Specific High Affinity Interactions of Monomeric Endotoxin·Protein Complexes with Toll-like Receptor 4 Ectodomain

Abstract

Potent Toll-like receptor 4 (TLR4) activation by endotoxin has been intensely studied, but the molecular requirements for endotoxin interaction with TLR4 are still incompletely defined. Ligandreceptor interactions involving endotoxin and TLR4 were characterized using monomeric endotoxin⅐protein complexes of high specific radioactivity. The binding of endotoxin⅐MD-2 to the TLR4 ectodomain (TLR4 ECD ) and transfer of endotoxin from CD14 to MD-2/TLR4 ECD were demonstrated using HEK293T-conditioned medium containing TLR4 ECD ؎ MD-2. These interactions are specific, of high affinity (K D < 300 pM), and consistent with the molecular requirements for potent cell activation by endotoxin. Both reactions result in the formation of a M r ϳ 190,000 complex composed of endotoxin, MD-2, and TLR4 ECD . CD14 facilitates transfer of endotoxin to MD-2 (TLR4) but is not a stable component of the endotoxin⅐MD-2/TLR4 complex. The ability to assay specific high affinity interactions of monomeric endotoxin⅐protein complexes with TLR4 ECD should allow better definition of the structural requirements for endotoxin-induced TLR4 activation.Essential arms of the innate immune system are the Tolllike receptors (TLRs).2 These receptors link recognition of unique microbial molecules to activation of host defense effector systems by rapidly triggering pro-inflammatory responses (1). Potent host responses toward many Gramnegative bacteria (GNB) are mediated by recognition and response to unique glycolipids (lipopoly-or lipooligosaccharides LOS, endotoxin) of the GNB outer membrane by TLR4. TLR4 does not function alone but requires the accessory protein MD-2, which binds non-covalently to the N-terminal ectodomain of TLR4 (2-6). Maximally potent endotoxin-induced cell activation also requires the extracellular lipopolysaccharide-binding protein (LBP) and membrane (m) or soluble (s) extracellular CD14 (4, 7-9). The sequential action of LBP, CD14, secreted or TLR4-associated MD-2, and TLR4 confers the extraordinary sensitivity of mammalian cells to many GNB endotoxins. This ordered action implies differences in endotoxin binding specificity, with LBP having the highest affinity for endotoxin organized at lipid/water interfaces (e.g. purified endotoxin aggregates and endotoxin in the GNB outer membrane), CD14 for LBP-modified endotoxin-rich interfaces, MD-2 for monomeric endotoxin⅐CD14 and TLR4, apparently, for endotoxin presented as a monomeric complex with MD-2 (8). Together, these proteins can convert one GNB (containing ϳ10 6 endotoxin molecules) to 10 6 TLR4-activating monomeric endotoxin⅐protein complexes (i.e. endotoxin⅐CD14 or endotoxin⅐MD-2), greatly amplifying host responsiveness to endotoxin. At pM concentrations, monomeric complexes of endotoxin⅐CD14 or endotoxin⅐MD-2 activate, respectively, mammalian cells expressing MD-2/TLR4 or TLR4 alone, triggering robust cell activation through engagement of Ͻ10 3 TLR4 molecules.Despite the ability of endotoxin⅐CD14 and endotoxin⅐ MD-2 to activate cells at pM concentrations (half-maximal cell acti...

Isolation of Monomeric and Dimeric Secreted MD-2

“…1A) or in generating monomeric LOS⅐MD-2 (9,24). Comparison of the two conditioned media by SDS-PAGE/immunoblot under nonreducing conditions demonstrated a significant difference in the amount and physical state of sMD-2 in the two media under these conditions.…”

“…HEK293 Cell-derived Recombinant Polyhistidine-tagged MD-2 and FLAG-TLR4 Ectodomain-Expression vectors containing DNA for production of FLAG-TLR4 ECD (amino acids 24 -634; pFLAG-CMV-TLR4) and MD-2-FLAG-His 6 (pEF-BOS-MD-2-FLAG-His 6 ) have been previously described and characterized (9,19). Briefly, HEK293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum to ϳ80% confluency in T75 flasks and transfected with 12 g of DNA using PolyFect reagent (Qiagen).…”

“…E⅐MD-2, but not E⅐sCD14 or purified endotoxin aggregates, can bind to TLR4 ECD with picomolar affinity and activate cells expressing TLR4 without MD-2 (e.g. HEK/TLR4 cells) at picomolar concentrations (4,9,14). Addition of as little as 1 l of this conditioned medium (representing ϳ1-2 ng of sMD-2; ϳ500 pM final concentration of sMD-2) together with 200 pM LOS⅐sCD14 (1 ng LOS/ml) produced maximum activation of HEK/TLR4 cells (Fig.…”

“…In contrast to the very stable and potently bioactive properties of the monomeric E⅐MD-2 complex (4, 18), recombinant MD-2 expressed and secreted from mammalian cell cultures, such as human embryonic kidney (HEK) 293 cells, is recovered mainly as inactive multimers with little monomer detected (19 -22). The recovery of active secreted MD-2 (sMD-2) from mammalian cell cultures is further compromised by the instability of sMD-2 at 37°C in serum-free culture medium (9,23,24). In contrast to MD-2 secreted from mammalian cell cultures, nearly 50% of MD-2, produced by insect cells after infection with baculovirus containing MD-2 cDNA, is monomeric * This work was supported, in whole or in part, by National Institutes of Health, United States Public Health Service Grants PO144642 and AI59372 (to J. P. W.).…”

“…This sensitivity depends upon an ordered series of endotoxin-protein and protein-protein interactions that include the host proteins LPS-binding protein (LBP), membrane-bound or soluble (s)CD14, secreted or TLR4-associated MD-2, and TLR4 (6 -8). Engagement of endotoxin-rich membranes or isolated endotoxin aggregates by LBP facilitates the extraction of endotoxin monomers by CD14 to form monomeric E⅐CD14 complexes that are the most efficient substrate for transfer of endotoxin to MD-2 (4,6,9). The monomeric E⅐MD-2 complex is necessary and sufficient to induce TLR4-dependent cell activation by endotoxin (4,6,10).…”