GLIADIN P31-43 PEPTIDE CAN DELAY EGFR (EPIDERMAL GROWTH FACTOR RECEPTOR) DECAY INTERIFERING WITH THE ENDOCYTIC PATHWAY

Background and aim: Entrance and sub-cellular localization of gliadin peptides in enterocytes is still debated. We have employed Caco 2 cells to verify whether gliadin peptides can enter the cells and where they localize. We show that both the toxic (P31-43) and the immunogenic peptide (P56-68) labelled with fluorochromes (lissamine and Cy3) enter Caco2 cells and interact with the endocytic compartment, but present different localization during maturation of endocytic vesicles. Materials and Methods: CaCo2 cells pulsed for 30 min and chased for 3 h (lower panel) with lissamine-labeled peptides were used for time-lapse experiments.Co-localization analysis of lissamine-labeled peptides was obtained by immunofluorescence techniques in pulse and chase experiments. Analysis of EGFR and HRS was performed by immunoblotting and subcellular fractionation. Results: Staining of P31-43 and P56-68 carrying vesicles with specific markers that can highlight steps of the endocytic pathway indicates that P31-43 remains into early endocytic vesicles at times when P56-68 carrying vesicles are allowed to mature into late endosomes. Time lapse experiments of early endocytic vesicles carrying P31-43 show that they are delayed in the cytoplasm of live Caco2 cells. Gliadin toxic peptide P31-43 can delay the maturation of early endocityc vesicles carrying EGF-Alexa, dextran and EEA1, indicating that it can interfere with the endocytic pathway both in Caco 2 cells and in cultured biopsies from patients with coeliac disease. This interference can leave longer activated TKR (Tyrosine Kinase Receptors) carried by endocytic vesicles. Western Blot analysis of EGFR phosphorylation during endocytosis shows that the receptor is longer activated in presence of P31-43.Conclusion: Gliadin peptide P31-43 interferes with maturation of early endosomes and can prolong activation of TKR such as EGFR.