Specific high affinity interactions of monomeric endotoxin•protein complexes with Toll-like receptor 4 ectodomain
Specific high affinity interactions of monomeric endotoxin•protein complexes with Toll-like receptor 4 ectodomain
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DOI：
10.1074/jbc.m609400200
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发表时间：
2007-01-12
影响因子：
4.8
通讯作者：
Gioannini, Theresa L.
中科院分区：
文献类型：
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作者：
Prohinar, Polonca;Re, Fabio;Gioannini, Theresa L.
内毒素对 Toll 样受体 4 (TLR4) 的有效激活已得到深入研究，但内毒素与 TLR4 相互作用的分子要求仍不完全确定。使用高特异性放射性的单体内毒素(.)蛋白复合物来表征涉及内毒素和TLR4的配体-受体相互作用。使用含有 TLR4(ECD)+/- MD-2 的 HEK293T 条件培养基证明了内毒素(.)MD-2 与 TLR4 胞外域 (TLR4(ECD)) 的结合以及内毒素从 CD14 转移到 MD-2/TLR4(ECD)。这些相互作用是特异性的，具有高亲和力（K-D < 300 pM），并且符合内毒素有效细胞激活的分子要求。两个反应都会形成类似于 190,000 个由内毒素、MD-2 和 TLR4(ECD) 组成的复合物的 M-r。 CD14 促进内毒素向 MD-2(TLR4) 的转移，但不是内毒素(.)MD-2/TLR4 复合物的稳定成分。检测单体内毒素（.）蛋白复合物与 TLR4（ECD）的特异性高亲和力相互作用的能力应该可以更好地定义内毒素诱导的 TLR4 激活的结构要求。
Potent Toll-like receptor 4 (TLR4) activation by endotoxin has been intensely studied, but the molecular requirements for endotoxin interaction with TLR4 are still incompletely defined. Ligand-receptor interactions involving endotoxin and TLR4 were characterized using monomeric endotoxin(.)protein complexes of high specific radioactivity. The binding of endotoxin(.)MD-2 to the TLR4 ectodomain (TLR4(ECD)) and transfer of endotoxin from CD14 to MD-2/TLR4(ECD) were demonstrated using HEK293T-conditioned medium containing TLR4(ECD)+/- MD-2. These interactions are specific, of high affinity (K-D < 300 pM), and consistent with the molecular requirements for potent cell activation by endotoxin. Both reactions result in the formation of a M-r similar to 190,000 complex composed of endotoxin, MD-2, and TLR4(ECD). CD14 facilitates transfer of endotoxin to MD-2(TLR4) but is not a stable component of the endotoxin(.)MD-2/TLR4 complex. The ability to assay specific high affinity interactions of monomeric endotoxin(.)protein complexes with TLR4(ECD) should allow better definition of the structural requirements for endotoxin-induced TLR4 activation.