title: Molecular basis of reduced potency of underacylated endotoxins.
abstract: 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.
authors.affiliation: Inflammation Program, Department of Internal Medicine, Coralville, IA 52241, USA.
authors.forename: Athmane
authors.initials: A
authors.lastname: Teghanemt
authors.forename: DeSheng
authors.initials: D
authors.lastname: Zhang
authors.forename: Erika N
authors.initials: EN
authors.lastname: Levis
authors.forename: Jerrold P
authors.initials: JP
authors.lastname: Weiss
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Gioannini
mesh_terms: D000215:Acylation; D002460:Cell Line; D004731:Endotoxins / Q000037:antagonists & inhibitors* / Q000378:metabolism* / Q000633:toxicity; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000502:physiology; D008070:Lipopolysaccharides / Q000378:metabolism; D009345:Neisseria meningitidis / Q000378:metabolism
chemical_list: D004731:Endotoxins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides