Kobayashi Makiko, Saitoh Shin-ichiroh, Tanimura Natsuko, Takahashi Koichiro, Kawasaki Kiyoshi, Nishijima Masahiro, Fujimoto Yukari, Fukase Koichi, Akashi-Takamura Sachiko, Miyake Kensuke
Division of Infectious Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo 108-8639, Japan.
J Immunol. 2006 May 15;176(10):6211-8. doi: 10.4049/jimmunol.176.10.6211.
LPS, a principal membrane component in Gram-negative bacteria, is recognized by a receptor complex consisting of TLR4 and MD-2. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. MD-2 directly interacts with LPS, and the region from Phe(119) to Lys(132) (Arg(132) in mice) has been shown to be important for interaction between LPS and TLR4/MD-2. With mouse MD-2 mutants, we show in this study that Gly(59) was found to be a novel critical amino acid for LPS binding outside the region 119-132. LPS signaling is thought to be triggered by ligand-induced TLR4 clustering, which is also regulated by MD-2. Little is known, however, about a region or an amino acid in the MD-2 molecule that regulates ligand-induced receptor clustering. MD-2 mutants substituting alanine for Phe(126) or Gly(129) impaired LPS-induced TLR4 clustering, but not LPS binding to TLR4/MD-2, demonstrating that ligand-induced receptor clustering is differentially regulated by MD-2 from ligand binding. We further show that dissociation of ligand-induced receptor clustering and of ligand-receptor interaction occurs in a manner dependent on TLR4 signaling and requires endosomal acidification. These results support a principal role for MD-2 in LPS recognition.
脂多糖（LPS）是革兰氏阴性菌的主要膜成分，可被由Toll样受体4（TLR4）和髓样分化蛋白2（MD-2）组成的受体复合物识别。MD-2是一种细胞外分子，与TLR4的细胞外结构域相关联，在LPS识别中起关键作用。MD-2直接与LPS相互作用，并且已证明从苯丙氨酸（Phe）119到赖氨酸（Lys）132（小鼠中为精氨酸（Arg）132）的区域对于LPS与TLR4/MD-2之间的相互作用很重要。通过小鼠MD-2突变体，我们在本研究中表明，甘氨酸（Gly）59被发现是119-132区域之外LPS结合的一个新的关键氨基酸。LPS信号传导被认为是由配体诱导的TLR4聚集触发的，这也受MD-2调节。然而，关于MD-2分子中调节配体诱导的受体聚集的区域或氨基酸知之甚少。用丙氨酸替代苯丙氨酸（Phe）126或甘氨酸（Gly）129的MD-2突变体损害了LPS诱导的TLR4聚集，但不影响LPS与TLR4/MD-2的结合，这表明配体诱导的受体聚集受MD-2调节的方式不同于配体结合。我们进一步表明，配体诱导的受体聚集和解离以及配体-受体相互作用以依赖于TLR4信号传导的方式发生，并且需要内体酸化。这些结果支持MD-2在LPS识别中的主要作用。
