Screening for the Preferred Substrate Sequence of Transglutaminase Using a Phage-displayed Peptide Library

Abstract

Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPϕD(P), QxPϕ, and QxxϕDP; Factor XIIIa, QxxϕxWP (where x and ϕ represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa. Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPϕD(P), QxPϕ, and QxxϕDP; Factor XIIIa, QxxϕxWP (where x and ϕ represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa. Transglutaminase (TGase, 2The abbreviations used are: TGase, transglutaminase; Bio-Cd, 5-(biotinamido)pentylamine; CBB, Coomassie Brilliant Blue; Dansyl-Cd, monodansylpentylamine; DTT, dithiothreitol; DMC, dimethyl casein; Me2SO, dimethylsulfoxide; GST, glutathione S-transferase. 2The abbreviations used are: TGase, transglutaminase; Bio-Cd, 5-(biotinamido)pentylamine; CBB, Coomassie Brilliant Blue; Dansyl-Cd, monodansylpentylamine; DTT, dithiothreitol; DMC, dimethyl casein; Me2SO, dimethylsulfoxide; GST, glutathione S-transferase. 2.3.2.13) is an enzyme that catalyzes the formation of isopeptide cross-links between glutamine and lysine residues in a variety of proteins and also attaches other primary amines to peptide-bound glutamines (1Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (926) Google Scholar, 2Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (491) Google Scholar, 3Griffin M. Casadio R. Bergamini C.M. Biochem. J. 2002; 368: 377-396Crossref PubMed Scopus (843) Google Scholar, 4Lorand L. Graham R.M. Nat. Rev. Mol. Cell. Biol. 2003; 4: 140-156Crossref PubMed Scopus (1193) Google Scholar, 5Esposito C. Caputo I. FEBS J. 2005; 272: 615-631Crossref PubMed Scopus (150) Google Scholar). To date, eight human TGase isozymes (Factor XIII, TGases 1–7) have been identified, comprising a large protein family with unique tissue distributions and physiological roles. Among the isozymes, TGase 2 and Factor XIII are two major members, although their locations and regulation differ. TGase 2 is expressed ubiquitously and has been implicated in many biological processes, including apoptosis, stabilization of the extracellular matrix, and regulation of growth factors (6Chen J.S. Mehta K. Int. J. Biochem. Cell Biol. 1999; 31: 817-836Crossref PubMed Scopus (176) Google Scholar, 7Fesus L. Piacentini M. Trends Biochem. Sci. 2002; 27: 534-539Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). Plasma Factor XIII is synthesized as a zymogen that comprises two A and two B subunits (8Ichinose A. Thromb. Haemostasis. 2001; 86: 57-65Crossref PubMed Scopus (112) Google Scholar). The A subunit contains the catalytic domain and is converted by thrombin-dependent proteolysis into its active form during clot formation. Activated Factor XIII (Factor XIIIa) is involved in fibrin stabilization and wound healing (9Standeven K.F. Ariëns R.A. Grant P.J. Blood Rev. 2005; 19: 275-288Crossref PubMed Scopus (116) Google Scholar). Generally, TGase reactions involve a Ca2+-dependent acyl transfer via a double displacement mechanism. In the first step, a glutamine γ-carboxyamide group in the substrate binds to a cysteine at the active site, resulting in formation of a γ-glutamylthioester bond and release of ammonia. Formation of the covalent acyl enzyme intermediate is the rate-limiting step. The ϵ-amino group of a peptide-bound lysine, as a nucleophilic substrate, binds to the acyl enzyme intermediate and then attacks the thioester bond, thereby generating an intermolecular isopeptide ϵ-(γ-glutamyl)lysine cross-link. Primary amines can replace lysine in transamidation reactions and become incorporated into the substrate proteins. Additionally, water can act as a nucleophile, resulting in deamidation of the glutamine residue and formation of a glutamic acid residue. However, the mechanisms by which TGases recognize substrates remain poorly understood. During the first step, TGases are selective with regard to which glutamine residues in the substrate protein take part in the reaction; they are much less selective with regard to the lysine residue or primary amines. Although partial amino acid sequences have been identified from a variety of highly reactive substrates (10Ichinose A. Tamaki T. Aoki N. FEBS Lett. 1983; 153: 369-371Crossref PubMed Scopus (109) Google Scholar, 11Parameswaran K.N. Velasco P.T. Wilson J. Lorand L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8472-8475Crossref PubMed Scopus (54) Google Scholar, 12Lorand L. Parameswaran K.N. Velasco P.T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 82-83Crossref PubMed Scopus (33) Google Scholar, 13Zeeuwen P.L. Hendriks W. de Jong W.W. Schalkwijk J. J. Biol. Chem. 1997; 272: 20471-20478Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 14Schalkwijk J. Wiedow O. Hirose S. Biochem. J. 1999; 340: 569-577Crossref PubMed Scopus (170) Google Scholar, 15Pastor M.T. Diez A. Peretz-Paya E. Abad C. FEBS Lett. 1999; 451: 231-234Crossref PubMed Scopus (28) Google Scholar, 16Grootjans J.J. Groenen P.J. de Jong W.W. J. Biol. Chem. 1995; 270: 22855-22858Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 17Groenen P.J. Bloemendal H. de Jong W.W. Eur. J. Biochem. 1992; 205: 671-674Crossref PubMed Scopus (56) Google Scholar), fewer consensus sequences and structures have been identified around the reactive glutamine residues (5Esposito C. Caputo I. FEBS J. 2005; 272: 615-631Crossref PubMed Scopus (150) Google Scholar, 18Aeschlimann D. Paulsson M. Mann K. J. Biol. Chem. 1992; 267: 11316-11321Abstract Full Text PDF PubMed Google Scholar). Investigations using the TGase substrate protein data base have produced information regarding TGase substrate sequence preferences (19Ruoppolo M. Orru S. D'Amato A. Francese S. Rovero P. Marino G. Esposito C. Protein Sci. 2003; 12: 1290-1297Crossref PubMed Scopus (31) Google Scholar, 20Facchiano A.M. Facchiano A. Facchiano F. Nucleic Acids Res. 2003; 31: 379-382Crossref PubMed Scopus (25) Google Scholar). Additionally, in studies on celiac disease, in which an unusual gluten-derived peptide antigen is produced, the amino acid distribution around the glutamine residue has been investigated to determine the deamidation sites preferred by TGase 2 (21Vader L.W. de Ru A. van der Wal Y. Kooy Y.M. Benckhuijsen W. Mearin M.L. Drijfhout J.W. van Veelen P. Koning F. J. Exp. Med. 2002; 195: 643-649Crossref PubMed Scopus (315) Google Scholar, 22Fleckenstein B. Molberg Ø. Qiao S.W. Schmid D.G. von der Mulbe F. Elgstoen K. Jung G. Sollid L.M. J. Biol. Chem. 2002; 277: 34109-34116Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 23Sollid L.M. Nat. Rev. Immunol. 2002; 2: 647-655Crossref PubMed Scopus (787) Google Scholar, 24Qiao S.W. Bergseng E. Molberg Ø. Jung G. Fleckenstein B. Sollid L.M. J. Immunol. 2005; 175: 254-261Crossref PubMed Scopus (141) Google Scholar). Although several preferred substrate sequences have been suggested by these studies, no research has been performed to date using an unbiased peptide library for identification of favorable primary structures. To study the amino acid sequences around reactive glutamine residue(s) that are preferred by TGases, we used an unbiased phage display random peptide library. In this system, peptides are expressed as fusions with the M13 bacteriophage coat protein pIII, displaying the random peptide sequence on the phage surface (25Clackson T. Lowman H.B. Phage Display. 2004; (Oxford University Press, Inc., New York)Google Scholar, 26Sidhu S.S. Lowman H.B. Cunningham B.C. Wells J.A. Methods Enzymol. 2000; 328: PubMed Google Scholar). system has been used for of and of substrate sites for J. G. J. Mol. Biol. 1991; PubMed Scopus Google Scholar, M. J.A. A.M. J. 2002; PubMed Scopus Google Scholar, W. M. M. R.H. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). We a random peptide library using of phage clones that incorporated a biotin-labeled primary amine in catalytic reactions with TGase 2 or Factor XIIIa. In the peptide sequences on phage we a tendency a preferred primary sequence around the reactive identified peptide sequences were as recombinant fusion proteins with a modified GST peptides demonstrated a incorporation of primary amine and on cross-linking the of a screening system that is to the for substrate sequence preferences in other TGase 2 was by K. H. R. H. PubMed Scopus Google Scholar). XIII was activated by with to for phage display was by of The were confirmed using which the incorporated of primary amine into as Screening Using Phage M13 phage display system was used for screening of preferred substrate sequences in the TGase 2 and Factor catalytic or phage clones were at with TGases in DTT, and To as a activity in the TGase 2 and Factor were at of and In screening TGase was performed for and 2 in reactions for and The was by the of Phage were then by of and to in the of as a phage clones that incorporated were by using with and 2 and then with the phage clones were by The was used to and the were by for at The phage clones were by with and then used for in sequences the peptide of phage clones were with an using a of for GST GST with and sites was by using the with an as a and into which has at amino acid residues glutamine to was by to the for the the were synthesized to the sequences for glutamine from and and to The GST from the was between the and sites of the The resulting was used to modified GST fusion proteins with a at the phage was by and the The phage the peptide and peptide sequences were by the using to at the was with and which in the sequence of phage The and was then into The resulting was to the peptide at the In recombinant the peptide sequence for was at the of The fusion proteins with several peptide sequences from were also expressed and as the for fusion protein with the sequence from P.J. Bloemendal H. de Jong W.W. Eur. J. Biochem. 1992; 205: 671-674Crossref PubMed Scopus (56) Google Scholar), was using amino acid sequence the sequence for the were and into the and of In regard to the other were synthesized for the sequence for the (10Ichinose A. Tamaki T. Aoki N. FEBS Lett. 1983; 153: 369-371Crossref PubMed Scopus (109) Google Scholar), K.N. Velasco P.T. Wilson J. Lorand L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8472-8475Crossref PubMed Scopus (54) Google Scholar, 12Lorand L. Parameswaran K.N. Velasco P.T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 82-83Crossref PubMed Scopus (33) Google Scholar), and P.L. Hendriks W. de Jong W.W. Schalkwijk J. J. Biol. Chem. 1997; 272: 20471-20478Abstract Full Text Full Text PDF PubMed Scopus (52) Google M.T. Diez A. Peretz-Paya E. Abad C. FEBS Lett. 1999; 451: 231-234Crossref PubMed Scopus (28) Google Scholar), B. Molberg Ø. Qiao S.W. Schmid D.G. von der Mulbe F. Elgstoen K. Jung G. Sollid L.M. J. Biol. Chem. 2002; 277: 34109-34116Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), and of GST was with for fusion in was with and the recombinant proteins were from of the using the with The protein of the protein was by the of in Transglutaminase of recombinant fusion proteins were by incorporation of a The was DTT, of recombinant and in the of TGase 2 or Factor a of was used for The was at and then on A of the was on a to the was by the of the and with the In the of incorporation using primary was used at a of in the protein was with TGase 2 or Factor in and were on which were with acid and then on the was using a using peptides and were from in form and confirmed by and The peptides were in dimethyl at the of as a To substrate and specificity as was performed with C.S. Biochem. 1992; 205: PubMed Scopus Google Scholar, F. A. T. K. M. K. Eur. J. Biochem. 2002; PubMed Scopus Google Scholar). of was with peptide at the of in the of TGase 2 or Factor in and at the of the was to a of the was for at that in the was to The sites were with in was then with alone. was to and for at The were with and then of into peptide was by of and of was and the at was of of on and human were used as substrates for TGase 2 and Factor XIIIa, In the was in the substrate protein was with TGase 2 or Factor XIII in and in the or of the peptides and The were to by Coomassie Brilliant of a Screening Using a Phage of phage clones were with in the of TGase 2 or Factor XIIIa. catalytic phage clones that were by were to transamidation The TGase was with to increase the of Among the clones and phage clones were from reactions of TGase 2 and Factor XIIIa, The sequence the peptide by was and the amino acid sequences were A and of that of the clones in the TGase 2 screening a glutamine residue to the The that a glutamine residue was used as a in 2, as of primary amine into peptide-GST fusion proteins by Factor XIIIa. incorporation of into fusion proteins sequences in the screening with Factor XIIIa. and data were performed as in of for is as in of preferred sequences with TGase 2 and Factor XIIIa. fusion proteins were with for in the of TGase 2 or Factor of the was performed as in The of of on the is by a on the of the using as substrate is expressed as a is as in of the substrate sequences by TGase 2 and Factor fusion proteins of the peptide sequences and were for with in catalytic reactions for TGase 2 and Factor The incorporation was performed as represent the of of with the glutamine donor sequences from several substrate proteins. of into fusion proteins of several glutamine donor peptides from identified and fusion proteins with and are also fusion protein was by TGase 2 and Factor were performed as in sequences were in to the reactive glutamine residue. of the clones were to display sequences (where x a non-conserved amino The amino acid sequences the were into (where ϕ a hydrophobic amino QxPϕ, and sequences with no In reactions with Factor XIIIa, of clones glutamine most of which were in the from the of the clones in of the preferred peptide sequences into of the clones the sequence the or no of Using the peptide sequences as glutamine donor substrates, we used primary amine in the of TGase 2 or Factor and the of to the glutamine residue. Dansyl-Cd, a is used for transamidation activity incorporation into substrate proteins by can using We were to incorporation of into phage of the of substrate peptide for the the peptide sequences were produced in E. as recombinant GST fusion proteins. of were incorporated into GST, we modified the GST to its with the primary were performed that glutamine residues and in GST were with to of the the fusion proteins a modified GST as as a at the of the fusion sequences were from at group of fusion proteins that were expressed and A and marked with an were then with in the of TGase 2 or Factor XIIIa. The were by and then 2, TGase Factor XIIIa). was and and In transamidation was used as a and the were used for The fusion proteins that were in screening in the TGase 2 reactions and that glutamine residues were used as a 2, In TGase 2 the of incorporation of into the fusion proteins with of the of incorporation between fusion proteins that and incorporated less or which In in the with Factor XIIIa, most fusion proteins with sequences proteins of and which to the major group Among exhibited the Although and sequences with the sequence in the major also considerable of for TGase 2 and Factor the fusion proteins were in the of TGase 2 or Factor XIIIa. a the were A and respectively). Generally, most fusion proteins from substrate sequences preferred by TGase 2 exhibited a incorporation of the reactions were by TGase 2 by Factor XIIIa. In and higher for enzyme In several fusion proteins sequences preferred by Factor a of of which the and exhibited the specificity in the with Factor XIIIa. To the specificity of the preferred sequences for two sequences and were and investigated using in the of TGase 2 or Factor TGase 2 the incorporation of into the was for reactions by Factor XIIIa. sequences have specificity in the transamidation with from sequences have been as glutamine donor To substrates with the sequences identified in this fusion proteins several sequences at the were produced and and most fusion proteins favorable in the catalytic by TGase 2, fusion proteins with sequences from by Factor XIIIa. However, with and these fusion exhibited much higher in TGase 2 or Factor reactions of the and specificity of sequences and we the lysine donor proteins using peptides and The lysine residue in was with to peptides were with in an system using a of and were that these sequences can also act as glutamine donor substrates in a peptide form with the using the fusion exhibited of incorporation into the in TGase 2 catalytic reactions and incorporation in reactions by Factor XIIIa. or represent for TGase substrates, were in and fibrin cross-linking reactions with TGase 2 and Factor A and respectively). In the of a of in large unique which in the of In the of the and the in a the of the In the of Factor the of fibrin resulting in the of and which in the of of cross-linking reactions was between and and was with However, the of and were the by was less that of The of substrates by TGases is that are primary structures for between the enzyme and glutamine residues in the substrate, although their and structures are factors (5Esposito C. Caputo I. FEBS J. 2005; 272: 615-631Crossref PubMed Scopus (150) Google Scholar, 23Sollid L.M. Nat. Rev. Immunol. 2002; 2: 647-655Crossref PubMed Scopus (787) Google Scholar, C.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). has demonstrated that the of residues in the are highly in TGases (5Esposito C. Caputo I. FEBS J. 2005; 272: 615-631Crossref PubMed Scopus (150) Google Scholar). However, distribution the isozymes, and is that has unique preferred substrate of information in the TGase substrate data base have several sequences that to favored in these reactions A.M. Facchiano A. Facchiano F. Nucleic Acids Res. 2003; 31: 379-382Crossref PubMed Scopus (25) Google Scholar). However, has been no of an unbiased random peptide library to for preferred substrate Phage display has been used to peptides on the of sequences to to preferred sequences between the TGase and peptide on In system, we of peptides using as substrates for incorporation of primary we demonstrated that the modified phage display system the substrate sequence preferred by TGase 2 and Factor XIIIa. In we performed of using the incorporation of and Although several clones were two of their sequences exhibited a group distribution to the In several clones with sequences were we that were for of the phage clones displaying preferred identification and of the sequences that the preferred sequences were unique to TGase 2 and Factor XIIIa. that other of the TGase family also substrate primary sequence In that reactive glutamine residues are to the although the sequences preferred by differ. is with that an for reactive glutamine residues (5Esposito C. Caputo I. FEBS J. 2005; 272: 615-631Crossref PubMed Scopus (150) Google Scholar, 15Pastor M.T. Diez A. Peretz-Paya E. Abad C. FEBS Lett. 1999; 451: 231-234Crossref PubMed Scopus (28) Google Scholar, 18Aeschlimann D. Paulsson M. Mann K. J. Biol. Chem. 1992; 267: 11316-11321Abstract Full Text PDF PubMed Google Scholar). In the sequences displaying no glutamine residues were has been to have to peptide phage clones during K. 2003; PubMed Scopus Google Scholar). of celiac have identified highly reactive glutamine residues for TGase 2 in for preferred deamidation sites of the peptides of which are in (21Vader L.W. de Ru A. van der Wal Y. Kooy Y.M. Benckhuijsen W. Mearin M.L. Drijfhout J.W. van Veelen P. Koning F. J. Exp. Med. 2002; 195: 643-649Crossref PubMed Scopus (315) Google Scholar, 22Fleckenstein B. Molberg Ø. Qiao S.W. Schmid D.G. von der Mulbe F. Elgstoen K. Jung G. Sollid L.M. J. Biol. Chem. 2002; 277: 34109-34116Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 23Sollid L.M. Nat. Rev. Immunol. 2002; 2: 647-655Crossref PubMed Scopus (787) Google Scholar, 24Qiao S.W. Bergseng E. Molberg Ø. Jung G. Fleckenstein B. Sollid L.M. J. Immunol. 2005; 175: 254-261Crossref PubMed Scopus (141) Google Scholar). that glutamine residues in sequences are and to In these were also in sequences that were from an unbiased peptide library. Furthermore, Fleckenstein B. Molberg Ø. Qiao S.W. Schmid D.G. von der Mulbe F. Elgstoen K. Jung G. Sollid L.M. J. Biol. Chem. 2002; 277: 34109-34116Abstract Full Text Full Text PDF PubMed Scopus (195) Google the of amino acid on for transamidation by the partial sequence of P. A. Marino G. S. Esposito C. R. Biochem. Res. PubMed Scopus (25) Google Scholar). with and were less favorable for Additionally, they that hydrophobic residue is preferred in the to the reactive glutamine which is confirmed by of study demonstrated that phage display screening is a system for identification of preferred transamidation or deamidation a and were highly preferred and that this sequence is also by TGase 2 in transamidation or Additionally, a for and at the and from the glutamine was first identified in this the sequence was in the of Factor However, as in the of TGase 2, hydrophobic amino were at from the glutamine residue. In this QxxϕxWP was the most in the catalytic of Factor XIIIa, and represent the first identification of sequence for this Although this to the cross-linking sites of major proteins as studies have identified substrates for Factor H. Y. S. S. T. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. H. A. Y. U. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). on this a for is in of sequences as fusion proteins that sequences were favorable glutamine donor However, identified the of were in sequences that to the major sequence to or the in sequences In the for were of The is the in the of in sequence in to Among the amino acid sequences two and were identified as substrates for TGase 2 and Factor XIIIa, In several sequences exhibited unique the reactions with TGase 2 and Factor and were for TGase 2 cross-linking and less with Factor XIIIa. Although we two sequences and for further as peptides in this the other sequences also to of Furthermore, fusion proteins with these sequences to to with identified glutamine donor sequences of peptide sequences as the TGase of sequence specificity can also the and of and selective We used substrate to the peptides were for cross-linking reactions by exhibited of the Factor cross-linking the TGase 2 Although and considerable activity cross-linking reactions in TGase 2, Factor at the higher the peptide was the fusion was to with Factor at higher of these peptides with specificity and several inhibitory peptides of Factor and TGase 2 have been identified on partial amino acid sequences of substrates (10Ichinose A. Tamaki T. Aoki N. FEBS Lett. 1983; 153: 369-371Crossref PubMed Scopus (109) Google Scholar, 11Parameswaran K.N. Velasco P.T. Wilson J. Lorand L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8472-8475Crossref PubMed Scopus (54) Google Scholar, 12Lorand L. Parameswaran K.N. Velasco P.T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 82-83Crossref PubMed Scopus (33) Google Scholar, J.J. C.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and are highly to the identified their sequences exhibited as glutamine that peptides have inhibitory on the cross-linking peptides as research in of and substrate mechanisms of In using a random peptide library, we a screening system for identification of amino acid sequences preferred by TGase screening system is for of other TGase isozymes and and in of substrates and mechanisms of of the TGase this system to elucidate the by TGases and in formation of a in which by cross-linking proteins as and proteins M.T. M. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, E. R. G. Nat. Rev. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar, K. Eur. J. 2005; Google Scholar). In using this system for the of substrate specificity a for the of and form the of We K. of for TGase We also H. in for