Molecular Characterization of Covalent Complexes between Tissue Transglutaminase and Gliadin Peptides
Tissue transglutaminase (TG2) modifies proteins and peptides by transamidation or deamidation of specific glutamine residues. TG2 also has a central role in the pathogenesis of celiac disease. The enzyme is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides recognized by intestinal T cells from patients. Incubation of TG2 with gliadin peptides also results in the formation of covalent TG2-peptide complexes. Here we report the characterization of complexes between TG2 and two immunodominant gliadin peptides. Two types of covalent complexes were found; the peptides are either linked via a thioester bond to the active site cysteine of TG2 or via isopeptide bonds to particular lysine residues of the enzyme. We quantified the number of gliadin peptides bound to TG2 under different conditions. After 30 min of incubation of TG2 at 1 μm with an equimolar ratio of peptides to TG2, approximately equal amounts of peptides were bound by thioester and isopeptide linkage. At higher peptide to TG2 ratios, more than one peptide was linked to TG2, and isopeptide bond formation dominated. The lysine residues in TG2 that act as acyl acceptors were identified by matrix assisted laser desorption ionization and nanoelectrospray mass spectrometry and tandem mass spectrometry analysis of proteolytic digests of the TG2-peptide complexes. At a high molar excess of gliadin peptides to TG2 altogether six lysine residues of TG2 were found to participate in isopeptide bond formation. The results are relevant to the understanding of how antibodies to TG2 are formed in celiac disease. Tissue transglutaminase (TG2) modifies proteins and peptides by transamidation or deamidation of specific glutamine residues. TG2 also has a central role in the pathogenesis of celiac disease. The enzyme is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides recognized by intestinal T cells from patients. Incubation of TG2 with gliadin peptides also results in the formation of covalent TG2-peptide complexes. Here we report the characterization of complexes between TG2 and two immunodominant gliadin peptides. Two types of covalent complexes were found; the peptides are either linked via a thioester bond to the active site cysteine of TG2 or via isopeptide bonds to particular lysine residues of the enzyme. We quantified the number of gliadin peptides bound to TG2 under different conditions. After 30 min of incubation of TG2 at 1 μm with an equimolar ratio of peptides to TG2, approximately equal amounts of peptides were bound by thioester and isopeptide linkage. At higher peptide to TG2 ratios, more than one peptide was linked to TG2, and isopeptide bond formation dominated. The lysine residues in TG2 that act as acyl acceptors were identified by matrix assisted laser desorption ionization and nanoelectrospray mass spectrometry and tandem mass spectrometry analysis of proteolytic digests of the TG2-peptide complexes. At a high molar excess of gliadin peptides to TG2 altogether six lysine residues of TG2 were found to participate in isopeptide bond formation. The results are relevant to the understanding of how antibodies to TG2 are formed in celiac disease. Tissue transglutaminase (TG2 1The abbreviations used are: TG2, tissue transglutaminase; CD, celiac disease; CID, collision-induced dissociation; ESI, electrospray ionization; GST, glutathione S-transferase; MALDI-TOF MS, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry; Fmoc, N-(9-fluorenyl)methoxycarbonyl. ; EC 2.3.2.13) belongs to a family of structurally and functionally related enzymes that catalyze the formation of intra- or intermolecular Nϵ(γ-glutamyl)lysine bonds within their target proteins (1Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (493) Google Scholar, 2Grenard P. Bates M.K. Aeschlimann D. J. Biol. Chem. 2001; 276: 33066-33078Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). In this Ca2+-dependent transamidation reaction, an acyl residue, derived from the γ-carboxamide group of a peptide-bound glutamine (acyl donor), is transferred to an appropriate primary amine, most commonly the ϵ-amino group of lysine residues (acyl acceptor) (3Folk J.E. Adv. Enzymol. Relat. Areas Mol. Biol. 1983; 54: 1-56PubMed Google Scholar). A catalytic triad involving cysteine 277, histidine 335, and aspartic acid 358 forms the active site of TG2 (4Liu S. Cerione R.A. Clardy J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2743-2747Crossref PubMed Scopus (280) Google Scholar). In the first step, the side chain of a glutamine residue forms a thioester with the active site cysteine, and ammonia is released (acylation). In the following transamidation step, the activated acyl group is transferred to the acyl acceptor amine, forming an isopeptide bond (deacylation). Alternatively, hydrolysis of the thioester bond leads to deamidation, converting the glutamine into a glutamic acid residue. The food-sensitive enteropathy celiac disease (CD) is a chronic inflammatory disorder with a multifactorial etiology (5Sollid L.M. Annu. Rev. Immunol. 2000; 18: 53-81Crossref PubMed Scopus (567) Google Scholar, 6Schuppan D. Gastroenterology. 2000; 119: 234-242Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). In patients with CD, ingestion of wheat gluten and related proteins of barley and rye induces mucosal lymphocyte infiltration and villous atrophy. CD demonstrates a strong genetic association with the genes encoding for HLA-DQ2 and -DQ8 (5Sollid L.M. Annu. Rev. Immunol. 2000; 18: 53-81Crossref PubMed Scopus (567) Google Scholar). Gluten-reactive CD4+ T cells have been isolated from the small intestine of CD patients and are almost exclusively restricted by either of these HLA molecules (7Lundin K.E.A. Scott H. Hansen T. Paulsen G. Halstensen T.S. Fausa O. Thorsby E. Sollid L.M. J. Exp. Med. 1993; 178: 187-196Crossref PubMed Scopus (528) Google Scholar, 8Lundin K.E.A. Scott H. Fausa O. Thorsby E. Sollid L.M. Hum. Immunol. 1994; 41: 285-291Crossref PubMed Scopus (147) Google Scholar). Activation of such T cells is likely to be a critical event in the disease development (5Sollid L.M. Annu. Rev. Immunol. 2000; 18: 53-81Crossref PubMed Scopus (567) Google Scholar). The gluten-reactive T cells of the celiac lesion predominantly recognize modified gluten peptides in which distinct glutamines have been deamidated and thereby converted to glutamic acid residues (9Sjöström H. Lundin K.E.A. Molberg Ø. Körner R. McAdam S.N. Anthonsen D. Quarsten H. Noren O. Roepstorff P. Thorsby E. Sollid L.M. Scand. J. Immunol. 1998; 48: 111-115Crossref PubMed Scopus (231) Google Scholar). Accumulating evidence suggests that this deamidation and formation of gluten-derived T cell epitopes in the intestinal mucosa is mediated by TG2 (10Molberg O. McAdam S.N. Körner R. Quarsten H. Kristiansen C. Madsen L. Fugger L. Scott H. Noren O. Roepstorff P. Lundin K.E.A. Sjöström H. Sollid L.M. Nat. Med. 1998; 4: 713-717Crossref PubMed Scopus (984) Google Scholar, 11van de Wal Y. Kooy Y. van Veelen P. Pena S. Mearin L. Papadopoulos G. Koning F. J. Immunol. 1998; 161: 1585-1588PubMed Google Scholar, 12Molberg O. McAdam S. Lundin K.E.A. Kristiansen C. Arentz-Hansen H. Kett K. Sollid L.M. Eur. J. Immunol. 2001; 31: 1317-1323Crossref PubMed Scopus (153) Google Scholar). Although the transamidation reaction has a higher rate than the deamidation reaction at neutral pH (3Folk J.E. Adv. Enzymol. Relat. Areas Mol. Biol. 1983; 54: 1-56PubMed Google Scholar), the rate of the deamidation reaction is increased at lower pH (13Fleckenstein B. Molberg O. 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 (199) Google Scholar), such as in the proximal small intestine (14Evans D.F. Pye G. Bramley R. Clark A.G. Dyson T.J. Hardcastle J.D. Gut. 1988; 29: 1035-1041Crossref PubMed Scopus (961) Google Scholar). The sequence specificity of TG2 toward acyl donor substrates was recently elucidated (13Fleckenstein B. Molberg O. 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 (199) Google Scholar, 15Vader 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 (321) Google Scholar), and studies on the affinity of TG2 for various gliadin peptides suggested that TG2 is directly involved in the selection of gluten T cell epitopes (13Fleckenstein B. Molberg O. 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 (199) Google Scholar). Patients with active CD have autoantibodies to TG2 (16Dieterich W. Ehnis T. Bauer M. Donner P. Volta U. Riecken E.O. Schuppan D. Nat. Med. 1997; 3: 797-801Crossref PubMed Scopus (1794) Google Scholar), which are highly disease-specific and whose formation is dependent on ingestion of gluten. Notably, TG2 itself was described to be involved in covalent complex formation with gluten-derived peptides in the presence of calcium (6Schuppan D. Gastroenterology. 2000; 119: 234-242Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, 17Schuppan D. Dieterich W. Ehnis T. Bauer M. Donner P. Volta U. Riecken E.O. Ann. N. Y. Acad. Sci. 1998; 859: 121-126Crossref PubMed Scopus (33) Google Scholar). Those TG2-gliadin peptide complexes have been suggested to act as hapten-carrier complexes, which could explain the typical antibody response against TG2 in untreated CD patients (18Sollid L.M. Molberg Ø. McAdam S. Lundin K.E.A. Gut. 1997; 41: 851-852Crossref PubMed Scopus (245) Google Scholar). In this model the complexes are taken up after binding to the immunoglobulin receptor of TG2-specific B cells. Subsequently the gliadin peptide cargo is presented and recognized by gliadin-specific T cells, which give help to the B cells to secrete TG2-specific antibodies. However, little is known about the formation and the molecular nature of these TG2-gluten peptide complexes. The aim of the current study was to investigate the complex formation between TG2 and two immunodominant gliadin-derived T cell epitopes. Because these peptides contain glutamine but no lysine residues, TG2 will act as an acyl acceptor in these experiments. Our experiments allowed quantitation of the number of gliadin peptides covalently bound to TG2 under different conditions and demonstrated the chemical nature of this linkage. After proteolytic digestion of these complexes, the sites of the modified lysine residues within TG2 were identified by mass spectrometry. Definition and Synthesis of Peptides—The following peptides were synthesized: peptide QLQPFPQPQLPY (defined as αI, corresponding to α9-gliadin-(57–68)), the deamidated derivative α9-gliadin-(57–68) E65 (QLQPFPQPELPY, defined as αIE), the N-terminal-biotinylated analogues of peptides αI and αIE and the of peptide αI and peptide (defined as corresponding to were by peptide on a and G. A.G. Chem. 31: Scopus Google Scholar). A molar excess of was used for and of or both from was to the group of the peptides as The of peptides was by electrospray mass and was by high The at the residue of peptide αI was the T J. PubMed Scopus Google Scholar). of Tissue TG2 was as a in the as described T.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in this for the The of the was by the a modified as as the sequence TG2 was used as a of by peptides and were at with 1 μm TG2 at in pH for In one TG2 was by incubation with for min at incubation with The were to and at for and to with in the The proteins were a by After of binding in pH for the was with for min and by pH were and was by to a the study μm TG2 was at in with amounts of or with μm αI or After the complexes were from peptides on with and which was used as the the of the and were as described S. A. K. N. PubMed Scopus Google Scholar). by small were with in the and for in a were on of the and were by an within min by for in the and in the was by a The were converted to an number of gliadin peptides bound TG2 as number of of of In the peptide 1 μm TG2 was with different of peptide αI for 30 min at were either directly by experiments or with pH or pH deamidation of peptide αI, 1 μm TG2 was for 30 min with the peptide under conditions to in experiments. The deamidated was and quantified by and at as described (13Fleckenstein B. Molberg O. 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 (199) Google Scholar). were by into a in μm by and pH min in were at at and were from the to the experiments were at in of within of was against reaction pH with was in of reaction and of in was and for 30 The was against reaction and with the peptide 1 for After the of cysteine pH for 30 the modified was against and modified was with in for at and proteolytic were by MALDI-TOF and mass spectrometry. corresponding to complexes were from the and into The were by min and min and in a were by the in was and were by min at in the were and as described A digestion of of in was and were on for 30 min to of the with of The was by and digestion was at laser desorption ionization mass were on a MALDI-TOF ionization tandem mass were on a mass peptide were and on in mass were into nanoelectrospray as described G. M. Chem. 71: PubMed Scopus Google Scholar). were at toward a In collision-induced experiments were by the of by was for with μm gliadin QLQPFPQPQLPY The reaction was by the of and a step, and were to of the on the following in a distinct corresponding to the molecular of the The of the increased with higher peptide a at A was at no was at in peptide αI is known to be deamidated by TG2 H. R. Molberg O. Quarsten H. W. Kooy Y.M. Lundin K.E.A. Koning F. Roepstorff P. Sollid L.M. McAdam S.N. J. Exp. Med. 2000; PubMed Scopus Google Scholar). of that glutamine for a glutamic acid residue in a of the was used at The was TG2 was with incubation with to an of cysteine residues in TG2 of to formation was by a derivative of αI as a peptide were on a and peptides were from TG2-peptide complexes and the enzyme. The high molecular were exclusively in the as by The peptide was in the as by the in the and the After incubation of TG2 with peptide αI, in these two was and the molar of peptide in the was with the known of TG2, the number of peptides bound to a could be In a first TG2 was in the presence of calcium with a molar excess of peptide αI, and the number of peptides bound to TG2 was after different formation was found to After and about and αI peptides TG2 were A of was between 30 and min of incubation a but of this ratio was found a of after was found in the TG2 was by incubation with peptide αI TG2 was with a molar excess of peptide αI incubation of 30 min was to complex formation an equimolar an number of about was The a between μm and μm At a molar peptides were to be bound to TG2, with the from for an incubation and molar The a at molar a number of was and in In the TG2-peptide complexes described and peptide αI could be bound via a thioester bond to in the active site of the enzyme or via isopeptide bonds involving lysine of these two peptide TG2 was with peptide αI for 30 min by with at pH or The thioester but The to a of the number of peptides bound to TG2, as by analysis The between both to peptides bound via thioester bonds to At peptide between and 1 of TG2 about to of peptide αI linked via thioester of peptide αI the of peptides increased After incubation with 1 μm peptide αI, about of the bound that peptide αI is bound in amounts via thioester and isopeptide linkage. both thioester and isopeptide bond formation a the to reaction for the formation of isopeptide complexes is we the reaction by TG2 to isolated TG2-peptide αI complexes in a molar an of the reaction in as be from an to the deamidation reaction to complex in which peptide αI was by derivative were under conditions. were by Incubation for 30 min with μm in μm which increased at incubation However, at lower peptide the ratio between peptides thioester and and deamidated peptides At about μm peptides were in the and μm were At 1 the amounts of covalently bound and deamidated peptides were almost conditions of the bound peptides are linked via that μm of converted peptides were peptide isopeptide bond formation a event with thioester of within TG2 by we identified which lysine residues in the are involved in isopeptide bond formation to the glutamine residue in peptide After incubation of TG2 with peptide αI at molar of and complexes were by The corresponding to TG2-peptide complexes and TG2 are under the conditions was from the and to digestion by were by MALDI-TOF mass spectrometry in the and and were for for from to peptide were by of a lysine residue involved in isopeptide bond formation and by one site in the In the MALDI-TOF mass after peptide αI with TG2 in a molar six for the were found Because peptide αI an glutamine residue, six are by a typical to formation of that lysine residues of TG2 involved in covalent complex formation with two peptides The the of six TG2 peptides that are by isopeptide bonds to either peptide residues as acyl acceptors are and in and their number within the TG2 sequence is in the were by MALDI-TOF or peptides to gliadin of by MALDI-TOF of for mass after formation at the of peptide was for collision-induced was for collision-induced were in the the The mass after formation at the of peptide The was for collision-induced were in the the in a of the six was by six were as or in the in A and the of the and corresponding to and was by collision-induced of in the of the mass The of both the peptides and and the gliadin peptide αI could be elucidated from the and P. J. PubMed Scopus Google as and were by the of and which the isopeptide within peptide αI was to as in and the was the in also the site at which the reaction taken glutamine residues in peptide αI, is to lysine and lysine of the peptides and the from of the isopeptide bond itself were these conditions in the of peptide αI were as in the of peptides to peptide αI, are in of in The six peptides also either bound to peptide αI or to peptide were to CID, and were A the isopeptide and from within the peptide are by to an in a of the identified sites in the TG2-peptide complexes were with gliadin peptide conditions. of the by MALDI-TOF and the results for peptide of peptides to peptide were both in and analysis for peptides and were no were for peptide to the of the or peptide of in within TG2-peptide complexes at different molar and was as the results from MALDI-TOF and mass spectrometry for complexes with both the αI and gliadin a in the of the identified lysine residues in the reaction was At a a was for the of peptide more at a and also for peptides and the ratio to and for six peptides were with different However, no lysine residues within the were found to be involved in isopeptide bond formation a molar excess of peptides was complex sites in TG2 at different molar of TG2 to peptide αI The for the peptides covalently linked to peptide αI were found with or high in the MALDI-TOF and mass results were by peptide for complex formation. were by their acyl acceptor residue and to peptide itself and was to the sequence of acyl acceptor peptides to peptide ratio in a of within a the lysine residues most for chemical were corresponding to of lysine was with amounts of the and with the peptide The modified was by which also the peptide of and lysine residues are modified by a in a typical mass of mass of the corresponding The was by MALDI-TOF and and lysine residues were identified as TG2, and were found as (TG2 sequence by In the and were identified as modified sequence by of in from the of TG2 in the the six lysine residues in isopeptide bond formation to gliadin peptides are and are in the and and are in is at the of and in the of the than at the to be the lysine residues are to the of the In this study we covalent gliadin complexes in A was used to the number of gliadin peptides covalently bound to were either linked by thioester or isopeptide MALDI-TOF and tandem mass spectrometry were to directly the of the acyl acceptor sites within demonstrates the chemical nature and of covalent TG2-gliadin peptide complexes with two of the immunodominant T cell epitopes in CD αI and TG2-peptide complexes have been described (6Schuppan D. Gastroenterology. 2000; 119: 234-242Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, W. Ehnis T. Bauer M. Donner P. Volta U. Riecken E.O. Schuppan D. Nat. Med. 1997; 3: 797-801Crossref PubMed Scopus (1794) Google Scholar), but their molecular has Incubation of TG2 in the presence of calcium with the known peptide in complex formation between the peptide and TG2 The of and the presence of a covalent between the peptide and covalent complexes from thioester formation between in the active site of TG2 and the side chain of in Alternatively, TG2 itself could act as an acyl acceptor in isopeptide bond formation. The acyl acceptor sites in TG2 were identified by mass analysis of digests of the complexes. MALDI-TOF MS, MS, and six lysine residues were identified to be involved in isopeptide bond formation to in peptide αI peptide evidence for of at of these six lysine residues was The number of identified acyl acceptor sites for different were higher than from the from experiments and The results the number of peptides bound via isopeptide However, these peptides could be to different lysine residues in different TG2 The of peptides bound TG2 via isopeptide bonds to six lysine residues of TG2 identified as acyl that isopeptide bond formation is modified lysine residues in the TG2 lysine residues in the were found to be involved in isopeptide bond has been described as a of TG2 L. M. W. G. R. M. J. PubMed Scopus Google Scholar). The identified acyl acceptor residues in TG2 are from in the active the to reaction in the complexes is either intermolecular or TG2 a binding to peptide the lysine residues we the is more However, experiments in which TG2 was to complexes after the peptide to of the reaction in as could be the reaction is intermolecular L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The be that this reaction is to be under conditions. we that and complexes were against a as the involved in the to The specificity for the acyl acceptor residues is by by chemical of lysine residues within After a peptide via a and lysine residues were identified as of in the one of involved in was modified in these experiments the specific selection of these acyl acceptor In the within the lysine residues demonstrates the specificity The different of the lysine residues to the active site of a TG2 in an intermolecular acyl reaction or from in the between the lysine residues. In of the six peptides the lysine residue is by acid in In peptide an residue is in from the in which induces a in the A residue lysine residues. these a sequence of acyl acceptor peptides to the αI or mass at be be to by the of the complexes by MALDI-TOF in the be by as dependent on their to be also be more for molecules of higher molecular within the MALDI-TOF mass were in the and were by the the mass by either of these explain the typical antibody response against TG2 in untreated CD patients by TG2-gliadin complexes (18Sollid L.M. Molberg Ø. McAdam S. Lundin K.E.A. Gut. 1997; 41: 851-852Crossref PubMed Scopus (245) Google Scholar), the deamidated peptide be released after of the complexes. could be by the hydrolysis of either the thioester or the isopeptide The results in that such a of peptides is formation at a molar excess of peptide αI a but of the complexes was after 30 After min a of was with such a molar excess of peptide αI about of their peptides via thioester of the isopeptide bonds either an intermolecular of the from the modified lysine to in an reaction, a of TG2, as the lysine residues are from the active site The to by more TG2 to isolated complexes and the of and complexes that by hydrolysis of thioester bonds of isopeptide of the thioester bond the deamidation reaction and is at pH (13Fleckenstein B. Molberg O. 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 (199) Google Scholar), which be found in after of the complexes. these complexes are formed in and their will be in the in of an of TG2 and after a of complexes on their by TG2-specific B cells and on will on their for antibody against We for in peptide and and for the TG2