Gliadin T Cell Epitope Selection by Tissue Transglutaminase in Celiac Disease

Abstract

Tissue transglutaminase (TG2) can modify proteins by transamidation or deamidation of specific glutamine residues. TG2 has a major role in the pathogenesis of celiac disease as it is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides that are recognized by CD4؉ , DQ2-restricted T cells from the celiac lesions. Capillary electrophoresis with fluorescence-labeled gliadin peptides was used to separate and quantify deamidated and transamidated products. In a competition assay, the affinity of TG2 to a set of overlapping ␥-gliadin peptides was measured and compared with their recognition by celiac lesion T cells. Peptides differed considerably in their competition efficiency. Those peptides recognized by intestinal T cell lines showed marked competition indicating them as excellent substrates for TG2. The enzyme fine specificity of TG2 was characterized by synthetic peptide libraries and mass spectrometry. Residues in positions ؊1, ؉1, ؉2, and ؉3 relative to the targeted glutamine residue influenced the enzyme activity, and proline in position ؉2 had a particularly positive effect. The characterized sequence specificity of TG2 explained the variation between peptides as TG2 substrates indicating that the enzyme is involved in the selection of gluten T cell epitopes. The enzyme is mainly localized extracellularly in the small intestine where primary amines as substrates for the competing transamidation reaction are present. The deamidation could possibly take place in this compartment as an excess of primary amines did not completely inhibit deamidation of gluten peptides at pH 7.3. However, lowering of the pH decreased the reaction rate of the TG2-catalyzed transamidation, whereas the rate of the deamidation reaction was considerably increased. This suggests that the deamidation of gluten peptides by TG2 more likely takes place in slightly acidic environments.The food-sensitive enteropathy celiac disease (CD) 1 is a chronic inflammatory disorder with a multifactorial etiology (1, 2). The disease is precipitated by dietary wheat gluten and related proteins in barley and rye. The ingestion of such proteins induces mucosal lymphocyte infiltration and villus atrophy. Subjects with active disease have autoantibodies specific for the enzyme tissue transglutaminase (3). CD shows a strong genetic association with the genes encoding for HLA-DQ2 and -DQ8 (1). Gluten-reactive CD4 ϩ T cells isolated from the small intestine of CD patients are almost exclusively restricted by either of these HLA molecules (4, 5), and activation of such T cells is probably a critical event in the disease development (1). Interestingly, the gluten-reactive T cells of the celiac lesion predominantly recognize gluten peptides in which certain glutamines are converted to glutamic acid by deamidation (6). Evidence indicates that tissue transglutaminase (TG2) can mediate this deamidation in vivo (7-9). TG2 is best known for its ability to catalyze an acyl transfer reaction in which the carboxamide group of a p...

Isopeptidase activity of human transglutaminase 2: disconnection from transamidation and characterization by kinetic parameters

“…The actual pH can influence the ratio of the different type of transglutaminase reactions: deamidation and isopeptidase type of transglutaminase activities prefer acidic pH opposed to transamidation which favours alkaline pH (Fleckenstein et al 2002;Adamczyk et al 2013). The effect of pH was also tested on the isopeptidase and transamidase activities of TG2-I and TG2-T mutant enzymes, respectively, and both activities showed responsiveness similar to the wild type enzyme (Fig.…”

Proteomics Identification of Acyl-acceptor and Acyl-donor Substrates for Transglutaminase in a Human Intestinal Epithelial Cell Line

“…A model was postulated on the basis of the finding that tTG is the main target of immune response in CD (16) and that gliadin is able to act as a tTG substrate (17,18). This model suggests that tTG catalyzes in situ the deamidation of specific glutamine residues present in immunodominant gluten peptides, which are recognized by CD4ϩ DQ2-restricted mucosal T cells (19,20). However, the mucosal environment should be without amine donors to allow the occurrence of such reactions in vivo.…”

Real-time kinetic method to monitor isopeptidase activity of transglutaminase 2 on protein substrate

“…FLpepT26-S100A4(GST) with unmodified S100A4(GST) were purified from the free, unbound FLpepT26 peptide by centrifugal concentrator filter (Amicon ultra, 10 kDa, Millipore, Billerica, MA, USA). Then the buffer was replaced by 20 mM MOPS buffer, pH 6.8 containing 0.5 mM EDTA, 150 mM NaCl, 5 mM DTT, 0.01% Tween 20 because isopeptidase activity prefers slightly acidic pH [19]. Due to co-purification of FLpepT26-S100A4(GST) and S100A4(GST) their ratio was calculated based on the total protein concentration (determined by Bio-Rad Protein Assay) and its fluorescein content (absorption at 493 nm) using 79600 M -1 cm -1 as molar extinction coefficient for fluorescein.…”

“…The reaction was stopped by the addition of 10 mM EDTA (final concentration) to prevent unwanted further modification of the crosslinked molecules during their separation from free FLpepT26 peptide and the buffer was replaced to MOPS buffer, pH 6.8 for optimal isopeptidase activity [19] (Scheme on Fig. 1A).…”