Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2

Abstract

Toll-like receptors (TLRs) are innate recognition molecules for microbial products, but their direct interactions with corresponding ligands remain unclarified. LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4. Although TLR4-MD-2 recognizes LPS, little is known about the physical interaction between LPS and TLR4-MD-2. Here, we demonstrate cell surface LPS–TLR4-MD-2 complexes. CD14 greatly enhances the formation of LPS–TLR4-MD-2 complexes, but is not coprecipitated with LPS–TLR4-MD-2 complexes, suggesting a role for CD14 in LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. A tentative dissociation constant (Kd) for LPS–TLR4-MD-2 complexes was ∼3 nM, which is ∼10–20 times lower than the reported Kd for LPS–MD-2 or LPS–CD14. The presence of detergent disrupts LPS interaction with CD14 but not with TLR4-MD-2. E5531, a lipid A antagonist developed for therapeutic intervention of endotoxin shock, blocks LPS interaction with TLR4-MD-2 at a concentration 100 times lower than that required for blocking LPS interaction with CD14. These results reveal direct LPS interaction with cell surface TLR4-MD-2 that is distinct from that with MD-2 or CD14.

Mice expressing high levels of soluble CD14 retain LPS in the circulation and are resistant to LPS‐induced lethality

“…Subsequent events involve interaction of CD14 with two other molecules, TLR4 and MD-2 [17]. All three molecules are indispensable for LPS responses and are believed to form a stable complex with one another [18]. Therefore, it was important to see whether hCD14 is capable of complementing other components of the LPS-recognition cluster in mice.…”

Cathelicidin Antimicrobial Peptides Block Dendritic Cell TLR4 Activation and Allergic Contact Sensitization

“…Cathelicidins are known to interact with bacterial membranes, and TLR4 function requires the membrane-dependent association of a receptor complex including, TLR4, CD14 and MD2 (23). We therefore investigated whether the ability of cathelicidin to block TLR4 activation involved alterations in the DC membrane that might interfere with the assembly of this receptor complex.…”

Mast cell‐mediated immune responses through IgE antibody and Toll‐like receptor 4 by malarial peroxiredoxin

“…Ba/F3 cells stably expressing mTLR4/mMD-2/CD14/NFjB/GFP or NFjB/GFP were kindly provided by Dr. S. Takamura-Akashi, University of Tokyo [31]. The mouse cDNA encoding TLR4, MD-2, and CD14 were cloned, and Ba/F3 cells were transfected with expression vectors of pMX-puromycin encoding mTLR4, pEFBOS encoding mMD-2, and pcDNA3 encoding mCD14.…”

“…Ba/F3 cells stably expressing mTLR2 or mTLR4/mMD-2 were established by transduction with the retroviral vector pMXpuromycin encoding these molecules, which were tagged with the flag epitope followed by His6 at the C terminus, as previously reported [31]. Cells (1 Â 10 8 in 10 mL medium) were incubated with PbAPrx (20 lg/mL) at 37 C for 2 h. After washing, cells were lysed on ice for 30 min in a lysis buffer containing 150 mM NaCl, 50 mM Tris/HCl (pH 7.6), 2 mM EDTA, 10 lg/mL aprotinin, 10 lg/mL leupeptin, 1 mM PMSF, and 1% Triton X-100.…”

“…Samples were subjected to SDS-PAGE and immunoblotted as described above [32]. PVDF membranes were probed with an anti-rabbit PbAPrx antibody to detect malarial Prx, or anti-mouse TLR4 or TLR2 antibody to detect TLR4/MD-2 or TLR2 [31]. Binding was detected using AP-conjugated anti-mouse IgG or anti-rabbit IgG antibodies (2:1000 dilution; KPL), and bands were visualized using NTB and BCIP reagents.…”