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 microm 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.
Fleckenstein
Burkhard
B
Institute of Immunology, Rikshospitalet University Hospital, N-0027 Oslo, Norway. burkhard.fleckenstein@labmed.uio.no
Qiao
Shuo-Wang
SW
Larsen
Martin R
MR
Jung
Günther
G
Roepstorff
Peter
P
Sollid
Ludvig M
LM
D000595:Amino Acid Sequence; D019921:Biotinylation; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D005903:Gliadin / Q000737:chemistry* / Q000235:genetics; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D011503:Transglutaminases / Q000737:chemistry* / Q000235:genetics
D010455:Peptides; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D005903:Gliadin; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; D008239:Lysine