SUSCEPTIBILITY TO DEAMIDATION BY TISSUE TRANSGLUTAMINASE AS A TOOL TO IDENTIFY IMMUNOGENIC GLIADIN PEPTIDES IN THE WHOLE GLIADIN EXTRACTS

Abstract

Aim: Celiac disease (CD) is an enteropathy of the small intestine induced by dietary gliadin. Tissue transglutaminase (tTG) plays a direct role in the pathogenesis of CD by deamidating specific glutamine (Q) residues of gliadin peptides and increasing their immunogenicity. Based on tagging with a monodansylcadaverine fluorescent probe and nanospray tandem mass spectrometry, we previously identified 6 peptides containing tTG-susceptible Q (Q) residues within a pepsin-trypsin digest of wheat gliadins (see Table) [1].TableThese six gliadin/glutenin peptides and the immunodominant 33-mer (α-gliadin 57-89) peptide were synthesized and tested for recognition by CD patients. Methods: Gliadin-specific T cells lines (TCL) were generated from intestinal mucosa of 4 adult CD patients. Both proliferation and cytokine (γ-IFN, IL-4, IL-2, IL-10) production were analyzed in response to either native or tTG-treated peptides. Results: The 18-mer, 25-mer and 14-mer-1 peptides were recognized by TCLs from all 4 CD patients. 25-mer peptide entirely contained the 18-mer and had a high sequence homology with the 33-mer. 18-mer, 25-mer, and 33-mer induced proliferation, γ-IFN and IL-10 productions in either native and deamidated forms, these latter being more potent in stimulating proliferation: (SI ± SD) 17.1 ± 5.4 vs 8.1 ± 9.7; 17.0 ± 4.2 vs 7.9 ± 9.1; 20.2 ± 3.2 vs 9.9 ± 11.4; and γ-IFN production: (net value ± SD) 3449 ± 1788.3 vs 402.5 ± 72.8; 3536 ± 1530 vs 607 ± 346.5; 3511 ± 1735 vs 765.5 ± 317.5pg/ml, respectively. By contrast, IL-2 and very low IL-4 were produced only in response to the tTG-treated peptides. 14-mer-1 peptide was active only after the tTG-treatment and exerted a lower stimulatory capacity compared to 18-mer and 25-mer. No responses were observed against the 14-mer-2, 19-mer, and 21-mer despite the tTG-treatment. Summary: A high percentage (3 out 6, 50%) of gliadin peptides naturally occurring in the extracts of whole gliadins and identified on the basis of their susceptibility to be tTG substrate, were recognized by CD patients. The tTG-treated peptides were largely more immunogenic than in native form. Conclusion: This study offers an important tool to detect within wheat cultivars and other edible cereals, the immunologically active gliadin peptides relevant for CD pathogenesis.