The Function analysis of MD-2 molecule about LPS recognition

MD-2分子对LPS识别的功能分析

基本信息

批准号：
13670460
负责人：
AKASHI Sachiko
金额：
$ 2.62万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670460/
关键词：
MD-2 TLR4 LPS CD14 LipdIVa E5531 RP105 MD-1

项目摘要

Lipopolysaccharide (LPS), a membrane constituent of Gram-negative bacteria, is the best-studied ligand that potently activates the immune system and induces endotoxin shock. Recently we discovered MD-2 molecule, which is associated with TLR4 extracellular domain, and we showed that TLR4/MD-2 complex is indispensable for LPS recognition and response, but it is not clear how TLR4/MD-2 recognizes LPS. Firstly to address the role played by MD-2 in vivo, we generated MD-2-/- mice. B cells, Macrophages, and Dendritic cells of MD-2-/- mice did not respond to LPS. And We showed that MD-2-/- mice did survive endotoxic shock but were susceptible to Salmonella typhimurium infection. We found that in MD-2-/- embryonic fibroblasts, TLR4 was not able to reach the plasma membrane and predominantly resided in the Golgi apparatus, whereas TLR4 was distributed at the leading edge surface of cells in wild-type embryonic fibroblasts. Next we showed that MD-2 directly regulates LPS recognition by TLR4. To address the issue, we took advantage of species-specific pharmacology of lipidIVa, an analogue of lipidA. LipidIVa acted agonistically on mouse(m)TLR4/MD-2 but not on human(h)TLR4/MD-2. LipidIVa antagonized the agonistic effect of lipidA on hTLR4/MD-2. We examined the chimeric complex consisting of mTLR4 and hMD-2 to ask whether species specificity is coinferred by TLR4 or MD-2. hMD-2 conferred on mTLR4 responsiveness to lipidA but not to lipidIVa. Moreover, lipidIVa acted as a lipidA antagonist on mTLR4 that is associated with hMD-2. Collectively, MD-2 directly influences the fine specificity of TLR4.

脂多糖 (LPS) 是革兰氏阴性细菌的膜成分，是研究最深入的配体，可有效激活免疫系统并诱导内毒素休克。最近我们发现了与TLR4胞外结构域相关的MD-2分子，并表明TLR4/MD-2复合物对于LPS识别和反应是不可缺少的，但目前还不清楚TLR4/MD-2如何识别LPS。首先为了解决 MD-2 在体内发挥的作用，我们生成了 MD-2-/- 小鼠。 MD-2-/-小鼠的B细胞、巨噬细胞和树突状细胞对LPS没有反应。我们还表明，MD-2-/- 小鼠确实能够幸存内毒素休克，但易受鼠伤寒沙门氏菌感染。我们发现在MD-2-/-胚胎成纤维细胞中，TLR4无法到达质膜并主要驻留在高尔基体中，而在野生型胚胎成纤维细胞中TLR4分布在细胞的前缘表面。接下来我们证明MD-2直接调节TLR4对LPS的识别。为了解决这个问题，我们利用了脂质IVa（脂质A的类似物）的物种特异性药理学。 LipidIVa 对小鼠 (m)TLR4/MD-2 具有激动作用，但对人 (h)TLR4/MD-2 没有激动作用。 LipidIVa拮抗lipidA对hTLR4/MD-2的激动作用。我们检查了由 mTLR4 和 hMD-2 组成的嵌合复合物，以探究物种特异性是否由 TLR4 或 MD-2 共同推断。 hMD-2 赋予 mTLR4 对脂质 A 的反应性，但对脂质 IVa 没有反应性。此外，lipidIVa充当与hMD-2相关的mTLR4的lipidA拮抗剂。总的来说，MD-2 直接影响 TLR4 的精细特异性。