Specific high affinity interactions of monomeric endotoxin.protein complexes with Toll-like receptor 4 ectodomain.
Potent Toll-like receptor 4 (TLR4) activation by endotoxin has been intensely studied, but the molecular requirements for endotoxin interaction with TLR4 are still incompletely defined. Ligand-receptor interactions involving endotoxin and TLR4 were characterized using monomeric endotoxin.protein complexes of high specific radioactivity. The binding of endotoxin.MD-2 to the TLR4 ectodomain (TLR4ECD) and transfer of endotoxin from CD14 to MD-2/TLR4ECD were demonstrated using HEK293T-conditioned medium containing TLR4ECD+/-MD-2. These interactions are specific, of high affinity (KD<300 pm), and consistent with the molecular requirements for potent cell activation by endotoxin. Both reactions result in the formation of a Mr approximately 190,000 complex composed of endotoxin, MD-2, and TLR4ECD. CD14 facilitates transfer of endotoxin to MD-2 (TLR4) but is not a stable component of the endotoxin.MD-2/TLR4 complex. The ability to assay specific high affinity interactions of monomeric endotoxin.protein complexes with TLR4ECD should allow better definition of the structural requirements for endotoxin-induced TLR4 activation.
Prohinar
Polonca
P
Inflammation Program, Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, and Veterans Affairs Medical Center, Iowa City, Iowa 52242, USA.
Re
Fabio
F
Widstrom
Richard
R
Zhang
DeSheng
D
Teghanemt
Athmane
A
Weiss
Jerrold P
JP
Gioannini
Theresa L
TL
D000215:Acylation; D002460:Cell Line; D004731:Endotoxins / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology; D008024:Ligands; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D012995:Solubility; D051197:Toll-Like Receptor 4 / Q000737:chemistry* / Q000378:metabolism*; D014316:Tritium
D004731:Endotoxins; D008024:Ligands; D018950:Lipopolysaccharide Receptors; D051218:Lymphocyte Antigen 96; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D014316:Tritium