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The structure and functions of occludin

occludin的结构和功能

基本信息

批准号：
08407006
负责人：
TSUKITA Shoichiro
金额：
$ 20.61万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin 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 occludin-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 occludin 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-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 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 TI 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

通过靶向破坏occludin基因的两个等位基因，产生了occludin缺陷胚胎干细胞（ES）。当这些细胞进行悬浮培养时，它们聚集形成简单胚状体，然后形成囊性胚状体（EBs），其形成过程与野生型ES细胞形成EB的时间相同。免疫荧光显微镜和超薄切片电镜显示极化上皮（内脏内胚层样）细胞分化，不仅从野生型分化出EBs，而且从occludin缺乏的ES细胞分化出EBs。冷冻断裂分析表明，野生型和occludin缺陷型上皮细胞之间TI链的数量和形态没有显著差异。这些发现表明存在尚未确定的可形成链结构的TJ积分膜蛋白。因此，我们重新检查了鸡肝脏中分离的连接部分，其中首次鉴定出occludin。在该组分中，通过4 M胍-盐酸提取、超声处理和逐步的蔗糖密度梯度离心，occludin条带仅检测到22 kD左右的宽银染色带。从宽频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，我们分离出两个全长cdna，分别编码相关的小鼠22-kD蛋白，分别由211和230 a.a组成。亲水性分析表明两者都有四个跨膜结构域，尽管它们与occludin没有任何序列相似性。免疫荧光和免疫电镜显示，这两种标记有FLAG或GFP的蛋白都被靶向并结合到TI链本身。我们将它们分别命名为“claudin-1”和“claudin-2”。虽然TJ链与claudin的精确结构/功能关系尚不清楚，但这些发现表明，具有四个假定跨膜结构域（occludin和claudin）的多个完整膜蛋白构成了TJ链。少