Ahlswede Lena, Siebenaller Carmen, Junglas Benedikt, Hellmann Nadja, Schneider Dirk
Department of Chemistry, Biochemistry, Johannes Gutenberg University Mainz, Mainz, Germany.
Institute of Molecular Physiology, Johannes Gutenberg University Mainz, Mainz, Germany.
Front Mol Biosci. 2022 Jun 27;9:908383. doi: 10.3389/fmolb.2022.908383. eCollection 2022.
Human Claudin-7 (Cldn7) is a member of the Claudin (Cldn) superfamily. , these proteins form tight junctions, which establish constricted connections between cells. Cldns oligomerize within the membrane plane (= -interaction), and also interact with Cldns from adjacent cells (= -interaction). Interactions of Cldns are typically studied and structural analyses of isolated Cldns are limited. Here, we describe heterologous expression in and purification of human Cldn7, enabling analyses of the isolated protein using detergent and model membrane systems. Cldn7 exists as a monomer, hexamer, and various higher oligomers in micelles. While only limited unfolding of the protein was observed in the presence of the anionic detergent sodium dodecyl sulfate, decreased ionic strength did affect Cldn7 -interactions. Furthermore, we identified two amino acids which mediate electrostatic -interactions and analyzed the impact of disturbed -interaction on -contacts via atomic force microscopy and monitoring Förster resonance energy transfer between fluorescently labeled Cldn7-containing proteoliposomes. Our results indicate that Cldn7 -oligomerization might not be a prerequisite for establishing -contacts.
人Claudin-7（Cldn7）是Claudin（Cldn）超家族的成员。这些蛋白质形成紧密连接，在细胞之间建立紧密连接。Cldn在膜平面内寡聚化（= -相互作用），并且还与相邻细胞的Cldn相互作用（= -相互作用）。Cldn的相互作用通常被研究，而对分离的Cldn的结构分析是有限的。在这里，我们描述了人Cldn7在 中的异源表达和纯化，使得能够使用去污剂和模型膜系统对分离的蛋白质进行 分析。Cldn7在胶束中以单体、六聚体和各种更高的寡聚体形式存在。虽然在阴离子去污剂十二烷基硫酸钠存在下仅观察到蛋白质的有限解折叠，但离子强度的降低确实影响了Cldn7的 -相互作用。此外，我们鉴定了介导静电 -相互作用的两个氨基酸，并通过原子力显微镜和监测荧光标记的含Cldn7的蛋白脂质体之间的Förster共振能量转移，分析了 -相互作用的干扰对 -接触的影响。我们的结果表明，Cldn7的 -寡聚化可能不是建立 -接触的先决条件。
