Analysis of Binding Affinity of the Zonulin Agonist (At1002) and Antagonist (At1001) to the Zonulin Intestinal Receptor

Background: Zonulin is a modulator of intestinal tight junctions (tj) permeability whose physiological role within the GI tract is to protect against proximal bowel contamination. We have found that Zonulin upregulation is involved in a series of autoimmune diseases, including type 1 diabetes and celiac disease (CD). We have also demonstrated that the effect of Zonulin on the cell cytoskeleton and tj permeability can be mimicked by its synthetic peptide agonist AT1002 and can be inhibited by the synthetic peptide AT1001. Both peptides seem to bind to the same Zonulin receptor through a specific binding motif. Aim: To analyze the binding affinity of Zonulin agonist and antagonist to Zonulin expressing Caco2 intestinal epithelial cells by fluorescent microscopy. Methods: CaCo2 cells were used to analyze the affinity of AT1001 and AT1002. AT1001 untagged and tagged with FITC, AT1002 untagged and tagged with FITC and scrambled peptide tagged with FITC was obtained. Cells were cultured and fixed on 8 chamber mounted on glass slide and incubated with FITC labeled peptide (AT1001 or AT1002) either in the presence or absences of unlabelled peptide. Slides were then analyzed in blind fashion with a flourescence microscope. Results: Both AT1001 and AT1002 bind to CaCo2 cells, while no detectable binding was observed with the scrambled peptide. FITC-AT1001 was displaced when untagged AT1001 was used at a concentration >100 times that of the tagged peptide. FITC-AT001 was also displaced by untagged AT1002 but at lower concentrations (75 times). Similarly, FITC-AT002 was displaced by untagged AT1002 and AT1001 but at higher concentrations (>150 times for untagged AT1002 and >200 for untagged AT1001). Conclusions: Our results demonstrated that both AT1001 and AT1002 bind to the same receptor with AT1002 showing higher affinity than AT1001. These findings will assist us to develop strategies to properly antagonize the zonulin pathway for the treatment of autoimmune diseases, including CD.