Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin.
Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin.
复制标题
DOI：
10.1083/jcb.141.7.1539
复制
发表时间：
1998-06-29
期刊：
影响因子：
--
通讯作者：
Tsukita S
中科院分区：
文献类型：
--
作者：
Furuse M;Fujita K;Hiiragi T;Fujimoto K;Tsukita S
紧密连接蛋白（Occludin）是目前已知唯一定位于紧密连接（TJ）的整合膜蛋白，但近期对紧密连接蛋白基因的靶向破坏分析表明，在紧密连接中还存在尚未被鉴定的整合膜蛋白。因此，我们重新检测了从鸡肝中分离出的连接组分，紧密连接蛋白最初就是从鸡肝中被鉴定出来的。在该组分的众多成分中，经过4M盐酸胍提取以及超声处理后再进行逐步蔗糖密度梯度离心，只有一条约22kD的宽银染带与紧密连接蛋白带一起被检测到。从该宽带的下半部分和上半部分获得了两个不同的肽序列，通过对数据库的相似性搜索，我们分离出了两个编码相关小鼠22 - kD蛋白的全长cDNA，它们分别由211和230个氨基酸组成。亲水性分析表明，这两种蛋白都具有四个跨膜结构域，尽管它们与紧密连接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，标记有FLAG或GFP的这两种蛋白都能靶向并整合到紧密连接链本身中。我们分别将它们命名为“克劳丁 - 1（claudin - 1）”和“克劳丁 - 2（claudin - 2）”。尽管克劳丁蛋白与紧密连接之间精确的结构/功能关系仍不清楚，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，即紧密连接蛋白和克劳丁蛋白，构成了紧密连接链。
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 ∼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.