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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缺陷胚胎干细胞是通过定向破坏occiudin基因的两个等位基因而产生的。在悬浮培养下，这些细胞聚集成简单的囊状胚体(EBS)，其形成过程与野生型ES细胞形成EB的过程相同。免疫荧光显微镜和超薄切片电子显微镜显示分化的极化上皮细胞(内脏内胚层样细胞)不仅可以区分野生型的EBS，还可以区分缺失oceludin的ES细胞。冷冻断裂分析表明，野生型和闭合素缺陷型上皮细胞在TI链的数量和形态上没有显著差异。这些发现表明，目前尚未鉴定的TJ完整膜蛋白(S)可以形成链结构。因此，我们重新检测了从鸡肝中分离出的连接组分，从其中首次鉴定出了occiudin。在该组分的众多成分中，…此外，通过4M盐酸胍提取、超声和蔗糖密度梯度离心法，只在22kD左右检测到一条银染的条带，并带有闭锁蛋白条带。从宽带的下半部分和上半部分获得了两个不同的肽序列，通过数据库的相似性搜索，我们分离到了两个编码相关的小鼠22-kD蛋白的全长cDNA，分别由211和230个氨基酸组成。亲水性分析表明，它们都有四个跨膜结构域，尽管它们没有显示出任何与occludin相似的序列。免疫荧光和免疫电子显微镜显示，两种标记有FLAG或GFP的蛋白质都以TJ链本身为靶标并被掺入。我们将它们分别命名为“claudin-1”和“claudin-2”。虽然Claudins与TI的确切结构/功能关系仍然不清楚，但这些发现表明，TJ链由具有四个假定的跨膜结构域的多个整膜蛋白组成，即occludin和claudins。较少