Kiyotaka Hitomi · Soichi Kojima Laszlo Fesus Editors Transglutaminases Multiple Functional Modifiers and Targets for New Drug Discovery Transglutaminases Kiyotaka Hitomi • Soichi Kojima • Laszlo Fesus Editors Transglutaminases Multiple Functional Modifiers and Targets for New Drug Discovery Editors Kiyotaka Hitomi Graduate School of Pharmaceutical Sciences Nagoya University Nagoya, Japan Laszlo Fesus Department of Biochemistry and Molecular Biology University of Debrecen Debrecen, Hungary Soichi Kojima Micro-Signaling Regulation Technology Unit RIKEN Center for Life Science Technologies (CLST) Wako, Saitama, Japan RIKEN Molecular and Chemical Somatology Tokyo Medical and Dental University Bunkyo-ku, Tokyo, Japan ISBN 978-4-431-55823-1 ISBN 978-4-431-55825-5 DOI 10.1007/978-4-431-55825-5 (eBook) Library of Congress Control Number: 2015958519 Springer Tokyo Heidelberg New York Dordrecht London © Springer Japan 2015 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made Printed on acid-free paper Springer Japan KK is part of Springer Science+Business Media (www.springer.com) Preface Posttranslational modification, which refers to covalent and generally enzymatic modification of proteins during or after protein biosynthesis, is a highly important subject of the next-generation, post-genome research Among various posttranslational modifications, enzymes that catalyze protein–protein cross-linking reactions are transglutaminases conserved from microorganisms to mammals, forming covalent isopeptide bonds between peptide-bound glutamine and lysine residues under strict regulatory conditions Transglutaminases also catalyze attachment of primary amine (transamidation) or replacement to glutamic acid residue (deamidation) at the glutamine residues In mammals, this enzyme family consists of eight isoforms (isozymes) differentially expressed in various tissues Although the first description on transglutaminase was made almost 60 years ago and numerous studies have been performed to characterize these enzymes and their physiological and pathological roles, there are many unresolved issues Newer and newer mysteries have evolved, and once the answers to these are found, the challenge of application of novel knowledge to practical use remains for the future Intriguing transglutaminase features include diverse functions and sometimes opposing activities of transglutaminase family members, their multiple and changing localization inside and outside of cells, their non-enzymatic interactions and scaffolding activity, the mechanism of their secretion, and so on By transglutaminase-catalyzed reactions the structure, function, and stability of the substrate proteins are altered, associating with a number of biological phenomena The unique catalytic reactions and multiple interactions of transglutaminases have fascinated many scientists during the last six decades so that they have achieved biological significance in a wide scientific area and applications in chemical, food, cosmetic, and pharmaceutical industries Because the aberrant activity or ectopic expression of the enzymes causes several diseases, inhibitory and regulatory molecules have been developed as promising new drugs Additionally, the dramatic advances in molecular life science technologies have brought much progress in all the transglutaminase research areas Considering current trends and advances, we planned to publish this review book to cover basic knowledge and novel findings in transglutaminase research We have v vi Preface attempted to review structures, expression, functions, and regulatory mechanisms from the scope of enzymology, biochemistry, physiology, pathology, pharmacology, chemistry, and applied bioscience Particularly, we focused on diseases and drug development related to the enzymes’ role in various pathologies At the Gordon Research Conference on Transglutaminases in Human Disease Processes held in Italy in 2014, we discussed with the authors, who have been engaged in prominent transglutaminase work, the purpose and scope of the book’s content and decided to devote more space to basic knowledge underlying the theme of each chapter, in addition to the new, cutting-edge findings, so that “newcomers” can obtain useful information and technical insight, and most importantly an interest in studying these enigmatic enzymes in the future The book could not have been achieved without the full dedication of each contributing author and the people who have supported us We hope that it will develop a further basis of new collaborations by stabilizing “cross-linking” of researchers and newcomers in the future Nagoya, Japan Wako, Japan Debrecen, Hungary Kiyotaka Hitomi Soichi Kojima Laszlo Fesus Contents Structure of Transglutaminases: Unique Features Serve Diverse Functions ´ Deme´ny, Ilma Korponay-Szabo, and Laszlo Fesuăs Mate A Control of TG Functions Depending on Their Localization Yutaka Furutani and Soichi Kojima 43 Preferred Substrate Structure of Transglutaminases Kiyotaka Hitomi and Hideki Tatsukawa 63 Insights into Transglutaminase Function Gained from Genetically Modified Animal Models Siiri E Iismaa 83 Transglutaminase in Invertebrates 117 Toshio Shibata and Shun-ichiro Kawabata A New Integrin-Binding Site on a Transglutaminase-Catalyzed Polymer 129 Yasuyuki Yokosaki Transglutaminase 2-Mediated Gene Regulation 153 Soo-Youl Kim The Role of Transglutaminase Type in the Regulation of Autophagy 171 Manuela D’Eletto, Federica Rossin, Maria Grazia Farrace, and Mauro Piacentini Transglutaminase and Celiac Disease 193 Rasmus Iversen and Ludvig M Sollid 10 Transglutaminase II and Metastasis: How Hot Is the Link? 215 Kapil Mehta vii viii Contents 11 Transglutaminases: Expression in Kidney and Relation to Kidney Fibrosis 229 Elisabetta A.M Verderio, Giulia Furini, Izhar W Burhan, and Timothy S Johnson 12 Transglutaminases in Bone Formation and Bone Matrix Stabilization 263 Cui Cui and Mari T Kaartinen 13 Transglutaminases and Neurological Diseases 283 Julianne Feola, Alina Monteagudo, Laura Yunes-Medina, and Gail V.W Johnson 14 Regulation of Transglutaminase by Oxidative Stress 315 Eui Man Jeong and In-Gyu Kim 15 Blood Coagulation Factor XIII: A Multifunctional Transglutaminase 333 Moyuru Hayashi and Kohji Kasahara 16 Inhibition of Transglutaminase 347 Jeffrey W Keillor 17 Substrate Engineering of Microbial Transglutaminase for Site-Specific Protein Modification and Bioconjugation 373 Noriho Kamiya and Yutaro Mori Index 385 Chapter Structure of Transglutaminases: Unique Features Serve Diverse Functions ´ Deme´ny, Ilma Korponay-Szabo, and La´szlo Fesuăs Mate A Abstract Understanding the diverse functions and pathologies of transglutaminases requires detailed analysis and interpretation of their structures This chapter is an attempt to describe in detail how these enzymes are folded into functional domains, what type of catalytic and scaffolding functions have been gained as the result of their evolution, how their regulation is achieved through unique Ca2+ and purine nucleotide binding sites, redox changes and specific proteolytic actions, and by influencing the equilibrium of open-close configurations The importance of structural motifs in pathologies is underlined by the celiac epitopes of transglutaminase 2, responsible for autoimmune reactions Keywords Domain organization • Crystallography • Catalytic mechanism • Regulation • Ca2+ • Purine nucleotides • Proteolysis • Redox • Open-close conformation • Substrates • Interactions • Celiac epitope 1.1 Introduction Transglutaminases (Tgases) are a large family of enzymes canonically responsible for amidation of protein and non-protein amines They are ubiquitous in higher organisms but have also been identified in lower life forms, including archea, bacteria, plants, worms and insects Their structural core and catalytic residues are strongly related to proteases and other hydrolases Their common ancestor evolved to utilize acyl acceptors other than water by changing the active site so that it would be less accessible for water by a mechanism that is perfected in the vertebrate enzymes The simple ancient Tgases, similar to the present microbial enzymes, acquired additional domains to serve regulatory, interacting and new enzymatic functions In some instances these additions rendered them zymogens, which show differential activity based on proteolytic activation They evolved to be Demeny L Fesuăs (*) M.A Department of Biochemistry and Molecular Biology, University of Debrecen, Debrecen, Hungary e-mail: fesus@med.unideb.hu I Korponay-Szabo Department of Pediatrics, University of Debrecen, Debrecen, Hungary © Springer Japan 2015 K Hitomi et al (eds.), Transglutaminases, DOI 10.1007/978-4-431-55825-5_1 376 N Kamiya and Y Mori Fig 17.2 Lys-specific protein labeling catalyzed by MTG with a small functional Gln-donor substrate 17.3 C-Terminal Modification of Z-QG 17.3.1 C-Terminal Modification of Z-QG for Protein Labeling In general, the C-terminal carboxylic acid of Z-QG is a feasible target for introducing new chemical entities with desired function (Fig 17.3) Given the fact that the recognition of Gln-donor substrates by MTG relies on the hydrophobic character of the N-terminus (Ando et al 1989; Ohtsuka et al 2000a), the C-terminal modification is reasonable to avoid steric hindrance upon the binding between MTG and a substrate The introduction of monodansylcadaverine at the C-terminal carboxylic group of Z-QG was first studied to obtain a fluorolabeled Z-QG (Pasternack et al 1997) In this context, C-terminal modification of Z-QG and Z-QQPL with fluorescent derivatives was explored for MTG- and human tissue transglutaminase-mediated protein labeling (Mindt et al 2008) 17.3.2 C-Terminal Modification of Z-QG for the Preparation of Protein-Nucleic Acid Conjugates We started from designing the C-terminal labeling of 5’-end amine-modified oligodeoxynucleotides (NH2-ODN) with Z-QG for the preparation of proteinODN conjugate (Fig 17.4) This was feasibly conducted by chemical activation of carboxylate of Z-QG with N-hydroxysuccinimide (NHS) The NHS-activated Z-QG (Z-QG-NHS) was mixed with an aminated ODN, and the resultant Z-QGODN was conjugated with recombinant enhanced green fluorescent protein (EGFP) and bacterial alkaline phosphatase (AP) A short substrate peptide comprising six amino acids (MKHKGS, K-tag) was fused to the N-terminus of these two model proteins, allowing site-specific labeling of the peptidyl tag with Z-QG-ODN 17 Substrate Engineering of Microbial Transglutaminase for Site-Specific 377 Fig 17.3 Design concept for a functional Gln-donor substrate of MTG for Lys-specific protein labeling Fig 17.4 Design of Gln-donor substrates for MTG-mediated preparation of protein-nucleic acid conjugates (Tominaga et al 2007) Protein-ODN conjugate is depicted as a tadpole molecule exhibiting dual functions derived from distinct functionalities of conjugated biomolecules A potential application of this type of bioconjugates is site-specific, oriented protein immobilization through the hybridization of ODN with the complementary ODN presented on solid surface The basic concept can be seen on a traditional in situ hybridization technique for the detection of either target DNA immobilized on membranes or mRNA on tissue sections However, a short ODN is not suitable for those applications, which led us to develop a new enzymatic strategy for the preparation of DNA-protein conjugates (Kitaoka et al 2011) The design of a new nucleotidyl substrate of MTG, Z-QG-dUTP, was also based on the simple NHS chemistry (Fig 17.4) However, in this case, we should consider the availability of the new substrate for DNA polymerase as well as MTG Fortunately, Z-QG-dUTP was well recognized by different families of DNA polymerases (such as KOD and Taq polymerases), and a new DNA scaffold, (Z-QG)n-DNA, was successfully obtained by the partial substitution of dTTP with Z-QG-dUTP in 378 N Kamiya and Y Mori Fig 17.5 Design of nucleic acid scaffolds with multiple Gln-donor sites for MTG-mediated labeling with a K-tagged enzyme polymerase chain reaction Subsequent labeling of (Z-QG)n-DNA with a K-tagged enzyme resulted in a DNA-(enzyme)n conjugate with 1:n stoichiometry (Fig 17.5) Upon the labeling of a thermostable alkaline phosphatase from Pyrococcus furiosus, the obtained bioconjugate with new molecular architecture enables the detection of a tiny amount of target nucleic acids having the complementary sequence in a dot blot hybridization assay under harsh hybridization conditions (Kitaoka et al 2011) Besides the preparation of DNA-enzyme conjugates, the synthesis of Z-QG-UTP yielded RNA-(enzyme)n conjugates, which are applicable to an in situ hybridization technique for the detection of mRNA on tissue sections (Kitaoka et al 2012) The number of enzymes labeled on the RNA scaffold impacted on the hybridization efficiency Importantly, the potential steric hindrance, caused by the increase in the number of labeled enzymes on RNA, was controlled by simply changing the molar ratio of Z-QG-UTP to UTP on in vitro transcription catalyzed by RNA polymerases The synthetic strategy was applied to assemble cellulases on fully doublestranded DNA (dsDNA) to create a potent artificial cellulosome for enhancing hydrolysis of cellulosic substrates (Mori et al 2013a) In the case of using dsDNA as a scaffold, insertion of a short ethylene oxide linker was critical for increasing the conjugation yields of DNA-(cellulase)n conjugates The concept has been extended to label single or multiple proteins to a DNA aptamer by combining MTG and terminal deoxynucleotidyl transferase (TdT) (Takahara et al 2013) The single protein labeling of ODN by the two-step enzymatic reaction with Z-QGddUTP may offer a feasible way to access tadpole molecules 17 Substrate Engineering of Microbial Transglutaminase for Site-Specific 17.4 379 N-Terminal Substitution of Z-QG 17.4.1 Design of Fluorescent Labeling Reagents One might imagine that the N-terminal substituent, benzyloxycarbonyl group (Z), could be a target for introducing new chemical entities (Fig 17.3) However, the substrate preference of MTG may limit the chemical property available for the replacement of the Z moiety: it has been known that the N-terminal side of reactive Gln residue is critical for the substrate recognition of MTG For instance, Z-QG is one of the most reactive Gln-donor substrates for MTG although GQG is not recognized as a substrate (Ando et al 1989) As mentioned in the preceding section, most researchers were interested in the C-terminal labeling of the core structure, Z-QG It is interesting for us that in spite of the preference of MTG for hydrophobic amino acids at the N-terminus of reactive Gln residue, it still shows rather broad substrate specificity Therefore, we explored the idea that the substitution of the Z moiety with a fluorescent group may open new avenues for MTG-mediated protein modification with small Gln-donor substrates In this design concept, the fluorescent group needs to play a dual role One is for the intrinsic probe, and the other is for the substrate recognition During the course of basic trials, we found that MTG accepts a wide range of fluorophores in the molecular size (i.e., fluorescein, dansyl and rhodamine derivatives) (Kamiya et al 2009) Importantly, the reaction was only realized upon the insertion of a short linker (e.g., β-alanine) between the N-terminus of reactive Gln residue and the functional groups (Fig 17.6), indicating the requirement of a linear flexible linker for the substrate recognition of MTG as was reported for primary amine substrates (Ohtsuka et al 2000a, b; Lorand et al 1979) The results suggest the potential utility of MTG that can accept a wider range of substrates than mammalian TGases (Sato et al 2001) Fig 17.6 The effect of a linker on MTG-mediated protein labeling with a fluorescent Gln-donor substrate 380 N Kamiya and Y Mori Fig 17.7 Design of Gln-donor substrates for MTG-mediated biotinylation of proteins of interest 17.4.2 New Biotinylation Reagents and Their Applications As another functional N-terminal substituent of the Z moiety, a small molecular ligand, biotin, was selected, which can bind to a tetrameric protein receptor, avidin The ligand-protein interaction between biotin and avidin has been widely employed in biotechnology and bioengineering, due to its high specificity and strong affinity (i.e., Kd ¼ 10À15) In the specific case of Z-QG modification, we first designed a small biotinylation substrate with the shortest linker, biotin-QG (Fig 17.7) In the structure of biotin-QG, the N-terminal biotin moiety gives hydrophobicity, and the C4 alkyl linker, between the binding moiety and the N-terminus of reactive Gln residue, provides flexibility, which in turn facilitates substrate recognition by MTG (Mori et al 2013b) Similarly, a larger biotinylation substrate, biotin-GGGGGLQG, was synthesized and its C-terminal LQG tripeptides showed positive reactivity in the presence of MTG (Pasternack et al 1997) Like other avidin variants, avidin from Streptomyces avidinii, streptavidin (SA), forms a tetramer More specifically, two SA dimers assemble, facing opposite directions Bis-biotin can bind to the SA dimer from one face so that it behaves differently from mono-biotin Therefore, we designed a bis-biotinylated substrate, bis-(biotin-GGG)-KGLQG, to control the vector of biotin-avidin interaction A proof-of-concept study was conducted by the directional assembly of AP as a model protein, because of its symmetric dimeric structure (Fig 17.8) Within a biotin-avidin system, the biotin-labeled position to a target protein should be an important factor for the formation of a supramolecular protein complex To assess the utility of MTG combined with a new biotinylated Gln-donor substrate, we explored the internal labeling of AP to which a MTG reactive Lys-containing peptide loop (IRINRGPGKAFVT, K-loop) was inserted (Mori et al 2011) The K-loop-inserted AP mutant maintained the original catalytic activity, and the Lys 17 Substrate Engineering of Microbial Transglutaminase for Site-Specific 381 Fig 17.8 Molecular design for assembling proteins by MTG-mediated internal biotinylation of dimeric protein residue in the loop was recognized efficiently by MTG without impairing enzymatic activity due to the insertion of the K-loop at a suitable site in AP After the loop-specific biotinylation with designed new biotinylation reagents, biotinylated APs were mixed with SA to form supramolecular protein complexes (Fig 17.8) As a result, several factors to assemble a protein complex with the biotin-avidin interaction were noticed: (1) use of a shorter biotinylation substrate and (2) arranging the position and direction of two biotins, as for example the labeled AP dimers, could prevent the intramolecular self-cyclization but promote the intermolecular interaction and the consequent enlargement of the AP-SA complexes; (3) with different types of biotinylation substrate, the shapes of protein molecular assemblies can be controlled (i.e., a spherical protein complex was formed with mono-biotin, and the bis-biotinylation of AP resulted in a one-dimensional stringy structure) (Mori et al 2013a, b) Altogether, through the optimal design of the biotinylation substrate and biotinylation sites in a protein, the formation (i.e., growth and shape) of supramolecular protein assemblies can be designed according to each specific application For the design of such selfassembled protein structures, protein-lipid conjugates are of interest as well, and the development of a Gln-donor substrate for MTG-mediated lipidation of a recombinant protein was demonstrated (Abe et al 2011) 17.5 Conclusion In this chapter, we exemplified that a small modification of the base structure of a substrate, X-QG-Y, greatly expands the utility of MTG-mediated protein manipulation in a range of biotechnological applications Our results demonstrated that fine-tuning of artificial substrates may find new biomolecular architectures with unique functionality, and are promising for further development of MTG-mediated creation of artificial biological products One of the next challenges would be the 382 N Kamiya and Y Mori design of MTG mutants, exhibiting the orthogonal substrate specificities, making it possible to create one-step multifunctional modifications of different proteins and/or target sites in situ On-going efforts in the development of new biotechnology should achieve the goal in the near future Acknowledgments We express our sincere gratitude to all colleagues who worked together with MTG We also appreciate Ajinomoto Co., Inc for providing us with the MTG sample and information on its basic characteristics This work was supported by a Grant-in-Aid for Scientific Research (Grant Number 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Biochemistry 35:13072–13080 Sato H, Hayashi E, Yamada N, Yatagai M, Takahara Y (2001) Further studies on the site-specific protein modification by microbial transglutaminase Bioconjug Chem 12:701–710 Strop P (2014) Versatility of microbial transglutaminase Bioconjug Chem 25:855–862 Takahara M, Hayashi K, Goto M, Kamiya N (2013) Tailing DNA aptamers with a functional protein by two-step enzymatic reaction J Biosci Bioeng 116:660–665 Tominaga J, Kemori Y, Tanaka Y, Maruyama T, Kamiya N, Goto M (2007) An enzymatic method for site-specific labeling of recombinant proteins with oligonucleotides Chem Commun 2007:401–403 Yokoyama K, Nio N, Kikuchi Y (2004) Properties and applications of microbial ransglutaminase Appl Microbiol Biotechnol 64:447–454 Index A Ackermann-Potter plot, 356 Actin, 45, 46, 93, 99, 221, 232, 301, 337 Active sites, 1, 3, 5–11, 14, 15, 17, 23–30, 34, 65, 66, 133, 197, 200, 201, 207, 248, 253, 321, 334, 357–359, 362, 363 Adhesion, 44, 45, 47–49, 93, 94, 103, 107, 134–136, 217, 219–222, 233, 234, 265, 268, 269, 271, 274, 284, 288, 297, 300, 301, 335, 336, 341 Adrenoceptor α1B binding site, 18, 30, 31 Alzheimer’s disease, 47, 54, 182, 284, 291–294 AMBRA1, 176, 177 Amyloid, 47, 49, 50, 131, 133, 141, 292–294, 317 Amyotrophic lateral sclerosis, 284, 295–296, 304–305 Anchorage-independent growth, 223 Anergy, 206 Angiotensin II type receptor (AT1), 131, 132 Angiotensin II type receptor (AT2), 132, 317, 318 Animal husbandry, 84–86, 91, 93, 98, 101, 103, 105, 108 guidelines, 84–86, 91, 93, 96, 98, 101, 103, 105 Animal models, 84, 91–107, 121, 156, 231, 298, 317, 318 Animal study design, 91 guidelines, 84–86, 91, 93, 96, 98, 101, 103, 105 Anopheles gambiae, 125 Antibodies, 31–35, 91, 130, 136, 138, 141, 145, 194, 197–203, 206–208, 248, 249, 251, 253, 293, 338, 375 Antigen presentation, 194, 197, 199, 203–206, 208 Antimicrobial peptides, 92, 123, 124, 158 Apoptosis, 44, 45, 47, 51–54, 94–99, 108, 122, 131, 132, 143, 154, 158–161, 164, 180, 183, 184, 206, 217, 223, 239, 274, 291, 301, 302, 316, 324, 338, 340 Arg-Gly-Asp (RGD), 130, 133, 134, 136, 234, 269–271 Arterial calcification, 99–101 Arterial remodelling, 102–103, 107 Arthropod, 10, 120 Assay, 34, 71, 75–77, 90, 132, 145, 159, 200, 224, 236, 237, 294, 335, 336, 350, 357, 358, 360–362, 364, 365, 375, 378 Astrocytes, 155, 284, 296–299, 301 AT1 See Angiotensin II type receptor (AT1) AT2 See Angiotensin II type receptor (AT2) Atherosclerosis, 99–101 Autoantibodies against transglutaminases, 91, 208 Autophagy, 171–191 Avidin, 73, 380 B Backcrossing, 86, 88, 103 B-cell receptor, 34, 105, 198 B cells, 34, 92, 93, 154, 194, 195, 197–200, 202–209, 318–320 BECLIN1, 176, 185 Bioconjugation, 373–382 Biomolecular engineering, 380 Biotin, 380, 381 © Springer Japan 2015 K Hitomi et al (eds.), Transglutaminases, DOI 10.1007/978-4-431-55825-5 385 386 Bone, 48, 93, 100, 101, 105–106, 135, 144, 207, 234, 237, 263–275, 335 formation, 101, 263–275 Brain, 70, 97, 174, 202, 284–286, 291–301, 303–305 C Cadaverine, 73, 74, 118, 120, 122, 123, 179, 302, 361, 375, 376 Calcium binding motifs, 3, 12–16, 21, 24–27, 253 Cancer, 49, 50, 107, 135, 154–156, 159–165, 172, 175, 180, 181, 183, 184, 215–225, 250, 251, 302, 316–321, 325, 327 Cancer stem cells (CSCs), 223 Caraxin, 119, 120 Cardiac hypertrophy, 99 Catalytic mechanism, 6–10, 27 CD See Celiac disease (CD) CD44, 48, 99, 134, 222 CE See Cornified envelop (CE) Celiac anti-TGM2 antibody epitopes, 33, 34 Celiac anti-transglutaminase antibody formation, 201 Celiac disease (CD), 31, 56, 69, 91, 102, 107, 156, 193–209, 225, 248, 317, 323 Cell adhesion, 45, 48, 49, 103, 134, 135, 233, 234, 268, 274, 288, 336 motility, 219, 220, 223, 224 signaling, 219, 220 spreading, 131 Central nervous system (CNS), 92, 93, 176, 284, 295–301 Chemoattractant, 130, 133, 134, 137, 140, 141, 145 Cheng-Prusoff equation, 350 Chitin, 120–122 Chronic kidney disease (CKD), 229–254 Cis peptide bonds, c-Jun N-terminal kinase (JNK), 131, 132, 143 CKD See Chronic kidney disease (CKD) Closed conformation, 15, 16, 20, 24, 30, 97, 220, 254, 322, 368 Closed form, 3, 5, 17, 24, 31, 44, 219, 322 Clotting protein (CP), 121 c-Met, 96, 131, 132, 143, 159 CNS See Central nervous system (CNS) Coagulation, 19, 44, 47, 95, 117–119, 121–123, 125, 131, 274, 333–343 Coagulin, 118, 119 Co-isogenic, 89, 90, 105 Index Collagen, 45, 48, 49, 93, 94, 102, 135, 219, 222, 232, 234–237, 240, 242, 243, 247, 264, 266–269, 317, 333, 339, 341–343 Commensal community, 124 microbes, 123, 124 Compensation by other transglutaminases, 90, 94, 247 Conditional inactivation, 2, 84 Conformational change, 2, 5, 142, 334, 348, 356 Conformational rearrangement, 2, 22–28 Congenic full, 86 incipient, 86 Cornea, 106–107 Cornified envelop (CE), 19, 50, 51, 68, 76 Crayfish, 121 Cross-linking, 30, 45–47, 49–52, 63–65, 68– 72, 77, 119, 120, 122–125, 153, 154, 157, 159, 161–164, 195, 206, 207, 334– 340, 342, 343, 368, 374 Crustaceans, 121 Cuticle, 120, 122 Cuticular formation, 120 morphogenesis, 121–122 proteins, 122 Cystamine, 238, 271, 292, 303–306, 327 Cytosol, 43–46, 117, 153, 156, 165, 238, 323, 325 D Database, 5, 11, 30, 72, 79 ddUTP, 378 Deamidation, 6, 8, 12, 21, 34, 53, 63, 65, 69, 194–196, 198, 200, 203–206, 208, 253 Dendritic cells, 92, 204, 205, 208, 233, 335 Dermatitis herpetiformis, 35, 70, 91 Detection, 64, 75–79, 236, 241, 270, 293, 341, 368, 377, 378 Diabetes, 35, 105, 108, 229–231, 237, 239, 317, 318 DLK See Dual leucine zipper-bearing kinase (DLK) DNA, 51, 83, 89, 95, 162, 177, 180, 184, 186, 302, 319, 374, 377, 378 Domains β-barrel, 334 β-barrel 2, 334 β-sandwich, 334 catalytic, 20, 23, 334 Index Drosophila, 52, 120–125, 158 Drug resistance, 160, 161, 217–219, 221, 225 Dual leucine zipper-bearing kinase (DLK), 46, 131, 132, 143 dUTP, 378 E EAE See Experimental autoimmune encephalomyelitis (EAE) ECM See Extracellular matrix (ECM) EGFR See Epidermal growth factor receptor (EGFR) ε-(γ-glutamyl)lysine (GGEL) antibody, 287 crosslinks, 235, 237, 241, 242 Endogenous inhibitor protein, 326 Endostatin, 45, 49 Epidermal growth factor receptor (EGFR), 161, 302 Epithelial to mesenchymal transition, 160, 219, 233 Evolutionary origins, 10–12 Experimental autoimmune encephalomyelitis (EAE), 93, 104 Extracellular matrix (ECM), 30, 44, 48, 94, 107, 130, 131, 133, 163, 194, 203, 219, 232, 254, 264, 267–269, 271, 274, 285, 293, 317, 318, 326, 337, 342 Extracellular matrix stabilization, transglutaminase, 263–275 Eye, 70, 106–107, 122 F Factor XIII calcium binding, 12–16 catalytic residues, cis-peptide bonds, heparin binding, 32 open-close conformational rearrangement, 22–28 proteolytic activation of, 19 rare earth metal binding, 13 structural models, targeted glutamine residues, 69 Fibronectin, 4, 30, 32, 33, 45, 48, 49, 51, 54, 67, 68, 72, 107, 130, 135, 164, 219, 221, 222, 232, 234, 235, 244, 265, 268–269, 297, 298, 301, 317, 336, 337, 339, 342, 343, 362 Fibronectin binding site, 4, 32, 33 Fibrosis, 49, 90, 93–95, 99, 107, 108, 154, 156– 159, 181, 222, 225, 229–254, 320, 324– 326 Flap-region, 25–27 387 Fluorescein, 379 Fluorescent labeling, 379–380 Fondue, 122, 123 Function, 1–35, 43–56, 64, 66, 68, 69, 79, 83– 108, 118–122, 130–145, 154, 160, 172, 173, 176, 178, 179, 182, 184, 186, 218– 223, 230–232, 234, 239, 241, 243, 254, 264, 266, 268–271, 273, 274, 284–286, 288, 289, 291, 294, 295, 302–306, 317, 336, 338, 339, 341, 343, 350, 351, 368, 374–377 G Gαh, 19 GDP/GTP-binding site, 18 Gene inactivation, 83 Genetically-modified mouse models, 83, 107 Germ-free, 124 Germinal centers, 203 GGEL antibody See ε-(γ-glutamyl)lysine (GGEL) antibody Glioma, 134, 300–302 Gluten, 32, 56, 67, 91, 193–200, 202–209, 322 Gluten ataxia, 35, 70, 202 H Hemocytes, 117–122, 158 Heparan sulfate binding site, 243 Heparan sulphate proteoglycans, 94 Hexamerin, 123 HIF1 See Hypoxia-inducible factor-1 (HIF1) HIF-1α See Hypoxia-inducible factor-1 alpha (HIF-1α) Homology models, 3, 11, 13, 15, 368 Homopolymer, 118, 135 Horseshoe crab, 117–121 Human leukocyte antigen, 194 Humoral response, 93 Huntingtin, 52, 182, 183, 285, 317 Huntington’s disease, 52, 103, 108, 180, 184, 284, 286–289, 303–304 Hypoxia-inducible factor-1 (HIF1), 162, 223, 299, 320–321 Hypoxia-inducible factor-1 alpha (HIF-1α), 154, 155, 162–165, 218, 221, 225, 319– 321 I IC50 parameter, 253, 349–352, 354, 358, 360– 362, 364–366 IgA See Immunoglobulins A (IgA) IgD See Immunoglobulins D (IgD) 388 IgG See Immunoglobulins G (IgG) IMD See Immune deficiency (IMD) Immune deficiency (IMD), 123, 124 Immune system, 91–93, 107, 194, 195, 204, 217 Immune tolerance, 52, 124 Immunoglobulins A (IgA), 31, 91, 198, 199, 202, 203, 207, 238, 239, 243 Immunoglobulins D (IgD), 207 Immunoglobulins G (IgG), 91, 198, 207, 208, 245 inactivation constant (kinact), 356–359, 363– 365 Inflammation, 21, 69, 93–95, 98, 99, 133, 154– 160, 194, 204, 206, 208, 217–222, 225, 230, 296, 297, 318, 325–327 Inhibition active-site directed, 357–358, 362 acyl-donor analogue, 360–361 competitive, 348–350, 356, 361 conformational effects, 368 DON derivative, 365–366 GTP analogue, 360 3-halo-4,5-dihydroisoxazole, 363 irreversible, 356–359 Michael acceptor, 363–365, 368 non-competitive, 352–354, 361 reversible, 348–356 slow binding, 355, 360 structure-based design, 367–368 suicide, 358–359 sulfonium, 366 tight binding, 355–356 time-dependent, 356, 357 uncompetitive, 348, 350–353, 361 Inhibition constant (Ki), 349, 352, 353, 355, 361, 363 In situ hybridization, 377, 378 Integrin, 45, 50, 53, 98, 99, 107, 129–145, 218, 221–223, 233, 269–272, 274, 297, 298, 301, 339, 341, 342 Interaction motifs, 30–31 Intermediate, 7–9, 25, 28, 45, 46, 64–66, 73, 131, 195, 206, 359 Intrinsically disordered regions, 31 Invasion, 100, 160, 216, 217, 219, 222, 321, 339 Ischemia, 97, 104, 298, 299 Isopeptidase activity, 30, 44 Isopeptide bonds, 29, 63, 65, 129, 141, 142, 145, 195, 293, 294, 334, 336 Index J JNK See c-Jun N-terminal kinase (JNK) K Ki See Inhibition constant (Ki) Kidney, 56, 94, 107, 158, 184, 229–254, 324 Kitz and Wilson conditions, 357, 358 Knock-in, 56, 83, 89, 90, 289, 304 Knockout, 46, 48, 83, 84, 86, 89, 90, 100, 105, 154, 181, 234, 247, 268, 269, 290, 335, 336, 339, 342 L Laminin, 45, 49, 130, 222, 232, 235, 269 LAP See Latency associated peptide (LAP) Latency associated peptide (LAP), 130, 233 Latent TGF-β1, 45, 130, 233, 241, 247 Latent TGF-β associated protein (LTBP-1), 130, 131 LC3 See Lipid-conjugated form (LC3) Lens, 70, 80, 106, 326 Lipid-conjugated form (LC3), 174, 176–178, 180–182 Lipid rafts, 341–343 Lipopolysaccharide (LPS), 92, 94, 117–120, 155, 158–160 Liver, 3, 46, 49, 52, 56, 71, 93–94, 96, 97, 108, 153, 154, 158–160, 182, 184, 245, 247, 268, 269, 286, 292, 321, 335, 364 LPS See Lipopolysaccharide (LPS) LTBP-1 See Latent TGF-β associated protein (LTBP-1) Lung, 94–95, 107, 159, 236, 242, 244, 245, 247, 324, 326 M Macroautophagy, 172–180 Malaria, 125 MAPK See Mitogen-activated protein kinase (MAPK) matrix metalloproteinases, 4, 135, 222, 240, 247 Matrix metalloproteinases (MMP-3), 134 Matrix metalloproteinases (MMP-7), 134 MDM2 See Mouse double minute homolog (MDM2) mesenchymal stem cell, 264–266, 273 Mesenteric lymph nodes, 197, 205 Index Metastasis, 107, 134, 155, 215–226 Michaelis-Menten equation, 8, 348, 349, 351–354, 357 Microbes, 118–121, 123, 124, 172 Microbial transglutaminase, 8, 10, 73, 374–382 Midkine, 45, 49, 131, 132 Migration, 107, 135–139, 194, 222, 233, 268, 274, 284, 288, 296–298, 301, 336, 339, 340 Mitochondria, 44, 52–53, 95–98, 104, 173, 175, 180, 182, 183, 185, 223, 285, 287, 294, 316, 319, 321 Mitogen-activated protein kinase (MAPK), 132, 143, 233, 271 Mitophagy, 52, 98, 173, 182, 183 MMP-3 See Matrix metalloproteinases (MMP-3) MMP-7 See Matrix metalloproteinases (MMP-7) Monoclonal antibodies, 141, 198, 254, 335 Mosquito, 125 Mouse double minute homolog (MDM2), 162 mRNA, 74, 90, 236, 253, 268, 286, 292, 293, 298, 305, 320, 377, 378 Multifunctional activities, 47 Multiple sclerosis, 92, 93, 108, 296–298 N NES See Nuclear export signal (NES) Neurodegenerative diseases, 56, 103–104, 183, 184, 284, 286, 291, 303, 317 Neurons, 46, 89, 92, 103, 104, 183, 264, 284, 286, 288–291, 294–299, 303, 304 Neutrophil, 46, 93, 131, 134, 137–141 NF-κB See Nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB) NF-κB inhibitor alpha (I-κBα), 155 NHS See N-hydroxysuccinimide (NHS) N-hydroxysuccinimide (NHS), 376, 377 NLS See Nuclear localization signals (NLS) Nuclear export signal (NES), 54–56 Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), 93, 154 Nuclear localization signals (NLS), 4, 54–56, 299 Nucleus, 43–45, 51–56, 86, 96, 143, 154, 163, 164, 218, 221, 274, 285, 288, 291 O OGD See Oxygen and glucose deprivation (OGD) Oligodeoxynucleotide, 376 389 Oligomer, 120, 132, 141, 164, 286, 296, 304, 317 Open conformers, 24–29 Open form, 3, 5, 24, 25, 27, 28, 44, 51, 219, 322, 367, 368 OPN See Osteopontin (OPN) Oral tolerance, 195, 204 Osteoarthritis, 48, 105–107, 134 Osteoblast, 49, 50, 234, 264–267, 269–274, 335, 343 Osteogenesis, 265, 268 Osteopontin (OPN), 48, 68, 130, 131, 133–140, 142–144, 145, 265, 272 Oxidative stress, 104, 106, 230, 315–331 Oxygen and glucose deprivation (OGD), 298, 299 P p53, 161–162, 164, 165, 180, 184–186 PAI-2, 45, 47–49, 70 Parkinson’s disease, 104, 108, 182, 284, 294–295, 304 Pathogens, 92, 118–121, 123, 158, 173 Peptide library, 69, 73–75, 336 Peptides, 21, 27–30, 32, 56, 64, 69–79, 91, 123, 124, 158, 194–200, 202–206, 208, 209, 248–250, 285, 286, 293, 374 Peptidoglycan, 123 Peptidyl tag, 376 Peroxisome proliferator-activated receptor-γ (PPAR-γ), 266 Phagocytosis, 98–100, 107, 158 Phospholipase C δ1 binding site, 19, 30 Plasma cells, 194, 197, 198, 200–203, 206–208 Plasma fibronectin, 343 Plasma membrane, 19, 43, 44, 47–51, 68, 174, 267, 272, 343 Platelets, 47, 217, 233, 273, 333–336, 339, 341–343 Plugin, 125 Polyglutamine, 285–291 Polymer, 71, 129–145, 156, 163, 292, 335 Polymerization-incompetent, 139–141 PPAR-γ See Peroxisome proliferator-activated receptor-γ (PPAR-γ) Proliferation, 92, 95, 96, 102, 135, 155, 162, 173, 197, 200, 203, 216, 217, 222, 233, 235, 239, 243, 264, 265, 268–270, 273, 288, 298, 300, 302, 316, 321, 327, 339, 340, 342, 362 Protein band ATP binding, 17 inactivity, Protein band 4.2, 2, 12, 17 390 Protein labeling, 375–379 Protein-lipid conjugate, 381 Proxin, 118 pVHL See von Hippel-Lindau tumor suppressor (pVHL) R Rb, 52, 131, 133, 143 RCC See Renal cell carcinoma (RCC) Reaction intermediates, Reactive oxygen species (ROS), 231, 316, 318–327 Rebers and Riddiford (R&R) consensus, 120 Regulation of activity by calcium, 12–16 by proteolysis, 19–20 by purine nucleotides, 16–19 by redox reactions, 21–22 Relish, 52, 123, 124 Remyelination, 93, 104, 288 Renal cell carcinoma (RCC), 155, 161, 162, 180, 181, 184 Renal fibrosis, 94, 234, 240, 242, 246, 253 RGD See Arg-Gly-Asp (RGD) Rhodamine, 379 RNA, 51, 219, 378 ROS See Reactive oxygen species (ROS) S S19, 131, 133 Secondary structure, 4, 10, 23, 63, 223 Site-specific conjugation, 373–382 Sp1, 51, 52, 96, 131, 132, 143, 159 Spinal cord, 285, 291, 293–296, 300 spinocerebellar ataxia, 291 Stablin, 118, 121 Streptoverticillium mobarense transglutaminase structural model, 3, 12 substrate specificity, 29 Stroke, 52, 104, 288, 298–300, 305–306, 317 Structural models, 6, 22 Subcellular localization, 44, 285, 299 Substrate binding, 14, 25–30, 348, 351, 352, 354, 356, 367, 368 specificity, 11, 12, 29, 35, 375, 379, 382 SUMOylation, 321, 322, 324–325 SVVYGLR, 134–136, 141, 142 Syndecan-4, 31, 48, 49, 94, 99, 242–244, 253, 271 Index Systemic lupus erythematosus, 208 T Targeted mutant mice, 83, 86, 88–90 Tau, 291–294 Tauopathies, 293–294 T-cell receptor, 195, 196 T cells, 34, 69, 70, 91–93, 159, 194–199, 202–209 TdT See Terminala´deoxynucleotidyl transferase (TdT) Tenascin-C, 135, 269 Terminala´deoxynucleotidyl transferase (TdT), 378 Tertiary structure, 2–6, 374 TGF-β See Transforming growth factor β (TGF-β) TGM1 See Transglutaminase (TGM1) TGM2 See Transglutaminase (TGM2) TGM3 See Transglutaminase (TGM3) TGM5 See Transglutaminase (TGM5) TGM6 See Transglutaminase (TGM6) TGM7 See Transglutaminase (TGM7) TG2-/-mice, 90, 92, 94–97, 99–101, 103, 297, 299 Thrombin, 2, 19, 20, 69, 134, 135, 139, 140, 142, 154, 334–336, 341, 342 Transamidation, 8, 10, 24, 43, 44, 51, 53, 54, 56, 63, 65, 195, 200, 234, 235, 241, 244, 253, 271, 274, 285–287, 289, 292, 299, 303, 304, 325, 327, 334, 341, 343, 365 Transcription repressors, 218, 221 Transforming growth factor β (TGF-β), 98, 231–234, 239–244, 247, 254 Transgenic, 46, 83, 88–89, 99, 104, 180, 183, 184, 209, 269, 270, 288, 294, 296, 339 Transglutaminase (TGM1) calcium binding, 15 GTP binding, 16 heparin binding, 31 inhibition by GTP, 16 proteolytic cleavage of, 5, 19 structural models, targeted glutamine residues, 69 Transglutaminase (TGM2) ATPase/GTPase activity, 17 ATP/GDP/GTP binding, 17, 18, 24, 31, 219, 220, 322 calcium binding, 12, 21 catalytic residues, 5, 8, celiac anti-TGM2 antibody epitopes, 31, 33, 34, 201, 206 Index cis-peptide bonds, FRET beacons, 24, 28, 368 G-protein function, 16, 17, 19 heparin binding, 45, 132, 254 inhibition by GTP, 17 interaction motifs, 30, 31 intrinsically disordered regions, 31 isopeptidase activity, 44 open-close conformational rearrangement, 22–28 redox regulation, 21, 323 secondary srtucture, 3, 165 structural models, 22 Transglutaminase (TGM3) ATPase/GTPase activity, 20 calcium binding, 3, 13–15, 26 catalytic residues, cis-peptide bonds, GMP/GTP binding, 16 heparin binding, 31 proteolytic activation of, 19, 125 structural models, targeted glutamine residues, 69, 70 Transglutaminase (TGM5) calcium binding, GTPase activity, 17 GTP binding, 16 inhibition by GTP, 17 proteolytic cleavage of, structural models, 16 Transglutaminase (TGM6) autoantibodies against, 91 calcium binding, GTP binding, 21 inhibition by GTP, 17 proteolytic cleavage of, redox regulation, 21 structural models, 16 391 Transglutaminase (TGM7) GDP/GTP binding, 16 structural models, 29 targeted glutamine residues, 73 Transient receptor potential vanilloid (TRPV), 131, 132 Trappin-2, 45, 48, 51 Trasnsglutaminase fold, 5, 11 Traumatic brain injury, 300 TRPV See Transient receptor potential vanilloid (TRPV) Tumor protein p53, 161–162, 164, 165, 180, 184–186 Tumors, 107, 131, 133, 134, 155, 162, 165, 174, 184, 185, 216–219, 221–223, 225, 226, 288, 300–302, 319, 321, 340, 363 U Ubiquitination, 162, 177, 183, 184, 321, 322, 324, 325 V vascular remodelling, 99–103, 108 VEGFR2, 47, 50 Vicinal disulfide, 22, 321–323 von Hippel-Lindau tumor suppressor (pVHL), 155, 162–165, 319, 320 W Wound repair, 120–121, 247 X X-ray crystallography models, 2, 11, 12 .. .Transglutaminases Kiyotaka Hitomi • Soichi Kojima • Laszlo Fesus Editors Transglutaminases Multiple Functional Modifiers and Targets for New Drug Discovery Editors Kiyotaka... laws and regulations and therefore free for general use The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate... at sites and serves to anchor together β-strands and and β-strands 12 and 13 of the core domain, respectively, with neighboring α-helices Clustering these structural elements together forms what