Transglutaminases: Family Of Enzymes With Diverse Functions (Progress in Experimental Tumor Research)

Transglutaminases

Progress in Experimental Tumor Research Vol. 38

Series Editor

Joseph R. Bertino

New Brunswick, N.J.

Transglutaminases Family of Enzymes with Diverse Functions

Volume Editors

Kapil Mehta Houston, Tex. Richard Eckert Cleveland, Ohio

39 figures, 7 in color, and 16 tables, 2005

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Singapore · Tokyo · Sydney

Progress in Experimental Tumor Research Founded 1960 by F. Homburger, Cambridge, Mass

Kapil Mehta

Richard Eckert

Department of Experimental Therapeutics The University of Texas M.D. Anderson Cancer Center 1515 Holcombe Boulevard Houston, Texas 77030 (USA)

Case School of Medicine 2109 Adelbert Road Cleveland, OH 44106-4970 (USA)

Library of Congress Cataloging-in-Publication Data Transglutaminases : family of enzymes with diverse functions / volume editors, Kapil Mehta, Richard Eckert. p. ; cm. – (Progress in experimental tumor research ; v. 38) Includes bibliographical references and index. ISBN 3-8055-7901-2 (hardcover : alk. paper) 1. Transglutaminases. [DNLM: 1. Transglutaminases–physiology. ] I. Mehta, Kapil, Dr. II. Eckert, Richard (Richard L.) III. Series. RC254.A335 vol. 38 [QP606.T73] 616.99
4 s–dc22 [612/.1 2004029529 Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and Index Medicus. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2005 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISSN 1660–8984 ISBN 3–8055–7901–2

Contents

VII Preface 1 Mammalian Transglutaminases: A Family Portrait Mehta, K. (Houston, Tex.) 19 Structure-Function Relationships of Transglutaminases – A Contemporary View Nemes, Z.; Cso´´sz, É.; Petrovski, G.; Fésüs, L. (Debrecen) 37 Transglutaminases and Their Substrates Facchiano, F. (Roma); Facchiano, A. (Avellino) 58 Type 2 Transglutaminase and Cell Death Piacentini, M.; Amendola, A.; Ciccosanti, F.; Falasca, L.; Farrace, M.G.; Mastroberardino, P.G.; Nardacci, R.; Oliverio, S.; Piredda, L.; Rodolfo, C.; Autuori, F. (Rome) 75 Transglutaminase and Cell-Survival Signaling Singh, U.S.; Pan, J. (Temple, Tex.) 89 Transglutaminases in Wound Healing and Inflammation Verderio, E.A.M. (Nottingham); Johnson, T.S. (Sheffield); Griffin, M. (Nottingham) 115 Transglutaminases in Epidermis Eckert, R.L.; Sturniolo, M.T.; Broome, A.-M.; Ruse, M.; Rorke, E.A. (Cleveland, Ohio) 125 Tissue Transglutaminase (TG2) in Cancer Biology Mangala, L.S.; Mehta, K. (Houston, Tex.)

V

139 Transglutaminases in Neurodegenerative Disorders Bailey, C.D.C.; Tucholski, J.; Johnson, G.V.W. (Birmingham, Ala.) 158 Tissue Transglutaminase and Celiac Disease Esposito, C.; Caputo, I. (Salerno); Auricchio, S.; Troncone, R. (Naples) 174 Transglutaminases as Biotechnological Tools Mariniello, L.; Porta, R. (Naples) 192 Extracellular Transglutaminase: Factor XIII Ichinose, A. (Yamagata) 209 Transglutaminases of Lower Organisms Rao, R.U. (St. Louis, Mo.); Chandrashekar, R. (Westbrook, Maine); Mehta, K. (Houston, Tex.) 223 Transglutaminases of Higher, Lower Plants and Fungi Del Duca, S.; Serafini-Fracassini, D. (Bologna) 248 Author Index 249 Subject Index

Contents

VI

Preface

This collection of articles is published as we approach the 50th anniversary of the discovery of the first of a large family of protein-remodelling enzymes by Heinrich Waelsch and his colleagues. An earlier book with a similar title appeared in 1984 and, since then, there has been quite an upsurge of interest in the subject of transglutaminases (TGs). It attracts researchers from around the globe representing different subspecialities in biology, medicine and, not surprisingly, also in biomedical engineering mainly because of a need for biocompatible polymers. Hundreds of participants attend special conferences held at frequent intervals; in recent years, a meeting in Ferrara, Italy in 2002, was followed by a symposium in Rome in 2003 and yet another one is scheduled for July 2005 as part of the FEBS-IUBMB meeting in Budapest, Hungary. The Ca2+-dependent TGs evolved from the papain family of proteases and, as such, operate with a Cys/His/Asp catalytic triad. However, the enzyme: substrate intermediate stabilizing function of a Gln residue in papain is replaced by the indole ring of a Trp in the active centers of all eukaryotic TGs, of which – excepting the catalytically inactive homologue of the band 4.2 protein – there are seven in the human genome. The TG name, coined by Waelsch, is somewhat of a misnomer because these enzymes do not react with the free amino acid of glutamine; rather, they target the -carbonylamide function in the side chain of Gln residues in protein substrates. Selection of a particular Gln depends less on the primary sequence surrounding it than on its location in the ternary structure of the protein. TGs seem to react best with Gln (acceptor) residues in unstructured flexible regions of proteins, often in the N and C terminal domains, but always in endo-positions.

VII

Modification of the -carbonylamide group of the Gln residue depends on the nature of the electron pair donating nucleophile as the second reactant that might be water (causing hydrolysis), alcohol (esterification), a small amine (transamidation with a biogenic amine, mono-, di- or polyamine) or the -amino group of a Lys side chain of a protein. The simplified scheme below illustrates the outlines for some of the possible exchange reactions. Unlike papain, which is also known to promote transamidation, TGs display a high degree of specificity (saturable behavior) with regard to the amine donor substrate and also in the selection of Lys side chains in proteins with which they react; the enzymereactive Lys’s, too, are usually located in flexible domains of proteins. 1) O   CH2CH2CNH2  H2NCH2CH2CH2CH2

O   CH2CH2CNCH2CH2CH2CH2 H

 NH3

2) O  CH2CH2CNH2  H2N

3)

O  
CH CH CNH  H NCH CH CH CH 2 2 2 2 2 2 2 2

O  CH2CH2CNH

 NH3

O  
CH CH CNCH CH CH CH 2 2 2 2 2 2 H

 NH3

Each reaction competes with all the others. Cross-linking of protein substrates can be blocked at the monomeric stage by the concomitant incorporation of extraneous amines (reaction 2) or short Gln peptides (reaction 3) into the participating protein partners. This proved to be an efficient way for probing reaction 1, often the focus of main interest in TG-catalyzed processes. Stabilization of human blood clots by thrombin-activated coagulation Factor XIII (FXIIIa) was the first such study where the small amines were shown to inhibit selectively only the covalent fusion of fibrin particles without interfering with their assembly into a clot network. By contrast, the same amines completely blocked the clotting of lobster blood and the formation of the copulation plug in rodents; the latter two are examples of nature’s simplest systems for the covalent polymerization of proteins where the relevant TG is sequestered into a compartment separate from its substrate. In fact, TGs may have been the earliest clotting enzymes in evolution; a TG isolated from the half billion year old species of Microciona (sea sponge) is efficient in clotting lobster plasma and any TG2 can stabilize the human fibrin clot, regardless whether it was derived from guinea pig liver or from human erythrocytes.

Preface

VIII

The introduction of N(-glutamyl)lysine side chain bridges by TGs – be it through stabilization of a pre-existing protein assembly or through the de novo direct polymerization of proteins – plays an important role in the organization of extracellular matrices. Similar, covalently linked membrane skeletal and cytoskeletal polymers are generated in cells when the latent TG becomes activated by the influx of Ca2+ (human red cells, keratinocytes) or by the action of some other signal (thrombin activation of platelets). Though a few intracellular TGs, just like Factor XIII, may require prior limited proteolytic processing, Ca2+ seems to be the universal trigger for the expression of TG enzyme activity. Much current research is focused on cross-linked polymers of cells with elevated Ca2+ contents, such as found in senescent cells, terminally differentiated cells and in cells undergoing pathological changes (Hb-Koln and sickle cell disease, cataract, neurodegenerative protein deposit diseases) and apoptosis. Reaction 1 in the scheme might be assumed to proceed with no change of free energy; nevertheless, this may be an essentially irreversible transition because the high molecular weight polymer probably forms a separate phase. Thus, efforts should be concentrated on preventing the TG-catalyzed maturation of pathological polymers with inhibitors rather than trying to reverse the process. TG inhibitory compounds might also be used to facilitate the lysis of blood thrombi, because stabilized clots are quite resistant to digestion by lytic enzymes. One of the most interesting members of the family of TGs is TG2, originally discovered by the Waelsch group. Even in this age of multifunctional proteins, one cannot help but marvel at the versatility of TG2. For example, in addition to its Ca2+-binding ability to assist the Cys/His/Asp/Trp catalytic unit in carrying out the enzymatic activities mentioned, this mid-size protein, along with several other TGs, binds GTP/GDP (the non-catalytic 4.2 band protein relative binds ATP). Nucleotide binding lowers the affinity for Ca2+ and inhibits the transamidating activity of the enzyme by cross-talk. Furthermore, TG2 can function as a G-protein (Gah) in signal transduction and binds to phospholipaseC. TG2 also forms a very tight complex with fibronectin and, at the interface of the cell with the extracellular matrix, this non-covalent unit influences cell migration. The medical relevance of research on TGs is well documented. Perturbations in the activities of these enzymes are known to give rise to inherited hemorrhagic and skin diseases (Factor XIII deficiency and TG1 deficiency in lamellar ichtyosis). Much new information is expected from gene knockout experiments, such as the finding reported at the recent Ferrara meeting that TG2 plays a critical role in normal wound healing. There are also autoimmune conditions where a TG is the autoantigen. Inhibitory antibodies to Factor XIII may appear in the circulation and cause potentially fatal hemorrhage. In celiac disease, characterized by debilitating intestinal and systemic manifestations,

Preface

IX

TG2 is the main target of autoantibodies, and symptoms in the related skin disease: dermatitis herpetiformis are caused by immune complex deposits of TG3. This selection of articles illustrates the breadth of current research on transglutaminases, and it is hoped that the volume will stimulate further interest in this important field. Laszlo Lorand Chicago, Ill.

References Lorand L: Transglutaminase: Remembering Heinrich Waelsch. Neurochem Int 2002;40:7–12. Lorand L, Conrad SM: Transglutaminases. Mol Cell Biochem 1984;58:9–35. Iismaa SE, Holman S, Wouters MA, Lorand L, Graham RM, Husain A: Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases. Proc Natl Acad Sci USA 2003;100:12636–12641. Lorand L, Graham RM: Transglutaminases: Crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 2003;4:140–156. Lorand L: Sol Sherry lecture in thrombosis: Research on clot stabilization provides clues for improving thrombolytic therapies. Arterioscler Thromb Vasc Biol 2000;20:2–9. Lorand L: Factor XIII: Structure, activation and interactions with fibrinogen and fibrin. Ann NY Acad Sci 2001;936:291–311.

Preface

X

Mehta K, Eckert R (eds): Transglutaminases. Prog Exp Tum Res. Basel, Karger, 2005, vol 38, pp 1–18

Mammalian Transglutaminases: A Family Portrait Kapil Mehta The University of Texas M. D. Anderson Cancer Center, Houston, Tex., USA

Introduction

Transglutaminases (TGs; EC 2.3.2.13) are a family of structurally and functionally related enzymes that catalyze Ca2
-dependent post-translational modification of proteins by introducing protein-protein crosslink (between specific glutamine and a lysine residue), amine incorporation, and site-specific deamidation (fig. 1). They are widely distributed enzymes and have been identified from unicellular organisms (chapter 13) to mammals and plants (chapter 14). Nine TG genes have been described from Homo sapiens (human) of which 8 code catalytically active enzymes. Some common features shared by each member of the TG family include the lack of glycosylation and disulfide bonds despite the presence of potential N-linked glycosylation sites and cysteine residues in these proteins. Moreover, all the TGs, including those that are secreted (such as Factor XIII) or associated with membrane (e.g., TG1) lack N-terminal hydrophobic sequence. Similarly, all members except a catalytically inactive homolog (band 4.2) require calcium for the catalytic activity. Table 1 summarizes important features of each member of the TG family. While the overall primary structure of TG enzymes appear to be different, they all share the same amino acid sequence at the active site (fig. 2). This review is an attempt to compile important features of all the nine TGs that have been described in humans. Whenever possible, the information has been included in the form of self-standing figures or tables to provide the beginners and even devoted followers in the field of TG a quick reference to know the facts about each member of the TG family. Figure 3 shows the genomic organization and table 2 summarizes general features of gene and protein structure for each member of the TG family.

O  II CH2CH2—C—NR
NH3 (1) H

P1 H2N-R Ca2+

 O II —CH2CH2C—NH2
TG2 —Cys

N(-glutamyl)lysine isopeptide

 H2N—CH2—CH2—CH2—CH2— Lysine

Glutamine P1

P2

 O  II —CH2CH2C—N—CH2—CH2—CH2—CH2—
NH3 H P1 P2 (2)

H2O  O II —CH2CH2C—OH
NH3

(3)

P1

Fig. 1. Biochemical reactions catalyzed by TGs. Transamidation reaction, which requires the presence of Ca2
, can result in (1) covalent incorporation of an amine (R-NH2, such as polyamines, histamines) into the glutamine residue of the acceptor protein (P1), (2) covalent cross-linking of proteins by introducing N(-glutamyl)lysine isopeptide bond between the lysine donor residue of one protein (P2) and the acceptor glutamine residue of another (P1), and (3) in some instances water can replace amine donor substrates leading to deamidation of the protein (P1)-bound glutamine residue.

Keratinocyte Transglutaminase (TGM1)

Nomenclature TG1, TGM1, TGK, transglutaminase epidermal type I, K polypeptide epidermal type I, particulate transglutaminase. Site of Expression Expressed primarily in stratified squamous epithelia of the integument, the upper digestive tract, and the lower female genital tract. Promoter Three AP2-like response elements approximately 0.5 kilobases (kb) from the transcription initiation site (fig. 4). Inducers and Suppressors Proteolytic cleavage, Ca2
and TIG-3 protein activate catalytic functions. Phorbol esters induce the expression while retinoic acid downregulates the expression.

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Table 1. Important features of TG proteins Protein

Main functions

Distribution

Disease

Alternate names

TG1

Cell envelope formation during keratinocyte differentiation

Membrane-bound in keratinocytes

Lamellar ichthyosis

TG1, TGK, keratinocyte TG, particulate TG

TG2

Apoptosis, cell adhesion, matrix stabilization, cellsurvival signaling

Widely distributed in many tissues; cytosolic, nuclear, membrane, extracellular

Autoantigen in celiac disease

Tissue TG, TGC, liver TG, endothelial TG, erythrocyte TG, Gh

TG3

Cell envelope formation during keratinocyte differentiation

Hair follicle, epidermis, brain

Expression downregulated in head and neck squamous cell carcinoma and in laryngeal carcinoma

TGE, callus TG, hair follicle TG, bovine snout TG

TG4

Reproduction especially in rodents as a result of semen coagulation

Prostate

Not known

Prostate TG, TGp androgen regulated major secretory protein, vesiculase, dorsal prostate protein 1 (DP1)

TG5

Cornified cell envelope formation during keratinocytes differentiation

Foreskin keratinocytes, epithelial barrier lining and skeletal muscular striatum

Secondary effect to the hyperkeratotic phenotype in ichthyosis and in psoriasis

TGX

TG6

Not known

Testis and lung

Not known

TGY

TG7

Not known

Ubiquitous but predominantly in testis and lung

Not known

TGZ

FXIIIa

Blood clotting, wound healing, bone growth

Platelets, placenta, synovial fluid, chondrocytes, astrocytes, macrophages

Factor XIIIa deficiency

Fibrin-stabilizing factor, fibrinoligase, plasma TG, LakiLorand factor

Band 4.2

Major component in erythrocyte skeletal network

Erythrocyte membranes, bone marrow, spleen

Spherocytic elliptocytosis

B4.2, ATP-binding erythrocyte membrane protein band 4.2

Mammalian TGs: An Overview

3

361 263 259 254 269 260 265 301 263

LSYLRTGYSV RWKNHGCQRV NWKKSGFSPV QQYYNTKQAV QWHATGCQPV KWLKGRYKPV QWSARGGQPV LEYRSSETPV QMLTGRGRPV

P YGQCWVFA K YGQCWVFA R YGQCWVFA CF GQCWVFA R YGQCWVFA K YGQCWVFA K YGQCWVFA R YGQCWVFA YD GQAWVLA

G A G G A G S G A

VTTTVLRCLG VACTVLRCLG TLNTALRSLG ILTTVLRALG VMCTVMRCLG VLCTVLRCLG VMCTVMRCLG VFNTFLRCLG VACTVLRCLQ

LATRTVTNFN IPTRVVTNYN IPSRVITNFN IPARSVTGFD IPTRVITNFD IATRVVSNFN VPTRVVSNFR IPARVITNYF IPARVVTTFA

SAHDTDTSLT TG1 SAHDQNSNLL TG2 SAHDTDRNLS TG3 SAHDTERNLT TG4 SGHDTDGNLI TG5 SAHDTDQNLS TG6 SAHNVDRNLT TG7 SAHDNDANLQ FXIIIa ŠAQGTGGRLL Bands4.2

Fig. 2. Alignment of the catalytic site region of the TG proteins. Amino acids that form the core fold consisting of the catalytic cysteine residue are shown in bold text.

TATA ATG Intron:

1

2

3

45 6 7 8

9

10 11 12 13

14

TGM1 Exon: I

II III

Intron: ATG

IV V VI VIIVIIIIX

1

X

2

XI XIIXIIIXIV

XV

3

4 5

6

7

8

9

10

11

12 TGM2

Exon:

I CAT TATA Intron: Cap ATG

II

III

IV

1

2 3

4

VI

VII

5

VIII

6

IX

7

X 8

XI

9

XII

10

XIII

11

12 TGM3

Exon: I

II III IV

Intron:

V

1

2

V

3

VI VII

4

5

VIII 6

7

IX

X

8

9

XI

XII XIII

10 11

12 TGM4

Exon:

I

II

III

Cap ATG Intron: 1

IV

2

V

3

4

VI VII 5

6

VIII 7

IX

8

9

X

10

XI XII

XIII

11 TGM5

Exon:

I

Intron:

II III 1

2

IV

V

3

4

VI

VII

5

6

VIII IX

7

X XI 8

9

XII XIII

10

11

12

13

14 F13A1

Exon: Intron:

I

II

III

IV 2

1

V VI 3 4

5

6

VII VIII 7

8

9

10

IX X 11

XI

XII

XIII

XIV

XV

12 EPB4.2

Exon: I

II

III IV V

VI VII

VIII IX X

XI

XII

XIII

Fig. 3. Genomic organization of TG genes. The human TG genes are represented with exons indicated in Roman numerals and vertical bars. The exons are separated by introns indicated by Arabic numerals.

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Table 2. Human TGs: genes and proteins Protein

Gene

Gene locus

Gene size (kb)

Number of exons

Amino acid residues

Molecular weight (kDa)

TG1 TG2 TG3 TG4 TG5 TG6 TG7 Factor XIIIa Band 4.2*

TGM1 TGM2 TGM3 TGM4 TGM5 TGM6 TGM7 F13A1 EPB4.2

14q11.2-13 20q11-12 20q11-12 3p21-p22 15q15.2 20q11 15q15.2 6p24-25 15q15.2

14 37 43 35 35 45 26 160 20

15 13 13 13 13 13 12 15 13

814 686 692 684 720 625 710 732 690

90 78 77 77 81 70 80 83 72

*Catalytically inactive homolog of TG. Except Band 4.2 all TGs require Ca2
for enzymatic activity.

ATG AP-2 AP-2 AP-2

TATA-like

TGM1 500


1

CAAT RARE

TRE

TATA NF-1 SP-1

AP-2

AP-1

SP1 SP1

634

183

56 45

ATG

TGM2 1700

868

Ets


1 NF-1 SP-1

SP-1

ATG

AP-1

TGM3 128

Cyclophilin pseudogene

91

ARE

SP-1

?

96

15
1

ATG

TGM4 1276

?

Ets


1

SP-1 AP-1 NFB

ATG c-Myb

TGM5 335

GATA-1

198 175 134

Ets-1 Ets-1 MZF-1 MZF-1 MZF-1 NF-1

9


1

ATG

F13A1 SP-1 756

554 482

SP-1

260 223

SP-1

92 39


1

Intron A

Fig. 4. Schematic representation of 5-flanking regulatory sequences in TG genes. The transcription and translational initiation sites are indicated by
1 and ATG, respectively. In case of TGM4 the exact binding site for the transcription factor, androgen receptor (androgen response element, ARE) has not been mapped.

Mammalian TGs: An Overview

5

TG1 Protein Linked to the plasma membrane via fatty acyl linkage in the N-terminal cysteine residue. Released from the membrane by proteolysis in form of 10, 33, and 66 kDa fragments. Contains 4 serine residues (S24, S82, S85, S92) that are susceptible to phosphorylation. Functions Responsible for crosslinking the epidermal proteins and formation of cornified envelopes in the stratum corneum. Disease Autosomal recessive lamellar ichthyosis results from mutations in TGM1 gene. The common mutations observed include a C- to -T change in the binding site for the transcription factor Sp1 within the promoter region; Gly143- to -Glu mutation in exon 3 and Val382- to -Met mutation within exon 7. Lamellar ichthyosis is a rare keratinization disorder of the skin characterized by abnormal cornification of the epidermis; generalized large scales and variable redness. The patients exhibit drastically reduced TG1 activity and an absence of detectable TG1 polypeptide. Accession Number BC034699

Tissue Transglutaminase (TGM2)

Nomenclature TG2, TGC, endothelial TG, erythrocyte TG, liver TGase, Gh Site of Expression Widely distributed in many tissues and cells. Promoter A retinoic acid response element located 1.7 kb upstream from the initiation site, interleukin-6 specific cis-regulatory element located within first 4.0 kb of the promoter and TGF- 1 response element (TRE; GAGTTGGTGC) located 868 base pairs upstream and two AP-2-like response elements located approximately 634 and 183 base pairs upstream from the transcription initiation site (fig. 4). Inducers and Suppressors Retinoic acid, vitamin D, TGF- 1, IL-6, TNF/NFB, EGF, phorbol esters, oxidative stress, and HOX A-7 all induce the expression.

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Integrin aa 1

AEELVLE

Fn

277 335 358

538

460

Catalytic core PKFLKNAG..

7

520 GTP

139

-sandwich

N 2

Catalytic triad

(28 kDa fragment)

200

C H D

214

BH3 motif

EGSEEERE.. 430 453 Ca2


538

-barrel 1 1 -adrenergic receptor

687

-barrel 2

C

LHMGLKLV 657 677

PLC 1

Catalytic core

-sandwich

-barrel 1

-barrel 2

Fig. 5. Functional domains and three-dimensional structure of human TG2 protein. The four structural domains are shown by different shades of cylindrical structures. Various functional regions along with their amino acid positions and sequences are indicated. Catalytic triad, Cys277-His335-Asp358, is indicated by triangles underneath the functional domains.

TG2 Protein Predominantly a cytosolic protein but also present in the nucleus, on plasma membrane, and extracellularly. Two transcript variants encoding different isoforms have been described for TG2. GTP inhibits the protein’s transamidation activity. Functions Figure 5 shows various functional domains of TG2 protein. In addition to transamidation reaction, TG2 catalyzes GTPase, ATPase, protein kinase, and protein disulfide isomerase activities. It can modulate the functions of other proteins by directly interacting or associating with them; some examples include phospholipase- 1, beta members of the integrin family, fibronectin, osteonectin, RhoA, multilineage kinases, and pRB. TG2 plays a role in biological processes such as apoptosis (fig. 6) (chapter 4), wound healing (chapter 6), and cataract formation. Recent work suggests that TG2 may serve as a signaling molecule (fig. 7) (chapter 5) and promote cell growth, drug resistance, and metastatic functions in tumor cells (fig. 8) (chapter 8).

Mammalian TGs: An Overview

7

Catalytic triad

139

520 Catalytic core

PKFLKNAG.. BH3 motif

C

H D

Catalytic core

EGSEEERE.. 430 453 Ca2


Ca2
Activated TG2 Intracellular Ca2


Bax/Bak

Strong stressors (osmotic stress, UV radiation, ROS)

Cross-linking (pRB, MLKs, SP1, IB etc.)

Apoptosis

Fig. 6. TG2 as a proapoptotic factor. Oxidative stress or other stresses (e.g. osmotic stress and UV radiations) can induce TG2 expression in cells. Under extreme stressful conditions (e.g. hypoxia, growth factor deprivation, or treatment with chemotherapeutic drugs) Ca2
homeostasis inside the cell may become perturbed. Sudden changes in the cytosolic Ca2
may transform TG2 into its cross-linking configuration resulting in cross-linking of some key proteins, such as pRB, multilineage kinases, or the transcription factor SP-1 and initiate the process of apoptosis. Continuous activation of TG2 can lead to extensive cross-linking of intracellular proteins, their polymerization, and formation of detergent-insoluble structures (shown in inset). The protein scaffolds thus formed can stabilize the structure of the dying cell before its clearance by phagocytosis, preventing the release of intracellular components and subsequent inflammatory response.

Disease Not known. TG2 knockout mice show no phenotype. Fibroblasts derived from TG2-/- mice, however, show defective attachment and motility functions. Finally, the TG2 protein serves as an autoantigen and is implicated in celiac disease etiology (chapter 10). Mehta

8

520 538 GTP TG2/TG3/TG5

-barrel 2

-barrel 1 Mg2



GTP

Ca2


1B-adrenergic receptor

C Gh

LHMGLKLV

657 677 PLC- 1

GhA1/TG2-GDP Thromboxane Oxytocin

Ligand

IP

GDP
Pi

Adrenergic R

Agonist

5 M

IP3 Transamidation

Gh or calreticulin GTP

DAG

Gh1/TG2-GDP PKC

Ca2


GDP Gh1/TG2-GTP

Signaling

Cell growth

Fig. 7. TG2 as a signaling protein. The GTP-binding ability of TG2 couples it to 1B adrenergic, thromboxane, and oxytocin receptors. Ligation of the receptor with an agonist induces exchange of GDP to GTP and dissociation of GTP-TG2/Gh from -subunit, a calcium-binding protein calreticulin. GTP-bound TG2/Gh then interacts with phospholipase 1, (PLC 1) resulting in the hydrolysis of phosphoinositide (IP) and increase in Ca2
within the cell. TG2/Gh, through its GTPase activity, can also regulate other signaling pathways.

2

Fn 7 AEELVLE.

N -sandwich

C TG2

Integrin 1,3,or 5 Integrin  subunit

Integrin

Signaling

Antiapoptosis Fibronectin Drug-resistance metastasis

Fig. 8. TG2 promotes integrin-mediated signaling. TG2 can locate to the cell membrane in association with members of the integrin family of proteins. Because of its high affinity for fibronectin, TG2 can promote the interaction between cell surface integrins and fibronectin, which can lead to the activation of focal adhesion kinase, an upstream event that leads to the activation of several downstream cell growth and cell survival signaling pathways.

Mammalian TGs: An Overview

9

Accession Number BC003551

Epidermal Transglutaminase (TGM3)

Nomenclature TGE, callus TG, hair follicle TG, bovine snout TG Site of Expression Keratinocytes, hair follicle, epidermis, brain. Promoter The sequences between 128 and 91 base pairs upstream from the transcription initiation site represent proximal promoter region. The binding of Sp1- and Ets motifs by their cognate binding factors is required for TGM3 transcription (fig. 4). Inducers and Suppressors Expression of protransglutaminase 3 mRNA is regulated by calcium. TG3 Protein Is an unusual member of the TG family in that the protein encoded by this gene consists of two polypeptide chains activated from a single precursor protein by proteolysis. Calcium-activated TG3 can bind, hydrolyze, and is inhibited by GTP. Functions Cross-links trichohyalin and keratin intermediate filaments to harden the inner root sheath, which is critical for hair fiber morphogenesis. Responsible for cell envelope formation during later stages of differentiation in the epidermis and hair follicle. TG3 knockout mice embryos fail to implant suggesting that this gene has a pivotal role during early developmental stages. Disease Expression is downregulated in esophageal and head and neck squamous cell carcinoma and in laryngeal carcinoma. Accession Number NM_003245

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Transglutaminase 4 (TGM4)

Nomenclature Prostate TG, TGp, androgen regulated major secretory protein, vesiculase, dorsal prostate protein 1 (DP1) Site of Expression Prostate, prostatic fluids, and seminal plasma. Promoter The region between positions 113 and 61 is essential for core promoter activity. The Sp1 binding motif at positions 96 to 87 is critical for transcriptional regulation. At positions 1276 to 563, the promoter harbors a cyclophilin pseudogene with 94% similarity to the cyclophilin A cDNA (fig. 4). Inducers and Suppressors Treatment with androgen induces upregulation of TG4 mRNA in a human prostate cancer cell line, PC346C. Expression of protransglutaminase 4 RNA is regulated by calcium. GTP inhibits TG4 activity. TG4 Protein In rats it is glycosylated protein and possesses a lipid anchor, which is retained during enzyme apocrine secretion. While these post-translational modifications are not essential for the enzymatic activity, the N-terminus end is responsible for both transglutaminase functionality and GTP-binding. Functions In rats the enzyme participates in the formation of copulatory plugs in the female genital track and plays a role in masking the antigenicity of the male gamete and suppressing immune response against the sperm cells. The exact function of TG4 in humans is not known. Disease Increased transcript levels have been observed in breast cancer cells in patients who died of the disease suggesting that TG4 may serve as prognostic factor in breast cancer with poor survival. Accession Number NM_004245

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Transglutaminase 5 (TGM5)

Nomenclature Transglutaminase X, TGX Site of Expression Expressed in foreskin keratinocytes, epithelial barrier lining and skeletal muscular striatum. The mRNA expression is ubiquitous with highest levels in the female reproductive system, skeletal muscle, and fetal tissues. Promoter TGM5 gene has TATA-less promoter but contains potential binding sites for several transcription factors such as C-Myb, activator protein-1 (AP-1), nuclear factor B, and NF-1 (fig. 4). Inducers and Suppressors GTP and ATP inhibit cross-linking activity of TG5 in vitro, and Ca2
reverses this inhibition. TG5 Protein The protein is acetylated at the N-terminal end. In addition to full-length TG5 protein, three alternatively spliced isoforms, 3 (deletion of exon 3),

11 (deletion of exon 11), and 3 11 (deletion of both the exons) have been described. Full-length TG5 and 11 isoforms are active, whereas 3 and

3 11 have very low activity. Full-length TG5 is very efficient in using specific epidermal substrates (loricrin, involucrin, and SPR3). The isoforms are associated with insoluble proteins. TG5 co-localizes with vimentin and it is able to cross-link vimentin in vitro. Also, it induces cell death when overexpressed intracellularly and contains GTP-binding domains similar to TG2 and TG3. Functions Contributes to the formation of cornified cell envelopes of keratinocytes. Disease Contributes as a secondary effect to the hyperkeratotic phenotype in ichthyosis (both vulgaris and lamellar) and in psoriasis. Overexpressed or totally absent in different areas of the Darier’s disease lesions. Accession Number NM_004245

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Transglutaminase 6 (TGM6)

Nomenclature Transglutaminase Y, TGY Site of expression Testis and lung. TG6 Protein Activated by proteolytic processing. Consists of two polypeptide chains and is synthesized as a precursor form of a single polypeptide. Functions Responsible for the later stages of cell envelope formation in the epidermis and the hair follicle. Accession Number NM_198994

Transglutaminase 7 (TGM7)

Nomenclature Transglutaminase Z, TGZ, TGMZ Site of Expression Ubiquitous but predominantly expressed in testis and lung. Also, the transcript level is elevated in breast cancer cells especially in patients with poor prognosis. Promoter Not characterized. Accession Number NM_052955

Factor XIII A1 Subunit (F13A1)

Nomenclature Fibrin stabilizing factor, FSF-A subunit, fibrinoligase, plasma transglutaminase, Factor XIII A subunit, Laki-Lorand factor

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Site of Expression Platelets, plasma, astrocytes, macrophage, dermal dendric cells, chondrocytes, placenta, synovial fluid, heart, eye, bone. Promoter The expression is predominantly regulated by myeloid-enriched transcription factor (MZF-1-like protein) and two ubiquitous transcription factors, NF-1 and SP-1. Two myeloid enriched factors GATA-1 and Ets-1 also enhance expression. Inducers and Suppressors F13A1 expression is increased with the female gender, smoking and age; the mechanisms for these alterations are not well understood. The enzyme activation is regulated by thrombin and calcium ions. F13A1 Protein Is the last zymogen to become activated in the blood coagulation cascade. Plasma Factor XIII is a heterotetramer composed of 2 each, A and B subunits. The A subunits have catalytic function while the B subunits are catalytically inactive but may serve as carrier molecules. Platelet Factor XIII is comprised of only two A subunits, which are identical to those of plasma origin. Upon activation by the cleavage by thrombin and in the presence of calcium ions, the plasma Factor XIII dissociates B subunits and yields the same active enzyme, Factor XIIIa, as platelet Factor XIII. In presence of Ca2
, the enzyme catalyzes cross-linking of fibrin molecules, stabilizing the fibrin clot. It also cross-links fibronectin to the  chains of fibrin. Functions Factor XIII is activated by thrombin and calcium ions to a transglutaminase that catalyzes the formation of -glutamyl--lysine cross-links between fibrin chains, thus stabilizing the fibrin clot during blood coagulation. Plays a role in inflammation and bone growth. Induces cross-linking of agonist-induced AT1 receptor homodimers which then display enhanced signaling and contribute to an enhanced monocyte adhesiveness in hypertensive patients thereby sustaining the process of atherogenesis chronic sensitization of circulating monocytes. Disease F13A1 deficiency is an autosomal recessive disorder characterized by a life-long bleeding tendency, impaired wound healing and spontaneous abortion

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in affected women. In addition to the common presentation such as subcutaneous and intramuscular haematomas, severe bleeding such as intracranial hemorrhages may occur. Accession Number NM_000129

Band 4.2 (EPB4.2)

Nomenclature B4.2, ATP-binding erythrocyte membrane protein band 4.2 Site of Expression Cytoplasmic surface of erythrocyte membranes, bone marrow, fetal liver, spleen. Band 4.2 Protein Band 4.2 protein shares homology with various transglutaminases. Within the 5 contiguous consensus residues of the transglutaminase active site, band 4.2 has an amino acid substitution (Cys-Ala), which leads to loss of transglutaminase activity (fig. 2). Two isoforms are produced due to alternative splicing, the shorter form is more abundant. Functions Band 4.2 is a major component in the erythrocyte skeletal network and plays an important role in the regulation of erythrocyte shape and mechanical properties. The major membrane binding site for band 4.2 is the cytoplasmic domain of the erythrocyte anion transporter, band 3. Disease Band 4.2 protein is partially or completely absent in Japanese patients with recessive spherocytic elliptocytosis. In patients with protein 4.2 deficiency ankyrin is much less firmly associated with the membrane skeleton than normal. Accession Number NM_000119

Mammalian TGs: An Overview

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Resources for TGs Research

Anti-TG2 Antibodies A. Calbiochem 1. Rabbit Reactivity: human 2. Rabbit Reactivity: human, mouse, rat B. Lab Vision 1. Mouse clone CUB 7402; clone TG100 Reactivity: human and guinea pig. Others-not known. 2. Rabbit Reactivity: human, mouse and rat C. Novus Biologicals 1. Rabbit Reactivity: human, guinea pig, rat, mouse, echinococcus parasite.

Anti-TG3 Antibody Immundiagnostik 1. Rabbit Reactivity: human

Anti-TG5 Antibody Orbigen, Inc. 1. Chicken

Anti-Factor XIIIa Lab Vision 1. Rabbit Reactivity: human 2. Mouse anti-factor XIIIa Ab-1 monoclonal antibody Reactivity: human

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Anti-Erythrocyte Membrane Protein Band 4.2 (EPB4.2) Antibody Orbigen, Inc. 1. Rabbit Reactivity: human Anti-Isopeptide Antibody Novus Biologicals 1. Mouse monoclonal Reactivity: unspecified

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Fesus L, Piacentini M: Transglutaminase 2: An enigmatic enzyme with diverse functions. Trends Biochem Sci 2002;27:534–539. Lorand L, Graham RM: Transglutaminases: Crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 2003;4:140–156. Chen JS, Mehta K: Tissue transglutaminase: An enzyme with a split personality. Int J Biochem Cell Biol 1999;31:817–836. Griffin M, Casadio R, Bergamini CM: Transglutaminases: Nature’s biological glues. Biochem J 2002;368:377–396. Dvorcakova M, Macejova D, Pallet V, Higueret P, Vasson M-P, Rock E, Brtko J: Transglutaminases and endocrine system. Endocr Regul 2002;36:31–36. Grenard P, Bates MK, Aeschlimann D: Evolution of transglutaminase genes: Identification of a transglutaminase gene cluster on human chromosome 15q15. J Biol Chem 2001;276:33066–33078. Lesort M, Tucholski J, Miller ML, Johnson GV: Tissue transglutaminase: A possible role in neurodegenerative diseases. Prog Neurobiol 2000;61:439–463. Griffin M, Verderio E: Tissue transglutaminase in cell death. Symp Soc Exp Biol 2000;52: 223–240. Akimov SS, Krylov D, Fleischman LF, Belkin AM: Tissue transglutaminase is an integrin-binding adhesion co-receptor for fibronectin. J Cell Biol 2000;148:825–838. Piacentini M, Colizzi V: Tissue transglutaminase: Apoptosis versus autoimmunity. Immunol Today 1999;20:130–134. Beninati S, Piacentini M: The transglutaminase family: An overview. Amino Acids 2004;26: 367–372. Karpuj M, Steinman L: The multifaceted role of transglutaminase in neurodegeneration. Amino Acids 2004;26:373–379. Mhaouty S: Gh/tissue transglutaminase 2: An emerging G protein in signal transduction. Biol Cell 2004;96:363–367. Verderio EA, Johnson T, Griffin M: Tissue transglutaminase in normal and abnormal wound healing. Amino Acids 2004;26:387–404. De Laurenzi V, Melino G: Gene disruption of tissue transglutaminase. Mol Cell Biol 2001;21: 148–155. Nanda M, Ismaa SE, Owen WA, Husain A, Mackay F, Graham RM: Targeted inactivation of Gh/tissue transglutaminase II. J Biol Chem 2001;276:20673–20678. Milakovic T, Tucholski J, McCoy E, Johnson JVW: Intracellular localization and activity state of tissue transglutaminase differentially impacts cell death. J Biol Chem 2004;279:8715–8722.

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18 19

20 21

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Mehta K, Fok J, Koul D, Miller F, Sahin AA: Prognostic significance of tissue transglutaminase expression in drug-resistant and metastatic breast cancer. Clin Cancer Res 2004;10:8068–8076. Antonyak MA, Miller AM, Jansen JM, Boehm JE, Balkman CE, Wakshlag JJ, Page RL, Cerione RA: Augmentation of tissue transglutaminase expression and activation by epidermal growth factor inhibits doxorubicin-induced apoptosis in human breast cancer cells. J Biol Chem 2004; 279:41461–41467. Jiang WG, Ablin R, Douglas Jones A, Mansel RE: Expression of transglutaminases in human breast cancer and their possible clinical significance. Oncol Rep 2003;10:2039–2044. Rodolfo C, Mormone E, Matarrese P, Ciccosanti F, Farrace MG, Garofano E, Piredda L, Fimia GM, Malorni W, Piacentini M: ‘Tissue’ transglutaminase is a multifunctional BH3-only protein. J Biol Chem 2004;279:54783–54792. AbdAlla S, Lother H, Langer A, Faramawy Y, Quitterer U: Factor XIIIA transglutaminase crosslinks AT1 receptor dimers of monocytes at the onset of atherosclerosis. Cell 2004;119: 343–354.

Dr. Kapil Mehta Department of Experimental Therapeutics UT M. D. Anderson Cancer Center Unit 422, 1515 Holcombe Blvd Houston, TX 77030 (USA) Tel.
1 713 792 8140, Fax
1 713 745 4167, E-Mail [email protected]

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Mehta K, Eckert R (eds): Transglutaminases. Prog Exp Tum Res. Basel, Karger, 2005, vol 38, pp 19–36

Structure-Function Relationships of Transglutaminases – A Contemporary View Zoltán Nemes, Éva Cso´´sz, Goran Petrovski, László Fésüs Departments of Biochemistry and Molecular Biology, Signaling and Apoptosis Research Group of the Hungarian Academy of Sciences, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary

The Inherent Chemical Reactivity of Carboxamido and Thiol Functional Groups in Proteins

Protein-bound asparagines and glutamines have a remarkable potency to engage in acylation reactions driven by the free energy of ammonia release. Accumulating data suggest that the ␤- and ␥-carboxamido bonds are intrinsically unstable and undergo spontaneous deamidation in aqueous solutions [1]. The hydrolysis, however, is greatly accelerated by acidic conditions (such as in the gastric juice) or by specific enzymes, such as deaminases, proteinases and transglutaminases. The replacement of a single glutamine or asparagine with a negatively-charged side chain may lead to drastic changes in protein conformation and, presumably, function. Deamidation by enzyme catalysis (e.g. by transglutaminases), therefore, must be under strict control. The juxtaposition of a labile cysteine or hypothetically, also serine proton within the protein may favor and accelerate the decay of the carboxamido bond by the formation of an intrinsic thioester or ester bond. The existence of such rearrangement has been demonstrated with the coagulation protein ␣2-macroglobulin. Within the ␣2-macroglobulin sequence Gln952 forms spontaneously a thioester bond with Cys949 enabling this protein to bind covalently to its protein targets by transferring the glutamyl moiety from the cysteinyl to a lysyl residue in various proteases, such as trypsin, plasmin and elastase, which are sterically inhibited by the covalently cross-linked bulky inhibitor [2].

The attack of the carboxamido bond requires only a cysteine or serine moiety, which can be made reactive by the electron-depriving effect of juxtaposed moieties, most commonly histidine imidazole side chain reciprocally protonating its imidazole nitrogen from the cysteine/serine residue, while the other imidazole nitrogen is hydrogen bonded to a ␤-Asx side chain carbonyl oxygen. This way a charge-relay system is formed, which is identical to that of the common proteases (fig. 1). Charge relaying through activated cysteinyl moieties are used by different types of enzymes – including proteases, transglutaminases and peptidyl-disulfide isomerases – to carry out nucleophilic attack on covalent bonds. Peptidyl-disulfide exchange requires exposed SH groups, no wonder transglutaminases show peptidyl-disulfide isomerase catalytic activity with suitable protein substrates [3] and the other way round, peptidyl-disulfide isomerases, such as human ER-60 protein and worm homologs, which are discussed in other chapters of this book, also reveal transglutaminase activity, at least under in vitro conditions [4]. Presumably, protein-bound asparagine could engage in the same type of reactions as glutamine, although, protein bound asparagine deamidation and transasparagination reactions have not been investigated yet. According to our present knowledge of the literature, transglutaminase-analogous ‘transasparaginase’ enzymes have not yet been identified, although we have no information on the extent and intensity of the scientific effort to rule out the existence of such type of enzymes.

Waterproof Activated Cysteines Make Transglutaminases – In Theory

Cysteine proteases, the prototype of which is papain, are phylogenetic relatives of the ‘classical’ (or, more exactly ‘nominal’) transglutaminases (TGases). TGases differ from them mainly in the rate of access by water or by other acyl-acceptor nucleophiles to the catalytic site. If water has free access to the acyl-enzyme thioester intermediate, other substrates have little chance to compete with the abundance and nucleophilicity of this solvent. However, if water is repelled from the catalytic area of the thioester bond, and nucleophilic, but uncharged moieties can approach it, the reaction will favor the transfer of the acyl residue onto the nucleophile (fig. 1). Such nucleophilic functional groups are not too manifold in biological systems. Only primary amines and primary alcohols need to be considered. Historically, the amine-incorporating activity into proteins was discovered first by Heinrich Waelsch, who first termed this phenomenon ‘transpeptidation’, and after he and his colleagues showed that the catalytic

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Fig. 1. Acid-base catalytic mechanism of transglutamination. Nucleophylic attacks on both the glutamine ␥-carboxamido as well as the glutamyl-enzyme thioester bonds are facilitated by the proton-sucking effects of the histidine imidazole nitrogens as well as the formation of a quaternary oxyanion intermediate around the ␥-carbon atom. Similar reaction mechanisms are possible for other nucleophilic acceptor substrates reacting by virtue of their R-OH (or HOH) functional groups.

activity uses the ␥-carboxamido group of peptide-bound glutaminyl side chains, the nomenclature was adjusted to ‘transglutamination’ [5]. Since the initial discovery, many proteins with transglutaminase activity were identified from prokaryotes to mammals; some are structurally and phylogenetically related to each other, others are not. The question arises: what makes a protein transglutaminase and what are the essential and salient features of such a catalytic activity? The exclusion of water from the catalytic site implicates that both the Gln-containing substrate (also known as the ‘acyl’- or ‘donor’-substrate) and the nucleophile (also known as ‘amine’- or ‘acceptor-substrate’) must access the catalytic cysteine across a channel surrounded by apolar residues, and this tube has to be at least as long as 2–3 times, and cannot be broader than twice the average radius of hydrogen bonding (6–10 ⫻ 6 Å) in order to achieve an efficient exclusion of solvent water. The ‘channeling’ of the substrate residues requires that the two substrates must approach and engage the catalytic site from two directions, therefore two such tunnels are needed, one for the acyl, the other for the amine substrate. If bulky substrates, such as proteins, are to

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Substrate protein 2

Lys TGase

Gln

Substrate protein 1

a

b

cross-link

c

Fig. 2. Three-dimensional scheme of a ‘minimal’ transglutaminase enzyme. (a) Two tunnels lock out the solvent water from the depth of the globular domain. (b) The glutamineand amine-substrate channels guide and position the substrate glutamine and lysine moieties to the catalytic center, where their ends can interact with the catalytic residues and one another. (c) After the formation of a ␥-glutamyl-␧-lysine isopeptide cross-link, both tunnels have to open up and form a continuous groove to release the cross-linked product. This opened conformation could not have been characterized from the solved structures of latent or idle TGases.

be cross linked, the glutaminyl- and amine-substrate tunnels need to open reasonably distantly from one another, practically, on opposite facades of the transglutaminase (fig. 2.). The small, but considerable likelihood of the appearance of water molecules in the hydrophobic niches of an enzyme implicates that the acylenzyme intermediate is unstable. However, to the best of our knowledge, the kinetic parameters for glutamine deamidation have never been compared to the reaction

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velocity of transamidation, or if were, these data were left unpublished. On the other hand, the hydrolysis of acetyl, ␣-alanyl- and ␥-glutamyl-p-nitrophenyl esters by guinea pig liver transglutaminase was routinely used in Jack Folk’s laboratory to compare kinetic parameters for amine substrates, as the better amine substrate resulted in a faster release of the nitrophenol chromogen [6]. These calculations subtracted the velocity of hydrolysis (as blank) from that of amine incorporation. From the slopes of published graphs, it can be estimated that the rate of hydrolysis is by two magnitudes of order lower than that of transamidation, if the amine substrates were given at near-saturating (0.05–2 millimolar) concentrations [7]. The effective ratio between deamidated and transamidated glutamines under in vivo conditions may depend largely on the availability of amine substrates. Whereas polyamines may reach concentrations as high as several hundred ␮moles in specialized dividing cells (especially in plants), ␧-amino groups of specific lysyl residues of specific substrate proteins are not likely to be in such abundance. Moreover, the rate of diffusion of protein-bound lysines in complex natural protein matrices, such as blood plasma, cytoplasm, pericellular space, might be by several magnitudes of order slower than those of small amines. Therefore, it is plausible to assume that the amine substrate protein should be attached to the transglutaminase and keep its lysyl ␧-amino group in the hydrophobic channel, even before the ␥-glutamyl-thioester bond is formed.

The Genes and Expression of Human TGases

The genes for the 9 members of the human TGase family (TGM1–7, F13A1 and EPB42, which codes a catalytically inactive protein) reveal high degree of similarity in the numbers, lengths and boundaries of exons. The TGM1 and F13A1 genes consist of 15 exons and 14 introns, whereas the other genes (TGM2–7 and EPB42) consist of 13 exons and 12 introns. Phylogenetical analysis revealed that an early gene duplication event of an ancestral TGM gene gave rise to these two groups and that the different exon number is a consequence of the acquisition of a noncoding first exon and the intersection of exon 9 by an intron sequence, which formed exons 10 and 11 of F13A1 and TGM1 (see ensuing chapters of this book for review and references). The conservation of TGase intron-exon boundaries reflects the modular assembly and similar domain structure of vertebrate TGases. Whereas the expression, biochemical and structural features and (patho) biological relevance of factor XIIIa and TGases 1–3 have been studied in remarkable depth, very little is known about the specific features of the other

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four TGases. TGase 4 is involved in the formation of the copulatory plug in rodents; however, a covalent protein cross-linking in prostatic or seminal secretes was not noted in other mammals. Nevertheless, the enzyme is expressed in human prostate where its function is questionable [8]. TGase 5 mRNA has been shown to be present in various organs [9]. The catalytic activities of TGase 6 and TGase 7 have not yet been demonstrated.

Regulators of TGases

Owing to the potentially disruptive effects of glutamine deamidation and transamidation, human transglutaminases are expressed and stored in zymogenic or otherwise inactivated forms and can be activated only in the presence of calcium levels which are not common and ubiquitous in living cells. TGase 1, TGase 3 and factor XIIIa, which typically function in highcalcium milieus of keratinocytes and blood plasma, are expressed and stored as zymogens and require enzymatic cleavage besides Ca2⫹ to attain active conformation. The transglutaminase activity of TGases 2, 3 and 5 is strongly inhibited by the regular intracellular concentrations of GTP/GDP/GMP and also by ATP [9–11]. TGase 4 also binds GTP, however its hydrolysis and transglutamination-inhibitory effects have not been shown [12]. This noncompetitive allosteric inhibitory effect is reciprocally regulated by Ca2⫹ ions; higher nucleotide phosphate levels elevate the Ca2⫹ concentrations needed for transglutaminase activity, and excess Ca2⫹ ions displace the inhibitor from its binding site. Low Ca2⫹ and high GTP concentrations represent double protection against inappropriate TGase 2 activation in the cytosol. It was shown that the magnesium ions, which are present in the cells at near-extracellular (millimolar) levels also compete for one of the Ca2⫹-binding sites and further increase the Ca2⫹-requirements for TGase 3 [13]. Moreover, when a Ca2⫹binding site is occupied by Mg2⫹, it is not only unable to activate TGase 3, but also a prerequisite for binding of the noncompetitive allosteric inhibitor, GTP to the enzyme [11]. TGase 1 (‘keratinocyte’ transglutaminase, TGK) is undoubtedly the most divergent isoform from the other TGase homologs. TGase 1 is a 100-kDa membrane bound protein with a well-documented role in the formation of the cornified envelope in keratinocytes of squamous epithelia. TG1 is anchored to the cell membrane by fatty acylation of cysteines at its 10 kDa N-terminal part. The enzyme undergoes proteolysis into three 10 ⫹ 30 ⫹ 60 kDa fragments, which increases its catalytic activity. The 60-kDA part eventually cycles off the membrane and acts as a cytosolic enzyme [14]. TG1 enzyme was shown to cross-link loricrin, SPRs and involucrin and to attach ␻-hydroxyceramides to

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involucrin, thereby forming the ‘lipid envelope’, a velcro-like linkage between the protein CE and the lipid layers between the cornified keratinocytes [15].

Enigmatic Secretion of TGases

Factor XIIIa, TGase 2 and also TGase 4 lack hydrophobic amino-terminal leader sequence, their mRNA is translated on free ribosomes, are N-terminally acetylated after the removal of initiator methionine and have no carbohydrate decoration. Nevertheless, these proteins enigmatically get out from the cells. Following the cleavage of its activation peptide by thrombin, the secreted factor XIIIa functions as a plasma-clotting factor and modulator of tissue assembly. An extracellular role for TGase 2-mediated cross-linking activity was demonstrated in various processes, such as cell-ECM interactions, enchondral ossification, wound healing, cytokine activation and reinforcement of the dermoepidermal junction by the cross-linking of anchoring fibrils. TGase 2 acts extracellularly as an integrin coreceptor for fibronectin and is the main endomysial antigen in celiac disease (see other chapters of this book for reviews and references).

The Solved TGases Structures

Thus far, three of the human TGase enzymes (factor XIIIa dimer, TGases 2 and 3) have been structurally modeled on the basis of X-ray diffraction coordinates. The protein models built on the basis of X-ray coordinates are available from public web domains (www.rcsb.org.pdb). Our current knowledge of transglutaminase structure is nevertheless very limited; inasmuch as the modeled structures give little hint that how the static crystal structures shall rearrange in the presence of both glutamyl donor and acceptor substrates and other putative regulatory proteins, in order to make the catalysis work. Human TGase 2 was crystallized in a GTP-bound, calcium-free protein at 2.8 Å resolution (available in the PDB at the accession code 1KV3, [16]). Human factor XIIIa was crystallized in a zymogenic dimer form with one tightly-bound Ca2⫹ ion at 2.5 Å resolution (PDB code 1EVU) [17, 18]. TGase 3 was crystallized in both zymogenic (1L9M) [19], latent with 2 bound Ca2⫹ and one Mg2⫹ ions (PDB: 1NUG) [13], then latent with 2 bound Ca2⫹, 1 Mg2⫹ and GMP, GDP, and a nonhydrolyzable GTP analog, GTP␥S (PDB: 1SGX, 1RLE and 1RLL) [11, 20] and activated forms (PDB: 1L9N and 1NUD) [19] at resolutions between 1.9 and 2.7 Å. Further, the crystal structure of the latent red sea

From Function to Structure of TGases

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␤-sandwich N-terminus

C-terminus

Active site c o r e

Barrel 2

GDP

Barrel 1

Fig. 3. Ribbon and barrel diagram of the structure of the inactive TGase 2 protein with one tightly-bound GDP and no Ca2⫹ ions. ␤-strands are shown by bands, ␣-helixes are represented by barrels. The positions of the mobile terminal and linking loops were not, or poorly identifiable by the X-ray diffraction pattern.

bream transglutaminase (The fish homolog of mammalian TGase 2) at 2.5 Å resolution has also been solved (PDB: 1GOD) [21]. The divergent functions and localizations make a mechanistic comparison between homologous and structurally similar TGase isoforms difficult. For the sake of simplicity, we will introduce TGase 2 as a prototype for other isoenzymes and characterize other TGases with regard to their similarity to, or differences from this protein.

TGase Domain Structure

Akin to other crystallized homologs, as well as sequence homology-based structural predictions on not yet crystallized TGases, TGase 2 is built from one N-terminal ␤-sandwich-, the (catalytic) core-, and two ␤-barrel-domains (fig. 3). These domains span amino acids 1–139, 140–454, 479–585 and 586–687 in TGase 2, respectively. The N-terminal ␤-sandwich domain consists of an initial flexible loop, a short 310 helix, an isolated ␤-strand (B1), followed by five antiparallel strands (B2–B6) tightly packed into a ␤-sandwich

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motif, and a short B7 strand, which interacts with the B1 strand. In the core domain, the peptide backbone folds into two additional ␤-strands (B8 and B9), which stretch along the surface of the core domain, reach into the pocket between the core and barrel 1 domains and continues in four additional ␤-strands (B10–B13) and four ␣-helixes (H1–H4). The first three ␣-helixes of the latter are triangularly arranged and center the catalytic Cys277 residue to the focus of the core domain, whereas the other two residues of the catalytic triad (His335 and Asp358) reach to the center of the domain from adjacent ␤-sheet strands. The last ␣-helix (H4) harbors the residues homologous to the sole Ca2⫹-binding site in factor XIIIa. The core and barrel 1 domains are connected by a flexible loop (amino acids 454–478) which might act as a hinge when barrel 1 releases the core domain to retrieve the latent transglutaminase activity. Barrel 1 comprises six ␤-strands and one ␤-turn, barrel 2 folds into seven antiparallel ␤-strands.

Structural Correlates of Transglutamination

Data from deletion studies revealed that the two barrel domains are not necessary for TGase activity – on the contrary, their loss increases catalytic rate – whereas the ␤-sandwich and the core domains are indispensable [22]. The catalytic site of TGase 2 is buried in the center of the core domain, where the charge-relay system detaches the proton from Cys277 and transfers it to the parting ammonia. The catalytic triad of TGase 2 involves His335, which relays one proton between the catalytic cysteine and Asp358 (fig. 1). In the crystal structure of the inactive TGase 2 (as well as all the other 3 crystallized human, and also the sea bream TGase), the barrel 1 also buries the access to the glutaminyl-substrate-binding surface of the core domain (fig. 2). Trp241 is essential for the exclusion of water from the catalytic site, and is conserved in all homologous TGase protein sequences, except for the inactive band 4.2 [23]. In papain-like proteases a Gln residue is present on the place of this Trp, which does not block the penetration of water. Ahvazi and Steinert [24] assumed that, at least in TGase 3, two conserved Trp moieties might surround the glutamine-substrate groove, one of which corresponds to Trp241, the other to Trp332 in TGase 2. The indole NH-s of these two Trp residues should interact with the quaternary oxyanion and amine intermediates, respectively. The interaction of Trp241 with the ␥-glutamyl carbonyl oxygen is obvious, though the positioning and activating effect of Trp332 towards the acceptor substrate is questionable. Nevertheless, it is possible that these and also other Trp residues are essential for the ‘waterproofing’ of the active site. Homologous TGases have ten highly-conserved tryptophans, nine of which are located in the core

From Function to Structure of TGases

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domain sequence. As assessed by fluorescence resonance transfer measurements, the indole moieties of five of these nine tryptophans in TGase 2 (Trp180, Trp241, Trp278, Trp332 and Trp337) are positioned in molecular proximity (within 20 Å) to the catalytic site so that these might have a role in locking out of the water from the proximity of the ␥-glutamyl-TGase thioester intermediate [23, 25]). The access of the glutamyl donor and acceptor substrates to the active center is possible from the two opposed sides of the TGases, and the angle of the two substrate channels is ⬃120⬚ [24]. The lockout of water from the direction of the access of the amine substrate is probably accomplished by an ion bond between two conserved residues (His305 and Asp306 in TGase 2) which keeps the groove shut until this ion bond is disrupted by the non-binding electron pair of the nucleophile [17]. Even if Asp306 (corresponding to Asp343 in factor XIIIa) and His305 might be important for positioning and deprotonation of the amines substrate group to the active site (fig. 1) [17, 26]; this pair of residues alone might just as well position water to the thioester linkage, indicating that further layers of water-damming hydrophobic residues are also indispensable for an efficient transamidation. The factor XIIIa and fish TGase equivalent of Cys336 in TGase 2 were suggested to engage the Cys277 in disulfide bridge formation in the active enzymes [21]. This residue is, however, not conserved in TGase 3 (Val331) and the formation of disulfide bond is not supported by structural data. Nevertheless, the possible formation and reversal of the putative Cys277–Cys336 disulfide bond would greatly favor the peptidyl-disulfide isomerase catalysis by TGase 2 [3].

Activation of Latent TGases

In either of the crystallized zymogenic TGases, the proton-relay system of the catalytic triad is kept inactivated by the detention of the active site SH proton through a hydrogen bridge formed with a tyrosine (Tyr516 of TGase 2) of barrel 1. The sterical hindrance as well as the hydrogen bonding by barrel 1 assures a ‘bomb-proof ’ inactivation of the latent enzymes. In TGase 2, either GTP or GDP is tightly bound into the groove between the core and barrel 1 and thereby fixes the inhibitory position of barrel 1 on the catalytic domain (fig. 2). In the factor XIIIa zymogene dimer, the activation peptide stoppers the niche between the core and barrel 1 domains of one monomer and the core ␣-helixes and ␤-sandwich domain from the opposite subunit. Given the over secured and tightly compacted rigid zymogene structures, several structural hypotheses have been generated to explain, how the zymogenic conformation could be remodeled to possible and plausible active conformations. Small-angle X-ray and neutron scattering experiments revealed that the power of Ca2⫹ binding

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per se might be sufficient to rearrange the structure of TGase 2 to the catalytically active (transglutaminating) conformation. As a result of the Ca2⫹ ion coordination, TGase 2 was found to release its nucleotide and dissociate the barrels from the ␤-sandwich-plus-core domains, which thereafter, might accommodate an elongated protein shape and rotate freely on the axis of the linker sequence between the two parts [27]. This model provides explanation for the wellestablished antagonism between calcium and GTP binding. Unfortunately, the Ca2⫹-binding sites for TGase 2 have not yet been clarified. Based on equilibrium dialysis, activated TGase 2 protein can bind 6 calcium ions, all of which bind with an apparent affinity constant of 90 ␮moles [28]. We do not know, how many of these are required for the TGase activity, and whether a gradual occupation of the six binding sites may create enzymes with different catalytic properties, or activation proceeds in an all-or-none fashion. By surface mapping and 43Ca-NMR Ambrus et al. [29] predicted numerous low-affinity Ca2⫹-binding sites on the surface of TGase 2, which were only saturated with Ca2⫹ at supraphysiological concentrations. Partial occupation of several Ca2⫹-binding sites may contribute to the structural plasticity and ‘lubricate’ the movement of domains by decreasing the energy hurdles between conformational states. The comparison with the crystal structures of other TGase homologs provides little hint to model the Ca2⫹-dependent activation dynamics of TGase 2. Both the zymogen and the active factor XIIIa contains one and the same bound calcium ion, which is coordinated by the residues of the loop connecting the core domain to barrel 1. The carbonyl groups coordinating the Ca2⫹ of factor XIIIa (␣-carbonyl of Ala457, as well as the side-chain carbonyls of Asn436, Asp438, Glu458 and Glu490) are also conserved in TGase 2 (␣-carbonyl of Ser419, as well as side-chain carbonyls of Asn398, Asp400, Glu447, Glu452). In the GTP/GDP-inhibited conformation of TGase 2, the ␣-carbonyl of Ser419 is pushed away by the nucleotide, and therefore, cannot coordinate Ca2⫹. However, the elimination of the above mentioned side-chain carbonyls by side-directed mutagenesis did not completely abrogate the TGase activity of TGase 2, which indicates that other Ca2⫹-binding sites might be equally, or more important for active conformation [30]. TGase 3 zymogen binds one Ca2⫹ after proteolytic cleavage of the hinge loop connecting the core and barrel 1 domains at Ser469. The 27 kDa and the (catalytic) 50 kDa fragments remain associated together as an active transglutaminase [31]. After the cleavage two further Ca2⫹-binding sites are formed: one is the structural homolog of that in factor XIIIa; the other Ca2⫹ recruits its chelating carbonyls by pulling Asp324 together with the loop sequence harboring this residue by ⬃9 Å aside, and thereby opens the inlet of the hydrophobic channel, through which the glutaminyl substrate can access the active site [19]. It was

From Function to Structure of TGases

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demonstrated by comparison of the TG3 structures binding 3 Ca2⫹ ions with the 2 Ca2⫹and 1 Mg2⫹ ion-bound form (the latter is more likely to form under the expected intracellular ion concentrations) that chelation of Mg2⫹ instead of Ca2⫹ does not pull away Asp324 (‘the lever of the channel lid’) due to the different radius and coordination geometry of the two ions. Thus Mg2⫹ inhibits TGase 3 transglutaminating activity by occupying the third Ca2⫹-binding site [13]. In spite of the elaborated and plausible mechanistic reasoning on the catalytic mechanism of TGase 3, it is unclear how the cross-linked enzyme product could leave this channel, as the opening of its lid does not detach the bonding or open up the angle between the core and barrel 1 per se (fig. 2). In order to cover the activation energy requirements for factor XIII, which does not bind further Ca2⫹ on activation, it was proposed that the relaxation of three nonproline cis-peptide bonds, by conversion into the energetically favored trans-conformation, might drive the remodeling of the zymogene into an active enzyme [18]. These three nonproline cis-peptide bonds were also noted in the core domains of TGase 2 and TGase 3 and sea bream TGase structures and show strict conservation between these TGases. One of these strained bonds was located between the catalytic Cys277 and Tyr274 in TGase 2. The mutation of Tyr274Ala mutation, which relaxes the bond was found to abolish TGase activity as well as the translocation of the protein into the extracellular space [32]. Nevertheless, the comparison of zymogene and activated TGase 3 structures did not reveal the relaxation of either of the nonproline cis-peptide bonds [19]. Therefore, Ahvazi et al. [24] proposed that the nonproline cis-bonds might rather serve the mechanical stabilization and tightening of the core domain instead of storing energy akin to a strained spring. Though the Ca2⫹-binding and the nonproline cis-peptide bond relaxation mechanisms might both contribute some free energy to the activation of the crystallized TGases, their estimated thermodynamical contribution is well below the overall energy necessary to tear off the tightly attached barrel 1 from the core domain. The missing fraction of energy should come from the binding of the inactive TGases to their glutamine substrate proteins. Even if the residues involved in catalysis and water shielding of the active site are highly conserved, the surface charge distributions of the core domains are significantly different in the crystallized TGases, which makes their protein substrate preferences divergent and difficult to predict.

Purine Nucleotide Binding and Hydrolysis by TGases

The nucleotide-binding pocket of TGase 2 is unique among GTP-binding proteins, inasmuch as TGase 2 does not use the structurally conserved

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a

b

c

Fig. 4. Amino acids involved in the binding and hydrolysis of GTP (a) in TGase 2, (b) TGase 3 and (c) TGase 5. Conserved residues are labeled with boldface letters, the labels for conservatively replaced residues are italicized.

GTP-binding motif present in all known mono- and trimeric G-proteins, elongation factors and dynamin. The hydrophobic nucleotide-binding groove of TGase 2 is formed by the niche between the core and barrel 1 domains, which opens to the surface opposite to the glutamine substrate binding site (fig. 4). The anhydride bond between the ␤- and ␥-phosphates of the ATP/GTP can be attacked by a water molecule, which may be positioned by hydrogen bonding to Lys173 (core) or Arg476 (barrel 1). These two basic residues might neutralize the two extra negative charges generated by the release of the ␥-phosphate. The negative charges of ␣- and ␤- phosphates are neutralized by Arg580 (barrel 1). Furthermore, the NH in Val479 connects to the ␤-phosphate through a hydrogen bond. The purine base is positioned by Lys173 and Phe174 from the core domain and Trp583 and Ser482 on barrel 1. These residues involved in the tethering of the GTP/ATP are conserved between mammalian TGase 2 sequences, however, other TGase homologs, which also bind purine nucleotides, have replaced most of these by different interacting residues located in nonidentical sequence positions (fig. 4.). TGase 3 and TGase 5 were also found to hydrolyze and be inhibited by GTP/ATP in a Ca2⫹-concentration-reversible manner, though the inhibitory constants seem to be several-fold higher than with TGase 2 [9, 33]. Interestingly, whereas TGase 2 hydrolyzes both ATP and GTP at similar pace [34], TGase 3 is a 100-fold

From Function to Structure of TGases

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faster GTPase then ATPase in its ATP/GTP-binding form (2 Ca2⫹ ⫹1 Mg2⫹/ protein) [11]. TGase 5 showed only GTP hydrolysis and no measurable ATPase activity [9]. The binding site of TGase 3 for ATP/GTP is similar to that of TGase 2, although this similarity comes rather from the mechanism of proteinnucleotide interaction than from evolutionally conserved binding residues. Basically, from all the 15 (TGase 2) or 16 (TGase 3) amino acids which were modeled to bind the nucleotide triphosphate, only two residues (Val479 and Arg580 in TGase 2, binding the ring and N-7 atom of the base, and coordinating the ␥-phosphate, respectively), were conserved (fig. 4) and only two residues were conservatively replaced (Arg474, Arg476 of TGase 2 and Lys485, Lys487 of TGase 3, all needed for the coordination and hydrolysis of ␥- and ␤-phosphate groups and neutralization of the resulting two extra anionic charges). The conservatism is notably higher among the amino acids not involved in GTP-binding among the 15, 11 and 7 amino acids that surround the ATP/GTP-binding pocket from the sides of the core domain and the first and last ␤-strands of barrel 1 (fig. 4). It is remarkable that phylogenetically very closely-related TGases use such variable sequences for the same function. A probable explanation may be a rapid functional diversification between these isoenzymes in order to function under different regulatory conditions, such as high and low nucleotide and calcium concentrations. TGase 5 requires ⬃6-fold higher levels of calcium for the same activation as TGase 2, whereas it retains ⬃20% of its full activity in the presence of usual intracellular GTP concentrations [20].

G-Protein Signaling by TGase 2

TGase 2 was identified as a GTP-binding transducer (Gh␣) protein [35], although the biological relevance of this activity is unclear. In cell transfection experiments, GTP-bound TGase 2 was shown to inhibit phospholipase C␦1 and this inhibition is suspended if the GTP is hydrolyzed to GDP upon receiving signal from ␣1B, ␣1D, adrenergic, oxytocin, or thromboxane A2 receptors [36]. The rapid degradation of GTP to GDP and consecutive GDP/GTP exchange by TGase2/Gh␣, a phenomenon measurable with pure enzyme in vitro, is decelerated by binding of TGase2/Gh␣ to a non-GTP-binding subunit (Gh␤, calreticulin) [37], which stabilizes the GDP-bound form [38]. Deletion and peptide-mapping analyses located the receptor-interacting surfaces of TGase2/Gh␣ to barrel 1 (547–561 residues) and barrel 2 (633–646 residues), both are located on the glutaminyl-substrate-binding site. The phospholipase C␦1-interacting surface was located to the C-terminal part of barrel 2 (665–672 residues) [39].

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The signaling activity of the protein was found to be unrelated to its potential transglutaminase activity, as cell transfections with the Cys277Ser active site mutant had the same effect on phospholipase C␦1 as the wild-type TGase2/Gh␣ protein. Further, the abrogation of GTP-binding by missense mutation of Ser171 was found not to interfere with the TGase activity of the protein [40].

Disulfide Exchange and Regulation Through Thiol Modifications

TGase 2 was shown to have peptidyl-disulfide isomerase activity [3]. TGase 2 harbors numerous cysteine residues, besides the catalytic one and all of these cysteines have to be in reduced (SH) state, otherwise the enzyme loses its TGase activity. Which of the numerous cysteines may participate in disulphide exchange reactions, and what the specificity and biological relevance of this catalysis might be, is yet unknown. Besides oxidation, nitrosylation may also reversibly switch off the transglutaminase activity of TGase 2 by modifying any of its 15 NO-reactive cysteine residues in a Ca2⫹-dependent manner [41].

NonCatalytic Interactions of TGase 2

Besides its various enzymatic activities, extracellular TGase 2 was shown to link fibronectin to ␣5␤1 and ␣␯␤3 integrin receptors in an Arg-Gly-Asp sequence independent (‘alternative’) manner and thereby increase the attachment and spreading of cultured fibroblasts on fibronectin-coated plates [42]. TGase 2 was shown to bind to the 42 kDa gelatin-binding domain of fibronectin [43]. The interacting surfaces on TGase 2 to ␤-integrins and to fibronectin were located to the amino-terminal 28-kDa ␤-sandwich domain [44]. Unfortunately, this domain contains mobile N-terminal and inter-␤-strand sequences invisible to crystallographic analysis. As concluded from experiments with catalytically inactive TGase 2 mutants, the strong binding of the protein to either ␤-integrin or fibronectin is not affected in catalytically inactive Cys277Ser and Tyr274Ala mutants, still, these proteins lacking transglutaminase activity did not get out from the cytosol and thus could not function as integrin coreceptors either [32].

Concluding Remarks

Intensive research efforts over the last decade solved the structure of three human TGases crystallized as zymogenes, or potentially active, but resting

From Function to Structure of TGases

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proteins. However, the crystal structures provided insufficient information about the molecular mechanics of the active enzymes and their interactions with protein substrates. A detailed understanding of TGase function will require further advancements in structural analysis, a more accurate knowledge of the relevant biological activities, recognition of interacting proteins and some further decades of meticulous TGase research.

Acknowledgments This work was funded by grants OTKA T92545,TS 044798, T 043083, ETT 115/03, EU-QLK3-CT-2002–02017.

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36 37 38 39 40

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Murthy SN, Lomasney JW, Mak EC, Lorand L: Interactions of G(h)/transglutaminase with phospholipase C␦1 and with GTP. Proc Natl Acad Sci USA 1999;96:11815–11819. Feng JF, Readon M, Yadav SP, Im MJ: Calreticulin down-regulates both GTP binding and transglutaminase activities of transglutaminase II. Biochemistry 1999;38:10743–10749. Baek KJ, Das T, Gray CD, Desai S, Hwang KC, Gacchui R, Ludwig M, Im MJ: A 50 KDa protein modulates guanine nucleotide binding of transglutaminase II. Biochemistry 1996;35:2651–2657. Feng JF, Gray CD, Im MJ: ␣1B-adrenoceptor interacts with multiple sites of transglutaminase II: Characteristics of the interaction in binding and activation. Biochemistry 1999;38:2224–2232. Iismaa SE, Wu MJ, Nanda N, Church WB, Graham RM: GTP binding and signaling by Gh/ transglutaminase II involves distinct residues in a unique GTP-binding pocket. J Biol Chem 2000;275:18259–18265. Lai TS, Hausladen A, Slaughter TF, Eu JP, Stamler JS, Greenberg CS: Calcium regulates S-nitrosylation, denitrosylation, and activity of tissue transglutaminase. Biochemistry 2001;40: 4904–4910. Akimov SS, Krylov D, Fleischman LF, Belkin AM: Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin. J Cell Biol 2000;148:825–838. Akimov SS, Belkin AM: Cell-surface transglutaminase promotes fibronectin assembly via interaction with the gelatin-binding domain of fibronectin: A role in TGF␤-dependent matrix deposition. J Cell Sci 2001;114:2989–3000. Gaudry CA, Verderio E, Jones RA, Smith C, Griffin M: Tissue transglutaminase is an important player at the surface of human endothelial cells: Evidence for its externalization and its colocalization with the ␤1 integrin. Exp Cell Res 1999;252:104–113.

Dr. Zoltán Nemes Departments of Biochemistry and Molecular Biology University of Debrecen Medical and Health Sciences Center PO Box 6, H–4012 Debrecen (Hungary) Tel. ⫹36 52416432, Fax ⫹36 52314989, E-Mail [email protected]

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Mehta K, Eckert R (eds): Transglutaminases. Prog Exp Tum Res. Basel, Karger, 2005, vol 38, pp 37–57

Transglutaminases and Their Substrates Francesco Facchianoa,b, Angelo Facchianoc a Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Roma, bLaboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata, IRCCS, Roma, and cIstituto di Scienze dell’Alimentazione, CNR, Avellino, Italy

Introduction

The family of enzymes called ‘Transglutaminase’ (TGase, EC 2.3.2.13), represents a fascinating field of study for protein biochemistry researchers. Transglutaminase was described for the first time in 1957 by Clarke et al. [1] and thereafter studied and characterized in more than 1,500 MEDLINE indicized publications having the term ‘transglutaminase’ in the title, the first published in 1959 [2]: from then, Medline citations for such enzyme have constantly increased reaching more than 3,600 as a whole. Since several synonymous or alternative names have also been used in literature [3–5] to describe the members of this family (as an example, the BRENDA database http://www.brenda. uni-koeln.de/ reports the following synonymous: Factor XIIIa, fibrin stabilizing factor, fibrinoligase, glutaminylpeptide ␥-glutamyltransferase, glutamyltransferase, glutaminyl peptide ␥-, Laki-Lorand factor, polyamine transglutaminase, R-glutaminyl peptide: amine ␥-glutamyl transferase, tissue transglutaminase, transglutaminase, transglutaminase C, TGC), the interest about this enzyme is surely larger than that the number of citations may suggest. Despite the large effort and interest on such molecule, many aspects regarding its function are still poorly understood. This is due to a number of reasons: (a) a complete vision of the function(s) of most members of TGase family is still unknown. This makes it difficult to frame the enzyme within a well-defined structural/functional classification; (b) most members of this family possess more than one or two enzymatic functions, only partially related;

(c) the number of family members is constantly growing. During the last years, new members have been discovered and characterized, adding further complexity to the field. Today, the members of this family are nine, but for some proteins showing high homology with one of the eukaryotic TGases, it is still unclear whether they may be considered part of the TGase family. Therefore, most probably the number of the family members will increase in the future and consequently the complete list of their substrates. Hence, grouping all members within a family allows unifying molecules otherwise different, which share the TGase activity, or at least one of the ‘canonical’ TGase’s functions, as a common feature. The observation that the substrates for a TGase member only partially overlap with those of another member, but some times they are characteristic for just one TGase member, makes even more complex in their classification. In order to clarify the complex scenario of TGase’s biological functions and substrates, we created an Internet web site (http://crisceb.unina2.it/what/) specifically devoted to improve and spread the knowledge on TGase. It was called W.H.A.T., an acronym standing for Worldwide Happening Around Transglutaminase. Until today, more than 15,000 web contacts to the web site confirm a large interest about this enzyme. The Members of TGase’s Family Aim of the present chapter is to list, present and discuss transglutaminase substrates. Therefore, the known substrates for each of their enzymatic functions will be reported and discussed. Several distinct TGase isoenzymes have been identified in mammals at the genomic level. Eight are structurally and functionally tightly related with the TGase’s functions, namely the TGase 1–7 and Factor XIII; an additional one, known as band 4.2, is highly homologous to the others, but lacks the catalytic site, therefore it is considered structurally but not functionally related to the others [3]. As shown in table 1, the nine members of this family are widely but differently expressed in cell compartments or tissues, as well as their biochemical features (like calcium- or GTP-dependent activity or protease-activation), although this kind of information is still missing for most of them. In addition to the above mentioned nine TGase members, recently other members have been identified from lower species, like a bacterial TGase from Streptoverticillium, from the filarial worm Dirofilaria immitis, or from the nematode worm Caenorhabditis elegans, whose correlation and evolutionary tree have been recently reviewed [3]. All mammalian members of this superfamily are homologous and are products of different genes arising from rearrangement, duplication and

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Table 1. Classification of TGases: related information about activation, localization, chemical reaction and possible involvement in human diseases, when available on published reports, have been enclosed Isoenzyme

Activation

Localization

Biological function(s)

Chemical reaction

Related disease

TGase 1

By proteolysis

Keratinocyte TGase, exists as membranebound and soluble forms

Cell envelope formation in keratinocytes differentiation

Transamidation [3–5]

Congenital autosomal recessive ichthyosis and other human epidermis diseases

TGase 2

By calcium, clostridial toxins [48]

Ubiquitous tissue, but also in extracellular space and nuclear

Programmed cell death, differentiation, cytoskeleton functions, cell motility and adhesion, signal transduction

Transamidation [3–5] (including serotonylation [28], pegylation [22]) G-protein/nucleotidebinding/hydrolyzing, ATPase [23–25], PDI [29–31], kinase [114] and deaminase [26, 27]

Celiac disease, neurological disorders, cataract, inflammation, possibly involved in diabetes mellitus and cancer

TGase 3

By calcium and proteolysis

Keratinocyte and hair follicle

Terminal differentiation of the keratinocyte, hair follicle

Transamidation nucleotidebinding/ hydrolyzing activity [55]

Human epidermis diseases

TGase 4

By calcium and phosphatidic acid [56]

Prostatic secretory

Reproduction and fertility in rodents [57]

Transamidation

Infertility

TGase 5

By calcium [58]

Epidermal differentiation

Transamidation nucleotide binding [58]

Several human epidermis diseases

Type 6

Not characterized

Not characterized

Not characterized

Transamidation

Type 7

Not characterized

Not characterized

Not characterized

Transamidation

Factor XIII

By calcium and thrombindependent proteolysis

Plasmatic, but also intracellular

Blood coagulation and wound healing

Transamidation

Erythrocyte membrane

Membrane functions

Band 4.2

TGs and Substrates

Coagulation disorders

Spherocytosis

39

chromosomal shifts [6]. They all belong to the papain-like superfamily of cysteine proteases [7] possessing a catalytic triad of Cys-His-Asp or Cys-His-Asn. Basically, function of an enzyme is to catalyze the reaction which modifies a specific substrate to obtain a product, therefore to better describe the TGase’s substrates we need to talk about the enzymatic function(s) before. Many enzymes possess more than just one enzymatic function, and some times the functional relationships among the different functions are not clear. In the case of TGase superfamily, these difficulties are much more evident and represent a fascinating field of study. As shown in table 1, the nine members of TGase superfamily possess many functions, some of them shared by more than one member, others by only a few members, others are unique functions. Since some of these functions are not well characterized at the moment, we will focus mainly our attention on the best known and most studied TGase enzymatic functions, and we will list and describe their substrates, although reporting, when available, some information regarding the other less known and studied functions and substrates. Hopefully in the near future, thanks to the new available technologies, more complete functional information for all TGase family members will be available. Table 1 shows also, when known, whether a clear relationship between a human disease and a TGase dysfunction has been shown: even in this case, future studies will be useful to complete the present table. It is noteworthy that, according to the enclosed table 1, the members of this enzyme family seem to catalyze at least seven different reactions, but such number may depend on how these reactions are considered different. Some of them may be considered chemically similar although with differences in terms of substrate specificity and kinetic features. Most of these functions are present in TGase 2. This may reflect a real multifunctionality restricted to this isoenzyme only, but most probably this is simply due to the fact that TGase 2 is ubiquitous, heavily involved in cellular processes like apoptosis and differentiation, and in human diseases like celiac disease. Therefore it is not surprising that TGase 2 is the most studied and it is possible that other TGase isoenzymes, less studied, are multifunctional enzymes too. The ability to hold a second job has been called ‘moonlighting’ and this ‘social-astrophysical’ concept has been applied to protein also. In fact, a number of proteins were classified as ‘moon-lighting proteins’ [8], like for instance crystallins, pinin or other proteins [9–11]. Looking at the different functions belonging to the TGase’s family members, TGase can be considered a bright example of moon-lighting protein. TGases Substrates These and other reasons make difficult a complete classification of all the substrates of TGase’s family, since a definitive classification of the enzymatic

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functions is still lacking. Nevertheless, in order to classify TGase’s substrates, we need to cluster them according to the following functions, reported in table 1. TGase is a Transamidating Enzyme: This is the most known and studied function, consisting of the catalytic post-translational modification of proteins by the formation of isopeptide bonds. Substrates of this reaction are divided into two main groups: i) proteins, and ii) other molecules containing primary amino groups reactive as amine group donors. Protein substrates may be divided in two main families: • protein substrates acting as acyl donor, i.e. possessing the reactive glutamine; • protein substrates acting as acyl acceptor, i.e. possessing the reactive lysine. An exception to this rule is the case of human protein synthesis initiation factor 5A, a protein that acts as protein substrate of transglutamination through the unique amino acid hypusine [12]. Some times, a protein TGase substrate may contain both reactive glutamine(s) and lysine(s) residue. The availability and the number of these reactive residues represent the biochemical features leading to dimer or polymer formation by cross-linking reaction catalyzed by TGase. To elaborate a complete list of known protein TGase’s substrates is a really difficult task: we included their name into a table, where we report also at least one relevant reference describing this substrate feature and, when available, in the case of transamidation reactions, whether it is modified on a glutamine or a lysine side chain. Further, we identify these proteins as ‘intracellular’ or ‘extracellular’ in order to facilitate a correct framing of the overall functions, at least at intra- or extra-cellular level. In some cases, it was possible to report also the subcellular localization for such substrate. Of course, protein secretion or translocation processes may move proteins from a compartment to another under particular conditions. Further, the peculiar localization of a TGase isoform within a specific compartment, like for instance the TGase 2 in cytosolic compartment and FXIII in plasmatic fluid, is today not more accepted as a dogmatic rule, since their localization in other compartments was demonstrated in several reports [13–16]. Therefore, the localization field of table 1 should be considered only indicative. In many cases it is not known whether a protein is a substrate for a specific isoform or not, but when such information is available, we highlighted it. Obviously, lacking a biochemical demonstration, we cannot rule out that a substrate for an isoform can be also modified by other isoforms. Of course, due to the complexity and the amount of the available data, this table should be constantly updated and improved. A database has been constructed aimed at identifying, through a bio-informatics approach, putative novel protein substrates (available at http://bioinformatica.isa.cnr.it/

TGs and Substrates

41

TRANSIT/) [17] and the transglutaminase’s researchers are encouraged to submit any more information about protein TGase substrates: therefore we try to keep this database updated as much as possible. The most comprehensive and complete list available for protein substrates of TGases has been reported in function/localization structuration in table 2. As above reported, the transamidating reaction may involve also molecules containing a reactive primary amine: among these, the most reactive substrates of TGase are: polyamines (putrescine, spermidine, spermine, cadaverine) [18], diamines [19, 20] or monoamines (like histamine or serotonin) [18, 21, 22], and they are also listed in table 2. TGase Possesses other Enzymatic Functions: In table 2, together with substrates of transamidating reaction, are listed so far identified substrates of other enzymatic functions of TGase family, namely GTP-/ATP-binding – hydrolyzing activity [23, 24], related to its G-protein function [25], deaminase enzyme [26, 27], protein serotonylation [28], protein disulphide isomerase (PDI) [29–31], intrinsic kinase activity [114], protein pegylation [22]. An interesting field of debate is still the specificity of the TGase’s substrate sites, which may help to identify novel substrate sites by means of computational/modeling/comparative methods. Many studies have been published [32–44], but no clear consensus has ever been identified, although some common features of substrate sites have been identified. For instance, a sequence consensus has been proposed for the deamidation reaction catalyzed by TGase. Different studies [45–47] aimed at the investigation of celiac disease involvement of TGase, reported a preference of deamidation for QXP sequences, while a QXXP sequence seems to be not suitable to deamidate the Q residue. This observation may represent an encouraging step towards the finding of similar consensus rules for other reactions catalyzed by TGase, as already under investigation. However, the reason for the unsuccess of such researches may be due to the limits of the simple primary structure and the need of the knowledge of the tertiary structure of substrates, so that a consensus should be searched among functional groups and Cartesian distances instead of residues and sequence positions. In this perspective, our structural/functional studies on known protein substrates of transamidating reaction [17, 48, 49] will be used as the starting knowledge for further studies in the three-dimensional vision of structural consensus among TGase substrates.

Conclusions

Looking at the whole set of substrates of TGase’s members family, their localization and sublocalization, we can draw a sort of ‘functional sketch’ for

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Table 2. Substrates of TGases Substrates

Isoenzyme

Reactive site

Localization

References

Acetylcholine esterase Actin

TGase 2 TGase 2

Intracellular Intracellular (cytoplasmatic)

[59] [60–62]

Aldehyde dehydrogenase

TGase activity of the nematode Caenorhabditis elegans

Glutamine Glutamine and lysine Lysine

Aldolase Amines (monoamines, diamines, polyamines): cadaverine and monodansylcadaverine, histamine, putrescine, serotonin, spermidine, spermine ␤-amyloid peptide

Intracellular Different isoenzymes

Act as amine donor

TGase 2

Glutamine and lysine

Androgen receptor

TGase 2

Annexin I (lipocortin I) ␣2-antiplasmin

TGase 2 FXIII better than TGase 2 TGase 2 and Factor XIII TGase of the nematode Caenorhabditis elegans Bacterial TGase

Aspartyl protease ATP synthase-␣ subunit

Bacteriorhodopsin Calgizzarin – S100C protein – MLN 70 – S100A11 Calpactin I light chain (S100A10) ␤ casein CD38 Cell adhesion molecule C (CAM) Clara Cell p10 Kda Chloroplast proteins Collagen ␣1(III) ␣␤-crystallin ␤A3-crystallin

TGs and Substrates

[63]

Glutamine Glutamine Glutamine and lysine Glutamine

[64] [18]

[65] Intracellular (nuclear receptor) Intracellular Extracellular?

[66]

Viral protein (HIV-1)

[69]

[67] [68]

[70]

TGase 1 and TGase 2

Glutamine and lysine Glutamine and lysine

[71]

TGase 2

Glutamine

Factor XIII TGase 2 TGase 2

Glutamine Lysine

[32] [74] [75]

Plant TGase(s) TGase 2 TGase 2 TGase 2

Glutamine Glutamine Lysine Glutamine

[76] [77] [78, 79] [80, 81] [82, 83]

Keratinocyte cornified envelope (CE)

[72]

[73] Secreted protein Intracellular

Extracellular Intracellular Intracellular

43

Table 2 (continued) Substrates

Isoenzyme

␤B3-crystallin ␤Bp (␤B2)-crystallin Cystatin Cytocrome C eIF5A (initiation factor 5A) ␤-endorphin Enolase

Erythrocyte anion transporter – Band 3 anion transport protein Fibrinogen A-␣ Fibrinogen-␥ Fibronectin FibN (peptide derived from the N-terminal sequence of fibronectin) Filaggrin linker segment peptide (FLSP) Galectin 3 Glucagon Glutamate dehydrogenase

Glutathione S-transferase

Gluten proteins (␣/␤-, ␥-gliadin and low molecular weight glutenin)

TGase 2 and possibly others TGase 2 TGase 2 and Factor XIII TGase 2 Transglutaminase of the nematode Caenorhabditis elegans Intrinsic TGase of human red blood cell TGase 2 and factor XIII Factor XIII Factor XIII

Reactive site

Localization

References

Glutamine Glutamine Lysine

Intracellular Intracellular

[82] [82] [84]

Glutamine Hypusine

Intracellular Intracellular

[85] [12]

Glutamine and lysine Glutamine

Endogenous opiates Intracellular (cytoplasmatic)

[86]

Glutamine

Intracellular

[87]

Glutamine and lysine Glutamine

Extracellular

[88–90]

Extracellular Extracellular Extracellular

[90] [91, 92] [93]

Glutamine

TGase 3 TGase 2 TGase 2 TGase activity of the nematode Caenorhabditis elegans TGase 2

TGase 2

Facchiano/Facchiano

[70]

[94] [95, 96] [97] [63]

Glutamine Lysine

Glutamine and lysine Fluorescein is covalently attached only to the N- or C-terminal site Glutamine deamidation

Intracellular

[98–100]

Extracellular

[45–47, 101, 102]

44

Table 2 (continued) Substrates

Isoenzyme

Reactive site

Localization

References

Glyceraldehyde-3phosphate dehydrogenase gp41

TGase 2

Lysine

Intracellular

[103, 104]

TGase 2

Glutamine and lysine Glutamine Serotonylation

Transmembrane

[105]

Viral envelope

[106] [28] [107]

gp120 Small GTPases Histidine-rich glycoprotein

TGase 2

H3 histone H4 histone H2A histone H2B histone Hepatitis C virus core protein ␣2 HS-glycoprotein (AHSG) Huntingtin Importin ␣3

TGase 2 TGase 2 TGase 2 TGase 2 TGase 2

Insulin A chain Insulin B chain Insulin-like growth factor-binding protein-1 (IGFBP-1) Insulin-like growth factor-binding protein-3 (IGFBP-3) Interleukin-2

TGase 2 TGase 2 TGase 2

Interacts with TGase 2 Glutamine Glutamine Glutamine

TGase 2

Kinase activity

[114]

Microbial transglutaminase (M-TGase) Keratinocyte TGase TGase 2 and TGase 3 TGase 2 and TGase 3 TGase 2 and TGase 3 TGase 2 and TGase 3 TGase 2

Glutamine

[115]

Involucrin Keratin, type II cytoskeletal 1 Keratin, type II cytoskeletal 2 epidermal Keratin, type II cytoskeletal 5 Keratin, type II cytoskeletal 6 ␣-ketoglutarate dehydrogenase

TGs and Substrates

FXIII

Glutamine Glutamine Glutamine Glutamine Glutamine

Expressed by the liver and secreted in plasma Intracellular Intracellular Intracellular Intracellular Viral protein

TGase 2 TGase 2 TGase 2

[108] [108] [108] [108] [109] [110]

Glutamine Lysine

Nuclear transport protein

[111] [112] [97] [97] [113]

Membrane

[116] [117]

Lysine

[117]

Lysine

[117]

Lysine

[117]

Lysine

Intracellular (mitocondrial)

[103]

45

Table 2 (continued) Substrates

Isoenzyme

Reactive site

Localization

References

␣-lactalbumin

Glutamine and lysine

Secretory protein

[118–120]

␤-lactoglobulin

TGase 2 and Streptoverticillium TGase (M-TGase) TGase 2 (?)

Secretory protein

[121, 122]

Laminin

FXIII

Glutamine and lysine Glutamine and lysine

Latent TGF-␤-binding protein-1 (LTBP-1) Lipoprotein a Loricrin

TGase 2

Myosin Nidogen (entactin) Nucleotide(s) binding/hydrolyzing Osteocalcin Osteonectin Osteopontin (extracellular matrix cell adhesion protein) Root and leaf pea proteins Periplakin Phosphoglycerate kinase Phospholipase A2 Phosphorylase kinase ␣2-plasmin inhibitor Plasminogen-activator inhibitor type-2 Proapoptotic kinase DLK

Extracellular

[123]

Glutamine and lysine

Cell envelopes

[124] [125]

Glutamine

Extracellular

[126] [127]

Glutamine Glutamine and lysine

Intracellular

[128] [129]

TGase 2 and FXIII

␣2-macroglobulin ␣2-macroglobulin receptorassociated protein Mellittin TGase 2 Microtubule-associated TGase 2 protein tau – isoform Tau-F (Tau-4) Midkine TGase 2 Monellin (analog of)

[165]

Glutamine

[96, 130, 131] [132]

Microbial transgluGlutamine taminase (M-TGase) and lysine TGase 2 TGase 2 TGase 3 TGase 5

Glutamine

Intracellular Extracellular

[133] [134] [23–25, 55, 58]

TGase 2 TGase 2

Glutamine Glutamine

Extracellular Extracellular Extracellular

[135] [136] [110, 137, 138]

Glutamine and lysine

[139]

TGase 2 TGase 2

Glutamine Glutamine

FXIII TGase 2 and FXIII

Glutamine Glutamine

TGase 2

Oligomerization

Facchiano/Facchiano

Intracellular? Extracellular Intracellular Extracellular?

[140] [141] [142] [143] [144] [145, 146] [147]

46

Table 2 (continued) Substrates

Isoenzyme

Reactive site

Procarboxypeptidase U (EC 3.4.17.20) plasma procarboxypeptidase B Protein disulfide isomerase (PDI)

TGase 2 and factor XIII

Glutamine

[148]

TGase activity of the nematode Caenorhabditis elegans

Amine donor

[63]

Retinoblastoma protein Rho A Ribonuclease A S100 calcium-binding protein A7 – psoriasin (S100A7 or PSOR1) Semenogelin I Semenogelin II Seminal vesicle secretory protein IV Bone sialoprotein (BSP) Soybean proteins S-peptide

Substance P Synapsin I ␣-synuclein Thrombospondin Thymosin-␤4 Troponin T Tubulin Uteroglobin Vasoactive intestinal peptide – VIP Vimentin Vinculin Vitronectin Von Willebrand factor Whey proteins

TGs and Substrates

Localization

References

[149] [150] [29] [73]

Intracellular TGase 2 TGase 2

Factor XIII Factor XIII TGase 2

PDI activity Glutamine

Glutamine and lysine (lower reactivity)

TGase 2 Microbial transglutaminase (M-TGase) from Streptomyces mobaraensis TGase 2 TGase 2 TGase 2 Factor XIII

TGase 2 FXIII ? TGase 2 TGase 2 Factor XIII Factor XIII Factor XIII Microbial transglutaminase (M-TGase)

[151] [151] [152]

Extracellular

Bone matrix Extracellular

[110] [153] [154]

Glutamine Glutamine

Extracellular Intracellular

Lysine

Intracellular (cytoplasmatic) Intracellular Intracellular Extracellular

[155] [53] [156] [157] [62]

Glutamine and lysine

[158] [159] [160] [161]

Glutamine and lysine Glutamine and lysine Glutamine Glutamine

[162] [163] [164] [165] [119]

Extracellular Extracellular

47

this enzyme, still complex. This opens the main question about how many of the indicated functions/substrates are really relevant in biological cell functions, and three main conclusions can be reached: (a) the number of functions, substrates and isoforms, indicates overall that this enzyme family is still a very complex one, requiring a further effort to better understand its biological function; most of the reported substrates were identified through in vitro studies only, therefore a more complete understanding of their real involvement in biological functions as TGase substrates deserve further investigation, possibly through in vivo studies or more physiological approaches; (b) besides the ‘canonical’ roles in coagulation, membrane/cytoskeleton/ extracellular matrix protein stabilization, cells differentiation and the G-protein function, a limited number of functions is emerging more convincingly from published studies, namely, deamidation, disulphide isomerase, intrinsic kinase activity, membrane trafficking regulation. Most probably, starting from these recent studies and the identification of substrates for these functions, novel clues and information will be available in the future; (c) looking from a mechanicistic point of view, we can grossly cluster the different TGases enzymatic activities within two functions: (i) to modify the structure/assembly of proteins, by cross-linking them to other proteins or small reactive molecules, with the aim to make more rigid and resistant the final product (as reviewed [50]: this is true for extracellular proteins, cytoskeleton components, blood- or seminal-plasma proteins, as well as for proteins in food, plants, viruses, bacteria and nematodes. This mechanic function may be useful for many biological purposes, like blood coagulation, cell differentiation, cell envelope formation, condensation of apoptotic bodies and so on; (ii) to transfer a piece of information (information transducer function) across or along a pathway, in response to specific stimuli acting on a receptor (e.g. the ␣-adrenergic receptor, therefore playing its G-protein function), on an enzyme (e.g. phospholipase A2) but also by functioning as a calcium-sensor, or modifying the levels of other messengers like polyamines or monoamines, or acting as a kinase. Further, another important way to transfer an information may be to induce a conformational change in other proteins, by a folding-modifying action, modulating for instance the structure/function of other enzymes able to transfer the information along the pathway. Of course, the two main functions above listed are not reciprocally exclusive, since many times they may occur simultaneously and in a functional and inter-dependent way. Just as an example, one important and emerging biological function of TGase may be related to membrane-traficking events: cell endocytosis, phagocytosis and exocytosis were in the past, and more recently, shown to be directly or indirectly related to TGase functions [16, 28, 51–54]. Therefore, it is worth to note that many substrates reported in this chapter (table 2) are membrane or cytoskeleton

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components (mechanical players), or important signals or transducer of signals inside-outside the cell or across the membrane (information players): since TGase is able to functionally interact with both groups of players, it might be one of the referees in this match. Finally, to put new light on biological function(s) of TGase family of enzymes, we need to study it both under the classical point of view, i.e. the Sun-Light, of its canonical functions, and under the Moon-light deriving from the different, still poor studied, activities recently identified for this enzyme, with a careful evaluation of biological aspects emerging also from clinical studies focused on abnormalities of TGases and its substrates’ expression or functions.

References 1 2 3 4 5 6

7 8 9 10

11 12

13

14

15

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17 18 19 20 21 22 23 24 25 26

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40

41 42 43 44 45

46

47

48 49 50

51

52 53 54 55

56

57

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Coussons PJ, Price NC, Kelly SM, Smith B, Sawyer L: Factors that govern the specificity of transglutaminase-catalysed modification of proteins and peptides. Biochem J 1992;282(Pt 3): 929–930. Bruce SE, Bjarnason I, Peters TJ: Human jejunal transglutaminase: Demonstration of activity, enzyme kinetics and substrate specificity with special relation to gliadin and coeliac disease. Clin Sci (Lond) 1985;68:573–579. Folk JE: Mechanism and basis for specificity of transglutaminase-catalyzed epsilon-(gammaglutamyl) lysine bond formation. Adv Enzymol Relat Areas Mol Biol 1983;54:1–56. Lorand L, Parameswaran KN, Stenberg P, Tong YS, Velasco PT, Jonsson NA, et al: Specificity of guinea pig liver transglutaminase for amine substrates. Biochemistry 1979;18:1756–1765. Pincus JH, Waelsch H: The specificity of transglutaminase. II. Structural requirements of the amine substrate. Arch Biochem Biophys 1968;126:44–52. Pincus JH, Waelsch H: The specificity of transglutaminase. I. Human hemoglobin as a substrate for the enzyme. Arch Biochem Biophys 1968;126:34–43. Vader LW, de Ru A, van der Wal Y, Kooy YM, Benckhuijsen W, Mearin ML, et al: Specificity of tissue transglutaminase explains cereal toxicity in celiac disease. J Exp Med 2002;195: 643–649. Mazzeo MF, De Giulio B, Senger S, Rossi M, Malorni A, Siciliano RA: Identification of transglutaminase-mediated deamidation sites in a recombinant alpha-gliadin by advanced massspectrometric methodologies. Protein Sci 2003;12:2434–2442. Mamone G, Ferranti P, Melck D, Tafuro F, Longobardo L, Chianese L, et al: Susceptibility to transglutaminase of gliadin peptides predicted by a mass spectrometry-based assay. FEBS Lett 2004;562:177–182. Facchiano F, Luini A: Tetanus toxin potently stimulates tissue transglutaminase. A possible mechanism of neurotoxicity. J Biol Chem 1992;267:13267–13271. Facchiano F, Benfenati F, Valtorta F, Luini A: Covalent modification of synapsin I by a tetanus toxin-activated transglutaminase. J Biol Chem 1993;268:4588–4591. Rao RU, Mehta K: Transglutaminases, thioredoxins and protein disulphide isomerase: Diverse enzymes with a common goal of cross-linking proteins in lower organisms. Indian J Exp Biol 2004;42:235–243. Szondy Z, Sarang Z, Molnar P, Nemeth T, Piacentini M, Mastroberardino PG, et al: Transglutaminase 2-/- mice reveal a phagocytosis-associated crosstalk between macrophages and apoptotic cells. Proc Natl Acad Sci USA 2003;100:7812–7817; Epub 2003 Jun 16. Teshigawara K, Kannagi R, Noro N, Masuda T: Possible involvement of transglutaminase in endocytosis and antigen presentation. Microbiol Immunol 1985;29:737–750. Facchiano F, Valtorta F, Benfenati F, Luini A: The transglutaminase hypothesis for the action of tetanus toxin. Trends Biochem Sci 1993;18:327–329. Abe S, Yamashita K, Kohno H, Ohkubo Y: Involvement of transglutaminase in the receptormediated endocytosis of mouse peritoneal macrophages. Biol Pharm Bull 2000;23:1511–1513. Ahvazi B, Boeshans KM, Steinert PM: Crystal structure of transglutaminase 3 in complex with GMP: Structural basis for nucleotide specificity. J Biol Chem 2004;279:26716–26725; Epub 2004 Apr 14. Esposito C, Pucci P, Amoresano A, Marino G, Cozzolino A, Porta R: Transglutaminase from rat coagulating gland secretion. Post-translational modifications and activation by phosphatidic acids. J Biol Chem 1996;271:27416–27423. Dubbink HJ, Verkaik NS, Faber PW, Trapman J, Schroder FH, Romijn JC: Tissue specific and androgen-regulated expression of human prostate-specific transglutaminase. Biochem J 1996; 315(Pt 3):901–908. Candi E, Paradisi A, Terrinoni A, Pietroni V, Oddi S, Cadot B, et al: Transglutaminase 5 is regulated by guanine-adenine nucleotides. Biochem J 2004;381(Pt 1):313–319. Hand D, Dias D, Haynes LW: Stabilization of collagen-tailed acetylcholinesterase in muscle cells through extracellular anchorage by transglutaminase-catalyzed cross-linking. Mol Cell Biochem 2000;204:65–76. Takashi R: A novel actin label: A fluorescent probe at glutamine-41 and its consequences. Biochemistry 1988;27:938–943.

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Nemes Z Jr, Adany R, Balazs M, Boross P, Fesus L: Identification of cytoplasmic actin as an abundant glutaminyl substrate for tissue transglutaminase in HL-60 and U937 cells undergoing apoptosis. J Biol Chem 1997;272:20577–20583. Safer D, Sosnick TR, Elzinga M: Thymosin beta 4 binds actin in an extended conformation and contacts both the barbed and pointed ends. Biochemistry 1997;36:5806–5816. Madi A, Hoffrogge R, Blasko B, Glocker MO, Fesus L: Amine donor protein substrates for transglutaminase activity in Caenorhabditis elegans. Biochem Biophys Res Commun 2004;315: 1064–1069. Lee KN, Maxwell MD, Patterson MK Jr, Birckbichler PJ, Conway E: Identification of transglutaminase substrates in HT29 colon cancer cells: Use of 5-(biotinamido)pentylamine as a transglutaminase-specific probe. Biochim Biophys Acta 1992;1136:12–16. Rasmussen LK, Sorensen ES, Petersen TE, Gliemann J, Jensen PH: Identification of glutamine and lysine residues in Alzheimer amyloid beta A4 peptide responsible for transglutaminasecatalysed homopolymerization and cross-linking to alpha 2M receptor. FEBS Lett 1994;338: 161–166. Mandrusiak LM, Beitel LK, Wang X, Scanlon TC, Chevalier-Larsen E, Merry DE, et al: Transglutaminase potentiates ligand-dependent proteasome dysfunction induced by polyglutamineexpanded androgen receptor. Hum Mol Genet 2003;12:1497–1506. Ando Y, Imamura S, Owada MK, Kannagi R: Calcium-induced intracellular cross-linking of lipocortin I by tissue transglutaminase in A431 cells. Augmentation by membrane phospholipids. J Biol Chem 1991;266:1101–1108. Lee KN, Lee CS, Tae WC, Jackson KW, Christiansen VJ, McKee PA: Cross-linking of wild-type and mutant alpha 2-antiplasmins to fibrin by activated factor XIII and by a tissue transglutaminase. J Biol Chem 2000;275:37382–37389. Beninati S, Mukherjee AB: A novel transglutaminase-catalyzed posttranslational modification of HIV-1 aspartyl protease. Biochem Biophys Res Commun 1992;187:1211–1218. Madi A, Kele Z, Janaky T, Punyiczki M, Fesus L: Identification of protein substrates for transglutaminase in Caenorhabditis elegans. Biochem Biophys Res Commun 2001;283:964–968. Seitz A, Schneider F, Pasternack R, Fuchsbauer HL, Hampp N: Enzymatic cross-linking of purple membranes catalyzed by bacterial transglutaminase. Biomacromolecules 2001;2:233–238. Robinson NA, Eckert RL: Identification of transglutaminase-reactive residues in S100A11. J Biol Chem 1998;273:2721–2728. Ruse M, Lambert A, Robinson N, Ryan D, Shon KJ, Eckert RL: S100A7, S100A10, and S100A11 are transglutaminase substrates. Biochemistry 2001;40:3167–3173. Umar S, Malavasi F, Mehta K: Post-translational modification of CD38 protein into a high molecular weight form alters its catalytic properties. J Biol Chem 1996;271:15922–15927. Hunter I, Sigmundsson K, Beauchemin N, Obrink B: The cell adhesion molecule C-CAM is a substrate for tissue transglutaminase. FEBS Lett 1998;425:141–144. Mantile G, Miele L, Cordella-Miele E, Singh G, Katyal SL, Mukherjee AB: Human Clara cell 10-kDa protein is the counterpart of rabbit uteroglobin. J Biol Chem 1993;268:20343–20351. Dondini L, Del Duca S, Dall’Agata L, Bassi R, Gastaldelli M, Della Mea M, et al: Suborganellar localisation and effect of light on Helianthus tuberosus chloroplast transglutaminases and their substrates. Planta 2003;217:84–95; Epub 2003 Mar 14. Bowness JM, Folk JE, Timpl R: Identification of a substrate site for liver transglutaminase on the aminopropeptide of type III collagen. J Biol Chem 1987;262:1022–1024. Orban JM, Wilson LB, Kofroth JA, El-Kurdi MS, Maul TM, Vorp DA: Crosslinking of collagen gels by transglutaminase. J Biomed Mater Res 2004;68A:756–762. Lorand L, Velasco PT, Murthy SN, Wilson J, Parameswaran KN: Isolation of transglutaminasereactive sequences from complex biological systems: A prominent lysine donor sequence in bovine lens. Proc Natl Acad Sci USA 1992;89:11161–11163. Groenen PJ, Bloemendal H, de Jong WW: The carboxy-terminal lysine of alpha B-crystallin is an amine-donor substrate for tissue transglutaminase. Eur J Biochem 1992;205:671–674. Berbers GA, Feenstra RW, van den Bos R, Hoekman WA, Bloemendal H, de Jong WW: Lens transglutaminase selects specific beta-crystallin sequences as substrate. Proc Natl Acad Sci USA 1984;81:7017–7020.

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83 Groenen PJ, Grootjans JJ, Lubsen NH, Bloemendal H, de Jong WW: Lys-17 is the amine-donor substrate site for transglutaminase in beta A3-crystallin. J Biol Chem 1994;269:831–833. 84 Zeeuwen PL, Van Vlijmen-Willems IM, Jansen BJ, Sotiropoulou G, Curfs JH, Meis JF, et al: Cystatin M/E expression is restricted to differentiated epidermal keratinocytes and sweat glands: A new skin-specific proteinase inhibitor that is a target for cross-linking by transglutaminase. J Invest Dermatol 2001;116:693–701. 85 Butler SJ, Landon M: Transglutaminase-catalysed incorporation of putrescine into denatured cytochrome. Preparation of a mono-substituted derivative reactive with cytochrome c oxidase. Biochim Biophys Acta 1981;670:214–221. 86 Pucci P, Malorni A, Marino G, Metafora S, Esposito C, Porta R: Beta-endorphin modification by transglutaminase in vitro: Identification by FAB/MS of glutamine-11 and lysine-29 as acyl donor and acceptor sites. Biochem Biophys Res Commun 1988;154:735–740. 87 Murthy SN, Wilson J, Zhang Y, Lorand L: Residue Gln-30 of human erythrocyte anion transporter is a prime site for reaction with intrinsic transglutaminase. J Biol Chem 1994;269: 22907–22911. 88 Doolittle RF, Watt KW, Cottrell BA, Strong DD, Riley M: The amino acid sequence of the alphachain of human fibrinogen. Nature 1979;280:464–468. 89 Kimura S, Aoki N: Cross-linking site in fibrinogen for alpha 2-plasmin inhibitor. J Biol Chem 1986;261:15591–15595. 90 Murthy SN, Wilson JH, Lukas TJ, Veklich Y, Weisel JW, Lorand L: Transglutaminase-catalyzed crosslinking of the Aalpha and gamma constituent chains in fibrinogen. Proc Natl Acad Sci USA 2000;97:44–48. 91 McDonagh RP, McDonagh J, Petersen TE, Thogersen HC, Skorstengaard K, Sottrup-Jensen L, et al: Amino acid sequence of the factor XIIIa acceptor site in bovine plasma fibronectin. FEBS Lett 1981;127:174–178. 92 Mosher DF, Schad PE: Cross-linking of fibronectin to collagen by blood coagulation Factor XIIIa. J Clin Invest 1979;64:781–787. 93 Sato H, Yamada N, Shimba N, Takahara Y: Unique substrate specificities of two adjacent glutamine residues in EAQQIVM for transglutaminase: Identification and characterization of the reaction products by electrospray ionization tandem mass spectrometry. Anal Biochem 2000; 281:68–76. 94 Takahashi M, Tezuka T, Katunuma N: Filaggrin linker segment peptide and cystatin alpha are parts of a complex of the cornified envelope of epidermis. Arch Biochem Biophys 1996;329: 123–126. 95 Mehul B, Bawumia S, Hughes RC: Cross-linking of galectin 3, a galactose-binding protein of mammalian cells, by tissue-type transglutaminase. FEBS Lett 1995;360:160–164. 96 Mahoney SA, Wilkinson M, Smith S, Haynes LW: Stabilization of neurites in cerebellar granule cells by transglutaminase activity: Identification of midkine and galectin-3 as substrates. Neuroscience 2000;101:141–155. 97 Folk JE, Cole PW: Structural requirements of specific substrates for guinea pig liver transglutaminase. J Biol Chem 1965;240:2951–2960. 98 Ikura K, Kita K, Fujita I, Hashimoto H, Kawabata N: Identification of amine acceptor protein substrates of transglutaminase in liver extracts: Use of 5-(biotinamido) pentylamine as a probe. Arch Biochem Biophys 1998;356:280–286. 99 Piredda L, Farrace MG, Lo Bello M, Malorni W, Melino G, Petruzzelli R, et al: Identification of ‘tissue’ transglutaminase binding proteins in neural cells committed to apoptosis. FASEB J 1999;13:355–364. 100 Taki M, Shiota M, Taira K: Transglutaminase-mediated N- and C-terminal fluorescein labeling of a protein can support the native activity of the modified protein. Protein Eng Des Sel 2004; 17:119–126; Epub 2004 Jan 12. 101 Fleckenstein B, Qiao SW, Larsen MR, Jung G, Roepstorff P, Sollid LM: Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides. J Biol Chem 2004; 279:17607–17616; Epub 2004 Jan 27. 102 Piper JL, Gray GM, Khosla C: High selectivity of human tissue transglutaminase for immunoactive gliadin peptides: Implications for celiac sprue. Biochemistry 2002;41:386–393.

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103 Cooper AJ, Sheu KR, Burke JR, Onodera O, Strittmatter WJ, Roses AD, et al: Transglutaminasecatalyzed inactivation of glyceraldehyde 3-phosphate dehydrogenase and alpha-ketoglutarate dehydrogenase complex by polyglutamine domains of pathological length. Proc Natl Acad Sci USA 1997;94:12604–12609. 104 Orru S, Ruoppolo M, Francese S, Vitagliano L, Marino G, Esposito C: Identification of tissue transglutaminase-reactive lysine residues in glyceraldehyde-3-phosphate dehydrogenase. Protein Sci 2002;11:137–146. 105 Mariniello L, Esposito C, Di Pierro P, Cozzolino A, Pucci P, Porta R: Human-immunodeficiencyvirus transmembrane glycoprotein gp41 is an amino acceptor and donor substrate for transglutaminase in vitro. Eur J Biochem 1993;215:99–104. 106 Mariniello L, Esposito C, Gentile V, Porta R: Transglutaminase covalently incorporates amines into human immunodeficiency virus envelope glycoprotein gp120 in vitro. Int J Pept Protein Res 1993;42:204–206. 107 Halkier T, Andersen H, Vestergaard A, Magnusson S: Bovine histidine-rich glycoprotein is a substrate for bovine plasma factor XIIIa. Biochem Biophys Res Commun 1994;200:78–82. 108 Ballestar E, Abad C, Franco L: Core histones are glutaminyl substrates for tissue transglutaminase. J Biol Chem 1996;271:18817–18824. 109 Lu W, Strohecker A, Ou Jh JH: Post-translational modification of the hepatitis C virus core protein by tissue transglutaminase. J Biol Chem 2001;276:47993–47999; Epub 2001 Oct 17. 110 Kaartinen MT, El-Maadawy S, Rasanen NH, McKee MD: Tissue transglutaminase and its substrates in bone. J Bone Miner Res 2002;17:2161–2173. 111 Zainelli GM, Ross CA, Troncoso JC, Fitzgerald JK, Muma NA: Calmodulin regulates transglutaminase 2 cross-linking of huntingtin. J Neurosci 2004;24:1954–1961. 112 Peng X, Zhang Y, Zhang H, Graner S, Williams JF, Levitt ML, et al: Interaction of tissue transglutaminase with nuclear transport protein importin-alpha3. FEBS Lett 1999;446:35–39. 113 Sakai K, Busby WH Jr, Clarke JB, Clemmons DR: Tissue transglutaminase facilitates the polymerization of insulin-like growth factor-binding protein-1 (IGFBP-1) and leads to loss of IGFBP-1’s ability to inhibit insulin-like growth factor-I-stimulated protein synthesis. J Biol Chem 2001;276:8740–8745; Epub 2000 Dec 19. 114 Mishra S, Murphy LJ: Tissue transglutaminase has intrinsic kinase activity: Identification of transglutaminase 2 as an insulin-like growth factor-binding protein-3 kinase. J Biol Chem 2004; 279:23863–23868; Epub 2004 Apr 5. 115 Sato H, Hayashi E, Yamada N, Yatagai M, Takahara Y: Further studies on the site-specific protein modification by microbial transglutaminase. Bioconjug Chem 2001;12:701–710. 116 Simon M, Green H: The glutamine residues reactive in transglutaminase-catalyzed cross-linking of involucrin. J Biol Chem 1988;263:18093–18098. 117 Candi E, Tarcsa E, Digiovanna JJ, Compton JG, Elias PM, Marekov LN, et al: A highly conserved lysine residue on the head domain of type II keratins is essential for the attachment of keratin intermediate filaments to the cornified cell envelope through isopeptide crosslinking by transglutaminases. Proc Natl Acad Sci USA 1998;95:2067–2072. 118 Lee DS, Matsumoto S, Matsumura Y, Mori T: Identification of the epsilon-(gamma-glutamyl)lysine cross-linking sites in alpha-lactalbumin polymerized by mammalian and microbial transglutaminases. J Agric Food Chem 2002;50:7412–7419. 119 Truong VD, Clare DA, Catignani GL, Swaisgood HE: Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase. J Agric Food Chem 2004;52: 1170–1176. 120 Nieuwenhuizen WF, Dekker HL, de Koning LJ, Groneveld T, de Koster CG, de Jong GA: Modification of glutamine and lysine residues in holo and apo alpha-lactalbumin with microbial transglutaminase. J Agric Food Chem 2003;51:7132–7139. 121 Coussons PJ, Price NC, Kelly SM, Smith B, Sawyer L: Transglutaminase catalyses the modification of glutamine side chains in the C-terminal region of bovine beta-lactoglobulin. Biochem J 1992; 283(Pt 3):803–806. 122 Nieuwenhuizen WF, Dekker HL, Groneveld T, de Koster CG, de Jong GA: Transglutaminasemediated modification of glutamine and lysine residues in native bovine beta-lactoglobulin. Biotechnol Bioeng 2004;85:248–258.

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123 Verderio E, Gaudry C, Gross S, Smith C, Downes S, Griffin M: Regulation of cell surface tissue transglutaminase: Effects on matrix storage of latent transforming growth factor-beta binding protein-1. J Histochem Cytochem 1999;47:1417–1432. 124 Borth W, Chang V, Bishop P, Harpel PC: Lipoprotein (a) is a substrate for factor XIIIa and tissue transglutaminase. J Biol Chem 1991;266:18149–18153. 125 Hohl D, Mehrel T, Lichti U, Turner ML, Roop DR, Steinert PM: Characterization of human loricrin. Structure and function of a new class of epidermal cell envelope proteins. J Biol Chem 1991; 266:6626–6636. 126 Mortensen SB, Sottrup-Jensen L, Hansen HF, Rider D, Petersen TE, Magnusson S: Sequence location of a putative transglutaminase crosslinking site in human alpha 2-macroglobulin. FEBS Lett 1981;129:314–317. 127 Rasmussen LK, Ellgaard L, Jensen PH, Sorensen ES: Localization of a single transglutaminasereactive glutamine in the third domain of RAP, the alpha2-macroglobulin receptor-associated protein. J Protein Chem 1999;18:69–73. 128 Perez-Paya E, Thiaudiere E, Abad C, Dufourcq J: Selective labelling of melittin with a fluorescent dansylcadaverine probe using guinea-pig liver transglutaminase. FEBS Lett 1991;278: 51–54. 129 Murthy SN, Wilson JH, Lukas TJ, Kuret J, Lorand L: Cross-linking sites of the human tau protein, probed by reactions with human transglutaminase. J Neurochem 1998;71:2607–2614. 130 Mahoney SA, Perry M, Seddon A, Bohlen P, Haynes L: Transglutaminase forms midkine homodimers in cerebellar neurons and modulates the neurite-outgrowth response. Biochem Biophys Res Commun 1996;224:147–152. 131 Kojima S, Inui T, Muramatsu H, Suzuki Y, Kadomatsu K, Yoshizawa M, et al: Dimerization of midkine by tissue transglutaminase and its functional implication. J Biol Chem 1997;272: 9410–9416. 132 Ota M, Sawa A, Nio N, Ariyoshi Y: Enzymatic ligation for synthesis of single-chain analogue of monellin by transglutaminase. Biopolymers 1999;50:193–200. 133 Eligula L, Chuang L, Phillips ML, Motoki M, Seguro K, Muhlrad A: Transglutaminase-induced cross-linking between subdomain 2 of G-actin and the 636–642 lysine-rich loop of myosin subfragment 1. Biophys J 1998;74(2 Pt 1):953–963. 134 Aeschlimann D, Paulsson M, Mann K: Identification of Gln726 in nidogen as the amine acceptor in transglutaminase-catalyzed cross-linking of laminin-nidogen complexes. J Biol Chem 1992;267: 11316–11321. 135 Kaartinen MT, Pirhonen A, Linnala-Kankkunen A, Maenpaa PH: Transglutaminase-catalyzed cross-linking of osteopontin is inhibited by osteocalcin. J Biol Chem 1997;272:22736–22741. 136 Aeschlimann D, Kaupp O, Paulsson M: Transglutaminase-catalyzed matrix cross-linking in differentiating cartilage: Identification of osteonectin as a major glutaminyl substrate. J Cell Biol 1995;129:881–892. 137 Prince CW, Dickie D, Krumdieck CL: Osteopontin, a substrate for transglutaminase and factor XIII activity. Biochem Biophys Res Commun 1991;177:1205–1210. 138 Sorensen ES, Rasmussen LK, Moller L, Jensen PH, Hojrup P, Petersen TE: Localization of transglutaminase-reactive glutamine residues in bovine osteopontin. Biochem J 1994;304(Pt 1): 13–16. 139 Lilley GR, Skill J, Griffin M, Bonner PL: Detection of Ca2⫹-dependent transglutaminase activity in root and leaf tissue of monocotyledonous and dicotyledonous plants. Plant Physiol 1998;117: 1115–1123. 140 Aho S: Many faces of periplakin: Domain-specific antibodies detect the protein throughout the epidermis, explaining the multiple protein-protein interactions. Cell Tissue Res 2004;316:87–97; Epub 2003 Aug 16. 141 Coussons PJ, Kelly SM, Price NC, Johnson CM, Smith B, Sawyer L: Selective modification by transglutaminase of a glutamine side chain in the hinge region of the histidine-388––glutamine mutant of yeast phosphoglycerate kinase. Biochem J 1991;273(Pt 1):73–78. 142 Cordella-Miele E, Miele L, Mukherjee AB: A novel transglutaminase-mediated post-translational modification of phospholipase A2 dramatically increases its catalytic activity. J Biol Chem 1990;265:17180–17188.

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143 Nadeau OW, Traxler KW, Carlson GM: Zero-length crosslinking of the beta subunit of phosphorylase kinase to the N-terminal half of its regulatory alpha subunit. Biochem Biophys Res Commun 1998;251:637–641. 144 Tamaki T, Aoki N: Cross-linking of alpha 2-plasmin inhibitor to fibrin catalyzed by activated fibrin-stabilizing factor. J Biol Chem 1982;257:14767–14772. 145 Jensen PH, Lorand L, Ebbesen P, Gliemann J: Type-2 plasminogen-activator inhibitor is a substrate for trophoblast transglutaminase and factor XIIIa. Transglutaminase-catalyzed cross-linking to cellular and extracellular structures. Eur J Biochem 1993;214:141–146. 146 Ritchie H, Robbie LA, Kinghorn S, Exley R, Booth NA: Monocyte plasminogen activator inhibitor 2 (PAI-2) inhibits u-PA-mediated fibrin clot lysis and is cross-linked to fibrin. Thromb Haemost 1999;81:96–103. 147 Robitaille K, Daviau A, Tucholski J, Johnson GV, Rancourt C, Blouin R: Tissue transglutaminase triggers oligomerization and activation of dual leucine zipper-bearing kinase in calphostin C-treated cells to facilitate apoptosis. Cell Death Differ 2004;11:542–549. 148 Valnickova Z, Enghild JJ: Human procarboxypeptidase U, or thrombin-activable fibrinolysis inhibitor, is a substrate for transglutaminases. Evidence for transglutaminase-catalyzed crosslinking to fibrin. J Biol Chem 1998;273:27220–27224. 149 Oliverio S, Amendola A, Di Sano F, Farrace MG, Fesus L, Nemes Z, et al: Tissue transglutaminasedependent posttranslational modification of the retinoblastoma gene product in promonocytic cells undergoing apoptosis. Mol Cell Biol 1997;17:6040–6048. 150 Singh US, Kunar MT, Kao YL, Baker KM: Role of transglutaminase II in retinoic acid-induced activation of RhoA-associated kinase-2. Embo J 2001;20:2413–2423. 151 Peter A, Lilja H, Lundwall A, Malm J: Semenogelin I and semenogelin II, the major gel-forming proteins in human semen, are substrates for transglutaminase. Eur J Biochem 1998;252:216–221. 152 Porta R, Esposito C, Metafora S, Malorni A, Pucci P, Siciliano R, et al: Mass spectrometric identification of the amino donor and acceptor sites in a transglutaminase protein substrate secreted from rat seminal vesicles. Biochemistry 1991;30:3114–3120. 153 Larrè C, Chiarello M, Dudek S, Chenu M, Gueguen J: Action of transglutaminase on the constitutive polypeptides of pealegumin. J Agric Food Chem 1993;41:1816–1820. 154 Kamiya N, Tanaka T, Suzuki T, Takazawa T, Takeda S, Watanabe K, et al: S-peptide as a potent peptidyl linker for protein cross-linking by microbial transglutaminase from Streptomyces mobaraensis. Bioconjug Chem 2003;14:351–357. 155 Ferrandiz C, Perez-Paya E, Braco L, Abad C: Gln5 selectively monodansylated substance P as a sensitive tool for interaction studies with membranes. Biochem Biophys Res Commun 1994;203: 359–365. 156 Andringa G, Lam KY, Chegary M, Wang X, Chase TN, Bennett MC: Tissue transglutaminase catalyzes the formation of alpha-synuclein crosslinks in Parkinson’s disease. Faseb J 2004;18: 932–934; Epub 2004 Mar 4. 157 Lynch GW, Slayter HS, Miller BE, McDonagh J: Characterization of thrombospondin as a substrate for factor XIII transglutaminase. J Biol Chem 1987;262:1772–1778. 158 Gorza L, Menabo R, Vitadello M, Bergamini CM, Di Lisa F: Cardiomyocyte troponin T immunoreactivity is modified by cross-linking resulting from intracellular calcium overload. Circulation 1996;93:1896–1904. 159 Maccioni RB, Seeds NW: Transglutaminase and neuronal differentiation. Mol Cell Biochem 1986;69:161–168. 160 Manjunath R, Chung SI, Mukherjee AB: Crosslinking of uteroglobin by transglutaminase. Biochem Biophys Res Commun 1984;121:400–407. 161 Esposito C, Cozzolino A, Mariniello L, Stiuso P, De Maria S, Metafora S, et al: Enzymatic synthesis of vasoactive intestinal peptide analogs by transglutaminase. J Pept Res 1999;53: 626–632. 162 Clement S, Velasco PT, Murthy SN, Wilson JH, Lukas TJ, Goldman RD, et al: The intermediate filament protein, vimentin, in the lens is a target for cross-linking by transglutaminase. J Biol Chem 1998;273:7604–7609. 163 Asijee GM, Muszbek L, Kappelmayer J, Polgar J, Horvath A, Sturk A: Platelet vinculin: A substrate of activated factor XIII. Biochim Biophys Acta 1988;954:303–308.

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164 Skorstengaard K, Halkier T, Hojrup P, Mosher D: Sequence location of a putative transglutaminase cross-linking site in human vitronectin. FEBS Lett 1990;262:269–274. 165 Usui T, Takagi J, Saito Y: Propolypeptide of von Willebrand factor serves as a substrate for factor XIIIa and is cross-linked to laminin. J Biol Chem 1993;268:12311–12316.

Dr. Francesco Facchiano Dipartimento di Ematologia, Oncologia e Medicina Molecolare Istituto Superiore di Sanità Viale Regina Elena 299, IT–00161 Roma (Italy) Tel. ⫹39 06 49902059, Fax ⫹39 06 49387087, E-Mail [email protected]

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Mehta K, Eckert R (eds): Transglutaminases. Prog Exp Tum Res. Basel, Karger, 2005, vol 38, pp 58–74

Type 2 Transglutaminase and Cell Death Mauro Piacentini, Alessandra Amendola, Fabiola Ciccosanti, Laura Falasca, Maria Grazia Farrace, Pier Giorgio Mastroberardino, Roberta Nardacci, Serafina Oliverio, Lucia Piredda, Carlo Rodolfo, Francesco Autuori Department of Biology, University of Rome ‘Tor Vergata’ and National Institute of Infectious Diseases IRCCS ‘Lazzaro Spallanzani’, Rome, Italy

Transglutaminases (TGases) are a large family of calcium-dependent transamidation acyltransferases, catalyzing post-translational modification of proteins. In invertebrates, only a single transglutaminase gene is known, whereas nine genes, evolutionary related and probably evolved by successive duplications have been identified in mammals [1]. In the present article, we will focus on the role of type 2 (TG2) or ‘tissue’ transglutaminase, first identified in 1953 as the liver enzyme incorporating amines into proteins [2]. In particular, we will focus on the potential role(s) played by the enzyme in cell death both under physiological and pathological conditions. TG2-mRNA expression and protein levels increase significantly in a dose- and time-dependent manner in response to cell death induction both in tissues and cell culture (fig. 1); TG2 expression can be used as a trace marker for detection and quantification of apoptosis in vitro and in vivo [2, 3]. The TG2-dependent cross-linking activity represents together with the extensive proteolysis (i.e. due to the activation of cellular cysteine proteases), the most important event leading to the modification of the proteome of a dying cell.

‘Tissue’ Transglutaminase or Type 2

Tissue or type 2 transglutaminase (TG2) is the most ubiquitous isoform belonging to TGases family. TG2 is a versatile multifunctional protein

involved in a variety of biochemical functions at various cellular locations [2–4]. Depending upon the nature of the group entering in the reaction, TG2 Ca2⫹-dependent cross-linking activity is responsible for different related modifications such as incorporation of amine into proteins, protein-protein cross-linking as well as site-specific deamidation (if the entering group is a water molecule instead of an amine) [2]. Mainly under pathological settings, TG2 can also act extracellularly, it can be exposed on the external leaflet of the plasma membrane or released from cells, where it has been suggested to mediate the interaction between integrins and fibronectin with extracellular matrix (ECM) [5, 6]. In addition to the above mentioned activities, TG2 can act as G-protein binding and hydrolyzing GTP with an affinity and a catalytic rate similar to the ␣-subunit of large heterotrimeric G proteins and small Ras-type G proteins [7]. Under such circumstances, TG2 couples ␣1b and ␣1d adrenoreceptors, thromboxane and oxytocin receptors to phospholipase C (PLC-␦1) [8]. When the enzyme is in a GTP/GDP-bound form, it cannot act as transglutaminase [9]. The inhibition is suspended by Ca2⫹ which plays a role as molecular switch between these two functions [10]. Finally, based on in vitro observations, a Ca2⫹-independent protein disulfide isomerase (PDI) function has been proposed for this enzyme [11]. The PDI activity seems to rely on an independent active domain from that used for TGase activity, being the cysteine of this latter active site not responsible for PDI activity itself [11].

Role of TG2 in Cellular Functions

The above described TG2 activities together with its various subcellular locations and protein partners clearly suggest multiple functions for this enzyme, and although, extensive analysis has been carried out in different cellular populations under physiological as well as pathological settings lacked to provide a unified view [2]. In fact, some cell types (e.g. endothelial, mesangial and smooth muscle cells) express constitutive high TG2 levels [12] and other cell types do not, and its expression is induced by distinct signaling pathways which target specific response elements in the regulatory region of the gene. Retinoic acid (RA), TGF-␤, IL-6 and NF-kB responsive sites and regions have been functionally identified and all of them are related to induction of cellular defense mechanisms and cellular differentiation [13]. Once expressed, the regulation of TG2 activities is mediated by multiple heterogeneous factors. As a G-coupled protein, the role of TG2 in transmitting signals from seven transmembrane-helix receptors to phospholipase C has been clearly described [9], being phospolipase C activated following TG2 binding of

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GTP (fig. 2). High Ca2⫹ levels can induce the release of GTP/GDP molecules, inhibiting signaling and promoting the transamidating activity [4, 9]. The Ca2⫹ requirement for such an activity might be reduced by the interaction of TG2 with specific molecules, such as sphingosylphosphocholine, [14, 15]. Nitric oxide can also strongly influence TGase activity, since up to 15 of the 18 cysteine residues in the protein can be nitrosylated and denitrosylated in a Ca2⫹-dependent manner, inhibiting and activating the enzyme respectively [16]. Following activation by calcium, TG2 interacts and modifies major component of cytoskeleton [2–4]. After RA induction, the enzyme can modify RhoA, a member of Rho GTPases widely involved in cytoskeletal rearrangements [17, 18]. Such modification results in increased binding of RhoA to ROCK-2 protein kinase, autophosphorylation of ROCK-2 itself and consequent phosphorylation of vimentin, finally leading to the formation of stress fibers as well as increased cell adhesion [17]. Moreover, TG2 can interact with ␤-tubulin and with microtubule binding proteins, including tau, which can be eventually cross-linked by the enzyme [19, 20]. Cytoskeletal protein does not represent the only intracellular target for the transamidating TG2 enzymatic activity. The enzyme possesses nuclear localization sequences (NLS), TG2 can translocate, presumably with the help of importin- ␣3, into the nucleus (fig. 2) [21]. There, it can perform as a G-protein as well as transamidating enzyme, activated by nuclear Ca2⫹ signals to cross-link both histones and transcriptional factors [22, 23]. TG2 is selectively expressed in dying cells during programmed cell death in vivo (fig. 1 and 2). Its overexpression potentiates apoptosis and conversely, silencing through antisense technology or inhibition reduces the cell death onset [24]. The activation of TG2 transamidating activity in dying cells induces extensive polymerization of intracellular proteins, including actin, histones and retinoblastoma protein (Rb; 24–25) leading to the assembly of detergent insoluble structures [26]. These protein scaffolds stabilize the structure of the dying cell prior its clearance by phagocytosis [27]. TG catalyzed cross-linking limits, Fig. 1. In vivo and in vitro immuhistochemical localization of TG2 expression in cells undergoing cell death. a Rat uterus 4 h postpartum; b Involuting mouse thymus 24 h after glucocorticoid injection; c HIV-infected human lymph nodes showing TG2-positive dying syncytia; d 48 h retinoic acid-treated human neuroblastoma cells; e Macrophage-derived HIV-infected human syncytia; f Higher magnification of retinoic acid-treated human neuroblastoma cells. Note the specific expression of TG2 (red staining) in cells showing the features of apoptotic cells (i.e. nuclear and cytoplasm condenzation, chromatin margination and detachment from neighboring cells) as well as in dying syncytia. TG2 localization was detected both in 6 mm of paraffin-embedded tissue sections (a–c) and in cell lines (d, f ) using an affinity purified monospecific IgG raised in rabbits against human red blood cell soluble TG2.

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Fig. 2. Schematic view of the prosurvival and proapoptotic TG2 functions. The cross-linking inactive TG2/G␣h protein bound to various membrane receptors (R) can translocate into the nucleus where it protects Rb antiapoptotic transcriptional activity. The inhibitory effect of PI3K is inhibited by the growth factors-dependent continuous stimulation of the Ras-ERK pathway. As a consequence of various apoptotic stimuli the cross-linking active cytosolic TG2 post-translationally modifies DLK leading the activation of the JNK pathway that controls the translocation of BAX [74] on the mitochondria and consequently the activation of the intrinsic apoptotic pathway [74]. TG2 itself is also localized on mitochondria [28] and through the cross-linking of GST P1-1 [20] and of other mitochondrial substrates contributes by the alteration of the MMP and the formation of ROS to cell death. An apoptotic stimulus can lead to the detachment of Rb from TG2, its translocation to the cytosol where together with the other cytosolic protein, upon calcium activation, can act as proapoptotic enzyme.

the leakage of intracellular components, thus preventing the release of soluble, harmful and immunogenic cellular degradation products to avoid the occurrence of inflammatory and autoimmune reactions [27]. Finally, it has been recently reported that TG2 interacts with mitochondria; its localization on this organelle changes the intermembrane polarity status, shifting to a higher polarized one (fig. 2) [28]. Being mitochondria responsible for intracellular calcium buffering and being this ability related to mitochondrial

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membrane potential, it has been hypothesized that activation of TG2 crosslinking activity might be the outcome of the described alterations [15, 28]. Overexpression of TG2 in neural cells determines the unbalance of the redox status of the cells leading to the accumulation of reactive oxygen species (ROS) associated to a large depletion of GSH [28]. In keeping with these findings, GST P1-1 acts as a very efficient acyl donor as well as acceptor TG2 substrate both in cells and in vitro (fig. 2) [20]. The TG2-dependent polymerization of GST P1-1 leads to its functional inactivation and is effectively inhibited by GSH [20]. Many genes involved in apoptosis encode proteins that may generate or respond to oxidative stress [29]. Massive GSH depletion characterizes the early phases of apoptosis and the fact that GST P1-1 might be functionally inactivated by TG2catalyzed oligomerization indicates a potential proapoptotic role for TG2 in antagonizing the cytoprotective effect involving the elimination of ROS originating from oxidative metabolism [29, 30].

Effects of TG2 Deletion in Mice

Although it is now clear that TG2 biochemistry has multiple and unique cellular features, knockout (KO) mice carrying the homozygous deletion of TG2 gene on a mixed background do not exhibit an embryonic lethal phenotype; they are viable and born with Mendelian frequency [31, 32]. Under normal physiological conditions TG2 KO animals do not show major anomalities [31–32]. Moreover, no obvious alterations have been observed in the onset of apoptosis, in the ECM structure or in the heart function, in which TG2 G-protein activity is thought to be important [31, 32]. However, when cell death is induced in vivo in KO mice, the clearance of apoptotic cells by phagocytosis is defective in the thymus and in the liver and inflammatory as well as autoimmune reactions develop in the animals [33]. The most obvious explanation for the lack of lethal phenotypes is that other transglutaminases may exert a compensatory effect in mammalian tissues. However, such compensation is necessarily partial, since there are no evidences showing that the other mammalian transglutaminases can bind GTP acts as PDI or, with the exception of FXIIIa, be exposed out of the cell surface. Thus, it is reasonable to suppose that, at least under certain pathological or stressful conditions, evidences for organ alterations could be gained from more careful studies [34]. In fact, consistent with the described extra- and intracellular functions of TG2, decreased adherence of primary fibroblasts and impaired wound healing, related to altered cytoskeletal dynamics in fibroblasts, have been observed in TG2 KO mice [32]. Finally, it has been shown that TG2 deficient mice have impaired insulin secretion with consequent glucose intolerance and hyperglycemia, a phenotype

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strongly resembling a subtype of diabetes named MODY (for maturity-onset diabetes of the young) [35].

Is TG2 a Proapoptotic or Prosurvival Protein?

In the last 15 years more than 300 papers have clearly shown that TG2 acts as a proapoptotic enzyme. In keeping with this notion, TG2 KO mice show defects in the clearance and stability of apoptotic cells [33]. However, recently antiapoptotic activities for the enzyme have also been reported [36–38]. TG2 is mostly cytosolic, however the enzyme protein is also localized in the nucleus and associated with the plasma membrane (fig. 2) [38]. It has been proposed that both the intracellular localization and the transamidating activity of TG2 are important in modulating its effects on apoptosis [38]. In fact, cells transiently transfected with wild-type TG2 or mutants (which lack transamidating activity) targeted to different intracellular compartments confirmed the proapoptotic nature of cytosolic TG2 [38]. By constrast, the nuclear localization of cross-linking-inactive TG2 reduced apoptosis [38]. Membrane-targeted TG2 had neither pro- nor antiapoptotic functions, thus indicating that intracellular localization is essential in determining the effect of TG2 on cell death. Although, the early induction of TG2 in dying cells in tissue suggests regulatory function for the enzyme, the molecular mechanisms of its action are only recently being partially unrevealed (fig. 2). DAP-like kinase (DLK) is a nuclear serine/threonine-specific kinase, which has been implicated in apoptosis [39]. This protein belongs to a subgroup of serine/threonine protein kinases, referred to as the mixed-lineage kinases, that act as key regulators of the stress-activated c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase signaling pathway [40]. Induction of apoptosis by DLK requires its relocation to the cytoplasm, in particular its association with the actin cytoskeleton which is achieved through interaction with proapoptotic protein Par-4 [41]. DLK undergoes TG2-dependent oligomerization in cells undergoing apoptosis upon exposure to calphostin C (fig. 2) [39]. Overexpression of DLK in neural cells and in sympathetic neurons induces apoptosis through the mitochondrial-dependent death pathway; conversely, overexpression of a dominant-negative form of DLK in these cells prevents apoptosis, thus indicating that DLK is involved in the control of cell death [42]. Similarly with the suggested TG2 dual function, also nuclear DLK does not induce apoptosis and rather it has been implicated in transcription [43]. Interestingly, the TG2-dependent DLK oligomerization occurs early in the apoptotic response and significantly enhances the kinase activity of DLK and consequently its ability to activate the JNK pathway (fig. 2) [39]. Moreover,

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functional studies demonstrate that TG2-mediated oligomerization of wild-type DLK sensitizes cells to calphostin C-induced apoptosis, while cross-linking of a kinase-inactive variant of DLK does not [39]. These findings suggest that TG2 proapoptotic activity is at least partially mediated by the oligomerization and activation of the proapoptotic kinase DLK that in turn will activate the proapoptotic c-Jun N-terminal kinase pathway (fig. 2). As far as the antiapoptotic mechanisms of TG2 are concerned only recently some clues about the possible mechanisms at the basis of this prosurvival function have been published [36–38]. We first demonstrated that TG2 post-translationally modified the Rb, an important suppressor of apoptosis [22]. Recently, it has been suggested that the TG2 interaction with Rb increases significantly concomitant with an attenuation of apoptosis (fig. 2) [38]. Thus implying that TG2 might protect cells against apoptosis in response to stimuli that translocate the enzyme into the nucleus and do not result in increased transamidating activity [38]. The TG2/Rb interaction is emerging as an important aspect of the prosurvival effects of TG2. In fact, while in cells undergoing apoptosis Rb is degraded by caspases, this degradation is blocked when cells are pretreated with RA, an important transcriptional inducer of TG2 (fig. 2) [37]. Experiments performed with Rb(⫺/⫺) fibroblasts demonstrate that Rb is required for TG2 to exhibit antiapoptotic activity in response to RA treatment [37]. Thus suggesting that the ability of TG2 to modify Rb represents a key step for TG2 to provide protection against apoptotic insults and to ensure that cells remain viable. However, it is still controversial whether the transamidating activity of nuclear TG2 is required to bind and protect Rb from the degradation occurring during apoptosis [37, 38]. It is well known that RA consistently induces TG2 expression and activation [44]. It has been shown that RA-dependent TG2 expression protects NIH3T3 cells from apoptosis [45]. Interestingly, in this cell line RA stimulation activates ERK and phosphoinositide 3-kinase (PI3K); however, only PI3K activation is necessary for RA-induced TG2 expression (fig. 2). The exposure of cells expressing TG2 to the PI3K inhibitor reduces the ability of the enzyme to bind GTP, suggesting that PI3K might regulate the GTP binding activity of TG2 consequently, blocking its transamidating activity (fig. 2) [45]. Moreover, cell viability assays showed that incubation of RA-treated cells with LY294002 together with the TG2 inhibitor monodansylcadaverine convert RA from a differentiation factor to an apoptotic stimulus [45]. These findings suggest that PI3K activity is required for the RA-stimulated expression and GTP-binding activity of TG2, thereby linking the upregulation of TG2 with a well-established cell survival factor (fig. 2). In keeping with this notion, TG2 expression and its GTP-binding activity is very high in a number of transformed cell lines indicate that constitutively active TG2 may be a characteristic of certain cancer cells [46]. These findings suggest that

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TG2 may also act as a survival factor and its expression/activation requires the PI3K, but is antagonized by the Ras-ERK pathway. Interestingly, the switch between the prosurvival TG2 GTPase activity and the cell death transamidating one can be achieved through the Ras-ERK pathway [46]. Future studies should address as to how the different catalytic activities of the enzyme are involved in cell survival or as to whether this antiapoptotic function is simply achieved by protein-protein interactions and by which partners.

Is TG2 Involved in Autophagy?

Autophagy is a degradative mechanism mainly involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells [47]. Recently, genetic screenings have considerably increased our knowledge about the molecular mechanisms of autophagy identifying number of genes involved in fundamental steps of the autophagic pathway [48]. In yeast, autophagy is mainly involved in adaptation to starvation, but in multicellular organisms this route is emerging as a multifunctional pathway involved in a variety of additional processes such as autophagic cell death (ACD), removal of damaged organelles and development of different tissue-specific functions [49]. In addition, autophagy is involved in a growing number of pathological conditions, including cancer, myopathies and neurodegenerative disorders [47]. The existence of a mitochondrial-specific autophagic programme has been proposed [50], this is suggested by the observation that a Bcl-2 antisense oligonucleotide can trigger mitochondrial membrane permeabilization (MMP) and ACD [50]. In several paradigms of ACD induction, the so-called death-associated protein (DAP) kinase has been shown to be required [51]. Overexpression of constitutively active DAPkinase is sufficient to trigger ACD accompanied by MMP [50, 51], and Bcl-2 can prevent DAP-kinase-induced cell death, presumably through its capacity to interfere with MMP [50, 51]. These findings further indicate the existence of a crosstalk between autophagic and apoptotic cell death; recent findings indicate that multiple genes involved in apoptosis are also acting during ACD [52], supporting the notion that these two processes can utilize common pathways or pathway components. As previously reported, TG2 regulates the functions of DLK that is one of the member of the DAP-kinase family (fig. 2) [47]. Based on these findings and considering its pathogenic role in neurodegenerative diseases (see below) it is possible to hypothesize that, under some circumstances, TG2 could be one of the molecule participating in the physiological switch between ACD and apoptosis [53]. It is also relevant to mention that the persistence of the death stimulus and the impairment of the mitochondrial function in the presence of an apoptosome block can lead to death by an alternative pathway inducing

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autophagy [50]. In keeping with these notions, TG2 overexpression in various cell lines leads to their priming for cell death switching from a caspase-dependent into a caspase-independent type of death [20, 28], thus further suggesting its potential pro-ACD function. In fact, autophagic activity remained elevated in neurons treated with pan-caspase inhibitor, which inhibited morphological apoptosis but neither inhibit cytochrome C release nor prevent cell death [54]. Future studies should define as to whether TG2 is indeed involved in this cell death switch under physiological and pathological settings.

TG2 and Cell Death in Pathological Settings

Neurodegenerative Diseases As an active component of the cell death machinery, TG2 is involved in the pathogenesis of several diseases, the enzyme being activated in various disorders [2, 27, 33–35, 55]. TG2 cross-linking activity has been proposed to participate to protein aggregates formation occurring in the major neurodegenerative diseases, including Alzheimer, Parkinson and Huntington’s Disease (HD) [56, 57]. In particular, HD is a progressive autosomal-dominant neurodegenerative disorder (incidence 1 in 10,000 individuals), resulting from expanded polyglutamine tracts (beyond 35–40 residues) in the coding region of huntingtin (htt) [58]. The disease is associated with the formation of misfolded ubiquitinated protein aggregates, (neuronal intranuclear inclusions; NII), containing all or part of the mutant htt. These protein inclusions have predominantly been detected in the nucleus, but may also be observed in the cytoplasm of striatal and cortical neurons [58]. Several reports suggest that in vitro aggregation might occur both through polar zippers formation and/or protein cross-linking catalyzed by TG2 [56, 59]. On the other hand studies on cellular models showed that TG2 does not interact with mutant htt in vitro [60]. In order to elucidate the role of TG2 in HD, we produced a new animal model, by crossing a TG2 KO strain [31] with the R6/1 strain, transgenic for exon 1 of htt carrying 116 (CAG) repeats, which develops a HD resembling phenotype [61]. The transgene expression induces a marked increase in the ␧(␥-glutamyl)lysine bond levels, as compared to controls, thus indicating that the polyglutamine tracts are good TG2 substrates in vivo [62]. In keeping with these findings, the depletion of TG2 in HD transgenic mice results in a drastic reduction in ␧(␥-glutamyl)lysine bonds [62]. The drastic decrease (10-fold) of the isodipeptide levels, observed in the TG2-null mouse brain, represents the first in vivo evidence of the central role, among the various members of the transglutaminases family, played by TG2 in mouse brain [62]. Interestingly, ablation of TG2 did not produce a significant reduction in NII number, thus suggesting that TG2 cross-linking of the mutated ntt is not directly involved in NII assembly

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in vivo. By contrast, a significant increase in the NII/nuclei ratio in R6/1, TG2⫹/⫹ versus R6/1, TG2⫺/⫺ brains was observed, suggesting that TG2 gene deletion causes impairment in some protective mechanism preventing the assembly of these nuclear protein complexes [62]. As previously mentioned, TG2 gene induction characterizes cells undergoing programmed cell death in either physiological or experimental settings (fig. 2). In previous studies, other groups [63] and our own [53] described that cell death in the cortex and striatum of HD-transgenic mice is characterized by condensed neurons not displaying the classical apoptotic features, rather these dying cells have been shown to undergo ACD. Interestingly, a dramatic reduction (60–70%) in the number of these dying cells in the neocortex and striatum of HD transgenic/TG2 KO brains, as compared to HD transgenic animals, was observed [62]. It is important to note that the TG2 gene deletion ameliorates the neurodegenerative process observed in HD mice both in the symptoms and survival of these animals with a significant increase of their life span [62]. These evidences indicate that TG2 is the most relevant protein cross-linking enzyme among the various members of the TG family expressed in mouse brains and that TG2 plays an important role in the cell death process characterizing HD pathogenesis.

Liver Pathologies

TG2 has been shown to have an important role in the maintenance of tissue integrity following cell stress or injury [2, 64]. Its role in tissue repair, as a response to loss in tissue homeostasis following trauma, is well documented [64]. The participation of the enzyme has been proposed in various degenerative diseases leading to severe tissue damage characterized by cell death and accumulation of insoluble protein aggregates both at intracellular and extracellular level [64, 65]. These diseases include, between others, various types of liver damages. A liver pathology characterized by accumulation of intracellular aggregate is the alcoholic hepatitis. The characteristic of this disease is the formation of Mallory Bodies that are cytoplasmic deposits of cytokeratin proteins, and TG2 has been shown to participate in their cross-linking [66]. In hepatic diseases characterized by fibrotic tissue formation, TG2 has been shown to participate in ECM cross-linking. In acute hepatic failure, such as in Budd-Chiari syndrome and in chronic liver pathology (viral hepatitis especially HBV and HCV), an abnormal accumulation of TG2 has been described on hepatocytes localized on the areas adjacent to scar and/or fibrotic tissue [34, 55, 67, 68]. At present, the liver fibrogenesis is referred to a dynamic process, strictly related to the extent and duration of parenchymal injury [69]. TG2 has been

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suggested to play a role in the ECM organization either in normal or in pathological conditions [55]. A variety of ECM components, such as collagens, fibronectin, fibrinogen, laminin, nidogen and transforming growth factor-␣1 (TGF-␣1) act as TG2 substrates [5, 68, 70–72]. On the other hand, several inflammatory mediators, including TNF-␣, TGF-␤, IL-1␤, IL-6, involved in the fibrotic process and in apoptosis induction, are known to regulate TG2 expression in various biological settings [64]. The TG2 enzymatic reaction contributes to these pathologies by increasing the resistance of the deposited ECM to breakdown. Its ablation, in a mouse model, leads to an impaired liver regeneration after injury, associated with an increased inflammatory response, an abnormal tissue architecture and a reduced survival [34]. This evidence supports the hypothesis that the increased TG2 levels detected in early stages of HCV-induced liver damage plays a protective role, trying to maintain liver architecture [34]. The enzyme, by cross-linking extracellular proteins and/or by increasing cell-cell and cellECM adhesion, might also counteract the infiltration of inflammatory cells in the liver parenchyma. According with this view, TG2 induction must be considered as part of stress-induced damage and an important member of the hepatic tissue reaction to the progression of liver pathogenesis [34]. Another important function of TG2 during liver pathogenesis is its participation to apoptotic cell death. The abnormal accumulation of TG2 on hepatocytes in Budd-Chiari syndrome depends on the rapid induction of apoptosis that takes place in this disease [67]. The apoptotic cell death has been described in HCV-infected liver, but it is not a prominent phenomenon during this type of liver pathogenesis; in fact, when the liver activity increases, the HCV appears to have the ability to inhibit apoptosis advantaging its replication [73]. In general the enzyme has a protective and stabilizing role in hepatic damage, by eliminating the infected/altered cells and by contributing to tissue repair. However, under pathological condition, the uncontrolled activation of TG2 can turn its protective function to a pathological one. In some cases the stress response might be potentiated leading to pathological effects, in fact, excessive TG2 protein cross-linking can increase the resistance of ECM to breakdown and generate hepatic cirrhosis.

Conclusions

In conclusion, despite extensive investigations, the question of the physiological role played by TG2 and the relevance of the post-translational modifications of its substrates remained largely unanswered so far. As discussed in

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this review, compelling evidence demonstrated that the enzyme is involved in the regulation of cell death under physio-pathological settings [2]. However, several other questions remain to be addressed: is indeed the enzyme acting as PDI in vivo? Is this the physiological TG2 activity? Is this activity involved in the regulation of cell death? It will also be important to investigate as to how this potential enzyme activity complements the transamidating and G-protein TG2 activities. In fact, under stressful circumstances leading to increased free calcium concentration, such as those induced by many cell death stimuli, TG2 appears to switch its activity from the G-protein to cross-linking enzyme actively participating in apoptosis/autophagy (fig. 2). Does the PDI act on the same substrates modified by the TG2 during cell death? TG2 represents a striking example of a cell death-associated protein playing different functions according to the cellular context and needs.

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Dr. Mauro Piacentini Department of Biology, University of Rome ‘Tor Vergata’ Via della Ricerca Scientifica, IT–00133 Rome (Italy) Tel. ⫹39 0 672 594 234, Fax ⫹39 0620 23500, E-Mail [email protected]

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Transglutaminase and Cell-Survival Signaling Ugra S. Singh, Jing Pan Division of Molecular Cardiology, Cardiovascular Research Institute, The Texas A&M University System HSC, College of Medicine, Temple, Tex., USA

Introduction

TGase-2 is a member of the calcium-dependent family of transglutaminases that catalyze post-translational modification of proteins by incorporating polyamines or forming protein cross-links. This transglutaminase activity or transamidation activity involves formation of an isopeptide bond between the ␥-carboxamide group of peptide bound glutamine residue and the ␧-amino group of a lysine residue in a polypeptide chain or primary amino group of a polyamine [1]. Eight isoenzymic forms of mammalian TGases have been identified at the genomic level. However, only six have so far been isolated and characterized at the protein level [2]. Among them, ubiquitous TGase-2 is implicated in functions which result in intra- and/or extracellular structural alterations [3], such as modeling of the extracellular matrix [4], cell survival [5], glucose-stimulated insulin secretion [6], cell adhesion [7], neuronal differentiation [8, 9], tumor growth [10], type 2 diabetes [11], and platelet aggregation [12]. Although, many specific substrates and binding partners of TGase-2 have been identified, their roles in mediating particular effects of TGase-2 in many cases are not well defined. Present article is focused towards the delineating the mechanism(s) by which TGase-2 may be working as signal transducer for promotion of cell survival and differentiation.

TGase-2 as a cross-linker enzyme

The covalent linkage between the ␧-amino group of the peptide bound lysine and the ␥-carboxamide group of glutamine catalyzed by TGase-2

has been shown to contribute to post-translational modifications of proteins [13, 14]. The amino group donor could also be a polyamine, such as spermine, spermidine, putrescine, and/or a monoamine such as histamine. When normal human blood lymphocytes are treated with mitogens in the presence of [3H]-putrescine, label is incorporated into cellular proteins, indicating that polyamines are natural substrates of TGase-2 [1]. Since polyamines have been implicated in many important cell functions, covalent linkage to other proteins could assume special significance [15, 16]. One such example is that of phospholipase A2 where cross-linking of polyamines to the enzyme molecule is shown to induce its activity significantly [17]. Treatment of TGase-2 with iodoacetamide, a cysteine-directed reagent, causes a 94% loss of transglutaminase activity. Cysteine-277 is known to be selectively modified by iodoacetamide. A mutant-containing serine, instead of cysteine-277 does not have transglutaminase activity [18]. TGase-2 is formed with one N-terminal ␤-sandwich, an ␣␤ catalytic core and two C-terminally located ␤-barrels [19]. The catalytic triad (Cys-277, His-335, and Asp-358) located in the catalytic pocket of the core domain of TGase-2, is identical to other cysteine proteases, suggesting similarity in mechanisms of action [20]. Unlike the action of caspase, which leads to hydrolysis of the peptide bond in the presence of a water molecule, the transamidation reaction catalyzed by TGase-2 ends in the formation of an isopeptide bond [21, 22]. TGase-2 Activates RhoA by Transamidation RhoA is a member of Ras superfamily of small G-proteins [23]. Like all G-proteins, RhoA binds guanosine triphosphate (GTP) in the active state and following hydrolysis, returns to an inactive guanosine diphosphate (GDP)bound state [24, 25]. Two domains, switch 1 and 2 are involved in conformational changes during hydrolysis of GTP to GDP [26]. Switch 1 (RhoA amino acids 34–42) is involved in the activation of RhoA downstream effectors/ targets, and switch 2 (RhoA amino acids 63–79) is implicated in GTP hydrolysis [27]. It has been suggested that in contrast to Ras, the effector-binding domain of Rho extends beyond switch 1 [28, 29]. Another unique feature that distinguishes the Rho family from other Ras-related GTPases is the insert region (amino acids 123–137 in RhoA), shown to be essential for Rho kinase activation and morphological transformation in NIH3T3 cells [30]. Cytotoxic necrotizing factor-1, produced from uropathogenic strains of Escherichia coli [31], deamidates RhoA glutamine 63 to glutamic acid [32]. Glutamine 63 is a critical residue of the RhoA switch 2 domain for GTP hydrolysis [33]. By deamidating glutamine 63 of RhoA, cytotoxic necrotizing factor-1 inhibits, both intrinsic GTP hydrolysis and that stimulated by RhoGAP, resulting in constitutive activation [34], and in effect leading to increased stress fiber

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formation [35]. Cytotoxic necrotizing factor-1 has been shown to possess in vitro transglutaminase activity in the presence of primary amines and to transamidate RhoA [36]. RhoA is an in vivo substrate of TGase-2 in HeLa cells. After transamidation at glutamine 63, RhoA binds and activates ROCK-2 [37]. Retinoic acid (RA) promotes transamidation of RhoA in SH-SY5Y cells leading to its activation [8]. Studies using mass spectrophotometric analysis demonstrated that TGase-2 catalyze transamidation of RhoA at glutamines 52, 63, and 136 [36]. Cross-linking of a polyamine to glutamine 63 leads to activation of RhoA and promotes cytoskeletal rearrangement in response to RA [8, 37]. Glutamine 52, is in close proximity with the switch I domain (effector-binding region), and glutamine 136 is located in the insert region (critical for ROCK-2 activation and cellular transformation) [30, 33]. The transamidation of glutamine 136 promotes dissociation of RhoA from the membrane [34, 38]. However, the importance of glutamine 52 transamidation in terms of known RhoA functions remains unknown. In addition to promoting cytoskeletal rearrangement, transamidation of RhoA is also involved in the activation of MAP kinase pathway. This conclusion was drawn from a study in which RAinduced activation of p38␥ MAP kinase was blocked by monodansylcadaverine (an specific inhibitor of TGase-2), and C-3 exoenzyme (RhoA inhibitor) [8]. A downstream target of RhoA, p38␥ is known to have a role in cell differentiation and may be involved in mediating RhoA effects on gene expression in response to RA [39, 40]. RhoA transamidation is also known to be involved in platelet aggregation, especially in mediating exocytosis and cytoskeleton rearrangement [41]. ␣-granule secretion from platelets requires high cytoplasmic serotonin and Ca2⫹ concentrations. Under these conditions serotonin is cross-linked to RhoA. The cross-linking activity catalyzed by TGase-2 results in constitutive activation of RhoA, proposed to be playing an important role in ␣-granule exocytosis and platelet aggregation [12].

TGase-2 as G-Protein Signal Transducer

The purified preparation of TGase-2 possesses Mg2⫹-dependent GTPase activity (turnover number – 1 mole of [32P]Pi released per min, per mole of protein), and Ca2⫹-dependent transglutaminase activity. The transglutaminase activity is blocked by 5 ␮M of GTP (not by GDP), indicating a regulatory nature of GTP-binding/hydrolysis on transamidation function (fig. 1) [14]. TGase-2 is not the only member of transglutaminase family of proteins binding GTP and hydrolyzing it. Recently, TGase-3 and -5 are also shown to bind and hydrolyze

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Fig. 1. Transglutaminase activity of TGase-2 is tightly regulated by guanine nucleotides. Transglutaminase activity of TGase-2 is inhibited by GTP, but not by GDP, indicating that GTP-binding function of TGase-2 regulates transamidation function [14].

GTP, and structural data show that the GTP-binding pocket is conserved between TGase-2 and TGase-3 and -5 [42, 43]. Although the signaling pathways regulated by TGase-3 and -5 remain to be elucidated. The GTP binding and hydrolysis activity of TGase-2 is shown to couple to ␣1B- and 1D-adrenergic [44], thromboxane [45], and oxytocin receptors [46]. ␣1-adrenergic receptors (␣1-AR) are, however, the most studied TGase-2coupled receptors. Activation of ␣1-AR induces exchange of GDP to GTP and dissociation of GTP-TGase-2/Gh from ␤-subunit also called calreticulin [47]. GTP-bound TGase-2 interacts with downstream effector PLC␦1, thereby resulting in phosphoinositide hydrolysis and increase in Ca2⫹ level within the cells [48]. TGase-2 also mediate cell adhesion in fibroblasts by acting as a ␤1 and ␤3 integrin-associated coreceptor for fibronectin [49]. Integrin-mediated cell adhesion modulates many different signal transduction cascades and support cell survival, proliferation, and influences expression of differentiation-related genes [50]. Association of TGase-2 with integrin, that is independent of transamidation function, stimulates cell adhesion, spreading and migration of several cell types [7, 49, 51, 52]. TGase-2 is mostly cytoplasmic in distribution, with a small fraction present in the nucleus [14, 53]. Although TGase-2 does not contain any signal sequence for export to the cell surface, some amount of TGase-2 is found on the cell surface and extracellular matrix [54–56]. Cell surface TGase-2

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has been proposed to serve as an integrin-associated adhesion receptor that might be involved in extravasation and migration of monocytic cells into tissues containing fibronectin matrices during inflammation [7]. GTP binding and hydrolyzing properties of TGase-2 are regulated by RA in HeLa cells, indicating that TGase-2 may be undergoing post-translational modification, or interacting with another protein for regulation of its GTP-binding and hydrolyzing properties. After activation by RA, TGase-2 translocates to membranes and induces the activation of phospholipase C [57]. Further studies demonstrated that phosphatidylinositol 3-kinase (PI3K) is required for RA-induced activation of TGase-2 (GTP-binding activity) [58, 59]. In addition to GTP binding and transamidation activities, TGase-2 is also shown to exhibit kinase activity towards insulin-like growth factor binding protein-3 [60]. However, further studies will be required to establish the role of kinase function in mediating TGase effects on cell differentiation and apoptosis.

TGase-2 as Cell-Survival Factor

RA and various analogs are also known to have a role in affecting mammalian cell growth by promoting differentiation, or inducing apoptosis in nonneuronal cells. Treatment of the human leukemia cell line HL60 with RA results in cell differentiation. In contrast, N-(4-hydroxyphenyl)retinamide, a synthetic analog of RA, induces apoptosis in HL60 cells. However, pretreatment of HL60 with RA blocks N-(4-hydroxyphenyl)retinamide-induced cell death. It has been suggested that RA, by inducing the activation of TGase-2, provides protection against apoptotic stimuli required for the differentiation process [61]. Retinoblastoma protein (RB), a substrate of TGase-2, is implicated in cell survival functions. TGase-2 protects RB from caspase-induced degradation in a transamidation-dependent manner. Experiments performed with fibroblasts from RB⫺/⫺ mice further demonstrated that RB is required for TGase-2 to exhibit the antiapoptotic activity of RA [5]. In a related study, it was demonstrated that intracellular localization and activity state of TGase-2 differentially regulates cell death; cytosolic TGase-2 elicits proapoptotic, nuclear TGase-2 antiapoptotic, and membrane-bound TGase-2 neither pro- nor antiapoptotic functions. It was suggested that antiapoptotic activity of TGase is conferred by its interaction with RB that is independent of transamidation function [62]. Although, TGase-2 knockout mice were viable, phenotypically normal, and were born with the expected Mendelian frequency, primary fibroblasts isolated from TGase-2 ⫺/⫺ mice showed decreased adherence to the plate, and thymocytes had reduced viability compared with that of TGase-2 ⫹/⫹ mice

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[63]. These findings indicated that TGase-2 might be involved in wound healing that requires fibroblast adhesion. Further studies using stable cell lines of fibroblasts-expressing wild type, and C277S mutant (transglutaminase defective) demonstrated that TGase-induced cell adhesion is mediated by protein kinase C-␣ [64]. Eukaryotic initiation factor-5A (eIF-5A) was identified as a GDPdependent binding partner of TGase-2. But the precise function of eIF-5A interaction with TGase-2 is not defined. Presence of an unusual amino acid residue hypusine in eIF-5A has been shown to be essential for supporting methionylpuromycin bond formation [65, 66]. Studies have shown that eIF-5A may also be working as a shuttle protein for nuclear export of ribonucleic acid (RNA)/proteins [67]. During this process, eIF-5A binds to protein cofactors present in the nucleus containing nuclear export signals and whole cargo (RNA, nuclear export signals protein, and eIF-5A) is exported to cytoplasm [68]. The C-terminal domain of eIF-5A consisting of five ␤-strands, and resembling a cold shock domain present in bacterial cold shock protein A, suggests that it may interact with nucleic acids, particularly RNA. In support of this hypothesis, it has been demonstrated that eIF-5A is capable of binding to the Rev-response element and U6 RNA in vitro [69, 70]. RA promotes transcription of new genes and synthesis of RNA [71]. Newly transcribed RNA may be exported to the cytoplasm by binding with eIF-5A. Nucleotide sensitivity of eIF-5A binding with TGase-2 may thus function as regulatory mechanism for export of RNA during RAinduced neuronal differentiation. In a similar study, it was demonstrated that eIF-5A is an in vitro substrate of TGase-2. The hypusine residue at position 50 in eIF-5A is cross-linked to a polyamine by transglutaminase activity of TGase-2 [72]. Overexpression of TGase-2 in BALB/c 3T3 cells induced a 100-fold reduction in hypusine levels and 50% increase in ␥-glutamyl-␻hypusine formation. These effects were associated with a significant reduction in cell proliferation, suggesting that eIF-5A transamidation may be involved in regulation of cell growth [73]. TGase-2 Is Activated under Stressing Conditions Under the condition of oxidative stress or UV irradiation in situ TGase2 activity is elevated, resulting in the formation of water insoluble dimers or polymers of ␣␤-crystallin, ␤2-crystallin and vimentin. Formation of these cross-linked proteins has been suggested to play a role in cataractogenesis [74]. Another example where cross-linking activity has been suggested to play a role in apoptosis is N-methyl-D-aspartic acid induced excitotoxicity in cerebellar granular neurons. N-methyl-D-aspartic acid treatment of granular neurons is shown to induce expression of TGase-2, coupled with calcium influx and apoptosis [75, 76]. Increased calcium inside the granular neurons

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in response to N-methyl-D-aspartic acid treatment has been suggested to promote cross-linking activity of TGase-2, resulting in apoptosis of neurons. The signaling mechanisms involving cross-linking activity are further compounded by the presence of many other isoforms of TGase-2 that may be involved in mediation of differentiation and apoptosis [77, 78]. Transforming growth factor-␤ and several inflammatory cytokines such as interleukin1␤, interleukin-6, and tumor necrosis factor-␣ also induce expression and activation of TGase-2 in many cell lines and in response to tissue injury and inflammation [79–83], but the role of TGase-2 in mediating their effects remains unclear.

TGase-2 as Inducer of Neuronal Differentiation

Expression of TGase-2 is regulated by various agents including RA, shown to induce cell differentiation [8, 9, 61, 84]. Many studies on a variety of embryonic neuronal types have shown that RA can also stimulate both neurite number and length [85–88]. The cellular effects of RA are mediated through the action of two classes of receptors (RARs and RXRs), which are of three major subtypes (␣, ␤ and ␥) [71, 89, 90]. These receptors function as transcription factors and induce transcription of target genes. The promoter region of TGase-2 gene has response elements for binding of RA receptors/transcription factors [91]. There are some other examples such as that of vascular smooth cells and promonocytic U937 cell line where RA promotes cell death and induced expression of TGase-2 has been suggested to be playing an important role [92, 93]. Mechanism of TGase-2-induced neuronal differentiation is not yet well defined; however, TGase-2-mediated apoptosis in U937 cells is shown to involve cross-linking of RB [92, 93]. In fibroblasts the modification of RB by transamidation is shown to make the protein resistant to caspase-7-mediated proteolysis and provide protection against apoptosis [5]. Role of TGase-2 in promoting cell differentiation or apoptosis thus depends on cell line and the type of stimulus. For example, TGase-2 mediates neuronal differentiation in SH-SY5Y cells in response to RA, but promotes apoptosis in NIH3T3 cells by cross-linking and resulting in the activation of dual leucine zipper-bearing kinase in response to calphostin C [9, 94]. These reports clearly demonstrated that TGase-2 uses various signaling molecules for the regulation of cell differentiation and apoptosis. Neuronal differentiation in SH-SY5Y cells in response to RA is coupled with increased expression and activation of TGase-2 [95]. Using stable SHSY5Y cells overexpressing wild-type TGase-2 or transglutaminase defective

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mutant C-277S, it was demonstrated that TGase-2 is sufficient and transglutaminase activity is required for the promotion of neurite outgrowth [8, 9]. Further studies by Tucholski et al. [9] demonstrated that TGase-2 mediates forskolin-induced activation of adenylyl cyclase, resulting in the activation of c-AMP-response element-binding protein in SH-SY5Y cells. Previous studies by other investigators have demonstrated that c-AMP-response elementbinding protein plays an important role in neuronal differentiation, survival and plasticity [96, 97]. Whether c-AMP-response element-binding protein mediates TGase-2 effects on neuronal differentiation in SH-SY5Y cells remains to be determined. Various signaling molecules such as RhoA and MAP kinases (MAPKs) are activated in response to wild-type TGase-2 overexpression suggesting that TGase-2 may also impact gene transcription [8]. RA promotes transamidation and activation of RhoA, although RhoA activation is not involved in the induction of neurite outgrowth in SH-SY5Y cells [8, 37]. Rac1 is also activated in response to RA but activation does not involve transamidation. RA-induced activation of Rac1 is mediated by PI3K, probably due to phosphorylation of the p85 regulatory subunit by Src kinases. Overexpression of constitutively active PI3K or Rac1-V12 induces neurite outgrowth and promotes expression of TGase-2. In addition, RA-induced TGase-2 expression is partially inhibited by LY294002 or overexpression of Rac1-N17, suggesting that PI3K/Rac1 may require TGase-2 for the promotion of neuronal differentiation. These findings suggested that RA-induced neuronal differentiation in SH-SY5Y cells might be mediated by a pathway dependent on TGase-2 and PI3K functions. While all the signaling components of the TGase-2-induced pathway are not well known, the PI3K-induced pathway includes Rac1, PAK1 and MAPKs which are involved in the induction of neurite outgrowth and expression of neuronal markers (fig. 2) [Singh U.S., 2004. unpubl. data) . Studies performed in cerebellar granular neurons demonstrated that TGase-2 functions to stabilize the neurites. Inhibition of transamidation function of TGase-2 did not reduce the number of neurites, but resulted in an increase in neurite retraction and premature axon collateral formation/bifurcation. Two glial proteins involved in cell-extracellular matrix interactions, midkine and galectin-3, were identified as putative substrates for granule neuron TGase-2. The resulting covalent cross-link formation generated multimeric molecular forms of proteins, suggested to play a role in stabilizing newly formed neurites [98]. While the role of TGase-2 in mediating neuronal differentiation is being investigated, studies demonstrate that TGase-2 may also be involved in promoting neuronal degeneration [99, 100]. How the signaling events induced by TGase-2 are converted into apoptotic signals will be the focus of future investigations.

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Neurite outgrowth Cytoskeletal rearrangement

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Fig. 2. TGase-2 mediates part of retinoic acid effects on neuronal differentiation. Retinoic acid-induced neuronal differentiation in SH-SY5Y cells is mediated by TGase-2 dependent and PI3K-dependent pathway. TGase-2-dependent pathway includes RhoA, MAP Kinases (MAPKs) and some unknown signaling molecules that may be involved in the regulation of neurite outgrowth. PI3K-dependent pathway includes Rac1 and MAPKs. Overexpression of Rac1-V12 promotes expression of TGase-2, indicating that PI3K/Rac1 pathway may also require TGase-2 for mediation of neurite outgrowth and expression of neuronal markers.

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Ugra S. Singh Division of Molecular Cardiology, Cardiovascular Research Institute Building 205, 1901 South 1st S., Temple, TX 76504 (USA) Fax ⫹1 254 743 0165, E-Mail [email protected]

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Mehta K, Eckert R (eds): Transglutaminases. Prog Exp Tum Res. Basel, Karger, 2005, vol 38, pp 89–114

Transglutaminases in Wound Healing and Inflammation E.A.M. Verderioa, T.S. Johnsonb, M. Griffina a School of Biomedical and Natural Sciences, Nottingham Trent University, Clifton Lane, Nottingham, bSheffield Kidney Institute, Sheffield University Division of Clinical Science, Northern General Hospital, Sheffield, UK

Introduction

Wound healing is a complex and dynamic process occurring in all body tissue compartments and involves multiple cell types, a wide range of cytokines and also ‘novel’ mediators whose role is less understood [1]. In this chapter we have gathered past and recent evidence which suggests that transglutaminases (TGs) [2, 3] are involved in the repair, stabilization and general physiology of many tissue compartments (e.g. skin, kidney), and as a consequence we demonstrate how TGs may be regarded as a novel class of wound-healing mediators. We report substantial findings from our and other groups which have demonstrated that TGs are implicated in the repair process at many levels. In addition we show that in the presence of continuous tissue insult or abnormalities of the repair process, excessive TG activity can lead to the patho-physiology of fibrotic disorders.

Cutaneous Wound Healing

Wound healing whether in skin, which is the focus of this initial section, or in other tissues (e.g. kidney), as described later, is a complex process involving the interaction of a large variety of growth factors, proinflammatory molecules, components of the extracellular matrix (ECM) and different cell types, which participate in three main overlapping and inter-related events: inflammation, proliferation and remodeling (fig. 1) [1]. Hence, apart from the re-epithelialization

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phase, which in cutaneous healing involves the proliferation and migration of a specific cell type (keratinocyte), present understanding suggests that the complexity of the skin repair process is common to many other cell compartments. Role of Transglutaminases in Skin Morphogenesis and Cutaneous Wound Healing At least four members of the TG family comprising nine genes have been found to be expressed in the epidermis (and dermis) and have a function in skin morphogenesis and repair. Keratinocyte differentiation in the stratum corneum serves as an important barrier against the external environment and is regulated by members of the TG family. The stratum corneum is made by polyhedral corneocytes, which results from the final differentiation of keratinocytes, embedded in a matrix of lipid-enriched membranes. The corneocyte cytosol is covered by a chemically resistant and flexible protein shell known as the cornified cell envelope (fig. 1, small inset). Keratinocyte TG (TG1) has been proposed to participate in the ‘glueing’ of the numerous proteins e.g. loricrin, small proline rich proteins, involucrin, which forms the cornified cell envelope through formation of ␧(␥-glutamyl)lysine bonds. In addition to protein transamidation in the epidermis, TG1 is also known to catalyze esterification of glutamine residues Fig. 1. Schematic showing the basic structure of skin and the different stages of wound repair in relation to the multipoint actions of transglutaminases. a Following injury the wounded area is rapidly filled with a blood clot consisting mainly of cross-linked fibrin, this is followed by invasion of neutrophils, then monocytes and lymphocytes which trigger the inflammatory phase of wound repair. Various cytokines and growth factors are secreted by these cells and by the aggregated platelets in the fibrin clot and later by macrophages (PDGF, TGF-␤, FGF, VEGF) to mediate the transition between inflammation and tissue repair. b During re-epithelialization, epidermal cells undergo profound changes, including dissolution of desmosomes and hemidesmosomal links to permit cell movement into the wound space, between the collagenous dermis and the provisional fibrin matrix. Re-epithelialization is accompanied by the proliferation and migration of dermal fibroblasts into the wound area from the healthy adjacent tissue, where they participate in new tissue formation (granulation tissue) by synthesizing ECM leading to the progressive substitution of the temporary matrix with a collagen matrix. MMPs play a key matrix remodeling role, which involves degradation of existing matrix in/around the wound edge and creation of a path for cell migration while new matrix is deposited. Neovascularization occurs to provide the oxygen and nutrients required to sustain the proliferation of keratinocytes and fibroblasts and the formation of new tissue. c During the transition from granulation tissue to scar, fibroblasts assume a contractile phenotype (myofibroblast) that promotes wound contraction and collagen is continuously remodeled with the formation of large collagen bundles and intermolecular cross-links. The granulation tissue is gradually replaced by scar tissue, a relatively acellular matrix in which many cells and blood vessels are deleted by programmed cell death. The multipoint actions of TG2 and other members of the TG family in cutaneous wound healing are also shown. The inset shows the composition of the epidermis.

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of cell envelopes proteins with ␻-hydroxyl group of ceramides [4]. This posttranslational modification is critical for the attachment and organization of lipid layers, which underlie the barrier function of the epidermis. A time course of TG1 expression following incision on the dorsal skin of neonatal mice has provided insights on the role of TG1 in cutaneous healing [5]. By in situ hybridization, TG1 was found to be expressed in suprabasal keratinocytes near the wound edge 2-hours postwounding and its expression increased in the subsequent hours near the injury edge. TG1 expression was induced in the wound site well before leukocyte infiltration, suggesting that TG1 is required very early during wound repair in preparation for the remodeling of the stratum corneum. Interestingly, TG1 was also found to be highly induced in the leading edges of migrating keratinocytes 20-hour postwounding when re-epithelialization started to become evident, and returned to normal levels 4-day postwounding when the re-epithelization process was completed. In the migrating keratinocytes, TG1 expression appeared to parallel that of its cross-linking substrate involucrin on the plasma membrane, suggesting early colocalization in ‘premature cornified envelopes’ [5]. The role of TG1 in wound healing was further examined in skin of TG1⫺/⫺ mice. However, because of neonatal lethality of TG1-deficient mice (see next section), skin samples from TG1⫺/⫺ mice were grafted on athymic nude mice and examined for histological changes and expression of k6a, a marker of hyperprolific epidermis. Unlike skin grafts from normal neonates, which developed into adult-like skin 2 weeks from transplantation, skin grafted from TG1⫺/⫺ mice showed profound morphological alterations with a thick stratum corneum and signs of hyperproliferation. Injuries to the grafted skins confirmed the importance of TG1 not only in skin morphogenesis but also in wound repair, since a marked delay in regeneration of the epidermis was measured in TG1⫺/⫺ injured grafted skin over an 11-day period after wounding [5]. Another member of the family, epidermal TG (TG3) participates, together with TG1, in the maturation of the epithelium by catalyzing the specific cross-linking of a number of structural proteins in the cell envelope, including loricrin, small proline-rich proteins, and involucrin and also keratins, filaggrin, desmoplakin, and envoplakin or desmosomal proteins [6]. To date, no inherited disorder has been associated with TG3 deficiency, unlike TG1 deficiency [7]. TG3 is predominantly expressed in the granular layer of the epidermis and is thought to be involved in the stabilization of the provisional matrix during wound healing along with other family members [3]. A recently identified new member of the TG family, TG5 (also known as TGX) [8] is found to be induced during the early phase of keratinocyte

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differentiation and very efficiently cross-links epidermal substrates such as loricrin, involucrin and small proline-rich proteins, SPR3, in vitro [9]. Indirect immunofluorescence staining of TG5 has shown its expression in the upper layer of human epidermis where it may be associated with insoluble intermediate filament-like proteins [9, 10]. The role of TG5 in wound repair has yet to be investigated. Tissue TG (TG2) is another of the four TG members found to be expressed in the epidermis [2, 3, 7]. Unlike TG1, TG2 expression appears to be restricted to the proliferative basal layer. In an important study from Greenberg’s group [11], TG2 expression was monitored at all stages of rat skin wound healing. TG2 was found to be expressed in the keratinocytes involved in re-epithelialization, in macrophages lining the re-epithelialization border and in the provisional matrix, including the new invading blood vessels, at day 1 postwounding. Expression was however transient in the epithelial layer as it was reduced after re-epithelialization was completed. TG2 continued to be expressed at the dermoepidermal junctions, where it may serve to attach the epithelial layer to the dermo-epidermal junction in wound repair [12]. TG2 immunoreactivity, which increased significantly at day 1–3 postwounding, was also present in the granulation tissue during its maturation and slowly decreased to control levels between day 3 and 9 postwounding [11]. Interestingly, the early increase in TG2 immunoreactivity was mostly due to its fragmented forms (20, 50 and 55 kDa) and very little to the full length TG2, suggesting that TG2 is proteolytically degraded following wounding. The TG-mediated isopeptide bond was detected by immunohistochemistry from day 1 postwounding up to day 8 postwounding, suggesting a role of TG throughout the wound repair process. Indeed, earlier studies reported increased TG activity in rat wound healing [13] and demonstrated the binding of endogenous TG to the ECM after mechanical wounding of a monolayer of fibroblasts [14]. In the course of our studies, we have shown intense TG2-mediated transamidating activity following wounding of both a monolayer of Swiss 3T3 cells induced to overexpress TG2 and in an epithelial-like cell line ECV304 [15, 16]. The increased TG activity which occurred both in the intracellular and extracellular compartments could lead to in vivo stabilization of the wound area, given the wide range of intracellular and extracellular substrates described for TG2. Increased protein polymerization as a result of TG-mediated protein modification may serve to increase matrix valency resulting in the exposure of matricryptic sites [17], thus enhancing cell-matrix interactions in the wound area in support of the wound repair process. Consistent with this observation, nonreducible FN multimers have been reported to form following exposure of human dermal fibroblasts to a solarium ultraviolet A source [18].

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As shown in figure 1, soon after injury the wound is filled by a hemostatic plug composed mainly of cross-linked fibrin, which re-establishes hemostasis and represents the first healing remedy. In this process, the activated ‘A’ subunit of FactorXIII (FXIIIa) [3] plays an essential role, as its transamidating activity is essential for the stabilization of blood clots. It is activated by thrombin that is released by platelets in vascular injury, which catalyses the cleavage of its propeptide in response to Ca2⫹. Thrombin is also responsible for converting soluble fibrinogen into fibrin gel, which is the main target of the Ca2⫹-dependent cross-linking reaction catalyzed by FXIIIA, leading to fibrin stabilization and strengthening of the provisional matrix during wound healing [3]. In this context, FXIIIA does not only catalyze the formation of ␧(␥-glutamyl)lysine bonds between fibrin subunits but also incorporates inhibitors of fibrinolysis such as plasminogen activator inhibitor-2 and ␣2-antiplasmin, thus playing a wider role in blood coagulation [19]. TG2, like FXIIIA but unlike TG1 or TG3, is known to be externalized from cells, moreover it does not require proteolytic activation, as required instead by FXIIIA (thrombin activation), TG1 and TG3, and therefore, could continue to function as a matrix stabilizer when thrombin is removed from the injury site. The multiple functions of members of the TG family in skin wound repair are summarized in figure 1. Genetically Modified Mice for Investigating the Role of Transglutaminases in Wound Healing The availability of genetically modified mice e.g. gene knockout or in which TG is specifically overexpressed, has shed new light on the understanding of the molecular basis of TG involvement in wound healing. Data acquired with these animal models indicate that members of the TG family are not only important in skin biology, but they also play a critical role in wound repair. Moreover, they indicate that TGs are also involved in abnormal wound healing, which is characterized by the accumulation of extracellular matrix leading to fibroproliferative disorders [1] (see later section on ‘Wound healing and fibrosis in the kidney’). A summary of the effects on cutaneous wound healing resulting from modulation of TGs in mice is shown in table 1. Using an in vitro wound healing model which predicts the rate of wound closure, deficiency in the migration ability of TG2⫺/⫺ mouse embryonic fibroblasts (MEF) [22], compared to TG2⫹/⫹ MEF have confirmed the critical role for TG2 in wound repair. Moreover, it was noted that addition of exogenous purified guinea pig liver TG2 to monolayers of TG2⫺/⫺ MEF improved the stability of the sheet of cells and contributed to partially shift the altered cell migration pattern detected in TG2⫺/⫺ MEF following wound scratching,

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Table 1. Implication of transglutaminases in cutaneous wound healing – Studies with genetically modified mice Gene

Gene product

In vivo model

Viability/ Phenotype

Effect on wound healing

References

TGM1

TG1 (keratinocyte transglutaminase)

Knockout mouse

Die 4–5 hours after birth; ichthyosiform skin phenotype

Impaired skin barrier function; abnormal keratinization; delay in wound healing

[20, 5, 21]

TGM2

TG2 (tissue transglutaminase)

Knockout mouse

Viable, fertile, phenotypically normal; late development of type 2 diabeteslike phenotype

Impaired skin repair; defective phagocytic clearance of apoptotic cells and activation of active TGF-␤1; inflammation

[22–26]

Cardiac overexpression through myosin heavy chain (MHC) promoter

Mild hypertrophy, and diffuse interstitial fibrosis

Upregulation of COX-2, TxS

[27, 28]

Knockout mouse

Viable; impaired reproduction; reduced survival

Impaired clot stabilization; delayed bleeding arrest

[29, 30]

F13A1

FXIIIA (blood coagulation factor XIIIA)

towards the wild-type phenotype. This finding suggested a role for matrix/cell surface TG2 in wound migration during tissue repair [Verderio, Telci, Li, Melino, Griffin, unpubl. data]. Although the genetic approach of targeting inactivation of TG genes by homologous recombination in embryonic stem cells has revealed the in vivo involvement of TG1 and TG2 in wound repair and confirmed the importance of FXIIIA, it cannot be ruled out that some of the TG functions may be hidden by compensatory pathways occurring in the knockout mice, including the increased expression of other members of the TG family.

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General Role of TG2 in Cell-Matrix Interactions Significant in Wound Repair

TG2 appears to be involved in the wound healing process at multiple levels and in many body compartments thus reflecting its wide expression profile. For this reason we have dedicated to it a separate section which reflects its involvement in general tissue repair. Multiple Activities and Regulation of TG2 Unlike other TGs, TG2 is characterized by unique structural features, which leads to a wide range of biological activities and physio-pathological implications [2, 3, 7 and references therein]. Although some controversy and redundancy of information is present on the definitive functions of TG2, rapid advances have been made in the past few years and multiple biological activities have been ascribed to it which include: protein transamidation; GTPase; structural adhesive protein; protein disulphide isomerase and insulin-like growth factor-binding protein-3 kinase activities [2, 3, 7, 31]. Integrated with the Ca2⫹-regulated transamidase active site of TG2 is a GTP binding and hydrolysis site, which is responsible for the G-protein/ signal transduction function of TG2, and for this role is known as Gh␣. Binding of GTP negatively regulates the protein transamidation activity of TG2 by inducing a conformational change that blocks the access of substrates to the transamidating active site [32]. Reciprocally, the binding of Ca2⫹ inhibits the binding of GTP. The TG2-mediated protein transamidation is not only regulated by the Ca2⫹/GTP:GDP ratio but outside the cell also by matrix binding and the redox state of the Cys active site [33, 34]. Due to Ca2⫹ activation requirements it is generally believed that the transamidating activity is mostly latent intracellularly [35, 36]. More recently cross-linking-independent adhesive functions have also been ascribed to TG2, which would depend on and be regulated by its externalization and association with ECM FN [33, 37–39]. Externalization of TG2 during Cell Stress and TG2-Associated Cell Death TG2 is localized in three major cell compartments (cytosol, plasma membrane and nucleus) and it is also secreted into the ECM. The mechanism of secretion is unusual because TG2 lacks a signal peptide and is not secreted by a classical endoplasmic reticulum/golgi-dependent mechanism. It is known that TG2 secretion requires the active-state conformation of TG2 [39] and an intact N-terminal FN-binding site [40]. Due to its atypical secretion

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mechanism, TG2 is not efficiently released. However its externalization dramatically increases in situations of tissue damage and cellular stress [11, 14, 18, 41–43] when it accumulates in the ECM in complex with FN [18, 41]. For this reason TG2 may be regarded as a novel cell/tissue response modulator [2, 16]. Role of TG2 in Matrix Synthesis and Degradation Successful tissue repair much depends on an optimum balance between ECM synthesis (formation of granulation tissue and deposition of collagen) and matrix degradation. This is controlled by matrix metalloproteinases (MMPs) and plasminogen activators and further regulated by tissue inhibitors of proteases. Matrix synthesis is essential to allow cell migration, while degradation is important to create a migration path in the deposited ECM, which together with wound contraction stimulates wound closure. TG2 actively participates in the remodeling of the ECM and a variety of mechanisms have been proposed. TG2 may facilitate the stabilization of the ECM after synthesis. In this respect, the ability of TG2 to form nonreducible multimers of ECM FN leading to its stabilization is well demonstrated [18, 36, 44, 45]. Apart from FN, TG2 has also been shown to cross-link a number of different types of collagen (II, III V, VII and XI) [46 and references therein] and more recently collagen type I [47]. We have recently shown that TG2-mediated modification of type I and III collagen leads to an increased rate of fibril formation and to the formation of an improved substratum for cell adhesion of fibroblasts and osteoblasts in culture [48]. Collagen type I when cross-linked by TG2 also becomes increasingly stable to MMP-1, thus facilitating its accumulation [42]. Recent work has shown that TG2 deficiency in fibroblasts transfected with antisense TG2 cDNA leads to decreased collagen matrix tension and decreased activation of gelatinase (MMP-2), which correlated with reduced levels of MT1-MMP activity [49]. Collagen remodeling by TG2 may therefore lead to the formation of large bundles with the creation of new intermolecular cross-links, which give tensile strength to the scar. TG2 however may also affect matrix deposition indirectly by participating in the activation mechanism of latent TGF-␤1 [50, 51]. It is indeed well documented that TGF-␤1 plays a primary role in regulating proliferation and synthesis of ECM in fibroblasts, namely collagen synthesis. The involvement of TG2 with TGF-␤1 activation is further demonstrated by the recent finding that lack of TG2 leads to a deficiency in activation of TGF-␤1, which is related to a reduced rate of apoptotic thymocytes clearance by TG2⫺/⫺ macrophages [26]. In turn, TGF-␤1 can stimulate TG2 expression [52], possibly through a TGF-␤1/BMP4 response element in the TG2 gene [53], leading to a positive amplification loop. This finding is supported by the recent observation that

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neutralizing anti-TGF-␤1 antibodies significantly reduce TG2 expression in mice [26]. Matrix Structural Role of TG2 Our studies and those of other groups have confirmed that TG2 is a FN-binding protein in cultured cells, which tightly associates with cell surface and ECM FN [36, 37 40, 51]. TG2 is able to modulate and stabilize the FN matrix by forming nonreducible Ca2⫹-dependent ␧(␥-glutamyl)lysine cross-links [45, 36]. Reciprocally, FN binding protects TG2 from proteolytic degradation [54] and controls its transamidating activity [55], which otherwise would be unsustainably high and eventually detrimental to cells in the high Ca2⫹ containing extracellular environment. It is likely that once externalized and bound to FN, TG2 becomes gradually inactive as a protein cross-linker and assumes instead a structural role in complex with FN. In a recent study, we developed a model of TG2-rich FN matrix, which would form in response to tissue injury, when TG2 is upregulated and exported to the ECM. TG2 would either directly bind to FN fibrils or bind plasma FN, which is then deposited in the damaged area [36, 40, 55]. We demonstrated that matrices of FN in complex with TG2 have a distinctive adhesive role. In response to TG2-FN, various cell types could largely restore loss of cell adhesion following inhibition of the classical FN ArgGlyAsp (RGD)dependent adhesion pathway mediated by ␣5␤1 integrin receptors [33]. This process however was not linked to the intrinsic TG2 ability to modify the FN matrix by Ca2⫹-dependent transamidation, but is consistent with the previously described transamidating-independent role for TG2 in cell-matrix interactions [37, 39]. This matrix complex was sufficient to support the formation of focal contacts in the presence of RGD peptide, the assembly of associated actin stress fibers and activation of focal adhesion kinase. A PKC-␣ inhibitor (GO6976) negatively affected RGD-independent cell adhesion to TG2-FN but not normal cell adhesion to FN, suggesting the involvement of PKC-␣ [33]. At this stage, it is still not entirely clear how signals are conveyed from the extracellular TG2-FN. However, we demonstrated that treatment of human osteoblasts cells with heparitinase, an enzyme that catalyses the eliminative cleavage of heparin and heparan sulfate, greatly diminished the RGD-independent adhesion in response to TG2-FN, suggesting that cell-surface heparan sulphate proteoglycans may mediate RGD-independent cell adhesion to TG2-FN (see also fig. 4). Such a complex of TG2 and FN may be necessary to ensure adhesionmediated cell survival in situations of cell wounding or cell stress, where the increased expression of matrix-degrading metalloproteinases, would lead to fragmentation of the ECM and formation of matrix peptides which act as competitive inhibitors of the classic RGD-dependent cell adhesion [33].

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Role of TG2 in Cell Migration Participation of TG2 in cellular processes which are relevant to wound healing, such as cell migration, has been well described [39, 56, 49] although, controversy still exists on whether these activities depend on the TG-mediated protein modification as opposed to a TG matrix structural role [37] or both or an intracellular G-protein function [49]. Cell migration on FN has been shown to depend on the expression of TG2 and does not seem to require TG2 transamidating activity [39]. The ability of anti-TG2 monoclonal antibody Cub7402 to reduce either cell adhesion or migration on FN in a dose-dependent manner in many cell types [36, 39, 45, 57] is reminiscent of the modulation of cell-matrix interactions by antibodies directed against cell surface integrin receptors ␤1 and ␣5 and indicates that cell-surface TG2 is an important component in the migration of cells. Akimov and Belkin [56] have shown that TG2 is expressed on the surface of monocytic cells and it is also involved in the adhesion and migration of monocytic cells on a FN matrix. These authors also showed that TG2 might act as a coreceptor of ␤1 and ␤3 integrins by mediating cell adhesion to the gelatin-binding site of FN [37]. In contrast, migration of fibroblasts on collagen does not seem to be related to its role as a structural protein since unlike migration studies on FN matrices incubation of cells with antibody to TG2 does not modulate cell migration [49].

Role of TG2 in the Inflammatory Response

Soon after tissue damage the inflammatory phase starts with neutrophils and monocytes migrating into the wound tissue to destroy tissue debris and pathogenic microorganisms (fig. 1a). An increase in TG2 activity has also been associated with the inflammatory phase, with the majority of attention focused on macrophages, which mediate the transition between inflammation and repair [1]. The TG2 antigen was found to be particularly expressed in macrophages, adjacent to the re-epithelialization zone and in the provisional fibrin matrix during rat dermal wound healing [11]. It was also reported that inflammatory cytokines such as IFN-␥ in rat small intestinal cells, IL-6 or TGF-␤1 might induce TG2 expression, which in turn could also affect the adhesion/motility of white blood cells during inflammation [58 and references therein]. TG2 and Activation of Phospholipase A2 Some reports have clearly linked TG2 with the inflammatory response by demonstrating that TG2 enhances the activity of sPLA2s (secretory isoforms of phospholipase A2). This would occur through the formation of an isopeptide bond within sPLA2 either via its crosslinking or the incorporation of polyamines into sPLA2s [59, 60]. The activated sPLA2 would then enhance the release of

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arachidonic acid from the cell membrane during inflammation, which is the rate limiting step in the biosynthesis of eicosanoids by cyclooxygenase. Sohn et al. [59] have recently shown that new chimeric peptides which are derived from proelafin, a cornified cell envelope component discovered in hyperproliferative epidermis, and antiflammins, peptides originating from natural PLA2-inhibitory proteins, can inhibit sPLA2 and TG2 activity including TG2-mediated modification of sPLA2 and display strong in vivo anti-inflammatory activity. In vivo studies conducted in a transgenic mouse model where TG2 is specifically overexpressed in ventricular myocytes (table 1) [27, 28], have confirmed the in vitro prediction of a link between TG2 upregulation and cyclooxygenase-2. The consequent cardiomyocyte overexpression of thromboxane synthase (TxS) and the receptor for thromboxane (TxA2) results in cardiac failure [27]. TG2 and Inflammatory Conditions The implication of TG2 in inflammation is further supported by many reports showing the association of TG2 with various inflammatory conditions that are characterized by mucosal inflammation, such as celiac disease [61–63], other intestinal diseases such as Crohn’s disease and other autoimmune diseases affecting various tissues and associated with chronic inflammation (e.g. sporadic inclusion body myositis or SIBM) [3, 64 and reference therein].

Wound Healing and Fibrosis in the Kidney

As noted earlier in the introduction to cutaneous wound healing, there are a number of events in the wound healing process that are common to all tissues. For example the kidney, like all other tissues and organs is susceptible to frequent insult and wounding encountered in daily living, e.g. dietary components, drugs, environmental factors and disease. This triggers a programmed and controlled repair process that is not that dissimilar to that found in skin [65], which ultimately terminates and resolves. However, since the kidney is an internal organ both the damage and repair process go virtually unnoticed. In fact, the only time it becomes apparent that these processes are occurring is when the insult becomes chronic or severe, or if the repair process becomes aberrant. Aberrant Wound Healing in the Kidney Leading to Progressive Renal Scarring The highly efficient wound resolution within the kidney (as in all other major internal organs) remains largely unnoticed, however it is aberrations in this process that lead to greater than 99% of all cases of end-stage kidney failure requiring either dialysis or transplantation. The problems arise when the

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Glomerulosclerosis

Tubulo-interstitial fibrosis and scarring

Normal

Early scarring

Advanced scarring

Falling kidney function

End-stage kidney failure ⬍10% ⫽Dialysis

Fig. 2. Progressive scarring in the kidney – aberrant wound healing. The images are stained with Masson’s Trichrome which stains collagenous material blue/green, cell cytoplasm pink and nuclei purple. In normal glomeruli there is a thin discrete glomerular basement membrane (GBM), a clearly defined Bowman’s Space, the blood capillaries have good spatial orientation each supported by podocytes and mesangial cells. In progressive scarring the GBM progressively thickens, the Bowman’s space fills with collagenous material and mesangial cell and fibroblast proliferation becomes deregulated. This ultimately leads to podocyte death, capillary collapse and a remodeled glomerulus completely filled with ECM. In the normal tubulointerstitium there is intricate tight packing of tubules, each having a fine bore for optimal reabsorption. There is a small interstitial space with minimal fibroblasts detectable. Disease development is seen in both hypertrophy and flattening of the tubular epithelium leading to a distended lumen and tubular atrophy to epithelial cell loss. Interstitial fibroblasts proliferate and inflammatory cells invade the interstitium. The interstitial space widens as thickening of the tubular basement membrane (TBM) occurs due to excessive ECM accumulation. At end-stage kidney failure the tubulointerstitium is little more than a sea of collagenous material packed with fibroblasts with a few remaining tubules.

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a

b

c

d

e

f

g

h

i

j

k

l

p

m

n

o

q

Fig. 3. a–d Tissue transglutaminase (TG2) staining (red) in normal human cortical tubulointerstitium (a) and glomeruli (b) compared to that in renal scarring resulting from focal segmental glomerulosclerosis (c, d). Arrow 1 indicates increased staining in the expanded tubulointerstitium, arrow 2 the mesangial matrix/glomerular basement membrane and arrow 3 periglomerular. e–h ␧(␥-glutamyl)lysine staining (red) in biopsies from implanted human allografts [cortical tubulointerstitium (e) and Glomeruli ( f )] compared to that in scarred grafts resulting from chronic rejection in the glomeruli (g) and cortical tubulointerstitium (h). Arrows indicate as in panels a–d. i–l Transglutaminase in situ activity (red)

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insult becomes chronic rather than acute [65]. In the kidney, this can occur for many reasons. Worldwide, the largest cause of chronic kidney disease is diabetes (both type 1 and 2) leading to diabetic nephropathy in about 30% of diabetic patients [United States Renal Database System 2003 (USRDS)]. Other causes of chronic kidney damage can result from autoimmunity to the basement membrane resulting in glomerulonephritis, high blood pressure or drug toxicity. Once the insult becomes chronic, then the wound healing also becomes chronic leading to a nonresolving inflammatory response, fibroblast proliferation [66] and ultimately a massive overexpression and accumulation of ECM components leading to cell deletion and destruction of the kidney [67]. This remodeling once progressive leads to scarring and fibrosis of the kidney and eventually the subsequent deletion of specialized renal cells and the ultimate destruction of the kidney (fig. 2). The Role of TG2 in Wound Healing and Scarring in the Kidney Recent studies on progressive kidney scarring both in animal models and using human biopsy material have shown that there are considerable increases in the levels of TG2 and most importantly the ␧(␥-glutamyl)lysine isodipeptide cross-link during the scarring process suggesting that TG2 may have a role in progressive renal scarring (fig. 3) [42, 68, 69, 70, 72]. However, there is also a significant level of TG2 protein in a normal kidney [68]. In localized areas of damage, the cells e.g. tubular, epithelial, mesangial, within and surrounding the lesion stain far more strongly for TG2, but more importantly there is infinitely stronger staining for TG2 outside the cell in a pattern that would be consistent with TG2 localized within the ECM that forms the basement membranes; either glomerular or tubular depending on the location. These observations are highly significant in that they confirm first, that following stress/damage TG2 can move to the extracellular environment where the high Ca2⫹ and low nucleotide levels would lead to the activation of TG2 [32, 35]. Secondly, like the observations for cutaneous wound healing, that TG2 also has a normal role in the wound response as outlined in the earlier sections of this chapter. in normal rat kidney (i) and that from the 5/6th subtotal nephrectomy model of renel scarring (j–l). k excludes autofluorescent emissions (green) showing renal morphology. Arrow 4 shows activity is strongest peritubular in the tubular basement membrane and that TG2 in the expanded ECM loses most of its activity. m–q TG2 in situ hybridization (black) in normal rat kidney (m) and that from a 5/6th subtotal nephrectomy model of renal scarring (n, o). Arrow 5 shows that proximal tubular cells are the predominant source of TG2 in renal scarring; although arrow 6 indicates isolated patches of interstitial cells (macrophages or myofibroblasts) are also able to synthesize TG2. p shows a glomeruli from a patient with crescentic nephritis with TG2 synthesis within the scarring crescent (arrow 7) (myofibroblasts) and q shows mesangial cell synthesis in a patient with mesangial proliferative glomerulonephritis.

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TG2 in Progressive Renal Scarring In some renal conditions, most notably diabetic nephropathy, there is an initial (i.e. preclinical presentation) increase in ␧(␥-glutamyl)lysine levels independent of changes in overall renal TG levels as measured by the activation of total renal TG and in situ enzyme activity [69]. The ␧(␥-glutamyl)lysine dipeptide bonds are located within an expanding ECM and seem to result initially from export of existing levels of TG2, although the contribution of other isoforms cannot be excluded [69]. As the disease develops and moves into a progressive scarring phase, there is an increase of TG2 at the mRNA and protein level predominantly in tubular epithelial cells (proximal and loop of Henle cells mainly) and mesangial cells (fig. 3), irrespective of the disease type [70–72]. Most importantly, most of this increased production of TG2 in epithelial cells is passed straight out of the cell leading to a massive pool of TG2 in the extracellular environment where high Ca2⫹ and low nucleotide levels lead to its activation, an event that can be recreated in cell culture [73]. With increasing levels of enzyme secreted from cells, modulation of its activity either through the binding of ECM FN or through proteolytic degradation [54] becomes increasingly ineffective resulting in the cross-linking of any appropriate substrate. This results in both quantitative and qualitative changes to the local ECM affecting both its deposition and proteolytic clearance [18, 48, 74]. Analysis of diseased kidneys clearly shows that the TG2 antigen, TG activity and increased levels of ␧(␥-glutamyl)lysine are associated with expanding tubular and glomerular basement membranes indicating ECM components as the major target for the enzyme (fig. 3) [42, 65, 71, 72]. In vitro, the effect of growing renal cells (mesangial and tubular epithelial cells) in a high glucose environment is associated with an increased synthesis and accumulation of ECM and represents a basic model of diabetic nephropathy [75, 76]. These changes are thought to be driven through the production of various growth factors, most notably TGF-␤1 [77] through a PDGF loop [78]. Recently, using OK proximal tubular epithelial cells we have been able to demonstrate that increases in ECM and particularly collagen are associated with the increases in glucose levels [93] and that these changes lead to increases in TG2 synthesis and subsequent changes in TG2 and ␧(␥-glutamyl)lysine in the ECM [48]. To test if TG2 had any role in the accumulation of ECM or it is concomitant to this, similar studies have been performed in the presence of a specific site-directed TG inhibitor 1,3-dimethyl-2[(oxopropyl)thio] imidazolium [79, 80]. These studies categorically show that the level of ECM accumulated can be reduced if TG2 is blocked thus confirming a causative role in the accumulation of ECM associated with hyperglycemia [48]. Further, they clearly show that TG2 has a direct action on the ECM, as the effects of these inhibitors are independent of TGF-␤1 synthesis or activity in this cell line over the time frame used.

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In support of these results is the finding that ECM generated from proximal tubular epithelial cells transfected to give increased expression of TG2 has a higher collagen content with increased resistance to trypsin, MMP-1 and MMP-2 [74, and unpubl. data]. To determine the importance of TG2 in the aberrant wound healing leading to scarring, fibrosis and loss of renal function in the kidney, we have used mini osmotic pump technology to deliver a number of site-directed TG inhibitors to the kidney. As well as the well established site-directed inhibitor 1,3-dimethyl-2[(oxopropyl)thio] imidazolium [79, 80], an inhibitor based on a synthetic CBZ-glutaminyl glycine analogue molecule that has poor transfer across the cell membrane (thus effectively limiting its action to the extracellular compartment) [48] has been used. Using the 5/6th subtotal nephrectomy model of focal segmental glomerulosclerosis [81] and the streptozotocin model of type 1 diabetes mellitus and diabetic nephropathy (unpubl. data), we have been able to reduce the elevated levels of extracellular TG2 activity in excess of 50% for up to 8 months which results in an almost total prevention of the build up in ␧(␥-glutamyl)lysine that is associated with kidney scarring. Most importantly the consequence of the prevention of ␧(␥-glutamyl)lysine cross-linking is to dramatically reduce the degree of scarring that occurs in both these models of renal failure [81]. Anti-TG compounds therefore offer great potential not only in the treatment of kidney scarring, but the modulation of all types of tissue scarring due to the similarity between different tissues.

Dual Approach to Transglutaminase-Based Wound Repair: Transglutaminase as a Novel Molecular Target and as a Biocatalyst for Tissue Engineering

In general, wounds can be managed by a variety of approaches depending on the severity of the injury but treatments for severe and chronic wounds (e.g. skin burns, diabetic ulcers, critical-size bone fractures) and conditions leading to abnormal repair (e.g. fibroproliferative conditions) are still difficult to manage. Healing promotion by modulation of cytokines or other wound mediators is an attractive strategy e.g. neutralizing antibodies towards growth factors can be used to inhibit their function in cutaneous wounds and wound fluids [82]. However, their clinical usage has been limited by the complexity and range of cytokines and other wound mediators involved in cutaneous healing whose role in vivo is only partly understood. The use of tissue-engineered products both acellular, such as matrices that stimulate cell migration, angiogenesis and induce growth factor function, and cellular-based products has demonstrated to be a valid approach for wound repair and healing of chronic wounds [83, 84].

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Table 2. Examples of transglutaminase-based wound repair: Transglutaminase as a novel molecular target and as a biocatalyst for tissue engineering TG

Wound repair approach

Major biological effect

Potential therapeutic applications

Clinical trial

FXIIIA

Administration of plasma or recombinant FXIIIA preparations

Stabilization of blood clots

Treatment of postsurgical bleeding complication; Improvement in wound healing (topical treatment of ulcerative leg disease)

Phase I [85–87] (rFXIIIA)

FXIIIA

Covalent incorporation of bifunctional adhesive peptides or mutant growth factors in fibrin during coagulation

Enhancement of neurite extension in rats on fibrin modified with adhesive peptides. Enhanced endothelial proliferation on fibrinVEGF

Variety of clinical situations that require fibrin as therapeutic sealant with added specific bioactivity

TG2

Administration Competitive of competitive inhibition of amine substrate TG activity putrescine dihydrochloride (1, 4-diaminobutane dihydrochloride, Fibrostat)

Hypertrophic scarring

TG2

Administration of specific site directed inhibitors into the kidneys of rats to prevent renal scarring

Renal scarring

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Site directed irreversible inhibition of TG

References

[88–90]

Phase I [92–93] (fibrostat)

[48, 81]

106

Table 2 (continued) TG

Wound repair approach

Major biological effect

Potential therapeutic applications

TG2

Administration of chimeric peptides containing proelafin and the conserved core of anti-inflammins

Inhibition of both PLA2 and TG2 activity

Antiinflammatory activity in vivo

[58, 94]

TG2

Application of TG2 at cartilage interfaces

Increased adhesive strength at cartilage interfaces in vitro

Treatment of cartilage lesions

[95]

Increased proliferation of fibroblasts and differentiation of osteoblasts

Scaffolds for wound dressing and tissue repair

[96–98]

Microbial Modification of TG gelatin and collagen

Clinical trial

References

TGs represent a new class of wound healing mediators. By targeting this group of enzymes, new healing strategies are under development with promising applications in clinics. The use of TGs has been exploited in a number of different ways: some TG members in purified or recombinant form have been utilized to facilitate healing in a wide range of therapeutic contexts; different TG inhibitors have also been utilized to control fibroproliferative conditions; finally, TGs have also been utilized as biocatalysts, by virtue of the protein cross-linking activity, for the modification of natural polymers e.g. fibrin and gelatin for the development of scaffolds for wound repair using a tissue engineering approach. A short overview of some of the TG-based approaches to wound repair is presented in table 2.

Conclusions

In conclusion the information presented in this chapter indicates that a number of TGs may be associated with the events, which occur during both

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Fig. 4. TG2 – a multifunctional role in normal and aberrant wound repair. Exposure of certain cell types to various stress factors causes an upregulation of TG2. If the cell loses Ca2⫹ homeostasis TG2 becomes activated resulting in the cross-linking of cellular proteins and TG2-associated cell death. This is independent of chromatin cleavage and caspase-3 activity. If the TG2 is released, at the cell surface it may act as an integrin coreceptor in the binding of cells to FN, thus facilitating migration. In addition, it can be deposited into the ECM where once bound to FN it acts as an integrin-independent adhesive protein which binds to cell surface heparan sulphate. Both these roles are independent of transamidating activity. However in the extracellular environment high Ca2⫹ and low GTP/GDP levels will result in TG2 activation. Once activated it can participate in TGF-␤1 activation, deposit ECM components through protein cross-linking employing nonconventional deposition pathways

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normal and aberrant wound healing (fig. 4). Importantly, it confirms that TGs are indeed a new class of wound healing mediators that represent strategic therapeutic targets in the treatment of various diseases. In addition, it demonstrates the growing interest in these enzymes as applied biocatalysts for use as tissue glues, in the production of novel biomaterials for tissue engineering and for facilitating the repair of both soft and hard tissues after disease or injury.

Acknowledgments The authors would like to thank the EPSRC, the BBSRC, the Wellcome Trust, Diabetes UK, the National Kidney Research Fund for funding the work referred to in this review. They would also like to thank Shakthi Dookie for helping in its preparation.

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Dolynchuk KN, Ziesmann M, Serletti JM: Topical putrescine (Fibrostat) in treatment of hypertrophic scars: Phase II study. Plast Reconstr Surg 1996;97:117–123. Miele L: New weapons against inflammation: Dual inhibitors of phospholipase A2 and transglutaminase. J Clin Invest 2003;111:19–21. Jurgensen K, Aeschlimann D, Cavin V, Genge M, Hunziker EB: A new biological glue for cartilage-cartilage interfaces: Tissue transglutaminase. J Bone Joint Surg Am 1997;79:185–193. Ito A, Atsunori M, Takizawa Y, Shinkai M, Honda H, Hata K, Ueda M, Kobayashi T: Transglutaminase-mediated gelatin matrices incorporating cell adhesion factors as a biomaterial for tissue engineering. J Biosci Bioeng 2003;95:196–199. Broderick EP, O’Halloran DM, Rotchev YA, Griffin M, Collighan RJ, Pandit AS: Enzymatic stabilisation of gelatin based scaffolds. J Biomed Mat Res 2004, in press. Chau D, Collighan R, Verderio EAM, Griffin M: UK Patent: Application GB 0420091.1 Medical Implant Materials.

Dr. Martin Griffin School of Life and Health Sciences, Aston University Aston Triangle, B4 7ET, Birmingham (UK) Tel. ⫹44 121 204 3942, Fax ⫹44 121 359 2677, E-Mail [email protected]

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Mehta K, Eckert R (eds): Transglutaminases. Prog Exp Tum Res. Basel, Karger, 2005, vol 38, pp 115–124

Transglutaminases in Epidermis Richard L. Eckert a–d, Michael T. Sturnioloa, Ann-Marie Broomea, Monica Rusea, Ellen A. Rorkee Departments of aPhysiology and Biophysics, bOncology, c Dermatology, dBiochemistry, and eEnvironmental Health Sciences, Case School of Medicine, Cleveland, Ohio, USA

Introduction

The epidermal keratinocyte is the major cell type of the epidermis. These cells undergo a process of cell differentiation that results in the assembly of a multilayered stratified squamous epithelium that protects the organism from the environment. Transglutaminases (TG) are enzymes that catalyze the formation of covalent isopeptide protein-protein cross-links. Three TG enzymes are expressed in epidermis and play a role in the process of differentiation. This review discusses the role of these enzymes in constructing the epidermal surface.

Epidermal Differentiation

The epidermis is designed to provide protection. This is achieved by assembling a multilayered stratified squamous epithelium through a regulated process called differentiation. This process begins in the innermost epidermal layer, the basal layer, which contains the proliferative cells. Cells in this layer undergo regulated proliferation and give rise to nondividing daughter cells [1, 2]. These daughter cells then differentiate to form the suprabasal layers which include, from the innermost to outermost layers, the spinous, granular, transition and cornified layers [1, 3]. The spinous layer is characterized by the formation of desmosomes that provide intercellular connections that help stabilize the skin surface. Granular layer cells, in contrast, are distinguished by the presence of granules [4–8]. These granules contain lipids and proteins that are subsequently released and used as TG substrates during the terminal stages

of differentiation. Transitional cells comprise the border between the living and dead epidermis. Cells in this zone undergo remodeling which results in the proteolytic and nucleolytic destruction of the intracellular organelles (nucleus, mitochondria, etc.). The result of this process is the terminal cell type, the corneocyte, which consists of a network of keratin macrofibrils contained within an envelope of cross-linked protein called the cornified envelope. Cornified envelope assembly proceeds simultaneously with the destruction of intracellular structures. The cornified envelope is a protein/lipid structure that is deposited beneath the keratinocyte plasma membrane during the terminal stages in differentiation [9–12]. It is assembled from protein precursors [10, 13, 14] and is characterized by the presence of numerous covalent interprotein cross-links [9, 15–21]. These cross-links are formed by TG, enzymes that catalyze the formation of interprotein N6-(␥-glutamyl)lysine isopeptide bonds [22, 23]. These bonds link a lysine residue (the amine donor) and a glutamine (the amine acceptor) through an isopeptide bond. The extensive formation of these types of bonds provides the links that render the cornified envelope an efficient and abrasion-resistant barrier. Three TG present in surface epithelia are thought to participate in cross-link formation – TG1 [24, 25], TG3 [26] and TG5 [27–30] (table 1). These enzymes are discussed below.

Keratinocyte Transglutaminases

Transglutaminase Type 1 Transglutaminase Type 1(TG1) is expressed in the spinous and granular layers [31] and is unique among the TG in that it is anchored to the inner surface of the plasma membrane through myristate and palmitate linkages [32–34]. The myristate and palmitate modification of TG1 occurs at amino acids located in the N-terminus [32]. This modification is essential for anchoring of TG1 to the plasma membrane. In addition, a fraction of the membrane-associated TGase 1 undergoes proteolysis to release fragments [35]. These cleavage products increase in level during keratinocyte differentiation [34–37]. In vitro studies suggest that cleaved TG1 is more active than intact TG1 [37, 38]. Thus, this may be a mechanism whereby TG1 activity can be regulated. In addition, TGase 1 is phosphorylated on serine residues – predominantly Ser82 [39, 40]. The phosphorylated serine residues reside in close proximity to the amino acids that are modified by the addition of lipid membrane anchors. Phosphorylation is increased by the treatment of keratinocytes with agents that activate protein kinase C. This suggests that phosphorylation may influence the process of lipid anchor addition; however, there is no direct evidence supporting this idea.

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Table 1. Transglutaminases in surface epithelia Transglutaminase

Epidermal expression

Subcellular distribution

Cleavage modification

Activity regulators

TG1

Expressed in spinous and granular layers

Plasma membrane

Calcium and TIG3

TG3

Expressed in spinous and granular layers

Cytosol

Myristylation, phosphorylation, palmitylation, cleavage to release 10, 33 and 68 kDa products Zymogen to active form transition involves protease cleavage at a site in the flexible loop surrounding Ser469 followed by binding of calcium

TG5

Expressed in spinous and granular layers of epidermis, slight expression in the basal layer

Nuclear matrix and cytoskeleton – associates with vimentin filaments

Calcium, GTP and GTPase cycle

Calcium and GTP

The cornified envelope is composed of a layer of covalently cross-linked proteins and a layer of associated lipids [20, 41, 42]. Thus an important question is the mechanism whereby lipids are attached to the cornified envelope. TG1, when reconstituted with involucrin, a ␻-hydroxyceramide analog, and phosphatidylserine vesicles, promotes lipid ester formation of involucrin at Gln107,118,122,133 and 496 [43]. This pattern of labeling mimics the pattern observed in vivo [44]. Thus, TGase 1 may also function to cross-link lipids to the envelope. Regulation of TG1 Level and Activity Both TG1 level and TG1 activity are regulated. TG1 expression is differentiation dependent; that is, expression is confined to the suprabasal epidermal layers [31]. TG1 levels are also increased in cultured keratinocytes treated with differentiating agents such as calcium [15, 45–50]. These findings are not unexpected, since calcium is an important regulator of epidermal differentiation [51]. In addition to calcium, other agents also regulate TG1 level. In general, differentiating agents increase TG1 level while differentiation-suppressing agents

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reduce TG1 level [52–60]. In addition, calcium is a required cofactor for enzyme activity [15, 49, 61]. Recently, a protein, TIG3, has been described as that which triggers TG-associated cell death in the absence of increased extracellular calcium [62]. TIG3 overexpression in keratinocytes results in growth suppression and produces cells that resemble cornified envelopes [62]. TG mRNA and protein levels are little changed by TIG3 expression, but TG activity is markedly increased. Detailed studies reveal that TIG3 colocalizes with TG1, and that TG1 activity is specifically increased at locations of colocalization [62]. Additional studies will be required to identify the mechanism of this regulation; however, TIG3 appears to be a unique TG1 regulatory protein. Transglutaminase Type 3 The gene encoding TG3 is located on chromosome 20q11–12 [63]. Like TG1, TG3 expression is increased in calcium-treated epidermal keratinocytes by a transcriptional mechanism that involves ets and Sp1 transcription factor interaction with the TG3 gene promoter [64, 65]. Like TG1, TG3 is expressed in the upper epidermal layers [66]. However, unlike TG1, which is membrane associated, TG3 is localized in the cytoplasm. Recent structural studies have provided insight regarding the TG3 mechanism of cross-linking. The TG3 zymogen consists of four folded domains that share a common structure with other TG. The active site is buried in a cleft formed by two ␤-sheets of the catalytic core and the barrel 1 c-terminus [67]. In contrast to TG1, TG3 is produced as a zymogen that requires processing to become active. The role of calcium in regulating TG3 function has been carefully examined, since calcium is known to be a cofactor for the activation of this and other TG. The TG3 zymogen sequesters one calcium ion per protein monomer; this ion is retained in both the inactive and activated form and is thought to be required for stability. TG3 activation begins with proteolytic cleavage within the flexible loop region at Ser469. This cleaved form is not active, but, unlike the zymogen, it can be activated by calcium. In addition to the single calcium ion that it constitutively bound in the enzyme, activation requires binding of two more calcium ions. Binding of the second calcium ion produces a minimal change in protein active site conformation; however, binding of the third calcium ion produces a dramatic change in structure that opens the channel [68]. Thus, TG3 activation involves two sequential steps – proteolytic cleavage at Ser469 followed by binding of two calcium ions. Some years ago it was realized that TG2, another TG (tissue type) that is expressed in many tissues, had two activities – a calcium-dependent protein cross-linking activity and a GTPase activity [69]. Thus, TG2 functions in cross-linking and also in signal transduction. It has recently been reported that TG3 also interacts with and is regulated by guanine nucleotides [70]. Binding of

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GTP to TG3 is associated with substitution of Ca2⫹ with Mg2⫹ at the third (activating) calcium-binding site. This binding causes a conformation change that closes the active site channel and inactivates the cross-linking function. Subsequent hydrolysis of the bound GTP results in a reversion of the enzyme to the active (cross-linking) state. These findings have important implications for the regulation of TG3 function. It is supposed that high intracellular concentrations of calcium lead to increased TG3-dependent cross-linking. However, when GTP is bound, this activity is inhibited. Thus, it is likely that TG3 alternates between functioning as a cross-linking enzyme and as a signal transduction GTPase. However, the targets of the potential signaling function are not yet known. Transglutaminase Type 5 The third TG type that is present in epidermis is TG5. Like TG1 and TG3, TG5 is expressed in the spinous and granular layers [28]. In addition, keratinocyte differentiating agents, such as calcium, increase TG5 expression in cultured keratinocytes. Human keratinocytes produce the full-length TG5 mRNA and three alternate splice products in which exons 3, 11, or 3 and 11 are missing [29]. Cell free studies reveal that the TG5 isoforms that lack exon 3 are not active [29]. Recombinant TG5 can utilize involucrin, loricrin, SPR1 and SPR2 as substrates [71]. The TG5 isoforms have the expected reactivity towards these substrates – the full-length TG5 protein and the protein lacking exon 11 are active, while the forms lacking exon 3 are not active. TG5 can cross-link loricirin – both interprotein and intraprotein bonds are formed. The fact that loricrin multimers are formed suggests that TG5 can utilize both glutamine and lysine residues as cross-linking sites. As we have discussed for TG3, TG5 is also regulated by nucleotides – in this case GTP and ATP. These nucleotides inhibit the calcium-dependent crosslinking activity of recombinant TG5 [27]. It is presumed that calcium and nucleotides (GTP, ATP) have opposing effects on TG5 cross-linking activity. Thus, in the presence of high calcium, calcium is bound and the enzyme is active, but this activity is inhibited when GTP or ATP are bound to the enzyme. This regulation mirrors the inverse relationship between nucleotidedependent signaling activity and TG cross-linking activity observed for TG2 and TG3 [70, 72]. TG5 is expressed at low levels in cultured keratinocytes. Thus, TG5 subcellular localization and function has been studied using vector-expressed enzyme. Expressed TG5 is extractable by treatment with SDS, but is not extracted by nonionic detergent, reducing agent, or varied ionic strength or pH. These findings suggest that TG5 is insoluble. Cell fractionation studies reveal that TG5 is associated with the cytoskeleton, and confocal microscopic studies suggest that it localizes with vimentin [29].

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Transglutaminases in Skin Disease It is not surprising, given the important role that TG have in assembly of the skin barrier, that mutations in these enzymes have also been implicated in the genesis of skin disease. Lamellar ichthyosis is a disorder of cornification. These patients develop large plate-like scales with accompanying erythroderma, orthokeratotic hyperkeratosis and acanthosis [73]. A mutation of the TG1 enzyme that results in reduced TG1 activity has been identified in a large percentage of lamellar ichthyosis patients [74, 75], suggesting that reduced TG1 activity may be an underlying causative factor in the pathogenesis of this disease. Supporting this idea is the fact that TG1 knockout mice have an ichthyosis-like phenotype [76, 77]. TG3 has been implicated as having a role in autoimmune disease. Patients who develop a sensitivity to glutens frequently develop celiac disease, a chronic disorder of the small intestinal epithelium [78]. In certain individuals, development of celiac disease is associated with the development of dermatitis herpetiformis, a blistering skin disease. Dermatitis herpetiformis patients produce anti-TG3 antibodies, and IgA precipitates in the papillary dermis of dermatitis herpetiformis patients contain TG3 [79]. Thus, it has been proposed that TG3 may be an autoantigen in this disease. Less is known about a potential role for TG5 in disease. However, TG5 level is increased in several epidermal diseases, and it may contribute to the hyperkeratotic phenotype observed in ichthyosis [28].

Transglutaminase Function in Epidermis Several conclusions can be derived from the information presented in this brief review. First, there is a restriction on the expression of TG in epidermis, such that expression of each of the three isoforms is confined to the suprabasal layers. This makes sense, since there is presumably no reason to have these cross-linking enzymes expressed in the basal proliferative cell layer. Second, the presence of three TG in epidermal keratinocytes suggests that cross-linking is a multienzyme task. This is supported by our knowledge regarding subcellular localization. TG1 is anchored on the inner surface of the plasma membrane, TG3 is soluble and cytoplasmic, and TG5 is insoluble and associated with intermediate filaments. This differential subcellular distribution of TG suggests that each enzyme may have a specific function during the terminal differentiation. In addition, the differing use of substrates suggests that the three enzymes may cooperate to create the final pattern of inter- and intramolecular cross-links that is observed in envelopes prepared from epidermis [71, 80–83]. Finally, the available information suggests that TG, especially TG1, are essential for normal epidermal differentiation.

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Acknowledgments This work was supported by the National Institutes of Health and utilized the facilities of the Skin Diseases Research Center of Northeast Ohio (AR39750).

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Dr. Richard Eckert Case School of Medicine, 2109 Adelbert Road Cleveland, Ohio 44106–4970 (USA) Tel. ⫹1 216 932 1324, Fax ⫹1 216 932 5586, E-Mail [email protected]

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Tissue Transglutaminase (TG2) in Cancer Biology Lingegowda S. Mangala, Kapil Mehta The University of Texas M. D. Anderson Cancer Center, Houston, Tex., USA

Introduction

Most cancers are in essence caused by defects or dysfunction of regulatory circuits that govern cell cycle regulation and homeostasis [1]. Indeed, many oncogenes and tumor suppressor genes code for proteins that are either intrinsic components of the cell cycle circuit or indirectly influence cell cycle progression. Despite significant advances in the development of new therapeutic modalities, global burden of cancer has continued to increase. For example, in the year 2000, 5.3 million men and 4.7 million women developed a malignant tumor and 6.2 million died from the disease [2]. It is becoming apparent that long-term survival of patients is mainly dependent on early detection and treatment of the cancer. Unfortunately, most patients reporting to the clinic have advanced disease, which in many cases is inoperable. At the time of diagnosis, the initial tumor in many patients has progressed to a more malignant phenotype, characterized by invasive and metastatic foci, which becomes resistant to chemotherapeutic interventions. A common feature among advanced cancers is that they exhibit resistance to apoptosis [3]. This property endows the tumor cells with not only an increased ability to grow and survive in foreign tissue sites (i.e. metastasize), but also an ability to develop a drug-resistant phenotype [4]. It is commonly believed that certain cell adhesion molecules, such as integrins can strongly influence the ability of neoplastic cells to migrate, proliferate, and undergo apoptosis and mediate signal transduction, gene expression, invasion, and metastasis [5, 6]. Integrins are heterodimeric molecules composed of
and  subunits. Depending on the
-subunit pairing, they can interact with one or more

ligands in the surrounding extracellular matrix (ECM). Integrins differ from other cell surface receptors in that they bind to their ligands (ECM components) with a low affinity (106–109 l/mole) and must exceed certain minimal number at focal contacts to facilitate the binding of the cell to the ECM. When integrins are diffusely distributed over the cell surface, no adhesion occurs. However, in response to certain stimuli these integrins can cluster (in focal contacts), and their combined weak affinities give rise to a spot on the cell surface that then has enough adhesive capacity (avidity) to adhere to the ECM. Alternatively, certain transmembrane proteins can associate with integrins and enhance their affinity for ECM ligands and promote signaling [7, 8]. The first such interaction was identified between integrin 3 and integrin-associated protein (or CD47) [9]. More recently, a similar association between tissue transglutaminase 2 (TG2) and the 1 and 3 subfamilies of integrins has been observed [10, 11]. It is estimated that 40% of 1 integrins can form complexes with TG2 in a 1:1 ratio [12]. TG2, also referred to as the cytosolic, type II, or liver transglutaminase, is a unique member of the transglutaminase enzyme family, in that in addition to catalyzing the calcium-dependent post-translation modification of proteins, it can also bind and hydrolyze GTP with an affinity similar to those of the
subunits of large heterodimeric G proteins and small Ras-type G proteins [13–16]. TG2/Gh is involved in coupling the
1b- and
1d-adrenoreceptors, thromboxane, and oxytocin receptors to phospholipase C, mediating inositol phosphate production in response to the agonistic activation. In GTP/GDP-bound form, TG2 cannot catalyze protein cross-linking reactions, but Ca2 can reverse this inhibition and regulate the transamidation functions of the TG2 protein. Although TG2 is one of the most extensively studied members of the transglutaminase family, its precise physiologic function remains largely a matter of speculation. Calcium-dependent activation of TG2 has been implicated in diverse biologic functions, such as differentiation, receptor-mediated endocytosis, cell adhesion, and induction of apoptosis. Although, it is a predominantly cytosolic protein, TG2 can also be secreted outside the cell, where it can stabilize the ECM by making the matrix resistant to mechanical and proteolytic degradation [17]. Several ECM proteins, such as fibronectin, vitronectin, collagen, osteonectin, and osteopontin, can serve as substrates for TG2-catalyzed cross-linking reactions (see, Chapter 3). TG2-catalyzed cross-linking of ECM proteins also plays an important role in the deposition and stabilization of the ECM, which in turn help to promote the attachment and spreading of several cell types [15, 18]. Under certain conditions, with the help of importin protein, TG2 can translocate to the nucleus, where it can function as a G protein or as a transamidase activated by nuclear Ca2 to cross-link histones, retinoblastoma (pRb),

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and SP1 transcription factor proteins [13, 19]. Similarly, TG2 can also translocate to the cell membrane in association with integrins and promote the interaction between cell surface integrins and fibronectin.

TG2 as Antiapoptotic Factor

Many reports have supported the involvement of TG2 in apoptosis (see, Chapter 4). Cells undergoing apoptosis show induced expression of TG2. Its overexpression primes cells for suicide, and inhibition of its expression results in decreased cell death. What is becoming apparent is that under extreme stressful conditions (e.g. hypoxia, growth factor deprivation, or treatment with chemotherapeutic drugs) Ca2 homeostasis inside the cell may become perturbed. Sudden changes in the cytosolic Ca2 may transform latent TG2 into its active cross-linking configuration, resulting in extensive cross-linking of intracellular proteins, their polymerization, and formation of detergent-insoluble structures. The protein scaffolds thus formed can stabilize the structure of the dying cell before its clearance by phagocytosis, preventing the release of intracellular components and subsequent inflammatory response. Nevertheless, the expression of TG2 and apoptosis do not completely overlap. Direct evidence of this comes from ‘TG2(–/–)’ knockout mice, which show no phenotype suggestive of any perturbation in apoptosis [20, 21]. However, the possibility that some other isoform compensates for the loss of TG2 in these mice cannot be ruled out. Moreover, several rapidly dividing cancer cells that do not undergo apoptosis have been shown to express high levels of TG2 [22–24]. More recent studies have provided direct evidence and suggest that increased expression of TG2 can prolong cell survival by preventing apoptosis [25–27]. It has been proposed that proapoptotic and antiapoptotic effects of TG2 strongly depend on its location within the cell [27]. In the nucleus, TG2 can interact with the pRb protein and protect cells from apoptosis [27, 28]. Similarly, in the cell membrane, it can associate with integrins and provide a binding site for fibronectin [29]. TG2 exerts this function by simultaneously binding with  integrins and fibronectin through a gelatin-binding domain (42-kDa fragment, module I6II1,2,I7–9) [10–12].

The Connection between TG2, Drug Resistance and Metastasis

It is estimated that, depending on the cell type, 5–40% of the 1 integrin on the cell surface could form complexes with TG2, and TG2 is present on the cell surface as 1:1 complexes with integrins [10, 11]. This interaction of TG2

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with integrins occurs primarily at the extracellular domains of integrin  subunits. It does not require cross-linking activity [30] and facilitates adhesion, spreading, and motility of cells [11, 30, 31]. In view of this and previous findings that integrin-mediated signaling pathways play an important role in cell growth, cell survival and metastasis, it is tempting to speculate that TG2 expression in cancer cells promotes signaling events that could affect not only the adhesive, migratory, and invasive functions of tumor cells but also their growth and survival. Several years ago, we observed that macrophages collected from inflammatory sites or activated in vitro accumulate large amounts of TG2 [32–34]. An important property of activated macrophages is that they readily migrate to inflammatory sites and kill infectious agents. This killing of invaders by the activated macrophages involves the production and release of a large number of soluble mediators that exert cytotoxic effects on their targets [35]. However, the survival of the activated macrophages that produce these cytotoxic mediators is not appreciably compromised. We now believe that the induction of TG2 in activated macrophages might play a role in their migration to inflammatory sites and in protecting them from the toxic effects of soluble mediators that they produce. Indeed, two recent reports have documented the direct involvement of TG2 in promoting the migration of normal cells. The first report suggested that the inhibition of TG2 could effectively block transmigration of T lymphocytes across endothelial cells treated with interferon- and tumor necrosis factor-
[36]. The second report showed that the transforming growth factor- (TGF-)-induced increase in cell surface expression of TG2 was responsible for augmenting the attachment and migration of retinal pigment epithelial cells on fibronectin-coated matrices [37]. More importantly, in an attempt to identify metastasis-associated proteins by proteomic analysis, Jiang et al. [38] identified TG2 as one of eleven proteins that were selectively amplified in metastatic human lung carcinoma.

TG2 Expression in Drug-Resistant Cells

We have ample evidence that irrespective of their source or type, cancer cells selected for resistance against chemotherapeutic drugs exhibit inordinately high levels of TG2 expression when compared with the parental cell line from which they were derived [23, 39–42]. TG2 expression in drug-resistant cancer cells is upregulated at all three levels: mRNA, protein, and enzyme activity. A similar increase in TG2 expression was observed by Han and Park [22] in PC-14 lung cancer cells after their selection for resistance to doxorubicin (i.e. PC-14/ADR cells). These investigators further demonstrated that

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downregulation of TG2 by stable transfection with TG2-specific antisense or ribozyme rendered the drug-resistant PC-14/ADR cells sensitive to doxorubicin and other chemotherapeutic drugs, suggesting that TG2 plays a role in the acquisition of drug resistance. We have observed that TG2 in drug-resistant MCF-7 (MCF-7/DOX) cells exists as an inert or latent enzyme because of the deficient or defective intracellular calcium pools in these cells [39]. Nevertheless, activation of TG2 by elevating intracellular calcium pools with a pharmacologic agent, A23187, led to a massive and spontaneous apoptosis of MCF-7/DOX cells [40]. These findings clearly suggest that the development of resistance, at least against multidrug resistance-related drugs, is associated with an increased expression of TG2 and that TG2 exists as a latent enzyme in these cells and could serve as a potential therapeutic target for driving drugresistant cancer cells into apoptosis.

TG2 Expression in Metastatic Cancer Cells

The spread of cancer to distant sites (metastasis) is a multistep process and is the most common cause of cancer-related deaths in patients. Key steps in the metastatic process are: detachment of malignant cells from the primary tumor, their invasion through the tissue into the blood vessel lumen, and their proliferation in distant tissue sites. Invasion of ECM is crucial for a number of physiologic and pathologic processes, including metastasis, arthritis, embryo implantation, wound healing and early development. Invasion itself is a multistep process, which involves attachment of cells to the ECM, degradation of the structural components and migration through the ECM. Although most metastatic cells during migration or upon reaching the new microenvironments (distant tissues) die, yet, some become dormant or growth arrested and survive in these stressful microenvironments for extended time periods. They remain protected from death and, in fact, survive multiple rounds of chemotherapy administered for the very purpose of eradicating them. Some of these cells begin to proliferate years later and form secondary and tertiary metastatic tumors that become hard to treat and result in death of the patient. Although there is a general consensus that drug-resistance and metastasis represent different phenotypes, yet, there are several reasons to believe that they share some common pathways. For example, during advanced stages, cancer cells accumulate large number of genetic alterations that can render them more resistant to apoptosis [3]. This property endows the tumor cells with not only an increased ability to grow and survive in foreign tissue environments (i.e. metastasize), but also an increased likelihood of having a drug-resistant phenotype. Moreover, cancer cell lines selected in vitro for resistance to drugs

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are more metastatic in vivo, whereas cancer cells isolated from metastatic sites, in general, exhibit higher resistance to chemotherapeutic drugs. In view of these observations and the results reported by Aoudjit and Vuori [43] showing that culture of a highly metastatic breast cancer cell line MDA-MB-231 on fibronectin-coated surfaces rendered the cells resistant to vincristine and paclitaxel-induced apoptosis, we reasoned that metastatic cancer cells might have high basal expression of TG2. Because, TG2 has high binding affinity for fibronectin [29] and it can associate with cell surface integrins [11, 12], its presence can promote not only the binding and motility of cancer cells on fibronectin-coated surfaces, but also render them resistant to chemotherapyinduced apoptosis by upregulating cell survival and antiapoptotic signaling pathways. In support to this finding, we observed that metastatic breast cancer cell line MDA-MB-231 express high basal levels of TG2. Importantly, two clones derived from this cell line, MDA231/cl.9 and MDA231/cl.16, showed a 10- to 15-fold difference in TG2 level. TG2-deficient MDA231/cl.9 cells exhibited higher sensitivity to doxorubicin and were less invasive than the TG2-sufficient MDA231/cl.16 cells [44]. Similarly, several sublines derived from an immortal but normal breast epithelial cell line, MCF10A, representing various stages in the progression of breast cancer from benign hyperplasia to atypical hyperplasia to carcinoma in situ and fully malignant invasive tumors that can metastasize, showed increased TG2 expression as they advanced from noninvasive to an invasive phenotype [44]. Moreover, lymph node metastases obtained from patients with breast cancer showed significant higher levels of TG2 expression compared with the primary tumors from the same patients [44]. These results suggest that the development of drug resistant and metastatic phenotypes in breast cancer cells is associated with an increased expression of TG2 and that TG2 may play a role in conferring these phenotypes.

TG2 and Integrin-Mediated Signaling

It is likely that increased expression of TG2 in drug resistant and metastatic tumor cells is involved in protecting cells from chemotherapy- and stressinduced apoptosis by promoting interactions between integrins and fibronectin thereby activating cell survival and antiapoptotic signaling pathways. Indeed, factors that promote interaction between cell-surface integrins and their ECM ligands, including fibronectin, have been shown to affect signaling pathways that can influence not only the cell adhesive, migratory, and invasive functions, but also survival and proliferation in tumor cells [45–48]. For example, the
51 integrin that preferentially binds to fibronectin induces strong expression

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of the antiapoptotic protein Bcl-2, protecting cells from apoptosis and from stresses such as a lack of growth factors [49]. Similarly, activation of Sch in response to
51-fibronectin interaction modulates the adhesion and motility of MCF-7 breast cancer cells [50] and induces activation of AKT pathway that, in turn, confers increased ability to cancer cells for resisting apoptotic death [51]. More importantly, the culture of TG2-positive breast cancer cells on fibronectin-coated surfaces leads to the activation of focal adhesion kinase (FAK), an event that allows FAK to recruit SH2-containing proteins such as Src, Fyn, p85 subunit of PI3K, and phospholipase C. Recruitment of Src provides more SH2-docking sites by phosphorylating additional tyrosine residues on the FAK molecule and initiates signaling pathways, such as Ras/Erk, PI3K/Akt, and Crk/Dock180/Rac that render the cells resistant to apoptosis [52, 53]. Moreover, Src kinase activation has been known to play an important role in the metastatic spread of carcinoma cells [54]. A similar activation of FAK was observed by Akimov et al. [10] in TG2-transfected fibroblasts following their incubation on fibronectin or its gelatin-binding domain. Alternatively, TG2 expression in drug resistant and metastatic cancer cells can confer protection against apoptosis by interacting with pRb as previously described [27, 28]. An emerging paradigm in the field of carcinogenesis suggests that interaction of integrins with surrounding stroma plays important role in the process of tumorigenesis. For example, studies by Bissell and her group provide strong evidence that if integrins in the tumor’s stroma that drive the process of tumorigenesis are absent; breast cancer cells maintain normal breast acini architecture [55, 56]. Moreover, it is well known that inflammatory cytokine TGF- secreted by surrounding stroma cells, contributes substantially in the processes of tumorigenesis and metastasis [57, 58]. In this regard, it is worth noting that TGF- has been reported to regulate the expression of TG2 activity in several cell types [59]. Conversely, TG2 is involved in the regulation of TGF- activation by cross-linking latent TGF-1-binding protein to the ECM [60]. Therefore, it is plausible that TG2-mediated activation of TGF-1, which in turn can induce the expression of TG2, may play a role in conferring metastatic potential to cancer cells. In addition to TGF-, oxidative stress has been recently shown to induce TG2 expression [61]. Thus, oxidative stress induced in response to chemotherapeutic drugs or tumor’s intrinsic oxidative stress may help to promote chemoresistance in tumor cells because of their ability to upregulate TG2 expression. In another recent report, Antonyak et al. [62] observed that ligation of epidermal growth factor receptor (EGF-R) potently induced TG2 expression in several breast cancer cell lines. The EGF-induced TG2 expression protected the cells from doxorubicin-induced cytotoxic effects whereas exposure of cells

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Table 1. Known inducers of TG2 Agent 1. Cyclic AMP 2. Dexamethasone 3. Dimethyl sulfoxide 4. Interleukin-6 5. Retinoic acid

6. Sodium butyrate 7. Tumor necrosis factor 8. Phorbol esters 9. Transforming growth factor- 10. Epidermal growth factor (EGF) 11. Interferon-
and - 12. Hydrogen peroxide 13. Glucose 14. Statins 15. UV radiations

Target cell Cerebellar granule Hamster fibroblasts and fibrosarcoma Friend’s erythroblastoma Human hepatoblastoma Human myeloid Human neuroblastoma Rat superior cervical sympathetic ganglia Squamous carcinoma Human lung fibroblast Rat pheochromocytoma Human hepatoblastoma Human myeloid Human colon carcinoma Human retinal pigment epithelial cells Primary cultured hepatocytes Breast cancer cell lines Human non-small cell lung cancer 3T3 fibroblasts Kidney epithelial cells Endothelial cells Lens epithelial (HLE-B-3)

to a TG2 inhibitor or expression of a dominantly negative form of TG2 potently inhibited the EGF-mediated protection from doxorubicin-induced apoptosis. Other agents that are known to regulate TG2 expression are listed in table 1. On the basis of these observations, we propose that TG2 expression can likely confer drug resistant and metastatic phenotypes by constitutively activating antiapoptotic and cell-survival signaling pathways. A schematic representation of TG2’s involvement in cell survival and cell-death signaling pathways is shown in figure 1.

TG2 and Carcinogenesis

We believe that inactivation of TG2 by tobacco smoke components represents an important biochemical alteration induced by smoking. Besides lung cancer, smoking is linked with the development of larynx, oral cavity, pharynx, esophagus, pancreas, kidney, and bladder cancer. Tobacco smoke contains more than 4,000 individual compounds of which several are known carcinogens,

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ECM TG2 Inactive TGF-

Stress (Chemotherapeutic agents and UV radiations)

Active TGF-

Motility invasion

pFAK ROS hypoxia ER

Cell growth Cell survival TG2

Ca2

Metastasis Drug resistance

Importin-3 NOCHO-

Protein cross-linking (e.g. MLKs, SP1, pRb)

T-pRb G HDAC 2 E2F

Proapoptotic genes

Apoptosis

Fig. 1. TG2-mediated proapoptotic and antiapoptotic functions. Stressful conditions (e.g. UV radiations, chemotherapeutic agents) can generate reactive oxygen species (ROS) that results in the upregulation of TG2. Extreme stressful conditions can further trigger Ca2 release from the endoplasmic reticulum resulting in perturbation of intracellular Ca2 homeostasis, which in turn converts TG2 into a protein cross-linking configuration, resulting in cross-linking of some vital proteins, such as pRb, SP1, and MLKs, the event that may initiate the apoptotic process. In the nucleus, TG2 can interact with pRb and protect cells from apoptosis. Similarly, TG2 can be translocated on cell membranes in association with  integrins. In this form, TG2 can serve as a coreceptor for integrins and promote their binding to fibronectin. TG2-dependent interaction between integrins and fibronectin can lead to the activation of cell survival and antiapoptotic signaling pathways. In the extracellular environments, TG2 can transform latent TGF- into its active form, which in turn can upregulate the expression of TG2. Also, some free radicals (e.g. CHO– and NO–) can bind and damage the cysteine residue in the active site and render TG2 as an antiapoptotic factor.

mutagens and tumor promoters [63]. Similarly, water-soluble extracts of gasphase cigarette smoke have been shown to contain powerful oxidizing elements, which can induce TG2 expression and mediate oxidative damage to proteins in vitro and over several days to weeks. Each puff of smoke contains over 10 trillion free radicals, which may induce oxidative damage and thus initiate the process of carcinogenesis. Several thiol-containing enzymes have been shown to be susceptible to inactivation by smoke components [64, 65]. In this context, it is tempting to speculate that oxidative damage of the thiol group in the active-site cysteine residue (Cys277) of TG2 can render this enzyme catalytically inactive. Indeed, our preliminary results, using purified TG2 protein from guinea pig liver revealed that cigarette smoke condensate (CSC) could effectively inhibit the enzymatic activity of TG2 in a dose-dependent manner

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125 Enzymatic activity (% of control)

Ca2 (2.5mM) TG2

100

75

b

1

2

3

4

5

6

50

25

0

a

EDTA 0 0.01 0.1 1.0 2.5 5.0 CSC concentration (g/ml)

Fig. 2. Effect of cigarette smoke on TG2 activity and levels. a Aliquots (0.5 g each) of purified guinea pig liver TG2 were incubated with increasing amounts of cigarette smoke condensate (CSC) during a 15-min assay. The amount of radioactivity (CPMs due to 3Hputrescine) incorporated into dimethylcaseine in the absence of CSC reflects fully active (100%) TG2 under the conditions employed and was used for calculating % inhibition induced by CSC at indicated concentrations. The enzyme was completely inactive in the absence of calcium (EDTA). b At the end of incubation, samples (50 l each  0.25 g TG2 protein) were solubilized in 3X sample buffer and subjected to Western blotting using TG2-specific moAb.

(fig. 2). A similar inhibition of Factor XIIIa (plasma TG) was observed earlier by Roth et al. [66] and Galankis et al. [67]. On the basis of these observations, we believe that the inactivation of TG2 enzyme in alveolar epithelial cells can result in failure of cells to undergo apoptosis in response to smoke or carcinogen-induced injury. The failure of cells to undergo apoptosis can permit the accumulation of mutations and activate TG2-depedent cell-growth and cellsurvival signaling pathways (as discussed in previous section and fig. 1) leading to increased tumorigenic potential. A schematic representation of TG2’s involvement in cigarette smoke-induced carcinogenesis is shown in figure 3.

Conclusion

The development of resistance to multiple chemotherapeutic drugs and metastasis represent significant clinical problems in the treatment of cancer. Several events can trigger apoptosis in tumor cells; among these are chemotherapy- or radiation-induced DNA damage, hypoxia, loss of growth

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Epithelial cell

Toxic ‘free radicals’

Perturbed Ca2+ homeostasis

CSCs Nitrosamines and polycyclic aromatic hydrocarbons

‘Free radicals’ in CSC can render TG2 enzymatically inactive (latent) Apoptosis

Oxidative stress induces TG2 expression

Resistance to apoptosis promotes accumulation of mutations and tumor progression

Integrin

TG2

Fibronectin Latent TG2 by serving as coreceptor for integrins may promote cell attachment and cell survival signaling as shown in fig. 1

Fig. 3. Hypothetical model for cigarette smoke components (CSCs)-induced inactivation of TG2 and its role in carcinogenesis. CSCs have been shown to contain powerful oxidizing potential, which can lead to ‘free radical’ production and TG2 induction in bronchial epithelial cells. The intracellular antioxidant defenses, such as glutathione and catalase, can mitigate further oxidative damage to the cells. TG2- expressing cells undergo apoptosis through Ca2+-dependent activation of the enzyme. However, if intracellular oxidant load is capable of overcoming the intracellular antioxidant defenses, it can lead to oxidative damage and inactivation of thiol-containing enzymes, including TG2. The inactivation of TG2 can prevent the cells from undergoing apoptosis, on one hand, and induce cell survival and cell-growth signaling pathways by promoting interaction of cell-surface integrins with ECM, on other hand. The increased threshold of cells to undergo apoptosis can permit the accumulation of additional mutations in response to oxidative damage or other smoke-related carcinogens, thus increasing the tumorigenic potential of the cells.

factors, and loss of cell anchorage. Therefore, dysregulation of apoptosis can be a key determinant in rendering tumor cells highly malignant and chemoresistant. On the basis of our initial observations that drug-resistant and metastatic breast cancer cells express high levels of TG2 and the observation by others that TG2 can interact with integrins and fibronectin, we propose that TG2 by serving as a coreceptor for integrin-mediated binding of tumor cells to the ECM, can induce activation of cell survival and antiapoptotic signaling pathways thereby bestowing chemoresistant and metastatic phenotypes to the tumor cells. Understanding of TG2-mediated cell-survival signaling pathways and their contribution in the development of drug resistant and metastatic phenotypes may help to define the new targets and in developing effective approaches for treating aggressive tumors.

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Dr. Kapil Mehta Department of Experimental Therapeutics UT M. D. Anderson Cancer Center, Unit 422 1515 Holcombe Blvd., Houston, TX 77030 (USA) Tel. 1 713 992 81 40, Fax 1 713 792 41 67, E-Mail [email protected]

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Mehta K, Eckert R (eds): Transglutaminases. Prog Exp Tum Res. Basel, Karger, 2005, vol 38, pp 139–157

Transglutaminases in Neurodegenerative Disorders Craig D.C. Bailey, Janusz Tucholski, Gail V.W. Johnson Department of Psychiatry, University of Alabama at Birmingham School of Medicine, Birmingham, Ala., USA

Introduction

Transglutaminases (TGs) are a family of calcium-dependent enzymes that can catalyze various post-translational reactions including protein crosslinking, amine incorporation and deamination. Eight catalytically active TGs have been identified in the human genome along with one catalytically inactive member [1]. Some members of the TG family are expressed in specific tissues (e.g. factor XIIIA and TG4), while others are expressed more ubiquitously (e.g. TG5 and TG7) [1]. TG2 also is expressed ubiquitously and is the predominant TG in the mammalian nervous system [2–4]. However, depending on the species and brain region other TGs such as TG1 [3], TG3 [3] and TG5 [5] have been identified in brain and spinal cord albeit at lower levels than TG2 [1, 6]. Consequently, the majority of the studies on the role of TGs in neurodegenerative diseases are focused specifically on the TG2 isoform. In addition to its transamidation and deamination activities, TG2 can bind and hydrolyze guanosine-5⬘-triphosphate (GTP) [7], and may also have intrinsic kinase activity [8]. The binding of GTP to TG2 not only inhibits its transamidation and deamination activities but also likely plays a role in its ability to regulate signal transduction pathways [9]. Recently TG3 and TG5 have been shown to be GTPases as well, with GTP/guanosine-5⬘-diphosphate binding also negatively regulating their transamidating activity [5, 10, 11]. These findings suggest that although TG2 is the only TG that has been reported to be involved in signal transduction, a common characteristic of several of the TGs is their ability to bind, hydrolyze and be inhibited by GTP.

It has been clearly demonstrated that TGs in the brain are elevated in a number of neurodegenerative diseases including Alzheimer’s disease (AD), Huntington’s disease (HD), Parkinson’s disease (PD) and progressive supranuclear palsy [6, 12–14]. In addition, TG2 expression is elevated after spinal cord injury, traumatic brain injury and ischemia [15–17]. Therefore, there is significant interest in the role of TGs in neurodegenerative processes, especially as there is substantial evidence indicating that TG2 can facilitate the apoptotic process [18, 19] and thus could be detrimental to neuronal survival. Nonetheless there is also good evidence that TG2 plays important physiological roles in the cell such as contributing to neuronal differentiation events [20] and enhancing cyclic adenosine monophosphate (cAMP) production [21]. In this chapter the role of TGs in neuronal development and apoptosis will be briefly reviewed, followed by a discussion of its role in neurodegenerative diseases and neuronal injury.

TGs in Development of the Nervous System

Although the majority of research on TGs in the brain has focused on their role in neuronal pathology, significant work over the past two decades suggests that TGs may play an important role in the development of the nervous system. TG enzymatic activity increases significantly during mouse embryogenesis, where tubulin acts as an endogenous acyl donor, suggesting that TGs may contribute to the stabilization of cytoskeletal elements [22]. This role for TGs may be important for the production and stabilization of neuronal processes. For example, serum withdrawal in the N18 neuroblastoma cell line results in differentiation to a neuronal phenotype and extensive neurite outgrowth concurrent with a large increase in TG activity [23]. When SH-SY5Y neuroblastoma cells that stably overexpress TG2, but not the inactive mutant C277S TG2, are maintained in low (5%) serum media they spontaneously differentiate into a neuronal phenotype with extensive neurite outgrowth [20]. Treatment of naïve SH-SY5Y cells with retinoic acid leads to a pronounced elevation in TG2 expression [20, 24] and the development of a neuronal morphology [25]. However, in SH-SY5Y cells overexpression of an anti-sense TG2 construct that suppresses the expression of endogenous TG2 results in cells that are unresponsive to retinoic acid [20]. These findings and the report that treatment of SH-SY5Y cells with the TG inhibitor, monodansylcadaverine, inhibited retinoic acid-induced differentiation [26], provides evidence that TG2 is necessary for neuronal differentiation in this cell line. In primary rat cerebellar granule cells TG2 is externalized, likely promotes neurite outgrowth by stabilizing neurites during the initial outgrowth period and also contributes to

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axon collateral branching [27, 28]. Thus TGs, and TG2 in particular, likely play an important role in the formation and stabilization of connections between neurons in the nervous system. One mechanism by which TG2 may contribute to neuronal differentiation processes [20, 28] is by facilitating adenylyl cyclase activation resulting in enhanced cAMP production and subsequent cAMP-responsive-element-binding protein (CREB) phosphorylation. In SH-SY5Y cells stable overexpression of TG2 results in a robust enhancement of adenylyl cyclase activation and a significant increase in CREB-driven transcriptional activity, compared to vector or inactive mutant C277S TG2-transfected cells [21]. These findings are significant given the fact that the cAMP-CREB pathway has been shown to play a critical role in neuronal differentiation process [29–32]. Indeed, increasing cAMP levels has been shown to induce neuronal differentiation in several cell models [29, 30, 33]. Further, a critical role for CREB in neuronal differentiation has been clearly illustrated by the fact that overexpression of CREB in SK-N-BE cells leads to morphological alterations typical of neuronal differentiation [31]. By contrast, overexpression of a dominant negative CREB construct results in a poorly differentiated phenotype [31]. It has also been shown that in CREB-null mice axonal growth is severely compromised [34, 35]. Thus it is readily evident that any processes that result in sustained CREB activation are likely to contribute significantly to the neuronal differentiation process. Further studies are required to identify the mechanisms by which TG2 enhances adenylyl cyclase activity, however this likely is a mechanism by which TG2 contributes to neuronal differentiation.

TGs in Neuronal Apoptosis

The ability to regulate apoptosis has been the most studied role for TG2 within the cell. Early studies showed that in human neuroblastoma SK-N-BE(2) cells, induction of apoptosis by retinoic acid coincided with increased TG2 expression [36]. Furthermore, human neuroblastoma SK-N-BE(2) cells that overexpress TG2 show an increased apoptotic cell death [37]. TG2 also sensitizes other neuroblastoma cells (e.g. SH-SY5Y cells) [38], as well as nonneuronal cells (e.g. Balb-C 3T3 fibroblasts [39] or human embryonic kidney 293 cells [40]) to specific apoptotic stressors. Moreover, inhibition of TG2 expression with antisense TG2 constructs greatly attenuates apoptosis in SH-SY5Y [38], SK-N-BE(2) [37] and human promonocytic U937 cells [41]. Although these findings indicate that TG2 is a facilitator of apoptosis, this is not always the case. For example, induction of TG2 expression either by retinoic acid treatment [20, 24] or stable transfection with TG2 cDNA

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constructs [20] does not result in any changes in the rate of spontaneous apoptosis in SH-SY5Y cells. Treatment of normal SVC1 or ras-transformed Ki-SVC1 epithelial cells with retinoic acid or sodium butyrate results in elevated TG2 expression, growth arrest and differentiation without apoptosis [42]. It has also been demonstrated that retinoic acid-induced increases in TG2 expression in human leukemia HL60 and fibroblast NIH3T3 cells are associated with a suppression of N-(4-hydroxyphenyl) retinamide-induced apoptosis [43]. In vivo, TG2 knockout mice appear phenotypically normal and display no apparent deficits in the major apoptotic pathways [44, 45]. However, in one TG2 knockout mouse model there is a significant decrease in the viability of thymocytes after treatment with dexamethasone [45]. Taken together these results demonstrate that TG2 is not a necessary component of apoptotic pathways, and that depending on the context it can either facilitate or attenuate apoptosis. Given the duplicity of TG2’s effect on apoptosis, perhaps TG2 may be described as a ‘a cellular sentinel’ because depending on the context, cellular location, stimuli and which of TG2’s activities are increased TG2 can either increase or decrease apoptosis. In support of this hypothesis, TG2 facilitates apoptosis in response to stressors that also cause an increase in TG activity. However, when the stressors do not result in an increase in TG activity, TG2 strongly ameliorates the apoptotic response in the same neuronal-like cell model [38]. This role for TG2 in mediating apoptosis also is dependent on cellular localization in addition to TG activity. Active TG2 in the cytosol facilitates thapsigargin-induced apoptosis, while inactive mutant C277S TG2 in the nucleus attenuates thapsigargin-induced apoptosis. If TG2 is targeted to the membrane it neither facilitates nor attenuates apoptosis [40]. Although the mechanisms by which TG2 elicits its effects on apoptosis remain to be identified, the interaction of TG2 with retinoblastoma protein (Rb) in the nuclear compartment may play a role in TG2’s antiapoptotic function [40, 46], given that Rb is a key player in the regulation of apoptosis [47]. However, further studies are required to determine how TG2 may affect Rb function. Overall, these findings clearly demonstrate that the subcellular compartment, as well as the activity level of TG2 plays a role in determining whether it will increase or decrease apoptotic cell death.

TGs in Neurodegeneration

The most extensively studied role for TGs in the nervous system is their proposed involvement in neurodegenerative processes (table 1). TGs were first postulated to play a role in the pathogenesis of HD and AD because in vitro they can cross-link huntingtin [48, 49] and tau [50, 51], which are aggregated

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Table 1. Transglutaminase activity and expression in neurodegenerative conditions Neurodegenerative condition

Transglutaminase expression

Transglutaminase activity

Huntington’s disease

↑ TG2 protein in brain [74]

Alzheimer’s disease

↑ TG1 and TG2 mRNA and protein in brain [3, 87, 88] ↑ TG2 mRNA for short-splice variant in brain [12, 88] ↑ TG2 protein in CSF [90] ↑ TG2 mRNA and protein in brain [12, 108] ←→ TG2 protein in nmd mouse spinal cord [117]

↑ in brain [49, 74] ↑ in lymphocytes [73] ↑ in R6/2 HD mouse brain [75, 76] ↑ in brain [3, 87, 88]

Parkinson’s disease Amyotrophic lateral sclerosis

Spinal cord injury

↑ TG2 mRNA and protein [15] ↑ TG2 mRNA and protein for short-spice variant [15]

Forebrain injury

↑ TG2 mRNA and protein following traumatic brain injury [16] ↑ TG2 mRNA and protein for short-splice variant following traumatic brain injury [16] ↑ TG2 mRNA and protein following global ischemia [17, 120] ↑ TG2 mRNA for short-spice variant following global ischemia [17] ↑ TG1 mRNA following global ischemia [17, 120]

↑ in brain [12] ↓ in spinal cord [115] ↓ in CSF [116] ↑ in nmd mouse brain and spinal cord [117, 118] ↑ in injured vagus nerve [119] ↑ in ischemic spinal cord [115] ↑ following global ischemia [120]

in HD and AD, respectively. However, subsequent studies have provided evidence that cross-linking of these proteins in vivo does not contribute to the formation of aggregates in each disease [4, 52–54]. Indeed in HD mouse models TG2 appears to inhibit the formation of mutant huntingtin protein aggregates [4, 52]. Nonetheless great interest in the role of TGs in neurodegeneration remains because TG enzymatic activity, and for most cases TG2 protein, are increased in many neurodegenerative conditions. Moreover, TG2 has been

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demonstrated to contribute to the disease process in mouse models of HD [4, 52]. The inhibition of TG enzymatic activity therefore may prove advantageous to ameliorate or delay the onset of disease for numerous pathologies of the nervous system. Huntington’s Disease The strongest connections between TGs and neurodegeneration are those for HD. HD is a progressive, inherited neurodegenerative disorder that inevitably leads to the death of affected individuals. HD is characterized by a specific pattern of neuronal dysfunction and degeneration, predominantly in the striatum and at later stages of the disease in the cerebral cortex [55]. The gene that causes HD encodes for a protein of unknown function called huntingtin [56]. Mutation of the gene results in a pathological expansion of CAG repeats leading to an abnormally expanded stretch of glutamine residues near the N-terminal of the huntingtin protein [57–61]. In the nonaffected population this CAG domain ranges from 6 to 36 repeats, whereas subjects with more than 39 CAG repeats will develop the disease. HD is one of 9 identified neurodegenerative diseases that are caused by a polyglutamine expansion mutation in otherwise unrelated proteins [62]. One common neuropathological hallmark of HD and each of the other polyglutamine expansion diseases is the presence of insoluble intraneuronal aggregates that contain a ubiquitylated form of the disease-specific mutated protein [60, 63, 64]. It has been postulated that these huntingtin-containing protein aggregates in HD play a toxic role [65–68], although there also is data to suggest that they may be uninvolved in the etiology of HD or even beneficial to neurons [69–72]. Despite the knowledge of the genetic cause of HD, the mechanisms by which mutant huntingtin produces neurodegeneration remain elusive. The first link between TGs and HD was made when it was demonstrated that TG enzymatic activity is increased in HD lymphocytes [73]. TG enzymatic activity and TG2 protein content are upregulated within neurons in affected areas of HD brain [49, 74]. This upregulation of TG activity in HD occurs in a manner that is dependent on the stage of disease progression, indicating that TGs may be involved in the disease process [74]. TG activity also was found to be upregulated in the brain of R6/2 HD mice in whole cell lysates in one study [75] but only in the nuclear fraction in another study [76]. TGs were originally postulated to contribute to the HD disease process by mediating the formation of huntingtin protein aggregates. TG2 can cross-link polyglutamine domains [77] and polyglutamine-mutant huntingtin protein [48, 49] in vitro, suggesting that it can facilitate the formation of huntingtin protein aggregates in HD brain. This postulate is supported by the subsequent finding that the TG-cross-linked product ␥-glutamyl-␧-lysine (GGEL) is

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increased in HD cerebrospinal fluid [78]. Although these findings clearly indicate that TGs are more active in HD brain, resulting in increased protein cross-linking, there is now considerable evidence to suggest that TG2 does not contribute to the formation of huntingtin protein aggregates in vivo. In a cell culture model TG2 did not contribute to the formation of mutant huntingtin aggregates and TG2 protein actually was excluded from aggregates that did form [79]. Polyglutamine tracts can self-aggregate in vitro in the absence of TGs through the formation of antiparallel ␤-sheets (called ‘polar zippers’) that are stabilized by hydrogen bonds between glutamine main-chain amides and sidechain amides [80]. In addition, mutant huntingtin self-aggregation may be driven by the relative hydrophobicity of the polyglutamine stretch, regardless of the conformation of this sequence [81]. Although TG2 catalyzes the formation of intermolecular cross-links between proteins containing expanded polyglutamine tracts, this modification actually results in the formation of high molecular weight, soluble oligomers. Moreover, this TG2-catalyzed modification actually decreases the propensity for these oligomers to form mature, insoluble protein aggregates, most likely by disrupting the formation of polar zippers and by altering the hydrophobicity of the polyglutamine stretch [53]. Thus, these combined findings suggest that TG2 not only fails to contribute to the formation of insoluble huntingtin aggregates, but it likely acts to inhibit their formation. This postulate has been confirmed in vivo in two independent studies. Genetic deletion of TG2 in two mouse models of HD resulted in increased aggregate formation within the striatum and cerebral cortex [4, 52]. Since there is evidence to suggest that it is the soluble form of mutant huntingtin protein and not the formation of insoluble aggregates that is pathogenic in HD, the inhibition of aggregate formation by TG2 could accelerate disease progression. It is now clear that TG2 contributes to pathogenesis in HD through mechanisms other than aggregate formation. In cell culture models, TG2 polyaminates proteins that selectively interact with truncated mutant huntingtin, but not truncated wildtype huntingtin, and thus has the potential to alter the function of these unknown substrates [82]. TG2 also potentiates inhibition of the ubiquitin-proteasome system in cell culture that is caused by polyglutaminemutant androgen receptor (which is responsible for the polyglutamine disease spinobulbar muscular atrophy) in an aggregate-independent manner [83]. In the R6/2 HD mouse model genetic deletion of TG2 delays the onset of motor dysfunction and prolongs lifespan in an aggregate-independent manner [52]. Genetic deletion of TG2 in the R6/1 HD mouse model also has been demonstrated to prolong lifespan and ameliorate both brain and body weight loss [4]. Further, administration of the TG inhibitor cystamine delays the onset of pathogenesis in the R6/2 HD mouse [75, 76], although these results should be regarded with caution because cystamine also acts as an inhibitor of caspases and leads to

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increased glutathione production [84], which could provide beneficial effects in R6/2 HD mice. Nonetheless, these combined data demonstrate that TG2 is upregulated in HD and contributes to the disease process. Inhibition of TG activity may prove beneficial as part of a therapeutic strategy to delay or prevent the onset of HD. Alzheimer’s Disease AD is the most common cause of dementia in the elderly and is characterized by progressive neuronal degeneration that results in impaired memory and cognitive function [85, 86]. The neuropathology of AD is also characterized by the formation of intraneuronal neurofibrillary tangles (NFTs) that are composed of hyperphosphorylated tau protein in a paired-helical filament conformation, and of extracellular senile plaques that are composed mainly of the amyloid ␤-protein (A␤). TG activity and TG2 protein are increased in AD brain. Johnson et al. [87] first reported that both TG activity and TG2 protein are selectively upregulated in AD prefrontal cortex compared with age- and postmortem interval-matched controls. These findings have been confirmed in subsequent investigations [3, 88]. TG1 protein has been reported to be increased in AD cerebellum, although this brain region is devoid of significant pathology in AD [3]. TG2 mRNA is upregulated in AD cerebral cortex [12, 88], which is accompanied by the appearance of a short-splice variant of TG2 mRNA that encodes for TG2 lacking the GTP binding domain [12]. It has been postulated that this short-splice variant of TG2 may exhibit increased TG activity in AD brain resulting from decreased GTP inhibition of the TG reaction. However, the TG activity of this TG2 isoform has not been determined and in an analogous rat TG2 splice variant, TG activity is lower than the full length protein [89]. Both TG2 protein content [90] and the level of the TG-cross-linked product GGEL [91] are increased in AD cerebrospinal fluid. Interestingly, TG activity, in addition to TG1 protein and mRNA for the short-splice variant of TG2 are increased in the globus pallidus of patients diagnosed with the tauopathy progressive supranuclear palsy [14]. The first studies examining a link between TGs and the formation of NFTs in AD were in 1982. In these early studies human brain cytosolic fractions that contained endogenous TG activity caused the polymerization of human brain neurofilament fractions (containing neurofilament polypeptides, tubulin and actin) in vitro to create insoluble aggregates [92]. A later study also demonstrated that neurofilament and microtubule proteins are substrates for TG cross-linking in vitro [93]. Although the insoluble aggregates formed by TG cross-linking in these studies were not similar in structure to paired helical filament’s observed in AD brain, this was the first evidence that TGs are capable of cross-linking cytoskeletal proteins. Later it was found that TG2 can cross-link tau protein

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in vitro to form filamentous polymers [50, 94]. Although it is clear that tau is a substrate of TGs in vitro [51, 95] and likely in situ as well [96], there has not been a direct demonstration that TGs are involved in the formation of NFTs in vivo. The identification of TG2 protein in AD cerebrospinal fluid does not correlate with the presence of tau protein within the same subjects [90]. Also, although the amount of GGEL-containing insoluble material is increased in AD frontal cortex and hippocampus, the composition of such material is identical between AD patients and age-matched controls [54]. Moreover, although these aggregates contain ubiquitin, ␣-synuclein, parkin and heat-shock protein 27, no tau was present, suggesting that TG2-mediated cross-linking of tau may not be related to NFT formation in AD brain [54]. Because TG2 can be externalized [97, 98], the possibility that TGs may cross-link A␤ and contribute to senile plaque formation also has been investigated. Although TG2 can cross-link both a short version [99] and full-length versions [100–102] of A␤ there is no evidence for TG-catalyzed cross-linking of A␤ in situ or in vivo. Indeed the GGEL-containing insoluble material in AD brain does not contain A␤ [54]. Parkinson’s Disease PD is the second most common neurodegenerative disease after AD, and affects nearly 1% of the population over the age of 65 years [103, 104]. The onset of PD is predominantly sporadic although the mutations of 4 genes have been linked to rare genetic forms of PD [105–107]. The neuropathology of PD involves the degeneration of dopaminergic neurons within the substantia nigra pars compacta, which is accompanied by the formation of cytoplasmic protein aggregates known as Lewy bodies that contain ␣-synuclein. As with aggregates in HD and AD, neither the mechanism of Lewy body formation nor their role in the PD disease process has been elucidated. TG2 is upregulated in PD substantia nigra. TG2 mRNA is increased in PD substantia nigra compared with age-matched controls. Increased immunohistochemical staining for TG2 is observed in PD substantia nigra neurons and immunoblot analysis results in a trend towards increased TG2 protein content in the cytosolic fraction of PD substantia nigra [12]. There also is a trend towards increased TG activity in these same PD cytosolic fractions [12]. Results from a more recent study show that TG2 protein is increased in whole tissue lysates taken from PD substantia nigra when compared with age-matched controls [108]. TG activity has been implicated in the formation of Lewy bodies. The nonamyloid component is a fragment of ␣-synuclein that is found within Lewy bodies and is an excellent in vitro substrate for TG2 [109]. TG2 also cross-links ␣-synuclein in vitro and in COS-7 cells [13]. Moreover, this role for TG2 is dependent on its TG activity, as both wild-type TG2 in the

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presence of the TG inhibitor cystamine, and inactive mutant C277S TG2 fail to aggregate ␣-synuclein in COS-7 cells [13]. TG2 coimmunoprecipitates with ␣-synuclein when both are expressed in COS-7 cells and TG2 colocalizes with ␣-synuclein protein aggregates in human embryonic kidney 293T cells [13]. A slightly different story emerges when examining the role of TG2 in PD brain. TG2 and ␣-synuclein coimmunoprecipitate from PD substantia nigra tissue, but not from control tissue. Interestingly, although TG2 and ␣-synuclein colocalize in the cytosol of PD substantia nigra neurons, TG2 protein is excluded from Lewy bodies in vivo [108]. These data suggest that, as is the case for mutant huntingtin in HD and for tau protein in AD, TG2 is able to modify ␣-synuclein, but may not be responsible for the formation of Lewy bodies in vivo. It should be noted that there is co-localization of staining with antibodies against GGEL cross-links and ␣-synuclein at the level of the Lewy bodies in PD substantia nigra neurons [13, 108]. However, these studies need to be interpreted with caution due the finding that the antibodies against GGEL cross-links may not be specific for the TG-catalyzed reaction [110]. Further investigations are required to examine the possible role of TGs in the formation of Lewy bodies in PD. Amyotrophic Lateral Sclerosis Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease [111]. As with AD and PD, the incidence of ALS increases with age and the majority of cases of ALS are sporadic in origin. However, 5–10% of ALS cases have been linked to a genetic component, with the majority of these cases involving mutations in the gene encoding for copper-zinc superoxide dismutase (CuZnSOD). The neuropathology of ALS involves neurodegeneration of selective motor neurons including supraspinal motor pathways and their frontotemporal neurons of origin, in addition to brainstem and spinal neurons. Also observed in ALS is the formation of protein aggregates within these populations of susceptible neurons that contain CuZnSOD, neurofilament and ubiquitin [112–114]. The study of TGs in ALS thus far has been strictly observational. In both thoracic and lumbar spinal cord, TG activity is decreased to 47–69% that of age-matched non-ALS patients [115]. TG activity in the cerebrospinal fluid of ALS patients is slightly lower than that for non-ALS patients, although two ALS patients who were near the beginning of the disease process showed extremely elevated TG activity measurements, suggesting that there may be an early rise in TG activity followed by a decrease during progression of the disease [116]. TG activity and TG2 protein content also have been measured in the nmd mouse. This mouse has a spontaneous mutation resulting in an ALSlike phenotype including the formation of inclusion bodies. Motor impairment is observed in the first few weeks after birth with neuropathology evident by 6 weeks of age. TG activity was found to be increased throughout the brain

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(except for the cerebellum) of the nmd mouse at 8 weeks of age, although this increase was most pronounced within the spinal cord [117]. Subsequent analysis has shown that this increase in TG activity in nmd mouse spinal cord is evident as early as 3 weeks of age and peaks at 5 weeks of age [118]. The ability of TGs to polyaminate endogenous substrates within the spinal cord also is increased in the nmd mouse, although protein content for TG2 is not altered. This discrepancy between decreased TG activity in ALS patients and increased activity in the nmd mouse may reflect the different etiologies underlying pathogenesis. However, the potential increase in TG activity observed in cerebrospinal fluid of two ALS patients who were near the beginning of the disease progression [116] combined with the increased TG activity in the nmd mouse suggests that TGs may be involved with the early pathogenesis of ALS.

Injury of the Nervous System

Several groups have examined the effects of acute injuries to the nervous system on TG activity and TG2 expression. Within the peripheral nervous system crushing of the rat vagus nerve results in increased TG activity within one hour near the site of injury but not in a region more proximal to the nodose ganglion [119]. However, TG activity was increased within the nodose ganglion 48 h after injury [119]. Moderate contusion of the rat spinal cord results in a modest increase in TG2 mRNA content 8 h postinjury and a larger (approximately 3.5 times) increase 72 h postinjury [15]. This increase in TG2 mRNA is accompanied by an increase at both the mRNA and protein level of a shorttranscript variant of TG2 that is expressed in cultured astrocytes exposed to TNF-␣ and IL-1␤ [15, 89]. In addition to traumatic injury, ischemia of the spinal cord by occlusion of the dorsal spinal artery branch results in increased TG activity within the spinal cord that peaks at one day postsurgery [115]. This activity declines slowly over the first month postsurgery but remains greater than the nonaffected side of the spinal cord at 30 days postsurgery [115]. Injury to the forebrain also results in upregulation of TG2. Traumatic impact injury to the rat cerebral cortex results in increased TG2 protein expression compared with sham controls that peaks on the 5th day postinjury [16]. This upregulation of TG2 is accompanied by the expression of the same shorttranscript variant of TG2 that is observed following spinal cord injury described above. Protein content for full length TG2 also is increased in the ipsilateral hippocampus after traumatic impact injury, but to a lower degree than that observed for the cerebral cortex. mRNA content for both the full length and short variants of TG2 increase and peak at 3 days postinjury in the affected cerebral cortex and to a much lower extent in the ipsilateral hippocampus [16].

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Cytosol Nucleus

Physiological stimuli

Pathological cellular stress

TG2 Antiapoptotic Proapoptotic Cellular signaling

⫺ Aggregated protein

TG2 Soluble oligomers of disease-related protein Proteasome inhibition

Neurite outgrowth Cell death

Fig. 1. Depending on the context, TG2 mediates both physiological (dashed lines) and pathological (solid lines) events within neurons. TG2 localized to the nucleus can attenuate apoptosis [40]. TG2 is required for neurite outgrowth in the developing nervous system [20] and likely mediates intracellular signaling in response to physiological stimuli [7, 9]. In contrast, TG2 contributes to cell death during neurodegeneration. TG2 in the cytosol facilitates apoptosis in response to specific stimuli [38]. In models of polyglutamine disease (e.g. HD) TG2 protein content is increased [49, 74] and TG2 contributes to the disease process [4]. TG2 inhibits the formation of insoluble protein aggregates [4, 53] and increases the levels of soluble oligomers of the disease-related protein [53], which in turn mediate cellular pathology [57–61]. In addition TG2 may act together with the disease-related protein to cause cell death, as is the case for inhibition of the proteasome [83].

Two models of cerebral ischemia also resulted in increased TG2 expression. In the gerbil, occlusion of the common carotid arteries for 3 min results in increased TG activity in the hippocampus, and to a much lesser extent in the cerebral cortex, that peaks at 24 h postsurgery and returns to baseline within 48 h [120]. This study found that mRNA for TG1 and TG2, and protein content for TG2, are increased in the hippocampus 24 h postsurgery. Middle cerebral artery occlusion in the rat for one hour results in increased TG2 protein content in the cerebral cortex and hippocampus at days 3, 5 and 7 postsurgery [17]. mRNA content for both the full length TG2 and the shorter splice variant are increased in both the cerebral cortex and hippocampus in response to ischemia [17]. While it is clear that TG2 expression is increased following injury of the

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nervous system, the mechanisms responsible for this increase remain to be determined. Moreover, the precise effects of TG2 must be determined under these pathological conditions before pharmacological manipulation of TG activity is attempted to ameliorate associated pathology.

Conclusions

TGs likely play many roles throughout the nervous system (fig. 1). TG2 plays an important part in mediating the formation and survival of neural circuits within the developing nervous system. However, depending on the situation it is clear that TG2 also is capable of facilitating the neuronal death. Further, cellular studies suggest that this switch in TG function may be dependent on the specific stimulus and on the resulting subcellular localization of TG2 protein. A large body of research now demonstrates that TG activity and TG2 protein content are increased in the brain in many neurodegenerative conditions (table 1), suggesting that TGs may play a role in disease processes. The pharmacological inhibition of TG activity therefore may prove beneficial as part of a therapeutic strategy to delay disease progression. However, it should be noted that a direct role for TGs in contributing to pathogenesis has only been demonstrated for HD. Therefore the role of TGs within specific neurodegenerative processes (i.e. detrimental vs. beneficial) needs to be determined before they are targeted as part of a therapeutic treatment.

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Dr. G.V.W. Johnson Department of Psychiatry University of Alabama at Birmingham School of Medicine 1720, 7th Avenue South, SC 1061 Birmingham, AL 35294–0017 (USA) Tel. ⫹1 205 934 2465, Fax. ⫹1 205 934 2500, E-Mail [email protected]

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Tissue Transglutaminase and Celiac Disease Carla Espositoa, Ivana Caputoa, Salvatore Auricchiob, Riccardo Tronconeb a

Department of Chemistry, University of Salerno, Salerno, and Department of Pediatrics & European Laboratory for the Investigation of Food-Induced Diseases, University Federico II, Naples, Italy

b

General Concepts on Celiac Disease

Celiac disease (CD), or gluten-sensitive enteropathy, is a multifactorial permanent disease affecting approximately 1 out of 100 individuals in Europe and United States [1]. CD is triggered by the ingestion of gliadin (a component of wheat gluten) and of related prolamines of cereal proteins, as secalins in rye and hordeins in barley [2]. Rice and maize are nontoxic, while the toxicity of oats is still debated [3, 4]. CD is characterized by a dysregulated immune response at the gut level involving both innate and adaptive immune responses [5]; the latest is dominated by T cells of the Th1 type [2]. This abnormal mucosal immune response results in the enteropathy of the upper small intestine characterized by flattening of absorptive villi, crypt hyperplasia, and marked inflammatory cell infiltrate. The patients may have a wide spectrum of symptoms ranging from malabsorption with steatorrhea, fatigue, anemia and weight loss to extraintestinal involvement, such as neurological dysfunctions, to complete absence of symptoms. Until now, the only effective therapy for CD is a strict and lifelong diet excluding toxic grains. Nutritional deficiencies, osteoporosis, malignant diseases and secondary autoimmune diseases represent the predominant risks that CD patients not compliant with gluten-free diet are exposed to. There is a genetic predisposition to the disease; 95% of celiac patients express an almost identical histocompatibility leukocyte antigen (HLA)-DQ heterodimer. The alpha chain is coded by the DQA1*0501 allele, the beta chain by the DQB1*02. Although many patients present this heterodimer, around 5% of them do not express it. In the latter the

disease is associated with a different heterodimer coded by the genes DQA1*0301 and DQB1*0302, coding for a DQ8 molecule [6, 7]. HLA class II antigens bind into their peptide-binding groove, gluten fragments that are in this way presented to specific CD4⫹ T cells eventually leading to an inflammatory response. The central role of dietary grains in the initiation and maintenance of the celiac lesion is supported by the isolation of CD4⫹ memory T cells from the intestinal mucosa of CD patients, but not of nonceliac controls. Active CD is accompanied by the presence of serum antibodies to gluten, but also to self-components. In fact, a highly specific autoimmune response was observed against endomysium [8], a connective structure present in the extracellular matrix of intestinal mucosa, and serum titers of such autoantibodies fluctuate with exposure to dietary toxic grains. The demonstration of autoimmune phenomena in CD and the strict association with other autoimmune diseases have favored the inclusion of such pathology in the number of autoimmune diseases.

The Discovery that Tissue Transglutaminase is the Main Autoantigen in CD: New Tool for CD Diagnosis

The work of Dieterich et al. [9] in 1997 revealed the identity of endomysial autoantigens. Immunoprecipitation of proteins produced by metabolically labeled fibrosarcoma cells with IgA from CD patients allowed the identification of tissue transglutaminase (tTG) as the main if not sole autoantigen in CD. Such discovery has had a large diagnostic implication [10]; serological tests based on the research of anti-tTG antibodies have acquired a strong importance, in particular for the possibility to realize valid tools for a wide noninvasive mass screening. The great sensitivity and specificity of anti-tTG antibodies allows to recognize subjects with positive serum endomysial antibodies presenting normal histology which have a high chance to develop enteropathy in the following years [11, 12]. Recently, after first generation tests based on the use of guinea pig antigen, the most recent assays, based on the use of recombinant human enzyme as coating antigen in ELISA, have further improved the diagnostic efficacy allowing to discriminate very efficiently CD patients [13, 14]. We can expect further improvement by the definition of the epitopes of tTG recognized by celiac sera. In this respect, N terminal and central regions of tTG have been recently identified as the predominant target of immunoglobulins from CD patients [15, 16]. However two requirements are still necessary to make a certain diagnosis of the pathology: the finding of villous atrophy and crypt hyperplasia observed on a duodenal biopsy and a full clinical remission after a gluten-free diet [17, 18].

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tTG is Upregulated and Catalitically Active in CD Intestinal Mucosa

tTG, a member of the vast TG enzyme family, prevalently catalyzes the Ca2⫹-dependent formation of isopeptide linkages between the ␥-carboxamide group of protein-bound glutamine residue and the ␧-amino group of proteinbound lysine residue. When a suitable amine is absent the glutamine residue can be deamidated to glutamic acid [19]. Besides cross-linking activity, additional functions, such as GTP-hydrolyzing activity [20], disulphide isomerase activity [21] and kinase activity [22], have been recently attributed to tTG. The intriguing properties of this enzyme are relative also to its cellular and tissue distribution [23–25]. tTG is prevalently an intracellular enzyme mainly localized in the cytosol and, in some cell types, such as neuroblastoma cells, it is also found in the nuclear compartment [26]. Finally, tTG can be released through a still unclear mechanism by cells into extracellular space where it cross-links proteins of the extracellular matrix. Moreover, as a outside membrane-bound protein, tTG mediates the interaction of integrins with fibronectin in a catalytic independent manner. Through this interaction it seems to modulate cell adhesion and cell motility [27, 28]. Several observations have been published on the immunohistochemical localization of tTG in normal and CD patients [29–31]. In normal subjects, the enzyme is mainly expressed in the subepithelial region of the lamina propria and in the muscularis mucosae, while only a little amount is located within the epithelium (fig. 1a). In untreated celiac patients not only an increased expression of tTG proved by a slight but significant increase of tTG mRNA was observed, but also a different distribution pattern, as compared to treated patients and controls [31]. In fact, tTG is heavily expressed in the lamina propria, in particular in the subepithelial region just below the basal membrane. tTG appears concentrated in the extracellular matrix, as well as in the enterocytes, both at brush border and cytoplasm levels (fig. 1b). However, the source of the increased tTG levels is not clear since endothelial, fibroblast, macrophages, mononuclear cells as well as enterocytes may be the possible sources of the enzyme. It is known that there exists a complex but tight tTG gene regulation that is influenced by several factors including retinoids, transforming growth factor-␤ (TGF-␤), interleukin-6 [23, 32–34]. Elevations in interferon ␥, and to a less extent in interleukin-2, -4, -6 and tumor necrosis factor have been reported in duodenal biopsies from CD patients cultured in the presence of gliadin [35], thus suggesting that the increased expression of tTG observed in the small intestine of celiac patients could be due to the stimulation by proinflammatory cytokines.

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b

a Fig. 1. Immunohistochemical localization of tTG protein in duodenal sections from a control patient (a) and from a CD patient (b). Magnification 400⫻. Photos are taken from Esposito et al. [31] and reproduced with the permission of the Blackwell Publishing, Oxford.

A strict correlation between the pattern of tTG protein expression and in situ tTG activity was reported [31]. In the intestinal mucosa of CD patients there is an evident increase of the enzymatic activity measured in situ by using a biotinylated gliadin-derived peptide as probe for endogenous enzymatic activity. Signals were concentrated extracellularly in specific areas of the mucosa, particularly in the subepithelial region (fig. 2). These results were recently confirmed by analyses by confocal microscopy showing that tTG colocalizes with gliadin at the epithelial and subepithelial levels in active CD [36]. The presence of tTG within the extracellular compartment in bioptic fragments from CD patients compared to controls suggests its translocation from the intracellular to the pericellular environment. Since the consequence of the externalization is an increase in cross-link products in the extracellular space, it has been suggested that changes in both the expression and the location of tTG is part of a cellular stress response [37, 38]. Moreover, it has been demonstrated that in enterocytes undergoing apoptosis in the earlier stage of disease there is an increase of tTG expression. Such finding has allowed to hypothesize that the accumulation of active tTG in the enterocyte as well as its release in the extracellular matrix could be a consequence of the induction of apoptosis in the intestinal mucosa regions where gliadin-induced damage is more severe [30].

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b

a

c Fig. 2. In situ TG activity in duodenal section from a CD patient visualized by using biotinylated 31–43 A-gliadin peptide as probe (b, green). Superimposition of tTG (a, red) with activity shows a significant overlap of the two staining patterns (c, yellow). Photos are taken from Esposito et al. [31] and reproduced with the permission of the Blackwell Publishing, Oxford.

tTG Deamidating Activity Can Trigger Immune Response to Gliadin

The HLA DQ2 and DQ8 peptide-binding groove displays a preference for negatively charged residues at several positions [39]. Gliadin, that is characterized by a high content of glutamine (approximately 35 mol%) other than proline (15 mol%), presents a very low amount of negative charges; however these can be introduced by treatment at high temperature and low pH [40]. Under these conditions a nonspecific deamidation of glutamine residues to negatively charged glutamic acid occurs generating better binding of gliadin peptides to DQ2 and DQ8. Since it was well known that gliadin is a preferred acyl donor substrate of tTG in vitro [41, 42], it has been hypothesized that tTG might be responsible for the deamidation of specific glutamine residues in gliadin peptides prior to their recognition by gluten-sensitive T cells (fig. 3). As a consequence, gluten peptides

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Gluten protein Q QQ

Q QQ

Q Q Q

Q

Digestive enzymes Q QQ Q

tTG deamidation of specific Q

QQ Gluten peptides

E QQ E QE

Processing & presentation by mucosal APC of deamidated peptides

APC DQ2 E

CD4⫹

TCR T cell

Activation of DQ2-restricted T cells Generation of a Th1 response

Fig. 3. Schematic representation of tTG role in the induction of a Th1 response through deamidation of specific Q in gluten peptides. APC ⫽ Antigen presenting cell.

bind with an increased affinity to the HLA-DQ2 and HLA-DQ8 molecules and the activation of these normally silent CD4⫹ T-lymphocytes triggers a T-helper type 1 pattern of cytokine production leading to mucosal damage [2]. It is interesting to underline that while the nonenzymatic deamidation produced negatively charged residues randomly distributed in gliadin peptides, tTG selective deamidation involves only a few specific glutamines that are of primary importance to enhance the immunogenicity of gliadin peptides [43]. A lot of findings

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(see also next paragraph) support the concept that tTG play a crucial role in triggering CD by selective deamidation of specific glutamine in gluten, but there are only partial evidences that such deamidation occurs also in vivo [44]; in fact, in intestinal mucosa there are abundant lysine-containing proteins and polyamines that render the deamidation reaction unfavorable. A detailed study of pH influence on the transamidation versus deamidation reactions support the hypothesis that at low pH (pH 7.3) deamidation occurs even in the presence of an excess of primary amines [45]. Such reactions could occur both in the extracellular subepithelial region where pH is around 7.3 and where tTG expression is increased in CD patients, and/or at the brush border level where pH is about 6.6 and tTG is also enhanced during the disease. Furthermore, deamidation could be an intracellular phenomenon; in fact, the antigen presenting cells of the intestinal mucosa could internalize tTG together with free gluten peptides by endocytosis and the activity of the enzyme during the initial phase of pH decrease in early endosomes can provoke deamidation of specific glutamines [45]. Finally, it can not been excluded that the deamidation of gluten peptides could be attributed to a bacterial TG, eventually present in the gut lumen, that preferentially deamidates, instead of cross-linking, certain substrates [46].

Substrate Specificity of tTG Towards Gluten Peptides

Several researches for many years were performed to find which peptide in gluten and the other disease-activating dietary grains is the target of tTGmediated deamidation. Answers to questions as how many T cell epitopes are involved, if a hierarchy between them could be delineated, if different groups of patients (for example children and adults) could recognize different gluten peptides as antigen, are greatly relevant not only to further clarify the mechanisms of CD pathogenesis, but also to design active agents for blocking gluteninduced toxicity. Unfortunately, the achievement of this goal was particularly difficult since gliadin as well as glutenin proteins because of their peculiar amino acid composition, which is characterized by a high content of glutamine and proline, are excellent tTG substrate in vitro [41, 42]. So far a variety of peptides derived from gluten have been identified as epitopes for specific mucosal T cell, however only some of them appear to be more important as they are recognized by intestinal DQ2 and DQ8 T cells [47] (table 1). A common feature among these epitopes is the presence of many proline and glutamine residues, which made them particularly resistant to gastrointestinal proteolysis as well as good tTG substrates in which selected glutamine residues are deamidated. In fact, though no consensus sequence around the reactive glutamine residues was derived, the position ⫺1, ⫹1, ⫹2 and ⫹3 relative to glutamine

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Table 1. Gluten epitopes relevant for immune response in celiac disease Origin

Position

Sequencea

tTG deamidationb

Presentation

References

␣2-gliadin ␣2-gliadin ␣9-gliadin ␣-gliadin ␥-gliadin ␥5-gliadin ␥5-gliadin ␥5-gliadin ␥5-gliadin

62–75 67–75 57–68 206–217 141–150 228–236 66–78 102–113 60–79

PQPQLPYPQPQLPY PYPQPQLPY QLQPFPQPQLPY SGQGSFQPSQQN QPQQSFPQQQ IIQPQQPAQ FPQQPQQPYPQQP FSQPQQQFPQPQ LQPQQPFPQQPQ QPYPQQPQ

Required (E65) Required (E72) Required (E65) Required (E208, E216) Required (E148) Required (E232) Required (E68, E71) Required (E106, E108) Not required

DQ2 DQ2 DQ2 DQ8 DQ2 DQ2 DQ2 DQ2 DQ2

[50, 53] [52, 53] [50, 51] [56, 57] [40] [54] [52] [52] [52]

a

tTG targeted glutamines are underlined. Residues transformed in glutamic acid through tTG deamidation are indicated in parenthesis.

b

played a dominating role in the specificity of tTG and the enzyme preferred mostly QxP but not QP or QxxP [45]. According to the designed algorithms based on the spacing between targeted glutamine and neighboring proline, as well as the occurrence of other aminoacidic residues, Vader et al. [48, 49] hypothesized the existence of more than 50 putative activating sequences in gluten as well as in hordeins and secalins, but not in avenins that contain a lower percentage of prolines. In 2000 two independent studies identified two overlapping tTG-modified ␣-gliadin peptides (62–75 in ␣2-gliadin and 57–68 in ␣9-gliadin) both containing a sequence motif, PQPQLPY, as the dominant gliadin T-cell epitopes in adults. Authors reported that tTG deamidation at position Q65 was critical for T cell recognition of both peptides [50, 51]. With the contribution of a later study, three proline and glutamine-rich peptides were described as the main DQ2 restricted T-cell epitopes in ␣-gliadin [52]. Recently, Shan et al. [53] demonstrated that the in vitro digestion of recombinant ␣2-gliadin produced a digestion-resistant 33-mer peptide (residues 57–89) containing all three previously described epitopes. Moreover, the 33-mer product showed the highest specificity toward tTG-deamidation than that reported for any peptide studied thus far, therefore it has been indicated as the primary initiator of the inflammatory response to gluten in CD patients. When the picture of CD-activating peptides was studied in children, some different findings were found. Vader et al. [54] reported that in children with CD there were multiple gliadin or glutenin peptides that could bind to DQ2 or

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DQ8 and activate mucosal T cell populations and that in some cases tTG deamidation was not required for T cells activation. Moreover there was a wide variability in the response of different patients; in fact, many children did not show any reaction to gliadin epitopes that activated T cells in adults. Therefore, the authors concluded that the deamidation of peptides occurred as disease develops, but may not be required for the initial activation of mucosal T cells in the very early stages of disease. The potentiality of high molecular weight glutenins (large polymeric proteins present in gluten) to trigger an intestinal T cell response was also investigated. Recently Molberg et al. [55] demonstrated that high molecular weight glutenin-specific DQ2-restricted T cells were present in intestinal biopsies in 9 of 22 CD patients both adults and children. However T cells were activated only after tTG deamidation of glutenin protein.

tTG Transamidating Activity Seems to be Responsible for Autoimmunity in CD

The most evident expression of autoimmunity in CD is the presence of circulating mucosal (immunoglobulins A) autoantibodies to tTG in patients ingesting gluten [9]. Recently, Marzari et al. [58] demonstrated that it was possible to isolate anti-tTG antibodies from all intestinal lymphocyte libraries, but not from those obtained from peripheral lymphocytes. This is in contrast to antibodies against gliadin, which could be obtained from all libraries, indicating that while the humoral response against tTG occurs at the local level, that against gliadin occurs both peripherally and centrally. The mechanisms leading to autoimmunity are largely unknown; unmasking of cryptic epitopes has been hypothesized in the context of an inflamed environment where antigen processing and presentation may be more efficient. In accordance with the upregulation of tTG in intestinal inflamed sites, tTG may generate additional antigenic epitopes by cross-linking gliadin peptides to itself and/or to other protein substrates [9]. Sollid et al. [59] depicted an intriguing explanation to describe the role of tTG in the humoral autoimmune response in CD. tTG-gliadin complexes, by acting as hapten-carrier complexes, are recognized by B cells that express tTG-specific immunoglobulins on their membrane, are endocytosed and processed. This processing permits both gliadin and tTG to be presented by DQ2 tTG-specific B cells to gliadin-specific T cells which give the necessary help to induce an antitTG antibodies Th2 response [59–61] (fig. 4). Since the existence of tTGspecific T cells in the intestinal mucosa of untreated patients is not proven it is hypothesized that the production of anti-tTG antibodies is driven completely by intestinal gliadin-specific T cells. The observation that anti-tTG antibody titers

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Gluten protein Q Q Q QQ Q Q Q Q QQ

Digestive enzymes QQ QQ

Q Q

tTG transamidation

Gluten peptides

tTG

Q

Cross-linking with enzyme itself (or with tissue proteins)

Processing and presentation by tTG-specific B cells

tTG-gluten complex B cell

Y

Y

Y

Y

Y

Y

Y

Y

T cell

Y

TCR

Y Y Y

Y

CD4

Y

Y

Plasma cell DQ2

Activation of B cell by gluten-specific T cell and generation of a plasma cell secerning anti-tTG IgA (Th2 response)

Fig. 4. Schematic representation of the role of tTG cross-linking in the generation of a Th2 response against autoantigens.

fall and can become undetectable during a gluten-free diet suggests that B cell activity depends on persistent antigen presentation. The model described by Sollid et al. [59] could also explain the presence in CD patients of autoantibodies different from anti-tTG and antigliadin antibodies. The ability of tTG to cross-link gliadin peptides to other protein substrates supports the hypothesis that this event is responsible for the humoral autoimmune response in CD towards lysine donor protein substrates. In an enterocyte-like system, by a proteomic approach, more than 25 endogenous proteins, both lysine-donor and glutamine-donor, that may represent putative substrates of tTG have been identified. These targets include proteins involved in cytoskeletal network organization, as well as in a miscellaneous of metabolic functions [62]. An immune reaction against actin, a known cytoskeleton tTG substrate, was observed in

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both children and adults with CD [63]. Antibodies against calreticulin, desmin, and a 90 kDa dermal glycoprotein have also been described in CD patients [64–66]. Recently, three new autoantigens as ATP-synthase ␤ chain and two variants of enolase – ␣ have been identified by mass fingerprinting approach [67]. Interestingly most of these proteins are in some way correlated with tTG; for example, actin has been indicated as a tTG substrate in cells undergoing apoptosis [68] and both ATP-synthase and enolase are tTG substrates in Caenorhabditis elegans [69]. On the other hand calreticulin is an in vitro modulator of tTG transamidating and GTPase activities [70]. Increased prevalence of concomitant autoimmune diseases, such as collagen diseases, type I diabetes, dermatitis herpetiformis, autoimmune alopecia, have been described in CD patients with prolonged gluten exposure [71]. It is tempting to hypothesize that gliadin peptides can trigger a cascade of events leading to the inappropriate presentation of tTG and cross-linked substrates to the immune system contributing to the immune aspect of such pathologies.

Potential Role of Anti-tTG Antibodies in CD Pathogenesis

Given the strong disease-specific presence of tTG antibodies in CD, it would be very important to address the question if such autoantibodies have a role in the pathogenesis of the mucosal lesion typical of the disease [72, 73]. As far as mechanisms of damage are concerned, CD autoantibodies and the commercial antitTG antibody display a partial and dose-dependent inhibitory effect toward the transamidating activity of human tTG both in vitro and in situ [74, 75]. Therefore, tTG activity could be differently modified in the intestine of celiac patients by autoantibodies thus modulating the course of the disease. A study published in 1999, using a three-dimensional fibroblast – epithelial cell co-culture model mimicking the in vivo crypt-villus axis, provided an indirect evidence of a contribution of tTG autoantibodies to the mucosal transformation observed in celiac patients. In that study it was demonstrated that the TGF-␤-dependent differentiation of T84 crypt epithelial cells can be prevented by the addition of anti-tTG IgA as well as commercial anti-tTG monoclonal antibodies. Authors suggested that blocking tTG prevented the generation of the active form of TGF-␤ which relies on tTG for its maturation, thus affecting the differentiation of the celiac epithelium [76]. More recently, the same authors show that celiac disease-specific autoantibodies targeted against tTG interfere in fibroblast-extracellular matrix interaction by inhibiting fibroblast motility and collagen gel contraction, as well as by increasing type I collagen degradation in vitro. In view of this the authors suggest that celiac disease-specific IgA targeted against tTG decrease the level of matrix stabilization rather than increase matrix degradation per se (Halttunen T,

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pers commun). In fact, is well known that tTG plays a fundamental role in extracellular matrix stabilization [23]. The finding of IgA deposits on extracellular tTG in the liver, lymphnodes and muscles indicates that tTG is accessible to the gut-derived autoantibodies [77]. In celiac patients with IgA deficiency in vivo targeting of jejunal tTG could be mediated by immunoglobulins M [KorponaySzabó IR, unpubl. data]. Therefore, several intestinal and extraintestinal clinical manifestations of CD (e.g. liver, heart, nervous system) are possibly related to the presence of autoantibodies in situ. Furthermore, recent data suggest a function for the tTG autoantibodies, both commercially available and from CD patients, in the regulation of cytoskeleton rearrangement and the modulation of cell cycle in different cell lines [78]. Therefore, these results support the hypotheses that tTG autoantibodies could have an important role per se in the progression and maintenance of the hyperplastic atrophic mucosal lesion typical of CD.

Conclusions

The putative involvement of tTG in the pathogenesis of CD could be due to two distinct but interdependent pathways through gliadin-derived peptides deamidation/cross-linking reactions (fig. 3 and 4). The tTG-mediated deamidation of specific gliadin peptides transforms the ingested gliadin from nonstimulatory to efficient T cell antigens able to evoke a massive secretion of local cytokines, thereby leading to alterations in enterocyte proliferation and differentiation. The tTG-mediated cross-linking between gliadin peptides and the enzyme itself leads to the formation of tTG-gliadin complexes that may contain neoepitopes that trigger mucosal T cells with the production of autoantibodies against tTG and gliadin. Both reactions could be involved in the breakdown of tolerance and precipitation of disease. While tTG seems to plays a central role in the onset of CD, the function of celiac disease-specific anti-tTG antibodies has not been clarified. Recent data suggest an important involvement of these antibodies in the mucosal pathology through inhibition of the tTG catalytic activity. The growing understanding of the immune response to gluten can provide new prospective for future therapeutic treatments. In particular, full characterization of the tTG substrate specificity will allow to project specific inhibitors of tTG catalytic activity, that remains a key target in designing a therapeutic strategy for CD. Up to date a modified gluten peptide has been synthesized and characterized in both biochemical and cell-based assays. This proteolitically resistant peptide seems to be a more potent and specific inhibitor of other molecules identified so far [79]. Further in vivo studies are needed to assess the validity of such approach, but results appear very encouraging on the way of discovery new efficacious tools for CD treatment.

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Kim C-Y, Quarsten H, Bergseng E, Khosla C, Sollid LM: Structural basis for HLA-DQ2mediated presentation of gluten epitopes in celiac disease. Proc Natl Acad Sci USA 2004;101: 4175–4179. Molberg O, McAdam S, Lundin KEA, Kristiansen C, Arentz-Hansen H, Kett K, Sollid LM: T cells from celiac disease lesions recognize gliadin epitopes deamidated in situ by endogenous tissue transglutaminase. Eur J Immunol 2001;31:1317–1323. Fleckenstein B, Molberg O, Qiao SW, Schmid DG, von der Mulbe F, Elgstoen K, Jung G, Sollid LM: Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process. J Biol Chem 2002;277:34106–34116. Schmidt G, Selzer J, Lerm M, Aktories K: The rho-deamidating cytotoxic necrotizing factor 1 from escherichia coli possesses transglutaminase activity: Cysteine 866 and histidine 881 are essential for enzyme activity. J Biol Chem 1998;273:13669–13674. Koning F, Vader W: Gluten peptides and celiac disease. Science 2003;299:513–515. Vader LW, de Ru A, van der Wal Y, Kooy YM, Benckhuijsen W, Mearin ML, Drijfhout JW, van Veelen P, Koning F: Specificity of tissue transglutaminase explains cereal toxicity in celiac disease. J Exp Med 2002;195:643–649. Vader LW, Stepniak DT, Bunnik EM, Kooy YM, de Haan W, Drijfhout JW, Van Veelen PA, Koning F: Characterization of cereal toxicity for celiac disease patients based on protein homology in grains. Gastroenterology 2003;125:1105–1113. Arentz-Hansen H, Korner R, Molberg O, Quarsten H, Vader W, Kooy YMC, Lundin KEA, Koning F, Roepstorff P, Sollid LM, McAdam SN: The intestinal T cell response to alpha-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J Exp Med 2000;191:603–612. Anderson RP, Degano P, Godkin AJ, Jewell DP, Hill AVS: In vivo antigen challenge in celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope. Nat Med 2000;6:337–342. Arentz-Hansen H, McAdam SN, Molberg O, Fleckenstein B, Lundin KEA, Jorgensen TJD, Jung G, Roepstorff P, Sollid LM: Celiac lesion T cells recognize epitopes that cluster in regions of gliadin rich in proline residues. Gastroenterology 2002;123:803–809. Shan L, Molberg O, Parrot I, Hausch F, Filiz F, Gray GM, Sollid LM, Khosla C: Structural basis for gluten intolerance in celiac sprue. Science 2002;297:2275–2279. Vader W, Kooy Y, Van Veelen P, De Ru A, Harris D, Benckhuijsen W, Pena S, Mearin L, Drijthout JW, Koning F: The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides. Gastroenterology 2002;122:1729–1737. Molberg O, Solheim Flaete N, Jensen T, Lundin KEA, Arentz-Hansen H, Anderson OD, Uhlen AK, Sollid LM: Intestinal T-Cell responses to high-molecular-weight glutenins in celiac disease. Gastroenterology 2003;125:337–344. Molberg O, McAdam S, Korner R, Quarsten H, Kristiansen C, Madsen L, Fugger L, Scott H, Noren O, Roepstorff P, Lundin KEA, Sjostrom H, Sollid LM: Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med 1998;4:713–717. Van der Wal Y, Kooy Y, van Veelen P, Pena S, Mearin L, Papadopulos G, Koning F: Cutting edge: Selective deamidation but tissue transglutaminase strongly enhances gliadin-specific T cell reactivity. J Immunol 1998;161:1585–1588. Marzari R, Sblattero D, Florian F, Tongiorgi E, Not T, Tommasini A, Ventura A, Bradbury A: Molecular dissection of the tissue transglutaminase autoantibody response in celiac disease. J Immunol 2001;166:4170–4176. Sollid LM, Molberg O, McAdam S, Lundin KE: Autoantibodies in celiac disease: Tissue transglutaminase-guilt by association? Gut 1997;41:851–852. Schuppan D, Dieterich W, Riecken EO: Exposing gliadin as a tasty food for lymphocytes. Nat Med 1998;3:666–667. Molberg O, McAdam SN, Sollid LM: Role of tissue transglutaminase in celiac disease. J Pediatr Gastroenterol Nutr 2000;30:232–240.

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Orrù S, Caputo I, D’Amato A, Ruoppolo M, Esposito C: Proteomics identification of acylacceptor and acyl-donor substrates for transglutaminase in a human intestinal epithelial cell line. Implications for celiac disease. J Biol Chem 2003;278:31766–31773. Clemente MG, Musu MP, Frau F, Brusco G, Sole G, Corazza GR, De Virgilis S: Immune reaction against the cytoskeleton in coeliac disease. Gut 2000;47:520–526. Maki M: The humoral immune system in celiac disease. Baillieres Clin Gastroenterol 1995;9: 231–249. Krupickova S, Tuckova L, Flegelova Z, Michalak M, Walters JR, Whelan A, Harries J, Vencovsky J, Tlaskalova-Hogenova H: Identification of common epitopes on gliadin, enterocytes, and calreticulin recognised by antigliadin antibodies of patients with coeliac disease. Gut 1999;44:168–173. Teesalu K, Uibo O, Kalkkinen N, Janmey P, Uibo R: Increased levels of IgA antibodies against desmin in children with coeliac disease. Int Arch Allergy Immunol 2001;126:157–166. Stulík J, Hernychová L, Porkertová S, Pozler O, Tuckova L, Sanchez D, Bures J: Identification of new celiac disease autoantigens using proteomic analysis. Proteomics 2003;3:951–956. Nemes Z, Adany R, Balas M, Boross P, Fesus L: Identification of cytoplasmic actin as an abunant glutaminyl substrate for tissue transglutaminase in HL-60 and U937 cells undergoing apoptosis. J Biol Chem 1996;272:20577–20583. Madi A, Kele Z, Janaky T, Punyiczki M, Fesus L: Identification of protein substrates for transglutaminase in Caenorhabditis elegans. Biochem Biophys Res Commun 2001;283:964–968. Feng JF, Readon M, Yadav SP, Im MJ: Calreticulin down-regulates both GTP binding and transglutaminase activities of transglutaminase II. Biochemistry 1999;38:10743–10749. Collin P, Kaukinen K, Valimaki M, Salmi J: Endocrinological disorders and celiac disease. Endocr Rev 2002;23:464–483. Caputo I, D’Amato A, Troncone R, Auricchio S, Esposito C: Translutaminase 2 in celiac disease. Amino Acids 2004;26:381–386. Freitag T, Schulze-Koops H, Niedobitek G, Melino G, Schuppan D: The role of the immune response against tissue transglutaminase in the pathogenesis of coeliac disease. Autoimmun Rev 2004;3:13–20. Esposito C, Paparo F, Caputo I, Rossi M, Maglio M, Sblattero D, Not T, Porta R, Auricchio S, Marzari R, Troncone R: Anti-tissue transglutaminase antibodies from coeliac patients inhibit transglutaminase activity both in vitro and in situ. Gut 2002;51:177–181. Dieterich W, Trapp D, Esslinger B, Leidenberger M, Piper J, Hahn E, Schuppan D: Autoantibodies of patients with coeliac disease are insufficient to block tissue transglutaminase activity. Gut 2003;52:1562–1566. Halttunen T, Mäki M: Serum Immunoglobulin A from patients with celiac disease inhibits human T84 intestinal crypt epithelial cell differentiation. Gastroenterology 1999;116:566–572. Korponay-Szabo IR, Halttunen T, Szalai Z, Laurila K, Kiraly R, Kovacs JB, Fesus L, Maki M: In vivo targeting of intestinal and extraintestinal transglutaminase 2 by coeliac autoantibodies. Gut 2004;53:641–648. Caputo I, Barone MV, Troncone R, Auricchio S, Esposito C: Antibodies against Type 2 transglutaminase modulate actin rearrangements and cell cycle in several cell line. J Pediatr Gastroenterol Nutr 2004;39 (suppl 3):S775. Hausch F, Halttunen T, Maki M, Khosla C: Design, synthesis, and evaluation of gluten peptide analogs as selective inhibitors of human tissue transglutaminase. Chem Biol 2003;10:225–231.

Dr. Carla Esposito Department of Chemistry, University of Salerno Via S. Allende, IT–84081 Baronissi, Salerno (Italy) Tel. ⫹39 089 965298, Fax ⫹39 089 965296, E-Mail [email protected]

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Transglutaminases as Biotechnological Tools Loredana Mariniello, Raffaele Porta Department of Food Science, University of Naples ‘Federico II’, Naples, Italy

For their capacity to cross-link protein substrates, transglutaminases have always attracted a wide interest from both scientific and applied points of view. Since 1957, when Clark et al. [1] described a transamidating activity in guinea pig liver and introduced the term ‘transglutaminase’ (TGase), research on this class of enzymes has been growing, reaching a very consistent number of published reports (around 400 per year) during the last five years. The fields of research are very broad, from human health to industrial applications in different sectors. Such a large range of interest is related to the existence of different isoforms of TGase which are widely distributed in different organisms, such as bacteria [2], plants [3], invertebrates [4], vertebrates including amphibians [5], fish [6] and birds [7]. For most of them the physiological role has been established, while for others further studies are still needed. Moreover, some TGase isoforms have attracted a large interest as potential biotechnological tools because of their different substrate specificity and since they are easier to be manipulated outside of their natural environment. For example, keratinocyte TGase, which needs a complex posttranslational modification to be fully active [8], was never suggested for biotechnological applications. But, for its implication in lamellar ichthyosis [9], this molecular form of the enzyme was only proposed as a possible target for gene therapy. On the other hand, Factor XIIIa, despite the fact of being the first isoform to be used to modify protein and peptide substrates [10], is nowadays used as a therapeutic agent to treat severe pathology, as fatal bleeding, since its role in this disease has been well assessed. Also the so called ‘tissue’ TGase (type 2), the ability of which to modify biological properties of some peptides and proteins has been well established [11–21], is currently used in biomedicine

and specifically for the diagnosis of an autoimmune pathology like the celiac disease, where the enzyme was suggested to be the major autoantigen [22]. In the last decade Streptoverticillium mobaraense isoform, a TGase of microbial origin possessing wide substrate specificity, calcium independence and high thermostability, has been largely utilized as a biotechnological tool, but mostly in the food field [23, 24]. In this chapter we describe the main applications of the multiple TGase molecular isoforms in different sectors, from biomedicine to cosmetics, from food to leather and textile industries.

Biomedicine

The applications of TGase in biomedicine are directly related to the studies on the physiological role of each enzyme isoform, on the identification of new endogenous and exogenous substrates, and on the enzyme capability to modify the biological properties of the proteins able to act as acyl donor and/or acceptor substrates. Investigations on the physiological role of Factor XIII have allowed to design products for therapeutic use of this isoenzyme. As it is well known, coagulation Factor XIII (fibrin stabilizing factor) is a TGase that circulates in blood plasma as an inactive heterotetramer consisting of two catalytic A and two regulatory B subunits (A2B2), both of which are synthesized and secreted by the liver. Factor XIII is converted into the fully active enzyme (Factor XIIIa) by a thrombin-mediated proteolysis occuring in the final stage of the blood coagulation cascade. Once produced, Factor XIIIa cross-links fibrin aggregates both stabilizing them against mechanical stress and proteolytic degradation and incorporating proteinase inhibitors into the fibrin clot. Factor XIIIa activity suggested the design of products with blood clotting properties to be used for bleeding control during surgery. Thus, the aim to produce fibrin sealants was reached by different enterprises promoting new therapeutic preparations, like Tissucol kit (by Baxter) and Tisseel kit (by Immuno), both containing Factor XIII and used for the treatment of postsurgery hemostatis. Also the ‘tissue’ TGase has been proposed as biological glue because of its capability to cross-link proteins occurring in the extracellular matrix. Its use in promoting cell adhesion for the coating of medical implants has been, in fact, recently patented [25]. Other commercial products related to Factor XIII involve its administration in patients affected by Factor XIII deficiency, a very rare bleeding disorder either inherited or acquired [26] which is characterized by the production of defective and unstable blood clots in response to tissue damage. Replacement therapy in Factor XIII deficiency has been proved to be generally straightforward owing to

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the low levels of Factor XIII required to control bleeding. Nowadays two commercial products are available for substitutive therapy with plasma-derived Factor XIII (Factor XIII from Bio Products Laboratory and Fibrogammin P from Centeon). Further application of TGase in biomedicine is related to the therapeutic treatment of the ‘excessive or hypertrophic scarring’, a pathological state characterized by the occurrence of hypertrophic scars following a dermal insult, such as surgery, grafting, trauma and severe burns. Hypertrophic scars are characterized by being thick, red, painful and itchy and can cause functional deficits when they occur across a joint. In the wound healing process ‘tissue’ TGase plays a role in the production of ␧-(␥-glutamyl)lysine cross-links with the formation of insoluble collagen matrices. In hypertrophic scarring ‘tissue’ TGase is found to be overexpressed resulting in both an overhealing process and an excess of collagen deposition. After assessing the effectiveness of polyamines as alternative acyl acceptor substrates of ‘tissue’ TGase [27], Procyon Biopharma Incorporation developed Fibrostat®, a topical cream containing putrescine. The development status of Fibrostat® is currently in phase IIb of clinical trial to further evaluate the safety and the effectiveness of the product [28]. Moreover, the progress in the knowledge of the etiology of the celiac disease has pointed out the importance of ‘tissue’ TGase as an effective diagnostic tool. In fact, the identification of ‘tissue’ TGase as the major autoantigen of celiac disease, against which the endomysial antibody is directed [22], led to more extensive understanding of the pathogenesis of this immunologically mediated intolerance to wheat gliadins. These findings also allowed to change the technique by which the diagnosis of the disease was made. In fact, previous methods based on the identification of antiendomysium antibodies have been replaced by the immunological detection through ELISA tests of IgA autoantibodies against ‘tissue’ TGase. First generation tests, identifying IgA class anti‘tissue’ TGase antibodies, used the enzyme purified from guinea pig liver as antigen [29–31]. However, since several studies demonstrated significant differences in the performance of the guinea pig enzyme compared to the human isoform, human TGase antigen-based kits have been recently developed. Most of them contain a recombinant form of the enzyme, expressed either in E. coli or in Baculovirus/insect cell systems, together with antibodies raised against both types of the recombinant forms. Another pathology related to a member of the TGase family is the lamellar ichthyosis, a disfiguring skin disease characterized by an abnormal epidermal differentiation and a reduced cutaneous barrier function. Since it has been established that lamellar ichthyosis patients possess a defective keratinocyte TGase (type 1) gene [9], several studies have been carried out in the attempt to design gene therapy-based medical approaches to restore TGase 1 activity.

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Both retroviral and plasmid vectors have been used for delivering engineered molecules and two different procedures have been investigated to optimize cutaneous gene delivery. The first one involves grafting of primary keratinocytes derived from lamellar ichthyosis affected patients and cell transformation with normal TGase 1 [32]. The second, representing a less labor intensive approach, consists in the direct injection of the vector harboring normal TGase 1, as naked DNA, into intact skin [33]. The unsatisfactory results obtained with both systems indicate that further studies are needed to apply gene therapy to this kind of disease. A role played by TGase in some neurodegenerative disorders, such as Alzheimer’s, Huntington’s and Parkinson’s diseases, has been also suggested [34]. The involvement of the enzyme has been hypothesized since some proteins related to these pathologies have been proven to act as TGase substrates and an increase in TGase activity was observed. For example, ‘huntingtin’, the protein product of the mutated gene responsible for Huntington’s disease, is known to be characterized by the presence of polyglutamine stretches in its aminoacid sequence. In vitro studies demonstrated that this protein is able to act as substrate of ‘tissue’ TGase and that an elevated enzyme activity occurs in the affected cerebral regions [35]. It has been demonstrated that administration of the TGase inhibitor cystamine caused an improvement in patient survival as well as in the symptoms associated with neurodegeneration [36].

Cosmetics

Applications in this field are, in general, related to TGase ability to covalently bind specific compounds containing primary amino groups to keratinocyte proteins known to act as acyl donor substrates. Thus, a method to deliver a large variety of compounds (i.e. sunscreens, antimicrobials, either skin or hair conditioning agents, anti-inflammatory and antioxidants drugs, colorants, perfumes, insect repellents) has been patented [37]. Moreover, topical preparations containing TGase and one or more proteins occurring in the stratum corneum of the skin (like involucrin, loricrin, cornifin) have also been described. The effect of these products is related to the formation of a protective film useful for the care of hair, skin and nails [38]. Another topical preparation, containing an inhibitor of TGase and possessing the property to alter the rate of mammalian hair growth, has been patented. This kind of product could be of interest to remove undesired hair in specific parts of the human body [39]. Finally, microparticles containing TGase-substrate reactive groups, which can be cross-linked to the skin surface by either endogenous or exogenous TGase, represent an additional pharmaceutical proposal [40]. However, even

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though to our knowledge the products related to the aforementioned patents are not at the moment on the market, it is predictable that a large number of skin care preparations derived from investigations in this field will be available soon.

Food

Most preliminary studies addressed to the TGase-catalyzed modifications of proteins of food interest have been carried out with the enzyme purified from guinea pig liver or bovine plasma. The limited supply of these isoforms and the high costs of their production inhibited the development of technologies involving TGase in food processing to enhance texture and emulsion properties of protein-based foods. In 1989 a microbial TGase was isolated from Streptoverticillium sp. and its characterization indicated that this isoform could be extremely useful as a biotechnological tool in food industry [41]. In fact, microbial TGase was shown to be active over a wide range of temperatures and stable between pH 5 and 9, as well as to possess a calcium-independent activity [42]. This isoform has been ‘Generally Recognized As Safe’ and its use is allowed as food additive. Ajinomoto Incorporation actually produces several preparations of microbial TGase that are commercialized with different names. They differ in stabilizer composition in relation to the type of food for the production of which they have been designed. For example, Activa WM, a powder which contains 1% TGase and 99% maltodextrins, was described to enhance the texturizing properties of meat-based foods and successfully used to prepare novel dairy products [43]. Conversely, Activa MP, which contains lactose besides maltodextrins as stabilizer, was suggested to be used in modifying milk protein-based foods as cheese or yogurt. To date, even though many typical oriental foods are already produced in the presence of TGase as adjuvant, the use of the enzyme is certainly destined to be spread out worldwide in the future. In agreement with the European legislation (Directive 89/107/EC), Ajinomoto declared that TGase could be considered as a processing aid and, thus, its presence does not need to be indicated in the finished products. Seafood Products The use of TGase in food industry started in Japan to prepare surimiderived products (fish paste). One of this, kamaboko, is thought to date as far back as 1,100. To become surimi, fish is skinned, boned, repeatedly rinsed to eliminate any fishiness and pigments and ground into a paste. This odorless white paste is then mixed with a flavor concentrate made from real shellfish.

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The paste is then formed, cooked and cut into the desired shapes. Since the presence of endogenous TGase has been detected in species of pollack, fish used for surimi [44], several experiments have been carried out to assess the effect of TGase on the physical properties of surimi gels [45–48]. There are no doubts that both endogenous fish TGase and exogenous microbial TGase are able to improve the texture of the raw materials by catalyzing the formation of ␧-(␥-glutamyl)lysine cross-links between fish proteins. In particular, the addition of exogenous microbial TGase is certainly responsible for increasing elasticity and firmness of the surimi gel. Meat Products Also the use of TGase to prepare meat products is based on studies showing that several meat-related proteins are able to act as substrates of the enzyme. In fact, Factor XIIIa-catalyzed cross-link formation between fibrin and fibronectin, fibrin and actin, myosin and fibronectin, and myosin and actin was previously demonstrated [49]. Further studies reported the capability also of guinea pig ‘tissue’ TGase to modify meat proteins [10]. Finally, most recent investigations demonstrated the effectiveness of the microbial isoforms of the enzyme to produce different types of meat-based foods by using successfully beef, poultry and pork to prepare restructured products. For example, it has been described that the texture of chicken sausages, which originally showed a gel strength weaker in comparison with the pork sausages, was significantly improved by TGase. Thus, the enzyme offers the possibility of creating new poultry products with improved textural characteristics. The most significant advantage in the utilization of the enzyme in meat processing is due to the ability of TGase to efficiently substitute salts and phosphates, generally used in the traditional procedures of meat binding and texturing. In the alternative process involving TGase, a mixture of the enzyme and sodium caseinate, added to the meat pieces, allows the formation of cross-links among the casein molecules and the resultant meat product can then be cooked without breaking apart [50]. Nowadays, this process is extensively used to restructure meat from many different sources and to produce corresponding food products that visually and texturally meet consumer demands. Dairy Products Modifications of milk proteins by TGase have been extensively studied. The ability of milk proteins to act as TGase substrates was preliminary investigated by using both the enzyme purified from guinea pig liver and the blood coagulation Factor XIII, while more recent studies involved the utilization of the microbial isoform of the enzyme. Experiments carried out using single milk proteins allowed to establish that caseins are effective substrates for

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TGase, even if ␣s1-, ␤- and ␬-casein react differently with the enzyme depending on the isoform used. In fact, Ikura et al. [51] reported a lower reactivity of ␬-casein, compared to ␣s1- and ␤-caseins, with the guinea pig liver enzyme, whereas ␣-casein was shown to act less effectively as acyl donor substrate for the blood coagulation enzyme in comparison with both ␤- and ␬-caseins [52]. More recently, Cozzolino et al. [43] demonstrated no significant differences among the different caseins by using microbial TGase, the preferred isoform both to produce new protein ingredients and to change the texture of food products. Most studies have also demonstrated the effectiveness of TGase to prepare novel yogurts. Kuraishi et al. [24] reported that a yogurt made in the presence of TGase possesses improved gel strength and viscosity as a consequence of its enhanced water-holding properties. Moreover, microbial TGase has been recently proposed for producing whey protein-enriched cheeses by adding the enzyme during the manufacturing process [43]. The obtained new products showed an increased hardness and deformability, depending on the amount of the enzyme used, as well as increased protein content. Development of this kind of dairy production is desirable since it would represent a way for the re-utilization of dairy plant by-products, thus contributing to decrease the environmental pollution due to the whey protein disposal. Soy Products Soy proteins are of great interest in the world food industry since they are widely used as ingredients in a variety of western products, such as sausages and ham, as well as being the basic component of typical eastern foods. Their importance is related to their ability to undergo gelation after thermal treatment. It is well known that gelled proteins provide some useful textural properties to different foods. It has been extensively studied how TGase-mediated polymerization of both soy 7S and 11S globulins influences soy protein gel properties. Chanyongvorakul et al. [53] and Kang et al. [54] reported that TGase-induced 11S globulins gels are more rigid and elastic than the corresponding thermally induced gels. The authors proposed that rigidity might be due to an extensive cross-linkage, since it was possible to influence the protein textural quality by varying enzyme concentration. These molecular studies suggested the utilization of TGase in the manufacturing of tofu, the major soy product in Asia. The use of TGase, together with magnesium chloride acting as coagulant, provides a tofu with a smoother, firmer texture. These new properties depend on an enhanced breaking strength compared to that of tofu obtained in the absence of the enzyme. Tofu prepared with TGase also exhibits an increased water-holding capacity, probably because of the presence of more stable covalent cross-links that hold more water despite the temperature changes.

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Cereal-Based Products Wheat is known as one of the most important cereals for human nutrition and many reports indicate that wheat proteins are able to act as TGase substrates. Porta et al. [55] demonstrated the reactivity of different cereal proteins as acyl donor substrates for ‘tissue’ TGase and that wheat globulins, glutenins and gliadins were more effective than prolamins from oat, maize and rice. Different authors reported similar results by using the microbial isoform [56]. The rheological properties of gluten modified following the formation of ␧-(␥-glutamyl)lysine cross-links were also investigated. Viscoelasticity of TGase-treated gluten, as well as its sensitivity to thermal processing, was reduced compared to that of the unmodified gluten. Therefore, the enzymatic treatment was shown to cause a considerable reinforcement of the network. These studies have promoted the use of TGase to prepare noodles and pasta in Japan. TGase, added when flour and other ingredients are mixed, confers to both pasta and noodles a firmness higher than that of untreated products, indicating that this characteristic depends on the enzyme amount [24]. In addition, since the cross-links introduced by TGase are heat stable, firmness and elasticity of noodles and pasta are retained even after cooking. The use of the enzyme showed beneficial effects also on breadmaking. In fact, TGase improves dough elasticity and its utility in breadmaking is similar to that of the oxidizing improvers [57]. Finally, recent studies investigated the possibility to modify rice flour proteins with the aim to use this important cereal for breadmaking. In its natural conditions rice flour is used only to make unfermented baked products since rice proteins are unable to hold gas produced during fermentation. Conversely, Gujral and Rosell [58] have demonstrated that TGase-modified rice proteins provide a protein network effective in holding gas produced during fermentation. Edible Films An increasing interest toward edible films has been registered in the last few years mostly for their potential use in food industry. First of all, edible films can represent an alternative to the chemically synthesized polymeric films that are, nowadays, widely used for packaging. Since latter films are not environment friendly, development of films constituted by edible and biodegradable components is strongly advisable. Moreover, edible films have substantial possibilities to enhance stability and quality of foods. Their functional efficiency strongly depends on the nature of the components that can be, typically, hydrocolloids and/or lipids. Many scientific papers and patents refer to films constituted by different proteins, like collagen, casein, wheat gluten and whey and soy proteins, whereas most of the effects of TGase treatment on film properties have been studied using the guinea pig purified

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enzyme. Utilization of whey proteins for the production of packaging films was investigated by Mahmoud and Savello [59, 60]. Additional studies were carried out using ␣s1-casein [61], 11S globulin [62] and egg white proteins [63]. Cross-links introduced by TGase confer on films a precise network that influences mechanical and permeability properties, reflecting characteristics at microstructural level. Smoother surface and higher homogeneity are features found in microbial TGase-synthesized film made with whole soy flour and pectin [64]. Such films exhibit also an increased strength, are less flexible, and less permeable to oxygen, carbon dioxide and water vapour [65]. It is worthy to note that TGase-mediated polymerization modifies, among the proteins occurring in soy flour, the soybean vacuolar protein, known to possess allergenic properties and that can be neutralized also by the Maillard reaction [66]. Finally, edible films have also been proposed as vehicles to carry substances to monitor and/or to influence the quality of wrapped foods. In this way, edible films may represent an active packaging and its use might be extended outside the food sector if the vehiculated substances are specific drugs [10]. Nutritional Aspects of TGase-cross-linked Proteins The evaluation that so many applications of TGase exist in food processing raises questions about the nutritional value of the proteins containing isopeptide bonds. In this respect bioavailability and digestibility of the glutamine-lysine cross-links deserve to be considered. It has been established that a number of isopeptide bonds occur in many tissues of different animals that are commonly eaten. One explanation of this phenomenon is related to the presence of different endogenous TGase isoforms responsible for the cross-links formation in uncooked foods. But also the processed foods, including the Japanese kamaboko, ham, fried chicken, grilled pork, and hamburger, were found to contain ␥-glutamine-␧-lysine isopeptide bonds. In particular, cooked foods have an higher content of isopeptide bonds compared to raw food, probably because at the beginning of the process endogenous TGases become more active and are able to better catalyze the formation of cross-links. On the other hand, enzymes able to hydrolyze the ␥-glutamine-␧-lysine dipeptide, which is not susceptible to gastrointestinal proteolysis, have been described. In particular, kidney was shown to be provided with the enzyme ␥-glutamylamine cyclotransferase [67], while a different ␥-glutamyl transferase was demonstrated to be present mainly in intestinal brush-border membranes and blood [68]. Lysine, which is an essential amino acid, is generated from the cleavage operated by these two enzymes and is readily available and nutritionally beneficial. As a matter of fact, Seguro et al. [69] demonstrated that

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rats fed with TGase-cross-linked caseins grew as much as control rats fed with unmodified caseins.

Leather and Textile Industries

The use of TGase also is of interest for other industrial sectors. In the leather industry casein is used to coat leather through a process that involves hardening agents such as aldehydes, isocyanates or aziridine. Because of the high toxicity of these agents, for both operators and environment, new and different methods are demanded. In this respect the application of TGase-modified casein as a coating has been patented [70] and its wider use in leather manufacturing is to be desired. More recently, studies have been carried out demonstrating the importance of TGase also for the wool finishing industry [71]. To overcome both felting and shrinkage problems of wool fibers, chemical processes are currently used. Most of them involve acid chlorination of the wool goods or the application of permonosulphuric acid. Although these methods concur to confer a significant level of shrink-resistance to the wool, they are of high environmental impact due to the toxicity of the reagents used. On the other hand, the alternative technique based on the use of proteases to prevent shrinkage, a problem occurring after repeated laundering, results in an undesired reduction of wool fibre strength and weight. Cortez et al. [72] have demonstrated, by using both guinea pig liver and microbial TGases, that the enzyme increases tensile strength of the wool products, in some cases completely reverting the loss caused by a previous proteolytic treatment. Protease pre-treatment enhances the effect of subsequent TGase treatment since it causes an increase in accessibility of the fibres to protein cross-linking. Finally, even in the absence of exogenous TGase addition, fibre matrix is stabilized, beside the most abundant disulphide bonds, also by ␧(␥-glutamyl)lysine bonds catalyzed mainly by keratinocyte and epidermal TGases that normally confer resistance to hair and wool. Cortez et al. [72] have also demonstrated that both TGase isoforms may be used to incorporate the primary amine substrate fluorescein cadaverine into wool fibres. This result suggests a possible use of the enzyme also to incorporate functional agents as antimicrobials, water repellents and perfumes, as far as they are provided with an alkylamino side group [72]. It is worthy to note that these studies have underlined the different substrate specificities of microbial and tissue TGases towards wool proteins. In fact, microbial enzyme was used at a protein concentration 20 times higher than that of guinea pig liver TGase. Only the availability of recombinant ‘tissue’ TGase obtained at low costs, hence competitive with the microbial isoform,

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will allow its wider use as a biotechnological tool, becoming more popular in this field of interest.

Analytical Biotechnology Applications

Avidin-biotin technology attracts a great interest because of its ability to replace many tests employing radioactively labeled materials. Thus, this system is frequently utilized to identify proteins which have been biotinylated. It has been demonstrated that both acyl acceptor and donor TGase substrates are still able to be modified by the enzyme after they have been previously biotinylated. Often the biotinylated molecules are useful to demonstrate whether a peptide or a protein is a TGase substrate. Josten et al. [73] have used biotinylated compounds endowed with an acyl acceptor amino group to obtain biotinylated antibodies. In particular, by using microbial TGase for the biotinylation of a monoclonal IgG against the herbicide 2,4-dichlorophenoxyacetic acid, they demonstrated that the biotinylated antibody exhibited the appropriate biological activity. TGase has also been proposed to synthesize products useful for immunochemical assays. Testing of antibodies against small molecules, such as haptens, is routinely performed by ELISA techniques. For accurate results, it is essential to provide an efficient coupling of the hapten to a protein carrier in order to obtain conjugates to be used to coat the microplates in which immunoassays are carried out. As long as hapten is provided with an acyl acceptor group, it can be incorporated through TGase into an acyl donor substrate. Josten et al. [74] have demonstrated the effectiveness of microbial TGase to cross-link an aminofunctionalized hapten to casein, a typical acyl donor substrate for the enzyme, which is currently used to coat plates for immunoassays. Similar results were obtained by other authors by using ‘tissue’ TGase [61]. Enzymatic synthesis of the conjugates is advisable since chemical procedures are time and labor consuming and the degree of conjugation is hard to achieve. Conversely, enzymatic catalysis is highly reproducible and needs reduced times for conjugation. The use of microbial TGase for the development of biosensors has also been successfully exploited. In particular, the microbial enzyme was used to prepare a protein matrix, constituted by TGase-cross-linked casein or fibrinogen, onto which model enzymes like glucose oxidase or lactate oxidase were entrapped [75]. It was demonstrated that lactate enzyme sensors, obtained by microbial TGase-mediated immobilization, exhibited a higher storage and operational stability compared to sensors prepared by chemical cross-linking through

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glutaraldehyde. In contrast to other enzyme membranes, prepared by entrapment of enzymes in hydrogels, the TGase-mediated network remains homogeneous during drying in air with beneficial effects on the reproducibility of the enzyme sensors.

Other Applications

TGase was recently also proposed as a tool to synthesize glycosylated proteins to be employed in different sectors, from food industry to medical field. Thus, microbial TGase has proved to be effective to prepare trypsinoligosaccharide conjugates with improved stability properties of the proteolytic enzyme. Trypsin, widely used in food manufacturing and processing industry, is able to act as acyl donor for TGase and to incorporate different types of aminoderived cyclodextrins, compounds previously employed as physical additives to increase the solubility and the catalytic properties of various enzymes in organic media. In particular, TGase-synthesized trypsin-cyclodextrin conjugates have been shown to exhibit significantly improved both specific esterolytic activity and kinetic constants, besides being more resistant to autolytic degradation at alkaline pH and to heat inactivation [76, 77]. The effectiveness of glycosylation through TGase in the medical field was exploited to modulate the biological properties of interleukin-2, a lymphokine with important immunoregulatory functions [78]. In particular, polyethyleneglycol modified with an alkylamine straight chain was used as acyl acceptor substrate. Interleukin-2, acting as glutamine donor substrate, showed an improved capability to survive in the blood circulation of treated rats after TGase modification. In fact, unmodified interleukin-2 showed an half life of only 5.5 min, while TGase glycosylated lymphokine exhibited an half life almost 40 times higher [78].

Conclusions

In this chapter we describe a quite wide panorama on the most revelant biotechnological applications of TGases. The potentiality offered by this class of enzymes in creating many different products is shown in figure 1. Due to their capacity of synthesizing isopeptide bonds, homo- and heteropolymers may be formed with the consequence of obtaining products with new or improved features. Biological activities of proteins and peptides can be also influenced by incorporating polyamines and/or aminosugars. In addition, a different performance can be induced in a protein by its partial TGase-catalyzed deamidation

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185

es Product

Ho mo po ly

p o ly m e in

es

p ti d

p ro t

ein

X

ied

Prot

in te p ro

d if

in X

HO 2

De

am

ina

ted

p ro t

e in

A min

os

ug

Mo

d ate

ote

Pe

Pr

in e

m in

am

Pr

ein

ote

Prot

P o ly

Polya

in X

Y

ers

te in

te i n X

Immun ogl ob ul i

tero

e Prot zym ein En X or substr n o ate ld y c A

Pro

n

m er s

He

su bs tra te s

Pro bes tei nX so r Ab l acceptor substrate y s c A

Pro

Biotin-d eriv ed

en zy m

s

Biotiny late da nti bo dy

m Im

d ilize ob

ar

ly c o Neog

tei p ro

n

Fig. 1. Examples of different reactions catalyzed by TGase. The internal circle contains the acyl donor substrates, the central circle contains the acyl acceptor substrates, whereas the external circle contains the corresponding products.

converting one or more glutamine residues in glutamic acid(s). Finally, the use of the enzyme has been successfully exploited in analytical biotechnology with the aim both to synthesize effective biotinylated antibodies and to immobilize enzymes acting as biosensors. In many of these applications, the use of TGase resulted alternative to chemical modification methods that often give rise to toxicity problems for users and provoke environmental pollution. In conclusion, the reported studies indicate that TGase-based methodologies are destined to have further important applications in many different industrial sectors. Thus, the development of novel inexpensive sources of the enzyme represents a crucial point for future research studies. To date, the most convenient TGase as a biotechnological tool is the enzyme purified from Streptoverticillium mobaraense, even though this isoform seems to be useful mainly in the food sector. For different applications, the production in large

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amounts of other isoforms, i.e. Factor XIII or ‘tissue’ TGase, is needed. One promising approach to obtain these enzymes at low costs involves the genetic manipulation using heterologous hosts. By this way, the expression of recombinant human Factor XIII was obtained using both E. coli [79, 80] and yeast [81] systems as bioreactors. In the same manner, the recombinant ‘tissue’ TGase has been produced in E. coli [82, 83] and Baculovirus/insect cells [84] and used as reliable antigen for the diagnosis of celiac disease. However, the high costs of production suggest to find alternative systems. More recently, plants have been proposed as effective hosts for the biosynthesis of mammalian proteins [85]. Gao et al. [86] engineered tobacco plants producing Factor XIIIa with the aim to obtain a recombinant isoform which could replace Factor XIII occurring in the commercially available therapeutic products. Similarly, Nicotiana tabacum-derived cells have been recently investigated as bioreactor for the production of human ‘tissue’ TGase. A catalytically active form has been efficiently produced and the partially purified enzyme shown to be effective in recognizing anti-TGase antibodies present in celiac patient antisera [87]. Thus, the perspective that new transgenic sources may provide large amounts of the different TGase isoforms useful for various biotechnological applications should be considered at the moment more than a simple possibility.

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49 50 51 52 53

54 55

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84

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Lai T-S, Santiago MA, Achyuthan KE, Greenberg CS: Purification and characterization of recombinant human coagulation factor XIII A-chains expressed in E. coli. Protein Expr Purif 1994;5: 125–132. Bishop PD, Teller DC, Smith RA, Lasser GW, Gilbert T, Seale RL: Expression, purification and characterization of human Factor XIII in Saccaromyces cerevisiae. Biochemistry 1990;29: 1861–1869. Shi Q, Kim S-Y, Blass JP, Cooper ALJ: Expression in Escherichia coli and purification of hexahistidine-tagged human tissue transglutaminase. Protein Expr Purif 2002;24:366–373. Gillet SM, Roberto A, Chica A, Keillor JW, Pelletier JN: Expression and rapid purification of highly active hexahistidine-tagged guinea pig liver transglutaminase. Protein Expr Purif 2004;33:256–264. Osman AA, Richter T, Stern M, Conrad K, Henker J, Brandsch C, Zimmer KP, Mothes T: Production of recombinant human tissue transglutaminase using the Baculovirus expression system, and its application for serological diagnosis of coeliac disease. Eur J Gastroenterol Hepatol 2002;14:1217–1223. Giddings G, Allison G, Brooks D, Carter A:. Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 2000;18:1151–1155. Gao J, Hooker BS, Anderson DB: Expression of functional human coagulation factor XIII A-domain in plant cell suspensions and whole plants. Protein Expr Purif 2004;37:89–96. Sorrentino A, Schillberg S, Fischer R, Rao R, Porta R, Mariniello L: Recombinant human tissue transglutaminase produced into tobacco suspension cell cultures is active and recognizes autoantibodies in the serum of coeliac patients. Int J Biochem Cell Biol; in press.

Dr. Loredana Mariniello Department of Food Science, University of Naples ‘Federico II’ Parco Gussone, Portici, IT–80055 Naples (Italy) Tel. ⫹39 0817754867, Fax ⫹39 0817755116, E-Mail [email protected]

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Mehta K, Eckert R (eds): Transglutaminases. Prog Exp Tum Res. Basel, Karger, 2005, vol 38, pp 192–208

Extracellular Transglutaminase: Factor XIII Akitada Ichinose Department of Molecular Patho-Biochemistry and Patho-Biology, Yamagata University School of Medicine, Yamagata, Japan

Introduction

Transglutaminases (TGases) are enzymes that catalyze the formation of ␧-(␥-glutamyl)lysine bonds, in so-called protein cross-linking (XL) reactions, between a number of proteins. The XL reactions not only enhance the original functions of the substrate proteins, but also add new properties to the substrates, which subsequently change their functions. Protein XL is involved in many physiological and pathological reactions, such as hemostasis, wound healing, tumor growth, skin formation, apoptosis, etc. Accordingly, the XL reaction is one of the most important of the post-translational modification reactions, the others being proteolysis, phosphorylation, glycosylation, etc. TGases exist widely in many organisms, including bacteria, horseshoe crab, fish, mouse, and human [1]. Thus, they are one family of proteins/enzymes with fundamental biological reactions, and are likely to have appeared early in the evolutionary history. Recently, a total of 10 members of the TGase family were identified in the human genome project [2] (table 1). There used to be 9 known TGases: plasma TGase (the A subunit of factor XIII, XIIIA), tissue (liver, RBC, or endothelial) TGase, keratinocyte TGase, epidermal TGase, prostate TGase, TGases X, Y, and Z, and erythrocyte band 4.2 protein (table 2). Accordingly, an additional homologous gene, TGase 3L (TGM3L), has to be characterized in detail in order to understand thoroughly its nature and its interaction with the 10 family members. It is of note that although all TGases exist in the cytoplasm inside cells (intracellular TGase), XIIIA also circulates in blood (extracellular TGase) and works outside cells in the blood coagulation system. It is very likely that

Table 1. Genes and domains related to hemostasis (modified from [2]) Access number

Domain

Description

Human

Yeast

Capsell

PF00386 PF00200 PF00754

Clq Disintegrin F5_F8_type_C

24 18 15 (20)

0 2 5 (6)

PF01410

COLF1

10

PF00039

Fn1

PF00040

Fn2

PF00051

Kringle

PF01823

MACPF

PF00084

Sushi

PF02210

TSPN

PF01108 PF00594

Tissue_fac Gla

PF00147

Fibrinogen_C

PF00092 PF00093 PF00094

Vwa Vwc Vwd

Clq domain Disintegrin F5/8 type C domain Collagen C-terminal domain Fibronectin type I domain Fibronectin type II domain Kringle domain MAC/Perforin domain Sushi domain (SCR repeat) Thrombospondin N-terminallike domains Tissue factor ␥-carboxyglutamic A domain Fibronectin-␤ and -␥ chains C-terminal globular domain vWF type A domain vWF type C domain vWF type D domain ADAM/ADAMlTS Globin Matrix metalloprotease Serum amyloid A Serum paraoxonase/ arylesterase Serum albumin Transglutaminase

0 3 2

0 0 0

0 0 0

0

0

0

0

5 (18)

0

0

0

0

11 (16)

0

0

0

0

15 (24)

2

2

0

0

0

0

0

0

8 (45)

0

0

6 53 (191)

Fly

11 (42)

Worm

14

1

0

0

0

1 11

0 0

0 0

0 0

0 0

6

0

0

0 0 0 0 0 0

1 0 0 0 3 3

26

34 (58) 19 (28) 15 (35) 51 10 19

10 (11)

0 6 (11) 3 (7) 9 2 2

17 (19) 2 (5) 9 12 3 7

4 4

0 0

0 3

0 0

0 0

4 10

0 1

0 0

0 0

0 0

() ⫽ Number of domains.

Extracellular Transglutaminase: Factor XIII

193

Table 2. Chromosomal loci and expression patterns of TGase gene family F13A1 TGM1 TGM2 TGM3 TGM3L TGM4 TGM5

6p25-p24 14q11.2 20q11.2 20q11.2 20q11.2 3p22-p21.33 15q15

TGM6 TGM7 EPB42

20p12.2-13 15q15.2 15q15

monocyte, megakaryocyte/platelet keratinocyte Ubiquitous human foreskin unknown prostate female reproductive system, skin, skeletal muscle, and fetal tissues neuron? testis and lung red blood cell

the presence of the B subunit of XIII (XIIIB) stabilizes the unstable XIIIA in plasma [3] by forming a complex, as discussed later.

Gene and Expression of Factor XIII

The gene organization of TGase was first established for XIIIA in 1988 [4]. The gene for erythrocyte band 4.2 was obtained next, and then for keratinocyte TGase, and so on. These studies revealed that members of the TGase family also share gene organization. With some exceptions, their genes are coded by 15 exons, interrupted by 14 introns, and the exon/intron boundaries of these TGases occur in the same sites among the primary structures. A major difference between these genes is their size. The gene for XIIIA spans about 180 kb, while the keratinocyte TGase spans only 15 kb. The sizes of the introns in each TGase gene are responsible for this difference. The gene for XIIIA is localized to chromosome 6 at bands p24–25. The gene encoding keratinocyte (epidermal) TGase is localized to chromosome 14q11.2–13, and that for prostate TGase (type IV, TGM4) to chromosome 3p21.33-p22. Thus, gene loci for these TGases are dispersed throughout chromosomes; however, the remaining 4 and 3 TGases are clustered to chromosome 20q1 and 15q15, respectively (table 2). Recently, we have characterized the cell type-specific transcriptional regulation of the XIIIA gene [5]. Although XIIIA and other TGases share significant similarity in their gene organization, their nucleotide sequences in the 5⬘-flanking region differ from each other, and mechanisms for their gene regulation seem to be diverse as well. Accordingly, nucleotide elements for transcription factors in

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Factor XIII

GATA-1 Ets-1 Ets-1

⫺554 ⫺482

⫺756 Keratinocyte TGase AP-1 SP-1 SP-1

CRE

⫺1520 ⫺1506 ⫺1388

⫺462

Tissue TGase

MZF-1 MZF-1 MZF-1

⫺260

⫺39 ⫺92 ⫺223 SP-1 SP-1 SP-1 ⫹1 TATA-like

AP-2 ⫺634

ATG

Intron A

Androgenresponse

⫺??

ATG

Intron A ⫹1 ATG AP-1 SP-1 SP-1 NF-1 SP-1

NF-1 ⫺183 ⫺56 ⫺45 TATA CAAT ⫹1 ATG Ets SP-1 AP-1

Epidermal TGase

Prostate TGase

⫹1 NF-1

⫺118 ⫺100 ⫺15

SP-1

⫹1 ATG

SP-1

⫺96

Fig. 1. Schematic diagram of 5⬘-flanking regulatory sequences in the genes for XIIIA and other TGases. The transcription and translation initiation sites are indicated by ⫹1 and ATG, respectively.

one TGase gene are quite different from those in others (fig. 1). For example, the gene for XIIIA is driven mainly by a myeloid-enriched transcription factor (MZF-1-like protein) and two ubiquitous transcription factors (NF-1 and SP-1); two myeloid-enriched factors (GATA-1 and Ets-1) also work as enhancers. In contrast, in the human epidermal TGase gene, SP-1- and Ets-1-like motifs were required for the function of its promoter, and analysis of the keratinocyte TGase promoter indicated that one region containing AP-1- and SP-1-like motifs and another containing a CRE site are essential for its expression. These transcription factors play a major role in the cell type-specific expression of each TGase. Although tissue TGase exists in most tissues and organs, XIIIA is mainly limited to plasma and platelets: keratinocyte TGase to skin keratinocyte, epidermal TGase to skin and hair follicular cells, and prostate TGase to prostate. Northern blotting analyses of mRNA samples from placenta, macrophages, and megakaryoblastoid cells showed a single mRNA species (about 4.0 kb) for XIIIA, while little or no detectable hybridization signal was obtained from mRNA samples of normal liver, HepG-2 cells, and fibroblasts. These results indicate that placenta, macrophages, and megakaryocytes do synthesize XIIIA. It is of interest that XIIIA levels increase significantly with the female gender, age, and smoking; the mechanisms of these changes, however, are to be explored.

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The gene for XIIIB is about 28 kb in length [6]. It is composed of 12 exons interrupted by 11 introns, and each of the 10 Sushi domains is encoded by a single exon. This is also true of the genes for other Sushi domain-containing proteins, with few exceptions. The gene for XIIIB is localized to chromosome 1 at bands q32–32.1. The genes for several proteins containing multiple Sushi domains, such as factor H, the a and b chains of C4b-binding protein, complement receptors type I and II, membrane cofactor protein, and decay accelerating factor, are clustered at the same locus. Accordingly, these genes appear to be generated from a common ancestor gene by gene duplication and exon shuffling during the evolution. The site of synthesis for the XIIIB gene has been suggested to be the liver. HepG-2 and PLC/PRF/5 hepatoma cells secrete XIIIB, and cDNA clones coding for XIIIB have been obtained from a normal human liver library. In addition, the phenotype of the recipient’s XIIIB changed to the donor’s phenotype after liver transplantation, while that of XIIIA remained unchanged. Therefore, it is clear that the liver is the major site of synthesis for XIIIB. The regulatory mechanism of XIIIB expression has not been characterized as yet.

Structure of Factor XIII

XIII (fibrin-stabilizing factor) is a plasma TGase, and circulates in blood as a heterotetramer consisting of two catalytic XIIIA and two noncatalytic XIIIB (A2B2) subunits. The primary structure of TGase was first established in 1986 by a combination of amino acid sequencing analysis and cDNA cloning for XIIIA. XIIIA consists of 731 amino acid residues [7]. The molecular weight of the polypeptide portion of the molecule was calculated to be 83,150. In 1988, the sequence of guinea pig tissue TGase was determined and the homology between XIIIA and tissue TGase was recognized for the first time. Since then, the amino acid sequences of many TGases have been determined, and their homology and evolutionary relationships have been established [1]. Since erythrocyte band 4.2 shares homology with TGases, it is also added as a noncatalytic member of the TGase family, although its active site (Cys residue) is replaced by an Ala residue. When we cloned the cDNA for XIIIA, we first noticed that there is no signal peptide for secretion. We could not find any internal signal either. If any of the listed cells is the major source of the ‘plasma’ XIIIA, then there should be a unique mechanism(s) for its release into circulation, where the free XIIIB readily binds and forms an A2B2 tetramer. Results of the expression of XIIIA and/or XIIIB in a mammalian cell system suggest that XIIIA is not secreted

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through the conventional secretory pathway, but is released from the cells by cell damage [8]. In contrast, XIIIB is readily released from the synthesizing cells by the secretion pathway because it has a typical signal peptide. The three-dimensional structure of XIII was demonstrated previously by electron microscopic studies. XIIIA and XIIIB appear to be a globular particle and filamentous strand, respectively. XIIIA of placenta and recombinant proteins have been crystallized. X-ray crystallography demonstrated that XIIIA is composed of five distinct domains: an activation peptide, ␤-sandwich, central core, barrel 1, and barrel 2 regions [9, 10]. The carboxyl- (C-) terminal portion of XIIIA corresponding to the two ␤-barrels forms thermostable domains, while three thermolabile domains are formed by the amino- (N-) terminal ␤-sandwich and core domains. It is of interest that the activation peptide, upon cleavage by thrombin, has the same conformation and occupies the same position with respect to the rest of the molecule as it does in the zymogen structure; moreover, the activation peptide blocks the entrance to the catalytic cavity in the core domain [11]. Although Yee et al. [11] reported that the X-ray crystal structure of activated and nonactivated XIII showed no significant differences, Turner et al. [12] demonstrated that certain changes resulted from the exposure or sequestration of several cysteine or lysine residues after the activation of the enzyme. Recently, examination of the X-ray crystal structure of human ␣-thrombin bound to a decapeptide (amino acid residues 28–37) of XIIIA revealed detailed atomic-level interactions between the activation peptide and thrombin [13]. Calcium-binding sites were also identified in XIIIA by X-ray crystallography [14].

Sushi Domains in Factor XIIIB

XIIIB is composed of 641 amino acid residues with a calculated molecular weight of 73,183 [15]. The addition of 8.5% carbohydrate gives a molecular weight of about 79,700 for each XIIIB. This molecular weight is in agreement with those estimated by SDS-polyacrylamide gel electrophoresis. XIIIB contains 10 tandem repeats that have been designated as Sushi domains, GP-I structures, or short consensus repeats [1]. Homologous Sushi domains have been found in a total of 53 genes by the human genome project [2] (table 1). Sushi domains have also been identified in fruit fly and worm. Therefore, it may be a rather old protein module, which plays important roles in the biological interactions through binding to other proteins. To this author’s knowledge, the X-ray crystal structure of XIIIB has not been established as of this writing. NMR studies revealed that Sushi domains in complement factor H consist of double- or triple-stranded ␤-sheets and several

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␤-turns and cuboid forms [16]. This is consistent with the data obtained from the crystal structure of human ␤2-glycoprotein I [17], which is composed of 5 Sushi domains.

Functions of Factor XIII

XIII is a proenzyme activated by thrombin that is generated in the final stage of the blood coagulation cascade. XIII plays an important role(s) in hemostasis, wound healing, and maintenance of pregnancy. The enzyme promotes clot stability by forming covalent bonds between fibrin molecules and also by XL fibrin with several proteins including ␣2-plasmin inhibitor, fibronectin, and collagen. These reactions lead to an increase in the mechanical strength, elasticity and resistance to degradation by plasmin of fibrin clots, and promotion of wound healing by providing a scaffold for fibroblasts to proliferate and spread. Interactions of XIII with other cells, such as macrophages and platelets, are also suggested to play important roles in the above-mentioned physiological reactions. A number of other proteins are known to be substrates of XIII: plasminogen-activator inhibitor type-2, osteopontin, lipoprotein (a), platelet vinculin, factor V, thrombospondin, etc., are also reported to become substrates for activated XIII in vitro; however, the physiological implications of these reactions are unclear at present.

TGase-Related Diseases

As discussed above, there are 10 members in the human TGase-gene family. These TGases are expressed in quite different patterns among various tissues and cell types. Their substrates vary depending upon each enzyme, but also overlap between some TGases. Accordingly, deficiencies of these TGases cause various disease states. We have named these as ‘TGase-related diseases’. They include congenital lamellar ichthyosis caused by keratinocyte TGase deficiency, congenital spherocytosis by deficiency of erythrocyte Band 4.2 protein, and maturity onset diabetes in young caused by tissue TGase deficiency, as described in other chapters. Animal models of various TGase deficiencies have been generated by gene targeting techniques, as described in other chapters. In general, the TGasedeficient mice demonstrate symptoms similar to human patients; however, it is also the case that phenotypes of mice differ from those of human counterparts, and one must keep in mind, therefore, the profound differences that exist between human and mouse.

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Factor XIII-Related Diseases

XIII is known to be related to a number of disease states, such as inflammation, thrombosis, dementia, cancer, etc. It has been reported that a conditioned medium from type II human Tcell leukemia virus-infected T cells induces the conversion of endothelial cells to a Kaposi’s sarcoma cell-like phenotype, and that the endothelial cells grown by hepatocyte growth factor in the culture medium acquired the ability to express XIIIA [18]. These findings suggest that hepatocyte growth factor and XIII play a role in the initiation and maintenance of Kaposi’s sarcoma lesions. The expression of XIIIA may be induced by other cytokines and growth factors, as well. XL of cellular proteins such as lysosomal enzymes and histones by TGases may prevent the leakage of these proteins from apoptotic cells and protect surrounding tissues from possible inflammation. Nishiura et al. [19] found that S19 ribosomal protein is cross-linked by XIIIA and tissue TGase in apoptotic cells in rheumatoid tissues, and that the dimerized S19 ribosomal protein acquires chemotactic activity for monocytes. The apoptotic cells are removed by the infiltrated monocytes/macrophages. This reaction has been shown both by in vivo experiments as well as in vitro. Accordingly, TGases play important roles in the cleaning up of apoptotic cells. Recruitment of monocytes from circulation into the lesion may also enhance inflammatory reactions, which result in chronic and severe disease states. XIII has been reported to decrease in the plasma of patients with chronic inflammatory bowel diseases [20], and XIII replacement therapy is useful in the treatment of these diseases. It is very likely that XIII is consumed in the inflammatory intestine, which in turn results in impaired wound healing in the damaged tissue. XIII is also known to decrease in patients with Henoch-Schonlein Purpura [21], especially with abdominal complications. In such cases, the administration of XIII concentrates often leads to remission of the disease. The mechanism(s) of this XIII reduction is not known at present. It has been found that many XIIIA-positive microglias were associated with primitive senile plaques in the brains of patients with Alzheimer’s disease, while few or no XIIIA-containing microglias were found associated with classical plaques [22]. XIII may play a role(s) in the early phase of Alzheimer’s disease, since the tau protein is reported to be cross-linked by TGases in the affected brain tissue. Recently, Wozniak et al. [23] reported that XIII could protect endothelial barrier function and prevent the development of myocardial edema in children undergoing surgery for congenital heart disease. They observed a thrombin increase combined with a simultaneous decrease of XIII as a probable cause.

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Since preoperative XIII substitution showed a distinct effect in minimizing the incidence of myocardial swelling, they suggested that the clinical application of XIII might have a valuable therapeutic benefit in cases of capillary leakage syndrome during extracorporeal circulation in congenital heart surgery.

Polymorphisms of Factor XIII and Thrombosis

Using Agarose gel electrophoresis, several different allelic forms of XIIIA were identified in the normal population. The heterogeneity of these XIIIA was confirmed both by amino acid and DNA sequencing. All amino acid substitutions can be explained by single nucleotide polymorphisms in coding regions: These include: Val34Leu, Phe204/Tyr, Leu564/Pro, Val650/Ile, and Glu651/Gln [4, 24]. Although Arg77/Gly, Arg78/Lys, and Phe88/Leu polymorphisms have been described in literature, a series of genetic studies has failed to identify the corresponding nucleotide sequences [Ichinose et al. unpubl. data]. It has been reported that the Val34Leu polymorphism is associated with myocardial infarction and venous thrombosis [25]. A mechanism whereby Leu34 is protective against thrombosis has been investigated by several groups; however, their conclusions are inconsistent thus far. The activation rate of the Leu34 type by thrombin seems to be faster than that of the Val34 type, although both of them have the same specific activity when fully activated. It is of interest that early covalent XL of the fibrin clot by FXIII Leu34 reduced lateral aggregation of the fibrin fibers, leading to a reduction in fiber thickness from 121.0 ⫾ 23.9 nm to 75.7 ⫾ 11.3 nm and an alteration in rates of fibrinolysis of the fibrin clot [26]. The effect of Val34Leu on fibrin structure and function is dependent on fibrinogen levels and may alter the interaction with platelets. It is also possible that the Val34Leu polymorphism may be in a tight linkage with some unknown mutation(s) in the XIIIA gene, and this may prove to be the true cause of the protective effect against thrombosis. Recently, we developed a new detection method to determine the prevalence of this polymorphism by combining an amplification refractory mutation system with rapid automated capillary electrophoresis [27]. In contrast to previous studies, no healthy Japanese individuals (n ⫽ 103) were found to possess the polymorphism. This was also the case for 201 Japanese patients with ischemic heart disease or cerebrovascular dementia; therefore, this polymorphism cannot be a discriminative factor for thrombosis among Japanese. The polymorphism was detected neither in Korean (n ⫽ 100) nor in Chinese (n ⫽ 95) populations, while its gene frequency was 0.24 in Italians (n ⫽ 75). Accordingly, theVal34Leu polymorphism is another example of ethnic specificity of gene polymorphisms analogous to factor V Leiden and prothrombin G20210A.

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There are many SNPs in the 5⬘-flanking region of the XIIIA gene; however, none of them has a serious effect on XIIIA expression [Souri and Ichinose, unpubl. data]. The microheterogeneity of XIIIB was also classified into several alleles. Recently, we have identified several single nucleotide polymorphisms in coding regions that are responsible for differences in the amino acid sequence of XIIIB and its genotypes [Umetsu and Ichinose, unpubl. data], as well as several nucleotide substitutions in noncoding parts of the gene [28]. Dr. Grant’s group has suggested that some of the single nucleotide polymorphisms in coding regions, like His95Arg in the XIIIB gene, may be related to a decreased risk of myocardial infarction, especially in the presence of the Val34Leu polymorphism of XIIIA [29].

Clinical Features of Congenital Factor XIII Deficiency

More than 500 cases of XIII deficiency have been identified. In most cases, diagnosis of XIII deficiency is made by measuring the enzymatic activity of XIII, which represents the amount of functional XIIIA. XIII deficiency was originally classified by the presence or absence of antigens into two categories: Type I, characterized by the lack of both XIIIA and XIIIB; and Type II, by the lack of XIIIA alone. Based on our results from genetic analyses of XIII deficiency, we previously concluded that Type I deficiency results from genetic defects of XIIIB, and Type II from genetic defects of XIIIA [30, 31]. We then proposed a new classification of XIII deficiency at the DNA level: XIIIA deficiency (former Type II deficiency) and XIIIB deficiency (former type I deficiency), and a possible combined deficiency of XIIIA and XIIIB. This proposal was approved by the Scientific and Standardization Committee (SSC) in 1999 (http://www.med.unc.edu/isth/ 99FXIII.html). The genes for XIIIA and XIIIB are localized to 6p and 1q, respectively. Thus, XIII deficiency is inherited as an autosomal recessive trait and is caused by the absence of either subunit, as described above. In affected individuals, the first manifestation of a deficiency is usually bleeding from the umbilical cord after their birth [32]. Intracranial hemorrhage occurs in one-fourth of the patients and is the leading cause of death. Superficial bruising, and hematomas in subcutaneous tissue and muscle are common, and the bleeding at these sites may recur if not treated. Patients may bleed around joints after trauma, but have much less spontaneous hemarthrosis than do hemophiliacs. Deficiency of either XIII or ␣2 -plasmin inhibitor results in ‘delayed bleeding’ after trauma, while primary hemostasis in individuals with these

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traits is normal. The delayed bleeding is caused by premature lysis of hemostatic clots. Because of the absence of XL between ␣2-plasmin inhibitor and fibrin, these clots have decreased resistance to proteolytic degradation by plasmin. In addition to a lifelong bleeding tendency, abnormal wound healing in affected individuals and habitual spontaneous miscarriage in affected females are not uncommon. It is noteworthy that recurrent miscarriage has also been described in patients with congenital hypofibrinogenemia or afibrinogenemia. Moreover, abnormal wound healing and repetitive spontaneous miscarriage are reported in patients with congenital dysfibrinogenemia. These facts suggest the real functions of XIII and fibrin, and their importance in vivo. The definite diagnosis of a homozygote with congenital deficiency of XIII is based on the total pattern of inheritance, clinical symptoms, and laboratory tests. In addition to the typical umbilical cord bleeding after birth, the characteristic delayed bleeding after trauma strongly suggests this disorder. Deficiencies and molecular abnormalities of ␣2-plasmin inhibitor and fibrinogen should be ruled out before final diagnosis, since these disorders show symptoms similar to those described above. Screening tests for XIII deficiency are based on TGase activity. These include thromboelastography and, more specifically, a solubility test of the recalcified plasma clot in 5 M urea or 1% monochloroacetic acid. However, attention needs to be paid because these methods may overlook mild XIII deficiency. Visualization of a ␥-dimer or ␣-polymers of fibrin by SDS-polyacrylamide gel electrophoresis is useful in obtaining a rough estimate of the functional level of XIII in plasma or ammonia release. The TGase activity of XIII is quantitatively measured by amine incorporation assays. A standardized method for screening of XIII deficiency, however, needs to be established in order to ensure that patients of this disease are not to be misdiagnosed or completely overlooked. In addition, we have been collaborating with 23 other laboratories in a joint effort to establish the first international standard material for XIII in order to allow the direct comparison of its plasma levels between cases, laboratories, countries, and ethnic groups, etc. (factor XIII Standardization Working Group: http://www.med.unc.edu/isth/99FXIII.html). Immunological quantitation of XIIIA and XIIIB in plasma is essential in determining which subunit is primarily deficient, although the incidence of XIIIB deficiency appears to be rather rare. Concentrations of XIIIA and XIIIB are used routinely to be measured by the Laurell’s rocket electrophoresis method, although these measurements can now be determined more precisely by an ELISA test. Laboratory coagulation tests except for those related to XIII are within normal range.

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Diagnosis of a heterozygote, usually lacking symptoms of XIII deficiency, can be made only by the specific quantitative measurements of both the A and B subunits. Genetic diagnosis at the DNA level will help both in the prenatal detection of affected patients and in the determination of carrier states. It would be difficult, however, to perform genetic diagnosis for a new subject unless a type of mutation is known in at least one member of the patient’s family. This is because mutations causing XIII deficiency are highly heterogeneous as discussed below.

Molecular Defects in Congenital Factor XIII Deficiency

Mutations in the gene for XIIIA have been detected by in vitro amplification of DNA samples obtained from patients with XIIIA deficiency. These include a variety of missense and nonsense mutations, small deletions and insertions with or without out-of-frame shift/premature termination, and splicing abnormalities [31, 33]. A large deletion, exons IV–XI, has been reported in one case with XIIIA deficiency. The effects of these mutations on XIIIA biosynthesis have been confirmed in some cases in vitro. More than one-third of patients with XIIIA deficiency have point mutations that cause amino acid substitution. For example, rapid degradation of a novel Tyr283-Cys mutant has been ascribed to its instability characterized in an expression system employing megakaryoblastoid MEG01 cells that endogenously synthesize XIIIA [34]. Gel-filtration analysis revealed that the mutant was a monomer, while the wild-type formed a dimer. These results were consistent with the prediction by molecular modeling that the mutant molecules would be misfolded [3]. A deletion of 4 bp was observed in exon XI in one case. The deletion in this case leads to a premature termination at codon 464 [35]. RT-PCR analysis demonstrated that the level of mRNA was greatly reduced. Recombinant XIIIA bearing the mutation was also expressed in mammalian cells, showing that the mutant disappeared rapidly inside cells. Molecular modeling predicted that the dimer formation of the mutant XIIIA must be impaired by the truncation of residues 464–731. In the native XIIIA, this second part of domain III in one monomer contacts residues Pro383 and Asp384 directly in the counterpart monomer. Also found were a 20-bp deletion at the boundary of exon I/intron A, and an insertion of T in the invariant GT dinucleotide at the splicing donor site of exon IV/intron D in one patient [36]. RT-PCR analysis demonstrated that only one kind of mRNA without exon IV was detected, although its level was greatly reduced to less than 5% of normal. The other defective allele of the XIIIA gene containing the 20-bp deletion was not detected. Thus, both mutations impaired

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the normal processing of mRNA for XIIIA. Exon skipping has also been reported in several cases by other investigators. A rare case with complete deficiency of XIIIB was found in Japan and was characterized in 1990. This patient, who manifests a mild bleeding tendency, has no XIIIB and a significantly reduced level of XIIIA in plasma. The half-life of an infused placental concentrate (A2 dimer) in the patient was shorter than that in the plasma of patients with XIIIA deficiency; therefore, the lack of XIIIB most likely causes instability of XIIIA. Most recently, 2 additional Japanese patients were newly diagnosed as having XIIIB deficiency (former type I deficiency) [28]. Both patients have a previously described one-base deletion at the boundary between intron A/exon II in the XIIIB gene, heterozygously or homozygously. A founder effect was proposed for this mutation since 3 unrelated cases with XIIIB deficiency also share two 3⬘-polymorphisms. In 1 patient heterozygous for the above mutation, a novel mutation was also identified: a deletion of guanosine in exon IX (delG) of the XIIIB gene. To understand the molecular and cellular pathology of the deletion of guanosine in exon IX mutation, expression studies were performed using a cultured mammalian cell line. It was shown by pulse-chase experiments that a resultant truncated XIIIB remained inside the cells and could not be secreted into the culture medium. Furthermore, immunocytochemical examinations by epifluorescence, confocal, and electron microscopes indicated impaired intracellular transportation of the truncated XIIIB from the endoplasmic reticulum to the Golgi apparatus. No mutations in the gene for XIIIA were identified in this patient. Therefore, secretion of the truncated XIIIB must also be impaired in vivo, leading to a secondary XIIIA deficiency in the patient. These results support our previous conclusion that genetic defects of XIIIB are the basis for the former type I FXIII deficiency [30]. It is important to closely examine amino acid substitutions and the deletion/ insertion of peptide regions in order to understand the structure/function relationships of the XIII molecule, as well as its clinical implications in XIII deficiency.

Animal Model of Factor XIII Deficiency

We have identified a number of mutations in the XIIIA and the XIIIB genes in patients’ genomic DNA and also analyzed the molecular mechanisms using in vitro procedures. However, in order to completely understand the clinical pathological mechanisms of this disease, in vivo experiments are called for. Therefore, to generate its disease model and ascertain the role of XIIIB in

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vivo, we established XIIIB knockout (KO) mice by homologous recombination in embryonic stem cells. Both homozygous and heterozygous KO mice showed no marked difference from the wild-type mice in general appearance [Koseki and Ichinose, unpubl. data]. More recently, XIIIA KO mice were produced by German and Japanese groups and the phenotypes of these mice are under investigation [37] [Souri et al. unpubl. data]. A German group has established a transgenic XIIIA deficiency mouse model with an exon 7 deletion in the XIIIA gene. TGase activity in plasma was ⬍5% in homozygous XIIIA KO mice, and no ␥-dimerization of fibrin in the plasma of the XIIIA KO mice could be detected. The mortality rate was higher in the XIIIA KO mice compared to normal mice because of serious bleeding episodes (one-year survival: 70% in XIIIA KO mice vs. 100% in normal mice). When examined for the bleeding disorder in more depth, XIIIA-deficient mice were found to have an increased bleeding time, the precise mechanism of which is unclear. Thromboelastography experiments demonstrated impaired clot formation in the XIIIA KO mice: the maximal amplitude was decreased and premature clot destruction was observed. We have examined two separate lines of XIIIA KO mice, one line generated by the German group and the other by ourselves. Several types of breeding pairs were tested for fertility using XIIIA KO and wild-type mice. Although all XIIIA KO female mice were capable of becoming pregnant, most died due to excessive vaginal bleeding during gestation [38]. Abdominal incisions revealed that the uteri of the dead mice were filled with blood and that some embryos were much smaller than others within a single animal. Histopathological analyses of the small embryos revealed intrauterine fetal death in the dead XIIIA KO female mice. Furthermore, a series of histological examinations of the pregnant animals suggested that massive placental hemorrhage and subsequent necrosis developed in the uteri of the XIIIA KO mice on day 10.5 of gestation. This was true regardless of the genotypes of male mating partners or those of fetuses. These results are reminiscent of spontaneous miscarriage in human female patients with XIII deficiency, and indicate that the maternal XIII plays a critical role in uterine hemostasis and in maintaining the placenta during gestation. We also found that the XIIIA KO mice demonstrated abnormal wound healing as seen in human patients with XIII deficiency [Koseki and Ichinose, unpubl. data]. Artificial wounds became deep ulcers in XIIIA KO mice and the healing process was retarded when compared to the wild-type mice. Further analysis of these XIII KO mice will lead to an improved understanding of the physiological and pathological functions of XIII in vivo.

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Factor XIII Replacement Therapy

Both congenital and acquired XIII deficiencies have been treated successfully with XIII concentrates obtained previously from placenta and more recently from plasma [39]. Whereas placental XIII product consists of the A2 dimer alone, the plasma concentrate contains the A2B2 tetramer. Maintaining the level of plasma XIII at 10–20% of normal is sufficient to obviate symptoms, since bleeding occurs frequently in patients with less than 1% of normal, and levels of 1 and 10% of normal are all that are required for the in vitro ␥-dimerization and ␣-polymerization of fibrin, respectively. However, it is highly recommended that higher levels, such as 50–100% of normal, be maintained for replacement therapy in cases of intracranial bleeding or major surgery. The long half-life of XIII in plasma (about 10 days) and its minimum requirement for hemostasis are beneficial both for the treatment of acute bleeding and for prophylaxis. Prophylactic therapy by XIII concentrates would be desirable for severe cases who may otherwise bleed frequently [40]. Although the development of inhibitors to XIII following multiple infusions is rare, it must be considered when bleeding is uncontrollable by a therapeutic dosage of XIII. In such a case, immunosuppressive therapy may be required as well. The cloning of human XIII made it possible to prepare recombinant XIIIA as a therapeutic material that is free of viral contamination. The recombinant XIIIA is confirmed to be comparable to the native XIIIA protein with respect to structural and functional properties; it will thus provide a safe substitute for the previous placental and the current plasma XIII concentrates in transfusion therapy. XIII has also been introduced as an essential ingredient in ‘tissue glue’, which has been used widely for all types of surgery and for the treatment of trauma [41]. References 1 2 3

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Ichinose A, Bottenus RE, Davie EW: Structure of transglutaminases. J Biol Chem 1990;265: 13411–13414. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, et al: The sequence of the human genome. Science 2001;291:1304–1351. Souri M, Yee VC, Kasai K, Kaneshiro T, Narasaki K, Castaman G, Ichinose A: Novel Y283C mutation of the A subunit for coagulation factor XIII: Molecular modelling predicts its impaired protein folding and dimer formation. Br J Haematol 2001;113:652–654. Ichinose A, Davie EW: Characterization of the gene for the a subunit of human factor XIII (plasma transglutaminase), a blood coagulation factor. Proc Natl Acad Sci USA 1988;85:5829–5833. Kida M, Souri M, Yamamoto M, Saito H, Ichinose A: Transcriptional regulation of cell type-specific expression of the TATA-less A subunit gene for human coagulation factor XIII. J Biol Chem 1999; 274:6138–6147. Bottenus RE, Ichinose A, Davie EW: Nucleotide sequence of the gene for the b subunit of human factor XIII. Biochemistry 1990;29:11195–11209.

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Ichinose A, Hendrickson LE, Fujikawa K, Davie EW: Amino acid sequence of the a subunit of human factor XIII. Biochemistry 1986;25:6900–6906. Kaetsu H, Hashiguchi T, Foster D, Ichinose A: Expression and release of the a and b subunits for human coagulation factor XIII in baby hamster kidney (BHK) cells. J Biochem (Tokyo) 1996;119: 961–969. Yee VC, Pedersen LC, Le Trong I, Bishop PD, Stenkamp RE, Teller DC: Three-dimensional structure of a transglutaminase: Human blood coagulation factor XIII. Proc Natl Acad Sci USA 1994;91: 7296–7300. Weiss MS, Metzner HJ, Hilgenfeld R: Two non-proline cis peptide bonds may be important for factor XIII function. FEBS Lett 1998;423:291–296. Yee VC, Pedersen LC, Bishop PD, Stenkamp RE, Teller DC: Structural evidence that the activation peptide is not released upon thrombin cleavage of factor XIII. Thromb Res 1995;78:389–397. Turner BT, Jr., Sabo TM, Wilding D, Maurer MC: Mapping of factor XIII solvent accessibility as a function of activation state using chemical modification methods. Biochemistry 2004;43: 9755–9765. Sadasivan C, Yee VC: Interaction of the factor XIII activation peptide with alpha-thrombin: Crystal structure of its enzyme-substrate analog complex. J Biol Chem 2000;275:36942–36948. Fox BA, Yee VC, Pedersen LC, Le Trong I, Bishop PD, Stenkamp RE, Teller DC: Identification of the calcium binding site and a novel ytterbium site in blood coagulation factor XIII by X-ray crystallography. J Biol Chem 1999;274:4917–4923. Ichinose A, McMullen BA, Fujikawa K, Davie EW: Amino acid sequence of the b subunit of human factor XIII, a protein composed of ten repetitive segments. Biochemistry 1986;25:4633–4638. Barlow PN, Baron M, Norman DG, Day AJ, Willis AC, Sim RB, Campbell ID: Secondary structure of a complement control protein module by two-dimensional 1H NMR. Biochemistry 1991;30: 997–1004. Schwarzenbacher R, Zeth K, Diederichs K, Gries A, Kostner GM, Laggner P, Prassl R: Crystal structure of human beta2-glycoprotein I: Implications for phospholipid binding and the antiphospholipid syndrome. EMBO J 1999;18:6228–6239. Naidu YM, Rosen EM, Zitnick R, Goldberg I, Park M, Naujokas M, Polverini PJ, Nickoloff BJ: Role of scatter factor in the pathogenesis of AIDS-related Kaposi sarcoma. Proc Natl Acad Sci USA 1994;91:5281–5285. Nishiura H, Shibuya Y, Matsubara S, Tanase S, Kambara T, Yamamoto T: Monocyte chemotactic factor in rheumatoid arthritis synovial tissue. Probably a cross-linked derivative of S19 ribosomal protein. J Biol Chem 1996;271:878–882. Lorenz R, Olbert P, Born P: Factor XIII in chronic inflammatory bowel diseases. Semin Thromb Hemost 1996;22:451–455. Sano H, Izumida M, Shimizu H, Ogawa Y: Risk factors of renal involvement and significant proteinuria in Henoch-Schonlein purpura. Eur J Pediatr 2002;161:196–201. Yamada T, Yoshiyama Y, Kawaguchi N, Ichinose A, Iwaki T, Hirose S, Jefferies WA: Possible roles of transglutaminases in Alzheimer’s disease. Dement Geriatr Cogn Disord 1998;9:103–110. Wozniak G, Noll T, Akinturk H, Thul J, Muller M: Factor XIII prevents development of myocardial edema in children undergoing surgery for congenital heart disease. Ann NY Acad Sci 2001;936: 617–620. Ichinose A: Factor XIII: state of the art. Minerva Biotechnol 2002;14:121–128. Ariens RA, Lai TS, Weisel JW, Greenberg CS, Grant PJ: Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms. Blood 2002;100:743–754. Ariens RA, Philippou H, Nagaswami C, Weisel JW, Lane DA, Grant PJ: The factor XIII V34L polymorphism accelerates thrombin activation of factor XIII and affects cross-linked fibrin structure. Blood 2000;96:988–995. Okumura T, Yamada T, Park SC, Ichinose A: No Val34Leu polymorphism of the gene for factor XIIIA subunit was detected by ARMS-RACE method in three Asian populations. J Thromb Haemost 2003;1:1856–1857. Koseki S, Souri M, Koga S, Yamakawa M, Shichishima T, Maruyama Y, Yanai F, Ichinose A: Truncated mutant B subunit for factor XIII causes its deficiency due to impaired intracellular transportation. Blood 2001;97:2667–2672.

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Reiner AP, Heckbert SR, Vos HL, Ariens RA, Lemaitre RN, Smith NL, Lumley T, Rea TD, Hindorff LA, Schellenbaum GD, Rosendaal FR, Siscovick DS, Psaty BM: Genetic variants of coagulation factor XIII, postmenopausal estrogen therapy, and risk of nonfatal myocardial infarction. Blood 2003;102:25–30. Ichinose A, Izumi T, Hashiguchi T: The normal and abnormal genes of the a and b subunits in coagulation factor XIII. Semin Thromb Hemost 1996;22:385–391. Ichinose A, Souri M, Izumi T, Takahashi N: Molecular and genetic mechanisms of factor XIII A subunit deficiency. Semin Thromb Hemost 2000;26:5–10. Seitz R, Wolf M, Egbring R, Havemann K: The disturbance of hemostasis in septic shock: Role of neutrophil elastase and thrombin, effects of antithrombin III and plasma substitution. Eur J Haematol 1989;43:22–28. Anwar R, Miloszewski KJ: Factor XIII deficiency. Br J Haematol 1999;107:468–484. Souri M, Ichinose A: Impaired protein folding, dimer formation, and heterotetramer assembly cause intra- and extracellular instability of a Y283C mutant of the A subunit for coagulation factor XIII. Biochemistry 2001;40:13413–13420. Takahashi N, Tsukamoto H, Umeyama H, Castaman G, Rodeghiero F, Ichinose A: Molecular mechanisms of type II factor XIII deficiency: Novel Gly562-Arg mutation and C-terminal truncation of the A subunit cause factor XIII deficiency as characterized in a mammalian expression system. Blood 1998;91:2830–2838. Izumi T, Nagaoka U, Saito T, Takamatsu J, Saito H, Ichinose A: Novel deletion and insertion mutations cause splicing defects, leading to severe reduction in mRNA levels of the A subunit in severe factor XIII deficiency. Thromb Haemost 1998;79:479–485. Lauer P, Metzner HJ, Zettlmeissl G, Li M, Smith AG, Lathe R, Dickneite G: Targeted inactivation of the mouse locus encoding coagulation factor XIII-A: Hemostatic abnormalities in mutant mice and characterization of the coagulation deficit. Thromb Haemost 2002;88:967–974. Koseki-Kuno S, Yamakawa M, Dickneite G, Ichinose A: Factor XIII A subunit-deficient mice developed severe uterine bleeding events and subsequent spontaneous miscarriages. Blood 2003; 102:4410–4412. Brackmann HH, Egbring R, Ferster A, Fondu P, Girardel JM, Kreuz W, Masure R, Miloszewski K, Stibbe J, Zimmerman R, et al: Pharmacokinetics and tolerability of factor XIII concentrates prepared from human placenta or plasma: A crossover randomised study. Thromb Haemost 1995; 74:622–625. Anwar R, Minford A, Gallivan L, Trinh CH, Markham AF: Delayed umbilical bleeding – A presenting feature for factor XIII deficiency: clinical features, genetics, and management. Pediatrics 2002;109:E32 (31–37). Dunn CJ, Goa KL: Fibrin sealant: A review of its use in surgery and endoscopy. Drugs 1999;58: 863–886.

Dr. Akitada Ichinose Department of Molecular Patho-Biochemistry and Patho-Biology Yamagata University School of Medicine, Yamagata 990–9585 (Japan) Tel. ⫹81 23 628 5275, Fax ⫹81 23 628 5280, E-Mail [email protected]

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Transglutaminases of Lower Organisms Ramakrishna U. Raoa, Ramaswamy Chandrashekarb, Kapil Mehtac a

Washington University School of Medicine, St. Louis, Mo., bResearch and Development, IDEXX Laboratories, Westbrook, Maine, USA, cThe University of Texas M. D. Anderson Cancer Center, Houston, Tex., USA

Introduction

Bacteria, fungi, viruses and several eukaryotes have unique ability to survive and protect themselves under adverse physiologic conditions and environmental stress. They achieve this by synthesizing rigid, biochemically complex structures around them which are highly stable and resistant to mechanical and chemical exposures. Biochemically, these structures are composed of complex carbohydrates, such as chitin and heavily cross-linked with a scaffold of proteins to form complex structures such as exoskeletons (sheath, cuticle, cuticulins and epicuticle). Transglutaminases (TGs; EC2.3.2.13) are a family of enzymes that share catalytic function with thioredoxin (EC1.8.4.10) and protein disulphide isomerases (PDI; EC5.3.4.1) and catalyze protein cross-link reaction by establishing
(-glutamyl)lysine isopeptide bonds. The isopeptide bonds are of great physiologic significance because once formed, they cannot be hydrolyzed by known enzymes of the eukaryote system and they exhibit high resistance to reducing agents, detergents, and chaotropic agents. Therefore, protective structures such as sheath, cuticle, epicuticle, bacterial cell walls and viral core proteins synthesized by microorganisms likely involve the active participation of TGs. In this chapter, we describe nonmammalian TGs and their possible role in growth, development, and survival of small organisms. We give special emphasis to bacterial and filarial nematode TGs since they are the most well characterized enzymes, and their functions at the molecular level have been well studied.

TGs and Their Homologs Involved in Protein Cross-Linking

Similar to TGs, PDIs are a family of multifunctional endoplasmic reticulum (ER)-resident enzymes that belong to the thioredoxin superfamily of proteins. They can act as molecular chaperones, catalyze disulphide bond formation during protein folding [1, 2], and perform specialized functions as exemplified by  subunit of P4H [3]. PDIs contain 2 redox-active domains near N- and C-termini of the protein that are similar to thioredoxins and both can play a role in disulphide isomerase activity. A number of ER proteins differ from the major PDI isozyme in that they contain two (e.g. ERp60, ERp55) or three (e.g. ERp72) thioredoxin domains, but all of them exhibit PDI activity. Several recent studies have revealed that PDI and thioredoxin enzymes can catalyze transamidation reactions, which can be linked with thioredoxin motif present in these proteins. Thioredoxin is a multifunctional enzyme that participates in many processes, including DNA synthesis and DNA repair. Published data also suggest that these enzymes play a role in cell division in animals. Thioredoxins are mainly localized at the site of DNA replication and transcription. Thioredoxin is required for both meiotic cell division and embryo development in Drosophila. Moreover, thioredoxin is essential for generating sulfur through sulfate reduction and plays a role in reducing methionine for protein repair of oxidized side chains. Methionine is also reduced by thioredoxin in the assembly of proteins [4, 5]. Furthermore, thioredoxin systems involve two redox conditions of a dithiol/disulphide group and thioredoxin contains an active site that involves cysteine (Cys) residues. These residues reverse to from a dithiol (-SH HS-) group to a disulphide bridge (-S-S-). The oxidized protein is a disulphide with one bridge between two Cys, whereas the reduced protein is a dithiol with two Cys. These observations suggest that PDI, thioredoxins, and TG have a common biological role in regulating certain cellular and biological processes. TGs are widely distributed enzymes and have been characterized in a diverse group of organisms including, vertebrates, invertebrates, plants, and bacteria [6, 7]. The evidence published in the literature suggests that these enzymes play an important role in various biologic processes that are beneficial to their hosts. In mammals, particularly in humans, at least nine TGs have been described which differ in their specificity towards the substrate proteins. They are encoded by separate genes [8], and reveal a high degree of sequence homology as described in Chapter 1. In general, TGs require Ca2 for catalytic activity. However, in recent years, TG-like activity independent of Ca2 has been reported in eukaryotes and bacteria (table 1) and some of the TG-encoding genes in these organisms have been successfully cloned. Although, some TGs from these organisms have retained moderate to significant homology to the known mammalian TGs, others have shown major differences with little or no structural similarities to

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Table 1. Transglutaminases and their homologues from the lower-order organisms Molecular weight, kDa

Ca2 requirement

Algae Chlamydomonas reinhardtii

72

Yes

Fungi Candida albicans Phytophthora sojae

? 42

? Yes

Other eukaryotes and bacteria Bacillus subtilis Bacillus circulans Escherichia coli Physarum polycephalum Streptoverticillium spp. Streptomyces mobaraensis

28 45 110 96–101 38 ?

No Yes No Yes No Yes

Protozoans Giardia lamblia Plasmodium falciparum

26, 50, 13 ?

Yes No

Invertebrates Ciona intestinalis (Sea squirt) Limulus spp. (Horseshoe crab) Orconectes spp. (Crayfish) Grasshopper

80 86 86 97

Yes Yes Yes Catalytically inactive Yes

55 57 57

Yes Yes Yes

78

Yes

Source

Strongylocentrotus spp. (Sea urchin) Nematodes Brugia malayi Caenorhabditis elegans Dirofilaria immitis Other lower vertebrates Pagellus bogaraveo (Red sea bream)

mammalian counterparts. For example, TGs cloned from two bacteria (Bacillus subtilis and Streptoverticillium spp.) exhibit no homology to any of the known TGs or to each other [9]. Similarly, cytotoxic necrotizing factor-1 and -2 from Escherichia coli and dermonecrotic toxin from Bordetella pertussis both catalyze deamination of a specific glutamine residue in Rho GTPases. Both these proteins, though retain the catalytic Cys and histidine (His) residues and catalyze

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the transamidation reaction [10], do not exhibit similarity to mammalian TGs. In contrast, multiple proteins (in silico) from Mycobacteria spp., gene profiling data generated by PSI-BLAST program of sequenced genomes, contain sequences in three motifs that center around conserved Cys, His, and asparagine (Asp) residues that form the catalytic triad in the structurally characterized mammalian TGs [9]. The presence of multiple TG homologs in Mycobacteria spp. may imply that these proteins play a role in the development of rigid membrane structures that have been observed in these bacteria. Moreover, the similarity of the catalytic triad and the reaction mechanism indicates that TGs share the core structural fold with thiol proteases. On the basis of the experimentally demonstrated activity of the Methanobacterium phage pseudomurein endoisopeptidase [11], it was proposed that many, if not all, microbial homologs of the TGs are proteases and that the eukaryotic TGs have evolved from an ancestral protease. Therefore, it is likely that microbial homologs of TG may act as proteases. Interestingly, the virulence factor of Candida albicans is its hyphal wall protein 1 (HWP1) and hyphal wall protein1 is a TG substrate, and is used by C. albicans to adhere to the intestinal epithelium [12]. TG secreted from bacteria may play a role in cleaving the host proteins as a part of their intracellular life cycle, cell-to-cell migration or in the induction of apoptosis. These proteins can thus serve as new therapeutic targets. Similarly, the proteins that share homology to mammalian TG, but lack the active site domain (e.g. annulin in the grasshopper) may perform structural and proteinbinding functions similar to those of 4.2-band protein in red blood cells or alternatively may function as dominant-negative regulators of the active enzyme. Phylogenetic analysis of several known TGs shows that bacterial, arthropod, worm and mammalian enzymes are distantly related (fig. 1); and in general, both bacterial and worm TGs form different groups, separate from mammalian TGs suggesting their prokaryotic and invertebrate origin. Interestingly, few arthropod TGs were grouped close to the mammalian TGs and may have functional similarities. However, unlike TGs of other bacteria, fungi and worms, TG from Physarum spp. was located close to rat TG4 as reported earlier. Worm TG/PDI although exhibits dual functional activities, the analysis branch them into separate subgroups. More work is needed to understand the evolutionary significance of these TGs and their functional similarities.

TG in Growth and Development

TG-catalyzed post-translational modification of proteins has been implicated in several biological processes. For example, in vertebrates TG gene expression specifically characterizes cells undergoing apoptosis, the physiologic form of cell

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82 87

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Fig. 1. A representative phylogenetic tree constructed using TG amino acid sequences to show the relationship between several transglutaminases of lower organisms and mammals. Transglutaminase amino acid sequences were aligned using Clustal X multiple alignment software. Alignments were optimized and used to generate a phylogenetic tree using MEGA2 program. Neighbor-joining tree generated by using gamma distance model with pair wise deletion was tested by bootstrapping using 500 replications. GI numbers with lower organisms (Bac  bacteria; Fun  Fungi; Pro  Protozoan; Arth  Arthropod; Ins  Insect; Asc  Ascidian; Wor  Worms; Fis  Fish) and mammalian (TG 1, 2, 3, 4, 5, 7 and Band 4.2) shown here are from the different organisms used in the construction of phylogeny tree.

death that plays a critical role during reproduction and development [13]. In the early 1990s, we were the first to report that in simple multicellular organisms, such as filarial nematodes, TG-catalyzed protein cross-linking reactions are intimately involved in growth and differentiation process of early embryonic stages of these parasites [14]. The main function of fecund female worms in their vertebrate host is to produce millions of embryos that subsequently differentiate into first-stage larvae (microfilariae). This in utero differentiation of early

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a

b

c

Fig. 2. Effect of TG inhibition on the development and differentiation of embryos to first-stage larvae in filarial nematode, Brugia malayi. a, b Uterine contents from adult female worm show numerous embryos after 72 h of incubation in medium alone or (c) medium containing monodansylcadaverine (100 M). Magnification: a, 40 and b and c, 400.

embryos to mature microfilariae is accompanied by substantial post-translational modifications of proteins and rearrangement of cellular structures [15]. The evidence that TG is involved in the growth, development, and maturation of microfilariae and other larval stages is accumulating and is supported by the observations that the membranous structures, such as cuticle, epicuticle, and sheath of these organisms are highly enriched with TG-catalyzed
(-glutamyl)lysine isopeptides [16, 17]. Moreover, inhibition of TG activity by enzyme-specific inhibitors completely blocks the differentiation and production of microfilariae [14]. Examination of uterine contents from TG-inhibited Brugia worms revealed that TG activity is absolutely critical for in utero development and maturation of embryos. Female worms when treated with the enzyme inhibitors showed deformed embryos that had failed to differentiate into discrete stages (fig. 2). Further studies with Brugia and other nematodes demonstrated that the inhibition of TG can completely block protein cross-linking reactions of the host and parasite proteins, the biochemical process that is essential for the assimilation of the new sheath and cuticle in developing embryos and microfilariae [16–18]. The significance of TG-catalyzed protein cross-linking reactions in growth and development of larvae has been further supported by the observations that metabolically labeled proteins are effectively incorporated into the sheath and cuticle of live microfilariae only in the presence of enzymatically active TG [18]. Adult worms developed from the larval stages clearly show 3 basic components of collagenous cuticle (median and basal layers) and noncollagenous cuticle (external cortical layer). The collagens and other proteins formed in these layers are byproducts of protein cross-linking and are highly insoluble to any detergents. Moreover, the adult worms of several nematodes showed high levels of TG activity [19, 20]. TG activity can be detected in both male and female worms, although the enzyme levels in female worms are generally much

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higher. The TG protein expression is also evident in the gut epithelium and in developing embryos. In male worms, the expression of native TG can be observed in somatic tissues. High levels of active TG could be observed in developing embryos inside the female worms of human filarial nematode parasite (B. malayi) by probing in situ incorporation of the fluorescent enzyme substrate, monodansylcadaverine [18]. In addition, immunohistochemical studies reveal that TG protein in adult female worms of the canine heartworm nematode, D. immitis, is predominantly localized in the hypodermis and muscle cells, the site of high metabolic activity [21, 22]. Inhibition of transamidation activity affected the worm viability in several nematodes [23, 24] and microfilarial production of Brugia malayi in vitro [23]. Morphological and biochemical evidence strongly support the direct involvement of TG-catalyzed cross-linking reactions in molting process of the third-stage infective larvae of filarial nematodes [25, 26]. Molting of larvae and viability was also affected in nematode Nippostrongylus braziliensis when treated with a synthetic TG inhibitor N-benzyloxycarbonyl-D,L--(3-bromo-4,5-dihydroisoxazol5-yl)-alanine benzylamide (BOCAB) [27]. Similar to filarial nematodes, the free-living nematode Caenorhabditis elegans express a Ca2-dependent TG whose activity is associated with a 61-kDa protein [28]. Immunohistochemical studies reveal that the expression of TG protein is predominantly localized in the intestinal cells of adult worms. Higher expression of TG protein has also been observed in L1-stage larvae than in other larval stages. The increased TG activity in the L1 stage was associated with apoptotic death of cells during this stage. Interestingly, TG activity was lower in ced-3, ced-4, and ced-9 mutants when compared with the wild-type worms. It is noteworthy that mutations in ced-3, ced-4, and ced-9 genes confer survival phenotype to cells that otherwise will die through apoptosis. In contrast, the ced-2 and ced-5 mutants in which the cells die normally, but do not undergo phagocytosis, showed lower levels of the enzyme activity. The lower levels of enzyme activity in ced-3 mutant were not accompanied by a parallel decrease in
(-glutamyl)lysine isopeptide bonds. In the ced-5 mutant and ced-5/ced-7-double mutants, in which the cells undergo normal apoptosis but are not phagocytosed, levels of isopeptide cross-links were high and several immunopositive corps were detected in the head of the adult worms, where most neuronal cells normally die. These data suggest that the TG expression in C. elegans is either not linked with apoptotic events or other protein(s) with TG activity, may participate in these events. Interestingly, TG-catalyzed reactions also occur during fertilization and reproduction of small organisms. For example, in vivo cross-linking of nucleosomal proteins catalyzed by nuclear TG in starfish (Asterina pectinifera) sperm has been reported and the reaction was found to be triggered by egg jelly [29].

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Gene Sequence Analysis of TGs

Genes coding for various members of TG family have been characterized in vertebrates (see, Chapter 1) and invertebrates including ascidians, grasshopper and Limulus [21, 30–36]. Cloning of bacterial TG has been reported from Bacillus subtilis [37] and streptoverticillium spp. [38], but their protein sequences do not match either with each other or to other members of the TG family. The crystal structure of TG from Streptovercillium has been recently described [39] and reveals some interesting features. The overall structure of bacterial TG is completely different from the mammalian TGs. Nevertheless, Cys64, Asp255, and His274 of the bacterial TG perfectly superimpose with Cys, Asp, and His catalytic triad of the mammalian TGs, which is sufficient to confer transamidation activity. On the other hand, TG gene recently cloned from Physarum spp., demonstrates a remarkable similarity to mammalian TGs, including the conserved Cys, His, and Asp catalytic triad [40]. Moreover, similar to the mammalian tissue-type TG, a GTP-binding domain has been observed in the sequence of Physarum TG and the enzyme protein can catalyze GTP-hydrolyzing activity and GTP can inhibit the transamidation activity of the bacterial TG in a manner, similar to the tissuetype TG [40]. Although the TG activity has been described in C. elegans [28], no gene with significant homology to vertebrate or bacterial TG gene has been identified in its (nearly) complete genome sequence database. This suggests that a TG homolog is among the gene products that are not yet sequenced or that C. elegans nematode has a completely different TG that has no resemblance to other members of TG superfamily. In fact, the evidence obtained so far in other nematodes strongly supports the latter contention. PDI and other thioredoxin motif- containing proteins have been shown to catalyze the transamidation reaction in a calcium-dependent manner, similar to the mammalian TG. It is, therefore, likely that in C. elegans, these proteins could catalyze the protein cross-linking functions. Indeed, evidence is accumulating that proteins with thioredoxin motifs, such as Erp60 [41], PDI-3 [42], and Erp57 [43] from C. elegans can catalyze transamidation reaction and effectively cross-link proteins. Conversely, TGs of the mammalian origin (e.g. tissue-type TG) can also catalyze PDI activity [44]. We first purified TG protein to homogeneity from a filarial nematode, B. malayi based on its ability to catalyze calcium-dependent transamidation reaction [19]. The N-terminal amino acid sequence of this putative purified TG protein showed no homology with any of the known TGs or other protein sequences in the GenBank database. An antibody against the synthetic N-terminal peptide of B. malayi TG, recognized a 56-kDa protein in immunoblots

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of crude extracts from B. malayi and the dog parasite, Dirofilaria immitis [19–21]. Primers based on the N-terminal sequence of D. immitis TG cDNA was cloned. The nucleotide sequence or the deduced amino acid sequence of this cDNA clone showed no homology to any of the known TGs. The TG protein expressed in E. coli, however, was fully functional and like other TGs, recombinant D. immitis protein requires Ca2 for the catalytic activity. In addition, the activity of TG protein could be inhibited by TG-specific inhibitors, ammonia, primary amines, EDTA, and –SH group-blocking reagents [19]. Interestingly, the nematode TG shows significant homology to a PDI-related ER protein, ERp60. Another interesting feature of nematode TG is that it contains two distinct regions that are identical to the active site sequences of PDI/thioredoxin family of proteins [21]. More importantly, it exhibits the catalytic triad composed of Cys, His, and Asp residues suggesting that PDI and PDI-like proteins (such as Erp60) play an important role in catalyzing transamidation and other post-translational modifications during the growth and development of these parasites. Recently, a homolog of TG having significant homology to PDI and thioredoxin active sites was cloned and characterized at the protein level from nematode B. malayi [45]. Preliminary studies suggest that individuals living in areas, endemic for filarial infections (endemic normals) harbor high titers of anti-TG antibodies [45], implicating a possible role for TG in mounting protective immune response in the human host against filarial parasites. These initial observations warrant further evaluation of TG as a target for developing effective vaccine and/or chemotherapeutic agents for controlling infections caused by these nematode parasites. Three isoforms of PDI have been recently isolated from another eukaryote, Giardia lamblia, exhibited Ca2-dependent transamidation activity [46, 47]. In addition to having TG activity, these proteins can also catalyze PDI reaction as determined by their ability to reconstitute the scrambled RNase into its native form. Thus, identification of these novel homologs of TG with ‘thioredoxinactive sites’ suggests an interesting aspect of protein post-translational modification in primitive forms of living organisms. It is possible that TGs require PDI activity to promote catalysis of cross-linking reactions in these organisms and vice versa.

Amino Acid Residues Involved in Transamidation Activity of Thioredoxins and PDIs

PDIs are members of the thioredoxin family of enzymes. They catalyze the formation, isomerization, and reduction of disulphide bonds in the ER, and

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display chaperone activity. PDIs have a characteristic a–b–b –a –c domains, where domains a and a show sequence homology to thioredoxins, including the reactive CXXC thioredoxin motif and catalyze the isomerase activity. The c domain contains a putative calcium-binding site. Ca2-dependent TGs have a Cys residue in their active site and cross-link proteins using a Cys-His-Asp catalytic triad in an acyl transfer reaction where -carboxamide group of a protein-bound glutamine residue is the acyl donor and
-amino group of a lysine residue or a primary amine serves as acyl acceptor. As discussed in previous sections, D. immitis and B. malayi proteins with no sequence homology to any of the known TGs, display transamidation activity. However, they show significant homology to a PDI-related protein, Erp60, and the recombinant protein exhibits both PDI and TG activities. Similarly, three isoforms of PDI that have been isolated from the protozoan Giardia lamblia [46, 47], C. elegans PDI-1,2,3 [41–43], and a mammalian PDI [44] are all capable of catalyzing the transamidation reaction. Mutagenesis studies revealed that the second Cys in CXXC motif of the thioredoxin domain of C. elegans PDI-3 and His adjacent to it are critical for TG activity of this enzyme [41], suggesting that the crucial catalytic residues for transamidation activity are localized in the thioredoxin-like domains of PDIs. This indicates the possibility that thioredoxins themselves are able to accomplish TG-type catalytic reactions. Moreover, E. coli and human PDIs containing thioredoxin motifs have been found to display Ca2-dependent TG activity [44]. Analysis of known PDIs and thioredoxin sequences has revealed the presence of conserved Cys, His, and Asp residues required for TGs to catalyze the incorporation of primary amines into protein-bound glutamine residues. The three-dimensional structures of PDI and thioredoxin show that these residues are in close proximity to achieve transglutamination of substrate proteins. Evidence for this also comes from the fact that filarial worm thioredoxin sequences contain conserved Cys, His, and Asp residues. Interestingly, some of the known human and bacterial thioredoxins, containing catalytic triad, show TG activity and can be inhibited by blocking His residues [39]. Recent findings suggest that PDIs of both higher and lower organisms can function like TG cross-linking enzymes by catalyzing the formation, reduction and isomerization of disulphide bonds. In addition, evidence is accumulating that thioredoxins, like their functionally related proteins PDIs, can also serve as polypeptide-binding proteins and participate in protein-folding mechanism [48]. Dual functional activity has also been observed in G. lamblia PDI-3 [46–47]. Recently, several PDI orthologs have been discovered and Erp57, Erp60, Erp72 are a few of them that are widely distributed and have also been reported in several nematodes like Trichinella, Strongyloides, Haemonchus and Ostertagia spp [41]. PDI-protein sequence

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alignments with nematode Erp60 clearly show conserved catalytic triad of Cys, His, and Asp residues. Both PDI and TG are known to use Cys as an essential residue in the active site for transglutamination functions. The protein crystallography of C. elegans PDI and E. coli thioredoxins reveal that His, Cys, and Asp residues in these structures closely resemble the His, Cys, and Asp catalytic triad of other transglutmainases [42]. In summary, these observations suggest that the shared enzymatic activities among various members of the PDI, thioredoxin and TG enzyme families might have important biological implications, especially in small organisms whose main function is to reproduce and maintain the progeny.

Conclusion

Identification of novel TGs and other proteins with ability to catalyze transamidation reactions is likely to provide important leads in the areas of therapeutic development and industrial applications such as food processing. For example, TGs produced by microorganisms have been commercially used to replace meat products with vegetable proteins without compromising the taste and texture [49, 50]. Similarly, all the scientific studies show that TG-catalyzed reactions play a pivotal role in the growth, development, and survival of nematode parasites, and thus these enzymes may be an excellent biochemical targets for controlling debilitating conditions caused by these organisms in humans and animals. It is estimated that filarial nematodes alone afflict about 190 million people worldwide and continues to be a major cause of morbidity and suffering to the mankind for centuries. Furthermore, with the insurgence of HIV-infections, hepatitis and tuberculosis, dual infections with filarial worms in some of these individuals may increase the clinical outcome of the disease pathogenicity. Since TG-mediated posttranslational modification of viral, bacterial, and nematode parasite proteins has been well documented, these proteins may be suitable targets for the drug development. Modern drug discovery platforms should enable us to identify new and nontoxic inhibitors which could lead to potential nematicides targeting parasite TGs. Similar to nematodes, TG-mediated reactions play a role in the development and survival of two protozoan parasites, Plasmodium parasite [51] which is responsible for resurgence of malaria and Giardia [46, 47] that causes severe water-borne diarrheal infections in the tropics. Therefore, it is tempting to speculate that the inhibition of TG-catalyzed reactions may offer pathogen selective and therefore, less toxic agents to control and eradicate the debilitating and life-threatening parasitic infections.

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Acknowledgments The authors would like to thank Drs R. Singh, E. Devarajan and P. Kaliraj for their contributions to this project. We also wish to thank Dr. T. V. Venkatesh, Bioinformatics Division, Monsanto, St. Louis, MO for performing phylogeny studies.

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Wada F, Nakamura A, Masutani T, Ikura K, Maki M, Hitomi K: Identification of mammalian-type transglutaminase in Physarum polycephalum. Evidence from the cDNA sequence and involvement of GTP in the regulation of transamidating activity. Eur J Biochem 2002;269:3451–3460. Eschenlauer SC, Page AP: The Caenorhabditis elegans ERp60 homolog protein disulphide isomerase-3 has disulphide isomerase and transglutaminase-like cross-linking activity and is involved in the maintenance of body morphology. J Biol Chem 2003;278:4227–4237. Blasko B, Madi A, Fesus L: Thioredoxin motif of Caenorhabditis elegans PDI-3 provides Cys and His catalytic residues for transglutaminase activity. Biochem Biophys Res Commun 2003;303: 1142–1147. Natsuka S, Takubo R, Seki R, Ikura K: Molecular cloning and expression of Caenorhabditis elegans ERp57-homologue with transglutaminase activity. J Biochem 2001;130:731–735. Hasegawa G, Suwa M, Ichikawa Y, Ohtsuka T, Kumagai S, Kikuchi M, Sato Y, Saito Y: A novel function of tissue-type transglutaminase: Protein disulphide isomerase. Biochem J 2003;373: 793–803. Devarajan E, Mishra PK, Thirugnanam S, Mehta K, Chandrashekar R, Perumal K: Molecular characterization of a Brugia malayi transglutaminase. Parasitol Res 2004;93:145–150. Knodler LA, Noiva R, Mehta K, McCaffery JM, Aley SB, Svard SG, Nystul TG, Reiner DS, Silberman JD, Gillin FD: Novel protein-disulphide isomerases from the early-diverging protist Giardia lamblia. J Biol Chem 1999;274:29805–29811. Davids BJ, Mehta K, Fesus L, McCaffery JM, Gillin FD: Dependence of Giardia lamblia growth and encystation on novel transglutaminase activity. Mol Biochem Parasitol 2004;136:173–180. Freedman RB, Hirst TR, Tuite MF: Protein disulphide isomerase: Building bridges in protein folding. Trends Biochem Sci 1994;19:331–336. Zhu Y, Rinzema A, Tramper J: Microbial transglutaminase – A review of its production and application in food processing. Appl Microbiol Biotechnol 1995;44:277–282. Collighan R, Cortez J, Griffin M: The biotechnological applications of transglutaminases. Minerva Biotechnol 2002;14:143–148. Adini A, Krugliak M, Ginsburg H, Li L, Lavie L, Warburg A: Transglutaminase in Plasmodium parasites: Activity and putative role in oocysts and blood stages. Mol Biochem Parasitol 2001; 117:161–168.

Dr. Ramakrishna Rao Department of Internal Medicine, Infectious Diseases Division Washington University School of Medicine St. Louis, MO 63110 (USA) Tel. 1 314 454 8982, Fax 1 314 454 5392, E-Mail [email protected]

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Mehta K, Eckert R (eds): Transglutaminases. Prog Exp Tum Res. Basel, Karger, 2005, vol 38, pp 223–247

Transglutaminases of Higher, Lower Plants and Fungi Stefano Del Duca, Donatella Serafini-Fracassini Dipartimento di Biologia e.s., Università di Bologna, Bologna, Italy

Introduction

Among the different enzymatic functions that transglutaminases (TGases) fulfill in animals [this book and 1, 2], transamidation catalysis, which yields the covalent conjugation of polyamines (PAs) and other amine donors (among which lysyl-residues) to ␥-carboxamide groups of protein endoglutamine residues, is the activity which has been most extensively studied in plants. The three main PAs (putrescine, PU; spermidine, SD; spermine, SM) are ubiquitous growth factors in animals and plants and differ in both their number of positive charges exhibited at the cell physiological pH (2 in PU; 3 in SD and 4 in SM) and their backbone length (SM: 14.6 Å; SD: 11.12 Å; PU: 6.5 Å) (fig. 1a). Cadaverine, 1,5 diamino pentane, is a natural polyamine. The two terminal amino groups of PAs conjugate to one or two glutamyl residues giving rise either to mono-(␥-glutamyl)-PAs (mono-PAs) or bis-(␥-glutamyl)-PAs (bis-PAs) (fig. 1b). The additional positive charges introduced by proteinbound PAs may induce protein conformational changes. Moreover, bis-PA derivatives can form in proteins both inter- and intramolecular cross-links. The backbone length of the PAs determines the length of the cross-link it forms: bis-(␥-glutamyl)-SD (bis-SD) bridges, and even more so those involving bis(␥-glutamyl)-SM (bis-SM), span greater distances than those formed by bis-(␥-glutamyl)-PU (bis-PU). The link formed between glutamyl and lysyl residues is much shorter than those involving PAs (fig. 1c). Since several PAs can cross-link more proteins simultaneously, high molecular complexes may form. The binding is highly specific and is probably primarily dependent on substrate conformation [1]. The use of labeled PAs in both in vitro and in vivo plant experiments allows the identification of TGase-modified proteins

a

b

c

SD

PU

mono-(␥-glutamyl)-PAs

SM

bis-(␥-glutamyl)-PAs

glut-lys

Fig. 1. a The three main aliphatic polyamines present in plants, putrescine (PU), spermidine (SD) and spermine (SM), have different backbone length and positive charges. b Transglutaminases covalently bind PAs to a single protein glutamyl residue forming mono(␥-glutamyl)-PA derivatives or to two glutamyl residues, each located on the same or separate proteins forming bis-(␥-glutamyl)-PA derivatives. The ‘bridges’ produced in the latter case have a different length according to the molecular lenght of the PA involved. c Transglutaminases covalently bind glutamyl and lysyl residues of two proteins forming a very short cross-link.

without having to resort to the use of extraneous color or fluorescent dyes which might alter the conformation of the physiological products. Of course, the added PAs or dyes might compete with other natural amine donors (for example lysyl-residues). Mono-PA production is affected by PA concentration since high levels of PAs saturate the acyl donor residues of the substrate proteins preventing the formation of bis-derivatives. In this sense, the levels of PAs have a critical role in the modulation of the number of protein cross-links formed. Antibodies against gln-lys are also a good tool for the identification of cross-linked TGase substrates. Recently, animal substrates immobilized on microplates, improved by the use of a biotinylated exapeptide, have been successfully utilized in the detection of plant TGase activity [3]. However, they are not applicable to the evaluation of the endogenous activity of the enzyme on its natural substrates.

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Until very recently, research on plant TGases has been hampered by difficulties encountered in the purification and sequencing of these enzymes and to the lack of a significant amino acid sequence homology between TGases of animal origin and any of the polypeptides reported in plant databanks, even though at present these contain some fully sequenced plant genomes. However, several data confirm that plants too exhibit such an enzymatic activity. In fact: 1. typical products of its catalysis, viz. glutamyl-polyamine derivatives, have been found in all plants assayed and specific animal or synthetic protein TGase substrates undergo amine conjugation induced by plant extracts; 2. amine conjugation activity requires Ca2⫹, as supported by both its dose dependence and inhibition by EGTA or EDTA; 3. some protein bands of plant extracts separated by SDS-PAGE are immunorecognized by specific antibodies raised against TGases of animal origin; 4. specific TGases’ inhibitors inhibit plant activity; 5. the enzyme probably contains a Cys in its active center, its activity being DTT dependent and inhibited by specific inhibitors; 6. finally, a recombinant protein containing the Cys-His-Asp triad present in the TGase catalytic domain encoded by a putative N-glycanase gene of Arabidopsis thaliana L., showed a typical TGase activity. The majority of data reported above were obtained with higher plants even though some refer to algae and fungi. Algae, especially the green ones (chlorophyta), in which TGase activity has been detected are phylogenetically related to higher plants. On the contrary, fungi (myceteae) are not plants and do not represent a natural group. In phylogenetic analysis some fungi divisions, for example those including Physarum, are placed in an outgroup of animal-fungi clades.

Possible Roles of Plant TGases

Several papers have already been reviewed [4, 5], however, some of these contributions will be discussed in some detail here as they provide information which is helpful in defining the state of the art in this field of research. As the number of references per chapter is restricted, we apologize if several individual references are reported under quoted review articles. The different characteristics of TGase activity in various organs of the same plant suggest that probably more than one enzyme, or different forms of the same enzyme, are present in the same organ. In plants, TGases probably play a role similar to that exhibited in animal cells, but their location in specific

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compartments and the different substrates affected indicate that they might have additional roles. In table 1 TGase(s) locations in plant cells are reported together with the biological functions in which they are, or supposed to be, involved. The presence of these enzymes in plants was at first suggested by the detection of PAs tightly bound to proteins. Direct evidence that a TGase enzyme is responsible for such a conjugation was provided by the isolation of glutamylPAs. The characteristics of many plant TGase activities, together with their substrates, are reported in tables 2 and 3 for nonphotosynthetic and photosynthetic tissues or organisms, respectively. TGase was at first detected in dividing cells, like those in the etiolated apical meristems of stems and during the cell cycle (Icekson and Apelbaum, 1987 and Serafini-Fracassini et al., 1988 and 1989 referenced in [4]). During the synchronous cell cycle of parenchyma cells of Helianthus tuberosus L., the TGase activity was found to be low in the early G1 phase and to increase during the second half of G1 and during the S phase (Serafini-Fracassini et al., 1989 referenced in [4]). In parallel, also the PA conjugation to protein of high molecular weight increased as well as two bands of 58 and 90 kDa immunostained with antibodies against rat prostatic gland TGase (Del Duca et al., 2000 referenced in [5]). In the meristematic apices also leaflets are present, and TGase activity was in fact also found in leaves as reported in the following paragraphs. Lilley et al. [6] detected TGase activity in root and shoot tissues of dicotyledonous (pea and broad bean) and monocotyledonous (wheat and barley) plants. In all species, roots exhibited a specific activity higher than that of leaf tissue of the same age. A relationship was shown to exist between TGase activity and the age of the plant tissue. In the root, TGase activity was involved in the early growth and development but afterwards decreased, while in the leaf it was present in both developing and mature tissue. In both root and leaf, TGase exhibited Ca2⫹ dependence; Ca2⫹ concentration that affected the type of linkage produced. The incorporation of PAs into proteins was activated by 20 nM Ca2⫹; this means that the resting level of cytosolic Ca2⫹ could be responsible for the intracellular role of TGase i.e. the attachment of primary amines to proteins with consequent effects on their charge and/or conformation. On the other hand, the plant TGase protein cross-linking function, resulting in the formation of the ␧(␥-glutamyl)lys isodipeptide bonds, was activated by Ca2⫹ at millimolar concentrations. Thus TGase could catalyze protein cross-linking reactions in high Ca2⫹ environments. This paper, to our knowledge, is the first report on isodipeptide bond detection in plants. The calcium dependence of plant TGases has been questioned, because frequently, when crude plant extracts are assayed, Ca2⫹ has an inhibitory effect or no effect at all (tables 2 and 3). This phenomenon might be due to the leakage

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Table 1. Transglutaminase location and possible involvement in plant morpho/functional events Biological function

Organism

Organ/Tissue/ Organelle

References

Cell division

Helianthus tuberosus

Cycling parenchyma Meristematic apex

Dunaliella salina

Entire cell

Serafini-Fracassini et al., 1989 in [4, 5] Serafini-Fracassini et al., 1988 in [4, 5] [21, 22]

Apical growth

Malus domestica Candida albicans

Pollen Mycelium

[9] [11]

Development/ Differentiation

Helianthus tuberosus

Callus

Del Duca et al., 1993 in [4]

Zea mays Vicia faba Pisum sativum Hordeum vulgare Triticum aestivum Arabidopsis thaliana Physarum p. polycephalum Candida albicans Chlamydomonas reinhardtii Saccharomyces c. cerevisiae

Callus Leaf/Root Leaf/Root Leaf/Root Leaf/Root Growing plant Cell wall

[18] [6] [6] [6] [6] [23] [7]

Cell wall Vegetative cell and zygote cell wall Cell wall

[11] [10]

Beta vulgaris

Leaf

Medicago sativa Helianthus tuberosus

Floral buds Leaf chloroplast

Glycine max Helianthus tuberosus Helianthus tuberosus Dunaliella salina Zea mays Zea mays

Leaf/Seedlings Chloroplast Stroma/Thylakoids Chloroplast Chloroplast grana Purified LHC II

Signorini et al., 1991 in [4, 5] [14] Del Duca et al., 1994/95 in [4] Kang and Cho, 1996 in [5] [15] [16] [21, 22] Villalobos et al., 2001 in [5] [17]

Photosynthesis/ Photoprotection

[12]

Stress response

Dunaliella salina

Cell

[21]

Defence elicitor

Phytophthora sp.

Cell wall

[13]

Protein storage

Glycine max

Cotyledons

Kang et al., 1998 in [5]

Programmed cell death

Nicotiana tabacum Solanum tuberosum and Phaseolus aureus

Flower petals Mitochondria

[19] [20]

Transglutaminases of Plants

227

Table 2. Characteristics of transglutaminases of nonphotosynthetic tissues/organisms Del Duca/Serafini-Fracassini

Higher Plants

Fungi

Helianthus Malus Nicotiana tuberosus domestica tabacum L. Borkh. L.

Enzymes Immunostained bands or purified protein (kDa) Localization

Pisum Phaseolus Candida sativum aureus L. albicans L. and Berkhout Solanum tuberosum L.

Physarum polycephalum cephalum Schwein

Saccharomyces cerevisiae Gasp.

Phytophthora sojae Kaufm. and Gerd.

Etiolated sprout apex SerafiniFracassini et al., 1988 in [4]

Cycling cells SerafiniFracassini et al., 1989 in [4, 5]

Pollen [9]

Flower corolla [19]

Etiolated Roots stem [6] apex Apelbaum et al., 1988 in [4]

Mitochondria [20]

Yeastlike and mycelia [11]

Cells [12]

Spherules Plasmodia Plasmodia Cells and [24] [13] [7] spherules [8]

n.d.

90, 58

80

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

77

101

93

42

Entire cell

Entire cell

Entire cell

Entire cell

Entire cell

Entire cell

Mitochondria

Cell wall

Cell wall

Entire cell

Entire cell

Entire cell

Cell wall

228

Assay pH

7.8/8.4

7.6/8.4

8.0

8.5

8.5

7.8/7.9

7.2

7.4

7.4

7.5

7.5

8.0

5.2

Time dependence

Hyperbolic

Hyperbolic

Linear

n.d.

Linear

Hyperbolic

Linear

n.d.

Hyperbolic

n.d.

Linear

n.d.

Yes

Stimulation by Ca2⫹ supply

No

No

Yes

Yes

No/Yes

Yes

n.d.

n.d.

Yes

Yes

Yes

Yes

Yes

Transglutaminases of Plants

EDTA/EGTA

Inhib.

Inhib.

Inhib.

n.d.

Inhib.

Inhib.

n.d.

n.d.

Inhib.

Inhib.

n.d.

Inhib.

Inhib.

Sulphydrylreagent sensitivity

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

no

n.d.

n.d.

n.d.

Inhib.

n.d.

n.d.

Light dependence

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

Protein substrates N,N⬘-DMC

No

No

Yes

n.d.

Yes

Yes

n.d.

n.d.

n.d.

Yes

Yes

Yes

Yes

Endogenous (kDa)

High and low

High and 18

High and low, 43, 55, 66

n.d.

Yes

n.d.

Yes

High

High and low

43

High and 40, 43

n.d.

n.d.

Identified substrates

n.d.

n.d.

Actin ⫹ tubulin

n.d.

n.d.

n.d.

Catalase

Cell wall prot

Cell wall prot

Actin

Lav 1–2 ⫹ actin

n.d.

n.d.

GlutamylPAs or Gln-Lys

n.d.

n.d.

Glutamyl- Glutamyl- n.d. PAs PAs

GlnLys

n.d.

n.d.

Gln-Lys

n.d.

n.d.

n.d.

n.d.

229

Table 3. Characteristics of transglutaminases of photosynthetic tissues/organisms Del Duca/Serafini-Fracassini

Higher Plants

Algae

Medicago sativa L.

Beta vulgaris L.

Floral bud Kuhen et al., 1991 in [4] and [14]

Leaf Signorini et al., 1991 in [4]

Leaf and other organs Falcone et al., 1993 in [4]

Greening explant Del Duca et al., 1993 in [4]

Chloroplast Del Duca et al., 1994 in [5] and [15, 16]

39

n.d.

150, 58, 20

n.d.

150, 58

Localization

Entire cell

Entire cell

Entire cell

Entire cell

Assay pH

7.9

7.8

8.2

Time depenence

n.d.

Linear

Linear

Enzymes Immunostained bands or purified protein (kDa)

Helianthus tuberosus L.

Zea. mays L

Lupinus albus L.

Glycine max L.

Callus [18]

Chloroplast [17]

Seedling Siepaio and Meunier, 1995 in [5]

Leaf and Entire plant seedling Kang and [23] Cho, 1996; Kang et al., 1998 in [5]

Cell and Cell [10] chloroplast [21, 22]

58

39

n.d.

80

86

70, 50, 25

72

Thylakoids/ Entire Thylastroma cell/ koids chloroplast

Entire cell

Entire cell

Microsomal fraction

Chloroplast

Cell wall

7.8

8.5

8.3

8.5

7.5

7.6

7.5/8.5

8.5

7.4

n.d.

Biphasic

n.d.

n.d.

Yes

n.d.

n.d.

n.d.

Linear

Arabidopsis Dunaliella thaliana L. salina Teodor.

Chlamydomonas reinhardtii Dangeard

230

Transglutaminases of Plants

Stimulation by Ca2⫹ supply

No

Yes

No

n.d.

Yes

n.d.

Yes

Yes

n.d.

Yes

n.d.

Not strictly

EDTA/ EGTA

Inhib.

Inhib.

Inhib.

n.d.

Inhib.

n.d.

Inhib.

n.d.

n.d.

Inhib.

n.d.

Low inhibition

Sulphydryl- n.d. reagent sensitivity

n.d.

n.d.

n.d.

Yes

n.d.

n.d.

Yes

Yes

n.d.

n.d.

Yes

Light dependence

Stim. dependdence

n.d.

Stim.

Stim.

Stim.

Yes

Stim.

n.d.

n.d.

n.d.

Stim.

n.d.

Yes

Yes

No

n.d.

n.d.

Yes

Yes

Yes

Yes

Yes

n.d.

n.d.

Endogenous (kDa)

High and low ⫹ 52–57

n.d.

High and low

High and low, ⬍36, 50–60

High and low, 20–30, 55

High and low

20–30

High

Storage proteins

n.d.

⬎205, 68, 55, 29, 27

Outer wall saltsoluble glycoproteins

Identified substrates

Rubisco

n.d.

n.d.

n.d.

LHC ⫹ Rubisco

n.d.

LHC II

Exogenous globulins ⫹ Rubisco

Glycinin

n.d.

n.d.

GP1.5, GP2 GP3a, b

GlutamylPAs or Gln-Lys

n.d.

Glutamyl- n.d. PAs

n.d.

GlutamylPAs

n.d.

Glutamyl- n.d. PAs

(indirect evidence)

GlutamylPAs

n.d.

n.d.

Protein substrates N,N⬘DMC

231

of Ca2⫹ stores caused by cellular damage occurring during cell extraction or cell death, as discussed below. Structural Role in Cytoskeleton and Cell Wall In the plant cytoplasm, TGase fulfils a structural role by modifying cytoskeletal proteins. After a first report that actin is a TGase substrate in animals, actin was recognized as a TGase substrate also in Physarum polycephalum Schwein, a primitive fungus that shares several cell characteristics with animals. In this case, actin modification may be important for the conversion of microplasmodia to spherules which allows them to survive starvation or desiccation. In Physarum polycephalum, a native 77-kDa TGase gave rise to a 39-kDa form [7]. A TGase of a similar molecular weight was also found in Medicago sativa L. (Kuehn et al. 1991 referenced in [4]) and in Zea mays [17]. Mottahedeh and Marsh [8] reported an increase in TGase activity following the cell damage. LAV1–2, the major calcium-binding protein in Physarum polycephalum, is a TGase substrate and has a role in the formation of large aggregates possibly involved in the recovery from cellular damage. In higher plants, tubulin and actin are substrates of a TGase of the pollen tube during its rapid growth, which is sustained by a dramatic cytoskeletal re-arrangement [9]. Two [14C]-PU-labeled bands of a mass of about 43 kDa were identified by mouse monoclonal antibody against actin; a 55-kDa band matched with the mouse monoclonal anti-␣-tubulin antibody. The addition of exogenous actin and tubulin to a cell-free extract of ungerminated, but committed to germinate, and germinated pollen enhanced its activity and autoradiography of the SDS-PAGE of these samples clearly showed that both actin and tubulin were TGase substrates. Labeled aggregates of high molecular mass were formed in the presence of actin, especially in germinating pollen. Other TGase products were also identified in pollen and some of the labeled proteins again formed heavy aggregates, very difficult to solubilize, a typical product of TGase activity. Their location in the cytoplasm and pores of ungerminated grain, at the tip and along the newly formed wall of the pollen tube of germinated grain was immunorevealed by dansylcadaverine conjugation catalyzed by pollen transglutaminase (fig. 2). Cross-reactivity with two polyclonal antibodies against mammalian TGases led to the detection of a protein of about 80 kDa in both ungerminated and germinated pollen. It may be envisaged that TGases play a structural role also in the organization of the cell wall of higher plants, even though there is no direct evidence of their presence in this location. The digestion of parenchymal cells by cellulase and pectinase causes in fact the disaggregation of PA-conjugated proteins of apparent high mass, an event which suggests the existence of an interconnection between these proteins and some wall polysaccharides

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TGase substrates

Fig. 2. The modification of pollen proteins with dansylcadaverine catalyzed by transglutaminase is immunorevealed in pollen grain and tube apex.

(Dinnella et al., 1992 referenced in [4]). The presence of PAs in the cell wall is well documented. In the alga Chlamydomonas reinhardtii Dangeard, TGase activity has been shown to affect cell wall glycoproteins [10]. During its life cycle, the alga elaborates two distinct cell walls. The vegetative wall, which surrounds both vegetative and gamete cell, has salt-soluble glycoproteins located in its outer layers and salt- and detergent-insoluble components only in the inner layers. In the early zygotes, this wall is substituted by a desiccation resistant, insoluble zygote wall. Both walls lack abundant complex polysaccharides typical of higher plants’ extracellular matrices (cellulose, hemicellulose and

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pectins), which facilitate the investigation of the assembly of the structural wall proteins. Three distinct vegetative wall assembly events have been identified: (a) an early TGase catalyses cross-linking reactions which lead to the formation of a ‘soft envelope’; (b) this organizes the self-assembly of glycoproteins; and (c) finally, an oxidative cross-linking reaction (isodityrosine cross-linking) renders the wall insoluble. Chlamydomonas reinhardtii secretes an extracellular 72-kDa TGase, the maximal activity of which precedes the insolubilization of the assembled Hyp-rich glycoprotein. The addition of cadaverine, SM, SD and PU at high concentrations disrupts the normal process of wall assembly. In the cell wall, PAs are linked to several proteins and salt-soluble glycoproteins such as GP2 and GP3. However, the authors cast doubts on the physiological meaning of these linkages. The two above-mentioned glycoproteins carry potential sites for the covalent attachment of PU by TGase, but these sites are normally inaccessible. By contrast, the labeling of inner wall components is likely to be significant because TGase activity appears responsible for nucleating the assembly of the wall. TGase was also found to be localized in the cell wall of fungi. In the fungus Candida albicans Berkhout, TGase activity was detected by the incorporation of radioactive PU into TCA-precipitable material and most of the activity was present in the cell wall [11]. Cystamine, a competitor of the amine donor substrates, has been used to analyze a possible role of TGase in the organization of the cell wall of the fungus; cystamine delayed protoplast regeneration, increased the sensitivity of the protoplast to the osmotic shock and inhibited the yeast-to-mycelium transition. Cystamine also affected the cell morphology: cells grown in its presence appear as dwarf mycelia, pseudomycelia and budding yeast. Moreover, the incorporation of covalentbound high molecular weight proteins into the cell wall was inhibited. The authors suggested that TGase activity is important in the structural organization of the fungus possibly by establishing cross-links among structural glycoproteins. In Saccharomyces cerevisiae Gasp., the interpeptidic cross-link N-␧(␥-glutamyl)lysine was obtained by the incorporation of [14C]lysine prevalently in cell walls [12]. Like in Candida albicans, cystamine inhibited TGase activity and cell growth. Furthermore, it altered cell morphology and sensitivity to cell wall degrading enzymes. In another fungus (Oomycete) Phytophthora sojae Kaufm. and Gerd., a destructive plant pathogen, as well as in other Phytophthora species, a cell wall glycoprotein was identified as a Ca2⫹-dependent TGase. A surface exposed peptide fragment of this protein acted as an elicitor of defense responses in parsley and potato [13].

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TGases in Chloroplasts A considerable level of TGase activity was found in green tissues and a large body of evidence confirms that the enzyme is widespread in the chloroplasts of higher plants and algae (table 3). The immunorecognition, by antibodies raised against animal TGases, of a SDS band in extracts of isolated chloroplasts was the first indication of the cross-reactivity of this enzyme between plants and animals (Del Duca et al. 1994, referenced in [4, 5]). The protein most frequently immunorecognized in leaf extracts by polyand monoclonal antibodies raised against animal TGases has a molecular mass of 58 kDa. Its purification proved very difficult and it is still unclear whether it represents the enzyme active form. In Medicago sativa L., a 39-kDa TGase subunit recognizes the L subunit of Rubisco as a substrate and causes its assembly (Kuehn et al., 1991, referenced in [4]). The activity of plastidic TGases of Helianthus tuberosus L. parenchyma cells exhibits traits already described above: Ca2⫹-dependence, optimum at basic pH values and, in addition, a putative dependence on a cysteine residue located in the active site of the enzyme [15]. The covalent linkage of PAs (PU and SD) to protein glutamyl residues, which represents an unequivocal test of TGase catalysis, was shown to occur in Beta vulgaris L. leaf extracts (Signorini et al., 1991 referenced in [4]) and Helianthus tuberosus L. isolated chloroplasts (Del Duca et al., 1995, referenced in [4, 5]) (table 3). It is known that PAs are present in this organelle where their biosynthesis is controlled by white light and their concentration is correlated to chlorophyll biosynthesis and photosynthetic rate. In this organelle, PAs, through their binding to particular substrates, could affect regulatory mechanisms such as that which operates on the structure and function of the photosynthetic apparatus during photoadaptation. PAs, added to osmotically stressed leaves, preserved from degradation thylakoid proteins and the large Rubisco subunit. A possible molecular role of PAs is discussed below. Recently, TGase activity was detected both in thylakoid- and stromaenriched fractions of Helianthus tuberosus chloroplasts [16] (fig. 3). In the former, two electrophoretic bands (58 and 24 kDa) were recognized by antibodies raised against rat prostatic gland TGase while only one band (150 kDa) was recognized in the latter. The analysis of PA-glutamyl derivatives (prevalently bisconjugates) showed that PA conjugation to endogenous proteins through TGase catalysis occurred in both fractions [16]. These data indicate that the enzyme and its substrates are located in both chloroplast compartments. Furthermore, a synergism between the activities of stroma and thylakoids was observed during the TGase assay and was found to be markedly affected by light. A TGase activity, which shared many properties with that of the Helianthus tuberosus chloroplast, was observed in the Zea mays L. chloroplast and its

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Thylakoids Rubisco Chloroplast

150-kDa TGase Rubisco

Thyl TGase

Stroma LHC II Photosynthesis-Photoprotection?

LHC I

Growth-Senescence delay?

Fig. 3. In chloroplasts, some transglutaminases of different molecular mass have been found both in the stroma, where the substrates is Rubisco the carboxylase responsible for the atmospheric CO2 fixation, and in thylakoids where the substrates are the light harvesting complexes (LHC I and LHC II). The catalysis of the latter is strictly light dependent and possibly affects photosynthesis and/or photoprotection with a positive effect on growth. A delay of the chloroplast decay during senescence was also observed.

fractions [17, 18]. This activity was found to be prevalent in thylakoids, and bis-(␥-glutamyl)spermidine and bis-(␥-glutamyl)putrescine were the main polyamine conjugates formed [17]. Light stimulated the thylakoid activity. When the light harvesting complex of photosystem II (LHC II) was isolated and assayed for conjugation with putrescine, spermidine and spermine, different degrees of efficiency were observed, spermine being the polyamine most efficiently conjugated. The LHC II fraction, partially purified on a sucrose gradient, also contained a 39-kDa band which was immunorecognized by two anti-TGase antibodies (Ab-3 and rat prostatic gland-TGase). Two assay methods for TGase activity, one carried out in the presence of biotinylated cadaverine and the other of PAs labeled with radioactive isotopes and resulting in the isolation of glutamyl PAs, further confirmed that this thylakoid enzyme is indeed a calcium-dependent TGase (Thyl-TGase) [17]. Preliminary data (Di Sandro et al., pers. commun.) also suggest that the putative light harvesting complex of photosystem I (LHC I) has associated with an active TGase. More recently, it was reported that TGase activity is even more precociously enhanced during the light-induced greening of proplastids of cucumber endosperm when this is stimulated by cytokinin (Sobieszczuk-Nowicka et al., pers. commun.).

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TGases in Programmed Cell Death TGase activity has a great functional significance not only in differentiating and light-exposed differentiated chloroplasts, but also appears to be involved in the senescence of leaves and of flower petals, which are transformed leaves with a vexillar role for insects to favor pollination. The study of senescence is important for both basic and applied research. Freshly cut leaves which are utilized as food, like lettuce, and freshly-cut flowers have a short shelf life. Therefore, prevention of their rapid degradation is a major objective of applied research. Also in colored flowers, chlorophyll decreases in parallel with the progression of senescence. Chloroplasts are among the first organelles that breakdown: they become inactive and photosystem apoproteins are degraded together with bound chlorophylls. PAs are known antisenescence agents in leaves: they delay senescence temporarily to prevent membrane damage and retard the degradation of nucleic acids and proteins, including the chloroplast photosystems. Many events occur during flower petal senescence, such as nuclear blebbing, DNA laddering, cell wall modification, a decline in protein, water and pigment content, a decrease in membrane integrity and an increase in TGase activity, detected as an increase in glutamyl-PA production and amount of the protein modified [19]. This ‘senescence’ TGase activity could be related to the appearance of a new 58-kDa band immunodetected by anti-TGase antibodies. It has been observed that SM delays senescence and PCD of flower petals, retards DNA fragmentation and vacuole damage, prolongs chloroplast viability with visible preservation of chlorophyll content (fig. 4). This protective effect could be mediated by SM covalent binding to TGase substrates located in different cell compartments including those of the chloroplast. All these data support the view that in chloroplasts one or several TGase forms mediating PAs conjugation are involved in photosynthesis and photoprotection, and probably delay cell senescence and death. Votyakova et al. [20] reported the covalent attachment of PAs to proteins in plant mitochondria. Mitochondria were isolated and purified from potatoes (Solanum tuberosum L.) and mung beans (Phaseolus aureus Roxb.). The organelles were incubated with radioactive PAs and the radioactivity of the acid insoluble material, a significant amount of which was released by pepsin, was measured. The activity was temperature dependent and no incorporation occurred at 0oC. The activity in the matrix fraction did not increase as a function of time, whereas the membrane fraction showed a significant increase of radioactivity during the period of incubation. As detergent stopped the incorporation of radioactivity, the integrity of the membrane structure appears to be important and connected to PA incorporation, which occurs either co- or post-translationally. One of the PA-labeled proteins was identified as a

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SM

Young

Dead

Fig. 4. The delay of senescence of tobacco flowers caused by spermine (SM) could be mediated by a transglutaminase, the activity of which increases in treated flowers.

membrane protein with a catalase-like sequence (catalase is normally regarded as a soluble matrix protein). This finding led the authors to the hypothesis that these PAs linked possibly through TGase could have a role in the apoptosis oxidative metabolism. Light Sensitivity of TGase in Higher Plants Light response represents a novel aspect of the regulation of TGase activity. In the nineties, it was already reported that TGase activity is light stimulated in green leaves, or more precisely in the entire chloroplast and in its isolated subfractions, as described above. However, in animals, where TGases are more extensively studied, their regulation by light in organs like the eye or the skin is still to be verified. In differentiating chloroplasts, light stimulates the appearance of particular products of TGase activity probably dependent on an upturn in the synthesis of the enzyme. This is supported by the increase in immunostaining of a 58-kDa protein band observed in Helianthus tuberosus nongreen tuber tissue cultivated in vitro in light condition (Del Duca et al., 2000 referenced in [5]). In vivo light modulates the availability of some plant TGase substrates by regulating the expression of the genes of ␣- and ␤-tubulin and of several plastidial proteins like Rubisco, LHC I, LHC II and others. This could occur concomitantly with the maturation of the thylakoid structure, a process of which TGase seems to be part. A TGase form, characteristic of differentiated leaves, is inducible in a nongreen tissue when exposed to light. This effect was shown by growing in vitro the initially nonphotosynthetically committed explant (dormant tuber parenchyma of Helianthus tuberosus devoid of green plastid and organized thylakoids)

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exposed to hormonal conditions which either allow or prevent the differentiation of chloroplasts under light conditions (Del Duca et al., 1993 referenced in [4]). In addition, the light effect is also most probably related to phytochrome and hormone regulation of the enzyme activity. In fact, the TGase activity of in vitro maize cultures was reported to show a marked daily rhythm, but with changes of activity not coincident with the light/dark transitions [18]. TGase activities and PA content, the latter probably dependent on arginine decarboxylase activity, both increased in the subjective morning, when the light was on, decreased in the subjective afternoon (still in the light) and increased again during the subjective night; thus they were not strictly light regulated. The effect was most marked in differentiating calluses, but not so evident in growing ones. Thus, it would appear that a significant role must also be played by the hormonal system that regulates tissue differentiation versus growth. Despite these complex systems of regulation occurring in vivo, the TGase activity detected in the test tube is absolutely light dependent. In an assay performed by incubating labeled PU with both purified Thyl enzyme and LHC II, one of the enzyme main substrates, mono- and bis-glutamyl-PU were produced in similar amounts exclusively in light conditions, while only traces were detectable in the dark (fig. 3) [17]. This result allows one to speculate on the mechanism of direct light regulation. As reported by Della Mea et al. [17] the known light-induced conformational change of LHC II causes two specific glutamyl residues to modify their relative distance to a value close to the molecular dimensions of SM, which could then form a bis-glutamyl-SM bridge involving these residues, if in correct juxtaposition. In agreement with analyses carried out on other tissues, SM was in fact found to be the most efficient conjugate to LHC II apoproteins while PU was the least effective. Two further LHC II glutamyl residues could be possible PA-conjugation sites. However, in vivo they are positioned in a loop facing the grana lumen where the pH is lowered when photosynthesis is active, a condition that would not favor the activity of TGase that has a basic pH optimum [15]. The light independence observed when the animal TGases are tested suggests that not all glutamyl residues of LHC II are available to these enzymes. In fact, erythrocyte TGase and guinea pig liver TGase are mainly cytosolic in location and their hydrophilicity would hardly allow them to access the hydrophobic inner environment of LHC II. Their catalysis leading mainly to the formation of monoderivatives supports this view. By contrast, Thyl TGase produces considerable amounts of bis-derivatives, as usually observed in vivo in the entire chloroplast. The thylakoid enzyme, also due to its small size and specificity, could reach key glutamyl residues that, once cross-linked exert a structural role in complex stabilization. The data reported by Della Mea et al. [17] suggested that in the thylakoid membrane a TGase is localized closely to,

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or in association with the LHC II. The reported presence in LHC II of highaffinity sites for Ca2⫹, involved in membrane stacking occurring during photosynthesis, and of low-affinity sites could also be relevant for the Ca2⫹dependent catalysis of Thyl TGase in vivo. A similar light-sensitive activity was also detected in Helianthus tuberosus thylakoids [16]. Thus, the similarities observed in monocots and dicots lead to suppose that the presence of a TGase associated with LHC II is widespread. This thylakoid enzyme may itself be responsible for the light sensitivity; however, the reported light-induced conformational modification of LHC II probably plays an important role in the in vivo activity of the specific thylakoid TGase. In figure 3, it is summarized that in the chloroplast thylakoids and stroma of higher plants, the TGase catalysis is strictly light dependent and possibly affects photosynthesis and/or photoprotection with a positive effect on growth. A delay of the chloroplast decay during senescence was also observed, as reported above. Light and Salt Stress Effects on TGase Activity in Algae In Dunaliella salina Teodor., a green halophilic alga, the TGase activity was reported to be light- and salt-stress dependent [21]. Here, the enzyme appears to play a role in the adaptation to hypersaline stress. In Dunaliella salina, freshly subcultured in an optimal NaCl concentration medium, TGase activity increased only transiently. A similar increase was also observed during the cell cycle resumption in dormant cells of vascular plants after wounding [Serafini-Fracassini et al., 1989 referenced in 4]. However, in algae subjected to hypersaline stress under light, TGases present in chloroplasts as two units of 25 and 50 kDa undergo, in particular the latter, an immediate concentration change, coincident with variations in light-affected enzymatic activity. Under these conditions, the algal photosynthetic complexes are severely affected with loss of many components and especially of the active trimeric form of the LHC II, mainly after one day under stress. Some TGase substrates were found to be similar to those present in higher plants (thylakoid photosynthetic complexes and Rubisco). They were labeled by [1,4-14C] PAs, more intensely in the light than in the dark and to a greater extent in algae, already acclimated to hypersaline conditions than in those cultured in the optimal saline medium or subjected to stress. The concentration of some polypeptide substrates was particularly high in cells acclimated to high salt. It has been proposed that PA conjugation could have a role in the assembly of chloroplast proteins in cells affected by salt stress [21]. A Dunaliella salina PAs-deficient variant strain is characterized by a very low growth capacity and its TGase, chlorophylls and other chloroplast

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Dunaliella salina PA-variant strain

⫹ PU

Slow growth Few PAs Unbalanced PU/SD ratio Low Chl TGase activity Low chlorophyll content Low protein content Unbalanced chlorophyll a/b ratio

Growth resumption Increased PA content Normal PU/SD ratio Increased Chl TGase activity Increased chlorophyll content Increased protein content Normal chlorophyll a/b ratio

Fig. 5. A variant strain of Dunaliella salina Teodor, deficient in PAs, when treated with putrescine, resumes growth and several parameters are normalized in concomitance with a light-stimulated increase of chloroplast (Chl) transglutaminase activity.

parameters are also deficient (fig. 5). All of these are stimulated by the supply of PU, concomitantly with an upturn in cell growth [22]. Chloroplast TGase of these PU-treated algae increases its conjugating activity by 9-fold in the light and less than 3-fold in the dark, when compared to controls. The PA-deficient variant strain appears to be more severely affected by both salt and subculture stresses. Its recovery time is also longer. Its TGase activity increases after salt stress and is always higher in the light than in the dark, showing an additive stress effect of salt and light. In the PA-deficient variant strain acclimated to high salinity, or immediately after stress application, there is a considerable enhancement in the chloroplast content of chlorophyll a and b and in TGase activity with changes exhibiting almost overlapping behaviors. These observations validate the view that in green algae too TGase is implicated in the protection of the photosynthetic apparatus from stress.

Plant and Animal TGases Recognize Each Other’s Substrates

Della Mea et al. [17] reported that two purified TGases of animal origin, namely guinea pig liver TGase and erythrocyte TGase when incubated with

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LHC II catalyze the conjugation of PU with this chloroplast substrate, forming mainly monoderivatives, and that the light has no effect on this LHC II modification. By contrast, when LHC II is incubated with the chloroplast 39 kDa TGase (ThylTGase) both mono- and bis-PU are produced in similar amount, but mostly in the light condition. In the dark, in fact, very few derivatives are detected. Thus, the plant enzyme conjugates PA to its natural plant substrate and the catalysis is light dependent. In addition, a sucrose gradient fraction enriched in intergrana thylakoids containing LHC I, also containing a TGase, isolated from the photosystem I of Lactuca sativa L. efficiently catalyzed the conjugation of labeled SM. This catalysis was also light stimulated (Di Sandro et al., pers. commun.). Dimethylcasein (DMC) (the specific substrate for animal TGases) added to the incubation assay for Thyl TGase, is also recognized as substrate, in competition with the natural LHC substrate, but its modification is light insensitive [17]. Thus, although the plant enzyme is able to recognize as a substrate DMC, its catalysis is unaffected by light. DMC is also a substrate of other plant TGases (tables 2 and 3). This characteristic was utilized in the development of a colorimetric method for the detection of TGase based on its capacity to conjugate biotinylated cadaverine to immobilized DMC and to cross-link biotin-labeled casein to immobilized casein [6]. However, some plant crude extracts are unable to recognize this protein as a TGase substrate. Equally, bovine serum albumin is recognized and polymerized to high mass products only by some plant TGases (see next paragraph) [23]. These results, summarized in figure 6, suggest that plant and animal TGases can cross-recognize at least some of their substrates, thus suggesting a similarity in their specificity. However, they exhibit differences in their sensitivity to light conditions.

Thylakoid TGase recognizes animal substrates Its catalysis is not light regulated

Animal TGases recognizes plant substrates Its catalysis is not light regulated

Only the plant TGase/plant substrate catalysis is light regulated

Fig. 6. The activity of transglutaminases on substrates of different origin (plants and animals) has dissimilar light sensitivity.

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The First Plant TGase

The studies reported above have revealed the presence of TGase activities in plant chloroplasts, mitochondria, cell wall and cytoplasm similar to those present in animals and prokaryotes, and probably having mainly a structural role. However, although in several purified plant protein extracts a close association could be established between the TGase activities and some proteins, detected by SDS-PAGE, at present the identity of these proteins remains unknown. In addition, no DNA sequence sharing homology with animal TGases has been found in the databases of Arabidopsis thaliana, Zea mays, Oryza sativa L. and Solanum tuberosum. This makes it difficult or even excludes the possibility of identifying plant TGases by sequence in comparison with wellknown animal TGases. A recent computational analysis has shown the presence of a gene, AtPng1p in Arabidopsis thaliana, which encodes a putative N-glycanase. AtPng1p contains the Cys-His-Asp triad present in the TGases catalytic domain. In the Arabidopsis thaliana genome, only AtPng1p appears to have this catalytic triad. These glycanases are de-N-glycosylating enzymes involved in the degradation of misfolded proteins. As amidases, they have a Cys residue that acts as a nucleophile critical for their enzymatic activity. AtPng1p is a single gene expressed ubiquitously, but at low levels, as shown by nested RT-PCR mRNA accumulation in the entire plant, in different organs, growth stages, in all light-assayed conditions [23]. To elucidate whether AtPng1p encodes a TGase, its coding sequence was overexpressed in Escherichia coli, the recombinant protein was purified by Ni2⫹-affinity chromatography, and a 86-kDa band was immunodetected using three antianimal TGase antibodies [23]. Furthermore, Western blot analysis using antibodies raised against the recombinant AtPng1p protein have led to its detection in the Arabidopsis thaliana microsomal fraction. Other bands of lower molecular mass were identified in the cytosolic fraction, raising the question of whether these represent soluble fragments produced by the proteolytic degradation of AtPng1p protein, a usual occurrence with TGases of animal origin, like epidermal TGase. This is also in keeping with the presence of TGase activities and different immunorecognized bands in the extracts of several organs of the same plant (tables 2 and 3). The low amounts of AtPng1p protein present in the cell could explain why several laboratories have failed to isolate plant TGases over the years. To our knowledge, in addition to the partially purified 39-kDa protein from chloroplasts, only Kang et al. [1998, referenced in 4] succeeded in purifying homogeneity from soybean leaves, a 80-kDa protein, so far unsequenced, which exhibited TGase activity (see also table 3). The Arabidopsis thaliana recombinant protein [23] links PAs (spermine ⬎ spermidine ⬎ putrescine – and biotin-cadaverine) to DMC in a calcium-dependent

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manner. Analyses of the ␥-glutamyl-derivatives revealed that the AtPng1p gene product acts as a TGase. The Ca2⫹- and GTP-dependent cross-linking activity of the AtPng1p protein can be visualized by the polymerization of bovine serum albumin, obtained, like the commercial TGase, at basic pH and in the presence of dithiotreitol. This is the first plant protein, isolated and characterized at molecular level, which displays a TGase activity, the parameters of which so far agree with those typically exhibited by animal TGases. Therefore, the hypothesis was put forward that this protein, despite having an amino acid sequence difference, with the exception of the triad of the active site, from those of known animal TGases, but sharing with these immunological, biochemical properties and possibly an overall similar conformation, is a true TGase, probably with a different but convergent phylogenetic history.

The First Mammalian-Type TGase of Slime Molds

While several TGase genes of lower vertebrates and of some invertebrates have been cloned and appear to have DNA sequences similar to those of mammals, no homologous gene has been found in lower eukaryotes. In Physarum polycephalum, a cDNA clone encoding a TGase was isolated by Wada et al. [24] on the basis of partial amino acid sequences of the purified enzyme. The primary structure deduced from the cDNA sequence appeared to be significantly similar to those of mammalian TGases. The middle region of the sequence appeared to be significantly conserved and was characterized by a GTP-binding region, an active site with the typical catalytic triad residues and a Ca2⫹-binding region. It was 40–50% identical to those of human TGase 2 and of several invertebrate TGases. At variance with mammals, but in keeping with some invertebrates, a long amino-terminal region was found to be present, but its function is unknown. The purified Physarum polycephalum TGase also exhibited the hydrolyzing activity of GTP, which inhibited its transamidating activity, as in the case of TGase 2. Physarum polycephalum is the lowest organism in which a mammalian-type TGase was found.

Biotechnological Aspects

Animal TGases can be expressed by plants. An important applied use of TGases rests on their cross-linking function which produces large supramolecular protein networks. The prevalent utilization of the enzyme is in the food industry. First used to improve the gelling characteristics of fish paste; TGases are now added to a variety of different foodstuffs, like meat, fish, bread and

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dairy products to improve their texture by cross-linking proteins to achieve a higher breaking strength and better viscoelastic properties. This widespread use of TGases has increased the demand for an inexpensive, efficient and safe source of recombinant enzyme. The bacterium Streptoverticillum mobaraensis [1] is currently the most widely used source of the recombinant protein. However, it should be noted that proteins produced in bacteria are not glycosylated (often leading to structural instability or loss of biological activity) and form disulphide bridges at a significantly slower rate than in eukaryotes. This results in protein misfolding and precipitation in the form of inclusion bodies. Therefore, bacterial cultures are suitable for the production of simple and unmodified animal proteins, but not for large, complex glycoproteins. Some of these problems associated with protein modification can be overcome using yeast cultures, but there remain significant differences in glycosylation patterns between fungi and higher eukaryotes, making animal or plant expression systems necessary. Traditionally, most recombinant human proteins have been produced in mammalian cell lines. These have the advantage of carrying out authentic post-translational modifications, but the costs of setting up and running industrial-scale mammalian cell cultures can be prohibitive, particularly for industrial proteins required in bulk. A further disadvantage associated with all animal systems is the possible contamination of the recombinant protein product with an endogenous pathogen. Molecular farming in plants offers a unique combination of advantages in terms of cost, safety, productivity and convenience. Higher plants are attractive hosts for the production of recombinant macromolecules for pharmaceutical, animal feed and nutraceutical applications. Plant-based production systems are inherently safer than alternative expression platforms and significantly more economical. An important additional advantage of plants is that they can carry out appropriate post-translational modifications of recombinant proteins. Cereal crops offer several additional advantages as expression systems. Recombinant proteins can be stored safely in protein bodies of seed endosperm cells, thereby escaping proteolysis in the cytosol, and subsequent programmed cell death during the final stages of cereal grain maturation. A wide diversity of high value recombinant proteins with diagnostic or therapeutic potential has been expressed in plants. Recombinant proteins can be targeted to storage organs, such as seeds, where they remain stable for months even at ambient temperatures, reducing the costs of distribution and storage. Moreover, plants do not contain human pathogens. The potential yield of recombinant proteins in transgenic plants is extremely high. For industrial proteins, plant material can be added directly to the industrial process, eliminating the costs of extraction and purification.

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The use of plant-based systems for the production of TGases was thus explored by Claparols et al. [25]. Mature rice embryos were engineered by bombardment with a rat prostate TGase, driven by the strong constitutive maize-1 ubiquitin promoter and its first intron. The plants were shown to express the recombinant enzyme at mRNA and protein levels. The Ca2⫹ dependence of the recombinant enzyme was confirmed by the biotin-labeled cadaverine incorporation in vitro assay. The result is consistent with an accumulation of the enzyme mostly in an inactive form. It may be that endogenous biochemical mechanisms in the plant cell(s) sequester TGase in an inactive form to protect the plant from dramatic alterations that would result from the indiscriminate and uncontrolled cross-linking of cellular proteins by the enzyme. This is in agreement with the properties of prostatic gland TGase that is normally inactive inside the cell, and becomes active only when secreted. The molecular and biochemical characterization of transgenic plants expressing rat prostatic gland TGase indicate that rice plants could be a viable alternative to mammalian or prokaryotic expression systems for the production of TGases. Acknowledgments We are particularly indebted to Prof Augusto Serafini-Fracassini Emeritus of the St Andrews University, for scientific and language suggestions. We acknowledge MURST (PRIN2003) and FIRB for supporting our work on TGase.

References 1 2 3

4 5 6

7 8 9

Griffin M, Casadio R, Bergamini CM: Transglutaminase: Nature’s biological glues. Biochem J 2002;368:377–396. Lorand L, Graham RM: Transglutaminases: Crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 2003;4:140–156. Trigwell SM, Lynch PT, Griffin M, Hargreaves AJ, Bonner PL: An improved colorimetric assay for the measurement of transglutaminase (type II)-(gamma-glutamyl)lysine cross-linking activity. Anal Biochem 2004;330:164–166. Serafini-Fracassini D, Del Duca S, Beninati S: Plant Transglutaminases. Phytochemistry 1995;40: 355–365. Serafini-Fracassini D, Del Duca S: Biochemistry and function of plant transglutaminases. Minerva Biotechnol 2002;14:135–141. Lilley G, Skill J, Griffin M, Bonner PL: Detection of Ca2⫹-dependent transglutaminase activity in root and leaf tissue of monocotyledonous and dicotyledonous plants. Plant Physiol 1998;117: 1115–1123. Klein JD, Guzman E, Kuehn GD: Purification and partial characterization of transglutaminase from Physarum polycephalum. J Bacteriol 1992;174:2599–2605. Mottahedeh J, Marsh R: Characterization of 101-kDa transglutaminase from Physarum polycephalum and identification of LAV1–2 as substrate. J Biol Chem 1998;273:29888–29895. Del Duca S, Bregoli AM, Bergamini C, Serafini-Fracassini D: Transglutaminase-catalyzed modification of cytoskeletal proteins by polyamines during the germination of Malus domestica pollen. Sex Plant Reprod 1997;10:89–95.

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Waffenschmidt S, Kusch T, Woessner JP: A transglutaminase immunologically related to tissue transglutaminase catalyzes cross-linking of cell wall proteins in Chlamydomonas reinhardtii. Plant Physiol 1999;121:1003–1015. Ruiz-Herrera J, Iranzo M, Elorza MV, Sentandreu R, Mormeneo S: Involvement of transglutaminase in the formation of covalent cross-links in the cell wall of Candida albicans. Arch Microbiol 1995;164:186–193. Iranzo M, Aguado C, Pallotti C, Canizares JV, Mormeneo S: Transglutaminase activity is involved in Saccharomyces cerevisiae wall construction. Microbiology 2002;148:1329–1334. Brunner F, Rosahl S, Lee J, Rudd JJ, Geiler C, Kauppinen S, Rasmussen G, Scheel D, Nurnberger T: Pep-13, a plant defense-inducing pathogen-associated pattern from Phytophthora transglutaminases. EMBO J 2002;21:6681–6688. Margosiak SA, Dharma A, Bruce-Carver MR, Gonzales AP, Louie D, Kuehn GD: Identification of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase as a substrate for transglutaminase in Medicago sativa L. (Alfalfa). Plant Physiol 1990;92:88–96. Del Duca S, Dondini L, Della Mea M, Munoz de Rueda P, Serafini-Fracassini D: Factors affecting transglutaminase activity catalyzing polyamine conjugation to endogenous substrates in the entire chloroplast. Plant Physiol Biochem 2000;38:429–439. Dondini L, Del Duca S, Dall’Agata L, Bassi R, Gastaldelli M, Della Mea M, Di Sandro A, Claparols I, Serafini-Fracassini D: Suborganellar localisation and effect of light on Helianthus tuberosus chloroplast transglutaminases and their substrates. Planta 2003;217:84–95. Della Mea M, Di Sandro A, Dondini L, Del Duca S, Vantini F, Bergamini C, Bassi R, SerafiniFracassini D: A Zea mays 39 kDa thylakoid transglutaminase catalyses Light Harvesting Complex II by polyamines in a light-dependent way. Planta 2004;219:754–764. Bernet E, Claparols I, Dondini L, Santos MA, Serafini-Fracassini D, Torné JM: Changes in polyamine content, arginine and ornithine decarboxylases and transglutaminase activities during light/dark phases of initial differentiation in maize calluses and their chloroplasts. Plant Physiol Biochem 1999;37:899–909. Serafini-Fracassini D, Del Duca S, Monti F, Poli F, Sacchetti G, Bregoli AM, Biondi S, Della Mea M: Transglutaminase activity during senescence and programmed cell death in the corolla of tobacco (Nicotian a tabacum) flowers. Cell Death Differ 2001;9:309–321. Votyakova VT, Wallace HM, Dunbar B, Wilson SB: The covalent attachment of polyamines to proteins in plant mitochondria. Eur J Biochem 1999;260:250–257. Dondini L, Bonazzi S, Del Duca S, Bregoli AM, Serafini-Fracassini D: Acclimation of chloroplast transglutaminase to high NaCl concentration in a polyamine-deficient variant strain of Dunaliella salina and in its wild type. J Plant Physiol 2001;158:185–197. Dondini L, Bonazzi S, Serafini-Fracassini D: Recovery of growth capacity and of chloroplast transglutaminase activity induced by polyamines in a polyamine-deficient variant strain of Dunaliella salina. J Plant Physiol 2000;157:473–480. Della Mea M, Caparros-Ruiz D, Claparols I, Serafini-Fracassini D, Rigau J: AtPng1p: The First Plant Transglutaminase. Plant Physiol 2004;135:1–9. Wada F, Nakamura A, Masutani T, Ikura K, Maki M, Hitomi K: Identification of mammalian-type transglutaminase in Physarum polycephalum. Evidence from the cDNA sequence and involvement of GTP in the regulation of transamidating activity. Eur J Biochem 2002;269:3451–3460. Claparols MI, Bassie L, Mirò B, Del Duca S, Rodriguez-Montesinos J, Christou P, SerafiniFracassini D, Capell T: Transgenic rice as a vehicle for the production of the industrial enzyme transglutaminase. Transgenic Res 2004;13:195–199.

Dr. S. Del Duca Dipartimento di Biologia e.s., Sede Botanica Università di Bologna, Via Irnerio 42, IT–40126 Bologna (Italy) Tel. ⫹39 0512091292, Fax ⫹39 051242576, E-Mail [email protected]

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Author Index

Amendola, A. 58 Auricchio, S. 158 Autuori, F. 58

Farrace, M.G. 58 Fésüs, L. 19 Griffin, M. 89

Bailey, C.D.C. 139 Broome, A.-M. 115 Caputo, I. 158 Chandrashekar, R. 209 Ciccosanti, F. 58 Cso´´sz, É. 19 Del Duca, S. 223 Eckert, R.L. 115 Esposito, C. 158 Facchiano, A. 37 Facchiano, F. 37 Falasca, L. 58

Pan, J. 75 Petrovski, G. 19 Piacentini, M. 58 Piredda, L. 58 Porta, R. 174

Ichinose, A. 192 Johnson, G.V.W. 139 Johnson, T.S. 89

Rao, R.U. 209 Rodolfo, C. 58 Rorke, E.A. 115 Ruse, M. 115

Mangala, L.S. 125 Mariniello, L. 174 Mastroberardino, P.G. 58 Mehta, K. 1, 125, 209

Serafini-Fracassini, D. 223 Singh, U.S. 75 Sturniolo, M.T. 115

Nardacci, R. 58 Nemes, Z. 19

Troncone, R. 158 Tucholski, J. 139

Oliverio, S. 58

Verderio, E.A.M. 89

248

Subject Index

Algae transglutaminases light and salt stress effects 240, 241 overview 210–212 Alzheimer’s disease (AD) neuropathology 146 transglutaminase activity and expression 143, 146, 147 Amyotrophic lateral sclerosis (ALS) neuropathology 148 transglutaminase activity and expression 143, 148, 149 Apoptosis cancer resistance 125 plant transglutaminases in programmed cell death 237, 238 tissue transglutaminase cell death role DAP-like kinase as substrate 64, 65 expression 61 knockout mouse studies 64 neuron apoptosis 141, 142 research prospects 70 retinoic acid induction 65, 66, 79 cell survival signaling cancer 127 eukaryotic initiation factor-5A interactions 80 retinoblastoma protein as substrate 65, 79 stress-induced activation 80, 81, 96, 97

Autophagy, tissue transglutaminase role 66, 67 Bacterial transglutaminases, overview 210–212 Band 4.2 antibody sources for research 17 deficiency 15, 198 functions 15 isoforms 15 nomenclature 15 regulators 24 tissue distribution 15 Biotechnology plant transglutaminases 244–246 transglutaminase application prospects 184, 185 Brain injury, transglutaminase activity and expression 143, 149–151 Cancer apoptosis resistance 125 epidermal transglutaminase expression 10 prevalence 125 tissue transglutaminase role cigarette-smoke-induced carcinogenesis 132–134 drug resistance and metastasis role drug-resistant cell expression 128, 129 metastatic cell expression 129, 130 overview 127, 128

249

Cancer (continued) tissue transglutaminase role (continued) extracellular matrix interactions 126 inducers in cancer 132 integrin interactions 126, 130–132 prospects for study 134, 135 transglutaminase 4 expression 11 Carboxamido group, inherent reactivity in proteins 19, 20 Celiac disease heredity 158, 159 prevalence 158 T helper response 158, 166 tissue transglutaminase autoantibodies in pathogenesis 168, 169 autoantigen discovery and diagnostics 159, 176 autoimmunity role 166–168 deamidating activity and triggering of gliadin immune response 162–164 intestinal mucosa expression and activity 160, 161 prospects for study 169 substrate specificity towards gluten peptides 164–166 Cell migration, tissue transglutaminase role in wound healing 99 Collagen, remodeling by tissue transglutaminase 97 Cosmetics, transglutaminase application prospects 177, 178 Cysteinyl residue catalysis in transglutaminases 20–23 thiol group inherent reactivity in proteins 19, 20 DAP-like kinase (DLK), tissue transglutaminase substrate in apoptosis 64, 65 eIF-5A, see Eukaryotic initiation factor-5A Epidermal transglutaminase (TG3) activation 29, 30 antibody sources for research 16 cancer expression 10 functions 10

Subject Index

gene regulation 10 GTP binding and hydrolysis 118, 119 mechanism 118 nomenclature 10 posttranslational processing 10, 118 purine binding and hydrolysis 31, 32 regulators 24, 29, 118 skin disease defects 120 tissue distribution 10, 139 wound healing role 92 X-ray crystal structure 25 Epidermis differentiation 115, 116 transglutaminase functions 120 Eukaryotic initiation factor-5A (eIF-5A), tissue transglutaminase interactions 80 Factor XIIIA activation 30 antibody sources for research 16 functions 14, 198 gene regulation and structure 14, 194–196 knockout mice 95 nomenclature 13, 37 pathology congenital deficiency clinical features 201–203 knockout mouse model 204, 205 molecular defects 203, 204 deficiency 14, 15, 199 diseases 199, 200 polymorphisms and thrombosis 200, 201 recombinant protein expression 187, 206 replacement therapy 206 structure 14, 175, 196, 197 Sushi domains 197, 198 therapeutic applications 174–176 tissue distribution 14, 139, 195 trafficking 25 wound healing role 94 therapeutic targeting 106 X-ray crystal structure 25 Fibronectin, tissue transglutaminase interactions 98, 127

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Fibrosis kidney aberrant wound healing 100, 103 tissue transglutaminase role progressive renal scarring 104, 105 wound healing and scarring 103 therapeutic applications of transglutaminases 176 tissue transglutaminase role in liver disease 100 Food science, transglutaminase application prospects cereal products 181 dairy products 179, 180 edible films 181, 182 meat products 179 nutritional aspects of cross-linked proteins 182, 183 overview 178, 219 seafood products 178, 179 soy products 180 Fungal transglutaminases overview 210–212 structural role in cytoskeleton and cell wall 232–234 Gliadin, deamidation and immune response triggering in celiac disease 162–164 Gluten peptides, tissue transglutaminase substrate specificity 164–166 Glycosylated proteins, synthesis using transglutaminases 185

Invertebrate transglutaminases growth and development role 216 overview 210–212 Keratinocyte transglutaminase (TG1) functions 6, 24, 25 gene regulation 2 gene therapy for lamellar ichthyosis 176, 177 knockout mice 92, 95 membrane association 6, 24 mutations and disease 6, 120 nomenclature 2 posttranslational processing 6, 24, 116 regulators 24, 116–118 tissue distribution 2, 116, 139 wound healing role 91, 92, 95 Lamellar ichthyosis, keratinocyte transglutaminase defects 6, 120 gene therapy 176, 177 Leather manufacturing, transglutaminase application prospects 183 Light sensitivity, see Plant transglutaminases Meat processing, see Food science Mitochondria, tissue transglutaminase function 62, 63

Haptens, transglutaminase application prospects in biotechnology 184 Huntington’s disease (HD) tissue transglutaminase role 67, 68, 144–146 therapeutic targeting 177 transglutaminase activity and expression 143–146

Nematode transglutaminases growth and development role 213–215 overview 210–212 sequence analysis between species 216, 217 Neuronal development tissue transglutaminase role 140, 141 transglutaminases in apoptosis 141, 142 transglutaminases in differentiation 81, 82, 140

Inflammation, tissue transglutaminase role enzyme induction 99 pathology 100 phospholipase A2 activation 99, 100 Integrins, tissue transglutaminase interactions 126, 130–132

Parkinson’s disease (PD) neuropathology 147 transglutaminase activity and expression 143, 147, 148 Phospholipase A2 (PLA2), activation by tissue transglutaminase 99, 100

Subject Index

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Plant transglutaminases Arabidopsis thaliana enzyme 243, 244 assays 223, 224 biotechnological aspects 244–246 calcium dependence studies 226, 228, 231, 232 chloroplast enzymes 235, 236 compartmentalization and putative functions 226, 227 light and salt stress effects in algae 240, 241 light sensitivity in higher plants 238–240, 242 mammalian enzyme expression in recombinant plants 244–246 mammalian-type enzyme in slime molds 244 nonphotosynthetic tissue enzyme features 228, 229 overview of features 225 photosynthetic tissue enzyme features 230, 231 polyamines conjugation 223 protein conjugation 223, 224 types 223 programmed cell death role 237, 238 structural role in cytoskeleton and cell wall 232–234 substrate specificity 229, 231, 241, 242 Polyamine conjugation, see Plant transglutaminases Protein disulfide isomerase (PDI) functions 210 structure and mechanism 210 tissue transglutaminase activity 33 transamidation catalysis 217–219 transglutaminase sequence homology 216, 217 Protozoan transglutaminases overview 210–212 therapeutic targeting 219 Retinoblastoma protein (Rb), tissue transglutaminase substrate in cell survival 65, 79 Retinoic acid (RA) neuronal differentiation induction 81, 82

Subject Index

tissue transglutaminase induction 65, 66, 79 RhoA, activation by tissue transglutaminase 76, 77 Scarring, see Fibrosis Smoking, see Cancer Spinal cord injury, transglutaminase activity and expression 143, 149 Streptoverticillium mobaraense transglutaminase, biotechnology applications 175, 186 Textiles, transglutaminase application prospects 183, 184 TG1, see Keratinocyte transglutaminase TG2, see Tissue transglutaminase TG3, see Epidermal transglutaminase TG4, see Transglutaminase 4 TG5, see Transglutaminase 5 TG6, see Transglutaminase 6 TG7, see Transglutaminase 7 Thiol group, inherent reactivity in proteins 19, 20 Thioredoxin, transamidation reactions and catalytic residues 210, 217–219 Tissue transglutaminase (TG2) activation 28, 29 adhesion molecule interactions 33 antibody sources for research 16 apoptosis role, see Apoptosis autophagy role 66, 67 functions 7, 9, 59, 61–63, 75, 76, 96, 126 gene regulation 6 G-protein signaling 32, 33, 59, 61, 77–79 GTP binding and hydrolysis 30, 31, 77, 78, 96 inflammation role, see Inflammation knockout mouse phenotypes cell survival 63, 64, 127 wound healing 94, 95 mitochondrial function 62, 63 neuronal differentiation induction 81, 82 nomenclature 6 pathology cancer, see Cancer celiac disease, see Celiac disease gene mutations and disease 8

252

liver disease 68, 69 neurodegenerative diseases 67, 68, 142–149 peptidyl-disulfide isomerase activity 33 regulators 24, 28, 29 RhoA activation 76, 77 subcellular distribution 7, 25, 78, 79, 96, 97, 126, 127 tissue distribution 6, 139 trafficking 25, 78, 79, 126, 127 wound healing role, see Wound healing X-ray crystal structure active site 27, 28 calcium-binding site 28, 29 domain structure 26, 27 overview 25, 26 purine-binding site 30–32 Transforming growth factor beta-1 (TGF-␤1), activation by tissue transglutaminase (T62) 97, 98, 131 Transglutaminase, see also specific enzymes active-site sequence homology 1, 4 barriers to research 37, 38 biochemical reactions 1, 2, 39–42, 185, 186 catalytic reaction 20–23 classification 1, 3, 5, 38–40, 192 discovery 37 functional overview 48, 49, 192 genomic organization 1, 4, 23, 192, 194 sequence analysis between species 216, 217 substrate-binding sites 42 substrate specificity 40–47, 164–166, 229, 231, 241, 242 Transglutaminase 4 (TG4) cancer expression 11 functions 11, 24 gene regulation 11 nomenclature 11 posttranslational processing 11 tissue distribution 11, 139 trafficking 25 Transglutaminase 5 (TG5) antibody sources for research 16 epidermal distribution 119 functions 12

Subject Index

gene regulation 12 nomenclature 12 pathology 12 posttranslational processing 12 purine binding and hydrolysis 32, 119 regulators 24, 119 subcellular distribution 119 tissue distribution 12, 139 wound healing role 92, 93 Transglutaminase 6 (TG6) functions 13 gene regulation 13 nomenclature 13 posttranslational processing 13 tissue distribution 13, 139 Transglutaminase 7 (TG7) nomenclature 13 tissue distribution 13, 139 Transglutaminase X, see Transglutaminase 5 Transglutaminase Y, see Transglutaminase 6 Transglutaminase Z, see Transglutaminase 7 Wool, transglutaminase applications in finishing 183, 194 Wound healing, see also Fibrosis cutaneous healing overview 89, 91 kidney 100, 103–105 therapeutic targeting of transglutaminases in wound repair 105–107 transglutaminases in skin morphogenesis and wound healing epidermal transglutaminase 92 factor XIIIA 94 keratinocyte transglutaminase 91, 92, 95 prospects for study 107, 109 tissue transglutaminase cell migration role 99 functions 96 matrix structural role 98 matrix synthesis and degradation role 97, 98 overview 93 stress response 96, 97 transgenic mouse studies 94, 95 transglutaminase 5 (T65) 92, 93 Yogurt, transglutaminases in preparation 180

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