Sensing Bacterial Flagellin by Membrane and Soluble Orthologs of Toll-like Receptor 5 in Rainbow Trout (Onchorhynchus mikiss)*
Sensing Bacterial Flagellin by Membrane and Soluble Orthologs of Toll-like Receptor 5 in Rainbow Trout (Onchorhynchus mikiss)*
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DOI：
10.1074/jbc.m407634200
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发表时间：
2004-11
影响因子：
4.8
通讯作者：
Tadayuki Tsujita;Hironobu Tsukada;M. Nakao;H. Oshiumi;M. Matsumoto;T. Seya
中科院分区：
文献类型：
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作者：
Tadayuki Tsujita;Hironobu Tsukada;M. Nakao;H. Oshiumi;M. Matsumoto;T. Seya
虹鳟鱼（Onchorhynchus mikiss）具有两个基因编码假定的富含亮氨酸重复序列（LRR）的蛋白，类似于人类的TLR5。这两个LRR蛋白的分子克隆表明存在tlr5样膜形式（rtTLR5M）和可溶性形式（rtTLR5S）。本文阐明了这些鱼类TLR5基因的主要结构和独特的组合功能。rtTLR5S和rtTLR5M的LRR区域与人类TLR5 （huTLR5）的细胞外结构域同源性较高（分别为35.6%和33.7%）。因此，在鱼类中有两个不同的基因编码TLR5同源物，其中一个具有一致的细胞内结构域（TIR）。为了测试它们的功能，我们构建了rtTLR5S （s -嵌合体）或rtTLR5M的LRR区域与huTLR5 （m -嵌合体）的TIR的融合蛋白。在HeLa或CHO细胞中表达的S-和m -嵌合体标志着鳗弧菌鞭毛蛋白的存在，导致NF-κ b活化。rtTLR5M在肝脏中普遍表达，而rtTLR5S主要在肝脏中表达。在虹鳟鱼RTH-149肝癌细胞系中，用鳗弧菌或其鞭毛蛋白刺激rtTLR5M可使rtTLR5S上调。鞭毛蛋白介导的NF-κB活化在rtTLR5S存在或同时表达时更为显著。因此，鞭毛蛋白反应发生在鱼类宿主对细菌感染的防御中：(a)鞭毛蛋白首先通过膜TLR5诱导NF-κB的基础活化，促进可溶性TLR5和最小急性期蛋白的产生；(b)可诱导的可溶性TLR5以正反馈的方式放大膜TLR5介导的细胞反应。
Rainbow trout (Onchorhynchus mikiss) possess two genes encoding putative leucine-rich repeat (LRR)-containing proteins similar to human TLR5. Molecular cloning of these two LRR proteins suggested the presence of a TLR5-like membrane form (rtTLR5M) and a soluble form (rtTLR5S). Here we elucidated the primary structures and the unique combinational functions of these fish versions of TLR5. The LRR regions of rtTLR5S and rtTLR5M exhibited 81% homology and relatively high (35.6 and 33.7%) homology to the extracellular domains of human TLR5 (huTLR5). Thus, two distinct genes encode the TLR5 orthologs in fish, one of which has a consensus intracellular domain (TIR). In order to test their functions, we constructed fusion proteins with the LRR region of rtTLR5S (S-chimera) or that of rtTLR5M and the TIR of huTLR5 (M-chimera). The S- and M-chimeras expressed in HeLa or CHO cells signaled the presence of Vibrio anguillarum flagellin, resulting in NF-κB activation. rtTLR5M was ubiquitously expressed, whereas rtTLR5S was predominantly expressed in the liver. In the hepatoma cell lines of the rainbow trout RTH-149, stimulation of rtTLR5M with V. anguillarum or its flagellin allowed the up-regulation of rtTLR5S. Flagellin-mediated NF-κB activation was more significant in the presence of or simultaneous expression of rtTLR5S. Therefore, a two-step flagellin response occurred for host defense against bacterial infection in fish: (a) flagellin first induced basal activation of NF-κB via membrane TLR5, facilitating the production of soluble TLR5 and minimal acute phase proteins, and (b) the inducible soluble TLR5 amplifies membrane TLR5-mediated cellular responses in a positive feedback fashion.