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.

Potent TLR4-dependent cell activation by Gram-negative bacterial endotoxin depends on sequential endotoxin?protein and protein?protein interactions with LBP, CD14, MD-2 and TLR4. LBP and CD14 combine, in an albumin-dependent fashion, to extract single endotoxin molecules from purified endotoxin aggregates (E(agg)) or the bacterial outer membrane and form monomeric endotoxin:CD14 complexes that are the preferred presentation of endotoxin for transfer to MD-2. Endotoxin in endotoxin:CD14is readily transferred to MD-2, again in an albumin-dependent manner, to form monomeric endotoxin:MD-2 complex. This monomeric endotoxin:protein complex (endotoxin:MD-2) activates TLR4 at picomolar concentrations, independently of albumin, and is, therefore, the apparent ligand in endotoxin-dependent TLR4 activation. Tetra-, penta-, and hexa-acylated forms of meningococcal endotoxin (LOS) react similarly with LBP, CD14, and MD-2 to form endotoxin:MD-2 complexes. However, tetra- and penta-acylated LOS:MD-2 complexes are less potent TLR4 agonists than hexa-acylated LOS:MD-2. This is mirrored in the reduced activity of tetra-, penta- versus hexa-acylated LOS aggregates (LOS(agg)) + LBP toward cells containing mCD14, MD-2, and TLR4. Therefore, changes in agonist potency of under-acylated meninigococcal LOS are determined by differences in properties of monomeric endotoxin:MD-2.

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,

Potent TLR4-dependent cell activation by Gram-negative bacterial endotoxin depends on sequential endotoxin?protein and protein?protein interactions with LBP, CD14, MD-2 and TLR4. LBP and CD14 combine, in an albumin-dependent fashion, to extract single endotoxin molecules from purified endotoxin aggregates (E(agg)) or the bacterial outer membrane and form monomeric endotoxin:CD14 complexes that are the preferred presentation of endotoxin for transfer to MD-2. Endotoxin in endotoxin:CD14is readily transferred to MD-2, again in an albumin-dependent manner, to form monomeric endotoxin:MD-2 complex. This monomeric endotoxin:protein complex (endotoxin:MD-2) activates TLR4 at picomolar concentrations, independently of albumin, and is, therefore, the apparent ligand in endotoxin-dependent TLR4 activation. Tetra-, penta-, and hexa-acylated forms of meningococcal endotoxin (LOS) react similarly with LBP, CD14, and MD-2 to form endotoxin:MD-2 complexes. However, tetra- and penta-acylated LOS:MD-2 complexes are less potent TLR4 agonists than hexa-acylated LOS:MD-2. This is mirrored in the reduced activity of tetra-, penta- versus hexa-acylated LOS aggregates (LOS(agg)) + LBP toward cells containing mCD14, MD-2, and TLR4. Therefore, changes in agonist potency of under-acylated meninigococcal LOS are determined by differences in properties of monomeric endotoxin:MD-2.