Monoclonal antibody 7H6 reacts with a novel tight junction-associated protein distinct from ZO-1, cingulin and ZO-2.

Abstract

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-aftiliated 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.

Human astrocytes/astrocyte-conditioned medium and shear stress enhance the barrier properties of human brain microvascular endothelial cells

“…Previous studies suggest that permeability of endothelial and epithelial cell is regulated by components of tight and adherens junction proteins, which include occludin [32] VE-cadherin [2,17,38], claudin [26], ZO-1 [29,62,71] ZO-2 [31], cingulin [16], 7H6 antigen [65,82] and symplekin [40]. We examined whether the permeability changes occurred in response to astrocyte/ACM and shear stress are correlated with the changes in expression of tight and adherens junction proteins (e.g., ZO-1 and β-catenin, respectively).…”

Enhanced paracellular barrier function of rat mesothelial cells partially protects against cancer cell penetration

“…We examined whether the treatment enhanced mesothelial barrier function, using TER and expression of 7H6 antigen, which preferentially localises at the tight junction (Zhong et al, 1993) as parameters. TER across a mesothelial monolayer grown on type I collagen-precoated Transwell filter at a confluent density without RA was low.…”

“…The 7H6 antigen is a 155-175 kDa tight junctionassociated protein discovered in our laboratory (Zhong et al, 1993), which has been shown to correlate closely with the barrier function of both epithelial cells and endothelial cells in vitro (Zhong et al, 1994).…”

Enteropathogenic Escherichia coli dephosphorylates and dissociates occludin from intestinal epithelial tight junctions

“…These proteins include ZO-1 (Stevenson et al, 1986), ZO-2 (Gumbiner et al, 1991), ZO-3 (Haskins et al, 1998), occludin (Furuse et al, 1993), junctional adhesion molecule (Martin-Padura et al, 1998), claudins Morita et al, 1999), cingulin (Citi et al, 1988), 7H6 (Zhong et al, 1993) and many signalling proteins (Mitic and Anderson, 1998). Although many tight junction-associated proteins have been identified individually, little is known about their specific functions and interactions.…”