Expression of Functional D299G.T399I Polymorphic Variant of TLR4 Depends More on Coexpression of MD-2 Than Does Wild-Type TLR4

Two missense variants (D299G and T3991) 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 TLR4(ecd) demonstrated high affinity (K-d similar to 200 pM) interaction of endotoxin.CD14 with MD-2.TLR4(ecd) and endotoxin.MD-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. The Journal of Immunology, 2010,184: 4362-4367.