Monoclonal antibody 7H6 reacts with a novel tight junction-associated protein distinct from ZO-1, cingulin and ZO-2.
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 canaliculus-rich 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 anti-cingulin 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.
Zhong
Y
Y
Department of Pathology, Sapporo Medical College, Japan.
Saitoh
T
T
Minase
T
T
Sawada
N
N
Enomoto
K
K
Mori
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D001648:Bile Canaliculi / Q000166:cytology* / Q000648:ultrastructure; D002460:Cell Line; D004285:Dogs; D015151:Immunoblotting; D007150:Immunohistochemistry; D007365:Intercellular Junctions / Q000648:ultrastructure*; D007668:Kidney; D008099:Liver / Q000737:chemistry / Q000166:cytology / Q000648:ultrastructure; D008565:Membrane Proteins / Q000032:analysis* / Q000276:immunology; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D010750:Phosphoproteins / Q000032:analysis*; D051381:Rats; D011916:Rats, Inbred F344; D062826:Zonula Occludens-1 Protein / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D010750:Phosphoproteins; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein