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 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.
Prohinar
Polonca
P
Department of Internal Medicine, Roy A and Lucille J Carver College of Medicine, University of Iowa, Iowa City, IA 52240, USA.
Rallabhandi
Prasad
P
Weiss
Jerrold P
JP
Gioannini
Theresa L
TL
D019943:Amino Acid Substitution / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D004306:Dose-Response Relationship, Immunologic; D004731:Endotoxins / Q000378:metabolism / Q000494:pharmacology; D014644:Genetic Variation / Q000276:immunology; D006239:Haplotypes; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism / Q000494:pharmacology; D051218:Lymphocyte Antigen 96 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D020125:Mutation, Missense / Q000276:immunology; D011110:Polymorphism, Genetic / Q000276:immunology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D017434:Protein Structure, Tertiary / Q000235:genetics; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D004731:Endotoxins; C495377:LY96 protein, human; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C023023:lipid-linked oligosaccharides