CLAUDIN-1 AND CLAUDIN-2 - NOVEL INTEGRAL MEMBRANE-PROTEINS LOCALIZINGAT TIGHT JUNCTIONS WITH NO SEQUENCE SIMILARITY TO OCCLUDIN

Occludin is the only known integral membrane protein localizing at tig ht junctions (TJ), but recent targeted disruption analysis of the occl udin gene indicated the existence of as yet unidentified integral memb rane proteins in TJ. We therefore re-examined the isolated junction fr action from chicken liver, from which occludin was first identified. A mong numerous components of this fraction, only a broad silver-stained band similar to 22 kD was detected with the occludin band through 4 M guanidine-HCl extraction as well as sonication followed by stepwise s ucrose density gradient centrifugation, Two distinct peptide sequences were obtained from the lower and upper halves of the broad band, and similarity searches of databases allowed us to isolate two full-length cDNAs encoding related mouse 22-kD proteins consisting of 211 and 230 amino acids, respectively. Hydrophilicity analysis suggested that bot h bore four transmembrane domains, although they did not show any sequ ence similarity to occludin. Immunofluorescence and immunoelectron mic roscopy revealed that both proteins tagged with FLAG or GFP were targe ted to and incorporated into the TJ strand itself. We designated them as ''claudin-1'' and ''claudin-2'', respectively. Although the precise structure/function relationship of the claudins to TJ still remains e lusive, these findings indicated that multiple integral membrane prote ins with four putative transmembrane domains, occludin and claudins, c onstitute TJ strands.