Gliadin peptide P31-43 interferes Hrs (hepatocyte growth factor-regulated tyrosine kinasesubstrate) localization to endocytic vesicles: implications for Celiac Disease (CD)

Maria Vittoria Barone1, Merlin Nanayakkara2, Delia Zanzi2, Sara Santagata2, ValentinaDiscepolo2, Maria ten Eikelder2, Giuliana Lania2, Salvatore Auricchio21Pediatric Department (ELFID), Università degli Studi di Napoli, Federico II, Napoli2Pediatric Department (ELFID), Università degli Studi di Napoli,Background and AimsWe previously observed that A-gliadin peptide P31-43 induces effects similar to Epidermal growthfactor (EGF) both in cultured cell lines and in enterocytes from celiac disease (CD) patients. Wealso showed that the effect is mediated by delayed EGF degradation and prolonged EGF receptor(EGFR) activation in endocytic vesicles. Here we address the molecular mechanisms underlyinggliadin peptide effects on trafficking and maturation of vesicles responsible for EGFR endocytosis.MethodsA sequence similarity search revealed that P31-43 is strikingly similar to a region of Hrs, a keymolecule involved in endocytic maturation. Western blot and immunofluorescence microscopy wereused to determine Hrs localization to endocytic vesicles and cytosol. Pulse and chase labelling intime-lapse experiments allowed to follow uptake and sub-cellular localization of gliadin peptides inCaCo 2 cells and enterocytes from CD patients and controls.ResultsA-gliadin peptide P31-43 interferes with Hrs localization to early endosomes. Both P31-43 and thecontrol P56-68 peptide enter CaCo 2 cells and interact with the endocytic compartment, but P31-43 is localized to vesicles carrying early endocytic markers at time points when P56-68-carryingvesicles mature into late endosomes. Dynamic analysis shows that P31-43 labelled vesicles areslowed down in time-lapse experiments. The effect is independent of the cargo they carry: dextrancontaining vesicles, behave similarly to EGFR containing ones. Markers of recycling pathwayTrasferrin receptor and Lamp, are increased on the surface of P31-43 treated cells together withEGFR. Gliadin peptide P31-43 enters epithelial cells and colocalize with EEA1 both in CD biopsiesand controls coltured for 24 hours. Pulse and chase experiments show a delay of trafficking of P31-43 peptide in EEA1 positive vesicles only in CD biopsies, but not in controlsConclusionsGliadin peptide P31-43 delays vesicle trafficking by interfering with Hrs mediated maturation to lateendosomes and also promotes the recycling pathway. As a consequence, in P31-43 treated cells,EGFR activation is extended, more transferrin and EGFR receptors find their way to the cellsurface. In CD biopsies trafficking of this peptide is delayed. This may help explaining the roleplayed by gliadin peptides in CD pathogenesis.NOTE: To