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)

The integral membrane protein WecA mediates the transfer of N-acetylglucosamine (GlcNAc) 1-phosphate to undecaprenyl phosphate (Und-P) with the formation of a phosphodiester bond. Bacteria employ this reaction during the biosynthesis of enterobacterial common antigen as well as of many O-specific lipopolysaccharides (LPSs). Alignment of a number of prokaryotic and eukaryotic WecA-homologous sequences identified a number of conserved aspartic acid (D) residues in putative cytoplasmic loops II and III of the inner-membrane protein. Site-directed mutagenesis was used to study the role of the conserved residues D90, D91 (loop II), D156 and D159 (loop III). As controls, D35, D94 and D276 were also mutagenized. The resulting WecA derivatives were assessed for function by complementation analysis of O-antigen biosynthesis, by the ability to incorporate radiolabelled precursor to a biosynthetic intermediate, by detection of the terminal GlcNAc residue in LPS and by a tunicamycin competition assay. It was concluded from these analyses that the conserved aspartic acid residues are functionally important, but also that they participate differently in the transfer reaction. Based on these results it is proposed that D90 and D91 are important in forwarding the reaction product to the next biosynthetic step, while D156 and D159 are a part of the catalytic site of the enzyme.
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PMID:Conserved aspartic acids are essential for the enzymic activity of the WecA protein initiating the biosynthesis of O-specific lipopolysaccharide and enterobacterial common antigen in Escherichia coli. 1183 20

We have studied the folding pathway of a beta-barrel membrane protein using outer membrane protein A (OmpA) of Escherichia coli as an example. The deletion of the gene of periplasmic Skp impairs the assembly of outer membrane proteins of bacteria. We investigated how Skp facilitates the insertion and folding of completely unfolded OmpA into phospholipid membranes and which are the biochemical and biophysical requirements of a possible Skp-assisted folding pathway. In refolding experiments, Skp alone was not sufficient to facilitate membrane insertion and folding of OmpA. In addition, lipopolysaccharide (LPS) was required. OmpA remained unfolded when bound to Skp and LPS in solution. From this complex, OmpA folded spontaneously into lipid bilayers as determined by electrophoretic mobility measurements, fluorescence spectroscopy, and circular dichroism spectroscopy. The folding of OmpA into lipid bilayers was inhibited when one of the periplasmic components, either Skp or LPS, was absent. Membrane insertion and folding of OmpA was most efficient at specific molar ratios of OmpA, Skp, and LPS. Unfolded OmpA in complex with Skp and LPS folded faster into phospholipid bilayers than urea-unfolded OmpA. Together, these results describe a first assisted folding pathway of an integral membrane protein on the example of OmpA.
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PMID:Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide. 1250 34

Tissue factor (TF) is an integral membrane protein that binds factor VIIa and initiates coagulation. The extracellular domain of TF is responsible for its hemostatic function and by implication in the dysregulation of coagulation, which contributes to death in endotoxemia. The role of the cytoplasmic domain of tissue factor in endotoxemia was studied in mice, which lack the cytoplasmic domain of TF (TF(deltaCT/deltaCT)). These mice develop normally and have normal coagulant function. Following i.p injection with 0.5 mg of lipopolysaccharide (LPS), TF(deltaCT/deltaCT) mice showed significantly greater survival at 24 hours compared to the wt mice (TF(+/+)). The serum levels of TNF-alpha and IL-1beta were significantly lower at 1 hour after LPS injection and IL-6 levels were significantly lower at 24 hours in TF(deltaCT/deltaCT) mice compared to TF(+/+)mice. Neutrophil recruitment into the lung was also significantly reduced in TF(deltaCT/deltaCT) mice. Nuclear extracts from tissues of endotoxemic TF(deltaCT/deltaCT) mice also showed reduced NFkappaB activation. LPS induced leukocyte rolling, adhesion, and transmigration in post-capillary venules assessed by intravital microscopy was also significantly reduced in TF(deltaCT/deltaCT) mice. These results indicate that deletion of the cytoplasmic domain of TF impairs the recruitment and activation of leukocytes and increases survival following endotoxin challenge.
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PMID:The cytoplasmic domain of tissue factor contributes to leukocyte recruitment and death in endotoxemia. 1521 87

Tissue factor (TF) is an integral membrane protein essential for hemostasis. During the past several years, a number of studies have suggested that physiologically active TF circulates in blood at concentrations greater than 30 pM either as a component of blood cells and microparticles or as a soluble plasma protein. In our studies using contact pathway-inhibited blood or plasma containing activated platelets, typically no clot is observed for 20 minutes in the absence of exogenous TF. An inhibitory anti-TF antibody also has no effect on the clotting time in the absence of exogenous TF. The addition of TF to whole blood at a concentration as low as 16 to 20 fM results in pronounced acceleration of clot formation. The presence of potential platelet TF activity was evaluated using ionophore-treated platelets and employing functional and immunoassays. No detectable TF activity or antigen was observed on quiescent or ionophore-stimulated platelets. Similarly, no TF antigen was detected on mononuclear cells in nonstimulated whole blood, whereas in lipopolysaccharide (LPS)-stimulated blood a significant fraction of monocytes express TF. Our data indicate that the concentration of physiologically active TF in non-cytokine-stimulated blood from healthy individuals cannot exceed and is probably lower than 20 fM.
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PMID:Tissue factor activity in whole blood. 1560 22

The ABC transporter MsbA is an integral membrane protein involved in the transport of lipid A and lipopolysaccharides to the outer leaflet of the inner membrane in bacteria. Here, the critical role of the natural substrate lipopolysaccharide in the crystallization and diffraction quality of MsbA crystals is reported. Initial crystals grown in complex with ATP-vanadate alone diffracted to approximately 9 A. Screening of the natural substrate lipopolysaccharides led to the crystallization of MsbA in complex with ADP-vanadate and Ra lipopolysaccharide. The increased order within the crystal lattice allowed structure determination to 4.2 A.
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PMID:Lipopolysaccharide stabilizes the crystal packing of the ABC transporter MsbA. 1651 Nov 20

WecA is an integral membrane protein that initiates the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide (LPS) by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate (Und-P) to form Und-P-P-GlcNAc. WecA belongs to a large family of eukaryotic and prokaryotic prenyl sugar transferases. Conserved aspartic acids in putative cytoplasmic loops 2 (Asp90 and Asp91) and 3 (Asp156 and Asp159) were targeted for replacement mutagenesis with either glutamic acid or asparagine. We examined the ability of each mutant protein to complement O-antigen LPS synthesis in a wecA-deficient strain and also determined the steady-state kinetic parameters of the mutant proteins in an in vitro transfer assay. Apparent K(m) and V(max) values for UDP-GlcNAc, Mg(2+), and Mn(2+) suggest that Asp156 is required for catalysis, while Asp91 appears to interact preferentially with Mg(2+), possibly playing a role in orienting the substrates. Topological analysis using the substituted cysteine accessibility method demonstrated the cytosolic location of Asp90, Asp91, and Asp156 and provided a more refined overall topological map of WecA. Also, we show that cells expressing a WecA derivative C terminally fused with the green fluorescent protein exhibited a punctate distribution of fluorescence on the bacterial surface, suggesting that WecA localizes to discrete regions in the bacterial plasma membrane.
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PMID:Functional characterization and membrane topology of Escherichia coli WecA, a sugar-phosphate transferase initiating the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide. 1723 64

The term sepsis describes a potentially lethal clinical condition that develops as a result of a dysregulated host response to bacterial infection. The most common bacterial component implicated in initiating the septic syndrome is a cell wall molecule derived from Gram-negative bacteria, known as lipopolysaccharide (LPS) or endotoxin. Like all mammals, humans are equipped with an LPS-sensing machinery consisting, primarily, of LPS-binding protein (LBP), CD14, a glycosylphosphatidylinositol (GPI)-anchored monocyte differentiation antigen, and toll-like receptor 4 (TLR4), a signal-transducing integral membrane protein. Modest stimulation of TLR4 facilitates the elimination of invading microorganisms. Potent TLR4 stimulation, however, produces severe reactions in the host, often leading to multiple organ failure and death. The search for pharmaceuticals that reduce mortality in septic patients has been a painstaking process. Thus far, only a few compounds have been found to significantly reduce mortality rates. Perhaps one of the more promising therapeutic strategies currently pursued is based on the identification of synthetic or naturally occurring substances that neutralize LPS or inhibit LPS-mediated activation of host immune cells, such as monocytes and macrophages. Here, we describe a number of diverse molecular structures with a capacity to either enhance or blunt LPS-induced monocyte activation. The underlying molecular mechanisms are discussed.
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PMID:Targeting bacterial endotoxin: two sides of a coin. 1740 10

Membrane type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane protein that participates in the processing and degradation of cell surface proteins and the extracellular matrix (ECM). This enzyme regulates ECM turnover in wound repair, promotes cell migration and activates other MMPs, such as MMP-2, which is involved in angiogenesis, cell migration and tumoral metastasis. An increase in pro-inflammatory cytokine expression, such as gamma interferon (IFN-gamma), has been associated with chronic wounds in inflammatory bowel diseases. However, the extent to which cytokines modulate MT1-MMP has not been totally defined. In this report, the effects of the bacterial lipopolysaccharide (LPS) and ECM-bound IFN-gamma on MT1-MMP expression and MMP-2 activity were evaluated by Western blot, RT-PCR and zymography in isolated intestinal epithelial and cultured HT-29 cells. In the presence of LPS, ECM-bound IFN-gamma, but not soluble IFN-gamma, reduced the enterocyte MT1-MMP protein expression. In addition, the active form of MMP-2 was also decreased in the presence of both LPS and IFN-gamma, indicating that lower MMP-2 activity accompanied the decrease in MT1-MMP expression. These results suggest the possibility that endotoxin and ECM-bound IFN-gamma may affect matrix remodeling by modulating matrix metalloproteinase in enterocytes during wound healing.
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PMID:Modulation of membrane type 1 matrix metalloproteinase by LPS and gamma interferon bound to extracellular matrix in intestinal crypt cells. 1816 51

The WecA transferase is an integral membrane protein and a member of the polyprenyl phosphate N-acetylhexosamine-1-phosphate transferase superfamily. It initiates the biosynthesis of various bacterial cell envelope components such as the lipopolysaccharide O-antigen. We report on the first large-scale enzymatic synthesis, purification, and characterization of the undecaprenyl-pyrophosphoryl-N-acetylglucosamine product of the WecA transferase. This is an essential lipid intermediate for the biosynthesis of various bacterial cell envelope components. Its availability in a pure form will allow the biochemical and structural characterization of the various enzymes requiring it as a substrate for the synthesis of cell wall polymers.
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PMID:Preparative enzymatic synthesis of polyprenyl-pyrophosphoryl-N-acetylglucosamine, an essential lipid intermediate for the biosynthesis of various bacterial cell envelope polymers. 1944 46

Ectodomain shedding is a posttranslational modification mechanism, which liberates extracellular domains of membrane proteins through juxtamembrane processing executed mainly by the ADAM (a disintegrin and metalloprotease) family of metalloproteases. Shedding is a unique and effective mechanism for inducing multifaceted effects through the soluble extracellular domains released and/or the remaining membrane-bound portions; however, the physiological functions of shedding are not yet fully understood. In this study, we performed unbiased proteomic screening for shedding targets in a lipopolysaccharide (LPS)-stimulated macrophage cell line to elucidate a new immunological function of shedding. We identified VIP36 (36-kDa vesicular integral membrane protein), a lectin domain-containing transmembrane protein postulated as a cargo receptor for Golgi-to-endoplasmic reticulum transport, as a new target for shedding and found that the shedding of VIP36 occurs mainly on the cell surface. In addition, we demonstrate that the amount of VIP36 precisely regulates phagocytosis in macrophages and that the shedding of VIP36 is required for this regulation. These results substantially expand our knowledge of the immunological and cell biological functions of both the shedding process and VIP36 itself.
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PMID:VIP36 protein is a target of ectodomain shedding and regulates phagocytosis in macrophage Raw 264.7 cells. 2201 86


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