• The Vibrio Cholera-generated Zonulin occludens Toxin (Zot) N-terminal Cleavage Site Contains a Protease-activated Receptor Activating Peptide (PAR-AP) That Retains Biological Activity on Intestinal Tight Junctions

      Thakar, Manjusha; Vogel, Stefanie N.; Kao, Joseph P. Y.; Fasano, Alessio (2005)
      Background : We have previously demonstrated that Zonulin occludens toxin (Zot) elaborated by Vibrio Cholera anchors to the bacterial outer membrane through its single spanning domain and undergoes to a Vibrio-specific cleavage at amino acid residue 288. The resulting 12 Kda c-terminal fragment is then released in the intestinal micromilieu were exerts its permeating effect on intercellular tight junctions (tj). The N-terminus of th cleaved Zot fragment contains a conserved 6-mer protease activated receptor (PAR)-activating peptide (AP) motif. Aim: We have previously reported that Zot/Zonulin receptor is similar to PAR2, we elected to establish whether the six-mer motif (called AT1002) retains the Zot biological activity on tj. Methods: Rat small intestine was mounted in ussing chambers and AT1002-induced changes in transepithelial electrical resistance TEER) monitored Rat epithelial cells (IEC6) were used to study the intracellular signaling, including Ca2+ release,changes in tj protein-protein interactions, and phosphorylation of tj proteins. Results: Rat small intestine exposed to AT1002 showed a significant reduction in TEER as compared to the negative control starting at 30 minutes and reaching a plateau after 120 minutes. At a time point coincident with the effect con TEER (30 min) AT1002 induced an increment in ZO-1 phosphorylation that was associated to a decrease in ZO1-occludin interaction and an increase in ZO1-ZO2 interaction. Addition of AT1002 to IEC6 cells did not cause any increase in intracellular Ca2+, while the PAR-AP caused a dose-dependent increased in intracellular Ca2+ that reached a plateau at 50 fym. Conclusions: The 6-mer synthetic peptide At1002 retained the Zot biological activity an intercellular tj and caused a decrease in TEER. This effect was not associated to Ca2+ release, however was related to changes in protein-protein interaction of tj elements following Z)1 phosphorylation.