Identification and Analysis of Multivalent Proteolytically Resistant Peptides from Gluten:  Implications for Celiac Sprue

Abstract

Dietary gluten proteins from wheat, rye and barley are the primary triggers for the immunopathogenesis of Celiac Sprue, a widespread immune disease of the small intestine. Recent molecular and structural analyses of representative gluten proteins, most notably α-and γ-gliadin proteins from wheat, have improved our understanding of these pathogenic mechanisms. In particular, based on the properties of a 33-mer peptide, generated from α-gliadin under physiological conditions, a link between digestive resistance and inflammatory character of gluten has been proposed. Here we report three lines of investigation in support of this hypothesis. First, biochemical and immunological analysis of deletion mutants of α-2 gliadin confirmed that the DQ2 restricted T cell response to the α-2 gliadin are directed towards the epitopes clustered within the 33-mer. Second, proteolytic analysis of a representative γ-gliadin led to the identification of another multivalent 26-mer peptide that was also resistant to further gastric, pancreatic and intestinal brush border degradation, and was a good substrate of human transglutaminase 2 (TG2). Analogous to the 33-mer, the synthetic 26-mer peptide displayed markedly enhanced T cell antigenicity compared to monovalent control peptides. Finally, in silico analysis of the gluten proteome led to the identification of at least 60 putative peptides that share the common characteristics of the 33-mer and the 26-mer peptides. Together, these results highlight the pivotal role of physiologically generated, proteolytically stable, TG2-reactive, multivalent peptides in the immune response to dietary gluten in Celiac Sprue patients. Prolyl endopeptidase treatment was shown to abolish the antigenicity of both the 33-mer and the 26-mer peptides, and was also predicted to have comparable effects on other proline-rich putatively immunotoxic peptides identified from other polypeptides within the gluten proteome.

Celiac disease: pathogenesis of a model immunogenetic disease

“…The high proline content renders these proteins resistant to complete proteolytic digestion by gastric, pancreatic, and brush border enzymes in the human intestine, since those enzymes are deficient in prolyl endopeptidase activity (17,18). This can result in the accumulation of relatively large peptide fragments (as many as 50 amino acids in length) with a high proline and glutamine content in the small intestine (18,19). Nonetheless, the relatively poor digestion of these proteins alone is not sufficient to cause CD, and there is no known difference between healthy individuals and those susceptible to developing CD in their ability to digest these proteins.…”

HLA-DQ Molecules as Affinity Matrix for Identification of Gluten T Cell Epitopes

“…In contrast to the a33-mer peptide, the DQ2.2-glut-L1-and DQ2.2-glia-a1-related peptides were observed in many length variants and often without an intact 9-mer core. Some peptides were truncated adjacent to a serine residue, possibly as the result of elastase cleavage, as this enzyme is reported to cleave after serine, alanine, glycine, and valine residues (36). Testing of TCL reactivity and staining with tetramers indicate that there is a hierarchy among the three DQ2.2 epitopes, with DQ2.2-glut-L1 being the epitope recognized by most T cells.…”

The Genetic Variability of Wheat Can Ensure Safe Products for Celiac Disease Patients?

“…All peptides found to be active in celiac disease contained the sequences Gln-Gln-Gln-Pro and Pro-Ser-Gln-Gln; inactive peptides contained no such sequences [8,15,19,20]. Moreover, a 33-mer peptide resistant to intestinal proteases and containing three of the most immunogenic epitopes was identified as one of the main stimulators of the inflammatory response to gluten [10,21].…”