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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Abstract
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.