Host proinflammatory responses to minute amounts of endotoxins derived from many Gram-negative bacteria require the interaction of lipopolysaccharide-binding protein (LBP), CD14, Toll-like receptor 4 (TLR4) and MD-2. Optimal sensitivity to endotoxin requires an ordered series of endotoxin–protein and protein–protein interactions. At substoichiometric concentrations, LBP facilitates delivery of endotoxin aggregates to soluble CD14 (sCD14) to form monomeric endotoxin–sCD14 complexes. Subsequent interactions of endotoxin–sCD14 with TLR4 and/or MD-2 have not been specifically defined. This study reports the purification of a stable, monomeric, bioactive endotoxin–MD-2 complex generated by treatment of endotoxin–sCD14 with recombinant MD-2. Efficient generation of this complex occurred at picomolar concentrations of endotoxin and nanogram per milliliter doses of MD-2 and required presentation of endotoxin to MD-2 as a monomeric endotoxin–CD14 complex. TLR4-dependent delivery of endotoxin to human embryonic kidney (HEK) cells and cell activation at picomolar concentrations of endotoxin occurred with the purified endotoxin–MD-2 complex, but not with purified endotoxin aggregates with or without LBP and/or sCD14. The presence of excess MD-2 inhibited delivery of endotoxin–MD-2 to HEK/TLR4 cells and cell activation. These findings demonstrate that TLR4-dependent activation of host cells by picomolar concentrations of endotoxin occurs by sequential interaction and transfer of endotoxin to LBP, CD14, and MD-2 and simultaneous engagement of endotoxin and TLR4 by MD-2.
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...
Potent TLR4-dependent cell activation by Gram-negative bacterial endotoxins depends on sequential endotoxin-protein and protein-protein interactions with LPS-binding protein, CD14, myeloid differentiation protein 2 (MD-2), and TLR4. Previous studies have suggested that reduced agonist potency of underacylated endotoxins (i.e., tetra- or penta- vs hexa-acylated) is determined by post-CD14 interactions. To better define the molecular basis of the differences in agonist potency of endotoxins differing in fatty acid acylation, we compared endotoxins (lipooligosaccharides (LOS)) from hexa-acylated wild-type (wt), penta-acylated mutant msbB meningococcal strains as well as tetra-acylated LOS generated by treatment of wt LOS with the deacylating enzyme, acyloxyacylhydrolase. To facilitate assay of endotoxin:protein and endotoxin:cell interactions, the endotoxins were purified after metabolic labeling with [3H]- or [14C]acetate. All LOS species tested formed monomeric complexes with MD-2 in an LPS-binding protein- and CD14-dependent manner with similar efficiency. However, msbB LOS:MD-2 and acyloxyacylhydrolase-treated LOS:MD-2 were at least 10-fold less potent in inducing TLR4-dependent cell activation than wt LOS:MD-2 and partially antagonized the action of wt LOS:MD-2. These findings suggest that underacylated endotoxins produce decreased TLR4-dependent cell activation by altering the interaction of the endotoxin:MD-2 complex with TLR4 in a way that reduces receptor activation. Differences in potency among these endotoxin species is determined not by different aggregate properties, but by different properties of monomeric endotoxin:MD-2 complexes.
Studies with purified aggregates of endotoxin have revealed the importance of lipopolysaccharide-binding protein (LBP)-dependent extraction and transfer of individual endotoxin molecules to CD14 in Toll-like receptor 4 (TLR4)-dependent cell activation. Endotoxin is normally embedded in the outer membrane of intact Gram-negative bacteria and shed membrane vesicles ("blebs"). However, the ability of LBP and CD14 to efficiently promote TLR4-dependent cell activation by membrane-associated endotoxin has not been studied extensively. In this study, we used an acetate auxotroph of Neisseria meningitidis serogroup B to facilitate metabolic labeling of bacterial endotoxin and compared interactions of purified endotoxin aggregates and of membrane-associated endotoxin with LBP, CD14, and endotoxin-responsive cells. The endotoxin, phospholipid, and protein composition of the recovered blebs indicate that the blebs derive from the bacterial outer membrane. Proteomic analysis revealed an unusual enrichment in highly cationic (pI > 9) proteins. Both purified endotoxin aggregates and blebs activate monocytes and endothelial cells in a LBP-, CD14-, and TLR4/MD-2-dependent fashion, but the blebs were 3-10-fold less potent when normalized for the amount of endotoxin added. Differences in potency correlated with differences in efficiency of LBPdependent delivery to and extraction of endotoxin by CD14. Both membrane phospholipids and endotoxin are extracted by LBP/soluble CD14 (sCD14) treatment, but only endotoxin⅐sCD14 reacts with MD-2 and activates cells. These findings indicate that the proinflammatory potency of endotoxin may be regulated not only by the intrinsic structural properties of endotoxin but also by its association with neighboring molecules in the outer membrane.
Experiments utilizing endotoxin aggregates, lipooligosaccharides (LOS) isolated from metabolically labeledNeisseria meningitidis serotype group B, demonstrate that albumin is an essential component of lipopolysaccharide binding protein-(LBP) and sCD14-dependent 1) disaggregation of LOS and 2) LOS activation of human umbilical vein endothelial cells (HUVEC). Aggregates of LOS (LOS agg ) with an apparent M r > 2 ؋ 10 7 were isolated by gel sieving on Sephacryl HR S500 in buffered balanced salts solution plus albumin. Incubation of LOS agg with LBP and sCD14 promoted LOS agg disaggregation in an albumin-dependent fashion to complexes that contain LOS and sCD14, but no LBP, with an apparent M r ϳ 60,000 (LOS:sCD14) as determined by Sephacryl S200 chromatography. Isolation by gel filtration of LOS agg :protein aggregates formed by the interaction of LOS agg with either LBP or sCD14 alone revealed that the sequence of LOSprotein interactions as well as the step(s) at which albumin is necessary for the production of bioactive LOS: sCD14 were specific. Efficient generation of LOS:sCD14 required 1) interaction of LOS agg with LBP before interaction with CD14 and 2) the presence of albumin during the interaction of LBP with LOS agg . Activation of HUVEC by LOS agg , as measured by IL-8 production, required both LBP and sCD14 and was thirty times more potent in the presence of albumin. In contrast, LOS:sCD14 did not require additional LBP, sCD14, or albumin to activate HUVEC but depended on the presence of albumin for optimal solubility/stability once formed. The albumin effect is apparently specific, because neither ovalbumin nor gelatin substituted for albumin in facilitating LBP:sCD14-dependent disaggregation of LOS agg or activation of endothelial cells. These results indicate that albumin is an essential facilitator of LBP/sCD14-induced LOS disaggregation that is required for activation of endothelial cells by LOS agg .
Acyloxyacyl hydrolase (AOAH) is an eukaryotic lipase that partially deacylates and detoxifies Gram-negative bacterial lipopolysaccharides and lipooligosaccharides (LPSs orLOSsTissue invasion by even minute quantities of many Gramnegative bacteria (GNB) 2 initiates rapid mobilization of the innate immune responses of the host. In these circumstances, both GNB recognition and many responses depend upon activation of the exquisitely sensitive Toll-like receptor 4 (TLR4) by endotoxins, structurally unique and abundant glycolipids that occupy much of the outer leaflet of the GNB outer membrane (3). Maximal TLR4-dependent host responses to endotoxin are orchestrated through a sequential set of interactions of endotoxin with lipopolysaccharide-binding protein (LBP), membrane or soluble CD14, and soluble or TLR4-associated MD-2 (3-5). Although timely mobilization of host responses is essential, equally important is the regulation of the duration and intensity of host responses to endotoxin to prevent over-exuberant and sustained responses that can result in severe pathological consequences (6).One mechanism of dampening host responses to endotoxin is for the host to modify endotoxin itself, converting it from a potent TLR4 agonist to a much weaker agonist with antagonistic properties. To date, the best described host endotoxin-detoxifying enzyme is acyloxyacyl hydrolase (AOAH) (6). AOAH reduces endotoxin activity by catalyzing the release of secondary fatty acyl chains that are attached to primary 3-hydroxy fatty acyl chains within the bioactive lipid A region (7,8). AOAH thus converts hexaacylated endotoxin species that are potent TLR4 agonists to pentaacylated or tetraacylated forms that have reduced or no agonist properties (9 -13) and, at least in vitro, possess the ability to inhibit TLR4 activation by intact, hexaacylated species (10,11,13, 14). Recent studies indicate that hexaacylated and partially deacylated or underacylated endotoxin react similarly with LBP, CD14, and MD-2 to form monomeric complexes with MD-2 (13). However, only hexaacylated endotoxin-human MD-2 is a potent TLR4 agonist (13, 14).AOAH-dependent deacylation of endotoxin has been demonstrated in vivo (1,15,16) and in complex in vitro settings that roughly simulate the extracellular and intracellular conditions of inflammatory fluids (1, 2, 15,
Host response to invasion by many gram-negative bacteria depends upon activation of Toll-like receptor 4 (TLR4) by endotoxin presented as a monomer bound to myeloid differentiation factor 2 (MD-2). Metabolic labeling of hexaacylated endotoxin (LOS) from Neisseria meningitidis with [(13)C]acetate allowed the use of NMR to examine structural properties of the fatty acyl chains of LOS present in TLR4-agonistic and -antagonistic binary and ternary complexes with, respectively, wild-type or mutant (F126A) MD-2 ± TLR4 ectodomain. Chemical shift perturbation indicates that Phe(126) affects the environment and/or position of each of the bound fatty acyl chains both in the binary LOS·MD-2 complex and in the ternary LOS·MD-2·TLR4 ectodomain complex. In both wild-type and mutant LOS·MD-2 complexes, one of the six fatty acyl chains of LOS is more susceptible to paramagnetic attenuation, suggesting protrusion of that fatty acyl chain from the hydrophobic pocket of MD-2, independent of association with TLR4. These findings indicate that re-orientation of the aromatic side chain of Phe(126) is induced by binding of hexaacylated E, preceding interaction with TLR4. This re-arrangement of Phe(126) may act as a "hydrophobic switch," driving agonist-dependent contacts needed for TLR4 dimerization and activation.
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