Abstract
TLRs have been implicated in recognition of pathogen-associated molecular patterns. TLR4 is a signaling receptor for LPS, but requires MD-2 to respond efficiently to LPS. The purposes of this study were to examine the interactions of the extracellular TLR4 domain with MD-2 and LPS. We generated soluble forms of rTLR4 (sTLR4) and TLR2 (sTLR2) lacking the putative intracellular and transmembrane domains. sTLR4 consisted of Glu(24)-Lys(631). MD-2 bound to sTLR4, but not to sTLR2 or soluble CD14. BIAcore analysis demonstrated the direct binding of sTLR4 to MD-2 with a dissociation constant of K(D) = 6.29 x 10(-8) M. LPS-conjugated beads precipitated MD-2, but not sTLR4. However, LPS beads coprecipitated sTLR4 and MD-2 when both proteins were coincubated. The addition of sTLR4 to the medium containing the MD-2 protein significantly attenuated LPS-induced NF-kappaB activation and IL-8 secretion in wild-type TLR4-expressing cells. These results indicate that the extracellular TLR4 domain-MD-2 complex is capable of binding LPS, and that the extracellular TLR4 domain consisting of Glu(24)-Lys(631) enables MD-2 binding and LPS recognition to TLR4. In addition, the use of sTLR4 may lead to a new therapeutic strategy for dampening endotoxin-induced inflammation.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Antigens, Surface / genetics
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Antigens, Surface / isolation & purification
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Antigens, Surface / metabolism*
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Antigens, Surface / physiology
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Cell Line
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Extracellular Fluid / metabolism
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Humans
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Interleukin-8 / antagonists & inhibitors
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Interleukin-8 / metabolism
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Lipopolysaccharide Receptors / metabolism
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Lipopolysaccharides / antagonists & inhibitors
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Lipopolysaccharides / metabolism*
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Lipopolysaccharides / pharmacology
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Lymphocyte Antigen 96
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Membrane Glycoproteins / antagonists & inhibitors*
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / metabolism
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Membrane Glycoproteins / physiology*
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Molecular Sequence Data
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NF-kappa B / antagonists & inhibitors
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NF-kappa B / genetics
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NF-kappa B / metabolism
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Protein Binding / genetics
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Protein Binding / immunology
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Protein Structure, Tertiary / genetics
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Receptors, Cell Surface / antagonists & inhibitors*
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Receptors, Cell Surface / genetics
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Receptors, Cell Surface / metabolism
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Receptors, Cell Surface / physiology*
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Recombinant Proteins / metabolism
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Signal Transduction / genetics
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Signal Transduction / immunology*
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Solubility
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Surface Plasmon Resonance
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Toll-Like Receptor 2
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Toll-Like Receptor 4
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Toll-Like Receptors
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Transcriptional Activation / immunology
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Transfection
Substances
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Antigens, Surface
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Interleukin-8
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LY96 protein, human
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Lipopolysaccharide Receptors
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Lipopolysaccharides
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Lymphocyte Antigen 96
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Membrane Glycoproteins
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NF-kappa B
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Receptors, Cell Surface
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Recombinant Proteins
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TLR2 protein, human
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TLR4 protein, human
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Toll-Like Receptor 2
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Toll-Like Receptor 4
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Toll-Like Receptors