The functional and structural properties of MD-2 required for lipopolysaccharide binding are absent in MD-1
The functional and structural properties of MD-2 required for lipopolysaccharide binding are absent in MD-1
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DOI：
10.4049/jimmunol.174.1.340
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发表时间：
2005-01-01
影响因子：
4.4
通讯作者：
Kimoto, M
中科院分区：
文献类型：
--
作者：
Tsuneyoshi, N;Fukudome, K;Kimoto, M
MD-1和MD-2是与跨膜蛋白复合存在于细胞表面的分泌性糖蛋白。MD-1由辐射防护105（RP 105）锚定，MD-2与TLR 4相关。体内研究表明，MD-1和MD-2在对LPS的应答中起作用。虽然已经观察到MD-2与LPS的直接结合功能，但MD-1的生理功能仍然未知。在这项研究中，我们比较了MD-1和MD-2的脂蛋白结合功能。对于用TLR 4/MD-2转染的细胞，检测LPS与细胞表面复合物的结合。相反，未观察到RP 105/MD-1转染细胞的结合。当rMD-2蛋白在大肠杆菌中表达时，它在含有LPS的复合物中被纯化。相反，MD-1的制剂不含LPS。当在缺乏lpxM基因的突变株中制备rMD-2蛋白时，LIPS结合消失。因此，在这些条件下，MD-2识别LPS需要连接到由LpxM添加的（R）-3-羟基肉豆蔻酰基链的肉豆蔻酰基仲链。MD-2中由碱性和疏水性残基组成的两亲性簇被认为是LPS结合位点。我们特别关注两个Phe残基（119和121），它们可以与脂肪酸结合。在Phe（191）或Phe（121）处的突变强烈降低结合活性，并且在这些残基处的双突变阻止任何结合的发生。Phe残基存在于MD-2中而不存在于MD-I中。因此，RP 105/MD-1的LPS识别机制不同于TLR 4/MD-2的LPS识别机制。
MD-1 and MD-2 are secretory glycoproteins that exist on the cell surface in complexes with transmembrane proteins. MD-1 is anchored by radioprotective 105 (RP105), and MD-2 is associated with TLR4. In vivo studies revealed that MD-I and MD-2 have roles in responses to LPS. Although the direct binding function of MD-2 to LPS has been observed, the physiological function of MD-1 remains unknown. In this study, we compared the LPS-binding functions of MD-1 and MD-2. LPS binding to cell surface complexes was detected for cells transfected with TLR4/MD-2. In contrast, binding was not observed for RP105/MD-1-transfected cells. When rMD-2 protein was expressed in Escherichia coli, it was purified in complexes containing LPS. In contrast, preparations of MD-1 did not contain LPS. When rMD-2 protein was prepared in a mutant strain lacking the lpxM gene, LIPS binding disappeared. Therefore, the secondary myristoyl chain attached to the (R)-3-hydroxymyristoyl chain added by LpxM is required for LPS recognition by MD-2, under these conditions. An amphipathic cluster composed of basic and hydrophobic residues in MD-2 has been suggested to be the LPS-binding site. We specifically focused on two Phe residues (119 and 121), which can associate with fatty acids. A mutation at Phe(191) or Phe(121) strongly reduced binding activity, and a double mutation at these residues prevented any binding from occurring. The Phe residues are present in MD-2 and absent in MD-I. Therefore, the LPS recognition mechanism by RP105/MD-1 is distinct from that of TLR4/MD-2.