Milatz Susanne, Piontek Jörg, Schulzke Jörg-Dieter, Blasig Ingolf E, Fromm Michael, Günzel Dorothee
Institute of Clinical Physiology, Campus Benjamin Franklin, Charité-Universitätsmedizin Berlin, 12200 Berlin, Germany Institute of Physiology, Christian-Albrechts-University Kiel, Hermann-Rodewald-Straße 5, 24118 Kiel, Germany.
Institute of Clinical Physiology, Campus Benjamin Franklin, Charité-Universitätsmedizin Berlin, 12200 Berlin, Germany.
Biochem J. 2015 Jun 15;468(3):449-58. doi: 10.1042/BJ20150148. Epub 2015 Apr 7.
Claudins form a large family of TJ (tight junction) proteins featuring four transmembrane segments (TM1-TM4), two extracellular loops, one intracellular loop and intracellular N- and C-termini. They form continuous and branched TJ strands by homo- or heterophilic interaction within the same membrane (cis-interaction) and with claudins of the opposing lateral cell membrane (trans-interaction). In order to clarify the molecular organization of TJ strand formation, we investigated the cis-interaction of two abundant prototypic claudins. Human claudin-1 and claudin-3, fused to ECFP or EYFP at the N- or C-terminus, were expressed in the TJ-free cell line HEK (human embryonic kidney)-293. Using FRET analysis, the proximity of claudin N- and C-termini integrated in homopolymeric strands composed of claudin-3 or of heteropolymeric strands composed of claudin-1 and claudin-3 were determined. The main results are that (i) within homo- and heteropolymers, the average distance between the cytoplasmic ends of the TM1s of cis-interacting claudin molecules is shorter than the average distance between their TM4s, and (ii) TM1 segments of neighbouring claudins are oriented towards each other as the cytoplasmic end of TM1 is in close proximity to more other TM1 segments than TM4 is to other TM4 segments. The results indicate at least two different cis-interaction interfaces within claudin-3 homopolymers as well as within claudin-1/claudin-3 heteropolymers. The data provide novel insight into the molecular TJ architecture consistent with a model with an antiparallel double-row cis-arrangement of classic claudin protomers within strands.
紧密连接蛋白形成了一个紧密连接（TJ）蛋白的大家族，其特征为具有四个跨膜区段（TM1 - TM4）、两个细胞外环、一个细胞内环以及细胞内的N端和C端。它们通过同一膜内的同源或异源相互作用（顺式相互作用）以及与相对侧细胞膜的紧密连接蛋白的相互作用（反式相互作用）形成连续且分支的紧密连接链。为了阐明紧密连接链形成的分子组织，我们研究了两种丰富的典型紧密连接蛋白的顺式相互作用。在无紧密连接的人胚肾（HEK）-293细胞系中表达了在N端或C端与增强型青色荧光蛋白（ECFP）或增强型黄色荧光蛋白（EYFP）融合的人紧密连接蛋白-1和紧密连接蛋白-3。使用荧光共振能量转移（FRET）分析，确定了整合在由紧密连接蛋白-3组成的同聚物链或由紧密连接蛋白-1和紧密连接蛋白-3组成的异聚物链中的紧密连接蛋白N端和C端的接近程度。主要结果是：（i）在同聚物和异聚物中，顺式相互作用的紧密连接蛋白分子的TM1细胞质端之间的平均距离短于其TM4之间的平均距离；（ii）相邻紧密连接蛋白的TM1区段相互朝向，因为TM1的细胞质端比TM4与其他TM4区段更接近其他TM1区段。结果表明在紧密连接蛋白-3同聚物以及紧密连接蛋白-1/紧密连接蛋白-3异聚物中至少有两个不同的顺式相互作用界面。这些数据为分子紧密连接结构提供了新的见解，与链内经典紧密连接蛋白原聚体的反平行双排顺式排列模型一致。
