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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The structural determinants of lipopolysaccharide required for the induction of tissue factor in human umbilical vein endothelial cells were studied. Intact lipid A was essential for the induction of tissue factor whereas the incomplete lipid A precursors lipid IVA and lipid X, as well as monophosphoryl lipid A and acyloxyacyl hydrolase-treated lipopolysaccharide, were unable to induce tissue factor and tissue factor specific mRNA. However, the lipid A precursor, lipid IVA, was able to inhibit LPS-mediated induction of tissue factor; structural determinants distal to lipid A were found to be required for maximal induction of tissue factor activity and tissue factor mRNA. The presence of serum in the assay was found to amplify but was not obligate for tissue factor induction by LPS.
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PMID:Induction of endothelial tissue factor by endotoxin and its precursors. 811

The lipopolysaccharide of Rhizobium leguminosarum differs from that of other Gram-negative organisms. R. leguminosarum lipid A lacks phosphate groups, but it contains a galacturonic acid residue at the 4'-position and an aminogluconate moiety in place of the usual glucosamine 1-phosphate unit. R. leguminosarum lipid A is esterified with a peculiar long chain fatty acid, 27-hydroxyoctacosanoate, not found in enteric Gram-negative bacteria, and the inner core of R. leguminosarum contains mannose and galactose in place of heptose. Despite these differences, the biosynthesis of R. leguminosarum lipid A is initiated by the same seven enzyme pathway as in Escherichia coli (Raetz, C. R. H. (1993) J. Bacteriol. 175, 5745-5753) to form the phosphorylated precursor, (Kdo)2-lipid IVA, which is then processed differently. We now describe several novel Rhizobium-specific enzymes that recognize and modify (Kdo)2-lipid IVA. The 1- and 4'-phosphatases were detected using (Kdo)2-[1-32P]-lipid IVA and (Kdo)2-[4'-32P]-lipid IVA, respectively, as shown by release of 32Pi. In the presence of GDP-mannose and/or UDP-galactose, membranes of R. leguminosarum first transferred mannose and then galactose to (Kdo)2-[4'-32P]-lipid IVA. In addition, at least two hydrophobic metabolites were generated from (Kdo)2-[4'-32P]-lipid IVA in a manner that was dependent upon both membranes and a cytosolic factor from R. leguminosarum. These compounds are attributed to novel acylations of (Kdo)2-[4'-32P]-lipid IVA. E. coli membranes and cytosol did not catalyze any of the unique reactions detected in R. leguminosarum extracts. Our findings establish the conservation and versatility of (Kdo)2-lipid IVA as a lipid A precursor in bacteria.
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PMID:Lipopolysaccharide biosynthesis in Rhizobium leguminosarum. Novel enzymes that process precursors containing 3-deoxy-D-manno-octulosonic acid. 894 64

The lipopolysaccharide structure of the nitrogen-fixing bacterium Rhizobium leguminosarum differs from that of Escherichia coli in several ways, one of which is the sugar composition of the core. The E. coli inner core consists of 3-deoxy-D-manno-octulosonic acid (Kdo) and L-glycero-D-manno-heptose (heptose), while the inner core of R. leguminosarum contains 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo), mannose, galactose, and galacturonic acid. The two Kdo residues and their linkages appear to be identical in both species. The linkages of heptose in E. coli and of mannose in R. leguminosarum to Kdo are both alpha1-5. We now characterize a membrane-associated glycosyl transferase in R. leguminosarum extracts that incorporates mannose into nascent lipopolysaccharide, using Kdo2-lipid IVA as the acceptor and GDP-mannose (or synthetic ADP-mannose) as the donor. The mannosyl transferase is associated with the inner membrane. The apparent Km values for GDP-mannose and Kdo2-lipid IVA are 4.3 microM and 7.1 microM, respectively, in the presence of excess co-substrate. Extracts of E. coli do not catalyze GDP-mannose-dependent glycosylation of Kdo2-lipid IVA, but they are active when ADP-mannose is substituted for GDP-mannose. Given the structural similarity of ADP-mannose to ADP-heptose, we examined the possibility that heptosyl transferase I of E. coli (the product of the rfaC gene) catalyzes mannose transfer from ADP-mannose to Kdo2-lipid IVA. Extracts of E. coli mutants defective in the rfaC gene are unable carry out ADP-mannose-dependent glycosylation of Kdo2-lipid IVA. Plasmids bearing rfaC+ not only restore the missing activity but also direct its overexpression. Our assay using ADP-mannose as a substitute for ADP-heptose (which is not readily available) should facilitate the purification and characterization of heptosyl transferase I of E. coli. The GDP-mannose-dependent enzyme of R. leguminosarum may represent a functional equivalent of E. coli RfaC.
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PMID:Lipopolysaccharide core glycosylation in Rhizobium leguminosarum. An unusual mannosyl transferase resembling the heptosyl transferase I of Escherichia coli. 894 65

Heptosyltransferase I, encoded by the rfaC(waaC) gene of Escherichia coli, is thought to add L-glycero-D-manno-heptose to the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide core. Lipopolysaccharide isolated from mutants defective in rfaC lack heptose and all other sugars distal to heptose. The putative donor, ADP-L-glycero-D-manno-heptose, has never been fully characterized and is not readily available. In cell extracts, the analog ADP-mannose can serve as an alternative donor for RfaC-catalyzed glycosylation of the acceptor, Kdo2-lipid IVA. Using a T7 promoter construct that overexpresses RfaC approximately 15,000-fold, the enzyme has been purified to near homogeneity. NH2-terminal sequencing confirms that the purified enzyme is the rfaC gene product. The subunit molecular mass is 36 kDa. Enzymatic activity is dependent upon the presence of Triton X-100 and is maximal at pH 7.5. The apparent Km (determined at near saturating concentrations of the second substrate) is 1.5 mM for ADP-mannose and 4.5 microM for Kdo2-lipid IVA. Chemical hydrolysis of the RfaC reaction product at 100 degrees C in the presence of sodium acetate and 1% sodium dodecyl sulfate generates fragments consistent with the inner Kdo residue of Kdo2-lipid IVA as the site of mannosylation. The analog, Kdo-lipid IVA, functions as an acceptor, but is mannosylated at less than 1% the rate of Kdo2-lipid IVA. The purified enzyme displays no activity with ADP-glucose, GDP-mannose, UDP-glucose, or UDP-galactose. Mannosylation of Kdo2-lipid IVA catalyzed by RfaC proceeds in high yield and may be useful for the synthesis of lipopolysaccharide analogs. Pure RfaC can also be used together with Kdo2-[4'-32P]lipid IVA to assay for the physiological donor (presumably ADP-L-glycero-D-manno-heptose) in a crude, low molecular weight fraction isolated from wild type cells.
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PMID:Enzymatic synthesis of lipopolysaccharide in Escherichia coli. Purification and properties of heptosyltransferase i. 944 88

The lipopolysaccharide (LPS) core of the Gram-negative bacterium Rhizobium leguminosarum is more amenable to enzymatic study than that of Escherichia coli because much of it is synthesized from readily available sugar nucleotides. The inner portion of the R. leguminosarum core contains mannose, galactose, and three 3-deoxy-D-manno-octulosonate (Kdo) residues, arranged in the order: lipid A-(Kdo)2-Man-Gal-Kdo-[O antigen]. A mannosyltransferase that uses GDP-mannose and the conserved precursor Kdo2-[4'-32P]lipid IVA (Kadrmas, J. L., Brozek, K. A., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 32119-32125) is proposed to represent a key early enzyme in R. leguminosarum core assembly. Conditions for demonstrating efficient galactosyl- and distal Kdo-transferase activities are now described using a coupled assay system that starts with GDP-mannose and Kdo2-[4'-32P]lipid IVA. As predicted, mannose incorporation precedes galactose addition, which in turn precedes distal Kdo transfer. LPS core mutants with Tn5 insertions in the genes encoding the putative galactosyltransferase (lpcA) and the distal Kdo-transferase (lpcB) are shown to be defective in the corresponding in vitro glycosylation of Kdo2-[4'-32P]lipid IVA. We have also discovered the new gene (lpcC) that encodes the mannosyltransferase. The gene is separated by several kilobase pairs from the lpcAB cluster. All three glycosyltransferases are carried on cosmid pIJ1848, which contains at least 20 kilobase pairs of R. leguminosarum DNA. Transfer of pIJ1848 into R. meliloti 1021 results in heterologous expression of all three enzymes, which are not normally present in strain 1021. Expression of the lpc genes individually behind the T7 promoter results in the production of each R. leguminosarum glycosyltransferase in E. coli membranes in a catalytically active form, demonstrating that lpcA, lpcB, and lpcC are structural genes.
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PMID:Cloning and overexpression of glycosyltransferases that generate the lipopolysaccharide core of Rhizobium leguminosarum. 975 77

CD14 has been implicated as a receptor of lipoteichoic acid (LTA) and other bacterial components as well as lipopolysaccharide (LPS). Since the structures of LTAs from various gram-positive bacteria are heterogeneous, we analyzed the effects of LTAs on the secretion of interleukin-8 (IL-8) by high- and low-CD14-expressing (CD14(high) and CD14(low)) human gingival fibroblasts (HGF). While Bacillus subtilis LTA had an IL-8-inducing effect on CD14(high) HGF which was considerably weaker than that of LPS, Streptococcus sanguis and Streptococcus mutans LTAs had practically no effect on the cells. B. subtilis LTA had only a weak effect on CD14(low) HGF, as did LPS. S. sanguis and S. mutans LTAs at a 1,000-fold excess each completely inhibited the IL-8-inducing activities of both LPS and a synthetic lipid A on CD14(high) HGF. The effect of LPS was also inhibited by the presence of an LPS antagonist, synthetic lipid A precursor IVA (LA-14-PP), with a 100-fold higher potency than S. sanguis and S. mutans LTAs and by anti-CD14 monoclonal antibody (MAb). S. sanguis and S. mutans LTAs, LA-14-PP, and anti-CD14 MAb had no significant effect on phorbol myristate acetate-stimulated IL-8 secretion by HGF. These LTAs also inhibited the IL-8-inducing activity of B. subtilis LTA on CD14(high) HGF, as did LA-14-PP and anti-CD14 MAb. The antagonistic and agonistic functions of LTAs were also observed with human monocytes. Binding of fluorolabeled LPS to human monocytes was inhibited by S. sanguis LTA, although the inhibition was 100 times weaker than that of LPS itself, and anti-CD14 MAb inhibited fluorolabeled LPS and S. sanguis LTA binding. Binding of LTAs to CD14 was also observed with nondenaturing polyacrylamide gel electrophoresis. These results indicate that LTAs act as antagonists or agonists via a CD14-dependent mechanism, probably due to the heterogeneous structure of LTAs, and that an antagonistic LTA might be a useful agent for suppressing the periodontal disease caused by gram-negative bacteria.
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PMID:Lipoteichoic acid acts as an antagonist and an agonist of lipopolysaccharide on human gingival fibroblasts and monocytes in a CD14-dependent manner. 1008 95

Calcium may play a key role in the lipopolysaccharide (LPS)-induced release of calcitonin gene-related peptide in rat dorsal root ganglion neurons. Both calcium entry via voltage-activated calcium channels sensitive to either omega-conotoxin GVIA or omega-agatoxin IVA, and calcium release from ryanodine-sensitive calcium stores were involved in this process. Cyclic ADP-ribose antagonist affected neither the enhancement of cytoplasmic free calcium nor the release of calcitonin gene-related peptide induced by LPS. These findings underscore a pivotal role of calcium-induced calcium release in lipopolysaccharide-stimulated calcitonin gene-related peptide release from nociceptive neurons.
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PMID:Lipopolysaccharide evoked peptide release by calcium-induced calcium release. 1507 23

P388D1 cells release arachidonic acid (AA) and produce prostaglandin E2 (PGE2) upon long-term stimulation with lipopolysaccharide (LPS). The cytosolic Group IVA (GIVA) phospholipase A2 (PLA2) has been implicated in this pathway. LPS stimulation also results in increased expression and secretion of a secretory PLA2, specifically GV PLA2. To test whether GV PLA2 contributes to PGE2 production and whether GIVA PLA2 activation increases the expression of GV PLA2, we utilized the specific GIVA PLA2 inhibitor pyrrophenone and second generation antisense oligonucleotides (AS-ONs) designed to specifically inhibit expression and activity of GV PLA2. Treatment of P388D1 cells with antisense caused a marked decrease in basal GV PLA2 mRNA and prevented the LPS-induced increase in GV PLA2 mRNA. LPS-stimulated cells release active GV PLA2 into the medium, which is inhibited to background levels by antisense treatment. However, LPS-induced PGE2 release by antisense-treated cells and by control cells are not significantly different. Collectively, the results suggest that the upregulation of GV PLA2 during long-term LPS stimulation is not required for PGE2 production by P388D1 cells. Experiments employing pyrrophenone suggested that GIVA PLA2 is the dominant player involved in AA release, but it appears not to be involved in the regulation of LPS-induced expression of GV PLA2 or cyclooxygenase-2.
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PMID:PGE2 release is independent of upregulation of Group V phospholipase A2 during long-term stimulation of P388D1 cells with LPS. 1615 Aug 19

Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) is a prototypic sPLA(2) enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA(2) by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA(2) (cPLA(2)alpha) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA(2)alpha is required for de novo induction of sPLA(2). However, because of the broad specificity of several cPLA(2)alpha inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA(2)-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA(2) (iPLA(2)gamma), rather than cPLA(2)alpha, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA(2)-IIA in cPLA(2)alpha knockdown cells, in which cPLA(2)alpha protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA(2)gamma, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA(2), markedly reduced the cytokine-induced expression of sPLA(2)-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA(2)gamma inhibitor, suppressed the cytokine-induced sPLA(2)-IIA expression, whereas (S)-bromoenol lactone, an iPLA(2)beta inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA(2)-IIA expression was also abolished by knockdown of iPLA(2)gamma. These findings open new insight into a novel regulatory role of iPLA(2)gamma in stimulus-coupled sPLA(2)-IIA expression.
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PMID:A novel role of group VIB calcium-independent phospholipase A2 (iPLA2gamma) in the inducible expression of group IIA secretory PLA2 in rat fibroblastic cells. 1747 22

In Escherichia coli, FtsH (HflB) is a membrane-bound, ATP-dependent metalloendoprotease belonging to the AAA family (ATPases associated with diverse cellular activities). FtsH has a limited spectrum of known substrates, including the transcriptional activator sigma32. FtsH is the only known E. coli protease that is essential, as it regulates the concentration of LpxC, which carries out the first committed step in the synthesis of lipid A. Here we identify a new FtsH substrate--3-deoxy-D-manno-octulosonate (KDO) transferase--which carries out the attachment of two KDO residues to the lipid A precursor (lipid IVA) to form the minimal essential structure of the lipopolysaccharide (LPS) (KDO2-lipid A). Thus, FtsH regulates the concentration of the lipid moiety of LPS (lipid A) as well as the sugar moiety (KDO-based core oligosaccharides), ensuring a balanced synthesis of LPS.
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PMID:Dual role of FtsH in regulating lipopolysaccharide biosynthesis in Escherichia coli. 1877 15


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