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Monoclonal Antibody 7H6 Reacts with a Novel Tight Junction-Associated Protein Distinct from ZO-1, Cingulin and ZO-2

Yun Zhong, Tsuyoshi Saitoh, Takashi Minase, Norimasa Sawada, Katsuhiko Enomoto and Michio Mori
The Journal of Cell Biology
Vol. 120, No. 2 (Jan., 1993), pp. 477-483
Stable URL: http://www.jstor.org/stable/1615577
Page Count: 7
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Monoclonal Antibody 7H6 Reacts with a Novel Tight Junction-Associated Protein Distinct from ZO-1, Cingulin and ZO-2
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Abstract

The tight junction is an essential element of the intercellular junctional complex; yet its protein composition is not fully understood. At present, only three proteins, ZO-1 (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766), cingulin (Citi, S., H. Sabanay, R. Jakes, B. Geiger, and J. Kendrick-Jones. 1988. Nature (Lond.). 333:272-275) and ZO-2 (Gumbiner, B., T. Lowenkopf, and D. Apatira. 1991. Proc. Natl. Acad. Sci. USA. 88:3460-3464) are known to be associated with the tight junction. We have generated a monoclonal antibody (7H6) against a bile canaliculusrich membrane fraction prepared from rat liver. This 7H6 antigen was preferentially localized by immunofluorescence at the junctional complex regions of hepatocytes and other epithelia, and 7H6-affiliated gold particles were shown electron microscopically to localize at the periphery of tight junctions. Immunoblot analysis of a bile canaliculus-rich fraction of rat liver using 7H6, anti-ZO-1 antibody (R26.4C), and anticingulin antibody revealed that 7H6 reacted selectively with a 155-kD protein, whereas R26.4C reacted only with a 225-kD protein. Anti-cingulin antibody reacted solely with 140 and 108-kD proteins, indicating that the protein recognized by 7H6 is immunologically different from ZO-1 and cingulin. Immunoprecipitation of detergent extracts obtained from metabolically labeled MDCK cells with R26.4C coprecipitated a 160-kD protein, which corresponds to ZO-2, with ZO-1. However, 7H6 did not react with the 160-kD protein. These results strongly suggest that the 7H6 antibody recognizes a novel tight junction-associated protein different from ZO-1, cingulin and ZO-2.

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