Viriyakosol Suganya, Tobias Peter S, Kirkland Theo N
Veterans Affairs Medical Research Foundation and Department of Pathology and Medicine, University of California-San Diego, San Diego, California 92161, USA. sviriyakucsd.edu
J Biol Chem. 2006 Apr 28;281(17):11955-64. doi: 10.1074/jbc.M511627200. Epub 2006 Feb 8.
Toll-like receptor 4 and MD-2 form a receptor for lipopolysaccharide (LPS), a major constituent of Gram-negative bacteria. MD-2 is a 20-25-kDa extracellular glycoprotein that binds to Tolllike receptor 4 (TLR4) and LPS and is a critical part of the LPS receptor. Here we have shown that the level of MD-2 expression regulates TLR4 activation by LPS. Using site-directed mutagenesis, we have found that glycosylation has no effect on MD-2 function as a membrane receptor for LPS. We used alanine-scanning mutagenesis to identify regions of human MD-2 that are important for TLR4 and LPS binding. We found that mutation in the N-terminal 46 amino acids of MD-2 did not substantially diminish LPS activation of Chinese hamster ovary (CHO) cells co-transfected with TLR4 and mutant MD-2. The residues 46-50 were important for LPS activation but not LPS binding. The residues 79-83, 121-124, and 125-129 are identified as important in LPS activation but not surface expression of membrane MD-2. The function of soluble MD-2 is somewhat more sensitive to mutation than membrane MD-2. Our results suggest that the 46-50 and 127-131 regions of soluble MD-2 bind to TLR4. The region 79-120 is not involved in LPS binding but affects monomerization of soluble MD-2 as well as TLR4 binding. We define the LPS binding region of monomeric soluble MD-2 as a cluster of basic residues 125-131. Studies on both membrane and soluble MD-2 suggest that domains of MD-2 for TLR4 and LPS binding are separate as well as overlapping. By mapping these regions on a three-dimensional model, we show the likely binding regions of MD-2 to TLR4 and LPS.
Toll样受体4（TLR4）与MD-2形成了脂多糖（LPS）的受体，LPS是革兰氏阴性菌的主要成分。MD-2是一种20 - 25 kDa的细胞外糖蛋白，它与Toll样受体4（TLR4）及LPS结合，是LPS受体的关键部分。在此我们表明，MD-2的表达水平调节LPS对TLR4的激活作用。通过定点诱变，我们发现糖基化对MD-2作为LPS膜受体的功能没有影响。我们使用丙氨酸扫描诱变来确定人MD-2中对TLR4和LPS结合重要的区域。我们发现，MD-2 N端46个氨基酸的突变并没有显著减弱与TLR4和突变型MD-2共转染的中国仓鼠卵巢（CHO）细胞的LPS激活作用。46 - 50位残基对LPS激活很重要，但对LPS结合不重要。79 - 83位、121 - 124位和125 - 129位残基被确定对LPS激活很重要，但对膜MD-2的表面表达不重要。可溶性MD-2的功能对突变比膜MD-2更敏感。我们的结果表明，可溶性MD-2的46 - 50位和127 - 131位区域与TLR4结合。79 - 120位区域不参与LPS结合，但影响可溶性MD-2的单体化以及与TLR4的结合。我们将单体可溶性MD-2的LPS结合区域定义为125 - 131位的碱性残基簇。对膜MD-2和可溶性MD-2的研究表明，MD-2与TLR4和LPS结合的结构域是分开的但也有重叠。通过在三维模型上定位这些区域，我们展示了MD-2与TLR4和LPS可能的结合区域。
