Department of Internal Medicine, Roy A and Lucille J Carver College of Medicine, University of Iowa, Iowa City, IA 52240, USA.
J Immunol. 2010 Apr 15;184(8):4362-7. doi: 10.4049/jimmunol.0903142. Epub 2010 Mar 8.
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 endotoxinCD14 and endotoxinMD-2 complexes of high specific radioactivity to measure: 1) interaction of recombinant MD-2TLR4 with endotoxinCD14 and TLR4 with endotoxinMD-2; 2) expression of functional MD-2TLR4 and TLR4; and 3) MD-2TLR4 and TLR4-dependent cellular endotoxin responsiveness. Both wild-type and D299G.T399I TLR4(ecd) demonstrated high affinity (K(d) approximately 200 pM) interaction of endotoxinCD14 with MD-2TLR4(ecd) and endotoxinMD-2 with TLR4(ecd). 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.
两种 TLR4 的错义突变（D299G 和 T399I）在欧洲血统个体中紧密连锁，并且在许多测试系统中导致对内毒素的反应性降低。TLR4 外显子（ecd）内的这些变化如何影响 TLR4 功能尚不清楚。对于野生型和 D299G.T399I TLR4，我们使用高比活度的内毒素 CD14 和内毒素 MD-2 复合物来测量：1）重组 MD-2TLR4 与内毒素 CD14 和 TLR4 与内毒素 MD-2 的相互作用；2）功能性 MD-2TLR4 和 TLR4 的表达；3）MD-2TLR4 和 TLR4 依赖性细胞内毒素反应性。野生型和 D299G.T399I TLR4（ecd）均表现出内毒素 CD14 与 MD-2TLR4（ecd）和内毒素 MD-2 与 TLR4（ecd）的高亲和力（K(d)约 200 pM）相互作用。然而，当 D299G.T399I TLR4 与 MD-2 共表达时，功能性 TLR4 的水平降低了高达 2 倍，而当不表达 MD-2 时则降低了 10 倍以上，这与细胞内毒素反应性的差异相平行。D299G.T399I 单倍型对无 MD-2 表达的功能性 TLR4 表达的巨大影响表明，表达无 MD-2 的 TLR4 的细胞受这些多态性的影响最大。
