Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S
Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.
J Cell Biol. 1998 Jun 29;141(7):1539-50. doi: 10.1083/jcb.141.7.1539.
Occludin is the only known integral membrane protein localizing at tight junctions (TJ), but recent targeted disruption analysis of the occludin gene indicated the existence of as yet unidentified integral membrane proteins in TJ. We therefore re-examined the isolated junction fraction from chicken liver, from which occludin was first identified. Among numerous components of this fraction, only a broad silver-stained band approximately 22 kD was detected with the occludin band through 4 M guanidine-HCl extraction as well as sonication followed by stepwise sucrose 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 both bore four transmembrane domains, although they did not show any sequence similarity to occludin. Immunofluorescence and immunoelectron microscopy revealed that both proteins tagged with FLAG or GFP were targeted 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 elusive, these findings indicated that multiple integral membrane proteins with four putative transmembrane domains, occludin and claudins, constitute TJ strands.
闭合蛋白是唯一已知的定位于紧密连接（TJ）的整合膜蛋白，但最近对闭合蛋白基因的靶向破坏分析表明，紧密连接中还存在尚未鉴定的整合膜蛋白。因此，我们重新检查了最初从鸡肝中分离出的连接部分，闭合蛋白就是从这里首次被鉴定出来的。在该部分的众多成分中，通过4M盐酸胍提取、超声处理，然后进行逐步蔗糖密度梯度离心，只有一条约22kD的宽银染带与闭合蛋白带一起被检测到。从这条宽带的下半部分和上半部分获得了两个不同的肽序列，通过数据库相似性搜索，我们分离出了两个全长cDNA，它们分别编码由211和230个氨基酸组成的相关小鼠22-kD蛋白。亲水性分析表明，两者都有四个跨膜结构域，尽管它们与闭合蛋白没有任何序列相似性。免疫荧光和免疫电子显微镜显示，用FLAG或GFP标记的这两种蛋白都靶向并整合到紧密连接链本身中。我们分别将它们命名为“claudin-1”和“claudin-2”。尽管claudin蛋白与紧密连接的确切结构/功能关系仍然难以捉摸，但这些发现表明，具有四个假定跨膜结构域的多种整合膜蛋白，即闭合蛋白和claudin蛋白，构成了紧密连接链。
