ISOLATION AND PARTIAL PURIFICATION OF THE ZONULA OCCLUDENS TOXIN (ZOT) HUMAN RECEPTOR (1998)

77 Zonula Occuldens Toxin (Zot) is a protein elaborated by Vibrio cholerae that reversibly increase the intestinal permeability by acting on the intercellular tight junctions (tj). Zot effect involves the binding to a surface receptor, whose distribution varies within the intestine, being present only in the jejunum and distal ileum, but not in the colon, and decreasing along the villous-crypt axis. This distribution coincides with the regional effect of ZOT on tissue resistance, and with the preferential F-actin redistribution induced by the protein at the tip of the intestinal villi. Aim of the present study was to purify the human Zot receptor. To establish whether Zot interacts with human eukaryotic cells, binding experiments were conducted on both CaCo2 and T84 human intestinal cells. A Zot affinity column was also prepared by coupling 1.0 mg of purified Zot to a resin support at 4°C overnight. The column was then loaded with crude intestinal and brain cell lysates obtained from human cadavers. After 90 min of incubation at room temperature, the column was washed five times with PBS, and the proteins which bound to the Zot affinity column were eluted by step gradient with NaCl(0.3-0.5 M) containing 0.1% (v/v) Triton X-100 in PBS, pH 7.4. The fractions were subjected to 6.0-15.0% (w/v) gradient SDS-PAGE under reducing conditions and then transferred to a PVDF membrane for N-terminal sequencing. Binding experiments on human cell lines showed that Zot binds to CaCo2 cells (that resemble the mature enterocytes), but not to T84 (crypt-like cells), suggesting that the expression of the receptor is up-regulated during enterocyte differentiation. Fractions from both human intestine and brain cell lysates loaded onto the Zot affinity column yielded two protein bands with a Mr of 60 kDa and 45 kDa, respectively. The 60 kDa band N-terminal sequence from both brain and intestine resulted identical to tubulin. On the other hand, the N-terminal sequences of the 45 kDa band from the human intestine and brain resulted different. A comparison of the N-terminal sequence of the 45 kDa band from the human intestine (that may represent the Zot receptor) to the NBCI sequence database using BLAST revealed similarities to E-MAP-115, a novel microtubule-associated protein, and to verprolin, a protein isolated from Saccharomyces cervisiae involved in cytoskeletal organization. The N-terminal sequence of the human brain receptor resulted different from the sequence obtained from the intestine. In conclusion: 1. Zot interacts with a surface receptor that may represent the interface between its ligand and the intracellular signaling involving microtubule binding and cytoskeletal rearrangement; 2. Zot receptor seems to be expressed also in the human brain where may play a pivotal role in the regulation of the blood-brain barrier. 3. The distinct N-terminal sequences of the receptors isolated from the intestine and the brain may be responsible for the tissue-specificity of zonulin, a molecule that we have recently described and that probably represents the physiologic ligand of the Zot receptor.