sepharose and lipoteichoic-acid

The hypothesis that soluble peptidoglycan (sPGN, a macrophage-activator from Gram-positive bacteria) binds to CD14 (a lipopolysaccharide (LPS) receptor) was tested. sPGN specifically bound to CD14 in the following three assays: binding of soluble 32P-CD14 (sCD14) to agarose-immobilized sPGN, enzyme-linked immunosorbent assay, and photoaffinity cross-linking. sCD14 also specifically bound to agarose-immobilized muramyl dipeptide or GlcNAc-muramyl dipeptide but not to PGN pentapeptide. Binding of sCD14 to both sPGN and ReLPS (where ReLPS is LPS from Salmonella minnesota Re 595) was competitively inhibited by unlabeled sCD14, 1-152 N-terminal fragment of sCD14, sPGN, smooth LPS, ReLPS, lipid A, and lipoteichoic acid but not by dextran, dextran sulfate, heparin, ribitol teichoic acid, or soluble low molecular weight PGN fragments. Binding of sCD14 to sPGN was slower than to ReLPS but of higher affinity (KD = 25 nM versus 41 nM). LPS-binding protein (LBP) increased the binding of sCD14 to sPGN by adding another lower affinity KD and another higher Bmax, but for ReLPS, LBP increased the affinity of binding by yielding two KD with significantly higher affinity (7.1 and 27 nM). LBP also enhanced inhibition of sCD14 binding by LPS, ReLPS, and lipid A. Binding of sCD14 to both sPGN and ReLPS was inhibited by anti-CD14 MEM-18 mAb, but other anti-CD14 mAbs showed differential inhibition, suggesting conformational binding sites on CD14 for sPGN and LPS, that are partially identical and partially different.

Topics: Animals; Binding, Competitive; Cell Line; Escherichia coli; Gram-Positive Bacteria; Humans; Insecta; Kinetics; Lipopolysaccharide Receptors; Lipopolysaccharides; Peptidoglycan; Recombinant Proteins; Salmonella; Sepharose; Staphylococcus aureus; Streptococcus; Teichoic Acids; Transfection

The relative activities of lipoteichoic acid (LTA) from four Gram-positive bacteria were compared to different lipopolysaccharide (LPS) preparations for activation of arachidonic acid metabolism in mouse peritoneal macrophages. Total eicosanoid was determined in cultures labeled with [3H]-arachidonic acid. Prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) were determined by EIA analysis. The relative potencies of the different preparations were: smooth LPS from Salmonella abortus > or = Re-LPS from Salmonella minnesota (R-595) > or = LTA from Streptococcus pyogenes approximately Streptococcus faecalis approximately Staphylococcus aureus > or = monophosphoryl lipid A derived from the Re-LPS >> LTA from Bacillus subtilis. Activation of eicosanoid release was inhibited by staurosporin for all of the amphiphiles tested. Treatment of the macrophage cultures with LTA from S. pyogenes, S. faecalis, and S. aureus, either in the presence or absence of indomethacin, desensitized the cells to eicosanoid release on subsequent challenge with LPS. The desensitized cells remained responsive to the phorbol ester phorbol myristate acetate. LPS from Gram-negative bacteria has immunostimulatory and endotoxic activities which result, in part, from the release of eicosanoids and other mediators from activated macrophages. The similarities in the patterns of cell activation by LPS and LTA suggest that lipoteichoic acids might contribute to the pathogenicities of Gram-positive bacteria.

Topics: Animals; Arachidonic Acid; Cells, Cultured; Chromatography; Eicosanoids; Gram-Positive Bacteria; Lipopolysaccharides; Macrophage Activation; Macrophages, Peritoneal; Mice; Mice, Inbred ICR; Salmonella; Sepharose; Streptomyces; Teichoic Acids; Tetradecanoylphorbol Acetate

Analyzed were (I) the oligoglucosyl-, alanyl-substituted poly(glycerophosphate) lipoteichoic acid of Enterococcus hirae containing Glc(alpha 1-2)Glc(alpha 1-3)acyl2-Gro and a phosphatidyl derivative thereof as lipid anchor, (II) the poly(digalactosyl, galactosylglycerophosphate) lipoteichoic acid of Lactococcus garvieae containing the same glycolipid and an acyl derivative of it, and (III) the N-acetylglucosaminyl-, alanyl-substituted poly(glycerophosphate)lipoteichoic acid of Staphylococcus aureus with Glc(beta 1-6)Glc(beta 1-3)acyl2Gro as the sole lipid moiety. Hydrophobic interaction chromatography on octyl-Sepharose separated lipoteichoic acids I and II into two peaks according to the number of fatty acids. Within each peak further fractionation occurred in the order of decreasing length of the hydrophilic chain. A similar fractionation was observed within the single peak of lipoteichoic acid III. With lipoteichoic acid I and III the extent of glycosylation decreased with decreasing length of the hydrophilic chain whereas the content of alanine ester remained either constant or increased. Variations in the oligosaccharide pattern of lipoteichoic acid I and in the fatty acid composition of all lipoteichoic acids could also be observed. Collectively, the data provide for the first time a detailed picture of the complex polydispersity of lipoteichoic acids comprising the number of fatty acids, the length of the hydrophilic chain, the kind and extent of chain substitution, and the fatty acid composition. The procedure will also be applicable to the molecular analysis of lipopolysaccharides and bacterial lipoglycans. Moreover, the results are of physicochemical interest because they demonstrate for lipid macroamphiphiles an inverse relationship between hydrophobicity and the size of the hydrophilic headgroup.

Topics: Animals; Carbohydrate Sequence; Cardiolipins; Chromatography, Agarose; Enterococcus; Evaluation Studies as Topic; Fatty Acids; Glycosylation; Lactococcus; Lipids; Lipopolysaccharides; Molecular Sequence Data; Oligosaccharides; Sepharose; Staphylococcus aureus; Teichoic Acids

Hydrophobic interaction chromatography fractionated the lipoteichoic acid of Enterococcus faecalis into species of decreasing poly(glycerophosphate) chain length and decreasing extent of substitution with alpha-kojibiosyl residues (Glcp alpha 1----2Glcp alpha 1----). The chain length varied between 14 and 33 glycerophosphate residues per lipid anchor, the extent of glycosylation between 0.18 and 0.44 mol of alpha-kojibiosyl residues per mole of phosphorus, and, accordingly, the number of alpha-kojibiosyl substituents per chain between 3 and 15. Almost identical values were obtained when the same lipoteichoic acid was chromatographed on DEAE-Sephadex and concanavalin A, which separate molecular species according to increasing number of phosphate groups and alpha-kojibiosyl residues, respectively. Species from all three columns, which were identical in chain length and glycosylation, also had similar fatty acid patterns. These results prove the suitability of all three procedures for species analysis. One advantage of hydrophobic interaction chromatography over the other two procedures lies in its broader applicability since it is not dependent on negative charges or specifically binding oligosaccharide structures. Another advantage is the capacity of hydrophobic interaction chromatography to separate molecular species differing in the number of fatty acids [W. Fischer, H.U. Koch, and R. Haas (1983) Eur. J. Biochem. 133, 523-530] and render them accessible to molecular analyses.

Topics: Chromatography, Affinity; Chromatography, Ion Exchange; Concanavalin A; Enterococcus faecalis; Lipopolysaccharides; Sepharose; Teichoic Acids; Water