Structure and function of occludin in tight junctions : comparison with connexin gap junctions

紧密连接中occludin的结构和功能：与连接蛋白间隙连接的比较

基本信息

批准号：
09044290
负责人：
TSUKITA Shoichiro
金额：
$ 4.1万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Occiudin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occiudin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from oceludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TI strands between wild-type and occludin-deficient epithelial cells. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures. We therefore re-examined the isolated junction fraction from chicken liver, from which occiudin was first identified. Among numerous components of this fraction, … More only a broad silver-stained band around 22 kD was detected with the occludin band through 4 M guanidine-HCI 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 data bases allowed us to isolate two full-length cDNAs encoding related mouse 22-kD proteins consisting of 211 and 230 a.a., 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 TI still remains elusive, these findings indicated that multiple integral membrane proteins with four putative transmembrane domains, occludin and claudins, constitute TJ strands. Less

occiudin缺陷性胚胎（ES）细胞是通过靶向occiudin基因的两个等位基因的靶向破坏而产生的。当这些细胞受到悬浮培养的情况时，它们汇总形成简单，然后形成与野生型ES细胞相同的时间过程的囊性胚胎体（EB）。免疫荧光显微镜和超薄片段电子显微镜表明，极化上皮（内脏内胚层状）细胞不仅从野生型中，而且还从oculudin缺乏的ES细胞中分开了EBS。冻结分数分析表明，野生型和缺陷型上皮细胞之间TI链的数量或形态没有显着差异。这些发现表明，尚未确定的TJ积分膜蛋白可以形成链结构。因此，我们从鸡肝的许多组成部分中重新检查了从鸡肝的孤立连接分数，…更多的是，仅通过4 m鸟苷-HCI的提取，然后通过4 m的occludin band检测到大约22 kD的宽片染色带，然后进行超声处理，然后逐步进行了逐步的分量性梯度梯度。从宽带的下半部分和上半部分获得了两个不同的肽序列，数据库的相似性搜索使我们能够分别隔离两个编码相关小鼠22 kD蛋白的全长cDNA，分别由211 a.A.组成。亲水性分析表明，尽管它们没有显示出与塞链丁物的任何序列相似性，但两种均具有四个跨膜结构域。免疫荧光和免疫电子显微镜表明，标记为FLAG或GFP的两个蛋白质均针对并掺入TJ链本身中。我们分别将它们设计为“ Claudin-1”和“ Claudin-2”。尽管Claudins与Ti的精确结构/功能关系仍然难以捉摸，但这些发现表明，具有四个假定的跨膜结构域，Occludin和claudins的多个积分膜蛋白构成TJ链。较少的