Novel roles in human MD-2 of phenylalanines 121 and 126 and tyrosine 131 in activation of Toll-like receptor 4 by endotoxin.
Potent mammalian cell activation by Gram-negative bacterial endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). TLR4 activation requires simultaneous binding of MD-2 to endotoxin (E) and the ectodomain of TLR4. We now describe mutants of recombinant human MD-2 that bind TLR4 and react with E.CD14 but do not support cellular responsiveness to endotoxin. The mutants F121A/K122A MD-2 and Y131A/K132A MD-2 react with E.CD14 only when co-expressed with TLR4. Single mutants K122A and K132A each react with E.CD14 +/- TLR4 and promote TLR4-dependent cell activation by endotoxin suggesting that Phe(121) and Tyr(131) are needed for TLR4-independent transfer of endotoxin from CD14 to MD-2 and also needed for TLR4 activation by bound E.MD-2. The mutant F126A MD-2 reacts as well as wild-type MD-2 with E.CD14 +/- TLR4. E.MD-2(F126A) binds TLR4 with high affinity (K(d) approximately 200 pm) but does not activate TLR4 and instead acts as a potent TLR4 antagonist, inhibiting activation of HEK/TLR4 cells by wild-type E.MD-2. These findings reveal roles of Phe(121) and Tyr(131) in TLR4-independent interactions of human MD-2 with E.CD14 and, together with Phe(126), in activation of TLR4 by bound E.MD-2. These findings strongly suggest that the structural properties of E.MD-2, not E alone, determine agonist or antagonist effects on TLR4.
Teghanemt
Athmane
A
Department of Internal Medicine and The Inflammation Program, Iowa City, Iowa 52242.
Re
Fabio
F
Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163.
Prohinar
Polonca
P
Department of Internal Medicine and The Inflammation Program, Iowa City, Iowa 52242.
Widstrom
Richard
R
Department of Internal Medicine and The Inflammation Program, Iowa City, Iowa 52242.
Gioannini
Theresa L
TL
Department of Internal Medicine and The Inflammation Program, Iowa City, Iowa 52242; Veterans' Administration Medical Center, Iowa City, Iowa 52246; Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242.
Weiss
Jerrold P
JP
Department of Internal Medicine and The Inflammation Program, Iowa City, Iowa 52242; Department of Microbiology, Iowa City, Iowa 52242. Electronic address: jerrold-weiss@uiowa.edu.
D002460:Cell Line; D003470:Culture Media; D004731:Endotoxins / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D051218:Lymphocyte Antigen 96 / Q000737:chemistry* / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D012995:Solubility / Q000187:drug effects; D013329:Structure-Activity Relationship; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D014316:Tritium; D014443:Tyrosine / Q000378:metabolism*
D003470:Culture Media; D004731:Endotoxins; C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D050505:Mutant Proteins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C023023:lipid-linked oligosaccharides; D014316:Tritium; D014443:Tyrosine; D010649:Phenylalanine