MD-2 and TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor
MD-2 and TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor
复制标题
DOI：
10.1074/jbc.m109910200
复制
发表时间：
2002-01-18
影响因子：
4.8
通讯作者：
Ulevitch, RJ
中科院分区：
文献类型：
--
作者：
Correia, JD;Ulevitch, RJ
脂多糖（LPS）受体是一种多蛋白复合物，由至少三种蛋白质组成：CD 14、TLR 4和MD-2。由于这些蛋白质中的每一个都是糖基化的，我们已经研究了MD-2和TLR 4的N-连接的碳水化合物的功能作用。我们证明MD-2在Asn（26）和Asn（114）位置含有2个N-糖基化位点，而人TLR 4的氨基末端胞外域含有9个N-连接的糖基化位点。定点突变研究表明，MD-2的细胞表面表达不依赖于任何一个N-连接位点的存在，而相反，携带Asn（526）或Asn（575）取代的TLR 4突变体不能被转运到细胞表面。在交联测定中使用Re 595 LPS的UV活化衍生物（ASD-Re 595 LPS），我们证明了MD-2和TLR 4碳水化合物在LPS与LPS受体交联中的关键作用。还在瞬时转染的HeLa细胞中评价了各种糖基化突变体支持细胞活化的能力。MD-2的双突变体不能支持LPS诱导的白细胞介素-8（IL-8）启动子驱动的荧光素酶报告基因的激活以诱导IL-8分泌或激活氨基末端c-Jun激酶（JNK）。在缺乏三个或更多个N-连接糖基化位点的TLR 4突变体中观察到类似的结果。令人惊讶的是，由MD-2和TLR 4的Asn突变体的表达引起的活化降低可以通过与CD 14共表达而部分逆转。这表明LPS受体的功能完整性取决于至少三种蛋白质CD 14、MD-2和TLR 4的表面表达，并且MD-2和TLR 4的N-连接位点在维持该受体的功能完整性中是必不可少的。
The lipopolysaccharide (LPS) receptor is a multi-protein complex that consists of at least three proteins, CD14, TLR4, and MD-2. Because each of these proteins is glycosylated, we have examined the functional role of N-linked carbohydrates of both MD-2 and TLR4. We demonstrate that MD-2 contains 2 N-glycosylated sites at positions Asn(26) and Asn(114), whereas the amino-terminal ectodomain of human TLR4 contains 9 N-linked glycosylation sites. Site-directed mutagenesis studies showed that cell surface expression of MD-2 did not depend on the presence of either N-linked site, whereas in contrast, TLR4 mutants carrying substitutions in Asn(526) or Asn(575) failed to be transported to the cell surface. Using a UV-activated derivative of Re595 LPS (ASD-Re595 LPS) in cross-linking assays, we demonstrated a critical role of MD-2 and TLR4 carbohydrates in LPS cross-linking to the LPS receptor. The ability of the various glycosylation mutants to support cell activation was also evaluated in transiently transfected HeLa cells. The double mutant of MD-2 failed to support LPS-induced activation of an interleukin-8 (IL-8) promoter-driven luciferase reporter to induce IL-8 secretion or to activate amino-terminal c-Jun kinase (JNK). Similar results were observed with TLR4 mutants lacking three or more N-linked glycosylation sites. Surprisingly, the reduction in activation resulting from expression of the Asn mutants of MD-2 and TLR4 can be partially reversed by co-expression with CD14. This suggests that the functional integrity of the LPS receptor depends both on the surface expression of at least three proteins, CD14, MD-2, and TLR4, and that N-linked sites of both MD-2 and TLR4 are essential in maintaining the functional integrity of this receptor.