The structure and functions of occludin

occludin的结构和功能

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08407006/
• 关键词: tight junction; occludin; claudin; intercellular junction; cell adhesion; knockout; epithelial cells; 内皮細胞; アドへレンスジャンクション; ES細胞; 相同組換え; 上皮細胞; ドラッグデリバリー; 極性; ターゲッティング; 細胞極性; 自己抗体; ZO-1; ZO-2; 細胞接着; 接着分子

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

Occludin 缺陷型胚胎干 (ES) 细胞是通过靶向破坏 Occludin 基因的两个等位基因而产生的。当这些细胞进行悬浮培养时，它们聚集形成简单的，然后形成囊性胚状体（EB），其时间过程与野生型 ES 细胞形成 EB 的时间过程相同。免疫荧光显微镜和超薄切片电子显微镜显示，极化上皮（内脏内胚层样）细胞不仅从野生型而且从occludin缺陷的ES细胞中分化出EB。冷冻断裂分析表明野生型和occludin缺陷型上皮细胞之间TI链的数量或形态没有显着差异。这些发现表明，尚有尚未鉴定的 TJ 整合膜蛋白可以形成链结构。因此，我们重新检查了从鸡肝中分离出的连接部分，occludin 首次从中被鉴定出来。在该级分的众多成分中，通过 4 M 盐酸胍提取以及超声处理和逐步蔗糖密度梯度离心，仅检测到大约 22 kD 的宽银染带和 occludin 带。从宽带的下半部和上半部获得了两个不同的肽序列，并且数据库的相似性搜索使我们能够分离出两个全长cDNA，其编码分别由211和230个氨基酸组成的相关小鼠22-kD蛋白质。亲水性分析表明两者都具有四个跨膜结构域，尽管它们没有显示出与 occludin 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 的两种蛋白质都靶向并整合到 TI 链本身中。我们分别将它们命名为“claudin-1”和“claudin-2”。尽管紧密蛋白与 TI 的精确结构/功能关系仍然难以捉摸，但这些发现表明，具有四个假定跨膜结构域的多个整合膜蛋白（occludin 和 claudins）构成了 TJ 链。较少的

项目成果

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Shigenobu Yonemura：“ERM 蛋白与一组特定的整合膜蛋白结合，在其近膜胞质结构域中含有带正电荷的氨基酸簇。”

Yonemura,S.et al.: "ERM proteins bind to a specific group of integral membrane proteins containing a positively-harged amino acid cluster in their juxta-membrane cytoplasmic domain." J.Cell Biol.140. 885-895 (1998)

Yonemura,S.et al.：“ERM 蛋白与一组特定的整合膜蛋白结合，在其近膜胞质结构域中含有带正电荷的氨基酸簇。”

Saitou,M.,Fujimoto,K.,Doi,Y.,Itoh,M.,Fujimoto,T.,Furuse,M.,Takano,H.,Noda,T. and Tsukita,Sh.: "Occludin-deficient embryonic stem cells can diferentiate into polarized epithelial cells bearing tight junctions." J.Cell Biol.141. 397-408 (1998)

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Itoh, M., Nagafuchi, A., Moroi, S. and Tsukita, Sh.: "Involvement of ZO-1 in cadhherin-based cell adhesion through its direct binding to α catenin and actin filaments." J. Cell Biol.138. 181-192 (1997)

Itoh, M.、Nagafuchi, A.、Moroi, S. 和 Tsukita, Sh.：“ZO-1 通过直接结合 α 连环蛋白和肌动蛋白丝参与基于钙粘蛋白的细胞粘附。”138 .181-192 (1997)