Progress in Nucleic Acid Research and Molecular Biology, Volume 45

PROGRESS IN

Nucleic Acid Research and Molecular Biology Volume 45

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PROGRESS IN

Nucleic Acid Research and Molecular Biology edited by

WALDO E. COHN Biology Division Oak Ridge National Laboratory Oak Ridge, Tennessee

KlVlE MOLDAVE Department of Molecular Biology and Biochemistry University of Calqornia, Zruine Iroine, California

Volume 45

ACADEMIC PRESS, INC. Harcourt Brace & Company

Son Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @

Copyright 0 1993 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system,without permission in writing from the publisher.

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United Kingdom Edition pubfished by

Academic Press Limited 2 4 2 8 Oval Road, London NW17DX International Standard Serial Number: 0079-6603 International Standard Book Number: 0-12-540045-4

PFUNTEDW THE UNITED STATES OF AMERICA

939495969798

BB

9 8 1 6 5 4 3 2 1

Contents

ABBREVIATXONSAND SYMBOLS ........................................

ix

SOMEARTICLESPLANNED FOR F ~ U RVOLUMES E .......................

xi

Analysis of Rice Genes in Transgenic Plants Ray Wu, Xiaolan Duan and Deping Xu I. Analysis of Rice Genes ........................................ Methods for Gene Transfer in Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of Transferred Genes in Transgenic Plants . . . . . . . . . . . . . . . . IV. Concluding Remarks and Future Prospects ....................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. 111.

2 9 16 22 24

Immunoglobulin Gene Diversification by Gene Conversion Wayne T. McCormack, Larry W. Tjoelker and

Craig B. Thompson Evidence for Somatic Gene Conversion . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Mechanism of Somatic Immunoglobulin Gene Conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Enzymatic Activities Implicated in Immunoglobulin Gene Conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

29

11.

32 38 43 44

ADP-ribosylation Factors: Protein Activators of Cholera Toxin Joel Moss and Martha Vaughan I. 11. 111.

Biochemistry of ADP-ribosylation Factors . . . . . . . . . . . . . . . . . . . . . . . . Structure of ADP-ribosylation Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . Functions of ADP-ribosylation Factors in Animal Cells . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

49 55 60 63

vi

CONTENTS

Molecular Biology in the Eicosanoid Field Colin D. F u n k I. 11. 111. IV. V. VI. VII. VIII.

Lipoxygenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leukotriene A4 Hydrolase . . . . . . . . .. . Prostaglandin G / H Synthase ( ................... Prostaglandin-F Synthase . . . . . . . References

69 81

83

. . _ . . . . . . . . . . 89

. . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . ... .. . . .. . .. . . . . . .

93

Mamma Iian 6- Phosphofructo-2-kinase/fructose-2,6bisphosphatase: A Bifunctional Enzyme That Controls Glycolysis Guy G. Rousseau and Louis H u e I. PFK-2/ FBPase-2, A Bifunctional Enzyme That Synthesizes and Degrades Fructose 2,6-Bisphosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Molecular Characterization of PFK-2lFBPase-2 Isozymes . . . . . . . . . , 111. Characterization of PFK-2/ FBPase-2 Genes . . . . . . . . . . . . . . . . . . . . . . rV. Hormonal Control of PFK-Z/FBPase-Z Gene Expression . . . . . . . . . . . V. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

100 106 116 119 124 124

tRNA Structure and Aminoacylation Efficiency Richard Gieg6, Joseph D. Puglisi and Catherine Florentz

.. .

I. Structural Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 11. Phenomenology and Early Structural Results . . . . . . . . . . . . . . . . . . . . . 111. Complexes between tRNAs and Aminoacyl-tRNA Synthetases . . . . . . IV. tRNA Identity for Aminoacyl-tRNA Synthetases . . . . . . . . . . . . . . . . . . V. General Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

. .

131 149 159 166 192 195

Evolution of Caz+-dependent Animal Lectins Kurt Drickamer 1. Classes of Animal Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 11. CaZ+-dependent Carbohydrate-Recognition Domains . . . . . . . . . . . . . . 208 111. Organization of C-type Animal Lectin Genes . . . . . . . . . . . . . . . . . . . . . 216

VII IV. Evolution of Saccharide-bindingSpecificity . . . . . . . . . . . . . . . . . . . . . . . 220 V. History of Carbohydrate-Recognition Processes . . . . . . . . . . . . . . . . . . . 229 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 INDEX

.............................................................

233

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Abbreviations and Symbols All contributors to this Series are asked to use the terminology (abbreviations and symbols) recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN) and approved by IUPAC and IUB, and the Editors endeavor to assure conformity. These Recommendations have been published in many journals (I, 2) and compendia (3) and are available in reprint form from the Office of Biochemical Nomenclature (OBN); t h y are therefore considered to be generally known. Those used in nucleic acid work, originally set out in section 5 of the first Recommendations (I) and subsequently revised and expanded (2.3),are given in condensed form in the frontmatter of Volumes 9-33 of this series. A recent expansion of the one-letter system ( 5 ) follows. (5) SINGLE-LETTER CODERECOMMENDATIONS. Symbol

Origin of symbol

Meaning

G A TW) C

G A T(U) C

Guanosine Adenosine (ribo)Thym idine (Uridine) Cytidine

R

G or A T(U) or C A or C G or T(U) G or C A or T(U)

puRine pyrimidine aMino Keto Strong interaction (3 H-bonds) Weak interaction (2 H-bonds)

Y M K S

Wb

not not not not

or C or T(U) or T(U) or C or C or A or A or T(U)

G; H follows G in the alphabet A; B follows A T (not U); V follows U C; D follows C

D

A G G G

N

G or A or T(U) or C

aNy nucleoside ( i t . , unspecified)

Q

Queuosine (nucleoside of queuine)

H

B V

Q

‘Modified from h.Natl. Acad. Sci. U.S.A. 83, 4 (19E6). bW has been used for wyosine, the nucleoside of “base Y (wye). ‘D has been used for dihydrouridine (hU or H, Urd). Enzymes

In naming enzymes, the 1984 recommendations of the IUB Commission on Biochemical Nomenclature ( 4 ) are followed as far as possible. At first mention, each enzyme is described either by its systematic name or by the equation for the reaction catalyzed or by the recommended trivial name, followed by its EC number in parentheses. Thereafter, a trivial name may be used. Enzyme names are not to be abbreviated except when the substrate has an approved abbreviation (e.g.. ATPase. but not LDH, is acceptable).

ix

ABBREVIATIONS AND SYMBOLS

X

REFERENCES I . JBC241,527 (1966); &hem 5, 1445 (1966); /A/ 101. 1 (1966); ABB 115. 1 (1966). 129. 1 (1969); and e1smhere.t Gmeral. 2. EIB 15. 203 (1970); JBC 245. 5171 (1970); /ME 55, 299 (1971); and c1smhcn.t 3. "Handbook of Biochemistry" (G. Fasman, ed.), 3rd ed. Chemical Rubber Co., Cleveland, Ohio, 1970, 1975. Nuclcic Acids, Wls. I and 11, pp. 3-59. Nucleic acids. 4. "Enzyme Nomenclaturc" [Recommendations (1984) of the Nomenclature Committee of the IUB]. Academic Press, Ncw York, 1984. 5. EIB 150, 1 (1985). Nucleic Acids (One-letter system).? Abbreviotions of Journal Titles

Journals

Abbreviations used

Annu. Rev. Biochem. Annu. Rev. Genet. Arch. Biochem. Biophys. Biochem. Biophys. Res. Commun Biochemistry Biochem. J. Biochim. Biophys. Acta Cold Spring Harbor Cold Spring Harbor Lab Cold Spring Harbor Symp. Quant. Biol. Eur. J. Biochem. Fed. Proc. Hoppe-Seyler's Z. Physiol. Chem. J. h e r . Chem. Soc. J. Bacteriol. J. Biol. Chem. J. Chem. Soc. J. Mol. Biol. J. Nat. Cancer Inst. Mol. Cell. Biol. Mol. Cell. Biochem. Mol. Gm. Genet. Nature. New Biology Nucleic Acid Research Prcc Natl. h a d . M.U.S.A. Proc Soc Exp. Biol. Med. Progr. Nucl. Acid. Res. Mol. Biol.

ARB ARGen ABB BBRC Bchem BJ BBA CSH CSHLab CSHSQB EJB FP ZpChem JACS J. Bact. JBC JCS JMB JNCI MCBiol MCBchem MGG Nature NB NARes PNAS PSEBM This Series

tReprints available from the Office of Biochemical Nomenclature (W. E.Cohn, Director).

Some Articles Planned for Future Volumes Global Regulation of Mitochondrial Biogenesis in Saccharornyces cerevisioe

J. H. DE WINDEAND L. A. GRIVELL Adenoviral DNA Integration and Changes in DNA Methylation Patterns: A Different View of Insertional Mutogenesis

WALTERDOERFLER mRNA Binding Proteins in Eukaryotic Cells

TOMDONAHUE AND KEITH GULYAS Genomic Organization of T and W, A New Family of Double-Stranded RNAs from Sacchorornyces cerevisiae

ROSA ESTEBAN.NIEVES RODRIGUEZ-COUSIRO AND LUISM. ESTEBAN Enzymology of Homologous Recombination in Sacchorornyces cerevisiae

W. D. HEYERAND R. D. KOLODNER Regulation of mRNA Stability in Yeast

ALLAN JACOBSON AND

STUART h L T Z

Parallel-stranded DNA and RNA

THOMAS M. JOVIN,KARSTEN RIPPE, VITALY KURYAVYI AND ANGEL GARCIA Signal Transducing G Proteins: Basic and Clinical Considerations

MICHAEL A. LEVINEAND CHARLESEMALA Synthesis of Ribosomes

LASE LINDAHLAND

JANICE

M . ZENGEL

Drugs That Deplete Mitochondrial DNA in Vertebrates: Basic and Physiological Considerotions

REJEAN MORAIS DNA Polymerase II, the Epsilon Polymerase of Socchorornyces cerevisiae

ALAN MORRISONAND AKIO SUGINO Posttranscriptional Control of Lysogenic Pathway in Bacteriophage Lambda AMOS B. OPPENHEIM,DANIELKORNITZER, SHOSHY ALTUVIAAND

DONALDL. COURT Molecular Biology and Regulatory Aspects of Bacterial Glycogen and Plant Starch Synthesis

JACKPREISS xi

xii

SOME ARTICLES PLANNED FOR FUTURE VOLUMES

Mechanism of Action and Regulation of Protein Synthesis Initiation Factor 4E. Effects on mRNA Discrimination, Cellular Growth Rate, and Oncogenesis ROBERT E. RHOADS, SWATI JOSHI-BARVEAND CARRIERINKER-

SCHAEFFER Prosomes and Their Multi-catalytic Prateinase Activity: Relations to Messenger RNPs and the Cytoskeleton

KLAUS SCHERRER Regulation of Bacillus subtilis Gene Expression during the Transition from Exponential Growth to Stationary Phase

MARKA. STRAUCH The Egr Family of Immediate-Early Transcription Fodors

VIKASP. SUKHATME lntron Structure in Angiosperms VIRGINIAWALBOTAND KENNETH R.

LUEHRSEN

Analysis of Rice Genes in Transgenic Plants RAY Wu,' XIAOLANDUAN AND DEPING Xu Section of Biochemistry, Molecular and Cell Biology Cornell University fthaca, New York 14853

I. Analysis of Rice Genes ........................................... 11. Methods for Gene Transfer in Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Analysis of Transferred Genes in Transgenic Plants . . . . . . . . . . . . . . . . . IV. Concluding Remarks and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . References .....................................................

2 9 16 22

24

Rice is the most important cereal crop in the world because, for over two and a half billion people, it is the major source of food (1). In 1990, over 510 million tons of rough rice were produced, and together with wheat, these two major crops feed most of the worlds population (2). Although much research has been carried out in rice genetics, breeding, and physiology, practically no work had been carried out prior to 1985 on the analysis of rice genes at the molecular level. As of 1984, there were only a handful of laboratories working on rice genes, and only three papers had been published on this subject. In contrast, dozens of papers were published on the molecular analysis of wheat and maize genes. In early 1985, The Rockefeller Foundation launched a major program to promote research on rice molecular biology and biotechnology. With a clear vision, adequate funding, and superb management by The Rockefeller Foundation, the number of first-rate laboratories working on rice molecular biology increased to 20 in 1986 and to over 70 in 1992. Largely as a result of this program, there has been an explosion of published studies: to date (April 1992) there are over 100 publications on the molecular analysis of rice genes. Some of these studies are described in Section I of this article. In addition to cloning and analyzing rice genes, it is essential to develop efficient methods for transformation and regeneration of rice plants. Transgenic plants are needed for analyzing the regulatory sequences of genes in vivo, and for transferring agronomically important genes into rice plants. As

To whom correspondence may be addressed. 1

Progress In Nucleic Acid Research and Molecular Blology, Vol 45

Copynght 8 1993 by Academic Press, Inc All rights of repmductmn in any form resewed

2

RAY WU ET AL.

of 1988, at least two methods had been developed for transforming rice protoplasts or intact cells that can lead to the regeneration of fertile transgenic plants; more details on transformation and regeneration of rice plants are given in Section 11. With these advances, rice has become the preferred model system for the molecular analysis of genes in cereal crops, for it is much easier to produce transgenic plants with rice (3-8) than with other cereals such as maize (9, 10), wheat ( I l ) , barley, or oat. Another advantage is that the genome size of rice is relatively small, only 4.2 x lo8 base-pairs per haploid genome, whereas the genome size of maize is six times larger and that of wheat is 30 times larger (12). In this article, we present information on molecular analyses of rice genes by in uiuo and in uitro methods (Section I) and in transgenic plants (Section 111).We have limited our discussion to nuclear genes and selected only a small number of representative genes for more detailed description. This treatment is not meant to be exhaustive: rather than including molecular analyses of all known rice genes, we give selected examples of several important types of rice genes, particularly those about which we have first-hand information. Another consideration in choosing specific genes for discussion involves selection of those genes that are unique to plants: for example, genes related to seed storage proteins in rice and other monocotyledenous plants such as wheat and barley. The response of rice phytochrome genes to light is another such example of gene regulation unique to plants. Finally, the development of specific applications, such as attempts to produce insectresistant transgenic rice plants, is a unique and important application of plant biotechnology.

1. Analysis of Rice Genes In general, molecular analyses of animal genes are far more advanced than those of plant genes. This is directly related to the fact that the level of funding for animal (including human) research has been more than ten times that for plant research. Moreover, in plant research, work on dicotyledenous plants such as tobacco and Arabidopsis is more advanced than that on monocotyledenous plants such as rice and wheat because these dicots are easier to transform and regenerate more plants than monocots. In this section, examples of the molecular analyses of several rice genes are given in order to show what types of experiments have been carried out, and how much is known about rice genes. It should be mentioned that, even though the genes described here are unique to plants, many of the methods used for analyzing plant genes are similar to those used in research on animals or bacteria.

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

3

A. Rice Actin Genes Actin is an essential component of the cytoskeleton of eukaryotic cells in a number of animal systems. Although the function of actin is less well understood in higher plants than it is in animals, actin is believed to play an important role in plant cell shape determination, cytoplasmic streaming, organelle movement, and extensive growth (13). In higher plants, actin proteins are encoded by multigene families. Four different actin genes have been isolated from a rice genomic library (14); however, these four do not represent all actin genes in rice, because genomic blot analysis shows the presence of 8-10 hybridizing bands, and at least seven different rice actin cDNAs have been isolated (15). Each of the four genomic actin genes contains three introns in the coding regions, and codes for a protein of 376-379 amino acids. In the actin 1gene there is also a 5' intron separating the 5' noncoding exon from the first coding exon.

1. In Vim ANALYSIS OF RICE ACTIN GENEEXPRESSION To determine whether the expression of the four isolated members of the rice actin gene family are differentially regulated, their respective transcript levels were determined. The results show that RNA transcribed from individual rice actin genes displays either a constitutive or developmentally differentiated pattern of accumulation in different vegetative tissues. Moreover, members of the actin gene family differ from each other in tissuespecific abundance of their respective transcripts. Gel blots of total RNA from rice were hybridized with radioactive actin gene-specific probes to determine the transcript size and the abundance of each transcript. Each of the four actin genes encodes a unique transcript 1.5 to 1.7 kb in length. The steady-state mRNA levels of each gene were assayed using RNAs isolated from different tissues at varying times during early vegetative growth. The results indicate that the transcript level of the four actin genes differ by approximately 20-fold in 4-day-old shoots. The relative mRNA level of act1 : act2 : act3 : act7 was 100 :35 : 6 : 5. The transcript levels of act1 and act7 were relatively constant in shoots 2, 4, 7, 13, and 35 days after germination. In contrast, the transcript levels of act2 and act3 35-day shoots dropped to approximately 20% of the level observed in 2-day-old shoots (1S). Abbreviations and special names: a d , rice actin 1 gene; bar, phosphinothricin acetyltransferase gene; bialaphos, a phosphinyl-substituteddialanine; CAT, chlorarnphenicol acetyltransferase; DAF, days after flowering; glufosinate, 2-amino-4-(hydroxymethylphosphiny1)butanoate; GUS, bacterial P-glucuronidase;gus, bacterial 0-glucuronidase gene; hpt, hygromycin phosphotransferase gene; nos, nopaline synthase gene; PEG, polyethylene glycol; Pfr, active form of phytochrome; phosphinothricin, the S-isomer of glufosinate; phyA, phytochrome A gene; p h y B , phytochrome B gene; Pr, inactive form of phytochrome.

4

RAY WU ET AL.

81

R1

-450

E l 1 2 61

81

MUM of Construct

O w Erprorrlon Efficiency (X)

PACn-F

100%

PActl-G

50%

PACtl-L

28%

pAcnJ

4%

Xbl

C l

R1

81

R1

Xbl

t

+i

-45n

81

61

81

Bl

81

Xbl

I

-45s

+1

R1

81

Xbl

2% -450

0

+1

&18EodlnO -ton

W 1 5 ’ roglon non-codlng nons

nos 3 iorrn~~tor sqwnso

AM1 5’ lntron

FIG. 1. Activity of GUS enzyme in rice protoplasts transformed with Actl-Gus constructs. The results represent the average of four independent experiments assayed 20 days after transformation of protoplasts (16). The lengths of the different regions of the constructs are not to scale. The indicated restriction enzyme sites are abbreviated as follows: B1, BomHI; 82, BglII, RI, EcoRI; S1, Sstl; Xbl, XbaI; Xhl, XhoI.

2. TRANSIENT EXPRESSION ANALYSIS

OF

RICE ACTIN-1 GENE

Analysis of the 5’ region of the rice actin-1 gene (actl)was carried out by constructing plasmids containing different lengths of the 5’ region joined to the coding sequence of a gene encoding a bacterial P-glucuronidase (GUS). Transient assays using rice protoplasts showed that a region of 1,400 base pairs upstream from the act1 translation-initiation codon contains all of the S‘-regulatory elements necessary for high-level gus expression (construct pActl-F, Fig. 1).Deletion of 384 bp from the 5’-end of pActl-F gives pActlG, which was 50% as active as pActl-F. Starting from pActl-G, by deleting 133 bp between the acceptor splice site and the putative mRNA branchpoint splice site from its 5’ intron (pActl-L), lowered the GUS activity to 28% of that observed for pActl-F. Deletion of the mRNA branch-point splice site (vertical arrows in Fig. 1) and 3’-donor splice site from its 5’ intron (pActl-J) lowered GUS activity to 4% of that for pActl-F. Deletion of the

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

5

entire act1 5’ intron (pActl -H) resulted in a drop of expression level to 2%of that for pActl-F. These and other data suggest that the intron-mediated stimulation of gus expression is associated, in part, with an in vivo requirement for efficient intron splicing (16). Additional deletion analysis of the 5’ region of act1 was carried out in the region upstream from the transcription start site (designated + 1 position). Transient assay identified two distinct cis-acting regulatory elements in the promoter region. The first regulatory element, a 40-bp poly(dA-dT) region between positions -186 and -146 (Fig. 1, pAct1-F; the location is marked by a large dot), was a positive regulator of the Act1 promoter activity. Deletion of this sequence lowers gus expression by at least 3-fold compared to pActl-F. By gel-retardation and footprinting analyses, we identified a ubiquitous rice protein that specifically recognizes this poly(dA-dT) element (17). Similarly, a yeast poly(dA-dT)-binding protein, named “T-rich binding factor,” has been characterized, and this factor stimulates transcription from templates containing poly(dA-dT) elements (18). The second regulatory element, which serves as a negative regulator of the act1 promoter, consists of a CCCAA pentamer-repeat (between positions -297 and -265). A CCCAA-binding protein was detected in rice root extract but not in rice leaf extract. Furthermore, there are several additional potential regulatory elements within the act1 promoter. Deletion between nucleotides -152 and -37 decreases GUS activity to about one-half. There is also a putative TATA box within this region (17). Thus, these findings suggest that the act1 5‘ region contains multiple cis-acting elements, each of which interacts with either ubiquitous and/or tissue-specific trans-acting factors to confer the observed constitutive pattern of act1 promoter activity.

B. Rice Phytochrome Genes Phytochrome is a regulatory protein that functions as a photoreceptor in response to light signals. The photoreceptor molecule is reversibly interconvertible between two forms: red light converts the inactive form of phytochrome (Pr) to the active form (Pfr). The active phytochrome controls the expression of selected responsive genes, and ultimately results in altered growth and development appropriate for the light environment. Phytochrome constitutes an ideal system for analyzing the molecular processes involved in the light-signal perception, transduction, and transcriptional response phases of phytochrome-regulated gene expression (19). The genes encoding phytochrome from half a dozen plant species have been cloned and characterized. In some plants, there are several different phytochrome genes (reviewed in 20). Kay et al. (21, 22) cloned a rice phytochrome cDNA, encoding a type I phytochrome (PI) (also known as phytochrome A, phyA), and the corresponding genomic sequence. Further,

6

RAY WU ET AL.

Dehesh et al. (23) cloned a second rice genomic sequence of the phytochrome gene, p h y B , and found that it shares only 50% amino-acid identity with the rice p h y A .

1. I n Vivo ANALYSISOF RICE PHYTOCHROME GENEE ~ R E S S I O N Levels of phytochrome mRNA, isolated from rice leaves after different light treatments, were determined by using a portion of the rice cDNA clone as the probe. Slot-blot hybridization analysis of mRNA isolated from etiolated rice leaves shows that a one-minute red-light pulse, followed by four hours of darkness, results in a substantial decrease in phytochrome mRNA abundance. The decline in the level of mRNA is due to phytochrome itself because the effect is partially reversed when plants are illuminated with red light followed by a far-red light. These results suggest that rice phytochrome decreases the expression of its own gene. Furthermore, nuclear “run-on” experiments show that this autoregulatory response is exerted on transcription of the phytochrome gene. Kinetic analysis of the phytochrome-induced decrease in mRNA abundance shows a very rapid decline within 15 minutes following a red-light or white-light treatment. After 30 minutes, the phytochrome mRNA was barely detectable (21). Expression of the rice p h y B has also been studied in parallel experiments. The results indicate that, whereas p h y A is negatively regulated by its gene product (phytochrome) in rice seedling shoots in a manner typical of monocots, p h y B is constitutively expressed irrespective of light treatment (23).

2. In VitrO ANALYSIS

OF A

RICE PHYTOCHROME GENE

Gel-retardation analysis was carried out using a fragment from the 5’ upstream region of a rice genomic phytochrome gene ( p h y l 8 , also known as phyA). The results show that protein extracts from etiolated rice-leaf nuclei contain a factor tHat binds to the p h y l 8 5’ region, and this factor is similar to GT-1, shown previously to bind light-responsive elements of photophilic genes such as the pea rbcS-3A. Indeed, an expected GT-motif sequence (Fig. 2) was found in the rice p h y 2 8 (between positions -242 and -220). The -220 ---------

-242

Rice

TAGGTTAATTA-TTGGCGGTAT

Oat

TAGGTTMTCAATTTCAGGTTAAT -1 76 -1 53

.**a*****

0 .

* * a

FIG.2. Nuclear protein binding sequences in the upstream conserved regions of the n c e and oaf phytochrome genes (21,24).-=, identical nucleotides between rice and oat phyA,- on top of the sequence, GT1 binding site, --- on top of the sequence, GT2 binding site.

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

7

binding of the rice nuclear factor to phyl8 was effectively competed for by the pea rbcS-3A box-I1 sequence, but not by a mutated box I1 fragment in which the conserved GG sequence was changed to a CC sequence (21). 3. TRANSIENT EXPRESSION ANALYSISOF A RICE PHYTOCHROME GENE

Functional analysis of a rice phyA gene promoter was carried out by transient expression assays using microprojectile-mediated gene transfer into etiolated rice seedlings. Results from deletional analysis and linker-scan mutation show that a GT motif, GCGGTAATT (see Fig. 2, sequence marked with dotted line on top), closely related to elements in the promoters of a number of other light-regulated genes, is important for phytochrome gene expression. An oligonucleotide containing the tandem GT motifs from rice phyA promoter was used to isolate a cDNA clone encoding a protein (GT-2) that bound to these motifs. GT-2 from the over-expressing clone specifically binds to the rice phyA promoter, and it has domains related to certain other transcription factors. RNA blot analysis indicates that GT-2 mRNA levels decline in white light, although red and far-red light pulses are ineffective (24). The data indicate that phytochrome exerts autoregulatory control over the transcription of its own phyA genes in monocots. Conversion of this photoreceptor to its active Pfr form initiates repression of phyA transcription within 5-15 minutes of light signal perception by a mechanism that does not require new protein synthesis (20).

C. Rice Storage Protein Genes The average protein content of milled rice is lo%, whereas prolamins and globulins are minor components and glutelin is the major component, constituting 80% of the total endosperm protein (25). The mature glutelin protein is composed of two subunits, one acidic (around 32 kDa) and one basic (around 22 kDa), that are produced by the post-translational cleavage of a precursor molecule (26-28). In several studies, cDNA (28,29)and genomic DNA (29-32) sequences of rice glutelin genes have been observed. The genes have been classified into three subfamilies, Gtl, Gt2, and Gt3, according to their DNA sequences. Each subfamily is composed of several copies (28, 29). The coding regions of G t l and Gt2 subfamilies are closely related and show 94-96% DNA sequence identity to each other, whereas the Gt3 subfamily shows only 77-82% DNA sequence identity to the other two subfamilies (29). These three subfamilies together have been named “subfamily A” (30),and several new glutelin genes that belong to a new subfamily, named “subfamily B,” have been described. Two members of the subfamily B glutelin genes show only 60% DNA sequence identity to those in subfamily A.

8

RAY W U ET AL.

1. I n Viuo ANALYSIS OF GLUTELINGENEEXPRESSION Very little is known about the regulation of glutelin gene expression, except that mRNA levels are regulated differentially during seed development. RNA hybridization analysis was carried out using cDNA probes specific for Gtl and Gt3. With both probes, glutelin mRNA transcripts were detected at five days after flowering (DAF), but the accumulation patterns for Gtl and Gt3 transcripts differ during further seed development. Gtl transcripts continue to increase in level throughout seed development, reaching a maximal level at 25 DAF. In contrast, Gt3 transcripts reach the maximal level at 10 DAF and then steadily decline between 10 and 25 DAF. Accumulation patterns for Gt2 appear to be similar to those obtained for Gtl (29). The mRNA accumulation pattern of a subfamily B glutelin gene indicates that the mRNA is first detectable at six DAF, and the level gradually increases reaching a maximum at 14 DAF, when it begins to decrease (30).This pattern of glutelin mRNA accumulation is similar to that observed for Gt3 (29).

2. I n Vitro ANALYSISOF RICE GLUTELINGENE5' REGION

In uitro analysis of a rice glutelin gene, pGL5-1 of the Gt2 subfamily, gave the following information. The DNA sequence at the 5' region of pGL5-1 and two other Gt2-related clones show a high degree of sequence identity between position -213 and the transcription start-site (+ 1). A DNA fragment (Fragment A, between positions -224 and -45) from pGL5-1 was incubated with partially purified nuclear extract prepared from immature rice seeds. Four major retarded bands were observed in a gel-retardation assay. To localize the protein-binding regions more precisely, DNase-I botprinting analysis of the same glutelin DNA fragment (Fragment A) was carried out. Several protected regions (boxes I, 11, 111, and IV)were observed (Fig. 3). Two regions that show strong protection are Box 111 (positions -164 and -146) and Box I (centered around position -90) (31). The protein-binding study was extended using DNA fragments further upstream from Fragment A of the rice glutelin gene pGL5-1. Fragment B spanned the sequence between -515 and -224, and Fragment C spanned the region between -677 and -515. Gel-retardation assay indicates two retarded bands using Fragment C. In footprinting analysis one region (Box V) centered around position -585 was protected from DNase-I digestion. To confirm the results of the protein-binding studies, two sets of synthetic oligonucleotides were made. GT1-14 includes 14 nucleotides of Box I, and GT3-13 includes 13 nucleotides of Box 111. Each oligonucleotide was selfligated to produce multimeric units. Using GT1-14 for gel-retardation analy-

b b b 5 9

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS -677 Nsll

Box V

-224

Box IV

Box I1 Box 111

BOX I (-103 to -86)

ATATCATGAGTCACTTCA a

BOX 111 (-164 to -148) BOXII (-122 to -108)

Box I

* a

a a a

maam

am

ACAAATGATGTGTCAATTA CTTCCGTGTACCACA a

aaa

aaa

a

BOX IV (-206 to -189)

ATCATCCATPTCATATT

Consensus (I to IV)

T---GT_GTCA--T

BOXV (-595 to -575)

-

AAGTCATAACTGA

--

.=.,

FIG.3. Protein-binding sequences in the promoter region of a rice glutelin gene, pGL5-1 (31). identical nucleotides between different protein-bindingsequences, dashed lines under the sequence GTCA represent the most conserved portion of the five sequences; a solid line on top of the Box-I sequence represents a sequence identical to the binding motif found in transcription factors jun and GCN4, + + below the Box V sequence, 5 bp inverted repeat.

sis, several protein-DNA coinplexes were observed; however, with GT1-13, a single complex was detected. It is worth noting that the TGAGTCA binding motif, to which transcription factorsjun and GCN4 are known to bind, is located in the middle of the Box I and Box I11 sequences (Fig. 3, the sequence marked with a solid line on top) (31).

II. Methods for Gene Transfer in Rice Gene transfer refers to a process that allows a specific fragment of DNA to enter protoplasts or intact cells. The gene transfer technology has served plant scientists in at least three ways. First, it serves as a rapid and simple method for assaying transient expression of foreign genes. Second, gene transfer, followed by regeneration of transgenic plants, serves as a way for assaying gene expression after stable integration of the foreign gene into the plant genome. Tissue-specific or developmentally regulated gene expression can be analyzed in transgenic plants. Third, gene transfer, followed by regeneration of fertile transgenic plants, serves as a new and powerful method for introducing agronomically useful traits into crop plants.

RAY WU ET AL.

A. Introduction of DNA into Rice Protoplasts 1. FORTRANSIENT ASSAYOF GENEACTIVITY Efficient methods for transfer of a foreign gene into rice protoplasts were established several years ago (reviewed in 33). For promoter analysis, different lengths of the upstream regulatory sequence to be analyzed are often ligated to the gus reporter gene, and these gene constructs are transferred into rice protoplasts by either electroporation or by incubation with polyethylene glycol (PEG). Several days after gene transfer, regenerated cells are assayed for GUS enzyme activity. In this method, as long as the same batch of protoplasts are divided into aliquots and are used for the analysis of different gene constructs at the same time, transformation efficiency is relatively constant and the values of the GUS activity can be compared.

2. FORPRODUCTION OF TRANSGENIC PLANTS For efficient selection of transformed cells and calli, it is desirable to transform protoplasts with a construct that contains a selectable marker gene. Selectable genes that are currently widely used for rice transformation include hygromycin phosphotransferase gene (hpt) and phosphinothricin acetyltransferasegene (bar) (9, 34). The selectable marker gene can be used in a ca-transformation experiment with a second construct to be analyzed, such as a d - g u s . However, it is preferable that the selectable marker gene be incorporated into the same construct that is carrying the foreign gene to be analyzed. One study reports the recovery of several fertile transgenic plants after transforming rice protoplasts with an hph-containing construct, using PEG treatment. These plants transmitted their integrated hph gene into the second generation (35). Similarly, we co-transformed rice protoplasts with two plasmids: pin2 promoterlactl intronlgus (50 pg DNA/ml), and CaMV 35-S promoterlbar (20 pg DNA/ml). After regeneration of cells and calli, resistant calli were selected using ammonium glufosinate as the selective agent. We found that 60-70% of the resistant calli show GUS activity. In a typical transformation experiment starting with 4 x lo6 protoplasts and 20 pg of plasmid DNA containing the bar gene, over 500 calli resistant to ammonium glufosinate can be obtained. Approximately 100-150 transgenic plants can be produced, out of which between 5-30 plants are fertile. An advantage of the protoplast method is that a number of investigators successhlly regenerated fertile rice plants from protoplasts; expensive equipment is not needed. The disadvantage of this method is that, at present, less than a dozen rice varieties (out of hundreds of varieties grown in the

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

11

field) efficiently regenerate fertile plants from protoplasts. Thus, the potential for the direct application of this method to produce agronomically superior plants for farmers is limited. However, regardless of its value as a practical application, the protoplast system serves the purpose successfully in studying gene regulation in both transient assays and in transgenic plants.

6. Introduction of DNA into Intact Rice Cells by the Biolistic Method The biolistic method, also known as the particle-gun-bombardment method, was developed by John Sanford and his collaborators at Cornell University (36). In this process, high-velocity microprojectiles coated with DNA are used to penetrate the walls of intact cells. The major advantage of this method is that any type of intact plant cell or tissue can be used as a target for receiving foreign DNA Thus, one can use the technique to transform rice varieties that may not be readily amenable to plant regeneration from protoplasts. Furthermore, one can study tissue-specific and developmentally regulated gene expression by introducing gene constructs directly into distinct plant parts at any developmental stage.

1. BASICDESIGNOF

THE

BIOLISTICDEVICE

There are three major types of the biolistic device, differing mainly in the process of accelerating the microprojectiles, which are made of either tungsten or gold particles. The original design used gunpowder charges to accelerate DNA-coated tungsten particles (36),but the gunpowder has now been replaced by compressed helium (37). The second design uses an electrical arc-discharge device to accelerate the DNA-coated gold particles (38).The third design uses compressed nitrogen to accelerate microprojectiles (39). THE BIOLISTICMETHOD GENE EXPRESSION

2. APPLICATIONOF FOR STUDYING

One major application of the biolistic method is in the study of gene expression in intact cells or specific tissues. For example, rice, maize, and wheat suspension cells have been bombarded by a plasmid containing either a gus reporter gene or a chloramphenicol acetyltransferase (CAT) reporter gene (40).Two days after bombardment, the GUS or CAT activity was measured. These experiments were carried out to optimize the biolistic method and to compare the expression of specific gene constructs in the major cereal crops (40).Using intact tissues, and bombarding them with plasmids containing different promoters of rice genes, we found that a glutelin gene is specifically expressed in rice immature seeds (34,and an a-amylase gene is specifically expressed in the aleurone cells of mature rice seeds (41). It would be

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RAY WU ET AL.

very diacult to isolate protoplasts from the above mentioned cells or tissues for transient assays of different constructs that contain tissue-specific promoters.

BIOLISTICMETHOD FOR PRODUCING AGRONOMICALLY USEFULTRANSGENIC PLANTS The second major application of the biolistic method is to produce transgenic rice plants without the need to regenerate plants from protoplasts. In one study, a foreign DNA was biolistically introduced into intact cells of embryo sections dissected from mature seeds of rice. The aim was to hit the embryogenic cells in the shoot apex region. Seven varieties of rice were tested, including five indica varieties, from which plants are not readily regenerated from protoplasts. After bombardment with a GUS gene construct the embryo sections were cultured. Between 40-70% of embryo sections developed directly into plantlets 2 to 3 weeks later. GUS assay and DNA blot hybridization showed that the GUS gene was integrated into the rice genome in 20 transgenic plants. However, the GUS gene was not found in plants of the next generation (42). In another study, two different plasmids were introduced separately into rice immature embryos by electric discharge particle acceleration. Six varieties of rice were tested, including two varieties currently grown commercially in the United States, and four indica varieties. One plasmid, containing both a visible reporter gene (gus) and a selectable marker gene (bar),was used. At 12 to 15 days after pollination, rice immature embryos were isolated and bombarded with DNA-coated gold particles. The target was the scutellar region of the immature embryos. After particle bombardment, embryos were plated in suitable culture media to allow the development of embryogenic calli, and then plantlets, under both selective (with bialaphos which contains phosphinothricin) and non-selective conditions. Transformed embryos and regenerated plants were obtained, which expressed GUS activity and contained the foreign gene as detected by DNA gel blot hybridization. Fertile transgenic plants (% generation) were recovered and Mendelian segregation of foreign genes was observed in the progeny (R,progeny). On average, out of 1OO immature embryos bombarded, three transformed plants were recovered. Thus, engineering of rice through transformation of organized tissue can be accomplished by particle bombardment (43).

3.

APPLICATION OF THE

4. ALTERNATIVEMETHODS FOR

PRODUCING TRANSGENIC PLANTS

As discussed in the previous section, bombardment of immature rice embryos with plasmids can lead to the creation of first and second generations of transgenic plants. However, the frequency of obtaining pure lines of

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

13

transgenic plants is relatively low, mainly because of the high frequency of chimeric plant formation, which, in turn, is probably due to cross-protection of nontransformed cells by transformed cells that results in inefficient selection of pure transformants from bombarded immature embryos. An alternative method for producing transgenic plants is to bombard embryogenic rice suspension cells with plasmids, followed by the efficient selection of resistant calli. To test the efficiency of this system, small clumps of cells from embryogenic suspension culture were bombarded with an uctllbur plasmid. Five to seven days after bombardment, cells were cultured for six weeks in a medium containing ammonium glufosinate to select resistant calli. In a typical experiment, several hundred resistant calli were produced, and almost all were pure transformants (not chimeric). This is probably due to the fact that, with small clumps of suspension culture cells, there is no cross-protection of nontransformed cells by transformed cells. Transformed calli readily regenerate into plants. Furthermore, transgenic plants were resistant to spraying with a 1% solution of the glufosinatebased herbicide BASTA, whereas nontransformed plants died of ammonia toxicity in 5-10 days (44). DNA gel-blot analysis of rice plants resistant to ammonium glufosinate showed the expected hybridization bands using digerent restriction enzymes to digest rice genomic DNA. The results demonstrate integration of the input foreign gene into the rice chromosome. A high percent of the transgenic plants were fertile, and the second generation plants (R,) also gave positive results in DNA gel blot analysis. This study clearly demonstrates the feasibility of introducing agronomically useful traits into rice by bombardment of suspension cell culture, followed by plant regeneration.

C. Introduction of DNA into Intact Cells via the Pollen-Tube-Pathway Method A method for transferring DNA into recently pollinated rice florets, the “pollen-tube-pathway” method, has been developed (45),based on the success of producing transgenic cotton using a similar method (46). In the original study (4.57, total DNA from a variety of rice with purple-tip glumes and purple leaves (donor DNA) was transferred to a variety with normal green pigmented tissues (recipient plant) after cutting off the stigma of the floret and applying a drop of DNA solution to the cut end of the style. It is assumed that the DNA reached the ovule by flowing along the pollen tube and entering the egg cells or the zygote in the ovary. The cells of young embryos are either without or with partial cell walls, so foreign genes can enter the cells more readily. Mature seeds were collected and planted. The results show that some progenies, including those of the D,, D,, and D,

14

RAY W U ET AL.

generations, possessed glumes with purple tips and awns, similar to the phenotype characteristics of the donor plants. In addition, some glumes became completely purple (45). In a second study (43, total DNA of two rice varieties with high levels of resistance to bacterial leaf blight (Xanthomonas campestris pv. Oryzae) was used to treat the florets of nine sensitive rice varieties using the same pollentube-pathway method. A total of 2,400 florets were treated and 233 viable seeds (Do) were produced. These seeds were planted, and the plants (D, generation) were inoculated with the bacterial pathogen. After two weeks, development of disease on leaves was scored. One D, plant was highly resistant to bacterial blight. All second generation plants that came from this D, plant were also resistant to bacterial blight. The results indicated that disease resistance was genetically transmitted to the next generation (47). Experiments involving the addition of purified plasmid DNA (instead of total DNA) to the cut end of the styles of recently pollinated rice florets have been carried out by at least four groups of investigators. Their results are summarized below. (a) Zhou and Gong (48) constructed kanamycin-resistant plasmids containing different rice repetitive DNA sequences. These plasmids were then introduced into rice, and D, and D, generation plants were obtained. Among the D, plants, one plant showed strong NPTII enzyme activity, and DNA gel-blot analysis showed a hybridized band of the expected size. (b) Luo and Wu (49) treated a total of 259 rice florets (variety Fujisaka-5) with DNA containing 3 5 3 promoter and NPTII coding sequences as well as histone-3 coding sequences; 54 seeds matured. After germination of the seeds, root tips from 6-day-old plantlets and leaf tissues from 8-week-old plants were analyzed. Using p35S/NPTII DNA as the probe, genomic DNA from seven plants out of 54 gave positive signals in DNA slot-blot hybridization. When genomic DNA from four transgenic plants was digested with an enzyme that does not cut within the plasmid, different hybridization patterns were observed, as expected. These results suggest that the foreign gene had integrated into the rice genome. Assay for NPTII activity demonstrated that several transgenic rice plants gave positive results (49). (c) Hensgens and Schilperoort (SO) used the rice variety Taipei 309 and a plasmid (HH271) that contains a Gus and a hpt gene in their experiment. By using the pollen-tube-pathway method, a number of F, and F, plants were produced (F, and F, are the same as D, and D,). Approximately 20%of the F, plants displayed GUS activity, and two of them (out of 12 F, plants) showed a signal in genomic blot analysis. Several individual F, plants, derived from a single F, plant, gave strong signals in DNA blot hybridization, thus indicating DNA integration into the rice chromosomes.

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

15

To further substantiate the integration of the plasmid DNA into rice chromosome, F, seeds were analyzed by in situ hybridization. A low but significant number of cells displaying a hybridization site on a rice chromosome were detected, thus indicating DNA integration (SO). (d) Xie and Fan (51)treated 993 florets using a plasmid containing a deltaendotoxin gene of Bacillus thuringiensis (B.t.) var. aizawai 7-29. Out of the 387 D, plants, 11 gave positive results in DNA dot-blot analysis. Genomic DNA blot analysis was also carried out, and one plant (D,-73) gave a positive signal. Also, the root of this plant showed GUS activity in the histochemical GUS assay. Thirty-three mature seeds obtained from the plant D,-73 were planted. Ten out of 30 plants examined gave hybridization signals in DNA dot-blot analysis. Southern blot analysis showed that one of them (D,-73-19) gave a positive hybridization signal. An additional 400 D, seeds were obtained but have not yet been analyzed (51). (e)X. Duan and R. Wu (unpublished data) carried out experiments using different varieties of rice (Taipei 309, Lemont, and Labelle), different plasmid constructs (eight different constructs), different time intervals between the beginning of flowering and the time of adding DNA (varying between 1, 2, and 3.5 hours), and the concentration of DNA (100, 200, 300, 400 pg/ml) applied to the florets. Eight experiments were carried out and 181 to 557 florets were treated in each experiment. Approximately 470 first-generation plants (D,) were obtained, and 240 plants fiom florets treated with the Guscontaining plasmids were analyzed for GUS activity. The results indicate that 12 plants (5%)gave a positive GUS signal in leaves or roots of young plants three weeks after germination. However, in older plants, the percentage of GUS positive plants progressively decreases. DNA hybridization results using either the slot blot or the wick blot (52) procedure show that approximately 1% (five plants of 470 plants analyzed) of D, generation plants (younger than 6 weeks) gave positive results. Southern blot analysis using DNA from mature plants gave suggestive evidence for the presence of the input plasmid in rice genomic DNA. In conclusion, we now believe that the pollen-tube-pathway method is not as simple and efficient as it appeared to us several years ago. The method can give transgenic plants at low frequencies. However, it is difficult to reproduce because important variables have not yet been well-defined. In 1987, when we started to use the pollen-tube-pathway method (49), other methods for producing fertile transgenic rice plants had not been established. Now, both the protoplast method and the biolistic method are relatively well established; thus, there seems to be no compelling reason to use less well-established and less efficient methods such as the pollen-tubepathway method.

16

RAY W U ET AL.

D. Introduction of DNA into Intact Rice Cells via the Agrobacterium System The Agrobacterium system has been widely used to transfom dicot plants by co-cultivation (53). However, monocot plants, especially cereal plants, are generally considered d s c u l t to transform by this method. In one experiment, mature rice (var. Nipponbare) embryos were inoculated with a wide-host range supervirulent strain of A. tumefaciens (54).The tumorigenic callus tissue grew on hormone-free medium. The transformation of this tissue was confirmed by DNA hybridization analysis that showed the presence of T-DNA in the rice genome. Additional experiments were carried out using Agrobacterium harboring a plasmid that included hpt and Gus. Hygromycin-resistant calli were selected and plants generated. Plants from independent transformants contained an intact gus fragment, and 50% of the seeds obtained from the selffertile plants germinated successfully. Seeds obtained from two plant lines were tested for their ability to germinate in the presence of 40 p.g/ml hygromycin. Both lines gave rise to healthy seedlings, whereas control seedlings turned black and died (55). The results from these and other experiments suggest that the hpt gene had been transferred, integrated, and then expressed in the rice cells (54). In another study (56), A. tumfaciens was treated with phenolic chemicals to increase its host range and then co-cultivated with rice suspension cells. The results indicate that both NPTZZ and nos were transferred and expressed in cultured rice cells. Integration of foreign genes into the rice genome was confirmed by Southern blot hybridization.

111. Analysis of Transferred Genes in Transgenic Plants Information on promoter analysis obtamed in vitro or by transient expression assays in uiuo must be confirmed and extended by analysis using transgenic plants. This includes transient expression analyses on tissue specificity and developmental-stage specificity after biolistic bombardment of different tissues. Regulation of gene expression is expected to be different when the gene of interest is on a plasmid, which exists as an extrachromosomal entity from when the gene is integrated into the rice chromosome. Therefore, analysis in transgenic plants is essential for understanding the regulation of gene expression.

A. Rice Actin-1 Gene (act?) To determine the pattern and level of activity of the act1 5' region, transgenic rice plants containing the 5' region of act1 ligated to the gus

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

17

coding sequence were generated. In a transformation experiment, the act1 (5'-region)lgus (coding) plasmid was incubated with rice protoplasts in the presence of PEG. Among the large number of regenerated plantlets, 44 exhibited gus expression in root and leaf tissues. From these plantlets, two clonal plant lines, named T8-1 and T8-2, were selected for detailed analysis

(57). One goal of this experiment was to provide proof that integration of the input plasmid into the rice genome had occurred. DNA gel blot analyses of leaf genomic DNA showed that each transgenic plant derived from T8-1 contained four copies of the input plasmid. Integration of the input plasmid, uctI(5')lgus, into the rice chromosome was shown definitively by three gel blot analyses. (a) In undigested rice genomic DNA, the hybridizing band is in the 25-50 kb size range, which is the average size of the isolated genomic DNA fragments. (b) In DNA digested with NcoI restriction enzyme, for which there are no restriction sites within the input plasmid, the T8-1 plants gave three hybridizing bands. Since each band differs in size compared to the input plasmid, this indicates that this plasmid has integrated into the rice chromosome at three independent integration sites. (c)When genomic DNA was digested with HindIII, which cuts once within the plasmid, the T8-1 line clearly showed four hybridizing bands (57). The second goal of this experiment was to analyze the GUS activity and the GUS protein level quantitatively in transgenic rice plants. GUS enzyme activity was measured and the specific activity calculated. From the known specific activity of the purified bacterial GUS enzyme, the amount of GUS enzyme produced in the transgenic plants was estimated. Quantitative immunoblot analysis was also carried out to determine the amount of GUS protein accumulated in the transgenic plants. The results of these analyses show that, in the T8-1 plants, the GUS enzyme in the leaves amounted to 3.2% of total soluble leaf protein (57).

B. Expression of Rice Phytochrome Genes in Transgenic Dicots In order to investigate the mechanisms of phytochrome action in vivo, rice phytochrome gene was fused to a suitable promoter and transferred to tobacco or Arubidopsis. The behavior of the transgenic plants was then studied. (1)A full-length rice phytochrome cDNA was ligated to the CaMV 35-S promoter and transferred to tobacco, and the progenies of the transgenic plants were analyzed. Some of the transgenic plants contained large amounts of rice phytochrome mRNA in green leaves. Indeed, extracts prepared from these plants contain up to fivefold more phytochrome than extracts from control plants. By using species-specific, anti-phytochrome monoclonal antibodies, it was found that the rice phytochrome assembles with the chro-

18

RAY WU ET AL.

mophore and is photoreversible. Analysis of the circadian pattern of cab mRNA levels in transgenic plants versus control plants showed that the overproduction of rice phytochrome extends the duration of the free-running rhythm of cub gene expression (58). Additional experiments showed that rice type-I phytochrome regulates hypocotyl elongation in transgenic tobacco seedlings. It was also shown that the hypocotyl elongation phenotype is dependent on both light quality and on light intensity. Furthermore, successive pulse irradiation with red light elicited short hypocotyls similar to those obtained under dim white light, and the effect was reversed by immediate far-red light treatment. This provides a direct indication that the phenotype is caused by biologically active rice phytochrome gene in transgenic tobacco (59). (2)The rice phytochrome B gene was ligated to CaMV 35-S promoter and introduced into Arubidopsis. Transgenic plants that overproduced phytochrome B were analyzed: the over-expressed phytochrome B is spectrally active, undergoes redlfar-red-light-dependent conformational changes, and the protein is stable in light. Overproduction of this protein is tightly correlated with a short hypocotyl phenotype in transgenic Arabidopsis seedlings. This phenotype is strictly light-dependent, showing that phytochrome B is a biologically functional photoreceptor. Based on similarities to phenotypes obtained by over-expression of phyA, it appears that phytochromes A and B can control similar responses in plants (60).

C. Expression of Rice Glutelin Gene in Transgenic Tobacco Whenever possible, it is desirable to analyze rice gene expression in transgenic rice plants because the results are more meaningful when obtained from a homologous system that matches the physiological conditions. The match between a gene and the plants into which the gene is transferred is even more important in the case of analyzing a rice seed storage-protein gene such as a glutelin gene, because of the complex packaging of rice seed storage proteins into protein bodies. On the other hand, since fertile transgenic rice plants were not easily produced until several years ago, results obtained in transgenic tobacco can provide some useful information. A chimeric gene construct, composed of the rice Gt3 promoter and the CAT gene, was introduced into tobacco. Analysis of the transgenic tobacco seeds indicate that 980 bp of the 5’ flanking sequence of Gt3 is sufficient for the full promoter activity and directs a seed-specific expression (61). The 5‘ upstream region of a type I1 glutelin gene joined to a gus reporter was introduced into tobacco. The chimeric genes were expressed specifically in developing seeds of the transgenic tobacco. Histochemical analysis demonstrated that the GUS activity wss restricted to the endosperm tissue. A deletion series of the 5’ flanking region was generated from positions -1329

ANALYSIS OF RICE GENES I N TRANSGENIC PLANTS

19

to -74 relative to the transcriptional initiation site. After transformation and regeneration of transgenic tobacco, the results show that GUS activity in the seeds reaches maximum when the region between positions -441 and -237 is included (62).

D. The Protease Inhibitor Genes Until now, the improvement of major crops has depended mainly on conventional breeding methods. However, in certain situations, introduction of a given agronomically desirable trait into crop plants has been either impossible or prohibitively time-consuming. For example, certain agronomically useful traits found in some crop species cannot be introduced into other crop species by conventional breeding due to a species barrier in sexual crosses. In other cases, introduction of a new genetic trait, such as disease resistance, is a slow process because it takes many years to accomplish the transfer of the desired genetic trait through repeated backcrosses. Recently, gene-transfer technologies have provided new solutions to some of these problems. We use insect-resistant transgenic plants as an example to demonstrate how gene transfer technology can engineer an agronomically useful trait into rice plants.

1. NEED FOR EXPRESSING INSECT-RESISTANT GENESIN PLANTS The losses of agricultural products caused by insect pests are enormous. For example, in rice alone, the worldwide loss of yield due to insect damage is at least several billion dollars a year (63).Furthermore, several devastating viral diseases in rice are transmitted by insects (64). Conventional control of insect pests in rice cultivation often depends upon the use of chemically synthesized insecticides; thus, environmental damage has long been one of the major concerns regarding the long-term, massive use of these synthetic chemicals. Increasing pressure to use nonhazardous, environmentally compatible (safe) pest control measures has spurred interest in natural insecticides such as plant-derived proteinase inhibitors and Bacillus thuringiensis crystal proteins. Plant-derived proteinase inhibitors are of particular interest because they are part of the natural plant defense system against insect attack. Studies on the effects of dietary proteinase inhibitors, either artificially introduced into defined diets or those already present in plant tissues, have shown that these native proteinase inhibitors can interfere with the growth and development of insects by inhibiting their digestive enzymes. These proteinase inhibitors are easily inactivated by cooking, and they show no harmful effect on either humans or animals who eat proteinase-inhibitorcontaining plant products (see 65 and 66 for review). Several recent reports demonstrate that, when either a potato proteinase

20

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inhibitor I1 (pi&) or a cowpea trypsin inhibitor (CpTi) gene is joined to a suitable promoter and introduced into tobacco plants by Agrobacterium-mediated transformation, those transgenic tobacco plants that produced relatively high Ievels of the protease inhibitors become resistant to tobacco insect pests (67, 68). Our first approach to achieving high level expression in rice was to use a constitutive and efficient promoter of rice, the actl promoter, which was isolated in our previous studies. Both actl lpin2 and actl lCpTi constructs were made and introduced into rice using our transformation and selection system (3).Over 300 transgenic rice plants have been regenerated and some of them have been confirmed by Southern blot analysis. The levels of proteinase inhibitors in different tissues of transgenic rice plants were analyzed. The protein extract from leaf tissues expressing PIN11 protein exhibited inhibitory activity against both purified trypsin and chymotrypsin, whereas the protein extract from leaf tissues expressing CpTi protein showed strong inhibitor activity only against trypsin. These results suggest that (a) these two proteinase inhibitorswere correctly synthesized and functionally active in transgenic rice plants; and (b) with the availability of an efficient transformation system for rice or other monocot plants, genetic engineering for applied purposes can be quickly extended to the major cereal crops by introducing agronomically important genes.

2. AN ADVANTAGEOF USINGA WOUND-INDUCIBLE PROMOTER TO EXPRESSUSEFULFOREIGNGENESIN TRANSGENICPLANTS For the production of insect-resistant transgenic crop plants, one desires to synthesize high levels of the insecticidal proteins and also to be able to regulate the synthesis of these proteins. High level expression of insecticidal protein genes in all tissues during the entire life cycle of the plant may have the following undesirable effect. The synthesis of high levels of insecticidal proteins in plants may compete for energy and building blocks for RNA and protein synthesis with the synthesis of other essential cellular components. This may lead to a decrease in crop yield. This undesirable effect can be largely overcome by using a wound-inducible gene construct to transform plants that will produce high levels of the insecticidal proteins only after insect attack. It has been reported that the potato proteinase inhibitor I1 gene ( p i d ) is not expressed in leaves, stems, or roots of potato plants until the plants are mechanically wounded; thus it mimics the attack of chewing insects. Upon wounding, the production of proteinase inhibitor is induced, not only in the wounded leaves, but also in non-wounded upper and lower leaves, as well as in the upper part of the stem; thus, this represents a systemic, woundinducible gene activation (69). When a p i d - g u s construct was introduced

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

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into potato or tobacco plants, the expression of the gus reporter gene followed the same pattern of expression as the endogenous pin2 gene in potato (70). The unique property of the pin2 gene promoter, the systemic wound inducibility, makes this gene promoter a potentially useful tool for genetic engineering of insect resistance.

3. TRANSFORMING RICE WITH A CONSTRUCT USING THE WOUND-INDUCIBLE mv2 PROMOTER As a first step towards evaluating the dicot pin2 promoter for use in rice, we constructed several pin2lgus plasmids. At the outset, we had reason to believe that the dicot pin2 promoter might not function efficiently in monocot plants such as rice. We therefore incorporated the 5’-intron from the rice act1 into the 5’-transcribed region of the pin2-gus fusion construct because the act1 5’ intron had previously been shown to greatly enhance gene expression of a dicot gene promoter in transformed monocots. The functionality and activity of two constructs, pin2lgus and pin2lactl intronlgus, were analyzed in transgenic rice plants. In transgenic rice plants, the pin2tgus construct showed a very weak wound response, as measured quantitatively by the GUS activity. However, in plants harboring the pin2lactl intronlgus construct, wounding of a leaf dramatically increased the production of GUS by 10-15 fold. Moreover, the wound response is systemic because high levels of GUS activity were also found in non-wounded leaves. We carried out parallel experiments by measuring the gus mRNA level using the gus coding region as the probe. Similar results were found, showing that wounding greatly increases the level of gus mRNA in transgenic rice plants. Next, histochemical analyses of the transgenic rice plants were carried out. The results show that the pin2 promoter is generally active in rice leaves, roots, and stems. However, the highest level of expression of the pin2lactl intronlgus construct was associated with the phloem part of the vascuIar tissue in both wounded leaves and roots (Xu and Wu, unpublished results). This result is consistent with the previous observation made in transgenic potato transformed with a similar pin2 promoterlgus construct, and this supports the conclusion that the signals mediating the wound response are transported via the vascular system of the tissues (70). We can conclude that systemic wound inducibility of a typical dicot gene promoter, pin2, works equally well in transgenic rice plants. This observation suggests that the wound signal(s)and its transduction pathway are highly conserved between dicot plants and monocot plants. As described above, a major application of gene transfer technology is the introduction of agronomically useful traits into crop plants. Although there is still much room for further improvement of several established transforma-

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tion systems, we believe that with extensive efforts of scientists in this field, the gene transfer technologies for rice and other major cereal crops will eventually become routine. Currently, the challenge for plant scientists is the identification and isolation of agronomically useful genes, including those responsible for insect resistance, viral, and/or fungal pathogen resistance. It can be predicted that, with the availability of efficient transformation systems for rice or other monocot plants, genetic engineering for introducing agronomically important genes can be quickly extended to the major cereal crops.

IV. Concluding Remarks and Future Prospects A. Analysis of Monocot Genes in Transformed Rice Protoplasts, Cells, and Transgenic Plants For analysis of gene expression directed by promoters from monocot genes, rice is now the preferred system because transgenic rice plants can be produced relatively easily, much more easily than other cereal plants such as maize, wheat, barley, sorghum, and oat. Since these monocots are all in the Grumineae family, rice is a close relative to them and thus can be used as a suitable system for the analysis of genes from these cereal plants. Transformation systems for rice protoplasts are well established for both transient expression assays of promoter activity and for production of transgenic plants to study tissue-specific and developmentally regulated gene expression. In the analysis of different rice genes, as well as genes of other monocots or dicots, we have identified several promoters with different tissue specificities and regulatory properties for expressing useful foreign genes in transgenic rice. For example, we have shown that rice act1 promoter is highly and constitutively expressed in rice plants. The rice act1 5' intron can substantially enhance the expression of a potato pin2 gene promoter in transgenic rice without affecting the favorable property of wound inducibility of this promoter. Glutelin gene promoter is immature-seedspecific, and a-amylase gene promoter is mature-seed-specific. Other tissue specific promoters, such as leaf-specific, root-specific, or pollen-specific, have been described by other investigators. These promoters will be useful for expression of foreign genes in transgenic plants to improve different desirable agronomic traits of major cereal crops.

B. Production of Agronomically Useful Transgenic Rice Plants Biological insect control agents are now an attractive alternative to chemical pesticides. Biological control will probably cost less and will have fewer adverse effects on the environment as compared to the use of chemical insecticides. Success in producing insect-resistant dicots by transferring

ANALYSIS OF RICE GENES IN TRANSGENIC PLANTS

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genes that encode protease inhibitors (65-71) or B . t . toxins (72-75) into plants show that biological insect control is a feasible and practical approach. It can be predicted that within the next few years, transgenic rice plants transformed with genes encoding insecticidal proteins will be generated. Once insect-resistant transgenic rice plants are produced, the same approach can be used to transfer other agronomically important traits, such as disease resistance, into transgenic plants. Since each of the several known disease-resistance traits is coded for by a single gene based on genetic analysis, the chance of success will be high in generating disease-resistant rice plants. The large amounts of synthetic chemicals that have been used as herbicides have caused pollution of the environment. We believe that a superior alternative is to use insect-resistant crop plants to control weeds. For example, one can identdy the major weeds in a rice field and know which insects attack the weeds. Then, one can introduce one or two genes encoding insecticidal proteins into the rice plant so that these plants will be resistant to the insects and, thus, the insects will be forced to eat the weeds. Using a genetic engineering approach to produce transgenic rice plants that are tolerant to cold, drought, flood, or salt stresses is also possible as long as the genes encoding these useful traits are identified from different sources. Thus, identification and cloning of agronomically useful genes remains a challenge for plant scientists. Moreover, each of the above-mentioned traits is probably coded for by multiple genes instead of by a single gene, making the task of cloning genes more difficult. Nevertheless, with the rapid progress of plant molecular biology and plant biotechnology, the ultimate goals of identification and cloning a number of agronomically useful genes will be achieved. It is almost certain that many different varieties of transgenic rice plants will be grown in the fields around the world in the 21st century. Some transgenic rice plants will harbor several insect-resistant genes to protect them against insects, and other plants will carry several disease-resistant genes to protect plants against viral, fungal, and bacterial diseases. Some transgenic plants will include modified rice storage protein genes to increase the content of certain essential amino acids, and still other plants will include novel genes to make plants tolerant to low temperature, drought, flood, or salt stress. Thus, the future looks bright because the problem of food shortage can be largely solved by using superior transgenic plants to improve crop yield and quality.

ACKNOWLEDGMENTS This research was supported in part by a research grant RF 91001, #136, from the Rockefeller Foundation. D. Xu was supported by a predoctoral fellowship from the Rockefeller Foundation.

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40. Y. C. Wang, T. Klein, M. Fromm, J. Cao, J. C. Sanford and R. Wu, Plant Mol. Biol. 11,433 (1988). 41. J. K. Kim, J. Cao and R. Wu, MGG 232, 383 (1992). 42. J. Cao, Y. C. Wang, T. M. Klein, J. C. Sanford and R. Wu, in “Plant Gene Transfer” (C. J. Lamb and R. N. Beachy, eds.), pp, 21-33. Alan R. Liss, New York, 1990. 43. P. Christou, T. L. Ford and M. Kofron, BiolTechnology 9, 957 (1991). 44. J. Cao, X. Duan, D. McElroy and R. Wu, Plant Cell Rep. 11, 586 (1992). 45. X. Duan and S. Chen, China Agric. Sci. 3, 6 (1985). 46. G. Y.Zhou, J. Weng, Y. Zeng, J. Huang, S. Qian and G. Liu, in “Methods in Enzymology” (R. Wu, L. Grossman and K. Moldave, eds.), Vol. 101, p. 433. Academic Press, New York, 1983. 47. S. Chen and X. Duan, in “Rice Genetics 11: Proceedings of the Second International Rice Genetics Symposium.” Int. Rice Res. Inst., Manila, Philippines, p. 663,1991. 48. G. Y. Zhou and Z. Z. Gong, ”A Collection of Papers on Advances in Biochemistry for the Celebration of Professor Y. L. Wang’s Eightieth Birthday,” pp. 148-152. Chin. Publ. House Sci. Technol., Shanghai, China, 1987. 49. Z. X. Luo and R. Wu, Plant Mol. Biol. Rep. 6, 165 (1988). SO. L. A. M. Hensgens and R. A. Schilperoort, in “Gene Conservation and Exploitation” (R. Appels, P. Raven and J. P. Gustafson, eds.). Plenum, New York, in press, 1992. 51. X. Xie and X. L. Fan, Chin. Sci. Ser. B. 8, 830 (1991). 52. J. M. Irvine, J. V. Oakes, C. K. Shewmaker and A. Crossway Gene A d . Techn. Appl. 7,25 (1990). 53. S. G . Rogers, R. B. Horsch and R. T. Fraley, in “Methods in Enzymology” (A. Weissbach and H. Weissbach, eds.), Vol. 118, p. 627. Academic Press, Orlando, Florida, 1986. 54. D. M. Raineri, P. Bottino, M. P. Gordon and E. W. Nester, BiolTechnology 8, 33 (1990). 55. T. Ti, Y. Y. Bai, D. M. Raineri, M. P. Gordon and E. W. Nester, in “Fifth Annual Meeting of the International Program on Rice Biotechnology,”p. 46. Rockefeller Foundation, New York, 1991. 56. B. J. Li, Y. Xu, X. P. Xu, H. Shi, X. Ke and D. Yu, in “Fifth Annual Meeting of the International Program on Rice Biotechnology,” p. 61. Rockefeller Foundation, New York, 1991. 57. W. Zhang, D. McElroy and R. Wu, Pkmt Cell 3, 1155 (1991). 58. S. A. Kay, A. Nagatani, B. Keith, M. Deak, M. Furuya and N.-H. Chua, Plant Cell 1, 775 (1989). 59. A. Nagatani, S. A. Kay, M. Deak, N.-H. Chua and M. Furuya, PNAS 88, 5207 (1991). 60. D. Wagner, J. M. Tepperman and P. H. Quail, Plant Cell 3, 1275 (1991). 61. D. J. Leisy, Y. Hnilo, Y. Zhao and T. W. Okita, Plant Mol. Biol. 14, 41 (1990). 62. F. Takaiwa, K. Oono and A. Kato, Plant Mol. Biol. 16, 49 (1991). 63. E. A. Heinrichs, F. G. Medrano and H. R. Rapnsas, in “Genetic Evaluation for Insect Resistance in Rice,” p. 1. IRRI, Los Banos, Philippines, 1985. 64. K. C. Ling, in “Rice Virus Diseases,” pp. 11-33. IRRI, Los Banos, Philippines, 1972. 65. C. A. Ryan, Annu. Reu. Phytopathol. 28, 425 (1990). 66. C. A. Ryan, BioEssays 10, 20 (1989). 67. V. A. Hilder, A. M. R. Gatehouse, S. E. Sheerman, R. F. Barker and D. Boulter, Nature 330, 160 (1987). 68. R. Johnson, J. Narvaez, G. An and C. A. Ryan, PNAS 86, 9871 (1989). 69. H. Pena-Cortes, J. Sanches-Serrano, M. Rocha-Sosa and L. Willimitzer, Phnta 174, 84 (1989). 70. M. Keil, J. J. Sanches-Serrano and L. Willimitzer, EMBO J. 8, 1323 (1989).

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Immunoglobulin Gene Diversification by Gene Conversion WAYNET. MCCORMACK Department of Pathology and Laboratory Medicine Center for M a m d h n Genetics University of Florida College of Medicine Gainesville, Florida 32610

LARRYW. TJOELKER AND CRAIGB. THOMPSON' Howard Hughes Medical lnstitute Departments of lnternal Medicine and Microbiology lInimunalogy Uniuersity of Michigan Medical Center Ann Arbor, Michigan 48109 I. Evidence for Somatic Gene Conversion ........................ A. Somatic Diversity in Chicken Immunoglobulin Genes . . . . . . . . . . . . B. Gene Conversion in Chicken B Cell Tumors and Cell Lines C. Somatic Diversity in Rabbit V,, Genes .......................... 11. Molecular Mechanism of Somatic Immunoglobulin Gene Conversion . . . A. Gene Conversion vs. Double Homologous Recombination ......... B. Properties of Chicken IgL Gene Conversion ..... ..... C. Molecular Models for Immunoglobulin Gene 111. Enzymatic Activities Implicated in Immunoglobulin Gene Conversion . . A. Chromatin Structure and the Role of Transcription B. Initiation of Gene Conversion .................................. C. Bursa1 Expression of the Recombination-Activating Genes . . . . . . . . . D. Enzymes Involved in Homologous Recombination . . . . . . . . . . . . E. Heteroduplex DNA Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29 29 31 32 32 33 34 36 38 38 39 41 42 43 43 44

Genetic recombination mechanisms have evolved in vertebrate species that allow their immune systems to generate an extremely diverse antibody repertoire. It is estimated that this repertoire can include at least loy antibodies, each with distinct antigen specificity. Antibody molecules are heterotetramers consisting of two Ig heavy (H) and two Ig light (L) chains; it is

To whom correspondence may be addressed.

27 Progress in Nucleic Acid Research and Molecular Biology, Vol 45

Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any fnmm reserved.

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WAYNE T. MCCORMACK ET AL.

6

*a I

-- -- --

t5Y I

I

*%/I-so)

u,

I

4(

-

JM

I

I0

119

-.c,-- I

01

FIG. 1. Representative genomic organization of (A) a mammalian heavy chain gene locus and (B) the avian heavy chain locus as defined by Reynaud et al. (15).

the N-terminal regions of these chains that encode the antigen binding sites. Instead of relying on individual germline genes to encode the required antibody repertoire, during lymphoid development each B cell creates a functional immunoglobulin heavy and light chain gene by a site-specific DNA recombination process referred to as V(D)J2joining (reviewed in 1-3). During V(D)J joining a functional antigen-binding site is created by the somatic assembly of variable (V), diversity (D, in the case of heavy chains), and joining 0) gene segments. In humans, these gene segments are found within large clusters in the Ig loci (for example, in the IgH locus there are hundreds of V genes, twelve D genes, and four J genes; Fig. 1A). It is the random assortment of the individual gene segments that are assembled into a complete antigen-binding exon and the junctional diversity inherent to the V(D)J joining process that generate most of the diversity required to generate a primary repertoire. Most higher vertebrates, including teleost fish (4), the amphibian Xenopus (5), the reptile Caiman (6), and most of the mammalian species studied, appear to rely on these combinatorial and junctional mechanisms to generate the primary antibody repertoire. However, some vertebrate species utilize V(D)J joining not to generate diversity, but only to activate Ig gene expression in a B cell-specific fashion. In these species, diversity is generated subsequent to V(D)Jjoining by a second type of DNA recombination event, called gene conversion. Somatic gene conversion has been documented as the molecular mechanism used by the chicken (reviewed in 7-10) and a variety of other avian species (11).More recently, gene conversion has Abbreviations used: Ig, immunoglobulin; H, heavy chain; L, light chain; V, variable region or gene segment, D, diversity region or gene segment, J joining region or gene segment; V(D)J joining, recombination of Ig V, D, and J segments. ALV, avian leukosis virus; CDR, complementaritydetermining region, RAG, recombinase activating gene.

IMMUNOGLOBULIN GENE DIVERSIFICATION

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also been demonstrated to generate somatic immunoglobulin diversity in the rabbit (12),a mammalian species well known for the diversity of its immune response. Although less is known about the mechanism of gene conversion than that of V(D)J joining, recent work has begun to shed some light on the molecular mechanisms involved in immunoglobulin gene conversion. In this review, we focus on recent advances in the study of gene conversion, primarily in the chicken, and on current models for the molecular mechanism of this form of somatic gene conversion.

1. Evidence for Somatic Gene Conversion A. Somatic Diversity in Chicken Immunoglobulin Genes The chicken IgH and IgL loci are novel in that they each contain only single V and J gene segments (Fig. 1B) that are capable of undergoing V(D)J joining in B cell progenitors (13-16). In addition, V(D)J recombination in the chicken is not an ongoing development process (17, 18). Chicken B-cell differentiation begins with a single wave of Ig gene rearrangement between days 10 and 15 of embryonic development, primarily in the splenic anlage (reviewed in 19-22). V(D)Jjoining is complete by day 18 of embryogenesis, and chicken progenitor B cells then migrate to a specialized lymphoid organ, the bursa of Fabricius, which is a posterior invagination of the cloaca of avian species. Within the bursa of Fabricius, an initial population of about 3-5 x 105 B cells expands by proliferation to approximately 1-2 x lo9 mature B cells. The diversity of the rearranged Ig genes in the progenitor B cells entering the bursa is relatively restricted, due to the limitations of the V(D)J joining process, which can utilize only single V and J gene segments in the IgL and IgH locus. However, during the expansion of the B-cell population within the bursal microenvironment, sequence diversification occurs in the rearranged VL and V, regions by intrachromosomal gene conversion, using sequence information copied from germline variable region pseudogenes (qV) located 5’ of the functional V genes (14,23).Each B cell accumulates at least 4-10 gene conversion events within the rearranged V regions before migrating from the bursa to the peripheral lymphoid organs (14, 24). Only those B cells that have completed this gene conversion process leave the bursa. Gene conversion in the chicken IgL locus has been shown to be restricted to the rearranged V gene segment, and does not occur in the leader region, the J gene segment, nor within a germline IgL allele that has not undergone V-J joining (23). Restriction-site analyses of other genes expressed at high levels in bursal lymphocytes have been performed to determine whether gene conversion is specific for the Ig loci. For example, the

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WAYNE T. MCCORMACK ET AL.

FIG. 2. Genomic organization of the chicken immunoglobulinlight-chain gene locus. The organization depicted is for the light-chain locus of the S3 inbred strain as defined by W. T. McCormack et al. (unpublished observations) and is similar to that previously described by Reynaud et 02. (14) for the CB strain.

lymphoid-specific Ca5-tubulin gene has a genomic organization resembling the IgL locus. This gene is actively transcribed in bursal lymphocytes and is located just 3' of a pseudogene segment with an extensive open reading frame (25). Despite these similarities in organization to the Ig loci, the Ca5tubulin gene does not appear to undergo gene conversion in bursal lymphocytes (26). Similarly, clusters of the chicken histone H2b genes do not appear to undergo gene conversion during B-cell development (26). Taken together, these data support the hypothesis that the gene conversion process is specifically targeted to the rearranged V gene segments of the Ig loci. Nucleotide sequence analyses (14) of chicken IgL cDNA clones confirm that all rearranged chicken IgL genes are encoded by the single functional V, and JL gene segments. When sequences of cDNA clones derived from the bursa at different developmental times were compared, it was observed that the number of nucfeotide substitutions increases with time (14). Most importantly, these substitutions are clustered in blocks of sequences that exactly match sequence blocks within a cluster of 25-27 WLsegments situated in the -20 kb region 5' of the functional V,, gene (Fig. 2). Although homologous to the functional VL element, each of these YVL segments is truncated at the 5' end and therefore lacks the leader exon and promoter elements required for transcription, and is mutated or truncated at the 3' end, resulting in loss of the recombination signal sequence (heptamer / 12 or 23 bp spacer / nonamer) required for V-J joining (1-3). Therefore, the WL segments cannot participate in the formation of a functional Ig gene by joining to the J, segment. The observation that nucleotide substitutions in the rearranged gene are found in the VVL segments suggests that the *V, segments act as templates for the diversification of V,, via a somatic recombination process, now known to be gene conversion. The chicken IgH locus is organized similarly to the IgL locus, in that only one functional V, and JH gene segment is available for V(D)J joining (15) (Fig. IS). The amount of combinatorial and junctional diversity in the IgH locus is somewhat greater than in the IgL locus, due to the presence of approximately fifteen D, gene segments. However, just as in the IgL locus, the primary mechanism for generating somatic diversity of the V, region is through gene conversion, using a family of 90 WHsegments located in a

-

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31

60-80 kb region just 5’ of the functional V, gene as donors. Interestingly, gene conversions in rearranged IgH genes often extend into the D, region.

B. Gene Conversion in Chicken B Cell Tumors and Cell Lines Just as the availability of murine B cell lines, such as the Abelson murine leukemia virus-transformed pre-B cell lines, has facilitated study of the V(D)Jjoining process (2), the availability of chicken B cell lines is contributing to our understanding of the gene conversion mechanism of Ig gene diversification. Infection of susceptible chicken strains with the avian leukosis virus (ALV) induces a preneoplastic proliferation of lymphocytes in single bursal follicles to form a structure called the transformed follicle (27, 28). In most cases, integration of ALV adjacent to the c-rnyc proto-oncogene causes deregulation of c-myc expression in these transformed follicle cells (29). After a latency period of up to several months, undefined secondary transforming events cause malignant progression of transformed follicle cells to produce a B-cell lymphoma, which may later give rise to metastatic tumors (27, 28). Transformed follicle cells are capable of homing to the bursa of Fabricius and continuing proliferation .(30),suggesting that they are arrested by ALVtransformation at the bursal stem cell stage of differentiation, and should also be capable of undergoing gene conversion in their rearranged Ig genes. Restriction-site analyses of sites within the rearranged V,, gene demonstrate that restriction sites are progressively lost in these cells (23, 30), suggesting that gene conversion does indeed occur in them. One of the cell lines derived from an ALV-induced bursal lymphoma and tested by this assay, the DT40 cell line (31), undergoes gene conversion constitutively in uitro (30, 32, 33). Therefore, the DT40 cell line provides an in uitro model for the study of gene conversion in chicken Ig genes. For example, surface IgM-negative variants of DT40 that arise spontaneously in culture are due to gene conversion events that result in frameshifts due to nucleotide insertion or deletion (33). Reversion of these variants back to surface IgM expression occurs through the superimposition of additional gene conversion events on the frameshift mutations. Nucleotide sequence analyses of the rearranged IgL locus of DT40 cells passaged over a period of one year (32)revealed that most (39151) subclones of DT40 represent novel variants of the parental tumor, and that gene conversions could account for 2031220 nucleotide substitutions observed in the variants. DT40 is the only ALV-induced chicken B-cell line that has been found to undergo gene conversion in uitro. Chicken B-cell lines generated by infection with the v-rel oncogene-containing retrovirus, reticuloendotheliosis virus (34),also appear not to undergo gene conversion. The assays that have been used to analyze these cell lines include nucleotide sequencing of the

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WAYNE T. MCCORMACK ET AL.

rearranged IgL genes of v-re2 cell lines (32), and restriction-site analysis of the rearranged IgL alleles (34).There has been one report of a v-reE cell line undergoing a low frequency of sequence diversification as detected by the loss of restriction sites (35),but this observation has not been confirmed by nucleotide sequencing data.

C. Somatic Diversity in Rabbit VH Genes The rabbit V, family, consisting of a few hundred V, genes, is similar in size to that of other mammalian species (36, 37). The rabbit V, genes all appear to belong to a single subgroup, and as many as 40-50% appear to be pseudogenes (37-39). The average spacing of rabbit V, genes is only 3-7 kb (36,40),resembling the close spacing of chicken *V segments more than the 8-25 kb spacing of other mammalian V, genes (41). Surprisingly, studies of V, gene rearrangement in rabbit B lymphocytes revealed that only a single V, gene, the most D,-proximal V, gene (V,,), is rearranged in nearly all rabbit B cells (42,43). Most of the potential diversity available through V(D)J joining of multiple functional germline V, genes is bypassed. Therefore, the rabbit IgH locus is functionally very similar to the chicken IgH locus, which only has one functional germline V, gene. The observation that a single V, gene is rearranged in most rabbit B cells explained the Mendelian inheritance of allotypic markers expressed on rabbit V, regions (V,al, V,a2, and V,a& reviewed in M), because the rabbit V,, gene encodes the V,a allotype expressed on 7040% of all serum Ig molecules. However, the question arises as to how diversity is generated in rabbit immune responses, which are used by biologists in many fields to generate antisera. Nucleotide sequencing of random rabbit V, cDNA and genomic clones revealed that blocks of nucleotide sequence are shared in different cDNA clones (44,suggesting the possibility that gene conversion might occur in rabbit V, genes. However, sequence analyses of rearranged V,, genes (12) demonstrated conclusively that blocks of nucleotide substitutions, insertions, and deletions appear in the V,,, gene after V(D)J joining. Furthermore, potential gene conversion donors for many of these diversified regions were identified within other genornic V, genes andlor pseudogenes. These results suggest that, as in the chicken IgL and IgH loci, somatic gene conversion is a major mechanism used by rabbit B cells to generate somatic diversity of the rearranged vH1 gene.

II. Molecular Mechanism of Somatic Immunoglobulin Gene Conversion Because of the relatively simple organization and small size of the chicken IgL locus, it is ideal for studies detailing the regulation of somatic gene conversion. The chicken IgL locus spans only about 25 kb, including a

IMMUNOGLOBULIN GENE DIVERSIFICATION

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cluster of 25-27 q V , segments and single V,, J,, and C, genes (14, 16). Transcription of the chicken IgL locus is regulated by a promoter site consisting of a conserved octamer element (46) and a TATA box located 5' of the functional V,, gene. The nucleotide sequences of all 25 *VL segments of three inbred chicken strains have been determined, allowing studies of gene conversion donor relationships in rearranged V,, genes. Using this information, several laboratories have undertaken detailed descriptions of the products of the gene conversion process. Based on these studies, an understanding of the molecular mechanism by which blocks of nucleotide sequence are transferred from the pseudogene pool into the functionally rearranged V genes is emerging.

A. Gene Conversion vs. Double Homologous Recombination The sequence exchange events that are observed in the chicken V,, gene are compatible with two types of recombination event between the V,, gene and TV, segments. One of these is gene conversion, defined as a nonreciprocal exchange of DNA sequence from a donor gene (W,) into a recipient gene (V,J. The second type of recombination event compatible with the observed sequence diversification in the rearranged V genes is double homologous recombination, that is, reciprocal exchange between the ?VL gene segments and the V,, gene. To distinguish between these possibilities, Carlson et ul. (47) examined the products of sequence exchange events in an F, cross between two inbred chicken strains that differed in their IgL loci by several restriction-site polymorphisms and nucleotidesequence polymorphisms in the TV, segments. On the basis of the sequence polymorphisms between the V,, and VV, segments of the parental alleles, the nucleotide-sequence exchanges between the rearranged V,, gene and the 'PV, segments were shown to occur exclusively within a single allele, that is, intrachromosomally. The reciprocality of sequence exchange events was determined by analyzing the 'PV, segments in a panel of chicken B-cell lines, derived from the same strain by direct transformation with a v-rel-containing retrovirus. These clonal B-cell lines had undergone many rounds of sequence exchange between the V, gene and q V L segments, as evidenced by restriction-site loss within the rearranged V,, gene (47). For three cell lines in which gene conversion events appeared to be specifically derived from the 9 V L 7 segment, the WV,, segment was isolated from both alleles of the cell lines and sequenced. In all cases, the sequences of the 9 V L , segments matched the germline sequence and was not modified. As an additional test for unidirectional sequence exchange, and to exclude the possibility that homologous recombination occurs between a V,, gene and *V, segment on sister chromatids, extensive restriction-site analyses were performed on a large panel of v-re1 B cell lines. Whereas this assay does detect sequence modification in

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WAYNE T. MCCORMACK ET AL.

the rearranged V,, gene, there was no evidence for sequence modification in the W,cluster (47).

B.

Properties of Chicken IgL Gene Conversion

1. PSEUDOGENE DONORUSAGE

The frequency at which VV, gene segments are used as gene conversion donors appears to depend on three variables. The first of these is proximity to the functional V,, gene. Reynaud et al. (14)pointed out that the 9 V L segments located proximal to V,, are used more frequently than those distal to V,, and the same pattern was observed in a comprehensive analysis of gene conversion events in rearranged V,, genes isolated from the bursa of Fabricius at day 18 of embryogenesis (24). The second factor influencing pseudogene usage is the overall sequence homology of the VVL segment with the V,, gene, which is determined by the length of the 9 V L segment and the percent of nucleotide-sequence identity to V, (24). For example, truncated "V, segments are used less frequently than full-length VVLsegments. The third factor determining the frequency of "VL usage as a gene conversion donor appears to be the relative orientation of the q V L segment (24).The W gene segments that are situated in the inverted or antisense orientation with respect to the V,, gene are used as donors more frequently than PV, segments in the same orientation as VLl. This bias for the usage of inverted VV, segments is independent of location within the W,cluster and overall homology to V,,. As gene conversions begin to accumulate within the rearranged V,, gene, the newly acquired sequence may influence further gene conversion events. For example, during sequence analysis of gene conversion events in a bursal lymphoma cell line, DT40, which undergoes gene conversion in oitro, Kim et al. (32) observed that an early gene conversion event during the passage of this cell line involved a 15-bp insertion derived from the "vL8 segment. In later sublines of the parental DT40 cell line, the later gene conversion events observed in this region were highly biased toward the use of the *VLl8 segment, which shares a 15-bp insertion similar to that found in ?VM. This observation suggests that sequence alterations of early gene conversion events may alter the ability of other pseudogenes to act as efficient sequence donors. 2. LENGTHAND LOCATIONS OF GENECONVERSION EVENTS

Diversification of the rearranged V,, gene segment occurs by multiple serial gene conversion events within the V region. The number of events per clone isolated horn the bursa of Fabricius at day 18 of embryogenesis is one to four (14,24),and the average number of events per clone from the bursa at

IMMUNOGLOBULIN GENE DIVERSIFICATION

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three weeks of age is three to seven (24).Rearranged V,, genes isolated from later stages of development accumulate so many gene conversion events that it is impossible to identify discrete events, due to overlaps among the gene conversion events (W. T. McCormack and C. B. Thompson, unpublished data). These observations support the hypothesis that gene conversion events accumulate within the rearranged V,, gene until the lymphocyte leaves the bursa1 microenvironment. The exact length of gene conversion events is impossible to determine, because of regions of sequence identity between the q V , donors and the target V,, gene at the boundaries of gene conversion events. However, the minimum lengths of gene conversion events may be determined by the length of sequence between the 5’-most and 3’-most substitution derived from an individual gene conversion event. The minimum length of gene conversion events in day 18 cDNA clones is usually between 8 and 120 bp, with an average of 27 bp (14, 24). The longest gene conversion event observed in day 18 clones was at least 249 bp (24). When the locations of gene conversion events within rearranged V,, genes isolated from the bursa at 18 days of embryogenesis were compared, using their minimum lengths, there appears to be a paucity of events in the most 5‘ portion of V,, gene segment (24), even though donor sequences homologous to the leader portion of the V,, exon and part of the leader intron are present in the pseudogene pool. There are sequence polymorphisms in this region of the pseudogene donors that would allow detection of gene conversion events in that region (14). Gene conversions occur throughout the remainder of the V,, exon to the V,-J, junction. In addition, gene conversion events occur more frequently within the complementarity-determining regions (CDR), which are the regions of the Ig molecule that form the antigen binding pocket of the antibody molecule. 3. POLARITYOF GENE CONVERSION EVENTS

At the 3’ end of the rearranged V,, region, gene-conversion events have been observed that extend past the region of homology between the donor lIrV segment and the rearranged V,, gene segment. The marked difference in the frequency of gene conversion at the 5‘ and 3’ ends of the V region suggests the possibility that gene conversion may have a 5’-to-3’ polarity. Analysis of the ends of gene conversion events also revealed other differences in the 5‘ and 3’ ends of individual events. The 5’ ends of gene conversion events always lie within a region of nucleotide sequence identity between V,, and the q V L donor. In contrast, the 3’ ends of gene conversion events often end in regions of nonhomology, and may result in an increase or decrease in the overall length of the rearranged V,, segment due to imprecision in the joining of sequences derived from the gene conversion donor and

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WAYNE T. MCCORMACK ET AL.

V,, (24). These observations support the hypothesis that the mechanism of gene conversion exhibits 5'-to-3' polarity within the target V gene. One explanation for these observations is that gene conversion is initiated at its 5' end by homologous pairing of the nontranscribed V,, strand with a WL segment, and is followed by strand extension in the 3' direction. Observations that gene conversions sometimes extend in the 3' direction into the JL region (14,24), and that gene conversions in the chicken V,, gene often extend beyond the region of W, homology with V,, into the D, region (15, 48) support this hypothesis.

C. Molecular Models for Immunoglobulin Gene Conversion 1. SINGLE-STRAND BREAK-REPAIR

The polarity that appears to be inherent to the gene conversion process may be explained by a variation of the Meselson and Radding single-strand break-repair model for gene conversion (49). As illustrated in Fig. 3, gene conversion may be initiated by single-strand breaks in the nontranscribed

e -

7

~V~donor rearranged VL, gene

DNA nick strand transfer

6

strand extension

6

resolution

6

DNA ligation

6

heteroduplex DNA repair

FIG.3. A single-strand break-repair model for sequence transfer associated with immunoglobulin gene conversion. ’PV gene donor sequence, thick lines. Rearranged functional V gene segment, thin lines.

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strand of the V,, gene, which are made accessible during Ig transcription in

the bursa1 lymphocyte. The unwound 3' end may then invade the DNA duplex of a homologous q V L segment to create a "D loop" by homologous pairing, perhaps mediated by a strand transfer activity analogous to the recA activity of Escherichia coli (50). The relative paucity of gene conversion events in the 5' region of the V,, gene and apparent 5'-to-3' polarity of gene conversion (24)would then be explained by the requirement for this homologous pairing with the donor gene segment. For example, homologous pairing mediated by the recA protein requires 35-50 bp of homology between the two DNA strands (50). A D N A polymerase could then extend the 3' end of the invading strand using the *VL sequence as a template for DNA synthesis. At this point, resolution of the D-loop structure without a cross-over event would produce an extended strand that may overlap with the pre-existing DNA strand, depending on the extent of DNA synthesis and the possible action of exonucleases on the free 3' and 5' ends. The newly extended strand could be preferentially rewound into the V,, gene by the rewinding action associated with the RNA polymerase-I1 enzyme complex during continued transcription (51). Imprecision in the religation of the extended and the nascent strand could result in the codon loss and duplication observed at the 3' ends of gene conversion events (24). The final product is a V,, gene with a region of heteroduplex DNA, which may or may not be repaired before the cell divides. If DNA repair does not occur, one daughter cell will inherit the gene conversion event, while the second daughter cell will inherit the unmodified DNA strand. If heteroduplex DNA repair does occur, the final product will depend on whether there is any strand bias during the repair process. A strand bias for the repair of the transcribed strand, for example, would result in a geneconversion event in all cases. Examples of such strand bias in heteroduplex DNA repair have been reported for some forms of DNA damage in mammalian cells (52-54).

2. DOUBLE-STRAND BREAK-REPAIR The products of chicken immunogIobulin gene conversion are also consistent with the double-strand break-repair model proposed by Szostak and co-workers for gene conversion in yeast (55, 56), if the observed polarity can be accounted for. According to this model (Fig. 4), gene conversion may be initiated by breaks on both strands of the rearranged V,, gene, followed by strand invasion of the *V, donor molecule. Strand extension would then occur on both strands of the V,, gene, and resolution of the recombination intermediate without crossovers would result in heteroduplex DNA formation. The lower frequency of gene conversion events in the 5' region of the

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WAYNE T. MCCORMACK ET AL.

yV~donor rearranged VL1 gene

7 7 9

DNA nicks I strand transfer

y p ,strand extensions

-

9

resolution / DNA ligation

F

9

heteroduplex DNA repair

7

FIG.4. A double-strand break-repair model for transfer of sequence information between a YV gene donor sequence (thick lines) and rearranged functional VL1 gene segments (thin lines).

V,, gene would suggest that extensions in the transcribed strand are not recovered as efficiently in the final product. This may be due to an inability of the transcription complex to wind this strand in as a result of a nick or gap. Alternatively, strand bias during heteroduplex DNA repair could remove the donated sequence on the transcribed strand.

111. Enzymatic Activities Implicated in Immunoglobulin Gene Conversion

A. Chromatin Structure and the Role of Transcription One of the distinguishing features of the rearranged IgL locus, as compared to an IgL locus that remains in the germline configuration, is the presence of clusters of DNase-I-hypersensitive sites, which are expressed in a lymphoid-specific fashion (23).These DNase-I-hypersensitive sites are not present in the unrearranged V,, gene segments in bursa1 lymphocytes, nor in T lymphocytes. This observation suggests that the chromatin configura-

IMMUNOGLOBULIN GENE DIVERSIFICATION

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tion of the rearranged IgL allele is more “open” than the germline allele, and is perhaps more accessible to the enzyme activities involved in the gene conversion process. This may result from localized changes in D N A topology associated with the transcriptional enzyme complex (57-59). In addition to the difference in DNase-I hypersensitivity, two features distinguish the rearranged V gene that is a target for gene conversion from the pseudogene donors and the unrearranged V gene. The first of these is simply the fact that the rearranged V gene has been joined to the J L gene and its flanking sequence by the V-J joining event; the second is that transcription is activated by this rearrangement event. There are, therefore, two potential molecular signals which may direct gene conversion to the rearranged V,, gene. One is the physical juxtaposition of the V, and JL gene segments and/or the deletion of the region between V, and JL. The second possible signal is the passage of the transcriptional enzyme complex. The possible roles of gene rearrangement vs. transcription may be separated by study of gene conversion in the Muscovy duck IgL locus. In contrast to all other avian species examined, the Muscovy duck IgL locus consists of more than one functional VL gene in addition to a large number of P V , segments (11).Interestingly, some of the Muscovy duck *VL segments have retained the recombination signal sequence in their 3‘ flanking region, which is required for V-J joining. However, these PV, segments are truncated at their 5’ ends and lack the promoter regions required for transcription. Tjoelker and co-workers (60 and unpublished data) have demonstrated that although such PV, segments cannot be transcribed they do undergo joining to the JL gene, and may be recovered from developing Muscovy duck B cells due to a functional V, gene rearrangement on the other IgL allele (Fig. 5). These rearranged P V , segments do not undergo gene conversion events in the bursa of Fabricius, suggesting that joining to the J L gene segment is not sufficient to target the rearranged PV, segment as a recipient of gene conversion events. Furthermore, nonfunctionally rearranged V, genes that can be transcribed but do not produce a functional protein product due to an out-of-frame V,-J, junction or stop codon do undergo gene conversion (33, 60). Taken together, these results suggest that V-J rearrangement is not sufficient to activate gene conversion, and that an active transcription process is required for the initiation of gene conversion.

6. Initiation of

Gene Conversion

As described above, the gene-conversion process appears to be intimately tied to the transcription of the rearranged VL, gene. One mechanism by which gene conversion may be initiated by transcription is via the introduction of random nicks in the rearranged V,, gene by nonspecific endonucleases during the localized unwinding and rewinding of the gene during

40

WAYNE T. MCCORMACK ET AL.

-w-}L

VL

qJVL

-1-1L

"L

JL

JL

JL

+

Gene Conversion

+

No Gene Conversion

> -,

Gene Conversion

FIG.5. Susceptibility of various VJ recombination products to gene conversion. (*), stop d o n . As described in the text, transcribed VJ recombination products, whether or not they encode a hnctional product are susceptible to gene conversion, while a recombinationproduct that lacks a promoter is not susceptible to gene conversion.

passage of the RNA polymerase. Alternatively, nicks may be left by the incomplete strand religation of topoisomerases associated with the transcriptional machinery (61). However, such nonspecific nicking would not explain the unique targeting of the gene conversion process to the rearranged V genes. Other genes that are transcribed at high levels in bursal lymphocytes, and that have potential donor genes, do not undergo gene conversion events in bursal lymphocytes (26),suggesting an Ig-specificcomponent to the initiation of gene conversion. One mechanism for the specific introduction of nicks into the rearranged V,, gene could be via an Ig-specificendonuclease activity, similar to the HO endonuclease that targets the MAT locus for gene conversion during yeast mating-type switching (62).Although no evidence for specific single-strand or double-strand breaks in the rearranged V,, has heen presented, chromatin structural studies reveal the presence of three dominant DNase-Ihypersensitive sites that are expressed in a bursal-specific manner and are localized within the rearranged V ,, gene (23).As discussed above, the increased DNase-I hypersensitivity suggests that the rearranged V,, gene in bursal lymphocytes is at least in a chromatin configuration that may be more accessible to enzymatic activities. Sequence analyses have suggested that gene conversion events occur more frequently near the complementarity-determiningregions of the rearranged V , gene, at sites corresponding to the locations of sequences that resemble the conserved heptamer element of the recombination signal sequence required for V(D)J joining (24). It has been suggested that these

IMMUNOGLOBULIN GENE DIVERSIFICATION

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heptamer-like elements may be targets for a site-specific endonuclease activity similar to the one involved in V(D)Jjoining (9, 10). Consistent with a role for these heptamer-like elements within the V,, gene, a bursal-derived chicken B-cell line that constitutively undergoes gene conversion in oitro that has acquired a point mutation in one of these heptamer elements appears to have ceased to accumulate gene conversion events in the vicinity of that heptamer element (32). These observations suggest a possible relationship between the V(D)Jjoining mechanism and gene conversion, such that the component(s) of V(D)Jjoining that recognize the heptamer elements may be involved in targeting gene conversion to the V,, gene via recognition of heptamer-like sequences. For example, endonucleolytic activities have been reported that cleave at or near the recombination signal sequences (63). However, it is unlikely that the complete V(D)J recombinase activity is involved in gene conversion because ongoing rearrangement of IgH and IgL genes is not detected during the bursa1 stage of chicken B-cell development (18).

C. Bursa1 Expression of the Recombination-Activating Genes To investigate the possibility that gene conversion and V(D)Jjoining are functionally related, Carlson et al. studied the expression of the recombination-activating genes RAG-1 and RAG-2 during chicken lymphoid cell differentiation (64). The recombination-activating genes were described in experiments designed to identify genes encoding V(D)Jrecombinase activity (65, 66). Random genomic fragments were transfected into a fibroblast cell line harboring a selectable V(D)J recombination substrate. Genes encoding the V(D)Jrecombinase activity could be positively selected by the expression of the selectable marker, which could be expressed only after a chromosomal inversion via a V(D)Jrecombination event. A pair of linked genes (RAG-1 and RAG-2) was discovered by this assay, and their patterns of expression in lymphoid cells were determined. The co-expression of RAG-1 and RAG-2 is observed in all mammalian lymphoid cells of the B and T cell lineages that can rearrange V(D)Jrecombination substrates. The function of RAG-1 and RAG-2 is under investigation. It is unknown whether RAG-1 and RAG-2 encode components of the a V(D)J recombinase enzyme complex, or whether their expression regulates the expression of recombinase genes. The chicken homologs of RAG-1 and RAG-2 have been identified and sequenced, and the expression of chicken RAG-1 and RAG-2 in various lymphoid tissues during development has been examined by Northern blot analysis (64).In contrast to the coexpression of RAG-1 and RAG-2 in mammalian cells undergoing V(D)Jjoining, chicken B cells undergoing gene conversion in the bursa of Fabricius express high levels of RAG-2 in the absence of RAG-1 expression. This selective expression of RAG-2 is unique

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WAYNE T. McCORMACK ET AL.

to the bursal lymphocyte population, as chicken T cells developing in the thymus [and undergoing rearrangement of their T-cell receptor genes via V(D)Jjoining] were found to coexpress RAG-1 and RAG-2. Mature lymphocytes in the chicken spleen express neither RAG-1 nor RAG-2. These results suggest that the expression of RAG-2 alone, or of genes regulated by RAG-2 in the absence of RAG-1, may be involved in the regulation of somatic Ig gene conversion in bursal lymphocytes. To test this hypothesis, Takeda et al. (67) deleted the RAG-2 gene by homologous recombination from the DT40 cell line, which expresses RAG-&and undergoes gene conversion in culture as described above. RAG-2.’- DT40 cells were found to retain the ability to undergo Ig gene conversion, demonstrating that RAG-2 expression is not absolutely required for Ig gene conversion in this cell line. Therefore, the exact role of selective RAG-2 expression in developing bursal lymphocytes remains speculative.

D. Enzymes Involved in Homologous Recombination Many of the enzymatic activities that appear to be involved in somatic Iggene conversion are also likely to be involved in normal DNA replication and repair processes, and may not be unique to the gene conversion event. For example, strand extension of an invading V,, gene strand using the VVL segment as a template requires the activity of DNA polymerase, and the extended strand may be re-ligated to the pre-existing strand by DNA ligase activity. According to the models for immunoglobulin gene conversion, for gene conversion to be an efficient mechanism for the generation of sequence diversity in developing chicken B cells, these cells must be capable of promoting homologous pairing of the donor and target sequences. These observations suggest that chicken B cells may also be proficient at other recombination events that involve homologous pairing. Chicken B-cell lines display a dramatic increase in the ratio of targeted to random integration events compared to other vertebrate and chicken cell lines (68).This process was shown to be cell-type specific, occurring in a variety of B-cell lines, but not in non-B-cell lines. However, this homologous recombination process differs from gene conversion in several ways. Homologous recombination in chicken B-cell lines is not specific for the immunoglobulin locus, since targeted integration into the P-actin locus was also observed. In addition, transcription of the target locus is not required, as targeted integration occurs efficiently at the nontranscribed vitellogenin locus in chicken B-cell lines. Finally, although targeted integration events occur in the bursal lymphoma line DT40, which also undergoes constitutive gene conversion, a number of B-cell lines that do

IMMUNOGLOBULIN GENE DIVERSIFICATION

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not undergo gene conversion in vitro did exhibit the homologous recombination activity (68).These results suggest that avian B cells are competent at homologous recombination activities, and may use some of the enzymes involved in homologous recombination in the gene conversion process. An enzyme activity that may be required for both of these types of recombination events is the strand transfer activity, which may resemble the E . coli recA protein (50).Similar strand-transfer activities have been partially purified from a number of eukaryotic cell types, including the rec-1 activity from the fungus Ustilago (69), and from a variety of human cell types, including bladder carcinoma, fibroblasts, HeLa cells, and B lymphoblasts (70-72).

E. Heteroduplex DNA Repair The single-strand break-repair and double-strand break-repair models for immunoglobulin gene conversion both involve the formation of a heteroduplex region within the V,, gene. As described above, the polarity of gene conversion events in the day 18 bursa1 IgL clones (24) may be explained in part by strand bias during heteroduplex DNA repair (52-54). In fact, if a strong directionality in heteroduplex repair exists, one might argue for an alternative model of gene conversion that involves heteroduplex DNA repair within a D loop formed between the target V gene and donor WV segment, in the absence of strand breakage. However, the nucleotide sequence data for gene conversion events do not support this model. The observation that the 3' ends of gene-conversion events can be imprecise, with nucleotide loss or duplication, suggests that strand breakage, extension, and religation must be involved.

IV. Conclusions The study of the molecular genetics of chicken Ig light chain locus has provided insights into several types of DNA recombination events. For example, studies of V(D)J type joining in this locus provided the first evidence for specific nucleotide addition to coding ends during Ig gene rearrangement (16).However, the expressed chicken V-gene repertoire is generated not by the combinatorial and junctional diversification of V(D)J joining, but by somatic gene Conversion, utilizing families of variable region pseudogenes as a pool of donor sequence information (8, 10).Chicken B cells have also been shown to be capable of targeted integration events via homologous recombination (68).Taken together, these results suggest that the chicken B cell provides a good experimental model system in which to study the mechanism of a variety of somatic DNA recombination events in higher vertebrate cells.

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37. S. J. Currier, J. L. Gallarda and K. L. Knight, J. Immunol. 140, 1651 (1988). 38. W. T.McCormack, S. M. Laster, W. F. Marzluff and K. H. Roux, NARes 13, 7041 (1985). 39. K. H. Roux, P. Dhanarajan, V. Gottschalk, W. T. McCornlack and R. W. Renshaw, J. Zmmunol. 146, 2027 (1991). 40. K. E. Bernstein, C. 8 . Alexander and R. G. Mage, J . Zmmunol. 134, 3480 (1985). 41. T. Honjo, Annu. Reu. Zmmunol. 1, 499 (1983). 42. R. S. Becker, M. Suter and K. L. Knight, Eur. J. Zmmunol. 20, 397 (1990). 43. K. L. Knight and R. S. Becker, Cell 60, 963 (1990). 44. R. G. Mage, K. E. Bernstein, N. McCartney-Francis, C. B. Alexander, G. 0. YoungCooper, E. A. Padlan and G. H. Cohen, Mol. Zmmunol. 21, 1067 (1984). 45. W. T. McCormack, P. Dhanarajan and K. H. Roux, J. Zmmunol. 141, 2063 (1988). 46. T. Parslow, D. Blair, W. Murphy and D. Granner, PNAS 81, 2650 (1984). 47. L. M. Carlson, W. T. McCormack, C. E. Postema, C. F. Barth, E. H. Humphries and C. B. Thompson, Genes Deu. 4, 536 (1990). 48. R. Parvari, A. Avivi, F. Lentner, E. Ziv, S. Tel-Or, Y. Burstein and I. Schechter, EMBO]. 7, 739 (1988). 49. M. S. Meselson and C. M. Radding, PNAS 72, 358 (1975). 50. C. M. Radding, BBA 1008, 131, (1989). 51. H. 8. Gamper and J. E . Hearst, Cell 29, 81 (1982). 52. I. Mellon, G. Spivak and P. C. Hanawalt, Cell 51, 241 (1987). 53. P. C. Hanawalt, Enuiron. Mol. Mutagen, 14 (Suppl. 16), 90 (1989). 54. H. Vrieling, M. L. Van Rooijen, N. A. Groen, M. Z. Zdzienicka, J. W. Simons, P. H. Lohman and A. A. van Zeeland, MCBiol9, 1277 (1989). 55. J. W. Szostak, J. L. Orr-Weaver and R. J. Rothstein, Cell 33, 26 (1983). 56. H. Sun, D. Treco, N. P. Schultes and J. W. Szostak, Nature 338, 87 (1989). 57. L. F. Liu and J. C. Wang, PNAS 84, 7024 (1987). 58. B. I. Osborne and L. Guarente, Genes Deu. 2, 766 (1988). 59. S. J. Brill and R. Sternglanz, Cell 54, 403 (1988). 60. L. W. Tjoelker, W. T. McCormack, L. M. Carlson and C. B. Thompson, in“Abstracts of the 7th International Congress of Immunology,” p. 13. Fischer, Stuttgart, 1989. 61. B. J. Thomas and R. Rothstein, Cell 56, 619 (1989). 62. A. J. S. Klar, in “Mobile DNA” (D. E. Berg and E. E. Howe, eds.), p. 671. Am. Soc. Microbiol., Washington, D.C. 1989. 63. T. J. Hope, R. J. Aguilera, M. E. Minie and H. Sakano, Science 231, 1141 (1986). 64. L. M. Carlson, M. A. Oettinger, D. G. Schatz, E. L. Masteller, W. McCormack, D. Baltimore and C. B. Thompson, Cell 64, 201 (1991). 65. D. G . Schatz, M. A. Oettinger and D. Baltimore, Cell 59, 1035 (1989). 66. M. A. Oettinger, D. G. Schatz, A. Gork and D. Baltimore, Science 248, 1517 (1990). 67. S. Takeda, E. L. Masteller, C. B. Thompson and J.-M. Buerstedde, PNAS 89, 4023 (1992). 68. J.-M. Buerstedde and S. Takeda, Cell 67, 179 (1991). 69. E. B. Kmiec and W. K. Holloman Cell 29, 367 (1982). 70. P. Hsieh, S. Meyn and R. D. Camerini-Otero, CeU 44, 885 (1986). 71. D. Ganea, P. Moore, L. Chekuri and R. Kucherlapati, MCBiol 7, 3124 (1987). 72. E. Cassuto, L.-A. Lightfoot and P. Howard-Flanders, MGG 208, 10 (1987).

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AD P- ribosy lat ion Factors: Protein Activators of Cholera Toxin’ JOEL MOSS AND

MARTHA VAUGHAN Laboratory of Cellular Metabolism National Heart, Lung, and Blood lnstitute National lnstitutes of Health Bethesrkr, Maryland 20892 I. Biochemistry of ADP-ribosylation Factors ........................... 11. Structure of ADP-ribosylation Factors .............................. 111. Functions of ADP-ribosylation Factors in Animal Cells . . . . . . . . . . . . . . .

References

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49 55

60 63

Some microorganisms affect their hosts by the action of secreted toxins that disrupt regulatory or metabolic pathways. Cholera toxin, a secretory product of Vibrio cholerae, closely related Escherichia coli secretory products (known as heat-labile enterotoxins, or LTs), and a Bordetella pertussis protein (with the dual names of pertussis toxin and islet-activating protein) all appear to affect cell metabolism through effects on proteins that require guanine nucleotides for activity and are responsible for transmembrane signalling (1).The toxins alter the activity of these guanine nucleotide-binding proteins (G proteins) by catalyzing the transfer of the ADP-ribose moiety of NAD to a critical amino acid in the a subunit. This ADP-ribosylation reaction results in either activation (e.g., cholera toxin, E . coli heat-labile enterotoxin) or inactivation (e.g., pertussis toxin) of the G protein. The G proteins are heterotrimeric signal-transducing proteins responsiAbbreviations: ARF, ADP-ribosylation factor; sARF, soluble ARF from bovine brain; hARF, human ARF; bARF, bovine ARF; yARF, yeast ARF; CPS, chicken pseudogene; G proteins, guanine-nucleotide-bindingproteins; G, and G,, the inhibitory and stimulatory G proteins, respectively, of the adenylyl-cyclase system; G , or transducin, the G protein coupled to rhodopsin and involved in visual excitation; Go, the G protein found in high concentration in brain; G,, the a subunit of the G proteins; DMPClcholate, dimyristoylphosphatidylcholine plus cholate; CTA, cholera toxin A subunit; CTA1, the A 1 protein of cholera toxin; LT, E. coli heat-labile enterotoxin; LT-Ih, LT serogroup type I that is encoded by an E. coli plasmid isolated From a human; LT-IIa, LT serogroup type 11, variant a; LT-IIb, LT serogroup type 11, variant b; GTPyS, guanosine 5’-[y-thio] triphosphate; GDPPS, guanosine 5’-[P-thio]diphosphate; GPP(NH)P, guanosine 5 -[P,y-imido]triphosphate; APP(NH)P, adenosine 5 ’ 4 3 , ~ imido]triphosphate . 47 Progress in Nucleic Acid Research and Molecular Biology, Vol. 45

48

JOEL MOSS AND MARTHA VAUGHAN

ble for coupling cell-surface receptors to their intracellular effectors (2-4). Activation of a receptor occurs through its interaction with a specific ligand or agonist. Activated receptor in turn activates the G protein. In its inactive state, the G protein consists of a, @, and y subunits, with the nucleotidebinding site of the a subunit containing GDP (4). Activation of a receptor by a ligand is associated with (i) release of GDP from the a subunit followed by binding of GTP, and (ii) dissociation of a(GTP) from Py. In most instances, a(GTP) is responsible for alteration of effector activity, although Py subunits may be involved in the control of phospholipases and certain other enzyme activities (4, 5). In this regulatory pathway, signal termination occurs, in part, as a result of the hydrolysis of bound GTP to GDP by an activity intrinsic to the 01 subunit (2-4). The a(GDP) is believed to reassociate with @y. Reactivation of the inactive a(GDP)Py complex results from its reassociation with agonist-activated receptor. Cholera toxin and E. coli heat-labile enterotoxins catalyze the ADPribosylation of the a subunit of G,, the stimulatory G protein of the adenylylcyclase system that is also believed to regulate Ca2+ channels (1, 6). There appear to be four forms of G,, generated by alternative splicingof transcripts of a single gene (7 ). Pertussis toxin can ADP-ribosylate several highly similar G proteins (8). These include: the Gai family (Gail, Gu,e, and Gai3), which is involved in the inhibition of adenylyl cyclase and control of ion flux; G,, which may also be involved in the regulation of ion flux; and Gat, or transducin, which plays a critical role in visual excitation in the retina (8).There are at least three different G,, genes and two G,, genes, and one G, gene that yields two proteins with different carboxy termini through alternative splicing of mRNA (9). ADP-ribosylation by pertussis toxin of the G,,, G,,, and G,, proteins on a cysteine residue near the carboxy terminus interferes with their ability to interact hnctionally with cell-surface receptors, thereby interrupting transmembrane signalling (8). In addition to these proteins, there is a family of G, subunits that appear not to be substrates for any of these bacterial toxins (10). Cholera toxin-catalyzed ADP-ribosylation of the Gas isoforms leads to activation of the protein, as a result of inhibition of its GTP hydrolytic activity and consequent persistent subunit dissociation into active a(GTP) and f3-y components (11,12).Inhibition of GTP hydrolysis thus prolongs the life-time of the activated species. In addition, ADP-ribosylation may promote release of GDP, thus clearing the nucleotide-binding site and permitting GTP binding (13, 14). ADP-ribosylation occurs on an arginine residue located near the center of the OL subunit (15). In the adenylyl-cyclasesystem, activation of G, by ADP-ribosylation results in a GTP-dependent enhancement of cyclase activity and increased formation of cyclic AMP (I). Early studies showed that cholera toxin-catalyzed ADP-ribosylation of

49

ADP-RIBOSYLATION FACTORS

G,, and/or activation of adenylyl cyclase is stimulated by GTP or an analog, and by membrane and soluble factors from a variety of species and tissues (16-28). Pigeon erythrocyte cytosol contains a factor of 15-20 kDa that, in the presence of GTP, enhances toxin activity; the cytosolic factor was sensitive to trypsin (16, 17). Enhancement of the capacity of membranes to respond to toxin has been achieved by incubation in the presence of GPP(NH)P and cytosolic factor. Adenine nucleotides are only weakly effective. A trypsin-sensitive cytosolic factor of -13 kDa that enhances cholera toxin-catalyzed activation of pigeon erythrocyte adenylyl cyclase and ADPribosylation of a 43-kDa protein has also been identified in horse erythrocytes (18).

-

1. Biochemistry of ADP-ribosylation Factors A. Purification and Characterization ARFs from a number of tissues and species including rabbit liver and bovine brain have been extensively purified and characterized (23-25, 27). The ARFs isolated from rabbit liver membranes were two proteins of -21 kDa (23), with Stokes radius (R,) of 2.38 nm, S20,w (S) of 2.10, partial specific volume (ml/g) of 0.74, M , of 21,500, and frictional ratio, fl/fO, of 1.19 (23).The protein appeared to be both activated and stabilized by detergents; it was stable to N-ethylmaleimide, but thermolabile. ARF was also isolated from bovine brain membranes and soluble fractions (24). The membrane ARF had a sedimentation coefficient similar to that of the rabbit liver protein in the presence of detergent. Mg2+ increases its stability and altered its chromatographic behavior. Two soluble forms of ARF (termed sAAF I and sARF 11) isolated from bovine brain had similar properties (25, 27).

B. Activation of Cholera Toxin by ARF To determine the mechanism of activation of cholera toxin by ARF, advantage was taken of the fact that cholera toxin, in addition to catalyzing the ADP-ribosylation of G,,, can use simple guanidino compounds (e.g., arginine, agmatine) and proteins unrelated to G,, as ADP-ribose acceptors (29, 30). Included among these protein acceptors is CTA1, the catalytic fragment of the toxin (31, 32). Cholera toxin-catalyzed ADP-ribosylation of simple guanidino compounds is stereospecific and proceeds by an SN2-like reaction with inversion of configuration (33).P-NAD is the substrate and the product is a-ADP-ribosyl-arginine (33). ADP-ribosylation of G,, by cholera toxin is stimulated by a purified bovine brain soluble ARF (sARF 11) or membrane ARF (mARF) in the

ADP-RIBOSYLATION FACTORS

51

presence of GTP or GTP analogs such as GTPyS or Gpp(NH)p; GDP, GDPPS or adenine nucleotides are inactive (Fig. 1)(25, 27). The reaction is further enhanced by DMPC/cholate. Under similar conditions, cholera toxin also catalyzes the auto-ADP-ribosylation of the CTAl catalytic unit (Fig. 1). Optimal modification also requires GTP or poorly hydrolyzable GTP analogs; GDP, GDPPS, and adenine nucleotides are ineffective (Fig. 1). DMPC and cholate are not necessary and, in fact, inhibit ARF- and GTPdependent auto-ADP-ribosylation, although they slightly enhance basal (in the absence of ARF and guanine nucleotide) auto-ADP-ribosylation. ARF also enhances ADP-ribosylation of simple guanidino compounds such as agmatine, which is stimulated by GTP and nonhydrolyzable GTP analogs, but not by GDP, GDPPS, and adenine nucleotides (25,27,34,35). The apparent EC,, for GTP was influenced by certain phospholipids and detergents (35). In the presence of SDS, the EC,, is -5 pM; with DMPC/cholate, it is -50 nM (35).Thus, ARF appears capable of activating cholera-toxin ADP-ribosyltransferase activities under conditions where it exhibits either relatively high or low S n i t y for guanine nucleotides. With SDS, activation is observed at relatively low, submicellar concentrations (-0.003%), whereas at higher concentrations, both ARF-stimulated and basal ADP-ribosyltransferase activity is inhibited (35).Activation by DMPC/ cholate is likewise dependent on both the concentrations and the ratio of the two agents. Maximal rates of reaction occur without a delay in SDS. However, in the presence of DMPC/cholate there is a lag before optimal activity is achieved. The delay is eliminated by incubation of sARF I1 with GTP before initiation of the reaction. The effects of phospholipids and detergents on the GTP-dependent activation of cholera toxin by ARF are also reflected in their effects on guanine nucleotide-binding. As expected from the activity data, high-affinity binding by soluble ARF from bovine brain is not observed in SDS (35). In DMPC/cholate, Mg2+-dependent, high-affinity GTP binding is not significantly affected by components of the transferase assay (cholera toxin, NAD, or agmatine). ARF purified from bovine brain membranes binds GTP, GDP, and GTPyS but not adenine nucleotides (24). Optimal binding of guanine nucleotides requires DMPC, high salt, and Mg2+ with a maximal stoichiomeFIG. 1. Effect of dimyristoylphosphatidylcholine,ARF, and nucleotides on ADP-ribosylation of G,, and auto-ADP-ribosylation of the cholera toxin A1 protein. Assays contained [32P]NAD, cholera toxin A subunit, G,, and, as indicated, dimyristoylphosphatidylcholine (DMPC), a soluble ARF purified from bovine brain (sARF 11). and guanine or adenine nucleotides. Shown are four autoradiograms of results of SDS-PAGE. Mobilities of CTAl and G,, are indicated on the left. Data are from Tsai et al. (27).

52

JOEL MOSS AND MARTHA VAUGHAN

try of almost 0.9 molGTP per molARF. Unlike many other GTP-binding proteins, ARF does not hydrolyze GTP. Binding of GTP or GTPyS increases the intrinsic fluorescence relative to that observed in the presence of GDP. Consistent with a lack of GTP hydrolytic activity is the finding that the effects of GTP are similar to those of GTPyS and are stable with time (24). Recombinant ARF 1, synthesized in E. coli, bound GTPyS in the presence of DMPC, cholate, and MgCl, with a K , of -0.1 p M (36).ARF had a significantly higher affinity for GDP than it did for GTPyS at low Mg2+ concentrations. At millimolar Mg2+, the apparent affinities for GTPyS and GDP are similar. Nucleotide binding is inhibited by 0.2 to 0.8 M NaC1. The recombinant ARF does not hydrolyze GTP (less than 0.0005/min).

C. Guanine-Nucleotide-Dependent Complex Formation with Cholera Toxin From kinetic studies, it has been concluded that ARF directly activates CTAl (Fig. 2) (34). It appears not to stimulate the toxin-catalyzed reaction by binding to G,,, as had been proposed (23).To determine whether stimulation by ARF is associated with formation of a stable ARFCTA complex, the proteins were incubated in the presence of guanine nucleotides and SDS, and the sizes of the products determined by gel permeation chromatography (37).In the presence of ARF and GTPyS, but not GDPPS, an active ARFtoxin complex was formed that eluted near the void volume of a Sephadex G-75 column. Only a minor fraction of ARF formed such complexes with toxin. CTAl in the aggregate was much more effective in catalyzing autoADP-ribosylation than was toxin that did not complex with ARF. The presence of both toxin and ARF in addition to SDS and GTPyS were required to obtain an active aggregate. ARF in the aggregate stimulated the ADPribosylation by added CTA of G,, and other proteins, but was itself a poor substrate for CTA. Monomeric ARF and CTA are more effective than the aggregated proteins in ADP-ribosylating model protein substrates such as albumin. The substrate specificities of the toxin in the monomeric form and in complex with ARF are clearly different. With DMPC/cholate (instead of SDS) and GTPyS, an ARF-toxincomplex was not detected. Instead, some of the ARF appeared to form a GTPyS-dependent aggregate, independent of toxin. Similar to its behavior with SDS, only a minor fraction of ARF formed aggregates in DMPC/cholate; the effects of the aggregated and monomeric ARF on the substrate specificity of cholera toxin were different (37)*

D. Immunological Characterization of ARFs Both anti-ARF peptide and anti-ARF protein polyclonal antibodies have been used to evaluate distribution and cross-species conservation (38, 39).

53

ADP-RIBOSYLATION FACTORS

IGTP 1 Thiol

ARF-GTP*

A,-SH

ARF*GTP*A,-SH"

ADP-RiboseGS,

"-J

ADP-RiboseG sa .GTP*

'Inactive

L

-

1

ADP-RiboseGS; GTP VActiive

b

ATP

Cyclic AMP

FIG. 2. Participation of a guanine nucleotide-binding protein cascade in the activation of adenylyl cyclase by cholera toxin. ARF is activated by GTP (or, in oitro, by a nonhydrolyzable GTP analog). ARF-GTP then complexes with and activates CTA1. Cholera toxin, composed of one A subunit consisting of A 1 and A2 fragments linked through a single disulfide bond, and five B subunits, is activated by thiol, generating an active A 1 protein. Activated CTAl catalyzes the transfer of ADP-ribose from NAD to G,,, which, in the presence of GTP, stimulates adenylyl cyclase catalytic unit, increasing the conversion of ATP to CAMP. Model is from Tsai et al. (27).

Rabbit anti-bovine brain sARF I1 antibodies react on immunoblots of bovine brain proteins with two soluble forms of ARF that behave electrophoretically like sARF I and sARF I1 and a membrane form of ARF, mARF (39). The antibodies do not react with a subunits of the heterotrimeric G proteins, G,, (transducin) from bovine retina, Go from bovine brain, or G,, and G,, from rabbit liver. Rabbit anti-sARF I1 antibodies react with -20-kDa ARF-like proteins in bovine, rat, and frog tissues (39). ARF appears to be present in highest concentrations in brain extracts. In rat brain, as in bovine brain, the predominant immunoreactive species is sARF 11. In peripheral tissues, the major immunoreactive species is sARF I. Anti-peptide antibodies detected ARF immunoreactivity in Saccharomyces cerevesiae and Dictyostehm discoidium, but not E . cob (38).Between 50 and 90% of ARF immunoreactivity was found in the soluble fractions of animal cells and tissues.

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JOEL MOSS AND MARTHA VAUGHAN

E. Developmental and Hormonal Regulation of ARF In rat brain, as in bovine brain, most of the ARF is cytosolic (39). Immunoreactivity showed two soluble ARF species, corresponding in electrophoretic mobility to bovine brain sARF I and sARF I1 (39). It should not be inferred that these are identical to sARF I and I1 purified from bovine brain cytosol. There is no increase with age in the protein corresponding to sARF I (39). On postnatal day two, the amounts of sARF I1 and sARF I in rat brain were equivalent. By the tenth postnatal day, sARF I1 was predominant. The relative increase in immunoreactive sARF I1 over sARF I was associated with an increase in ARF activity, quantified by its ability to stimulate cholera toxin ADP-ribosyltransferase activity, following partial purification by gel permeation chromatography from a soluble fraction (39). Some ARF mRNAs appear to be under hormonal control. Chronic corticosterone administration increases the amounts of mRNAs for ARF 1 and ARF 3 and increases A R F immunoreactivity in rat cerebral cortex (40). Consistent with possible physiological control of ARF mRNAs by corticosteroids, bilateral adrenalectomy decreases the amounts of mRNAs for ARF 1 and ARF 3.

F. Activation of E. coli Heat-Labile Enterotoxins by ARF Escherichia coZi heat-labile enterotoxins (LTs) are responsible in part for “travelers diarrhea” (41). The LTs include two serogroups, LT-I and LT-11, and two variants of the LT-I1 serogroup, LT-IIa and LT-IIb, have been analyzed in detail (1, 42-45). The LTs are similar to cholera toxin in that their effects on cells result from ADP-ribosylation of G,, (46-50). The LTs, however, do have some structural differences from CT. LT-I is most similar to cholera toxin in amino-acid sequence, enzymatic activity, and immunoreactivity, whereas LT-IIa and LT-IIb appear to represent a different branch of the toxin family tree (45, 51, 52). The A subunits of cholera toxin and all the LTs, however, share a significant degree of amino-acid homology (51, 52). Both purified bovine brain ARF and recombinant ARF synthesized in E . coli stimulate the ADP-ribosyltransferaseactivities of cholera toxin and the LTs (SO). In the presence of ARF, DMPC, and cholate, ADP-ribosylation of G,, by cholera toxin and LT-I is slightly greater than that observed with LT-IIa or LT-IIb. In all instances, the ARF effect is dependent on GTP (50). Like cholera toxin, the LTs catalyze the ADP-ribosylation of agmatine, a simple guanidino compound (SO). In the presence of ARF and GTP, but not with either alone, there is a significant increase in toxin-catalyzed ADPribosylagmatine formation (Fig. 3). SDS further enhances GTF- and ARFstimulated activity of all four toxins (Fig. 3).Significant differences in specific activities are observed when agmatine is used as the ADP-ribose acceptor.

55

ADP-RIBOSYLATION FACTORS

CT

LT-lh

LT-lla

LT-llb

FIG.3. Effects of ARF, GTP, and SDS on ADP-ribosylation of agmatine catalyzed by cholera toxin and E. coli heat-labile enterotoxins. Cholera toxin, and E . coli,heat-labile enterotoxins, LT-I, LT-Ila, and LT-IIb, were assayed for their ability to catalyze the ADP-ribosylation of agmatine in the presence of buffer (open bars), purified sARF I1 (a soluble ARF from bovine brain) (cross-hatched bars), GTP (double cross-hatched bars), or sARF I1 plus GTP (speckled bars). SDS was present as indicated. In the panels describing LT-IIa and LT-IIb, the presence of 103 on the ordinate indicates that the specific activities were multiplied by 1 8 to obtain numbers equivalent to those observed with cholera toxin. Data are from Lee et al. (50).

NAD:agmatine ADP-ribosyltransferase activities of cholera toxin and LT-I are over 100 times those of LT-IIa and LT-IIb. The differences in specific activities are evident both with and without ARF. It appears that the catalytic sites of LT-IIa and LT-IIb have a much more restricted substrate specificity and use “model” substrates with much lower efficiency than do cholera toxin and LT-I (50).

II. Structure of ADP-ribosylation Factors To identlfy the structural features of ARFs responsible for guanine nucleotide-binding, activation of cholera toxin, and reconstitution of yeast mutants, deduced amino-acid sequences of ARFs and ARF-like proteins were determined through the isolation of cDNA and genomic clones and the generation of cDNAs by reverse transcription and the polymerase chain reaction (53-61). At present, six mammalian, two yeast, and two Giardin ARF clones have been isolated (53-61). The mammalian ARF genes can be grouped into three classes based on deduced protein size and amino-acid sequence (Fig. 4) (58). Class-I ARFs, which include ARFs 1-3, contain 181 amino acids and differ from each other

56

JOEL MOSS AND MARTHA VAUGHAN

hARFl hARF3 bARF2 BARF4 hARF5 hARF6 y ARF hARFl hARF3 bARF2 hARF4 hARF5 hARF6 yARF hARFl BARF3 bARF2 hARF4 hARF5 hARF6 yARF

FIG. 4. Comparison of deduced amino-acid sequences of mammalian and yeast ARFs. All ARFs are compared to hARF 1 (55). Asterisks (*) indicate amino-acid identity with ARF 1, whereas hyphens (-) represent gaps introduced to optimize sequence alignment. Human ARFs 1, and 3-6 are abbreviated as hARF 1(55). hARF 3 (SS),hARF 4 (SS), hARF 5 (58),and hARF 6 (58), respectively. Bovine ARF 2 and yeast ARF 1 are indicated as bARF 2 and yARF I, respectively. Not shown in the figure are deduced amino-acid sequences for bovine (53) and rat ARF 1 and rat ARF 3 (Price et al., unpublished data), which are identical to those of the human counterparts. Data are, in part, from Tsuchiya et al. (58).

only near the amino and carboxy termini (Fig. 4) (58).Class-I1 ARFs (ARFs 4 and 5) contain 180 amino acids and differ from Class-I ARFs near the amino terminus and in the carboxy half of the proteins (Fig. 4).The single known member of Class I11 (ARF 6) has only 175 amino acids and differs from ClassI and -11ARFs most extensively near the amino terminus and in the carboxy half of the protein, but there are differences in amino-acid sequence throughout the protein. Separation of the mammalian ARFs into three classes based on similarities in size and amino-acid sequence is supported by phylogenetic analysis of the ARF coding region nucleotide sequences. In this scheme, yeast ARF is clearly differentiated from the six known mammalian proteins (58). The division of mammalian ARFs into three classes is also evident when coding region nucleotide and deduced amino-acid sequences are compared (Table I). Class-I ARFs are 95-96% and 80-84% identical in deduced aminoacid and nucleotide sequences, respectively (58). Class-I1 ARFs (human ARFs 4 and 5), are only 79-802 identical in amino acid sequence to Class-I

57

ADP-RIBOSYLATION FACTORS

6

5

4

3

2

1

ARF 2b

cDNA

ARF-Specific Probes

FIG. 5. Hybridization of rat brain poly(A)+ RNA with ARF-specific cDNA and oligonucleotide probes. Rat brain poly(A)+ RNA was hybridized with a coding region cDNA probe generated from a bovine retinal ARF 2 clone (ARF 2h) and oligonucleotide probes specific for ARFs 1 to 6. Data are from Moss et d.(69).

TABLE I COMPARISONS OF ARF-DEDUCED AMINO-ACID AND CODING-REGION NUCLEOTIDE SEQUENCES" hARFl hARFl bARFl hARF2 hARF3 hARF4 hARF5 hARF6 CPSl yARF

91 79 84 67 75

68 69 64

bARFl

hARF2

hARF3

hARF4

hARF5

hARF6

CPSl

yARF

100

96 96 -

80 80 80 79

80 80 80 79 90 65 65 64

68 68 69 68

67 67

80 84 68 73 69

96 96 95

-

77 77 77 76 72 69 65 65

59

-

68 66

80 68 71 64 63 66

-

71 73 66

68 65

77 60 60 67

64 64 89 60

68 68 64 64 99

0 Comparisons of the deduced amino-acid sequences are above the diagonal, and those of nucleotide sequences are compared below the diagonal. Comparisonsare expressed as percentage identity. Sources of the ARF sequences are noted in Fig. 4. Data are from Tsuchiya et 01. (58). Abbreviations are listed on page 47.

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JOEL MOSS AND MARTHA VAUGHAN

ARFs, and 64% identical to Class-111 ARF; however, they are 90% identical to each other. At the nucleotide level, the similarities are less obvious. ClassI1 ARFs are 67-75% identical to Class I and 60-65% identical to Class 111, but they are only 77% identical to each other. Human ARF 6 (Class 111)is 60-69 and 64-69% identical to ARFs 1-5 in coding region nucleotide and deduced amino-acid sequences, respectively. Despite the differences in deduced amino-acid sequences among Classes I, 11, and 111, all ARFs contain the conserved amino-acid signature sequences believed to participate in guanine-nucleotide binding and GTP hydrolysis (62). The phosphate-binding/GTP hydrolysis region (GX,X,X,X,GK; amino acids 24-30 in human A R F 1;Fig. 4) differs from other ras-like GTP-binding proteins in the presence of aspartate at position X, (63-65). In the ras proteins, this substitution, or the presence of almost any amino acid other than glycine at this site, is associated with a marked reduction in GTPase activity (62). One of the sequences involved in the coordination of Mg2+, believed by analogy to ras to be involved in the binding of the GTP p and y phosphates, bears more similarity to the 01 subunits of the heterotrimeric G proteins than to the cognate regions of other members of the ras superfamily of -20 kDa proteins (e.g., ras, rap, rho, rac, ral, rub) (62). This region, DVGGQ (amino acids 67-71 in human ARF 1; Fig. 4), is conserved among all of the ARFs. Similarly, the CAT sequence (amino acids 159-161 in human ARF 1; Fig. 4), which has been implicated in interaction with the guanine ring, is identical to a sequence in the 01 subunits of the heterotrimeric G proteins. It differs from those in the ras superfamily, in which only the alanine is present (62).The NKXD sequence (amino acids 126-129 in human ARF 1; Fig. 4) is believed to participate in binding of the guanine ring. In some members of the ras superfamily, such as rac, this sequence is TKXD (62). The ARFs are highly conserved among mammalian species, and between mammalian species and lower organisms, such as Saccharomyces cerevesiae and Giardia lamblia (53, 58, 59, 61, 62). In Saccharomyces cerevesiae, two ARF genes have been described, encoding two proteins that are 96% identical (61).There is an ARF-like gene (termed arl) in Drosophila melanogaster; the deduced amino-acid sequence i s -55% and -70% identical to the mammalian and yeast ARFs, respectively (66). The arl gene, encoded by a 1.05kb mRNA, was essential for viability. Use of one of two potential initiation codons gives rise to an open reading frame of 540 bp, encoding a 180 aminoacid protein of -20 kDa. The ARL protein reacted with an antibody made against a peptide representing amino acids 22 to 35 of ARF 1. It bound guanine nucleotides and, in contrast to ARFs, hydrolyzed GTP. It did not exhibit ARF activity in a cholera toxin ADP-ribosyltransferase assay and did not suppress ARF mutations in yeast (66). It thus appears that the ARF

ADP-RIBOSYLATION FACTORS

59

family includes related proteins similar in sequence but devoid of cholera toxin-stimulating activity. Among mammalian ARFs, human ARF 1 is 100% identical in deduced amino-acid sequence to rat and bovine ARF 1, and human ARF 3 is 100% identical to rat ARF 3 (53,55; S. R. Price, M . S. Nightingale, J. Moss and M. Vaughan, unpublished data). Human ARF 6 differs from a chicken ARF pseudogene only at position 158 where serine replaces threonine (58, 67). Conservation of amino-acid sequence is associated with conservation of coding region nucleotide sequence. Human ARF 1 nucleotide sequence is 91% identical to that of bovine ARF 1; those of human ARF 6 and the chicken pseudogene are 89% identical (58, 67) (Table I). This degree of identity is somewhat remarkable for cross-species comparison to a pseudogene. Yeast ARF 1is 65-77% and 60-67% identical to the mammalian ARFs in deduced amino-acid and coding region nucleotide sequences, respectively (53, 58) (Table I). ARF 2 mRNA, although present in bovine and rat tissues, has not been detected to a significant extent in human or monkey tissues by Northern analysis, by amplification of ARF-specific domains using the polymerase chain reaction, or by screening of human cDNA libraries (68; 1. M. Serventi, E. Cavanaugh, J. Moss and M. Vaughan, unpublished observations). Some ARFs, thus, may either diverge greatly in nucleotide sequence or be expressed in a tissue- or developmentally-specific manner. Expression of ARF 2 in rat brain, for example, appears to be developmentally regulated (39). There is considerable conservation of both mRNA size and sequence among the mammalian ARFs. A coding region probe excised from a bovine retinal ARF-2 cDNA with EcoRI and PvuII hybridized with multiple bands in poly(A)+-RNA from human, bovine, and rat tissues (68) (Fig. 5). ARFspecific hybridization was obtained by using specific oligonucleotide or cDNA probes under conditions of high stringency. In hybridization with rat brain poly(A)+-RNA, it was found that ARF 1corresponds to a 2.1-kb band, ARF 2 to one of2.6 kb, ARF 3 to bands at 3.8 and 1.3kb, ARF 4 to one at 1.8 kb, ARF 5 to a band at 1.3 kb, and ARF 6 to bands at 4.2 and 1.8 kb (Fig. 5) (69). Based on gene structure, it appears that the two mRNA species observed for ARF 3 result from the use of alternative polyadenylation sites (70). The human genes for ARFs 1and 3 have five exons (70, 71), four introns, and similar positions of exon-intron boundaries; a similar structure has been observed for the bovine ARF-2 gene (Fig. 6) (72). For all three Class I ARFs, exon 1 is not translated, exon 2 contains the GX,X,X,X,GK regions, exon 3, the DVGG, and exon 5, the CAT; the NKQD is divided, with NKQ in exon 4 and the aspartate in exon 5. For ARFs 1 and 3, the promoter regions are consistent with those of housekeeping genes and contain neither a TATA nor

60

JOEL MOSS AND MARTHA VAUGHAN GLDMQK

DVGG

NKQ

D CAT

FIG. 6. Structural comparison of human and bovine class I ARF genes. Exons and introns of the bovine ARF 2 (bARF 2), human ARF 1(hARF l),and human ARF 3 ( U R F 3) genes are indicated by boxes and solid (-) or broken ( I / )lines, respectively. All three genes are drawn to scale, with -//- indicating deletions in introns. Darkened boxes represent coding regions, with letters above the bARF 2 coding exons indicating, in single letter code, those amino acids believed to be involved in guanine nucleotide-binding and GTP hydrolysis (phosphate binding). Nucleotides shown below the non-coding exons in the 3'-untranslated regions indicate potential polyadenylation signals. Data are from Lee ct 01. (74, Serventi et al. (72), and Tsai et at. (70).

a CAAT box (70, 71).In both genes, there are potential Spl sites (GC boxes). Consistent with the promoter structure is the evidence from primer extension analysis and S1 and mung-bean nuclease mappings that there are multiple transcription initiation sites (70, 71).

111. Functions of ADP-ribosylation Factors in Animal Cells Although ARF proteins were initially identified by their ability to enhance the ADP-ribosyltransferase activity of cholera toxin in uitro, it is unclear what role they have in toxin action on cells and there is only very limited evidence that they influence endogenous ADP-ribosylation in animal cells (73). ARFs purified fiom soluble and membrane fractions of bovine brain have no effect on the activity of NAD:arginine transferases purified from turkey erythrocytes (J. Moss and S. J. Stanley, unpublished observations). Recently, however, four ADP-ribosyltransferasesthat modify non-muscle actin were purified fiom rat-brain extract and used to assess the effects of two soluble ARFs purified from rat brain by the procedure described earlier for bovine brain (73). Each of the enzymes incorporated a maximum of -1 mol of ADP-ribose per mole of G actin; F actin is apparently not a substrate. ADP-ribosylation of G actin prevented its polymerization. The enzymes had similar K,s for NAD (12-31 pM) and pH optima of -8, but different abilities to modify certain proteins, different responses to four phospholipids, and different responses to sARF I and sARF I1 (73). sARF I stimulated activity of transferase 11, inhibited transferase 111, and had no effect on the

ADP-RIBOSYLATION FACTORS

61

other transferases, whereas sARF I1 activated transferases I and IV, inhibited 111, and had no effect on transferase 11. These observations, albeit intriguing, are somewhat puzzling as there is no indication that GTP was present in the transferase assays, although ARF activation of toxin requires GTP (or an analog). ARF proteins have been identified as components of the non-clathrincoated vesicle system of the Golgi complex that transports newly synthesized proteins in eukaryotic cells (74).In NIH 3T3 cells, immunoreactive ARF is largely localized in the cytoplasmic surface of cis-Golgi membranes (60). Affinity-purified antibodies used in these studies were prepared against a peptide representing a sequence in bovine ARF 1 that is identical in mammalian ARFs 1 to 5 and has only one difference (leucine vs. valine) in ARF 6 (60).

Saccharomyces cerevisiae has two ARF genes encoding proteins of 181 amino acids that are 96%identical (61). ARF 1 represents -90% of the total ARF protein, ARF 2 only -10%. Disruption of the ARF 2 gene caused no detectable alteration in phenotype, whereas disruption of ARF 1 resulted in slowed growth, cold sensitivity, a defect in the secretion of invertase, and sensitivity to fluoride at concentrations that do not kill wild-type cells, perhaps due to the major reduction in ARF content. The effects of ARF-1 gene disruption were overcome by increasing the expression of ARF 2. They were also overcome in spontaneous revertants, some of which contained a functioning ARF-1 gene resulting from recombination with the ARF-2 gene. Overexpression of ARF also interfered with growth (61).It is possible that the inhibition of maturation of Xenopus oocytes produced by microinjection of ARF is related to this kind of effect (75).Deletion of both ARF genes was lethal (61). Such mutants could be rescued by the products of human ARF-1 and ARF-4 genes, from which it may be inferred that functions of human and yeast ARFs are, at least to some extent, conserved (76). In homogenates of RINm5F cells, a -20-kDa protein(s) that binds GTP on nitrocellulose blots was found almost entirely in the cytosol (13,000 x g, 30 min supernatant) (77). After incubation of permeabilized cells with GTPyS, the amount of the 20-kDa GTP-binding protein (and also others) in the particulate was markedly increased. GPP(NH)P (100 p,M) was somewhat less effective than GTPyS at the same concentration, and other nucleotides (GTP, GDP, GDPyS, ATP) were without effect. Further subcellular fractionation revealed that the increased binding was confined to plasma membrane- and Golgi-enriched fractions, whereas fractions enriched in lysosomes, mitochondria, and secretory granules contained no 20-kDa GTPbinding protein (77). Based on size, immunoreactivity, and ADP-ribosylation catalyzed by the C3 enzyme from Clostridium botulinum, which modifies certain rho pro-

62

JOEL MOSS AND MARTHA VAUGHAN

teins, rho, rub 3, and kreu 1were ruled out, and it was concluded that ARF is the GTP-binding protein that is translocated from cytosol to membranes in cells exposed to GTPyS (77). After electrophoretic separation, proteins in crude membrane and cytosol fractions from RINm5F cells were transferred to nitrocellulose and reacted with antibodies against ARF. Subsequently, the filter was stripped and used to assess GTP-binding. It is stated (77)that, after two-dimensional (as well as one-dimensional) electrophoresis, labeling by antibody and by GTP “coincided exactly”. In addition, distribution of ARF between the two fractions, and transfer of ARF from cytosol to membranes after exposure of permeabilized cells to GTPyS, was similar whether quantified by immunoreactivity or by GTP binding. Thus, there is clear circumstantial evidence of GTP binding, and that recombinant ARF immobilized on nitrocellulose also binds GTP (77). These observations may settle earlier disagreement about whether ARF immobilized on nitrocellulose does or does not bind GTP. Incubation of homogenates of PC12 cells with non-hydrolyzable GTP analogs resulted in accumulation of immunoreactive ARF from cytosol in membrane fractions; GTP, GDP, GMP and ATP were inactive (78).ATPyS was partially effective, presumably as a result of formation of GTPyS, catalyzed by nucleoside diphosphate kinase from endogenous GDP. Cytosolic ARF also associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in the presence of GTPyS (78). Similarly, incubation of purified recombinant ARF 1 with GTPyS or GTP, but not GDP, and dimyrisoylphosphatidylcholine resulted in association of ARF with the phospholipid vesicles (79).This system provides additional evidence for the view that ARF (like other “small” GTP-binding proteins) is not activated by AlFg, which does not support phospholipid binding by GDP-liganded ARF (79). Data relevant to a role for ARF in the structure and hnction of Golgiderived coated vesicles have been accumulating rapidly. Many of these observations are brought together in the recent report of Serafini et al. (74)that ARF is a major component of these so-called COP-coated vesicles, with an estimated approximately three molecules of ARF per molecule of a-COP. aCOP (-160 kDa) is one of four relatively large (61-160 kDa) proteins associated with three smaller proteins in a cytosolic complex referred to as a “coatomer”(80).In the model of Serafini et a2. (74),ARFaGDP in the cytosol would bind to an ARF-specific nucleotide exchange protein in the donor Golgi membrane. The resulting ARFvGTP could then itself interact with the adjacent phospholipid membrane to provide a site for assembly of coatomers and formation of a COP-coated vesicle that would be stable until interaction of the ARF with a specific GAP (GTPase-activatingprotein) in the acceptor membrane. There, GTP hydrolysis would produce ARF-GDP, which would

63

ADP-RIBOSYLATION FACTORS

dissociate, followed by disassembly of the coat structure and release of coatomers separately into the cytosol. This model accounts, of course, for the effect of GTPyS that would stabilize ARF in the GTP-liganded conformation, preventing disassembly of the coat and fusion of the uncoated vesicle with the target membrane, and thus interrupting vesicular transport. In principle, a specific nucleotide-exchange protein could be replaced by an ARF-specific nucleoside diphosphate kinase, which could catalyze the synthesis of GTP from GDP bound to the ARF molecule (81). The mechanism of generating ARF-GTP, whether by nucleotide exchange or by kinase action, would appear to be of secondary importance. The specificity is, however, critical, so that only when the ARF molecule is at the correct membrane is it activated by conversion to ARF*GTPand enabled to initiate vesicle budding. Likewise, the specificity of the ARF-GAP interaction ensures delivery of the Golgi-derived vesicle and its contents to the proper site. As pointed out by the authors (74), it was not proven that the 20-kDa protein in the Golgi-derived vesicles that bound GTP immobilized on nitrocellulose blots was, in fact, the immunoreactive ARF that was also on the blots, and there is still some lack of agreement concerning GTP binding by ARF on membranes. Nevertheless, it seeins probable that the model proposed is fundamentally correct. It has the added virtue of providing potential roles for the several ARFs that have now been identified, perhaps more than six in a single species. Whether each of these functions with its own specific nucleotide exchange protein (or nucleoside diphosphate kinase) and GAP, or whether at least some of the ARFs utilize the same exchange protein and GAP, i. e., operate between the same donor and acceptor compartments but are expressed only in specific cells or at specific times in development and/or differentiation, remains to be determined.

REFERENCES 1 . J. Moss and M. Vaughan, Ado. Entymol. R e h t . Areas Mol. B i d . 61, 303 (1988). 2. L. Birnbaumer, J. Codina, R. Mattera, A. Yatani, N. Scherer, M.-J. Tor0 and A. M. Brown, Kidney Znt. 32 (Suppl. B),514 (1987). 3. A. G. Gilman, ARB 56, 615 (1987). 4. L. Birnbaumer, R. Mattera, A. Yatani, J. Codina, A. M. J. VanDongen and A. M. Brown, in “ADP-ribosylating Toxins and G Proteins: Insights into Signal Transduction” (J. Moss and M. Vaughan, eds.), p. 225. Am. SOC.Microbiol., Washington, D.C., 1990. 5. D. R. Manning, in “ADP-ribosylating Toxins and G. Proteins: Insights into Signal Transduction” (J. Moss and M. Vaughan, eds.), p. 349. Am. Soc. Microbial., Washington, D.C., 1990. 6. R. Mattera, M. P. Craziano, A. Yatani, Z. Zhou, R. Graf, J. Codina, L. Birnbaumer, A. G. Gilman and A. M. Brown, Science 243, 804 (1989). 7. P. Bray, A. Carter, C. Simons, V. Guo, C. Puckett, J. Kamholz, A. Spiegel and M. Nirenberg, PNAS 83, 8893 (1986).

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8. M. UI,in “ADP-ribosylating Toxins and G. Proteins: Insights into Signal Transduction” (J. Moss and M. Vaughan, eds.), p. 45. Am. SOC.Microbiol., Washington, D.C., 1990.

9. Y. Kaziro, in “ADP-ribosylating Toxins and G. Proteins: Insights into Signal Transduction” (J. Moss and M. Vaughan, eds.), p. 189. Am. Soc. Microbiol., Washington, D.C., 1990. 10. M. I. Simon, M. P. Strathmann and N. Gautam, Science 252,802 (1991). 11. D. Cassel and Z. Selinger, PNAS 74, 3307 (1977). 12. R. A. Kahn and A. G. Gilman, JBC 259, 6235 (1984). 13. D. L. Bums, J. Moss and M. Vaughan, JBC 258, 1116 (1983). 14 D. L. Bums, J. Moss and M. Vaughan, JBC 257, 32 (1982). 15. C. Van Dop, M. Tsubokawa, H. R. Bourne and J. Ramachandran, JBC 259, 696 (1984). 16. K. Enomoto and D. M. Gill, J. Supramol. Struct. 10, 51 (1979). 17 K. Enomoto and D. M. Gill, JBC 255, 1252 (1980). 18. H. Le Vine LII and P. Cuatrecasas, BBA 672, 248 (1981). 19. K. Enomoto and T. Asakawa, Biomed. Res. 3, 122 (1982). 20. M. 0. Pinkett and W. B. Anderson, BBA 714, 337 (1982). 21. L. S. Schleifer, R. A. Kahn, E. Hanski, J. K. Northup, P. C. Sternweis and A. G. Gilman, JBC 257, 20 (1982). 22. D. M. Gill and R. Meren, JBC 258, 11908 (1983). 23. R. A. Kahn and A. G. Gilman, JBC 259,6228 (1984). 24. R. A. Kahn and A. G. Gilman, JBC 261, 7906 (1986). 25. S.-C. Tsai, M. Noda, R. Adamik, J. Moss and M. Vaughan, PNAS 84, 5139 (1987). 26. D. M. Gill and J. Coburn, Bchem 26, 6364 (1987). 27 S.-C. Tsai, M. Noda, R. Adamik, P. P. Chang, H.-C. Chen, J. Moss and M. Vaughan, JBC 263, 1768 (1988). 28. D. A. Bobak, S.-C. Tsai, J. Moss and M. Vaughan, in “ADP-ribosylating Toxins and G Proteins: Insights into Signal Transduction” (J. Moss and M. Vaughan, eds.), p. 439. Am. SOC. Microbiol., Washington, D.C., 1990. 29. J. Moss and M. Vaughan, JBC 252, ,2455 (1977). 30. J. Moss and M. Vaughan, PNAS 75, 3621 (1978). 31. J. B. Trepel, D -M. Chuang and N. H. Neff, PNAS 74, 5440 (1977). 32. J. Moss, S . J. Stanley, P. A. Watkins and M. Vaughan, JBC 255, 7835 (1980). 33. N. J. Oppenheimer, JBC, 253, 4907 (1978). 34. M. Noda, S.-C. Tsai, R. Adamik, J. Moss and M. Vaughan, BBA 1034, 195 (1990). 35. D. A. Bobak, M. M. Bliziotes, M. Noda, S.-C Tsai, R. Adamik and J. Moss, Bchem 29,855 (1990). 36. 0. Weiss, J. Holden, C. Rulka and R. A. Kahn,JBC 264, 21066 (1989). 37. S.-C. Tsai, R. Adamik, J. Moss and M. Vaughan, Bchem 30, 3697 (1991). 38. R. A. Kahn, C. Goddard and M. Newkirk, JBC 263, 8282 (1988). 39. S.-C. Tsai, R. Adamik, M. Tsuchiya, P. P. Chang, J. Moss and M. Vaughan, JBC 266,8213 (1991). 40. R. S . Duman, S. M. Winston, J. A. Clark and E. J. Nestler, J. Neurochem. 55, 1813 (1990). 4 1 . M. Field, Am. J. Clin. Nutr. 32, 189 (1979). 42. B. E. C . Guth, C. L. Pickett, E. M. Twiddy, R. K. Holmes, T. A. T. Gomes, A. A. M. Lima, R L. Guerrant, B. D. G. M. Franc0 and L. R. Trabulsi, Infect. Zmmun. 54, 587 (1986). 43. R. K. Holmes, E. M. Twiddy and C. L. Pickett, Infect. Immun. 53, 464 (1986). 44. B. E. C. Guth, E. M. Twiddy, L. R. Trabulsi and R. K. Holmes, Infect. Immun. 54, 529 (1986). 45. C. L. Pickett, E. M. Twiddy, B. W. Belisle and R. K. Holrnes, J. B u t . 165, 348 (1986). 46. J. Moss and S. H. Richardson, J. Clin. Inoest. 62, 281 (1978)

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47. D. M. Gill and S. H. Richardson, J. Infect. Dis. 141, 64 (1980). 48. J. Moss, J. C. Osborne, Jr., P. H. Fishman, S. Nakaya and D. C. Robertson, JBC 256, 12861 (1981). 49. P. P. Chang, J. Moss, E. M. Twiddy and R. K. Holmes, Infect. Zmmun. 55, 1854 (1987). 50. C.-M. Lee, P. P. Chang, S.-C. Tsai, R. Adamik, S. R. Price, B. C. Kunz, J. Moss, E. M. Twiddy and R. K. Holmes, J. Clin. Znoest. 87, 1780 (1991). 51. C. L. Pickett, D. L. Weinstein and R. K. Holmes, 1. Bact. 169, 5180 (1987). 52. C. L. Pickett and R. K. Holmes, in “Advances in Research in Cholera and Related Diarrheas” (R. B. Sack and Y. Zinnaka, eds.), Vol. 7, p. 165. KTK Sci. Publ., Tokyo, 1990. 53. J. L. Sewell and R. A. Kahn, PNAS 85, 4620 (1988). 54. S. R. Price, M. Nightingale, S.-C. Tsai, K. C. Williamson, R. Adamik, H.-C. Chen, J. Moss and M. Vaughan, PNAS 85, 5488 (1988). 55. D. A. Bobak, M. S. Nightingale, J. J. Murtagh, S. R. Price, J. Moss and M. Vaughan, PNAS 86, 6101 (1989). 56. L. Monaco, J. J. Murtagh, K. B. N-M, S.-C. Tsai, J. Moss and M.Vaughan, PNAS 87, 2206 (1990). 57. 2. Peng, I. Calvert, J. Clark, L. Helman, R. Kahn and H.-F. Kung, Biofactors 2, 45 (1989). 58. M. Tsuchiya, S. R. Price, S.-C. Tsai, J. Moss and M. Vaughan, JBC 266, 2772 (1991). 59. J. J. Murtagh, Jr., MI R. Mowatt, C.-M. Lee, F.-J. S. Lee, K. Mishima, T. E. Nash, J. Moss and M. Vaughan, JBC 267, 9654 (1992). 60. T. Stearns, M. C. Willingham, D. Botstein and R. A. Kahn, PNAS 87, 1238 (1990). 61. T. Stearns, R. A. Kahn, D. Botstein and M. A. Hoyt, MCBiol 10, 6690 (1990). 62. S. R. Price, A. Barber and J. Moss, in “ADP-ribosylating Toxins and G Proteins: Insights into Signal Transduction” (J. Moss and M. Vaughan, eds.), p. 397. Am. SOC.Microbiol., Washington, D.C., 1990. 63. J. B. Gibbs, I. S. Sigal, M. Poe and E. M. Scolnick, PNAS 81, 5704 (1984). 64. J. P. McCrath, D. J. Capon, D. V. Coeddel and A. D. Levinson, Nature 310, 644 (1984). 65. R. W. Sweet, S. Yokoyama, T. Kamata, J. R. Feramisco, M. Rosenberg and M. Cross, Nature 311, 273 (1984). 66. J. W. Tamkun, R. A. Kahn, M. Kissinger, B. J. Brizuela, C. Rulka, M. P. Scott and J. A. Kennison, PNAS 88, 3120 (1991). 67. C. R. Alsip and D. A. Konkel, NARes 14, 2123 (1986). 68. M. Tsuchiya, S . R. Price, M. S. Nightingale, J. Moss and M. Vaughan, Bchem 28, 9668 (1989). 69. J. Moss, M. Tsuchiya, S. R. Price, S.-C. Tsai and M. Vaughan, 26th U.S.-Jpn. Cholera Con5 Kyoto, Japan in press (1990). 70. S.-C. Tsai, R. S. Haun, M. Tsuchiya, J. Moss and M. Vaughan, JBC 266, 23053 (1991). 71. C.-M. Lee, R. S. Haun, S.-C. Tsai, J. Moss and M. Vaughan, JBC 267, 9028 (1992). 72. I. M. Serventi, E. Cavanaugh, J. Moss and M. Vaughan, JBC in press (1993). 73. S. Matsuyama and S. Tsuyama, 1. Neurochem. 57, 1380 (1991). 74. T. Serafini, L. Orci, M. Amhedt, M. Brunner, R. A. Kahn and J. E. Rothman, Cell 67, 239 (1991). 75. T. D. Bahnson, S.-C. Tsai, R. Adamik, J. Moss and M. Vaughan, JBC 264, 14824 (1989). 76. R. A. Kahn, F. G. Kern, J. Clark, E. P. Gelmann and C. Rulka, JBC 266, 2606 (1991). 77. R. Regazzi, S . Ullrich, R. A. Kahn and C. B. Wollheim, BJ 275, 639 (1991). 78. M. W. Walker, D. A. Bobak, S.-C. Tsai, J. Moss and M. Vaughan, JBC 267, 3230 (1992). 79. R. A. Kahn, JBC 266, 15595 (1991). 80. M. G. Waters, T. Serafini and J. E. Rothman, Nature 349, 248 (1991). 81. P. A. Randazzo, J. K. Northup and R. A. Kahn, Science 254, 850 (1991).

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Molecular Biology in the Eicosanoid Field’ COLIND . FUNK Department of Pharmacology Division of Clinical Phannacology Vanderbilt University Nashuille, Tennessee 37232

I. Lipoxygenases . . . . . . . . . . . . . . A. 5-Lipoxygenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. 12-Lipoxygenase . . . . . . ...................... C. 15-Lipoxygenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................... 11. Leukotriene & Hydrolase . . . . . 111. Prostaglandin G/H Synthase (Cy IV. Thromboxane-A Synth V. Prostaglandin-D Synthase .................................... VI. Prostaglandin-F Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Eicosanoid Receptors VIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

69 69 74 78 81 83 86 87 89 89 93 93

Nearly 30 years have passed since the discovery of the biotransformation of arachidonic acid to prostaglandins ( I , 2). Since this original finding, the number of oxygenated arachidonic acid metabolites discovered has multiplied exponentially; collectively, these compounds are referred to as eicosanoids. For some of the eicosanoids, including thromboxane A,, prostacyclin, and leukotrienes, biological roles have been reasonably well delineated, whereas for other eicosanoids, like lipoxins and hydroxyeicosatetraenoic acids (HETEs), functions have not yet been elucidated (3-5). Eicosanoid involvement in the pathophysiology of inflammation and many other disorders has been the focus of research efforts worldwide. Since the biosynthetic pathways, biochemistry, and pathophysiological implications of prostaglandins and leukotrienes have been covered in detail in many excellent reviews (6-11) these topics are not addressed here to any great extent. Rather, I direct attention (see Fig. 1) to the most recent research on the molecular biology of the main enzymes and receptors involved in eicosanoid formation and signal transduction. Molecular biology made its main entrance to the eicosanoid field in 1987 when two groups simultaneously reported the cloning of the cDNA for leukotriene A, hydrolase (12,13).Subsequently, the field has expanded considerably to include many Abbreviations. PG, prostaglandin, LT, leukotriene, Tx, thromboxane, H(P)ETE, hydro(pero)xyeicosatetraenoicacid, FLAP, 5-lipoxygenase activating protein, COX, cyclooxygenase.

67 Progresa in Nucleic Acid Research and Molecular Biology, Val. 45

Copyright 0 1993 by Academic Press, Inc All rights of rrproductron In any form reserved

COOH

1

LEUKOTRIENE A,

mDRccAsE

PGGdH,

1

OH

\

OH COOH

FIG. 1. Metabolic transformation of arachidonic acid. Only those transformations discussed in the text are shown. Enzymes catalyzing the primary transformations are boxed.

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

69

interesting findings with the lipoxygenases, prostaglandin-synthesizing enzymes, and prostanoid receptors.

1. Lipoxygenases Lipoxygenases catalyze the incorporation of molecular oxygen into cis, cis-pentadiene-containinglipids to form the primary hydroperoxide reaction products (14). These enzymes contain a non-heme iron atom and they are widely distributed in the plant and animal kingdoms. Lipoxygenases differ in their specificity of molecular oxygen insertion. Within mammalian tissues, there are three major forms, the 5-, 12-, and 15-lipoxygenases, that catalyze the incorporation of dioxygen into positions 5, 12, and 15, of arachidonic acid, respectively.

A. 5-Lipoxygenase 5-Lipoxygenase (arachid0nate:oxygen 5-oxidoreductase, EC 1.13.11.34) catalyzes the first step in the formation of the biologically important leukotriene mediators. A hydrogen atom is abstracted &om position 7 of arachidonic acid and oxygen is inserted at C-5 to form 5-hydroperoxyeicosatetraenoic acid (15). A dehydrase activity of this enzyme catalyzes the subsequent rearrangement to the unstable allylic epoxide, Ieukotriene (LT) A, (16,lfl. The enzyme was first found in polymorphonuclear leukocytes (PMN) and was purified to homogeneity from human and porcine PMN and rat basophilic leukemia cells (18-21). To clone the human 5-lipoxygenase cDNA, polyclonal antibodies to the enzyme were used to immunoscreen a hgt-11 lung cDNA library (22).Purified human enzyme was also subjected to proteolytic cleavage with the lysine-specific protease from Achromobacter lyticus to generate peptide sequence information. Several clones were plaque-purified and one clone having a 0.4-kb insert, when sequenced, matched a 5-lipoxygenase peptide sequence. Using the 32P-labeled 0.4-kb insert, two clones were isolated from a human hgt-11 placenta cDNA library. From the sequence of clone pl5BS (excluding a 51-bp artifact), it was possible to deduce the complete primary sequence of 5-lipoxygenase (22).The cDNA sequence encodes a mature protein of 673 amino acids (674 including the initiator methionine) with a molecular weight of about 78,000. Although Ca2+ and -4TP are required for maximal activity, potential binding sites could not be predicted with high certainty from the primary sequence. The human 5-lipoxygenase cDNA was also cloned from a dimethyl sulfoxidedifferentiated HL-60 cell library using oligonucleotide probes based on peptide data from the purified human leukocyte enzyme (23). The placentaderived and HL-60 sequences were identical. Human 5-lipoxygenase is significantly related to other lipoxygenases

70

COLIN D. FUNK

(Table I). It is nearly identical to the rat 5-lipoxygenase (24) and it displays about 40% identity to other mammalian lipoxygenases (25-29) and 27% to the plant homologues (30-33). There are no significant homologies with other known proteins, with the exception of a short segment found in lipoprotein and hepatic lipases within the so-called interface binding domain. At present there are at least 10 cloned lipoxygenase sequences, and it has become increasingly evident that there is a core region in the central portion of the molecule that is highly conserved that probably cmtains the iron-binding site (Fig. 2). The motif His-X,-His-X,-His-XI,-His-X,-His is present in all sequences; included within this region are several interspersed conserved acidic and basic residues. There is a second region with another conserved histidine and glutamine residue, closer to the carboxy terminus and found in all 10 sequences, and a third region with a histidine, found in mammalian but not in plant lipoxygenases (Fig. 2). Mutagenesis studies have been carried out with 5-lipoxygenase to examine the role of many of these conserved residues with respect to enzyme activity (34-36). The results are summarized in Table 11. His-367, His-372 within the main core region and His550 in the second region are essential to enzyme activity and may be required for coordination of the iron atom. Studies with the soybean enzyme indicate that there are at least four imidazole ligands (37);however, similar experiments have not been performed TABLE I COMP~RISONS OF LIPOXYCENASES Lipoxygenase" h5LX r5LX hl2LX p12Lx hl5LX rbl5LX sbLXl sbLX2 sbLX3 PeLX

Amino acids

Molecular weight

mRNA length (kb)

Identity to h5LX

Identity to hl2LX

Identity to hl5LX

674 670 663 663 662 662 838 865 857 851

78,097 77,982 75,764 75,047 74,809 75,424 94,260 97,260 96,851 97,743

2.7 2.6 2.4 3.4 2.8 2.7b 3.0b 3.0b 3.0 3.0b

-

42 39

39 38 65 86 81 26 26 27 27

92 42 40 39 39 26 27 28 26

65 65 61 26 27 28 27

ah5LX, human 5-lipoxygenase (22, 23); r5LX. rat 5-lipoxygenase (24); hlBLX, human platelet 12-lipoxygenase (25, 26, 70); p12LX. porcine leukocyte 12-lipoxygenase (27); hl5LX, human 19lipoxygenase(28),rblBLX, rabbit 15lipoxygenase (29);sbLX, soybean lipoxygenase isozymes 1, 2, and 3 (3&32); peLX, pea seed lipoxygenase (33). Sequences retrieved from GenBank were analyzed by the software of Intelligenetics. bSizes of mRNA were not quoted in the original references and are estimates based on the cDNA clones obtained.

362

h5LX r5LX h12LX p12LX hl5LX rbl5LX sbLXl sbLX2 sbLX3 peLX

351 351 344 345 344 344 482 511 502 506

h5LX r5LX hl2LX pl2LX hl5LX rbl5LX sbLXl sbLX2 sbLX3 peLx

427 427 420 421 420 420

367

390

372

399

401 401 394 395 3 94 394 532 561 552 556 439 439 432 433 432 432

542 542 532 533 532 532 681 710 701 705

561 561 551 552 551 551 700 729 720 724

FIG. 2. Lipoxygenase sequence homology. The alignment of ten lipoxygenase sequences (one-letter amino-acid code) is shown in the putative iron-binding core region (top), and in two regions closer to the carboxy terminus (bottom). Conserved histidine, acidic and basic residues are boxed. The five histidines on top are numbered with respect to the 5-lipoxygenase (see Table 11). An arrow points to an essential glutamine residue. h5LX, human 5-lipoxygenase (22, 23); r5LX, rat 5-lipoxygenase (24);hllLX, human platelet 12lipoxygenase (25, 26, 70); p12LX, porcine leukocyte 12-lipoxygenase (27);hl5LX, human 15-lipoxygenase (28):rbl5LX, rabbit 15lipoxygenase (29);shLX, soybean lipoxygenase ixozymes 1, 2, and 3 (30-32);peLX, pea seed lipoxygenase (33).(Figure is adapted from Ref. 36.)

TABLE I1 SITE-DIRECTEDMUTACENESIS OF HUMAN~LIPOXYGENASE Enzyme activity"

A

Mutation

B

C

++

H362C H362K H362N H362Q H362S H367A H367C H367K H367N H367Q H367S H372N H372Q H372Sb H390A H390N H390Q H390S H399A H399N H399Q H399S H432A H432N H550A HSSOL H550N H5W D358N V374A v374s E376Q Y383F Y383S F393S F393W M435L Q557E COOH-6'

-

+ +++

++

+ +++ -

-

Lo

+

+++

+

++

-

++ +++

+++ ++ -

-

+ + +++ + + + +

+++

The results of in uitm mutagenesis studies from three research groups: (A) Refs. 35 and 38; (B) Ref. 34; and (C) Ref. 36. Enzyme activity is classified as: +, 67-1004 of native enzyme, , 34-66% of native enzyme; +, 1 4 3 % of native enzyme; -, no detectable activity; Lo, enzyme activity was detectable but expression was very low for accurate Q

++

++

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

73

with mammalian Iipoxygenases. It is also clear that Glu-358 and Gln-552, as well as the six residues at the carboxy-terminal are essential to activity and/or structural features of the molecule. It remains to be tested whether these mutant enzyme forms still bind iron. The 5-lipoxygenase enzyme has been expressed in several heterologous systems including baculovirus-infected insect cells, an osteosarcoma cell line, COS-M6 cells, Escherichia coli and yeast (38-43). The enzyme appears to retain all the essential characteristics of the native leukocyte enzyme in these systems, such as possession of both 5-lipoxygenase and leukotriene (LTA) synthase activities and dependence on Ca2+ and ATP for maximal activity. Studies performed &th the baculovirus/insect cells expressed enzyme have verified the presence of an iron atom (1.1 mol iron per mol enzyme) that is tightly bound and that is released after exposure of the enzyme to oxygen (44).No iron could be detected in the inactivated enzyme. Also, leukocyte microsomal and cytosolic proteins are not required for maximal enzyme activity as previously thought, but rather, they cause an enhanced stability of the enzyme in the in vitro assay (39). The 5-Lipoxygenase enzyme resides in the cytosol of unstimulated cells. Upon activation of leukocytes, H L 6 0 cells or rat basophilic leukemia cells with the calcium ionophore A23187 (calcimycin), the enzyme is translocated to a membrane fraction (45, 46). The activation is associated with a burst in leukotriene formation, and the enzyme found in the membrane apparently has undergone a “suicide-type’’ inactivation. The translocation step as well as the formation of leukotrienes in intact cells is blocked by the drug MK886 that binds to an 18-kDa membrane protein (47, 48). This small protein has been termed “5-lipoxygenase activating protein” (FLAP) (49). Experiments with osteosarcoma cells transfected with a 5-lipoxygenase expression vector revealed that these cells cannot make 5-lipoxygenase products from endogenous substrate when activated (40). However, when a vector expressing FLAP was co-transfected, activated cells synthesized leukotrienes; this synthesis was blocked by MK886 binding to FLAP (49).

TABLE I1 (Continued) assessment of activiv. Mutants are classifiedby the 1-letter code, e.g., H362C = histidine at position 362 mutated to cysteine. Expression studies were carried out in E. coli and in bacul-s-infected insect cells. H372S was originally reported to possess activityin the bacdovirus system (38). The explanation for this may have been an accidentalcontaminationof native5-Iipxygenasebaculwirus stockwith the mutant virus. c COOH-6 refers to deletion of the six carboxy-terminal residues.

74

COLIN D. FUNK

There is no solid evidence for a direct interaction of FLAP with 5lipoxygenase, so FLAP’S mechanism of action remains obscure (50). Interestingly, FLAP co-localizes with 5-lipoxygenase in leukocytes, HL60 cells, and several myeloid-derived cell lines, yielding credence to their mutual involvement in leukotriene formation (51). In the rat alveolar macrophage, the 5-lipoxygenase apparently resides in an active form within membrane fractions of unstimulated cells (52). It is possible, therefore, that some cell-type variability exists in the mechanism of formation of leukotrienes. Northern-blot data has revealed the presence of a predominant 2.7-kb mRNA transcript in human leukocytes, lung, placenta, and differentiated HL60 cells (22, 23). A band of similar size was observed in samples from rat cells and tissues, including basophilic leukemia cells, macrophages, lung, intestine, stomach, and epidermis (24). However, the presence of infiltrating white cells or resident mast cells could not be excluded as the source of 5lipoxygenase mRNA in some of these organs. A well-documented evidence for non-myeloid 5-lipoxygenase expression comes from immunohistochemical studies that located the enzyme in porcine pancreatic acinar cells (53). The human 5-lipoxygenase gene has been isolated from a series of genomic bacteriophage and cosmid clones (54).The gene spans at least 82 kb of DNA and is divided into 14 exons. There was one gap in the very large (>26 kb) third intron, so the actual size of the gene is unknown. The gene resides on human chromosome 10 distinctly separated from other lipoxygenase genes (55). All the members of the lipoxygenase gene family appear to be organized in the same exon/intron format (54-56). However, the 5-lipoxygenase gene appears to be much larger than other lipoxygenase genes. The site of transcription initiation was mapped to a thymidine residue 65 bp upstream of the start codon. The promoter region is rich in G-C boxes and lacks canonical TATA and CCAAT motifs. The sequence -179 to -56, which includes 5 tandemly arranged G-C boxes, is essential for transcription and from gel-shift analysis the Spl transcription factor can bind within this region (57). There was also evidence for two negative and two positive upstream transcription regulatory elements. Although there are many interesting features within the 5-lipoxygenase promoter region, very little is known about potential regulatory agents, and cis- and trans-acting factors that could control the expression of this gene and the resultant formation of leukotrienes. Obviously, there is still much to be learned in this area.

B.

12-Lipoxygenase

12-Lipoxygenase (arachid0nate:oxygen 12-0xidoreductase, EC 1.13. 11.31),found in platelets, was the first discovered mammalian lipoxygenase,

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

75

in 1974 (58).It catalyzes the stereospecific incorporation of molecular oxygen onto C-12 of arachidonic acid to yield 12(S)-hydroperoxyeicosatetraenoicacid (12-HPETE). 12-HPETE is reduced by cellular glutathione peroxidases to 12 (S)-hydroxyeicosatetraenoicacid (12-HETE). Although 12-HETE is synthesized in greater amounts than the cyclooxgenase-derived arachidonate metabolite, thromboxane A,, by various types of platelet stimuli, little is known about the biological relevance of this pathway. The enzyme is cytosoliclocalized, and it appears to undergo a translocation to a membrane fraction when (rat) platelets are activated by thrombin (59). 12-Lipoxygenase has been characterized from other sources, including porcine and bovine leukocytes, bovine tracheal epithelial cells, human uterine cervix, and human and mouse epidermal cells (60-66). The uterine cervix and epidermal cell enzymes are located in the microsomal fraction, and they may or may not be distinct from cytochrome P-450 enzymes (63-66). Much evidence has been obtained for the presence of 12-lipoxygenase isoforms (67), the two major forms being classified into “platelettype” and “leukocyte-type” 12-lipoxygenases. 12-Lipoxygenase from porcine leukocytes has been purified to apparent homogeneity, and several peptide sequences have been determined (27). The cDNA for this enzyme was cloned by hybridization screening with two sets of oligonucleotides based on one peptide sequence (27). On the other hand, human platelet 12-lipoxygenase cDNA was isolated by a polymerase chain reaction (PCR) homology approach (25). Oligonucleotide primers derived from conserved regions of 5- and 15-lipoxygenaseswere used to amplify a short 0.26-kb fragment from reverse-transcribed platelet RNA (25). This short fragment was radiolabeled and used to screen a human erythroleukemia (HEL) cell library to obtain the complete cDNA. Both the porcine leukocyte (662 amino acids) and human platelet/erythroleukemia cell (663 amino acids) 12-lipoxygenases encode = 75-kDa proteins. They share 65% amino-acid identity. The leukocyte-type enzyme exhibits more relatedness to 15-lipoxygenase than to the platelet 12-lipoxygenase. Additionally, the two enzymes have different substrate specificities and product profiles (68). The leukocyte 12-lipoxygenase will metabolize both arachidonic acid and linoleic acid whereas the platelet enzyme metabolizes virtually no C-18 fatty acids. The former enzyme forms small amounts of 15-lipoxygenase products, but the platelet enzyme forms exclusively lS-H(P)ETE (43). More recently, a bovine-trachea epithelial-cell 12-lipoxygenase cDNA was cloned by use of a 15-lipoxygenase probe from human reticulocytes (69). Its sequence is 89%identical to the porcine 12-lipoxygenaseand 86%identical to human 15-lipoxygenase. The relatedness of epithelial cell 12-lipoxygenase and human 15-lipoxygenase has been confirmed further by the fact that they share antigenic cross-determinants (69). Both the bovine trachea

76

COLIN D. FUNK

epithelial and porcine leukocyte 12-lipoxygenases,as well as 15-lipoxygenase have a Cys-X3-Cys-X3-His-X3-His zinc-finger motif (amino acids 532-544) which is not found identically in the platelet 12-lipoxygenase or 5-lipoxygenase sequences. The significanceof this motif is unknown. Two distinct 12lipoxygenase cDNAs have not yet been obtained from the same species, so the molecular nature of 12-lipoxygenase isoforms is still not clearly established. Even less clear is the biological significance of different 12-lipoxygenase isoforms. Northern-blot studies have revealed the presence of a single platelettype mRNA transcript in HEL cells and platelets (2.4, 3.0, or 3.1 kb sizes have been reported by three different groups) (25, 26, 70).Phorbol ester treatment of HEL cells increases this mRNA about 2-3 fold (25, 26). This mRNA is in the low abundance category being present at about five copies/cell(71). The mRNA has not been detected in other human tissues such as lung, placenta, adrenal gland, and liver by Northern-blot analysis. However, using reverse transcriptase (€IT)-PCR analysis, platelet-type 12-lipoxygenase mRNA was detected in small amounts in most tissues, probably due to platelets remaining within these tissues (55). Platelet-type 12-lipoxygenase was definitely present in cultured human umbilical vein endothelial cells (passage 4) since platelets would not represent a source of contamination in the PCR assay (55). The leukocyte-type 12-lipoxygenasemRNA (3.4 kb) was detected in several porcine tissues by Northem-blot analysis (27). The greatest amount was found in leukocytes followed by substantial amounts in pituitary gland and lung. Small amounts were detected in jejunum and spleen. No cross-hybridization on RNA blots, using the human platelet and porcine leukocyte 12lipoxygenase probes could be observed (70). Immunohistochemical studies have provided direct evidence for the presence of leukocyte-type 12-lipoxygenase in the parenchymal cells of porcine anterior pituitary and in porcine neutrophils and monocytes (72, 73). Other tissues positive by Northern blot appeared to contain 12-lipoxygenaseonly in resident mast cells or infiltrating granulocytes (73). Both forms of 12-lipoxygenasehave been expressed in heterologous systems (E. coli, baculovirus-infected insect cells, COS cells, and human embryonic kidney 293 cells) (25, 26, 43, 7 3 ~ )In. E . coli, the platelet-type 12lipoxygenase has been expressed as a large = 100-kDa fusion protein that retains activity, using the pGEX2T vector (74).The fusion moiety is a 26-kDa glutathione S-transferaseattached to the amino terminus of 12-lipoxygenase, which allows efficient affinity purification on a glutathione-agarose column. Sf9 insect cells infected with a recombinant platelet-type 12-lipoxygenase baculovirus can generate milligram amounts of enzyme from lo8 cells (73~).

77

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

A sequence coding for six histidines included after the initiator codon provides a simple one-step purification by Ni2 -NTA-agarose affinity chromatography. The native platelet enzyme has not previously been purified to homogeneity by conventional protein purification procedures. The expressed enzyme is highly active [up to 10 Fmol 12-H(P)ETE/mg p r o t e i d l 0 min incubation], is not dependent on any cofactors, and can be stored at -70°C in the presence of glycerol with retention of 7040% activity after 4 weeks. In oitro mutagenesis experiments show that the same histidine residues essential to catalytic activity for 5-lipoxygenase (His367, His372, and His550) are also essential to platelet-type 12-lipoxygenase activity (His360, His365, and His540) (73a).The nine N-terminal residues are also important to structure and/or function of the enzyme, as activity is abolished in their absence. The recombinant platelet-type 12-lipoxygenase synthesizes exclusively 12H(P)ETE from arachidonic acid. However, change of three adjacent amino acids Lys-416/Ala-417/Val-418 to Gln-416/11e-417/Met-418 (residues found in 15-lipoxygenases) enables the mutant recombinant enzyme to alter the specificity of 0, insertion to arachidonate substrate. 15-Lipoxygenase products [ lSH(P)ETE]account for = 25% of the total products synthesized from arachidonate by the mutant enzyme. These changes are discussed further in Section 1,C. The human platelet-type 12-lipoxygenase gene (12-lipoxygenase gene-1) has been isolated on several overlapping genomic cosmid and bacteriophage clones (55).The gene spans = 17 kb and exodintron junctions are analogous to the human 5-lipoxygenase gene. The putative promoter region has a “TATA-like” box (position -96 with respect to the ATG start codon), no CCAAT motif, and three G-C boxes in a 578-bp region from the ATG start. There were two sites that could account for the potential induction of transcription by phorbol esters at positions -531 and -511. However, this region requirw further characterization with transfection experiments using promoter-reporter gene constructs. Part of a second “12-lipoxygenase” gene (exons 7-8 and 10-11) was isolated on a series of overlapping bacteriophage clones from the same human genomic libraries (55). This gene (12-lipoxygenase gene-2) is most likely a pseudogene (Fig. 3) because: (i) it is not expressed in several tissues by RT-PCR analysis; (ii) it lacks two of the histidines in the translated exon-8 sequence, one of which is critical to lipoxygenase activity; (iii) there is no evidence for an exon-%like sequence; and (iv) there is an abnormal splice acceptor site before exon 8. The 12lipoxygenase gene-2 is = 85%identical to the 12-lipoxygenase gene-1 within exon sequences and both genes are located in human chromosome 17 (55).A leukocyte-type 12-lipoxygenase gene has yet to be found in humans. +

78

COLIN D. FUNK

394 hl ZLX-2

SWILKLRFQL

FIG.3. Alignment of the translated 12-lipoxygenase gene sequences within the putative iron-binding region coded by exons 8 and 9. hl2LX-1, human 12-lipoxygenase gene-1, corresponding to amino acids 344-394 of plateletlerythroleukemiacells enzyme (25, 55); h12LX-2, human 12-lipoxygenase gene-2. h12LX-2 is almost certainly a pseudogene (see Section I, B for details) and the amino-acid sequence, therefore, is hypothetical. h12LX-2 is lacking conserved residues (boxed) found in all lipoxygenases (see Fig. 2). The circled tyrosine residue would replace a conserved histidine that is essential to 5- and 12-lipoxygenaseactivity and would give rise to a nonfunctional enzyme (C. D. Funk, unpublished). An exon 9 sequence not present in h12LX-2 is denoted by dashes.

C. 15-Lipoxygenase 15-Lipoxygenase was first purified and characterized from rabbit reticulocyte-rich anemic blood cells (75). It was subsequently purified and characterized from rabbit and human polymorphonuclear leukocytes and human eosinophil-rich leukocytes (76-78). The enzyme is in the cytosol like other lipoxygenases, and there are no reports of translocation upon cellular stimulation as for the 5- and 12-lipoxygenases. The 15-lipoxygenasepathway is also present in lung, airway epithelial cells, and keratinocytes. Little is known about the function of t h i s pathway in these various cell types. The most plausible role for the reticulocyte enzyme lies with its ability to oxygenate lipid membranes directly (79, 80). Thus, it probably participates in the intracellular organelle degradation during maturation to the erythrocyte and possibly in the reconstruction and remodeling of the plasma membrane. The complete .15-lipoxygenase cDNA has been cloned from a human reticulocyte cDNA library using oligonucleotide probes based on the purified eosinophil sequence (28). The open reading frame encodes a 662aminoacid polypeptide with a molecular weight of 74,600. The cDNA from human tracheal epithelial cells encodes an identical protein (81).The rabbit reticulocyte 15-lipoxygenasecDNA has also been cloned and is 81% identical to the human homologue (29). Relationships to other lipoxygenases are shown in Table I. The rabbit cDNA contains a long 3’ untranslated region with ten copies of the tandemly arranged repeat sequence C,RC,TCTTC,AAG. This region has been proposed to be involved in the translational regulation of the mRNA. An issue yet to be resolved is whether there are isoforms of 15-lipoxygenase. One group has reported the purification of two 15-lipoxygenases from human leukocytes (82). Both forms have the same molecular mass (74 kDa), appear to be equally active with different substrates, and have the

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

79

same N-terminal sequence as the cloned human reticulocyte 15-lipoxygenase. The two forms were clearly separated by cation-exchange chromatography and their relative amounts dif€ered markedly between leukocytes of normal donors and from an individual with eosinophilia (82). Immunological evidence from another group has established the relatedness of 15-lipoxygenases from different cell-types (83). Specific localization experiments tend to indicate 15-lipoxygenase immunofluorescence only within the eosinophil population of leukocytes (83). In human trachea, 15-lipoxygenase is in both basal and ciliated epithelial cells. Mature reticulocytes, but not early or late stage reticulocytes, contain the enzyme (83). In another study, evidence for 15-lipoxygenase expression was found in both the cytosol and nuclei of human myometria (84). Southern-blot genomic DNA analysis apparently revealed the presence of a single copy gene both in rabbits and humans (56, 81). A major 2.7-kb mRNA for 15-lipoxygenase has been detected in human leukocytes, tracheal epithelium, and reticulocytes by Northern-blot analysis (81). Interestingly, the 15-lipoxygenase mRNA and protein have been detected in macrophage-rich human atherosclerotic lesions by in situ hybridization and immunofluorescence experiments (85). Moreover, these products co-localized with epitopes for oxidized low-density lipoprotein, and it has been proposed that the macrophage-induced modification of low-density lipoprotein may be mediated by 15-lipoxygenase, thus contributing to atherogenesis (86). In a more recent study, it was found that normal monocyte/macrophage populations do not contain the 15-lipoxygenase mRNA, but when induced with interleukin-4 they express 15-lipoxygenase mRNA and protein that is capable of oxygenating membrane lipids (87).Interferon y and hydrocortisone inhibit the interleukin-4 induction. These findings provide an interesting link between 15-lipoxygenase function and the immune/inflammatory response. The human 15-lipoxygenase has been expressed in E . coli, osteosarcoma cells, and COS-M6 cells (43, 88, 89). The recombinant enzyme retains the original characteristics of the protein isolated from eosinophil-rich leukocytes. Both 15-H(P)ETE and 12-H(P)ETE are synthesized in a ratio of 9 : 1. A comparison of three 12-lipoxygenase and two 15-lipoxygenase sequences showed that there are four conserved differences between the two lipoxygenases. A study to investigate the parameters controlling the specificity of molecular oxygen insertion at position 15 or 12 was carried out (go), based on mutagenesis of the conserved sequences. Change of a single methionine at position 418 of human 15-lipoxygenase to valine (found in 12-lipoxygenases) caused a shift in product profile with nearly equal amounts of 12-HETE and 15-HETE being formed. Further change of the two adjacent residues (Gln-416 to Lys-416, and lle-417 to Ala-417) to platelet 12-lipoxygenase resi-

80

COLIN D. FUNK

dues induced an even greater shift in the formation of 12-HETE accounting for over 90% of the products. Data from previous studies with the reticulocyte and soybean lipoxygenases (91)and these mutagenesis results provide the basis for a model that explains at least some of the parameters that control positional specificity of oxygen insertion (Fig. 4). Arachidonate fits into a substrate-binding cleft and the side-chain at position 418 of 15-lipoxygenase, and residues immediately adjacent control the deepness of penetration into the binding pocket. The different side chains in this region of 12-lipoxygenase apparently allow a deeper binding that consequently shifts the substrate relative to the site of hydrogen atom abstraction (C-10 for 12-lipoxygenation and C-13 for 15lipoxygenation). The complete rabbit 15-lipoxygenase gene and promoter sequence have been elucidated (92). Like the 5- and 12-lipoxygenase genes of humans, it is divided into 14 exons having the same exodintron format (Fig. 5). The gene is 8-kb long, the shortest of the mammalian lipoxygenase genes characterized so far. Transcription initiation starts 27-bp upstream from the ATG start codon. The 150-bp upstream segment of the start acts as a functional promoter in both erythroid and non-erythroid cell-lines. There is a “TATAlike” motif 30 bp upstream from the transcription initiation site, but no

,, Human enzyme

: 13

1,’

15-Lipoxygenase

‘...‘. 10

7

2

-;)‘Human

platelet

FIG.4. Determinants of positional specificity of molecular oxygen insertion in human 12and 15-lipoxygenases. The active site (&), presumably involving the non-heme iron atom, abstracts a hydrogen atom primarily at position C-13 in 15-lipoxygenase and exclusively at position C-10 in human platelet 12-lipoxygenase.The positioning of the arachidonate substrate relative to the active site could he controlled to some extent by residues 416-418 in 15lipoxygenase and 416-417 in platelet 12-lipoxygenase. R1 is CH3(CH2)4, Rz is CHzCH = CH(CHa),COOH. (Data are based on Refs. 73~2,90 and 91.)

81

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD I 3 kb

h5LX

I

I

h 12LX- 1

I

It

I

II

>82kb

> I

14

17 kb

14

1

h l2LX-2

. .

1

14

FIG. 5. Exonlintron structures of lipoxygenase genes. h5LX, human 5-lipoxygenase gene

(M), hl2LX-1, human 12-lipoxygenase gene-l which codes for platelet 12-lipoxygenase (55), h12LX-2, human 12-lipoxygenase gene-2, part of a probable pseudogene apparently lacking exon 9 (55),rblSLX, rabbit 15-lipoxygenase gene (56). Horizontal lines indicate gene loci and the vertical bars depict exons. Exons 1 and 14 are labeled for each gene, except for h12LX-2 where exons 7,8, 10, and 11 are shown. All genes are compared to the same scale except for five introns within the 5-lipoxygenase gene.

CCAAT-box is present in the correct position. The human 18lipoxygenase gene, like the 12-lipoxygenase genes-1 and -2, has been localized to chromosome 17 (55).It remains to be established if these three genes are linked to each other.

II. Leukotriene A, Hydrolase Leukotriene A, hydrolase (EC 3.3.2.6)catalyzes the conversion of leukotriene A, (LTA,) to the leukocyte chemotactic compound leukotriene B,. The hydrolysis reaction catalyzed by this enzyme is very specific for the LTA, epoxide substrate. The enzyme was originally found and purified from human PMN (93)but it has become increasingly evident that the enzyme is quite ubiquitous (94). cDNA clones encoding LTA, hydrolase have been isolated by immunoscreening with a polyclonal antibody, and by hybridization screening with a single 48-mer based on a purified PMN peptide sequence, from human lung and placenta, and spleen cDNA libraries, respectively (12, 13). The cDNAs from both sources were identical, covering an open reading fi-ame of 1833 bp encoding a mature 610 amino-acid protein of molecular weight = 69,000. More recently, cDNAs have also been isolated from mouse spleen and rat kidney cDNA libraries (95, 96). All three sequences are more than 93% identical. The 2.2-kb mRNA is widely distributed in virtually all tissues tested, although the highest relative amounts appear to be found in lung and leukocytes. From quantitative immunological

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COLIN D. FUNK

studies, small intestine epithelial cells are one of the richest sources of the enzyme (97). After the original cloning of the human sequences, data-base searches indicated that LTA, hydrolase is a unique epoxide hydrolase, unrelated to liver microsomal epoxide hydrolases and other proteins (12, 13).Two years later, however, a weak homology between LTA, hydrolase and aminopeptidase was reported (98). Independent observations revealed that LTA, hydrolase has a consensus motif found in zinc metalloproteinases (aminopeptidase N, thermolysin, endopeptidase, and collagenase are some of these enzymes) (99,100) (Fig. 6). After these observations, two groups experimentally verified the presence of a zinc atom in leukotriene A, hydrolase and found that the enzyme is bifunctional, displaying aminopeptidase activity, as well as LTA, hydrolytic activity (100-104). The endogenous peptide substrate, if one exists, has yet to be identified. The aminopeptidase homology lies within the central portion of the molecule (residues 233-340) adjacent to the most hydrophobic region (residues 190-205) of the protein. This hydrophobic region contains a cysteine at position 199, originally postulated to be a potential active-site residue within the LTA, substrate binding domain, LTA, hydrolase undergoes covalent inactivation during catalysis. However, from in uitro mutagenesis studies Cys-199 does not appear to be essential to activity. LTA, hydrolase with full activity is expressed in E . coli (95, 105).Mutagenesis studies with the expressed human and mouse enzymes have revealed that His-295, His-299, and Glu-318 constitute the three Zn atom coordination points (Fig. 6) since the mutated proteins were basically lacking zinc (106). Both LTA, hydrolase and aminopeptidase activities were reduced to nearly undetectable levels in the mutant proteins. The results verified the essential nature of the zinc atom to catalysis, but not to enzyme structural characteristics, as chromatographic and immunological properties were largely unaffected in the mutant proteins. LTA, hydrolase is inhibited by the aminopeptidase inhibitor bestatin and by the angiotensin-convertingenzyme inhibitor captopri1(104,107).Studies

human LTA 265 F P Y G G M E N P C L - T F V T P T L L A - G D - - - - - - K S - L S ~ I ~ I S ~ ~ G ~ 325 ~~T~H~~G mouse LTA 265 t t t * t * . f t * t _ t t * * * t t t * - * * - - - - - - * * * * * * * * * H * * * ~ ~ * * ~ * ~ * ~ * * * * ~ * * * *325 *~~~****~ rat LTR 264 t t * f * t t ~ t t t - t * f * t t t f * - * ~ - - - - - - * ~ * ~ * * ~ ~ ~ ~ * ~ ~ ~ * * * * ~ * * *324 * * * ~ * * * * M I N O P E P 349 ~NA*A*~*wG*v*YRENS**FDPLSSSSS"R"***B*FAS*V* 418

FIG. 6. Comparison of leukotriene A4 hydrolases (12, 13, 95, 96) and human aminopeptidase N (176) within the highly conserved zinc-ion binding region. Identity to the leukotriene A4 hydrolase sequence is indicated by *. Caps for alignment are shown by dashes. The two histidines and glutarnic acid residues that constitute the three zinc-binding ligands are boldfaced.

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

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with the latter drug indicate that the sulfhydryl group is an important determinant for inhibitory activity and that there is a differential susceptibility of the two activities for captopril. The fact that LTA, hydrolase is so widely distributed, yet the first enzyme (5-lipoxygenase) in the synthesis of LTA, is restricted to only a few tissues suggests that the alternative functional role (aminopeptidase) could be very important. These inhibitors could help define more clearly the biological role of LTA hydrolase and the significance of LTA, transcellular metabolism.

111. Prostaglandin G/H Synthase (Cyclooxygenase) The release of arachidonic acid from membrane glycerophospholipids by various stimuli leads in many cell types to the formation of prostaglandins via the enzyme prostaglandin G/H synthase (also known as prostaglandin endoperoxide synthase and commonly referred to as cyclooxygenase; EC 1.14.99.1)(10). This enzyme has been characterized most extensively from the ram seminal vesicle, its richest source. The enzyme apparently functions as a homodimer (subunit molecular weight of = 72 kDa). It is a glycoprotein in the endoplasmic reticulum that contains one heme group/subunit. The enzyme carries out the bisoxygenation of arachidonic acid to form PGG, (cyclooxygenase activity), as well as the subsequent reduction of the 15hydroperoxy moiety of PGG, to yield PGH, (peroxidase activity). PGG/H synthase has received widespread attention over the past two decades since it was discovered that this enzyme is the main target site for aspirin and nonsteroidal anti-inflammatory drug action (11, 108, 109). The cDNA for the cyclooxygenase from ram seminal vesicle was cloned independently by three groups in 1988 (110-112). It encodes for a 600aminoacid protein with a 24-residue signal peptide and four potential Nlinked glycosylation sites, three of which contain high-mannose carbohydrate (113). Adjacent to the signal peptide sequence is an EGF-like domain, and the aspirin acetylation site (Ser-530) is found close to the carboxy terminus (114, 115). Partial and complete cDNAs from human sources (human umbilical vein endothelial cell, platelet/erythroleukemia cell, embryonic lung fibroblasts) have been reported as well as the complete mouse sequence (116-119). The cyclooxygenases from the different species are closely related (= 90% identity) and have the same basic features as the ram seminal vesicle cyclooxygenase. Collectively, these sequences are referred to as cyclooxygenase-1 (COX-1). Although there were earlier indications for the presence of cyclooxygenase isoforms (120), it was not until recently that this was realized at the molecular level. The serendipitous finding of a second form of cyclooxygenase (COX-2) arose independently from two groups not working in the

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COLIN D. FUNK

eicosanoid field who were investigating the nature of immediate-early response genes induced by the Rous sarcoma virus (src) in chick embryo fibroblasts and by phorbol esters in Swiss 3T3 mouse fibroblasts, respectively (121, 122). When the primary sequences were scanned with nucleotide data bases, it was found that these primary response genes were = 60% identical at the amino-acid level with COX-1 and around 80% similar when conservative substitutions were taken into account. The COX-2 cDNA encodes a 603 (chick)/604(mouse)amino-acid protein. The major differences between the COX-1 and COX-2 forms are at the two termini (Fig. 7). At the N-terminus, a 17-aminoacid stretch within the signal peptide of COX-1 is not present in COX-2. In contrast, COX-2 contains an 18-aminoacid region at the carboxy terminus not present in COX-1. All the other major features found in COX-1, like the aspirin-sensitive serine residue, EGF-like domain,

4++H++& 1 2 3 4 5 6-9

10

1

mRNA 64 n t

ATTTA

AmA

2 kb

124 n t

H1s2093 1 I 390(heme Interactlon)

His295 3 7 4 Asn53 130

eF5 16

protein

N signal peptide

OH peroxidase homology

602 amino acids

NH EGF-like

Tyr371 peroxidase homology

604 amino acids

FIG. 7. Comparison of mouse cyclooxygenase genes, mRNAs, and proteins. Top: cyclooxygenase gene loci. The two mouse cyclooxygenase genes are compared on the same scale. Vertical bars represent exons. Middle: cyclooxygenase mRNA forms. The start of transcription is indicated by the arrow. The coding region is represented by the open box and the sizes of the 5’ and 3 untranslated regions are noted by the thin lines. COX-2 mRNA contains 11 copies of the sequence ATTTA, a sequence found in mRNAs that turn over rapidly. Bottom: mouse cyclooxygenase isoforms. COX-1 and COX-2 differ primarily at the two ends (darkened boxes). Other salient features (EGF-like domain, N-linked glycosylation sites, potential heme interacting histidine residues, catalysis-essential tyrosine, and aspirin-sensitive serine residues) are shared between COX-I and COX-2. Numbering begins with the first rnethionine. Mouse COX-1 numbering differs from the sheep COX-1 (602 v5. 600 amino acids). The latter enzyme has been studied extensively and the features depicted here are based on the sheep sequence.

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

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homology to peroxidases, and residues essential to catalytic activity, are present in COX-2. There are five potential sites for N-glycosylation and the three sites experimentally verified to have carbohydrate within COX-1 are conserved. COX-1 mRNA has been found in numerous tissues from various species, and is present as a = 2.8-kb transcript. The mRNA can be increased by many factors, such as phorbol ester, interleukin-1, progesterone, platelet-derived growth factor, EGF, CAMP, and serum, to mention a few (123-129). Glucocorticoids, aspirin, and heparin-binding (acidic fibroblast) growth factor-1 decrease COX-1 gene expression (130-132). The glucocorticoid effect is complex, probably mediated at several levels, and is cell-type specific (130, 133-135). The COX-1 3’-untranslated region (750-915 bp) is highly conserved among different species (= 50-70%) and this region is potentially involved in the regulation of expression of the gene (117, 118, 136). COX-2 mRNA transcripts are larger (= 4 kb); they appear to be expressed in very low amounts under basal or normal nonproliferating conditions, and are expressed in a tissue-type restricted fashion (121, 122). EGF, forskolin, phorbol ester, and serum induce a large and rapid increase in COX-2 mRNA levels in mouse fibroblasts. Glucocorticoids reduce the cycloheximide-induced increase in COX-2 mRNA levels (137). Luteinizing hormone/human chorionic gonadotropin (LH/hCG) induces a 40-fold induction of cyclooxygenase enzyme in rat granulosa cells of preovulatory follicles (138). The increase was not associated with a change in the 2.8-kb COX-1 mRNA. The specific cyclooxygenase induced by the treatment was partially purified from these cells and the N-terminal sequence was determined (139). The sequence is 96% identical to the mouse COX-2 indicating that LH/hCG specifically induced COX-2. Current data suggest, therefore, that there are two forms of COX mRNA and protein, each encoded by separate genes (see below), the one (COX-I) being more ubiquitously expressed and subject to low-level regulation (usually 2-4 fold changes in mRNA levels) and the other (COX-2), found in nearly undetectable levels by Northern-blot analysis, that is highly regulatable by various factors. COX-1 from ram seminal vesicles and human platelet/erythroleukemia cells, as well as Swiss 3T3 fibroblast-derived COX-2, have been expressed in COS cells (116, 117, 119, 140). So far, there have been no reports of expression of the heme-containing COX protein in E . coli, but recently it has been seen to be expressed in a baculovirus/insect cell system ( 1 4 0 ~ Exten). sive in uitro mutagenesis experiments have been performed with the expressed COX-1 enzyme (117, 119, 141-143). Replacement of Ser-530 with alanine does not markedly affect either cyclooxygenase or hydroperoxidase activity, indicating that the aspirin-sensitive residue is not essential to catalytic activity or substrate binding (119).

86

COLIN D. FUNK

The mutant enzyme, however, did not undergo irreversible inactivation by aspirin. Replacement by asparagine, though, abolished cyclooxygenase activity. Apparently, acetylation of Ser-530 causes an interference with substrate binding by introduction of a bulky side-chain. Mutagenesis of all 13 histidines in COX-1 to glutamine revealed the essential nature of His-207, His-309, and His-388 (141). It is likely that the imidazole group of His309 is an axial heme ligand and that His-388 is the distal ligand (143). His-207 may interact with a heme carboxyl side chain via ionic linkage. Site-directed mutagenesis has also shown the essential nature of Tyr-385 to cyclooxygenase, but not to peroxidase, activity (142). This residue probably lies at the active site and forms a tyrosyl radical during catalysis which is associated with the suicide-inactivation of the enzyme but not with the initial hydrogen abstraction step (143). Prostaglandin G/H synthase is a very complex enzyme and there is still much to be learned about its structurelfunction properties. The human and murine COX-1 genes have been cloned (144,145). They share the same exodintron boundaries and are divided into 11 exons spanning about 22 kb of genomic DNA. Characterization of the mouse COX-1 promoter has been performed (145). The murine COX-2 gene has been cloned and characterized (140). It contains 10 exons and spans 8 kb. The COX-2 gene is lacking an exon found in COX-1 (exon 2) that encodes the hydrophobic leader sequence. The eight internal exons are highly conserved between the COX-1 and COX-2 genes, with junctions located in analogous positions (Fig. 7). A 1.0 kb sequence upstream from the transcription initiation site conferred phorbol ester and serum inducibility in transfection studies. The human COX-1 gene is in chromosome 9 (117). The chromosomal position of the COX-2 gene is unknown at present.

IV. Thromboxane-A Synthase Thromboxane-A synthase (EC 5.3.99.5) catalyzes the conversion of PGH, to thromboxane A2 (TXA,). The enzyme is a ferrihemoprotein that undergoes inactivation during catalysis (146). TXA, is a potent platelet aggregator and a vasoconstrictor of vascular and respiratory smooth muscle. It has been implicated in the pathophysiologic expression of several disorders such as myocardial infarction, stroke, and atherosclerosis (147). Thromboxane-A synthase cDNAs have been isolated from human platelet and lung reverse-transcribed RNA by PCR using oligonucleotide primers based on direct peptide sequences (148, 149). Additional clones were isolated by hybridization screening of a human lung cDNA library. The platelet and lung enzymes appear to have identical 534 (or 533; there are two adjacent Met residues at the N-terminus) amino-acid sequences (TX synthase-I, with M, of = 60,684).However, the lung may have an alternative

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

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form, as cDNA clones lacking 163bp near the 3' end of the translated region were obtained. The deduced sequence of the shorter enzyme, TX synthase11, would have 460 amino acids with an M, of 52,408. Two distinct thromboxane-A synthase species have been detected previously in platelets and WI-38 lung fibroblasts (150, 151). It is possible, therefore, that the two forms arise from alternative splicing and/or a variable extent of glycosylation. There are five potential N-linked sites and a highly hydrophobic segment at the Nterminus that could serve to anchor the protein in the endoplasmic reticulum. A single 2.1-2.2 kb mRNA can be detected by RNA blot analysis in human platelet, HEL, and THP-1 (a human monocytic leukemia cell line) cells (148, 149).Phorbol esters increase its expression in HEL cells. Thromboxane synthase has certain features characteristic of cytochrome P-450s (Fig. 8). When aligned with these enzymes, it is evident that thromboxane synthase is related especially to the P-450s of family IIIA (== 35%), with human nifedipine oxidase (cytochrome P-450IIIA4)showing the highest similarity. P-450s have conserved Cys residue in the carboxy-terminalregion that is thought to be the proximal ligand for the heme moiety, as well as a helix-heme-helix structure (152). Both the cysteine residue (Cys-480) and the helical structure are conserved in thromboxane synthase-I but not in thromboxane synthase-11. A more detailed characterization of the structurelfunction properties of this enzyme awaits expression and in vitro mutagenesis experiments.

V. Prostaglandin-D Synthase Prostaglandin-D synthase (prostaglandin-H, D-isomerase, EC 5.3.99.2) catalyzes the transformation of the cyclooxygenase-derived intermediate PGH, to PGD,. This prostaglandin is a major arachidonate metabolite in the

TxSyn-I 391 L P Y L D M V I A E T L R M Y P P A F R F T R E A A Q D C E V L G Q R I P A G A P S P E TxSyn-11 391 ________________________________________-----P450-IIIA4 353 ME-----VN----LF-I-M-LE-VCKK-V-IN-MF--K-V-~IPSY---R--KY-TE-TxSyn-I 451 TFNPERFTAEARQQHRPFTYLPFGAGPRS~LGVRLGLLNKLTLLHLHKFRFQACPETQ TxSyn-I1 451 ------YRCS* ~ 4 5 0 - I I I A413 ~ K-L----SKKNKDNID-YI-T---S---NCI-M-FA-MNM--A-IR--QN-S-KP-K--TxSyn-I TxSyn-I1 P450IIIA4

511 VPLQLESKSALGPKNGVYIKIVSR* 473 I--K-SLGGL-Q-EKP-VL-VE--*

FIG. 8. Alignment of carboxy-terminal regions of human thromboxane synthases and human cytochrome P450IIIA4. The cysteine believed to be the proximal heme ligand is boldfaced. Dashes indicate identity to the thromboxane synthase-I. The end of the coding sequence is shown by *. (Adapted from Refs. 148 and 149.)

88

COLIN D. FUNK

brain, where it exerts several central actions related to sleep, regulation of body temperature, and modulation of pain response (153-154). PGD, is also a major metabolite of mast cells and spleen (155, 156). PGD, shows several pharmacological activities such as inhibition of platelet aggregation, pulmonary vaso- and bronchoconstriction, and peripheral vasodilation. A polyclonal antibody against rat brain PGD synthase was used to isolate cDNA clones encoding the enzyme (157). An open frame of 564 bp codes for a 188 amino acid protein with a calculated molecular weight of 21,232. The purified protein has a molecular weight of approximately 26,000 and can be deglycosylated to two smaller protein bands of 23 and 20 kDa. Consequently, it is believed that there are two N-linked glycosylation sites at Asn-51 and Asn-78. The first 20 amino acids probably act as a signal peptide. The enzyme is apparently associated with the rough endoplasmic reticulum and outer nuclear membranes but is not integral to the membrane since there are no strongly predicted membrane spanning domains and detergents are not required to solubilize the enzyme (158). cDNAs of glutathione-independent PGD synthase have been isolated from human brain (259). The complete cDNA encodes a 190-residue protein and there appears to be significant genetic heterogeneity as 16 differences were observed between two different overlapping cDNA clones. Southernblot analysis of genomic DNA indicate a single gene. Thus, the many differences suggest the presence of allelic variants. The human and rat aminoacid sequences are approximately 80% identical and they share the same N-linked glycosylation sites. Homology searches reveal that glutathioneindependent PGD synthase is a member of the lipocalin “superfamily”(260). This group comprises various small (160-190 residues) secretory proteins that share a common feature for binding and transport of small hydrophobic molecules. There are at least 16 members of this superfamily. PGD synthase is most similar to a,-microglobulin (29% identity; 52% similarity) and, together with the y-chain of C8 complement component, these proteins form a distinct cluster in the phylogenetic organization of this superfamily. Based on analysis of other members of this superfamily, it is postulated that PGD synthase displays a similar tertiary structure consisting of an a-helix and an eight-stranded hydrophobic anti-parallel P-barrel, which probably binds the PGH, substrate. Cysteine at position 65, within this pocket, has been postulated to be an active-site residue. Upon Northern-blot analysis, a single 850-900 bp transcript is detected in human and rat brain and in rat spinal cord and epididymis, but not in rat spleen, liver, and kidney. These results tend to indicate that brain PGD synthase is different from “spleen-type” PGD synthase or g1utathione-Stransferases, which can also catalyze the formation of PGD,. Based on several immunological and biochemical observations mast cells contain the spleen-type PGD synthase (162). The relatedness of the brain and spleen/

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

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mast cell types awaits the determination of the primary sequence of the latter.

VI. Prostaglandin-F Synthase Prostaglandin-F synthase (EC 1.1.1.188), an aldo-keto reductase, converts the endoperoxide intermediate PGH, to PGF,, and also converts PGD, to 901, IlP-PGF, on different active sites within the same molecule (162). This enzyme was initially purified to apparent homogeneity from bovine lung; it is a monomeric protein of M , 30,500 (163). Using two sets of short oligonucleotides of sequences based on aminoacid sequences, several cDNA clones were isolated from a bovine lung cDNA library (164). The clone with the longest insert (1.2 kb) had a 969 bp open reading frame coding for a 323 amino-acid polypeptide with a calculated molecular weight of 36,666. Sequence analysis revealed a strong similarity (65%) with human liver aldehyde reductase and rat lens aldose reductase and 77% similarity to frog lens p-crystallin, suggesting that these proteins form a gene family. The latter proteins share some of the enzymatic and cofactor features of PGF synthase but they do not share significant crossreacting antigenic determinants. Most striking is the exact homology of PGF synthase and p-crystallin in a 21-aminoacid stretch (residues 178- 198) with the exception of Pro-195. The dual functionality of PGF synthase is partially lost by mutagenesis of Pro-195 to Val-195 (found in crystallin) which suggests that this residue may be related to one of the active sites of the enzyme (165). By immunological studies and RNA blot analysis PGF synthase appears to be selectively distributed in lung and spleen (162). A 1.4-kb transcript has been observed in these tissues. The immunostained protein was localized primarily in alveolar interstitial cells, to a lesser extent in nonciliated epithelial cells of the bronchiole, and in both white and red pulp of the spleen (most likely in histiocytes and/or dendritic cells).

VII. Eicosanoid Receptors Prostaglandins and leukotrienes are known to transduce their signals via receptors linked to G-proteins. Distinct receptors for thromboxane A,, prostacyclin/PGE,, PGE (possibly three subtypes referred to as EP1, EP2, and EP3), PGF,, , LTB,, and LTD, present in various tissues have been described. Some of the receptors have been solubilized and partially purified (166-168) and the human platelet TXA, receptor has been purified to apparent homogeneity (169). The purified thromboxane receptor exhibited a very broad band on a SDS-polyacrylamide gel centered at = 57 kDa. Enough protein was obtained for partial sequence information. Oligonucleotide probes were used to screen a human megakaryocytic cell

90

COLIN D. FUNK

line (MEG-01)cDNA library (170).A partial length clone was obtained that, when sequenced, was found to encode the carboxy half of a putative Gprotein linked receptor. This clone was then labeled and used to screen a human placenta library. One full-length (= 2.9 kb) clone contained extensive 5’ and 3’ noncoding regions and a 1029 bp open reading frame coding for a 343 amino-acid protein of M , = 37000. The predicted sequence displays the characteristics of seven transmembrane G-linked receptors including two Nlinked glycosylation sites (Asn-4 and Asn-16) in the putative extracellular amino terminal tail (29 residues), conserved Cys residues in extracellular loops 1 and 2 (Cys-105 and Cys-183), and several other conserved residues within transmembrane regions, with the exception of the Asp residue found in transmembrane 3, known to be essential for receptors with small aminecontaining ligands (171). The sequence has a very short predicted third intracellular loop (27 residues). This portion of the molecule could possibly couple to the G-protein (Gs or larger G-protein) responsible for interacting with phospholipase C and causing subsequent changes in calcium ion flux (172, 173). The coding region for the thromboxane receptor is extremely G + C-rich (70%). It was nearly impossible to isolate this cDNA from placenta or platelet reverse-transcribed RNA under normal conditions of denaturation (94-95°C) with Taq polymerase (C. D. Funk, unpublished). However, a shifi of the denaturation temperature to 98°C and use of Vent polymerase (New England Biolabs, Beverly, MA) enabled amplification of the complete cDNA. The thromboxane receptor has been expressed in Xenopus oocytes. Apparently, it can couple with endogenous signal transduction components to elicit a calcium-activated C1- channel recorded by electrophysiological measurement (170).Binding studies have been performed with COS-1 cell membranes transfected by thromboxane receptor cDNA using the ligand S-145 (170). We have also shown high affinity binding of the thromboxane antagonist SQ-29548 in human embryonic kidney 293 cells and membranes transfected with thromboxane-receptor cDNA with maximal binding of 2-3 pmol/mg protein (C. D. Funk, unpublished). This level of expression is at least 5-10 times higher than in platelet membranes. On a per-cell basis assuming a 10% transfection efficiency, we estimate = 106 binding sites/ transfected cell as compared to = 1300 sites present on a platelet (174). Northern-blot analysis revealed the presence of a 2.8-kb band in the MEG-01 cell line, placenta, and lung. The mRNA is probably in the lowabundance category, based on the reported long exposure time (12 days) and amount of poly(A)+ RNA loaded (20 p,g) to see detectable signals. An approach to the isolation of other eicosanoid receptor genes by homology screening was taken, with the assumption that these receptors are related in primary structure (1 75).Primers were designed from the third and

MOLECULAR BIOLOGY IN THE EICOSANOID FIELD

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sixth transmembrane domains of the thromboxane receptor and used to amplify a 418-bp clone from reverse-transcribed total RNA from mouse lung. This clone had 78% homology to the thromboxane receptor in the corresponding region. This clone was 32P-labeled and used to screen mouse lung, and subsequently, mouse mastocytoma cell cDNA libraries. Overlapping clones ML64 and MP660 were isolated; they showed 38% relatedness to the human thromboxane receptor (49% within transmembrane domains). The open reading frame encoded a 365-aminoacid polypeptide with an estimated M , of 40,077. There are two potential N-glycosylation sites, one at the amino terminus (Asn-16) and one in the second extracellular loop (Asn-193), and there are nine serine and threonine residues at the carboxy terminus that are potential phosphorylation sites. In order to identify the endogenous ligand of this receptor, clone MP660 was expressed in COS-1 cells and membranes of the transfected cells were prepared to perform binding studies. [3H]PGE, bound specifically and with high affinity (Kd = 2.9 nM) and binding was inhibited by unlabeled prostaglandins in the rank order E, = E, > iloprost (a PGI, analog} > F,, > D,. In Chinese hamster ovary cells stably expressing the receptor, PGE, and a subtype specific agonist induced a potent inhibition of forskolin-induced CAMP accumulation (175). Taken together, these results indicated that a mouse PGE (subtype 3; EP3) receptor had been isolated. Northern-blot analysis revealed the presence of 2.3-kb band in several tissues where PGE, exerts biological effects and where binding sites have been reported, with kidney showing the highest mRNA levels. A 7.0-kb band was also observed in kidney, uterus, brain, and P815 inastocytoma cells. We have also used the homology screening approach to isolate a related eicosanoid receptor (C. D. Funk, L. Furci and G. A. FitzGerald, unpublished). Using a 0.3-kb thromboxane receptor fragment that covers most of the transmembrane 5-7 region of this receptor, a 1.4-kb cDNA clone (PGQ) encoding a 402-aminoacid putative receptor was isolated from a human erythroleukemia cell cDNA library. PGQ has two potential N-linked glycosylation sites (Asn-8 and Asn-25) and is extremely rich in basic (mainly arginine) and serine residues in the predicted third intracellular loop and the carboxy-terminal tail. At present, the endogenous ligand of the PGQ receptor is unknown. It is clear from sequence alignments that the thromboxane, PGE, and PGQ receptors form a related subfamily within the superfamily of G-protein coupled receptors superfamily (Fig. 9). They are about 40% identical to each other, and the areas of highest homology are in transmembrane 7 and part of transmembrane 4 extending 10 residues into the second extracellular loop. The molecular biology of eicosanoid receptors is still in the infancy stage

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COLIN D. FUNK t

1

t

TXR

MSPCDPLNLSLAO~A’ITCAAPWVPNTSAVPPSGASPALPIFSMTLGAVSNLLALALLGR~RRRSATT 72 t t I I I I1 I M W P f f i S S ~ P C F R P T N I T L E E R R L I A S ~ F ~ S F C W G L A R - - S62 S

PGE

64

PGQ

t I I I II I MA~APEHSAEAHSNLSSTTDDCGSVSVAFPITMMVTGFVGNALAM-LLVSRSYRRRESKRKKS

2

PGQ

3

FLLFVASLLATDLAGHVIPGLVLRLrPAGRAPAGRAPAGG-----ACHFLGCMVFFGLCPLLLGCGMAVERCVGV 139 II I II I I I l l I Ill II I I I I FLTFLCGLVLTDFMLLVTGTIWSQHAALFEWHAVDPGCRLCR~~IFFGLSPLLLGAAMASERYLI134 II I It I I I l l I Ill II Ill1 F L L C I O W L A L T D L V G Q L L T S P W I L W L S Q R R W E Q L D P S G R ~ ~ ~ L ~ G L S S L L V A S136 ~~RA~I

TXR

PGE

-

4

PGQ TRPLLHAARVSVARARLALAAVAAVALAVALLP~VGRYE~YPGTWCFIGLGPPGGWR---QALLAGLFA208 t

I

II

I

II

Ill

Ill1

I I I I I I

I

I

TXR T R P F S R P A V A S Q R R L ~ ~ G L L P L L G V G R197G I I II I I1 I l l Ill1 I I I I I I 1 * I PGE -RAPHWYASHMRFRATPVLLGVWLSVLAFALLPVLGVGRYSVQh’PGTWCFISTGPAGNETDPAREPGSVAFA 207

PGQ TXR

PGE 6

PGQ GGSRSSGSARRARAHDVEMVGQLVGIMWSCI~SPULVLVALAV----------GGWSSTSLQRPLFLAVR 338 I

I I I I I I

I I I I

I

TxR ------AAQQRPRDSEVEMMAQLLGIMWASVCWLPLLVFIAQTVLRNPPAMSPAGQLSRTTE-XELLIYLR PGE

I

295 I I1 I l l 1 II 1 I __---QSSAQ~RITPETAIQLMGIMCVL~CWSPLLIMMLKMIFNQMSVEQCKTQMGKEKECNSFLIAVR 309

I

-7

PGQ LASWNQILDPWVYILLRPAVLRQLLRLLPP~G~GGPAGLGLTPSAWEASSLRSSRHSGLSHF

TxR

PGE

I IIIIIIIIIII II VATWNQILDPWWILFRRAVLRRLQPRLSTRPRSLSLQPQLMRSGLQ I IlIt1l111II II LASLNQILDPWVYLLLRKILLRKFCQIRDHTNYASSSTSLPCPGSSALMWSD2LER

402 343 3 65

FIG. 9. Comparison of prostanoid receptors contained within a subfamily of the seven transmembrane G-protein coupled receptor superfamily PGQ, a putative human prostanoid receptor (C. D. Funk, L. Furci and C . A. FitzCerald, unpublished); TxR, human thromboxane receptor (170); PGE, mouse prostaglandin E (subtype 3) receptor (175). The putative seven transmembrane regions are overlined. Vertical bars indicate identity between the three sequences. Potential glycosylation sites are indicated by *.

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and it is obvious that much remains to be learned about eicosanoid signalling transduction, mechanisms of receptor desensitization, and residues important for ligand binding. In addition, many members of this family including the leukotriene receptors remain to be cloned.

VIM. Concluding Remarks The molecular biology of the eicosanoids has come a long way since its inception in 1987. It is clear, however, that much remains to be learned in this area. For instance, a clear biological role for the 12- and 15-lipoxygenases needs to be defined. Availability of the genes for these enzymes should allow targeting strategies to discern potential functions. Clear information on the regulation and significance of leukotriene formation by 5lipoxygenase, 5-lipoxygenase activating protein, and leukotriene A, hydrolase is still lacking. The recent discovery of the second form of cyclooxygenase has reopened a wide interest in the academic and pharmaceutical communities in prostaglandin research. The differential regulation of the two cyclooxygenase forms and the development of selective isoform inhibitors will be areas of intensive research. Cloning of the different members of the eicosanoid receptor family should facilitate our unravelling of the mechanisms involved in the action of this interesting class of arachidonate metabolites known as eicosanoids. ACKNOWLEDGMENTS These studies were supported in part by NIH grants HL46017 and HL30400 and a Research Career Development Award HL02710.

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Mammalian 6Phosphofructo-2 kinase/fructose-2,6bisphosphatase: A Bifunctional Enzyme That Controls Glycolysis’ GUYG. ROUSSEAUAND

LOUISHUE HonnAne and Metabolic Research Unit Department of Biochemistry and Cell Biology University of Louvain Medical School and International Institute of Cellular and Mokcukzr Pathology Brussels, Belgium

I. PFK-S/FBPase-2, A Bifunctional Enzyme That Synthesizes and Degrades Fructose 2,6-Bisphosphate . . . . . . . . . . . . . . . A. Liver PFK-2lFBPase-2 .................................. B. PFK-2/FBPase-2 Isozymes . . . . . . . . . . . . . . . . . . C. Short-Term Control of PFK-2/FBPase-2 ......................... 11. Molecular Characterization of PFK-&/FBPase-2 Isozymes . . . . . . . . . A. Characterization of Several PFK-Z/FBPase-Z mRNAs . . . . . . .

A. Glucocorticoids . . . . . .

...............................

C. Insulin and Glucagon . D. Growth Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V. Concluding Remarks . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

100 100 101 104 106 106 110 116 119 119 120 121 123 123 124

124

Glycolysis is the process by which glucose is converted first to pyruvate and then either to lactate or to ethanol and CO,. It is an amphibolic pathway, as it can provide the cell with energy from glucose catabolism and can also

Abbreviations: FBPase-2, fructose-2,6-bisphosphatase(EC 3.1.3.46); Fru-6-P, fructose 6phosphate; Fru-2,6-P~,fructose 2,6-bisphosphate; GRE, glucocorticoid response element; IRE, insulin response element; ORF, open reading frame; PCR, polymerase chain reaction; PFK-1, 6-phosphofructo-1-kinase (EC 2.7.1.11); PFK-2, 6-phosphofructo-2-kinase (EC 2.7.1.105); PKA, CAMP-dependent protein kinase; PKC, protein kinase C. 99 Progress in Nucleic Acid Research

and Molecular Biology, Vol. 45

Capyngfit 5 1993 by Academic Press, Inc.

All r i s t s of reproduction in any form reserved.

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GUY G . ROUSSEAU AND LOUIS HUE

serve an anabolic hnction by yielding C , precursors for the synthesis of amino acids, fatty acids, and cholesterol. The first committed step of glycolysis is the conversion of fructose 6-phosphate (Fru-6-P)and ATP into fructose l,6-bisphosphate catalyzed by 6-phosphofructo-l-kinase(PFK-1). The most potent stimulator of PFK-1 is fructose 2,6-bisphosphate(Fru-2,6-P2),present in all eukaryotes, discovered in 1980 (I,2). Unlike other regulatory ligands of PFK-1, which act at millimolar concentrations, Fru-2,6-P, stimulates PFK-1 as an allosteric effector active in the micromolar (physiological)range. It does so by increasing the affinity of PFK-1 for Fru-6-P and by relieving the inhibition caused by ATP (3).Thus, Fru-2,6-P2exerts a strategic control over glucose utilization. Aside from PFK-1, present in all cells, three other enzymes are controlled by Fru-2,6-P2, but these are not ubiquitous. The gluconeogenic enzyme fructose-l,6-bisphosphatase,present mainly in liver and kidney, is inhibited by Fru-2,6-P2(4,5).In trypanosomatihe, pyruvate kinase is stimulated by Fru-2,6-P2 (6). Plants and a few microorganisms contain a pyrophosphate-dependent PFK-1, which is also stimulated by Fru-2,6-P2(7, 8). These effects of Fru-2,6-P2 have been reviewed elsewhere (9-11). The aim of this paper is to focus on the enzyme system that catalyzes the biosynthesis and biodegradation of Fru-2,6-P,. The characterization of this system and of its regulation is a prerequisite for understanding how fluctuations in Fru-2,6-P2 concentration integrate the hormonal and metabolic signals that control glycolysis. We limit ourselves to the mammalian enzymes and emphasize the recent input of molecular biology.

1. PFK-2/FBPase-2, A Bifunctional Enzyme That Synthesizes and Degrades Fructose 2,6-Bisphosphate The concentration of Fru-2,6-P2in a given tissue depends on the balance between the activity of 6-phosphofructo-2-kinase(PFK-2),which synthesizes Fru-&,6-P2,and of fructose-2,6-bisphosphatase(FBPase-2), which hydrolyzes it. The properties of this system were studied first in rat liver and are summarized below for comparison with those of other tissues. It has not been possible to separate PFK-2 &om FBPase-2 by any purification procedure; indeed, the two activities belong to a single homodimeric protein composed of SS,OOO-Al,. subunits. Evidence for distinct catalytic sites on each subunit of this bifunctional enzyme has been obtained from chemical modification and proteolytic treatment, and is reviewed elsewhere (10).

A. Liver PFK-2/FBPase-2 PFK-2 catalyzes the synthesis of Fru-2,6-P2 from Fru-6-P and MgATP. By contrast with PFK-1, PFK-2 is not inhibited by physiological concentra-

MAMMALIAN

PFK-2/FRU-2,6-P2

101

tions of MgATP. In the presence of 5 mM Pi, a stimulator of PFK-2, the K , for Fru-6-P is about 50 FLM, a concentration also in the physiological range. PFK-2 is inhibited by citrate, phosphoenolpyruvate, and sn-glycerol3-phosphate at concentrations present in hepatocytes. Product-inhibition studies suggest that the reaction for Fru-2,6-P2 synthesis involves the formation of a ternary complex in an ordered sequential mechanism with a direct in-line transfer of the y-phosphate group of MgATP to the hydroxyl group of the C-2 of Fru-6-P. The hydrolysis of Fru-2,6-P2 to Fru-6-P and Pi is catalyzed by a specific phosphatase, FBPase-2, which is stimulated by Pi, sn-glycerol %phosphate, and nucleoside triphosphates. The K, for Fru-2,6-P2 is less than 0.1 p M , which is only & of the total concentration of Fru-2,6-P2 in livers of fed rats. Fru-6-P, one of the reaction products, inhibits FBPase-2 with an apparent & of 20 p M . As both the K , of PFK-2 and the K , of FBPase-2 are similar to the concentrations normally found in the liver, any change in Fru-6-P concentration will cause opposite changes in the two activities. The FBPase-2 reaction follows a “ping-pong” mechanism involving a phosphorylated enzyme intermediate. Incubation of the bifunctional enzyme with [2-32P]Fru-2,6-P2leads to the specific and transient phosphorylation of a histidine residue; the rate of phosphorylation is about 100 times the overall rate of hydrolysis, and thus is not rate-limiting. Vanadate, like Fru-6-P, inhibits FBPase-2 by binding to the phosphorylated intermediate (12). Interestingly, the phosphorothioate analogue of Fru-2,6-P2 is a better substrate than Fru-2,6-P2 itself (13). Liver PFK-2/FBPase-2 is a substrate of the CAMP-dependent protein kinase (PKA), which inactivates PFK-2 and activates FBPase-2 by phosphorylation. This results mainly from a change in their respective V,, values. The dephosphorylation of PFK-21FBPase-2 is catalyzed by phosphoprotein phosphatases [which correspond to phosphatases 2A and 2C in the classification of Cohen (13a)l.A more detailed account of the properties of liver PFK-2/FBPase-2 is given in Refs. 9 and 10.

B. PFK-2/FBPase-2 lsozymes Evidence for tissue-specific isozymes of PFK-2/FBPase-2 was first obtained in our laboratory when the properties of PFK-2IFBPase-2 purified from rat liver were compared with those of the bovine heart enzyme. The properties of the PFK-2/FBPase-2 purified from pigeon breast muscle and rat hindlimbs were also found to differ from those of the liver (reviewed in 14 and 15).The liver, heart, and skeletal muscle PFK-2IFBPase-2 have been termed the L, H, and M isozymes. They differ in molecular mass, kinetic properties, and response to protein kinases. All the mammalian PFK-2/ FBPase-2 isozymes can readily be purified by chromatography on anion-

102

GUY G . ROUSSEAU AND LOUIS HUE

exchange columns followed by affinity chromatography on either Blue Sepharose or phosphocellulose.

1. HEARTPFK-2/FBPASE-2 The H isozyme has relatively low FBPase-2 activity compared to the L and the M isozymes (Table I). The activity ratio of PFK-2 to FBPase-2 is about 100 for the H, 1 for the L, and 0.1 for the M isozyme. These ratios were obtained when FBPase-2 activity was measured at a physiological concentration of Fru-2,6-P,, i.e., in the micromolar range, which is & or less of the K, (40 pM) of the H isozyme for Fru-2,6-Pz. When the FBPase-2 activity is measured under V,, conditions, the PFK-2IFBPase-2 activity ratio decreases considerably. Another difference in the kinetic properties between the L and H isozymes is the poor PFK-2 sensitivity of the H isozyme to inhibition by sn-glycerol %phosphate, and its greater sensitivity to inhibition by citrate. These properties have been used as criteria to identify isozymes that differ from the L isozyme. Two forms of bovine heart PFK-2/FBPase-2 exist with subunit M,s of 54,000 and 58,000. Recent data (16)show that these forms result from alternative splicing of transcripts from the same gene (see Section II,A,3). Like the L isozyme, the H isozyme is a substrate for PKA. However, the phosphorylation sites differ, as does the response to phosphorylation. In the L isozyme, Ser-32 is phosphorylated and this inactivates PFK-2 and activates FBPase-2. In the H isozyme, the phosphorylation site has been identified in the Cterminal end at Ser-466 (164. Phosphorylation results in a slight stimulation of PFK-2 due to a decrease in K,,,for Fru-6-P. However, there is no effect on FBPase-2 activity. Unlike the L isozyme, the H isozyme is a substrate for protein kinase C (PKC) which phosphorylates Thr-475 without affecting the activity. The 54,000-M,form of the H isozyme does not contain these sites but is still phosphorylated by PKA and PKC, suggesting that other phosphorylation sites are present. The additional phosphorylation site for PKC has been TABLE I BIOCHEMICAL CHARhCrERISTICS OF PFK-2/FBPASE-2 ISOZYMES

PFK-2/FBPase-2 Phosphorylation by PKA PFK-2 FBPase-2 Phosphorylation by PKC Subunit M, ( x 10-3)

L

M

H

1 Yes

0.1 no

100

4

Yes

no

t

J.

-

t

no 55

T

no 53

Yes

Yes 55

MAMMALIAN

PFK-21FRU-2,6-P2

103

identified as Ser-84. No additional phosphorylation site for PKA has been identified so far. The multifunctional calcium/calmodulin-dependentprotein kinase phosphorylated the H isozyme, probably at the same sites as those phosphorylated by PKA. The phosphorylation and activation of PFK-2 by this kinase may explain the increase in Fru-2,6-P2 concentration that is observed when the workload applied to working hearts is increased (unpublished data &om this laboratory). The existence of two forms of the H isozyme and of multiple phosphorylation sites raises interesting questions concerning the possible formation of heterodimers and the effect of phosphorylation on enzyme activity. The PFK-2/FBPase-2 present in rat fetal (17) and regenerating (18)liver and in rat brain (19)have properties similar to those of the H isozyme. The low FBPase-2 activity of the H isozyme toward physiological concentrations of Fru-2,6-P, raises questions about the role of this enzyme in the degradation of Fru-2,6-P2. The FBPase-2 activity in heart, about Ajof that of the PFK-2, is 0.01 nmol/min and is clearly insufficient to account for the decrease in Fru-2,6-P, concentration observed in hearts p e h s e d with ketone bodies (20).In rat hepatoma H E cells, a specific FBPase-2 has been separated from PFK-2 and partially purified. It displayed a high K , for Fru-2,6-P2 (> 1mM) and, like the FBPase-2 of the bifunctional enzyme, was inhibited by Fru-6-P (21).The presence of such a high-&, FBPase-2 in other tissues has not been reported. In plants, yeast, and trypanosomes, monofimctional FBPase-2s have been separated from PFK-2 (9, 22). 2. SKELETAL MUSCLEP F K - ~ / F B P A s E - ~ The M isozyme displays a PFK-2/FBPase-2 ratio of about 0.1 and, as such, it resembles the L isozyme phosphorylated by PKA. However, the M isozyme is not a substrate for PKA and it has a smaller M , (Table I). The M isozyme was also detected in H E cells (23).On the other hand, the skeletal muscles of pigeon (24) and rat (24a) contain a small amount of PFK-21 FBPase-2 (subunit M , of 55,000), which is phosphorylated by PKA and recognized by antibodies specific for the L isozyme. 3. TESTISP F K - ~ / F B P A s E - ~ Rat testis contains some PFK-2/FBPase-2 activity that has physicochemical and immunological properties similar to those of the H isozyme (25). However, the bulk of the activity has been assigned to the so-called T isozyme (25). This isozyme, which has been cloned (see Section 11,A,4), is phosphorylated by PKC but not by PKA and exhibits other differences with respect to the isozymes described above (Table I).

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4. OTHERPFK-WFBPASE-2

Unlike the L isozyme, PFK-2 activity present in chick embryo fibroblasts (26), rat peritoneal macrophages (27), and FTO2B hepatoma cells (see Section II,A, 5), mouse B-lymphocytes (28), and human colon adenocarcinoma HT29 cells (29) is insensitive to inhibition by sn-glycerol 3-phosphate or PKA. The assignment of the PFK-2 in these tissues to a particular isozyme awaits further characterization.

C. Short-Term Control of PFK-2/FBPase-2 PFK-2/FBPase-2 activity is controlled by the concentration of its substrates and regulatory ligands as well as by covalent modification (Fig, 1). The existence of various isozymes which differ in kinetic properties and response to phosphorylation by protein kinases allows a tissue-specificregulation of metabolism. Phosphorylation of PFK-2/FBPase-2 by PKA in response to glucagon allows the liver to switch from glycolysis to gluconeogenesis by inactivating PFK-2 and activating FBPase-2 (30,31).Such a control does not exist for the skeletal and cardiac muscles, which have no gluconeogenic capacity. In the heart, by contrast, epinephrine increases Fru-2,6-P, and stimulates glycolysis, probably by stimulating glycogenolysis, which increases the concentration of hexose 6-phosphate (32, 33). The slight activation of the H isozyme resulting from phosphorylation by PKA may also participate in this regulation (16, 34). In the heart, insulin administration increases Fru-2,6-P2 by activation of PFK-2 (35).The molecular mechanism of this activation has not been elucidated. In the same tissue, phosphorylation by the calcium/ calmodulin-dependent protein kinase could also explain the increase in Fru-2,6-P2 concentration that is observed after a workload (see Section I,B, 1). Among the regulatory ligands of PFK-2, the inhibitor citrate is of particular interest as it also inhibits PFK-1, thereby providing a double mechanism to block glycolysis. The concentration of citrate increases in skeletal and cardiac muscle when these glycolyzing tissues are given alternative oxidizable substrates such as fatty acids, ketone bodies, or lactate. These “preferred substrates inhibit glycolysis by decreasing Fru-2,6-P2concentration. Therefore, Fru-2,6-P2can be regarded as a glycolytic signal that is turned on by glucose availability and switched off by alternative fuels. Variation in the hepatic concentration of sn-glycerol 3-phosphate, another inhibitor of PFK-2, which is also a stimulator of FBPase-2, may account for the fall in Fru-2,6-P2 concentration after ethanol administration or anoxia. The fact that heart and HTC cell PFK-2 is less sensitive to this inhibitor could explain why Fru-2,6-P2is barely affected by anoxia in these tissues. In

I glycolytic state I

gluconeogenic state gluconeogenic precursors, ethanol

free fatty acids

fast, diabetes, glucagon

I

glucose, a-adrenergic agents

insulin

I

I

sn-glycerol3-phosphate

hexoses-6-P

1

,U, Fr~-2,6-P,

high 6-10 phi)

\

glucose

t

lactate

I

Q

I

glucose

t

Q

t

lactate

FIG. 1. Role of fructose 2,6-bisphosphatein the short-term control of liver glycolysis and gluconeogenesis. For explanations, see Section 1,C.

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GUY G . ROUSSEAU AND LOUIS HUE

any case, the large stimulation of glycolysis by anoxia cannot be due to Fru-2,6-P2but rather to a fall in ATP and an increase in AMP. These inverse changes in adenine nucleotide concentration concur to stimulate PFK-1. An exhaustive review of this topic can be found elsewhere (14, 15).

II. Molecular Characterization of PFK-2lFBPase-2 lsozymes A. Characterization of Several PFK-2/FBPase-2 mRNAs 1. L-TYPEMRNA In view of the rarity of PFK-2/FBPase-2, the easiest way to elucidate the molecular basis for the PFK-2/FBPase-2 isozymes was to clone the corresponding cDNAs (Table 11). We obtained a partial rat liver cDNA by screening an expression library with polyclonal antibodies directed against purified rat liver PFK-2/FBPase-2 and by hybridization with a 47-mer oligonucleotide based on the microsequence of this purified enzyme. Further screening of another library with this partial cDNA (36) yielded two fully-coding poly(A)-tailed cDNA clones called 22c and 5c. The cDNA 22c was 1859 bp long, including a 29-bp poly(A) tail. It encompassed the full amino-acid sequence obtained by Pilkis and co-workers (37)from purified liver PFK-2/FBPase-2, and it confirmed a partial rat liver cDNA cloned by the same group (38).cDNA 22c therefore corresponds to the mRNA encoding the L isozyme. Primer extension showed that the corresponding mRNA in rat liver is 77 bases longer than 22c on the 5’ side. In Northern-blot analysis of poly(A)-rich RNA from rat liver, this cDNA probe showed a major band of 2.1 kb, consistent with the presence of a poly(A) tail of about 200 bases (39). No such band was seen with RNA from heart, brain, kidney, and testis (39, 40). Lange et al. cloned cDNAs fully coding for human (41) and bovine (42) liver PFK-S/FBPase-2 and corresponding to 470-residue polypeptides whose sequence was 95% and 97% identical, respectively, to that of the rat L isozyme. The second cDNA, 5c, is described below. 2. M-TYPEMRNA The cDNA 5c was 1750 bp-long. It was identical to clone 22c except for the nucleotide sequence upstream from the codon corresponding to aminoacid 33 of the L isozyme (39).This unique 126-bp sequence encoded an open reading frame (ORF) containing two ATGs, the second one being in a more favorable context for translation initiation. This predicted a unique N-terminal peptide of either 30 or, more likely, 9 amino acids in frame with the 438-

TABLE I1 CLONEDPFK-~IFBPAsE-~ MRNAs

Length in nucleotidesa Type

Species

5' leader

Coding

3' trailer

Total

(kb) ~

Liver (L) Muscle (M) Heart (Hl) (H2) (H3) Testis (T)

Rat Rat Bovine Rat Bovine Rat

249* 119d 26e n.p.f 268 34e

1413 1344 1593 1551 1413 1407

24Sd 245d

216' n.p. 216' 332e

Predicted peptide

Sizeb

1907 1708 1835 n.p.

1655 1773

2.1 1.9 4.0 n.d.8 n.d. 2.0

Length (aa)~

Mass (Da) ~

~~~~

470 447 530 516 470 468

Ref. ~~

54,570 51,880 60,679 59,267 53,891 54,023

Excluding the poly(A) tail. From Northern blot analysis. c Excluding the initial methionine. Determined by primer extension and S1 nuclease mapping (5' end) or from the presence of a poly(A) tail in the cDNA (3' end). p Actual limits of cloned cDNA. f n.p.. not published. c n.d., not determined.

36 39 44 25 45 25

108

GUY G . ROUSSEAU AND LOUIS HUE

amino-acid sequence common to the two cDNAs. Since this common sequence contained both the PFK-2 and the FBPase-2 domains, clone 5c most likely encoded a novel isozyme. We called it M (muscle) isozyme for the following four reasons. First, Northern blot analysis of rat poly(A)-rich RNA with probes corresponding to the unique sequence of clones 22c and 5c yields signals of 2.1 and 1.9 kb, respectively. The 2.1-kb signal (Ltype mRNA) is more prominent in liver, while the 1.9-kb signal is predominant in muscle. Primer extension analysis with muscle RNA shows that the corresponding mRNA is 21 bp longer than cDNA 5c on the 5' side (Table 11). Second, the smaller size of the peptide predicted for the M isozyme (448 residues) than of the peptide in the L isozyme (470 residues) is in keeping with the smaller M, of muscle as compared to liver PFK-2/FBPase-2 (see Section I, B,2). Third, the absence in the predicted M isozyme sequence of the site of phosphorylation (Ser-32) by PKA is consistent with the lack of effect of PKA on muscle PFK-21FBPase-2 (see Section I,B,2). Fourth (43), sequences of tryptic peptides of PFK-2IFBPase-2 purified from rat muscle and liver are identical in the two tissues except that the muscle enzyme lacks a peptide corresponding to residues 14-28 of the L isozyme and contains a peptide corresponding to residues 31-52 of the L isozyme but for the first two residues (Gly-Ser (P)), which are replaced by Thr-Ma, as predicted from the sequence of cDNA 5c. The amino-acid composition of the muscle enzyme is nearly identical to that of the liver enzyme, except for a lesser Arg and Ser content and a greater Ala content (43).This fits with the replacement of the 32 N-terminal residues of the L isozyme by a nonapeptide of different composition (see Section 11,B). Initiation of translation at the first AUG of the M-type mRNA would encode a longer form (468 residues) of the M isozyme. A comparison of the properties of the long and short forms expressed in Escherichia coli with those of muscle PFK-2/FBPase-2, and experiments with antibodies that distinguish these two forms, confirmed that it is the short one that is expressed in muscle (24a). 3. H-TYPEMRNA By screening a bovine heart cDNA library with a partial cDNA for human liver PFK-2/FBPase-2, a partial cDNA was obtained (44)that, when used as a probe, yielded a fully coding PFK-2/FBPase-2 cDNA. This cDNA (HI in Table 11)predicted a 530-residue peptide, identified as a H isozyme. Indeed, the deduced amino-acid sequence contained four tryptic peptides obtained from purified heart PFK-2/FBPase-2. One of these peptides, located near the C-terminus, contained sites for phosphorylation by PKA and

MAMMALIAN

PFK-2/FRU-2,6-P2

109

PKC, of which heart PFK-2/FBPase-2 is a known substrate. Moreover, this cDNA hybridized to a 4-kb mRNA in bovine heart (44).This mRNA population was in fact heterogeneous. Polymerase chain reaction (PCR) amplification of reverse-transcribed bovine heart RNA identified (16, 45) a smaller mRNA species that lacks a 180-bp sequence that includes the C-terminal region of the H isozyine containing the phosphorylation sites for PKA and PKC. The peptide encoded by this smaller H-type mRNA (H3 in Table 11)would account for the 54,000 M, isoform of heart PFK-2/FBPase-2 (see Section I,B,l). A third isoform ( H 2 in Table 11), corresponding to a cDNA characterized in the rat (25), would encode a peptide identical to the H1 isoform, except that the 20 C-terminal residues are replaced by an unrelated hexapeptide. This isoform is also present in bovine heart (unpublished data from this laboratory). Whether the 58,000-M, heart PFK-2/FBPase-2 (see Section I,B,l) is accounted for by the H1 and (or) H2 isoform(s) remains to be established.

4. T-TYPEMRNA By using the same human liver probe and strategy as for cloning the Htype mRNA, Sakata et al. (25) reconstituted a fully coding rat testis PFK-2/FBPase-2 cDNA for the T isozyme (Table I). This assignment was based on Northern-blot analysis and on the physicochemical properties of the recombinant enzyme expressed in E . coli.

5. OTHERTYPESOF MRNA AND PFK-2/FBPASE-2 ISOFORMS Rat hepatoma FTO2B cells reportedly (46) contain a PFK-2/FBPase-2 mRNA, which appears to be related to the L and M-types mRNA because it hybridizes with a probe common to these types. Although this mRNA hybridizes with an M-specific probe but not with an Gspecific probe, it is longer (2.2 kb) than the M-type mRNA (1.9kb). These cells contain a 55,000M , bifunctional isozyme whose PFK-2/FBPase-2 activity ratio, about 4, differs from that of the other isozymes (46). An Ltype mRNA probe also revealed a 6.8-kb mRNA in rat heart and muscle (39) and in rat brain (19). Rat hepatoma ( H E ) cells contain a PFK-2 that differs from the L isozyme and resembles the H isozyme. However, it is not sensitive to PKA and it has no detectable associated FBPase-2 activity (21).By screening an HTC cell library with probes derived from cDNA 22c we obtained, besides Gtype cDNA clones, clones corresponding to the M-type mRNA (23).Expression of the latter in E . coli yielded a protein that had the kinetic properties expected for the M isozyme. Several of these properties, e.g., insensitivity to PKA, were also typical of the H K enzyme. However, the recombinant protein had a high FBPase-2/PFK-2 activity ratio and an M, of 53,000, based on

110

GUY G . ROUSSEAU AND LOUIS HUE

covalent labeling and immunoblotting, as described for the M isozyme. This contrasted with the HTC enzyme that lacks associated FBPase-2 activity and has an M, of 56,000 (23). One interpretation of this discrepancy is that the PFK-2 of H E cells, albeit a product of the M-type mRNA, is modified posttranslationally so that it exhibits such peculiar properties. Another possibility is that the PFK-2 actually expressed in HTC cells is one of the H isoforms or derives from yet another mRNA that remains to be characterized.

B. Structure of the lsozymes The amino-acid sequence of several isozymes has been established either directly from the purified protein (liver) or from the corresponding cDNAs (Fig. 2). The overall structure of the various PFK-2lFBPase-2 isozymes is similar. A catalytic core containing the two distinct active sites is flanked by regulatory domains at both ends. The L and M isozymes are identical except for the N-terminal regulatory domain. The H isozyme differs from the L and M isozymes at both ends. The molecular basis for this difference is explained by the existence of at least two genes, one coding for the L and M isozymes, the other for the H isozyme (see Section 111). The N-terminal regulatory domain of the L isozyme (residues 1-32) includes Ser-32, the phosphorylation site for PKA. As said above (Section II,A,2), this regulatory domain is replaced in the M isozyme by a unique sequence of 9 amino acids that contains no phosphorylation site. This explains why the M isozyme is not controlled by PKA. The rest of the M isozyme sequence is identical to that of the L isozyme. The PFK-2 domain (33-249) contains four cysteine residues at positions 107, 160, 183, and 198, which maintain the configuration of the binding site for Fru-6-P, and which were identified after alkylation by radioactive iodoacetamide. Cys-160 is conserved in all the isozymes. The binding site of ATP has not been identified but a consensus sequence for a nucleotidebinding fold has been identified at residues 48-54 and is remarkably conserved. Recent studies using chemical modification of the protein by groupspecific reagents and site-directed mutagenesis have allowed identification of several amino acids essential for PFK-2 activity. The choice of amino acids to be mutated was made either from the results of chemical modification of residues, or according to a sequence alignment of the PFK-2 domain on the three-dimensional structure of bacterial PFK-1 as proposed by Bazan et al. (47). According to these authors, several residues involved in substrate binding, catalysis, and subunit interaction would have been conserved in PFK-1 and PFK-2. However, this study should be viewed with caution, as illustrated by the following example. Asp-127 in the active site of bacterial PFK-1

MAMMALIAN

PFK-2I FRU-2,6-P2

111

acts as a general-base catalyst and is essential for PFK-1 activity. In liver PFK-2, this Asp corresponds to Cys-160, which is one of the four cysteine residues essential for activity. However, the chemical properties of Cys-160 are such that it cannot act as a base catalyst. Moreover, mutation of Cys-160 to Asp, thus restoring the “original” PFK-1 active site, did not increase PFK-2 activity nor did it transform PFK-2 into PFK-1. However, it did decrease the affinity for the substrate, thereby confirming earlier studies with iodoacetamide (37). Therefore, the amino-acid residue(s) essential for PFK-2 catalysis and acting as (a) general base catalyst(s) remain(s) to be identified. The fact that the k,,, of PFK-2 is several orders of magnitude lower than that of PFK-1 suggests that a weak base might be involved in catalysis. On the other hand, recent studies using chemical modification of the enzyme and site-directed mutagenesis of the L isozyme have allowed the identification of several amino acids important for substrate binding. Liver Arg-195 (48)and Arg-225 (49) might be involved in Fru-6-P binding, whereas Arg-230 and -238 (48) could bind MgATP. As expected, mutation of Ser-32 into Ala abolishes phosphorylation by PKA, and replacement of Ser-32 by Asp mimics the effect of phosphorylation on activity probably by introducing one negative charge (50). The FBPase-2 domain (residues 250-470 in the L isozyme) contains His-258 in the active center (51).The sequence around His-258 (RHGESE) is very well conserved in all mammalian isozymes, but in the yeast a serine residue replaces histidine, thereby explaining the very low bisphosphatase activity of the yeast enzyme (52).A second histidine residue (His-392) participates in the FBPase-2 reaction and is conserved in all the isozymes. His-392 acts as a general-acid catalyst by donating a proton to the leaving group. Site-directed mutagenesis of His-392 to Phe, Lys, or Asp abolishes FBPase-2 activity (53).A consensus sequence for the start of a nucleotidebinding fold (274-280) is present in the L isozyme and may correspond to the binding site of nucleoside triphosphates, which are known stimulators of FBPase-2. The bisphosphatase domain of PFK-2lFBPase-2 shares structural (37) and functional (54)homology with the phosphoglycerate mutase family. The RHG sequence around His-258 is indeed conserved in this family, and His-258 of the liver PFK-2/FBPase-2 can be phosphorylated by 1,3bisphosphoglycerate. The FBPase-2 domain has been modeled on the threedimensional structure of yeast phosphoglycerate mutase (47). This has confirmed that His-258 is close to His-392 in the catalytic center. The C-terminal regulatory domain contains a consensus sequence for tyrosine-specific protein kinases. However phosphorylation of Tyr-359 remains to be demonstrated. The C-terminal end of the H isozyme difFers &om

bE418 ra48O y 651

R P R N Y S V G S R P L Q P L S P L R A . . . . . . . . . . . . . . . . . . . . P

bH 518 rH 520

H R L P S P A P P T S P S 530 P G Y R T S T E S P G V C K W T 535

L D T Q E G A D Q P K T Q A E T S R A A * * M * * * * * * * * * * p G p A * V S

FIG.2. Amino-acid sequence of PFK-2/FBPase-2 isozymes. r, rat; b, bovine; h, human; y, yeast, L, liver; M, skeletal muscle; H, heart (H, isozyme), T, testis. Dashes indicate identity with the corresponding residue in rL. Asterisks indicate identity with the corresponding residue in bH. Gaps and their length are indicated by numbers between parentheses. Italics (bottom lines) correspond to the residues that are identical in all sequences. References are as follows: rL (36, 37); bL (42);hL (41);rM (39); bH (44); rH (25, 59); rT (25); Y (52).

116

GUY G. ROUSSEAU AND LOUIS HUE

that of the L and M isozymes and contains phosphorylation sites for PKA (Ser-466) and for PKC (Thr-475).

111. Characterization of PFK-2/FBPase-2 Genes Examination of the nucleotide sequences of the digerent PFK-2I FBPase-2 cDNAs shows that at least three genes are required to encode the corresponding mRNAs, one (gene A) for the Ltype and possibly the M-type mRNA, one (gene B) for the three H-type mRNAs, and one (gene C) for the T-type mRNA. We have characterized genes A and B. No data on gene C are available so far.

A. Gene A By screening a rat genomic library with probes derived from cDNA 22c, we obtained seven overlapping clones that originated from a 55-kb gene (55). This gene contains 15 exons that encode the L and M-type mRNAs by using either one of two promoters separated by exon 1(Fig. 3). The Ltype mRNA is derived from a primary transcript that starts at exon 1’ and, after splicing, gives rise to a mRNA containing exons 1’ to 14. The L isozyme is translated from an AUG in exon 1’. The M-type mRNA is derived from a longer primary transcript that starts at exon 1 and matures by splicing of all the exons from 1 to 14 except exon l’, the last 13 exons (2-14) being common to the two mRNAs (Fig. 3). The M isozyme is translated from an AUG in exon 1

PY

A

I I

C

I

’

tkb

B PH

cI

PL

I

1 I

Ill1

1

I

I 1

I

IIY

(h) Xq27-28 (r) XqZ2-3t

i I 2

I

ATG

FIG. 3. Comparison of the PFK-Z/FBPase-2A and B genes. The exons are indicated by the M, L, and H promoter, respectively. The chromosomal localization in the rat (r) and in the human (h)is given at the right. For other explanations, see Section II1,A and B. vertical bars. PM, PL, and PH refer to

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which encodes the N-terminal peptide unique to this isozyme (see Section II,A,2). Exon 1’ encodes the 32-residue N-terminal peptide containing the Ser-32 phosphorylated by PKA in the L isozyme. Sl-nuclease mapping allowed the localization of the transcription initiation sites and confirmed the primer extension data (Table 11). These sites are located about 30 bp downstream from a TATA box (55). The cis-acting sequences in the first 2.7 kb of the L promoter have been delineated by transiently transfecting mouse and rat hepatoma cells with constructions containing this region and deletants thereof cloned in front of a reporter gene. Maximal promoter activity was contained within 360 bp upstream from the transcription initiation site. DNase I footprinting experiments with tissue extracts and purified proteins revealed, in this 5’ flanking fragment, binding sites for several proteins, some ubiquitous, some liverspecific (56).Further work on the cis-acting elements and trans-acting factors that control the L and M promoters is in progress. The way in which gene A gives rise to the M and L isozymes is reminiscent of the organization of the genes for glucokinase (57)and pyruvate kinase (58). Different primary transcripts encode isozymes that share the same catalytic core but differ at the N terminus which corresponds to alternative exons. Expression of the latter reflects the operation of tissue-specific promoters that can respond differently to control signals.

B. Gene B When screening the rat genomic library as described above, four overlapping clones yielded restriction fragments that were incompatible with the map of gene A. Their sequence showed six ORF (exons 9-14 of gene B) that have the same length as, and are similar to, exons 8-13 of gene A, followed by one ORF (exon 15 of gene B) that contains the C-terminal region of the H isozyme phosphorylated by PKA and PKC. The 5’ moiety of the gene was characterized by screening the genomic clones with a probe constructed by PCR on rat heart poly(A)-richRNA and with an oligonucleotide based on the sequence of a tryptic peptide from beef heart PFK-2/FBPase-2 located in exon 5 (59). While this work was in progress, the sequence of the bovine H-type mRNA became available (44). This enabled us to demonstrate that gene B encodes the H isozyme and to assign to its exons 2 to 8 the remaining ORFs, exon 1 being noncoding (Fig. 3). Beyond exon 15 of gene B, there was an ORF (ORF 16 in Fig. 3) ending by a stop codon and followed by a polyadenylation signal. This exon-intron organization accounts for the production of the three H-type mRNAs described in Table 11. These isoforms appear to be identical, except for the sequence beyond exon 14 and for the first coding exon (exon 2) (Fig. 2). In the H1 isoform, exon 15is spliced with a C-terminal

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exon that has not yet been identified in the gene B we have cloned. The H3 isoform is identical to the H1 isoform except for the loss of exon 15(16).As to the H2 isoform, it ends with an extended version of exon 15. N o mRNA containing a sequence that codes for ORF 16 has been found so far.

C. Comparison of the Two Genes A striking difference between genes A and B is the smaller size (22 kb) of gene B as compared to gene A (55 kb). Because the number and size of exons is similar in the two genes (Fig. 3), this difference corresponds to intronic material. Otherwise, the two genes are organized on the same pattern. There is a catalytic core containing six exons for the PFK-2 domain (exons 27 of gene A) followed by six exons for th6 FBPase-2 domain (exons 8-13). These twelve exons are homologous to exons 3-14 of gene B, with 52-80% nucleotide identity and 54-88% amino-acid identity for these exons between the two genes. Moreover, the conservation and relative position of the residues that are crucid for enzymatic activity is remarkabIe (Fig. 2). Th’ IS common core is flanked by regulatory exons that differ between the two genes as expected from the different properties of the isozymss. Interestingly, one finds between exons 14 and 15 of gene B an 80-bp noncoding sequence that corresponds, except for insertions and deletions, to a vestigial 5' end of exon 14 of gene A. This phenomenon, together with the future characterization of gene C, could shed some light on the evolution of PFK-2lFBPase-2 genes. Their origin most likely involves the hsion of two ancestral genes (31, 55), one that encodes the PFK-2 domain and one that encodes the FBPase-2 domain and is akin to the (bis)phosphoglyceratemutase genes (see Section II,B). The model proposed should also take into account the similarities of regions of the PFK-2IFBPase-2 genes with serine proteases, papilloma virus E l ORF and mouse polyoma middle T antigen (60).

D. Chromosomal Localization of Genes A and 5 On the basis of Southern blots of DNA from somatic cell hybrids, gene A was assigned to the X chromosome in the rat (55) and in the human (62).I n situ hybridization of probes generated by PCR showed (62) that gene A maps at the end of the long arm of this chromosome, namely Xq22-q31 in the rat and Xq27-q28 in the human, a region that includes many morbid loci. Gene B was assigned by the same techniques to 13q24-q25 in the rat and to lq31 in the human, which are homologous regions (62). The assignment of gene A to the X chromosome was at odds with our identification of Fru-2,6-P2in sperm (63),since the X chromosome is reportedly inactive in these cells (64). This paradox has now been solved by the discovery of gene B and its assignment to an autosome, and by the finding that

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the T isozyme is encoded by yet a third gene which, if expressed in sperm, must also be on an autosome. A similar situation has been described for phosphoglycerate kinase (64).

IV. Hormonal Control of PFK-2/FBPase-2 Gene Expression A. Glucocorticoids Adrenalectomy of the rat produces in liver a fall in Fru-2,6-Pz concentration, in PFK-2/FBPase-2 activity and content, and in PFK-2/FBPase-2 mRNA concentration, Administration of triamcinolone to these animals normalized Fru-2,6-P, concentration and PFK-2/FBPase-2 within 24 hr (65), confirming earlier work (66). The concentration of PFK-2/FBPase-2 mRNA was restored by 2 hr of treatment, reached 4 times the normal value by 8 hr, and returned to normal by 72 hr. Run-on assays and measurement of heterogeneous nuclear RNA indicated that the glucocorticoid stimulated transcription (65).There was also a 3- to 4-fold increase in muscle PFK-2IFBPase-2 mRNA after triamcinolone treatment (67). The effect of glucocorticoid hormone on mRNA was reproduced in rat hepatocytes exposed to dexamethasone added 12 hr after plating. The concentration of PFK-2/FBPase-2 mRNA which had nearly disappeared at 12 hr was back to normal after 4-6 h r of dexamethasone treatment, to reach twice this value after 8 hr (67).The stimulatory effect of dexamethasone seen after 24 hr was antagonized by insulin, while it was potentiated by dibutyryl CAMP (68). By 5 days of culture, however, the stimulatory effect of dexamethasone was potentiated by insulin or by thyroxine. This effect of dexamethasone alone or in combination was not accompanied by a change in the half-life (3-5 hr) of PFK-2/FBPase-2 mRNA. It was blocked not only by actinomycin D, but also by cycloheximide, suggesting a requirement for ongoing protein synthesis (68). Whether the PFK-2lFBPase-2 expressed after 5 days was still the L isozyme is not clear, the more so as neither Fru-2,6-P2 concentration nor PFK-2IFBPase-2 activity was determined in these experiments. Genes whose transcription is stimulated by glucocorticoids contain cisacting sequences, called glucocorticoid-response elements (GRE), that bind and are activated by the glucocorticoid receptor (69). Transcription of the socalled primary response genes (e.g . , tyrosine aminotransferase) is rapidly stimulated as a result of receptor binding to the GRE and this is insensitive to inhibitors of protein synthesis such as cycloheximide. Transcriptional stimulation of secondary response genes (e. g., trl-acid glycoprotein) is rapid but is blocked by cycloheximide presumably because receptor action at the GRE requires labile factor(s).

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The GREs in these two types of genes are made of two palindromic hexameric half-sites. Stimulation of the delayed secondary response genes (e.g., a2u-globulin) occurs via an ill-defined mechanism after a lag period and is sensitive to cycloheximide. The GREs of the a%-globulin gene are unusual and heterogeneous and they occur in the transcribed sequence (70). GRE-like hexameric half-sites occur in both the L and the M promoters. Although the effect of glucocorticoids on PFK-21FBPase-2 in long-term cultures of hepatocytes is reminiscent of that on secondary response genes, it may not reflect the situation in intact liver. More studies are needed to clardy this issue. As a rule, the liver enzymes that are induced by glucocorticoids are gluconeogenic. These enzymes are also induced by the CAMP-dependent pathway (31, 71). PFK-ZlFBPase-2 catalyzes not only the synthesis but also the degradation of Fru-2,6-P2. However, the glucocorticoid induction of the bifunctional enzyme is accompanied by an increase in Fru-2,6-P2concentration (65). Indeed, liver FBPase-2 is poorly active compared to PFK-2, due to its inhibition by physiological concentrations of Fru-6-P and because of the low phosphorylation state of the enzyme. Induction of liver PFK-2lFBPase-2 by glucocorticoids is therefore unexpected, because Fru-2,6-P2 inhibits gluconeogenesis. This paradoxical situation may result in increased substrate cycling in glycolysislgluconeogenesis. Rat FTO2B hepatoma cells contain a PFK-S/FBPase-2 isozyme and mRNA that differ from those in rat liver (see Section II,A,5). They also respond to dexamethasone within 2 hr by an increase rate of transcription of this mRNA, as demonstrated by run-on assays. This resulted in a 10-fold increase in mRNA concentration within 6-10 hr, an effect that was not potentiated, but instead was blocked, by dibutyryl CAMP (46). In rat hepatoma H E cells, which express yet another isoform of PFK-2lFBPase-2 (see Section II,A,5), dexamethasone increases PFK-2 activity, and this is inhibited by actinomycin D (72). In these cells, however, the glucocorticoid did not modify PFK-2lFBPase-2 mRNA concentration, as if it induced a protein that increases the catalytic activity of the enzyme (23).

B. Thyroid Hormones Liver PFK-2 activity is markedly decreased in hypothyroid rats and restored after 3 days of treatment with triiodothyronine. These changes are accompanied by parallel changes in PFK-2/FBPase-2 content and mRNA (73). Yet thyroxine did not increase the quasi-undetectable level of PFK-2dFBPase-2 mRNA in cultured rat hepatocytes, even after 5 days of culture in the presence of the hormone (68). Still, thyroxine potentiated the stimulatory effect of dexamethasone on this mRNA seen under these conditions, as mentioned above. The stimulation of liver Iipogenesis by thyroid

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hormones requires C , units. These might be provided through increased glycolysis, by virtue of the hormonal effects described here. Despite a report that PFK-2 decreases in the hearts of hypothyroid rats (74),we found no consistent change in this parameter with or without triiodothyronine treatment, whether the rats were hypothyroid or not (73).

C. Insulin and Glucagon As discussed in Section I,C, decreasing the ratio of insulin to glucagon in rats by fasting or by inducing diabetes rapidly inactivates PFK-2 and activates FBPase-2 in the liver as a result of phosphorylation. In addition, these treatments provoke long-term changes. Fasting for 72 hr led to an 80% decrease in liver PFK-2/FBPase-2 content (40,75)without change in Ltype mRNA concentration. The same situation prevailed in alloxan (75)or streptozotocin (40)induced diabetes. This was interpreted as the result of a decreased efficiency of mRNA translation or a decreased stability of the bifunctional enzyme, or both. However, refeeding starved rats with a standard or carbohydrate-rich, but not a high fat, diet for 48 hr, or treating diabetic rats with insulin for 24 hr not only normalized the hepatic PFK-BIFBPase-2 enzyme content, it also provoked a 3- to 6-fold increase in mRNA concentration above control values (40). In skeletal muscle, PFK-2/FBPase-2 activity (76)and mRNA concentration (40)were not affected by dietary manipulations, and PFK-2 activity did not change after insulin treatment (77). In severe streptozotocin-induced diabetes, L t y p e mRNA concentration decreased in rat liver; this was restored to normal not only by insulin, but also by oral vanadate administration (78). A stimulatory effect of insulin on Ltype mRNA could not be reproduced in primary cultures of rat hepatocytes (68).In contrast, the PFK-S/FBPase-2 mRNA present in the FTO2B cell line (see Section II,A,5) is inducible by insulin added in the culture medium, provided glucose is present. After a 4hr lag period, there was a 10-fold increase in mRNA concentration within 10 hr after hormone addition; this was prevented by dibutyryl CAMP(46).This induction results mainly from an increased rate of gene transcription. Indeed, the increase in PFK-2/FBPase-2 mRNA concentration was prevented by actinomycin D, insulin had no effect on mRNA stability, and insulin increased the ability of nuclei to incorporate precursors into PFK-S/FBPase-2 mRNA in run-on transcription assays (46). Both the significance and the mechanism of the induction of PFK-2/FBPase-2 mRNA by insulin remain to be clarified. While this effect was observed in FT02B cells, it was not seen in isolated hepatocytes, perhaps because these cells express different isozymes whose corresponding mRNAs might be controlled differently. One drawback of the published

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work on isolated hepatocytes is the lack of data on Fru-2,6-P2 concentration and PFK-2lFBPase-2 activity.and content. Another explanation for this discrepancy is that the requirement for glucose to elicit the insulin effect was satisfied in intact liver (40) and in FT02B cells (46), but not in isolated hepatocytes (68). Indeed, the genes whose transcription is stimulated by insulin fall into two categories (79). One includes genes that respond within minutes, such as glucokinase (80, 81), and glyceraldehyde-3-phosphatedehydrogenase (82), presumably through a direct effect of the hormone. The other category includes genes such as Ltype pyruvate kinase (83) and S14 (84).These genes respond to insulin after a lag period, and only in the presence of carbohydrate such as glucose or fructose. The effect of insulin appears to be indirect, perhaps via a glycolytic inteimediate. The experiments with FTO2B cells assign the gene that encodes their PFK-2/FBPase-2 to this second category. Several types of insulin-responsive cis-acting sequences (IRE) have been described in genes whose transcription is increased or decreased by insulin. The IRE-A present in the glyceraldehyde-3-phosphatedehydrogenase gene CCCGCCTC binds a protein related to the testis-determining factor SRY (85). We did not find this sequence on either strand within 1.8 kb of the PFK-2/FBPase-2 M promoter or in the L promoter. However, a sequence similar (2mismatches) to that (TGGTGTTTTG) which mediates the rapid transcriptional inhibition by insulin of the phosphoenolpyruvate carboxykinase gene is present at -293 of the L promoter. A similar sequence is present in the genes for glucokinase and malic enzyme whose transcription is stimulated by insulin (86).The significance of this finding for the induction of PFK-2/FBPase-2 mRNA by this hormone remains to be investigated. The genes of intermediary metabolism whose transcription is stimulated by insulin catalyze reactions that promote glycolysis and lipogenesis. Despite the fact that PFK-2/FBPase-2catalyzes both the synthesis and degradation of Fru-2,6-P,, its induction is expected to increase the steady-state concentration of the latter (see Section IV,A). Indeed, insulin treatment (87) or refeeding (40) in rats does lead to an increase in hepatic Fru-2,6-P2 concentration. This increase should participate in the stimulation of glycolysis and lipogenesis by insulin or carbohydrate. Similarly, in chick liver, glycolysis and lipogenesis from glucose are stimulated upon hatching. In cultured prenatal chick hepatocytes, insulin increased PFK-2 activity and Fru-2,6-P, concentration (88). In cultured fetal rat hepatocytes exposed for 3 hr to glucagon or to a CAMPanalog, total PFK-2 activity was not changed. However, based on pH sensitivity in oitro, these treatments appeared to promote the appearance of

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the L isozyme at the expense of the fetal isozyme. This unexpectedly rapid effect was partially prevented by cycloheximide or insulin (89).

D. Growth Factors In the experiments described above, insulin acted at concentrations (0.1 nM for half-maximal effect) at which it controls glucose uptake and intermediary metabolism. Mitogenic concentrations (100-fold higher) of insulin doubled PFK-2 activity and increased Fru-2,6-P, concentration and glycolysis in chick embryo (26) and human (90) fibroblasts, and in human colon adenocarcinoma HT29 cells (29). In human fibroblasts these effects of insulin were mimicked by serum; they required the presence of glucose and were insensitive to cycloheximide. In chick embryo fibroblasts, they were mimicked by epidermal growth factor (91). In HT29 cells, these effects of insulin were blocked by inhibitors of RNA and of protein synthesis. These experiments point to a role of PFK-2 in the increased glycolytic rate seen upon mitogenic stimulation. As discussed above (Section, I,B, l), regenerating liver contains a PFK-2/FBPase-2 that differs from the L isozyme and may be the same as that present in fetal liver. This isozyme switch might be due to humoral factors such as glucagon or hepatocyte growth factor, which increase transiently (92) following partial hepatectomy.

E. Phorbol Esters and Oncogenes Phorbol 12-myristate 13-acetate (PMA) and its active analogs, which stimulate PKC by mimicking the natural agonist diacylglycerol, increase glycolysis, PFK-2 activity, and Fru-2,6-P2 in chick embryo fibroblasts (26) and in human HT29 cells (29), but not in chick embryo hepatocytes (88).This effect of PMA was additive to that of insulin and it occurred after a lag period; it was blocked by inhibitors of RNA and of protein synthesis (29,91). Similar changes were seen following transformation of chick embryo fibroblasts by retroviruses carrying the v-ST-c or v-fps oncogenes, which encode tyrosine-specific protein kinases, but not the v-mit or v-myc oncogenes (93). Stimulation of PFK-2 activity by a thermosensitive pp6Ov-src at permissive temperature followed the same time-course as after phorbol ester treatment, was not additive to the latter, was accompanied by changes in the biochemical and physicochemical properties of the enzyme, and was prevented not only by actinomycin D but also by inhibition of PKC (94). This suggests that PKC activation stimulates the expression of a gene whose product activates PFK-2 or is another PFK-2 isozyme, and that pp6Ov-src can stimulate this pathway. These observations point to a novel mechanism for the stimulation of glycolysis by tyrosine-specific protein-kinase oncogenes.

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V. Concluding Remarks The molecular biology of PFK-2IFBPase-2 has already contributed significantly to our understanding of the control of Fru-2,6-P,. It has revealed the unexpected complexity of the system, with the identification of several genes and isozymes. It has also provided tools for tackling unsolved problems. The structureiactivity relationships of this bifunctional enzyme i s now being approached by site-directed mutagenesis. Thanks to the cloning of the isozymes, the comparison of their sequences will facilitate the identification of residues that are cruciai for catalysis and for control by allosteric effectors and by covalent modification. A challenging question is how the several kinases that phosphorylate the PFK-2/FBPase-2 protein at both ends have different effects on the separate catalytic sites of the various isozymes. The expression and biochemical characterization of the different recombinant forms of the H isozyme should shed some light on their physiological significance. The preferential expression of PFK-2iFBPase-2 isozymes in certain tissues offers models to study cell-specific promoter control by trans-acting factors. Such investigations are in progress. Other fields of interest are the ill-understood mechanisms of the long-term hormonal regulation of PFK-2/ FBPase-2 and the characterization of the PFK-2/FBPase-2 expressed in transformed cells. Advances along these lines pertain to the physiopathology of carbohydrate metabolism and to the basis for the abnormal glycolysis of cancer cells.

ACKNOWLEDGMENTS We thank ‘ I Lambert and V. Henry for typing and M. H. Rider for a critical reading of the manuscript. Work on this topic in the authors’ laboratory was supported by the F. N. R. S., the F.R.S.M., the Belgian State Prime Minister’s Office (Science Policy Programming), the Interuniversity Poles of Attraction, the “Caisse Generale dEpargne et de Retraite,” and the “Association Contre le Cancer’‘ (Belgium).

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52. M. Kretschmer and D. G. Fraenkel, Bchem 30, 10663 (1991). 53. A. Tauler, K. Lin and S. J. Pilkis, JBC 265, 15617 (1990). 54 A. Tauler, M. R. El-Maghrabi and S. J. P u s , JBC 262, 16808 (1987). 55. M. I. Darville, K. M. Crepin, L. Hue and G. G. Rousseau, PNAS 86, 6543 (1989). 56 F. P. Lemiugre, S. M. Durviaux and G . G. Rousseau, MCBiol 11, 1094 (1991). 57 M. A. Magnuson, J. Cell. Biochern. 48, 115 (1992). 58. G . L. Tremp, D. Boquet, M. A. Ripoche, M. Cognet, Y. C. Lone, J. Jami, A. Kahn and D. Daegelen, JBC 264, 19904 (1989). 59. M. 1. Darville, M. Chikri, E. Lebeau, L. Hue and G. G. Rousseau, FEBS Lett.288,91 (1991). 60. G . 6 . Rousseau and L. Hue, in “Activation of Hormone and Growth Factor Receptors” (M. N. Alexis and C. E. Sekeris, eds.), NATO AS1 Ser., Vol. 295, p. 93. Kluwer Academic Publ., Dordrecht, The Netherlands, 1990. 61 S. Olson, K. Uyeda and 0. W. McBride, Somatic Cell. Mol. Genet. 15, 617 (1989). 62 C. E. Hilliker, M. I. Darville, M. S. Aly, M: Chikri, C. Szpirer, P. Marynen, G. G. Rousseau and J. J. Cassiman, Genomics 10, 867 (1991). 63. 5. Philippe, G . G. Rousseau and L. Hue, FEBS Lett. 200, 169 (1986). 64. J. R. McCarrey and K. Thomas, Nature 326, 501 (1987). 65. A. J. Marker, A. D. Colosia, A. Tauler, D. H. Solomon, Y. Cayre, A. J. Lange, M. R. ElMaghrabi and S. J. Pilkis, JBC 264, 7000 (1989). 66 C. Schubert, H. J. Boehme and E. H o h a n n , BioSci. Rep 6, 513 (1986). 67. A. J. Lange, L. Kummel, M. R. El-Maghrabi, A. Tauler, A. D. Colosia, A. J. Marker and S. J. Pilkis, BBRC 162, 753 (1989). 68. L. Kummel and S. J. Pilkis, BBRC 169, 406 (1990). 69. M. Muller and R. Renkawitz, BBA 1088, 171 (1991). 70. 6. C. K. Chan, P. Hess, T. Meenakshi, J. Carlstedt-Duke, J. A. Gustafsson and F. Payvar, JRC 266, 22634 (1991). 71. D. K. Granner and S. J. Pilkis, JBC e65, 10173 (1990). 72. A. M. Loiseau, G . G. Rousseau and L. Hue, Cancer Res. 45, 4263 (1985). 73. S. R. Wall, M. F. van den Hove, K. M. Crepin, L. Hue and G. G. Rousseau, FEBS Lett. 257, 211 (1989). 74. A. Gualberto, P. Molinero and F. Sohrino. B] 244, 137 (1987). 75 K. M. Crepin, M. I. Darville, L. Hue and G. G. Rousseau, FEBS Lett.227, 136 (1988). 76 Y. Yamada, T. Shimizu, N. Hara, I. Mineo, M. Kawashi, H. Kiyokawa, K. Yamada, S. Fujioka, Y. Matsuzawa, N. Kono and T. Seiichiro, J . Biochern. 104, 576 (1988). 77. M. Carreras, A. M. Basso~o,J. Carreras and F. Climent, B i o c h . Int. 17, 359 (1988). 78 M. Miralpeix, E. Carhallo, R. Bartrons, K. M. Crepin, L. Hue and G G. Rousseau, Diabetologia 35, 243 (1992). 79. J. F. Decaux, 0. Marcillat, A. L. Pichard, J. Henry and A. Kahn, JBC 266, 3432 (1991). 80. P. Iynedjian, D. Jatterland, T. Nouspikel, M. Asfari and P. R. Pilot, JBC 264, 21821 (1989). 81. M. A. Magnuson, T. T. Andreone, R. L. Printz, S. Koch and D. K. Granner, P N A S 86, 4838 (1989). 82. N . Nasrin, L. Ercolani, M. Denaro, X. F. Kong, I. Kang and M. Alexander, PNAS 87, 5273 (1990). 83. J. F. Decaux, B. Antoine and A. Kahn, JBC 264, 11584 (1989). 84. K. S. Thompson and H. C. Towle, JBC 266, 8679 (1991). 85. N. Nasrin, C. B u s s , X. F. Kong, J. Carnazza, M. Goebl and M. Alexander-Bridges, Nature 354, 317 (1991). 86. R. M. O’Brien and D. K. Granner, BJ 278,609 (1991).

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127

87. P. Neely, M. R. El-Maghrabi, S. J. Pilkis and T. H. Claus, Diabetes 30, 1062 (1981). 88. M. J. Hamer and A. J. Dickson, BJ 269, 685 (1990). 89. P. Martin-Sanz, M. Cascales and L. Bosca, BJ 257, 795 (1989). 90. P. Bruni, P. Vasta and M. Farnararo, BBA 887, 23 (1986). 91. C. G. Rousseau, Y. Fischer, M. A. Gueuning, M. J. Marchand, X. Testar and L. Hue, Prog. Cancer Res. Ther. 35, 158 (1988). 92. R. Zamegar, M. C. D e Frances, D. P. Kost, P. Lindroos and G. K. Michapoulos, BBRC 177, 559 (1991). 93. L. Bosca, M. Mojena, J, Ghysdael, G. 6. Rousseau and L. Hue, BJ 236, 595 (1986). 94. M. J. Marchand, L. Maisin, L. Hue and G. G. Rousseau, BJ 285, 413 (1992).

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tRNA Structure and Aminoacylation €fficiencyI RICHARD GIEGI?, D. PUGLISI~ AND CATHERINEFLORENTZ

JOSEPH

Unit6 “Structure des Macromolicules Biologiques et Micanismes de Reconnaissance” Institut de Biologie MoUculaire et Cellulaire du Centre National de la Recherche Scient$fique F-67084 Strasbourg-Cedex, France

I. Structural Background ... ......................... A. tRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Noncanonical tRNAs and tRNA-like Structures C. Aminoacyl-tRNA Synthetases . . . . . . . . , . , . . . . . . . . . . . . . . . . , . . . . . . 11. Phenomenology and Early Structural Results . . . . . . . . . . . . . . . . . , . . . . . A. Functional Observations and Structural Implications B. Toward the Concept of Kinetic Specificity . . . . . . . . . . . . . . . . . . . . . . . C. Other Mechanistic Aspects . . . . D. Producing and Studying Altered 111. Complexes between tRNAs and Aminoacyl-tRNA Synthetases . . , , . . . . . A. Studying Complexes in Solution . . B. Crystallization and Functional C C. Three-dimensional Structures . IV. tRNA Identity for Aminoacyl-tRNA Synt . .. . .. ... .. A. General Considerations . . . . . , . B. In Vivo and in Vitro Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Major Positive Identity Nucleotides in Canonical tRNAs . . . . . . . . . . D. Mini- and Macro-RNA Substrates for Aminoacyl-tRNA

6. Evolution and Relation bet

. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .

131 131 142 147 149 149 151 155 156 159 159 163 164 166 166 167 169 180 186 189 190 192 195

1 We dedicate this review to the memory of Professor Jean-Pierre Ebel (1920-1992), who devoted his scientific life to understanding RNAs and RNA-protein interactions. H e stimulated and encouraged studies on tRNAs and aminoacyl-tRNA synthetases from both structural and functional viewpoints. Present address: Massachusetts Institute of Technology, Department of Chemistry, Cambridge, MA 02139-4307.

129 Progress In Nucleic Acid Research and Molecular B~olow,Vol 45

Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

130

RICHARD CIEGE ET AL.

Recognition and specific aminoacylation of tRNAs by aminoacyl-tRNA synthetases (aminoacid:tRNA ligases) is a key step that governs the accuracy of protein synthesis. The genetic code links DNA information content with its expression in protein structures; the tRN A-synthetase interaction is crucial for this expression. During translation, amino acids corresponding to the gene information are selected through the codon-anticodon interaction between messenger RNA and tRNA in a way that is independent of the nature of the amino acid charged to the tRNA (I).Charging of tRNAs by synthetases must be extremely specific, since errors in tRNA aminoacylation can lead to false incorporations of amino acids into proteins. To fulfill this requirement, nature had to solve an intricate problem of macromolecular recognition and of enzyme accuracy involving twenty different aminoacylation systems related by the chemical nature of the acylation reaction and the relative structural similarity among the various tRNAs and twenty synthetases. Certainly, structural elements in both the tRNA and synthetase mediate aminoacylation. These elements may be contiguous nucleotides in tRNA that would be recognized by a set of contiguous amino acids in the proteins. Because anticodon nucleotides already serve as code words to decipher messenger codons, it was soon proposed that they could also be involved in synthetase decoding (2). Generalizing this idea, the concept of a second genetic code was hypothesized (3-6a). This simplistic but conceptually important view is probably not correct for present day tRNAs; a more complex and likely scheme involves recognition of structural elements scattered over the entire structure of tRNA. Thus, synthetases would recognize chemical groups within a precise conformational hame of each tRNA. Whatever the case, one may define the identity of a tRNA or a synthetase by a set of structural elements, called “identity determinants.” As we show in this review, identity determinants within tRNAs are being deciphered. Because of the chemical similarity of aminoacylation systems, synthetases can recognize false tRNAs and amino acids. To minimize the deleterious effects of such nonspecific recognition, evolution has selected protection mechanisms. Structural elements (“antideterminants”) within tRNAs or synthetases may prevent incorrect recognition. In addition, competition and kinetic effects may also contribute to the overall specificity of the various aminoacylation systems; the balance between the concentration of tRNAs and synthetases would be essential for ensuring optimal specificity. According to this view, individual aminoacylation systems do not work at their optimal chemical efficiency, but work instead to assure optimal discrimination among the different aminoacylation systems. Such a balance may be perturbed under certain physiological or pathological conditions. Finally, it may be questioned whether evolution selected a unified solution to the specificity problem of tRNA aminoacylation systems throughout living systems, or if alternative solutions have been retained.

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131

The problem of tRNA aminoacylation has long fascinated molecular biologists. Numerous reviews have already covered the subject, from either enzymological, structural, or genetic viewpoints (4, 7-16). In recent times, the field has evolved rapidly. Many of these novel developments, such as those derived from the partition of aminoacyl-tRNA synthetases into two classes (17), from the description of crystal structures of tRNA/synthetase complexes (18-19a) or the concept of tRNA identity (20),have already been critically discussed (21-24b). Although of fundamental nature, research on tRNA aminoacylation finds extensions in applied fields. Correlations have been discussed at the phenomenological level between alterations in tRNA structure or function and cellular disorders such as cancer (e.g., 25, 26) or aging (27). Direct correlations have been discovered between human mitochondrial diseases and mutations in tRNA genes (e.g., 28,28a and reviewed in 29, 29a). Certainly, knowledge of the molecular functioning of tRNA will assist understanding of such pathologies linked to the most basic mechanisms of cellular life. In the present review we concentrate on the role of tRNA structure in the recognition process with synthetases and on the implications for aminoacylation efficiency. Many examples are taken from our own research on several specific aminoacylation systems (e.g., aspartate, histidine, valine), but concepts are presented more globally with reference to the complete set of aminoacylation systems. We also emphasize the importance of tRNA-like structures (30) for understanding the interaction of canonical tRNAs with synthetase. Although tRNA-like molecules found in some plant viral RNAs do not participate in protein synthesis, they represent interesting natural mutants to be compared to canonical tRNAs. This is also the case of tRNAlike structures found in some messenger RNAs (31) as well as of bizarre tRNAs from mitochondria (32-34). Finally, we compare recent results with previous observations, and show how old concepts established phenomenologically can now be tested more explicitly.

1. Structural Background A. tRNAs 1. SEQUENCES AND TWO-DIMENSIONAL FOLDING Transfer RNAs are a structurally homogeneous family of molecules folded in a "cloverleaf' secondary structure. Yeast tRNAA1"was sequenced in 1965 (35);now the sequences of more than 1700 tRNAs that originate from more than 130 organisms, including viruses, archaebacteria, eubacteria, eukaryotes, and their organelles are known, either from RNA or gene sequencing (36, 37). Complete sets of tRNAs from one organism, including at least

132

RICHARD GIEGg ET AL.

one isoacceptor species for each of the 20 amino acids, are known for several eubacteria (Mycoplasmu capricolum, Bacillus subtilis, Escherichia coli), yeast (Saccharomyces cereuisiae), and chloroplasts (Euglena gracilis, Marchantia polymorpha, Nicotiana tabacum) or mitochondria (Torulopsis glabra, rat) (37). Most of these sequences were deduced from gene sequencing. Their canonical cloverleaf fold comprises three stem-and-loop regions, a variable region, and a 3’ single-stranded NCCA end to which the amino acids become attached (Fig. 1). The stems corresponding to the different domains are the same length in all tRNA species; seven base-pairs in aminoacid-acceptor stems, five base-pairs in T-stems and anticodon-stems, and three or four base-pairs in D-stems. Many tRNAHlSspecies possess an additional 5’ “minus-1” nucleotide (e.g., 38) that sometimes base-pairs with the fourth 3’-terminal residue; in contrast, certain initiator tRNAs have the first base-pair in the amino-acid-accepting stem unpaired (e.g., 39). The absence of this 1.72 base pair is responsible for the initiator identity in prokaryotes, since a single base change at position 1 or 72 restoring a Watson-Crick pairing enables initiator tRNA to act as an elongator (40). Stem regions are also characterized by the frequent occurrence of noncanonical G*U base pairs that can serve as specificity signals in certain aminoacylation systems (41-43) (see Section V). Anticodon and T-loops (with the exception of some noncanonical tRNAs; see below) are seven nucleotides long in all tRNAs, whereas D-loops vary in length from four to more than ten nucleotides, and show variability in the location of the conserved G,, and G,, residues, making the c1 and p regions variable (37). In addition to these two Gs, 21 other residues are conserved or semiconserved in almost all sequences [U,, A,,, A,, (often G in leucine-specific tRNAs), U, G,,, T-, Vs5, C, A%, C,,, C,4, C,, and A,, for conserved residues and Y,,, R,,, R, ,,Y R3,, Y48, R5,, and Y, for semiconserved residues] (R = purine and Y = pyrimidine). Other tRNA residues are highly variable, although not random (44, 45). Finally, the length of the variable region distinguishes the two families of tRNAs, those of class I with short variable sequences of four to five nucleotides and class 11 with a variable region of ten or more nucleotides, as well as a 13-23 purine-purine conserved pair in the D-stem, instead of a classical Watson-Crick pair in class-I tRNAs (see Table I). This latter class comprises only leucine, serine, and prokaryotic tyrosine-specific tRNAs. tRNAs contain many modified nucleotides (46, 47). Some are common to almost all species, such as dihydrouridine in D-loops and ribothymidine in T-loops; others are characteristic of specific tRNAs, such as the hypermodified wybutosine residue (a derivative of guanosine, originally called Ybase) at position 37 in tRNAPhe or the queueosine derivatives (also analogs of guanosine, originally called Q or modified Q) at the first anticodon position

TRNA

133

STRUCTURE AND AMINOACYLATION EFFICIENCY

a i n o ac acceptor stem

D-stem

p region

w w

1

@-@ @--@ @-@

@

@

Variable region

Anticodon stem FIG. 1. Cloverleaf folding of canonical tRNAs emphasizing the different domains of the molecule as well as the position ofconserved and semiconserved residues (encircled in bold) and the tertiary interactions in which they participate. Tertiary interactions are indicated (special features of class-I1 tRNAs are indicated in Table I). Location of modified nucleotides is indicated (grey or hatched cirles); hatched positions are those where modifications present the greatest variability. The numbering system and the data are taken from the tRNA data bank (37).

of certain tRNAAsn,tRNAAsp, or tRNATyr species. The chemical structures of 53 such residues are known (37);a recent example is a nucleoside extensively modified at its ribose moiety, 0-ribosylphosphate-adenosine,which occurs at position 64 in the T-stem of yeast initiator tRNAMef(48). Another (mnm5se2U),the isolog of example is 5-methylaminomethyl-2-selenouridine

134

RICHARD GLEGE ET AL.

TABLE I VARIABLE TERTIARYINTERACTIONS IN THE CORE OF E . coli (a) AND S. cereuisiue (b) CANONICAL tRNAs, AND CONFORMATIONAL FEATURES OF D-LOOPS AND VARIABLE REGIONS Levin pau

tRNA0

15.48

a

B

V

GA

4

2

5

A C

4 3

2 3

5 4

A 4

4 3

3 3

5 5

A 4

4

2

5

AG @A

4 5

3 2

5 5

2644

W23.12)

da

Uul

45.(10.25)

(13.22P46

@A @A

AG

4

2

5

A.G

5

2

5

@A

4 4 4

2 2 2

5 5 5

AC

4 4

2 3

5 5

c*u

4

2

5

GG A*A A G

C1.u IIRNAs : c13 His(GUG)

G.C

CIm I1 tRNAs : A73 ~U(A*AA) (CAG)&(GAG)

A-U

A C

A.u

G G G

(C*G)

(UG) ( G C ) (CG) (GC) /U/C.GI /C-C)

G U

A.U

G.U A*C

(@A) (@A)

fUAGI&lCAAl

/A.AI

G

4 4 4

3 3 3

15 15 13

TYRGUA)

G-C

A€

U

(GG)

(UG)

(CG)

(@A)

U

3

4

13

Class 11 1RNAs : 673 SeRCGA) (GCU)&(GGA) (UGA)

0.C

A*A

GU AC

G G G

(CG) (W)

(AG) (A-A)

(C-G) (GA) (GRICIA) (@A) (G.C) (@A)

C

G.C G C

3 3 3

4 4 4

18 21/16 16

(CG) (CG)

(GC) (UG)

U U

(continued)

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STRUCTURE AND AMINOACYLATION

135

EFFICIENCY

TABLE I (Continued) ~

(b) Saccharomyces cerevisiae

CI- I tRNAr : A73 AINAGC) & (UGCJ

Gc

Gc Gc

4

2

5

3 3

2 2

4

3At

G.C

A*A

4

2

4

G.CS G.C

AG U-C

4 3

3 3

5 5

G.C.3 G.CS

G4.A G4.A

4 3

2 2

5 5

Gc

G4.A

4

2

5

GCS

G4.A

4

2

5

A-U

F-G

3

2

5

GCS

G4.A

4

4

5

GC G.C

A*A G2.A G4.A

4 3 3

3 3 4

5 5 5

AC A-C

A'A A.A

A-(C.G) A*(C-G)

G.(G.C) G.(C.C)

PUPA (U-Uj-A

3

3

4

3

3

4

GCS

G4.A

A-(A.U)

W(G2-C)

(CN3.07

4

2

5

Gc

LYs(U'Uu)

CG CG

= N4-acety1-C;CS = msC = 5-methyl :F = Y' = pseudo-U ; GI = mlG = 1-methy1-G : G2 = mZG = Nz-methylG: G4 = rn% = N2,NZdimethyI-C;G7 = mlG = 7-methyl-G ; U3 = Urn = 2'-O-methyl-U. N* = m d i i e d nucleotides ;A = residue 73 cat detemined ;A t is a missing nuclmtide in tRNA sequence. but C in gene sequence. For class II tRNAs. tertiary interactions m given according to the model of yeast tRNAW :e.g., no (13*22).46 and 903.1 2) triples. but freeresidue 47, (5'-adjacent to 48) and freeresidue 9. as well as base-pair (4547,.1) (71). The length of a and Li regions in D-loops. and of variable region (V)(see Fig. 1) are listed ;the conserved triple interaction of A21 with the U8-AI4reversed Hwgsteen pan and the WatsonCrick Gt9€56 pair are not included in the Table. Sequences w a e taken from the tRNA dab bank (37);data from gene sequences are in italics. a tRNAs are indicated by their amino acid specificity and anticodon. CI = a&

136

RICHARD GIEGE ET AL.

the sulfur-containing mnm5s2U found at position 34 of E. coli and other bacterial tRNAGIu and tRNALys (49). The seleno-tRNAs coexist with the thio-tRNAs; however, their biosynthesis appears to be a specific enzymatic process in which sulfur is lost and selenium incorporated (50, 50a). Modified nucleotides are located at 61 different positions in tRNAs, mainly in loop regions (Fig. 1).The number and amount of them in tRNAs increases with evolutionary complexity, and can be as high as 20% in tRNAs from higher organisms. Although many tRNA transcripts deprived of modified nucleotides retain their full aminoacylation ability (23),some modified residues, for example, a lysidine residue in the anticodon of a minor tRNA”“ species from E. coli (51) and modified residues in yeast tRNAAsp (probably m’G,,), are directly involved in the aminoacylation process (52) (see Section IV). Chemical fragility, due to the presence of the 2’-OH group on ribose, is an intrinsic property of RNA. Backbone cleavage is often observed at YpA sequences. It is dependent on the conformation of the RNA and is enhanced at alkaline pH or in the presence of divalent cations. This phenomenon has been observed in “footprinting” experiments of tRNA (53,s)and tRNA-like molecules (55) and within tRNA crystals (56-58). Although of small amplitude, these cleavages are intense enough to interfere with structural biochemical experiments, and most likely explain the difficulty in crystallizing tRNAs (59). Interestingly, cuts within a tRNA molecule do not necessarily hamper its aminoacylation ability (see Section 11,D), and in some cases can be induced by interaction with synthetases (60-64).This effect can detect conformational changes in tRNAs upon binding with synthetases, as in the glutamine system from E. coli (64). 2. THREE-DIMENSIONAL STRUCTURE a. Crystallographic Studies. The existence of only several crystallographic structures of elongator tRNAs at atomic resolution, all from Saccharomyces cerevisiae, refiects the difficulty in obtaining good tRNA crystals (59). The structure of tRNAPhe (65-70) shows the characteristic L shaped folding of the molecule with the amino-acid-acceptor CCA group and the anticodon at both ends of the Lfold and the T- and D-loops (Fig. 2A). This structure demonstrates the architectural significance of conserved and semiconserved residues of tRNA (37) (see Table I), including that of most conserved residues in class-I1 tRNAs (71), which cluster mainly near the junction of the arms of the Lconformation (Fig. 1). In addition to these constant elements, one finds variable nucleotides (residues 16, 17, 20, 59, and 60)clustering in the hinge region of the tRNA Lstructure. They form a variable pocket that could constitute a recognition site for synthetases (72) (Fig. 2A). This functional proposal was verified recently (see Section IV,C). A

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STRUCTURE AND AMINOACYLATION

EFFICIENCY

137

A

f-

f

FIG.2. Stereo-views of the three-dimensional structure of yeast tFWAPhe with positions constituting the variable pocket highlighted (A) and yeast tRNA*Sp (B) (courtesy E. Westhot). For original references, see Section 1,A.

forgotten study on the overall shape of tRNAs by X-ray small-angle scattering predicted an Glike structure for these molecules, long before the first Xray structure was solved (73). The structure of tRNAAsp(74-76) mimicks the conformation of a tRNA interacting with mRNA (Fig. 2B), as a result of the crystal packing, which involves anticodonlanticodon (GUC/GUC) interactions between two tRNA molecules. This interaction mediates conformational changes in the D-/Tloop region, in particular the opening of the G,,*C,, base-pair; solution studies with tRNAs forming dimers confirm this fact (58, 77). Another characteristic feature of tRNAAspis the different relative positioning of the two branches forming the Gshaped structure that are more open by about 10" than in tRNAPhe(74).It is not clear whether this opening is a consequence of the anticodonlanticodon interactions in the crystals or relates to sequence peculiarities of tRNAASp. The structures of the initiator tRNAs from E . coli and yeast are also known (78, 79) and, for the yeast species, the resolution has recently been

138

RICHARD GIEGE ET AL.

markedly improved (80). These initiators present Lshaped conformations resembling those of elongator tRNAs, which is not unexpected as they possess essentially the same frame of conserved residues. However, for the eukaryotic species, two special features are apparent: the cluster of tertiary interactions involving the conserved nucleotides A, A, and &, which are characteristic of eukaryotic initiators, and the hypermodificationat position A, that is placed in the minor groove and exposed to the solvent. These features may represent molecular signals that prevent elongation. This viewpoint was verified with a wheat-germ initiator tRNA, which could serve as an elongator tRNA upon removal of the modification at position 64 (81).

b. NMR Studies. Nuclear magnetic rgsonance provides insights into both the structures of tRNAs and their dynamics. A large number of NMR studies of tRNA have been performed (82,83).These studies have generally made use of resonances that arise from exchangeable protons attached to nitrogen. These protons normally exchange rapidly with solvent, but are protected from rapid exchange upon base-pair formation. Complete assignments of exchangeable proton resonances have been made for certain tRNAs, like yeast tRNAPhe(84) or tRNAAsp(85). These studies have shown the presence of a complete set of both secondary and tertiary pairings for tRNAs in solution. The exchange properties of these protons is correlated with the local kinetics of base-pair opening. Upon heating, base-pairs are disrupted in different regions of the tRNA at different temperatures. This allowed characterization of the thermal unfolding of tRNAs (84). In general, tertiary structure and D-stem base-pairs are disrupted at the lowest temperature; other stem regions denature at higher temperature. Studies have recently been performed on tRNA transcripts that lack modified bases; these transcripts have a lower thermal stability than their modified counterparts (86, 87). The NMR spectrum of yeast tRNAPhetranscripts show that similar tertiary interactions form, but the resonances are generally broader for the transcript than for the modified tRNA (88), which suggests a more labile structure. This view is in agreement with investigations on thermophilic tRNAs indicating that the high temperature stability of these molecules is partly due to the presence of methylated and thiolated nucleotides (89-91). It agrees also with the observation that posttranscriptional modifications rigidify the conformation of specific pyrimidine residues in tRNA ( 9 1 ~ ) . The effects of adding ligands can be monitored by changes in the NMR spectrum. Addition of Mg2+ significantly changes the NMR spectrum of the yeast tRNAPhetranscript, indicating some change in structure of the tRNA (88). Modified nucleotides offer convenient spectroscopic markers that can easily be observed in the NMR spectrum (92). Changes in anticodon en-

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vironment have been observed upon addition of oligonucleotides that correspond to the codon (93). The availability of isotopically labeled tRNAs should extend the NMR technique. Labeling of tRNA with 15N has been performed (94, 95). 19Fhas been incorporated into tRNAValas 5-fluorouracil(96). Single I9F resonances are observed for each uracil, and these can be used as probes for changes in conformation of regions of the tRNA, in particular upon addition of synthetase. Experiments in which tRNAs are labeled with 13C may allow the acquisition of more detailed NMR data on tRNA structure (97). Labeling will allow the application of multidimensional NMR methods so that the large number of proton resonances can be resolved. The availability of labeled tRNA molecules will be extremely valuable in the study of complexes with aminoacyl-tRNA synthetases, since NMR of these complexes is hampered by the large sizes of the tRNAs and synthetases. The large number of residues contributed by a synthetase could selectively be filtered out so that only those of the tRNA would be observed. Thus conformational changes of the tRNA can be characterized (96, 98). c. Other Solution Studies. Structural data on tRNAs can be obtained by computer modeling using data derived from chemical mapping in solution on the unknown tRNAs. Models incorporate known structural motifs from crystallographic or NMR studies. Three chemical probes, whose reactivity can be correlated with the known crystallographic structures, are particularly useful: (i) ethylnitrosourea, a phosphate-alkylating reagent (53, 99); (ii) dimethylsulfate (100, 101), specific for the N, of G and the N, of C residues; and (iii) diethylpyrocarbonate (100, 101) specific for the N, of A residues. Other probes, specific for standard or modified bases that can map the surface accessibility of tRNA, are also useful (11, 102, 103). Using the chemical mapping approach, a model for the three-dimensional structure of yeast tRNASer, a tRNA of class I1 with a large variable region, has been proposed (71). In this model, the variable arm is not far from the plane of the common Lshaped structure. Similarly, conformational features of: tRNAG1",elongator tRNAMet,tRNAPhe(99), initiator tRNAfMef(99, 104) and tRNAThr (105) from E. coli; of yeast tRNAPhe and tRNAVa'; of beef tRNATrp (61); and of a series of tRNAL'" species (62) were obtained by ethylnitrosourea probing. These experiment showed in particular the intrinsic conformation of the T-loop that exists when it is removed from the context of the interdigitated D-loops (106). Mapping of Watson-Crick positions (107) as well as the recent development of photoactivable probes (108, 109) will be useful for better probing tRNA structures. Polyamines derivatized with an aryldiazonium group will target helical regions (108) and transition metal complexes will probe tertiary structures (109).

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Rapid assays of tRNA conformation are often needed in studies on tRNA variants. Pb2 ions preferentially cut the ribose-phosphate chains in regions near its binding site (56, 110, I l l ) , and conformational differences between variants of tRNAPhe and tRNAASptranscripts (112, 113) or modified and unmodified tRNAAspmolecules have been detected (87). In particular, the lead cleavage patterns of yeast tRNAPhe with or without myosine differ (111). Structural mapping of these tRNAs by the rhodium complex Rd(phen),phi3 yielded similar conclusions (109). Lead cleavage was used recently to investigate the folding of circularly permuted tRNAs; the results suggested that tRNA folding motifs can occur internally within other RNA sequences (114), a conclusion consistent with the finding of tRNA-like domains within mRNAs (see Section 1,B). Using an in uitro selection method, it was possible to obtain two tRNAphe analogs that undergo, as canonical tRNAPhe, autolytic cleavage by Pb2+ ions at U,, (115). These “pseudotRNAs” are lacking the two conserved G,, and G,, residues but present D-T loop interactions, and thus may be relevant models for the structure of mitochondria1 tRNAs lacking G,, and G,, (115). tRNA conformation and dynamics in solution have been monitored by many biophysical methods other than NMR (116-118). The direct demonstration of the similarity between crystal and solution structure of tRNA came from a comparative laser Raman spectroscopic study on yeast tRNAPhe in the crystalline state and in aqueous solution (119). Raman spectroscopy (120)and recent electro-optical measurements (121)indicate modulations in the tertiary structures of tRNAs. Related to function is the question of tRNA conformation upon aminoacylation. Large conformational changes have been proposed in the past on the basis of light-scattering experiments (122)but reexamination of the problem emphasizes the tendency of tRNA to aggregate (123). +

+

d , Architectural Correlations. The network of tertiary interactions in the core of tRNA responsible for conformation includes most conserved residues found in tRNAs as well as semiconserved and variable nucleotides (Fig. 1and Table I). Thus, even if all tRNAs possess the same overall threedimensional Gshaped structure, this variability implies the existence of subtle conformational differences among them. This variability can lead to changes in the relative positioning of tRNA domains (e.g., anticodon region vs. CCA end or D-loop vs. T-loop) and consequently to different presentations of nucleotides to synthetases or other proteins (see Section IV). It is therefore interesting to search for tRNA families that present the greatest conformational similarity (or difference) on the basis of sequence correlations between residues involved in tRNA architecture (see also 124). Such structural correlations may be found in Table I for E. coli and yeast tRNAs.

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Because the aim of this review is discussion of the relationship between structure and aminoacylation function, we have also classified tRNAs according to a sequence feature, the nature of the discriminator residue 73 that is correlated with aminoacylation (see Sections 11, 111, and IV), but clearly not with tRNA architecture. Class-I and class-11 tRNAs differ from each other by the way the variable region is integrated within the tRNA architecture. This leads to differences in the (13*22).46triple that is essential in class-I tRNAs and cannot form in the class-11 species (71).Interestingly, the 13.22 pair is exclusively a purine. purine pair in class-I1 tRNAs, while it is a predominantly a classical WatsonCrick pair in class-I tRNAs, with the exception of a few tRNAs (e.g., E . coli tRNACys and tRNAGln, and yeast tRNAAsn, tRNAThr, and tRNATyr) that probably possess cryptic class-I1 sequences (notice the case of tRNATyr which is class I1 in E . coli and class I in yeast). Within class-I tRNAs, the strongest correlations appear among species with A,3, especially in E . coli. Moreover, when comparing tRNAs with a = 4, p = 2, and V = 5, it appears that they have essentially the same network of tertiary interactions [G15*C48; t$-(A,,-U,,); G,,*(GloC,,); and (C,,.G,,).G,,]. The length of alp sequences in the D-loop modulates the positioning of this loop respective to the T-loop via the G,,*C,, constant tertiary Watson-Crick pair. Interestingly, one finds in yeast tRNAs with OL = 4, p = 2, and V = 5, a quasiconserved modification pattern in their conserved architectural nucleotides, namely mZ2G,,, m2G,,, and m7G,,; probably their common conformation represents an identity element for the modification methylases. It is not yet well understood whether these structural features are related to aminoacylation function, or just reflect evolutionary relationships between tRNAs. Several observations seem to favor the view that sequence variability in tertiary interactions is unrelated to aminoacylation. For instance, in vitro transplantation of such interactions does not affect charging of yeast tRNAPhe (125), and functional tRNAA1"species can be selected in vivo that possess alterations in architectural nucleotides (126) (see Section IV, E). Noticeable, however, are the correlations between structural frame and (i) mischarging properties of tRNAs (see Table 111), or (ii) efficiency of identity element transplantations (see Table VII and Section IV, E). In conclusion, it appears that compensatory sequence effects or conformational changes during function may minimize the intrinsic effects of a variability in the structural frame of tRNAs. This variability, however, has to be taken into account when engineering new tRNA molecules or when trying to decipher the evolutionary relationship between tRNAs (see Section IV, H). Finally, it is not excluded that, in some tRNAs, nucleotides belonging to the tertiary network are identity elements, as already found for the Gl,*Uz5 basepair in yeast tRNAAspthat belongs to the 45.(10.25) triple (43)(see Fig. 12).

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B. Noncanonical tRNAs and tRNA-like Structures 1. AMINOACYLATABLE STRUCTURES

tRNA structures that vary considerably in both their overall size (sometimes less than 60 nucleotides)and the sequence of D- and T-stems and loops (that can be lacking) have been found in the mitochondrial genome of metazoa, especially in those of nematode worms (33,34,127) (Fig. 3A, B) and of the harbor seal (127a).Some of these tRNA genes have been be transcribed, and transcripts are of the same size as the respective genes (34).As they are difficult to isolate, very little is known about the biochemistry and conformational properties of these bizarre tRNAs. However, in the case of a bovine heart mitochondrial tRNASerlacking the D-arm (Fig. 3C), only the mitochondrial synthetase, and not those from E- co2i or yeast, charge this tRNA (32). A three-dimensional model of bovine and human mitochondrial tRNASer mimicking canonical tRNA based on chemical modification data has been

B

A A A -T C-G A -T G-C A -T T -T

C

T A-T A-T A-T C-G T -A A-T T-G -T I

T A

GAGGT

____ ___ -.TT A .-----T

A ---.T T -G A-T T -A G-C T-A T A T A TG A

A C C G G-C A-U A-U A-U A-U A-U GAu G-FFylp$I A C ~ C A U A ~ ~ ~ A

2

uG________cu C C---- - G A- UA A- U G- C

A- U

CA- 'A 'G

uA7

FIG.3. Three selected examples of bizarre mitochondrial tRNA sequences from nematodes (obtained from gene sequencing) (34) (A, B) and bovine heart (128) (C). These sequences emphasize structural alterations that have been found in mitochondrial tRNAs as for instance a missing T-arm in tRNATrp (57-nt) from Caenorhabditis ekgans (A), a missing D-arm and an unconventional T-arm in tRNASer(53-nt) isoacceptor from C. ekgans (B), and a missing D-arm as well as an 8 n t T-loop in bovine heart tRNASer (C). Notice the classical size of the amino-acid-accepting stem (seven base-pairs) and of the anticodon arm (five base-pairs and a 7nt loop). Notice also the high content of A and U (or T) residues in these tRNAs, which is characteristic for mitochondrial tRNAs in general. Putative base pairings in B and C are indicated by dashed lines. A7 = t6A = N-[(9-B-~-ribofuranosylpurine-6-yl) carbamoyl] threonine; C5 = m5C.

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proposed (129). Similarly, using enzymatic probing, a novel cloverleaf fold(130)characing was proposed for mammalian mitochondrial tRNASer(UCN) terized by a unique nucleotide between the canonical acceptor and the D-stems, an unusually short D-loop and variable region of five and three nucleotides, and a six-base-pair anticodon stem. Unexpectedly, this structure, supported by experimental evidence, differs from the more classical folding that was proposed after simple inspection of the gene sequence; this emphasizes the necessity of experimental verification of secondary structures of unusual RNAs. For bovine mitochondrial tRNAPhe lacking the canonical sequence features of D- and T-loops, thermal melting profiles suggest a less-stable higher-order structure, presumably due to altered or absent D/T-loop interactions (131). Selenocysteine-inserting tRNAs (tRNASec)have recently been found in many species (132, 133). These tRNAs are recognized and charged with serine by SerRS. (RS = RNA synthetase.) Serine is then converted to selenocysteine while on the aminoacylated tRNA and is incorporated in particular proteins at positions coded by a UGA stop codon in the mRNA (134). Their structure deviates from that of canonical serine-specific tRNAs. The selenocysteine tRNA from E. coli contains 95 nucleotides (135) and is the longest tRNA known to date, with an acceptor stem of eight base-pairs and a 22-nucleotide-long variable region; other peculiar features of this tRNA are the distribution of the conserved residues that govern the three-dimensional folding of tRNA and the presence of a hypermodified i6A residue adjacent to its UCA anticodon (Fig. 4A). These structural differences probably explain the 100-fold reduced aminoacylation activity of this molecule as compared to canonical tRNASer (137). Selenocysteine-inserting tRNAs have probably been optimized for the recognition by the specific enzymes involved in selenocysteine biosynthesis rather than for recognition by SerRS. Threedimensional models based on solution probing have been proposed that emphasize their peculiar structural features (137u,b). Another tRNA not involved in protein synthesis is Staphylococcus epidermidis tRNAC'y, which participates in cell-wall synthesis and does not possess the characteristic D- and T-loop features of classical tRNAs (136) (Fig. 4B). Interestingly, its sequence features are found in the D-loop equivalent domain in the TYMV tRNA-like structure (59, also not involved in protein synthesis (138). Although the 3'-ends of viral tRNA-like molecules can be aminoacylated efficiently by certain synthetases (see Sections I1 and IV), they lack several structural features of canonical tRNAs, such as strategic D- or T-loop sequences or conserved residues, and modified bases. Furthermore, they often present additional stem-and-loop structures and consequently cannot be folded into a canonical cloverleaf (Fig. 5). Despite these apparent differences in secondary structure, the tRNA-like molecules probably contain

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RICHARD GIEGE ET AL. 3’

3’

A

B

E 5'

G-C C-G G-C 70

G-C G-U

30 U-A 40

C U

C-G

G

u ccc

FIG.4. Cloverleaf structures of E . coli selenocysteine-inserting tRNASeC (135) (A) and bacterial tRNAG'y from S . epidermidis involved in cell-wall synthesis (136)(B), emphasizingthe noncanonical features of these molecules (in bold and boxed for the extra base-pair of tRNASeC [notice also the extralong variable region and two additional base-pairs in the D-loop of this tRNA (I37u)l. A4 = PA; D = hU; F = ? = pseudo-U; T = m5U; U4 = S ~ U ,

structural domains mimicking tRNA conformations that are recognized by the aminoacyl-tRNA synthetases. For the simplest tRNA-like structures, turnip yellow mosaic virus (TYMV) RNA and the related tymoviral RNAs, a three-dimensional conformation mimicking tRNA was proposed that contained, in the amino-acidaccepting arm, a new RNA folding principle, the pseudoknot (139)(Fig. 5A). A pseudoknot involves base pairing between residues in a hairpin loop with a single stranded region of the RNA that gives rise to a quasihelix mimicking an RNA double-helix (140-142).Chemical mapping combined with melting and NMR studies gave support to this model, and showed in addition the great sensitivity of this structure to solvent conditions (143,144). Pseudoknot foldings have been found in a variety of other plant viral tRNA-like molecules (30, 145, 146);they are an original alternative conformation for the interaction with components of the translation apparatus (147,147~). The pseudoknot structure is central to the structural mimicry of tRNAlike molecules. The amino-acid-accepting arm of the TYMV tRNA-like domain contains 12 stacked base-pairs as in canonical tRNA despite the fact that this structure is constructed with the 3'-end sequence of the tRNA-like domain and integrates the pseudoknoted conformation (141)(Fig. 6). More complex three-dimensional foldings have also tentatively been proposed for

A

B

C

CCCA 3‘ A GGGG UU CG U

4

u U U G A

I

C- G AG-CC G- C I A- U G-C GAU U __ 5’ G UU-A C-G U-A G-C U-A C -G

c

c

-

GAAA u U -A G-C U -A A-U U -A A-U U A

U U Ac

GG u U C

FIG. 5. Secondary and three-dimensional folding of several tRNA-like structures found at the 3’-end of plant viral RNAs from TYMV (A), TMV (B), and BMV (C) RNAs; for original references, see Section 1,B.

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RICHARD GIEGE ET AL.

FIG. 6. Computer model stereo view of the tRNK-like molecule from TYMV RNA based on chemical mapping experiments (141).The model corresponds to the 86 last nucleotides of the viral RNA (courtesy E. Westhof).

tobacco mosaic virus (TMV) and brome mosaic virus (BMV) tRNA-like domains (30, 145, 148) (Fig. 5B, c).The different domains in the BMV structure must still be defined (see Section 11,D). The 12 base-pair stack in the amino-acid-acceptor arm is not an absolute requirement for the interaction of tRNA-like molecules with synthetases, since the TMV tRNA-like structure charged by HisRS contains only 11 base-pairs (Fig. 5B).

2. NONAMINOACYLATABLE STRUCTURES tRNA-like structures occur in the regulatory region of certain messenger RNAs, such as the attenuator of the E. cali histidine operon (149), and the regulatory region of E. cob ThrRS (150) and MetRS mRNAs (151). The folding of the tRNA-like domain of ThrRS mRNA was established on the basis of chemical probing experiments (152). Despite its poor overall homology with tRNAThr (Fig. 7), this region of mRNA mimicks tRNAThrat the three-dimensional level, since it interacts specifically with ThrRS (153, 154). This property is responsible for the regulation of the ThrS mRNA. The mimicry of this structure with canonical tRNA was elegantly confirmed after mutation of the putative threonine identity positions in its pseudoanticodon, which resulted in a loss of translational control (153). Certain group-I introns are autocatalytically spliced in uitro. However, efficient splicing in vivo requires the presence of proteins. Splicing in uitro of a mitochondrial rRNA gene from Neurospora crassa requires TyrRS (155, 156). Similarly, yeast mitochondrial LeuRS is involved in RNA splicing (157). This suggests the presence of a tRNA-like domain within these introns that interacts with these synthetases (158). Finally, tRNA-like structures have been discovered in the short in-

TRNA

147

STRUCTURE AND AMINOACYLATION EFFICIENCY

C A A A U A A U A U A G A G

-50-

U U

-1.20

U

I

L G

c

u- A u U-A U-A G-C U - G A-U

u

-90

u c c A u G UGU u u A c--.

5'

h h b bJ A !I A A A I I I ....coding region of ThrRS....3’ A

4"= +1

C CA--20

G -C U-A -40-G -C A-U U - G C-G u- G

u

u

FIG. 7. Secondary folding of the tRNA-like structure present in the regulatory region of the messenger RNA of E . coli ThrRS (153).Numbering of the RNA is relative to the initiation codon AUG. Sequence features specific to E . coli tRNAThrare boxed.

terspersed repeated sequences that occur in eukaryotic genomes (159). For instance, sequences that can be folded in the classical tRNA cloverleaf and that share a strong homology with tRNALys have been found in the human Alu-family (160). Whether the transcription products of such sequences interact with synthetases or with other tRNA-recognizing enzymes remains unknown. The existence of tRNA-like structures has also been suggested in cis-acting elements involved in the replication of enterovirus and rhinovirus RNAs (161).

C. Aminoacyl-tRNA Synthetases Aminoacyl-tRNA synthetases are functionally homogeneous but structurally extremely heterogeneous with sizes ranging in E. coli from 51 kDa [CysRS (162, 163)]to 384 kDa [AlaRS (164)]; these correspond to a,a2,a*,

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RICHARD GIEGE ET AL.

or azpzquaternary structures, and polypeptide chain-lengths from 334 amino acids in TrpRS (165) to 1112 in PheRS (166).Their properties have been extensively discussed (9, 15, 22, 267-169). Here we recall only their partition into two separate classes (17) (Table 11).This partition reflects two different sequence organizations with two types of signature motifs: the HIGH (167) and KMSKS (170) sequences in class-I synthetases and three characteristic motifs with rather degenerate sequences in class-I1 synthetases, but containing one conserved arginine residue in motifs 2 and 3 (17)that contribute to the formation of the catalytic cavity in yeast AspRS (19). The separation of synthetases into two classes is also reflected at the three-dimensional (see Section III,C) and mechanistic levels (see Section 11,C) (the distinctions of class-I and -11 synthetases are not related to the distinctions of class I and I1 tRNAs; see Section 1,A). As found by X-ray crystallography in B . stearothennophiZus TyrRS (171, 172), E . coli MetRS (173), and E. coli GlnRS (18),all class-I enzymes probably possess a classical Rossman-like nucleotide binding domain and charge tRNAs on the 2'-OH position of the terminal CCA ribose (174). Class-I1 synthetases have a different ATP-binding domain, as observed in E. coli SerRS (175) and yeast AspRS (19), and charge amino acids on the 3'-OH position of terminal ribose (see TABLE 11 PARTITIONOF AMINOACYL-tRNA SYNTHETASES INTO Two CLASSES~ Class I

Class

HIGH + KMSKS sequences "minorgroove rtxognition"b

Motifs 1.2 and 3 "majorgroove recognition"

ArgRS (a)2'-0H CysRS (a)2'-0H GlnRS (a)2'-OH GluRS (a) .?'-OH IleRS (a)2'-OH LeuRS (a)2'-0H

AlaRS (Q) 3'-OH AsnRS (a21 3'-OH AspRS (a21 ? GlyRS (a2$2)3'-0H HisRS (a2)3'-OH LysRS (ad 3'-OH PheRS (a2p2) 2'-0H ProRS (a2)3'-OH SerRS (a2)3 ' - 0 H ThrRS (az)3'-OH

MetRS (a2) 2'-0H TyrRS (a2) ? TrpRS (a2) 2'-0H ValRS (a)2'-OH

II

"The table indicates the oligomeric structure of the synthetaes and the site of aminoacylation on the 3'-terminal ribose. Data are adapted from (17); bas found in the structure of the glutamine and aspartic acid complexes (see Section 111,C); 'only motif 3.

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Section II,C). The different folding topology in synthetases leads also to different interaction patterns with the tRNAs (see Section 111).This partition may reflect an early evolutionary divergence among synthetases (17, 176180b).

II. Phenomenology and Early Structural Results A. Functional Observations and Structural Implications 1. TRNA MISCHARCINC

Both the activation of amino acids (13) and the recognition between tRNAs and aminoacyl-tRNA synthetases (181) are not very specific, and significant mischarging of many tRNAs can be catalyzed by a variety of synthetases (182-186). As examples, Table 111 lists the tRNAs that can be most easily mischarged by several synthetases of various origins. (Here we consider only those mischarging reactions involving misrecognition of the tRNA and, accordingly, where the activation of the amino acids is specific.) Mischarging reactions were first found mainly in heterologous systems in the presence of such particular reaction conditions as acidic pH, low ionic strength with high Mg2+ /ATP ratios, and sometimes in the presence of such organic solvents as ethanol or dimethylsulfoxide. However incorrect aminoacylations have also been detected in homologous systems and in the presence of standard assay conditions, but mischarging levels were generally lower (184, 193, 194). Consequently, it was proposed that structural correlations exist among tRNAs recognized and charged by the same synthetase and that the correlations would be most meaningful if these tRNAs and synthetases originated from the same organism. Following these lines, sequence correlations between tRNAs were proposed. For instance, the tRNAs most easily mischarged by a given synthetase have essentially the same discriminator base at position 73 (184, 195) (see Table 111); also such comparisons suggested the importance of anticodon residues for valine charging (185). However, interpretation of mischarging data must proceed with caution. Strong sequence homologies between tRNAs aminoacylated by the same synthetase do not necessarily reveal recognition or identity sites. Homologies in the Dstem exist among tRNAs mischarged by yeast PheRS (see Table 11) that cannot be responsible for phenylalanylation since yeast tRNAMet3,which possesses the same features, is only poorly mischarged by PheRS (196). The above mischarging reactions were all detected in uitro. Such reactions can occur in uiuo, as for instance on suppressor tRNAs (see Section 11,D and IV,B) or can be catalyzed by mutated synthetases (197, 198). In-

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TABLE I11 TRANSFER RNAs MOSTEASILYMISCHARGEDin Vitro BY SEVERALAMINOACYL-tRNA SYNTHETASES N73

(cognate RNA)

Syntherases

mischarged tRNA speciesa spsinc for aq, with (N73)

aha ag-nu

agmmw

S.a(E.coliJ (G)> G&-3

ArgRS fE.srear0.J ad

references

(E.coliJ 0.

(187)

G>A>X

0.

(52)

ArgRS (yeast)

G.C

AspRS (yeast)

G

always G

His (E.coliJ (0 > Glu (E.coliJ (G) > Gln (E.coliJ (G) > Ser (E.coliJ (G)> Asn (Exofij (G)> ._.

GLnRS (E.coliJ

G

G>bU

(t)$&z

SerRS fB.stear0.J nd

&(yeast)

always G

>a (E.coliJ (A) = &-I

(Ec&J (0)

= k - 3 (Ecoli) (G) > -2

(EcoliJ (A).

Lkvl (E.cdiJ (A) > L e y 2 (E.cofiJ (A).

f1W

(E.coli) (A)

(186)

(1871

VaES (i3.steam.J A

ValRS(yean)

A

When underlined. experiments were conductedon pne tRNA @es 20%dimethyl sulfoxide

;(t)detectablemischargingonly in the presence of

terestingly, some wild-type synthetases mischarge tRNAs, as is the case for barley chloroplast G h R S that efficiently aminoacylates both the homologous tRNAC1" as well as tRNAC1" species (199, 200). Similarly, GluRS in some Gran-positive bacteria mischarges tRNAG1" with glutamate, which is subsequently converted to Gln-tRNAGIn (201).

2. AMINOACYLATIONOF VIRAL TRNA-LIKEMOLECULES The genomic RNAs of a number of RNA plant viruses can be aminoacylated at their 3'-CCA termini by host or heterologous aminoacyl-tRNA synthetases (138, 202): TYMV RNA is valylated (203, 204), BMV RNA is

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tyrosylated (205, 206), and TMV RNA is histidinylated (207, 208). These tRNA-like structures (209)show the limitations of sequence comparisons and highlight the importance of three-dimensional structure that would bring recognition signals of the RNA into a proper orientation to be specifically recognized by synthetases. Furthermore, the studies on tRNA-like structures naturally deprived of modified nucleotides first indicated that these residues are not major elements for specifying aminoacylation in the three systems (histidine, tyrosine, and valine) involving these RNAs.

B. Toward the Concept of Kinetic Specificity 1. THERMODYNAMICS OF TRNABYNTHETASE INTERACTION The thermodynamic stability of complexes between tRNAs and aminoacyl-tRNA synthetases has been characterized by techniques such as fluorescence and filter-binding assays. The binding constants for the interaction of a tRNA with its cognate synthetase is generally in the range of 106-1@ M-' (12);these affinities are much lower than for complexes of many sitespecific DNA-binding proteins with their recognition site. This relatively weak binding affinity facilitates the turnover of tRNA in the aminoacylation process. The discrimination among cognate and noncognate tRNA molecules through binding affinity is rather weak; noncognate tRNAs can have binding constants only Q to that of their cognate tRNA, even though the noncognate tRNA is poorly aminoacylated (210). Thus, kinetic discrimination dominates in the aminoacylation process. Like most protein/nucleic acid complexes, the a f h i t y of tRNA-synthetase interactions depends strongly on solution conditions. Affinities are increased at acidic pH values [e.g., at pH 5.2-5.5 (181, 211)], but both thermodynamic and kinetic discrimination are worse at these pH values. Thus, lower p H favors both nonspecific binding of tRNA to synthetase, as well as misacylation. However, higher specificity occurs in some cases at more alkaline pH values [as in the yeast phenylalanine system (190)] under conditions where synthetases exhibit their catalytic optima [e.g., as in the glutamine, glutamate, and valine systems (212-214)]. The salt dependence of tRNA-synthetase a n i t i e s emphasize the electrostatic nature of the interaction. An estimate indicates that electrostatic interactions contribute 4060% of the total energy of interaction in the complexes with yeast tRNAVal and tRNAPhe (215). However, complexes are stabilized by high concentrations of ammonium sulfate (216, 217), suggesting the existence of additional hydrophobic interactions (see Section III, B). For tRNA-synthetase complexes that have been studied in detail, complex formation is generally entropy-driven (210, 218). Measured enthalpy values are close to zero, but will probably be strongly temperature-depen-

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RICHARD GIEGE ET AL.

dent. This probably results from solvent release upon interaction of charged residues and removal of hydrophobic residues from the interaction surface (219). Favorable energetic contributions are used to drive unfavorable processes necessary for specific recognition of tRNA, such as conformational changes of the tRNA. Similar behavior has been observed for DNA/protein interactions (220) and it will be interesting to compare the thermodynamic properties of DNA- and RNA-protein interactions. Noncognate complexes are characterized by a strong positive enthalpy (210, 218). This was explained by an alteration in the postulated two-step interaction mechanism. While the first step remains diffusion-controlled as in cognate complexes, the second one is modified and no longer involves the conformational change of tRNA required for specific interaction (218, 221). This phenomenological scheme is supported by recent footprinting data on yeast tRNAAspmolecules interacting with AspRS that indicate the existence of conformational changes within wild-type tRNA upon complexation and their absence in complexes with variants mutated at identity positions (222) (see Section 111,A). Despite this qualitative view of tRNA/synthetase interaction, more rigorous thermodynamic studies are needed. The availability of a large number of sequence and structural mutants of tRNAs, as well as more advanced techniques for measuring thermodynamic stability should allow a more detailed understanding of binding specificity of tRNA and synthetase. 2. KINETIC EFFECTS Comparison of steady-state kinetic (Michaelis-Menten) parameters for canonical aminoacylation reactions with those of many mischarging reactions shows in almost all cases a predominant effect on the catalytic rate constant k,,, (or on Vma) (185,189,190,192,223) (Table IV). Whereas an increase in K, values could be 10- to 60-fold, the effects on aminoacylation rates were normally 102- to 104-fold more important. This results in a reduction of catalytic activity (kcat/&) for mischarging reactions by factors of 102 to 3 x 105; this represents AAG3 values of about 3 to 7 kcal/mol. Consequently, specific tRNA aminoacylation reactions are governed more by kinetic effects than by the binding of tRNA to synthetase (184, 224). Interaction between a tRNA and a synthetase first involves global recognition, mainly governed by nonspecific electrostatic interactions, followed by a second step that allows the perfect adaptation of the two macromolecules in order to favor the activation step of the aminocylation reaction. This mutual adaptation must be dependent on structure and sequence, and its effects are more reflected at the kinetic level (transition state) than on AAG = AG (wild-type reaction) minus AG (mischarging reaction).

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TABLE IV KINETIC PARAMETERS OF SEVERALHETEROLOGOUS AND MISCHARCINC REACTIONS OF PUREtRNA SPECIES tRNA wgaate

resctian

V-

Km

VmKm

(relative)

(rclauve)

(reMve)

100

1

Ar&xyl-tRNA synihttase

Asp(yeast)

(S. cerevisiae)

0.09

12

Phcnylnlanyl-IRNA synthetarc (S.

1

1

nlerrnas

Mgz+IATP = 6 ;Tris-Hc1 pH 7.5

7.9~10.'

cerevisine)

L

12700

(52)

Mg2+/ATP = I6 ,Tns-HCl pH 7 5

Phe (wheat germ) 118 0.7 0.6 1.1 20 7.0 0.028 Phe (E.coli) 35 Val-1 (E.coliJ 10 4.5 45 Ala-I (E.coliJ 4.9 5.1 (190) 104 Val-2(E.coli) 138 1.3 1.8 f-Met (E.coli) 174 0.69 1.2 219 0.73 1.6 Ile (E.coli) 0.0041 Ala-2 (Scoli) 241 1.7 4.1 0.0040 252 2.5 6.3 Lys (E.coli) .,.,,,,..,.,..,....,,....,,,,,...,.....................,....,....,..................,...............,.....,...,,....,,........................... MgZ*/ATP = 16 ;umdylllc pH 6.0

Val-I (E.coli) Lys (Exoli) f-Met (E.coli)

19.8 206 28

3.5 5.1 10.1

5.7 0.4 0.03

11.7

6.67~10.'

30

0.34

0.17 2.5 36

(190)

MgZ+/ATP = 1.3 ; MES pH 7.0

Val-l (E.coli)

0.78

1500

(223)

MgZ+/ATP = 1.3 ;MES pH 5.5

Val-1 (E.coli)

292

3

(223)

M ~ ~ + / A T=P15 ;T ~ ~ ~ - H p~C 8.5 I

Met-3 (yeast) Val-1 (E.coli)

1.54 14.4

VdyI-IRNA qnthetnat

Val (E.coli) Val(yeast) Phe (yeast) Meti (yeast) Ile (E.coli) f-Met (E.coJiJ

1.25 1.00 2.58 2.33 1.75 1.25

0.58 0.25 0.0082 0.0047 0.024 3.7~10'

13 12

2.4x10-' 1.8110-5

(E. coli)

0.031 0.022

178 29

(1%)

1.7 4 I22 209 414 2710

(185)

Mgz+/ATP = I5 ;TrkHCl pH 8.5

Vnlyl-IRNA aynthetaae (S.

cerevisine)

0.5 5 0.02 0.01

58 63 12

Meti (yeast) Ala (yeast) Phe (yeast)

0.0056 0.034

(8. stearothermophilus) Mg2+/ATP = 3 ;HEPES pH 7.6

72 25 2.1 1.1 0.43 0.05

VIIyI-1RNA synthrtsar

Ala (yeast)' Phe (yeast)

2.75 4.2

41700 55000

(192)

Mg2+/ATP = 1.55 ;TricHCl pH 7.5

9.5a10-' 3.2~10.~ 8.3~10-6

10500 31500 12oooO

(189)

All aminoacylationswere done at 3OOC.* Aminoacylation with 20% dimethyl sulfoxide L is the loss of catalytic activity defined as the inverse of (V-K&ell.

154

RICHARD CIEGE ET AL.

binding. However, the structural differences the synthetases will sense are likely subtle and lead to conformationalchanges needed to trigger the activation of the reaction. This view (184)is complementary to conclusions arising from the thermodynamic studies discussed above and is supported by many recent results. In the case of the mischarging reactions catalyzed by PheRS, the experimental values of specificity losses of many of the noncognate tRNAs listed in Table IV are consistent with predicted values calculated on the basis of kinetic data obtained for tRNAPhevariants mutated at identity positions (225) (see Section IV,C for details). Despite the potential of tRNAs to be mischarged, the in uivo level of erroneous acylations, and accordingly of errors in protein biosynthesis, remains low [e.g., 1:3000 for valine incorporation instead of isoleucine into proteins (226)l.This is due to correction &d proofreading mechanisms (12, 227-233), but also to competition (184)and preferential binding of tRNAs with their cognate synthetases that prevent the wrong reactions. Intracellular levels of tRNAs or synthetases influence these competitions and favor or disfavor misaminoacylation reactions, as demonstrated recently by in uiuo (234-236a) and in uitro (236-238) experiments. In E. coli, the intracellular level and regulation of synthetases (239) is such that tRNAs are mostly fully sequestered by their cognate synthetases, thus favoring specificity of tRNA aminoacylation, except for GlnRS, the enzyme known to mischarge many mutant tRNAs in oiuo (20,235). Considering also other effects influencing aminoacylation specificity (such as certain post-transcriptional modifications) it appears likely that perturbations in the biosynthesis of tRNA or synthetases may induce biological effects as a result of tRNA misacylations. Such effects may become significant when overproducing synthetases and it is tempting to hypothesize that they could occur during aging, cancer, or other pathologic processes. One might expect that the kinetic properties of tRNA-like aminoacylation resemble those of mischarging reactions. Surprisingly, however, the kinetic discrimination between aminaocylation of canonical tRNAs and tRNA-like molecules is much less pronounced than the one between specific and erroneous charging of tRNA (209),despite the important structural difference between tRNA and tRNA-like molecules (see Section 1,B). This suggests that the overall three-dimensional conformation of a tRNA does not play the preponderant role in speci6ing the identity of a tRNA; more likely, crucial nucleotides presented in a proper orientation to the synthetases govern the specificity of aminoacylation (see Section IV). The kinetics of heterologous aminoacylation reactions involving a synthetase from one organism and the cognate tRNA from another organism are related to the evolution of tRNA aminoacylation systems. Two examples are given in Table IV: whereas ValRS from E . stearothemphilus efficiently

TRNA STRUCTURE AND

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155

aminoacylates heterologous tRNAValspecies from E. coli and yeast, PheRS from yeast behaves differently, since charging of E. coli tRNAPheis only as efficient as that of homologous yeast tRNAPhe. This indicates that the molecular signals on tRNA needed for valylation are better conserved in evolution that those for phenylalanylation. This conclusion is supported by the present view on valine and phenylalanine identities (see Section IV,C and Fig. 13).

C. Other Mechanistic Aspects The charging of tRNA is a two-step event, with first, the activation of the amino acid, and second, the transfer of the activated amino acid to the CCA end of the tRNA (232). This general mechanism is valid even for those synthetases (ArgRS, GlnRS, and GluRS) requiring tRNA for the PP,-ATP exchange reaction (213).There are subtle mechanistic differences, however, such as the stability of the (aa-AMP)/synthetase complex, which in most systems is stable and can be isolated, but which sometimes dissociates into free aminoacyl-adenylate and enzyme when tRNA is absent, as found with S. cereuisiae AspRS (240). Another difference, related to the partition of synthetases into two classes (17) (see Section I,C), concerns the site of primary esterification of the amino acids, either on the 2'- (class I) or 3'-hydroxyl residue (class 11) of the 3'-terminal ribose of tRNA, before trans-esterification leading to the significant 3'-aminoacyl-tRNAs (174, 241-244). This mechanistic difference seems to be clearly related to different binding modes of ATP in the two synthetase classes (180). The rate-limiting step of the aminoacylation reaction is not necessarily the charging of the tRNA, as for tRNAPhe(245),and can be the release of the aminoacylated tRNA from the enzyme, as observed with S. cereuisiae ArgRS (246)or ValRS (247).The rate-limiting step can be changed after alteration of the tRNA structure or sequence, as observed for glutaminylation of tRNAGln modified in its anticodon (248).This point is of particular importance for the mechanistic understanding of reactions involving mutants of tRNA (or synthetase). Finally, it should be recalled that charging of a tRNA may be incomplete (18.3,as the level of aminoacylation reflects the balance between the aminoacylation rate and the rates of the deacylation reactions (194, 249) (spontaneous and enzymic deacylations of aminoacyl-tRNAs and reverse of the aminoacylation reaction). Many studies on the mechanism of tRNA aminoacylation have been interpreted in terms of conformational changes on both partners of the reaction (218, 250, 251). The catalytic centers of synthetases can be activated by tRNAs lacking their 3' ends, which led to the aminoacylation of 3'-terminal CCA fragments, or even of adenosine (191,252).This activation can only be explained by conformational changes on the protein induced by the interact-

156

RICHARD GIEGE ET AL.

ing tRNA. Conversely synthetases mediate such changes on the tRNA side, and in particular in the anticadon region (248, 251). This has been demonstrated explicitly by the crystallographic studies on the glutamine and aspartic acid complexes (see Section 111,C). As demonstrated in the case of tRNAGIU,a conformational change of the tRNA is required for the specific binding of the amino acid to the synthetase (253).

D. Producing and Studying Altered tRNA Sequences 1. EARLY STUDIESON

SUPPRESSOR TRNAs

The discovery of suppressor tRNAs with altered in uiuo specificities was another keystone in tWA-synthetase recognition research. The first examples were amber suppressor tRNATyr mutants h m E. coli that translate the UAG stop codon as glutamine as the result of a low level of mischarging by GlnRS, while still being chargeable by TyrRS (254-258). Mutations in these suppressor tRNAs are all located in the two last base-pairs of the amino-acidaccepting stem and at the discriminator position 73. Similarly, a tryptophanspecific tRNA from E. coZi was converted to a glutamine acceptor by a single mutation of C, to U, that switched the tryptophan to an amber CUA anticodon (259). This suggested the existence of signals in these parts of tRNA that govern specific recognition of the mutants by E. coli GlnRS, a conclusion confirmed by recent investigation on glutamine identity (24) (see Section IV,C). For a long time, the suppressor approach could not be generalized for a systematic search of identity positions. H o m e r , recent methodological developments have permitted production of suppressors with mutations at any desired location (20, 260-262) (see Section IV%B). 2. TRNA DISSECTION

Dissection experiments provided the first approach for locating residues in tRNA important for its arninoacylation. An early observation by Nishimura and Novelli showed that tRNAs can be altered by ribonuclease treatment but retain their ability to accept amino acids (263);later, Bayev et al. showed that the valylation activity of yeast tRNAVa’is not suppressed when the tRNA is cleaved at position I, (264).A number of experiments show that aminoacylation activity is restored when fragments that lack some nucleotides are reconstituted. Applying this rationale to yeast tRNAVd, it was shown that removal of a pentanucleotide from the 5’-end of the 3’-CCA half-molecule destroys aminoacylation, demonstrating that the anticodon region is involved in valylation activity (265). Similar experiments were conducted on a series of other tRNAs using combinations of half- and quartermolecules (4, 266-268) or single RNA fragments (269, 270). It is interesting to discuss the results of dissection experiments in light of

TRNA STRUCTURE

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our present knowledge on tRNA structure and identity (see also Section IV). With several combinations of yeast tRNAA1"fragments, partial charging activity by AlaRS is retained after annealing fragments corresponding to the amino-acid-acceptor stem of the tRNA (4, 271). Since the major identity element of tRNAAIais the G,.U,, base-pair in the acceptor stem (41,42),it is understandable that charging activity is restored by fragments encompassing this base-pair. Similarly, reconstitution experiments on yeast tRNAPhe showed that activity is maintained when the anticodon- and D-loop domains are not excessively perturbed (268), in agreement with the recent findings that anticodon nucleotides and residue G,, from the D-loop are part of the identity set of tRNAPhe (272). Phenylalanylation activity was unexpectedly restored for an inactive fragment corresponding to a three-quarter molecule lacking the doublestranded amino-acid-acceptor stem after removal of the modified m7G,, residue in the variable region of the tRNA (270).This three-quarter molecule contains the complete set of phenylalanine identity nucleotides, but it is not recognized by the synthetase; relaxing its conformation by excision of the modified m7G,, involved in maintaining the tertiary structure of the tRNA allowed recognition and aminoacylation. Similar results were obtained with halves or three-quarter tRNA fragments generated from yeast tRNASer that became chargeable by SerRS to levels up to 40%, but only after specific renaturation processes (269). Reconstitution experiments using heterologous combinations of tRNA fragments provide insights into tRNA recognition. These chimeras were designed after the observation of sequence similarities within the aminoacid-acceptor stems and the anticodon stems of yeast tRNAAIaand E . coli tRNAVal.The aminoacylation of reconstituted tRNA molecules yielded the highest alanine charging (6 to 13%)for the combination containing a 5'-pGhalf derived from tRNAA1", and highest valine charging (3 to 13%)for the reverse combination with a 5'-pG-half derived from tRNAVal (269). It is interesting to note that the heterologous combination with the G,.U,, alanine identity pair (41,42)formed between the 5'-half of tRNAValand the 3' complement from tRNAA1" is less well alanylated (3%) than the reverse combination with a G,.C, base-pair (up to 13%),suggesting that important molecular signals needed for AlaRS recognition are present in the 5' half of tRNAA1". Taken together, these experiments show that synthetases do not require recognition of intact tRNA molecules to catalyze aminoacylation, and emphasize the importance of conformational features in this process. They suggest further that identity elements are contained in different regions of tRNA that are not the same from one species to another. This excludes the existence of a simple molecular code in tRNAs for their recognition by

158

RICHARD GIEGE ET AL.

synthetases. Finally, it appears that identity elements in tRNA act independently.

3. ENZYMATIC MICROSURGERY OF TRNAs In contrast to the dissection method, in uitro microsurgery produces intact tRNA molecules with sequence alterations. It is based on the use of nucleases such as T4 RNA ligase and T4 polynucleotide kinase to dissect and join tRNA fragments (273).Microsurgery is particularly well-suited to obtain variant molecules of the anticodon loop, although it has been used in a few cases to engineer variants of other regions of tRNA [e.g., in the T-stem and loop of tRNAASp(106, 274) and elsewhere in other tRNAs (275, 276)].The practical limitation of this approach is the difficulty of dissecting the tRNA at internal or double-stranded regions, and in producing reconstructed molecules in sufficient amounts (273);this drawback is now being overcome by in uitro transcriptional systems (see Section IV,B). In contrast to newer methods, the enzymatic microsurgery approach generates molecules containing modified nucleotides. A few selected results on the role of the anticodon loop in aminoacylation are worth mentioning here. In the yeast phenylalanine system, impaired charging of anticodon loop variants differing in sequence (277,278)or length (six nucleotides instead of seven) (279) suggests that the loop size and in particular the anticodon sequence are of central importance for efficient aminoacylation by PheRS. In contrast, replacement of the constant , U residue by seven different substitutions (including modified nucleotides), which interfere with the interactions involving U,,, did not reduce the aminoacylation capacities of these variant tRNAPhe molecules (280). The above observations can easily be rationalized in light of crystallographic results on the aspartate and glutamine complexes (see Section 111,C). For the aminoacylation function, the canonical stacked conformation of the anticodon residues (needed for mRNA interaction) is no longer required, and accordingly there is no structural necessity for the presence of a U residue at position 33 to stabilize this conformation. In agreement with this view, U ,, is absent from tRNAs not involved in protein synthesis, such as the tRNA-like structure of TYMV (202). In the methionine system from E. coli, both the size of the anticodon loop and the anticodon sequence affect aminoacylation (281, 282). In tRNAfMef,substitutions in the wobble position reduce aminoacylation efficiency by at least (282) in contrast to tRNAPhe,where substitutions in the anticodon have only moderate effects. Aminoacylations by yeast TyrRS of a series of tyrosine-accepting RNAs deserve particular attention. In S. cereuisiae tRNATyr,positions G , and Y ,, of the anticodon seem to be important for tyrosylation (283) in contrast to what happens in tRNATyr from the yeast Torula utilis, in which anticodon

TRNA

STRUCTURE AND AMINOACYLATION EFFICIENCY

159

nucleotides 34-36 can be removed without eliminating aminoacylation (284). More intriguing, in the BMV tRNA-like structure, the domain mimicking the anticodon arm of canonical tRNA found in contract with TyrRS (148 and Felden et ul., unpublished results) contains no tyrosine anticodon. These apparently contradictory facts can be rationalized if TyrRS recognizes similar functional groups properly located in these RNAs by a common structural frame. A last example concerns manipulation of a minor E. coli tRNA1le in which "lysidine" in the anticodon was replaced by C34. This tRNA provided the first experimental evidence for a direct and strong involvement of a modified nucleotide in tRNA identity (51) (see Sections IV,C and IV,F).

111. Complexes between tRNAs and Aminoacyl-tRNA Synthetases A. Studying Complexes in Solution The structural aspects of tRNA binding to synthetases have been studied by a variety of approaches (13, 82). Here we have selected a few biochemical topics that are complementary to recent crystallographic results.

1. Uv CROSSLINKING BETWEEN TRNAs AND SYNTHETASES Ultraviolet light-induced crosslinking of tRNA to synthetases was the first direct approach that defined the contact regions between the two macromolecules (211, 2 8 4 ~ )The . method, pioneered by Schimmel and his coworkers on the E. coEi tRNA1Ie-IleRS and tRNATYr-TyrRS complexes, as well as the yeast tRNAPhe-PheRS complex (211, 285), led to a schematic model of interaction between yeast tRNAPhe and its cognate synthetase in which the binding site for the synthetase is along the diagonal side of the tRNA structure that contains the acceptor-, the D-, and the anticodon stems (286). Crosslinking data obtained on the tRNAVa'-ValRS and tRNAMetMetRS complexes from yeast led to similar conclusions (287, 288). In summary, these observations indicate that cognate and noncognate complexes are similar in the orientation of tRNAs on synthetases.

2. FOOTPRINTING OF TRNAISYNTHETASE COMPLEXES Contact regions of tRNAs with interacting synthetases were further defined by enzymatic and chemical mapping experiments. Digestion of complexed tRNAs by nucleases revealed, in all systems thus far studied, contacts of the anticodon and amino-acid-acceptor stems with synthetases (289-293). The most straightforward results were obtained with the phosphate-alkylating reagent, ethylnitrosourea. Protected phosphates were found to be scat-

160

RICHARD GIEGE ET AL.

tered throughout the tRNA molecules with stretches located in the acceptor and anticodon stems as well as in the variable and the phosphate P,, regions (53, 60-63, 105, 294). Comparing these footprinting data, quite different pictures emerge for different tRNAs (Fig. 8). For tRNAAsPp,the strongest contiguous protections are found in the PI, region and at the 5’ side of the anticodon stem, which corresponds to an interaction model in which tRNA is approaching the synthetase by the face comprising the variable region. This model is consistent with recent crystallographic data (19). For tRNAPhe,the protections are more scattered, which gives rise to a different interaction model (53)in agreement with that derived from UV crosslinking (286). Despite these differences in protections, similarities are apparent for several aminoacylation systems (Fig. 8). These may reflect similar binding modes of tRNAs with synthetases of the same oligomeric nature (53,295). This fact may also be correlated with the partition of synthetases into two classes as similarities are observed between protection patterns of tRNAs complexed to the monomeric class-I enzymes, and those of tRNAs complexed to the dimeric class-I1 enzymes. Differences between classes are apparent for the binding modes of tRNALeUand tRNASer, in particular at the level of their large extra-loops where protections are given by class-I1 SerRS (63),but not by class-I LeuRS (62). Proximities of bases in the anticodon region with synthetases have been found in several systems (e.g., aspartate, methionine, threonine, tryptophan) using dimethyl sulfate as the specific probe of N, and N, positions in cytosine and guanine (61, 105, 292,296). In the E. colt methionine system, the data indicate a conformational change of the anticodon arm (292), consistent with the present view on tRNA/synthetase interaction. 3. NOVEL FOOTPRINTING RESULTS A novel footprinting technique using phosphorothioate-containingRNA transcripts has been developed by Eckstein and his colleagues for identifying contacts between tRNAs and synthetases (297). Transcriptions with T, RNA polymerase are performed with nucleoside triphosphates complemented by 5% of the corresponding 5 -O-(l-thiotriphosphate).The phosphorothioate groups of these transcripts are readily cleaved by iodine. Footprinting is achieved by performing the same reaction with a phosphorothioatetRNA/synthetase complex. Another method takes advantage of the rapid cleavage of RNA by photoactivatable aryldiazonium derivatives (108). In both methods, probing of the RNA is done by limited amounts of the chemical probes and cleavage of the RNA is achieved in a very short time; this contrasts with other footprinting methods using, for instance, ethylnitrososurea (99) where the concentration of reagent is much higher and the time required to achieve reasonable levels of cleavage much longer.

aaRS

tRNA species/ origin

Ethylnitrosourea footprinting patterns

cli3sLI

Bean LeuRS (a)

Yeast ValRS

(a)

FIG. 8. Comparative protection patterns of phosphate alkylation by ethylnitrosourea in tRNAs complexed to their cognate synthetases. The length of each arrow indicates the strength of protection (weak, moderate, or strong). Nontested regions at both extremities of tRNAs are hatched. For yeast tRNA*bP, the regions in proximity to AspRS as seen by X-ray crystallography are boxed. For original references, see Section III,A.

162

RICHARD GIEGE ET AL.

Iodine was first used to probe the complex of E. coli tRNASerwith SerRS (297)(Fig. 9A). Positions in tRNASerin contact with SerRS are located in the acceptor stem (C, to U,,J in the T-stem (As2, G5J and in the stem of the large extra-loop (A4&. Except for U,,,, these contacts do not coincide with the minimal set of identity elements in this tRNA identified in the accepting stem and the D-stem (20, 261, 298). This contrasts with the results obtained in the aspartate system from yeast (Fig. 9B). Here major contacts coincide with identity nucleotides in the anticodon region, discriminator position 73, and the base-pair G,,.U, in the D-stem. Other protections were found at nonidentity nucleotides, but at positions in contact with AspRS as determined by crystallography. Protections as well as enhanced reactivities from iodine cleavage were observed in regions of tRNA clearly not in contact with AspRS. These can be interpreted as dffecting conformational changes in complexed tRNA (222). The iodine or aryldiazonium footprints on yeast tRNAAspgive similar regions of protections as ethylnitrosourea mapping, while emphasizing different specific phosphates. This can be explained by intrinsic methodological differences between the three techniques. Iodine reacts only with a single position on the phosphorothioate groups, the sulfur atom, whereas ethylnitrosourea or the aryldiazonium derivatives react with both oxygens of

A

B

FIG.9. Cloverleaf structures of E . coli tRNASer (A) and of yeast tRNA*v (B) with protection patterns of phosphates against iodine modification in the tRNAs mmplexed to their cognate synthetases (222, 297). The size of each arrow is proportional to the extent of protection; stars indicate enhancements of iodine cleavages (moderateor strong) that are indicative of mnformational changes. In tRNA*Sp, nontested regions are indicated by lines.

TRNA STRUCTURE

163

AND AMINOACYLATION EFFICIENCY

phosphate groups; also, the reaction time with ethylnitrosourea is much longer than with the two other probes. Therefore we believe that the protections that are observed by the iodine technique represent a minimum set of phosphates in possible contact with a synthetase (222).

B. Crystallization and Functional Consequences Because the interaction between nucleic acids and proteins involves electrostatic interactions (215, 299, 300), early crystallization attempts on tRNAsynthetase complexes were carried out at low ionic strength. Neutron smallangle scattering and fluorescence titration indicate complex formation between yeast tRNAAspand its cognate AspRS in the presence of ammonium sulfate (216). Stable complexes were observed in the presence of 1.6 M salt with association constants of about lo7 M - l (216) and crystals grown (301) under conditions sensitive to the tRNA-synthetase stoichiometry (302).This crystallization did not require polyamines as for tRNA crystallization (59). Fine-tuning of crystallization conditions permitted growth of the highly diffracting crystals (303) used for the structure determination of the aspartate complex. The tRNAAsp-AspRS complex is specific in high concentrations of ammonium sulfate. Aminoacylation of tRNA is efficient under these conditions (Fig. 1OA) with K , values similar to those measured at low ionic strength and only a 5-fold reduction in kcat (216). Complex formation and catalytic activity

120

:bc .h/\

A

la 100

r 2 1

20

40

20

0

-0

0

1

2

O

0

1

2

I

0

1

2

AmmoniumSulfare Molarity FIG. 10. Vdylation of yeast tRNAVa’(A and C) and of TYMV tRNA-like structure (B) by yeast ValRS as a function of increasing ammonium sulfate concentrations (adapted from 217). Valylation initial rates were normalized with values obtained for the tRNA or the tRNA-like structure in the absence of ammonium sulfate. For further explanation see Section III,B.

164

RICHARD GIEGE ET AL.

at high ionic strength is restricted to ammonium sulfate: no complex formation was observed at high concentrations of NaCl or other salts containing either ammonium or sulfate ions. This specific ammonium sulfate effect was also observed for the interaction of charged tRNA in the ternary complex with EF-Tu and GTP (304) and led to the crystallization of the E. coli tRNAG1"-GlnRS (305) and tRNAAsp-AspRS (306) complexes, as well as to the tRNASer-SerRS (307) and tRNAPhe-PheRS ( 3 0 7 ~complexes ) from T. themphilus. Although the specific action of ammonium sulfate on tRNA-synthetase complexes is yet not well understood, it is possible to propose a qualitative interpretation. The interaction between tRNAs and synthetases involves a balance between electrostatic and hydrophobic forces. At high concentration, ammonium sulfate favors hydrophobic interactions between the tRNA and the synthetases, while at low ionic strength its effect would be mainly electrostatic. If so, one would expect an ammonium sulfate dependence on aminoacylation rates (which are, in a first approximation, proportional to the strength of the tRNA-synthetase interaction) with a trough: first a decrease of the rate reflecting the progressive loss of electrostatic interactions followed by an increase of rate corresponding to the establishment of the hydrophobic interactions. This view was verified on the system formed by the tRNA-like structure of TYMV and yeast ValRS. The affinity between the tRNA-like structure and the synthetase (as estimated by &) is reduced by a factor of about 10 as compared with the canonical tRNA-synthetase interaction (209)and as anticipated, it was possible to uncouple the electrostatic and hydrophobic effects (217) (Fig. 10B). This is usually not possible in canonical tRNA-synthetase systems because the electrostatic effect dominates the hydrophobic effect and, as a consequence, only a decrease of aminoacylation rate is seen. However, by decreasing the strength of the electrostatic interactions by addition of NaCl to the aminoacylation medium, it is possible to obtain the same biphasic variation of tRNA aminoacylation rates as a function of ammonium sulfate concentration (Fig. 1OC). In conclusion, three-dimensional structures obtained from tRNA-synthetase crystals grown from ammonium sulfate solutions represent active conformations. However, because of these particular solvent conditions, it is possible that certain contacts of electrostatic nature, which would exist under physiological conditions, would be lost in the crystalline state. To date, this remains an open problem that deserves investigation.

C. Three-dimensional Structures The determination of the crystal structures of the tRNAG*"-G1nRS (18) and tRNAAsp-AspRS (19)complexes was certainly one of the major achievements for understanding tRNA/synthetase and more generally RNA-protein

TRNA STRUCTURE

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recognition at the molecular level. The two structures are examples for complexes involving a synthetase from class I (GlnRS) and class I1 (AspRS); they are known at equivalent resolutions of less than 3 A, so that useful comparisons can be made (Fig. 11).In both complexes the tRNA spans the enzyme from the acceptor stem to the anticodon region with tight contacts of these two domains with the proteins. The two tRNAs retain an overall conformation as seen in free tRNAAspor tRNAPhe. However, important conformational changes occur in the complexed tRNAs. In tRNAGlnthe terminal base-pair of the acceptor stem is disrupted, the 3'-CCA end is distorded and kinked, and the anticodon loop conformation is altered (18),with unstacking of the anticodon bases and the extension of the anticodon stem by two non-Watson-Crick 2'mU,.Y3, and U,.m2A3, base-pairs (308).A similar opening of the terminal base-pair of the aminoacid-accepting stem seems also to occur in initiator tRNAMetinteracting with class-I MetRS, as deduced from fluorescence energy transfer studies (309). In tRNAAsp, the 3' CCA-end remains in helical continuity with the aminoacid-accepting stem; however, the anticodon stem and loop change conformation. This conformational change starts beyond base-pair G,,*U,, and results in a disruption of the canonical U-turn at position 33 that leads to the unstacking of the anticodon bases (19). It is interesting to note that base-pair 30.40 is the point where the anticodon stems of tRNAAspand tRNAPhediffer from each other and where the helical continuity of anticodon stems are perturbed in several tRNAs as detected by chemical mapping in solution

FIG. 11. Three-dimensional structures of the tRNAGJn-GlnRS (18)(A) and tRNAAspAspRS (19) (B) crystalline complexes. (Adapted from 180.)

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RICHARD GIEGE ET AL.

(310).The distance between the positions of the phosphate group of position in free and complexed tRNAAspis close to 20 A, resulting in a more closed structure for the tRNA, with a smaller angle between the two edges of the L (19). The unstacking of anticodon bases, which are involved in the aminoacylation identity of both tRNAs (see Section IV,A), will favor their interaction with the synthetases. This viewpoint is supported by the refinement of the Gln and Asp complexes that clearly show distinct binding pockets for the three anticodon bases of tRNAG'" (308)and tRNAAsp( 1 9 ~ ) . The most global difference between the two complexes is the orientation of the two synthetases with respect to the tRNA. Whereas GlnRS approaches the tRNAGlnfrom the minor groove of its helical amino-acid-accepting stem, the opposite situation occws in the aspartate system in which AspRS approaches the major groove of the tRNA. This gives two quasisymmetrical binding schemes (Fig. 11).This may be a consequence of the structural difference between the two classes of synthetases (180).Confirmation of this proposal, although it is partially supported by footprinting experiments (see Fig. 8) and model building (31I ) , awaits further crystallographicanalysis of other complexes. The binding modes of tRNAs to dimeric synthetases may differ. The two tRNAs in the structure of the tRNAhp-AspRS complex bind to the enzyme at crystallographically equivalent positions (i.e., both have undergone the same conformational change) and each tRNA interacts with only one enzyme subunit (19). The tRNA bound to TyrRS from B . stearothemphilus is proposed to span the two subunits of the enzyme (312, 313). This conclusion is derived from an extensive mutational analysis of TyrRS combined with computer-modeling studies of the docking of a tRNA model on the known structure of the synthetase (171, 172). In conclusion, one of the major results arising from X-ray crystallographic studies concerns the conformationalchanges of tRNAs upon specific binding to synthetases. Such changes, also detected by other methods, seem to be a general property in tRNA aminoacylation systems (e.g., 64,96, 98, 222).

U ,

IV. tRNA Identity for Aminoacyl-tRNA Synthetases A. General Considerations Structural studies have identified nucleotides in tRNAs either in contact or proximity with synthetases. These nucleotides can be considered as recognition elements, but do not necessarily define the aminoacylation identity of a tRNA. Identity elements are probably a subset of the recagnition nucleotides and thus their definition may be more subtle. It is conceivable that nucleotides not in contact with a synthetase may act indirectly by correctly

TRNA

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positioning other nucleotides to interact with the protein. The concept of identity determinant (or antideterminant) should be refined in terms of chemical groups interacting with the synthetases. Thus, different nucleotides carrying the same chemical group could hlfill the same functional role, provided that their orientation permits the correct presentation of these groups for interaction with their counterparts on the protein. Finally, this concept implies that peculiar chemical groups carried by modified nucleotides could act as specific recognition signals for synthetases. The definition of tRNA identity for synthetases may differ in vitro and in vioo. tRNA identity can be defined in oitro from a strict physico-chemical point of view. Studies of variants of canonical tRNA, or of mini- and macrosubstrates that mimick canonical tRNA, will probe how the synthetases recognize and aminoacylate natural or man-made RNA or even DNA structures (314).Nucleic acid structures designed following this rationale may not be of direct biological relevance, but will represent molecular tools to understand the tRNA-synthetase recognition mechanism. In contrast, in vivo identity may b e influenced by the cellular context, where tRNAs behave as multifunctional molecules interacting and competing with a variety of other macromolecules. As a consequence, potential tRNA substrates for synthetases in vitro may not be substrates in vivo for a variety of reasons. These RNAs may be catabolyzed or incorrectly processed (315)and thus cannot be tested, or their aminoacylation ability may be estimated incorrectly, especially when measured by suppression assays. Indeed, suppression assays may simply reflect the way a tRNA variant passes through the various steps of the protein synthesis machinery after aminoacylation and not the aminoacylation function itself. As a consequence, identity nucleotides (or structural elements) defined by in vitro experiments on purified aminoacylation systems will represent features strictly correlated with the physico-chemical tRNA-synthetase recognition process, while those defined by the in vivo approach are more restrictive features compatible with the multiple interactions occurring during the overall biological process. The two approaches will clearly yield similar answers in many instances; however, they can yield quantitatively and qualitatively different results, especially when conformational features related with aminoacylation identity are concerned. In what follows, we first present briefly the novel RNA engineering methods and than illustrate the above concepts by showing how data originating from in oivo and in vitro approaches complement each other.

6. In Vivo and in Vitro Approaches Two methodologies have recently allowed the rapid determination of nucleotides within tRNAs that are important for their aminoacylation capaci-

168

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ty (reviewed in 16, 20, 21, 316). One is an improvement of the suppressor approach, and permits study of the specificity of tRNA variants in uiuo; the second permits classical biochemical analysis of variants obtained by in uitro transcription of synthetic genes by bacterial phage RNA polymerases. The first method consists in de nouo synthesis of suppressor tRNA genes, which are inserted into a proper plasmid, expressed in viuo and screened. The aminoacylation specificity of the suppressors is checked after analysis of the amino acids inserted at position 10 of E. coli dihydrofolate reductase synthesized from a gene carrying an amber mutation at codon 10 (261,317). In the second method, artificial genes containing a phage promoter next to the 5' start of the sequence encoding the tRNA are generally created by ligation of synthetic oligonucleotides. These genes are amplified after cloning and transformation of E. coli cells. Alternatively, DNA can be directly used for transcription and only the promoter region needs to be in doublestranded form (318).The in uitro approach was validated with studies on the aminoacylation ability of BMV tRNA-like (319) and yeast tRNAPhe variant transcripts (86, 272). A precedent of this approach used pure E. coli polymerase (320). Although these methods are extremely powerful and have yielded a wedth of new information, they have certain drawbacks. The tRNAs obtained by in vitro transcription do not contain modified nucleotides. Fortunately, in most cases they represent good substrates for synthetases (23). This drawback can be overcome by total chemical synthesis of tRNA molecules, as pioneered for the synthesis of yeast tRNAAla(321) and much improved recently, allowing preparation of functional molecules on automated DNA synthesizers (322-325). Unfortunately, the chemistry for the preparation of modified precursor nucleotides i s not yet sufficiently developed to allow systematic use of this method; however, improvements in RNA chemistry have recently permitted the total synthesis of a highly active tRNAAla from E. coli containing all its modified residues (326, 3 2 6 ~ )A. second drawback concerns the necessity for polymerases to recognize proper sequences at the 5' end of the gene, and transcription of sequences starting with non9'G nucleotides (52)or unconventional sequences (327)may be difficult. Furthermore, transcriptions do not generally yield homogeneous RNA populations terminated by 3'-CCA sequences; polyacrylamide gel electrophoresis is presently the only method to resolve biologically significant molecules from transcription mixtures. The major limitation of the suppressor approach, where tRNAs are produced in uiuo with anticodons mutated to triplets complementary to stop codons, is the inability to test their anticodons. However, methods that bypass this limitation are being deveioped (328-329~).Changes in the posttranscriptional modification patterns of variant tRNAs could also interfere

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with the interpretation of in vivo data. Finally, it should not be forgotten that mutations, beside their effects on aminoacylation, could affect the other steps of protein synthesis. Regardless of the method, the major problem will be the adequate choice of mutations for designing variant tRNA molecules. Because the possibilities are enormous, especially for multiple mutations, the choice should be directed by rational information. Such could originate from independent structural results on tRNA-synthetase complexes (see Section 111) or from computer-aided sequence comparisons (330-331a).

C. Major Positive Identity Nucleotides in Canonical tRNAs Here we discuss the major identity elements defined to date in canonical tRNAs; these normally correspond to nucleotides not primarily involved in the architecture of tRNAs (e.g., conserved or semiconserved residues). Anticodons are obvious candidates for aminoacylation identity elements of tRNAs, because of the biological correlations between anticodon sequences and amino acids. This assumption was soon verified in several cases (2). However, this concept cannot be generalized to all aminoacylation systems. The degeneracy of the genetic code (e.g., six codons each for arginine, leucine, and serine) implies the existence of isoacceptor tRNAs, daering by their anticodon sequences for reading the sets of degenerate codons (e.g., four, five, and five different anticodons have been found for E. coli tRNAArg, tRNALeU, and tRNASer, respectively, with two or three degenerate positions). Suppressor tRNAs are chargable by a variety of amino acids, while possessing anticodons complementary to one of the stop codons (UAA, UAG, and UGA).

1. TRNA IDENTITIES I N SYSTEMS

THE YEAST

ASPARTATEAND E . Cold GLUTAMINE

Identity nucleotides in the two tRNAs for which crystallographic structures of the tRNA-synthetase complex are known (see Section II1,D) are well defined. For yeast tRNAAsp, six nucleotides are essential for aspartate identity (43):the anticodon residues G, U,, and C,,, the discriminator base G, and the G,o.U,5 base-pair in the D-stem (Fig. 12A). They were identified by studying the in vitro aminoacylation of a series of tRNA transcripts. Mutations in tRNA essentially result in decrease of kcat, except for the G,,-U,, pair, where changes into G * Cor A-U pairs change &,Single mutations have rather moderate effects on the specificity constants of the aspartylation reaction (kcat/& is reduced by factors of 10 to 530),and at a given identity position, the decrease in k,,l& is dependent upon the nature of the mutation, indicating that specificity is governed by the nature of the chemical groups carried by the nucleotides (43).When multiple iden-

170

RICHARD GIEGE ET AL.

A

B

A-U

FIG. 12. Cloverleaf sequences of yeast tRNAASP(332) (A) and E . coli tRNAG1"(333) (B) with major identity nucleotides (boxed) (24, 43, 338).

tity positions are mutated together, the effect on k,,lK, has been found to be additive, cooperative, or anti-cooperative, depending on the mutated couple 0. Piitz, J. D. Puglisi, C. Florentz and R. GiegC, unpublished observation). In E. coli tRNAGIn,identity elements have been determined using both the in vitro and in vivo approaches (24, 334-338) (Fig. 12B). A set of 11 nucleotides located at both extremities of the molecule are important elements for glutamine identity: five residues in the anticodon region (the anticodon itself and the two adjacent 3'-residues) and five residues near the amino-acid-accepting end, including the discriminator G,, residue and the second and third base-pairs in the stem. As in the aspartate system, mutants at identity positions express their loss of specificity essentially in k,. However, specificity constants in this system are decreased by a greater factor (up to 103-105) than in the aspartate system. An interesting effect is related to the opening of the U,.A,, base pair in the complex (18):each mutation that strengthens this pair leads to decreased aminoacylation, and conversely, mutations that weaken it (or even that eliminate it) are without effect (336). 2. OTHERTRNA IDENTITIES

Beside E. coli tRNACln, major identity nucleotides responsibIe for aminoacylation specificity have been determined in 15other tRNA families from E. coli, namely those specific for alanine (41, 42, 339, 340), arginine (341343), asparate (344a),cysteine (344b),glycine (345),histidine (346,347),isoleucine (348),lysine (342a),methionine (343,349),phenylalanine (350a),ser-

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ine (20, 261, 298, 351), threonine (352, 353), tryptophan (354, 359, tyrosine (298),and valine (96,348,356).Fewer data are available for eukaryotic tRNAs. In addition to tRNAAsp,major identity determinants are known for four other yeast tRNAs, namely those specific for histidine (357), methionine (358), phenylalanine (225, 272), and tyrosine (see Section II,D and below). Major identity nucleotides have been described for Bombyx mori and human tRNAAla(359)and rabbit liver cytoplasmic elongator tRNAMet(3594. For phenylalanine identity, recent data reveal additional nucleotides in the anticodon stem of yeast tRNAPhe,necessary for charging by heterologous human PheRS (360). For heterologous charging of yeast tRNAPhe by T. thermophilus PheRS, however, major identity residues are restricted to the three anticodon nucleotides and to the discriminator base and do not include position 20, which is required for the reaction with E . coli, yeast, and human PheRSs ( 3 6 0 ~ ) . The results clearly show that the major sites determining the aminoacylation identity of tRNAs correspond to small sets of nucleotides (Table V). As anticipated, anticodon residues are essential in many aminoacylation systems. In the valine and methionine systems from E . coZi, the anticodon alone is sufficient to confer upon tRNAVal or tRNAmet their identity (349), although additional effects are not excluded, as found in the methionine systems from yeast (358) and E . co2i (360b,c, 362) and for valylation of the TYMV tRNA-like structure (see Section IV,D) or E . coli tRNAVd (356). In other systems, like yeast phenylalanine (225,272),additional nucleotides are required. In contrast, anticodon residues are not required in some systems, such as the alanine system (41, 42), and in those systems involving tRNAs recognizing highly degenerate codons (e.g., those specific for serine) (20, 351). At the 3' end of the tRNA molecule, residue 73, the so-called discriminator nucleotide, is important for the aminoacylation of many tRNAs (43, 272, 298, 336, 343, 344, 346, 354, 356, 362), in agreement with earlier proposals based on indirect evidence (184, 195). Interestingly, the discriminator position is without effect for the identity of E . coli tRNAThr(353).

3. IMPLICATIONS: PERMUTATION OF TRNA SPECIFICITIES

A consequence of the concept of identity determinants is the possibility of changing the specificity of a tRNA by transplantation of its identity set into other tRNA species. This prediction has been verified in several systems and, in fact, successful transplantations represent the explicit demonstration of tRNA identity (Fig. 13).For instance, the importance of the G,-U,o basepair in confering alanine identity was verified by transplanting it from the E . coli tRNAA1" to tRNACys or tRNAPhe, which makes these tRNAs alanine acceptors (41, 42). Similarly, switching the anticodons in valine and methionine specific tRNAs switches their specificities (349),and substituting two

172

RICHARD CIEGE ET AL.

TABLE V DETERMINANTS FOR AMINOACYLATION SERIESOF E . coli AND YEAST tRNAs

MAJOR IDENTITY IN A

tRNA Alanine Arginine Asparagine Aspartic acid Cysteine Glutamine

Escherichia coli [G3*U70]ab

[A20;Q5 ; A or G7318.b +

Saccharomyces cerevisiae (includes G30U70)C

importance of variable pocket*

-

[G73]b

[GlO*UZ ;@4u35€36 ;G73]b

[U73 ;!34C35A36la G2<71 ;G3C70 ;GI0 &34Y35G36 ;A37 ;U38 ;G7@

Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine

a 3 5 ;A731 E34A35Y36]ab [u20 ;G27<43 ;G 2 8 C 4 2 ; ;U45 ;U59 ;U60 ;A731a

044

K34A35U36 ;A731b [GZO ;@4&5&6 ;A73]b

(Importance of vanabIe pocket*)

Proline Serine h n i n e Tyrosine Tryptophan Valine

Anticodon nucleotides are underlined. Identity sets that were confirmed by transplantation experiments are in brackets : (a) after in vivo, (b) in v i m experiments ; (c) identity nucleotides determined on the basis of tRNA dissection or microsurgery experiments. * For sv~ctural details see Section.1.A ; ** only partial identity switch when transplanted into yeast tRNAW (357);*** identities that q u i r e specific conformational features (correct orientation of the variable arm in E. coli tRNASU)1298). References : $ (256);$$ (236); t (361) ; tt (283) ; other referencesare given in the text (Section IV).

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base-pairs in the acceptor stem changes a serine suppressor tRNA to an efficient glutamine acceptor (335). Identities can be derived from mischarging data without direct mutational search on the cognate tRNA. Certain mutants of E . coli initiator and suppressor tRNAMefare aminoacylated with tyrosine by yeast extracts. Inspection of the sequences of these mischarging mutants and of yeast tRNATyr suggests that discriminator residue A,,, as well as base-pair C,.G,, are important for recognition of yeast tRNATyr by cognate TyrRS (361), in addition to anticodon nucleotides identified previously (283) (see also Section 11,D). The puzzling prediction of this work (361) is that yeast TyrRS should approach the 1-72 base-pair of tyrosine-accepting tRNAs by the major rather than the minor groove, in a way that would be typical for class-I1 synthetases while TyrRSs are class-I enzymes (see Table 11). Histidine identity in yeast tRNAH” (mainly expressed by residues G-1) was also characterized by mischarging experiments after progressively changing yeast tRNAAspinto an efficient histidine acceptor (357). Studies on mutants of the E . coli initiator tRNA mischarged by E . coli GlnRS confirm the importance of discriminator base 73 and of bases 1 and 72, which should form a weak or a disrupted base-pair, as well as of the two following base-pairs, 2-71 and 3.70 for glutamine identity (361a, 361b). Except for the mutant with a U,-A, base-pair, there was a good correlation between in vitro mischarging assays and in vivo aminoacylation levels as well as suppression activities of an amber codon (361b). 4. MECHANISTICASPECTS

a. Strength of Zdentity Nucleotides. Identity nucleotides can affect aminoacylation reactions differently. This becomes apparent when comparing among different systems the effects on the catalytic specificity constants of mutations located at similar positions in the tRNA. The differences between the eukaryotic aspartate and phenylalanine systems and various prokaryotic systems are striking. Whereas in eukaryotes the loss of specificity (estimated as the ratio between the catalytic specificity constants k,,,/Y, of wild-type and variant tRNAs) after single mutations does not exceed 103, and often is only about 10 (43, 225, 360), they can reach values higher than lo5 in prokaryotes (23). Further, within the same tRNA, differences in strength between identity nucleotides are observed. For instance, in tRNAAsp,specificity drops by factors of 8 to 530. In general, it appears that the higher the number of identity nucleotides required to impart specificity to a tRNA, the less the importance of their individual contributions. Thus, discriminator nucleotides at position 73 are weak determinants and always are part of large identity sets. On the contrary, when the number of identity nucleotides is low, their strength is large. This fact becomes particularly evident for the

ooo~

d

o

Om O

E. coli tRNAAla E. coli tRNA*%

0-0 "0-0" 0-0 0 0 0 0 0.0

E. cofi tRNAGln 0 0

0

E. coti tRNAGly 0

0 0

0

0-•

Yeast tRNAPhe

E. coli tRNAser

FIG.13. Schematic representation of the location of major identity determinants for homologous charging (in black dots) in the secondary folding of a selection of tRNAs. For original references, see Section IV,C.

E. coti tRNAcYS Yeast tRNAMet

Yeast tRNAhP

I

33 0-0

oo-o

0

0

0

(0

0 0

*.* E. coli tRNAPhe

-0.

E. coli tRNAMe1 E. coli tRNAThr 0

0 0

0-6

0-0 0-0

0-0

0-070

0-070

0-0

0-0

0-0

mo-oa

0-0 0 0 0 0

0

0..

0

0..

E. coli tRNATrP

E. coli tRNAVal

FIG. 13. (Continued) 175

176

RICHARD GIEGE ET AL.

identity of E. coli initiator tRNAMef(23) and the valylatable TYMV tRNAlike structure (363)(see Section IV, D) in which anticodon residues are strong determinants. How are these differential effects of identity nucleotides expressed? It is probable that optimal ,k is mediated by the nucleotides in contact with the synthetases. The tightest specific contacts are possible for residues in loop regions, especially in the anticodon loop after conformational changes that optimally expose them for interaction with the amino-acid counterparts on the protein (see Section III,C, the crystallographic data for tRNAASpand tRNAGIn).Mutating these determinants disrupts specific contacts and thus perturbs catalysis, because transfer of chemical information to the catalytic site of the enzyme is altered. Weaker determinants probably act in synergy with major determinants by enhancing their action. Such synergies are certainly needed for strong determinants in the anticodon of certain tRNAs that cannot alone be responsible for specificity and thus are potentiated by modulating effects brought about by the sequence context or the discriminator nucleotide. Other determinants may act by more indirect mechanisms by conferring on the tRNA the conformation for optimal presentation of strategic chemical groups to the synthetases.

b. Sequence Context. Even if the existence of identity sets is supported by transplantation experiments, additional residues may contribute to identity. Besides residues important for conformation (see Section IV, E), other nucleotides (or chemical groups) that are in common between wild-type and transplant sequences could contribute to identity. In other words, the sequence context must be taken into account. This has been illustrated in the E. coli alanine system, where additional sites to the major G,.U,, determinant contribute to alanine identity. These sites are located at the discriminator position (A,,), in the acceptor helix (G,C,,), and in the variable pocket (G2J of the tRNA (339, 364). Alternatively, sequence change in the antican compensate for alteration of the major codon stem (C,,*G,,+ U,,C,,) alanine determinant (G,*U,,-* G34&) (364a). c. Contacts of Identity Determinants with S ynthetases. An important question concerns the relationship between identity nucleotides and their counterparts on synthetases. Identity nucleotides probably contact synthetases directly, although indirect effects by allosteric-type mechanims are not excluded. The assumption of direct contact is demonstrated explicitly in the aspartate and glutamine systems by crystallography (18, 19) and is reinforced for the aspartate system by footprinting (53, 108, 222, 296). Indeed, when mutating one of these residues, the variant tRNA still interacts with AspRS, but the contacts are lost at the positions of the mutations, and certain conformational changes in the tRNA, mediated by the interaction, are no longer observed (222). Interestingly, many positions in direct contact with

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AspRS do not influence aspartylation, as for instance the first base-pair U,. A, which can be changed to G,.C,,; in contrast, this base-pair, present in tRNAGln, plays a crucial role in glutamine identity. In the glutamine system, amino acids from GlnRS interacting with identity nucleotides on tRNAGInhave been identified. For instance, U,, interacts with R341, QSl7, and R520, and G,, interacts with K,ol and R402 (308). The functional role of the contacts between the 0 - 4 and 0-2atoms of U,, and G,, and amino-acid counterparts in GlnRS were established by a mutational analysis of tRNAGln anticodon (see Table VI). Other contacts with identity elements concern the G3mC70 base-pair (18). Changing the contact with this G3*C70identity pair by a mutation at D235 relaxes the specificity of GlnRS (197). For the alanine (293) and phenylalanine (53) systems, the proximities of tRNA identity elements to synthetase is also strongly supported by footprinting experiments. Contacts between the CAU anticodon of tRNAMet with MetRS are suggested by a number of experiments in both E . coli (23, 365, 365a, 366) and yeast (367, 368, 368a) systems. Further information on the role of amino-acid residues on synthetases, interacting with tRNAs or involved in aminoacylation activity, have been discussed elsewhere (e.g., 19a, 23, 369, 370). However, in the E . coli serine system, footprinting experiments suggest that positions in tRNA protected against iodine cleavage by SerRS do not coincide with identity nucleotides

(297).

d . Iclenttjiying Strategic Chemical Groups on tRNA. It is clear that crystallography is the most straightforward method to identify the chemical nature of the contacts between tRNAs and synthetases. But crystallography does not permit unambiguous discrimination between the contacts specifying the amino-acid identity of the tRNA and the less specific ones responsible for the overall binding with the synthetase. Thus, additional information arising from functional analysis of tRNA variants is needed. More generally, analysis of sets of variants obtained by mutagenesis for a given identity position can yield useful information, even without the support of structural data. Typical results concerning discriminator and anticodon positions are given in Table VI. They can be summarized as follows. (i) For the glutamine system, the ranking of tRNA variants in terms of aminoacylation efficiency is well-explained by crystallography (18, 24). (ii) For a given wild-type position in various tRNA species, this ranking is variable, suggesting different interaction schemes with synthetases (e.g., between AspRS or TrpRS with G,,, or between yeast AspRS or E , coli GlnRS with Us); however, the way both E . coli and yeast AspRSs recognize G73 appears to be the same. This is also a possibility for recognition of A,, by E . coli AlaRS and ValRS. (iii) The approach can reveal existence of blocking groups (e.g., N-6 of A, which hinders interaction between AspRS and noncognate tRNAs with A7J.

178

RICHARD GIEGE ET A L .

TABLE VI SEARCHOF CHEMICAL GROUPSIN tRNA INVOLVED IN AMINOACYLATION IDENTITY: A FEWSELECTEDEXAMPLES Ammoxylation

wild-type

Arninoq4&mdGciimcy

SySICUl

position

of tRNA variants

Ala (E. cofi)

(G3.Um) A73

G20

(CrU) # (1.U) # (2-AP-U) (324) (*) unpaired N-2 atom of G is required in nunor groove (340) N-6 of A required A>C=U>G (04of U 8t 0-6of G would be negative dmfats) G>>A>U=C purine nng and N-2 of G impoltpnc

Asp(E.mfiJ

GI3

G>>U2A2C

(344) 0-6 of G muired ;N-6 of A, and C hinder refognition

Asp (yeast)

G73

G > U >> A 2 C

(43)

GY

G>ALU>C

U3J

U>G>C>A C>USGwA

CM

Cmunentsand intapretations

ref.

(**) N-2 & 0-6ofG required ; 0-4of U. N-6 of A.

nndc hinder refognition (**) pxine ring imporfant (N-7 position ptected against dimethylm~lfate alkylltion in the complex with AspRs (2%) (**) 0-2 of U required (N-2of 0bderxcognition) (**)~~enngrequired(witb0-2mC&U)

U35

G 2 A >> C w U C>>A U>->A>C

036

G>U>A

ne (E. mli)

XY

0 %=W

Metr (E. cofi)

A13

A>ULG2C

Met-m (E. mfi)

CY

C >> U > G ;(A : nt) (349) (***)0-2 of C required .._.___...

Phe(yeast)

A13 GY A35 A36

A>C>U G>U>C=A A>C=U;(G:m) A>U>C;(G:nt)

Thr(E.coli)

An GI-CIZ CZG? I

A=G=U=C no effect of discriminator base 0 - C >> A-U 2 C G ;(l-A. a) rrcognition of tbe central part of the major groove is likely (tt) C-G >> U-A = G S > A-U (3531 recOgnitianof the centnlpad of the major groove is likely (tt)

Trp(E.coli)

GI3

G>C>A=U

(3WJ (onthe basis of aminoacylation plateuls) N-2 of 0 probably required

Tyrfyeast)

GY

G>U=C>A Y > U > C > A :( 0 :m)

(283) (***)moderateeffects ;as OM of R e (yeast) (*I*)moderate effects ;weak effect of Y, pyrimidine ring

Gln (E. cofi)

GI3 CY

(336) purine ring required (**) 0-2 of c required (**) 0-4of U required (N-6 of A & N-4 of C would be negative elemeats for GlnRS) (**) 0-6 of G imporIan1(= 04of U) @I-6of A would be a negative signal)

(t)

(348) taut0meric forms of L and G identitcpl at N-2 of G and C (262) modaateeffecls:haeGispNlaredtobe a negative b e n t for MetRS (23)

_--_

~

~

735

~

~

--.-..

~

(225) modem& effects ;N-6 of A importaot(rN-4 of C) (278)

(*+*)moderate effects ; 0-6of G importvlt (= 0-4of U) (***) m&ak

effects ;N-6 of A required

(***) moderate effects

neanstobeimportant

Val(E.coli)

An

A>C>U>G

(356) as f aAla identity

~

U not specifically uxhcated ranking of mutational dferU M bmed on qxcifrciIy conslant &/K,) delcrminations. (*) With RNA nunisuhstratcs p p d by chanical synthesis and having ioeapornted nucleotides analogs (= Ih i n e ;2-AP = 2-minopurme) ; (**) intupn3aIion of biocbemicd data continued by X-raynysUUopphy for h e aspartaw ( 1 9 4 and glutamine(l8,308)systems ;(***)

.

with C R N A s p Y by,enzymaticminosurgery ;(t)compeiron oftRNAllC isnaccepm (tt)according to prcdrcuonsdiscussed by Setmanelo (371) n1 noltesled.

TRNA STRUCTURE AND AMINOACYLATION

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Chemical synthesis of an RNA that introduces appropriate chemical alterations at identity positions is another powerful approach to study the role of chemical groups in tRNA identity. Important conclusions came from activity studies on tRNA*Ia minihelices in which inosine (I) or 2-aminopurine (2-AP) residues were introduced in place of identity nucleotide G, (324). These minihelices contain noncanonical base-pairings mimicking features of G-U or G C pairs: the wobble pair I,.U,, is similar to a G-U pair but lacks the N-2 group of G; the 2-AP,*U7, pair resembles a G C pair with an Hbond between N-2 of 2-AP and the 0 - 2 of U. The inactivity of the two variant minihelices demonstrates the necessity of a free 2-amino group at position G, in the accepting stem of tRNAAlafor specific alanylation (324).Functional 2’-hydroxyl contacts, clustered within a few angstroms of the critical 2’amino group, have also been pointed out (325). Using recombinant RNA technology, variants of E . coli tRNAGInwere prepared in which guanosines were replaced by inosine residues. Functional assays revealed the importance of G,, G,, and GI, N-2 groups for tRNA discrimination by GlnRS (338).

e. Multiple, Overlapping, and Partial Identities. Multiple identities can appear when removing antideterminants (see Section IV,F). They can also be detected in vivo when studying suppressor tRNA variants, or in vitro when studying tRNA mischarging (see Tables I11 and IV). In many cases, multiple identities imply overlap of identity elements; this potential occurs frequently because identity elements are often located at similar positions in tRNA (e.g., at discriminator position, variable pocket, and anticodon regions). Alternatively, multiple identities can appear when transplantation of new identity nucleotides leaves (some or all) identity positions of the parent molecule unchanged. For example, certain variants of E . coli su+III suppressor tRNATyr, with mutations at discriminator position and the two first basepairs in the amino-acid-acceptor stem, exhibit a dual tyrosine and glutamine specificity (256). Further, an E . coli suppressor tRNALys in which seven identity nucleotides from E . coli tRNAPhewere transplanted, inserted phenylalanine and small amounts of lysine and tyrosine into suppressed dihydrofolate reductase (350).Finally, yeast tRNAPhetranscripts in which the complete yeast aspartate identity set has been transplanted, beside becoming an efficient aspartate acceptor (43). retains partial phenylalanine acceptance because of the presence of the phenylalanine G , identity nucleotide and overlapping Phel Asp anticodon identity nucleotide G,, (J. Puglisi and J. Putz, unpublished results). The expression of partial identities is correlated with the existence of mischarging reactions: indeed tRNAs that can be mischarged by a noncognate synthetase contain part of the identity set of the cognate tRNA for this synthetase. The consequence of this assumption is that the kinetic specificity

180

RICHARD GIEGE ET AL.

of mischarging reactions can be predicted, provided the effects brought by identity nucleotides are independent (this is not always the case as, e.g., in the aspartate system from yeast). With this assumption, Sampson et al. (225) predicted the kinetic specificity (relative kcat/&) of mischarging reactions catalyzed by yeast PheRS (see Section 11,B). For those tRNAs where predicted and experimental values were best correlated, the structural frame was closest to that of yeast tRNAPhe (compare structural characteristics in Table I with kinetic data in Table IV).

5. ROLE OF MODIFIED NUCLEOTIDESIN POSITIVEDISCRIMINATION

Considering the efficient aminoacylation of tRNA-like molecules or of tRNA transcripts, all deprived of modified nucleotides, it could be prematurely concluded that post-transcriptional modifications are unrelated to aminoacylation. However, some old observations indicate that modifications may modulate aminoacylation [e.g., m2G,, in yeast tRNAPhe (372) or mnm5s2U,, in E. coli tRNAGlU(248)charging]. Relevant to this last observation is the strongly impaired aminoacylation (140-fold decrease of catalytic specificity) of an unmodified E. coli tRNALys transcript compared to the wild-type molecule that contains a mnm5s2U residue at position 34 (343). Also, catalytic-site activation of yeast PheRS by cognate tRNAPheor noncognate tRNATyr requires the presence of modified Y,, or i6A in the anticodon loop of the activating tRNA (191).The importance of modified nucleotides in identity is now reinforced by the finding that the isoleucine identity of the minor E. coli tRNA1le(LAU) is due to the presence of “lysidine” (L), a hypermodified cytosine residue. Indeed, replacement of L,, by C,, results in a 10-fold decrease of the isoleucylation extent (51).

D. Mini- and Macro-RNA Substrates for Aminoacyl-tRNA Synthetases 1. AMINOACYLATION OF TRNA MINI- AND MICROHELICES AND INTERACTION WITH SYNTHETASES The symmetric architecture of tRNA, with the quasiequivalent aminoacid-accepting and anticodon branches of its Lshaped structure, allows dissection of the molecule into its two components, which correspond to double-stranded helical structures closed by the T- or anticodon loops. These domains can be prepared easily by in oitro transcriptional methods or by chemical synthesis. Identity determinants of aminoacylation that are located in the amino-acid-accepting domain and in the anticodon loop (see Section IV,C) are conserved in these domains upon dissection. Moreover, for the alanine and histidine systems, the major determinants are exclusively comprised in the CCA-containing domain.

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With these considerations in mind, Schimmel et al. hypothesized the occurrence of aminoacylation for RNA minihelices mimicking an amino-acidacceptor stem and containing identity determinants (373). The prediction was first verified for alanine identity where mini- or even microhelices (with a shortened helical domain or minimalist RNA substrates containing tetraloops instead of seven-nucleotide canonical loops) containing the G,. U, determinant retain their capability to be charged by AlaRS (374-376). Similar results were obtained for the charging of histidine on mini- or microhelices containing an extra G-1 residue at their 5’-end (347) (Fig. 14). Interestingly, alanylation or histidinylation activities are retained after transplantation of the respective identity elements into minisubstrates derived from tRNAs of other specificities (346,374,375).Moreover, enzymatic alanylation of single-stranded RNA derived from the 3’ end of E . coli tRNAA1”is possible, provided that aminoacylation is conducted in the presence of complementary 5’-ribo- or deoxyribo-oligomers that hybridize with the accepting strand and restore the G,*U,, identity base-pair (377). This charging is even possible with limiting amounts of the 5’ oligomer that creates transient duplex substrates with the 3’-accepting oligomer (377). As observed with full length tRNAs, the aminoacylation efficiency of these RNA minisubstrates is modulated by the sequence context. Thus, discriminator nucleotide A,, and base pair G,.C,, contribute to the alanine identity of mini- and microhelices (364, 378); similarly, the two base pairs U,*A7, and G,-C,o participate in the histidine identity of microhelices (364) or minimalist RNA tetraloops (376).Interestingly, a minihelix comprising a pseudoknot (see Section 1,B) and corresponding to the acceptor arm of the TYMV tRNA-like structure, is a very efficient substrate for yeast HisRS (379) (see below, for the interpretation of the mimicry between tRNA-like and canonical tRNA structures for histidine identity). Aminoacylation of minihelices does not necessarily require the presence of major identity nucleotides. This fact was recently demonstrated with minihelices derived from tRNAs in which the discriminator base is a minor determinant, as in yeast tRNAVal(327) and tRNAASp(M. Frugier, C. Florentz and R. GiegC, unpublished results), and in E . coli initiator tRNAMet (376, 380) (Fig. 14). In such minihelices, aminoacylation levels are low but specific; in the valine minihelix, mutation of the weak identity nucleotide abolishes valylation (327), and minihelix methionylation is only possible on the structure derived from tRNAMe‘and not on noncognate sequences (380). Related to these observations is the moderate charging by E . coli GlyRS of a microhelix derived from tRNAG1yin which identity positions are located at both the amino-acid-acceptor stem and the anticodon (364, 376). Interestingly, a tRNAMetmicrohelix is charged by a deletion mutant ofE. coli MetRS in which the anticodon-binding site is missing ( 3 8 0 ~ This ) . sug-

I 1

Alanine (E.coZi) Minihelix Ala

I Histidine (E.coZi and TYMV) I I Minihelix His

I

I I I Microhelii Ala

Tetraloop

1 -1

55

I

Mcrohelix His

I

Pseudoknpntted minihelix His*

t------------’ I Glycine (E.col0

I I I

I

Minihelix Met

60

65 70 76 A~UF$F~;I~F~$YFFACCAT UGGAC$qCAUCGG9 49 7 1

cgug

S T 23G-C 12 G-U 25 C -G010 -44 C-G U-A 30G-C40 UC-G C

5’ G 3' U-A G-G G-C 30G-C40 cG-C A

$--A"

w

U

"CA 35

Anticodon hairpin Val

35

I

Anticodon hairpin Met

FIG. 14. Minihelices derived from the amino-acid-acceptor branch and the anticodon branch of tRNAs. The figure indicates the amino-acid specificity and the origin of the parental tRNA (or tRNA-like) as well as the location of the major identity nucleotides (boxed). *, This minihelix derives from the TYMV tRNA-like structure which is valine-specific. Its structure emphasizes the stacking of Uzz over Uzl (see text); note that this minihelix is not valylable. For references, see Section IV,D.

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gests that the specific catalytic function of MetRS is independent of the structure of this site, which contains the recognition elements of the major identity nucleotides of tRNAMet. It favors also the view that synthetase structures could have evolved from simplified versions just encompassing the amino-acid activation and tRNA acceptor-helix binding domains. The aminoacylation of the valine minihelix can be stimulated by the addition of a second minihelix corresponding to the anticodon stem and containing the major identity nucleotide responsible for efficient aminoacylation of tRNAVal(Fig. 14) (327). This suggests that information originating from the anticodon stem-loop can be transmitted to the active site of ValRS by the core of the protein. In the E . coli methionine system, an anticodon stem-loop minihelix derived from tRNAfMet and carrying the methionine CAU anticodon binds tightly to MetRS, but loses this ability when CAU is changed to the amber CUA anticodon (381).Interestingly, this binding is also abolished by a mutation at W,,, in MetRS (381)that is known to alter the binding of tRNA to MetRS (366).These experiments show that minihelices could become valuable models for structural studies on the interaction of anticodon loops with synthetases in systems where anticodon nucleotides are strong identity determinants.

2. AMINOACYLATIONIDENTITY OF VIRAL TRNA-LIKESTRUCTURES The aminoacylation identity of viral tRNA-like structures has been investigated by mutational analysis and footprinting experiments. An extensive mutational analysis has been performed on the tRNA-like structure of BMV (319, 382). It revealed that alterations at the 3’ terminus, the pseudoknot, and in two stem regions [which might be compared to the anticodon branch of tRNATyr (148)] lead to severe losses in the tyrosylation ability of the transcripts. The positions of these alterations overlap with those found protected by TyrRS at topologically similar positions in the strategic residues in yeast tRNATyr (148)(Fig. 15A). For TYMV RNA, the sequence corresponding to the Lshaped folding (see Fig. 5A) represents the minimal structure acylatable by ValRS (383), a fact partially corroborated by comparative footprinting data that indicate protections of the tRNA-like structure by ValRS at positions homologous to those protected in tRNAval, in particular in the anticodon region (384). However, regions upstream from the Lfold are also protected by ValRS, and the longer RNA molecules have better valylation properties than the minimal structure, suggesting either that the extra domain improves the binding of the RNA to the synthetase (385)or that alternative conformations are found in the shortened RNA that can change aminoacylation kinetics (202). In vitro studies on TYMV variants have emphasized the importance of the middle position of the anticodon as a major valylation determinant, and of the discriminator base as a minor determinant for yeast ValRS (363) (Fig.

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B

A

L2 3' U

L1

U U G A

G-C A- U G-C GAU U -5' G UU-A C-G U-A G-C U-A C-G

C C

C

FIG. 15. Location of major identities (boxed) in viral tRNA-like structures for charging of BMV RNA by wheat germ TyrRS (A) and of TYMV RNA by pure yeast ValRS (B).

15B). Further studies with wheat-germ ValRS confirmed this view, but showed in addition the contribution of the 3'-anticodon nucleotide, as well as of the 3'-most anticodon loop residue (386).Aminoacylation of TYMV RNA is also sensitive to local conformations in the RNA (387);however, as in canonical tRNAs, many structural mutations do not affect valylation. An extensive study on variants mutated in the pseudoknotted region showed that as long as the pseudoknot can form, there is no dramatic change in the valylation of the tRNA-like fragment. However, if the pseudoknot cannot be formed, valylation is suppressed (388). The identity nucleotides of the third type of viral tRNA-like structure, the tRNA from TMV that is charged by HisRS of various sources (but not E. coli), have not been characterized. The minimal length required for histidinylation (389)is in accordance with the size (95 nucleotides)of the model of this tRNA-like molecule (see Section I). Footprinting studies show that the putative anticodon is protected by yeast HisRS (390).A novel finding is the efficient mischarging of TYMV RNA by yeast HisRS. This mischarging is possible on variants mutated at valine-determinant positions, thus showing that valine and histidine identities do not overlap, and more specifically that the middle position of the valine anticodon does not participate in histidine

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identity (379). The efficient histidinylation of a TYMV RNA-derived minihelix containing a pseudoknot (379) convincingly showed that the major histidine determinants are located in the amino-acid-accepting arm. Probably, in this molecule as in canonical tRNAH*”from E . coli (346) and in minihelices derived from this tRNA (347), an additional nucleotide at position -1is required for identity. This nucleotide would be the first nucleotide of loop L, (see Figs. 14 and 15)from the pseudoknot in the case of the tRNAlike molecule (379).

3. AMINOACYLATIONIDENTITY

OF

SHORT MITOCHONDRIAL TRNAs

Little is known concerning the aminoacylation specificity of mitochondria1 tRNA species lacking structural characteristics of canonical tRNAs. A recent study on bovine mitochondria tRNASer(see Fig. 3) showed that the Tloop region is responsible for the aminoacylation by the homologous mitochondrial SerRS (391). The discriminator base is not involved in identity, in contrast to what was found for E . coli tRNASer(see Fig. 13).Considering also the special properties of mitochondria1 synthetases (131),different evolutionary origins of serine-specific tRNAs may be postulated (391). 4. AMINOACYLATIONOF CANONICAL TRNAs WITH ALTERED SIZE

Interesting structure/function relationships arise from studies on canonical tRNAs with longer or shorter sequences. With yeast tRNAA5p,a 15nucleotide long extension at its 5’ terminus does not affect its aminoacylation (392). The crystallographic structure of the tRNAASp-AspRScomplex clearly shows that continuation of the nucleotide chain at the 5’ side of the tRNA does not hamper interaction with the synthetase. If this 5’ extension is allowed to base-pair with the acceptor stem, a significant reduction in aminoacylation occurs 0. Puglisi and R. GiegC, unpublished results). Thus, the presence of a full complement of identity nucleotides is not sufficient for aminoacylation if the tRNA adopts alternate conformation. Less variability is tolerated at the 3’-CCA terminus of tRNA. In E . coli tRNAfMet,methionine acceptor activity is lost when the 3’ terminus is shortened by removal of the discriminator base A,,, or when it is lengthened by insertion of a CA dinucleotide between the discriminator base and the CCA sequence; if only one A residue is inserted, the aminoacylation activity is maintained with similar K , and k,,, reduced only by a factor of 3 (393).This suggests a conformational rearrangement of the amino-acid-accepting domain of the tRNA upon complexation to MetRS, in agreement with the present view on complexes with class-I synthetases. Considering the different binding of tRNAs to class-I1 synthetases, it would be interesting to see if a greater effect on aminoacylation is observed with a similar 3‘-lengthened transcript.

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Anticodon loop size has different effects on the aminoacylation ability of tRNAs. In tRNAs where anticodon residues that contact synthetases are not identity nucleotides, the size of the loop can be varied without affecting the aminoacylation kinetics [e.g., in yeast tRNAAla,this size was varied from six to eight nucleotides (394) and in bean tRNALeu(C*u) from seven to eleven nucleotides (395)].This is not the case for yeast tRNAPhe,where anticodon residues participate in identity (see Section 11,D).An interesting observation concerns frameshifting by an E. coli tRNAG1ymutant that contains an additional C in the CCC (glycine) anticodon (396). This mutant is charged by GlyRS at a slightly reduced rate, despite the structural alteration of the anticodon loop in which C,, is a glycine identity element (345). Little is known of the consequences of altering the T-loop size (seven nucleotides in canonical tRNAs) and amirioacylation activity. A study on a glycine frameshift mutant that has a nucleotide insertion between U, and C,, suggests that changing the T-loop structure of this tRNAG1ydoes not affect too severely its charging by GlyRS (397).Variations in the length of the variable region in bean tRNALeUhas a severe effect on its aminoacylation, although this region seems not to be in contact with LeuRS (395). Also, insertion of two nucleotides into the variable stem of E. coli tRNATyr generates serine-charging activity (298). The effect on alterations in the stem size of tRNA on aminoacylation is not well-known, and only fragmentary information arising from studies on tRNAs with unconventional structures is available. Selenocysteine tRNASec, possessing an extra base-pair in the amino-acid-accepting stem, is charged by SerRS at the catalytic efficiency of tRNASer,whereas removal of this base-pair improves the charging; interestingly, shortening the extra-loop stem also strongly impairs serine charging (137).

E. Effects of Conformational Features on Aminoacylation Efficiency

The relationship between conformation of canonical tRNAs and aminoacylation efficiency has often been addressed but has seldom been subjected to direct experimentation. The simplest method to evaluate this relationship is to study quantitatively the biological activity of tRNA variants containing a defined set of identity nucleotides with various alterations in their conformational frames. Most recent experiments on these lines have been conducted on a series of tRNA transcripts specific for aspartate and phenylalanine in S. cereoisiae. A possible role of nucleotides involved in maintaining the tertiary folding of these tRNAs could not be clearly established. For instance, inverting the constant G,,-C, tertiary base-pair in tRNAPheto a C ,,*G, pair (125),or the semiconserved Levitt pair A,,*U48 to

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U,,*A,, in tRNAASp(398) did not significantly affect the kinetic specificity constants of the variants. A more drastic alteration, the deletion of residue C,, in the T-stem of tRNAAsp,led only to a 17-fold decrease of k,,,lY, (398);this rather moderate effect was solely due to an increase of &, k,, being unaffected by the deletion. Mutations that affect the stability of the D-stem of tRNAAsp include certain conserved tertiary interactions (e.g., A,,.U9) (3984 These mutations resulted in a disturbed tertiary structure and significantly reduced the aspartylation of the corresponding transcripts. However, aspartylation activity was considerably improved upon further mutation to stabilize the D-stem secondary structure in which the G,,.U,, base-pair is an identity element (43). This shows the importance of tRNA tertiary structure for maintaining the base-paired conformation of the RNA, not only a precise three-dimensional folding. To study more global effects, one must introduce conformational features from one tRNA into another one. The structure of yeast tRNAPhehas been modified by transplantation of foreign tertiary interactions. These transplantations had little effect on the steady-state aminoacylation kinetics catalyzed by PheRS (125).In the case of yeast tRNAAsp,its conformation was altered by transplantations of structural features present in tRNAPhe. These transplantations were based on the differences in length of the variable region (four nucleotides in tRNAAsp and five in tRNAPhe) and in the sequence arrangement of the D-loops around the two conserved G , , and GI, residues (three or four, and three or two residues in the a or p regions in tRNAASpand tRNAPhe, respectively). As a consequence, the precise location of the D-loop with respect to the T-loop, which is governed by the G,,.C,, pair, is different in these two tRNAs. As anticipated, tRNAAFpmutants in which these tRNAPhestructural features were progressively introduced, showed progressive conformational alterations, as estimated by Pb2+ mapping (113).These variants were all efficiently recognized and aminoacylated by yeast AspRS, indicating that this synthetase can tolerate structural variability of its specific substrate (113, 398). The relation between conformation and expression of identity can be studied by the effects of permutation of identity nucleotides between tRNAs. Again, because of their conformational differences, experiments were conducted on yeast tRNAASpand tRNAPhe(113).The kinetic data summarized in Table VII indicate that (i) transplantation of identity sets is not suf€icient to confer to the host tRNA an optimal new specificity, (ii) synthetases are variably sensitive to the conformation of their tRNA substrates (PheRS has more stringent prerequisites for tRNA charging than AspRS), and (iii) manipulation of the tRNA conformation can overcome some of the constraints that

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TABLE VII EFFECTS OF PERMUTATIONS OF tRNA SPECIFICITIES ON THE KINETICS OF TRANSPLANTED tRNAs tRNAS

kcat

Km

(m-9

(nM)

bfim)rel

OF AMINOACYLATION

L

m l a u o n bv veast AS0RS tRNA4 tRNA‘4-e

0.5 0.4

phenvldanvlationbv veast PheRs tRNAm 2.0

50 450

1 0.086

1 12

1 111

tRNAMVw.l)*

0.053

360 1025

1 0.009

tRNAhHVw.2-

0.3 0.19

1600 150

0.030

30

tRNAhP(Vx3wk

0.045

22

tRNAhP(Vw.4-

0.50

880

0.1

10

...........................................................................................................

Data are taken from (113). In variant 1, phenylalanineidentity nucleotide G20 replaces C2od in the tRNAm sequence (a=3;p=3) and is located between U20 and A21 ;in variant 2, G20 is between GI9 and C2od; in variant 3, residue U20 is deleted after (319 and transplanted 5’ of G18 so that to have an a 4 ,j3=2 D-loop arrangement l i e in tRNAphe;furthennore U47 is inserted in the variable region ;in variant 4. is as variant 3, but with replacement of u13 by c13 and of A46 by G46 thus changing the (13.22)46 triple from (U13-G22)&.6 in tRNAM to (C13’G22)-G46as in tRNAh (see Table I). L is the loss of catalytic activity defined as the inverse of (k&K&el.

attenuate the efficiency of the specificity switch (for instance in the Asp -+ Phe specificity switch by changing in tRNAAspthe triple interaction (13.22). 46, which involves two identical purine residues at positions 22 and 46 in most cytoplasmic tRNAs possessing a five-nucleotide variable region (see Table I), from (U,,*G22).A46 to (C,,*G,)*G46 as in tRNAPhe).A similar conclusion could be drawn when converting yeast tRNATyr into an efficient substrate for yeast PheRS (272): efficient phenylalanylation requires a combination of the tertiary structure and of specific effects mediated by the contacts of the identity nucleotides with PheRS. Finally, since conformational changes of tRNA are a necessity for expression of identity (see Section 111),it could be postulated that any mutation that hinders or prevents the conformational adaptation of a tRNA to its cognate synthetase would have a detrimental effect on aminoacylation. This concept has been verified experimentally in the glutamine system in the case of base-pair 1.72 (336).

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F. Identity Antideterminants The concept of negative discrimination for ensuring specific aminoacylation is well-accepted and may be a biological necessity because of the structural and functional similarities in aminoacylation systems. Whereas identification of positive identity determinants is straightfonvard, that of antideterminants is more subtle, Conceptually, any structural feature in a tRNA can play the role of a blocking element for recognition by a noncognate synthetase. It is simplest to assay the role of modified nucleotides, because comparative aminoacylation assays can be performed on wild-type fully modified tRNAs and on the corresponding in uitro transcripts deprived of the modifications. Detection of negative discrimination may be directed by the knowledge of mischarging reactions (see Section 11,A). Following this rationale, it was found that yeast tRNAAsp transcripts have a very high propensity to be mischarged by noncognate ArgRS, while keeping their aspartylation properties unchanged (52).This clearly demonstrates that post-transcriptional modifications in this tRNA contribute to negative selection by preventing tRNAASp from being recognized by noncognate ArgRS. Inspection of tRNAAspand tRNAArgsequences suggests that the methyl groups of mlG,, or m5C,, could be good candidates for preventing productive interaction of tRNAAspwith ArgRS. Similarly, a single post-transcriptional modification in E . coli tRNATle, changing C,, to L,, prevents this tRNA from being mischarged by MetRS (51).Interestingly, in that last case, the lysidine modification acts as well as a positive determinant for IleRS (51). Only limited and indirect evidence is available on standard nucleotides in tRNAs that could act as antideterminants [e.g., G,, in noncognate tRNAs would prevent their recognition by E . coli MetRS (23)];similarly, nucleotides in the amino-acid-accepting stem of noncognate tRNAs hinder methionylation, since minihelices derived from such tRNAs are inactive (380). From a general point of view, antideterminants may be detected by structural comparison of different tRNAs in which a same identity set has been transplanted and that exhibits differential aminoacylation efficiencies. This approach has not yet been explored systematically. It should also be noticed that interpretation of experiments may be difficult, and that a clear distinction between negative and positive discrimination is not straightforward. Architectural elements of tRNA may act as antideterminants. This is illustrated by the improved valylation of the TYMV RNA when its tRNA-like structure is well accessible to ValRS: mutating residue U,, to C,,, at the junction of the amino-acid-accepting and anticodon branches of the tRNAlike domain, drastically impairs valylation capacity of the variant because it base-pair that brings the remaining viral allows formation of a new G,,.C,,

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RNA closer to the tRNA-like domain and consequently hinders interaction with ValRS (387). Transcripts of TYMV RNA with 5’ extensions of various lengths have lower aminoacylation activities than the minimal tRNA-like domain, suggesting that these extensions introduce steric hindrance for ValRS recognition (385).Transcripts of yeast tRNAAspwith a 5‘ extension are less arginylated than canonical transcripts, indicating that the extension is an antideterminant for ArgRS (392).Similarly, the large extra arm of class-11 tRNATyr containing a G,.U,, pair specific for alanine, has been proposed to act negatively in interactions with AlaRS (339).

G. Evolution and Relation between tRNA Aminoacylation Systems Because of the structural diversity of synthetases and identity sets within tRNAs, it appears at first glance that aminoacylation systems are idiosyncratic. This may only be apparent; it may be the result of an evolution that has disrupted an underlying primordial unity, but has led to the best compromise allowing tRNAs and synthetases to fulfill their present-day diverse biological functions. If so, one should find close relationships between aminoacylation systems, and, as a consequence, one should consider the possibility of a recognition “code” between tRNA and synthetases. This view was defended recently by de Duve (5, 399), but was rejected by others, presumably because such a “code” could not have the esthetic simplicity of the three-letter genetic code that is based on complementary nucleic acidnucleic acid interactions in a double-helix conformation. Such simplicity cannot exist in tRNA-synthetase recognition because RNA-protein recognition necessitates more sophisticated interaction schemes. However, whatever the semantic meaning of the word “code,” we propose that functional relationships between tRNA aminoacylation systems reflect the existence of related structural recognition mechanisms (taken in a broad sense) that for most have yet to be deciphered. Experimental and theoretical arguments support this view. First, the existence of mischarging reactions on wild-type tRNAs and on suppressor tRNAs that often exhibit multiple amino-acid specificities in viva indicate relationships between various identity sets. The existence of such relationships is illustrated by specificity changes of certain tRNAs that can occur after a single mutation [e.g., in the Trp * Gln switch (259)] or by the functional role of discriminator nucleotides at position 73 (see Section 11). Second, the evoIutionary conservation of tRNA structure and, more important, that of variable (and semiconstant) positions (400) that correlate with identity positions for aminoacylation (Fig. 16)statistically imply that identity sets for a given amino-acid specificity, and accordingly the relationships between aminoacylation systems, should be conserved during evolution. Conservation of identity sets is explicitly demonstrated for alanine (359) and in part for phenylalanine (360)identities. However, deviations from this

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3' Amino acid acceptor stem

T-stem

D-stem

Anticodon stem FIG. 16. Evolution of tRNAs and aminoacylation identities. The figure displays the location of constant (in black), semiconstant (in grey), and variable (in white) purine or pyrimidine positions in the cloverleaf structure of canonical tRNAs participating in protein synthesis. It emphasizes the location of identity nucleotides (indicated by crosses, X) mainly at variable and semiconstant positions. Adapted from Eigen et al. (400, copyright 1989 by the AkAS) and updated with recent data on tRNA identities. The figure indicates position -I (in brackets), which is an identity position for histidine-specific tRNAs; it is recalled that D-loops are of variable size and that the large variable region in class-11 tRNAs (see Fig. 1) does not contain constant or semiconstant residues.

trend have been reported. An E . coli tRNATyr is a leucine-specific tRNA in S . cerevisiae, suggesting that the determinants for tRNATyr identity are not conserved between yeast cytoplasm and E . coli (401) and likewise a mutant of E . coh initiator tRNAMetis a tyrosine-specific tRNA in yeast (361).However, such nonconservation of identities in evolution could arise from negative discrimination mechanisms mediated by antideterminants that may hide expression of cryptic positive signals. The evolution of tRNAs (49, 400, 402), synthetases (17, 176-180b), and more generally the origin of protein synthesis (403-405) is complex. The ranking of synthetases into two distinct classes based on structural dif-

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AL.

ferences in the CCA recognition domain suggests two distinct evolutionary origins of these enzymes. The recent finding of an aminoacyl esterase activity of the Tetrahymena ribozyme, opens the possibility that the first synthetase could have been an RNA molecule (406). Multiple evolutionary origins of their RNA substrates is also a likely possibility. Canonical tRNAs involved in protein synthesis are probably derived from a common ancestor. The fact that RNA minihelices can be specifically aminoacylated (373) suggests that simplified RNA structures could represent such an ancestor. However, nature has found other structural solutions as efficient RNA substrates for synthetases. This is the case of the viral tRNA-like domains and of the tRNA-like structures present in certain messenger RNAs. One can thus wonder whether other structural solutions for aminoacylation systems would be possible. This question can be approached experimentally for the RNA substrates of synthetases by taking advantages of in uioo or in uitro selection methods (407-409). Experiments have recently been reported for both in viuo (126) and in uitro (115) approaches, showing that functional tRNAs possessing noncanonical structural features can be selected in E. coli cells from a degenerated tRNA gene (126) or from an in vitro selection procedure based on Pb2+ cleavage (115). The evolution of mitochondrial tRNAs and their relation to canonical tRNAs raises interesting and as yet unsolved questions. Indeed, many mitochondrial tRNAs deviate markedly from canonical tRNA and exhibit a large sequence variability at the level of the conserved residues required for their tertiary structure, or even are lacking entire domains (see Section 1,B). It is not clear whether the simplified versions of mitochondrial tRNAs contain vestigial features of ancient tRNAs or are derived from canonical tRNA. Another unsolved problem concerns the lack of some tRNA genes in mitochondrial genomes. It has been shown in several cases that to overcome this drawback, mitochondria import cytoplasmic tRNA (410).We suggest that they could use an additional way to increase the informational content of their genomes. By epigenetic processes, two specificities could arise from a unique tRNA species, depending upon its post-transcriptional modification content. As found in E. coli, bean mitochondrial tRNA"' can be charged by IleRS or MetRS, depending on the presence or absence of lysidine at position 34 of its anticodon (411).

V. General Conclusions and Perspectives The present view on the specificity and efficiency of tRNA aminoacylation, especially when it is seen from the tRNA side, can be summarized as follows. (i) The specificity of interaction between tRNA and synthetases is not absolute. Binding energy is due to electrostatic and hydrophobic forces. In

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specific complexes, tRNAs undergo substantial conformational changes. Such changes occur frequently in nucleic acids interacting with proteins (412). (ii) The way a tRNA approaches and contacts synthetases seems to be related to the synthetase class as deduced from the structures of the glutamine and aspartate complexes. The recognition of the amino-acid-acceptor tRNA helix would occur through the minor groove side for class-I synthetases and the major groove side for class-I1 synthetases. (iii) Specific tRNA aminoacylation is more governed by kinetic (kcat) than by binding (K, or Q)effects. Thus, discrimination is best achieved during the transition state of the aminoacylation reaction. However, aminoacylation accuracy is also linked to intracellular levels of tRNAs and synthetases, as well as correction mechanisms. (iv) A limited number of nucleotides (the determinants) contribute to tRNA identity. These often include the anticodon triplet and the discriminator residue at position 73 (413). In some systems (e.g., alanine) the anticodon is not involved in aminoacylation. Identity nucleotides express their action differently: this is reflected by differences of several orders of magnitude in kcat/& values when comparing the effects provoked by mutations at identity nucleotides. These effects are modulated by the nature of the nucleotide introduced at the mutation point. Tests show identity nucleotides in contact with synthetases, but all contacts are not identity signals. Major identity elements seem to be conserved in evolution (as for alanine identity), but this may not be a general rule (as for tyrosine identity). (v) Identity switches occur by transplantation of identity determinants. Permutations of identity sets between tRNAs normally recognized by synthetases belonging to class I and class I1 are possible. (vi) Negative signals, the antideterminants, can prevent aminoacylation of a tRNA by a noncognate synthetase. These signals may be linked to the presence in tRNA of modified nucleotides, to conformational features, or just to canonical nucleotides in which specific chemical groups act as blocking elements. (vii) In many systems, modified nucleotides are not directly involved in the aminoacylation function. In particular, this is the case in viral tRNA-like structures, which have no such nucleotides. (viii) Aminoacylation efficiency is sensitive to the precise conformation of a tRNA. However, severe variations in tRNA architecture do not necessarily prevent aminoacylation. (ix) The complete Lshaped structure of a tRNA is not required for aminoacylation, provided that identity signals are present in minisubstrates bearing a CCA-accepting end. This view was strengthened by the recent finding that a polyU terminating with a CCA sequence is chargeable by LysRS (414). In this artificial RNA substrate, the 5‘ end of the ployU tail mimicks the tRNALys anticodon determinants.

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(x) Evolution has retained several structural solutions for the RNA substrates of synthetases (canonical tRNAs, bizarre mitochondria1 tRNAs, viral and messenger tRNA-like structures, etc.). For RNAs aminoacylated by a given synthetase, identity determinants are located at topologically similar positions within the nucleic acid structure. (xi) No clear correlation has emerged to date between identity sets and synthetase classes. However, a functional difference, which correlates partly with synthetase classes, exists between tRNAs with identity elements in the amino-acid-acceptor stem (where the anticodon is not, or only moderately, involved in identity) that are charged by class-I1 synthetases, and tRNAs for which anticodon nucleotides are strong determinants and are charged by class-I synthetases. This correlation was first perceived by Schimmel, based on arguments coming from minihelix amihoacylation experiments (179), and is confirmed by the more recent studies on minihelices (see Section IV,D). Another correlation is related to aminoacylation of free adenosine or CCA upon conformational activation of synthetases by the core of tRNA lacking the CCA (191): while class-I ArgRS and ValRS charge only CCA, class-I1 AspRS and PheRS are able to aminoacylate both free adenosine and CCA, thus suggesting different recognition mechanisms of the acceptor end of tRNA by class-I and class-I1 synthetases. What are the perspectives for future research on tRNA aminoacylation? It is clear that complete sets of identity nucleotides for all amino-acid specificities should be identified. With the increasing number of studies on E. coli tRNAs, this will probably soon be achieved for a prokaryotic organism. It should also be done for an eukaryote, probably yeast, and an archaebacterium, a phylum not yet explored. Studies on tRNA identities should have their counterparts on the protein side. This will be more difficult, but is already under way on a few systems, especially those for which crystallographic data are or will be available. Likewise, the mechanisms of negative discrimination should be studied more thoroughly. As for tRNA, research on synthetases should permit design of minienzymes containing only the catalytic and tRNA recognition sites. It can then be expected that general rules will emerge demonstrating the relationship between tRNA aminoacylation systems and their evolutionary origin. The conceptual frame on which future work will be based will certainly rely on the dichotomy between class-I and class-I1synthetases, and an important issue will be to understand this partition in relation to tRNA structure and tRNA specific aminoacylation. ACKNOWLEDGMENTS We thank all our colleagues from Strasbourg and elsewhere for many discussions over the years on tRNA structure and recognition by synthetases. This work was supported by grants

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from the Centre National de la Recherce Scientifique (CNRS), the Minist6re de la Recherche et de 1’Espace (MRE), Universite Louis Pasteur (Strasbourg, France), and the Human Frontier Science Program.

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350u. E. Tinkle-Peterson and 0. C. Uhlenbeck, Bchem 31, 10380 (1992). 351. J. Normanly, T. Ollick and J. Abelson, PNAS 89, 5680 (1992). 352. L. H. Schulman and H. Pelka, NARes 18, 285 (1990). 353. T. Hasegawa, M. Miyano, H. Himeno, Y. Sano, K. Kimura and M. Shimizu, BBRC 184, 478 (1992). 354. H. Himeno, T. Hasegawa, H. Asaham, K. Tamura and M. Shimizu, NARes 19, 6379 (1991). 355. M. Pak, L. Pallanck and L. D. H. Schulman, Bchem 31, 3303 (1992). 356. K. Tamura, H. Himeno, H. Asahara, T. Hasegawa and M. Shimizu, BBRC 177, 619 (1991). 357. J. Rudinger, thsse d e I'Universit6 Louis Pasteur, Strasbourg, France, 1992. 358. B. Senger, L. Despons, P. Walter and F. Fasiolo, PNAS 89, 10768 (1992). 359. Y. M. Hou and P. Schimmel, Bchem 28, 6800 (1989). 359a. T. Meinnel, Y. Mechulam, G. Fayat and S. Blanquet, NARes 20, 4741 (1992). 360. I. A. Nazarenko, E. Tinkle-Peterson, 0. D. Zakharova, 0. I. Lavrik and 0. C. Uhlenbeck, NARes 20, 475 (1992). 360a. N. A. Moor, I. A. Nazarenko, V. N. Ankilova, S. N. Khodyreva and 0. Lavrik, Biochimie 74, 353 (1992). 360b. C.-P. Lee, M. R. Dyson, N. Mandal, U. Varshney, B. Bahramian and U. L. RajBhandary, PNAS 89, 9262 (1992). 36012. T. Meinnel, Y. Mechulam, C. Lazennec, S . Blanquet and 6. Fayat, J M B 229, 26 (1993). 361. C. P. Lee and U. L. RajBhandary, PNAS 88, 11378 (1991). 361a. B. L. Seong, C. P. Lee and U. C. RajBhandary, JBC 264, 6504 (1989). 361b. U. Varshney, C. P. Lee and U. L. RajBhandary, JBC 266, 24712 (1991). 362. H. Uemura, M. Imai, E. Ohtsuka, M. Ikehara and D. So11, NARes 10, 6531 (1982). 363. C. Florentz, T W. Dreher, J. Rudinger and R. Gieg6, EJB 195, 229 (1991). 364. C. Francklyn, J.-P. Shi and P. Schimmel, Science 255, 1121 (1992). 364a. Y.-M. Hou and P. Schimmel, Bchem 31, 10310 (1992). 365. T. Meinnel, Y. Mechulam, F. Dardel, J. M. Schmitter, C. Hountondji, S. Brunie, P. Dessen, G. Fayat and S. Blanquet, Biochimie 72, 625 (1990). 365a. 6. Gosh, H. Pelka and L. Schulman, Bchem 29, 2220 (1990). 366. T. Meinnel, Y. Mechulam, D. LeCorre, M. Panvert, S . Blanquet and G. Fayat, PNAS 88, 291 (1991). 367. A. Garcia, These de I'Universit6 Louis Pasteur, Strashourg, France, 1990. 368. L. Despons, P. Walter, B. Senger, J. P. Ebel and F. Fasiolo, FEES Lett. 289,217 (1991). 368a. L. Despons, B. Senger, F. Fasiolo and P. Walter, J M B 225, 897 (1992). 369. D. Fourmy, Y. Mechulam, S. Brunie, S. Blanquet and G . Fayat, FEBS Lett. 292, 259 (1991). 370. Y. Mechulam, F. Dardel, D. LeCorre, S . Blanquet and G. Fayat, J M B 217, 465 (1991). 371. N. Seeman, J. M. Rosenberg and A. Rich, PNAS 73, 804 (1976). 372. B. Roe, M. Michael and B. Dudock, Nature 246, 135 (1973). 373. C. Francklyn, K. Musier-Forsyth and P. Schimmel, EJB 206, 315 (1992). 374. C. Francklyn and P. Schimmel, Nature 337, 478 (1989). 375. Y. M. Hou, C. Francklyn and P. Schimmel, TZBS 14, 233 (1989). 376. J.-P. Shi, S . A. Martinis and P. Schimmel, Bchem 31, 4931 (1992). 377. K. Musier-Forsyth, S. Scaringe, N. Usman and P. Schimmel, PNAS 88, 209 (1991). 378. J. P. Shi, C. Francklyn, K. Hill and P. Schimmel, Bchem 29, 3621 (1990). 379. J. Rudinger, C. Florentz, T. Dreher and R. Gieg6, NARes 20, 1865 (1992). 380. S. A. Martinis and P. Schimmel, PNAS 89, 65 (1992). 380a. S. H. Kim and P. Schimmel, JBC 267, 15563 (1992).

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T. Meinnel, Y. Mechulam, S. Blanquet and G. Fayat, JMB 220, 205 (1991). T. W. Dreher and T. C. Hall, ] M B 201, 41 (1988). S. Joshi, F. Chapeville and A. L. Haenni, NARes 10, 1947 (1982). C. Florentz and R. Gieg6, JMB 191, 117 (1986). T. W. Dreher, C. Florentz and R. Gieg6, Biochimie 70, 1719 (1988). T W. Dreher, C.-H. Tsai, C. Florentz and R. Gieg6, Bchem. 31, 9183 (1992). R. Gieg6, J. Rudinger, T. Dreher, V. Perret, E. Westhof, C. Florentz and J.-P. Ebel, BBA 1050, 119 (1990). 388. R. M. W. Mans,M. H. Van Steeg, P. W. G. Verlaan, C. W. A. Pleij and L. Bosch, JMB 223, 221 (1992). 389. R. L. Joshi, F. Chapevilee and A. L. Haenni, NARes 13, 347 (1985). 390. F. Garcia-Arenal, Virology 167, 201 (1988). 391. T. Veda, Y. Yotsumoto, K. Ikeda and K. Watanabe, NAAes 20, 2217 (1992). 392. V. Perret, C. Florentz and R. Gieg6, FEBS Lett. 270, 4 (1990). 393. T. Doi, H. Morioka, J. Matsugi, E. Ohtsuka and M . Ikehara, FEBS Lett. 190, 125 (1985). 394. Y. Jin, M. Qiu, W. Li, K. Zeng, J. Bao, P. Gong, R. Wu and T.P. Wang, Anal. Biochem. 161, 453 (1987). 395. I. Small, L. Mar&hal-Drouard, J. Masson. G. Pelletier, A. Cosset, J.-H. Weil and A. Dietrich, EMBO J. 11, 1291 (1992). 396. D. L. Riddle and J. Carbon, Nature N B 242, 230 (1973). 397. D. J. O’Mahony, B. H. Mims, S. Thompson, E. J. Murgola and J. Atkins, PNAS 86,7979 (1989). 398. R. Gieg6, C. Florentz, A. Garcia, H.Grosjean, V. Perret, J. D. Puglisi, A. Th6obaldDietrich and J. P. Ebel, Biochimie 72, 453 (1990). 398a. J. D. Puglisi, J. Pirtz, C. Florentz and R. GiegB, NARes 21, 41 (1993) 399. C. de Duve, “Construire une Cellule: Essai sur la Nature et I’Origine de la Vie.” InterEditions, Brussels, 1990. 400. M. Eigen, B. F. Lindemann, M. Tietze, R. Winkler-Osawatitsch, A. Dress and A. Von Haeseler, Science 244, 673 (1989). 401. H. Edwards, V. Tr6z6guet and P. Schimmel, PNAS 88, 1153 (1991). 402. M. Eigen and R. Winckler-Oswatitsch. Naturwissenschoften 68, 217 (1981). 403 F. H. C. Crick, S. Brenner, A. H u g and G. Pieczenik, Origins Lfe 7, 389 (1976). 404. A. Danchin, Prog. Bwphys. Mol. B i d . 54, 81 (1990). 405. J. T.-Z. Wong, Origins Lafe Evol. Biosphere 21, 165 (1991). 406. J. A. Piccirilli, T S. McConnell, A. J. Zaug, H. F. Noller and T. R. Cech, Science 256, 1420 (1992). 407. G . F. Joyce, Gene 82, 83 (1989). 408. A. D. Ellington and J. W. Szastak, Nature 346, 818 (1990). 409. C. Turek and L. Gold, Science 249, 505 (1990). 410. A. Dietrich, J. H. Weil and L. Markhd-Drouard, Annu. Rev. Cell B i d . 8, 115 (1992). 411. F. Weber, A. Dietrich, J.-H. Weil and L. Markhal-Drouard, NARes 18, 5027 (1990). 412. T. A. Steitz, Q. Reo Bbphys. 23, 205 (1990). 413. M. Shimuzu, H. Asahara, K. Tamura, T. Hasegawa and H. Himeno, J. Mol. E d . 35,436 (1992). 414. A. M. Khvorova, Yu. A. Motonn, A. D. Wolfion and K. L. Gladilin, FEBS Lett 314,256 (1992).

381. 382. 383. 384. 385. 386. 387.

Evolution of Ca2 dependent Animal Lectins’ +

KURT DRICKAMER Department of Biochemistry and iWolecular Biophysics Columbia University New York, New York 10032

I. Classes of Animal Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

................ ....................

11. Ca2+ -dependent Carbohydrate-Recognition Domains

A. Groups of Caz+-dependent Lecgns . . B. Shared Structural Features of C-type C Domains . . . . . . . . . . . . . ..... ...................... C. Divergence of Carbohydrate-Recognition Domains . . . . . . . . . . . . . . . 111. Organization of C-type Animal Lectin Genes . . . . . . . . . .......... A. Positions of Introns ................... .............. B. Exon Shuffling and Lectin Evolution ....................... IV. Evolution of Saccharide-binding Specifity A. Experimental Analysis of Sugar Bindin B. Sequence Correlates of Saccharide-Binding Specificity . . . . . . . . . . . . C. Convergent Evolution to Achieve Related Binding Specificities ....................... ...................... D. Specificity for Complex Oligosaccharides ........................ V. History of Carbohydrate-Recognition Processes . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

207 208 208 212 214 216 216 218 220 220 222 225 227 229 230

1. Classes of Animal Lectins Compared to the relatively clearly defined roles of proteins and nucleic acids, the functions of carbohydrates are not well understood. It is certainly true that, like proteins, they serve diverse roles in multiple contexts. Largely homopolymeric polysaccharides such as glycogen, cellulose, and chitin provide energy storage and structure. GIycosaminoglycans, consisting of slightly more complicated arrays of sugars, endow cartilage and other tissues with important physical properties such as the ability to retain water. Complex carbohydrates, composed of five or more types of rnonsaccharides, are often found as conjugates with proteins and lipids. Diverse oligosaccharides are exposed at the cell surface attached to both proteins and lipids, and a similar variety of sugars are found on secreted proteins destined for the extracelluIar matrix and extracellular fluids such as serum (I). The precision with which these polymers are assembled in the endoplasmic reticulum and Golgi apparatus (2)suggests that their precise structures must be important 1

Abbreviations: CRD, carbohydrate-recognition domain; EGF, epidermal growth factor.

207 Progress in Nucleic Acid Research and Molecular B~ology,Vol 45

CopyriKht 0 1593 by Academic Press, Inc All rights of reproduction in any b r m reserved

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KURT DRICKAMER

and that these polymers must be regarded as potential carriers of information. The effects of carbohydrates on proteins to which they are conjugated can be divided into two classes. In some cases, the attachment of sugars has a direct effect on the activity of a protein (3).An important example of this type of effect is modulation of the activation rate of tissue plasminogen activator by its carbohydrate moiety (4). However, in many cases, recognition of the saccharide by a sugar-binding protein (lectin) is necessary for the ultimate effect of the glycosylation to be manifest. Animal lectins have been identified in various contexts, both as a result of direct searches for proteins with selective sugar-binding activities, and less systematically in the course of investigation of biological recognition processes. In spite of the enormous diversity of animal lectins, analysis of their primary structures reveals that most fall into relatively few categories (5).In all cases, sugar-binding activity is associated with discrete protein modules, of 115-140 amino acids, termed carbohydrate-recognitiondomains (CRDs). Three major groups of animal lectins (P, S, and C-types) contain CRDs with distinct sequence motifs. This grouping has been discussed in detail (6). Animal lectins, grouped together based on structural considerations, also share certain important functional properties. The mannose-6-phosphate receptors (P-type lectins) and thiol-dependent p-galactoside-bindinglectins (Stype lectins) each have relatively restricted specificity for a particular type of sugar structure (7, 8). The C-type lectins, in contrast, are characterized not by a common type of sugar ligand, but by a shared dependence on Ca2 for sugar-binding activity (6). The C-type lectins are also the most diverse in overall organization, since C-type CRDs are found in association with many other protein domains. The diversity in C-type lectin structure and function raises intriguing questions about how such a multiplicity of proteins came to share a similar structural motif (the C-type CRD) and how the CRDs have diverged to react with digerent sets of sugars. It is the purpose of this review to explore our current understanding of the evolution of C-type CRDs as individual protein modules and as components of larger lectin molecules. Our knowledge of Ctype lectins also leads to some insights into the evolution of carbohydrates as recognition markers. +

11. Ca*+-dependent Carbohydrate-Recognition Domains A. Groups of Caz+-dependent Animal Lectins The structures of some C-type lectins are summarized in Fig. 1. In each case, carbohydrate-binding activity can be attributed to the presence of Ctype CRDs. However, the proteins differ widely in overall organization.

EVOLUTION OF

GROUP I\ (GROUP V)

ca2+ -DEPENDENT ANIMAL LECTINS GROUP IV

GROUP VI

209 GROUP 111

GROUP I

8

FN-\I

NNN

...

NNN

C

C

HA

N FIG. 1. Summary of the structures of several groups of C-type animal lectins. Representative structures for four groups of membrane-associated lectins are shown. Groups I1 and V are represented by the chicken hepatic lectin, the homolog of the mammalian asialoglycoprotein receptor. Group IV consists of the selectin cell-adhesion molecules, such as L-selectin. The rnacrophage mannose receptor is the only lectin in Croup VI. Two groups of water-soluble lectins are also depicted, Group 111 lectins (collectins), such as mannose-binding protein, are found in extracellular fluids. Group I CRD-containing proteins are proteoglycans of the extracellular matrix. Other domains present in these molecules include: EGF, epidermal growth factor-like domains; CR, complement-regulatory repeats; FN-11, fibronectin type-I1 repeats; COL, collagen-like sequences; GAG, glycosaminoglycan attachment sites; and HA, hyaluronic acid-binding domains. Reprinted from 9, with permission.

Their biological functions, described in greater detail elsewhere (6), are summarized in Table I. A few salient features of each of the major groups of C-type lectins are indicated here. Group I C-type animal lectins are core proteins of proteoglycans secreted into extracellular matrix by fibroblasts and chondrocytes ( 1 0 , I I ) . The C-type CRD in each of these molecules appears in electron micrographs as a

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KURT DRICKAMER

TABLE I SUBGROUPSOF C-TYPEANIMALLECTINS Functions Endocytic receptors

Evolutionary group I1

V VI Adhesion molecules Hurnod defense

Proteoglycans

IV 111

I

Location

Organization

Ligands

Examples

Plasma mem- Type I1 trans- Endogenous Asialoglycoprotein brane membrane receptor Endogenous Macrophage galactose receptor Endogenous? Kupffer cell receptor Endogenous Chicken hepatic lectin Endogenous Lymphocyte IgE F, receptor Placental mannose Exogenous receptor Exogenous? Natural killer cell receptors Macrophage manPlasma rnem- Type-I trans- Exogenous nose receptor brane membrane Plasma mem- Type-I trans- Endogenous Selectins brane membrane Mannose-binding Extracellular Soluble col- Exogenous proteins lagenous Pulmonary surfactant apoproteins Matrix Extended Endogenous Aggrecan Versican

globular domain located near the carboxyl terminus, sometimes preceded by epidermal-growth-factor- (EGF)-like domains and followed by complementregulatory repeats (12, 13).It is possible that sugar-binding activity of these core proteins plays a role in organization of the matrix, but evidence in support of this idea has not been presented. Group I1 C-type lectins are anchored to cell membranes by an aminoterminal, internal signal-anchor sequence, giving them a type I1 transmembrane orientation. The prototype for this group is the asialoglycoprotein receptor, a hepatic cell-surface protein that binds terminal galactose residues exposed upon partial desialylation of circulating glycoproteins (14).This receptor directs turnover of serum glycoproteins, leading to their internalization and delivery to lysosomes via an endocytic pathway, Other lectins in this group also mediate endocytosis, although their specificity for particular sugars and the cell types in which they are found differ. Most analogous to the hepatic asialoglycoprotein receptor are a galactose-binding homolog in per-

EVOLUTION OF

Ca2+ -DEPENDENT ANIMAL

LECTINS

211

itoneal macrophages (IS), an avian homolog that binds glycoproteins with terminal N-acetylglucosamine [the chicken hepatic lectin (Is)], the Kupffercell fucose receptor (17), and a mannose receptor recently isolated from placenta (18). The dominant features of the Group I11 C-type lectins (collectins) are amino-terminal collagen-like domains and carboxy-terminal CRDs. Several of these lectins, including the mannose-binding proteins, are found in serum, while others are secreted as part of the surfactant that lines alveoli in the lung (19, 20). At least some of these lectins provide a primitive immune response in which “nonself” is distinguished from “self‘ based on the presence of high concentration of certain sugars on the surface of potentially pathogenic microorganisms. Serum mannose-binding protein fixes complement and acts as an opsonin, thus invoking some of the effector functions of the immune system in an antibody-independent manner (21-23). A mutation in the collagenous domain of this protein results in susceptibility to recurrent, severe infections in childhood (24). The recently described selectin cell-adhesion molecules constitute the group IV C-type animal lectins (25-28). These molecules, located on the surface of T-lymphocytes and cells of the vascular endothelium, mediate the initial phase of extravasation of a variety of leukocytes. In this phase, complementary saccharide ligands cause the circulating leukocytes to adhere loosely and roll along the epithelial surface (29). Extravasation follows consolidation of the interaction by integrin-type cell-surface receptors. Lselectin on T-lymphocytes recognizes sugar ligands on high endothelial cells of lymphatic venules, while E-selectin and P-selectin are expressed on endothelial cells at sites of inflammation and bind to saccharides on the surface of neutrophils and monocytes. The only C-type lectin that contains multiple CRDs is the mannose receptor of macrophages [group VI (30)]. This receptor, like the soluble mannose-binding proteins, mediates first-line defense against pathogens, in this case by direct phagocytosis (31-33). The mannose receptor can also mediate uptake of soluble proteins, suggesting that it may have a role in scavenging lysosomal hydrolases that have escaped from cells and lost their mannose-6-phosphate recognition markers (32). Group V C-type CRDs are found in proteins on the surface of natural killer T-cells. They resemble the group 11lectins in overall organization, but have not been shown to have saccharide-binding activity (34, 35). C-type CRDs are also found without accessory domains. Some of the vertebrate protein that consist simply of isolated CRDs form the Group VII C-type lectins. Such proteins, only some of which bind sugars, occur in snake venom (36, 37) and pancreas (38, 39). Other isolated CRDs known to have saccharide-binding activity are found in invertebrate body fluids such as the

212

KURT DRICKAMER

hemolymph or coelomic fluid (40-44). In many cases, they are believed to serve a protective function, although a mechanism for such protection has not been described.

B. Shared Structural Features of C-type Carbohydrate-RecognitionDomains Several experimental definitions of C-type CRDs have been established. At the protein level, the domains are extremely protease-resistant in the presence of calcium, so protease can be used to trim away regions of the parent polypeptide not required for sugar-binding activity (45, 46). Alternatively, truncated segments of the lectin cDNAs can be expressed in uitro, so that activity of lectin fragments can be examined by affinity chromatography on immobilized sugars (47). The results of these experiments are in good agreement. The minimum fragments of the major subunit of the rat asialoglycoprotein receptor and rat serum mannose-binding proteins that display full activity are 134 and 115 amino-acids long, respectively. Comparison of the sequences of C-type CRDs reveals that the only portions similar in structure for all of the lectins shown in Fig. 1are the 115 to 134 amino acids that correspond to the experimentally defined CRDs. Amino-acid residues at 14 positions are completely conserved in 30 C-type CRDs that bind carbohydrate ligands (5, 6). An additional 18 residues are conserved in character. This sequence motif is summarized in Fig. 2(a). From structural analysis of the C-type CRD from rat serum mannosebinding protein, a clearer understanding of the role of the conserved residues of the C-type CRD sequence motif has emerged (48). The structure of this CRD was determined by X-ray crystallography of a fragment of the mannose-binding protein produced in a bacterial expression system. The CRD was complexed with holmium in place of calcium in order to obtain phase information needed to deduce the structure (49).As illustrated in Fig. Z(b), the CRD is characterized by a modest amount of regular secondary structure, all of which is located in the lower two-thirds of the domain. In addition to two a-helices, there are several segments of p-structure; however, only two of these @3 and p4) are paired for an extended distance to form a small P-sheet. The upper one-third of the structure, consisting of irregular loops, is stabilized by the two bound cations. Comparison of the C-type CRD sequence motif with the structure reveals that most of the conserved residues have critical roles in establishing the fold of the CRD. These roles are illustrated in Figs. 2(c) and (d). They include: (i) formation of two disulfide bonds by four cysteine residues, (ii) ligation of the bound cations by aspartate, asparagine, and glutamate residues, (iii)facilitation of turn formation by proline and glycine residues, and (iv) establishment of an organized hydrophobic core by aliphatic and aromatic side-chains.

L4

n

C

f===%

/ FIG.2. Structure of C-type CRDs. (A) Sequence motif characteristic of C-type CRDs is shown below the sequence of the CRD from rat mannose-binding protein A. Absolutely conserved residues are indicated by the one-letter amino-acid code, while residues conserved in character are indicated by 0, oxygen-containing; a, aromatic; 0 , aliphatic; and aromatic or aliphatic. Ligands for Caz+ 1 and 2 are denoted 1 and 2, respectively. (B) Ribbon diagram showing secondary structure elements of the mannose-binding CRD. Spheres 1and 2 represent calcium ions. In (C) and (D), the side chains of residues conserved in all of the C-type CRDs are shown along with the a-carbon backbone of the entire polypeptide. Conserved calcium-ion ligands and disulfide-bonded cysteines are shown in (C), while other, largely hydrophobic sidechains are shown in (D). Parts (B)-(D) adapted from 9, with permission.

a,

214

KURT DRICKAMER

Congruence between the comparative sequence analysis and the structural results strongly indicates that protein domains characterized by the Ctype CRD motif will have similar overall structures. The organization of the upper portion of the domain also explains why the domain is resistant to proteolysis in the presence of calcium, but sensitive to digestion in the absence of divdent cation: the presence of the cation is essential for holding the domain in a compact configuration. Loss of d n i t y for Ca2 , which can be induced by lowering the pH, results in partial unfolding of the domain and consequent loss of affinity for sugar (SO). This sequence of events plays a critical role in the function of the endocytic receptors that contain C-type CRDs, since they must release their ligands in the mildly acidic luminal environment of endosomes (51). +

C. Divergence of Carbohydrate-Recognition Domains Comparison of the C-type CRDs in approximately 50 C-type animal lectins reveals that the sequences of these domains are between 20 and 60% identical. Evaluation of the aligned sequences by cluster analysis (52) leads to a dendrogram such as that shown in Fig. 3. The groups of lectins arrived at, based on the overall degree of sequence similarity within the CRDs, correspond exactly to the groups obtained if the proteins are classified according to their overall structural organization (Fig. 1 and Table I). The dendrogram suggests that all of the proteins in a single group are descended from a common ancestor, and that the common ancestor of each group already had the distinctive organization of domains that characterizes the present day members of the group. The CRDs compared in Fig. 3 are almost all derived from proteins that are known to be lectins, that is, proteins for which selective binding of sugars has been demonstrated. However, the specific sugars bound by different CRDs vary greatly. The relationship between sugar binding and lectin primary structure is explored in more detail in Section IV. At this point it should be noted that a number of proteins containing domains with C-type CRD sequence motifs have not been shown to bind sugars. For example, studies of the lymphocyte low-&nity receptor for the F, portion of IgE (also designated CD23) have failed to detect inhibition of receptor-antibody binding by sugars, whereas competition with peptide mimics of the F, domain i s effective (%,a). These results suggest that the presence of the C-type CRD sequence motif may not necessarily imply that a protein will have a saccharide-binding site. However, the fact that tunicamycin treatment of a B cell line prevents binding of CD2Scontaining liposomes indicates that saccharide may form at least part of some ligands for this receptor (55}, suggesting that the domain may have both sugar- and peptide-specific binding sites. Such dual recognition is discussed in more detail in Section IV,D,2. We are left with a slight ambiguity in the definition of a C-type CRD. In

EVOLUTION OF

Ca2+ -DEPENDENT

215

ANIMAL LECTINS

CARTILAGE FIBROBLAST

I

PROTEOGLYCANS

KUPFFER CELL tFucI

- -

t

1

MACROPHAGE (Gall ASGP (Subunit 1 I ASGP (Subunit 21

TYPE II RECEPTORS

II

CHICKEN (GlcNAc) PLACENTAL (Man)

PANCREATIC PROTEINS VENOM LECTIN

FREECROa VII

VENOM BINDING PROTEIN CRD-4 CRPS CRDB CRDB CAD-2

MANNOSE RECEPTOR

VI

CRD-1

IuI I

CRO-3 CRD-7 MANNOSE-BINDING PROTEINS CONGLUTININ COLLECTINS

111

SP-D PULMONARY SP-A ]SURFACTANT

I IV

NK CELL ANTIGENS

V

FIG. 3. Dendogram summarizing sequence similarity between various C-type CRDs. Similarities were determined based on comparison of amino-acid sequences. The CRDs of the mannose receptor have been segregated from the other proteins for clarity. The horizontal scale cannot be used to infer specific times of evolutionary divergence.

some cases it is convenient to restrict use of the term to protein domains that bind sugars in a Ca2+-dependent manner. At other times in may be more useful to broaden the term to include all domains that share the C-type CRD sequence motif, which are probably similar in tertiary structure to the CRD of the C-type lectins. Fortunately, it is usually clear from the context which meaning is intended.

216

KURT DRICKAMER

111. Organization of C-type Animal Lectin Genes A. Positions of lntrons The sequences of at least the protein-coding portions of approximately 10 C-type lectin genes have been established. In Fig. 4,the positions of introns within genes for representative members of five of the groups of C-type animal lectins are compared with the protein domain boundaries. Except in the macrophage mannose receptor gene, introns delimit each CRD-coding region. A similar correlation of intron and boundaries for other domains in

GROUP I (CHICKEN PROTEOQLVCAN CORE PROTLIN)

EXON

A C D

E F

GROUP I1 (RAT ASIALOOLYCOPROfEIN RECEPTOR1 TM

DOMAIN EXON

T

1234587 8

9

GROUP 111 (RAT AND HUMAN YANNOSE-UNDINQ PROTEINS1 COLLAGEN

EXON

1 2 3

4

GROUP IV (HUMAN 1-SELECTIN)

EGF CH-2 SS UID 1CH-1 JTMTAIL

DOMAIN EXON

1 2

3

4 S 8 78

9

GROUP VI (HUMAN MANNOSE RECEPTOR) CVS-RICH

EXON

1

2

3

4 5 6 7 8 9 1 0 1 1 1 2 1 3 ~ 1 5 T 1 7 t 1 9 2 0 2 1 2 2 1 2 42 5 2 6 t 2 8 T 14 16 18 23 27 29

30

00 0

FIG.4. Comparison of domain and exon organization. Protein domains (above thick line) and exons (below thick line) are aligned relative to the receptor mRNA for typical genes in five of the subgroups of C-type lectins. Untranslated portions of the mRNA are stippled. TM, transmembrane domain. SS, signal sequence. FN-11, fibronectin type-I1 repeat. CH, complement-homology (regulatory) repeat. A scale in nucleotides is provided at the bottom.

EVOLUTION OF

C@+-DEPENDENTANIMAL

LECTINS

217

the lectins is evident. Examples of domains encoded by discrete exons include signal sequences, EGF-like domains, complement-regulatory repeats, and fibronectin type-I1 repeats. In addition to introns that divide CRD-coding regions from other portions of lectin genes, introns interrupt the coding sequences for some of the CRDs. Two introns are found at exactly corresponding positions within the CRD-coding regions of all of the group-I1 genes that have been described, including the genes for the major subunit of the asialoglycoprotein receptor (56),the chicken hepatic lectin (57), the Kupffer-cell fucose receptor (58),and the IgE F, receptor (59). Two introns are found at nearly these same positions in the single group-I gene that has been analyzed, that for the chicken cartilage proteoglycan core protein (60).In contrast, CRDs are encoded by single, uninterrupted exons in the group 111genes for rat and human serum mannose-binding proteins (61,62) and pulmonary surfactant apoprotein (63), as well as all of the group IV selectins (64-67). Correspondence of the intraCRD intron pattern with the classification of lectins based on overall domain organization and sequence comparisons within the CRDs provides additional, independent evidence that each of these groups arose from a single, distinct ancestor. It is difficult to discern a relationship between the positions of introns within CRDs in groups I and 11and the molecular structures of the domains. However, it is interesting to note that the final exon in each of these genes encodes portions of the proteins that are involved in forming the binding sites for Ca2+ #2 and the saccharide ligand. This subdomain includes the two paired P-strands (p3 and p4) and loops 3 and 4 (Fig. 2b). This segment of the protein may have been a somewhat independent, primordial CRD. As noted above, there is no clear exon-domain correspondence for the macrophage mannose receptor (group VI). Distinct exons encode the signal sequence, the NH2-terminal cysteine-rich domain, the fibronectin type-I1 repeat, several spacer regions, and the membrane anchor and cytoplasmic tail (68).However, while CRDs 1, 7, and 8 are each separated from adjacent domains by introns, individual exons encode portions of CRDs 2 and 3, CRDs 3 and 4, and CRDs 5 and 6. Further, there is no conservation of relative intron positions within the various mannose receptor CRDs or when compared to the intron positions in the CRDs of group I and I1 lectins. These observations can be explained by at least three different hypotheses. At one extreme, it is possible that the primordial C-type CRD gene contained roughly 20 introns, and that different exons have been lost in different descendent families (69, 70). A second possibility is that the primordial gene contained a few introns, but the positions of these introns have shifted gradually, by an ill-defined mechanism. Finally, at least some of the introns may have arisen more recently by insertion of DNA by genetic transposition

218

KURT DRICKAMER

(71). It is not possible to distinguish among these possibilities from the currently available evidence. An important consequence of any of these scenarios is that duplication of CRDs that led to the generation of the macrophage mannose receptor must have been an early event, occurring at roughly the same period as the duplications that led to the progenitor CRDs for each of the other groups of C-type lectins. This early divergence is also reflected in the sequence divergence indicated in Fig. 3.

B. Exon Shuffling and Lectin Evolution Although the origin of introns within CRDs is enigmatic, it is clear that

the presence of introns at the boundaries between CRDs is consistent with the suggestion that the lectin genes were assembled by a process of exon shuf€ling (72). It has been proposed that one prerequisite for this type of reorganization is that exons to be joined begin and end in the same positions relative to the reading frames of the amino-acid codons (73). Indeed, many of the introns that correspond to domain boundaries in the C-type lectins are located between the first and second bases of codons, as are the delimiting exon boundaries of many other extracellular protein modules. The fact that this rule does not hold for many of the introns within CRDs is consistent with the hypothesis suggested above that some of the introns, such as those within domains, may have a history distinct from those found between domains, perhaps having arisen from insertional events. An interesting structural analog of the idea that the ends of exons must be compatible in order to give an open reading-frame is the fact that domains that are shuffled must be able to assemble together in structurally compatible as well as functionally useful ways. The extracellular domains of proteins seem to fall into two general categories in this respect: those in which the polypeptide is folded so that the beginning and ending of the sequence are in the same place in space, and those in which the polypeptide starts on one side of the domain and ends on the other side. The C-type CRDs are an excellent example of the former type of organization, since the NH,- and COOH-terminal strands are very close to each other (Fig. Zb). An important consequence of this arrangement is that CRDs at the amino-terminal end of type I transmembrane proteins (i.e., the group IV selectins) and those at the carboxyl-terminal ends of type I1 transmembrane proteins (i.e., the group I1 and V lectins) can be displayed at the cell surface in very similar orientations in spite of the topological differences between these proteins. Although the shuffling events that led to various arrangements of CRDs observed in the present groups of C-type animal lectins occurred relatively early in the evolution of these proteins, some additional shuffling has occurred within the subgroups. For example, different selectins contain differ-

EVOLUTION OF

Ca2+ -DEPENDENT ANIMAL LECTINS

219

ent numbers of complement-regulatory repeats, each encoded by a separate exon (64-67). The number of these repeats must have changed after the selectins diverged as a group from the other C-type lectins. It is possible that the common precursor to the selectins contained no complement-regulatory repeats, and that variable numbers of them were introduced into each member of the group after they diverged from each other. However, it seems more likely that at least one repeat was present in the precursor, and that it was duplicated to different extents in the individual selectin genes. Divergence of the C-type lectins in group I1 is not so easily traced. All the members of this family are characterized by CRDs encoded by three exons, and amino-terminal cytoplasmic tails and internal signal-anchor sequences encoded by two exons. However, there are important differences between various members of the group. Most prominently, the structures of the neck regions between the membrane anchors and CRDs vary widely. The necks encoded by three exons in the asialoglycoprotein receptor (56), one exon in the chicken hepatic lectin (57)and the Kupffer-cell fucose receptor (SS), and four exons in the IgE F, receptor (59) do not bear obvious sequence similarities to each other. Only the neck of the F, receptor shows evidence that at least some of the multiple exons arose by duplication. These observations, combined with the topology of the dendrogram in Fig. 3, suggest that additional exons have been generated or recruited since the time of establishment of the basic type-I1 transmembrane organization and divergence from the other groups of C-type lectins. The most recent evolution of the animal lectins occurred during radiation of the animal kingdom. However, within the mammalian class, there is no evidence for the establishment of distinct organizations for C-type lectins within any subgroups of species. Stated another way, for any C-type lectin found in one mammalian species, homologs with closely similar domain arrangements have been identified in all other mammals investigated. Although lectins in other vertebrates have been less thoroughly investigated, lectins from groups I, 11, and IV have been characterized in birds, indicating that these families were probably established in the common reptilian progenitor of both mammalian and avian vertebrates. As noted above, many invertebrate C-type lectins consist of CRDs alone. The one example of a multidomain C-type lectin from an invertebrate, Limulus clotting factor C, has a unique combination of EGF-like, complement regulatory, and protease domains not observed in any known vertebrate proteins (74. In some cases, the presence of distinct exons encoding different protein domains allows multiple forms of lectins to be produced by alternative splicing of mRNA. One example of this phenomenon is observed in the group I lectins. Three distinct forms of the mRNA for the human cartilage proteoglycan core protein have been described. One form contains a C-type

220

KURT DRICKAMER

CRD flanked by an EGF-like domain on the amino-terminal side and a complement-regulatory repeat on the carboxyl-terminal side. Other forms lack either or both of the EGF domain and complement regulatory repeat (11).Interestingly, no form lacking the CRD has been observed, although the globular “G3” domain that corresponds to the CRD is removed proteolytically as cartilage matures (12). Differential splicing generates alternative forms of P-selectin containing variable numbers of complement-regulatory repeats (66). This protein can also be tethered to the membrane by a carboxyl-terminal stop-transfer sequence or released in soluble form, depending on 3’-exon usage. Alternative use of exons is also observed in the asialoglycoproteinreceptor, in which variant forms of the minor subunit with alternative splicing of both the cytoplasmic tail and the extracellular neck region have been described (75).

IV. Evolution of Saccharide-binding Specificity A. Experimental Analysis of Sugar Binding Examination of the three-dimensional structure of the C-type CRD from mannose-bindingprotein does not immediately indicate how it may interact with sugars. Extensive mutagenesis reveals that residues over much of the surface of the domain can be changed without altering its sugar-binding properties, suggesting that the binding site does not occupy a large area of the protein surface (76). Although changes in the interior of the hydrophobic core of the molecule do reduce sugar binding under physiological conditions, they do so by reducing the affinity of the domain for Ca2+. These results suggest that positioning of the loops in the upper third of the CRD is critical for optimal binding of Ca2 , The fact that raising the concentration of Ca2f compensates for suboptimal orientation of the loops resulting from changes in the lower portion of the CRD indicates that the sugar- and Ca2 binding sites are closely linked. Recent crystallographic analysis of a complex between the mannosebinding protein CRD and a high mannose oligosaccharide reveals that the association between Ca2+ and mannose is indeed very intimate, as hydroxyl groups 3 and 4 of the sugar are direct coordination ligands for Ca2 #2 of the CRD (77). As illustrated in Fig. 5, this complex is stabilized by four sidechains of the protein, two asparagine and two glutamate residues. Each of these side-chains forms a hydrogen bond with one of the hydroxyl groups and also serves as a coordination ligand of the Ca2 . The vital role played by these side-chains is illustrated by the phenotype of a mutant in which AsnIs7 is changed to Asp (76, 77). Although the affinity of the domain for calcium is undected, because the carboxyl group can still form the necessary coordina+

+

+

+

EVOLUTION OF

Ca2 + -DEPENDENT

22 1

ANIMAL LECTINS

H %

GLU U 193

FIG. 5. Structure of an oligosaccharide-CRD complex at calcium ion #2. The calcium ion is shown as a light grey sphere. White, dark grey, and black spheres represent carbon, nitrogen,

and oxygen, respectively. Ca2+ coordination bonds are denoted by long thick dashes, while short dashes represent hydrogen bonds. Numbers on the mannose carbon atoms represent ring positions. The a-glycosidic bond to the next sugar of the oligosaccharide (at carbon 1)has been cut off for clarity. Reprinted from Ref. 9, with permission.

tion bond with Ca2+, the change results in complete loss of sugar-binding activity, since one of the the hydrogen bonds to hydroxyl-3 of mannose cannot be formed in the absence of the amide group to serve as hydrogen donor. Using the structure of the mannose complex as a guide, it is possible to model how other sugars might also bind in the same site. Glucose and N acetylglucosamine share with mannose the equatorial arrangement of 3- and 4-hydroxyl groups, and would thus be expected to bind in the same relative orientation. Modeling studies indicate that these sugars can be accommodated without steric hinderance caused by the equatorial C-2 substituents (77). The other known ligand of the mannose-binding CRD is L-fucose. Because of the inverted stereochemistry of this sugar, it is possible to superimpose its 2- and 3-hydroxyl groups on the 3 and 4 positions of mannose, and

222

KURT DRICKAMER

thus achieve binding in a similar way. Galactose and N-acetylgalactosamine would not, however, be expected to bind because the only pair of equatorial hydroxyl groups in this sugar (at the 2 and 3 positions) are twisted with the opposite handedness of the mannose 3- and 4-hydroxyls.

B. Sequence Correlates of Sacchande-Binding Specificity The evidence presented in Sections I1 and I11 is consistent with the evolution of C-type animal lectins having occurred in the distinct groups illustrated in Fig. 3. However, it is striking that these groups, defined by sequence similarity of CRDs, overall protein structure, and genomic organization, do not define unique saccharide-binding characteristics. Although the proteins in group IV share the ability. to bind the sialyl-Lewis x epitope (78), the proteins in groups I1 and 111have much more divergent spectra of saccharide ligands. For example, the asialoglycoprotein receptor and its closest homologs bind galactose and N-acetylgalactosamine, but not mannose or N-acetylglucosamine, while the chicken hepatic lectin and placental mannose receptor have almost exactly the opposite behavior, in spite of the fact that all of these proteins fall in group 11. Knowledge of the importance of certain residues of the mannose-binding CRD in the binding of sugar makes possible a more informed analysis of the comparative sequences of CRDs with distinct saccharide-binding activities. As shown in Fig. 6, the pattern of residues at the positions of the five ligands of Ca2+ #2 is particularly interesting. The upper portion of the figure illustrates that residues corresponding to GluIg3, AmzE, and Aspzos in the mannose-binding CRD are essentially invariant in the group I to IV proteins. However, the sequence Glu-Pro-Asn (residues 185-187 of the mannose-binding protein) is found only in those CRDs known to bind mannose or glucose derivatives, that is, sugars with equatorial 3- and 4-hydroxyl groups (mannose-type binding). This sequence is also found in the two domains of the macrophage mannose receptor believed to be primarily responsible for ligand binding by this protein. In contrast, CRDs that bind galactose and N-acetylgalactosamine (3-hydroxyl equatorial, 4-hydroxyl axial; galactose-type binding) are characterized by the sequence Gln-Pro-Asn. These results suggest that the amino-acid side-chains on either side of Pro186 are critical determinants of ligand binding on the protein, and confirm the previously noted importance of hydroxyl groups 3 and 4 on the sugar ligand (79). Confirmation of the importance of the Glu-Pro-Asn sequence in the mannose-binding CRD is provided by analysis of a mutant in which this sequence has been changed to Gln-Pro-Asp (80).As expected from the considerations discussed above, this mutant now binds preferentially to galactose (Fig. 7), although it still shows significant interactions with mannose. In overall properties, the behavior of the mutant is actually closest to the CRDs

(nu)

185 187 193 2 0 5 206 1 1 1 11 URPNQPDNFFM----GEDCVI---UHEKGEUNDVPCN-YHLPFTC

FIBROBLAST PG (Hu)

YRPNPPDSFFSA----GEDCWII---UHENCPUIIDVPCN-YHLTYTC

M+ Gal-REC

(Ra)

UAPKPPDNLnGHGLGGGEDCAHFT-----SDCRVm)DVCO-RPYRWC

HEP LEC-1

(Re)

VRPGPPDDWGHGLGGGEDCAHFT-----TDGH~DVCR-RPYRWC

HEP LEC-2/3

UAfTPPDNYPGHEEGGSEDCAEIL-----SDELVLIDI

KUP FW-REC

(Re) (Re)

PLC Man-REC

(Hu)

1 1 1 11 U)(RGEPNNVGE------EDCAEFS------GNGUNDDKCN--LA~lC

HEP LEC

(Ch)

UKEGEPNNRGF-----NEDCAHVU-----TSCPUNDWCT-YECYYVC

MBP-A CWGLUTININ

(Ra)

UKKDEPNDHGS-----GEDCVlIV-----DNGLUNDlSCO-ASHTAVC

(Bo)

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(nu)

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(Mo)

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(Hu)

UGAGEPYNKKS~~--~KEDCVEIYIKRERDSGKUNDDACH~KRKMLC 1 UAPGEPWNRQK-----DEDCVEIYIKREKDVGMUNDERCS-KKKLALC GROUP I V UADNEPNNKRN-NEDCVEIY IKSPSAPGKUIIDEHCL-KKKHALC J

M i ManR CRD-4 M+ ManR CRD-5 M+ ManR CRD-2 Mi ManR CRD-7 M+ ManR CRD-8

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(Hu) (HU)

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(Hu)

UAADEPKLSKA----

CARTILAGE PG

Ga 1

Man GLC

FUC

I I I I I 1 I I

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(HU)

(HU)

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-

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(Hu)

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NK Ly49A

(Mo)

UIDNRPKLALN*YNlRDGGCMLLS-

- - - -SKTCLGLE--KETDFRKWNIYCG-WNPFVC

VENW X / I X BP (Sn) VENW LEC (Sn)

WKNPPDHYQN- - -- -KEFCVELV- -SLTGYRL~DPVCE-SKDAFLC

ACORN BARNACLE 2

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ACORN BARNACLE 3

USSNNPNNEN-------ODCGWN--YDTVTGPUDDDDCN-KNRNFLC

SEA URCHIN FLESH FLY

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TUNICATE

I I I I I I

GROUP V I

’

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GROUP V

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GROUP V I 1

USPNEPSNP-----PSYPLCVOIU----SKYNLLDDVGCG-GARRVIC WDSIPPDNA-----GGNENCGWH-----PNGGLNOIPCP-UKLPFVC

FIG. 6. Alignment of COOH-terminal portions of C-type CRD amino-acid qequences. Arrows indicate five ligands for calcium ion #2 shown in Fig. 5. PG, proteoglycan; REG, receptor; LEC, lectin; MBP, mannose-binding protein; SP, pulmonary surfactant apoprotein; ManR, mannose receptor; f i c , fucose; Ma, macrophage; NK, natural killer T cell; HEP, hepatocyte; KUP, Kupffer cell; PLC, placeneta; ST, stone; BP, binding protein; Hu, human; Ra, rat, Ch, chicken; Mo, murine; Bo, bovine; Do, dog; Sn, snake. Citations for most of the sequences may be found in 57, with additional data derived from 18, 30, 35-44.

224

KURT DRICKAMER

. .

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A QPD MUTANT

0.8

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.

0.2

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0.01 1 10 100 MONOSACCHARIDE CONCENTRATION (mM)

FIG. 7. Monosaccharidecompetition for binding of radioiodinated neoglycuprotein ligands to immobilized wild type and mutant CRDs. In the QPD mutant, the sequence Gin-Pro-Asp has been substituted for the native Glu-Pro-Am in mannose-binding protein. The reporter ligands used are indicated in each panel. Reprinted with permission from Nature (Ref. 80). Copyright 1992 Macmillm Magazines Limited.

EVOLUTION OF

Ca2+ -DEPENDENT

ANIMAL LECTINS

22s

in group I C-type lectins, as it binds only weakly to saccharide-containing resins, and has some a n i t y for sugars with either axial or equatorial 4hydroxyl groups (13).Further mutagenesis is required to define additional residues in the CRD that are necessary for higher affinity and more exclusive binding of galactose and N-acetylgalactosamine. It is also interesting to note that proteins that bind unusually broad varieties of sugars, such as pulmonary surfactant apoprotein SP-A (20). and those not known to bind sugars at all, such as the pancreatic stone protein and natural killer cell surface antigens, usually have other variant sequences in place of Glu-Pro-Asn (Fig. 6). The IgE F, receptor presents an interesting case, since the murine form does contain the Glu-Pro-Asn sequence, but Asn is substituted by Thr in the human receptor. As noted above, the role of saccharides in ligand binding by this receptor remains unclear. In addition, CRDs of the macrophage mannose receptor other than 4 and 5 are believed to contribute only weakly to binding of polyvalent ligands (81), and these CRDs also contain variant sequences. Finally, some invertebrate lectins that bind galactose contain the Gln-Pro-Asp sequence, although others have more divergent structures (40-44). Thus, it must be possible to construct a galactose-specific site in a number of different ways.

C. Convergent Evolution to Achieve Related Binding Specificities From the results in the previous section, it appears that CRDs in groups I, 11, and VII have evolved to achieve selective binding of galactose, using at least partially similar mechanisms, while CRDs in groups 11, 111, and VI have evolved to bind mannose and N-acetylglucosamine in similar ways. This raises the following questions: what did the CRDs in the precursors to each group bind, and what was bound by the common precursors of multiple groups? In attempting to answer these questions, the primary criterion for evaluating various possible pathways of evolution must be parsimony. The most likely route followed would be the one that involves the fewest changes in specificity in all the lineages taken in aggregate. The scheme shown in Fig. 8 accommodates the present-day specificity distribution with only four changes in specificity from mannose-type binding to galactose-type binding (denoted as paths 1 and 2). A common technique in evolutionary biology is to root a comparative tree using an outlying structure. In a qualitative way, this approach can by applied to the I, 11, 111, VI, and VII CRDs using the group IV lectins as the outlier. Since the selectins contain the Glu-Pro-Asn sequence, it is predicted that this sequence, and preferential mannose binding, were characteristics of the original saccharide-binding CRDs. This basic arrangement persists in all of the group-I11 lectins, in group-VI CRDs that retain the ability to bind sugars, and some of the group-I1 CRDs. It is also

226

KURT DIUCKAMER ManlGlcNAclFuc

~

(1I

- GallFuc

111 - GaVFuc

- Gal (41 - GbNAC (2)

ManlGlcNAclFuc

ManlGlcNAclFuc

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4 € ::: z::zl

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PROTEOGLYCANS

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KUPFFER CELL RECEPTOR ASIALOGLYCOPROTEIN RECEPTOR CHICKEN HEPATIC LECTIN

II

PLACENTAL MANNOSE RECEPTOR IgE F, RECEPTOR RATLESNAKE VENOM LECTIN PANCREATlClVENOM PROTEINS

I

VII

MACROPHAGE MANNOSE RECEPTOR

VI

MANNOSE-BINDING PROTEINS

111

SELECTINS

IV

FIG.8. Possible scheme for evolution of C-type CRD saccharide-bindingspecificity. This tree should be compared to the dendrogram shown in Fig. 3. Points at which switches in specificity from mannoselN-acetylglucosamme/fucose(Man/GlcNAc/Fuc)to gdactoselN-acetylgalactosamine (Gal) are proposed to have occurred are denoted by paths (1)and (2). Changes representing a narrowing of specificity are indicated by paths (3) and (4). In the case of the group-IV selectins, this occurs by addition o f a secondarybinding-site so that ligands containing both terminal fucose and sialic acid (Fuc + NeuNAc) are bound. Evolution to generate specificity for protein ligands is indicated by the path labeled (5).

presumed that the common precursor to groups I, 11, and VII, as well as the divergent precursors to each of the individual groups, also bound mannose. Independently, the ability to bind galactose using the Gln-Pro-Asp sequences was achieved in the group I lineage, in the sublineages of group I1 leading to the asialoglycoproteinreceptor and the Kupffer-cell receptor, and in the sublineage of group VII leading to the rattlesnake-venom lectin. In this sense, galactose-binding using the Gln-Pro-Asp motif is a result of convergent evolution of these three subgroups, since they share a common mannose-binding precursor, but not a common galactose-binding progenitor. The fact that group-I1 proteins that display galactose-type binding are more selective and exhibit higher affinity than those in group I is consistent with the suggestion that, although both groups derive affinity for galactose from the presence of the Gln-Pro-Asp sequence, additional features important for selective galactose binding have been incorporated in the group-I1 galactose-binding lineages. It is interesting that cartilage proteoglycan core protein and the Kupffer-cell receptor share the ability to to bind L-fucose as well as D-galactose. The structural basis for this multispecificity is unclear, since it is not possible to superimpose any vicinal hydroxyl groups of fucose

EVOLUTION OF

Ca2+-DEPENDENTANIMAL

LECTINS

227

onto the critical 3- and 4-hydroxyls of galactose. The tree in Fig. 8 suggests that CRDs in proteoglycans and in the Kupffer-cell receptor achieved this shared multispecificity through evolutionarily independent but parallel paths, both of which are marked (1). Since the asialoglycoprotein receptor and its closest homologs bind galactose (and N-acetylgalactosamine) but not fucose, a separate evolutionary path (marked (2)) has resulted in a distinct type of galactose binding. Domains that have evolved through either path (1) or (2) both use the Gln-Pro-Asp motif, but the other structural features that distinguish these two evolutionary routes remain to be elucidated. The evolution of C-type CRDs in lower vertebrates has not been studied extensively, but the structures of two group VII proteins isolated from snake venom provide an interesting parallel to the evolution seen in mammals and birds. The rattlesnake venom lectin and the coagulation factor IX/X-binding protein of Habu venum have been isolated from relatively closely related snakes in the Crotalinae subfamily of Viperidae (36, 37). The rattlesnakevenom protein is a galactose-specific lectin, and contains the expected GlnPro-Asp sequence. However, the Habu protein is not known to bind sugars, and is quite divergent in sequence at the specificity-determining sites. In summary, it appears that the ability of the C-type CRD motif to support binding of multiple types of ligands has been independently exploited on multiple occations, but that when galactose is a ligand, the GlnPro-Asp sequence is usually selected.

D. Specificity for Complex Oligosaccharides 1. ROLE OF CLUSTEREDCRDs Although our understanding of the structural basis for monosaccharide binding to individual CRDs has advanced to the atomic level, the way in which intact animal lectins achieve high affinity and great specificity for larger oligosaccharides remains to be explained. It is certain that the formation of oligomers of C-type CRDs plays an important role in the binding of multivalent ligands. The multiplicative effect achieved by combining several relatively weak interactions can be quite substantial, so that binding sites with intrinsic affinities in the millimolar range can combine to produce efficient binding at the nanomolar level. All the C-type animal lectins in groups 11, 111, and VI contain clusters of CRDs, either by virtue of oligomerization of polypeptides each with a single CRD, or because of multiple CRDs in a single polypeptide (Fig. l), so the effect of clustering of binding sites is likely to be important. Such effects have been demonstrated experimentalIy for the chicken hepatic lectin (50, 82, 83) and the asialoglycoprotein receptor (84), although they are less pronounced for the mannose-binding proteins (85). In those cases in which multiple CRDs must participate in forming a

228

KURT DRICKAMER

binding site, it is likely that geometry as well as simple proximity of clustered sites is important. Unfortunately, no structural information on how domains are arranged relative to each other is yet available, so a precise molecular description is not yet possible. The fact that a string of five CRDs from the mannose receptor are required for high-affinity binding of a natural ligand such as yeast mannan, while two domains are enough to bind tightly to mannosylated serum albumin, provides important evidence that the arrangement of CRDs may be an important determinant of complex ligandbinding (91). Selective binding of different branches of oligosaccharides by the asialoglymprotein receptor is also dependent on an appropriate combination of different CRDs, in this case present in distinct polypeptides (92). Given the current state of structural understanding of the way CRDs are clustered, it is too soon to be able to begin to try and understand the evolution of how clustering has occurred. It is intriguing that C-type CRDs are often found in groups of three, but the way the clusters are stabilized appear to be different in different cases. For example, the neck region of the collectins is enough to produce a trimer that is further stabilized by the triple-helical collagenous domain (49). In contrast, the neck of the chicken hepatic lectin is not sufficient to stabilize a trimer (46, 93). In this case, regions of the polypeptide in and adjacent to the membrane are necessary. These observations allow the tentative suggestion that oligomer formation may have arisen independently at several times in the different groups of C-type animal lectins, although some aspects of the basic shape of the CRD itself may be responsible for the propensity of the domains to form trimers.

2. SECONDARYBINDINGSITES IN CRDs Since the selectins are not known to be oligomeric, yet display highaffinity binding to oligosaccharides with paired terminal fucose and sialic residues (78), it seems likely that individual CRDs in these molecules have multiple sugar-binding sites. It has been suggested that the terminal fucose residue probably occupies a site analogous to the mannose-binding site of the mannose-binding CRD (77), while mutagenesis and antibody inhibition data indicate that a second site, near the loop connecting strands p3 and p4, is a possible sialic-acid-bindingsubsite (86). Evolution of restricted specificity for fucose plus a second terminal sugar is designated path (4) in Fig. 8. The stoichiometry of monosaccharide binding to some other C-type CRDs also suggests that they may interact with more than one sugar at a time (87),thus also achieving greater specificity through contacts with multiple sugar residues in an oligosaccharide. In a similar way, specificity can also be increased if a second contact site interacts with a nonsaccharide portion of a ligand. The possibility that the IgE F, receptor binds to both sugar and protein has already been discussed.

EVOLUTION OF

Ca2+-DEPENDENT

ANIMAL LECTINS

229

In addition, it appears that pulmonary surfactant protein SP-A interacts with both the saccharide and lipid portions of glycolipid ligands (88). Restriction of specificity appears to have been achieved in a slightly different way in the chicken hepatic lectin (path 4 in Fig. 8). While this CRD, like those in the mannose-binding proteins, binds mannose and fucose more strongly than galactose, its affinity for N-acetylglucosamine is significantly higher than its affinity for mannose (89, 90). The fact that N-acetylglucosamine also binds much more tightly than glucose suggests that the increased d n i t y results from interaction of the Z-acetamido group with a secondary site on the chicken hepatic lectin that is not present in the mannose-binding protein CRD studied by crystallography. Therefore, it appears that restricted specificity of binding to a single CRD can be achieved by simultaneous binding of two ddlerent terminal sugars in an oligosaccharide, a terminal sugar and a second (protein or lipid) portion of a complex ligand, or two portions of a single sugar residue.

V. History of Carbohydrate-Recognition Processes Examination of the biosynthetic pathway for complex carbohydrates attached to asparagine residues in proteins suggests that mannose-containing structures appeared early in the evolution of these conjugates. Specifically, the complex sugar structures are created by modification of an initial highmannose oligosaccharide (2), suggesting that the mechanism for synthesis of the high-mannose form probably was in place first, and that products of this initial pathway later became substrates for novel trimming and transferase reactions needed to create more complex structures. The fact that lower eukaryotes as well as plants utilize distinct terminal processing pathways is consistent with this proposal. The observation that mannose was probably present as an early potential recognition marker has an interesting parallel in the evolution of saccharidebinding specificity of the C-type CRDs discussed in the preceding section. The arguments presented suggest that the ability to recognize mannose and N-acetylglucosamine was a characteristic of the earliest forms of the C-type CRDs. It can be speculated that the need for more diverse recognition markers as eukaryotes evolved into multicellular organisms may have been a driving force for the simultaneous evolution of more complex sugar structures and modified CRDs with the ability to recognize them. The ease with which the specificity of the CRDs can be altered by changes in just a few amino acids (80)provides a model for understanding how such changes could have come about. Similar observations for transferases responsible for synthesis of the A and B blood-group structures (94) indicate how the biosynthetic machinery could have changed in parallel with the recognition mechanism.

230

KURT DRICKAMER

The preponderance of simple, repetitive sugar polymers in the outer walls of prokaryotes, lower eukaryotes, and plants suggests that a very early Function of cell-surface carbohydrates was structural, and that the potential information content of the sugars came to be exploited subsequently. It is possible that C-type CRDs originally evolved as sugar-recognitionmolecules in order to exploit the information content of cell surface saccharides. In this case, it would be argued that these domains have subsequently become diversified to act as Ca2+-dependent recognition domains capable of interacting with proteins and perhaps other ligands as well. However, it seems more likely that the earliest form of the CRD-like domains were general Ca2+-dependent structures capable of binding a variety of ligands, and that some descendants of this pool were recruitFd to become specialized sugarrecognition modules. If it turns out that the large family of natural killer cell proteins with C-type CRDs (group V) are in fact an early form of immune receptor, it could be that evolution of CRDs from generalized receptors to specific cell adhesion molecules is parallel to evolution of cell adhesion molecules as specialized forms of the general immunoglobulin domain (95). ACKNOWLEDGMENTS Work in the author’s laboratory is supported by Grant GM42628 from the National Institutes of Health and a faculty salary award from the American Cancer Society. I thank Maureen Taylor for comments on the manuscript, and Bill Weis for help with the figures.

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Index

A

sequences, 222-225 sugar binding, 220-222 Carbohydrate-recognition domains, calciundependent animal lectins and, 208-215, 229-230 Cholera toxin, ADP-ribosylation factors and, see ADP-ribosylation factors Chromatin, immunoglobulin gene diversification and, 38-39 Chromosomes, 6-pliosphofruct0-2-kinasel fructose-2,6-bispIiospliatase and, 118119 Crystallization, tRNA structure and, 163-

Actin-1 gene, rice, 16-17 Actiii genes, rice, 3-5 ADP-ribosylation factors, 47-49 biochemistry, 49-55 function in animal cells, 60-63 structure, 55-60 Agrobacteriunt, rice genes and, 16 Amiiioacy~ation,tRNA structure and, see tRNA structure, aminoacylation and Aminoacyl-tRNA synthetases, 147-149 wmplexes, 159-166 identity, 166-169 antideterminants, 189-190 conformational features, 186-188 evolution, 190-192 nucleotides, 169-180 RNA substrates, 180-186 Animal lectins, calcium-dependent, see Calcium-dependent aninxal lectins

I64 Cyclooxygenase, 83-86

D DNA iniinunoglol~ulingene diversification and, 43 rice genes and, Agrobacteriurn, 9-15

B B cells, inimunoglol~ulingene diversification and, 31-32 Biolistic method, rice genes and, 11-13

C Calcium-dependent animal lectins, 207-208 carl,ohydrate-recognition domains, 208215 history, 229-230 C-type genes exons, 218-220 in trons , 216-2 18 saccharide-binding specificity convergent evolution, 225-227 oligosaccharides, 227-229 233

E Eicosanoids, 67-69, 93 cyclooxygenase, 83-86 leukotriene A, hydrolase, 81-83 lipoxygenases, 69 5-]ipoxygenase, 69-74 12-lipoxygena..e, 75-78 15-lipoxygenase, 78-81 prostaglandiii-D synthase, 87-89 prostaglandin- F synthaw, 89 prostag1andin G/H synthase, 83-86 receptors, 89-93 thrnmboxane-A synthase, 86-87 Enterotoxins, ADP-rihosylation factors and, 54-55 Enzymes, glycolysis and, see 6-Phospho-

234

INDEX

Enzymes (cont.) fructo-2-kinase/fructose-2,6-bisphosphatase Escherichia coli, ADP-ribosylation factors and, 54-55 Exons, calcium-dependent animal lectins and, 218-220

H Heteropduplex DNA repair, immunoglobulin gene diversification and, 43 Homologous recombination, mmunoglobulin gene diversification and, 33-34, 42-43 Hormones ADP-ribosylation factors and, 54 6-phosphofructo-2-kinase/fmctose-2,6bisphosphatase and, 119-123

F Fructose-2,6-bisphosphatase,see 6-Phosphofructo-2- kinase/fructose-2,6-bisphosphatase

G Gene conversion, immunoglobulin gene diversification by, see Immunoghhulin gene diversification Gene expression, 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphataseand, 119123 Genes calcium-dependent animal lectins and, 216-220 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase and characterization. 116-119 chromosomal localization, 118-119 rice, see Rice genes Glucagon, &phosphofructo-Z-kinase/ fructose-2,6-bisphosphataseand, 121123 Glucocorticoids, 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphataseand, 119120 Glutelin gene, rice, 18-19 Glycolysis, control of, see &Phosphofmcto-2-kinase/fructose-2,6-bisphosphatase Growth factors, 6-phosphofructo-Zkinase/fructose-z, 6-bisphosphatase and, 12.3 Guanine-nucleotide-dependentcomplex, ADP-ribosylation factors and, 52

I

Immunoglobulin gene diversification by gene conversion, 27-29, 43 enzymes, 38-43 molecular mechanism, 32-38 somatic genes, 29-32 Immunological characterization, A DPrihsylation factors and, 52-53 Initiation, immunoglobulin gene diversification and, 39-41 Insulin, 6-phosphofructo-2-kinase/fructose-2.6-bisphosphatase and, 121-123 Introns, calcium-dependent animal lectins and, 216-218 Isozymes, 6-phosphofructo-2kinase/fructose-2,6-bisphosphatase and, 101-104 mRNAs, 105-110 structure, 110-116

K Kinetic specificity, tRNA structure and, 151-155

L Lectins, calciuin-dependent, see Calciumdependent animal lectins Leukotriene A4 hydrolase, 81-83 Lipoxygenases, 69 Slipoxygenase, 69-74 l%lipoxygenase, 75-78 15-lipoxygenase, 78-81

235

INDEX

Liver, 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase and, 100-101

M

Prostaglandin-F synthase, 89 Prostaglandin GIH synthase, 83-86 Protein, ADP-ribosylation factors and, see ADP-ribosylation factors Protein genes, rice, 7-9 Protoplasts, rice genes and, 10-11. 22

Monocot genes, rice, 22

R

N Nucleotides, tRNA structure and, 169-180

0 Oligosaccharides, calcium-dependent aiiiinal lectins and, 227-229 Oncogenes, 6-phosphofructo-2-kinase/ fructose-2,6-bispliosphatase and, 123

P Phorbol esters, 6-phosphofructo-2kinase/fructose-2,6-bisphosphataseand, 123 6-Phosphofructo-2-kinase/fructose-2,6bisphosphatase, 99-100, 124 gene expression glucagon, 121-123 glucocorticoids, 119-120 growth factors, 123 insulin, 121-123 oncogenes, 123 phorbol esters, 123 thyroid hormones, 120-121 genes characterization, 116-119 chromosomal localization, 118-1 19 isozymes, 101-104 mRNAs, 105-110 structure, 110-116 liver, 100-101 short-term control, 104-106 Phytochrome genes, rice,, 5-7, 17-18 Pollen-tube-pathway method, rice genes and, 13-15 Prostaglandin-D synthase, 87-89

Recombination, immunoglobulin gene diversification and, 33-34, 41-43 Rice genes, 1-2, 22-23 actin, 3-5 gene transfer, DNA, 9 Agrobucterium, 16 biolistic method, 11-13 pollen-tube-pathway method, 13-15 protoplast s, 10- 11 phytochrome, 5-7 storage protein, 7-9 transgenic plants, 16, 22 actin-1 gene, 16-17 agronomic uses, 22-23 glutelin gene, 18-19 phytochrome genes, 17-18 protease inhibitor genes, 19-22

s Saccl~aride-bindingspecificity, cdciuindependent animal lectins and, 220-230 Sequences calcium-dependent animal lectins and, 222-225 tRNA structure and, 156-159 Storage protein genes, rice, 7-9 Sugar, calcium-dependent animal Iectins and, 220-222

T Thromboxane-A synthase, 86-87 Thyroid hormones, 6-phosphofructo-2kinase/fructose-2,6-l~isphosphataseand, 120- 121 Tobacco, rice genes and, 18-19

236

INDEX

Transcriptidn, immunoglobulin gene diversification and, 38-39 Transgenic plants, rice genes and, 16-23 tRNA structure, aminoacylation and, 130141, 192-194 aminoacyl-tRNA synthetases, 147-149 complexes crystallization, 163-164 in solution, 159-163 three-dimensional structures, 164-166 functional observations, 149-151 identity, 166-169

90051

antideterminants, 189-190 conformational features, 186-188 evolution, 190-192 nucleotides, 169-180 RNA substrates, 180-186 kinetic specificity, 151-155 mechanisms, 155-156 noncanonical tRNAs, 142-147 sequences, 156-159 Tumors, iininunoglobulin gene diversification and, 31-32