which contains a poly(Ga1pal-6Gal pal -3(Galpd-2)-sn-Gro-l-P) chain [I 581, is another type of LTA differing from the classical poly(g1ycerophosphate) type in having hexose repeating units. One end of the hydrophilic chain of LTA is joined by a phosphodiester bond to position 6 of the outer hexosyl moiety of the constituent glycolipid (refs. 31, 160, and W. Fischer, personal communication), except in LTA from S. luctis Kiel (Table 2.2.5(6); Fig. 2.3C). In most cases, the “lipid anchor” of LTA is a Hex,DAG. which is the major membrane neutral GGroL. In addition to Hex,DAG, in Streptococcus
A
OH
0
CH~OCR’ II
X = -H X:
-CO-R
5%
95%
Fig. 2.3. The structure of lipoteichoic acids from streptococci. ( A ) Srreprococcus herno/r.ricusD-58 ( S . pyogenes. type 3) [141]; ( B ) S. lacus NCDO 712 [155];
X. -H. 58%; X. -CO-R, 42%;(C) S. larris 42172 [158]; X, - H . 5 4 : X. -CO-R. 95R..
121 lucris strains, its 6-0-acylated derivatives (Table 2.2.5(5-6, 11)) [31,141,158] are used. And in strains of Streptococcus fuecium the Ptd-derivatives (Table 2.2.5( 1-2)) serve as the anchor [155,161,162]. In Luctobucilluceae, Hex,DAG and its 6-0-acyl derivatives (Table 2.2.5(11-13)) are preferred [141,150,154] (c.f., Section 2.2.2). Amphiphilic capsular polysaccharide of meningococcus is different from classical capsular polysaccharide (Table 2.2.2(5)) [251]. It contains (NeuAca2-8) polymer as the charged hydrophilic moiety and Ptd-group as the lipophilic anchor. Succinylated lipomannan does not contain glycerol, but is also one of the highly acidic amphiphiles containing many carboxyl groups [252] (c.f., Section 3.6). Several liponucleotide analogs of CDP-DAG containing the 1-P-D-Araf moieties have been chemically synthesized for the purpose of evaluation of the antitumor activity of these compounds [163]. Multispecies Ara-CDP-DAG, which contains mixed fatty acyl chains obtained from phosphatidylcholine of egg yolk, was more active than 1-P-D-Araf cytosine, an anticancer drug clinically used against leukemia and an ascites carcinoma of mouse [164]. 2.2. LIPOPHlLlC D O M A I N
GGroLs are anchored with two “legs” in the lipid bilayer of cell surface membranes. These “legs” of DAG are replaced, in glycosphingolipids, by ceramide: a sphingoid base and a fatty acid linked to the sphingoid through an amide bond (Sections 5.1, 2.1 and Ref. 511). As shown in Table 2.3, GGroLs usually have two ester-linked long-chain fatty acids and a Gro molecule forming a lipophilic domain: sn-l,2-DAG (Fig. 2.lb, Table 2.3(1)), of which the position sn-3 of Gro is occupied with carbohydrate (the head). DAG is less polar than ceramide, and GalDAG and GalDAG sulfate migrate faster than GalCer and GalCer sulfate, respectively, on silica gel TLC. There are also less common types of legs, in which an alk-1-enyl (in alk-1-enyl-2-0-acyl-sn-Gro) or an 0-alkyl group (in l-O-alkyl-2-O-acyl-sn-Gr0, Fig. 2.la) is substituted for an acyl ester at position 1. These are called the plasmenic acid (plasmalogen) type (Table 2.3.4) or alkylacyl type GGroLs, respectively (Table 2.3(2, 4) and Fig. 2.4, Table 2.4) [525]. The di-0-alkylGro structures have been shown to be one of the marker compound of archaebacteria [501,508] (Table 2.3(5-8)) [12,13]. The above classification is in parallel to that of “phosphoglycerolipids”, by Hanahan and Brockerhof [84]. An arm, the third long-chain 0-acyl group, on the position-6 of internal hexose [201,233] (Table 2.3(15)) has often been observed in neutral HexDAGs as well as in LTAs of various gram-positive bacteria, especially in Luctobacilluceue [85]. The extra, third fatty acid can reside also on the amino group of hexosamine bound through an amide bond, as in LPS (Table 2.3(14)). Such long chain N-acyl linkages are distributed in some thermophilic or thermoacidophilic bacteria (94,2391. Phosphatidyl group (Fig. 2.lb) donate two extra arms, the third and fourth fatty acids, to some GroP-GGroLs or LTAs (Table 2.3(9)) [33,78,161]. Glycosides of phospholipids such as phosphatidylglycerol (PtdGro) (Table 2.3(10)) and diphosphatidylglycerol (diPtdGro = cardiolipin) (Table 2.3( 11)) with two or four fatty
122 acids, respectively, also occur in gram-positive bacteria. Ptd-Glc, DAG and Glc-diPtdGro have a common structural feature, that of a hydrophilic bridge which connects two lipophilic ends of the molecule [78]. In these cases, three or four fatty acids together make lipophilic groups to anchor GGroLs and LTAs very tightly on the outer surface of the plasma membrane. Such hyperacylation” on carbohydrate domains has been rarely observed in glycosphingolipids. except for the acylated cerebrosides of brain [304] (see Sections 2.3 and 4). Karlsson [250,289] has discussed the differences in physical properties of ceramides and DAGs (see also Section 5.1.1). “
2.2.1. Plant The lipophilic domains of the GGroLs in plant tissues are exclusively DAGs, except TABLE 2.3 Lipophilic domains of glycoglycerolipids Chemical structure (other name)
Source
(1) 1.2-Diacyl-sn-Gro (diacylglycerol. DAG)
Plant [328], eubacteria 1151 (Figs. 2.1, 2.3). animal brain [I 1,43,56] Brain (441, mammalian testis 1451. mammalian secretions [SO] Mung bean [248] Eubacteria [167,214] Archaebacteria [112] Archaebacteria I5081
(2) 1-O-Alkyl-2-O-acyl-sn-Gro (alkylacyl-
glycerol = AAG) a (3)I-0-Acyl-2-0-alkyl-Gro (4) 1 -Alk-l-enyl-2-O-acyl-sn-Gro a (5) 2,3-Di-O-phytanyl-sn-Gro (6)2-O-sesterpanyI(2,6,10,14,18-pentamethyleicosane)-3-O-phytanyl-sn-Gro (7)sn-2.3-Di-O-alkyl-Gro combined as a cyclic diether with two C-40 isopranoid ether (dibiphytanylglycerol-tetraether) (8) Carditol-glyceryl-tetraethers (9) 1.2-Di-O-acyl-sn-Gro-3-P (phosphati-
dyl = Ptd) a ( 10) 1 -sn-GroP-3-sn-diacylglycerol
Methanogenic and thermophilic archaebacteria [271.299,331] (Fig. 2.4) Thermoacidophilic archaebacteria [508, 526,5371 Streptococcus [30,31.78] mycobacteria 131, meningococcus (251) Bacilli and pseudomonads [34,35,68]
(phosphatidylglycerol = PtdGro) ( 11) 1’.3’-bis(l,2-Diacyl-sn-Gro-3-P)Gro
Streptococcus [4]
(cardiolipin, diPtdGro) ( 12)2,3-Di-O-phytanyl-sn-Gro-l-P-l’-sn-Gro ( 13)2.3-Di-O-phytanyl-sn-Gro-l -P-3’-sn-Gro ( 14) Long chain N-acyl ( 15)O-acyl on
C-6 of hexose
( 16)O-acyl on C-2 and C-3 of internal Gal/ ( 17) Monoacyl ethyleneglycol ( 18)AcylatedD( -)glyceric acid
Methanogenic archaebacteria [2711 Methanogenic archaebacteria 12711 Thermophilic eubacteria (94.2711 Gram-positive bacteria [145,146.233]. wheat flour [201] Bifidobacterium [183] Plant 12101 Nocardia 13321
According to IUPAC-IUB recommendation [1,2] I-0-alkyl-2-0-acyl- and I-alk-I-enyl-2-O-acyl derivatives of 3-phosphorylglycerol are called plasmanic and plasmenic acid, respectively.
a
123 an alkylacyl-oligoglycosylAAG (Table 2.3(3)) from mung bean ( Phaseolus mungo) [248]. MonoacylGros (MAG) have been described only from a few sources [91]. In general, triacylglycerols and phospholipids of photosynthetic tissues and cells, contain a high amount of polyunsaturated fatty acids [13,14,90,290,494] (Table 2.5). Also, the fatty acid component of GalDAG and Gal,-DAG in green algae, such as chlorella [178], and in the leaves of higher plants, such as runner bean (18 : 3 plant) [go], is almost exclusively 9,12,15-octadecatrienoicacid (a-linolenic acid = a-Lnn) [ 13,1421. In other photosynthetic cells, including cyabobacteria (blue-green algae), however, the situation is different (Table 5). In most cases, the proportion of palmitic acid (16 : 0) increases with increasing carbohydrate chain length, when Gal-, Gal,- and Ga1,DAGs from the same tissue are compared [91,291]. But in plants of lower taxonomic positions and spinach (16: 3 plant), GalDAG has more 7,10,13hexadecatrienoic (16 : 3) acid than Ga1,DAG [14,284]. This acid is restricted at the sn-2-position of GalDAG [195,291]. Exceptionally high amounts of 18 : 4 [6,9,12,15] and 20 : 5 [5,8,11,14,17] acids in the total lipid [297] as well as in GalDAG fractions [293], have been observed in red TABLE 2.4 Compositions of glycerylether and acyl ester in glycoglycerolipids Source (Ref.)
Glycolipid a
Composition (%)
Rat brain 68 day (102)
GalDAG = 46%+ GalAAG = 54% Sulfates of GalDAG = 86%+ GalAAG = 14%
I-Alkyl: (1) 14:O = 49, (2) 16:O = 44,(3) 15:O = 1 Acyl ester (C1+ C2) ': (1) 16 :0 = 32, (2) 18 : 1 = 30. (3) 2O:O = 7, (4) 20: 1 = 5 l-Alkyl:(l)16:1=81.(2)18:0=5,(3) 1 5 : 0 = 4 Acyl(Cl+C2): ( 1 ) 1 8 : 1 = 3 9 , ( 2 ) 1 8 : 0 = 2 5 , ( 3 ) 16:0=24,(4)20:0=5
Calf brain stem (1871
GalDAG + GalAAG
I-Alkyl: (1) 1 6 : 0 = 6 7 , ( 2 ) 1 4 : 0 = 1 1 Acyl(1 +2): (1) 16:O = 40, (2) 18: 1 = 20.0
Cod brain (51.521
GalDAG = 97% Sulfo-GalDAG = 98%
Acyl (C1 +C2): (1) 18: 1 = 41, (2) 16:O = 37, (3) 16: 1 = 13 Acyl (C1 +C2): (1) 18: 1 = 45, (2) 16:O = 29, (3) 16: 1 = 12
Human testis (471
Sulfo-Gal AAG = 100%
I-Alkyl:(1)16:0=98,(2)18:0=1.(3)14:0=1 2-Acyl: (1) 16:O = 97, (2) 14:O = 3
Human gastric content 1491
Sdfo-Glc,AAG
l-Alkyl:(1)16:0=37,(2)20:0=30,(3) 1 8 : 0 = 2 0 2-Acyl: (1) 16:O = 33, (2) 20:O = 15, (3) 18: 1 = 13
Mung bean (2481
RhamGal AAG
I-Acyl: (1) 16 :0 = 41, (2) 14 :0 = 22, (3) 18 : 1 = 14 2-Alkyl: (1) 16:O = 61, (2) 12:O = 28, (3) 14:O = 3
Composition of diacyl and alkylacyl forms (%). The figures in parenthesis are the order of abundance. Acyl ester (C1+ C2) means the overall composition including fatty acids on both C1 and C2 hydroxyls of glycerol. DAG, diacylglycerol; AAG, alkylacylglycerol. a
124
and brown algae [142]. On the other hand, GlcCer, the major glycosphingolipid of runner bean leaves, contained a-hydroxy fatty acids and trihydroxysphinganine [80]. Such diversity in different species appears to arise from evolutionary divergence [294] and may simply indicate the rout of descent on the phylogenetic tree. 6-SulfoquinovosylDAG (SQ-DAG) [195] and especially Gal ,DAG of whole leaf, envelopes and thylakoid membrane of chloroplast of rice plant are more saturated than GalDAG [174,175,336](c.f.. Table 2.7). In the spinach envelope, DAG-species of all Gal-lipids form a uniform family, pointing to a common biosynthetic sequence, with the exception of Ga1,DAG which shows reversed proportions of
TABLE 2.5 Fatty acid compositions of plant tissues and organella Species
Glycolipid
Fatty acids (in the order of % abundance)
Ref.
Runner bean leaf Spinach leaf Spinach homogenate '
GalDAG Gal DAG Gal DAG SQ-DAG GalDAG acyl-GalDAG acyl-Gal DAG GalDAG Gal DAG Gal DAG acyl-GalDAG GalDAG Gal, DAG GalDAG Gal DAG GalDAG SQ-DAG GalDAG Gal DAG SQ-DAG Ptd-Gro GalDAG Gal DAG Gal DAG
(1) a-Lnn, 96; (2) Lin, 2; (3) 16:O. 2 (1) a-Lnn, 93; (2) Lin, 1; (3) 16:O. 5
333
(1)18:3,88;(2)16:0,6; (3)16:3.2 (1)18:3,45;(2)16:0,39: ( 3 ) 1 8 : 2 , 6 (1)18:3,74; ( 2 ) 1 6 : 3 . 2 3 ; ( 3 ) 1 8 : 2 , 2 (1) 18:3, 81: ( 2 ) 16:3, 10; (3) 16:0, 5 (1) 18:3,79; (2) 16:3, 11; (3) 16:O. 6 (1) 18:3, 81; (2) 16:3,14; (3) 1 8 : 2 , 2 (1)18:3,73;(2)16:0. 10;(3)16:3,6 (1)18:3,76;(2)16:3,7 (1)18:1,74;(2)16:3,11 (1) y-Lnn, 62; (2) 18: 4 (6,9,12,15),29 (1) y-Lnn. 76; (2) I 8 : 4, 10 (1) 20:5, 51; (2) 16:0, 18; (3) 18:l. 7 (1)16:0,38;(2)20:5.35;(3)18:1,9 ( 1 ) 1 8 : 1 , 4 5 ; ( 2 ) 1 4 : 0 , 3 6 ; ( 3 ) 1 6 : 0 ,15 (1)20:5.42; (2)16:0, 16: (3)22:6, 14 (1)18:3,53:(2)16:4,30;(3)18:2,16 (1) 16:O. 55; (2) 18:3, 26; (3) 18:2, 10 (1) 16:O. 80; (2) 18:3, 11; (3) 18:2, 5 (1) 16: 1 42: (2) 18: 3,23; (3) 16:O. 15 (1)18:2,59:(2)18:3,40;(3)16:0,1 ( 1 ) 1 8 : 2 . 4 7 : ( 2 ) 1 8 : 3 , 2 5 : ( 3 ) 1 6 : 0 14 , (1) 18 : 2.43; (2) 18 : 3, 12; (3) 16 : 0,25
49 1
Spinach envelope
Chick weed Red algae Diatom Halotoleran t alga
Potato tuber
,
,
203 205 229 81
313 293 180
5 20
',
176 177
' For positional distribution, see Ref. 175. Carbohydrate-linked fatty acid. Isolated at pH 8.5. For the pattern of labeling by ['4C]02.see Ref. 336. The contents in the total lipid (moI%) for GalDAG. Gal,DAG. Ga1,DAG and acyl-GalDAG were: 37.4, 25.1, 1.0 and 0.8. Sfellaria media. 'Batrachospermum munilijorme, a fresh water alga. Nirrschia a h a , a non-photosynthetic marine diatom. DunalieNa bardawil. a halotolerant unicellular alga. d-trans-hexadecenoic acid. a-Lnn. a-linolenic acid; y-Lnn, y-linolenic acid = 18 : 3 (6.9, 12); Lin. linoleic acid.
'
125 18 : 3/16 : 3 and 18 : 3/16 : 0 species [335] (c.f., Table 2.5). In contrast, SQ-DAG, PtdGro and Ptd-choline from envelopes were separated from galactosylDAGs in their fatty acid pattern [174]. Thus the difference in fatty acid compositions in Gal,-DAG or SQ-DAGs raised a question concerning the biosynthesis of these compounds, not only the possible conversion of GalDAG to Ga1,DAG but also concerning acyl exchange and desaturation of acyl groups after the formation of glycerol ester linkage [14] (Section 4.1.2). In non-photosynthetic tissues, such as potato tuber (Table 2.5) and rice bran (Table 2.7), the fatty acids of GGroLs were more saturated, and linoleic acid (Lin) was a predominant fatty acid. Difference in DAG species in acidic glycerolipids (e.g., Ptd-inositol) and neutral glycerolipids (e.g., Ptd-choline and GGroLs) may be related to the fact that the former include CDP-1,2-DAG as a direct precursor (c.f., Section 4.1.1, Reaction (19)), whereas the latter is biosynthesized directly from 1,2-DAG [91]. Here again, Gal,-DAG, SQ-DAG and Gal,-DAG were much more saturated with higher amounts of 16 : 0 and less of 18 : 3 (n-3 = a-Lnn) than the GalDAG [91,176]. GlcDAG of rice bran showed an exceptionally high content of 16 : 0 but was devoid of 18 : 0 [296]. Photosynthetic bacteria contain exclusively saturated and monounsaturated fatty acids, a characteristic which they share with nearly all non-photosynthetic bacteria [181,320]. A non-photosynthetic marine diatom, Nitzschia alba contained neither 18 : 2 nor 18 : 3 fatty acids (Table 2.5). N. alba and other diatoms, however, have high proportions of 20 : 5 and 22 : 6 acids, which are concentrated in SQ-DAG [180] (Section 5.2). In contrast, the component fatty acids of Gal-DAG and Ga1,DAG in this organism were very similar to each other, the major acids being 18 : 1, 14: 0 and 16 : 0. Lyso-SQ-DAG of this organism had a fatty acid composition similar to that of the faster migrating band of SQ-DAG, except that the former had a much higher content of 22 : 6 acid (54%). In all materials, the fatty acid composition of GGroLs differs from that of triacylglycerol, which is also derived from 1,2-DAG. DAG is derived from phosphatidic acid (see Section 4.1.1, Reaction 3; for reviews see Refs. 21, 68, 142, 297, 399, 494. 525) and its fatty acid compositions are much different from those of acidic phospholipids, the direct precursor of which is CDP-1.2-DAG.
2.2.2. Eubacterium and archaebacterium Whereas plant and some animal tissues contain both GGroLs and glycosphingolipids, the types of lipophilic moieties in the gram-positive eubacteria are almost exclusively DAGs. The component fatty acids, however, are mostly saturated and extremely diverse in comparison to the saturated fatty acids found in plants and animals, depending on the families [496]. For instance, the major fatty acids are: branched iso- and anreiso- types in Micrococcaceae; cyclopropane acids in Lactohacillaceae and sero-group D, E, N of Streptococcaceae; and cyclohexyl fatty acids in a thermoacidophilic bacterium (Tables 2.6 and 2.7) [5,13,23,28,66,508]. Compared to higher photosynthetic organism, however, polyunsaturated fatty acids, even diunsaturated, are rare in microorganisms, including some photosynthetic bacteria.
126 Glycolipids of most of eubacteria including Staphylococcus aureus [ 161, S. epidermidis [139], Lactobacillus casei (Table 2.6) [97], and Streptococcus hemolyticus [97,135] are similar in their fatty acid compositions to those of the phospholipids or total lipid. As shown in Table 2.6, fatty acids of neutral GGroLs and GroP-Glc,DAG of S. hemolyticus (Landgraf, R.H., thesis, University of Erlangen-Nhrnberg), as well as GlcDAG and Gal(acy1-6)GlcDAG and LTA of L. casei [85,154], were similar to each other. In S. hemolyticus, fatty acid compositions of the Ptd group and DAG in Glc(Ptd)GlcDAG were identical with those of Glc,DAG, PtdGro or total lipid. Similarly, fatty acid compositions of GlcDAG and GlcUDAG of Alteromonas ruhescens were essentially identical [109].
Membrane models in archaebacteria
Alkalophlllc halophiles
E xtrerne halophi les
d
Methanogens
Thermoacidophiles
Fig. 2.4. Proposed archaebacterial membrane types. (b) “Zip” membrane formed by C,,, C,, and C2,C,, diether lipids proposed for alkalipiphilic halophiles. (d) Rigid monolayer membrane structure formed by C,, C, tetraether lipids including cyclized forms proposed for the thermoacidophiles Suljolobus and Thermoplasma. (a) Bilayer membrane formed by the C,,, C,, diether lipids proposed for Halobacrerium and Halococcus sp. and some methanogens. (c) Mixed membrane structure formed by a mixture of C,,, C,, diethers and C,, C, tetraethers proposed for some methanogens. By the courtesy of Dr. M. De Rosa from Ref. 508.
127 In contrast to glycosphingolipids in mammalian tissue, e.g., GalCer and GalCer sulfate or GlcCer and ganglioside [511], variation of the lipophilic moieties concomitant with the elongation of carbohydrate chain or with sulfation, has rarely been observed in bacterial GGroLs. As described above, the fatty acid composition is only slightly distinct from that of phospholipid, including the Ptd residue derived from diPtdGro (Section 4.1.1., Reaction 18),whch in turn is derived from CDP-DAG (Section 2.1.1, Reactions 13, 14). The long-chain fatty acyl residue linked through amide linkage to the glucosamine of GGroLs, the third 0-acyl ester or a Ptd group on C-6 of internal hexose of LTA, or cardiolipin with its four fatty acids (Table 2.3) (Section 2.2), serve to anchor these GGroLs tightly on the lipid bilayer of plasma membrane and are closely related to the in vitro LTA-carrier activity (Section 4.1.4). A similar function had been postulated for the “extra” acyl on mannose or inositol residues of phosphatidylinositol mannosides from mycobacteria [270,298]. Urkingdom Archaebacteriae, the progenote of which is considered to have been born 3.5 billion years ago on Earth, contains several unique types of lipophilic domains (Table 2.3, Fig. 2.4). For instance, di-0-phytanylether, linked to glycerol through an exceptional sn-2,3 configuration in addition to normal sn-1,2 type, has been isolated from halobacteria [112,239] and other archaebacteria (Table 2.3(5)) [299]. An sn-2,3-glyceryldiether combined as a cyclic diether with two C,, isopranoid residues (dibi-phytanyl-glycerylether) was also identified as a lipophilic component of the complex lipids of thermoacidophile, Thermoplasma acidophilum [239] and a methanogenic bacterium [271,299](Section 3.2.1; Table 2.3(7)). Also, an asymmetric C,,,C,,-diether (2-0-sesterpanyl-3-0-phytanyl-sn-Gr0,Table 2.3(6)) and about 20 species of nonitol (cardito1)glyceroleteraethers (Table 2.3(8)) were isolated from an alkaliphilic red halophile [508] and Sulfolobus [508,518,537], respectively. GalDAG of Treponema hyodysenteriae [167] was shown to contain 88.3% of 1-alk-1-enyl-2-acyl-sn-Gro. Alk-1-enyl ether (Section 2.2) are present in the phospholipids of myelin, blood platelets, some protozoa [300] and clostridia [28]. The high amount of alk-1-enyl lipids might reflect the metabolic state of the tissue, i.e., supply of NADPH to favor acyl dihydroxyacetone phosphate (ADHAP) pathway in the tissues of animals or habitat of bacteria, e.g., rumen (see Section 4.1.1, Reaction 9). In photosynthetic tissues, the gradient in oxygen tension at the subcellular level might affect the degree of unsaturation. The fatty acids of most treponemas are similar to those of the medium, since the organisms require long-chain fatty acids for growth due to their inability to synthesize them. In contrast, fatty acids of T. hyocllysenteriae were drastically unlike those of the medium, indicating that the organism is able to synthesize long-chain fatty acids. 2.2.3. Animal The lipophilic domains of the GalDAG fraction from brain of mammals were [44,82], in addition reported to contain from 10 to 20% of I-0-alkyl-2-0-acyl-X-Gro to DAGs. However, as pointed out by Rumsby and Rossiter [82], Norton observed that the alkylacyl component became enriched, during the preparation procedure,
from about 50 to 80% of total galactolipid. In these papers, however, the variations according to age were not considered. Later, the proportions of alkylacyl form in GalDAG and GalDAG sulfate fractions were found to range between 50 and 90%, depending on age [71,102,125,301] (Table 2.4, c.f., Section 3.3.2). The ratio of alkylacyl and diacyl forms of Gal-GGroL in frog was 19 : 81 in brain and 38 : 36 in sciatic nerve [539]. Ether lipids had been previously identified in many lipid classes of various species [302]. Comparative studies of testicular sulfoglycolipids [47,55,56] revealed that the structures of the carbohydrate domain are always Gal-3-sulfate or lactose-3'-sulfate. In contrast, lipophilic domains are different depending on the classes of animals. Only in mammalia, was the lipophilic domain AAG, while in other vertebrates studied, it was ceramide [56]. In mammalian testis, more than 90% of the molecular TABLE 2.6 Fatty acid composition of glycoglycerolipids in microorganism Species
Glycolipid
Fatty acid (in the order of Sg abundance) a
Streptococcus hemolyticus
GlcDAG I, GlcNAcDAG Glc,DAG Glc,DAG PtdGro Glc(Ptd)GlcDAG Ptd residue GroP-Glc, DAG
(1) 16 : 0,42; (2) 18 : 1, 30 '; (3) 16 : 1.24 (1) 16:O. 41; (2) 18:l.32; (3) 1 6 : l . 22
GlcDAG Gal(acyl)GlcDAG Glc-linked Gal (acyl)GlcDAG Glc-linked LTA
(1) 18:1, 51; (2) 16:0,29; (3) 19cyd, 10 ( I ) 18: 1.48; (2) 16:0, 29; (3) 19cy. 10 (1) 1 8 : l . 37; (2) 16:0, 29; (3) 19cy. 19
Bacillus acido caldarrus
GlcGlcNAcylDAG
(1) 17-cyclohexyl,42; (2) 17brg. 29; (3) 19-cyclohexyl, 19
238
Treponema hyodvsenteriae
GlcDAG
C-1 (alk-1-enyl): (1) 14:O. 47: (2) iso15:O. 22; (3) 16:0,22 C-2(acyl): (1)16:0.55; (2)is0-15:0,23 (3) anterso-15:0, 10
167
Luctobacillus case1
,
Ref.
(1)18:1,40;(2)16:0.36;(3)16:1,20 (1)18:1.42;(2)16:0,35;(3)16:1,19 (1) 16:0,40; (2) 18: 1, 28; (3) 16: 1. 21 (1)16:0,38;(2)16:1,26;(3)18:1,24 (1) 16:O. 34; (2) 1 6 : l . 30; (3) 1 8 : l . 30 (1)18:1,41;(2)16:0,40
85
(1) 1 8 : l . 47; (2) 16:O. 25; (3) 19cy, 14
(1) 18:1,45; (2) 16:0, 30; (3) 19cy. 25 ( 1 ) 1 8 : 1 , 4 4 ; ( 2 ) 1 6 : 0 , 2 8 ; ( 3 ) 1 4 : 0 .12
145
~~
Weight%. Lendgraf, H.R. (1976) thesis, University of Erlangen-Nbrnberg. ' 18 : 1 fraction contains significant proportions of cis-vaccenic acid 13371. Cyclopropane. ' Ester-linked to the C-6 of internal glucose. Preparation 2, similarly GroPGlcGlcDAG and GroPGlcGal(acyl-0-6)GlcDAG from L. casei contained the same fatty acids in similar proportions as the membrane GlcGalGlcDAG, GlcGal(acyl-0-6)GlcDAG and the LTA from the same organism [145]. g Branch.
129 species of seminolipid is 1-O-palmitoyl-2-O-palmityl (16 : 0/16 : 0, Table 2.4) [45,46,122]. In the testis of various ages of humans, 93.7-100% and 98.3-1008, respectively, of fatty acids and alkylglyceryl ethers are straight chain saturated [47]. Such simplicity and the saturated nature of the lipophilic domain are in striking contrast to the fatty acids at position-2 of choline and ethanolamine phosphoglycerolipids [253] in testis, which contain mainly polyenoic fatty acids, e.g., 20 : 5 and 22 : 6. Such uniformity of lipophilic domains has been observed only in several examples: the dipalmitoyl Ptd-choline of lung alveoli, choline- or ethanolamine phosphoglycerolipids of bovine epididymal spermatozoa [253], or in a tetraglycosyl AAG of germinating mung bean [248]. Although less pronounced, also in alkylacyl moieties in GGroLs of secretions, 1-O-palmityl-2-O-palmitoyl (16 : 0/16 : 0) or 2-O-stearoyl (16 : 0/18 : 0) species predominated (Table 2.4) [52,71,102,125]. However, GlcGGroL in human saliva contained up to about 50% of 2-hydroxy long-chain acids of 18-26 carbon numbers 1501. In the bovine [187], cod [52], and whale brains [304], the fatty acid composition of GalDAG is similar to that of esterified fatty acid of cerebroside esters (acyl-6GalCer). Reversed-phase high-performance liquid chromatography of the benzoylated derivatives of GalDAG and acylGalDAG also showed a strikingly similar pattern [187]. It is thus conceivable that these two lipids can substitute for one another in myelin and other membranes. Although Gal-GroL and its sulfate ester locate in myelin sheath, the compositions of acyl ester are rich in 16 : 0 and 18 : 0 acids, in striking contrast to those of major myelin components such as GalCer sulfates and GalCers, which contain predominantly C-24 and 2-hydroxy acids in rat brain, and 24 : 1 acid in cod fish brain [52]. The GalDAG fraction of the brain of frog [51,539], tadpole [307] and sheep [82], or spinal cord of beef [loll and pig [190] also showed similar fatty acid compositions. The alkylglycerylether compositions were predominantly 16 : 0 in all species examined. Tissue-specific lipid components of animals, such as sulfoglycolipids of myelin, show usually little or no species variability both in the structure of carbohydrate moiety and in the composition of lipophilic domains. This has been attributed to common functional requirements of these components in myelin (Section 3.6). 2.2.4. Positional distribution of fatty acids At the beginning of this Section, it should be briefly stated that there have been several methods for the determination of fatty acid distribution: ( I ) differential hydrolysis with lipase [175,308-310,322,3561;( 2 ) electron-impact direct-inlet (EI-DI) mass spectrometry of monoacetate of DAG, which is obtained by 2% H,SO, hydrolysis [311]; ( 3 ) to obtain the pattern of the Ptd-residue, treatment with phospholipase A followed by cleavage of phosphodiester bond by hydrolysis with acetic acid (98%) [78]; ( 4 ) EI-DI mass spectrometry of the acetates of intact glycolipid [85,175,229]; ( 5 ) removal of carbohydrate by glycosidase to obtain DAG, then gas chromatography-mass spectrometry or thin layer chromatography [102]. These methods were reviewed by Myrhe [194] and Douce and Joyard [142]. One
130 should only be careful to judge the results, because there are possibilities that the method of determination may influence the results owing to difficulties inherent to each technique. Roughly speaking, most of phospholipids and GGroLs of the biosphere contain unsaturated, more polar, branch, cyclopropane or shorter chain fatty acids at their position-2. On the contrary, the acid linked to position-1 is more saturated because acyl-CoA:2acylGroP-O-acyltransferaseshows specificity for saturated fatty acylCoAs [494]. 2.2.4.1. PIanr Analysis of the fatty acid distribution of GalDAG and Ga12DAG from plant (Table 2.7) might permit a similar generalization. 16 : 2, 16 : 3, 18 : 4 and C-20 polyenoic acids are concentrated on the C-2 position throughout the plant kingdom [13]. In contrast, 18 : 3 acids predominate in the C-1 position [142,308]. The distribution of fatty acids on the 1- and 2-positions of Gro moiety of Gal-DAG and Gal,DAG from Artemisia princeps (wormwood) was studied by Noda and Fujiwara [309] using commercial pancreatic lipase (Table 2.7). The most of GalDAG in this typical leaf of higher plant was C-1 (a-linolenic (a-Lnn)) and C-2 (a-Lnn). On the other hand, Safford and Nichols [312] concluded that, in Chlorella vulgaris, the distribution in GalDAG showed preference for C-16 acids at position-2, i.e., chain length specificity, whereas the specificity in higher plants showed preference for unsaturated fatty acids. Rullkotter et al. [232] indicated that 16 : 0 does not show a marked specific distribution, but observed in general that, the lower the taxonomic position, the higher the proportion of 16 : 0 in the C-2 position, and vice versa [142]. Later it was explained that plants forming 16 : 3 (16 : 3 plants) incorporate this acid mainly into GalDAG [313]. Actually, C16 : 3 Gal,DAGs of spinach, is concentrated at the sn-2 position of glycerol [291]. This may be explained, however, by specific desaturation of fatty acids attached at C-2 of GalDAG [314] (Section 4.1.7, Reactions 33 and 34). Also 16:O acid was found in appreciable proportions at sn-2 position of typical chloroplast lipids of spinach such as Gal, DAG, SQ-DAG, DAG and PtdGro [175,315,316] (Table 2.7). Exceptionally, in SQ-DAG of rice bran C-1 and C-2 positions are both rich in saturated acids [91]. Also in SQ-DAG of leaves, 16 : 0/16 : 0 combination occurs in high percentage [195,291] (Section 5.5). The fatty acid composition of 6-0-acyl of acyl-GalDAG and acyl-Gal DAG, examined by mass-spectrometric analyses of acyl-Gal fragments, showed that the composition was greatly different from that of the glycerol part (Table 2.5) being rich in 16 : 3. This result pointed to GalDAG as the acyl donor, because this lipid in spinach is characterized by its high content of 16 : 3 at C-2 of Gro [229] (Section 4.1.1, Reaction 12). In Streptococcus lactis the 6-0-acylated derivative accounted for about 1%of the total Glc,DAG, whereas it rose up to 39 and 58%, respectively, in GroP-Glc, DAG and (Crop),-Glc, DAG fractions [233]. The carbohydrate-linked from S. lactis [233] and Lactobacillus fatty acids of Glcal-2(acyl-6)GlcalUI-3DAG casei [85,154] were enriched in short chain or cyclopropane acids (Table 2.6). 2.2.4.2. Bacteria Similarity in positional distribution of fatty acids among microbial glycerolipids was first shown in phosphatidylethanolamine and Glc, DAG from
131 Bacillus cereus [235] using phospholipase A and the lipase preparation isolated from a mold, Rhizopus delemar, respectively. In both lipids, shorter-chain fatty acids were located at position-2 (Table 2.7). Similar distribution profiles have also been found with S. faecalis [76], S. lactis NCDO 712 and Staphylococcus epiderrnidis [139]. The Glcal-3DAG, Glc2a1-3DAG, PtdGro, Lys-PtdGro, diPtdGro and Glcal-2diPtdGro of group B streptococci [76,308] showed a very similar composition of fatty acids, and the positional distribution to each other. Saturated fatty acids (16 : 0, 18 : 0) are
TABLE 2.7 Positional distribution of fatty acids in glycoglycerolipids Species
GI ycolipid
Position a and composition
Ref.
W orm-wood ( Artemisia princeps)
GalDAG
309
Thylakoid membrane (Orvra saiiua)
GalDAG Gal DAG
Rice bran
GalDAG
C-I: (1) 18:3,92; (2) 18:2.4; (3) 16:O. 1 C-2: ( 1 ) 18:3.95; (2) 18:2.5 C-1; (1) 18:3. 31; (2) 16:0, 27; (3) 18:2, 15 C-2 ( 1 ) 18:3. 81; (2) 1 8 ~ 213; . (3) 1 6 1 0 . 4 (1)18:3/18:3,89;(2) 16:0/18:3.1 (1)18:3/18:3,66;(2) 16.0/18:3. 1 3 e ; ( 3 ) 16:0/18:2.8 ( 1 ) 16:0/18:3,63;(2) 16:0/16:0,29; (3) 16:0/18:2,8 ( 1 ) 18:2/18:2, 15; (2) 16:0/18:2. 14; (3) 16:0/18:3,12; (4) 18:1/18:2,12 ( 1 ) 18:2/18:2, 16; (2) 16:0/18:3. 10; (3) 18:3/18:3.10;(4)18:2/18:3.9 ( 1 ) 16:0/16:0,38; (2) 18:2/16:0.19; (3) 18:1/16:0,12; (4) 16:0/18:2,9 C-1: (1) 17br 49; (2) 15br. 19; (3) 16:O. 15 C-2: (1) 15br. 30; (2) 13br, 26; (3) 14br. 12 C-1: ( 1 ) 1 6 : 0 , 4 2 ; ( 2 ) 1 8 : 1 , 2 9 ; ( 3 ) 1 6 : 1 , 2 1 . C-2: ( 1 ) 1 8 : 1 , 4 7 ; ( 2 ) 1 6 : 0 . 3 1 ; ( 3 ) 1 6 : 119 C-1: (1)18:1.72;(2)19cyJ, 1 4 ; ( 3 ) 1 6 : 1 , 5 C-2: (1)16:0.48; (2)18:1,22;(3)16:1.21
,
Gal DAG
,
SQ-DAG
,
Gal DAG SQ-DAG Bacillus cereus g Streptococcus hemolvricus ' Sirepiococcus Jaecalis I
Glc, DAG GroP-Glc, DAG Glc, DAG
',
174 31 1
296
235 308 308
" T h e symbol, e.g.. 16:0/18:2 means position 1 and 2 of glycerol is acylated with 16:O and 18:2. respectively. The values are weight% of fatty acid or molecular species. Pancreatic lipase. ,I By gas chromatography-mass spectrometry of monoacetyldiacylglycerols131I]. the positional specificity of 16: 0 in glycolipids at C-2 determined by pancreatic or Rhiropus lipase is contrary to these values [174]. in the same thylakoid membrane preparation the most abundant molecular species of PtdGro were 16:O/delta-3-rrans-16:1. 16:0/16:0 and 18:2/16:0+ 18:3/16:0. 'In spinach envelope. also GalDAG and Gal,DAG, 18:3/16:3 (or 16:'3/18:3) and 18:3/16:0 (or 16 :0/18 : 3) were the major species. Pancreatic Iipase. Rhiropus delemar lipase. Branch, contains both iso- and onreiso- types. ' Rhiropus delemar lipase. I Cyclopropane acid.
' '
132 accumulated at position-1. Shorter-chain and unsaturated fatty acids (12 : 0, 14 : 0, 14 : 1, 16 : 1 and 18 : 1) were preferentially linked to C-2. Positional distribution profiles in S. faecalis were studied in great detail [77,78]. Longer-chain fatty acids (18 : 0, 18 : 1, 19-cyclopropane) were enriched in position-1 and shorterchain fatty acids (14 : 0, 16 : 0, 16 : 1) in position-2 as described above. The DAG moieties of all polar lipids, including Ptd- and GroP-GGroLs, showed an identical fatty acid make up with the exception of varying amounts of C19 cyclopropane fatty acid and C18 : 1. the sum of which, however, turned out to be identical to each other. Taking into account that cyclopropane acids are formed from the corresponding monoenoic acids at the level of preformed lipids [399], the differences found may be the results of varying affinities of the individual lipids to cyclopropane synthetase. These observations favored the hypothesis that the DAG portions of all membrane lipids are derived from a common phosphatidic acid precursor with negligible postsynthetic modification of the constituent fatty acids, except cyclopropanisation in S. faecalis. Also GalDAG and Ga1,DAG isolated from cyanobacteria, showed identical DAG makeups [291]. Direct-inlet mass spectrometry in electron impact mode of acetylated DAG obtained from Hex,- to Hex,-DAGs of L. casei also confirmed that each GGroL contains the same sets of DAG species in similar proportions. The most abundant species were 16 : 0/18 : 1 and 16 : 0/19cyclopropane [85]. The distribution in a gram-negative bacterium [264] was similar to that in gram-positive bacteria. The 1-position of the Ptd moiety of diPtdGro, Ptd-ethanolamine, PtdGro and GroP-GGroL was mainly associated with 16 and 18 carbon saturated acids (50-70%) with a smaller amount of 16 and 18 monoenoic acid. The position-2 was occupied by 17 carbon saturated and C17 cyclopropane acids (60-75%). According to Langworthy [238], the distribution of esterified and amide-linked fatty acids in a diglycosylDAG of Bacillus acidocaldarius was similar, being composed primarily of branched 17 : 0, 11-cyclohexylundecanoic and 13-cyclohexyltridecanoic acids.
3. Distribution 3.1. PLANT
3. I . 1. Tissue distribution Sastry [13] extensively surveyed literature on the distribution of galactolipids in various parts of plants. In short, the leaves of various species contained 2.0-14.6 pmol/g tissue (or 4.3-25.5% of total lipid) of GalDAG and 0.4-9.9 pmol/g (4.0-20.5% of total lipid) of Ga1,DAG. The molar ratio of GalDAG to Ga1,DAG ranged from 5.1 to 0.6 [13] (see also Table 2.1.1, 2.1.2). Usually, in the photosynthetic bacteria [181] and higher plants as well as in myelin of animals, the concentration of GalDAG is higher than that of Ga1,DAG. In contrast, the nonphotosyn-
133 thetic tissues of plants, most unicellular algae and most gram-positive bacteria show a preponderance of Hex, DAG [13], although GalDAG/Gal, DAG ratios may change depending on the age of cells [14]. (c.f., Section 3.1.3). The amount of SQ-DAG found in the leaves of higher plants is invariably less than that of individual galactolipids (0.01-18.3% of total lipid). The ratio of GalDAG to SQ-DAG varies somewhat from species to species [195]. The content of GGroLs in relatively differentiated unicellular eukaryotes, green algae [319] and diatoms [180,257], are usually a few times higher than those of leaves, accounting for up to 80%of the total lipid in the former, and 8.8 mg/g dry weight or 33% of total lipid in the latter. In the lipids of photosynthetic microorganisms, PtdGro, GalDAG, Gal DAG and SQ-DAG tend to predominate: indeed some photosynthetic blue-green algae (cyanobacteria), which are the only prokaryotes photosynthesizing as do green plants, contain only these four classes [290] and lack Ptd-choline [316]. Among the photosynthetic prokaryotes, Gal DAGs are confined to species, which possess a type I1 reaction center, that is cyanobacteria [320]. The fact that, however, within the eubacterial kingdom, many of the nonphotosynthetic species have clearly come from the photosynthetic ones [501], can probably explain the presence of GGroLs in gram-positive bacteria. The fruits and roots or tubers contained much less GalDAG (0.015-0.12 pmol/g or 1.3-13.6% of total lipid) and Ga1,DAG (0.05-0.34 pmol/g or 4.7-14.2% of total lipid [13]). The bran of rice contained about 3% of GGroLs in total lipid, including Gal,DAG, GalMAG and Ga1,DAG in addition to GalDAG and Ga1,DAG [91]. In general, seed oils are very rich in triacylglycerol [321]. GGroLs have never been isolated from animal viruses. The phage, infecting thermophilic bacteria, showed a similar GGroL pattern, on thin-layer chromatography, to that of the host [244]. Relatively little information is available on the GGroLs of fungi [322]. GalDAG and Ga1,DAG have been detected in Saccharomyces cerevisiae and Blastocladiella emersonii, respectively.
,
3.1.2. Photosynthetic membrane
Wintermans [12] compared the contents of various lipids in the leaves and isolated chloroplasts of spinach or beet. The results with beet leaves and chloroplasts (20%of leaf volume) showed that galactolipids are located specifically in chloroplasts, in concentrations up to, and even exceeding lop 2 M (mol/l). On the contrary cytoplasm was devoid of glycolipids. 3.1.2.1. Chloroplast of higher plant Each photosynthetically active cell from leaves contains about 200 chloroplasts, which have their own DNA coding the enzymes necessary for their ribosomal RNA and tRNA. Chloroplasts of Rhodophyta and Cryptophyta apparently arose from cyanobacteria [501] and those of Chlorophyta and higher plant were probably derived from non-cyanobacterial oxygenic-photosynthetic prokaryotes [387]. Green chloroplasts were found to be enriched in PtdGro, GalDAG and Gal ,DAG [12]. Spinach leaf chloroplasts contained GalDAG to Ga1,DAG in the ratio of 60 : 30 : 5 : 1 [246]. Today it is generally believed that all the GGroLs and PtdGro of leaves belong to the chloroplast [14,72,142,195].
134 The outer chloroplast membrane is the envelope. The lipid content of spinach chloroplast was about 0.6 pmol/mg protein and in the envelope it was 20 pmol galactolipid/mg envelope protein [81,173], although envelope lipid represents only 1-2.5% of the total chloroplast lipid [356]. In envelope lipid of spinach the mol% values of Gal-DAG, Gal,DAG and SQ-DAG were 13.2, 21.6 and 7.5%,respectively [175]. The pattern of GGroLs from the envelope isolated at pH 8.5, instead of the conventional pH value of 7.2, was significantly different, probably reflecting the presence of interGGroL galactosyl transfer [327] (Section 4.1.2, Reactions 23 and 24). The envelopes isolated at pH 8.5 have a lipid composition much more in line with the general plastid pattern, i.e., Gal-DAG/Gal, DAG ratio higher than unity, low concentrations of DAGs (see also Section 4.1) and higher homologs (see Table 2.5). The inner chloroplast membrane, thylakoid, is a light-transducing membrane, where photosynthesis takes place. The proteins comprize approximately 60% of thylakoid membrane, and polar lipids and pigments for the remaining 40% [142,324,325]. GalDAG, Gal,DAG, SQ-DAG as well as PtdGro [305] are the characteristic polar lipid components of thylakoid [71,504]. The wt.% contents of thylakoid lipids in total lipid were: GalDAG, 52; Gal,DAG, 26; SQ-DAG, 6.5; and PtdGro, 9.5 [174,324] (Section 5.1). GGroLs in this membrane were shown to occupy up to 85% of the total lipid [174]. Such high content of GGroLs has been only found in membrane of some streptococci, archaebacteria (Section 3.2.1.2) or mycoplasmas (Section 3.2.4) (Table 2.1). It is explained that the enzymes of glycosylation of DAGs reside in the outer envelope (Section 4.1.6.2) and the products, Gal-DAG, Gal ,-DAG and SQ-DAG are concentrated in the inner thylakoid [504]. 3.1.2.2. Microorganism In cyanobacteria, photosynthetic membrane containing galactolipids are classified into two types [490]. Intracytoplasmic type membrane is a bilayer of galactolipid involving bacteriochlorophyll. The second chlorobium type consists of sack-like structures with a monolayer of galactolipids surrounding light-harvesting proteins. Green, brown and red algae as well as cyanobacteria contain SQ-DAG [195,535]. The plasma membrane fractions of non-photosynthetic marine diatoms seem to have less GGroLs than their Golgi or endoplasmic reticulum fractions [257]. It is believed that diatoms phylogenetically branched from photosynthetic brown algae about one billion years ago, losing the ability of photosynthesis but still containing the characteristic GGroLs of their precursors [257]. In the green alga, Chlamydomonas reinhardtii it has been suggested that about 70% of SQ-DAG, but only about half of its galactolipid, is localized in the thylakoid [319]. 3.1.3. Differentiation
The study on the change of glycolipids and phospholipids in greening barley (Hordeum) seeds showed that both GalDAG and Ga1,DAG increased with the formation of thylakoids [493]. In maize leaves, the ratio of GalDAG/Gal ,DAG increased continuously from 1.25 to 1.80, corresponding to increasing physiological ages [14]. These changes, as well as the increasing ratio of galactolipids to sulfonoli-
135 pid, correlated well with the development of thylakoid structure [14,338.481,494].In the case of soy bean from 9 to 97 days after flowering, the proportion of glycolipid decreased from 29.2 to 1.6 wt.%, although the concentration of neutral lipids (mainly triacylglycerol) gradually increased [297,321]. Kondo [519] studied the changes in the lipid compositions during germination of mung bean (black grams for bean sprout) in the dark. GalDAG and Ga1,DAG increased greatly (to 14 and 17% of total GGroL) within one day of germination. A tetraglycosylAAG appeared 3 days after germination and the content increased until the seventh day. Thus, this ether lipid appeared to function in the earlier period of germination in plants. In senescent leaf tissue, loss of chloroplast lipids is accompanied by an increase in free fatty acid levels [321]. Also during ripening of fruits, losses of polar lipids were observed. Not only activities of the enzymes in chloroplasts for individual steps of GGroL synthesis, but also basic enzyme constitution of organelles, may change in relation to developmental stages [291]. 3.2. E U B A C T E R I A A N D A R C H A EBA C T E R I A
Taxonomy of the Kmgdom Prokaryotae has been revised repeatedly. Current bacterial classification is still far from being phylogenetically valid [501]. Cell-wall compositions are more reliable parameters for classification. Chemical taxonomy has been proposed [13,18,21,23]according to the structure of GGroLs of cell membrane. Most of these conventional taxonomical evaluations involved, however, only the structure of neutral GGroLs. Here we discuss also the relation of these neutral GGroLs with the newly discovered GroP-GGroLs, Ptd-GGroLs and LTA, as well as archaebacterial glycolipids. 3.2.I . Whole cell of microorganisms As previously postulated for fatty acids and phospholipids by Kates [5], Asselin%u [23] and Shaw and Baddiley [20,28] showed that the grouping of eubacteria (grampositive) on the basis of the carbohydrate structures of the neutral GGroLs, such as Glal-2Glca [30], GlcPl-6GlcP [31], Manal-3Mana residues, etc., results in a scheme similar to the traditional taxonomy [496]. However, the same GGroLs can be found in eubacteria of different families. On the other hand, in an Arthrobacter species both Ga12DAG and Man,DAG occurred [28]. 3.2.1.1. Qualitative distribution In 1978, a systematic study on 33 species of gram-positive bacteria revealed that GroP-GGroLs are as wide-spread as LTA [243]. Usually, the plasma membrane of a species contains a series of neutral and GroP GGroLs. They have the identical repeating structures of carbohydrates with respect to configuration, anomeric forms, and points of attachment (Tables 2.1 and 2.2), and very similar lipophilic domains (Tables 2.3 and 2.6). For instance, Streptococcus hemolyticus ( S . pyogenes type 3) contains Glcal-3DAG, Glcal-2Glcal-3- (kojibiosyl-)DAG, (Glcal-2),Glcal-3- (kojitriosy1)DAG [36] and GroP-6Glca1-2Glca13DAG [135]. Another example is Lactobacillus casei which contains Glca1-3DAG, Gal a1 -2Glcal-3DAG, GlcPl-6Galal-2Glcal-3DAG,(GlcPl-6),Gal a1 -2Glcal-
136 3DAG, as well as Galal-2(acyl-6)Glcal-3DAG, and GlcPl-6Galal -2(acyl-6)Glcal3DAG [85]. The glycolipid composition of a typical streptococcus is shown in Table 2.8. Among streptococci, the lipid pattern of group B is unique in both the presence of Glc-diPtdGro and a large amount (20% of polar lipid) of GlcDAG. Another remarkable feature of gram-positive bacteria is the limited occurrence of Ptd-containing GGroLs. GGroLs with an accessory Ptd residue on the position-6 of internal glucose have been isolated only from S. hemolyticus and group D streptococci (faecalis) (78,1371, and absent in other streptococci and the species belonging to Lactobacillaceae, Bacillaceae and Micrococcaceae [243]. On the contrary, GroP-GGroLs are ubiquitous among gram-positive bacteria, except Micrococcus luteus and an alkaliphlic B. subtilis A007 [530]. Micrococci seem to have arisen as degenerate forms of the arthrobacteria (locked into the coccoid stage of the arthrobacterial life cycle) [501]. In S. aureus, B. licheniformis and B. subtilis, the major membrane GGroLs are the precursors of the GroP-GGroL [42,145]. The occurrence of Glcal-2Glcal-3DAG and Glcal-2(acyl-6)Glcal-3DAG in both GroP-GGroL and LTA seems to be characteristic for group N streptococci, whereas GlcPl-6Gal a1-2Glca1-3DAG together with GlcPl-6Gala1 -2(acyl-6)Glcal3DAG for Lactobacillaceae [42,243]. On the other hand, Glcal-2Glcal-3DAG together with Glcal-2(Ptd-6)GlcaluI-3DAGare typical for Streptococcus faecalis strains. Furthermore, in group N streptococci, LTA is the group-specific antigen due to the substitution with aGal residues [339] (c.f., Section 5.3.1). A subgrouping was proposed because the LTA contains either a galactosylated poly(g1ycerophosphate) or a poly(Galal-6Galal-3(Galal-2)-sn-Gro-l-P) chain. These two types of carbohydrate chains are also reflected in the GroP-GGroL patterns [158,243] (Table 2.5). Both Bacillus subtilis W23 and strain Marburg have different teichoic acid structures. However, they can be grouped together on the basis of their LTA which is identical with respect to lipid anchor, chain structure and chain substitution (Fischer, W., personal communication). 3.2.1.2. Quantitative distribution The quantitative distribution of neutral GGroLs varies widely among gram-positive bacteria, although usually t h s class of glycolipids accounts for only about 1-2% of the total lipids in many species. In the following eubacteria, the neutral GGroLs are the major lipid components: Streptococcus hemolyticus (44 wt.% of total lipid) [36], Microbacterium lacticum (46%) [237], Flavobacterium stearothermophilus (70%) [94,244], Anaeroplusma, a thermophilic bacteria (43.5%) [340], Bacillus acidocaldarius (64%) [238], Acholeplusmu modicum (45.7%) [249], Lactobacillus cusei (58 mol% of the total polar lipid) [85], Spirochaeta aurantia (85 weight% of polar lipid) [166]. Generally in archaebacterial urkingdom (methanogenic, extremely halophilic and acidothermophilic bacteria) [501], the content of both neutral and acidic glycolipids is unusually high. For instance, sulfolobus [239,341,342] contain high amounts of GGroLs (60-80% of total lipid). A methanogen, Methunospirillum hungatei, contained as the only simple phospholipid, PtdGro, in only 5% of total lipid [271]. On the other hand, GGroLs and glycophospholipids occupied 94% of total lipids. The occurrence of several types of isopranyl esters (Section 2.2.2, Table 2.3) in a group of
137 archaebacteria is also unique from the taxonomical point of view [299,537]. These observations indicated that GGroLs, together with glycoproteins [502], of membranes of living cells were already a class of important constituents of the most early unicellular cells on Earth. However, a very unique exception was reported in an alkaliphdic Bacillus sp. A-007. This organism was shown to contain neither neutral glycolipid nor phosphoglycolipid or other glycolipids in the total lipid extract [467]. A decrease of the lipid galactose content of cells and membranes was observed in Bifdobacterium bifidum cultured in a medium without milk [182]. This change resulted both from a decrease of the amount of all glycolipid and from a shift in the ratio of hgher GGroLs to GalDAG mainly at the expense of the Ga1,DAG. The alteration was explained by a more limited disposal of UDP-Gal for glycolipid synthesis as a consequence of an increased cell wall polysaccharide synthesis. 3.2.1.3. Gram-negative microorganism Neutral glycosylDAGs were regarded as more typical of gram-positive bacteria than gram-negative [28], but they have been isolated from several gram-negative genera [ 108,4961. GlcU-DAGs can be the major [lo91 but expendable I1071 lipids in the atypical Pseudomonas (now Alteromonas) rubescens. The genera of the order Pseudomonadales (the photosynthetic bacteria (see Section 3.1.2.2); the sulfur bacteria, thiobacilli; the pseudomonads: and the obligate halophilic bacteria) (c.f.. Section 5.5) have very characteristic GGroL compositions [28]. The composition of GGroLs, remarkably different in comparison to the other species of the genus, has been described in Treponema hyodysenteriae [167], Pseudomonas diminuta and P. vesicularis [108]. Especially since no lipid composition comparable with the last two species has been reported for any other gram-negative species, the taxonomic position of these species is questionable [28,108]. 3.2.2. Cytoplasmic (plasma) membrane The envelope of a eubacterium is a composite structure, made of two different membranes: the outer membrane, including the peptidoglycan layer: and the inner or protoplasmic membrane [343]. The striking feature which emerges from comparative studies of “cell walls” of gram-positive and -negative bacteria is that the walls of gram-negative bacteria have high contents (up to 26%) of lipids. More precisely, gram-negative microorganisms have an “outer membrane”, and the amphiphilic components are localized in the outer leaflet of this membrane [41]. In contrast, walls of gram-positive bacteria have little or no lipids (3441. The lipid content of cytosol is also negligible [345]. For instance, in Streptococcus jaecalis [345], Bacillus subtilis [346], Streptococcus pyogenes [337] and Staphylococcus aureus [347], most of lipids were recovered from membrane fractions. Recently, GGroLs were shown to constitute mainly LTA of membrane as the anchor, rather than in their free form as the component of the membrane. I t was recently also shown that a gram-negative bacterium has capsular polysaccharide containing dipalmitoyl PtdGro [251]. In gram-positive bacteria, lipids and proteins make up at least 70%.and in some cases almost all, of the weight of the membrane [344]. Generally, the lipids constitute 15-30% of the dry weight of the membrane of gram-positive bacteria, whereas L-form membranes contained 25-36% 13471. The bulk of the remaining weight is
138
accounted for by protein, although in some cases 1-20% of carbohydrates are present [15,17,65]. Micrococcus futeus [501] contains neither LTA nor lipopolysaccharide. Instead, succinylated lipomannan is present [287] and may perform the functions of surface acidic and amphiphilic polymers [33]. GGroLs were isolated from the wall fraction of pseudomonads (gram-negative) [109]. Cell membranes, produced by the use of a phage-induced lysin, contained 15.3%lipid as compared with 35.6% found in membranes from the L-form. 3.2.3, Lipoteichoic acid LTA containing glycerol (Section 2.1.2.4, Table 2.2.5) could be isolated from the cell membrane of nearly all gram-positive bacteria [ 31,40,140]. GroP-GGroLs have been also found in the membrane of nearly all gram-positive genera. In all cases studied, GroP-GGroLs constituted the lipophilic domain of the corresponding LTA, presumably serving to anchor the molecule on the membrane surface (Fig. 2.3). The reversed phase elution profiles of LTAs or their partially deacylated derivatives (lyso-LTAs) from the column of octyl-Sepharose impressively illustrate the differences in the affinity of these molecules to lipophilic environment [534]. The hydrophilic domain, the glycerol phosphate polymer, is probably extending away from the membrane surface, in certain organisms, through the wall [33] (Section 5.3.3). The relative abundance of GroP-GGroLs ranged between 1 and 17 mol% of the polar lipids. In most cases, however, only small amounts of GroP-GGroLs were present, pointing to metabolic intermediates rather than to membrane constituents (Table 2.8) (Section 4.1.5). Studies on sections of cells of Lactobacillus ferrnenti in the electron microscopy, using ferritin-labeled antibodies, demonstrated the presence of LTA (the group antigen of this organism) in the cell wall [348] (see Sections 5.2.3 and 5.3.1). It was suggested that the poly(g1ycerophosphate) chains extended from the outer surface of the membrane into the cell wall matrix. With Streptococcus mutans BHT, the amount
TABLE 2.8 Molar composition of glyco-amphiphiles in the membrane of Streptococcus luctis NCDO 712 Compound
Mol%
DiPtdGro (cardiolipin) PtdGro (phosphatidylglycerol) Glcal-3DAG Glcal-2Glcal-3DAG GroP-6Glcal-ZGlcal-3DAG and GroP-6Glcal-Z(acyld)GIcal-3DAG (GroP),-6Glc al-2Glc a1-3DAG and Gal al-6Gal a1-3(Gal a1-2)GroP6Glc a1 -2(acyl-6)Glcal-3DAG LTA (lipoteichoic acid) a
1 43 6
29 I 4 10
a LTA was measured as hydrogen fluoride-releasable glycerol o f lipid-depletedcells, and calculated as mol on the basis of 1 mol LTA containing 25 mol glycerophosphate. Table kindly provided by Dr. W. Fischer and taken, with permission, from Ref. 31.
139 of extracellular LTA was some 8-9-fold greater than the cellular LTA at pH 6.0 [41]. From this fact, transient existence of LTA as a cell surface component could be envisaged. 3.2.4. Mycoplusmu und L-form The mycoplasmas are the smallest and simplest self-replicating procaryotes. Virtually all mycoplasma lipids are located in the cell membrane. The membrane of mycoplasma can be obtained easily, and has been extensively used as a model for a biological membrane [100,349-3511 comparable to erythrocyte membrane: the earliest studied pure animal plasma membrane I3521 (Sections 5.1, 5.2). Mycoplasmas are gram-negative owing to the absence of a normal cell wall, but their lipid composition as well as taxonomic position is akin to that of gram-positive bacteria [28,353]. The GGroLs of Acholeplusmu luidluwii were identified as Glcal-3(1)DAG and Glcal-2Glccul-3(1)DAG [97]. These same GGroLs have been characterized in Streptococcus fueculis and S. pyogenes [ 18,3371. The GGroLs of staphylococcal L-forms [347] and Mycoplusmu neurolyticum [223] are principally Glc, DAGs with pl-6-linkage between the sugar residues, namely P-gentiobiosyl linkage. In other words, the GlcDAG and GroP-GGroLs of A. luidlawii contain glucose chains linked through an d - 2 linkage, identical to those found in streptococci. Also M. neurolyticum contains Glc units attached by a Pl-6 linkage, identical to those found in the neutral GGroLs and GroP-GGroLs of staphylococci and bacilli [223]. In conclusion, there is more similarity between species of mycoplasmas in terms of GGroL structure [355]. The glycolipids of M . mycoides are Galf-containing DAGs, primarily Galf-DAG [354]. M. pneumoniue contains Gal-DAG and Glc-Gal-DAG [354]. Lactobacilli and pneumococci, both of which give rise to L-forms, also have mixed glycosylDAGs, containing Glc and Gal [20]. Usually, monoglycosyl GGroL predominates in contrast to bacteria [351]. In some of these mycoplasmas, also related compounds with more extended oligosaccharide chains were found [505]. Thermoplasma acidophilum is a mycoplasma, which requires a combined high temperature and low pH for growth and reproduction. This mycoplasma contains unique tetraether lipophilic moiety typical for archaebacteria (Section 2.2.2) [351]. The membrane of A. luidluwii contains 25-30% of simple phospholipids (PtdGro and diPtdGro), about 60% of GGroLs (GlcDAGs and GroP-GlcDAGs), and less than 10% of simple glycerides [loo]. The molar ratio of GlcDAG/GI%DAG is determined by membrane viscosity property [350,351] (c.f., Sections 5.2 and 5.4). The high contents of both GGroLs and “lipopolysaccharides” (the higher analog of GGroL) in mycoplasmas suggest that they can be viewed as an amphiphile akin to the lipopolysaccharides found in the surface structure of gram-negative bacteria [351]. On the other hand, the GGroL content of the lipid of wild type (11.3% carbohydrate in total lipid) was one half that of the L-form (22.2% carbohydrate) [337]. Similar results were obtained by the comparison of the cell membrane from Staphylococcus uureus and its L-form [347,357].
140 Specific binding of certain lectins by various mycoplasmas suggests that sugar residues are located at or near the surface of mycoplasmas [99]. Also, by lactoperoxidase-mediated radioiodination of both intact cell and isolated membrane from A. laidlawii, it was postulated that GlcDAG and Glc,DAG are located almost exclusively in the outer half of the bilayer, whereas the GroP-GGroLs, GroPGlc,DAG and GroP-GlcDAG were almost equally distributed in the outer and inner halves of the membrane [348]. The excretion of LTA from gram-positive bacteria has been clearly established. Extracellular LTA may consist of fully acylated LTA and deacylated LTA (dLTA) in various proportions. The enhanced release of LTA from streptococcus strains in the presence of penicillin has been reported [359]. The growing protoplast of S. fuecium ( faecalis ATCC 9790), made under valine-starvation in medium, has been known to excrete “extracellular deacylated LTA” [360,543] (see also Section 4.2.3). 3.2.5. Growth stage Fatty acid compositions of bacterial lipids are distinctly dependent on the age of culture, similar to those of plants (c.f., Section 3.1.3). In the case of a gram-negative bacterium [264], the composition varied with the growth stages dramatically. The younger cells contained more C-16 and C-18 monoenoic acids, while the cells of late logarithmic stages contained C-17 and C-19 cyclopropane acids up to 10 and 508, respectively. The relative proportion of Glc, DAG in the total lipid of Bacillus cereus increased depending on the age of culture, whereas that of Ptd-ethanolamine decreased, probably due to phosphate starvation [306]. In the mid-logarithmic growth phase, and at the beginning of the stationary phase of Lactobacillus casei, acylated Hex,DAG and Hex,DAGs could be detected, but Hex ,DAG predominated and Hex,DAG was present in smaller amounts. The increase of Hex,DAG and Hex,DAG was the characteristic pattern of GGroLs in the stationary phase [85]. By inhibition of protein synthesis in Streptococcus faecium after exposure to chloramphenicol or valine deprivation, electrophoretic mobilities of LTA decreased (i.e., increased negative charge) and glucose substitution at C2 of glycerols also increased. In A. laidlawii the total glycolipid concentration remained approximately constant throughout the culture period, although the ratio of GlcDAG/Glc, DAG increased from about 0.7 at 10 h of culture age to 2.6 at 32 h [97]. The presence of high concentration of the GlcDAG in A. laidlawii is unusual. The GroP-Glc,DAG (although the sn-3-GroP structure proposed by the authors should be now sn-1-GroP [30]) occurs in A. laidlawii at all cultural ages and constitutes almost half of the phospholipids in exponentially growing cells. Ptd-Glc, DAG occurs only in aged cultures [274]. In S. Luctis GroP-Glc,DAG together with its 6-0-acyl derivative, as well as Galal-2Gro-l-P-6Glc(acyl-6)Glcal-3(1)DAG(Fischer number XV. Table 2.2.4(8)) and Gala1-3Gro-l-P-6Glc(acyld)GlcDAG (XVI) did not appear before the end of the logarithmic growth phase, whereas Galal-3, Galal-2-sn-Gro-l-P-6Glc(acyl6)GlcDAG (XVII) and Gala1-6Gal a1-3-sn-Gro-1-P-6Glc( acyl-6)GlcDAG (XVI I I)
141 were found throughout in relatively constant proportions. On the contrary, Gro-l-P6GIc/?l-6Glc/?l-3(1)DAG [243] was detectable in B. licheniformis at all stages of growth in relatively high concentration. The GGroL pattern of group B streptococci is unique. Even in the stationary phase GlcDAG was present in large amounts and accounted on a molar basis for approximately 20% of the polar lipids. In group D and other gram-positive organisms GlcDAG was less abundant during logarithmic growth and was dramatically diminished at the beginning of the stationary phase. This suggested that GlcDAG functions in these organisms mostly as an intermediate in the biosynthesis of the more abundant Glc,DAG, whereas in group B streptococci it should be viewed as a membrane constituent [4]. 3.3. NERVOUS SYSTEM
The GGroL constitution of nervous system, more exactly speaking, of myelin sheath, as well as of germ cells of mammals is tissue-specific, i.e., variations among species are minimal. 3.3.1. Phylogenetic distribution The GGroLs in the central nervous systems of various mammalian species constitute up to about 20% of total brain glycolipids, with the exception of that reported in 14-day-old mouse cerebellum (42%) [540]. The brain of some fishes (gadiformes and lophiformes), however, contains much higher concentrations of GGroLs [51]. For instance, the whole brain of Pacific cod contained 1.15 pmol/g (16.0 mol%) and 0.84 pmol (11.7 mol%), respectively, of GalDAG and its sulfate (seminolipid) in the glycolipid fraction [52], whereas in carp brain the content of GalDAG was only 122.4 nmol/g or 6.6 mol% of total glycolipids. The value reported by Ishizuka et al. [lo21 on the content of sulfo-GalGroL in the whole brain of North Sea cod (62.3 nmol/g) is much lower probably due to experimental conditions. It was also shown that the sphingomyelin content was less than 1%of the total phospholipids in the gadiforme brain. These findings indicated that in the gadiforme brain, sphingolipid expression is regulated at the minimum level, while glycerolipid content is maintained at the highest level [51] (see also Section 5.1.1). The presence of GalGroLs also in the brain of Amphibia (frog) [307,539] and Crossopterygii (coelacanth) [447] as relatively minor components indicates the importance of these molecules for the biochemical evolution of nervous system. Sulfated GalDAG ( AAG) of chicken retina [256] was present at 0.62-1.75 nmol/retina, i.e., less than 35 nmol/g.
+
3.3.2. Regional distribution GalDAG was found to be concentrated in the white matter of the bovine spinal cord [loll and calf brain stem [187]. These regions consist mainly of myelinated axons. In rat brain [193], the area richest in myelinated fibers, such as corpus callosum and brain stem, had the hghest content of GGroLs as well as their sulfate. Glycolipids of various regions of sheep brain, determined by high performance liquid chro-
142 matography, showed that the concentrations of myelin-related glycolipids, including Gal-GroLs, was highest in the brain stem at all ages and reached a plateau the earliest of all brain parts tested. The maturational changes in these lipids were slowest in cerebellum, the developmentally oldest regon of brain [ 1931. Throughout development of mouse, the concentration of GalGroL in cerebellum (950 nmol/g tissue at 30 days) is higher than in the cerebrum [540]. This value may be even higher, because about half of sulfo-GalAAG cannot be determined by the methods used [364]. 3.3.3. Developmental variations The content of sulfo-GalGroL in brain of rat [102,125,301]increased with the onset of myelination, and then decreased dramatically after 68 days of age. In contrast, other typical myelin components such as GalCer, sphingomyelin and triphosphoinositides continued to increase. The survey of the brain of various ages of rats revealed that GalDAG [93,188] and GalDAG GalAAG [193], as well as the sulfates of GalDAG and GalAAG [102,125,301],increased parallel to the progress of myelination, similar to the increase of the other myelin components. The concentration, in whole rat brain, of GalDAG [93] or GalDAG GalAAG [193] as well as sulfo-GalDAG and sulfo-GalAAG [ 102,1251 was barely measurable before 10 days of age. GalDAG (3 nmol/brain at 5 days; 22 nmol/brain at 10 days [93]) and sulfo-GalGroL (7.5 nmol/brain at 14 days [102]) increased sharply after 14 or 16 days of age (GalDAG, 90 nmol/brain at 14 days, 103 nmol at 20 days; sulfo-GalGroL 248 nmol at 48 days). The concentration of sulfo-GalGroLs in rat brain after the peak of myelination until 72 days of age, was found to be fairly constant (0.3-0.4 pmol/brain) [125]. In the case of Sprague-Dawley rats, whose body size is larger than that of Wistar strain rats, the content of sulfo-GalGroL in total sulfoglycolipids ranged between 16%(21 day) and 8.4%(70 day) [125]. In Wistar strain rats after 18 days of age, the molar concentration of sulfo-GalGroL varied only between 7.2 and 2.1%of total sulfolipids [54b,102]. The increase of sulfo-GalCer and sulfo-GalGroL of chicken retina was started already at the 11th and 18th embryonal days, respectively, and the former continued to increase until about 70 days of age [256], whereas the latter remained more or less constant from hatching to adulthood. The ratio of sulfo-GalCer to sulfo-GalGroL increased from 4.1 to 13.9 during the development [256]. As described above, GalDAG and GalAAG have been shown to be characteristic glycolipids of immature rat brain. This was confirmed later [193]. These glycolipids were not detected in significant amounts in the subcellular fractions of the white matter of adult sheep brain nor in adult rat brain. Therefore, it was considered that higher concentrations of GalGroLs are characteristic of immature brain [ 1931. These results agree with the results obtained on rat brain [93,301], tadpole and frog myelin [307].
+
+
3.3.4. Turnover of lipophilic domain The slow turnover (c.f., Section 4.1.7) of whole brain sulfo-GGroLs, especially the
143 alkylacyl form until 68 days of age, reflects the metabolic stability of myelin [301]. The gradual increase of the precursor, the AAG form of GalGroL, in rat brain from 40% at 31 days to 65% at 175 and 310 days, may partially explain the above observation [361].The report by Wells and Dittmer [188], that GalDAG of rat brain behaved similarly to GalCer up to 330 days of age, has not been confirmed. In the case of sulfate esters, however, DAG form and AAG form began to decrease after 29 and 68 days, respectively, and both forms were not detectable at 310 days of age [301]. The contents of GalGroLs in myelin of 31- and 52-day-old rat brains were 67.6 and 23.6 nmol/mg protein, respectively. The AAG form occupied 22 and 35%, respectively [189]. T h s indicated that total GalGroLs also decreased depending on age as confirmed in rat brain by Nonaka and Kishimoto [193] and the fraction of AAG form increased, just like in the case of sulfo-GalGroLs [301]. DAG form of sulfo-GalGroL leveled off after 25 days and the AAG form after 68 days. It was in good contrast to sulfatide and ganglioside 13-NeuAcGalCer, which continue to increase until 10 months [533,540]. The ratios in sulfo-GalGroL fraction of AAG/DAG, which were about 1 : 1 until 31 days of age [71,102], increased to 6 : 1 at 68 days and 13 : 1 at 175 days [301]. Although the group of Pieringer obtained quite different values on rat brain [125] and mouse embryonal brain culture [192], the difference appears to reside on methods for isolation. The ratio of AAG/DAG in GalDAG in calf brain stem was 9 : 11 [187]. 3.3.5. Location in myelin By direct examination of the subcellular fractions of adult rats, it was also supported that GalDAG [93], GalGroLs [193] as well as the sulfates of GalDAG and GalAAG [125,189,365] in rat brain are exclusively localized in the fraction containing myelin, a differentiated membrane shielding axon. The specific radioactivity of the 35S-labeled lipid in a myelin fraction from the cerebrum of 19-day-old rats, at 48 h after the intraperitoneal injection of [ 35S]sulfate,enriched to about 15-fold of the radioactivity in the homogenate [365]. The relative specific activity of [ 35S]sulfolipidin myelin was about 50, whereas those from other fractions, i.e., nuclei, synaptosomes, mitochondria and cytosol were less than 1.0 [189]. The incorporation into sulfo-GGroLs (seminolipid) of myelin was 16.4%of the total sulfolipids. This value was very close to the incorporation into sulfo-GGroLs fraction of the whole brain from 18-day-old rats [102]. These results suggested that sulfo-GGroL was most enriched (17.8-fold on protein basis) in myelin of the central nervous system. In rat before 20 days of age, when myelin structure is not yet completed, the greater part of the GalDAG appeared in the microsomal fraction [93]. Th'IS was especially evident in the case of myelin deficient mutant (jimpy) mice [191], in which brain microsomes contained 168 nmol/g of GalDAG, whereas the myelin fraction contained only 155 nmol/g. The greatly reduced amount of GalDAG in the brain of jimpy mice agree with the myelin location of GGroLs [125,191]. On the other hand, the content of sulfo-GGroLs (8.7%) in total sulfolipids from microsomes of normal 19-day-old rats was close to the value obtained on the whole brain at 22 or 23 days of age [189]. This result may suggest that sulfo-GGroLs were synthesized in the
144 endoplasmic reticulum of oligodendroglia, and deposited as the integral structural component of myelin after several days. Deshmukh et al. [366] obtained direct evidence that the oligodendroglial cells are the primary site of the synthesis of myelin GalDAG. The activity of galactosyltransferase was about 20-fold higher than that of neuronal fractions, and these cells accumulated 2-4-fold more GalDAG than the astroglial and neuronal fractions. The DAG form in Gal-GGroL continuously increased in myelin from one to about 40 nmol/mg protein over the period from 16 to 29 days. However, the content of DAG-form in sulfo-GalGroL of rat myelin decreased from 77.7% at 31 days to 64.8% at 52 days [361] (Sections 4.1.8.1, 4.2.2). Thus, GalDAG and GalAAG as well as their sulfate esters may be a good marker for myelination, at least until the peak of myelination (in the case of the sulfate esters in rat brain, 68 days at the latest). The fact that the concentrations of nonsulfated Gal-GroLs are usually 4-5-times higher, is characteristic for the myelin of mammals in contrast to the plasma membrane of germ cells, where sulfated forms predominate. 3.3.6. Hormonal regulation Thyroid hormone possesses a differentiation-promoting effect. There was a 40% reduction in the concentration of GalDAG in the brain of 20- and 23-day-old rats, which were made hypothyroid at birth [367]. The embryonal mouse (15 days) brain cells grown in the presence of hypothyroid calf serum, which contained very low levels of thyroid hormone compared to normal serum, showed a diminished synthesis of myelin-associated glycolipids (sulfo-GalDAG and GalDAG) from H [ 35S]0, or [3H]Gal. This reduced activity could be restored to normal (3-4-fold increase) by including triiodothyronine (T3), the active form of thyroid hormone, in the medium (precocious myelination) [192]. In the system of cultures of dissociated brain cells from embryonic mice, T3 did not alter the synthesis of sulfated mucopolysaccharides, which share 3’-phosphoadenosine-5’-phosphosulfate(PAPS) as a common precursor, with sulfolipids. This observation argues against the hormone altering the entry of sulfate or the synthesis of PAPS. Rather, T3 acted, by inducing the PAPS:glycolipidsulfotransferase(s), in direct proportion to the concentration of T3 in the growth medium [192] (Section 4.1.8.1). 3.4. GERM CELL
3.4.I . Distribulion Seminolipid (sulfo-GalAAG) is the major glycolipid in mammalian testis. The tissue concentration was about 2.9% of the total lipid (more than 90% of the sum of neutral and sulfo-glycolipids) in boar spermatozoa. In boar testis, sulfo-GalAAG comprized about 0.88 of total lipid [45,123]. The tissue level, about 1 pmol/g (1 mM) in spermatozoa, is twice as high as the GalDAG content in the brain of rat. Murray [56] listed the distribution of sulfo-GalAAG and other sulfoglycolipids of 10 species of mammals, two and three species, respectively, of birds and fishes, as well as turtle
145 (Reptilia) and bull frog (Amphibia). Sulfo-GalAAG was found in all mammals [53,57,368], but not in the other species. Instead of sulfo-GalAAG, the testis of non-mammals contained sulfo-GalCer and/or sulfo-LacCer [55] (c.f., Section 3.6.2). 3.4.2. Location in germ cell membrane Since sulfo-GalAAG was found to be more than three times enriched in spermatozoa than in the testis of boar [45], evidence has been accumulated for the germ cell nature of GGroLs in testis of various mammalian species [56,228]. That sulfo-GalAAG was present only in very low concentration (10% of control) in hereditary aspermatogenic mice, also supported the germ cell location of sulfoGalAAG [46,56]. The isolated late spermatocytes from immature rat testes were shown to be highly enriched in seminolipid (5-times the level in whole rat testis) [369]. Klugerman and Kornblatt [ 3701, by the comparison of sucrose density gradient behavior with several marker enzymes (method of De Duve), showed that GalGroLs are the constituents of plasma membrane obtained from rat seminal tubules (mainly spermatocytes and spermatids). Sulfo-GalAAG was enriched 33.1-fold, together with 5’-nucleotidase in the plasma membrane fraction of adult rat testis, with low contamination of Golgi membrane [371]. Sulfo-GalAAG was found to be distributed both in head and tail portions of bovine spermatozoa [56]. However, arylsulfatase A preparation from boar testis [372], which is very active when isolated sulfo-GalAAG is used as the substrate, could not desulfate sulfo-GalAAG in intact bovine spermatozoa [56]. This was the case also in the myelin-bound GalCers [373]. Isolated GalAAG and sulfo-GalAAG are such poor substrates for galactose oxidase that the labeling of cell surface GalGroLs by galactose oxidase-sodium borotriitide method is not possible [374]. Recently, Morre [375] could stain sulfo-GalAAG specifically using phosphotungstic acid at low pH. The plasma membrane vesicles of dictyosome-like structures of sperm, thick cisternae of Golgi apparatus of spermatid and spermatocytes from guinea pig, were also stained supporting the concept that, generally, endoplasmic reticulum “flows” to the plasma membrane via the Golgi complex [376]. Immunological staining showed that GalAAG on the surface of rat testicular germinal cells is mobile within the plane of the membrane, undergoing ligand-induced “ patching” and occasional “capping” [460]. 3.4.3. Spermatogenesis and aging In rat testis at birth, sulfo-GalAAG is absent but there is a dramatic increase in the amount of this lipid between 15 and 22 days of age, although the total lipid concentrations remain relatively constant during this period [46]. The contents of sulfo-GalAAG in the testis of humans of various ages were studied [47]. Neither sulfo-GalAAG nor GalAAG was detected in testes of infants or a child of prepubertal age [47,228]. Sulfo-GalAAG was most concentrated in the adult testis (159 nmol/g) and was much lower in the aged (70 years, 25.3 nmol/g). The total lipid and phospholipids of seminal tubules is also increased in adult, and decreased in aged, humans [377,378]. Also, immature or prepubertal mice [70] or rats [56,128,228]
146 have neither GGroLs in detectable amount nor sulfotransferase activities. Highly non-differentiated (malignant) tumor (seminoma), originating from primordial germ cell, does not contain GGroL at all [199]. Instead, they contained sulfo-LacCer, which is the component of the mature testes of lower vertebrates. The origin of germ cell is believed to be the wall of the yolk sac. A cell from the extraembryonal endoderm migrates into the embryo by ameba-like movements. I t is not yet established whether the Schwann cell or oligodendrocyte, whch synthesizes myelin, originates from the similar extraembryonal cell [536]. Germinal cells within the seminiferous epithelium reside in two compartments: ( 1 ) a basal compartment containing spermatogonia and early (preleptotene) spermatocytes; and ( 2) an adluminal compartment containing late spermatocytes and spermatozoa [371]. Spermatogenesis involves the differentiation of the least mature germinal cells, the diploid spermatogonia, through the spermatocyte and spermatid stages, into highly differentiated, independently motile, haploid spermatozoa. Letts et al. [369] found that primary spermatocytes appeared to be the earliest spermatogenic cells to contain high levels of sulfo-GalAAG [56]. A dramatic increase (about 50-fold) of the activity of the sulfotransferase involved in the biosynthesis of sulfo-GalAAG, concomitant with glycoprotein-glycosyltransferases, also occurred when spermatocytes first began to appear in rat testis [128]. The rise in sulfotransferase activity preceded by several days a marked rise in the amount of sulfo-GalAAG at the stage prior to the late spermatocyte stage [369]. Thus, sulfoGalAAG, like GalCer or sulfo-GalCer of myelin [189] and renal tubules [127,131], is a specific marker substance of differentiated germ cells of mammals. Since the only germinal cells that synthesize DNA are spermatogonia and preleptotene spermatocytes, the incorporation of [ 3H]thymidine can be used as an internal reference for the timing of spermatogenesis in the animal. The 35Sradioactivity appears in the epididymis on day 32 and that of 3 H at day 37 or 38 after administration of the radioisotopes [197]. The mature spermatozoa, however, still underwent changes in constituent phospholipids in epididymis [253] (Section 4.1 3.2). 3.4.4. Regulation of the expression of seminolipid In the seminal tubules of hypophysectomized rat, differentiation to spermatocyte stage is not inhibited as reflected in relatively high content of sulfo-GalAAG, whereas spermatides and spermatozoa are reduced greatly in number [56]. In hypophysectomized rat, normal secretion of lutropin (LH) by the stimulation of gonadoliberin (LH-RH) from hypothalamus does not occur, resulting in the loss of stimulation of the Leydig cell to produce testosterone, the target of which is the germ cell. Follitropin (FSH) is also necessary for the maturation of spermatocyte. Examination of the levels of sulfo-GalAAG in the testes of hypophysectomized [46] or vitamin A-deficient [380] rats, which became aspermatogenic, showed that sulfoGalAAG was reduced to 70 and 138, respectively, of the control. Rats, fed for 35 weeks or more on a diet deficient in essential fatty acid or deficient diet supplemented with linolenate, develop testicular atrophy (5211, whereas rats given linoleate show normal testicular development. Analysis of the total lipids of the testes of these
147
essential fatty acid deficient and linolenate-supplemented rats revealed that there were reduced proportions of decosapentaenoic acid and of sulfo-GalAAG. 3.5. SECRETION OF ANIMAL
All the previously described GGroLs of plants, microorganisms and animals are the components of membranes. GGroLs of mammalian secretions, although as yet described only by Slomianys' group [50,522], are a unique feature of all glycolipids including glycosphingolipids (c.f., Tables 1 and 2; Section 5.1) [56]. The isomaltose series (Glcal-6).-Glca-) of oligosaccharides linked to AAG and their sulfated derivatives have been isolated from human gastric contents [48,49] and saliva [221], as well as from rabbit [222] and human [506] alveolar lavages. Glycerol-type glycolipids have been reported to constitute about 30-50% of the total lipid of human, dog and rat gastric secretions [50]. These compounds are present not only in the soluble portion of gastric secretion (dissolved much), but also in the gastric mucous barrier (8.2 pg/mg perfusates) and in the preformed intracellular mucous contained within the secretory granules of the epithelial cells [218]. The contents of neutral GGroLs in the mucus lining and the intracellular preformed mucus of the antral portion of the stomach were 4-5-times greater than those obtained from forestomach and body of the stomach [103]. The contents were reduced by feeding ethanol or hypertonic saline to rats and in patients with gastritis or peptic ulcer [50]. The recent studies [50] on the origin of glucoglycerolipids in the saliva indicated that these compounds were liberated by the parotid and submandibular glands, and that their levels were elevated in the salivary secretions derived from individuals with a high rate of salivary calculus formation. Extracellular glycolipids of respiratory tract were extracted from acellular material lining the alveoli of mammalian lungs [221,222]. The carbohydrate component associated with lipids consisted exclusively of glucose. About 60% of glucose was associated with neutral glycolipids and 40% with sulfoglycolipids [2223. These lipids form a unique protein-lipid mixture, which may function to help the exchange of oxygen at the surface of alveolar membrane [50,506]. 3.6. MOLECULAR EVOLUTION OF GLYCOGLYCEROLIPIDS
3.6.1. The concept of molecular evolution The conventional taxonomy for animal and plant kingdoms (the natural system) has been elaborated by many investigators, including Aristotle and Linne, over thousands of years. The major parameter was the morphological expression of genes. Since cells and microorganisms were discovered in 1667 by Van Leeuwenhoek, the Kingdom Prokaryotae has also been classified mainly on the basis of morphology. For microorganisms, which have few morphological markers, metabolic parameters must be included. The taxonomical system adopted in Bergey's Manual of Determinative Bacteriology has been repeatedly revised to up-date the increases in physiological parameters. The recent developments in analytical methods of chemi-
148 cal compositions in micro-scale has enabled so-called “chemical taxonomy”. The representative parameter has been the base sequence of 16 s ribosomal RNAs, which have about 1540 bases [501]. The applications of these values in the form of “numerical taxonomy” has occurred in parallel with the availability of minicomputers. Taxonomy, at the same time, enabled the construction of phylogenetic tree. Evolution can be expressed as “descent with modification of biological molecules”. The word “evolution” also means the divergence in the gene pool of population of organisms. In mammals, mutations, which are neutral (in natural selection), occur at the rate of roughly OS%/year/gamete [382]. This value is 100-1000-times higher than those conventionally assumed. In addition, transfer of domains (exons) and introns of DNA also occur frequently. These result in selection of genetic variations, and also in random genetic drift [383]. Functionally less important molecules or parts of a molecular are modified (in terms of mutant substitutions) faster than more important ones. As a result, selective elimination of definitely deleterious mutants and random fixation of selectively neutral or very slightly deleterious mutants, occur far more frequently in evolution than positive Darwinian selection of definitely advantageous mutants [384]. 3.6.2. Evolutionary convergence and adaptive divergence GGroLs are distributed in all kingdoms, eubacterial, arachaebacterial, fungal, plant and animal. Glycosphingolipids, on the other hand, are distributed mainly in plant and animal. The adaptive divergence of glycolipid molecules in biospheres produced variations of structures in both carbohydrate and lipophilic domains. For instance, the evolution of the molecular species of sialic acid in various phyla of coelomata has been discussed repeatedly [529,532]. Some pathogenic bacteria such as streptococci, meningococci and E. coli apparently have acquired adequate enzyme systems for the metabolism of sialic acids from the host [532]. In Neisseria meningitidis, the combination of imported sialic acid-synthesizing system and sialyltransferase coupled with the conventional phosphatidic acid acceptor might have resulted in a new anionic amphiphile, polysialosyl phosphatidic acid. This type of gene transfer, between cells belongmg to different kingdoms, to modify the structure of acidic amphiphiles, may have occurred frequently. The composition of acidic lipids in halotolerant Staphyfococcus epidermidis in various concentrations of NaCl (see Section 5.5) [500],as well as the distribution of SQ-DAG and chlorosulfolipids in algae of sweet water and sea water [535], might indicate evolutionary or adaptive “convergence at molecular level” of acidic amphiphiles in cell membranes [500]. It is tempting to expand this idea to all anionic groups of acidic amphiphiles: carboxyl group of uronic and sialic acids, esters of phosphate and sulfate or phosphono groups (in invertebrates) [548] may serve a similar function [475,449]. The most frequently discussed type of “adaptive divergence” in deuterostomia is the postulated phylogenetic variations in sialo-oligosaccharide structure of brain ganglioside [531]. The appearance of a new device in ganglioside structure, i.e., the simultaneous appearance of a set of glycosyltransferases and the specific lysosomal
149 degradative enzyme, was interpreted to correspond to the time when a significant number of gene duplications, involving many different structural genes, occurred during a few specific periods of vertebrate evolution [531]. Two sulfoamphiphiles, seminolipid and sulfatides in testis and myelin, respectively, of vertebrates, may possibly have worked as the definitely advantageous compound in natural selection. Moreover, the origin of these two anionic lipids may be ascribed to a common primordial cell [536] (see also Section 5.3). Also, the presence in mouse intestine of Gg,Cer-IV3-sulfate instead of sulfo-GalCer [528] is a good example of expression of sulfotransferase activity on a different glycoconjugate structure, when the precursor, GalCer, is absent in the tissue. In the terminology of animal evolution, these anionic amphiphiles might be examples of “molecular convergence” for germ cell and nerve conductivity functions, which have yet to be elucidated [368,533]. 3.6.3. Phylogenetic divergence of domains in glycoglycerolipids The differences in the nature of the lipophilic domains, including fatty acid compositions, expressed in various organelles and species, do not appear to be of any significance [294]. The lipophilic domains of these sulfoamphiphils, i.e., diacylglycerol, alkylacylglycerol and ceramide, may be the results of “neutral” divergence or “random drift” of the structure of molecules. The dialkylglycerol and diglyceryltetraether residue in archaebacteria turned out to be a conspicuous marker of their descent and is very useful for classification [537]. Also, the species-specific and non-tissue-specific variations of carbohydrate chain structures, such as those in extraneural gangliosides in deuterostomia and neutral glycosphingolipids of tissues of coelomata, appear to agree with the neutral mutation-random drift theory. It has even been postulated that species-specific variations of surface carbohydrates are ontogenically paleo-functional; that is, they have lost their functions after development like selfish DNA [387]. The concomitant progress in the studies in molecular evolution of chloroplasts, mitochondria and microtubules revealed that even the eucaryotic cells are the chimella of unicellular organisms (symbiotic theory), which appeared 2.0- 1.6 billions of years ago when the content of oxygen in the atmosphere was increasing [353,386,387,501].On the other hand, numerous phenomena and facts in relation to ontogenesis and aging have been accumulated. However, the mechanisms of differentiation on the biochemical level still remain obscure [388].
4. Metabolism The principles of carbohydrate chain elongation of GGroLs are similar in eubacteria, plant and animal tissues. There are, however, numerous modifications of this process, e.g., 6-0-acylation of hexose (plant and eubacteria) and phosphatidyl and glycerophosphate transfer (eubacteria). The site where these reactions take place, the specificities of the enzymes to substrates, the requirements for divalent cations, etc.,
150 are different depending on tissues and organisms. Biodegradation of the carbohydrate domain follows stepwise removal of sugars by specific hydrolases. 4.1. BIOSYNTHESIS
It is now generally accepted that biosynthesis of the lipophilic domain of GGroLs proceeds according to the pathways similar to biosynthesis of phosphoglycerolipids. The next step is the transfer of a carbohydrate to the hydroxyl group of lipophilic moieties such as sn-1,2-DAG. It is followed by sequential addition of another polar group including hexose (Section 4.1.2), sn-Gro-1-phosphate (Section 4.1.5) and sulfate (Section 4.1.3). Modifications by epimerization (Section 4.1.2) or addition of nonpolar groups such as fatty acid or Ptd group (Section 4.1.2) also occur. Most of these groups are supplied from appropriate high energy compounds such as nucleotide hexose, PtdGro [406], and 3’-phosphoadenosine-5’-phosphosulfate(PAPS). In fact, the lack of supply of UDP-Glc in a phosphoglucomutase-deficient mutant of B. licheniformis results in complete absence of glycolipids and LTA [281]. The participation of a dolichyl-phosphate system, recently proposed to occur in the biosynthesis of GlcCer [389], has never been reported. 4. I.1. Synthesis of lipophilic domain 4.1.1.1. Synthesis of diacylglycerol Synthesis of the most common lipophilic moiety ( = DAG) proceeds stepwisely as shown below (glycerol phosphate pathway); sn-Gro-3-P+ acyl-CoA + I-acyl-Gro-3-P (MAG-P) +CoA-SH
(1)
MAG-P + acyl-CoA + phosphatidic acid (DAG-P) +CoA-SH
(2)
DAG-P + diacylglycerol (DAG) +Pi
(3)
Reactions 1 and 2 are successive transfer of two fatty acid esters on sn-1 and 2 positions, respectively, of sn-Gro-3-P from fatty acyl-CoA (all living cells) [335] or acyl-acyl carrier protein (E. coli, green algae, and chloroplast [142]) to form first lysophosphatidic acid (1-acyl-sn-Gro-3-P) and then phosphatidic acid (1,2-diacyl-snGro-3-P). The minor diglyceride kinase pathway to form phosphatidic acid from DAG has also been reported [399]. Reaction 3 is hydrolysis of the phosphate to yield sn-l,2-DAG. In gram-positive eubacteria, a large excess of DAG is formed on LTA synthesis by pathways analogous to Reaction 27-1 or 27-2, and can readily be used for GGroL synthesis [545] (W. Fischer, personal communication). 4. I.1.2, Synfhesis of alkenylacyl- and alkylacylglycerol Unsaturated alkenylacylGro (“ plasmalogen” or, according to IUPAC-IUB recommendation, “ plasmenic acid” type) and saturated alkylacylGro (AAG) originate from a common precursor, dihydroxyacetone phosphate [300,525] as shown below: Dihydroxyacetone phosphate (DHAP) + acyl-CoA
+ 1-0-acyl-DHAP
+ CoA-SH
(4)
I-acyl-DHAP+ R-OH + I-0-alkyl-DHAP+ R-COOH
(5)
I-0-alkyl-DHAP+ NADPH,
(6)
+
I-O-alkyl-sn-Gro-3-P+NADP
151
+
1 -alkyl-sn-Gro-3-P R-CO-CoA + 1-alkyl-2-acyl-sn-Gro-3-P + CoA-SH
I-alkyl-2-acyl-sn-Gro-3-P (AAG-P) + H,O
--*
AAG + P,
(7)
(8)
I-alkyl-2-acyl-Gro-3-P (AAG-P) + NADP (or NAD) + 1-alkenyl-2-acyl-sn-Gro-P +NADPH, (or NADH,) 1-alkenyl-2-acyl-sn-Gro-3-P+ H,O
(9) +
1-alkenyl-2-acyl-sn-Gro + Pi
(10)
The I-acyl moiety of DHAP esterified by acyltransferase (Reaction 4) can be exchanged with an alkyl group (Reaction 5), then the 0x0 group at C-2 of glycerol is reduced by H, from NADPH, (Reaction 6), followed by acylation (Reaction 7), giving rise to I-alkyl-2-acyl-sn-Gro-3-P. AAG is formed from the last compound by hydrolysis of phosphate (Reaction 8) but when dehydrogenated beforehand at position 1 of the alkyl chain (Reaction 9) I-alkenyl-2-acyl-sn-Gro results (Reaction 10) [300,397]. The biosynthetic pathway of lipophilic domains in archaebacteria are predominantly those for isoprenoid synthesis, leading to glycerol ether formation, and the fatty acid pathway is operative only at low levels [517]. The pathway of the synthesis of the glycerylether back bone of the testicular seminolipid remains to be elucidated. In view of the hghly restricted alkyl and acyl composition (16:0/16:0) of the lipid, it would be of interest to determine the substrate specificities of the enzymes involved in formation and transfer of these building blocks [56,253] as was indicated for the lactoneo-series of glycosphingolipids [511]. 4.1.1.3. Acyl exchange The possibility that the final fatty acid composition of GalDAG in chloroplast is determined by an acyl exchange (remodeling) mechanism, operating in the chloroplast envelope was suggested first by the incorporation studies using a green alga, Chlorellu uulguris [14,312,391]. This process begins with the removal of a single acyl group from GalDAG or Ga1,DAG. The free OH- is then replaced by fatty acids from acylthioesters or Ptd-choline (Reaction 11) supplied from the endoplasmic reticulum (Section 4.1.7): GalDAG + 18 : 2-Ptd-choline + 18 : 2-GalDAG + Ptd-choline
(11)
Here, 18 : 2-Ptd-choline represents Ptd-choline with C18 : 2 fatty acid at position-C2 of glycerol. This process is analogous to the partial turaover by “Land’s cycle” of acyl exchange between phospholipids [434,525].However, this pathway was not operative in a cyanobacterium [316]. Acylation of GalMAG [393] and SQ-MAG (2-acyl isomer) [394] from acyl-CoAs was also studied. In spinach [315] and broad bean [404], 18 : 2-Ptd-choline desaturated from 18 : I-Ptd-choline in endoplasmic reticulum, appeared to be degraded to 18:2-DAG, which in turn is transferred to chloroplast (eucaryotic pathway). 4.1.1.4. Postsynthetic modifcution Evidence for the modification of the fatty acid residues by desaturase after formation of GalDAG (Reaction, 31) was presented by
152 the Nichols’ group using the system of a green alga (C. vulgaris) [392]. In this system, the quantity of Ptd-choline involved was so small that this lipid was only detected by radiochemical means [391]. On the other hand, cyanobacteria do not contain a detectable quantity of Ptd-choline. This prompted Appleby et al. [316] to clarify whether similar or different pathways to 18 :2 do in fact occur in green algae and cyanobacteria. In a cyanobacterium ( Anabaena oariabilis), the Ptd-cholinemediated pathway to linoleate observed in C. vulgaris was absent [316]. Later, Heinz [336] proposed the desaturation of 18: 1 to 18: 3 in GalDAG (Section 4.1.7, Reactions 31, 32), based on the time course of tracer (e.g., [I4C]acetate) incorporation into spinach and broad bean (Vicia faba) leaves. Substrate specificity of desaturase may possibly explain the association of more unsaturated fatty acids with GalDAG (18 : 3/18 : 3) and the virtual exclusion of 16 : 3 acid from Ga1,DAG (major species = 18 : 3/16 : 0). Actually, in broad bean leaves fed with [‘4C]02,the fatty acids of Gal,DAG did not appear to undergo further desaturation [404]. SQ-DAG also did not act as a substrate for desaturation [336] (Section 2.2.1). Cyclopropane ring of phospholipids of gram-positive bacteria is formed by the action of cyclopropane synthetase to transfer a methyl group from S-adenosylmethionine to monounsaturated fatty acid. However, there have been no reports on the cyclopropanization of fatty acids bound to GGroL. 6-0-acyl derivatives of GalDAG [323], GalMAG [205] and Ga1,DAG [229,396] were formed by the homogenization of plant leaves or even during the preparation (which took 7 h) of the chloroplast envelope fraction [81]: 2GalDAG
+ acyl-0-6GalDAG
+ GalMAG
(12)
GalDAG has been proposed as the acyl donor in each case. 4.1.1.5. In oivo and in vitroproducts are different As noted above, in vitro systems do not necessarily result in fully natural products [326]. Neufeld and Hall [59], studying the formation of galactolipids with acetone powder of spinach chloroplasts and UDP-[14CIGal, obtained a compound behaving chromatographically as Gal ,Gro after alkaline methanolysis of the labeled lipids. They reported the following. “However, only 17%of the terminal Gal was hydrolyzed with a-galactosidase, the remainder being susceptible to P-galactosidase”. Siebertz et al. [335] also found that isolated chloroplast envelope fraction of spinach incorporated UDP-[I4C]Gal into completely different patterns of galactolipid molecular species, as compared to the in vivo labeling pattern. Higher galactolipid homologs were produced in unusual proportions. These results pointed to either changed properties of this membrane system or to a loss of regulatory system after isolation. 4.1.1.6. Synthesis of phosphoglycerolipid domain In eubacteria and mitochondria, activation of DAG to CDP-DAG in the presence of GroP leads to the synthesis of PtdGro: CDP-DAG + GroP + PtdGroP + P,
(13.1)
PtdGroP+ H,O
(13.2)
-
PtdGro+ Pi
PtdGro, in turn, is the parent compound of Ptd residue in diPtdGro (Reactions 14,
153 15), Ptd-GGroLs (Reactions 16, 17) and GroP-GGroLs (Reactions 26, 27): PtdGro + CDP-DAG + diPtdGro + CMP
(14)
2PtdGro -+ diPtdGro + Gro
(15)
It was suggested that radiochemically pure Glc,DAG is the precursor of the new glycoglycerophospholipid, the structure of which was later proved to be a Glc( Ptd)GlcDAG [78,137] in Streptococcus faecium. This was confirmed by the discovery of “ transphosphatidylation” reaction in Pseudomonas diminuta [259]: PtdGro + GlcDAG + Ptd-6GlcDAG + Gro
(’6)
and S. faecium [161,389] as shown below: PtdGro + Glc, DAG
+
Glc( Ptd-6)GlcDAG
DiPtdGro + Glc, DAG -+ Glc( Ptd-6)GlcDAG + PtdGro
(17) (18)
These reactions are analogous to the biosynthesis of cardiolipin (diPtdGro) in bacterial system: condensation of two molecules of PtdGro and release of free glycerol (Reaction 15). In the P. diminuta [259] and Bifidobacterium [275] systems diPtdGro did not function as the substitute for PtdGro. The biosynthesis of Man-inositol-P-DAG (phosphatidylinositol mannoside) is different from the above schemes in that the Ptd group is first transferred from CDP-DAG on myo-inositol to form Ptd-inositol [399,494]: CDP-DAG + inositol --t Ptd-inositol+ CMP
(19)
4.1.2. Transfer of reducing carbohydrates 4.1.2.1. Plant and microorganism Mazliak [400], Mudd and Garcia [14], Sastry
[13], Heinz [291], Douce and Joyard [324,142] have already reviewed the development of the glycosyl transfer reactions of plant GroLs in detail. Here we make some efforts to correlate parts of these data with the biosynthetic pathways in microbial and animal GGroLs. It was shown by in vitro experiment that, in Chlorella pyrenoidosa, GalDAG and Gal DAG were biosynthesized by the transfer of Gal from UDP-Gal successively on sn-1,2-DAG as follows [60,401]: DAG + UDP-Gal -+ Galal-3DAG + UDP
(20)
Gal/31-3DAG+ UDP-Gal
(21)
-
Galal-6Gal/31-3DAG+ UDP
Gal al-6Gal/31-3DAG+ UDP-Gal + Galal-6Galal-6Gal/31-3DAG + UDP
(22)
I t has not been finally settled [326] if the enzyme catalyzing Reaction 22 is the same as the enzyme of Reaction 21, but it appears to be catalyzed, by different proteins
154 [291]. Subsequently, the following microorganisms were shown to catalyze similar transfer reactions: chloroplast [59,401]; Pneumococcus type XIV (Galal-2Glcal3DAG) [63]; Micrococcus luteus (Manal-3Manal-3DAG) [61]; Acholeplasma laidlawii (Glcal-2Glcal-3DAG) [402]; Streptococcus faecium (Glcal-3DAG and Glcal2Glcal-3DAG) [64]; and Bifidobacterium bifidum (GalP1-3DAG) [183]. The transfer of glucuronic acid (GlcU) from UDP-GlcU to DAG or GlcDAG occurred also in a halophile [110] and Pseudornonas diminuta [334]. These glycosyl transfer reactions in almost all enzyme systems were shown to require Mg2+ [212,227], and anionic or nonanionic detergents [212], for the optimum reaction. The syntheses of GalP1-3DAG and Gala1-6Galfil-3DAG by spinach chloroplasts were optimal at pH 7.2, and the proportion of GalP1-3DAG decreased while that of Gal,DAG increased as the pH was lowered [403]. A relatively high ionic strength was favorable for the glucosyl transfer system of A. laidlawii [402]. The sulfhydryl nature of the galactosyltransferase was established with spinach chloroplast preparations [14]. Mercuric salts were strongly inhibitory. The spinach preparation was shown to be resistant to elevated temperature and showed maximum activity at 45 OC [403].The ratio of GalDAG to Ga1,DAG was greater at the higher reaction temperatures. Recently, the group of Wintermans proposed a new pathway for the biosynthesis of Gal,DAG and of higher homologues in chloroplasts. This pathway, as shown below, does not require UDP-Gal: ZGalp1-3DAG + Galal-6Gal~1-3DAG + DAG
(23)
GalB1-3DAG + Galal-6GalB1-3DAG + Galal-6Galal-6Gal/31-3DAG + DAG
(24)
These reactions, “interlipid galactosyl transfer”, proceeded maximally at a pH of about 6, and very slowly at pH 8 and higher [81,324,326,446]. A cyanobacterium, Anabaena uariubilis, has a pathway entirely different from that in other photosynthetic organisms. The label from NaH[ I4C]O3 was first incorporated mainly into a GlcDAG, which was subsequently transformed into a GalDAG in 10 h, probably by the epimerization of the hydroxyl group at C-4 of galactose [498,4991. 4.1.2.2. Animal tissue The in vitro synthesis of GalDAG in an animal tissue was studied for the first time by the group of Pieringer, using the lyophilized and acetone-dried fractions from rat brain as an enzyme source [69], UDP-Gal as the Gal donor and DAGs as the acceptor, in analogy to the bacteria and plant pathway (Reaction 20). No kinetic studies were performed because the true concentration of dipalmitin in the assay mixture was difficult to assess. A Ga1,DAG with a terminal cu-linkage was also synthesized by the rat brain enzyme [226,367]. The galactosyltransferase activities could not be demonstrated with rat testicular enzyme preparations using sn-l,2-DAG or sn-1-alkyl-2-acylDAG as the acceptor (561. Labeled glycerol and dihydroxyacetone added to the incubation medium were readily incorporated into lipids of epididymal sperm, such as Ptd-choline, DAGs
155 and phosphatidic acid, under both aerobic and anaerobic conditions [253]. However, no radioactivity was found in alkylacyl lipid including seminolipid (sulfo-GalAAG) or alk-1-enyl glycerolipids showing slow turnover of these lipids. These results supported the idea that the ether lipids may provide stable structural components of sperm membrane, while the diacyl analogs undergo degradation and resynthesis similar to the glycolipids in the brain of young rat [301] (c.f., Section 3.3.3). The activities of P-galactosyltransferase to 1,ZDAG in Sprague-Dawley rat brain, were low or nonexistent before about 10 days of age [69,226], i.e., before the onset of myelination. They increased sharply, especially after 16 days up to about 20 days of age, and then decreased rather quickly to adult values [93]. The activities of a-galactosyltransferase for the synthesis of a Gal ,DAG [191] had also a relatively sharp peak (0.49 nmol/mg protein/h) at 19 days. The myelin-deficient Jimpy mice had little ability to synthesize GalDAG and Ga1,DAG [191]. UDP-Ga1:DAG-Pactivigalactosyltransferase as well as UDP-Gal:GalDAG-d-6-galactosyltransferase ties was markedly diminished. However, the activities of degradative enzymes, P-galactosidase and lipase, were the same as in normal mice [191], probably reflecting the failure of oligodendrocyte differentiation. An activity involving transfer of N-acetylneuraminic acid to DAG was demonstrated in the microsomal fraction of mouse brain [488]. This sialyltransferase was specific to GalP- structure. 4.1.3. Transfer of sulfate
Plants do not contain sulfoglycolipids, and no studies have yet been performed on the sulfate transfer reactions on microbial sulfoglycolipids. Since the discovery of phosphoadenosine phosphosulfate (PAPS) in 1949 by Lipmann, the synthesis of sulfo-GalCer [419] and sulfo-LacCer [54b] has been extensively studied in central nervous system and kidney. 4.1.3.1. Properties of SuIfotrunsferase The studies on GGroL-sulfotransferase were initiated on the basis of knowledge obtained with animal sulfotransferase (for review see Refs. 53, 57, 126,419). The Triton X-100 extract of the crude membrane fraction from boar testis [70], and the Golgi-rich fraction obtained from rat testis [128], catalyzed the transfer of sulfate from 35S-labeled PAPS to the acceptor GGroL, “desulfoseminolipid” (GalP1-3-sn-AAG, Fig. 2.la): GalAAG
+ PAPS
--t
HS0,-0-GalAAG
+ APS
(25)
The optimal pH was 6.8-7.0. K , for the acceptor GalPl-3AAG was 1.7 x lo-’ M [70]. Triton X-100 (5 mg/ml) and EDTA (5 mM) stimulated the transfer reaction [70]. Subsequently, Pieringer [124,254] demonstrated the presence in rat brain of a sulfotransferase activity capable of sulfating GalDAG. Some properties were different from the activities reported for GalCer-sulfotransferase of brain, kidney and cultured kidney epithelial cell lines [330,419]. The most interesting problem is the discrepancy on the effect of ATP to the sulfotransferase activity. Sulfate transfer in boar testis [70], rat brain [254]and rat gastric mucosa [523] to GalCer was stimulated by a few mM of ATP and Mg*+, but that to GalDAG or GalAAG was inhibited.
156 4.1.3.2.Acceptor specificity The sulfate transfer activity of boar testis enzyme was specific for glycolipids with a terminal P-Galp residue [70] in agreement with the substrate specificities of other glycolipid-sulfotransferases[lo]. From the resuhs of substrate competition studies, it was suggested that, primarily, one sulfotransferase was involved in the sulfation of various glycolipid substrates [70,254]. It is not clear at present why the terminal P-galactose is replaced by a-glucose in the di- and triglycosyldiether of Halobacterium marismortui [245,500]. Also, sulfated glycolipids are absent in these species. The absence of terminal galactose may account for the lack of sulfated GGroL, because in the sulfated glycolipids studied so far the sulfate group is located on a C3 hydroxyl group of P-Gal [lo] or P-GalNAc residue [131]. Further support for the consequence of the lack of precursor (e.g., GalCer) in a tissue was demonstrated in mouse intestine [528]. In a species of extreme halophiles from salt flats in Spain a sulfated diglycoGro) syldiether (with structure: HS03-O-6Manpal-2Glcal-1-sn-2,3-di-O-phytanyl replaces the more common sulfo-GGroL (HSO,-O-3Gal~l-6-Manal-2Glcal-1,2,3di-O-phytanylglycerylether). The corresponding desulfated diglycosyl diether is present as a minor glycolipid in almost all species studied [500]. A sulfotransferase activity, catalyzing the transfer of the sulfate ester group from PAPS to (Glcal6Glc),all-3(1)AAG or -MAG was demonstrated in cytosol fraction of rat gastric mucosa [516]. The sulfate ester formed was on the position-6 of the terminal a-linked glucose. 4.1.3.3. Ontogenesis and activity It became obvious that the sulfotransferase activity in rat brain increased rapidly with the onset of myelination, reached a peak during myelination, then decreased gradually [71,254]. At the age of 70 days, it occurred at a rate of about 1/6 of the maximum rate in the young animals; the activities of sulfatases were highest during the active period of myelination in mice brain [57,126]. In germ cells, the activities of sulfate transfer is regulated by the hypothalamus-hypophysis-Leydigcell axis. The activities of sulfotransferase coincided with the appearance of secondary spermatocytes [55,56,128]. In the retina and brain of chick during ontogenesis, sulfotransferase activities on protein bases were highest in the 19-day-old embryo, decreased gradually until about 15 days after hatching to 1/3 to 1/4 of the peak, then stayed practically constant through adulthood [256]. Pritchard [129] compared the sulfotransferase activities of the homogenate of testis and submandibular gland of rat (Section 2.1.2.2). The submandibular gland showed endogenous incorporation of [ 35S]sulfate into lipid fractions a few times higher than that of testicular homogenates. 4.1.3.4. Sulfono group The pathways of the synthesis of sulfono group in SQ-DAG are rather complex. The suspension of Chlorella pyrenoidosa, cultured in a low-sulfate (ca. M total sulfate) medium and 1 mCi of carrier-free [35S]sulfate, incorporated the radioactivity into a sulfur-containing lipid [9]. The biosynthesis of sulfonoGGroLs in plant has been extensively reviewed [10,14,195.294,305]. 4.1.4. Specificity of transferase to lipophilic domain 4.1.4.1. Diacylglycerol of microorganism and animal The stereochemistry of the
157 glycerol moieties in the acceptor DAGs was investigated in the synthesis of Glcal3DAG in Streptococcus faecium [64]. The synthesis of GlcDAG was stimulated by the addition of sn-l,2-DAG but not by sn-2,3-DAG. Such stereospecificity for glycerol had already been well established with various phospholipids. Mannosyltransferase of the particulate fraction from Micrococcus luteus showed a high specificity for an exogenous 1,2-DAG, especially for DAGs containing branched chain fatty acids, which are found in vivo [61]. Also, as a detergent for stimulating the reaction, the Na salt of C-15 branched-chain fatty acid was most effective. For enzymes from a streptococcus [64], bifidobacterium [212], and rat brain [69], dipalmitoyl-Gro (16 : 0/16 : O-DAG) was a better substrate than, e.g., diolein (18 : 1/18 : 1-DAG), whereas for the enzymes from a halotolerant bacterium [110] and Micrococcus luteus [61], it was a very inefficient acceptor. The most conspicuous example of selection of a specific lipophilic domain was observed on testicular seminolipid [45,253] (Table 2.4). It has not been settled yet if this selection occurs on the stage of sulfation [56]. GalCer with hydroxy fatty acids was better than that with nonhydroxy fatty acids in the acceptor activities in vitro, supporting the above possibility [70]. Later, preference for 1,2-DAG from the homologous organism as the acceptor of carbohydrate, was also noted for the glucosyltransferase of streptococcus [64], galactosyltransferase of C. uulgaris [403]; and A. laidlawii [402]. In conclusion, glycosyltransferase involved in the formation of glycosylDAGs from various organisms differ greatly in preferring different exogenous DAGs [291]. 4.1.4.2. Diacylglycerol ofplant Ongun and Mudd [401], using the acetone powder of chloroplasts, demonstrated that diolein (18 : 1/18 : 1-DAG) was an efficient acceptor for the biosynthesis of GalP1-3DAG, but not for the Ga1,DAG. In contrast to bacterial and animal brain enzyme, with the enzyme preparation from plant, DAGs with a high degree of unsaturation (e.g., 18 :2/18 : 2 species) served as the most efficient acceptors [13,335,395,402,404].The rates of galactosyl transfer in vitro increased with increasing numbers of double bonds in the fatty acids of exogenous GalDAGs [335,395], but beyond six double bonds (18 : 3/18 : 3 or 18 : 3/16 : 3) a decline was observed (e.g., 18 : 4/16 : 4) [291]. The fatty acids can undergo further desaturation after galactosylation (e.g., to 18 : 3/18 : 3-GalDAG from 18 : 2/18 : 2-GalDAG) (Section 4.1.1.4). The large proportion of hexaene DAG and GalDAG in isolated envelopes of spinach may be an in vitro artifact [175,335]. Rather, it appears that they are desaturated after galactosylation. 18 : 3/16 :O-GalDAG molecules are selectively utilized in the biosynthesis of Ga1,DAG in plants [13]. In other words, most of the Ga1,DAG and Ga1,DAG [335] are made de novo from oligoene DAG via GalDAG, which had no 16 : 3 fatty acid, excluding the use of a separate pool of inert hexaene GalDAG. Such “baton pass” mode of carbohydrate chain elongation of globoside (Gb,Cer) by multienzyme system was reported in a Golgi membrane of mammalian tissue [405] (Section 4.1.8). In eucaryotic membranes, the compartmentation of these precursor lipids is highly organized. sn-l,ZDAG, which is the most common glycosyl acceptor of GGroLs, exists usually in negligible quantity in animals, and is probably localized in
158
endoplasmic reticulum in equilibrium with DAG residues of phospholipids (e.g., 18:2-Ptd-choline). In contrast, DAGs amount to 15% of the lipids from isolated spinach envelopes, and contain C-16 : 3 and C-18 : 3 acids, which are characteristic for galactolipids [175,291](Section 4.1.6.1). 4.1.4.3. Phosphatidyl group In microorganisms such as streptococci, the fatty acid composition of DAG and Ptd portions of glycosylDAGs, GroP-glycosylDAGs and Glc(Ptd)DAGs suggested that both the DAG and Ptd portion of these polar lipids are derived from a common pool of phosphatidic acid, the precursor of DAG (Section 4.1.1.1, Reaction 3) [76] (see Table 2.2.3 and 2.2.5). In the transphosphatidylation reaction to form PtdGlcDAG in Pseudomonas diminuta [259], an acceptor, Glcal-3DAG from S. fuecium, was about 1/5 as active as the Glcd-3DAG from P. diminuta. These two GlcDAGs differed only in their fatty acid compositions. PtddGlca1-3DAG, a minor component in S. faecium, was not incorporated into lipoteichoic acid (LTA) [162]. The difference in structures of the glycolipid anchor portions of LTAs had no effect on their activity as LTA carrier (LTC) (Section 4.1.5.2) or as the acceptor of ribitolphosphate from CDP-ribitol in vitro. Because the loss of fatty acids led to a loss of LTA carrier activity, it was concluded that a nonspecific lipophilic portion is necessary for the appropriate orientation of LTA, probably as mixed micelles at the lipid-water interphase [141] (c.f., Section 5.1.6). 4.1.5. Transfer of sn-glycerol-1-phosphate and ribitol 1-phosphate The history of this topic was recently reviewed by Pieringer [406] and Fischer [31]. 4.1.5.1. sn-Glycerol-1-phosphateIn the early 1970s, the only known compound capable of donating an sn-Gro-1-P [135] in GroP-Glc,DAG was PtdGro, which is abundant in the membrane of gram-positive eubacteria [243]. If CDP-Gro or CDP-DAG is involved, an sn-glycerol-3-phosphate moiety or its 1,2-di-O-acylated analog (Ptd group) would be introduced. Actually, evidence for the incorporation of sn-Gro-1-P from PtdGro into GroP-Galf-DAG of Bifidobacterium was obtained [212] and later this precursor-product relationship was confirmed in vivo [276] and in vitro [275] by pulse-chase experiments: PtdGro+ (acyl)Gal/Bl-3DAG
+
GroP-6(acyl)Galf/l1-3DAG
+ DAG
(26)
A metabolic relationship between glycerophosphoglycolipids (GroP-GGroL) and LTA had been suggested because of the similarity of chemical structure [77,139]. Puke-chase experiments in vivo by Glaser and Lindsay [156] using Staphylococcus aureur, and by Emdur and Chiu [157] using Streptococcus sanguis, demonstrated that PtdGro, as in previously described experiments with other bacteria, had the highest turnover of any lipids. Radioactivity was rapidly transferred from PtdGro to a water-soluble, high molecular weight compound with properties similar to LTA. This was subsequently confirmed in vitro with particulate enzyme preparations from S. sanguis [407,408] and S. faecium [162]. Recently, a dynamic model for in vivo
159
biosynthesis of LTA, including Ptd-Gro and DAG, was presented [545]. These two components occupy 55 and 20 mol%, respectively, of the membrane lipid of S. aureus. Reduced PtdGro synthesis (Reaction 13) by inhibitory effect of 3,4-dihydroxybutyl-1-pyrophosphonate (CH20HCHOHCH2CH2P(02H)OP0,H,, an analog of sn-Gro-3-P) resulted in the inhbition of the formation of the putative LTA precursor, sn-Gro-l-P-6Glc/3l-6Glc/31-3DAG (analogous to Reaction 26) and of LTA itself in B. subtilis [409]. When the putative precursor phosphoglycolipids are not available, DAG serves as the anchor portion of LTA [281] (examples of change of lipophilic domains are listed in Section 3.6.3). 4.1.5.2. Lipoteichoic acid Ganfield and Pieringer [161], analysing the membrane GGroL fraction from S. faecium labeled with I4C specifically in glycerol moieties [410], found that PtdGro donated GroP to Glc(Ptd)GlcDAG with the formation of nascent LTA. In conclusion, LTA of S. faecium appeared to be formed by the following reaction: nPtdGro + Glc( Ptd)GlcDAG + (GroP),Glc( Ptd)GlcDAG + nDAG
(27-1)
Polyglycerophospho-GGroLof Staphylococcus aureus is also synthesized by sequential addition of sn-Gro-1-P group from PtdGro: nPtdGro+GroP-GlczDAG + (GroP),+,-Glc,DAG+ nDAG
(27-2)
An enzyme that catalyzes the transfer of ribitolphosphate to an LTA carrier (LTC) has been purified from Staphylococcus aureus H [151,411,412]: LTA + 30 CMP-ribitol + LTA-( P-ribitol),,
+ 30 CMP
(28)
S. aureus H was found to contain a ribitol-containing LTA, the structure of which was tentatively assigned as oligo(ribito1-P)-poly(GroP)Glc,DAG [413]. This LTA could be an acceptor of Glc and GlcNAc from the UDP-derivatives catalyzed by the membrane preparation of S. aureus H. The problem of LTA and LTA carrier was recently settled by the finding of Fischer [141,510], that LTA carrier and LTA were identical entities. It was concluded that, in LTA, a nonsubstituted (GroP), segment is necessary for recognition and a terminal GroP for acceptor function to poly(ribito1-ph0sphate)polymerase. It was suggested that the space between the phosphate groups is important to fit poly(ribito1phosphate)polymerase.This distance is 0.75 nm in poly(glycer0-phosphate). In contrast, the P to P distance of the LTA from Streptococcus lactis Kiel (Table 2.2.5(6)) was 1.65 nm and this polymer acted neither as an acceptor nor as an inhbitor. GroP-GGroL with more than three unsubstituted GroP units serve as the substrate for poly(ribito1phosphate)polymerase of S. aureus. The optimum activity was displayed by LTA with poly(GroP) chains of 20-25 units in length. The
160 (GroP),-4GlcNAc-l-P- moiety of the “linkage unit” of teichoic acid has the same length as the (GroP), segment of LTA. The four phosphate groups of both compounds appeared to fit into the same putative binding sites of the enzyme from S. aureus [141]. 4.1.5.3. Addition of D-alanine Substitution with D-alanine on the position-2 of the Gro of the hydrophilic chain in the LTA of S. lactis Kiel caused a marked decrease in the acceptor activities for ribitol phosphate [150]. Glycosyl (Gal) residues of streptococcal LTA showed a behavior intermediate between that of alanyl residues and their derivatives without positive charge [510]. None of the D-alanine-substituted LTAs, which are carefully prepared from various gram-positive organisms in mild acidic conditions (Table 2.2.5), could be used by the poly(ribito1-P)polymerase from S. aureus H. A single alanyl residue, anywhere on this sequence, was apparently sufficient to prevent binding to the enzyme [510].These findings strongly suggested that LTAs are physiologically incapable of functioning as LTA carrier in the biosynthesis of wall teichoic acids. The problem recently became complicated by the observations that in S. aureus the alanine ester substitutions of LTA decreased from 73 to 33% with increasing NaCl concentrations in the growth medium [155,159] (Section 5.5). Similar to other bacteria, the highly-substituted LTA displayed no LTA carrier activities [42]. However, when alanyl residues were chemically converted to uncharged lactyl or Nacetylalanyl derivatives, this inhibition of binding was relieved, and the activity required only a single unsubstituted terminal Gro residue [510].Furthermore, the LTA of Micrococcus uarians lacks any substituent [155] and consequently shows full LTA carrier activity [42]. Therefore, despite the recent progress in this field, further studies are necessary to find out whether, and in which organism, LTA functions as LTA carrier in vivo (Fischer, W., personal communication). 4.1.6. Location of enzyme activity The site, where lipophilic domains of mammalian GGroLs are constructed, is endoplasmic reticulum. In plant tissues, mitochondria and the chloroplast envelope are also capable of DAG synthesis [414,504]. The site of most active glycosyl transfer is the chloroplast [59], more exactly, the chloroplast envelope [142,325], particulate (crude membrane) fraction of gram-positive bacteria [64,66] and Golgi membrane of mammals (1281. 4.1.6.1. Microorganism and plant In Pneumococcw type XIV, both particulate and soluble enzyme preparations showed galactosyl- and glucosyltransferase activity [63]. In Micrococcus luteus [61] and Streptococcus faeciurn [64], however, formation of mono- and dihexosylDAGs were catalyzed by crude membrane fractions of bacteria. Since 1974, it has been shown that galactolipids, which form the lipid matrix of the thylakoid membranes, are not synthesized in thylakoids, but that the galactosylation from UDP-[14C]Gal (Reactions 20 to 22) is performed in the chloroplast envelope [ 142,324,325,5041. The activity was enriched 20- to 100-fold, at UDP-Gal concentrations around a K, value of 10-50 pM, which seems quite plausible in vivo. The rate of GalDAG synthesis could be 1.0-2.0 pmol/mg protein/h [142]. An
161 alkaline phosphatidic acid-phosphatase (Reaction 3) to form DAG was also demonstrated in the chloroplast envelope of spinach. This is in agreement with the discovery that this membrane contains high levels of DAGs (24% of the total envelope lipid, c.f., Section 3.1.2.1) [324,325], the lipophilic acceptors of GalGroLgalactose [335,415]. However, synthesis of DAG in mitochondria and subsequent transport, for instance as phospholipids by a phospholipid exchange protein [414], cannot be excluded. Thus, the envelope is an important interface, where precursors and intermediates, coming from the cytoplasm and the stroma [142], meet to be transformed into thylakoid membrane lipids [291,324,356,415]. Widely varying activities have been presented for these enzymes in envelope preparations responsible for the transfer of galactose (from 3 to 45 000 pmol/min/mg) [291,324]. K, values for UDP-Gal covered a range from 2.2 X M. Problems on acyl transfer and to 3.3 X desaturation will be discussed in the section of “in vivo” biosynthesis (Section 4.1.7.2) because these reactions occur in endoplasmic reticulum, envelope and thylakoid. Whether these enzymes are synthesized by protein-synthesizing machinery of chloroplast awaits future studies. 4.1.6.2. Animal Deshmukh et al. [366] showed that the oligodendroglia cells are the primary site for the synthesis of myelin GalDAG. The activity of galactosyltransferase was about 20-times enriched in this cell population. The sulfotransferase activity in mature rat testis [128] was markedly (17-47-fold) enriched in a Golgi apparatus fraction. The Golgi preparation from immature (23-dayold) rats, however, yielded preparations of poorer quality [ 3691. Galactosyltransferase and glycolipid sulfotransferase were also enriched about 10-15-fold and 18-fold, respectively. The rate of appearance of radioactivities of intraperitoneally injected [ 3SS]sulfate in various subcellular fractions of rat [125] and mouse [540] also suggested that there could be a precursor/product-relationship among sulfoglycolipids of microsomes and myelin. In other words, AAG or DAG are synthesized in the endoplasmic reticulum of oligodendroglial cells, then galactose and sulfate are added on the Golgi membrane. Completed GGroLs are subsequently transported to plasma membrane to be deposited and finally rolled along the axon (c.f., also Section 4.1.8.1). Alternatively, Burkart et al. [477] proposed that sulfo-GalGroL may be transported to myelin in vesicles associated with lysosome. The Golgi apparatus of testis, isolated within an hour after intratesticular injection of radioactive Na,SO, to rat, was highly enriched in [ 35S]sulfo-GalAAG [56]. Within 24 h, newly sulfated lipid left the Golgi and moved to the plasma membrane fraction [197] (c.f., Section 3.4.2). This was also suggested by Morre by histochemical studies [375]. Brain sulfotransferase to form sulfo-GalCer is also enriched in Golgi membrane but no studies have yet been undertaken on the subcellular location of sulfate transfer activities to GalDAG or GalAAG [419]. 4.1.6.3. Comments on compartmentation Problems arose in the biosynthetic studies using broken cell systems of animals, in which either the mixing of cell population occurred or the compartmentation of cell organella was displaced [ 1281. Also both
162 enzymes and substrates responsible for glycosyl or sulfate transfer, were extracted from the lipid bilayer by, e.g., Triton X-100 [70]. The transferases cannot distinguish the physiological substrate from the artificial one. In consequence, the formation of anomalous products such as sulfo-GalCer, which is not detected in the in vivo labeling experiment [131,329,417], occurred in testicular homogenates [197]. Such discrepancies have also been observed for plant organella (Section 4.1.1.5) and bacterial systems (Section 4.1.5.2). 4. I . 7. I n oivo biosynthesis in plant 4. I . 7.1. Photosynthesis Detached barley leaves incorporated about 1 pmol each of [I4C]acetate per gram of leaves into GalDAG and Ga1,DAG [418]. The pattern of ['4C]acetate-labeled lipids was different from that expected on consideration of total lipid assays. In short-time labeling studies, it was repeatedly observed that various chloroplast preparations synthesized GalDAG and Gal DAG containing a high proportion of [I4C]O2-or ['4C]acetate-labeled 16 : 0 and 18 : 1 fatty acids, whereas on the mass basis 18 : 3 acid occupied about 80% [142]. With increasing lengths of time, more label appeared in unsaturated species, and labeling patterns showed a slow adjustment toward the mass patterns. With developing pea seeds, the incorporation was considerably elevated by the ATP synthesis stimulated by illumination, reflecting the differentiation of thylakoid [338]. Chloroplasts. obtained from both photoauxotrophic and photoheterotrophic Euglena gracilis (a green-alga), are essentially devoid of such enzyme activities. This suggested that the photosynthetic condition is essential in E. gracilis for the biosynthesis of glycolipids [ 131. A genetic block in chlorophyll synthesis in barley, caused by mutation in the xan-f locus, led to a repression of the formation of chloroplast membranes and of acetate incorporation into GalDAG and Gal ,DAG (9-20% of wild type) 14201. 4.1.7.2. The role of organelle The production of GGroLs, involving desaturation of component fatty acids, is a more complex process in eucaryotes, such as green-algae and higher plants. In animals, long-chain acyl-CoA is the substrate of desaturase in endoplasmic reticulum. Phospholipids synthesized de novo in endoplasmic reticulum, are subsequently exchanged with those in plasma membrane and mitochondria1 membranes. On the contrary, in plant, synthesis of endoplasmic reticulum phospholipids occurs in the endoplasmic reticulum. However. synthesis of thylakoid phospholipids is carried out in the envelope. Phosphatidic acid (Section 4.1.1.1, Reaction 2 ) , DAG (Reaction 3) [414] and GalDAG (Section 4.1.2.1, Reaction 20) are synthesized in the chloroplast envelope [504]. The membrane glycolipids are subsequently exported unidirectionally into the thylakoids [356]. Phospholipid (PtdGro) is imported directly from endoplasmic reticulum to thylakoid (c.f., Section 4.1.1) [356]. From labeling experiments with whole leaves [404,421,422]or Chlorellu vulgaris [391], it was predicted that GalDAG normally synthesized from unsaturated 1,2-DAG derived from microsomal Ptd-choline. It has been suggested that Ptd-choline may be a substrate for oleate desaturation in endoplasmic reticulum [422,492] on evidence from algae [391]
163 (Section 4.1.1.3): 18 : 1-Ptd-choline + 18 : 2-Ptd-choline
(29)
a-Linoleic acid (a-Lnn), thus formed, is then exchanged with the fatty acid of GalDAG (Reaction l l ) , further desaturated to 18 : 3/16 : 0 or 18 : 3/18 : 3 species [314], and is in turn galactosylated as follows: 18 : 3/16:0-GaIDAG
+ UDP-Gal
-+
18 : 3/16:O-Ga12 DAG + UDP
(30)
as demonstrated in green-algae [401] and broad bean [404]. These ideas have now been extended so that Ptd-choline in endoplasmic reticulum is proposed, not only as the site of desaturation, but also as a donor of linoleic acid to the galactolipids of the chloroplast [421], shuttling long-chain polyunsaturated fatty acids between cellular compartments. Siebertz et al. [335], however, indicated that the envelope system, in vitro, could not use 18: 3/16:0 species of GalDAG as the acceptor of Gal to synthesize Gal,DAG. Also, the examination of the labeling pattern of ['4C]lipids from isolated envelope and thylakoid suggested that Ptd-choline could not function as the acyl donor in galactolipid biosynthesis (356,5041. Anyway, the acquisition of DAGs containing highly unsaturated fatty acids from the cytoplasm (host) by the chloroplast (symbiont) would be a major evolutionary advantage [404]. 4.1.7.3. Further desaturation The major process of desaturation of 18 : 1-GalDAG to 18 : 2-GalDAG in the photosynthetic lamellae of cyanobacteria [316] has now been shown also in higher plants. Roughan [314] suggested that 18 : 1/16 : O-GalDAG was a substrate for desaturase in the envelope of chloroplasts: 18: 1/16: O-GalDAG'+ 18 : 2/16: O-GalDAG
(31 1
In maize leaves [422] and spinach chloroplasts [315], this molecular species was further desaturated : 18 : 2/16:0-GalDAG
+
18 : 3/16:0-GalDAG
(32)
and in spinach chloroplast [335], and in the chloroplast of 1 6 : 3 plants [142], to 18 : 3/16 : 3 species: 18: 3/16:0-GalDAG
+
18: 3/16 : 3-GalDAG
(33)
On the other hand, the 16:O fatty acid on position-2 of glycerol of 18: 1/16:0GalDAG can be also desaturated [314]: 18 : 1/16 : O-GalDAG
-
18 : 1/16 : 3-GalDAG
(34)
The fatty acid labeling pattern of SQ-DAG was discussed by Harwood [195]. On the other hand, the pulse-labeling studies with a cyanobacteria showed that
164 the primary products of lipid biosynthesis were the 18 : 0/16 : 0 species of GlcDAG, PtdGro and SQ-DAG [499]. In GlcDAG, 18 : 0 was desaturated rapidly to oleic acid and further to linoleic acid, whereas the 16:O acid was hardly desaturated. The 18 : 0/16 : 0, 18 : 1/16 : 0 and 18 : 2/16 : 0 species of GlcDAG, thus formed, were in turn converted into GalDAGs [499]. In conclusion, fatty acid skeletons, built up de novo in the chloroplast [142], are exported to the endoplasmic reticulum in the form of fatty acyl-CoA (e.g., oleoylCoA), and are associated with Ptd-choline for further desaturation [419], after which they migrate back to the chloroplast envelope (e.g., as linoleoyl Ptd-choline) [356]. Now partially unsaturated, they are incorporated into GalDAG by exchange, exported to thylakoid, and finally polyunsaturated (e.g., to linolenoyl GalDAG) while they are in ester linkage in galactolipids. In cyanobacteria, which lack organized mitochondria and endoplasmic reticulum, photosynthetic lamellae are probably the only major site of fatty acid and lipid accumulation. 4.1.8. In oivo biosynthesis in animal 4.1.8.1. Central nervous system The intraperitoneal[329], and/or intracerebral [71] administration of [ 35S]sulfuricacid-labeled sulfo-GalGroL of rat brain has already been described (Section 3.3.3). The formation of [ '5S]sulfo-GalGroL in brain parallels the rate of myelin formation until the peak of myelination, which is around 14 days in mouse [477,540] and about 18 and 20 days of age in Wistar [lo21 and in Sprague-Dawley strain rats [ 1251, respectively. At the age of maximal incorporation, the radioactivity in sulfo-GalGroL is 17-20% of the total sulfo-glycolipid [102,124], in agreement with the value reported for cultured mouse brain cells (15520%)[192] and mouse brain [540]. This incorporation rate is very high, because the concentration of sulfo-GalCer in whole brain reaches 4% of brain dry weights [126] (c.f., Section 3.3.2). The rate of incorporation per brain, or per gram of rat brain, however, declined after 48 days of age [102,301]. In 300 g rat, it was remarkably low [75,417], which is also reflected in the tissue level. At 67 days, only 6.3% of total sulfolipid was sulfo-GalGroL, and at 10 months it could no longer be detected [301]. Myelin was isolated from the cerebrum of 31- and 52-day-old rats, previously injected with ["S]sulfuric acid at 16 days of age. The specific radioactivities in myelin on protein basis increased from 13.6 to 17.8-fold those of whole brain. The incorporation into sulfo-GalGroLs was 11.2 and 9.3% of total sulfolipid at 31 and 52 days, respectively. These values are significantly lower than the value with the whole brain at 18 days (17-20%), suggesting that the turnover rate of sulfo-GalGroLs is faster than that of sulfo-GalCer [131,365] (for the function c.f., Section 5.2.2). Also, in actively myelinating mouse brain, the newly synthesized sulfo-GalGroL showed a very large turnover rate, 40-8054; being degraded within 1 day [540]. Thus. the turnover behavior of sulfo-GalGroL in myelin is in good contrast to that of sulfo-GalCer of myelin or sulfo-GalAAG (seminolipid) of germ cell, which are quite inert components of these tissues [197]. 4.1.8.2. Germ cell The incorporation of intratesticularly injected [ ''C]galactose
165 into lipids showed an interesting time course [56]. ['4C]galactose was rapidly incorporated into GalP1-3AAG (the peak of specific activity was 2 h). The sulfated form of this glycolipid, however, reached a peak specific activity at 72 h. Although ejaculated spermatozoa of boar did not incorporate [ 35S]sulfate into lipid fraction [45], incorporation of [14C]fatty acids into the lipids of bovine epididymal spermatozoa was observed [253]. Previously, endogenous fatty acids, derived from plasmenic acids, were shown to support the respiration of bovine spermatozoa in the absence of glycolytic substrates by Hartree and Mann [390]. I t was notable that DAG contained myristic acid (1,2-DAG, 14 : 0 = 708, 16 : 0 = 23%) as the major component, and this acid was also prominent in phosphatidylinositol (14 : 0 = 14.496, 16 : 0 = 43.7%) [118]. It is well known among radiologists, that the testis is one of the main biological targets of sulfate. It is now established that seminolipid (sulfo-GalAAG) is the compound which incorporates [ 35S]sulfate [370,371]. Intraperitoneal [70] and intratesticular [46] administration of [ "S]sulfate to rat and mice, respectively, resulted in the labeling of single sulfolipid (seminolipid) [417]. The plateau of radioactivity of [ '5S]sulfuric acid in seminolipid was reached after 24 h in 6-week-old mice [70,131]. Studies with slices of guinea pig testis gave similar results [122]. The quantity of label incorporated into the lipid (lower phase of Folch) fraction of testis was more than 85% [197] of the radioactivities which remained in the testes. Suzuki et al. [122] and Selivonchick et al. [253] showed that spermatozoa (ejaculated or epididymal) possessed little or no capacity to incorporate [ "S]sulfuric acid or [2-'4C]glycerol into seminolipid. Studies on the incorporation of [ 'SS]sulfuric acid into the isolated late spermatocytes indicated that late spermatocytes were not the primary site of synthesis of seminolipid [46]. It was postulated that the early spermatocyte is the last cell species in which the last stage of seminolipid synthesis (sulfation, Reaction 25), took place [369,370]. Seminolipid is sulfated at a spermatocyte cell stage prior to the late (pachytene or diplotene) spermatocyte stage. More precisely, early pachytene spermatocytes were the last cells to synthesize seminolipid [370]. The ' 5 S disappearance rate (TI,,, 24.5 days) was identical to cell death (disappearance of [3H]thymidine, T,,,, 24.4 days). It was suggested that in the germ cells, surviving at the end of 5 weeks after the pulse labelling, seminolipid was completely stable [I971 (c.f., Section 3.4.3). Mouse embryos were cultured from the 2-cell stage to the morula stage in a medium containing [U-'4C]glucose. The major glycolipids synthesized were cerebrosides; however, evidence was obtained for the presence of a GGroL behaving similar to GalDAG on TLC [423]. 4.1.8.3. Other tissues The analytical data on parotid and submandibular gland for the presence of sulfated and non-sulfated isomaltose-series AAGs [50] appear to be consistent with the results of earlier studies on the biosynthesis of carbohydrate-containing substances in the salivary glands of mice (4241, in which stimulation with isoproterenol (a P-adrenergic agonist) increased the synthesis of glycolipid of GGroL type. Also, studies on sulfolipid formation [129] in rat submandibular glands, and demonstration of a sulfotransferase activity to form SO, H-6(Glcal-6),Glcal-3( 1)-
166 AAG [516], suggested the presence of a sulfotransferase catalyzing the transfer of labeled sulfate from PAPS to an endogenous lipid acceptor (Section 4.1.3.2). The intraperitoneal injection of [ 35S]sulfuric acid, and examination of tissue lipids by thin layer autoradiography, showed that the intestine appeared to contain at least several glycerol-type sulfolipids, but the stomach did not [417]. 4.2. BIODEGRADATION
Enzymatic degradation of GGroLs occurs in a stepwise manner by the removal of carbohydrates, sulfate, etc., from the nonreducing termini. Participation of endoglycosidase is not known. 4.2.I . Glycosidase The origin and properties of glycosidases from various sources have been extensively reviewed by Li [425], and those from plants by Sastry [13]. In eukaryotic cells these enzymes are contained in lysosomes. Acetone powder or lyophilized “microsomal fraction” from rat brain was used for the assay of GalPl-3DAG-~-galactosidase[226,227]. Microsomal and crude mitochondria1 preparations, which contained intact lysosomes, were used as a source of enzyme for the catabolism of [U-’4C]GalPl-3DAG in assays carried out at various pH values. The release of water-soluble radioactivity from the GalPDAG was observed to occur optimally at pH 4.4 (major activity) and 6.9 (smaller activity) with lysosome [227,367].The P-galactosidase activity toward GalDAG was generally higher in enzyme preparation of younger rats [227]. Krabbe’s disease (galactosyl leukodystrophy) is a rare genetic disorder of the central nervous system arising from a deficiency in lysosomal B-galactosidase specific to GalCer. The activity of GalDAG-0-galactosidase was extremely low in brain, liver and skin fibroblasts from patients who died of Krabbe’s disease [426]. Further studies on this enzyme in control brain tissue demonstrated that GalDAG is a potent competitive inhibitor of GalCer-P-galactosidase and that GalPDAG-Pgalactosidase is competitively inhibited by both GalCer and psychosine (GalPl-1sphingosine). This inhibition was not observed with LacCer, ganglioside I13NeuAcGg4Cer, GlcCer or 4-methylumbeliferyl-P-galactoside.The activity was normal in the liver of a patient with generalized gangliosidosis and also in the brain of Jimpy mice [191]. 4.2.2. Sulfatase
Farooqui [57.419.427], Dulaney and Moser [54a], Kolodney and Moser [54b], Radin
[533] and Mulder [546] recently reviwed the nature of various sulfatases from animals. Sulfatases catalyze the hydrolysis of sulfate esters according to the following scheme: R-0-SO;
+ H *O
+
R-OH + SO:
~
.
(35)
167 It was proved that seminolipid (sulfo-GalAAG) (Fig. 2.la) is another physiological substrate of arylsulfatase A (EC 3.1.6.1), in addition to sulfo-GalCer, sulfo-LacCer and ascorbic acid sulfate, using the enzyme preparation from boar testis [372], human urine [429] and human placenta [57,427]. The kinetic parameters for the hydrolysis of these sulfate esters were identical for sulfo-GalCer, sulfo-GalAAG and galactosylsphingosine sulfate, showing that arylsulfatase A recognizes the 3-sulfate ester of P-galactopyranoside residue. Further, both sulfo-GalAAG and psychosine sulfate competitively inhibited the hydrolysis of sulfo-GalCer with a Ki value similar to the K , value of sulfo-GalCer. This observation suggested that the above substrates occupy the same site on the enzyme, and that the chemical nature of the lipophilic domain is not essential [57,429]. Hatanaka et al. [329] purified an arylsulfatase from the liver of a mollusc (marine gastropod), Churoniu lumpus and could separate it from another sulfatase, which was formerly known as “glycosulfatase” (EC 3.1.6.3) and which had been believed to cleave sulfate esters at position-6 of glucose. The preparation of arylsulfatase could hydrolyze the sulfates of GalCer, GalAAG and ascorbate. This substrate specificity is similar to that of mammalian arylsulfatase. However, “glycosulfatase” could not cleave the sulfate ester of sulfo-GalAAG or sulfo-GalCer. Now the nomenclature of glycosulfatases must be revised, because, in spite of its name, “glycosulfatase” is inactive on natural sulfo-GGroLs (3291. Also, the Churoniu lumpus enzyme cannot be classified together with mammalian arylsulfatases because the former enzyme is not inhibited by inorganic sulfate [57,546]. Sulfatidosis (metachromatic leukodystrophy) is an autosomal recessive, lysosomal deficiency of arylsulfatase A [54a,b]. Histologically, metachromasia of accumulated sulfolipids with staining by Azure A or toluidine blue is observed. In the brain of two cases of a late infantile form of sulfatidosis, the activities of arylsulfatase A to paranitrocatechol sulfate, sulfo-GalCer and sulfo-GalAAG were markedly low (1-58 of control activities) (4281. It has not yet been determined whether sulfo-GalAAG can accumulate in the testes of adults with late developing forms of sulfatidosis [56]. Nor has it been established whether sulfo-GalAAG and sulfo-GalDAG can accumulate in human brain in this condition; two studies using the brain from the infantile form of this disease failed to demonstrate their presence [71,102]. The hydrolysis of sulfo-GalCer by arylsulfatase A (sulfatidase) in buffers of physiological ionic concentrations requires detergents or a specific lysosomal activator protein [303]. As shown with sulfo-GalCer, mixed micelles of detergent and lipid substrate or activator-lipid substrate complexes are the “true substrates” for the enzyme under these conditions (Section 5.1.5). Lysoseminolipid was hydrolyzed even in the absence of activator protein or detergents. Micelles of “lysoseminolipid” itself are the “substrate” for sulfatase A. The estimated critical micellar concentration value was below 0.1 mM [303]. The small activating effect for lysoseminolipid by the activator may be explained by the formation of activator-lipid complexes which are more suitable as a substrate for the enzyme than are pure lysoseminolipid micelles [303] (Section 5.1.5). Myelin GalDAG [69] and sulfo-GalGroL, especially the diacyl form [71,301], are
168 rapidly degraded after 25 days of age, probably by the action of both arylsulfatase A and lipases. The ratio of DAG/AAG forms of sulfo-GalGroL in rat brain is about 1 : 1 until 22 days of age, then it declines to 1 : 6 at 68 days of age, and 1 : 13 at 175 days [301] (c.f., Section 3.3.4). Most probably the DAG form of sulfo-GalGroL is more susceptible to lipase, resulting in a higher turnover rate [125] (Section 4.2.3). In mouse cerebellum, the biodegradation of sulfo-GalGroLs was low (ca. 20 nmol/g in 24 h) at the 6th day, rose to a peak (ca. 130 nmol/g) at 14 days of age, and then decreased again to low values at 20 days [540]. The rapid turnover, especially of GalPDAG and sulfo-GalpDAG in adult rat brain is in sharp contrast to other components of myelin, glycolipids of other types of nervous system cells (glia and neuronal cells), and sulfo-GalGroL of epididymal spermatozoa [253]. In chcken brain and retina, the activities of arylsulfatase A or sulfatidase rise sharply during embryonic life, reach a peak at the 18th day of embryonic age, and drop slowly after hatching. It is interesting to note that the developmental profile of PAPS-GalCer sulfotransferase is more or less similar to the developmental curve of sulfatidase in chicken brain and retina. Both enzymes showed the highest activity around the hatching period [256] (Sections 3.3.3, 4.1.3.3). 4.2.3. Lipase Homogenization of plant tissues triggers various enzymatic alterations of lipids [13,205]. In potato tubers, the enzyme is so active that most of the endogenous membrane-bound polar lipids are destroyed immediately when the tissue is disrupted, even at O°C [396,430]. Loss of fatty acids results in deacylated phospho- and glycolipids [333,430]. Acyl transfer reactions from Gal DAG to GalDAG cause the appearance of acyl GalDAG (Section 4.1.1.4, Reaction 12). Trans-phosphatidylation can result in various new phospholipids, such as Ptd methanol, which are not observed in intact cells [205] (Section 4.1.1 S ) . According to Galliard [396,430], the classification of the lipase (acyl hydrolase) of plants acting on polar lipids is in confusion. Depending on the substrates chosen for assays, various acyl hydrolase activities in plant tissues have been described as phospholipases, galactolipases, monoglyceride lipases, lipases and esterases. Further, a given lipolytic enzyme will show marked differences in properties and specificity under different assay conditions. By analogy to the degradation of phospholipids, Benson [72] postulated that the acyl esters of 6-sulfoquinovosyldiacylglycerol(SQ-DAG) in chlorella were cleaved by “sulfolipases” in a stepwise manner, first from position-2 and then from position-1 of glycerol, although the enzyme was not purified. The enzyme “sulfolipase” [195] was later shown to display broader specificity for other polar lipids. For example, this enzyme can also hydrolyze phospholipids, at least to the lyso-phospholipid level (i.e., phospholipase A,-type activity) [430]. Physiological degradation of GGroLs occurs most actively during germination of seeds and senescence (Section 3.1.3). Sastry and Kates I3331 presented the scheme of removal of fatty acids from GalPDAG catalyzed by an enzyme preparation from
169 broad bean. First, two fatty acids are removed, then Gal is removed to yield glycerol. Although this hypothetical pathway was based on the results obtained with the homogenates of leaves, it was confirmed later by Helmsing [431] using spinach leaves. The enzyme from Phaseolus (runner bean) leaves and a known galactolipase (EC 3.1.1.26) show a higher specific activity, with the lysophospholipids and monoacylglycerols as substrates. These enzymes are probably identical to non-specific lypolytic acyl hydrolase [ 396,4301, but are distinct from triacylglycerol lipase. The variation in both 1-acyl and 2-acyl galactolipase activities during storage of the enzyme preparation at 4OC, and the pH optima of the reactions for the GalDAG and Gal,DAG, were observed. Of a range of surface-active agents studied, unsaturated fatty acids were the most active [396]. Lysoglycoglycerolipids were detected in rice bran [91,296] or in mammalian testes [45,47], suggesting the presence of the activities of such lipases in these tissues. Reiter et al. [295] found that secondary lysosomes from rat liver contained not only arylsulfatase A, but also a lipase activity that could act to deacylate at position-2 of the glycerol moiety of sulfo-GalAAG. Under the conditions used, more product was formed by the action of the lipase on sulfo-GalAAG than by the action of arylsulfatase A. Although an enzyme activity hydrolyzing 0-alkylglycerol to glycerol and fatty aldehyde was present in rat liver microsome [432], no studies have been performed on the degradation of alkylglycerol of glycolipids. The activity of the lipase of rat brain microsome plotted against age revealed that the galactolipase activity, with either GalDAG or Gal,DAG as substrate, increased with age (up to at least 40 days), except during the period of most active myelination (approximately 10-20 days), at which time the galactolipase activity is notably depressed [227]. This may explain the rapid turnover in vivo of GalDAG and sulfo-GalDAG (Section 3.3.4). In Streptococcus faecium and group A streptococci, cell-bound lipase, which deacylates LTA and causes the excretion of the deacylated LTA to the culture fluid, was recently discovered [130] (c.f., Section 3.2.4). Because many other species excrete fully acylated LTA, the physiological role of this energy-wasting mechanism remains to be elucidated (W. Fischer, personal communication).
5. Biological property Many attempts to correlate the chemical structure and physical properties of GGroLs of cell membrane, ranging from the plasma (protoplast) membrane of procaryotes to the plasma (cytoplasmic) membrane of mammalian cells, have been performed. This section includes examples of such challenges by biochemists to elucidate the major functions of GGroLs. Efforts are made to assess the hypotheses in relation to the earlier works, and also to cross-reference the valuable information scattered in publications within the various branches of lipid or glycoconjugate biochemistry.
170 5.I. BIOPHYSICAL PROPERTY
5. I . I . Lipophilic domain The chemical groups specific to sphingolipids, as far as the intermolecular interactions among lipophilic groups are concerned, are the amide linkages. The ceramide portion of glycosphingolipids anchors them in the membrane. The corresponding region in the molecule of the typical GGroLs is the ester carbonyl region of DAG (Section 2.2). According to Karlsson [250,289], the bilayer region of the surface membrane of the cell can be divided into three layers: the lipophilic zone, intermediate zone (hydrogen belt) and polar head group [352]. In bilayer conformation, the glycerol “core” of, e.g., Ptd-choline, which forms the intermediate zone, is the most constrained region, whereas the mobility increases towards the terminal methyl of fatty acids as well as towards the quaternary amine group of choline residue [362]. The intermediate zone is distinctly different in sphingolipid and glycerolipid. In the latter, only acceptors of hydrogen bonding exist, while sphingolipid has both acceptors and donors [250,289]. Thus, the membrane consisting of sphingolipid forms a more rigid hydrophobic surface [378]. The hydroxyl groups (C3 and C4 hydroxyls of sphingoid and 2-hydroxyl of fatty acid), plus the amide moiety of sphingolipids, can form an extensive stabilizing hydrogen-bonding network [ 142,3781. Liposomes of sphingolipids are more ordered, less permeable, and have higher transition temperatures than those of phosphoglycerolipids, and indeed are in the gel state at physiological temperatures. This may be partly due to the greater length and saturation of the fatty acids generally found in sphingolipids compared with those in glycerophospholipids [378]. However, at least in erythrocyte membrane of ruminants and in endoplasmic reticulum of bovine liver, Ptd-choline (choline-P-DAG) is completely substituted by sphingomyelin (choline-P-ceramide) [376] (for an example of reversed substitution see Section 3.3.1). 5.I . 2. Micelles Amphiphdic lipids take different conformations in aqueous solutions: water-soluble micelles and liposomes. For instance gangliosides exist as hexagonally packed rod-like structures over the hydration range of 18-56%. Liposomes take either rod-like (hexagonal) form or lamellar structures, in which the molecules are arranged in bimolecular layers: an L,, high temperature, liquid form; and an L, form at lower temperature. These lamellae are separated by thin water layers. The application of [ 3’P]NMR, X-ray and freeze-fracturing techniques to fully hydrated preparation of individual membrane lipids, has shown that, as a rule, either the hexagonal ( H , , ) phase or the bilayer phase is preferred. Important examples of H,, phase lipids include Ptd-ethanolamines, GalDAG [349,395], as well as phosphatidic acid and diPtdGro in the presence of Ca2+. In addition, lipids such as cholesterol and unsaturated fatty acid can induce the formation of hexagonal phases from bilayer systems [379]. Shipley et al. [318] have concluded that GalDAGs, similar to GlcDAG from acholeplasma [349,395]. form, in their fully hydrated forms, a hexagonal (H,, )
171 type structure [538] in which about 20% (w/w) of water is incorporated into the lipid matrix in the form of cylinders of 3 nm diameter. Ga1,DAG [416], on the other hand, appears to form a lamellar structure in water over a wide concentration and temperature range. Also, GlcDAG of acholeplasma, which forms hexagonal phase in an excess of water, forms only a lamellar structure in the presence of an equimolar quantity of egg Ptd-choline at 4OC, as shown by [” PINMR, due to the bilayer-stabilizing ability of Ptd-choline. Further, when the temperature is elevated above 60 O C, virtually all Ptd-choline molecules undergo nearly isotropic (intermediate between bilayer and H l l ) motion, as observed by NMR. Freeze-fracture electron microscopy showed string-wise organized particles and pits (60-120 A in diameter) [395]. The particle size was about 55 A in diameter. The hexagonal structure of GlcDAG here is considered to be of the reversed H I , type [435]. Similar phase appearances, i.e., reversed hexagonal and lamellar, were observed for the GalDAG and Ga1,DAG from Pelargonium (geranium) leaves [318]. These systems possibly consist of a honeycomb network of vesicles, in which the lipidic particles are located on the nexus of intersecting bilayers [379]. In methanol, polar lipids exist in a monomeric form and tumble freely in solution (3621. Evidence for the formation of micelle of GroP-Glc,DAG in C’HCI, and Ptd-sulfocholine has been obtained by NMR spectroscopy [224,257]. Such behavior of polar lipids to form “inverted micelles” in the lipophilic environment has actually been demonstrated in lipid vesicle systems and biological membranes. 5.1.3. Unsaturation of lipophilic domain
[‘jC]NMR of GalDAG with saturated and unsaturated fatty acids showed that the mobilities of fatty acids increased with the introduction of a double bond. This increase in the rate of motion only occurs at carbon atoms beyond the first double bond in an acyl chain [437]. In agreement with the earlier report of Oldani et al. [438], it was found that monomolecular films of unsaturated GGroLs, which are the major lipid components of mycoplasmal and thylakoid membrane, closely resembled to those of unsaturated phospholipids, the major component of mammalian plasma and mitochondria1 membrane. Actually, it was reported that unsaturated GalDAG, but not saturated GalDAG, was required for the full activity of the CF,-CF,-ATP synthetase system of the thylakoids [538]. The transition temperature for highly unsaturated Ga1,DAG and GalDAG from spinach, is -50 and -30°C, respectively [439,440]. In contrast to GalDAGs, both the saturated and unsaturated Ga1,DAGs were shown to behave in a manner typical of Ptd-choline. Both of them formed closed lamellar structures [416] in aqueous systems. Also, the temperature at whch the distearoyl derivative undergoes a phase transition (about 51O C) is similar to the value of 54.2OC reported for distearoyl Ptd-choline [416]. The contribution of the uncharged glycolipids to the properties of the membrane surface [349] in A. laidluwii, a mycoplasma devoid of cell wall, has also been studied (Section 5.4). By the use of pure molecular species of GalDAG and Ga1,DAG for the studies with X-ray diffraction and freeze-fracture electron microscopy, GalDAG was shown
172 to form a hexagonal-type structure when unsaturated [416]. In contrast, the saturated glycolipid was arranged in a lamellar configuration. Thus, the phase behavior of GalDAG depends critically on the degree of unsaturation and saturation [538]. The biological rationale for the diversity of unsaturation in GalDAG molecules still remains unclear [437]. 5. I . 4. Electric charge
Lipids such as Ptd-inositol and the glycolipids form strong dipole-dipole interactions with water ihrough the numerous hydroxyl groups of carbohydrate residues. However, the polar interaction of most membrane lipids is dominated by the presence of an ionizable group giving rise to charge-dipole interactions with water molecules [376]. Among the lipids of the membrane of A. laidlawii, the charged lipids such as PtdGro and GroP-Glc, DAG, appeared to have larger hydration capacities than the neutral glycolipids such as GlcDAG and Glc,DAG [349]. Interestingly, there is no variation in hydration between the GlcDAG and Glc,DAG of A. laidlawii. Gulik-krzywicki [497] showed, by incorporating varying amounts of ionizable lipids in non-ionizable lipids, that the hydration behavior of liquid-water phases may fall between the extremes of systems, such as the “infinite swelling” Ptd-serine. and the systems which reach a limiting hydration level (6.9-10.1 mol/mol lipid for Glc, DAG) [ 3491. 51.5. Seminolipid
Fischer et al. [303] studied the state of lysoseminolipid (deacylated seminolipid) of mammalian germ cell in aqueous solution. It was shown that lysoseminolipid [122] penetrated an acrylamide gel gradient of up to 15% acrylamide concentration [303]. In contrast to lysoseminolipid, seminolipid, like sulfo-GalCer, barely penetrated the lowest acrylamide concentration. Also, sedimentation velocity measurement with sulfo-GalCer demonstrated the formation of large aggregates [303]. In the presence of activator protein of lysosomal membrane, however, a part of the seminolipid migrated together with the ‘activator’ near the front of the gel [303]. This had been already shown for sulfo-GalCer, which is transported by the activator in a one-to-one ratio (Section 4.2.2). With the methods described above, the critical micellar concentration of seminolipid was estimated to be about 1-4 X lo-’ M [303]. In contrast to its acylated (intact) form, therefore, lysoseminolipid formed apparently smaller micelles, comparable in size to the mixed micelles of taurodeoxycholate and sulfatides or the activator-lipid complexes. Such alignment of sulfolipids can be “substrates” for sulfatase (see Section 4.2.2). 5.1.6. Macroglycolipid
Wicken and Knox [39] observed that LTA was eluted earlier than nucleic acids from the column of Sephadex or 6% agarose. They assumed the micelle form of LTA analogous to lipopolysaccharide (LPS) or capsular macroglycolipid of gram-negative bacteria [39,251]. The solution of S. faecalis LTA, which had, e.g., 4 : 1 ratio of fatty acids to poly(g1ycerophosphate) chain in citrate buffered saline (pH 7.1). behaved
173 like a large molecule on centrifugation [282].It gave sedimentation coefficients 11.4and 14.1 s depending on the concentrations (0.2-1.0%),indicating micelle formation. However, only one peak of 2.0 s was observed in the presence of 1.5% SDS. That the glycolipid moiety survived periodate oxidation also supported the rricelle structure hypothesis [141]. Regarding the indispensability of a nonspecific lipophdic region for the activity of ribitol phosphate acceptor (Section 4.1.5,Reaction 28), it was speculated that ribitolphosphate transferase binds to LTA, which is then inserted into the micelles of Triton X-100. The concentration of Triton in the reaction medium was 0.1% (4.6-fold above critical micellar concentration) (Section 4.1.5.2). The partially deacylated LTA molecule (0.7 s ) was no longer capable of micelle formation [282].This was also confirmed by comparing the elution profile on a column of Sepharose 6B [158]or agarose [448].The hydrophobic (lipophilic) region of LTA may serve to keep the carbohydrate chain in an appropriate orientation for binding with ribitol phosphate at the lipophilic/hydrophilic boundary. Also, the LPS from Mycoplasma acidocaldarius, a glyceryldiether lipid with 24 mannose and 1 glucose (Table 2.1.6(1)) is highly aggregated (1200000 Da)) in aqueous solution. In the detergents, cetyltrimethylammonium bromide and SDS (0.5%), t h s LPS exhibited permeation chromatographic behavior indicative of a molecular weight of approximately 67 000, corresponding to 10-12 subunits [98]. On the contrary, trimethylsilylated LPS did not aggregate and behaved as the monomer in chloroform. This LPS appeared to be a more choice selection for specific antigens than the smaller GGroLs [442].The fine structure of LPS from Thermoplasma acidophilum, examined by electron microscopy, revealed long, ribbon-like structures with some branching. Treatment of the LPSs with 0.5% SDS resulted in the dissociation of the ribbon-like structures, suggesting that the LPS of this organism is morphologically similar to macroamphphiles isolated from gram-negative bacteria [ 351,4431. ( s ~ ~between . ~ )
5.2. MEMBRANE AS M A T R I X
Current models of biological membranes postulate a bilayer organization of the lipid components with the possible exception of those in some archaebacteria (Fig. 2.4). Complex lipids provide a fluid medium for proteins forming an amphiphilic compartment [379].Clustering of some special lipids into specific domains, especially around a functional protein (boundary lipid), affects the kinetic properties of enzyme and receptors. 5.2.1. Integration of membrane The thylakoid of chloroplast is a membrane extremely rich in polar lipids reaching about 40% of dry weight (Section 3.1.2). If the protein particle consists of four proteins, and it requires lipid molecules to surround each protein completely, about 25% of the thylakoid lipid will be in contact with protein [440]. Also lipid-protein associations, in the chromatophore of cyanobacteria, immobilize about 60% of the
174 negatively charged lipids [490]. On the other region of the same membrane, a high rate of lateral lipid diffusion, in the order of 3 pm/s or 5 x l o - ” cm2/s, is also taking place. Space-filling models of I-linolenoyl-2-palmitoyl SQ-DAG showed very good potential interactions (both ionic and hydrophobic) with the chlorophyll model [195]. According to the physical properties of plant GGroLs, it is suggested that GalDAG, Ga1,DAG and SQ-DAG may play similar structural roles in plant thylakoid membranes to Ptd-ethanolamine, Ptd-choline and Ptd-serine, respectively, of eukaryotic organelles. The latter three phospholipids are the major lipid components of animal cell membranes [318] (Section 5.1.3). More polar GGroLs of plant, such as GalMAG and Gal,MAG, are believed to be included in the amylose structure of amyloplasts, and thus constitute internal lipids [77,296,444]. Partially purified rice starch contains single carbon-chain lipids (and some non-monoacyl lipids). These probably interact with protein outside the amylose structure and might be called “external” lipids. I t has been established that glycosylDAGs are components of the cell membrane of gram-positive bacteria (Section 3.2.2). The loss of the cell wall, i.e., the inability to synthesize a rigid cell wall, has been reported to affect the lipid composition of the membrane (Section 3.2.4). In two strains, Streptococcus pyogenes [337] and Staphylococcus aureus [347], the contents of Glc,DAG, the total lipid and cis-vaccenic acid in the membrane of corresponding L-forms were 2-3-times higher than those in wild type membrane. The content of phospholipids was correspondingly lower in the L-forms. In these reports it was discussed whether the membrane of L-form is stronger than that of wild type against the osmotic pressure of the medium or treatment with sonication, and whether the loss of cell wall is compensated in part by the increase in glycosylDAG. However, Kanemasa et al. [436] showed that in the autoplast of S. aureus 209P, the increase of diPtdGro is rather important as the complementary compound to the lost cell wall. The control mechanism regulating membrane fluidity was reviewed in detail [351]. Anyway, when a part of the membrane lipids of mycoplasma is in the fluid phase, the elasticity of the cell membrane enables the cells to swell and behave as a good osmometer. However, when membrane lipids are in the gel phase, the cells are unable to swell and will lyse in a slightly hypotonic medium [351]. Earlier, it was postulated that the triglycosyldiether glycolipid sulfate (Table 2.2.1(4)) was necessary for formation of stable bilayer of Halobacterium cutirubrum polar lipids, probably as a result of its large carbohydrate head group [112]. Later this major glycolipid was found to be replaced by nonsulfated triglycosyldiether in H. marismortui (2451. The absence of sulfo-GGroL in H. marismortui appeared to be compensated by two polar lipids: sulfo-PtdGro to maintain a high surface negative charge (as in mammalian transporting epithelia [127]) and the triglycosyldiether lipid to provide a large carbohydrate head-group. In the membrane of methanogenic archaebacteria, the molecular size and the asymmetric structure of two glycophospholipids with dibiphytanylglycerylethers (Sections 2.2.2, 3.2.1.2) (Table 2.2.3(9,10)) suggested that they may behave as covalently bonded, cross-linked (or sealed) bilayers and are the equivalent of an amphiphilic lipid “monolayer” [517]. This
175 possibility was first suggested by Langworthy [239,299,331]for the lipids of thermoacidophilic archaebacteria. It was postulated that this rigid cyclic diether structure was required to overcome the membrane-disrupting effect of high concentrations of methane [271]. 5.2.2. Ion trap LTAs are acidic amphiphiles possessing poly(glycero1)chains linked by phosphodiesters (Section 2.1.2.4). It is believed that this polyanionic character permits the bacteria to maintain a high concentration of divalent ions, especially of Mg2+,in the region of the plasma membrane and cell surface [40,453]. This activity is similar to that with the LPS of gram-negative bacteria or sulfo- and sialo-glycoconjugates in mammalian cell surface [127]. 10 mM or more of Mg2+ ions are needed to maintain the integrity of the plasma membrane of animals. Mg2+ is also required in fairly high concentration and in bound form for the activity of enzymes involved in the synthesis of bacterial cell wall compounds [454]. LTAs are probably largely responsible for the net negative charge (Zeta potential) on most gram-positive bacteria [31,40,455].Disaggregation of the cell wall and death of the EDTA-sensitive Pseudomonas sp., on treatment with EDTA, could be caused by the removal of such bridging ions (1071. The reduction of LTA synthesis to 70%of control in B. subtilis resulted in a slight inhibition of cell growth [409]. Other interesting activities of LTA: binding to eukaryotic cells and complements, mitogenicity to lymphocytes and macrophage, stimulation of osteolysis, and production of Schwarzman reaction in rabbits, were reviewed by Wicken and Knox [41]. Van Deenen's group found that the concentration of lysyl PtdGro and GlcNPtdGro in Bacillus megaterium increased to about 30%of total phospholipids when the cells were cultured in a medium of pH 5.0 [34]. These two phospholipids were not detectable when the bacterium was cultured at pH 7.0. These authors suggested that the concentrations of these lipids are regulated to maintain the ion environment at the surface of the cell. However, Marinetti [452] found that GlcN-PtdGro of acholeplasma is located, together with the another amino-amphiphile, aminoacyl PtdGro, at the inner surface of the cytoplasmic membrane, rendering the above hypothesis less probable. Recently, Kakinuma et al. [513] compared the capacity of the activation of the oxygen radical-generating system of leukocytes by various sulfo-amphiphiles. The potassium salt of seminolipid or sulfo-GalCer had no effect on H,O, generation, in marked contrast to sodium or ammonium salts of sulfo-GalCer. Farooqui [475] suggested that the removal or alteration of terminal sulfate residue of seminolipid and steroids, as well as hydrolysis of glucuronic acid and sialic acid of glycoconjugates, may be an important step in the process of sperm capacitation. 5.3.INTERACTION WITH PROTEIN
The glycolipids from simple glycosylDAGs [450], in the membrane of chloroplast, to
176 most complex LTAs [39,41,339] of gram-positive bacteria, are recognized by their terminal sugar(s) as the immunodominant group [456]. 5.3.I . Bacterial antigens Serotype classifications have been frequently applied for the typing of bacteria including gram-positive microorganisms of the families Streptococcaceae and Lactobacillaceae. The specificities of the antigens have been believed to reside in the cell-surface carbohydrate chain of the complex glycoconjugates (Section 3.2.3). As lipopolysaccharides (LPS) are known to induce polyclonal B-cell activation with predominantly IgM humoral response, LTAs appear to be T-cell-dependent immunogens, and their humoral response gives rise to both IgM and IgG antibodies [41,3391. The group-specific antigen of the group D streptococci described by Lancefield in 1933, is now recognized as LTA [39,140,283]. Frequently, in gram-positive bacteria, the group-specific antigens are LTAs [339]. Although the poly(g1ycerophosphate) chains are themselves immunogenic, the group-specificity resides in the glycosyl substituents. a-D-Glc [286] and a-D-Gal of membrane LTA are the determinants in group A and F antigens, respectively, of lactobacilli. Glcal-2Glc, a-D-Gal and 8-D-Gal are group-specific determinants in group D and N streptococci and Streptococcus mutans, respectively [150,339]. The lipid anchor portion of LTA in group D streptococci (faecalis) is unique in that it contains a Ptd group (Glcal2(Ptd-6)Glcal-3DAG) [78]. The structure of the carbohydrate portion is that of a poly(GroP), to which are attached kojibiosyl (Glcal-2Glc-) or kojitriosyl groups in a-configuration and D-alanine residues [283]. The determinant group of S. lactis group N antigen, was found not to be the originally suggested galactose phosphate but a-D-Galp- residue [150]. In several lactobacilli including L. case;, the phospho-GGroL portion of membrane LTA shows antigenicity, whereas LTA without lipid anchor portion (lost by trichloroacetic acid extraction) does not [39,140].The strong modulating influence of alanine ester substitution on immunological properties was recognized earlier [285]. The extent to which a “membrane component” can act as a surface antigen will depend on mainly (a) wall thickness and density in terms of packing of the peptidoglycan, and (b) the length of LTA and its conformation within the wall [33]. The deacylated extracellularly excreted LTA of streptococci may provide a “sink” for antibodies against this surface polymer of pathogenic bacteria [360]. The group-specific meningococcal C-polysaccharide was recently characterized as a polysialosyl phosphatidic acid by methods including H F degradation [251]. The delipidated polysaccharides were shown to have a much smaller apparent molecular weight, but retained complete antigenicity. Smith [223] found that 8-gentiobiosylDAG is contained in Mycoplasma neurolyticurn. Since glycolipids appear to impart some degree of antigenic specificity to mycoplasmas [354], Smith concluded that GGroLs containing 8-gentiobiosyl residue is the basis for their serological distinction. The lipids and LPSs of five mycoplasmas were examined for complement-fixing activity to antimembrane rabbit sera [442].
177 The total glycolipid fractions and the aqueous phenol fractions (LPSs) from the membrane of Acholeplasma laidlawii, A . modicum, A . axanthum and M . neurolyticum exhibited significant antigenic activity. The pure glycolipids and phosphoglycolipids of the acholeplasma and mycoplasma species also exhibited significant complement-fixing activities. Acylation of the sugar residues, e.g., a-glucose acylated on C6, of these GGroLs reduced or negated complement-fixing activity. On the other hand, double cross-reactions between the GGroLs of A. laidlawii and A . modicum appeared to be due to the presence of both GlcDAG and Glcal-2GlcaDAG of identical structure. Also, the GroP-6Glcal-2Glca1-3DAG of A. Iaidlawii exlubited the ability to react in this system. Specificities of glycolipid structure were noted by the absence of cross-reactions between A . laidlawii and M . neurolyticum. The glycolipids of these two strains differ only in the nature of the glucose linkages, Glcal-2GIc (kojibiose) and GlcPl-6Glc (gentiobiose), respectively [442]. Studies using lectin binding and other methods agreed on the surface location of both GGroL and GroP-GGroLs of mycoplasmas [351]. 5.3.2. Immunogenicity of glycoglycerolipids A specific, complement-dependent glucose release from liposomes containing galactolipids was found with all antibody preparations produced by injecting several GGroLs in rabbit, although high titer antiserum against GalDAG was not obtained (451,456). The anti-Ga1,DAG was the only crude antiserum with an apparent high degree of specificity, and it did not cross-react with either GalCer or gangliosides. Inversely, the affinity-purified anti-GM1 rabbit serum did not cross-react with GalCer, Gb,Cer and Ga1,DAG [451,456](see aso Section 2.3.1). Rabbit antibody against GalP1-3DAG from treponema [185] cross-reacted with the mixture of galactolipids obtained from bovine spinal cord (the mixture of GalPl-3DAG, GalP1-3AAG and GalP1-ICer) [186]. It was speculated that some characteristics of these haptens and their antibodies are related to those of diPtdGro (cardiolipin), another lipid hapten common to treponema and human tissues [1861. The presence of a low titer natural antibody against GalAAG was demonstrated [460]. The sera obtained from patients with multiple sclerosis (a slow progressing demyelinating disease of the central nervous system) frequently contained antibodies against Galal-6Gal/31-3DAG [457]. Flynn and Hillman [423] examined the incorporation of ['4C]Glc into GGroLs of mice embryos (see also Section 4.1.8). Also, the synthesis of GalDAG and sulfoGalDAG was demonstrated in the culture of fetal mouse brain [192]. Moreover, NS-4 cell surface antigen which is specific for both brain and sperm cells, was present on pre-implantation mouse embryos [487]. Since GGroLs appear to be limited to the same tissues as the NS-4 antigen, a functional [423] or developmental [536] relationship may exist between these glycolipids. Glycolipids have been recognized generally as poor immunogens. Classical immunization by intramuscular injection of an emulsion in Freund's complete adjuvant, in the presence of a heterologous protein carrier, usually resulted in low titer
178 antibodies. Variable results were obtained from one animal to another when compared with protein immunogens. This may relate to the fact that the glycolipid is often normally present in the tissues of the immunized animals. Therefore the formation of antibody might be considered autoimmune [458]. Also, the wide-spread occurrence of natural antibodies [485] against simple glycolipids such as Gal DAG [457] causes confusion in analyzing specificities produced by experimental immunization. Previous studies on the immunogenicity of sulfo-GalCer have reported the production of high [459] and low [486] titer complement-fixing antibodies. Lingwood produced an antibody against seminolipid (4581 and located sulfo-GalAAG on the surface of rat germ cells [460]. However, the latter antibody was a basic IgG, which reacted non-specifically with acidic compounds at pH 7.2. Therefore, immunofluorescence studies to localize seminolipid on germ cell were performed at pH 8.6 to eliminate nonspecific staining [458]. Hakomori found that C-fixing anti-sulfoGalCer-antibodies could not be detected by Ouchterlony double diffusion [486]. The affinity purified anti-sulfo-GalAAG antibody, also could not be detected by this method. Lingwood et al. confirmed the finding by Alving and Richards [461] of complement-fixing activity against GalCer in the serum of unimmunized rabbits [458]. However, they could detect no C-fixing activity against sulfo-GalAAG in normal serum, probably because sulfo-GalAAG is immunologically isolated by the blood-testis barrier. The sulfo-GalAAG-liposome column demonstrated the affinity of the immune serum for sulfo-GalAAG [458]. Virtually all the complement-fixing activity was removed by the column, whereas no activity was bound to a control column. An autoantibody against a plasma membrane component of guinea pig sperm was demonstrated [509]. One of the antigens showed glycolipid characteristics. The properties of glycolipids on germ-cell surface may possibly be related to the spermicidal activity of cationic or nonionic detergents [ 5421.
5.3.3. Interaction with lectins and other proteins The synthetic glycolipids containing 2 or 3 units of 1-4-linked D-glucose residues in the head group, as well as a-glycosyl and a-kojibiosylDAG (from A . laidlawii) in phospholipid monolayer, interacted with 1 3 ' I-labelled concanavalin A (ConA). Since C o d is a plant lectin, which interacts with a-D-glucose and a-D-mannose. this system provides a convenient tool for studies of the interactions of protein and artificial lipid layer [33,462]. The interaction of ConA and monolayers of GlcDAG and Glc, DAG supported a parallel head configuration [ 3491. The physical dissimilarities of GlcDAG over Glc,DAG in the membrane of an A . laidlawii mutant lacking in Glc,DAG seem to make these cells resistant to reactive complement lysis [463]. (Manal-2Manc~l-2Manc~1-3),Glc-dibiphytanylglycerylether (Table 2.1.5(4)) appeared to be located on the surface of the cell because the cell binds ConA [239]. Evidence has been obtained that some phages adsorb specifically to the glycosyl substituents of wall teichoic acids [33,464]. Membrane lysin of Streptococcus zyrnogenes is also bound on LTA [42]. Inhibition of autolysin of pneumococcus by ribitol-containing LTA (F-polysaccharide) has also been reported [149]. When
179 side-chain D-alanine was removed from LTA, the enzyme was activated and resulted in autolysis of the cell. Autolysin prepared from autolyzed cells of B. subtilis ( N-acetylmuramyl-D-alanine amidase) binds to poly(g1ycerophosphate) in the presence of divalent cations [40]. Mammalian lysozyme was similarly inhbited by LTA [411. Fischer et al. [155] studied the inhibitory activities of Staphylococcus aureus LTA to autolysin. The inhibitory activities of LTAs, with Ala/P molar ratios of between 0.23 and 0.71, against extracellular autolysin from S. aureus decreased exponentially with increasing alanine content, approaching zero at substitutions of more than 0.6. The inhibitory activity of alanine-free LTA was highest at about 15 pM. On the contrary, glycosylation of LTA up to the extent of 0.5 did not depress the inhibitory activity. Neither the glycolipid carrying a single glycerophosphate residue nor possessing the hydrophilic chain (Galalul-6Gala1-3(Galal-2)GroP),nor neutral phospholipids showed inhibitory activity. It was concluded that autolysin, similar to ribitolphosphate polymerase, recognizes the negative charges of phosphodiesters in glycerophosphate units appropriately spaced (P-Gro-P = 0.75 nm). Other topics on the biological activities of LTA, especially pathological ones, were extensively reviewed by Wicken and Knox [41] in relation to LPSs. The activator proteins of lysosomal degradative enzymes such as sulfatidase also bind to sulfoGalAAG (3031. The specificities of these proteins to GGroLs remain to be established (Sections 4.2.2, 5.1.5). 5.4. ADAPTATION TO HIGH TEMPERATURE
Adaptation of phospholipid metabolism according to temperature shift has probably been most extensively studied [465], but such studies on GGroLs are relatively scanty at the present. The possible biological roles of complex lipids in the membrane of thermophilic bacteria [244,466,467,508], plants [440], and animals (poikilotherms) [468] have been extensively reviewed. The thermostability of the cell membrane is a factor determining thermophilic growth. 5.4.I. Photosynthetic organism The combination of both decrease in unsaturation (a-linolenic acid) and increase in GGroL content in a thermophilic cyanobacterium [469] has been reported. The proportion of saturated acid is relatively high in all lipids of the thermophilic algae, Cyanidium, ranging from about 1/3 in GalDAG to 3/4 in SQ-DAG [489]. The degree of unsaturation is dependent not only in light or nutrients [399,473], but also on temperature, e.g., in Anabaena uariabilis (a cyanobacterium, family Nostocaceae) (4701. The high content of polar GGroLs and phospholipids as well as the composition of different types of fatty acids renders the membranes, e.g., thylakoid membrane of cyanobacterium [470], to high phase-transition temperatures. However, the transition temperature is not sharp due to the heterogeneity in the composition of the membrane so that domains of gel and liquid-crystalline phases exist simultaneously. In some cases only phospholipids appeared to be involved in temperature adaptation [ 3991.
180
The unsaturation of 16 :0 at position-2 of GalDAG (possibly by direct unsaturation and via deacylation-desaturation and reacylation as reported for the phospholipids in rat liver [471,472](Section 4.1.7)) proceeds rapidly in cold adaptation of spinach. The desaturation of 18 : 0 at position-1 occurs comparatively slowly. 18 : 2GalDAG is also converted directly to 18 : 3-GalDAG (Section 4.1.7, Reaction 32) [291]. 5 4 . 2 . Mycoplasma and archaebacterium A decrease of viscosity was observed by the incorporation of oleic acid in the
membrane lipid [350]. With A. laidlawii, higher viscosity in membranes stimulated synthesis of GlcDAG at the expense of Glc,DAG. Synthesis of GroP-GGroLs was affected as well. Temperature shift-down from 37 to 17 " C resulted in an immediate synthesis of GlcDAG accompanied by an increased incorporation of unsaturated fatty acids in this lipid. The adaptation to higher temperature in thermophilic bacteria was demonstrated in lipophilic domains, i.e., the elongation of the chain, and lower unsaturation [466] or cyclization [508], less extensive branching and decreased diether/tetraether ratio [517,537]. Also, change in polar head groups, e.g., the accumulation of polyglycosylDAGs up to 70% of the total lipid, occurs [94]. In acholeplasma, the monoglycosyl/diglycosylDAG ratio decreased [350]. The content of highly polar tetraglycosylDAG with a long-chain N-acyl group in the membrane of Thermus thermophilus increased with increasing culture temperature [244]. In thermoplasma and sulfolobus, the sum of neutral and acidic GGroLs occupied 83.4 and 89.3% of the total lipid, respectively [239]. Chain elongation of fatty acids (thermus contains no unsaturated fatty acids) strengthens van der Waals' force between membrane components [376] to elevate the transition temperature of membrane or to maintain the gel domain [244]. However, because the attractive forces between the adjacent alkyl chains are weak, the bilayer portion is fluid in normal environments [440]. The amounts of glycolipids in a sulfolobus-like organism, as determined by a flame ionization detector on thin layer chromatography [474], were higher at optimum growth temperatures (49% of total lipid at 75 " C ) [342]. Usually the transition temperature is a few degrees centigrade higher than the culture temperature, corresponding to the phase-separation temperature [244]. This is an important feature of the lipid bilayer: to undergo a reversible phase transition from a thermotropic gel to a liquid-crystalline phase (Section 5.1.2). This so-called " viscosity" change is rapidly adapted by the regulation of the synthesis of various lipids [350]. The mycoplasmas will not grow below the transition temperature, when only about 10%of membrane lipids remain fluid [351]. 5.5. ADAPTATION TO IONIC ENVIRONMENT
5.5.1. Interaction with cations
Phospholipids bear at least one potential negative charge on the phosphate group. Other charged groups on membrane lipids include sulfate, carboxyl and amino
181
groups. Divalent ion bridging between adjacent binding sites [533] was reported for calcium binding to phospholipid and sulfolipid monolayers [200,376]. Ca2+ and Mg2+ have a hgh affinity for acidic lipids, and bind to lipids with one negative charge in a 1 : 2 cation/lipid ratio [533], and to lipids with two net negative charges (e.g., phosphatidic acid) in a 1 : 1 cation/lipid ratio [376]. The binding of Mg2+ to the lipomannan from M. Iuteus (Table 2.1.6(2)) was measured at various concentrations of Mg2+ under constant ionic strength (10 mM Na+) [287]. The binding isotherm showed that the compound progressively binds more Mg2+ ions as the concentrations of applied Mg2+ increased, even though Na' ions were present in a 10-100 molar excess. The total number of available binding sites, obtained from the intercept of the Scatchard plot, was equivalent to one Mg2+ ion bound per 3-4 succinic acid residues. The apparent association constant, was 1.47 X lo3 M-' at pH 7.0. This value was of the same order as that calculated for the binding of Mg2+ to LTAs (2.7 x lo3 M-', at pH 5.0) [476]. The dominating membrane lipids of A. faidluwii, GlcDAG and Glc,DAG, take up only limited amounts of water (bound + trapped), i.e., up to 13%(w/w). On the other hand, the phospholipids and phosphoglycolipids have much larger hydration capacities [277,349]. Addition of Mg" and Ca2+,but not Na', to the Glc,DAG, increases the hydration capability. This increase was accompanied by the formation of a metastable liquid crystalline phase, indicated by the existence of a reverse hexagonal phase structure for the GlcDAG and lamellar structure for Glc,DAG and other membrane lipids. Both GlcDAG and Glc,DAG have similar properties, with temperature, to Ptd-ethanolamine [349]. 55.2. Phosphate deficiency
Ellwood and Tempest [464] described the drastic change of the compositions of cell wall teichoic acid and teichuronic acid, as well as a mucopolysaccharide containing GalNAc and GlcU, in B. subtilis depending on the concentration of salt in the media. It was possible to reversibly replace the wall teichoic acid with teichuronic acid, by growing B. subtilis in a chemostat with a phosphate-limiting medium [464] (c.f., adaptive convergence, Section 3.6.2). In this case the synthesis of wall teichoic acid is stopped by inactivation of the first enzyme in the synthesis of linkage units [479]. The continued synthesis of LTA under the condition of phosphate-limitation [40,480] led to the idea that LTA plays an essential part in bacterial cell physiology. GlcU-DAGs are only produced when pseudomonads are grown on agar slopes; they are completely absent from the lipids of organisms grown on liquid culture [107]. Moreover, the organisms grown on solid media are very low in phospholipid contents. Growth in solid media is probably an effective way of stimulating phosphate-limiting growth conditions. After the first few cell divisions, the supply of phosphate for phospholipid synthesis may well be exhausted and the organism replaces the essential anionic phospholipids by similarly charged GlcU-GGroLs. This process is directly analogous to the replacement of teichoic acids in the wall of B. subtilis by teichuronic acids when grown under phosphate-limiting conditions [464,495], and also increased synthesis of lysyl-PtdGro under the accumulation of a
182
dihydroxybutyl-pyrophosphonate analog of PtdGro in the membrane (Section 4.1.5.1) [409]. When B. suhtilis W 23 [481] or Sraphylococcus aureus was grown chemostatically in PO:--deficient media, cell wall teichoic acid was not synthesized. The walls of the phosphate-limited bacteria contained no detectable teichoic acid. However, LTA of cytoplasmic membrane, which is the receptor of phage SP 50, is formed at a normal level. These phenomena are also supportive of the hypothetic role of LTA to trap Mg2+ (Section 5.2.2). Tevini [482]examined the lipid composition of Impatiens balsaminu (touch-me-not) and Hordeum vulgare (barley) under conditions of phosphate deficiency. In both cases there was a marked decrease in all phospholipids. However, the chlorophyll content was not changed and the amounts of GalDAG, Ga1,DAG and SQ-DAG were only a little increased. 5.5.3. High osmolarity The variability of alanine ester substitution on the hydroxyl of glycerol was studied by growing S. aureus in media of low and high NaCl, which were known to cause extremely high and low Ala ester substitution of the wall teichoic acid [480]. The alanine ester content of LTA was influenced in a similar way, dropping from 0.73 to 0.33 when NaCl was increased from 0.03 to 1.7 M [150,155,159]. This appears to regulate the Mg2+ binding capacity of LTA. The salt dependence of halophilic bacteria was ascribed to the need for screening negative charges on cellular proteins (including sulfated glycoproteins) which have high proportions (acid/base ratios of about 10) of acidic amino acids and few nonpolar ones (433,4671 (c.f., Section 5.4.2). When salt was removed from the medium of Halobacterium cutirubrum, the membrane broke down, probably due to electrostatic repulsion between negatively charged groups in the outer membrane. PtdGro increased, also by loss of cell wall in S. aureus [357], to increase the rigidity of cell wall (Section 3.2.4). Ohno et al. [264] also suggested that the occurrence of anionic phospholipids in high concentration in a moderately halophilic bacterium (Pseudomonas halosaccharolytica), grown between 0.5 and 4 M NaCI, may contribute to the regulatory mechanism of the permeation barrier in cationic environments. The changes of cellular phospholipid composition. and increase in PtdGro and decrease in Ptd-ethanolamine as well as a decrease in alanine ester of LTA [155], also support this hypothesis. The extreme halophiles, requiring over 3 M of NaCl for growth, contain similar levels of intracellular salt, but with K + predominating [467,478]. On the other hand, ha'lotolerant algae of the genus Dunaliella accumulate glycerol intracellularly in concentrations which are iso-osmotic with the extracellular NaCl [520). Cells grown in the presence of 4 M NaCl show an intracellular concentration of around 5 M glycerol. Among various halophilic or halotolerant bacteria, Halobacterium cutirubrum and H . halohium, extreme halophiles requiring 4 M NaCl for optimal growth, had a cell wall structure lacking a muramic acid-containing peptidoglycan layer in common
183 with other archaebacteria [353]. The lipophilic outer membrane of a moderate halophile [264] also contained a high concentration of acidic amino acids as in the thermophiles, also characteristic for archaebacteria. The lipophilic moieties of the membrane lipids were composed entirely of long-chain branched dialkyl ethers instead of acyl esters [113,299,483,500] (Fig. 2.4). Kates suggested that the membrane lipids contribute to structural stability, as the diether analog of PtdGro phosphate has a strong affinity for M g z + .On the other hand, Kushner and Onishi [433] concluded that the requirement for high salt concentrations in extreme halophiles is due to mutual repulsion between negatively charged groups on proteins, rather than to repulsion between negative charges on the lipids. Several reports, in addition to Ohno et al. [264] concerned with the lipids of moderately halophilic bacteria have been already published. The major lipids of these cases were diester mixtures of phospholipids and GGroLs [134]. The extractable lipids from a moderate halophile, 101-W3, contained abundant acidic phospholipids and GGroLs [264]. The Tietz’s group reported the occurrence of GlcPtdGro [484] and GlcUDAG [110] as the major components, respectively, when grown either in 1.5 M NaCI, or 3.5 M NaCl in addition to 0.5 M KCI. Chlorosulfolipids were detected only in fresh-water algal species [535]. On the contrary, the marine species, which lack chlorosulfolipids, contain only SQ-DAG. This phenomenon might also be related to the adaptation to salinity. Also, in halotolerant Staph-ylococcusepidermidis [139], the proportion of PtdGro (as percent of total polar lipid) decreased from 67 to 50% as the NaCl level was increased from 0 to 4 M. In contrast, the proportion of diPtdGro (which is known as the cation-ionophore) and GroP-GGroL (GroP-6Glcpl-6Glc~l-3DAG)increased from 0.5 to 11% and 5 to 15%, respectively. As a result, the average number of negative charge/mol phospholipid increased from 1.01 to 1.14, most of these changes occurring between 2.5 and 4 M NaCl in the medium. The composition of neutral GGroLs remained fairly constant. The levels of neutral GGroLs were only slightly changed. These phenomena might indicate evolutionary “convergence” of acidic amphiphiles in cell membranes (Section 3.6.2). Each lipid component had essentially the same fatty acid composition, namely, anteiso-15 : 0 (60-75%). anteiso17 : 0 (18-24%), iso-17 : 0 (8-lo%), and small amounts of palmitic and stearic acids (2-5%). The fatty acids were nonrandomly distributed in PtdGro, the shorter chain anteiso-15 : 0 fatty acid being exclusively esterified to the 2-position and the longer chain anteiso- and iso-17:0 fatty acids at the 1-position (Section 2.2.4). The fatty acid composition was not affected by increasing NaCl content in the medium in the range from 0 to 2.5 M, but the proportion of anteiso-15 : 0 increased greatly when the salt concentration was increased to 4 M.
A ckno wledgment We are especially thankful to Dr. Werner Fischer of the University of Erlangen/Niirnberg for many interesting discussions, supply of materials and read-
184 ing of the manuscript. We are grateful to Drs. Yoichi Tamai, Mieko Oshima. Hiroshi Kaneko and Toshihiro Itoh of Kitasato University, Drs. Ichiro Tadokoro and Toyozo Takahashi of the Yokohama City University, Dr. Masuo Nakano of Obihiro Veterinary University, Dr. William R. Mayberry of the East Tennessee State University, as well as Ms. Keiko Aritomi-Tadano, Drs. Kichitaro Kawaguchi, Toshiaki Abe. Yoshiro Miura and Norio Suzuki of the University of Teikyo and to the father of 1.1. for extensive discussions of the material in this paper. We also wish to thank Ms. Mikiko Miura, who handled secretarial work with efficiency and good nature. This study was supported by Grants-in-Aid from the Ministry of Education, Science, and Culture of Japan, Naito Memorial Fund and Alexander von Humboldt St i f tung.
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Wiegandt (ed.) Glycolipids 0 I985 Elseoier Science Publishers B. K (Biomedical Division)
CHAPTER 3
Gangliosides HERBERT WIEGANDT Department of Biochemistry, School of Medicine, Philipps University, Marburg an der Lahn, F.R.G.
I . Introduction Gangliosides are distinguished from other glycosphingolipids in that they contain one additional characteristic carbohydrate constituent, namely sialic acid *. It was indeed this sugar amino-acid that by its colour formation upon heating with p-dimethylaminobenzaldehyde/HCl (Ehrlich’s reagent) or orcinol/H,SO, (Bial’s reagent) led to the detection of these lipids [l]. In 1935, E. Klenk characterized a new type of acidic glycolipid, “substance X”, from the brain of patients suffering from amaurotic familial idiocy [2]. G. Blix, in 1938, recognized that such a glycolipid could also regularly be found in a normal brain [3]. Further work from the laboratory of Klenk in 1942 showed that “substance X” was concentrated in the brain grey matter, and it was suspected of being localized in ganglia1 cells. “Substance X” was therefore termed a “Ganglioside” [4]. Work on the characterization of gangliosides and the elucidation of the chemical structures of many of their components was mostly performed during the sixties in the laboratories of Ernst Klenk, Richard Kuhn and Lars Svennerholm (for reviews, see Refs. 5, 200). Since their discovery, the gangliosides have for several reasons elicited much interest. The concentration of complex gangliosides in membranal elements of the brain is suggestive of a functional role in the central nervous system. Furthermore, certain lipidoses that affect the nervous system, such as Tay-Sachs disease are characterized by a ganglioside accumulation within cells of the brain. An additional indication for an involvement of gangliosides in nervous function, was their interaction with the neuro-toxin of Clostridium tetani. This toxin is specifically fixed by brain tissue, a property believed to relate to its ganglioside content [6,7]. During the elucidation of the chemical nature of the sialic acids, it also became evident that they have numerous specific biological properties for which gangliosides frequently are the glycoconjugate carrier. * Sialic acid is the generic term designating all N-acylneurarninic acids.
200
Gangliosides also occur outside the nervous system. It is recognized now that they are important constituents of the surface membrane of most, perhaps all, cells of animals that belong to the phyla of the Deuferosfomiu. Before embarking on current concepts of the biological behaviour and significance of gangliosides, it will be necessary first to describe their chemical composition and physicochemical properties.
2. Chemistry, physics and methods of preparation and analysis 2.1. GENERAL PRINCIPLES OF CHEMICAL CONSTITUTION
Gangliosides are acidic glycosphingolipids due to their content of one or more sialic acid residues in the carbohydrate moiety. In common with other glycosphingolipids they consist of a long-chain base, a “sphingoid” that is linked to fatty acid by an amide bond thus forming the lipophilic “ceramide” portion. The carbohydrate is linked to the sphingoid’s primary hydroxyl by a glycoside. Acyl. N H
I
CH,. (CH2 )12.CH=CH .CH(OH) * C H * C H ~ . O * G I ~ C O S ~ I
Similar to other lipids, the lipophilic portion of the ceramide shows microheterogens ity with regard to the sphingoid and the fatty acid composition [8,9]. In most instances ganghoside components are distinguished and characterized on the basis of their sialo-oligosaccharide (for review, see Ref. 10). In general, glycosphingolipids can be classified into several series that may reflect their presumed biogenic relations, i.e., the sequential addition of monosaccharides to ceramide, and a growing sugar chain via glucosylceramide or galactosylceramide (Fig. 3.1). Of all glycosphingolipids indicated in Fig. 3.1, sialic acid-substituted components with one or two hexose units have been structurally characterized carrying glucose, galactose or lactose. All higher gangliosides so far identified are derived either from the gunglio-, lacto- or globo-series. The ceramide moiety of gangliosides frequently is similar to the fatty acid and sphingoid composition of other neutral glycosphingolipids that are derived from the same cellular source (for reviews, see Refs. 70, 71). It has, however, also been observed that the ceramide composition of gangliosides differs from that expected on the basis of the biogenic relation of their carbohydrate portion. An example of this is the fatty acid composition of ganglioside G,,J (“ hematoside”) which may be quite different from that of lactosylceramide of the same tissues [72], or else that of the human erythrocyte gangliosides IV3NeuAcnLc4Cer as compared to IV6NeuAc-nLc4Cer [582]. Similarly, with increasing sialic acid content, brain gangliosides show an increasing proportion of eicosa-sphing-4enine (Cz0:,)over sphing-4-enine (C18:l)[73-751. The way in which the relation between the composition of the ceramide to that of
201
INVERTEBRATES
/
Man-Man-Glc - C e r
Man-Glc- C e r
/ '
GIc-Cer
GlcNAC-Man-GIC-Cer
/
GaINAc-Gal-GIc-Cer /Gal-Gal-Gal-Glc-Cer
Cer
(lactosyl-Cer )
~GalNAc-Gal-Gal-Glc-Cer
F l
GlcNAc-Gal-Glc-Cer VERTEBRATES
Gal -Cer-Gal-Gal
-Cer (galabiosyl -Cer )
Fig. 3.1. Carbohydrate series as a base for classification of glycosphingolipids.
the carbohydrate portion is regulated is as yet unknown. The presence of sialic acid in ganglioside is an indication that these lipids are typical cell surface constituents (for sialic acid reviews, see Refs. 11, 12). Whereas free sialic acid in aqueous solution is present as its P-anomer, in gangliosides it is linked by an a-ketoside [13-151. With the exception of gangliosides discovered in starfish [16], sialic acid substitution always takes place in branching or terminal positions of the oligosaccharide. Sialic residues in ganglioside may form lactones under acidic conditions. Such lactone formation occurs with particular ease in the terminal sialic acid that is linked to another sialic acid by an a2 + 8-ketoside [16]. It is tempting to speculate that such lactonization-delactonizationof gangliosides may also occur under in vivo conditions. This would result in changes of electric charge of membranes that may be of functional significance. This speculation becomes all the more plausible since it was discovered that, in the brain, some sialic acid residues of gangliosides can be reduced by sodium borohydride as would be expected for lactone linkages [457]. The sialic acid of all gangliosides of human origin is N-acetylneuraminic acid or 9-0-acetyl-N-acetyl-neuraminicacid * [18]. The brain gangliosides of other vertebrates, except for trace amounts of N-glycolylneuraminic acid [19], also contain N-acetylneuraminic acid or N,O-diacetylneuraminic acid [20,615]. Teleost fish brain gangliosides are particularly rich in the latter diacetyl-derivative [Zl].
* There is some immunological indication that N-glycolylneuraminic acid might be a tumor-associated antigen in humans I458.6761.
202 Interestingly, the gangliosides of the bovine neurohypophysis, that in their component distribution are similar to those of the cerebral cortex, contain a high proportion of N-glycolylneuraminic acid [228]. This sialic acid is otherwise more typical for gangliosides met outside the central nervous system. In particular, gangliosides of horse and cat erythrocytes are rich in N-glycolylneuraminic acid. It appears that N-acetyl- and N-glycolylneuraminic acids can replace each other since, structurally, no positional preferences have yet been observed. The component profile of the gangliosides generally is quite typical for their orign, and there are certain trends for the occurrence of gangliosides containing lactose or those of the gungfio-, gfobo- and lucto-oligosaccharide series. But insufficient data have been recovered to obtain a clear picture of the biological significance of a particular ganglioside pattern. As an example of the complexity of their occurrence, one may compare gangliosides of the central nervous system and those of the red blood cells of different animals. Whereas brain gangliosides of vertebrates are almost exclusively of the gungfio-series, those of erythrocytes may vary considerably depending on the species. Some of the latter gangliosides may contain galactose (mouse), others lactose (man, pig, horse, cat, dog), or they may belong to the gunglio-series (rat), the fucro-series (cattle) as well as to the gfobo-series (man) [636,637]. 2.2. CHEMICAL COMPOSITION
Gangliosides of simplest chemical structure, G,,J and G,,,l, derived from glucosylor galactosylceramide (cerebroside) are NeuAca2-6Glcp-Cer and NeuAca2-3GalplCer for the gametes of the sea urchin [23]. The latter ganglioside, however, may not be restricted to this animal, since in one report it was also described as occurring in pig platelets [24]. Ganglioside Ggall,besides G,,,l, was identified as a typical and major component of oligodendroglial myelin of the brain [25,28]. Apart from human and chicken brain myelin, G,,,l, besides G,ril, also was detected as a major ganglioside of mouse erythrocytes [29], chicken-embryonic liver [459] and chicken egg yolk [30]. In the latter material, Gg,,l occurs together with the next higher, the lactose-derived gangliosides, G ,,J, NeuAccu2-3Galpl-4Glc/3-Cer and G lac 2, NeuAca2-8NeuAca23Gal/ll-4Glc/l-Cer [30]. Gangliosides G with one (G J), two (G,,,2) or three (Gl,,3) sialic residues, are among the most abundant extraneural gangliosides in vertebrates. In brain, however, where higher hexosamine-containing gangliosides predominate, G laclis oniy a minor constituent [31-34]. Ganglioside G luul with predominantly N-glycolyl-neuraminic acid was isolated in 1951 from horse erythrocytes by Yamakawa and Suzuki [35]. This was the first ganglioside extracted from extraneural material. In order to distinguish it from the brain “gangliosides”, it was named “ hematoside”. Hematosides G lac indeed are the major ganglioside components in erythrocytes of many animal species [36]: these include man [37], rabbit [42], cattle [39], giant panda
203 [36], horse [35], and the Cunidue, dog [40],jackal, dingo and racoon dog with Glacl, as well as the Fefidue, cat [38,42], lion and the closely related hyaena [36] with G,,,2. The latter ganglioside G,,,2 also occurs as a major component of mammalian retinal gangliosides [234,44]. Except for hematoside from human erythrocytes, with only N-acetylneuraminic acid, the gangliosides G,,J and Gl,,2 from other mammalian red blood cells may also contain N-glycolyl-neuraminic acid. The occurrence of these two sialic acids, NeuAc and NeuGc, shows a typical species and interspecies distribution: horse, cat (except Persian cat), racoon dog and giant panda hematosides contain exclusively N-glycolylneuraminic acid. In the european dog the hematoside is of the N-acetyltype, whilst in some oriental dogs it is N-glycolyl-hematoside [36]. Gangliosides from the invertebrate starfish Asterina pectinifera also contain ceramide-linked lactose, substituted by N-glycolylneuraminic acid residues [45-471. In this instance, however, the sialic acid carries additional carbohydrate- and 0-methyl residues, i.e., Arab1-6Galfil-4[ 80Me]NeuGcol2-3Gal~1-4Glcfil-Cer
Ara~l-6Gal~1-4NeuGca2-3Gal~l-4Glc~l-Cer and Arab1 -6Ga1/31-4[GalPl-8]NeuGca2-3Gal~l-4Glcfi-Cer.
The “classical” gangliosides, i.e., the predominant species in the brain of higher animals (deuterostomia), contain carbohydrate moieties of the gunglio-series: Gangliotriaose, GalNAc/31-4Gal~1-4Glc Gangliotetraose, Gal~1-3GalNAc~l-4Gal~l-4Glc Gangliopentaose, GalNAc~l-4Gal~1-3GalNAc~l-4Gal/31-4Glc
In this oligosaccharide series, substitution with single or multiple, a24inked sialic acids takes place at the galactose in 3-position and/or of the N-acetylgalactosamine residues in 6-position. Sialic acid residues are linked to one-another by an a2-8 ketoside (Fig. 3.2). Additional substitution by fucose may occur in 2-position of terminal galactose of the gangliotetraose (see Table 3.2). Other gangliosides, discovered in the fat body of frogs are derived from monosialogangliotetraosylceramide by substitution of the terminal galactose residue in 4
--
pent tet
*
tri lac
P
g
l
c
GolNAc-Gal-GolNAc-GoI- Glc- C e r
I
NeuAc N~uAc
I I NeUAC (b)
2b. 3b 4 b
NeUAC I
3 c . 4c
NeuAc ( a )
(C)
1. 2a
3a
5c
Fig. 3.2. Structure of gangliosides of the gmgh-series.
204 3-position by mono-, di- and trigalactopyranosyl units [53,54] (see Table 3.2). A pentasialogangliotetraosylceramide,isolated from fish brain, is the most highly sialylated ganglioside structurally characterized thus far [ 100,565]. In this species the major tetrasialoganglioside has the structure 1V monosialo-,I1 trissialoganghotetraosylceramide (i.e., “C” class in Fig. 3.2). Different from fish brain, the major tetrasialoganglioside from human and chicken brain is a IV 3bisialo-,I13bissialogangliotetraosylceramide(class “B’in Fig. 3.2) [56-581. The gangliosides of the gunglio-series are listed in Table 3.2. A ganglioside discovered by Watanabe et al. [59] in human erythrocytes has the Since it contains Nunique structure, NeuAccu2-3Gal~l-3GalNAc~l-4Gal~l-Cer. acetylgalactosamine substituted in 3-position by galactose, it shows some similarity to gangliosides of the gunglio-series. The gangliosides of the lacto-series occur more typically in extraneural sites. They contain, as neutral carbohydrate core unit, a lactoneotetraose linked to ceramide, [48,59,60-62,461,462,464,6541. i.e., Gal~l-4GlcNAc/3l-3Gal/3l-4Glc~l-Cer Further gangliosides of the lucto-series are derived from the core unit of lactoneotetraose extended with N-acetyl-lactosamine residues in the 3-position of the terminal galactose, in a linear position, to lactoneohexa-, octa-, etc., osylceramide [63]. Additional LacNAc residues are found branching in 6-position of galactose as, for instance, in lactoneo-IV6kladohexaosylceramide * or lactoneo-IV6kladooctaosylceramide of human red blood cells [64-67,636,637,5951: Gal/3l+ [4GlcNAc/31+ 3Gal/31+ ] , 4 G l c ~ l +Cer (lactoneotetra-,hexa-, octa-, etc., osylceramide)(631 Gal/3lJGlcNAc/31\ Gal B1-4Glc/31-Cer Galal-4GlcNAc/31/ (lactoneo-I16kladohexaosylceramide) [64] * Gala1 --4 4GlcNAcjIl\
Gal/31-4GlcNAc/31-3Gal/31-4Glc/31-Cer Gala1 + 4GlcNAc/31/ ( lactoneo-IV6kladooctaosylceramide) [69,595,637]
By analogy to the gangliosides of the gunglio-series, those of the lucto-series may in addition to sialic acid, carry fucose residues at branching or terminal positions e.g., as in human kidney ganglioside: IV3NeuAc-,1113Fuc-nLc,Cer(681 or in a branched-chain fuco-ganglioside identified in human erythrocytes: V13NeuAc-,
IV6[Fucal-2Gal/31-4GlcNAc]-nLc,Cer. A ganglioside of the lucfo-series with a P-N-acetylgalactosamine was discovered in [59]. human erythrocytes, i.e., IV3[NeuAccu2-3GalNAc~l]-nLc,Cer Gangliosides of the globo-oligosaccharide series have recently been discovered.
* This
oligosaccharide was named lacto-“nor”-hexaose. “Nor” the antipode of “homo”, however, designates a compound differing from the parent term by a minus of one carbon unit. Therefore it is suggested that instead of “nor” to apply the syllable “k1ado”-branch, (twig, greek, ~ X d l 8 0 l )for branched chain oligosaccharide isomeres, i.e., lacto-klado-hexaose.
205 From chicken skeletal muscle [462] or human teratocarcinoma cells [672] gangliosides were isolated that contain one or two sialic acid residues linked to globopentaose, i.e., NeuAccu2-3Gal~l-3GalNAc~l-3Galc~1-4Gal~l-4Glc~l-Cer and NeuAccu2-8NeuAccu2-3Gal~l-3GalNAc~l-3Galal-4Galpl-4Glc~l-Cer. A ganglioside from rat intestinal nonepithelial tissue has an isoglobopentaose as neutral carbohydrate core, i.e., NeuAca2-3Gal/3l-3GalNAc~l-3Gald-3Gal~l-4Glc/3l-Cer ~31. 2.3. NOMENCLATURE OF GANGLIOSIDES
A nomenclature of gangliosides was recommended by the Commission on Biochemical Nomenclature of the International Union of Biochemistry (1977). It follows that for neutral glycosphingolipids, which is based on trivial names for specific oligosaccharides (see also Fig. 3.1). Accordingly, the gangliosides are named sialo-Xosylceramide, where X stands for the root name of the neutral oligosaccharide, to which the N-acetyl- or N-glycolylneuraminosyl residue is attached. The position of the sialic acid group may be indicated by a Roman numeral for the number of the monosaccharide residue to which the sialic acid is linked, and with an Arabic numeral superscript indicating the position within that residue, to which sialic acid is attached. The neutral oligosaccharides are represented by symbols, in which the number of monosaccharide units is indicated by Ose,, preceded by two letters giving the trivial name of the oligosaccharide. To conserve space, Ose can be omitted: Gg, ganglio; Lc, lacto; nLc, lacto-neo, etc. (see also Fig. 3.1). For the sake of brevity in this chapter, short hand notations are used for gangliosides that contain as neutral carbohydrate core glucose (glc), galactose (gal), lactose (lac) or the oligosaccharides of the ganglio-series, i.e., gangliotriaose (tri), gangliotetraose (tet) and gangliopentaose (pent). G stands for ganglioside; the index, e.g., Gle,, is the abbreviation for its neutral sugar moiety. To this is added the number and, if necessary, the nature, e.g., NeuAc or NeuGc of the sialic acid residues, a, b to distinguish between positional sialo-isomers, e.g., GI,, 2a (Fig. 3.2). 2.4. CHEMICAL AND ENZYMATIC ALTERATIONS
A number of chemical as well as enzymatic reactions have been applied to ganglio-
sides. The products obtained were often instrumental in the elucidation of physicochemical or biological properties of these sialo-glycolipids. 2.4. I. Alteration of the ceramide Removal of part of the lipophilic ceramide portion yields " 1yso"-ganglioside analogues with properties similar to other amphipathic lyso-lipids. Ceramide cleavage can be achieved by strong alkaline hydrolysis of the long-chain fatty acid followed by re-N-acetylation [76-781 OH
OH
206 R, sialo-oligosaccharide
The secondary hydroxyl-group of the sphingosine, due to the carbon-to-carbon double bond in allylic position can be oxidized to the keto group. OH
n
R, sialo-oligosaccharide DCDCB, dichloro-dicyano-benzoquinone
In order to protect the sensitive sialic acid residues of the ganglioside from oxidative degradation by the dichloro-dicyano-benzoquinone,the reaction is performed with inverted mixed ganglioside/Triton X-100 micelles in toluene [464]. The product " keto-ganglioside" can be conveniently reduced to the parent ganglioside, e.g., with boro-tritiide. This then allows for radioactive labeling of the ganglioside in the ceramide moiety with retention of the carbon-to-carbon double bond but racemisation at carbon 3 [464]. Treatment of the keto-ganglioside with alkali leads to a liberation of the intact free, reducing oligosaccharide moiety from the glycolipid [465]. A point of any easy selective chemical attack at the ceramide of gangliosides or neutral glycosphingolipids, is the carbon-to-carbon bond of the sphingoid. Oxidative cleavage of ganglioside by ozone [86] or osmium tetroxide/periodate [87] yields aldehydic products that are used for further reactions: OH
R, glycosidic residue
207 (a) Mild alkaline fragmentation liberates the total intact sialo-oligosaccharide moiety of the gangliosides [88,89]. (b) Reduction of the ceramide C=C double bond-oxidation product with borohydride yields "lyso" ganglioside analogues, that still have the region of attachment of carbohydrate to the lipophilic molecular portion intact [77,79]. (c) The intermediate of oxidative C=C-cleavage can alternatively be further oxidized to contain a carboxyl function. A direct oxidation cleavage of the -C=Cdouble bond to carboxylic acids can be performed with peracetylated gangliosidesialic acid methyl esters in organic solvent using permanganate in the presence of dicyclohexyl-18-crown-6ether [606]. 0AC
P
o
l
c H 0 2 C q C H 2 . 0 , R (peracetyl)
.YNH 0 Such a fragment-ganglioside derivative can conveniently be used for coupling to matrices such as amino-alkyl-agarose. Prior to oxidation, the carboxyl group of sialic acid must be protected, e.g., by esterification [347,81]. The carbon-to-carbon double bond of the sphingoid can be reduced by catalytic hydrogenation. Using tritium in the presence of palladium, the gangliosides can be radioactively labeled in the ceramide moiety without apparent hydrogenolytic decomposition [82-851. Ganglioside analogues that contain a sialo-oligosaccharide moiety linked to fatty acid have been synthesized. Such derivatives carrying a fluorescent or electron paramagnetic resonance label were used as probes to investigate the properties of sialo-glycolipids in membrane systems [466-4721. To obtain such sialoglycolipids, a ganglioside-derived sialo-sugar is reduced with cyanoborohydride in the presence of ammonia. For tritium labeling, radioactive NaCNB3H3 can be used. The reductaminated oligosaccharide can then be coupled to a fatty acid of choice by an amide. A sialo-glycolipid is directly obtained when reductamination is performed with a long aliphatic hydrocarbon chain amine [90].
R, sialo-glycosidic residue R' and R", aliphatic hydrocarbon chain
208 Ganglioside analogues with two long aliphatic hydrocarbon chains have also been obtained from reductaminated sialo-sugars by the following reaction sequence [91]: R.CH2*NH2 + (p)02N.O,O.CO.CHN3.(CH2)n R .CH2 .NH * CO .CHNJ .(CH2 )n 'CH3
H2/P t
R.CH2 .NH.CO.CH.(NHr).(CH2)n.C~3
.CH3
-
tR'
[email protected] (P)
R .CHz.NH . C O . C H .( CH> ) n .CH3
I
N H . C O * R'
R, sialo-glycosidic residue R, aliphatic hydrocarbon chain 2.4.2. Alteration of the carbohydrate moiety Chemical as well as enzymatic reactions involving the sialo-sugar moiety of gangliosides, frequently aim at an introduction of a chemical or radioactive label. Furthermore, enzymes that specifically degrade gangliosides can be used as valuable tools in the elucidation of their chemical constitution. 2.4.2.1. Oxidation of terminal galactose by galactose oxidase Terminal galactopyranosyl, as well as terminal N-acetylgalactosaminyl, groups are oxidized in 6-position to the aldehyde by galactose oxidase (D-Galactose: oxygen 6-oxidoreductase, EC 1.1.3.9) from Dactylium dendroides, frequently misnamed as Polyporus circinatus [92,204,246,487,608,574].Reduction of the aldehyde with tritium can be used for the introduction of the radiolabel [93,94]. Alternatively, the 6-aldehyde is reacted with "S-labelled methioninehydrazide to yield the corresponding hydrazone [176]. 2.4.2.2. Oxidation of terminal sialic acid residues A label can also be introduced into ganghosides after oxidation of their terminal sialic acid residues with a low concentration of periodate. Model studies showed that, by choosing appropriate conditions, sialic acid of brain ganglioside is converted to its 8-C analogue with only negligible destruction of other monosaccharide residues [96]. At the stage of the aldehyde, the oxidation product may be reacted with dinitrophenylhydrazine to yield a colour-labeled ganghoside derivative [587], or else reduced with borotritiide for the introduction of a radioactive label. Another method of chemical alteration of sialic acid residues is by exchange of the usual N-acetyl or N-glycolyl for a N-trifluoroacetyl residue [422]. Interestingly, the obtained N-trifluoroacetylneuraminyl-ganglioside is very inhibitory to cell membrane sialidase, indeed more so than other sialidase inlubitors, such as siastatin, i.e., 2-acetamido-3,4-dihydroxy-5-carboxypiperidine [423] or 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (4241. Galactose-oxidase treatment as well as sialic acid-periodate oxidation, both have been applied successfully for the labeling of gangliosides in intact cells [97,98,587]. 2.4.2.3. Cleavage of terminal sialic acid by sialidase (see Table 3.1) It was the sequential enzymatic removal of sialic acid residues from the higher brain gangliosides by sialidase (N-acetyl-neuraminate glycohydrolase, EC 3.2.1.1 8) that provided the first clue for an understanding of their structural relationships (for review, see
209
Ref. 99). Sialidases of viral or bacterial origin show specificity with regard to the nature of the sialic acid and its linkage to the aglycon (Table 3.1). Sialidases have successfully been used to obtain information on the linkage position of sialic acid at the neutral core oligosaccharide [lOo]. 4-0-substituted sialic acids are not cleaved by the known sialidases. Sialic acid linked to the 3-position of TABLE 3.1 Specificities of sialidases Linkage type Gal c
t3
SV
VC
CP
FPV
NDV
AU
+
+
+
+
+
+
+
-
+
-
-
+
NeuAca2 GalNAc/ll+ 4Gala 3
+
4Glc
t
NeuAca2 GalNAcal+ 4Gala1 + GlcPl+ Cer 3
(cholate)
t
NeuAca2 GalNAcal+ 4Gal/31+ 4[1-amino-sorbitol]
-
+
t3
NeuAca2 Gala --* 3Ga lNAcBl+ 4Gala + 4Glca + Cer 2
(trace)
r3 NeuAca2
t
laFuc Gala <
+
-
+
+
+
+
-
+
r6
NeuAca2
GalNAcfl< 3
t
Neu Ac a 2 GlcNAc[3 + ] a 6 t NeuAca2 NeuAca < 8
+ -
+
t
NeuAca2 SV, Sendai virus neuraminidase (151; VC, Vibrio cholerae neuraminidase; CP. Clostridiurn perfringens neuraminidase; FPV, chicken fowl plague virus neuraminidase; NDV, New Castle disease virus neuraminidase [2.10-12); AU, Arthrobacfer ureafuciens neuraminidase [13,14]. These data are from References: Kuhn et al., 1961 [104]; Drzeniek and Gauhe. 1970 (1051; Suttajit et al., 1971 [loo]; lshizuka and Wiegandt, 1971 [loo]; Cassady et al., 1965 [107]; Wenger and Wardell, 1973 [101,102]; Rauvala, 1976 [108]; Schauer et al., 1980 [109]; Itoh et al., 1981 [llo]; Drzeniek et al., 1966 [ l l l ] ; Drzeniek, 1967 [112]; Huang and Orlich, 1972 [113]; Uchida et al., 1976 [114]; Sugano et al., 1978 [115]; Suzuki et al., 1980 [473].
210 internal galactose that is substituted in 4-position by N-acetylgalactosamine, as in gangliotetraose or gangliopentaose, is characteristically resistant to many sialidases. This has much facilitated structural investigation of gangliosides [293]. The reason for such resistance is as yet not well understood. Ganglioside G,,,l, resistant against Cl. perfringens sialidase, is cleaved, however, by this enzyme in the presence of cholate [101,102]. Surprisingly, monosialogangliotetraose, the free sialo-sugar of this ganglioside resists attack by this sialidase in the presence or absence of cholate. In contrast, N-acetylneuraminic acid is cleaved after reductamination of the monosialogangliotetraose to the parent amino-alditol. I t could therefore be speculated that the sialidase resistance of gangliosides Gtr,l, G,,la, G,,,la, Gpen,lu. G,,,2a and IV2F~~-,113NeuAc-Gg,Cer could be due to “steric hindrance”, caused by an arrest of the sialic acid residue to neighbouring N-acetamido group of N-acetylgalactosamine. This arrest may be released in a ganglioside-cholate mixed micelle. In such a lipid arrangement, cholate-carboxyl may be placed in a sterically favoured position to unlock the sialic acid block. Introduction of a neighbouring amino-group may have a similar effect. Sialidases have been used to probe the localization of ganglioside sialic acid in biological membranes. The results of such studies, however, are rather equivocal (for review, see Ref. 103). A recent analysis of N-18 mouse neuroblastoma cells shows that 728 of ganglioside G,,J and 85% of ganglioside Gte,2aare cleaved by Vibrio cholerae sialidase (5831. Interestingly enough, 50-80% of the sialidase-labile sialic acid residues of the neuronal perikaryon membrane were protected from added sialidase, whereas those of the synaptosomes were found to be accessible to the enzyme [474]. 2.4.2.4. N-Deacetylation-reacetylution Hydrazinolysis of gangliosides under anhydrous conditions leads to a preferential cleavage of N-acetyl residues from sialic acid and N-acetylhexosamine [546]. Specific N-re-acetylation of the product with radioactive acetic acid anhydride allows for a high radio-labelling of the parent ganglioside compound [546]. 2.5. P R E P A R A T l O N A N D A N A L Y S I S
2.5.1. Prepararion
Gangliosides, even though often freely soluble in aqueous media, are not extracted from biological membranes by water, or aqueous 1M KCI and 1 m M ethylenediaminetetraacetic acid [374]. They are, however, solubilized by detergents, e.g., by deoxycholate, and in part by the addition of Triton X-100. For their separation from proteins, it is best to use organic solvents. Gangliosides are extracted from biological material with aqueous chloroformmethanol or tetrahydrofuran [116]. Isolation of the sialo-glycolipid is then achieved by partition against an aqueous phase (“Folch partition”) [117-120,486,519], followed by Sep-Pak@purification [675]. Alternatively, gangliosides can be separated directly from total lipid extracts by sequential elution from anion exchange matrices [121-124,126,1271. The gangliosides
211 can thereby be separated into groups according to the number of sialic acid residues. Single species are then obtained by chromatography on silica [128] or silica-Kieselgur [192]. Based on these techniques, Iwamori and Nagai have devised a useful method for the “mapping” of gangliosides [125]. The application of a liquid partition technique for the separation of gangliosides has also been reported [476]. Gangliosides, can successfully be separated into components by high pressure liquid chromatography. This is performed either with the perbenzoylated derivatives [619,628] or in the underivatized state [620,621]. 2.52. Structural identification and analysis (see Table 3.2) A preliminary identification of gangliosides is achieved by thin layer chromatography [130,474,486,519,629,656]. When an anti-serum is available, specific detection on thin layer chromatograms can be performed by radioimmunoassay [592-5941. For quantitation of gangliosides, thin layer chromatography may be combined with colorimetric determination of single components [28,614]. Microestimations using ‘‘C-acetylation of glycosphingolipids [131]. or of sphingoid obtained after hydrolysis [32], have also been described. The structural characterization of gangliosides comprizes: (a) identification of the nature and number of sialic acid residues and their position along the neutral oligosaccharide core; (b) the neutral oligosaccharide moiety: and (c) the sphmgoid and the fatty acid composition of the ceramide. In recent years, the elucidation of the chemical constitution of gangliosides was greatly advanced by the introduction of newly developed gas chromatographic techniques, particularly in combination with mass spectrometry (for review, see Refs. 133, 134). An initial information of the sialic acid content of a ganglioside as mono-, di-, etc., sialocompound may be derived from its elution profile during anion exchange chromatography [125]. A further step in the characterization is by attempting to cleave sialic acid with Vibrio cholerae sialidase [loo]. Partial hydrolysis of oligosialogangliosides with this enzyme will thereby reveal intermediates in the degree of sialic acid substitution [293]. Dialysis or anion-exchange chromatography separates the liberated sialic acid from the neutral glycosphingolipid for further identification [85]. In the case of the sialic acid proving resistant to enzymatic cleavage, either sialic acid is present as 4-0-Me-substituted derivative or in a “sterically hlndered” configuration (see Section 2.4.2.3). Early structural studies of the total sialooligosaccharide portion of gangliosides were mostly performed by partial hydrolysis followed by identification of the fragment products (for review, see Ref. 99), by Smith periodate degradations [136] or by permethylation [ 1371. The long-chain base sphingoid composition was analyzed by gas-liquid chromatography of the trimethylsilyl derivatives after hydrolysis of the ganglioside under special conditions [ 1491. More recently, mass spectrometry of glycosphingolipids was employed that yielded information on the constituent sugars as well as the ceramide constitution [ 138- 140,4631.
h) CI
h)
TABLE 3.2 Structure
Gangliosides of the gonglio-series G a l p l + 4Glcfi1+ Cer 3
Designation according to
Short notations
IUPAC-IUB Recommendations
Wiegandt Svennerholm
I1 NeuAc-Lac-Cer
Refs.
M3
t
2aNeuAc
'
I1 NeuAc2-Lac-Cer
G a l p l + 4Glcfil- Cer 3
G D3
t
2aNeuAc8 + 2aNeuAc Gal/?l+ 4Glc/31+ Cer 3
I1 NeuAc3-Lac-Cer
T3
II 'NeuAc-GgOse,-Cer
MZ
2
t
2aNeuAcS + 2aNeuAc 8
T
2aNeuAc G a l N A c b l 4 4Gal/?1+ 4Glcp1 + Cer 3
t
2 aNeuAc GalNAcbl + 4 G a l a l + 4GIcbI + Cer 3
113NeuAc,-GgOse,-Cer
G D2
I1 'NeuAc,-GgOse,-Cer
Gl-2
+I
2aNeuAc8 + 2aNeuAc GalNAc@l+ 4Gal/31+ 4Glcp1+ Cer 3
t
2aNeuAc8 + 2aNeuAc 8
t
2aNeuAc Gal/.?l+ 3GalNAcpl+ 4Galp1+ 4Glcp1+ Cer 3 t
II NeuAc-GgOse,-Cer
1
4 4
2 d m’ N
* N
4
0
LL
L
N
c
N
213
TABLE 3.2 (continued) Structure Gal/3l+ 3GalNAc/31+ 4Gal/31- 4Glc/31+ Cer 3
Designation according to IUPAC-IUB Recommendations
Short notation
Refs.
11'NeuAc2-GgOse4-Cer
Gte~2b
GDlh
5
IV3NeuAc-,I13NeuAc-GgOse4-Cer G1,,2a
G Dla
6
Wiegandt Svennerholm
t
2uNeuAc8 + 2aNeuAc G a l S l - 3GalNAc/?l- 4Gal/31+ 4Glc/31+ Cer 2 3 t f laFuc 2aNeuAc8 + 2aNeuAc Gal,% 3
3GalNAcPl- 4Gal/31 3
+
t
-
4Glc/31+ Cer
IV2 Fuc-,I1 NeuAc,GgOse,-Cer
f
2aNeuAc
2aNeuAc
2aNeuAc
1
6 Gala1 + 3GalNAcP1- 4Gal/31- 4Glcj3 + Cer 3
IV NeuAc-,IIl6NeuAc-Gg0se,-Cer
26
t
2aNeuAc Gal/3l+ 3GalNAc/31- 4Gal/31- 4Glc/31+ Cer 3 f 2aNeuAc8 + 2aNeuAc 8
11 NeuAc,-GgOse,-Cer
Gtet3c
G n
6
t
2aNeuAc G a i S l + 3GalNAcBl- 4GalPl- 4Glc/31+ Cer 3 3 f f 2aNeuAc 2 aNeuAc8 + 2 aNeuAc
IV3NeuAc-,I13NeuAc,-GgOse4-Cer GI,, 3b
GTlb
7
Gala1 3
IV3NeuAc2-.I13NeuAc-GgOse4-CerG,,,3a
G,,,
8
t
-
3GalNAc/31
,-..
1-0.
7
-
4Gajl/31+ 4GlcPl- Cer
..
.,..
t
.1
X T .
.
2aNeuAc8 + 2aNeuAc
1
6 Gala1 + 3GalNAcPl+ 4GalS1 + 4GlcP1- Cer 3
IV NeuAc-,III6NeuAc,-GgOse4-Cer
26
IV NeuAc2-.1116NeuAc-GgOse.,-Cer
26
t
2aNeuAc 2aNeuAc 1 6 Gala1 + 3GalNAcPl+ 4Galfi1+ 4Glca1 + Cer 3
t
2aNeuAc + 2aNeuAc G a l P l + 3GalNAcal+ 4GalS1- 4Glc/31- Cer 3 3
t
IV3NeuAc,-,I13NeuAc2-GgOse4-Cer GI,,4b
GQlh
t
2aNeuAc8 + 2aNeuAc 2aNeuAc8 + 2aNeuAc
Gala1 3
-
2aNeuAc8 + 2aNeuAc
1
6 3GalNAcSl- 4Gal/31+ 4Glc/3l
+
Cer
IV NeuAc,-,111 NeuAc,-GgOse,-Cer
t
2aNeuAc8+ 2aNeuAc Galfil+ 3GalNAcSl- 4GalB1- 4GlcS1- Cer 3 3 f t 2aNeuAc 2aNeuAc8 + 2aNeuAc 8
GalSl 3
t
-
IV3NeuAc-.I13NeuAc3-GgOse4-cer G,,&
GQ,,
6
IV3NeuAc,-I13NeuAc,-GgOse4-Cer Glel5c
G,,,
6
t
2aNeuAc 3GalNAc/31+ 4Gal/31+ 4Glc/31+ Cer 3
t
2aNeuAc8 + 2aNeuAc 2aNeuAc8 + 2aNeuAc 8
t
2aNeuAc GalNAc/31- 4GalS1+ 3GalNAcSl- 4Gal/31+ 4GlcSl- Cer 3
t
2aNeuAc
I1 3NeuAc-GgOse,-Cer
9
TABLE 3.2 (continued) Structure
GalNAcPl -t 4Gals1- 3GalNAcPI -. 4Gal/31-. 4Glc/?1- Cer 3
Designation according to I UPAC-IU B Recommendations
Short notations
IV NeuAc-GgOse,-Cer
G,,,lw
23
IV3NeuAc-,II'NeuAc-GgOse5-Cer
GFn, 2a
10
Refs.
W iegand t
f
2aNeuAc GalNAcSl- 4GalPl- 3GalNAcPl- 4GalPI + 4GlcP1+ Cer 3 3
t
-
GalPl 3GalNAcBl 3 f 2aNeuAc
-
-
2aNeuAc
t
2aNeuAc 20
4GlcPl- Cer
Gangliosides of the loeto-series
4GlcNAcpl+ 3GalPl + 4GlcP1- Cer
IV NeuAc-nLcOse4-Cer
11-13
2aNeuAc Galpl 4GlcNAc/31+ 3GalB1- 4GlcPl- Cer 6
IV6 NeuAc-nLcOse4-Cer
3. 20
IV NeuAc-.Ill Fuc-nLcOse,-Cer
16.20
IV NeuAc,-nLcOse4-Cer
29.32
I V NeuAc3-nLcOse4-Cer
29
Galpl 3
r
r
2 aNeuAc G a l P l - 4GlcNAcpl- 3GalB1- 4GlcB1- Cer 3 3
t
2aNeuAc
r
laFuc
Gal@l- 4GlcNAcPl- 3GalP1- 4GlcPI + Cer 3
t
2aNeuAcS + 2aNeuAc Gala1 -. 4GlcNAcfil- 3GalP1- 4GlcB1+ Cer 3
t
2aNeuAcS -.2aNeuAcS + 2aNeuAc
m
8 t
t
N Wl
U
5
t
U
6
t
b
5 t
PL
t
9
PL
9
9
4
9 5
t
5
t
c
3 9t
c
g 3 t
9
c
t + 3
xt
L
s
eb 3
t
5
0
8 t
s
2
5
0
t
s 5 c
9t
xt
3 t
t 0
t
a
G
217
2 00 TABLE 3.2 (continued) Structure
Gangliiiaes of tbe gbbo-series Gal/Il+ 3GalNAcbl+ 3Galal+ 4Galpl 3 f 2uNeuAc
+
4Glcp1 + Cer
Galpl- 3GalNAcSl- 3Galal- 4Galp1+ 4Glcb1- Cer 3 f 2aNeuAc8 + 2aNeuAc
Designation according to IUPAC-IUB Recommendations
Refs.
V NeuAc-GbOse,-Cer
28. 31
V NeuAc,-GbOse5-Cer
28
V 3NeuAc-.V6NeuAc-GbOse5-Cer
32
V 3NeuAc-iGbOse,-Cer
27
2aNeuAc
1
6 Gal/3l+ 3GalNAc/31+ 3Galal+ 4Gal/31+ 4Glc/31+ Cer 3 f 2aNeuAc Galfil+ 3GalNAcPl+ 3Galal- 3Gal/31+ 4Glcfi1+ Cer 3 f 2aNeuAc
Key to literature references: 1, Kobata and Ginsburg, 1972 14521; 2, Ohashi and Yamakawa, 1981 1453); 3, Wiegandt, 1973 14541; 4, Ghidoni et al., 1976 [455]; 5, Sonnino et al., 1978 14561; 6, Ishizuka and Wiegandt, 1972 14571; 7, Kuhn and Wiegandt, 1963 14581; 8, Ando and Yu,1977 14591; 9, lwamori and Nagai, 1978a 12851; 10, Svennerholm et al., 1973 16731; 11, Wiegandt and S c h u k , 1969 1611; 12. Uemura et al., 1978 16091; 13, Li et al.. 1973 1621; 14, Rauvala, 1976 [68]; 15, Wiegandt, 1974 [63]; 16, Keranen, 1976 16091; 17, Watanabe et al., 1978 1691; 18, Watanabe et al., 1974 1641; 19, Suzuki et al., 1975 1491; 20, Watanabe et a]., 1979 11981; 21, Ohashi, 1979b 14711; 22, Hirabayashi et al., 1979 12833; 23, Itoh et al., 1981 11101; 24, Watanabe et al., 1979 11981; 25, Iwamori and Nagai, 1980 14511; 26, Ohashi, 1981 1541; 27, Breimer et al., 1982 14621; 28, Hogan and Chien, 1981 [462]; 29, Murakami-Murofushi et al., 1983 16541; 30, Homes and Hakomori, 1982 15911; 31, Kannagi et al., 1983 16361; 32, Kundu et al., 1983 16371; 33, Watanabe and Hakomori, 1979 1591; 34, Kannagi et al., 1983 [595].
219
For t h s end, glycosphngolipids are permethylated with methyl iodide in presence of dimethylsulfinyl carbanion [640] and hydrolyzed [641] or methanolyzed [642]. After reduction and peracetylation, the partially methylated alditol acetates can be analyzed by gas chromatography-chemical ionization mass spectrometry. This “mass chromatography” yields information on the ratio of the constituent monosaccharides and their linkage positions [148]. Direct probe mass spectrometry of permethylated glycosphingolipids produces ions that are characteristic for the sequence of monosaccharide residues, as well as for the ceramide portion [141-147,463,643-6451. More recent developments of mass spectrometry using negative ion fast atom bombardment, allow the structural elucidation of underivatized gangliosides. With this method, molecular weight, monosaccharide constituent sequence and the molecular species of the ceramide portion can directly be analyzed [648,649]. More specifically, for sequence analysis of the monosaccharides, exoglycosidases can be employed that will also distinguish the anomeric linkages. The anomeric configuration of glycosides can also be assayed by chromium trioxide procedure [646] or with no destruction of the glycosphngolipid sample by proton nuclear magnetic resonance spectroscopy [645,647]. Chemical structures of the gangliosides that have been established until 1983, are summarized in Table 3.2. 2.6. PH YSICOCHEMICAL CHARACTERIZATION (SEE TABLE 3.3)
2.6.1. General Resulting from the molecular combination of hydrophilic carbohydrate that, under physiological conditions, carries a negative electric charge, with a lipophilic moiety of comparable size (the ceramide) the gangliosides are highly polar lipids (for dipole moments, see Ref. 174 and Table 3.3). In addition, the unique properties of both moieties confer complex physicochemical characteristics on these lipids that may not be unrelated to their function in biological membranes. Gangliosides appear as highly rigid structures that allow for tightly packed arrangements, i.e., reducing the mobility of neighbouring hydrocarbon chains [485,581]. On the other hand, gangliosides have molecular properties that apparently promote the contrary effect, i.e., a loosening of lipid phase molecular configurations. It is speculated that the adaptation of such properties to environmental requirements occurs in vivo, by modification of the ceramide constituents (e.g., increase in hydroxyl content) as well as through changes in the carbohydrate portion (e.g., varying the degree of sialylation). The molecular arrangement of the ceramide has some special features that appear to contribute to the physicochemical chariicteristics of gangliosides. Due to the rigidity of the planar amide group, the hydrocarbon tails adopt a preferentially parallel conformation [155]. Ths, in addition to the presence of the trans carbon-tocarbon double bond in sphing-4-enine, promotes condensation of the ceramides into a closely packed arrangement. A further condensation of molecular packing is effected by groups in the
220 ceramide that can partake in hydrogen bonding, e.g., the secondary hydroxyl group of the sphngoid, and the hydroxyl of 2-hydroxy-fatty acids, if present. Hydrogen bonds in the region between the lipophilic and hydrophilic moieties of the gangliosides may contribute to a general hydrogen belt located at the level of the estercarbonyl groups of membrane phospholipids [156]. There is evidence that ganglioside-ceramide in a phospholipid mixture intercalates with the lipid portion of the latter [ 1571. Head group interactions also appear possible, e.g., between ganglioside and phospholipid resulting from a partial matching of opposing electrical dipole vectors [478]. 2.6.2. Gangliosides in solution All gangliosides are soluble in more polar organic solvents, such as alcohols, tetrahydrofuran, dimethylformamide, dimethylsulfoxide or in mixtures of chloroform methanol, with addition of a small amount of water. With the exception of the smaller components Ggall,GIaJ and GJ, the higher gangliosides are lipids that are also freely soluble in purely aqueous media. Their extreme amphipathic nature is influenced very sensitively by the environment provided by the solvent and additional complexing molecules. Thus, in the presence of mono- or divalent cations or protein, gangliosides may have different solubility properties, e.g., in a partitioning system of solvents of different polarity [152,153,476]. In organic solvents gangliosides form monomeric solutions [160,161]. In aqueous systems, however, gangliosides exist as micelles (Table 3.3). The micellar size of gangliosides thereby, in a subtle way, depends on the ratio of C2,,- to C,,-sphingoid, as well as on the sialooligosaccharide [ 1621. The aggregation properties of gangliosides are also influenced by their electric charge, i.e., environmental p H [490] or the presence of metal counter ions [163]. Corti et al. in 1980 [164], using laser lightscattering methods, observed some change in the micellar size of gangliosides at concentrations between and M. Reported values for brain ganglioside micelles range from approximately 200000 to 500000 M , [165] (Table 3.3). The micellar shape is globular and in the form of an oblate ellipsoid. Yohe and Rosenberg [166] suggested that the inner part of the ganglioside micelle may be partly permeable and house penetrating molecules, as shown, e.g., for the triiodide ion. Various physicochemical methods have independently revealed that gangliosides undergo a structural transition in aqueous solution at a concentration range of 10-4-10-5 M. This was interpreted as the critical micellar concentration, below which the gangliosides were assumed to be in a monomeric dispersion [161,162,166-1681. Further evidence for such a transition was obtained by the observation that gangliosides G,,1 and G,J are markedly hydrolyzed by Clostridium perfringens neuraminidase below concentrations that correspond to these previously reported “critical micellar concentrations” [108,169,170].Other data obtained from direct studies in the ultracentrifuge and by gel permeation chromatography, however, make it appear likely that the critical micellar concentration, e.g., for ganglioside G,J (I13NeuAc-Gg4Cer), is in a much lower concentration range, i.e., in the
221
order of lo-’ M [171-173,4901 (Table 3.3). I f t h s indeed were the case, it may be speculated that the unexpectedly low critical micellar concentration results from strong intermolecular non-covalent bonding within the region of the sialooligosaccharide moieties. Such interactions may perhaps become possible because the strong hydrophobic forces in the lipid region orient the sugar portions, and in this way promote micellar aggregate formation. 2.6.3. Gangliosides in membranes and at interfaces Much about the properties which gangliosides show in natural membranes, can be learned from their behavior in artificial model systems (e.g., at the air-water interface (see Table 3.3, for review, see Ref. 479)) or in phospholipid liposomes. According to Czarniecki & Thornton [158,159], gangliosides embedded with their ceramide in a lipid phase have their carbohydrate moiety stabilized by solvation as a network of intermolecular hydrogen bonds “anchored” in solution by sialic acid residues. Thereby the sialic acid neighbouring N-acetylgalactosamine, as, e.g., in gangliosides G,,,l or G,J, shows structural effects that are different from sialic acids positioned elsewhere as revealed by I3C-NMR spectroscopy [611]. This sialic acid residue may adopt a preferred position of its ring being perpendicular to that of the neighbouring N-acetylgalactosamine [157]. It is interesting to note that, according to Maggio et al. [281], the contribution of the sialic acid residues in gangliosides G,J, Glrilor G,J to the molecular dipole moment is opposite to that of a second or t h r d sialosyl group in di- or trisialogangliosides. From electron paramagnetic resonance studies it was suggested that gangliosides are randomly distributed and have their sugars protruding from phospholipid bilayers moving homogenously and comparably unrestricted about 2.5 nm above the interphase [483,665]. Ganglioside sugars, however, under such conditions were reported to be less mobile as compared to the carbohydrate of a glycoprotein such as glycophorin [484]. The surface properties of gangliosides could be studied with films formed with these lipids at the air-water interface. Depending on the charge of the molecules, brain gangliosides show different surface requirements in such films [ 1741 (see Table 3.3). The electric charges of ganglioside molecules due to repulsion and a concomitant larger area requirement of the head group, also induce an increase in fluidity of lipid films or dispersions [ 1741. Fluorospectroscopic studies revealed that indeed the hydrophobic region is influenced by the presence of sialic acid residues linked to the core oligosaccharide, resulting in an increased mobility of lipophilic probing molecules [165]. The lytic properties of gangliosides may also be reflected in their ability to induce membrane fusion in erythrocytes [177] or disruption of the Sendai virus envelope [179]. A role for ganglioside Gte12ain the fusion process of myoblasts to myotubes was also postulated [570]. Ganglioside molecules in artificial membranes tend to cooperate via hydrogen-bonding with clustering, particularly in the presence of physiological concentrations of Mg2+ or Ca2+ [179,483,665]. Lectins may disrupt such clusters of ganglioside molecules [482]. An aggregation of gangliosides around membrane glycoproteins was suggested
TABLE 3.3 Physicochemical properties of gangliosides
Ganghoside
G lac1 GI,2 Gtn1 GI,J
S,.
Stoke's radius (A)
7.8 [2] 6.6 [2] 7.6 121
60[2]
9.7[2] 10.3 [ S ]
60[2]
10191
63[11]
60[2]
M,
CMC
Dipole moment
( X lo3)
(MI
( c . (mD))
322*24[2] 281 *23 [2] 281 i-21 [2] 300k 15 141 337k22 [2] 257 (309)[S]
2 ~10-~[2] 1 X lo-' (21 5 X10-'[2] 7.5 x 10-5 [31 2 X10-8[2] 7 X [9] (sedimentation) 8.5X10-9[9] (gel film.)
Charged
532*50[ll] 244 171 34O+N) [4]
450 [8]
8.5x10-5 [3] 2.8x10-5 [lo] (50 Ac (4.6)) a 8.2X 10-5 [lo]
Surface potential (Wm2/moIecuIe)
Uncharged
Surface area
(A2)
Charged
70111
280[1]
0.95 [l]
1.15 [l]
120[1]
340111
l.lO[l]
1.26111
67 [l] 105 [6] 70[1]
40[1]
270[1]
0.85 [l]
1.02 [l]
75 [l]
Uncharged
60 [l]
GI,, 2 GI,, 2a
9.3 [5] 6.2 [2]
Gl,l2b
5.6 [2] 4.5 [2]
Gm3b
57 [2] 59[11] 57 [2] 55 [2]
257 [5] 181k15 [2] 470540 300 f 30 [4] 160514[2] 113*12[2] 250f25 [4]
1 x10-5[12] 2 X10-6 [2]
1 X10-6 [2] 1 X10-5[2] [3] 1 X
240 [I]
310 [l]
1.59 [l]
1.19 (11
95 [61 103 [l]
71 [l]
500[1]
320[1]
2.44[1]
1.26 [l]
105 [l]
90[1]
* 50Ac (4.6) = Na-acetate buffer, 50 rnM, pH 4.6. Key to references: [I] Maggio, B., Curnar, F.A. and Caputto, R. (1978) Biochern. J. 171, 559-565. [2] Ulrich-Bott, B. and Wiegandt, H. (1984) J. Lipid Res. 25, 1233-1245. [3] Yohe, H.C. and Rosenberg, A. (1972) Chem. Phys. Lipids 9, 279-294. [4] Yohe, H.C., Roark, D.E. and Rosenberg, A. (1976) J. Biol. Chern. 251, 7083-7087. [5] Gammack, D.B. (1963) Biochern. J. 88, 373-383. [6] Halser, H.and Dawson, R.M.C. (1967) Eur. J. Biochern. 1, 61-69. [7] Yedgar. S., Barenholz, Y. and Cooper, V.G.(1974) Biochern. Biophys. Acta 363, 98. [8] Tornasi, M., Roda, L.G., Ausiello, C., DAguolo, G., Venerando, B., Ghidoni, R., Sonnino, S. and Tettamanti, G. (1980) Eur. J. Biochem. 111, [9] [lo] [ll] [12]
315-324. Formisano, S., Johnson, M.L.,Lee, G., Aloj, S.M. and Edelhoch, H. (1979) Biochern. 18, 1119-1124. Rauvala, H. (1979) Eur. J. Biochem. 97, 555-564. Corti, M., DeGiorgio, V., Ghidoni, R., Sonnino, S. and Tettamanti, G. (1980) Chem. Phys. Lipids 26, 225-238. Howard, K.E.and Burton, R.M. (1964) Biochim. Biophys. Acta 84, 435-840.
from studies employing electron paramagnetic resonance spectroscopy [ 1791. In biological membranes, a portion of the ganglioside, strongly bound to integral protein, appears to be present. This is deduced from the fact that some integral proteins, e.g., spectrin-actin-depleted rat erythrocyte cytoskeletal elements, in the presence of neutral detergents tenaciously retain sphingolipids, including gangliosides [491]. In a different approach to studying the behaviour of sialoglycolipids in natural membranes, the lateral diffusion of a fluorescent ganglioside analogue was examined by using the method of fluorescence recovery after photobleaching [469]. With an apparent diffusion constant in the order of DDirr5 X cm2/s, and a fractional fluorescence recovery of R 80-loo%, the fluorescent ganglioside analogue shows an unrestricted lateral diffusion not much slower than that of a typical freely diffusing lipid probe [468,677]. 2.6.4. Cation binding to ganglioside Because of their acidic nature and their concentration in neuronal membranes that are probably active in ion fluxes, the gangliosides were, from early on, suspected of playing some role in special cation binding and transport or release mechanisms [180,5271. Gangliosides bind divalent cations [ 1821. Ca2' thereby complexes to gangliosides twenty-times more effectively than Mg2+. Ca2+ binding to ganglioside depends drastically on the position of the sialic acid residue along the neutral oligosaccharide M, chain. The sialic acid in G,,,1 complexes Ca2' above a concentration of whereas in the terminal position, e.g., in Gtet2ait is above l o p 6M. In contrast, sialic acid linked to sialic acid, as in ganglioside Gl,,2b, binds very little Ca2+ [82,478]. The degree of Ca2' complexing influences the structures of ganglioside aggregates in solution, and possibly also in membranes [613]. This was concluded from changes that occur with increasing Ca" concentration in the Ca2'-binding stoichiometry [183,184], as well as in the solvent and surface-labelling properties [153,613]. Solvent partition experiments have also shown that Ca2+ may complex gangliosides to protein [185]. The Ca2+ binding to gangliosides in turn is influenced by various other cations. Notably, tubocurarine was found to be very effective in the displacement of Ca2' from ganglioside [186]. '3C-Nuclear magnetic resonance spin lattice relaxation data revealed that metal ion binding to ganglioside G,,,l occurs via carboxyl- and the "glycerol" side chain of the sialic acid. Additional ligands are donated by the N-acetylgalactosaminyl-pyranoside and the terminal galactose residue [159,187-1891.
3. Distribution of gangliosides 3.1. ANIMAL SPECIES
Gangliosides occur, as far as available data show, only in animals of the Deuterostomia. whereas they have not been detected in Protostomia.
225
More detailed data that allow for a comparison of the gangliosides of different animal species are available for the brain [75,190,488,489]and, to a lesser extent, for the sphngolipids of erythrocytes [36] and the thymus [190]. Mammals, birds, amphibians and teleost fish, all have similar brain gangliosides. These gangliosides are of the ganglio-series with, predominantly, gangliotetraose as neutral core oligosaccharide, though with a varying degree of sialylation. There are, however, phylogenetic differences in the concentrations of brain gangliosides: the lower vertebrates (reptiles amphibia, fish) having 110-500 pg; and the higher vertebrates (birds, mammals) with 500-1000 pg lipid-bound sialic acid per gram brain fresh weight [489]. Among the species, variabilities in the concentrations of brain gangliosides are again found, e.g., fish with 160-390 pg lipid-bound NeuAc/g tissue fresh weight [492]. In contrast to the brain, the erythrocytes show drastic species-dependent qualitative, as well as quantitative, differences in their ganglioside components. Thus, typical major gangliosides of the red blood cells of various animals are: mice, G,J and Gtil; rats, Gtet2a;man, dog and pig, GIJ; cats, Gl,,2; and cattle, NeuAcnLc,Cer. For the gangliosides of the thymus, pronounced species dependence of the component distribution, although different from that of erythrocytes, were reported. It appears, however, that thymus gangliosides of all the species investigated are derived mostly from the lacto-series [190,192,495]. For the brain gangliosides, an evolutionary trend to lower animals seems to be reflected in a shortening of the average neutral oligosaccharide chain length. Whereas gangliotetraose is the core sugar predominant in mammals, birds, teleost fish and ganoids (sturgeon), lactose and gangliotriaose, respectively, are the neutral core saccharides of the brain gangliosides of elasmobranchs, ray and cartilaginous fish [192,193]. The gangliosides from the starfish, Asterina pectinifera, contain ceramide-linked lactose substituted by sialic acid residues [194], whilst the sea urchin has gangliosides derived from only glucose [45-471. Another evolutionary parallelism concerns the ceramide composition of brain gangliosides. In their progressive evolution, the brain gangliosides of vertebrates show an increase in the degree of fatty acid saturation and the relative content of C,,-sphngoid [192,194]. In addition to the systematic position of the species, environmental factors, such as temperature of the dwelling of the animal, affect the composition of brain gangliosides. Thus, changes in the degree of sialylation and in fatty acid composition of brain gangliosides appear to be involved in temperature-adaptive mechanisms (for review, see Ref. 493). Compared to warm-blooded animals, the brain gangliosides of poikilotherms are more highly sialylated, promoting a membrane fluidisation, which was interpreted as an adaption to cold environmental conditions [193,195,196,494] (see Section 4.3.3).
226 3.2. TISSUE DISTRIBUTION OF GANGLIOSIDES
3.2.1. General distribution Gangliosides are present in most, if not all, mammalian tissues [197]. The highest concentration of total gangliosides is found in brain grey matter [200]. Whereas the gangliosides of the central nervous system are mostly derived from the gunglio-series, those of peripheral nerves (approx. 0.11 pmol Neu per g wet weight [1971) and of extraneural tissues (approx. 0.1-0.35 pmol Neu per g wet weight [200]) contain a high proportion of gangliosides of the lacto- [88,461,464,496] or globo-series [462,463]. Ganghosides of the gunglio-series have, however, also been detected at extraneural sites. If, indeed, the presence of ganglioside G,,J confers sensitivity of a cell towards cholera toxin (see Section 6.4) then a ganglioside of the gunglio-series must be present rather ubiquitously. 3.2.2. Central nervous system A detailed analysis of the brain ganglioside component distribution of various animal species was given by Ando et al. in 1978 [28], and by Hilbig and Rahmann in 1980 [489] (for review, see Ref. 209). Expressed as per gram tissue fresh weight, mammalian grey matter has roughly three-times as much ganglioside as white matter [ 129,201,2021. The predominant gangliosides of brain grey matter of mammals are Glell,2a, 2b, 3b and 4b, whereas the major component of brain wlute matter in primates and chicken is G,,,1 with variable amounts of G,,,l [28,203,630]. Amphibia and fish myelin contain no ganghoside GJ [630]. The gangliosides, G,,J and Gtell,are characteristic constituents of differentiated mature oligodendroglial myelin [27]. The component pattern of oligodendroglial perikarya is more complex and contains, in addition to Ggalas major ganglioside, Gla,l, G,,,2 and G,J [205,206], and also Glell, 2a, 2b and 3b [27]. The latter four gangliosides are typical components of neurones and neuronal processes. These, as well as astrocytes, probably do not contain the ganghoside Ggall.Higher sialylated ganghoside components, G,,,4b and G,,,4c as well as hexa- and heptasialogangliosides are typical of early embryonic chicken brain [461]. 3.2.2.1, Cells ofthe nervous system in culture Cells derived from the central nervous system have been cultured and used as a model system of minimal complexity in the study of the presence of gangliosides and regulation of their biosynthesis (for reviews, see Refs. 220, 221, 566). There are, of course, certain constraints on conclusions drawn from the observation of gangliosides in cultured cells with regard to the tissue of origin. Thus, established cells of neuronal or glial origin, that indeed retain many of the electrophysiological and enzymatic characteristics associated with neurones or glial cells, show in general a glycosphingolipid profile not very typical of fresh brain tissue-derived cells, e.g., in their having a neutral tetrahexosyl-ceramide and a lack of tugher oligosialogangliosides (no G,,,3 and 4, only traces of G,,,2b) [221-2231. In addition, such cells do not synthesize the higher gangliosides that are typical for neurones of various types in the central nervous system even in the
227 presence of differentiation inducers and neurite outgrowth [224]. Astroblasts in primary culture (96% Gla,l, 4% G,,,2) or other glial tumor cells show only G,,,1 and 2 [207,208]. Coculturing of established neuronal and glial cells, however, induces synthesis at low levels of the gangliosides G,,,2b and G,,,3 that persist after reisolation of clones [221]. Neuroblastoma cells grown in vivo also produce ganghoside G,,,3. Primary cultures of isolated neurones or glial cells possess ganglioside patterns very close to the tissue cell fractions [567,568]. The homogeneity of cell types in culture allowed a reliable comparison of gangliosides of neurones and of glial cells. Thereby, the gangliosides G,,,2b, 3 and 4 were found to be higher in primary neurones than in ghal cells, even though the latter also contained appreciable amounts of gangliosides G,,,2b, 3 and 4 [221]. As with tumor cells, coculture of primary cells, astroglia and neurones change the expressed ganglioside pattern [2211. 3.2.3. Peripheral nerves Gangliosides have been identified in peripheral nerve tissue, including the neuroand adenohypophysis, the adrenal medulla and the visual tract. The gangliosides of the peripheral nervous system are rather different from those of the central nervous systems. They consist of ganghosides, mostly Gla,l, G,,,2 and NeuAc-nLc,Cer. Whereas, e.g., G,,J and G,,,2 are the major components of trigeminal nerve, ganglioside NeuAc-nLc,Cer was found to be localized mainly in the peripheral nerve myelin [670]. Even though more work on peripheral nerve gangliosides has been reported, they still appear insufficiently characterized: human femoral nerve [1971, rabbit sciatic nerve [226], rat sciatic nerve [227]. The neurohypophysis of cattle contains a high concentration of gangliosides, similar to those of the central cortex, except for a high proportion of N-glycolyl- in addition to N-acetylneuraminic acid [228]. In contrast, the adenohypophysis shows a decisively different ganglioside profile with a high proportion of gangliosides G,,JNeuGc and G,,lNeuAc. Similar to the adenohypophysis, with a component pattern resembling more that of extraneural sources, the ganghosides of the adrenal medulla consist of up to more than 90% of G,,J [229] *. Notably, in this case, in the membrane of an intracellular structure the adrenal chromaffin granules were shown to contain ganglioside [230]. The gangliosides of the visual system, including lens, retina, optic nerve fibres and the tectum opticum, have been studied in detail by several groups. The optic pathway offers certain advantages for investigations of this part of the central nervous system, such as structural simplicity and its light excitability [231]. The structural elements of the optic pathway provide more evidence that the higher gangliosides G,,,l, 2a, 2b and 3 are characteristic for ganglion cells. Lens and iris have some 90% of their ganglioside content as G,,,1 [232,233], whereas mammalian retina consists mostly of G,,,2 ( 3 5 4 6 % of total ganglioside) *
Characterization of ganglioside G,,,3b from bovine adrenal medulla has also been reported [671].
228 (234,2351. All other gangliosides of the retina are the same as those found in the brain. Gangliosides of retina show, however, species specificity. Thus, chcken retina - retinal ganglion cells as well as their presynaptic terminals [499] - contains, in addition to G,,1, predominantly Gl,,2a [231,236]. In frog photoreceptors two gangliosides were detected, tentatively identified as G,,,2a and 3 [237]. From subcellular fractionation experiments there is an indication that gangliosides G,,1 and 2 of the mammalian retina are predominantly localized in the rod outer segments of the photoreceptor, whereas the hexosamine-containing gangliosides may be located in other retinal elements [235,238-2411. It is probable that the hexosamine-containing gangliosides in this location are characteristic for the retinal ganglion cells. In support of this, in mammalian optic nerve, i.e., the axons of retinal ganglion cells, no ganglioside G1,,2 was detected [239]. Instead, the four major brain gangliosides Gl,,l, 2a, 2b and 3 constitute more than 90% of the glycolipid. 3.3. CELLULAR LOCALISATION
As with other glycosphingolipids, it is generally assumed that the gangliosides occur on the outer side of the cellular plasma membrane [250,252-256,6051. In the case of neurones, this includes elements of the nerve endings [257]. This location of gangliosides in conjunction with their complex physicochemical character, in particular with regard to calcium ion binding, has stimulated hypotheses for a functional role of gangliosides in synaptic transmission and memory formation [498]. It is not yet clearly established whether or not gangliosides are evenly or unevenly distributed on the outer neuronal cell surface (for review, see Ref. 10). Whereas, the neuronal cell bodies have a ganglioside content that is lower than that of whole brain [211,212], fractions containing nerve endings show enriched levels of sialoglycolipids [213-217,5691. This, however, might be explained by surface area considerations. Synaptic junctions from adult humans are particularly rich in the higher gangliosides G,,,2b, 3b and 4b [218]. On the other hand, Morgan et al. [219] have isolated synaptic junctions almost devoid of gangliosides. Engel et al. [569], on the contrary, have observed an enrichment of cholera toxin peroxidase labeling of ganglioside G,,J in the synaptic cleft region. Perikaryal as compared to synaptosomal gangliosides show a differential accessibility to bacterial sialidase [474] (see Section 2.4.2.3). Low levels of ganglioside * have also been localized intracellularly in membranal structures of the Golgi apparatus and the endoplasmic reticulum, and also possibly in lysosomes [72,242-2451. It is still an open question, as to whether or not the intracellular ganglioside merely belongs to a transient pool of membrane constituent recycling or is in the process of metabolism. This could also be the case for the soluble ganglioside in the cytosol [72,245,247,652]. However, the ganglioside that was detected in other intracellular structures, the chromaffin granules [230] or in the sacroplasmic reticulum [249], may well serve some special biological function.
*
Matyas, Morr.6 and Keenan (1982). however, report that their experiments indicate that less than 65% of the total ganglioside of rat liver is in the plasma membrane 16331.
229
4. Metabolism 4.1. BlOS YNTHESIS
The biosynthesis of gangliosides proceeds by stepwise addition of monosaccharide units onto the growing carbohydrate chain of the glycosphingolipid, and biodegradation by removal of single monosaccharide residues (for reviews, see Refs. 258-263). Gangliosides are comparatively long lived lipids, those of retinal cells all having similar turnover rates, with half lives of 34-38 days [499]. The transer of nucleotide-activated monosaccharides is primarily controlled by glycotransferases. Some evidence indicates that they may be multienzyme complexes as was postulated by S. Roseman in 1970 [263] and R. Caputto in 1971 [264,301]. According to this view, two pools would exist: one, a very small transient pool containing gangliosides in the process of biosynthesis, e.g., on multienzyme systems; and, the other, a ganglioside end-product pool predominantly at the plasma membrane [226,583]. The latter gangliosides cannot be returned to the transient pool, e.g., for repair synthesis. The biosynthesis of gangliosides on specific multienzyme systems could also help to explain why there is a connection between the constitution of the sugar moiety, e.g., the degree of sialylation of a ganglioside and that of the ceramide, e.g., content of C,,-sphingoid. The subcellular site of biosynthesis of gangliosides may largely be located in the Golgi membranes [242,267,268] (for reviews, see Refs. 269, 270). After intracellular synthesis, the gangliosides reach their destination at the cell surface within some 30 min, perhaps participating in a transport mechanism common to other plasma membrane constituents [584]. In nerve cells the primary site of ganglioside biosynthesis appears to be in the neuronal perikarya [271]. It is speculated that, from the cell soma, the gangliosides may then be translocated to the nerve endings, where further sialylation possibly takes place [272] *. The key steps in the biosynthetic pathway to higher sialylated gangliosides of the ganglio-series (pathway I) is the sequential transfer of sialic acid to lactosylceramide [273]: followed by addition of N-acetylgalactosamine to ganglioside G
- -
Pathway I Lac-~er
CMP-NeuAC Lac-Cer UDP-GalNAC GalNAc-4'LaC-Cer 3' step 1
t
NeUAC
Step
2
(Giocl)
1'
NeuAc (Gtr, 1 )
Pathway II
UDP-GolNAcGalNAC-4'Lac-Cer UDP-GalGal-3GalNAc-4'Lac-Cer
-
Lac-Cer
CMP-NeuAc
*
NeUAC-3Gal-3GalNAc-4' L a c - c e r ( G tet 1 w )
Dreifus et al., (1981) report the presence of ecto-glycosyltransferase (NeuAc, Gal, Fuc) on chick embryonic neurone primary cells [579].
230 Omitting the sialic acid transfer (Pathway I) other glycolipids of the ganglio-series could be formed by Pathway 11. Such compounds include: the neutral glycosphingolipids, gangliotriaosylceramide from guinea pig erythrocytes [274] (also detected as a characteristic constituent of neuroblastoma NB41A cells [20]. KIMSV tumour cells in Balb/c mice [275] and mouse lymphoma (LS 178c 127) cells [282]) or gangliotetraosylceramide found in immune-cells of rat and mice [276-2811. There also exist extraneural gangliosides originating by biosynthesis via Pathway I I, such as: G,,,lw (IV3NeuAc-GgOse,Cer) present in human erythrocytes [198], rat hepatoma cells [283], and mouse lymphoma L 5178; Gpentlw(IV3NeuAc-GgOse,Cer) [284] or the gangliosides discovered by Ohashi [460] in the frog brain (e.g., IV3NeuAc-. 1116NeuAc-GgOse,Cer). Little is known of the in vivo signals received by the cell that may regulate biosynthesis of gangliosides. Examples of such regulation are the enhancement effect of cocultivation of neuronal and glial cells on the biosynthesis of higher gangliosides. and similarly the increased amounts of gangliosides present when cells are grown as a solid tumor as compared to single cells in culture. The major route for biosynthesis of the higher sialylated classical brain ganglioside proceeds via pathway I a, b, and c: G a l - G l c - C e r 4 Gal-Glc-Cer ----o keUAc
I
GcllNAc-Gal-Glc-Cer bUAC
I
I
Gal-Glc-Cer t+UAc NeUAC
-
GalNAC-G?l-GlC-Cer ?WAC yeuAc NWAC
GalNAc-Gal-Glc-Cer (JeuAc NeuAc
I
I I
GVI-Glc-Cer YeuAC YeuAc NeUAC
Gal-GalNAc - Gal-Glc-cer keunc
Gal-GalNAc- Gal-Glc-Cer t+Ac NeUAC
Gal-GalNAc- Gpl-Glc-Cer YWAC yUAC NeuAC
Gal-GalNAc- Gal-Glc-Cer keUAC keuAc
G:I-GalNAc-G?l-Glc-Cer NeUAC YeuAc NeuAc
Gpl-GalNAc- GaCGlc-Cer NeuAc @UAC YeuAc NeuAc
I
I
I
4.2. BIODEGRADATION AND STORAGE DISEASES
In the past, particular attention was paid to the enzymes and enzyme activators participating in ganglioside biodegradation because of their involvement in certain hereditary diseases (for reviews, see Refs. 305, 306, 565). Apparently, all gangliosidoses are caused by deficiencies in the activity of degrading hydrolases. The reason for this may be missing or faulty enzyme or activator proteins that bind the ganglioside and present it to the enzymes. Table 3.4 is a classification of gangliosidoses according to the basic defects. “G ,,-gangliosidosis” with an accumulation of ganglioside Gt,,l is believed to result from a structural gene mutation causing the biosynthesis of a P-galactosidase
231 with a much decreased activity. This was concluded because near normal levels of P-galactosidase protein could be demonstrated in this disease [307]. Classical Tay-Sachs disease B and 0 variants are caused by a reduction or absence of ganglioside hexosaminidase. An explanation for the interrelationship of the gangliosidosis variants can be derived from the structural constitution of these enzymes [309,310]. Hexosaminidase A consists of subunits a2 and /I2, each containing two peptide chains linked by a disulfide. Hexosaminidase B has a P2P2 configuration with only one type of peptide. Hexosaminidase S is another enzyme found only in trace amounts. It consists of a2a2subunits only. This explains why hexosaminidase A never occurs without concomitant expression of hexosaminidase B [311]. It is concluded that in variant B of Tay Sachs disease, the a-chain, and in variant 0 the P-chain, of the hexosaminidase are inactive or missing. The degradation of gangliosides by specific hydrolases is greatly enhanced by low molecular weight ( M , , 22 000-25 000 [503]) glycoprotein activators, first isolated from human liver [312,313,504]. The activators are more or less specific for the gangliosides to be hydrolysed. Thus, one activator has been isolated that enhances the hydrolysis of ganglioside G,,J to G,ril by human P-galactosidase [313], and another for degradation of ganglioside G,J to G,,J by P-hexosaminidase [314]. It appears that the specificity requirements of the latter activator glycoprotein are also met when a sulfate group replaces the sialic acid residue, as in the sulfatide I13S0,-GgOse3Cer. This compound, present in rat kidney [472], shows increased hydrolysis by sulfatase in the presence of G,,,l-specific P-hexosaminidase activator (Li and Ishizuka, personal communication). A possible mechanism for the action of glycosphingolipid hydrolase activator protein has been suggested [316,317]. According to this view glycolipid monomers are complexed by the activator protein [503]. For enzymatic hydrolysis, the glycoTABLE 3.4 Ganglioside storage diseases Gangliosidosis
Defect
Storage product
G ,,-gangliosidosis
8-Galactosidase
II'NeuAc-Gg,Cer
Tay-Sachs disease (Variant B)
Hexosaminidase A, S (defect a-chain)
I13NeuAc-Gg,Cer a
Tay-Sachs disease (Variant 0)
Hexosaminidase A, B (defect 8-chain)
11'NeuAc-Gg,Cer Gg'Cer, Gb,Cer
Tay-Sachs disease (Variant AB) New type AB-variant (502)
Activator protein for hexosaminidase /3-Hexosaminidase
I1 'NeuAc-Gg3Cer
Mucolipidosis I1 (I-cell disease)
Acid neuraminidase
113 NeuAc-Gg,Cer
In Tay-Sachs brain, increased amounts of the following gangliosides were also detected: &,l, GF,,lw and Gpen,2a[110,308].
a
232
lipid as well as the activator simultaneously bind to the respective enzyme [316]. This may help to explain why the activator also determines the specificity of a hydrolase towards a glycolipid [317]. Thus, hexosaminidase B is more active towards GgOse,Cer than hexosaminidase A. However, only hexosaminidase A activity against substrate ganglioside GJ, and not that of B-enzyme, is enhanced by activator [318]. This could be the reason for the storage of ganglioside Gtrilin the B-variant of Tay-Sachs disease. Whereas in one type of Tay-Sachs disease AB-variant (AB variant = phexosaminidases A and B, both are present), the accumulation of ganglioside Gtrilis caused by a defect in the enzyme P-hexosaminidase A [502], another type of AB variant shows only a deficiency of an activator for this hydrolase [ 3191. Other sphingolipid storage disorders that are caused by a deficiency of catabolic enzymes not quite directly related to the ganglioside metabolism, by way of a derangement of glycolipid turnover, can also produce an increase of gangliosides, as shown in the case of Gaucher’s disease [602]. 4.3. CHANGES I N I N VIVO COMPOSITION
The component distribution of the gangliosides for a given cell is not always constant. It may change considerably as a result of the interplay of the varying activities of metabolizing enzymes. Occurring ganglioside pattern changes are, however, within the respective cell-specific sialooligosaccharide series. The factors that regulate such changes are not known. The ganglioside profile, significantly and sensitively, depends on the developmental state of the cells and their growth condition. From studies on the changes in the ganglioside pattern it is hoped that perhaps yet unknown functional aspects of these membrane constituents might be revealed. Other changes of gangliosides under pathological conditions, e.g., in the central nervous system, may signal a metabolic derangement with the disappearance of certain cell types by destruction, perhaps even aggravated by autoimmune events, as seen, e.g., in multiple sclerosis [538] (see Section 5.2). 4.3.1. Developmental changes Developmental alterations of ganglioside composition were investigated mostly with embryonal, postnatal, adult and senescent brain, but also with other tissues [510] or with cultured cells. As for sialic acid-containing glycoconjugates, it appears possibly to be a general trend that during embryonal until postnatal life the degree of substitution with sialic acid residues decreases (for reviews, see Refs. 320, 509). Gangliosides are already prominent constituents of the central nervous system at foetal stages of the brain development. Thereby, the quantity of gangliosides increases particularly during periods of rapid outgrowth of dendrites, axons and the formation of neuronal interconnections. But the ganglioside pattern also changes, as has been shown in mammals [321-3231, birds [324-326,248,609,507,569,6691, fish [327] and amphibians [329,669].
233 The available data from different laboratories are not always unequivocal, but justify the following generalizations. At “ very early stages” of development of the embryonal nervous system, gangliosides may function in the transition of neuroblasts into functionally mature neurones. Whereas premitotic cells have a high proportion of G ,ac2,this ganglioside is replaced by higher gangliosides, e.g., Gle12a during the formation of synaptic and dendritic membranes. Gangliosides G,,,3 and G,,,4 are formed during synaptogenesis and thereafter. At “early foetal stages”, the brain gangliosides of birds and mammals show a preponderance of comparably more highly sialylated compounds with Gle13a,3b and 4b more common than Gle12a and 1. Similarly, in embryonic extraneural tissues, chick thigh and leg muscles, the prenatal ganglioside G,,,2 and a GlcNAc-containing di-sialo-component are predominant, whereas the less sialylated ganglioside becomes the major sialo-lipid postnatally [510]. G During “later” foetal brain development, the tri- and tetrasialogangliosides, Gle13a,3b and 4b, decrease in relative amount in favour of Gle12aand 1. At hatching or birth, ganglioside G,,2a is the major brain ganglioside over G,,,3b, 2b and 1. A timely increase in the level of ganglioside G,,J appears to reflect myelination during embryogenesis [609]. The high foetal multisialoganglioside component distribution of birds is similar to that of the adult fish brain [329,330,334,609]; this may be suggestive of certain links between phylogenesis and mammalian brain ontogenesis [348]. Indeed, the brain ganglioside composition during ontogenetic development appears to reflect a phylogenetic recapitulation. During phylogeny to higher species an increase in brain ganglioside concentration is accompanied by a decrease in the more highly sialylated molecular components [508]. At present, a general interpretation of the developmental changes of brain gangliosides is difficult. They may represent alterations in morphology, but equally also reflect differentiation and maturation of membrane structures. Ganghosides Gte,3b and 4b perhaps are typical of early completed connections of foetal brain, whereas ganglioside Glel2a might reflect a later stage and newly completed structures. Gangliosides G,,J and G,,J are typical of mature myelination. A change in cerarmde composition with increasing age was shown for individual human brain gangliosides [332]. Until the age of ten, the ratio of C,, to C,,-sphingoid increases rapidly. It then levels off with 60-70% C,,-sphingoid after 30 years. The fatty acids of ganglioside-ceramide also change with age. At birth, 93% of the ganglioside fatty acids is stearic acid, whereas at age 98 only 78% of this C,,-fatty acid is present. At the same time C,,-fatty acids increase from 3 to 9%. 4.3.2. Changes after nerve stimulation Studies have been reported that deal with the effects of physiological stimulation on ganglioside composition. Thus, the rod outer segments of calf retina show a 40% increase in ganglioside content after stimulation with light, with no change in their component distribution [231]. Behavioural stimulation with rats that were forced to swim in a deep water tank, caused an increase of brain ganglioside G,,,3 with
234 concomitant decrease in G,,,2b [333]. When the weak electric tapir fish was stimulated with a tapir fish dummy, an increased ganglioside biosynthesis could also be observed [511]. 4.3.3. Temperature-adaptive changes in the brain Rahmann and co-workers [512] discovered that the polarity, i.e., the degree of sialylation of brain gangliosides of poikilothermic animals is correlated with the climatic temperature of their habitat. Such animals have increased proportions of more highly sialylated gangliosides the lower their environmental temperature [493]. Thermal adaptation by changing temperature appears possible. Thus, during hibernation, animals have more polar brain gangliosides as compared to those under non-hibernating conditions [513]. Rahmann also speculated that, during the neonatal heterothermic development of birds and mammals, their gangliosides are still correlated with t hermoregulation [493,4941. 4.3.4. Changes in disease The destruction of certain cell types in the central nervous system by disease may be reflected in a quantitative as well as qualitative alteration of the content of ganghoside components. A preferential elimination of nerve cells in Creutzfeld-Jacob subacute spongiform encephalopathy results in a drastic decrease of gangliosides. Thereby, those components are preferentially affected that are typical for neurones, i.e., those with a high content of the C,, long-chain base [635]. In multiple sclerosis, the gangliosides of the central nervous system also show abnormalities. Sclerotic plaques have a complete loss of ganglioside Gga!l and a decrease of G,J and GJ as compared to normal white matter. Interestingly, an elevation of the disialo- and higher oligosialogangliosides in plaque was reported [538]. Tumor cells shed constituents of their plasma membrane. Also among these are ganghosides typical for the respective type of malignant cell of origin. This may explain the increased levels of malignant melanocyte-specific gangliosides in the circulation of tumor bearing individuals [657]. 4.3.5. Changes at the cellular level 4.3.5.1. Normal growth conditions The glycosphingolipid composition of cells, including that of the gangliosides, changes according to the cellular growth phase. Reduction in cell growth or cell arrest, e.g., by cell contact, serum deprivation or induction by drugs, may cause alterations in glycosphingolipid profiles, with an increase in chemical quantity [335,336,340,653]. Furthermore, the mode of growth, in monolayer as compared to suspension culture, can influence the ganglioside pattern in a direction similar to that observed in resting versus rapidly dividing cells [334]. In addition, developmental changes may also be paralleled by an increase in ganghoside. Thus, during differentiation of a cloned rat myoblast cell line to myotubes a three-fold elevation in a ganglioside, tentatively identified as G,,, 2a, was
235 observed [570]. During cell aging, as shown in the case of human fibroblasts, higher gangliosides are drastically decreased with concomitant relative increase in G lacl [631]. In Balb/c 3T3 mouse fibroblasts, the synthesis and quantity of ganglioside G,,J and 2a is particularly h g h at early stages of cell contact; in parallel with a decrease in sialidase activity at the touching phase of the cells [338]. In contrast to this, in plasma membranes obtained from transformed oncogenic cells, as compared to their normal parental untransformed cells, an increase in sialidase activity was observed [339]. In C1300 mouse neuroblastoma cells also, sialidase activity increased in confluent cultures with a concomitant reduction of total ganglioside, but no change in ganglioside composition [653]. The cell contact phenomenon, with an increase in ganglioside G ,aclor G,J concentration in 3T3 mouse fibroblasts, can apparently be mimicked by adding antiganglioside G laclor G1,,1 antibody (Fab-fragment) to the cells [440]. An increase in cell proliferation, however, is not always accompanied by a decrease in the biosynthesis of higher, more complex gangliosides. When stimulation of cell division is paralleled by the expression of differentiation properties, such as in lectin-induced changes in lymphocytes, an elevated biosynthesis of gangliosides can be observed [277,341,342,344]. As yet unrelated to such ganglioside changes as described above, it was reported that treatment of human platelets with thrombin caused a two-fold increase in concentration of G,,J within 10 min [344]. 4.3.5.2. Changes related to cell transformation Oncogenic transformation results in the loss of growth regulation mechanism such as the cell-to-cell contact-dependent inhbition of growth. This loss is generally paralleled by an irreversible reduction in the levels of more complex neutral glycosphingolipids and gangliosides. A reversible phenomenon in a similar direction may be seen in vitro with normal, non-transformed cells that are in a state of rapid division as compared to the resting state, and in vivo with foetal as compared to adult tissues [345] (for reviews, see Refs. 260, 347-349,371). The change in ganglioside biosynthesis in cells undergoing transformation could in many instances be correlated to reduced activities of corresponding glycosyltransferases, thus explaining increases in neutral precursor glycosphingolipids or less complex gangliosides [351,517,5201. There are, however, exceptions where, instead of a decrease, an increase in apparently more complex ganglioside (G,ril)was observed after transformation with an oncogenic virus [518,519]. Infectious processes or drugs that inhibit protein biosynthesis may also interfere with the production of biosynthetic enzymes. The result is a change in ganglioside component profile similar to that generally observed after cell transformation [286,288,352,353,419]. 4.3.5.3. Drug-induced ganglioside changes Since little is known of how ganglioside biosynthesis is regulated by cell factors, drugs that have an influence on ganglioside metabolism may possibly help to shed light on the regulatory mechanism involved in glycosphmgolipid metabolism.
236
An inhibition of ganglioside biosynthesis was observed, when opiate receptorpositive rat neuroblastoma cells were treated with P-endorphin, enkephalins and opiates [354,501,625]. Since these agents effect an alteration in the level of cAMP in the cells with an initial decrease in concentration, it was speculated that ganglioside biosynthesis is somehow regulated by this nucleotide, and hence via a CAMP-mediated kinase system. This hypothesis is supported by the finding that agents which raise cAMP levels, such as cholera toxin, prostaglandin E, , phosphodiesterase inhibitors and dibutyryl- or 8-bromo-CAMP derivatives, also stimulate ganglioside biosynthesis [501]. On the other hand an involvement of protein kinase C in the stimulation of ganglioside biosynthesis is suggested by the action of phorbol esters on cells [638,639]. Other reports on the stimulation of ganglioside biosynthesis by dibutyryl-CAMP are, however, controversial [225,337,354,355]. It is also not clear, whether or not in cases where effects of dibutyryl-CAMP were observed, these were due either to the drug itself or its products of hydrolysis, cAMP or butyrate. An increase of cellular ganglioside biosynthesis is observed following the addition of short-chain fatty acids, including propionate, butyrate and pentanoate. The effect of butyrate could be related to an induction of a CMP-sialic acid Aactosylceramide sialyltransferase, while other glycosyltransferases remain unaffected [286-2881. Norepinephrine, which increases the cAMP level in cells, also stimulated ganglioside biosynthesis in C 1300 mouse neuroblastoma cells due to increased activity of a UDP-GalNAc: G GalNAc-transferase [3561. 4.3.5.4. Changes by uptake of exogenous-ganglioside It is still largely unknown, which particular biological properties of a membrane result from the presence of ganghosides. To solve this problem with a different approach, attempts were made to artificially incorporate exogenous gangliosides into cells. Gangliosides are taken up from the incubation medium by cells or isolated cellular membranes [357]. This uptake is saturable and may depend on the presence of calcium [528]. The cell-associated ganglioside is detected at the cell surface by galactose oxidase-labeling or susceptibility to neuraminidase [586]. Ganglioside uptake varies with the cell type and cell growth conditions, e.g., monolayer versus suspension cells [359,360].In addition, a quantitatively different uptake of exogenous ganglioside is observed with cells in M-phase as compared to randomly growing cells [360,514]. Exogenous ganglioside becomes membrane-associated by different modes of binding. Part of the added sialoglycolipid may be released from the cells by mild trypsinization or incubation with serum or serum albumin [514].During longer incubation with ganglioside (a few hours at 37 "C),the serum- and trypsin-stable portion increases, and a true insertion of the glycolipid into the lipid bilayer of the biological membrane is achieved. Sialoglycolipid incorporation into the membrane lipid phase could be shown with fluorescent or electron paramagnetically labeled ganglioside analogues [470,472,5 1 4 315,5641. When ganglioside is added to preformed phospholipid liposomes, the sialoglycolipid is rapidly incorporated into the membrane bilayer [361-363,5601. Such liposome-associated ganglioside is not removed by incubation of the liposomes with
237 serum, serum albumin or chase-ganglioside. There is no indication that ganglioside incorporated into the outer lipid bilayer leaflet undergoes “flip-flop” to the inner leaflet or that it may disrupt the bilayer integrity of the liposome [560]. Incubation of intact cells with exogenous sialoglycolipids results in their slow lysosomal degradation and also in an increased biosynthesis of more complex gangliosides [291,666]. Thus, free ganglioside added to primary cultures of chick embryonal neurons was a substrate for glycosyltransferases of the cells catalyzing the reactions [579]: G,,,1
+ UDP-(14C)Gal
-
G,,,l and
G,,,I+ CMP-( I4C)NeuAc + GIeI2a
Cell accumulation of exogenous ganglioside may be paralleled by various biological alterations. Most generally, after association of ganglioside, cells display an increased adhesiveness to the substrate [603], and a reversible inhibition of proliferation [358,599,616,627]. Under these conditions, a prolongation of the generation time, and of all phases of the cell cycle, was seen with astrocytoma cells [655]. The mechanism of ganglioside-induced growth inhibition is not yet known. There are, however, indications that a modulation of growth factor receptors by ganglioside could be involved [585]. On the other hand, a shielding effect for surface receptors by exogenously added ganglioside may explain its inhibitory effect for the mitogenic response of lymphocytes to concanavalin A (6161 or rosette formation with autologous erythrocytes [651]. Ganglioside association to neuronal cells may cause an outgrowth of neuritic processes that is interpreted to represent a cell differentiation phenomenon [526,578,580,650]. In addition, synapse formation is accelerated at neuromuscular junctions [525]. Involvement of gangliosides in these processes is also indicated by the observations that antiganglioside antibodies can inhibit neuritic outgrowth [596,612]. In primary neurons, added ganglioside effects a considerable extension of their survival time [573]. In vivo, ganglioside is able to stimulate reinnervations after seizure of peripheral nerve [42]. This regenerative property may involve the immune system, since ganglioside antibodies are produced following nerve injury [607]. Exogenous ganglioside taken-up by the cell may act as a functional receptor or mediator for signals from effector molecules, viruses or cells. This was shown towards cholera toxin [521], lymphokines (MIF/MAF) [529] or infection by Sendai virus [522]. An instance where exogenously incorporated ganglioside might function in the cellular reception of signals from other cells was reported for immune cells. Splenocytes, after incubation with ganglioside Gt,,4b, could be induced to transform in a system with autologous lymphocytes [559]. In explaining the biological activities of exogenous ganglioside, it must be
238 considered that its cell or cellular membrane association may also result in alterations in the activity of certain enzymes. Some of these are membrane-bound as in the case of an activation of adenylate cyclase * [524,576] and of (Na+,K+)-ATP-ase [523,575]. In other instances soluble (cytosol Ca*+-dependent nucleotide phosphodiesterase [525,575]) and solubilized ((Mg'+)-ATPase [526]) enzymes, are activated by added ganglioside.
5. Immuno-properties of gangliosides 5.1. GENERAL
Several reviews have in the past comprehensively dealt with the general immunological properties of the glycosphingolipids, including gangliosides (Rapport and Graf (1969) [364], Marcus and Schwarting (1976) [365], Alving (1977) [388], Yogeeswaran (1980) [622] and Marcus and Kundu (1980) [389]. Gangliosides contribute to the immunological expression of cells. Antibodies to ganghosides have therefore been used as markers for the specific immunological distinction of cell types of the immune [531,532] and nervous systems [367,368,533, 6171. They have further been applied to ultrastructural localization and in neurophysiology. Another example of the cell-typical expression of gangliosides is their detection by monoclonal antibodies, in particular as prominent cell surface antigens in certain tumor tissues, such as colon carcinoma [535] and human melanoma [536,664-6681. The immunological expression of a ganglioside may involve complexing proteins. An example for this was shown with ganglioside G,J which binds to protein that can be coextracted from bovine red blood cell membranes. This protein displays Paul-Bunnel antigen specificity, which is strongly enhanced by the presence of the ganglioside [369]. Gangliosides by themselves are poor immunogens. However, with particular immunization procedures specific antisera can be raised that have widely been used for the study and biological characterisation of gangliosides. Ganglioside antibodies can be induced by injection of lipid micelles and methylated bovine serum albumin [370] (for reviews on methodology, see Refs. 366, 371, 388). With this immunization procedure high titre antisera could be raised against gangliosides G,,,1 and G,,, 2b [373]. Gangliosides with terminal sialic acid residues, however, produced only negligible levels of specific antibodies. In another approach effective production of antiganglioside antibodies was induced by immunization with sialoglycolipid coupled, through their carboxyl groups, to a protein carrier [390,534]. With this technique, IgG-antibodies were obtained against ganglioside G ,==2.Antibodies that are directed against the sialooligosaccharide moiety of a ganglioside can also be produced with a sialooligosac* An inhibition of this enzyme by ganglioside was also recently reported [674].
239 charide-protein conjugate prepared from the liberated ganglioside sugar portion [391,392]: R . CH=O + H 2 N CH 2 C H 2 . $J.N H 2 +matrix - - N=C=S b
NaBH,
R .CH 2 . N H,CH 2 . @ .N H 2
R . C H 2 . N H . C H , . @ . N H . C S .NH-+matrix
The specificity of anti-ganglioside G ,aclNeuGcantibodies was shown to be directed mainly against the N-glycolylneuraminic acid residue [3741. The immunogenicity of this sialic acid also appears to be involved in the immunopathology observed in humans, i.e., “serum sickness”. In patients with t h s condition, who have received animal sera, heterophilic antibodies were found directed against gangliosides with terminal N-glycolylneuraminic acid residues [375]. Another example of the occurrence of anti-sialic acid antibodies was the isolation of a monoclonal IgM (agglutinin MKV) from a patient with Waldenstrom’s macroglobulinemia. The macroglobulin was reported to specifically cold-agglutinate erythrocytes by binding to N acetylneuraminic acid containing glycoconjugates including ganglioside G ,acl[376]. Antisera to gangliosides exhibit a number of biological actions in cellular systems that reflect the possible involvement of gangliosides in cell surface membrane-mediated processes of cell regulation. Thus, ganglioside G,,,l-antibodies are able to stimulate mitosis in rat thymocytes [377]. Monovalent Fab-fragments of ganglioside G ,,,lNeuAc- and G,,,l-antibodies decrease cell growth of Balb/3T3 and Nil fibroblasts and effectively inhibit the expression of transformed phenotypes in virus-transformed cells [378,350]. 5.2. INVOLVEMENT IN DISEASE
There is reason to believe that brain gangliosides can induce experimentally pathological reactions in nervous tissue that closely resemble encephalomyelitis, with concomitant extensive degeneration of peripheral nerves [372,471,537]. 5.2.1. Animal models
An autoimmune neurological syndrome similar to experimental allergic encephalomyelitis could be induced in rabbits by immunization with gangliosides G,J and G,,,2a [658]. Induction of experimental allergic neuritis by myelin protein fraction P2 is enhanced when applied together with ganglioside [659]. A multiple sclerosis-like central chronic neurological disease with no peripheral damage could also be observed in rabbits immunized with ganglioside [543,632]. 5.2.2. Anti-ganglioside immune activities in human pathology Sera from patients with a wide variety of disorders - schizophrenia, brain tumours and amyotrophc lateral sclerosis - show the presence of anti-ganglioside activities [663]. Sera from animals that were immunized, with ganglioside derivatives of
240
gangliotetraosylceramidefrequently show cross-reactivity with this neutral GSL. The presence of antibodies against gangliotetraosylceramidewas found in lupus erythematode patients [660] and in autoimmune thyroid disorders [661]. Certain cold agglutinins, in particular Pr, Gd, Sa and Fe antibodies, are directed against sialic acid-containing determinants, whereby the latter antibody reacts with a branched chain ganglioside of the structure NeuAccr2-3Gal~l-4GlcNAc~1-3[Fucal-2Gal~l4GlcNAcP1-6]Gal/31-4GlcNAc/31-3Gal/31-4GlcPl-Cer [5951. Antibodies against gangliosides, however, were also demonstrated in apparently normal healthy subjects [662]. Multiple sclerosis (MS) is one other neurological disorder where immunoreactions against brain gangliosides appear to be involved. Peripheral lymphocytes of MS patients show sensitization against MS-brain extracts when measured by an active erythrocyte-rosette formation test [539,540]. In this test system, gangliosides as well as cerebroside could produce a rise in active erythrocyte rosettes [541]. The most effective gangliosides thereby were tri- and tetrasialo species [542]. Total gangliosides from MS patients were more effective in the stimulation of active erythrocyte rosetting of MS lymphocytes than normal brain gangliosides, a fact that might be related to a higher content of ganglioside G,,,4 in MS brain [538]. In another assay system, i.e., by immune lysis of ganglioside-containing liposomes, it was also found that sera of MS patients contained a humoral serological activity directed against gangliosides such as Gga,l,G,,1 and G,,,1 [544,545].
6. Ligand-binding properties of gangliosides 6.1. GENERAL
It can be expected that whatever the biological significance of gangliosides will be, they will function in conjunction with other molecules - possibly proteins - that are able to more or less tightly interact with them. Due to their highly amphiphilic nature and their capacity to carry an electrical charge, the gangliosides are very “sticky” molecules. In an aqueous environment, gangliosides aggregate either with themselves or with other complexing molecules in the medium. Such interactions may thereby involve only lipophilic forces via the ceramide, or only hydrophilic binding via the sugar moiety. Complexing to the sialooligosaccharide will show the greater degree of specificity and allow third partner binding to the ceramide. Several instances of ganglioside-ligand binding, however, may involve both the lipophilic, as well as the hydrophilic, portion of their molecule. 6.2. GANGLIOSIDE COMPLEXING WITH LlGAND PROTEIN
Albumin is known to have a preferential binding capacity for anionically charged lipids and, as such, strongly binds to gangliosides. This phenomenon was therefore studied as an example of an interaction between ganglioside and protein that may have relevance to other similar, more biological instances [172,363,546,547,610].Gel
241 permeation chromatography, as well as ultracentrifugation studies, revealed the existence of two types of ganglioside-albumin complexes depending on the molar ratio of the ligands. Both complexes are dispersed by 0.1% Triton X-100 an indication of the comparatively nonspecific nature of the hydrophobic interactions. At a high ganglioside ratio, the complex is made up of ganglioside micelles that contain one or more molecules of albumin, whereas, at a low ratio, the ligands bind in a molar one-to-one fashion. The latter complex consists of a ganglioside monomer bound to albumin. It appears that albumin may associate with the ganglioside micelle, then again leaving this lipid aggregate carrying a molecule of ganglioside with it. Serving as a carrier for ganglioside in a one-to-one molar ratio, albumin seems to behave similarly to the purified lipid transfer protein from beef liver [561] or the /3-hexosaminidase activator protein [363,503]. In contrast to the latter two proteins, however, albumin was not shown to be able to extract single ganglioside molecules from liposomal phospholipid membranes. 6.3. INTERACTION WITH LECTINS
Since gangliosides contain complex carbohydrate they are able to bind to lectins of various origins. Wheat germ agglutinin has a specific N-acetylglucosamine binding site. This site obviously is responsible for interaction also with sialic acid and sialic acid-containing glycolipids [379]. Another lectin, limulin (Limufus polyphemus) agglutinates preferentially horse erythrocytes known to contain a high amount of ganglioside G JNeuGc [380]. It was found that limulin specifically binds to the N-glycolylneuraminic acid of this ganglioside [379]. Recently, a perhaps similar lectin was isolated from Carcinoscorpius rotunda cauda, named carcinoscorpin [420]. This lectin specifically binds NeuAca2-6[2-deoxy-2-N-acetamido-arabitol]. The lectin RCA, of Ricinus communis specifically complexes terminal /3-galactopyranoside. It therefore also interacts with gangliosides having this terminal group, as, e.g., in ganglioside G,,,l [381,382]. A case where perhaps specific cellular adhesiveness is mediated by a cell-surface lectin recognizing terminal N-acetyl-galactosamine of ganglioside G,,1 was reported by Marchase for chick neural retinal cells and surfaces of intact optic tecta [548]. 6.4. INTERACTION WITH TOXINS, HORMONES, INTERFERON AND CELL GROWTH AND
DIFFERENTIATION FACTORS
The pioneering work of W.E. van Heyningen opened up one of the most exciting chapters of ganglioside research, namely their interaction with the exotoxins of Clostridium tetani and Vibrio cholerae (for reviews, see Refs. 383-386,616). It was felt that the interaction between gangliosides and bacterial toxins deserved special interest, because this phenomenon indeed showed many of the characteristics of the binding of physiological effectors, such as hormones, to cell-surface receptors. The following toxins were reported to bind to ganglioside (References are initial reports): the Clostridia neurotoxins, tetanus toxin [393-3951; botulinus toxin [396];
242 the exo-enterotoxins, cholera toxin [397]; Escherichia cofi toxin [398]; Staphylococcus a-toxin [399]; the haemolysins of Streptococcus parahemolyticus [400]; and that of the sea wasp [401]. The toxins most thoroughly investigated with regard to ganglioside interaction are those of tetanus and cholera. Tetanus toxin in its extracellular form consists of two peptide chains ( M , approx. 100000 and 40000) linked by a disulfide bond [404] (for review, see Ref. 601). The heavy chain contains the ganglioside binding site [402,403]. There is no strict specificity on the side of the gangliosides. Several of them belonging to the ganglio-series are able to fix tetanus toxin. There is, however, some selectivity of binding, i.e., G,,,2b = G,,,3b >> G,,,1 = G,,,2a >> Glril [405-4081 with a preference for gangliosides that carry two sialic acid residues at the non-terminal galactose of gangliotetraose. In one assay system, employing Sephadex-adsorbed tetanus toxin, however, all gangliosides tested, including G,J, G,ril, G,,,l, 2b and 3b as well as unrelated synthetic sialoglycolipids, bound the toxin in a 1 : 1 molar ratio with high affinity, and to a comparable extent [403]. It can at present not be excluded that this preference for binding to certain ganglioside structures is influenced by matrix molecules used in the assay systems, e.g., a cerebroside preparation [405,407], ganglioside-containing liposomes [408] or native membranes of brain [409,410]. A prerequisite for binding to tetanus toxin appears to be the presence of a lipophilic moiety in the sialoglycoconjugate. Ganglioside-derived free sialooligosaccharide could not be shown to bind to tetanus toxin (Wiegandt, unpublished observations). Whereas at the molecular level the specificity of binding of tetanus toxin to certain isolated ganglioside species is less pronounced, its association with cells shows a high degree of selectivity. Cells of central neuronal origin, preferentially bind tetanus toxin [604]. T h s may be related to an exposure of the central neuron-typical di- and trisialogangliosides G,,,2b and 3b at the surface of these cells. It is speculated that, after its fixation to ganglioside centers, the tetanus toxin may be translocated and sequestered by other membranal structures that provide for its further intraaxonal transport to presynaptic terminals [634]. There is obvious parallelism for the binding of tetanus toxin and thyrotropin (thyroid-stimulating hormone) to membranes of the thyroid gland [413]. Ganglioside binding to thyrotropin shows an efficiency similar to that observed for tetanus toxin: G,,,2b >> G,,,3b > G,,,1 > G,ril = CI,,,~> G,,,2a [412,416,417].It was therefore postulated that gangliosides are involved as receptors for both the hormone and the toxin [412,414,415]. Speculations that only gangliosides act as cell receptors for tetanus toxin and thyrotropin are contradicted by the observation that neuroblastoma C 1300 cells, pretreated with neuraminidase and fl-galactosidase, still are able to fix tetanus toxin by a mechanism that may be unrelated to ganglioside [411]. Both effectors also specifically bind to a glycoprotein component from thyroid gland [418]. Perhaps both ganglioside and a specifically binding membrane glycoprotein may be involved
243 in the mechanism of reception, and the mediation of effector information [412]. Further doubt was cast on the assumption that gangliosides might serve as thyroid-stimulating hormone receptors by the finding that neurarninidase treatment of thyroid cells converting more complex gangliosides to ganglioside G,,,1 did not change binding of the hormone. Furthermore, down regulation of its receptors by thyrotropin has no effect on the distribution of gangliosides [549]. Cholera toxin binds specifically and multivalently to ganglioside G,J. The ganglioside G,,,2b binds some ten-times less strongly to the toxin as compared to G,,,1 [626] (for reviews, see Refs. 425, 385, 426, 486, 550, 616). Of the two cholera toxin promoter subunits, the A-protein, carrying ADP-ribosyltransferase activity, and the pentamer B-protein, only the latter binds to ganglioside [427]. In the binding, only part of the monosialogangliotetraose, the carbohydrate moiety of ganglioside G,,,l, is specifically involved [428-430,6281. Alteration of the 113-monosialogangliotetraose by substitution of the terminal galactose by fucose in 2-position, or reduction of the sialic acid-carboxyl group, as well as removal of galactose or sialic acid lead to a loss of binding capacity for cholera toxin. Integrity of the glucose residues appears not to be necessary for the toxin binding. Detailed studies showed that with cholera toxin only the sugar moiety of ganglioside G,,,1 is involved, whereas the ceramide provides an anchorage for the toxin at the cell membrane [429,431]. At present the mechanism of the ganglioside-dependent transmembrane events induced by the cholera toxin are not yet known in detail. After attachment to a cell-surface membrane the disulfide bond between the two subunits ( M , , 24 000 and 5400) of the A-protein is broken, and the larger peptide, that is hydrophobic, penetrates deeply into the lipid bilayer [551,623,624]. Specific disulfide bond reduction therefore appears necessary for the choleragenic action of the toxin. Since cholera toxin can induce redistribution of membrane constituents that are believed to be connected with the cytoskeletal system, it is also speculated that ganglioside membrane protein interactions could be involved [432-434,552,5531. A possible role of membrane ganglioside as receptor for interferon has also been suggested [435,437,554,555].Similar to tetanus toxin or thyrotropin, the specificity of binding to interferon is not unequivocal and restricted to one ganglioside, but decreases in effectiveness in the following order; G,,1 >> G,,,3b > G,,,l >> Gte12a> G,aJ *. Interferon-ganglioside interaction, however, appears not to be a general property of all types of interferon. Of the two interferon species detected in mouse fibroblasts, only one (type I) binds to ganglioside [438,555,572]. There is some indication that gangliosides may possibly function as receptors for certain cell growth and differentiation factors. One example for this is an L-cellderived factor that can stimulate the clonal growth of granulocyte macrophage ~~
* See, however, Ref. 555: mouse interferon type 1 is neutralized in the following order: G,,,3b. G1,,2b >> Glr,l: no binding by G,J or neutral glycosphingolipid. Also Ref. 554: Glr,l, G , J and Lac-Cer, all neutralize the antiviral action of interferon.
244 progenitor cells. Tlus factor is fixed by ganglioside in the following order of efficiency: G,,,1 > G,,1 > Gtet2a> G,,,3b [439]. Other findings are also in support of a possible receptor function of gangliosides for growth factors. Culturing cells in media that have been passed over a gangliotetraosylceramide or a ganglioside GJ-affinity column do not support the growth of 3T3-mouse fibroblast cells [440]. In addition, pretreatment of 3T3-cells with monovalent antibodies to gangliotetraosylceramide or to ganglioside G,,,l inhibits growth stimulation by serum. This might be interpreted as a masking of serum growth factor reception sites [440]. Another interesting example of the possible role of gangliosides as receptors for factors that influence cell behavior may concern lymphokine action on macrophages [556]. The macrophage migration inhibition factor (MIF) and macrophage activation factor (MAF) activities of culture supernatants of concanavalin A-stimulated lymphocytes can be abolished with a total brain ganglioside fraction [529]. Further indication of the involvement of ganglioside is the report that macrophages show an enhanced responsiveness to MIF after incubation with ganglioside-containing liposomes [529]. Even though the putative ganglioside receptor has not yet been characterized, it is believed to carry a terminal a-fucose residue, since this sugar is inhibitory for the MIF [557,558]. Sialooligosaccharide structures at the cell surface present receptors for viruses, such as paramyxovirus, influenza virus, encephalomyocarditis virus and Sendai virus [597,598]. A specific function of ganglioside in the cell reception for a virus was described in the case of Sendai virus [522]. Whereas sialidase treatment of cells makes them resistant to infection, incubation with ganglioside carrying a NeuAc-Gal-GalNActerminus restores susceptibility for the virus [522]. Due to their highly amphiphilic nature, gangliosides can act as rather sticky molecules. It is for this property that “receptor” functions may be observed for gangliosides that indeed are of no true biological significance. An example for this perhaps is reflected in the ability of gangliosides to inhibit a fibronectin-mediated cell attachment to collagen- or fibronectin-coated substrates in a nonspecific manner [5 88- 5901. 6.5. INTERACTION WITH NEUROTROPIC AGENTS
Wolley and Gommi [441,442] originally observed that the serotonin sensitivity of a neuraminidase-treated fundus preparation could be restored by adding ganglioside, in particular G,,,2. The involvement of sialic acid conjugates in the serotonin transport system could also be demonstrated in rat brain synaptosomes [445]. The question, however, whether or not gangliosides constitute serotonin tissue receptors has not yet been answered unequivocally (for review, see Ref. 383). Serotonin not only binds to gangliosides but also to other sialoglycoconjugates, e.g., fetuin [443]. Whereas the ion permeability of ganglioside-containing liposomes was not changed [443], release of glucose could be effected with serotonin and other biogenic amines
245
[a]. Tamir et al. [559]could see no binding of gangliosides to serotonin at relevant concentrations. These authors, however, made another interesting observation that may shed more light on the heretofore equivocal subject. In the presence of other lipids, e.g., lecithin and Fe2+, ganglioside, especially G ,ac2, strongly enhances the fixation of serotonin by the serotonin-binding protein [559]. It was speculated that possibly the interaction of ganglioside with serotonin-binding protein may regulate the concentration of the biogenic amine in the synapse. Other drugs that bind to ganglioside are d-tubocurarine [446], chlorpromazine [447] and colchiceine [448]. 7. Concluding remarks It is obvious that gangliosides appear to be involved in an embarrassing multitude of biological phenomena. Still, it is not yet possible to name clearly one universal role played by gangliosides in the life of cells, singly or in a tissue. The same holds true for the neutral members of the glycosphingolipid family, for which also no unified explanation of their biological significance can be offered at present. Even though t h s review attempts to keep the gangliosides “ under surveillance”, it may perhaps not be justified to consider the possible physiological function of the sialic acid-containing species only. However, considering the ubiquity of distribution of these plasma membrane constituents, future research of gangliosides is encouraged by the intriguing possibilities inherent in the complexity of these molecules as an expression of cell differentiation properties.
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CHAPTER 4
Glycosyl phosphopolyprenols FRANK W. HEMMING Department of Biochemistry, Queen’s Medical Centre, The University of Nottingham, Nottingham NG7 2 UH,England
I . Introduction Glycosyl phosphopolyprenols (see Section 3.1 for nomenclature of phosphorylated derivatives) differ from other glycolipids in a number of ways that are significant biochemically. The polyisoprenoid moiety has distinctive properties important in the interactions of these compounds with membranes and with specific glycosyl transferases. The lipid is also clearly derived by a very different biosynthetic route sharing parts of the pathway leading to sterols and polyisoprenoidquinones and hence is sensitive to some of the factors that control steroidogenesis. The linkage between sugar and lipid by a phosphate bridge is unique in glycolipids. Since t h s involves a sugar-l-phosphate bond, the transfer potential is comparable with that of the nucleotide diphosphate sugars. This leads to a difference in function: whereas the glycolipids are seen primarily as end products of metabolism, or as intermediates solely as acceptors of further sugar residues, the glycosyl phosphopolyprenols readily donate their glycosyl residues to some appropriate acceptor and have earned the term lipid-linked intermediates. This coenzymic role of phosphopolyprenols to some enzyme systems concerned with the transfer of sugars from nucleoside diphosphate sugars to form some glycosylated polymers is found in all living systems investigated so far. The type of polymer formed via lipid-linked intermediates differs in different kinds of organisms. In bacteria phosphopolyprenols are involved in the formation of the complex polysaccharides of the cell wall. In all eukaryotic cells the process of protein N-glycosylation is mediated by this type of coenzyme. In yeasts and fungi the coenzyme also functions in protein 0-mannosylation while in green plants their role also encompasses the formation of glucans. As yet a function in animal cells other than that in protein N-glycosylation has not been observed. Before discussing the details of the coenzymic role of phosphopolyprenols it is necessary to consider their chemical nature, for it will be seen that subtle differences of structure of these compounds, dependent upon their biological source, may be significant to their function. Acquaintance with phosphorylated derivatives of polyprenols will lead naturally into an account of their formation and metabolism;
aspects that will be particularly relevant to an appreciation of the discussion of the control of protein N-glycosylation towards the end of the chapter.
2. Polycis-isoprenoid alcohols 2.1. CHEMISTRY
2.1.1. General structures, nomenclature and methods
The chemistry of polyisoprenoid alcohols has been reviewed in detail by the author previously [l].The main features will be discussed here supplemented with more recent information. Polycis-isoprenoid alcohols possess the general structure 1 in which n varies from 5 to 24 depending upon the natural source. From any one source the range in the numerical value of n is relatively small giving rise to a family of isoprenologues spanning a difference in size of four or five residues. Most of the isoprene residues have cis-substituted double bands, and only two or three at the wend of the chain are in the trans configuration giving rise to the term di trans- (or tri trans-)polycis-isoprenoid alcohol. In this respect they differ from those polyprenols, such as solanenol, which are precursors of the side chains of plastoquinones, ubiquinones and menaquinones and are all-trans. Some polycis-isoprenoid alcohols contain one or a small number of saturated isoprene residues. Dolichols have a saturated a-residue (i.e., are 2,3-dihydro-polycis isoprenoid alcohols). They are also usually very hydrophobic molecules in which n is towards the upper end of the range (Greek SoXiKoa (dolikos): long). The structure of individual polyprenols has been established by using a combination of data from mass and infra red spectrometry, proton magnetic resonance, thin layer chromatography, hydrogenation and ozonolysis [ 11. Shibaev [2] has reported that ''C nuclear magnetic resonance allows assignment of the stereochemistry of each individual isoprenoid residue in the chain and this has since been taken further [2al. The term polycis isoprenoid alcohol is often abbreviated to polycis prenol or polyprenol depending on the context. The most sensitive and straightforward assay for unesterified polyprenols is by high performance liquid chromatography using a UV detector set at 210 nm [3]. This wavelength is a compromise between the peaks of absorption of the isolated double bonds and the absorption characteristics of appropriate solvents. Accurate assay of less than 100 pmol of mammalian dolichols is possible. In principle the method is also directly applicable to determination of glycosylated and phosphorylated derivatives. The concentration of polycis-prenols in tissues does not necessarily reflect the concentration of the functional form, the monophosphate. It may be more a measure of the phosphatase activity or the rate of metabolism of the liberated polyprenols. Nevertheless, the Occurrence of polycis-prenols can generally be taken either as positive evidence of the presence of the monophosphate and of phosphatase activity, or of a dietary source. It also indicates a potential to form the phosphate derivative using a polyprenol kinase, a phenomenon that will be discussed later.
263 2.1.2. Prokaryotic polycis-prenols All bacteria appear to contain ditrans, polycis-prenols containing between ten and 12 isoprene residues. The main component, undecaprenol, is sometimes called bactoprenol. The presence of phosphoundecaprenol and its glycosylated derivatives in baceria has been described on many occasions. The accumulation of unesterified bactoprenol to appreciable quantities has been described in only a few bacteria, notably Staphylococcus aureus [4] and various Lactobacilli [5]. In S. aureus over 90% of the undecaprenol (0.03 rnol g weight of cells) is unesterified at stationary phase. 2.1.3. Eukaryotic polycis-prenols 2.1.3.1. Ficaprenols and betulaprenols. Green leaves of higher plants contain tritrans-polycis-prenolsin which n (Fig. 4.1) usually varies from ten to 13 with the major components of the mixture containing eleven or twelve isoprene residues. Some of the earliest samples were obtained from Ficus elastica, giving rise to the trivial names ficaprenol-11 and ficaprenol-12 [6]. Small quantities of leaf polycis-prenols containing fewer than three trans residues have been observed to accompany the tri trans-polycis prenols. The concentration of these alcohols usually rises as the leaf ages, most of the increase occurring in osmiophilic globules of the chloroplast [7] with some also in the cell wall [ 81. Pine needles of several conifers accumulate tritrans, polycis-prenols ranging in size from 13 to 21 isoprene residues [9-111, and mainly as acyl esters. Non-photosynthetic tissue of hlgher plants sometimes yields slightly different polycis-prenols. These are ditrans, polycis-prenols comprising six to nine isoprene residues and are found mainly as fatty acyl esters. For example, the wood of silver birch, Betula oerrucosa, contains betulaprenols-6 to -9 [9]. 2.1.3.2. Dolichols. Most eucaryotic cells contain 2,3-dihydro, polycis-prenols which have been called dolichols. Most of these carry two trans residues at the wend of the chain. The distribution of dolichols among vertebrate animals is summarised in Table 4.1. Only human and rat tissues have been observed in detail. It can be seen that concentrations of dolichol vary markedly from one tissue to another. Human tissues appear to be a much richer source of dolichols than are rat tissues. Also, whereas the best human sources are testis, pituitary and adrenal, the richest rat tissues are spleen, pancreas and liver. The composition of the family of dolichols varies slightly from one species to another but in the rat the variation from one tissue to another is relatively small. Most of the unesterified dolichol in pig liver cells was found, in early work, to be associated with a subcellular fraction rich in mitochondria, whereas the major part of that esterified to fatty acids was located in a crude nuclear fraction [19]. More CH3
I
CH2-C=CH-CH2-
1 '
CH3
-
-CH2An 2
w - residue
Fig. 4.1. Polyisoprenoid alcohol - general structure
C=CH-CH2-OH
u-residue
264 TABLE 4.1 Distribution of dolichols in some vertebrate tissues Source
Concentration
Main components
Esterified % of total
Refs.
130- 170 200
20,19,18 ' ND
25 5
10,11 12
> 0.5 182 164- 316
ND ND ND
ND ND ND
13 13 15
ND ND ND 20,21, 19 ND ND ND ND ND ND ND
20 ND <5 11 ND 20 < 10 ND ND 3 5
15 15 15 15 15 15 10 15 15 15 15
69-129
19,20,18
52-63
17.18
10-40
18, 19.17, 20
ND
e
ND ND 18. 19, 17 18, 19, 17 18, 19, 17 18, 19, 17 18, 19, 17 18, 19, 17 ND ND ND 18, 19, 17 18,20,19
ND ND ND ND 12 ND ND ND ND ND ND 25 ND
14 14 14.16 16 14,16 16 14,16 16 14 14 14 14.16 16
( P g / g wet wt)
Bovine pituitary thyroid Chicken blood liver oviduct Human adrenal heart kidney liver lung pancreas pituitary prostate spleen testis thyroid Pig liver Rabbit liver Rat blood bone marrow brain heart kidney liver lung muscle pancreas skin small intestine spleen testis
1273 262 240 1226 82 943 1400 268 161 3 226 1145
<2 <2 17 8 13 23 14 4 26 <2 14 106 11
Value of n in Fig. 4.1. in order of concentration, highest first. Percentage esterified to fatty acid. ' Also contains small quantities of decaprenol [ll]. Also contains small quantities of dolichol-11 [18]. Pawson, S. and Hemming, F.W. (1979) unpublished results. a
265 recently, when [ 3H]dolichol was administered by i.v. injection to rats much of it was recovered in the liver and of this most was found in the mitochondria1 outer membrane [20]. These interesting observations justify a careful reinvestigation of the subcellular distribution of endogenous dolichol using modern methods. Studies with synthetic bilamellar membranes show that dolichol tends to aggregate in the lateral plane of the membrane [21]. The evidence suggested also that it does not flip from one lamella to the other at a significant rate. In marine invertebrates dolichol(s)-19 and -20 are probably the major isoprenologues present [22]. Dolichols of a similar size have been found in higher plants [23]. In both groups of organisms studies have been less extensive than in vertebrates. Yeast (Saccharomyces cerevisiae) contains dolichols with 14-16 isoprene residues, dolichol-15 predominating [3]. The dolichols of some filamentous fungi differ in three respects: they are longer, frequently consisting of 21-23 isoprene residues they differ stereochemically being derived from tetratrans, polycis-prenols and they carry substituents. For example in Aspergillus fumigatus both the o-isoprene residue and that adjacent to it are saturated, each possessing two extra hydrogen atoms [24]. In Aspergillus niger substitution goes further in the form of an exo-methylene group in the saturated w-residue 111. The relevance of these differences to the biological role of these compounds is not clear but it is possible that they affect their binding to hydrophobic areas of membrane proteins. 2.2. METABOLISM OF POLYCIS-PRENOLS
2.2. I . Formation and hydro&sis ofphosphoryl derivatives Both monophosphoryl and diphosphoryl derivatives of polycis-prenols have been recognised in natural sources. The non-glycosylated forms derive from three sources (Fig. 4.2). They can be formed via the usual biosynthetic route (see Section 2.3), by recycling from an involvement in glycosylation reactions (see Section 3) or by phosphorylation of unesterified prenol (discussed later in this section). Biosynthests I
I I I
Fatty Acyl Prenol
t PP-Prenol
I
Monoglycosylation
-p
t
- r:
+ATP(CTP*) P-Prenol
/
A
-P
.
.:
Oligoglycosylat ion
\
\
I
PP-Prenol
_..
Prenol
,. .w;
.
..
.. ,
i Catabolism
Exodenous source
Fig. 4.2. Pathways of metabolism of dolichol and its phosphorylated derivatives. * CTP in animal systems.
266
The ease of acid hydrolysis of phosphoprenols is related to the presence or absence of the 2,3 double bond [l].The presence of this double bond gives rise to an allylic phosphate and renders the prenol-phosphate linkage extremely labile to acid. On the other hand alkyl phosphates such as phosphodolichols which lack the 2,3 double bond are very resistant to acid hydrolysis. For example exposure to pH 2 at 100"C for 1-2 min completely hydrolyzes fully unsaturated phosphopolyprenols but has no effect on phosphodolichols. Unfortunately, rearrangement reactions and dehydration occurs during acid hydrolysis resulting in a mixture of polyprenol, the corresponding tertiary alcohol and the corresponding hydrocarbon. This contrasts with the products of hydrolysis by an appropriate phosphatase which liberates cleanly the polycis-prenols (including dolichols) from the monophospho-derivatives. The sugar-l-phosphate bond in monosaccharide and oligosaccharide phosphopolyprenols is also acid labile, complete hydrolysis being achieved by 10 min exposure to pH 2 at 100°C. Thus, exposure of obgosaccharide diphosphodolichol to these conditions yields diphosphodolichol. Further treatment at pH 1 and 100°C for 30 min will then release monophosphodolichol. Exposure of a lipid extract containing derivatives of phosphodolichol to this regime of acid treament will result in all of these derivatives being converted to monophosphodolichol. An appreciation of the different rates of hydrolysis is important in understanding the experimental approach to identification and analysis of these different derivatives. Discussion of further niceties of technique involving variation of solvent and use of phenol for acid hydrolysis is beyond the scope of this review. All of these bonds are fairly stable at neutral and moderately alkaline pH, a fact which makes it possible to use mild alkaline treatment (pH 13, 37OC, 15 min) to deacylate most fatty acyl O-esters in a lipid extract without damaging glycosyl phosphopolyprenols. This stability to mild alkali and lability to mild acid has been a useful factor in identifying, and purifying, these compounds. More severe treatment with alkali (e.g., 1 M NaOH at 50 O C) can lead to cleavage of glycosyl phosphopolyprenols, although the rate of hydrolysis depends upon the nature of the sugar and the anomeric configuration of the sugar-l-phosphate linkage. Preparative separation of phosphopolycis-prenols from neutral compounds, including polycis-prenols and their fatty acyl derivatives, has usually been achieved most readily by chromatography on silica gel or DEAE-cellulose (see, e.g., Ref. 25). Ion exchange chromatography (on DEAE-cellulose) has been particularly popular for separating monophospho- from diphospho-derivatives. This method also seems to achieve a partial separation of the diphospho-derivatives carrying oligosaccharides of different size. Identity of chromatographic mobility with authentic compounds in a number of thin layer chromatographic systems has also proved a valuable tool in identification of phosphopolycis-prenols and their derivatives. The amount of monophosphopolyprenols in most natural sources is very small. It has been estimated that the concentration of monophosphodolichol in pig liver is approximately 0.1 pM [26]. Enzyme assays whereby monophosphodolichol is assessed as an acceptor for glucosyl or mannosyl transferases have been used but. while very sensitive, they are at best semiquantitative because frequently other lipids
267
accompanying phosphodolichol in lipid extracts modulate the enzymic activity. However, using this approach Dallner has observed monophosphodolichol of rat liver to be at highest concentration in nuclear, Golgi and endoplasmic membranes [27]. Other promising methods of assay include hplc and formation of a chromophoric derivative [27a]. Although it is thought that most short chain phosphopolyprenols ( n > 4 in Fig. 4.1) are derived primarily by de novo biosynthesis, evidence that at least some of them can be derived by phosphorylation of exogenous polyprenols has been available for some time [26]. Direct evidence that large molecules ( n > 11 in Fig. 4.1) could be phosphorylated came from studies with Staphylococcus aureus. This organism was shown by Strominger’s group [28] to contain an ATP-dependent polyprenol kinase that had quite wide specificity to polyprenols, but was most active with bactoprenol forming the monophospho-derivative. The kinase is very hydrophobic, and once solubilized and purified (in butanol) its activity is dependent upon the presence of phospholipids (especially cardiolipin and phosphoglyceride) and detergents [29]. A similar enzyme has been described in Luctobacillus plantarum [30] and in Acetobacter xylinum [31]. In S. aureus and L. plantarum most of the undecaprenol present is unesterified. The proper balancing of the activities of the kinase and of the monophosphopolyprenol phosphatase has been proposed by Strominger as a method of controlling the concentration of monophospho-undecaprenol, and hence of the rate of bacterial wall polymer biosynthesis [32]. A membrane-bound diphosphatase activity capable of converting diphospho-undecaprenol to the monophospho-derivative present in S. aureus has been described in detail by Strominger and his colleagues [33]. Presumably, all bacteria contain both phosphatase activities, unesterified polyprenol being derived in two steps from diphospho-undecaprenol both from the biosynthetic route and from that linked to oligosaccharide (Fig. 4.2). As yet no evidence has been reported for the presence of a diphosphatase capable of removing the diphosphate moiety in one step. In mammalian cells Allen has shown that dolichol kinase is CTP-specific [34]. The enzyme is capable of catalyzing the phosphorylation of both exogenous and endogenous dolichol, and the monophosphodolichol so formed can function in glycosylation reactions [35,36]. Non-specific phosphatases of calf intestine and potato will, at relatively h g h concentrations, hydrolyze monophosphodolichol to an appreciable extent [34,37]. This activity has also been described in several other mammalian tissues and in yeast [38-401. The presence of polyprenol kinase activity has been observed in insects and, although ATP was shown to act as a phosphoryl donor, the specificity to nucleoside triphosphates has not been explored [41]. Keenan has observed that the fatty acids of fatty acyl dolichol (Fig. 4.2) in mammalian tissues are derived from phospholipids, especially lecithins, assisted by transacylase activity [42]. The enzyme showed a preference for short-chain polyprenols and for a-saturated, over unsaturated or multiply saturated versions [43]. Enzyme activity is highest in liver where it is found in all membrane fractions, a microsomal fraction being the richest source. Fatty acyl dolichol esterases have not been described although if, as seems possible, these compounds act as storage forms
268 of dolichol, such enzymes must exist. Early subcellular studies showed that in pig liver the fatty acyl dolichols were associated mainly with a crude nuclear fraction [19]. A similar distribution was observed in Aspergillus fumigatw [44], and in this fungus a much higher proportion of unsaturated fatty acids was found than among those esterified to ergosterol. The catabolism of polycis-prenols has not been studied. Possibly the degradation of short-chain prenols by bacteria is a guide [45]. 2.3. BIOSYNTHESIS OF POLYCIS-PRENOLS
2.3.1. Prokaryotic cells That bacterial polycis-prenols are formed from mevalonate was established some years ago in Lactobacilli by Thorne and Kodicek [46,47]. In fact it was as an essential growth factor of Lactobacilli that mevalonic acid was discovered [46]. Probably the need to form both phosphopolycis-prenol (to facilitate synthesis of several wall polymers) and also phosphopolyfrans-prenol (required for ubiquinone biosynthesis) explains the dependence upon a supply of this growth factor. A partially purified polycis-prenol synthetase of Lactobacillus plantarum normally forms mainly the undecaprenol isoprenologue. Detailed studies in Allen’s laboratory [48] suggested that the chain length of the completed polycis-prenol may be dictated by a chain termination site on the enzyme that is approximately seven cis isoprene units distant from the catalytic site, and which is capable of recognising the trans residues and one cis residue at the wend of the chain. The chain is assembled sequentially by addition of isoprene residues (steps 4-6 in Fig. 4.3), the isoprene residues being derived from acetyl CoA via P-hydroxy ,&methyl glutaryl coenzyme A (HMGCoA) and mevalonate (steps 1-3). Steps 7 and 8 give rise to the polyisoprenoid side chain of the quinone of the organism (ubiquinone or menaquinone in bacteria). In prokaryotic organisms steps 9 and 10 do not occur. The subcellular location of steps 1-8 in prokaryotes is not clear. There is evidence (491 that in Salmonella newingfon polycis-prenol synthetase (step 5, Fig. 4.3) is a membrane-bound activity, whereas polytrans-prenol synthetase (step 7) is located in the soluble fraction. In Lactobacillus casei undecaprenol appears to be formed by both mesosomal and plasma membrane fractions [50]. Probably steps 5 and 7 use the same pools of isopentenyl pyrophosphate and farnesyl pyrophosphate although there is no direct evidence on this point.
2.3.2. Eukaryotic cells In eukaryotic cells the available evidence suggests that mitochondria are active in the biosynthesis of both ubiquinone and polycis-prenols. Steps 7 and 8 have been shown by Rudney to be located in the inner membrane of liver mitochondria [51], whereas in the same organ dolichol synthetase (step 5), although present in several membrane fractions, has been reported by Pont-Lezica and his colleagues to be of highest activity in the outer membrane of mitochondria [52]. The same group report this latter activity in pea and the alga Prototheca zopfii also to be located primarily in the
269 mitochondria [53,54]. It is well established that steps 9 and 10 occur in smooth endoplasmic reticulum, and that the farnesyl pyrophosphate utilized by step 9 is formed in the cytoplasm and involves only one membrane bound step - step 2. Whether or not steps 5 and 7 use farnesyl pyrophosphate from the same site as that used in step 9 is not established. Indeed, steroid biosynthesis is so dominant that investigating the formation of farnesyl pyrophosphate in mitochondria for dolichol and ubiquinone biosynthesis, without risk of serious contamination from cytoplasmic and microsomal activities, is very difficult. It is important to realize that the stereochemistry of the isoprene residues of polyprenols is determined by the properties of the enzymes catalyzing steps 4, 5 and 7 in Fig. 4.3. Fig. 4.4 summarizes the result of the differences in mechanism of the reaction catalysed by a trans-prenyl transferase (Reaction a) and a cis-prenyl transferase (Reaction c). Isopentenyl pyrophosphate carries two hydrogen atoms on carbon 2. It was established by Cornforth, Popjak and collaborators in studies on the biosynthesis of allrrans-squalene and polycis-rubber [55,56] that the 2 pro S-hydrogen is retained, and 2 pro R-hydrogen eliminated, during chain elongation by addition of a trans residue, and that the converse (retention of 2 pro R-hydrogen, elimination of 2 pro S-hydrogen) occurs during the addition of a cis residue. In other words an isoprene residue that retains the 2 pro S-hydrogen (but not the 2 pro R-hydrogen) of IPP can be described as biogenetically trans, and a residue that retains the 2 pro R-hydrogen (but not the 2 pro S-hydrogen) can be termed biogenetically cis. When the biosynthesis of polycis-prenols was studied it became
1-
acetyl COA
'I
t
HMG Cop.
t
1-
mevalonote
.I
c--isopentenyl
pyrophosphate
trans
1( n-3)
(x-3) larnesyl pyrophosphate
71diphosphoolltrans-prenol-x
91 squolene
I
I
polyisoprenoid qutnone - x
1 dlphosphopolycfs-prenol -n I
8'
t
.
61
l0I steroids
t glycosylated derivatives
Fig. 4.3. Biosynthesis of diphosphopolycis-prenolsand relationship to some other isoprenoid biosynthetic pathways ( x and n relate to the number of isoprene residues in each molecule).
270 clear that those residues shown to be trans by physicochemical methods were also biogenetically trans, while physicochemically cis-residues were also biogenetically cis [57]. Some residues such as the w-residue or saturated residues are physicochemically neither cis or trans. Studies showed that w-residues are biogenetically trans (see b, Fig. 4.4) and also that a-saturated residues are biogenetically cis, whereas saturated residues at the w-end of the chain, such as found in Aspergilfi, are biogenetically trans [57]. It follows from these observations that cis:trans isomerism does not occur in polycis-prenols, and that the stereoisomeric pattern dictated by steps 4, 5 and 7 (Fig. 4.3) remains permanent in the completed polycis-prenols. It should be noted that, rarely, in some organisms (e.g., Aspergilfi) step 4 utilizes four molecules of IPP to form geranylgeranyl pyrophosphate, to which cis-isoprene residues are then added via step 5. The stage at which the a-residue of dolichols is saturated is uncertain. there is evidence from work with oviduct preparations for the formation of monophospho2,3-dehydrodolichol (i.e., possessing an unsaturated a-residue) from farnesyl pyrophosphate and isopentenyl pyrophosphate [58]. This would be consistent with the hydrogens being added to the a-residue after hydrolysis of the allylic pyrophosphate to the monophosphate. The observation in Chojnacki's laboratory, of the presence in pig liver of small amounts of dolichol-11 containing three internal trans-isoprene residues at the w-end of the chain [18], raises the possibility of saturation of the a-residue of a dietary source of unphosphorylated leaf ficaprenol-11 (see Section 2.1.3.1). Further work is needed in this area. A further understanding of aspects of the synthesis of polycis-prenols that are
/ FH3
IPP
DMAPP
CH3
, cI ~ @ ,
cis-isoprene residue
CHZ-0-P-P
Fig. 4.4. The incorporation of isopentenyl pyrophosphate (IPP) into diphosphopolyprenol as a trotis-isoprene residue (a). as a cis-isoprene residue (c), and as an w-residue via dimethyl ally1 pyrophosphate (DMAPP)(b). R. hydrogen of the pro-R position (Le., if replaced by a heavier isotope of hydrogen the configuration around C-2 of IPP becomes R): S, hydrogen of the pro-S position. C, carbon originally C-4 of IPP.
271
relevant to their activity in glycosylation reactions should benefit from work with solubilized and purified synthetases. In this respect, studies on pyrophosphoundecaprenol synthetase of Lactobacillusplantarum are of interest [59]. It appears that the enzyme requires a fluid-lipid bilayer and an optimal negative surface charge for maximum activity. Of the phospholipids of L. plantarum only cardiolipin was capable of stimulating the enzyme.
3. Phosphopolycis-prenols in glycosyl transfer 3.1. NOMENCLATURE
A decision on the most appropriate method of describing phosphopolyprenols and their glycosylated derivatives is complicated by analogies of structure, biosynthesis or function with compounds such as farnesyl pyrophosphate, nucleoside diphosphate sugars, polysaccharides and glycoproteins. The analogy of structure and donor function of monosaccharide derivatives with nucleoside diphosphate sugars encouraged the author, and several others in the past, to use terms such as dolichyl monophosphate sugars as a model [l].This leads to terms such as dolichyl diphosphate oligosaccharide, which is acceptable, but this nomenclature becomes particularly clumsy when one wishes to describe details of the oligosaccharide moiety. Using the accepted method, either with words or structures, for describing the sequence of sugars in an oligosaccharide, i.e., the reducing end of the chain is always on the right, one ends up with the sugar residue actually linked to the phosphate being furthest away from it in the description. In view of the great interest in oligosaccharides, and the need to avoid confusion in describing their fairly complicated structures, it now seems that maximum advantage and least disadvantage is to be obtained by extending the approach to oligosaccharides using the term glycosyl phosphopolyprenol as a model. The term phosphopolyprenol rather than phosphoryl polyprenol is in accord with recommendations in 1976 of the IUPAC-IUB Commission on Biochemical Nomenclature on Nomenclature of Phosphorus-containing compounds of Biochemical Importance. Even in oligosaccharide structures terminology varies. It has been accepted here that, since all glycosyl bonds involve the enantiomeric carbon of the named sugar, there is no need to include the numeral 1 (or 2 for sialyl bonds) when describing a glycosidic linkage (e.g., galactosyl-4 glucose, not galactosyl 1-4 glucose, for lactose). It also appears logical to describe the enantiomeric configuration of the sugar residue, rather than the glycosyl bond, by putting the a or as a prefix to the name of the sugar (e.g., 8-galactosyl-4 glucose, not galactosyl 8-4 glucose, for lactose). 3.2. GENERAL PRINCIPLES
Phosphopolycis-prenols function as coenzymes in several glycosylation reactions [for general reviews, see Refs. 1, 25, 601. The enzymes concerned are membrane-bound glycosyl transferases, and the system at its simplest and in general terms is shown in
272 Fig. 4.5. The sugar to be transferred is presented in the form of the usual water soluble donor, the nucleoside diphosphate sugar, from the cytoplasm to the membrane-bound transferase a which catalyzes its transfer to monophosphopolycis-prenol, also associated with the membrane. Since this reaction involves exchange of one sugar-1-phosphate bond for another, this reaction is readily reversible and the transfer potential of the sugar phosphate is retained in this lipid-linked intermediate. Glycosyl transferase b uses this hydrophobic donor as a substrate catalyzing the transfer of the sugar residue to a specific acceptor to be linked glycosidically (i.e., through C-1) and releasing monophosphopolycis-prenol to act again as a substrate of transferase a. Step b is not readily reversible, equilibrium being in favour of formation of the glycosylated acceptor. The latter may be also membrane-bound, or a component not directly associated with the membrane. Membrane-free acceptors are usually the opposite side of the membrane from the nucleoside diphosphate sugars but are not necessarily always so. Thus, although the net result may be transfer of a sugar residue across the membrane, the monophosphopolycis-prenol should not be seen as a carrier across the membrane. The arrows in Fig. 4.5 have no vectorial significance, but simply indicate the direction of the chemical reactions concerned. The vectorial fate of the sugar residues is the result of stereochemical properties of the reactions concerned and of the orientation of their enzymes and substrates. A more complex type of involvement is shown in Fig. 4.6. In this process of glycosylation an oligosaccharide (G, ) is built up on the diphosphopolycis-prenol by stepwise addition of monosaccharides to the non-reducing end of the growing +
-membmne
___c
-
,
- - - - -e
Fig. 4.5. The involvement of a monophosphopolycis-prenol as a coenzyme in glycosylation. A general scheme for transfer of a monosaccharide. G. any glycose. NDP, nucleosidc diphosphate.
GPP polycis-pronol
NDPG
NDP (or P prenol 1
P. polycis-prenol
p\
G,
, PP polycir-prenol
+
PP polycrs-prenol
4-acceptor
G,
, acceptor
Fig. 4.6. The assembly of oligosaccharide on diphosphopolycis-prenol and its subsequent transfer to acceptor. A general scheme showing the coenzymic role of monophosphopolycrs-prenol.
273
chain, and is then transferred en bloc to the acceptor (step e). In all reactions involving oligosaccharide assembly and transfer a diphosphopolycis-prenol is involved, in contrast to the monophospho-derivative concerned with monosaccharide transfer (Fig. 4.5). The diphospho-derivative results from the reversible transfer of sugar-1-phosphate to monophosphopolycis-prenol (step c). The monosaccharide donor in step d is either the usual nucleoside diphosphate sugar or, in some special cases, is the monosaccharyl monophosphopolycis-prenol. In the latter case, step d (Fig. 4.6) is a particular example of reaction b (Fig. 4.5). The diphosphopolycis-preno1 produced in step e has to be hydrolyzed by a phosphatase acting at step f before the cycle can be completed by the availability of monophosphopolycis-prenol again for step c. Examples of both schemes can be found in prokaryotic and eukaryotic systems. It appears that usually the concentration of monophosphopolycis-prenol is low and the glycosylation concerned responds to changes in its concentration. It may help to put the following into proper perspective to observe, at this point, that there is no evidence for the involvement of lipid-linked intermediates in the formation of nutrient polysaccharides, or in the glycosylation of glycolipids derived from glycerides or ceramides, in either prokaryotic of eukaryotic cells. Further aspects of the specificity of the process will be raised in the following pages. 3.3. PHOSPHOPOLYCIS-PRENOLSIN PROKAR YOTlC GLYCOSYL TRANSFER
3.3.1. General aspects A coenzymic role for phosphopolycis-prenols has been suggested in the formation of several complex polysaccharides of bacterial walls and exo-polymers. Not all of these systems will be discussed in detail here. Examples have been chosen to illustrate certain features of the involvement of the coenzyme, progressing from simple to more complex systems. More detailed descriptions will be found elsewhere (e.g., Refs. 1, 60). In most cases the polycis-prenol concerned is mainly undecaprenol (occasionally decaprenol). 3.3.2. Formation of peptidoglycan Peptidoglycan is a major component of the cell wall of gram-positive bacteria and occurs at a lower concentration in the wall of gram-negative bacteria. It provides an essential rigidity in the wall and inhibition of its synthesis renders gram-positive bacteria vulnerable to osmotic shock and can prove lethal. Several antibiotics interfere with the process. The simplified generalized structure of peptidoglycan (Fig. 4.7) shows polysaccharide chains of repeating disaccharides of the sugars N-acetylglucosamine and N-acetylmuramic acid-linked 81-4. These chains are crosslinked by short peptide bridges between N-acetylmuramic acid residues of adjacent chains. Inter-species differences occur primarily in the nature and frequency of these peptide bridges. The biosynthesis of this polymer was elucidated, mainly in Strominger’s laboratory, using membrane preparations of Staphylococcus aureus and Micrococcus
274 lysodeikticus [63].It has been confirmed subsequently in several other gram-positive and -negative bacteria. The involvement of phosphopolycis-prenol (Fig. 4.8) in the assembly and transfer of the disaccharide units is a particular version of Fig. 4.6. Here in step 1 the donor is UDPN-acetyl (pentapeptido) muramic acid and in step 2 is UDPN-acetylglucosamine. The pentapeptide chain is modified in a species-specific I I
I
I Glc NAc-MurNAc-
I
- - _GlcNAc -MurNAc-
I
I
peprde
peptide
I
- - -GlcNPr-MurNAc-GlcNAc-MurNAc-
I
GIcNAc-MUrNAc-GlcNAc-MirNAc-
--
I
I
peptide
---
--
P4jtidC -
peptide I
I
peptide I I
I
I
-
Fig. 4.7. A simplified representation of the structure of peptidoglycan: MurNAc, N-acetylmuramic acid; GlcNAc, N-acetylglucosamine.
&
/
GlcNAc-tprNAc PPpmnol
I
P prenol
P
45
Pep
GlcNAc -MurNAc PPprenol I Pep'
PPprenol
h
GICNAc-MurNAc-GIcNAc-Mu~NAc--
I
Pep'
6
I
I
GIcNAc-MhNAc--I
peptide cross linking
pept idoglycan
Fig. 4.8. The role of phosphoundecaprenol (P prenol) in the assembly of disaccharide units and their transfer to preformed peptidoglycan in the chain lengthening of the polymer. Pep. peptide.
275
manner in step 3, prior to transfer of the pentapeptidyl disaccharide unit to preformed peptidoglycan in the wall (step 4). Cross-linking by formation of a further peptide bond occurs later by a transpeptidation in the wall (step 6). 3.3.3. Formation of 0-antigen determinants and capsular exopolysaccharides Cell walls of a number of gram-negative bacteria contain lipopolysaccharides, the terminal oligosaccharide chains of which carry the antigenic determinants by which the bacteria can be identified serologically. The 0-antigen determinants of Salmonella newington comprise a repeating tri- or tetrasaccharide unit attached to a lipopolysaccharide core (Fig. 4.9). The trisaccharide may carry a further sugar X, such as abequose, or Y, such as glucose. Specificity of their antigenicity is determined by the sugars present and the value of n . Their formation has been studied in detail in the laboratory of Robbins and, later, of Wright [64,65] and shown to involve a variation on Fig. 4.6, whereby a trisaccharide is built up on diphosphoundecaprenol (Fig. 4.10). Sugar X is added by an extra step in the cycle after step 3, and analogous to steps 2 and 3, whereas glucose (sugar Y) is presented as glucosyl monophosphopolyprenol, the whole process being an example of a combination of Figs. 4.5 and 6. The precise stage at which the oligosaccharide chain is glucosylated is still uncertain. An important difference between Figs. 4.10 and 6 is the detail of the transfer of assembled tri- (or tetra-) saccharide to oligosaccharide. In the case of the 0-antigen the oligosaccharide is a preformed chain of repeating tri- or tetrasaccharide units still attached at the reducing end of the chain to diphosphoundecaprenol. As each tri- or tetrasaccharide is completed it remains attached to diphosphoundecaprenol and receives, at the non-reducing end, oligosaccharide donated from oligosaccharyl diphosphopolyprenol. In this way the chain of repeating tri- or tetrasaccharide units is lengthened from the reducing end. When the chain reaches the appropriate size it is transferred en bloc to the lipopolysaccharide core (step 6). Heteropolysaccharides outside the cell wall are a result of polysaccharide biosynthesis of bacteria of several genera. These exopolysaccharides may be present as a discrete capsule or they may take the form of an extracellular slime unattached to the bacterial surface [66-681. An early model for studies of the biosynthesis of these compounds was Klebsiella aerogenes which forms a capsule. One strain of the
rx
Yl
n
Fig. 4.9. Tri- and tetrasaccharide units of: (a) 0-antigenic lipopolysaccharides of Sulmoneh newingron in whih one of X, usually abequose, or Y , usually glucose, may also be present; (b) and (c) of Klehsiellu uerogettes. Rha, rhamnose; Gal- galactose: GlcUA, glucuronic acid: Glc. glucose.
276 organism studied by Heath’s group produces a polysaccharide chain of repeating units of structure (b) in Fig. 4.9 [69], and a second strain, investigated by Sutherland, forms repeating units of structure (c) in Fig. 4.9 [67]. Both of these tetrasaccharide units appear to be built in a manner analogous to that of the 0-antigen trisaccharide shown in Fig. 4.10. The growth sequence of structure b is
Gal
GlcUA
-.
I
ManGal+ ManGal
-
GlcUA
I
Gal ManGal
The fourth sugar of both structures (b) and (c) (galactose in (b) and glucuronic acid in (c)) are added by an extra step after step 3 which is analogous to steps 2 and 3. The value of n while the oligosaccharide chain is still attached to the diphosphoundecaprenol is uncertain. Its minimum value is two. A number of mutants of K. aerogenes deficient in early steps in the assembly of the tetrasaccharide have been isolated [70]. Colanic acid is an exopolysaccharide produced by strains of Escherichia coli, Salmonella and Aerobacter, and in some respects resembles the other exopolysaccharides in this section. It is a polymer containing fucose, glucose, galactose and glucuronic acid. The formation of a lipid-linked intermediate containing glucose and fucose has been described [68].
TDP Rha
/k-=’<
GalPPpronol
RhaGalPPprenol
!iI17
UDPCol
ManRhaGaIPPprenola
pprek ManRhaGaq Ppprenol n-’ b, I
P
I
p
p
p
r
e bn R h o G la g yn PPPrenol’---’
I
-
6 PPPAn0l0 u - - -~e-xI
/
0’
,, --
-
,I
\
llpopolysaccharide core
b a n ~ h a ~ a llipopolysacc ) haride core
Fig. 4.10. The role of phosphoundecaprenol in the assembly of trisaccharide units of the 0-antigen determinant of Salnionellu. Steps 1-5 are repeated until the appropriate value of n is reached. Then step 6 occurs.
211
3.3.4. Formation of teichoic acids and related compounds The teichoic acids and teichuronic acids are another group of important bacterial wall polymers. Gram-positive bacteria contain teichoic acids in both walls and plasma membranes. Several simple structures made up of repeating sugar-l-phosphate units form the backbone of the polymers (Fig. 4.11). One of these structures (a, Fig. 4.11) consists of repeating units of glycerol-1-phosphate linked head to tail. Another (b, Fig. 4.11) is made up of repeating units of ribitol-1-phosphate, while a third structure is built up from N-acetylglucosamine-1-phosphate(c, Fig. 4.11). The other hydroxyl groups of the sugar units frequently carry a substituent. Glycerol, for example, may carry at position-2 an alanine or a glucose residue. More complex teichoic acids containing mixtures of sugar phosphates also exist. Baddiley's group reports that the polymer backbone is assembled via a lipid-linked intermediate [71,72] containing a diphosphate bridge by a process (Fig. 4.12) which is a variation on the scheme in Fig. 4.5. A similar variation of the scheme in Fig. 4.6 is involved in building up disaccharide units of mixed teichoic acids (see, e.g., Fig. 4.13). In neither scheme (Fig. 4.12 nor 13) is a phosphatase required to regenerate the monophosphopolycis-prenol. In fact, the lipid phosphate has not been characterized chemically but competition experiments [73] suggest it is the same as that involved in peptidoglycan biosynthesis, namely phosphoundecaprenol. The teichoic acids are linked to peptidoglycans in the wall through a linkage unit containing N-acetylglucosamine-1-phosphatewhich is linked through the phosphate to C-6 of N-acetylmuramic acid of the peptidoglycan. It appears that the linkage unit is built up by a modification of Fig. 4.13 in which step b occurs three times
NHAC
NHAc
(C )
cMpxx
Fig. 4.11. The backbone structures of teichoic acids, made up of repeating units of: (a) glycerol-l-phosphate; (b) ribitol-1-phosphate; and (c) N-acetylglucosamine-I-phosphate. G I ~ -PPpolycrs-prenol C
CDpGlYC
P polycis-prenol
GIYC-P,
., acceptor
GIyc- P, acceptor
Fig. 4.12. The role of phosphopolycis-prenol in the formation of the poly-glycerophosphate backbone of teichoic acids. Glyc, glycerol.
278
successively to form a triglycerolphosphate, N-acetylglucosamine phosphate derivative. Onto t h s unit is then built the teichoic acid chain as indicated before, i.e., it acts as acceptor for schemes analogous to Figs. 4.12 and 13. Having achieved an appropriate chain length the whole polymer is then transferred to a residue of N-acetylmuramic acid of peptidoglycan [74]. The teichuronic acids are hybrids of teichoic acids in that they normally contain chains made up of sugar-1-phosphate residues alternating with uronyl residues. Bacillus licheniformisyields a polymer of repeating phosphodisaccharide units namely glucuronyl-N-acetylgalactosamine-1-phosphate. The disaccharide phosphate is built up by a scheme analogous to that in Fig. 4.13, in which galactosamine-1-phosphate is transferred in step a and only glucuronic acid (no phosphate) is transferred in step b [74]. The chain is built up from disaccharide units added at the reducing end of the chain as in the synthesis of 0-antigen oligosaccharide chain (step 4, Fig. 4.10), except that a phosphate is transferred with each disaccharide and monophosphoryl polycis-prenol is released. The completed chain is then transferred to peptidoglycan acid linkage, and forming an N-acetylglucosamine-l-phosphate-6-N-acetylmuramic liberating monophosphoryl polycis-prenol. 3.3.5. The formation of other bacterial polysaccharides Acetobacter xylinum exports cellulose into the growth medium. In fact it was in the formation of this polymer that preliminary observations over 30 years ago led to the first postulate involving lipid-linked intermediates [75]. The work was taken further relatively recently [76], and it appears that glucosyl and cellobiosyl diphospholipids are formed. The nature of the lipid has not been established unequivocally but it is probably a polyprenol[77]. A scheme analogous to that in Fig. 4.6 can be envisaged involving transfer of glucose-1-phosphate at step c and glucose (once only) at step d. Although it was observed that the cellobiose residue was incorporated into cellulose, details of this part of the process are still lacking. Colominic acid is a bacterial homopolysaccharide of N-acetylneuraminic acid. In
i
Fig. 4.13. The role of phosphopolycis-prenol in the formation of mixed teichoic acids.
279 E. coli it is formed at the expense of CMP N-acetylneuraminic acid via a monophospholipid intermediate [7 81. Halobacterium species differ from other bacteria in that they have an unusual cell wall. In Halobacterium salinarium the major wall component is a glycoprotein, each molecule of which contains several di- and trisaccharide units linked 0-glycosidically to protein and one oligosaccharide chain linked N-glycosidically to an asparagine residue in the protein chain. Mescher and Strominger [79] have established that the oligosaccharide resembles that of eukaryotic systems in that it contains N-acetylglucosamine, mannose, galactose and glucose, although the structures differ in detail. The assembly of at least the proximal part of this oligosaccharide appears to involve a cyclic process in which N-acetylglucosaminyl diphosphopolyprenol, mannosyl monophosphopolyprenol and glucosyl monophosphopolyprenol are formed [80,81]. The process has several features characteristic of eukaryotic systems (see Section 3.4 and Fig. 4.15). However, the polyprenol is probably shorter (& or C,s) and the saturation of the a-residue is not established. It seems likely that the process occurs at the periphery of the bacterial cells. Surprisingly, in view of the numerous examples of polysaccharide synthesis described so far, the production of the exopolysaccharide alginic acid, composed of residues of mannuronic acid and guluronic acid, by Azotobacter vinelandii does not appear to involve a lipid-linked intermediate [82]. 3.4. PHOSPHOPOLYCIS-PRENOLSIN EUKARYOTIC GLYCOSYL TRANSFER
3.4.1. General Glycosyl transfer leads to a large variety of important products in eukaryotic cells. These range from simple glycosides such as glucuronides, sterol glucosides and lactose to those compounds containing oligosaccharide or polysaccharide chains. These more complex polymeric products include nutrient storage glucans such as starch and glycogen, various wall polysaccharides of plants, exoskeletal polysaccharides of invertebrates and glycosphingolipids, glycoproteins and proteoglycans of mammalian cells. A coenzymic function of phosphopolycis-prenols in eukaryotic cells appears to be restricted to the glycosyl transfer involved in the formation of two specific groups of glycoproteins, and possibly also of some plant wall polysaccharides [ 1,25,60,83-87b]. Chemically, two main types of glycoprotein occur. There are those in which an oligosaccharide is linked by a glycosidic bond to the amido nitrogen of an asparagine residue of the peptide chain. The other type has an oligosaccharide linked by a glycosidic bond to the side chain oxygen of a serine, threonine or, more exceptionally, of a hydroxylysine or hydroxyproline residue in the peptide chain. The oligosaccharide chains are often described as being either N-linked or 0-linked. In animal systems a coenzymic function for phosphopolycis-prenol has been established only in N-glycosylation of proteins. Yeast and fungi appear to employ this lipid coenzyme in both N-glycosylation and in a particular type of O-glycosylation of proteins. The latter concerns the linkage of a mannose residue to a serine or
280
threonine residue of a protein chain. Both N-glycosylation and 0-mannosylation of proteins in green plants is mediated in this way, in addition to the synthesis of some polysaccharides. Although higher green plants appear not to utilize phosphopolycisprenols in cellulose biosynthesis there is evidence that this phenomenon does occur in a green alga. In most of these systems the evidence suggests that the lipid phosphate concerned is not allylic and closely resembles phosphodolichol. However, the amount of lipid involved is usually very small, and in only a few cases has sufficient evidence been adduced to establish the structure unequivocally. In fact it is possible that, in at least one plant system, a lipid phosphate other than phosphodolichol is involved [88]. In mammalian systems retinol phosphate has been implicated as a carrier in glycosyl transfer (see Section 3.5). 3.4.2. N-Glycosylation of proteins in animals 3.4.2.1. The mechanism. If a protein contains, in a p-loop, the triplet Asn-XSer(Thr), where X is an undefined amino acid, it is possible that the asparagine residue will carry an oligosaccharide residue linked N-glycosidically to its amido group [89,90]. Most oligosaccharide structures found attached in this way are closely related to either one or the other of the two types shown in Fig. 4.14. Structure A in this figure is usually referred to as the simple, high mannose or oligomannosyl type and structure B is known as the complex or lactosaminyl type (lactosamine = PGal4GlcNAc). Both A and B contain as a proximal core the identical pentasaccharide made up of three residues of mannose and two of N-acetylglucosamine. It is now established that the whole of the oligomannosyl oligosaccharide and the proximal core of the lactosaminyl oligosaccharide is assembled on diphosphodolichol. The details of this assembly are summarized in Fig. 4.15. It can be seen
A- Man uMan-ZuMan/i
''
I
I \
\6
uNeuNAc-6pGal-4pGlcNAc-,
p Man-4pGlcNAc-4pGlcNAc-
r---- - 2 uMQn ~
~
-
~
----I
l 6! ? ./ Man-4pGlcNAc-44/3GlcNAc-
I
I
__---
i-ASn
/3
__
~ N ~ ~ N A c - ~ P G ~ ~ - ~ P G I C N A_ C- - L ~ ~- ~ ~
~
~
I Asn
I !
..
~
- - -__-_ !
B
Fig. 4.14. The basic structure of oligosaccharides !inked N-glycosidically to asparagine residues of proteins. A, simple or oligomannosyl type. B, complex or lactosaminyl type. The rectangular broken lines enclose the proximal core common to both types of structure. NeuNAc, N-acetyl neuraminic acid.
281
that this is in fact a hybrid form of Figs. 4.5 and 6. This phosphodolichol cycle has been established in mammalian cells primarily as a result of work in Leloir's laboratory. A small but critical technical development in that laboratory was to observe that oligosaccharyl diphosphodolichols, especially the products of steps 4 and 5 (Fig. 4.15) were almost insoluble in a 2 : 1, v/v, mixture of chloroform and methanol, but dissolved readily in a 10 : 10 : 3, by vol, mixture of chloroform, methanol and water. The method of employment of this knowledge and of other developments in the Buenos Aires department in the course of investigating this cycle has been reviewed by Behrens and Tabora [91]. Step 1 of the cycle was first observed by Molnar and his colleagues in 1970 [92] using liver microsomes. Several other workers confirmed this finding in other tissue preparations, but it was not until Warren and Jeanloz [93] compared the product of pancreatic microsomes with the authentic, chemically-synthesized compound that the structure of the product of step 1 was firmly established. This step is specifically inhibited by tunicamycin, an antibiotic that contains a residue of both N-acetylglucosamine and fatty acid (see Section 4.2.2). The enzymic formation of N,N'-diacetylchitobiosyl diphosphodolichol (step 2) in liver microsomes was first described UwGicNAc
GlCNPr2P-P-DOl
f
GICNACP-PjDOI /
Y~GD \
4 GDPMan
Man,GlcNAc,P-P\
4'
Do1
UDPGICNAC
A + P-DO1
Glc 3 Mang~lcNAc2P-P-Dol
P-P-
'
D
+
F
r
o
t
e
i
n
Glc 3Mang Glc NAC2 Protein
I
lo
Mon9GIcNAc2 Protein I
i''
GlcNAcManj GlcNAcz Protein I
I12
I
NeuNAc2Ga12GlcNAc2Mon3GlcNAc2 Protein
Fig. 4.15. The phosphodolichol cycle summarizing the assembly of oligosaccharide on diphosphodolichol and its transfer en bloc to an asparagine residue of protein. The subsequent modification of the oligosaccharide chain is also indicated.
282 in Leloir’s laboratory [94],and the nature of the product confirmed later by chemical synthesis in Jeanloz’s department [95].The enzyme catalyzing this step has been solubilized by Elbein’s group [96]. The first mannose residue in the oligosaccharide is &linked and is derived directly from GDP mannose (step 3). The laboratories of Leloir [97]and Jeanloz and Strominger [98]observed this activity in 1974 in liver and lymphocyte preparations, respectively. One [99]of several other subsequent reports describes the solubilization of the enzyme. Further mannosylation directly from GDP mannose occurs at step 3, whereby four additional mannose residues are added one at a time all a-linked. Evidence for this conclusion rests on work with solubilized enzymes [100,101].the use of the antibiotic amphomycin (see Section 4.2.4)and observations made on mutant cell lines unable to carry out step 8 (and hence step 4) in Fig. 4.15 [102]. Inspection of Fig. 4.16B suggests that al-2,al-3 and al-6 mannosidic linkages are forged at this stage, indicating the involvement of at least three enzymes. Step 8 was one of the first of the cycle to be observed in a preliminary way in A detailed study on this activity in liver was made almost animal systems [103,104]. simultaneously by Hemming [105,106]and by Leloir [lo71 and their co-workers. The former group, and others, confirmed the nature of the lipid moiety, finally comparing it with material synthesized in Jeanloz’s laboratory [108-1101.In many tissues this step is one of the most active in the cycle. Initially, it was commonly accepted that the transfer of all of the mannose from GDP mannose to oligosaccharyl diphosphodolichol in microsomal preparations involved the intermediacy of mannosy1 phosphodolichol [18,111-1131. Subsequent reports from several laboratories, that some direct transfer from GDP mannose to oligosaccharyl diphosphodolichol occurred, was finally established as indicated above. That mannosyl phosphodolichol donates the last four mannose residues of the oligosaccharide (step 4) was shown in Kornfeld’s laboratory, using membrane preparations of mutants that lack step 8 [104].Only when exogenous mannosyl phosphodolichol was presented to .Man-PaMan
\
6 uMan
.Man-PaMan
/3
6 ‘ pMan-4~GlCNAC-4aGlCNAC-P-P-DOl 3
/
Glc-2aGlc-3aGk- 3aMan- 2aMan-2aMan
A
a Man
\6
pMan-4pGlcNAc-4aGlcNAc -P-P-DO1
i
Glc-2aGlc-3aGlc-3aMan-PaMan-2aMan
B
Fig. 4.16. The full structure of the oligosaccharyl lipid that functions as oligosaccharide donor to protein in step 6 of Fig. 4.15. A. in normal cells; B. in mutant cells lacking the enzyme for step 8 of Fig. 4.15. The configuration of the glucosidic bonds is a preliminary assignment.
283 membrane preparations was the mannosylation of the oligosaccharide moiety completed. Comparison of structure A with structure B in Fig. 4.16 suggests that al-2, al-3 and al-6 linkages are formed, implying the involvement of at least three enzymes. Identification of the lipid part of the products of steps 3 and 4 relies in part upon the use of N, N’-diacetylchitobiosyl diphosphodolichol as substrate for step 3, and also upon the chromatographic properties of the lipid released by treatment with mild acid (1141. The transfer of glucose by liver microsomes to monophosphodolichol (step 9,Fig. 4.15)was studied in detail in Leloir’s laboratory [115]and did, in fact, produce the first detailed description of the involvement of phosphodolichol in glycosyl transfer. The further transfer of the glucose residue to an acceptor was also recognized at that time, but it was only after the discovery of the peculiar solubility characteristics of oligosaccharyl diphosphodolichols [ 1161 that the nature of the immediate acceptor of step 5 (Fig. 4.15)was appreciated. The full structure of the product of step 5 (Fig. 4.16)is based primarily on work in the laboratories of Leloir [117],Spiro [118,119] and, more recently, of Robbins [120]and Kornfeld [121].Whether or not each glucosylation is catalyzed by a different enzyme is not yet known, although the fact that the final linkage is 1-3 and the other two are 1-2 suggests that at least two enzymes are involved. Transfer of glucosylated oligosaccharide to protein from a lipid-linked intermediate (step 6,Fig. 4.15)was first observed by Leloir’s group [122]in preparations of rat liver. Since then the process has been observed in many animal systems. The enzyme catalyzing this step appears capable of using any of the products of steps 2, 3, 4 or 5 in Fig. 4.15 as a donor but works most efficiently with the fully glucosylated compound (Fig. 4.16A)[ 1231.In vivo the complete oligosaccharide (Fig. 4.16A) is the major oligosaccharide transferred to asparagine residues of peptide chains. Pyrophosphatase activity capable of carrying out step 7 (Fig. 4.15) has been demonstrated in liver and lymphocyte preparations [39,40].However its function in the phosphodolichol cycle is not established without doubt. Belief in the cyclic nature of the pathway is based upon: (a) the existence of activity for step 6;(b) the high rate of protein N-glycosylation; (c) the low concentration of monophosphodolichol in most tissues; (d) the low rate of synthesis of monophosphodolichol in most tissues; (e) the existence of analogous cyclic pathways in prokaryotic systems (see, e.g., Section 3.3.2and 3). 3.4.2.2. The trimming and further glycosylation of the oligosaccharide chain while attached to the protein. In order to give rise to the oligomannosyl type of N-linked oligosaccharide (Fig. 4.14A), the product of step 6 (Fig. 4.15) must submit to glucosidase attack which removes the glucose residues one at a time (step 10, Fig. 4.15).The glucosidases concerned have been isolated and are being studied in detail [124].Probably two enzymes are involved. This trimming process may be extended by a-mannosidase activity as a necessary preliminary to the formation of the lactosaminyl-type of oligosaccharide (Fig. 4.14B).Step 11 (Fig. 4.15)summarizes this process. Liberation of all of the mannose
284 residues attached by al-2 linkage is followed by addition of N-acetylglucosamine to the mannose residue linked al-3 to the first mannose residue (i.e., that attached to N-acetylglucosamine). This is an essential prerequisite to removal of the two mannose residues linked directly to that mannose-linked al-6 to the first mannose residue. The mannose residue linked a1-6 then accepts a residue of N-acetylglucosamine to give the product of step 11 (Fig. 4.15).Schachter has discussed steps 10, 11 and 12 in detail [125].The lactosaminyl-type of oligosaccharide (Fig. 4.14B) is then completed by sequential addition of galactosyl and N-acetylneuraminyl residues to each terminal N-acetylglucosaminyl residue. These distal glycosylations (steps 11 and 12 of Fig. 4.15)proceed without any involvement of phosphodolichol or of any other lipid-linked intermediate. 3.4.2.3. Distribution of the phosphodolichol cycle within the cell. In cells producing glycoproteins for secretion or for location in the plasma membrane there is good evidence [126-1321 that the protein is synthesized by ribosomes of the rough endoplasmic reticulum, and that N-glycosylation proceeds as follows. The addition of the oligosaccharide (step 6, Fig. 4.15)to the protein occurs during, or immediately after, assembly of the protein chain. The glycoprotein with completed protein chain, now either in solution in the lumen or bound in the membrane with its oligosaccharide(s) at the luminal face, becomes exposed to the glucosidases, mannosidases and N-acetylglucosaminyl transferases of steps 10 and 11 of Fig. 4.15 as it proceeds to the smooth endoplasmic reticulum and the Golgi apparatus. In the Golgi, the galactosyl residues are added and here, as well possibly as in vesicles approaching the plasma membrane, sialyl transferase activity completes the fashioning of the lactosaminyl type of oligosaccharide. Consistent with this general picture were the reports that in vitro step 6 (Fig. 4.15) occurs in both rough and smooth microcomes of rat liver [133] and hen oviduct [134];in the latter tissue the specific activity being higher in the rough than in the smooth membranes. Although in most tissues N-glycosylating activity is relatively high in rough and smooth endoplasmic reticulum, it has also been reported at significant levels in plasma membrane [135-1371,Golgi [138],mitochondria1 [139,140] and nuclear membrane [141].There have also been several other reports of other enzyme activities of the phosphodolichol cycle in these cell fractions. The physiological significance of these observations is not yet clear. Possibly, although the enzymes have been transported to these other sites during the course of membrane rearrangements, they are bereft of substrate and therefore do not function in vivo. Alternatively, it must be recognized that, although the model systems used so far (i.e., cells specialized for secretion of glycoproteins such as oviduct, pancreas, myeloma or for production of plasma membrane glycoproteins such as cells infected with myxoviruses and lymphocytes) have yielded valuable information they are less informative regarding the biosynthesis of glycoproteins destined for other sites and functions. It is conceivable that the glycosylating activity of the other internal cell sites is essential for the biosynthesis, or repair, of glycoproteins not destined for secretion or for the plasma membrane. The activity at the plasma membrane may be important in cell interactions. Indeed, the evolutionary pressure that has resulted in the concentration
285 of enzymes at the plasma membrane, which are capable of hydrolyzing nucleoside diphosphate sugars [ 1421, implies that glycosyl transferases located there may not always be devoid of substrate. It is relevant to note at this stage the observations made in Beaufay’s laboratory [143,144], that rough microsomes of liver do not fully express their capacity to catalyze the assembly and transfer of oligosaccharides to asparagine residues of endogenous proteins unless the ribosomes are partially removed, and the membranes then incubated in the presence of guanosine triphosphate. The enhancement of activity by GTP, which is accompanied by the coalescing of the microsomal vesicles, is not understood. However, it should be appreciated that most other studies of glycosylating activity have been carried out without the benefit of this stimulation. The topological orientation of some of the enzymes of the phosphodolichol cycle in the rough endoplasmic reticulum of hen oviduct cells has been studied in detail in Lennarz’s laboratory [145,145a]. It was demonstrated that, in rough microsomal vesicles, newly synthesized N, N’-diacetylchitobiosyl diphosphodolichol was at the luminal face and that in this site it was able to act as a substrate for step 3 when GDP mannose was presented to the cytoplasmic face of the vesicles. It is possible, then, that the enzymes for steps 1, 2 and 3 are transmembrane proteins. Further, it was shown that newly glycosylated protein, the product of step 6, was also located at the luminal face of the vesicles. All this is in keeping with the previous discussion of the cell biology of protein N-glycosylation in secretory cells. Examination of the arrangement of activities in the endoplasmic reticulum of liver cells [87b,138,140, 146,146bI suggests that much of the glycosyl transferase activity to phosphodolichol (steps 1, 8 and 9, Fig. 4.15) is at the cytoplasmic face. Step 5 also appears to be susceptible to proteolytic attack from this face of the membrane. Possibly a small amount of transfer of sugars to protein at the cytoplasmic face is concerned primarily with glycosylation of protein that is destined to remain this side of the membrane [146b]. 3.4.2.4. Exogenous substrates for in vitro studies. A characteristic feature of studies of the phosphodolichol cycle has been the difficulty of using exogenous substrates with microsomal preparations. In view of the distribution of the activity to catalyze step 6 (Fig. 4.15) across the membrane discussed above it is clearly necessary to solubilize the enzyme if its activity is to be studied in detail. In fact, solubilization of this enzyme has been reported recently [147]. Exogenous peptide acceptors have been developed. These are usually small synthetic peptides containing the sequence Asn-X-Thr(Ser) [148-1501 or natural peptides dissociated and lacking tertiary structure following chemical treatment [ 151,1521. Studies on the sidedness of membrane activities cannot be conducted with exogenous peptide acceptor. They must depend on the presence of traces of endogenous protein acceptor in the microsomal preparation, or on the synthesis of acceptor by supplementing it with mRNA and a peptide-synthesizing system [130]. Exogenous supplies of the other substrate for step 6 (Fig. 4.15), the oligosaccharyl diphosphodolichol, depend upon enzymic synthesis, for the compound has not yet been synthesized chemically. Only small quantities of this substrate are available and if it is synthesized from radioactive precursors it is
286
usually of uncertain specific radioactivity due to the presence of endogenous substrates in the preparation of membrane-bound enzyme used. It is also relevant that this, like the other dolichol-linked components of the cycle, are insoluble in water and need the aid of detergent to present a solubilized form. Similar difficulties beset detailed studies of the other steps in the cycle although chemically synthesized substrates have been used to study reactions 1, 2, 3, 7, 8 and 9. The synthesis of dolichol bound sugars [93] and the solubilization of enzymes have been, and will be, important prerequisites to detailed enzymological studies in this area. 3.4.2.5. Direct N-glycosylation of peptide using UDP N -acetylglucosamine. The evidence that all transfer of oligosaccharides to asparagine residues in peptides is mediated by the phosphodolichol cycle is now very strong. The fact that in many systems the presence of tunicamycin, a specific inhibitor of step 1 of the cycle (see Section 4.2), blocks protein N-glycosylation points to the obligatory nature of the cycle to this process. Nevertheless, Marshall and his colleagues have reported that it is possible to transfer a single residue of N-acetylglucosamine directly to asparagine (in position 34) of pancreatic ribonuclease from UDP N-acetylglucosamine with the help of enzymes from liver, human serum and yeast [153-1551. This reaction appears to be the only case of an N-glycosylation that does not require phosphodolichol as a coenzyme. 3.4.3. N-Glycosylation of proteins in plants In the plant kingdom the role of phosphodolichol is probably best understood in the yeast Saccharomyces cereuisiae. This organism produces several proteins that are extensively mannosylated. The mannose residues are present either as part of a large oligosaccharide linked to an asparagine residue N-glycosidically via a bridge of N,N’-diacetylchitobiose (Fig. 4.17A) or as part of a short oligomannan linked to a serine or threonine residue 0-glycosidically (Fig. 4.1 7B) [ 1531. These polymannan structures are found in the wall mannan and similar structures occur in vacuolar carboxypeptidase Y and in extracellular invertase and phosphatase. There is good evidence, primarily from the laboratories of Tanner, Boer, Parodi, Nakayama and Palamarczyk that the proximal portion of the inner core of the structure represented by Fig. 4.17A is built up via the phosphodolichol structure as in animal systems (Fig. 4.15) as far as the product of step 10. Further mannosylation directly from GDPmannose accounts for the rest of the oligosaccharide. There is no evidence for the presence of sialic acid or of the lactosamine type of oligosaccharide (Fig. 4.14B) in plant tissues. Fungi produce glycoproteins carrying the oligomannosyl-type of N-linked oligosaccharide, for example glycosidases of Aspergillus niger [158]. Although the inhibition by tunicamycin of the N-glycosylation of these proteins [159] (see Section 4.2) implies the involvement of the phosphodolichol cycle none of the enzyme activities, other than step 8 (see Section 3) of the cycle have been demonstrated directly in fungi. Demonstration that the phosphodolichol cycle occurs in higher green plants has
287 proved more difficult than in animals and yeasts. However, Elbein’s group using preparations from cotton fibres, and several others using membrane preparations of seedlings, have shown the presence of most of the enzymes and formation of intermediates to support the scheme shown in Fig. 4.15 with the exception of steps 5, 6, 9 , l O and 11, and with some variance in the number of mannose residues added at step 4 [160-162). The process occurs in the rough endoplasmic reticulum with further mannosylation taking place in the Golgi or smooth endoplasmic reticulum [163-1651. A similar scheme has been shown to operate in at least one green alga [166]. Elbein’s group has suggested that the lipid carrier of the oligosaccharide (but not of mannose or glucose) may be a phosphopolyprenol different from phosphodolichol [ 1671. 3.4.4. 0-Glycosylation in plants Several glycoproteins of yeast carry short oligomannosyl groups linked O-glycosidically to serine or threonine (Fig. 4.17B). Early studies by Tanner and his colleagues [168] on mannosylation in yeast constituted the first reports of lipid-mediated glycosylation in eukaryotic systems. Subsequent elegant work [169] established that 7
7
uMan
6 ‘ .Man-PaMan
6 ‘ uMan-PaMan-PaMan
6‘
Xn
aMan-brMan-2aMan-2ln
k
[
amn-brMan-2aMan-P~n uMan-3aMan-gMan 6 ‘ aMan- 2uMan
6 ‘ sMan-3aMan
6‘ uMan-3aMan-2uMan A
6‘ uMan-3aMan-2aMan-3pMan-4pGlcNAc- 4pGlcNAc-Asn
1
aManaMan-PaMan-
B
uMan-2uMan-PaManaMan- 3uMan-2uMan- PaMan
Ser ( T h r )
Fig. 4.17. The structures of the oligosaccharide portions of mannoprotein of Succhuroniyces cererwiue [1531. t i = 6-10: A, N-linked oligosaccharide; B. 0-linked saccharide.
288 the mannose residue attached to the serine (threonine) residue is donated by mannosyl phosphodolichol having originated from GDP mannose in a manner similar to that in Fig. 4.5. Further mannosylation of the protein-bound mannose residue occurs directly from GDP mannose, one at a time until the appropriate chain length is achieved. The process is located in the rough endoplasmic reticulum [170]. In fact, in yeast, this mannosyl transferase system of cell-free preparations is much more active than is the phosphodolichol cycle. Less detailed evidence supports the view that 0-mannosylation may also be the main function of phosphodolichol in filamentous fungi [171]. 0-glucosylation of proteins in green plants has been described by Pont Lezica and his colleagues [161]. An oligoglucosyl diphosphodolichol acted as the precursor of the oligoglucan linked 0-glycosidically to pea isolectins. A similar intermediate was formed in the green alga Prototheca zopfi which gave rise to a glucoprotein that then acted as a cellulose primer according to the scheme in Fig. 4.18. Although there is no evidence for a similar scheme in higher plants Northcote and his colleagues [172] have proposed glycosylated phosphodolichol to mediate glucosylation of oligoglucans and polyglucans related to callose. 3.4.5. Miscellaneous glycosyl phosphodolichols The formation of several other glycosyl phosphodolichols, the function of which is not yet understood, has been described. Radioactive glycosyl monophosphodolichol has been described as a product of incubation of UDP[14C]- or [3H]galactose with
I
I
reticulum Golgi
GIC, protein
I i ,- GDPGIC I/
I I
t
cellulose
Fig. 4.18. The function of phosphodolichol as a coenzyme in the assembly and transfer of an oligoglucan to protcin in Prororhecu zopfii and thc utilization of this as a primer for cellulose synthesis ( r i approximates to 5) [161].
289 cell-free membrane preparations of several tissues, especially if the sugar nucleotide is protected from pyrophosphatase degradation [173]. The further transfer of galactose from phosphodolichol to oligosaccharyl lipid, containing 7-8 sugar residues, but not to protein has been reported by the same group of workers [174]. However, it has been suggested [25] that the radioactive sugar attached to phosphodolichol is not galactose but is glucose, presumably resulting from the presence of a 4-epimerase acting on the radioactive UDP galactose. The synthesis of xylosyl monophosphodolichol in a microsomal preparation from hen oviduct when incubated with UDP xylose was observed in Lennarz’s laboratory [175]. This glycosylated lipid could act as a donor of xylose residues both to an oligosaccharyl lipid and to protein. The significance of this observation is not clear, although the possibility has been raised that this transfer reflects the inability of the transferases to distinguish completely between glucose and xylose, since they differ only in substitution on C-5. The xylosyl transferase involved in the formation of the xylosyl serine bond as the first step in building the linkage region of proteoglycans has been purified to homogeneity [176]. Xylosyl phosphodolichol appears not to be involved in this step. The transfer of glucuronic acid from UDP glucuronic acid to N-acetylglucosaminyl diphosphodolichol to form ~-glucuronyl-4,N-acetylglucosaminyl diphosphodolichol by microsomal preparations of fibroblasts and of fibrosarcomas was reported simultaneously by the laboratories of Dorfman [117] and Heath [178]. The same product can be isolated labeled with tritium following administration of [ H]glucosamine to lung fibroblasts. These cells also produce heparan sulphate which contains the repeating unit P-glucuronyl-4, N-acetylglucosamine but there is no evidence that the disaccharyl lipid is a precursor of this polymer. Another product of N-acetylglucosaminyl diphosphodolichol identified after incubation with liver microsomes is the N-acetylmannosaminyl derivative [179]. The evidence suggests a 2-epimerization of the sugar while attached to diphosphodolichol. The function of such a metabolite is not known. 3.5 P H O S P H O R E T I N O L IN GLYCOSYL T R A N S F E R
Retinol is an allylic isoprenoid alcohol (Fig. 4.19). It is known to be a vitamin, and abnormalities in glycoprotein synthesis are one of the consequences of its absence from the diet. De Luca, Wolfe and their colleagues [180,181] have demonstrated that
\A
O O H
b
Fig. 4.19. The structure of (a) retinol and (b) retinoic acid.
290 retinol is readily phosphorylated in liver and that the phosphoretinol formed can be mannosylated, GDP mannose being the donor. Glycosylated derivatives of phosphoryl retinol are very unstable and have different solubility properties from comparable derivatives of phosphodolichol. This may explain why experiments designed to follow the latter type of compound sometimes give confusing and conflicting results for retinol derivatives. However, it appears that both retinol and dolichol derivatives are formed simultaneously in liver and there is evidence that different mannosyl transferases are involved for each lipid acceptor [182,182a]. De Luca’s group also report [183] that cell-free preparations are able to catalyze the direct transfer of mannose from mannosyl phosphoretinol to protein, under conditions in which transfer of oligosaccharide from oligosaccharyl diphosphodolichol to protein was negligible. There is no evidence for transfer of mannose from mannosyl phosphoretinol to oligosaccharyl diphosphodolichol. Some of the symptoms of vitamin A-deficiency can be relieved by administration of retinoic acid (Fig. 4.19), and a role for a metabolite of retinoic acid in mannosyl transfer has been suggested [184]. That this may, in transformed fibroblasts, be relevant to changes in cell adhesivity through changes in mannosylation of a cell surface glycoprotein has also been proposed [185]. However, it is clear that this metabolite is not phosphoretinol, for retinol cannot be formed from retinoic acid. The formation of galactosyl phosphoretinol by microsomal preparations of mouse mastocytoma when incubated with UDP galactose has been described [ 1861. Further transfer of the galactose to protein was reported. An involvement of phosphoretinol in galactosyl transfer has not been observed in other tissues.
4. The control of phosphopolyprenol-mediatedglycosylation 4.1. THE SIGNIFICANCE OF CONTROLLING THE PROCESS
The biological role of some polysaccharides and glycoproteins is well understood, but that of others still presents a serious challenge. The part played by the oligosaccharide in the overall function of glycoproteins is still being investigated from several different angles. Currently a function in recognition phenomena has been established for the oligosaccharide part of several glycoproteins. The hepatic elimination of serum glycoproteins is dependent upon the appropriate specific structure of their oligosaccharide chains [187]. The address of some glycosidases destined for lysosomes appears to be written in terms of mannose-6-phosphate residues attached to N-linked oligosaccharides [ 1881. An involvement of the oligosaccharides of glycoproteins in cell-cell interactions (and possibly in intracellular membrane interactions) through lectin-like compounds and in antigenic phenomena is also suggested by several experiments. It is also hghly likely that the presence or absence of the strongly hydrophlic oligosaccharide moiety of a glycoprotein will have significant consequences for its conformation and on its orientation, if present in a membrane. There is obviously a need to understand how the rate or degree of
291 glycosylation of both polysaccharides and glycoproteins may be controlled. Already several aspects of this have been explored in the case of phosphopolyprenol-mediated processes and will be discussed in the final section of this chapter. A more detailed discussion may be found elsewhere [188a, b]. 4.2. M A N I P U L A T I O N B Y A D M I N I S T R A T I O N OF A N T I B I O T I C S A N D O T H E R I N H I B I T O R S
4.2.1. Bacitracin T h s antibiotic is a small cyclic peptide which can bind irreversibly with diphosphopolyprenols by virtue of chelation through Zn2+ with the phosphates and hydrophobic bonding with the polyprenyl chain. The diphosphate so bound is no longer available as a substrate for phosphatase activity (e.g., step b, Fig. 4.6; step 5, Fig. 4.8; step 5, Fig. 4.10). The consequential interruption of peptidoglycan synthesis due to a shortage of monophosphopolyprenol provides one explanation for the antibacterial activity of this compound towards gram-positive bacteria. In medicine, only topical application of the compound is recommended. If taken internally bacitracin is toxic, probably partly because it will bind precursors (such as farnesyl pyrophosphate) of both sterol and ubiquinone biosynthesis. It has also been shown to inhibit protein glycosylation in several eukaryotic systems. Its interruption of protein glycosylation in Halobacterium salinarium following binding of diphosphopolyprenol has been used as evidence of the cyclic nature of the assembly and transfer of the oligosaccharide [80,81] (Section 3.3.5, c.f., Fig. 4.15). The mode of action in vitro on protein glycosylation in eukaryotic systems is not clear for the results of its presence in incubations seem to be quite varied. With pancreatic microsomes it inhibits step 1 of Fig. 4.15 and causes slight accumulation of mannosyl phosphodolichol and glucosyl phosphodolichol [ 1901. Hen oviduct membranes respond by accumulating mannosyl N,N’-diacetylchitobiosyl diphosphodolichol[191]. On the other hand, when bacitracin is added to yeast membranes, only step 2 (Fig. 4.15) is blocked [192]. In incubations of membranes of higher plants, and at higher concentrations of antibiotic, both steps 1 and 8 (Fig. 4.15) are inhibited [193]. 4.2.2. Tunicamycin Tunicamycin, first isolated by Tamura’s group [192] from Micrococcus lyso/ superificus, has been shown to be a powerful inhibitor of peptidoglycan biosynthesis, of the linking of teichoic acids and teichuronic acids to peptidoglycan, and of protein N-glycoslyation. The action is apparent at low concentrations of tunicamycin with whole cells and with cell-free membrane preparations of many different organisms and cell types. A common feature of these processes is that the first step in the phosphopolyprenol cycle involved is the translocation of a derivative of an N-acetylhexosamine-1-phosphate residue from the uridine diphosphate sugar to monophosphopolyprenol (step 1, Fig. 4.8; step a, Fig. 4.13; and step 1, Fig. 4.15). In fact the primary inhibitory action of tunicamycin at low concentrations appears to be totally specific to this type of translocation [74,193-1951. The compound is made
292
up of a residue of N-fatty acyl galactosamine, which acts as a bridge between residues of uridine and N-acetyl glucosamine [196]. It acts as a structural analogue of the substrate and hydrophobic product of the enzyme action and binds irreversibly to the translocase [197]. Because of its action on peptidoglycan synthesis tunicamycin is a powerful antibiotic against gram-positive bacteria. Its strong antiviral activity to those envelope viruses growing in cultured cells [198] depends on the arrest of N-glycosylation of the proteins destined for the viral envelope, and hence a loss of infectivity of the viruses released from the host cells. Despite these antimicrobial activities tunicamycin has no therapeutic use as it is too toxic to animal cells, presumably because of its inhibiting N-glycosylation of essential glycoproteins. The use of tunicamycin with whole animal cells in culture has proved a valuable tool in investigations on the biological significance of N-glycosidically linked oligosaccharides of several proteins. Unfortunately the effect of this compound has not always been clear cut. Possibly it varies in different cell types. Certainly biological activities of different glycoproteins respond differently. The sensitivity of cells from different sources to the antimetabolite varies markedly. The production of transferrin and VLDL apo-B-protein (both normally N-glycosylated) by hepatocytes was unchanged when their glycosylation was blocked by tunicamycin [199]. On the other hand, the antibiotic caused a marked drop in the activity of the low density lipoprotein receptor, normally a glycoprotein, of cultured fibroblasts [200]. The effect on another receptor, that in fibroblasts for lysosomal enzymes, is also to cause it to be produced in smaller quantities [201]. Interpretation of results was complicated by the absence of the recognition marker on the enzymes when tunicamycin was present. Recently, it has been reported that the regulation of the synthesis of the peptide moiety of thyroglobulin is coupled to the degree of N-glycosylation of the protein [202]. This conclusion was based on the indirect inhibitory effect of tunicamycin on thyroglobulin synthesis, the degree of glycosylation of the reduced amount of product being unchanged, and the evidence being against an accumulation of nascent peptide or an increased proteolysis of non-glycosylated protein. It may well be that a regulatory link also exists between protein synthesis and N-glycosylation in trophoblast cells of early mouse embryos. Here again inhibition by tunicamycin of N-glycosylation is accompanied by a fall in protein synthesis [203]. By contrast the inner cell mass of the same embryos was relatively insensitive to the antimetabolite. The authors suggested that trophoblast cells resembled transformed cells in their high degree of sensitivity to tunicamycin. Embryo development was arrested if it was presented to embryos during the two cell to eight cell stage of development. Kornfeld’s group (2041 observed that tunicamycin inhibited secretion of both IgG and IgA, whereas Williamson’s laboratory [205] found no effect on IgA but observed a reduction in the formation of IgM. The IgM result was explained not by a reduction in rate of biosynthesis but as a consequence of changes in conformation in the assembled non-glycosylated IgM on the rate of release. The effect of tunicamycin on the production of fibronectin is also a matter of dispute. Olden’s group [206] found no change in its production or biological activity when cultured
293 cells were exposed to the compound, whereas Duksin and Bornstein reported decreased production [207]. The latter group also observed that, although the glycosylation of procallagen is blocked by tunicamycin, it is still formed at the normal rate. However the rate of its conversion to collagen is reduced [208]. In a recent report the growth cycle of cells was observed to be arrested at the G1 phase if tunicamycin was added to the growth medium [209]. Tunicamycin is also effective in blocking N-glycosylation in invertebrates [210], and in the sea urchin it blocks embryonic development at a specific stage [211]. Inhibitory effects on the production of glycoprotein enzymes have been observed in higher plants [212], yeasts [213] and fungi [214]. In the latter morphological changes also follow [215]. These effects of tunicamycin are summarized in Table 2. A more detailed discussion appears elsewhere [188a, b]. TABLE 4.2 Summary of observed consequences of inhibiting protein N-glycosylation by tunicamycin A. Marked interference in cell biological phenomena: Cell cycle Embryonic development, mouse Cell division, Tetrahymena Embryonic development, sea urchin Cell spreading, morphology Fungal morphology Histogenesis, pancreas Differentiation: adipocyte kidney Mating, Tetrahymena myoblast Metastasis, lung fusion Nutrient. entry into cells odontoblast B. Marked interference in biochemical phenomena (usually reduction in concentration of substances or in activities listed): ACTH-endorphm, precursor IgA ’, IgG a secretion IgM Collagen. from procollagen Laminin, kidney Complement, component 4 Lipase, heart cells Fibronectin a Phosphatase, mouse L-cells Glycoprotein secretion; Receptors: acetyl choline hepatocytes egf plant cells IgM Glycoprotein, chick embryo PM insulin Glycoproteins. viral envelope LDL Glycosidases, lysosomal Thyroglobulin HCG a unit Hydrolases, fungal and plant C. No marked effect on concentration or activity of Apolipoproteins B. E, C Interferon CS granulocyte macrophage Rhodopsin Fibronectin a Thyroid-stimulating hormone Glycophorin Transferrin Human leukaemia antigen Trypanosome surface G P IgA ’. IgG ’, IgD
-
a
Disputed.
294
4.2.3. 2-Deoxyglucose This and some related sugar derivatives effectively inhibit the multiplication of envelope viruses [216] and the growth of yeasts and fungi. Schwarz and his co-workers have demonstrated that the primary effect is at the level of phosphodolichol derivatives. Although both UDP and GDP deoxyglucose are formed when 2-deoxyglucose (dGlc) is administered to cell cultures, it is the GDP derivative that mediates the inhibitory effect.This compound will act as a substrate for step 3 (Fig. 4.15) to form dGlcGlcNAc,PPDol which will not act as a substrate for the next reaction (step 4, Fig. 4.15). This causes a total block in the phosphodolichol cycle and hence stops protein N-glycosylation. In fact, relatively high concentrations of 2-deoxyglucose are required to achieve this effect and it can be relieved by increasing the concentration of mannose in the medium. 2-Deoxyglucosyl phosphodolichol is also formed (reaction 8, Fig. 4.15) and this can lead to the formation of a range of oligosaccharyl diphosphodolichols (via step 4, Fig. 4.15), each with a terminal residue of 2-deoxyglucose and incapable of further glycosylation of that particular chain of sugar residues. In yeast, 2-deoxyglucosyl phosphodolichol is also formed and donates its sugar residue to a serine residue of protein. However, the glycosylated protein is then unable to accept further sugar residues and so the elaboration of the 0-glycosidically linked oligosaccharide (normally oligomannoside, see Section 3.4.4) ceases [217]. 2-Deoxy-2-fluoroglucose and 2-deoxy-2-fluoromannose behave similarly to 2-deoxyglucose, the mechanism of behaviour being very similar [218]. Glucosamine also inhibits protein N-glycosylation by interfering with the phosphodolichol cycle but the mechanism is not understood. 4.2.4. Other antibiotics Elbein’s laboratory has reported the activity of several antibiotics on glycosylation reactions [218a]. Streptomyces produce a number of antibiotics that effect changes in this area. Streptovirudin and mycospocidin are probably closely related to tunicamycin chemically. The inhibitory action of all three is quite similar [219]. At high concentrations these compounds also inhibited glucosyl transfer to phosphodolichol. Streptomyces showdensis elaborates a different antibiotic called showdomycin. This is a nucleoside which also has antitumour activity. In membrane preparations (at 10 pg/ml) it inhibits glucosylation of phosphodolichol but has little effect on either mannosyl transfer, unless detergent is present, or the transfer of N-acetylglucosaminyl phosphate [220]. Bacillus cereus forms the polypeptide amphomycin which, at a slightly higher concentration (25 pg/ml), inhbits the transfer of mannose to phosphodolichol as well as to oligosaccharyl diphosphodolichol and glycoprotein in membrane preparations of aorta [221). A more recent report suggests that this antibiotic binds to phosphodolichol rendering it unavailable for glycosylation [221a]. It also reduced the transfer of mannose to phosphopolyprenol when added to preparations of Mycobacterium smegmatis. In plant membranes at higher concentrations (100 pg/ml) it blocks both steps 1 and 8 (Fig. 4.15) and in cultures of carrots it caused reduced formation of both oligosaccharyl diphosphodolichol and of glycoprotein [222].
295 The transfer of N-acetylglucosaminyl phosphate of phosphodolichol can also be reduced by an unknown compound called antibiotic 24010 [219]. Another step in the phosphodolichol cycle (step 8, Fig. 4.15) is sensitive to diumycin [223] and flavomycin [223a]. None of these steps appear to be affected by the antibiotics ristocetin, enduracidin and vancomycin although it has been known for some time that all three inhibit peptidoglycan synthesis at step 4, Fig. 4.8. 4.2. VARIATION I N THE CONCENTRATION OF PHOSPHOPOLYPRENOL
4.3.I . General The low concentration of phosphopolyprenols in living cells was discussed in Section 2.2.1. There, also, the importance of balancing the activities of polyprenol kinases and of phosphopolyprenol phosphatases was stressed. This is essential to the activity of phosphodolichol-mediated glycosylations. It has been shown that addition of more native phosphopolyprenol to a membrane preparation usually markedly stimulates the glycosylation process [1,25]. This has also been demonstrated when phosphodolichol has been added to whole cells (Section 4.3.4) and to tissue slices [223b]. In the latter case exogenous phosphodolichol appears to increase the extent of N-glycosylation of partially N-glycosylated sites on protein molecules. If phosphopolyprenols are removed from membrane preparations by careful extraction the drop in glycosylation activity can be restored by addition of the native coenzyme [224]. In most cells the main source of phosphopolyprenols is probably the biosynthetic route (Section 2.3). 4.3.2. Control of the biosynthesis of phosphopolyprenols
Changes in the rate and extent of biosynthesis of polyprenols and their phosphorylated derivatives have been investigated primarily in mammalian cells. Initially, Mills and Adamany in 1978 [225] observed that the presence of small quantities of 25-OH-cholesterol in the medium of cultured smooth muscle cells caused a reduction in biosynthesis of both cholesterol and phosphodolichol due to inhibition of HMGCoA reductase (step 2, Fig. 4.3). A fall in the concentration of phosphodolichol followed the lowered rate of synthesis and brought about a marked reduction in protein N-glycosylation. A fall in the extent of biosynthesis of dolichol consequent upon inhibition of step 2 has been confirmed in several lines of cultured cells [226]. Interaction of factors controlling cholesterol biosynthesis upon the biosynthesis of dolichol and its derivatives in whole animals has also been observed. A high level of dietary cholesterol in rats causes, first of all, a depression in the activity of hepatic HMGCoA reductase (step 2, Fig. 4.3) only, but an inhibitory effect at step 9 (squalene synthetase) steadily increases in the liver with time until, after several days, this further block becomes a major factor [227,228]. It may be that variation in the relative extent of inhibition of these two steps is responsible in part for conflicting observations on the effect of a high dietary cholesterol on dolichol biosynthesis. The author and lus collaborators [16,229] have observed that, if this regime is maintained
296 for 2 weeks with rats and rabbits, an increased synthesis of phosphodolichol occurs, and hence a stimulation of hepatic protein N-glycosylation follows. The evidence suggests that under these conditions HMGCoA reductase is still sufficiently active to provide more than sufficient precursor for the normal rate of synthesis of phosphodolichol, and that the block at step 9 (Fig. 4.3) makes most of this available for that biosynthetic route (step 5, Fig. 4.3). Assuming that this route (step 5) is not saturated with substrate a greater flux of metabolite through to phosphodolichol will follow. These authors also observed [16] that in several tissues other than liver the concentration of dolichol fell. In mice on a cholesterol-enriched diet Kandutsch has observed that hepatic dolichol synthesis, as judged by incorporation of [ l4 Clacetate, was depressed after both 1.5 and 14 days on the diet but this had no effect on protein N-glycosylation [230]. These studies also revealed an inhibitory effect of fasting on dolichol biosynthesis and a diurnal variation in the process. The lower rate during the day confirmed that at this time dolichol synthetase (step 5, Fig. 4.3) was not saturated with substrate in contrast to the situation observed in cells cultured under normal conditions [231]. The relationship of the biosynthesis of the isoprenoid side chain of ubiquinone to that of cholesterol has been studied in some detail in the laboratories of Brown and Goldstein [228,232] and of Rudney [233]. This work with fibroblasts in culture indicated that the presence of LDL-cholesterol in the culture medium switches off both steps 2 and 9 (Fig. 4.3). This allows a greater rate of incorporation of exogenous mevalonate into ubiquinone primarily, it appears, due to the endogenous pool of mevalonate being reduced consequent upon inhibition of step 2. The effect of inhibition of step 2 on the incorporation of exogenous mevalonate into cholesterol is less clear but it certainly is less marked than its effect on the incorporation into ubiquinone. Whether or not the biosynthetic pathways for cholesterol, dolichol and ubiquinone share the same pools of farnesyl pyrophosphate and mevalonate (as implied in Fig. 4.3) has still to be established. 4.3.3. The association of phosphopolyprenols with glycosyl transferase complexes The system of enzymes involved in the formation of any one of the polysaccharides or glycoproteins described in Section 3 probably exists as a multienzyme complex in which the phosphopolyprenol is an essential coenzyme or carrier. In any one bacterial cell several of these complexes may be present. It has been argued by Baddiley [73] that, initially, phosphoundecaprenol is available for any of the enzyme complexes but that once it becomes associated with one of these it remains with it until synthesis of that polymer is completed. Sutherland [67] has suggested that, in the initial distribution of phosphoundecaprenol, the peptidoglycan synthetase complex has priority over the enzyme complexes for the synthesis of O-antigen and capsular exopolysaccharide. This would be consistent with the observed synthesis of peptidoglycan mainly during the early log phase of growth, and the synthesis of O-antigen and capsular exopolysaccharide primarily in late log phase. It has been demonstrated using liver microsomes that detergent will separate phosphodolichol
297 from mannosyl and glucosyl transferases more readily than from phospho-Nacetylglucosaminyl transferase [140]. However, the results of competition experiments in eukaryotic cell-free systems in which different nucleotide sugars compete as donors for endogenous phosphodolichol are conflicting [232,233]. 4.3.4. Changes in concentration of phosphodolichol during development Protein N-glycosylation is stimulated in several developmental changes, and in two of these an increase in the concentration of phosphodolichol has been suggested as the immediate cause of the stimulation. Lucas and Levin [234] observed a marked increase in protein N-glycosylation in hen oviduct following stimulation of its development by oestrogen. Evidence of an increase in the concentration of phosphodolichol was, necessarily, indirect. (No satisfactory direct assay of phosphodolichol was available.) Waechter’s group [235] identified similar changes during myelination in developing brain. In addition Breckenridge and Wolf [236] have observed an increase in concentration of phosphodolichol and a fall in the activity of mannosyl and glucosyl transferases in the brains of chick embryos as they develop. Explanations of the mechanism whereby these changes might be brought about have not been proposed. Compactin specifically inhibits HMGCoA reductase (step 2, Fig. 4.3, Section 4.3.2). When it is included in the culture medium of early embryos of the sea urchin, Lennarz’s laboratory report [237] that development is halted at a specific stage in gastrulation. This is accompanied by a reduction in the synthesis of phosphodolichol and in protein glycosylation. The process reverts to normal if exogenous phosphodolichol is presented to the medium. The inference is that an appropriate concentration of phosphodolichol is essential to the proper development of the sea urchin. 4.4. CHANGES IN PHOSPHOPOLYPRENOL- MEDIATED GLYCOSYLATION I N MUTANT CELL
LINES
Changes in the activity of the phosphodolichol cycle have been observed in some mutant animal cell lines which have abnormal plasma membranes. b a g [238] has observed a deficiency of the glucosyl transferase that normally catalyzes step 5 (Fig. 4.15) in a mutant cell line resistant to concanavalin A, a lectin that binds to mannose or glucose residues of oligosaccharides at the cell surface. In a mutant lymphoma laclung the class E Thy-1 antigen (a major glycoprotein of the plasma membrane), the groups of Trowbridge and Kornfeld [239] have observed a lack of the mannosyl transferase which normally operates at step 8 (Fig. 4.15). This led to the lack of the antigen and several other glycoprotein defects, one of which was the formatiop of an oligomannosyl oligosaccharide with only five mannose residues (compare A, Fig. 4.14). A similar shorter oligosaccharide has been observed in a double mutant (PHARConAR)of CHO cells [240]. Some mutant strains of bacteria resist the effects of bacitracin. Sutherland [67] has suggested that these bacteria have an increased concentration of phosphobactoprenol. The mechanism by which this might be achieved has not been studied.
4.5. THE EFFECT OFANALOGUES OF NATURAL PHOSPHOPOLYPRENOLS ON GLYCOSYLA-
TION REACTIONS
It has been known for several years that, in processes of the type summarized in Fig. 4.5, transferase a will accept a fairly wide range of phosphopolyprenols as acceptor. On the other hand, transferase b was much more selective in the range of polyprenyl chains that make up a satisfactory glycosyl phosphopolyprenol donor [106]. More recently Chojnacki's group [241,242] has confirmed and extended these observations.
They found that glycosylation of phosphopolyprenol in membranes of prokaryotic cells requires the &-isoprene residue to be unsaturated and the polyprenol to be reasonably long. There was also a slight preference for a polycis-isoprenoid chain. Mammalian cell membranes exhibited a preference for a saturated a-residue, a long molecule and a poly-cis configuration in the phosphopolyprenols that it would use as glycosyl acceptors. Yeast membranes showed the same specificity in step 1 of Fig. 4.15 and in 0-mannosylation of protein (see step b, Fig. 4.5). In contrast to the high degree of specificity of step 1 of Fig. 4.15 for the native yeast phosphodolichol as acceptor [243], steps 3, 4, 5 and 6 had a low degree of specificity to the precise structure of the polyprenyl part of the lipid carrier of the oligosaccharide being built UP. Detailed knowledge of the specificity for the polyprenol structure of each step in Fig. 4.15 is important to understand fully what factors control the rate of the phosphodolichol cycle and possibly also allow manipulation of the process.
5. Summay Phosphopolycis-prenols function as coenzymes in several glycosyl transfer reactions. Those reactions leading to the formation of bacterial wall polymers are understood at a fairly detailed level. Of the eukaryotic processes, N-glycosylation of proteins in animals has been scrutinized closely and can now be described quite fully, although a number of aspects remain to be elucidated. 0-Mannosylation in yeasts is also well understood and, although the mechanism of N-glycosylation of proteins in plants has proved more difficult to unravel, it appears to resemble closely the phosphodolichol cycle of animal cells. There is a great deal of interest in cell biological aspects of these processes including for example its distribution in animals both among cell organelles and topologically within the membranes of these. A need to understand methods of control and manipulation of the process has also stimulated much research. The sites of action of inhibitors have been elucidated and, in the case of tunicamycin, the knowledge has proved invaluable in exploring the importance of N-glycosidically linked oligosaccharides in glycoproteins. Inevitably the experiments have not yielded a simple solution, but they have demonstrated that their significance to the formation and function of the (g1yco)protein varies from one glycoprotein, and from one cell type, to another. It may well be that the consequences of interactions between
299
the biosynthetic pathways of phospho-polycis-prenols and those of cholesterol and ubiquinone may be equally complex. The biological advantage to be gained from the evolution of the complex processes described in this chapter is still not clear. Perhaps the ability to control phosphopolyprenol-mediated glycosylation independently from others will be seen to be important. In the case of protein N-glycosylation the presence of preassembled oligosaccharide in a form ready for immediate co-translational donation to a peptide chain, and for rapid conversion to the oligomannosyl type, may also prove to be critical in some key cell biological phenomena. This will be better understood in eukaryotic systems when the function of the oligosaccharide chains on proteins is more fully defined.
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Subject index
AAG alkylacylglycerol 104 ABO blood group system 60 Acanthamoeba castellani 43 2-acetamid0-3.4-dihydroxy-5-caboxypiperidine 208 Acetobacter xylinum 267, 278 Acholeplasma 111, 175 granularum 118 laidlawii 106, 118, 139, 140, 179, 180 modicum 177 oxanthum 177 Activator protein 55 Acyl exchange 151 Adenohypophysis 227 ADHP, dihydroxyacetone phosphate pathway 127 ADP-ribosyltransferase 243 Adrenal chromaffin granules 227 Adrenal medulla 227 Aerobacler exopolysaccharide 276 Algae, blue-green 133 Alginic acid 279 Alk-1-enyl ether phospholipids 127 Alkenylacylglycerol synthesis 150 Alkylacyl GGroLs 121 Alteromonas rubescens 126 Alveolar membrane 147 Aminoalkylagarose 207 Amphomycin 282, 294 Amyloplasts 174 Amyotrophic lateral sclerosis 239 Anabaena variabilis 152, 154, 179
Anaeroplasma 111, 136
Anomeric configuration-chromium trioxide procedure 219 Antibiotic 24010 295 Anti-Gal2DAG 54 Anti-Gal a1-6GalS1-3DAG 177 Antigangliotetraosylceramide73 Antiglobopentaosylceramide(anti-IV’GalNAcaGb,Cer) 20 Antiglobotriaosylceramide 67 Antilactosylceramide 72 Antisulfo-GalAAG 178 Antisulfo-GalCer 178 Aplysia kurodai 43 Arabinose 42 Arabinosyl cytosine 121 Ara-CDP-DAG 121 Archaebacteria 112, 121, 125, 127, 134, 180 classification 125 Artemisia princeps 130 Arthrobacter 135, 136 Arthro series 42 Arylsulfatase 54 Arylsulfatase A (EC 3.1.6.1) 167 Aspergillus fumigatus 268 Aspergillus niger 43, 286 Asterina pectinifera 203 Atotobacter uinelandii 279 Bacillus, acidocaldarius 132, 136 cereus 131, 140, 294 licheniformis 278
308 megaterium 117, 175 subtilis 136, 137, 175, 179, 182 phosphate limiting medium 181 Bacitracin 291, 297 Bacteria, gram-positive, chemical taxoncm y 102 Bacteriochlorophyll 134 Bacterium licheniformis 150 B-active glycolipid, rabbit 25 Bactoprenol 263 Bacterial antigen 176 Betulaprenol 263 Betula uerrucosa 263 Bial's reagent 199 Bifidobacterium 137. 153, 154, 158 Blastocladiella emersonii 133 Blastocysts, mouse 68 Blastocysts, murine 68 Blood group A 59 Blood group B 50 Blood group systems 60 Botulinus toxin 241
Callose 288 Cancer-related glycosphingolipids 78 Carboxypeptidase 286 Carcinoma cells, embryonal, mouse 69 Carcinoscorpin 241 Cardiolipid 121, 127 Cardiolipin, biosynthesis 153 Cardiolipin, glucosylated 117 Cell aging 235 Cellobiosyl diphospholipid 278 Ceramidase, 51 Ceramide 200 Ceramide, alteration 205 Ceramide, biosynthesis 111 Ceramide phosphorylglycerol 106 Cerebroside 202 Cerebroside sulfate 27 Charonia lampas 167 Chicken egg yolk 202 Chicken embryonic liver 202 Chlamydomonas reinhardtii 134 Chlorellq pyrenoidosa 153, 156 uulgaris 130, 151, 162 Chlorobium 134 Chloroplast membrane 134 Chloroplasts 133 Chlorosulfolipids 183 Chlorpromazine 245 Cholera toxin 237, 243 Chromium trioxide oxidation 7
I3C-NMR spectroscopy 13 Colanic acid 276 Colchiceine 245 Colominic acid 278 Compactin 297 Concanavalin A 178 Creutzfeld-Jacob subacute spongiform encephalopathy 234 Cyanidium 179 Cyanobacterium 106 Cyclohexyl fatty acids 125 11-Cyclohexylundecanoic acid 132 13-Cyclohexyltridecanoic acid 132 Cyclopropane acids 125 Cyclopropane synthetase 152 Cytolipin H 72 Cytolipin K 21 Cytolipin S 23, 70 DAG, diacylglycerol 104
2,3-Dehydro-2-deoxy-N-acetylneuraminicacid 208 2-Deoxy-2-fluoroglucose 294 2-Deoxy-2-fluoroma~ose294 2-Deoxyglucose 294 2-Deoxyglucosyl phosphodolichol294 Detergents, spermicidal activity 178 Deuterostomia 224 Diacylglycerol synthesis 150 Diatoms 133 Dibi-phytanyl-glycerylether 127 Dibutyryl-CAMP 236 Dichloro-dicyano-benzoquinone206 Dictyostelium discoideum 43 Differentiation, immune cell 71 Digalactosyl ceramide 46 Digalactosyl diacylglycerol 101 Dihydrosphingosine 15
3,4-Dihydroxy-butyl-l-pyrophosphate159 Dimannosyl ceramide 41 Dinitrophenylhydrazine 208 Di-0-akylGro 121 Di-0-phytanylether 127 Diphosphopolyprenols 291 Direct inlet mass spectrometry 9 Di trans-polycis-prenols, bacteria 263 Diumycin 295 Docosapentaenoic acid 147 Dolichol kinase 267 Dolichols 262, 263 Dunaliella 182
309 Ehrlichs reagent 199 Eicosasphingenine 15 Electron impact mass spectrometry 9 Embryonal carcinoma cells, murine 68 Embryonic stage specific substances 68 Embryos, mouse 69, 165 Endo-/%galactosidase7, 95 Epididymis 146 Erythrocytes, human 203, 204 Erythrocytes, mouse 202 Escherichia coli, exopolysaccharide 276 toxin 242 Euglena gracilis 162 Exoglycosidases7 Experimental allergic encephalomyelitis 239 Fabry’s disease 21, 54 Farnesyl pyrophosphate 270 Fast atom bombardment mass spectrometry 10 Fatty acid, composition, age dependence 140 Fatty acid, distribution, GalDAG in plant 130 Fatty acid methyl esters 6 Fatty acids 16 cyclopropanisation 152 distribution in bacteria 130 positional distribution 129 short chain 236 6-Fatty acyl-glucosylceramide13 Fibronectin 244, 292 Ficaprenol263, 279 Ficus elastica 263 Field desorption mass spectrometry 10 Flavobacterium stearothermophilus 136 Flavomycin 295 Fluorescence recovery after photobleaching 224 Folch partition 4, 210 Forssman antigen, antigenic determinant 68 Forssman-antigenic F-polysaccharide, pneumococcus 119 F-polysaccharide 178 FSH, follitropin 146 Fucolipids 28 Fucosamin 106 Fucosidase 7, 55 Fucosidosis 55 Fucosyltransferase 48 Fungi 133 Galabiaosylceramide 19 Galactocerebroside esters 19 Galactolipid, distribution 132 Galactose oxidase 14, 208, 236
Galactosidase 7, 52, 54, 55 Galactosylceramide 16 biosynthesis 45 sulfate 27 sulfotransferase 50 Galactosyldiacylglycerol101 Galactosyl glycerolipids 103 Galactosyl lactoneotetraosyl-ceramide65 Galactosyl phosphoretinol290 Galactosyltransferase 50 Galacturonic acid 106 Gala series 16 GalMAG 174 Ganglioside, sialidase resistance 210 Ganghopentaose 203, 205 Gangho series 23,200, 202 Ganglioside, aggregation properties 220 alteration, chemical, enzymatic 205 albumin complexes 241 analogue, lateral diffusion 224 analogues 207 electron spin resonance labelled 236 biodegradation 230 biosynthesis 229 brain, adaptive divergence 148 poikilotherms 225 cation binding 224 cell growth inhibition by 237 cell uptake 236 cellular localisation 228 central nervous system 226 changes in disease 234 chemical constitution 200 chemistry 200 colon carcinoma 238 critical micellar concentration 220 developmental alterations 232 dipole moment 221 distribution 224-226 erythrocytes 224, 225 frog, fat body 203 G l a d 200 Glac2 203 hydrazinolysis,N-deacetylation-reacetylation 210 hydrogenation 207 localisation 210 mapping 211 melanoma, human 238 molecular packing 219 neurohypophysis 202 nomenclature 205
310
N-tnfluoroacetyl-neuraminyl208 oxidative cleavage 206 peripheral nerve 227 phospholipid, match of dipole vectors 220 physicochemical characteristics 219 rat, intestinal nonepithelial tissue 205 retina 203, 228 skeletal muscle, chicken 205 starfish 225 surface requirements 221 syndrome 73 temperature adaptive changes 234 visual system 227 Gangliosidoses 230 Gangliotetraose, 203, 205 Ganghotetraosylceramide23, 70, 229, 240 Ganghotetraosylceramide-disulfate28 Ganghotriaose 32, 82, 203, 205 Gangliotriaosylceramide23, 230 Ganghotriaosylceramide-did fate 28 Ganghotriaosylceramide-sulfate28 Gas chromatographic techniques 211 Gas chromatography 6 carbohydrates 6 Gastrointestinal mucosae 63 Gaucber's disease 20, 53, 56, 57, 232 Gentiobiose 177 Gentiobiosyl-DAG 176 G,,,l 202 GGroLs, animal 106 chemical synthesis 112 glycoglycerolipids 104, 121 plant 106 G lac 2 202 Glc-DAG 154 Glc-Glc-DAG 154 GlcU-DAG 181 GlcU. glucuronic acid 154 Globoisotriaosylceramide21 Globopentaose 205 Globopentaosylcerarnide9. 23 Globo series 20. 47, 78. 200 Globoside 3, 21, 47, 69 Globoside acid sepharose 47 Globotetraosylceramide11, 65, 69 Globotriaosylceramide20, 46,65 Glucocerebroside-ester20 Glucosaminyl-phosphatidylglycerol102 Glucosidase 7, 53 Glucosylceramide20 Glucosylceramidebiosynthesis 46 Glucosyl-diacylglycerol 102
Glucosyl-glycerolipids103 Glucosyl monophosphopolyprenol 279 Glucuronic acid 42. 289 /3-Glucuronyl-4,N-acetylglucosaminyl diphosphodolichol 289 Glucuronylceramide 41 Glycerol sn-2.3 127 Glycerophospho-GGroLs118 Glycerophosphoglycoglycerolipid102 Glycoglycerophospholipids,mycobacteria 117 Glycolipid acid 14 Glycophorin 60 Glycosphingolipid,non-vertebrate 38 biodegradation 51 biosynthesis 43 cell density dependence 77 changes 73 classification 2 constituents 6 extraction 4 hydrolase activator protein 231 in malignancy 73 in transformed cells 73 isolation 5 mass spectrometry 219 nomenclature 2 preparation 4 series 2 storage diseases 59 tumor markers 77 Glycosyl phosphopolyprenols 261 Glycosyl transfer 273 Glycosyltransferases,cell surface 77 GM, gangliosidosis 52 G M2 gangliosidosis 56 Gonadoliberin 146 Gram-positive eubacteria 135 Graves disease 73 Gro, glycerol 105 GroP-GGroL, glycerophosphoglycoglycerolipid 105
GroP, glycerophosphaie 106 growth cycle of cell, arrest at G1 phase 293 Gle,l 202 Gl,il 202 Guluronic acid 279 Halobacteria 127 Halobacterium,cutirubrum 174, 182 halobium 182 rnarismortui 156, 174 salinarium 278, 291
31 1 Halophilic bacteria, salt dependence 182 Hanganutziu-Deicher (H-D)antibodies 68 Hematoside 202 Heparan sulfate 289 Hexosaminidase A 231 Hexosaminidase activator protein 241 Hexosaminidase B 231 Hexosaminidase S 231 High performance liquid chromatography 5 HMGCoA, hydroxy-methyl-glutarylcoenzyme A 268 HMGCoA reductase 295, 296 Hordeum 134, 182 Horse radish peroxidase 14 Hydroxy acid esters 6 25-Hydroxycholesterin 295 Hydroxy fatty acids 13, 16 Hydroxysphinganine 15 Iatrobeads 5 Ii blood group system 63 Impariens balsamia 182 Infrared spectroscopy 13 lnositolphosphoceramides 42 Interferone-ganglioside interaction 243 lnvertase 286 Isogala series 42 Isoglobopentaose 205 Isoglobo series 20 lsomaltose 112, 147 Isopentenyl pyrophosphatase 269 Isopentenyl pyrophosphate 270 Keto-ganglioside 206 Klebsiella aerogenes capsule 275
Kojibiose 177 KojibiosylDAG 118, 178 Krabbe's disease 52 Lactalbumin 46 Lacrobacillus 121, 136, 263 casei 106. 130, 135, 136, 140, 269 fermenri 138 group A, F antigens 176 plantarum 267, 268, 271
Lacto-klado-hexaose 204 Lactoneohexaosylceramide 204 Lactoneotetraosylceramide24 Lacto-nor-hexaose 204 Lactose 46 Lacto series 24, 200 Lactosylceramide 20, 46
Lactosylceramide sulfate 27 Lactotetraose 204 Lactotetraosylceramide 25 Lactotriaosylceramide 24 Lactotriaosylceramide sulfate 27 Lands cycle 151 Lectins 178 Lewis blood group system 62 LH, lutropin 140 LH-RH, gonadoliberin 146 Limulin 241 Lipid bilayer 180 Lipid metabolism of bacteria, reviews 103 Lipomannan 110, 138 binding of Mgz+ 181 Lipophosphonoglycan 43 Lipoproteins, high-, low-density 63 Lipoteichoic acid 40,118. 119, 127,137, 138, 158, 159 carrier 119 extracellular, deacetylated 140 mitogenicity 175 stimulation of osteolysis 175 T-cell dependent immunogen 176 Lipoteichoic polyglycerophosphogIycoglycerolipid 102 binding to eukaryotic cells and complements 175 Lnn, linolenic acid 130 Low density lipoprotein cholesterol 296 LPS, lipopolysaccharide 176 LTA, lipoteichoic acid 118 LTC, lipoteichoic acid carrier 119 Lung alveoli 129 Lupus erythematodes 240 Lutropin 140 Lymphokines 237 Lysoganglioside 200 Lysoglycolipids 59 Lysoseminolipid 172 Macroglycolipids 36 Macrophage activation factor (MAF) 244 MAG, monoacylglycerol 105 Maize leaves 134 Malabiosylceramide 41 Membrane fusion 221 Man-inositol-P-DAG 153 Man-Man-DAG 154 Man-myo-inositol-phosphate117 Mannoglucosylceramide 106 Mannoheptose 106
312 Mannosylceramide 41 Mannosyl monophosphopolypreno1279 Mannosyl-N, N-diacetylchitobiosyl-diphosphodolichol 291 Mannosylphosphatidylinositol 101 Mannosyl phosphodolichol282 Mannosyl phosphoretinol290 Mannuronic acid 279 Man-p-undecaprenol 118 Marine diatoms 134 Mass spectrometry 9, 211 Meconium 25, 29 human 78 Megaloglycolipids 36 Meningococcal C-polysaccharide 176 Meningococcus 121 Metachromatic leukodystrophy 27, 57, 167 Methanospirillum hungutei 136 Methioninehydrazide 208 Methylation analysis 9 Mevalonic acid as growth factor 268 Micelles 220 Microbacterium lacfinum 136 Micrococci 136 Micrococcaceae 125 Micrococcus, lureus 110, 118, 138. 154, 157, 160 lysodeikticus 274 lysosuperificus 291 uarians 160 Migration inhibition factor (MIF) 244 Mollu series 42 Monoclonal antibodies 15. 71 Monoclonal antibodies, tumor-associated 72 Monophosphopolyprenols 266 Monosaccharide transfer 273 Monosialo-gangliotetraose243 Morulae. mouse, 69 Multiple sclerosis 177. 234, 240 Mung bean 129,135 Mycobacteria 127 Myco&acierium smegmaris 294 Mycoplasma, 134, 139. 180 ucidocaldarius 173 mycoides 106, 139 neurolyticum 139. 176, 177 pneumonioe 139 Mycospocidin 294 Myelination 142 Myelination marker 144 Myelin. oligodendral 226 Myeloid leukemia cells, mouse 71 Myoblasts 221 Myotubes 221
N-Acetylgalactosaminetransferase236 a-N-Acetylgalactosaminidase55, 7, 54 IV 3-N-Acetylgalactosaminyl-~-neolactotetraosylceramide 67 N- Acetylgalactosaminyltransferase 47, 48 a-N-Acetylgalactosaminyltransferases (A enzyme) 62 N-Acetylglucosaminyl diphosphopolyprenol 279 P-N-Acetylhexosaminidase7, 53 N-Acetylmuramic acid 277 natural killer (NK) cells 70 Neisseria meningitidis 148 Neogala series 42 Neolacto series 24 Neolactotetraosylceramide 46 Neolactotetraosylceramide sulfate 27 Neurite outgrowth 227 Neuroblastoma 227 Neurohypophysis 227 Neuronal perikaryon membrane 210 Neurospora crassa 43 N-glycolyl-neuraminic acid 68, 241 N-glycosylation, plants 286 proteins 279, 285 stimulation by oestrogen 297 NIL cells 77 Nifrschia alba, marine diatom 125 N, N-diacetylchitobiose 38 N. N’-diacetylchitobiosyl diphosphodolichol 281, 283 NS-4 cell surface antigen 177 0-antigen determination 275 0-glucosylation, proteins 288 0-glycosylation, plants 287 proteins 279 Oligosaccharide transfer 273 0-mannosylation, proteins 280 Osmium tetroxide-periodate oxidation 14 Ozonolysis 14 PI-active trisaccharide 65 P,-glycolipid 26 P-antigen 22, 60 Paragloboside 24 Paul Bunell antibodies 68 Penicillin 140 Pen tasialo-gangliotetraosylceramide 204 Peptidoglycan 273. 277, 278 Perikarya, oligodendroglial 226 Periodate oxidation 6. 9 Permethylation 211 Phosphatidyl-GGroLs 102, 118
313 Phosphatidyl-glucose 118 Phosphatidylinositol mannoside 127, 153 Phosphobactoprenol297 phosphodolichol cycle 281 block 294 distribution 284 enzymes, topological orientation 285 Phosphoglycosphmgolipids38, 42 Phospholipid, hydration capacities 181 Phospholipids, hydration 181 Phosphonoglycosphingolipids 38, 43 Phosphopolycis-prenol, nomenclature 271 Phosphopolycis-prenols, preparative separaticJn 266 Phosphopolyprenol phosphatases 295 Phosphopolyprenols, biosynthesis, control 295 Phosphoretinol 289 Phytoglycolipid 42 Phytosphingosine 15, 16, 42 P k-antigen 60 P k-erythrocytes 65 Plasmenic acid 121 Pneumococcal polysaccharide type XIV 25 Pneumococcus 106 autolysin 178 type XIV 154, 160 Polycis-isoprenoid alcohols, chemistry 262 Polycis-prenols, biosynthesis 268 metabolism 265 Polyclonal antibodies 71 Polymannan 286 Polyprenol kinase 267, 295 Poly(ribitol-P) polymerase 159, 160 Polysialosyl-phosphatidic acid 148, 176 cis-Prenyl transferase 269 Procollagen 293 Prostaglandin E, 236 Protein kinase C 236 Proton NMR spectroscopy 11, 219 Protostomia 224 Proioiheca zopJi 268, 288 Pseudomonas. diminuta 137, 153, 154, 158 halosaccharolytica 182 ooalis 117 rubescens 112 oesicularis 137 Ptd-GGroL. phosphatidylglycoglycerolipid 105 PtdGro, phosphatidylglycerol 117 Pyrophosphoundecaprenol synthetase 271
Q, quinovose 106 Radiolabelling 14
Reductamination 207 Retinol phosphate 280 Rhamnose 106 Rhizopus delemar 131 Rice bran 106, 133 Ricinus communis lectin (RCA1) 241 Ristocetin 295 Saccharomyces cereoisiae 133, 286 Saliva, human 129 Salmonella, exopolysaccharide 276 newington 268 0-antigen determinants 275, 276 Sandhoff-Jatzkewitzdisease 23, 53 Schizophrenia 239 Sea wasp toxin 242 Secondary (or sputtered ion) mass spectrometry 10 Seminal tubules 145 Seminolipid 103, 112, 129, 141, 144, 165, 167, 172 antibody 178 critical micellar concentration 172 synthesis, glycerylether backbone 151 Seminoma 146 Sendai virus 237 Serotonin binding protein 46, 245 Serotonin, binding of gangliosides to 46. 245 Serum sickness 239 2-0-Sesterpanyl-30-phytanyl-sn-Gro 127 Showdomycin 294 Sialic acid 199 evolution 148 lactone formation 201 periodate oxidation 208 Sialidase 76, 208 Sialidases, specificities 209 Sialyltransferase. CMP-sialic acid; lactosylceramide 236 Siastatin 208 Smith periodate degradation 211 Solvolysis, sulfate esters 117 Spermatocytes 103, 106, 112. 145, 156, 165 Spermatogonia 146 Spermatozoa, epididymal, bovine 129 Sperm, capacitation 175 guinea pig, autoantibody 178 Sphinganine 15 Sphingoid bases 6 Sphingoid biosynthesis 44 Sphingoid composition, gangliosides 21 1 Sphingolipidoses, characteristics 58 Spinal cord 111. 129
314 SQ-DAG, 6-sulfoquinovosyl-diacylglycerol 105 Squalene 269 SSEA-1, stage specific embryonic antigen 27, 69 SSEA-3, stage specific embryonic antigen-3 69 Staphylococcus, aurew 36, 137, 158, 159, 174, 182, 263, 267, 273 epidermis 126, 131, 148, 183 faecalis 131, 132 a-toxin 242 starfish 201 stereospecific numbering 105 Streptococci, group A 169 Streptococci, group B 117, 131, 136 Streptococci, group D 136, 176 Streptococci, group N 136 Streptococcus, faecium 106. 118, 121, 136, 139, 140, 154, 157, 158, 160. 169 group N antigen 176 hemolyticus 117. 118, 126. 135, 136 luctis 119, 130, 159 mutans 138, 176 parahemolyticus 242 pyrogenes 137, 174 pyrogenes, group A 119 sanguinis 158 showdensis 294 zymogenes 178 Streptovirudin 294 Sulfatidosis 57, 167 Sulfatid, sulfo-GalCer, immunogenicity 178 Sulfoglycolipids, reviews 117 Sulfoglycolipids. specific assay, azure A complexing 112 Sulfo-LacCer 146 Sulfolipase 168 Sulfolobus 127 Sulfoquinovosyl-diacylglycerol101, 168 Sulfotransferase 155 Sulphatide 13 Sulphoglycosphingolipids13 Synaptogenesis 233 Synaptosomes 210 Systemic lupus erythematodes 73
Tay-Sachs disease 23, 53, 231 Teichoic acid 102, 178, 181 Teichoic acid, formation 277 Teichuronic acid 181, 278 Teratocarcinoma cells, mouse 68, 69 Testis, boar 112 human 106 rat 112 Tetanus toxin 241 Thermoacidophilic bacterium 125 Thermoplmma ucidophilum 106, 127, 139 Thermus thermophiium 106, 180 Thin layer chromatography 21 1 Thylakoid membrane, phase transition temperature 179 Thylakoids 134, 160 Thymus T-cells, mouse 70 Thyroid hormone 144 Thyroid stimulating hormone 242 Thyrotropin 242 Trarrsacylase 267 Transfer protein 55 Transphosphatidylation 153, 158 Transprenyl-transferase 27, 137 Trimannosylceramide 41 Tri trans-pol yeis-prenols 263 Trophoblast cells 292 tuberculosis 101 d-Tubocurarine 43. 224. 245 Tumor-associated antigens 63 Tunicamycin 281, 286, 291 Undecaprenol263 Vaccenic acid 174 Vancomycin 295 Virus receptor 46 Wheat germ agglutinin 241 Xylose 106 Xylosyl monophosphodolichol 289
