Zonula occludin toxin, a microtubule binding protein.

AIM:To investigate the interaction of Zot with microtubule.METHODS:Zot affinity column was applied to purify Zot-binding protein(s) from crude intestinal cell lysates. After incubation at room temperature, the column was washed and the proteins bound to the Zot affinity column were eluted by step gradient with NaCl (0.3molcenter dotL(-1) -0.5molcenter dotL(-1)). The fractions were subjected to 6.0%-15.0% (w/v) gradient SDS-PAGE and then transferred to PVDF membrane for N-terminal sequencing.Purified Zot and tau protein were blotted by using anti-Zot or anti tau antibodies. Finally, purified Zot was tested in an in vitro tubulin binding assay.RESULTS:Fractions from Zot affinity column yielded two protein bands with a M(r) of 60kU and 45kU respectively. The Nterminal sequence of the 60kU band resulted identical to beta-tubulin. Zot also crossreacts with antitau antibodies. In the in vitro tubulin binding assay, Zot coprecipitate with Mt, further suggesting that Zot possesses tubulin-binding properties.CONCLUSION:Taken together, these results suggest that Zot regulates the permeability of intestinal tight junctions by binding to intracellular Mt, with the subsequent activation of the intracellular signaling leading to the permeabilization of intercellular tight junctions.