The toxic alpha‐gliadin peptide 31–43 enters cells without a surface membrane receptor

Abstract

Alpha-gliadin peptide 31-43 is considered to be the main peptide responsible for the innate immune response in celiac disease patients. Recent evidence indicates that peptide 31-43 rapidly enters cells and interacts with the early endocytic vesicular compartment. However, the mechanism of its uptake is not completely understood. Our aim is to characterize, isolate and identify possible cell surface proteins involved in peptide 31-43 internalization by Caco-2 cells. In this study, we used a chemical cross-linker to block peptide 31-43 on cell surface proteins, and pulled-down peptide-proteins complexes using antibodies raised against peptide 31-43. Through this experimental approach, we did not observe any specific complex between cell proteins and peptide 31-43 in Coomassie-stained denaturating gels or by Western blotting. We also found that type 2 transglutaminase was not necessary for peptide 31-43 internalization, even though it had a regulatory role in the process. Finally, we demonstrated that peptide 31-43 did not behave as a classical ligand, indeed the labeled peptide did not displace the unlabeled peptide in a competitive binding assay. On the basis of these findings and of previous evidence demonstrating that peptide 31-43 is able to interact with a membrane-like environment in vitro, we conclude that membrane composition and organization, rather than a specific receptor protein, may have a major role in peptide 31-43 internalization by cells.

Structural conformation and self‐assembly process of p31‐43 gliadin peptide in aqueous solution. Implications for celiac disease

“…By CD and TEM, we showed that p31‐43 is able to form oligomers in the multinanometer scale . Since a cell surface receptor for p31‐43 has not been identified , we hypothesize that structural features of p31‐43 may be of key importance in the induction of different biological effects.…”

“…Though biological effects triggered by p31‐43 (LGQQQPFPPQQPY) are observed very rapid upon treatment, the surface cell receptor or the mechanism of peptide entry has not been identified . In our previous study, we showed by CD and transmission electron microscopy (TEM) that p31‐43 presents a polyproline II (PPII) structure and forms oligomers which may promote the activation of the inflammasome platform .…”

Structural insights on P31‐43, a gliadin peptide able to promote an innate but not an adaptive response in celiac disease

“…33‐mer shows six overlapping copies of three different epitopes for DQ2 . This peptide, once de‐amidated by TG2 at specific Gln sites, as that of the epitope PQLP changed to PELP, increases its affinity to DQ2 and plays the main role in inducing the activation of adaptive response in CD . The other undigested gliadin peptide, the 25‐mer P31‐55, not de‐amidated by TG2, induces the innate immune response typically associated to mucosal damage of gut in CD .…”

“…P31‐43 cooperates with a viral ligand to activate the TLR7 pathway by interfering with endocytic trafficking . Furthermore, it was shown that P31‐43 enters intestinal epithelial cells (Caco‐2 cells) by endocytosis, apparently without requiring either TG2 on the cell membrane or other membrane receptors . Very recently, Vilella and co‐authors have shown that P31‐43 inhibits the function of cystic fibrosis trans‐membrane conductance regulator (CFTR), an anion channel involved in the epithelial adaptation to environmental stress.…”

Translational Chemistry Meets Gluten‐Related Disorders

“…Also, in competitive binding experiments the peptide could not displace bound peptide, which suggests nonspecific binding. On the basis of this result, the authors hypothesize that no receptor might be involved in the trafficking of the 13‐mer peptide …”