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

Previous research in this laboratory, using photoactivatable radioiodinated lipopolysaccharide derivatized with sulfosuccinimidyl-2-(p-azidosalicylamide)-1,3'-dithiopropionate (125I-ASD-LPS), has resulted in the identification of a specific LPS receptor with a molecular mass of approximately 73 kDa on murine lymphocytes and splenic macrophages. The experiments presented in this report investigated whether a similar LPS-binding protein was also expressed on human peripheral blood populations, including monocytes, lymphocytes, neutrophils, platelets, and erythrocytes. Each cell population was incubated with 125I-ASD-LPS, UV irradiated, washed, reduced, and solubilized, and the cell lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. On all of the cell populations, except erythrocytes, a similar 73-kDa LPS-binding protein was present. In addition, each population also expressed lower-molecular-weight secondary LPS-binding proteins, some of which were conserved among the populations. Binding of the photoactivatable LPS probe was found to be both time and temperature dependent. These data support the concept that the 73-kDa LPS-binding protein is conserved on multiple cell types from a variety of species.
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PMID:Identification and characterization of lipopolysaccharide-binding proteins on human peripheral blood cell populations. 137 70

A radioiodinated, photoactivatable derivative of Salmonella minnesota Re595 lipopolysaccharide (LPS) was used to label LPS-binding proteins in 70Z/3 cells. The labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. 125I-Labeled-2-(p-azidosalycylamido)1,3'-dithiopropionamide S. minnesota Re595 LPS (125I-ASD-Re595) labeled a limited number of proteins. The most prominent of these had a apparent molecular mass of 18 kDa. Less prominent labeling of 25- and 28-kDa proteins was also seen. Labeling was saturated by 5 micrograms/ml 125I-ASD-Re595 and was inhibited by a 10-100-fold excess of unlabeled LPS or lipid A. Labeling was maximal within 30 min at 37 degrees C; much less labeling occurred at lower temperatures. The proteins labeled with 125I-ASD-Re595 appear to be on the surface of the cell, since they can be digested by trypsin and were found in the membrane fraction of the cell but not in the cytosol. Studies with competitive inhibitors suggested that the proteins bind to the lipid A region of the LPS molecule. Biologically inactive lipid A analogs were poor inhibitors of labeling, suggesting that the LPS-binding proteins could discriminate between active lipid A and inactive analogs. These studies suggest that the 18- and 25-kDa proteins bind specifically to the lipid A region of the LPS molecule and should be considered as candidates for a functional LPS receptor.
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PMID:Identification of lipopolysaccharide-binding proteins in 70Z/3 cells by photoaffinity cross-linking. 234 80

125I-ASD photoaffinity-labeling derivatives of pertussis toxin (125I-ASD-PT) or lipopolysaccharide (125I-ASD-LPS) labeled similar 70-kDa proteins in Jurkat cells, a cell line derived from human CD4+ T lymphocytes. Labeling of this 70-kDa protein by 125I-ASD-PT was inhibited by underivatized PT but not by underivatized LPS. However, an immunoglobulin M monoclonal antibody with specificity for the p73 LPS receptor in murine splenocytes (S. W. Bright, T.-Y. Chen, L. M. Flebbe, M.-G. Lei, and D. C. Morrison, J. Immunol. 145:1-7, 1990) inhibited 125I-ASD-PT labeling of the 70-kDa species in Jurkat cells. Our results suggested that PT may bind to the same 70-kDa protein as LPS does in Jurkat cells but that PT and LPS bind to different sites on this receptor candidate. 125I-ASD-PT photoaffinity labeling of the 70-kDa protein was also inhibited by underivatized glycoproteins to which PT has been shown to bind, and this inhibition correlated with the relative binding affinities of the glycoproteins for PT. 125I-ASD derivatives of two sialic acid-specific plant lectins, Maackia amurensis leukoagglutinin and Sambucus nigra agglutinin, with oligosaccharide binding specificities similar to those of PT also labeled a 70-kDa protein in Jurkat cells. This suggests that the 70-kDa PT receptor candidate in Jurkat cells likely contains sialooligosaccharide sequences to which PT, M. amurensis leukoagglutinin, and S. nigra agglutinin bind. The cross-reacting epitope recognized by monoclonal antibody 5D3 in this 70-kDa species might overlap the PT- and LPS-binding sites.
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PMID:The 70-kilodalton pertussis toxin-binding protein in Jurkat cells. 751 75

Using a photoactivable, radioiodinated lipopolysaccharide probe, [125I]ASD-LPS (derivatized from purified E. coli 0111:B4 S-LPS), we earlier reported the presence of a 73-kDa (p73) predominant LPS-binding protein on mouse lymphocytes and macrophages with specificity for the lipid A region of LPS. Both Re-LPS from Salmonella minnesota and purified lipid A will inhibit the binding of LPS to the p73 LPS receptor. In the studies reported here, we have found that non-toxic diphosphoryl lipid A purified from Rhodo-pseudomonas sphaeroides has the capability to inhibit the binding of [125I]ASD-LPS to the p73 protein. However, using the same LPS probe and photoaffinity cross-linking techniques, our data suggest that a less dominant 38-kDa (p38) LPS-specific binding protein identified on mouse splenocytes, J774.1 macrophage-like cell line, and 70Z/3 pre B-cell line by SDS-PAGE is not inhibited by purified lipid A, even at a concentration in 50-fold excess of that of [125]ASD-LPS. The binding of the LPS probe to the 38 protein could be inhibited in a dose-dependent manner by underivatized native S. minnesota Re-LPS (composed only of Kdo and lipid A). We speculate that this p38 LPS-binding protein may manifest a specificity for inner core oligosaccharide determinants on LPS.
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PMID:Lipopolysaccharide (LPS) binding to 73-kDa and 38-kDa surface proteins on lymphoreticular cells: preferential inhibition of LPS binding to the former by Rhodopseudomonas sphaeroides lipid A. 769 Mar 43

Endogenous regulatory mechanisms exist in mammals that enable a rapid response to lipopolysaccharide (LPS, endotoxin) stemming from gram-negative bacterial infections. Serum proteins and cell surface receptors exist that bind LPS, and this interaction may either aid in nonpathogenic removal of LPS from the body or potentiate the effects of LPS. We have used a photoreactive, thiol-cleavable, radiolabeled derivative of E. coli 0111:B4 LPS [LPS-(p-azidosalicylamido)-1,3'-dithiopropionamide; 125I-ASD-LPS], to identify the presence of LPS-binding proteins (LBPs) in bovine serum. Ion exchange chromatography was used to fractionate bovine serum, and eluted protein was subsequently photoaffinity labeled using 125I-ASD-LPS. LBPs were identified by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. Several LBPs including three with apparent molecular masses of 65, 60, and 50 kDa were variably present within the chromatography pools. A 22-residue NH2-terminal amino acid sequence of the 60-kDa protein showed 77% homology with human LBP and 68% with rabbit LBP within this region. Further purification utilizing high-performance liquid chromatography yielded a protein fraction that contained the 60-kDa protein and was distinctly more active than whole bovine serum in LPS-dependent macrophage activation assays (up to 1600-fold on a weight/volume basis). The LPS-mediated macrophage activation in concert with chromatographically purified serum protein in tissue factor assays was inhibitable using anti-CD14 monoclonal antibodies. The results indicate that an LPS-binding protein exists in samples of pooled bovine serum and that this protein has features in common with human and rabbit LBP.
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PMID:Identification and characterization of a bovine lipopolysaccharide-binding protein. 799 53

Using radioiodinated, photoactivable, reducible cross-linker conjugated bacterial endotoxic lipopolysaccharide (125I-ASD-LPS), we have demonstrated that LPS selectively binds to the S2 subunit of pertussis toxin (PT). Since LPS also interacts with the S2 subunit of the B-oligomer of the toxin, the binding of LPS to PT is not A-protomer (S1 subunit) dependent. The binding can be inhibited with native underivatized LPS and with purified lipid A, suggesting that the binding is mediated through the lipid A moiety of the LPS molecule. The binding of PT to LPS can be inhibited by bovine fetuin glycoprotein. Since PT has been demonstrated to interact specifically with N-linked oligosaccharide side chains of fetuin, the interaction of LPS with the S2 subunit of PT may involve carbohydrate-dependent interactions of the disaccharide backbone of lipid A with S2. Additional studies have documented that LPS binding to PT may be competitively inhibited by lysozyme but not by polymyxin B. Sequence analysis has allowed identification of a high degree of amino acid sequence similarity between the S2 subunit of PT and hen egg white lysozyme at the N-terminal 80-residue regions. Shared N-terminal sequence similarity between lysozyme, PT-S2, and a third LPS-binding protein alpha-lactalbumin allows tentative identification of a second family of LPS binding proteins.
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PMID:Lipopolysaccharide interaction with S2 subunit of pertussis toxin. 841 48

Limulus amebocyte lysate, obtained from horseshoe crab (Limulus polyphemus) blood cells, contains a coagulation system which is activated by bacterial lipopolysaccharide (LPS). A chromatographic fraction of Limulus lysate, containing the endotoxin-sensitive factor(s) which initiates the coagulation cascade, was studied. We utilized a photoreactive, cleavable, radiolabeled derivative of Salmonella minnesota LPS, LPS-(p-azidosalicylamido)-1,3'-dithiopropionamide (LPS-ASD), to identify LPS-binding proteins. The lysate fraction was incubated with LPS-ASD, and LPS-binding proteins were identified by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. An 82-kDa protein, a major protein component of this fraction from Limulus lysate, was identified as a LPS-binding protein in a majority of lysates. Incubation of whole Limulus lysate with antiserum to this protein resulted in enhanced sensitivity of the lysate to LPS, suggesting that this 82-kDa protein is a negative regulator of coagulation. A minor 50-kDa protein component of lysate also was identified as a LPS-binding protein and is a candidate for the LPS-sensitive coagulation protein in L. polyphemus.
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PMID:Lipopolysaccharide-binding proteins of Limulus amebocyte lysate. 843 86

p73, a binding site for lipopolysaccharide (LPS) on human peripheral blood monocytes was identified using the radiolabeled photoaffinity cross-linker sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD). The 125I-labeled conjugate of SASD and LPS (125I-labeled ASD-LPS) was bound to monocytes and UV cross-linked, and the cellular extracts were analyzed with two-dimensional SDS-PAGE and autoradiography. In addition to the major binding site on human monocytes at 73 kDa, isoelectric point 5.95, there were multiple minor binding sites that recognized both smooth and rough LPS. Binding of 125I-labeled ASD-LPS to monocytes is concentration dependent, decreased in the absence of calcium and magnesium, and inhibited by either excess LPS or the low-molecular-weight soluble isolate of bacterial cell wall peptidoglycan (sPGN). However, sPGN only minimally stimulates tumor necrosis factor (TNF) secretion by human peripheral blood mononuclear cells. In contrast, the relatively insoluble high-molecular-weight peptidoglycan significantly stimulates TNF secretion.
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PMID:Lipopolysaccharide and peptidoglycan share binding sites on human peripheral blood monocytes. 851

We have previously shown that polymorphonuclear leukocytes (PMN) from cord blood of normal full-term infants have a decreased priming response to lipopolysaccharide (LPS) compared with PMN of adults. Because the reason for this difference is poorly understood, we compared LPS binding on PMN from adults and newborns by using a photoactivatable iodinated LPS (from Escherichia coli O111:B4), coupled to 2-(p-azidosalicylamido)-1,3'-dithopropionate (LPS-ASD) to covalently link LPS to the PMN membrane. We incubated 2 x 10(4) adult or neonatal PMN with 125I-ASD-LPS (100 ng/ml) together with unlabelled LPS (0 to 100,000 ng/ml) for 20 min at 4 degrees C. The maximum total 125I-ASD-LPS binding to newborn PMN (1,004 +/- 103 cpm) was lower than that binding to adult PMN (3,583 +/- 444 cpm; P < 0.01 with respect to newborn PMN). However, the concentration of unlabelled LPS that displaced 50% of the maximum specifically bound 125I-ASD-LPS was similar for PMN from adult and newborn infants (-4.85 +/- 0.04 and -5.13 +/- 0.14 log g of LPS per ml, respectively; P > 0.05). We further assessed the membrane binding of 125I-ASD-LPS to PMN by using membrane extracts analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. LPS binding proteins were found at approximately 73, 55 to 57, and 25 kDa in both adult and neonatal PMN. However, PMN from newborn infants had markedly lower membrane-associated 125I-ASD-LPS at the 55- to 57- and 25-kDa protein bands as indicated by the intensity of the autoradiograph. Binding of LPS at these bands was specific for the lipid A portion of LPS, since purified unlabelled lipid A displaced 125I-ASD-LPS in a dose-dependent manner. Thus, PMN from newborn infants bind less LPS than do PMN from adults, even though the sites for LPS membrane binding appear to be the same.
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PMID:Lipopolysaccharide binding proteins on polymorphonuclear leukocytes: comparison of adult and neonatal cells. 889 Feb 18

The structural features of some proteins of the innate immune system involved in mediating responses to microbial pathogens are highly conserved throughout evolution. Examples include members of the Drosophila Toll (dToll) and the mammalian Toll-like receptor (TLR) protein families. Activation of Drosophila Toll is believed to occur via an endogenous peptide rather than through direct binding of microbial products to the Toll protein. In mammals there is a growing consensus that lipopolysaccharide (LPS) initiates its biological activities through a heteromeric receptor complex containing CD14, TLR4, and at least one other protein, MD-2. LPS binds directly to CD14 but whether LPS then binds to TLR4 and/or MD-2 is not known. We have used transient transfection to express human TLRs, MD-2, or CD14 alone or in different combinations in HEK 293 cells. Interactions between LPS and these proteins were studied using a chemically modified, radioiodinated LPS containing a covalently linked, UV light-activated cross-linking group ((125)I-ASD-Re595 LPS). Here we show that LPS is cross-linked specifically to TLR4 and MD-2 only when co-expressed with CD14. These data support the contention that LPS is in close proximity to the three known proteins of its membrane receptor complex. Thus, LPS binds directly to each of the members of the tripartite LPS receptor complex.
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PMID:Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. transfer from CD14 to TLR4 and MD-2. 1127 65


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