title: Endotoxin-binding proteins modulate the susceptibility of bacterial endotoxin to deacylation by acyloxyacyl hydrolase.
abstract: Acyloxyacyl hydrolase (AOAH) is an eukaryotic lipase that partially deacylates and detoxifies Gram-negative bacterial lipopolysaccharides and lipooligosaccharides (LPSs or LOSs, endotoxin) within intact cells and inflammatory fluids. In cell lysates or as purified enzyme, in contrast, detergent is required for AOAH to act on LPS or LOS (Erwin, A. L., and Munford, R. S. (1990) J. Biol. Chem. 265, 16444-16449 and Katz, S. S., Weinrauch, Y., Munford, R. S., Elsbach, P., and Weiss, J. (1999) J. Biol. Chem. 274, 36579-36584). We speculated that the sequential interactions of endotoxin (E) with endotoxin-binding proteins (lipopolysaccharide-binding protein (LBP), CD14, and MD-2) might produce changes in endotoxin presentation that would allow AOAH greater access to its substrate, lipid A. To test this hypothesis, we measured the activity of purified AOAH against isolated, metabolically labeled meningococcal LOS and Escherichia coli LPS that were presented either as aggregates (LOSagg or LPSagg)+/-LBP or as monomeric protein (sCD14 or MD-2)-endotoxin complexes. Up to 100-fold differences in the efficiency of endotoxin deacylation by AOAH were observed, with the following rank order of susceptibility to AOAH: E:sCD14>or=endotoxin aggregates (Eagg):LBP (molar ratio of E/LBP 100:1)>>Eagg, Eagg:LBP (E/LBP approximately 1, mol/mol), or E:MD-2. AOAH treatment of LOS-sCD14 produced partially deacylated LOS still complexed with sCD14. The underacylated LOS complexed to sCD14 transferred to MD-2 and thus formed a complex capable of preventing TLR4 activation. These findings strongly suggest that LBP- and CD14-dependent extraction and transfer of endotoxin monomers are accompanied by increased exposure of fatty acyl chains within lipid A and that the acyl chains are then sequestered when LOS binds MD-2. The susceptibility of the monomeric endotoxin-CD14 complex to AOAH may help constrain endotoxin-induced TLR4 activation when endotoxin and membrane CD14 are present in excess of MD-2/TLR-4.
authors.affiliation: Inflammation Program, Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA. theresa-gioannini@uiowa.edu
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Gioannini
authors.forename: Athmane
authors.initials: A
authors.lastname: Teghanemt
authors.forename: DeSheng
authors.initials: D
authors.lastname: Zhang
authors.forename: Polonca
authors.initials: P
authors.lastname: Prohinar
authors.forename: Erika N
authors.initials: EN
authors.lastname: Levis
authors.forename: Robert S
authors.initials: RS
authors.lastname: Munford
authors.forename: Jerrold P
authors.initials: JP
authors.lastname: Weiss
mesh_terms: D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002460:Cell Line; D004731:Endotoxins / Q000378:metabolism; D006801:Humans; D007313:Insecta; D008055:Lipids / Q000737:chemistry; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D051218:Lymphocyte Antigen 96 / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D009345:Neisseria meningitidis / Q000378:metabolism; D010586:Phagocytes / Q000382:microbiology; D011485:Protein Binding; D051197:Toll-Like Receptor 4 / Q000378:metabolism
chemical_list: D004731:Endotoxins; D008055:Lipids; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008565:Membrane Proteins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C025357:endotoxin binding proteins; D002265:Carboxylic Ester Hydrolases; C048964:acyloxyacyl hydrolase