Interaction of soluble form of recombinant extracellular TLR4 domain with MD-2 enables lipopolysaccharide binding and attenuates TLR4-mediated signaling.
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.
Hyakushima
Naoki
N
Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo, Japan.
Mitsuzawa
Hiroaki
H
Nishitani
Chiaki
C
Sano
Hitomi
H
Kuronuma
Koji
K
Konishi
Masanori
M
Himi
Tetsuo
T
Miyake
Kensuke
K
Kuroki
Yoshio
Y
D000595:Amino Acid Sequence; D000954:Antigens, Surface / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D045604:Extracellular Fluid / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000037:antagonists & inhibitors / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D017434:Protein Structure, Tertiary / Q000235:genetics; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D012995:Solubility; D020349:Surface Plasmon Resonance; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D015533:Transcriptional Activation / Q000276:immunology; D014162:Transfection
D000954:Antigens, Surface; D016209:Interleukin-8; C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors