Claudin-2 Forms Homodimers and Is a Component of a High Molecular Weight Protein Complex
Claudin-2 Forms Homodimers and Is a Component of a High Molecular Weight Protein Complex
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DOI：
10.1074/jbc.m110.195578
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发表时间：
2011-02-04
影响因子：
4.8
通讯作者：
Anderson, James M.
中科院分区：
文献类型：
--
作者：
Van Itallie, Christina M.;Mitic, Laura L.;Anderson, James M.
紧密连接是一种多蛋白复合物，形成上皮细胞和内皮细胞之间的基本生理和解剖屏障，但关于其分子组织的信息很少。为了开始了解紧密连接的跨膜蛋白是如何组织成多蛋白复合物的，我们使用蓝色非变性PAGE（BN-PAGE）和交联技术来鉴定从MDCK II细胞和小鼠肝脏中提取的复合物。在非离子去污剂提取物从MDCK II细胞，紧密连接的膜蛋白claudin-2优先分离为同源二聚体，而claudin-4是单体。密蛋白-2和-4嵌合体之间的相互作用的分析与负责二聚化的密蛋白-2的跨膜结构域一致，并且随后交联的突变分析表明第二跨膜结构域在同源二聚体中紧密接近地排列。小鼠肝膜的BN-PAGE鉴定了相对离散的高分子量复合物，其至少含有封闭蛋白-1、封闭蛋白-2和闭合蛋白;培养的细胞和组织之间的蛋白质复合物大小的差异可能反映了紧密连接蛋白或脂质组成或翻译后修饰的差异。我们的研究结果表明，BN-PAGE可能是一个有用的工具，在了解紧密连接结构。
Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.