Prolamin (31-49)

Objective: Coeliac disease (CD) is a multisystemic autoimmune inflammation of the intestinal tract induced by wheat gluten and related cereals in human leucocyte antigen (HLA)-DQ2/8-positive individuals. The molecular mechanisms relevant to oral tolerance induction towards toxic cereals such as gliadin remain poorly understood. Enterocytes, which express predominantly HLA-DR proteins, are capable of processing, transcytosing and presenting food antigens from the intestinal lumen to T lymphocytes of the lamina propria. Methods: Epitope-specific monoclonal antigliadin antibodies are utilised to unravel the intraepithelial transport processes of gliadin peptides in human duodenal biopsy specimens from patients with CD and reconstitute the transepithelial and endocytic pathways of gliadin in intestinal epithelial HT29 cells. Results: The gliadin peptide AA 31-49 is segregated from the peptides AA 56-68 and AA 229-246 along the endosomal pathway. Thus, AA 31-49 bypasses HLA-DR-positive late endosomes in intestinal cells and in biopsy specimens of patients with untreated CD. Further, it is localised in early endosomes and consequently escapes antigen presentation at the basolateral membrane, unlike peptides AA 56-68 and AA 229-246 that reach HLA-DR-positive late endosomes. Strikingly, forms of gliadin peptide AA 31-49 conjugated to cholera toxin B are sorted into late endosomes of HT29 cells. Conclusions: Endocytic segregation of gliadin peptide AA 31-49 seems to be a constitutive process. It explains why this peptide cannot stimulate gluten-sensitive T cells. Presentation of gliadin peptides by HLA-DR proteins via late endosomes within enterocytes might induce a tolerogenic effect and constitutes a potentially promising therapeutic approach for induction of tolerance towards gliadin.

Work continues to progress in the unravelling of the molecular interactions involved in the pathogenesis of coeliac disease. The immunogenetics of the disease implicate certain HLA DQ alleles as necessary for subsequent disease development. These HLA molecules have been shown to be necessary in the binding and presentation of gliadin peptides to antigen-specific T cells. Current work is examining the precise HLA-antigen interaction that may lead to the development of antigen-blocking agents. The isolation of antigen-specific T cells has led to the confirmation of a toxic T-cell epitope of the gliadin protein (residues 31-49) and it would appear likely that additional toxic epitopes may be similarly characterized in the near future. No common TCR motifs have so far been detected, although these may become apparent as this work progresses. The gliadin peptide sequence, residues 31-49, has now been demonstrated to be toxic in vivo. Additional toxic T-cell epitopes may also be present within gliadins, but this identification of a toxic gliadin sequence for the first time raises the possibility of future manipulation of the wheat genome (and other toxic cereals) that could lead to the development of new graminae cereals with the properties of wheat, but which do not induce toxicity in patients with coeliac disease.

Celiac disease is a chronic enteropathy caused by the ingestion of wheat gliadin and other cereal prolamines. The synthetic peptides 31-43 (P31-43) and 31-49 (P31-49) from A-gliadin are considered to be model peptides for studying innate immunity in celiac disease. Our previous study demonstrated that P31-43 and P31-49 are encrypted within peptide 31-55 (P31-55), which is naturally released from gastropancreatic digestion and is not susceptible to hydrolysis by brush border membrane enzymes. Here, we analyzed the permeability of P31-55 through the epithelial cell layer of confluent Caco-2 cells using high-performance liquid chromatography, mass spectrometry, and fluorescence-activated cell sorting. Twenty-three percent of the P31-55 added to the apical chamber was transported to the basolateral chamber after 4 h of incubation without being degraded by hydrolysis. Treatment of Caco-2 cells with whole gliadin digests extracted from a common wheat cultivar increased the epithelial P31-55 translocation by approximately 35%. Moreover, we observed an atypical chromatographic profile consisting of a double peak. Chromatography using different column temperatures and circular dichroism highlighted the presence of more conformational structures around the amide bond of the two adjacent prolines 38 and 39. These findings confirm that P31-55 is gastrointestinally resistant and is permeable across a Caco-2 monolayer. Moreover, we hypothesize that the various conformations of P31-55 may play a role in the activation of innate immunity.