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.
Keywords: celiac disease; cell-penetrating peptides; chemical cross-linking; gliadin peptide 31-43; peptide internalization; type 2 transglutaminase.
© 2017 International Federation for Cell Biology.