N-linked glycosylations at Asn26 and Asn114 of human MD-2 are required for toll-like receptor 4-mediated activation of NF-κB by lipopolysaccharide
N-linked glycosylations at Asn26 and Asn114 of human MD-2 are required for toll-like receptor 4-mediated activation of NF-κB by lipopolysaccharide
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DOI：
10.4049/jimmunol.167.6.3354
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发表时间：
2001-09-15
影响因子：
4.4
通讯作者：
Tanamoto, K
中科院分区：
文献类型：
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作者：
Ohnishi, T;Muroi, M;Tanamoto, K
MD-2在物理上与toll样受体4 （TLR4）相关，是TLR4介导的LPS信号传递所必需的。Western blotting分析显示存在三种具有不同电泳迁移率的人(h)MD-2。表达hMD-2的细胞提取液经iv -糖苷酶处理后，只检测到迁移速度最快的单一形式。MD-2的两个潜在糖基化位点（Asn（26）和Asn(114)）中的任何一个发生突变都会导致最慢迁移形式的消失，在携带Asn（26）和Asn（114）突变的hMD-2中只检测到最快迁移形式。尽管这些突变体在细胞表面表达，并保持其与人TLR4相关的能力，但这些突变或tunicamycin处理大大损害了MD-2通过LPS补充TLR4介导的NF-kappaB活化的能力。LPS与表达CD14、TLR4和MD-2的细胞结合不受这些突变的影响。这些观察结果表明，hMD-2在Asn（26）和Asn（114）处经历了n链糖基化，这些糖基化对于tlr4介导的LPS信号转导至关重要。
MD-2 is physically associated with Toll-like receptor 4 (TLR4) and is required for TLR4-mediated LPS signaling. Western blotting analysis revealed the presence of three forms of human (h)MD-2 with different electrophoretic mobilities. After IV-glycosidase treatment of the cellular extract prepared from cells expressing hMD-2, only a single form with the fastest mobility was detected. Mutation of either one of two potential glycosylation sites (Asn(26) and Asn(114)) of MD-2 resulted in the disappearance of the slowest mobility form, and only the fastest form was detected in hMD-2 carrying mutations at both Asn(26) and Asn(114). Although these mutants were expressed on the cell surface and maintained its ability to associate with human TLR4, these mutations or tunicamycin treatment substantially impaired the ability of MD-2 to complement TLR4-mediated activation of NF-kappaB by LPS. LPS binding to cells expressing CD14, TLR4, and MD-2 was unaffected by these mutations. These observations demonstrate that hMD-2 undergoes N-linked glycosylation at Asn(26) and Asn(114), and that these glycosylations are crucial for TLR4-mediated signal transduction of LPS.