Expression of functional D299G.T399I polymorphic variant of TLR4 depends more on coexpression of MD-2 than does wild-type TLR4.
Expression of functional D299G.T399I polymorphic variant of TLR4 depends more on coexpression of MD-2 than does wild-type TLR4.
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DOI：
10.4049/jimmunol.0903142
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发表时间：
2010-04-15
期刊：
影响因子：
--
通讯作者：
Gioannini TL
中科院分区：
文献类型：
--
作者：
Prohinar P;Rallabhandi P;Weiss JP;Gioannini TL
TLR 4的两种错义变体（D299 G和T399 I）在欧洲血统的个体中共分离，并且在许多测试系统中导致对内毒素的反应性降低。TLR 4胞外域（ecd）内的这些变化如何影响TLR 4功能尚不清楚。对于野生型和D299G.T399I TLR 4，我们使用高比放射性的内毒素·CD 14和内毒素·MD-2复合物来测量：1）重组MD-2·TLR 4与内毒素·CD 14和TLR 4与内毒素·MD-2的相互作用; 2）功能性MD-2·TLR 4和TLR 4的表达;和3）MD-2·TLR 4和TLR 4依赖性细胞内毒素应答性。野生型和D299G.T399I TLR 4 ecd均显示内毒素·CD 14与MD-2·TLR 4 ecd和内毒素·MD-2与TLR 4 ecd的高亲和力（Kd ~ 200 pM）相互作用。然而，当D299G.T399I TLR 4与MD-2共表达时，功能性TLR 4的水平降低高达2倍，而当在没有MD-2的情况下表达时，功能性TLR 4的水平降低>10倍，这与细胞内毒素反应性的差异平行。D299G.T399I单倍型对不含MD-2的功能性TLR 4表达的显著影响表明，表达不含MD-2的TLR 4的细胞受这些多态性的影响最大。
Two missense variants (D299G and T399I) of TLR4 are cosegregated in individuals of European descent and, in a number of test systems, result in reduced responsiveness to endotoxin. How these changes within the ectodomain (ecd) of TLR4 affect TLR4 function is unclear. For both wild-type and D299G.T399I TLR4, we used endotoxin·CD14 and endotoxin·MD-2 complexes of high specific radioactivity to measure: 1) interaction of recombinant MD-2·TLR4 with endotoxin·CD14 and TLR4 with endotoxin·MD-2; 2) expression of functional MD-2·TLR4 and TLR4; and 3) MD-2·TLR4 and TLR4-dependent cellular endotoxin responsiveness. Both wild-type and D299G.T399I TLR4ecd demonstrated high affinity (Kd ~ 200 pM) interaction of endotoxin·CD14 with MD-2·TLR4ecd and endotoxin·MD-2 with TLR4ecd. However, levels of functional TLR4 were reduced up to 2-fold when D299G.T399I TLR4 was coexpressed with MD-2 and >10-fold when expressed without MD-2, paralleling differences in cellular endotoxin responsiveness. The dramatic effect of the D299G.T399I haplotype on expression of functional TLR4 without MD-2 suggests that cells expressing TLR4 without MD-2 are most affected by these polymorphisms.
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