Interaction of Soluble Form of Recombinant Extracellular TLR4 Domain with MD-2 Enables Lipopolysaccharide Binding and Attenuates TLR4-Mediated Signaling

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 Glu24-Lys631. 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 KD = 6.29 × 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-κB 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 Glu24-Lys631 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.

Mannan-binding lectin directly interacts with Toll-like receptor 4 and suppresses lipopolysaccharide-induced inflammatory cytokine secretion from THP-1 cells

“…43 sTLR4 protein (30 mg/ml), a molecule that may also function as an inhibitor of MBL, was pre-incubated with biotinylated MBL (10 mg/ml) at 37 uC for 30 min. HSA was used as a negative control.…”

Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2

“…sTLR4 and MD-2 cDNAs were described previously (37). sTLR4-His 6 was subcloned into pcDNA3.1(ϩ) (Invitrogen).…”

“…MD-2 binds to the extracellular TLR4 domain, and a complex of MD-2 and TLR4, but not TLR4 alone, can interact with LPS (37). POPG binds to MD-2 with high affinity.…”

Recombinant Soluble Forms of Extracellular TLR4 Domain and MD-2 Inhibit Lipopolysaccharide Binding on Cell Surface and Dampen Lipopolysaccharide-Induced Pulmonary Inflammation in Mice

“…sTLR4 consisting of the putative extracellular domain (Met 1 -Lys 631 ) and a 6ϫHis tag at the C-terminal end, sTLR2 consisting of the putative extracellular domain (Met 1 -Arg 587 ) and a 6ϫHis tag at the C-terminal end, and sMD-2 containing V5 tag and 6ϫHis tag were generated by the baculovirus-insect cell expression system, and the recombinant proteins were purified, as described previously (9,14). sTLR4 and sTLR2 start at Glu 24 and Glu 21 , respectively.…”

“…However, LPS beads coprecipitate sTLR4 and MD-2 when both proteins are coincubated, suggesting that the extracellular TLR4 domain-MD-2 complex, but not TLR4 alone, is capable of binding LPS. Addition of sTLR4 and sMD-2 attenuates LPS-elicited NF-B activation and IL-8 release in TLR4-expressing cells (9). One study has reported a 20-kDa naturally occurring soluble form of mouse TLR4 that is expressed by alternatively spliced TLR4 mRNA (10).…”