Abstract
ScopeCeliac disease is an autoimmune disorder caused by failure of oral tolerance against gluten in genetically predisposed individuals. The epithelial translocation of gluten-derived gliadin peptides is an important pathogenetic step; the underlying mechanisms, however, are poorly understood. Thus, we investigated the degradation and epithelial translocation of two different gliadin peptides, the toxic P31–43 and the immunogenic P56–68. As the size, and hence, the molecular weight of peptides might have an effect on the transport efficiency we chose two peptides of the same, rather short chain length.Methods and ResultsFluorescence labeled P31–43 and P56–68 were synthesized and studied in a transwell system with human enterocytes. Fluorometric measurements were done to reveal antigen translocation and flow cytometry as well as confocal microscopy were used to investigate cellular uptake of peptides. Structural changes of these peptides were analysed by MALDI-TOF-MS. According to fluorescence intensities, significantly more P31–43 compared to P56–68 was transported through the enterocyte layer after 24 h incubation. In contrast to previous reports, however, mass spectrometric data do not only show a time-dependent cleavage of the immunogenic P56–68, but we observed for the first time the degradation of the toxic peptide P31–43 at the apical side of epithelial cells.ConclusionConsidering the degradation of gliadin peptides by enterocytes, measurement of fluorescence signals do not completely represent translocated intact gliadin peptides. From our experiments it is obvious that even short peptides can be digested prior to the translocation across the epithelial barrier. Thus, the chain length and the sensibility to degradations of gliadin peptides as well as the integrity of the epithelial barrier seem to be critical for the uptake of gliadin peptides and the subsequent inflammatory immune response.
Highlights
With a prevalence of 1% celiac disease is one of the most common inflammatory disorders in Western populations; it is mainly triggered and maintained by gluten, storage proteins in wheat, rye, and barley [1, 2]
After 24 h incubation at the same concentration significant differences were observed depending on the peptide used, i.e., fluorescence intensity in the basal compartment was significantly higher after incubation of cells with P31–43 compared to P56–68 (2.2 vs. 1.7%, respectively) (Fig. 2B)
Toxic gliadin peptides (GP) like P31–43 are thought to induce mucosal damage by activation of the innate immune response whereas immunogenic GP like P56–68 contain epitopes which were shown to induce an adaptive immune response by stimulation of DQ2 restricted T-cell clones derived from celiac patients [6, 26]
Summary
With a prevalence of 1% celiac disease is one of the most common inflammatory disorders in Western populations; it is mainly triggered and maintained by gluten, storage proteins in wheat, rye, and barley [1, 2]. Up to now the only treatment is a life-long gluten-free diet to enable mucosal recovery and to minimize the risk for the development of enteropathyassociated T-cell lymphoma which has been seen to be markedly increased in patients with untreated celiac disease [2, 3]. In this context, gliadins as a fraction of gluten proteins [4] were identified as important inflammatory stimuli in celiac patients [5]. P31–43, a peptide sequence located at position 31–43 of a-gliadin represents a GP from the group of toxic peptides [12]
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