Isolation of monomeric and dimeric secreted MD-2. Endotoxin.sCD14 and Toll-like receptor 4 ectodomain selectively react with the monomeric form of secreted MD-2.
Potent cell activation by endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). MD-2 plays an essential role by bridging endotoxin (E) recognition initiated by lipopolysaccharide-binding protein and CD14 to TLR4 activation by presenting endotoxin as a monomeric E.MD-2 complex that directly and potently activates TLR4. Secreted MD-2 (sMD-2) exists as a mixture of monomers and multimers. Published data suggest that only MD-2 monomer can interact with endotoxin and TLR4 and support cell activation, but the apparent instability of MD-2 has thwarted efforts to more fully separate and characterize the individual species of sMD-2. We have taken advantage of the much greater stability of sMD-2 in insect culture medium to fully separate sMD-2 monomer from dimer by gel sieving chromatography. At low nanomolar concentrations, the sMD-2 monomer, but not dimer, reacted with a monomeric complex of E.sCD14 to form monomeric E.MD-2 and activate HEK293/TLR4 cells. The monomer, but not dimer, also reacted with the ectodomain of TLR4 with an affinity comparable with the picomolar affinity of E.MD-2. These findings demonstrate directly that the monomeric form of sMD-2 is the active species both for reaction with E.CD14 and TLR4, as needed for potent endotoxin-induced TLR4 activation.
Inflammation Program, Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.
Athmane
A
Teghanemt
Richard L
RL
Widstrom
Theresa L
TL
Gioannini
Jerrold P
JP
Weiss
D000180:Acrylic Resins; D000818:Animals; D002460:Cell Line; D003470:Culture Media; D017077:Culture Media, Conditioned; D019281:Dimerization; D004731:Endotoxins / Q000378:metabolism*; D006801:Humans; D007313:Insecta / Q000166:cytology; D018950:Lipopolysaccharide Receptors / Q000378:metabolism*; D051218:Lymphocyte Antigen 96 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009345:Neisseria meningitidis; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D051197:Toll-Like Receptor 4 / Q000378:metabolism*
D000180:Acrylic Resins; D003470:Culture Media; D017077:Culture Media, Conditioned; D004731:Endotoxins; D018950:Lipopolysaccharide Receptors; D051218:Lymphocyte Antigen 96; D011994:Recombinant Proteins; D051197:Toll-Like Receptor 4; C023049:sephacryl S 200