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.