Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have cloned a gene from a Salmonella typhimurium with the ability to complement the rfaC mutation (heptose-deficient lipopolysaccharide, sensitivity to rough-specific bacteriophages, and susceptibility to hydrophobic antibiotics). A 1018-base pair EcoRV-Tth111I fragment, subcloned into the pBluescriptKS+ vector to yield pKZ103, retains complementing activity. Nucleotide sequencing revealed an open reading frame corresponding to a protein of 317 amino acids (M(r) approximately 35,100). The plasmid pKZ103, which has a properly aligned T7 promoter, can overexpress a protein of M(r) = 31,000 when T7 RNA polymerase is supplied. An in vitro system was established for analysis of heptose addition to the precursor [4'-32P](KDO)2-IVA (Brozek, K. A., Hosaka, K., Robertson, A. D., and Raetz, C. R. H. (1989) J. Biol. Chem, 264, 6956-6966). Soluble fractions from wild-type or heptose-deficient rfa mutants were tested for their ability to convert [4'-32P](KDO)2-IVA to more polar substances. In wild-type extracts, these conversions required addition of ATP or ADP-heptose. In extracts of rfaC-, rfaD-, or rfaE-deficient strains, no polar products were observed with ATP. ADP-heptose restored synthesis in rfaD and rfaE but not rfaC extracts, indicating that rfaD and rfaE are involved in ADP-heptose formation. When the cloned rfaC gene was introduced into an rfaC-deficient mutant, extracts from such cells regained the ability to metabolize [4'-32P](KDO)2-IVA, showing that rfaC encodes the enzyme that attaches the proximal heptose to lipopolysaccharide.
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PMID:The rfaC gene of Salmonella typhimurium. Cloning, sequencing, and enzymatic function in heptose transfer to lipopolysaccharide. 152 14

The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene of Escherichia coli and plays a key role in lipopolysaccharide biosynthesis. It transfers Kdo from CMP-Kdo to lipid A or its tetraacyldisaccharide-1,4'-bisphosphate precursor, lipid IVA. Using a strain that overproduces the transferase approximately 500-fold, we have purified the enzyme to near homogeneity. The subunit molecular mass is approximately 43 kDa. Activity is stimulated by Triton X-100, is maximal at pH 7, but does not require Mg2+. The apparent Km values for lipid IVA and CMP-Kdo are 52 and 88 microM, respectively. Vmax is 15-18 mumol/min/mg when both substrates are added near saturation at pH 8. The purified enzyme transfers 2 Kdo residues to lipid A precursors or analogs bearing four to six fatty acyl chains and a 4'-monosphosphate moiety. Activity is inhibited by polymixin B and Re endotoxin. At low Kdo concentrations small amounts of the intermediate, (Kdo)1-IVA, accumulate. When this substance is isolated and incubated with purified enzyme in the presence of CMP-Kdo, it is converted to (Kdo)2-IVA. Formation of (Kdo)1-IVA is also observed when purified enzyme is incubated with (Kdo)2-IVA and 5 mM CMP, demonstrating that Kdo transfer is reversible. In summary, Kdo transferase consists of a single bifunctional polypeptide that incorporates the 2 innermost Kdo residues common to all lipopolysaccharide molecules in E. coli.
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PMID:Biosynthesis of endotoxins. Purification and catalytic properties of 3-deoxy-D-manno-octulosonic acid transferase from Escherichia coli. 157 28

We have investigated the aggregation behaviour of lipid IVA (a bioactive precursor of lipid A and the lipid anchor of lipopolysaccharide) in aqueous solutions in the physiological pH range using dynamic light scattering, nuclear magnetic resonance, fluorescence, surface pressure, electron microscopy and force field simulation studies. The sonication of lipid IVA in PBS, Tris and Hepes produces vesicles which are stable in the concentration range of 10(-3) - 10(-7) M, possibly even at lower concentrations. The vesicle size is not sensitive to the nature of the buffer, only to the pH and to some extent to the ionic strength. The long time stability of the small unilamellar vesicles as well as the structureless 1H-NMR spectra might be attributed to a rigid surface structure. This structure is also supported by the simulation studies. We have tentatively proposed a coexistence of micelles and/or other aggregates with the bilayered vesicles at higher lipid concentrations in order to explain some of the experimental observations.
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PMID:Aggregation behavior of lipid IVA in aqueous solutions at physiological pH. 1: Simple buffer solutions. 174 9

Lipid A is the active moiety of lipopolysaccharide (LPS, also referred to as endotoxin), a surface component of Gram-negative bacteria that stimulates macrophage activation and causes endotoxic shock. Macrophages can bind, internalize and partially degrade LPS, lipid A and its bioactive precursor, lipid IVA. We report here that lipid IVA binding and subsequent metabolism to a less active form by macrophage-like RAW 264.7 cells is mediated by the macrophage scavenger receptor. Scavenger-receptor ligands inhibit lipid IVA binding to, and metabolism by, RAW cells, and lipid IVA binds to type I and type II bovine scavenger receptors on transfected Chinese hamster ovary cells. Although in vitro competition studies with RAW cells indicate that scavenger receptor binding is not involved in LPS or lipid IVA-induced stimulation of macrophages, in vivo studies show that scavenger-receptor ligands greatly inhibit hepatic uptake of lipid IVA in mice. Thus, scavenger receptors expressed on macrophages may have an important role in the clearance and detoxification of endotoxin in animals.
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PMID:Recognition and plasma clearance of endotoxin by scavenger receptors. 185 9

Bacterial lipopolysaccharide (LPS) is a potent stimulus of cells, yet a target protein for LPS has not been defined. We used two approaches to define LPS-binding sites on cell surfaces: one assay measured binding of LPS-coated erythrocytes (ELPS) to cultured human cells, and a second measured binding of a radiolabeled probe, [32P]lipid IVA, to intact leukocytes. The first approach identified the CD11-CD18 family of integrins as lipid A-binding sites in human phagocytes, and the latter approach demonstrated saturable lipid A binding to intact murine macrophages, as well as to an approximately 95-kDa binding protein in purified membrane preparations. Because CD18 has a known molecular mass of 95 kDa, we sought to determine whether the [32P]lipid IVA-binding site was CD18. Binding of ELPS and [32P]lipid IVA to human macrophages was found to differ with respect to temperature, divalent cation dependence, cellular specificity, and susceptibility to competition by polyanions. To determine whether the previously described 95-kDa lipid A-binding protein was CD18, nitrocellulose-immobilized RAW264.7 membrane proteins were probed with [32P]lipid IVA and subsequently immunoblotted with a monoclonal antibody to murine CD18. The lipid A-binding protein has an electrophoretic mobility slightly different from that of CD18. Moreover, monoclonal antibodies and polyclonal antiserum to the CD11-CD18 family of proteins did not inhibit lipid IVA binding to intact human macrophages. Finally, mononuclear cells from two patients with CD18 deficiency failed to form rosettes with ELPS but bound [32P]lipid IVA normally. Thus, different LPS preparations may bind to cells in a CD18-dependent or -independent manner. Since ELPS is particulate and lipid IVA is a fine dispersion, the identity of the binding site may depend on the physical state of the LPS.
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PMID:Human phagocytes have multiple lipid A-binding sites. 197 20

An autoradiographic assay applicable to colonies immobilized on filter paper was developed for obtaining temperature-sensitive mutants of Escherichia coli defective in the transfer of 3-deoxy-D-manno-octulosonic acid (KDO) from CMP-KDO to a tetraacyldisaccharide 1,4'-bisphosphate precursor of lipid A, designated lipid IVA. Cell-free extracts from two mutants found in a population of 30,000 mutagen-treated cells showed normal KDO transferase activity when assayed at 30 degrees C, but almost no activity at 42 degrees C. The mutation was mapped by mating one of the mutants with different Hfr strains and analyzing genetic linkage of KDO transferase activity to selectable markers. The lesion was located to a position between 80 and 84 min on the E. coli chromosome. A plasmid from the Clarke and Carbon collection (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99), pLC17-24, known to contain genes from the rfa region (81 min), was shown to overexpress KDO transferase activity 4-5 times and to correct the mutation when the plasmid was conjugated into the mutant strains. The KDO transferase gene, designated kdtA, was subcloned from pLC17-24 into a multicopy vector. The resulting plasmid, pCL3, overproduced transferase activity approximately 100-fold. The kdtA gene was shown to code for a 43-kDa polypeptide, as judged by radiolabeling of minicells. Its DNA sequence was determined. The results demonstrate that overexpression of this single gene product greatly stimulates the incorporation of two stereochemically distinct KDO residues during lipopolysaccharide biosynthesis in extracts of E. coli.
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PMID:A gene coding for 3-deoxy-D-manno-octulosonic-acid transferase in Escherichia coli. Identification, mapping, cloning, and sequencing. 203 61

Tumor necrosis factor (TNF) released by lipopolysaccharide (LPS)-stimulated mononuclear phagocytes is a critical mediator of sepsis. We examined the capacities of rough mutant Salmonella typhimurium LPS (Rc) and LPS partial structures lipid A, monophosphoryl lipid A (MPLA), lipid IVA, and lipid X to induce production of TNF in whole blood. Rc LPS (0.0001-10 ng/ml) produced a dose-dependent release of TNF as determined by cytotoxicity of actinomycin D-sensitized L929 murine fibroblasts. Lipid A, MPLA, lipid IVA, and lipid X exhibited decreasing capacities to stimulate production of TNF in whole blood, respectively. Fractional deacylation of LPS by incubation with acyloxyacyl hydrolase isolated from human leukocytes produced a reduction in the capacity of LPS to induce TNF release in whole blood. Maximal enzymatic deacylation reduced activity of LPS by greater than 100-fold. Coincubation with lipid IVA inhibited TNF release induced by Rc LPS or lipid A, but not by phorbol ester. In contrast, MPLA, lipid X, and deacylated LPS failed to inhibit LPS-stimulated release of TNF. Corresponding to the inhibition of the release of TNF protein, lipid IVA also inhibited the accumulation of TNF mRNA in LPS-stimulated mononuclear cells. These results suggest that lipid IVA may act as a competitive antagonist of LPS, perhaps at the receptor level.
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PMID:Lipid IVA inhibits synthesis and release of tumor necrosis factor induced by lipopolysaccharide in human whole blood ex vivo. 219 1

A new class of antibacterial agents for Gram-negative bacteria, rationally designed to inhibit the incorporation of 3-deoxy-D-manno-octulosonate into lipopolysaccharide (LPS), was recently reported. In Salmonella typhimurium, where the lipid A species are well characterised, it was previously demonstrated that the addition of a compound which inhibits the enzyme 3-deoxy-manno-octulosonate cytidylytransferase (CMP-KDO synthetase; EC 2.7.7.38) leads to rapid accumulation of lipid A derivatives. The major lipid A species, IVA (O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1-6)-2-amino-2-deoxy-alpha-D - glucose, acylated at positions 2, 3, 2', 3' with beta-hydroxymyristoyl groups and bearing phosphates at positions 1 and 4'), was shown to be converted mainly to LPS by pulse-chase experiments in the absence of inhibitor. Labelled precursor (IVA) was also chased to other more polar lipid A derivatives. During chase in the presence of inhibitor, there was no conversion to LPS, while the major lipid A species was converted to the same polar lipid A derivatives as in chase without inhibitor. Our data indicate that despite the accumulation of several species of lipid A derivatives during inhibition of LPS synthesis, only IVA is destined for synthesis of mature LPS when LPS synthesis resumes. The more polar lipid A derivatives would thus represent aberrant side reaction products which occur when the pathway is inhibited.
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PMID:Accumulation of incomplete metabolic side products of lipid A in Salmonella typhimurium during inhibition of 3-deoxy-D-manno-octulosonate incorporation by a new class of antibacterial agents. 254 96

Previous studies in our laboratory led to the elucidation of the covalent structure of a tetraacyldisaccharide 1,4'-bisphosphate precursor of lipid A (designated lipid IVA), that accumulates at 42 degrees C in temperature-sensitive mutants defective in 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). Using [4'-32P]lipid IVA as the probe, we now demonstrate the existence of cytoplasmic KDO-transferases in Escherichia coli capable of attaching 2 KDO residues, derived from CMP-KDO, to lipid IVA. A partial purification has been developed to obtain a cytoplasmic subfraction that adds these 2 KDO residues with a 90% yield. The product is shown to have the stoichiometry of (KDO)2-IVA by fast atom bombardment mass spectrometry and NMR spectroscopy. The partially purified enzyme can utilize alternative lipid-disaccharide cosubstrates bearing five or six fatty acyl chains, but it has an absolute requirement for a monophosphate residue at position 4' of the lipid acceptor. When reincubated with a crude cytoplasmic fraction, a nucleoside triphosphate and Mg2+, (KDO)2-IVA is rapidly metabolized to more polar substances, the identity of which is unknown. The KDO-transferase(s) described in the present study should be very useful for the semisynthetic preparation of complex lipopolysaccharide substructures and analogs.
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PMID:Biosynthesis of lipopolysaccharide in Escherichia coli. Cytoplasmic enzymes that attach 3-deoxy-D-manno-octulosonic acid to lipid A. 265 35

Antibacterial agents which specifically inhibit CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase activity were used to block the incorporation of 3-deoxy-D-manno-octulosonate (KDO) into lipopolysaccharide. Lipopolysaccharide synthesis ceased, molecules similar in structure to lipid A accumulated, and bacterial growth ceased following addition of such agents to cultures of Salmonella typhimurium and Escherichia coli. Although four major species of lipid A accumulated in S. typhimurium, their kinetics of accumulation were different. The least polar of the major species was IVA [O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-(1----6)-2-amino-2-deoxy-a lph a- D-glucose, acylated at positions 2, 3, 2', and 3' with beta-hydroxymyristoyl groups and bearing phosphates at positions 1 and 4'], a molecule previously isolated from bacteria containing a kdsA mutation (C. R. H. Raetz, S. Purcell, M. V. Meyer, N. Qureshi, and K. Takayama, J. Biol. Chem. 260:16080-16088, 1985). Species IVA accumulated first and to the greatest extent following addition of the inhibitor, with other more polar derivatives appearing only after IVA attained half its maximal level. In contrast, only two major species of precursor accumulated in E. coli following addition of the inhibitor. One of these species was identical to IVA from S. typhimurium on the basis of chemical composition, fast atom bombardment mass spectroscopy, and comigration on Silica Gel H, and it also accumulated prior to a more polar species of related structure. We conclude that the addition of KDO to precursor species IVA is the major pathway of lipid A-KDO formation in both S. typhimurium LT2 and E. coli and that accumulation of the more polar species lacking KDO only occurs in response to accumulation of species IVA following inhibition of the normal pathway.
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PMID:Analysis of lipopolysaccharide biosynthesis in Salmonella typhimurium and Escherichia coli by using agents which specifically block incorporation of 3-deoxy-D-manno-octulosonate. 283 31


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