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1.
Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM). MALDI time-of-flight (TOF) mass spectrometry was used to characterize heterogeneity in the native LOS from Neisseria gonorrheae and N. meningitidis. A sample preparation methodology previously reported for Escherichia coli lipopolysaccharide (LPS) employing deposition of untreated LOS on a thin layer of a film composed of 2,4,6-trihydroxyacetophenone and nitrocellulose was used. Prominent peaks were observed corresponding to molecular ions and to fragment ions primarily formed by cleavage between the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and the lipid A (LA). Analyses of these data and comparison with spectra of the corresponding O-deacylated or hydrogen fluoride-treated LOS enabled the detection of novel species that apparently differed by the expression of up to three phosphates with one or more phosphoethanolamine (PEA) groups on the LA. We found that the heterogeneity profile of acylation and phosphorylation correlates with the induction of proinflammatory cytokines in THP-1 monocytic cells. This methodology enabled us to rapidly profile components of structural variants of native LOS that are of importance biologically. 相似文献
2.
Size heterogeneity of Salmonella typhimurium lipopolysaccharides in outer membranes and culture supernatant membrane fragments. 总被引:40,自引:11,他引:29
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Enterobacteriaceae cells growing in liquid media shed fragments of their outer membranes. These fragments, which may constitute a biologically important form of gram-negative bacterial endotoxin, have been reported to contain proteins, phospholipids, and lipopolysaccharides (LPS). In this study we compared the sizes of LPS molecules in shed membrane fragments and outer membranes from cells growing in broth cultures. Using conditional mutants of Salmonella typhimurium which incorporate specific sugars into LPS, we analyzed radiolabeled LPS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique revealed that S. typhimurium LPS are more heterogeneous than previously known; molecules possessing from 0 to more than 30 O-chain repeat units were identified in outer membranes, supernatant fragments, and purified LPS. The size distributions of LPS molecules in outer membranes and supernatant fragments were similar; supernatant fragments appeared to be slightly enriched in molecules with long O-polysaccharide chains. Our results indicate the LPS molecules of many sizes are synthesized, translocated to outer membranes, and released into culture supernatants. Since the hydrophilic O-polysaccharides extend from bacterial surfaces into the aqueous environment, our findings suggest that the cell surface topography of this bacterium may be very irregular. We also speculate that heterogeneity in the degree of polymerization of O-antigenic side chains may influence the interactions of the toxic moiety of LPS (lipid A) with host constituents. 相似文献
3.
Rough mutants of Salmonella typhimurium: immunochemical and structural analysis of lipopolysaccharides from rfaH mutants 总被引:10,自引:0,他引:10
Lipopolysaccharides, extracted by phenol/chloroform/petroleum ether, from two rough mutants of Salmonella typhimurium of class rfaH were studied by passive haemagglutination inhibition and by methylation analysis. The structural and immunochemical analyses showed that (i) formation of the galactose I unit of the core is defective, but the defect is not complete, and (ii) of those core chains which do receive the galactose I residue, many are not continued to form complete core, but instead terminate at intermediate points. This suggests that the rfaH gene, though involved in formation of the galactose I unit, is not the structural gene for the galactosyltransferase which adds this unit. The rfaH product may be a positive regulator for several rfa genes specifying glycosyltransferases, or it may be a protein needed for the efficient action of several such transferases. 相似文献
4.
Comparison of lipopolysaccharides from Agmenellum quadruplicatum to Escherichia coli and Salmonella typhimurium by using thin-layer chromatography. 总被引:5,自引:3,他引:2
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Lipopolysaccharide (LPS) was isolated from the unicellular blue-green bacterium Agmenellum quadruplicatum using the procedure of Westphal and Jann (1965). It was composed of a lipid A and polysaccharide region suggesting a similarity to other gram-negative LPSs. Chemical analyses demonstrated the presence of glucose, rhamnose, mannose, and xylose in the polysaccharide region, as well as 2-keto-3-deoxyoctonate, glucosamine, and phosphorous in the lipid A. Studies on the lipid composition revealed the presence of palmitic, behenic, and three beta-hydroxy fatty acids. A new procedure for thin-layer chromatography of bacterial LPSs was used to compare LPS from A. quadruplicatum to other gram-negative organisms. The method is capable of distinguishing between LPSs of different bacteria as well as between the wild-type organism and mutated forms unable to synthesize complete LPS. A comparison of LPS from A. quadruplicatum to Escherichia coli and Salmonella typhimurium demonstrated that, although the blue-green LPS was rather similar to that of the Enterobacteriaceae, distinct differences also existed. However, when several cell division mutants of A. quadruplicatum were compared chromatographically to the parent strain BG-1, no differences were observed. This suggests that cell division mutations in A. quadruplicatum are not associated with changes in the LPS. 相似文献
5.
K G Johnson M B Perry I J McDonald R R Russel 《Canadian journal of microbiology》1975,21(12):1969-1980
Cultures of eight non-pathogenic species of Neisseria grown in simple defined media released lipopolysaccharide (free lipopolysaccharide) by a process distinct from cellular autolysis. Analyses of the pure cellular and free lipopolysaccharides obtained from six species of Neisseria revealed that they were remarkably similar and were devoid of detectable O-antigen side chains. Three distinct types of core-oligosaccharides were demonstrated. Type I core-oligosaccharide was a branched structure of alpha-D-glucopyranosyl units (7 mol) terminated by a reducing end group of 3-deoxy-D-manno-octulosonic acid. Type II core-oligosaccharide contained D-glucose, 2-deoxy-2-amino-D-glucose, L-rhamnose, L-glycero-D-manno-heptose, 3-deoxy-D-manno-octulosonic acid, phosphate, and ethanolamine in a molar ratio of 3:2:1:1:1:1:1. Type III coreoligosaccharide was composed of D-glucose, L-glycero-D-manno-heptose, 3-deoxy-D-manno-octulosonic acid, and phosphate in a molar ratio of 3:3:1:1. Lipopolysaccharides of N. caviae and N. sicca contained type I core-oligosaccharides exclusively, while those of N. flava and N. perflava contained only type II core-oligosaccharide. Cellular lipopolysaccharide from N. cinerea contained core-oligosaccharides of types I and II in a ratio of 27:73, while the analogous preparation from N. flavescens contained core-oligosaccharide types II and III in a ratio of 21:4. Free lipopolysaccharides from these two organisms contained only one type of coreoligosaccharide. Lipid A components of all the lipopolysaccharide preparations were very similar being composed of about 25% by weight of dodecanoic acid, 3-hydroxy-dodecanoic acid, and 3-hydroxy-tetradecanoic acid. 相似文献
6.
Asymmetric localization of lipopolysaccharides on the outer membrane of Salmonella typhimurium. 总被引:11,自引:3,他引:8
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Treatment of intact cells of Salmonella typhimurium with galactose oxidase (EC 1.1.3.9) resulted in an extensive oxidation of the susceptible galactose residues in the lipopolysaccharides. Comparison with the extent of oxidation in isolated lipopolysaccharides indicated that most lipopolysaccharide molecules were located in the outer leaflet of the outer membrane in intact cells. 相似文献
7.
Cationic peanut peroxidase (CP) was isolated from peanut (Arachis hypogaea) cell suspension culture medium. CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185. ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans. Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS. The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS. The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site. Good agreement was found with the one glycan previously analyzed by (1)H NMR. This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry. 相似文献
8.
Protein glycosylation is critical since it connects complex metabolic pathways to diverse proteoforms, fine-tunes protein structures and exerts biological functions. Aberrant glycosylation on the other hand is associated with many diseases, including cancers, inflammation and metabolic disorders. By resolving monosaccharide residues on intact glycoprotein complexes, native mass spectrometry can shed light on glycan heterogeneity, glycoprotein structure and molecular recognition. Here, we focus on the two most prevalent forms of glycosylation, namely N- and O- linked, and discuss recent progress in native mass spectrometry for elucidating glycoprotein structural heterogeneity and relating specific glycan repertoires to glycoprotein interactions. 相似文献
9.
High-molecular-weight components in lipopolysaccharides of Salmonella typhimurium, Salmonella minnesota, and Escherichia coli. 总被引:32,自引:18,他引:14
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Lipopolysaccharide from smooth strains of Salmonella typhimurium, Salmonella minnesota, and Escherichia coli O111:B4, O55:B5, and O127:B8 was fractionated by gel filtration chromatography. All lipopolysaccharide samples separated into three major populations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions from S. typhimurium and S. minnesota indicated that the three peaks were made up of molecules with average O-antigen lengths of (i) 70 or more repeat units, (ii) 30 and 20 repeats units in the samples from S. typhimurium and S. minnesota, respectively, and (iii) 1 repeat unit. In contrast to the Salmonella samples, peak 1 from the E. coli samples was not detected on polyacrylamide gels and lacked detectable phosphate. This high-molecular-weight material had a sugar composition similar to that of O-antigen and was tentatively identified as capsular polysaccharide. Peaks 2 and 3 of the E. coli samples were analogous to those of the Salmonella isolates, containing lipopolysaccharide molecules with averages of 18 and 1 O-antigen repeat units, respectively. These lipopolysaccharide molecules did not completely dissociate during electrophoresis, and multimers were detected as distinct, anomalous, slow-migrating bands. Increasing the concentration of sodium dodecyl sulfate in the gels resulted in the dissociation of these multimers. 相似文献
10.
Characterization of the rho genes of Neisseria gonorrhoeae and Salmonella typhimurium. 总被引:4,自引:3,他引:1
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M Miloso D Limauro P Alifano F Rivellini A Lavitola E Gulletta C B Bruni 《Journal of bacteriology》1993,175(24):8030-8037
11.
Despeyroux D Walker N Pearce M Fisher M McDonnell M Bailey SC Griffiths GD Watts P 《Analytical biochemistry》2000,279(1):23-36
Electrospray mass spectrometry (ES/MS), capillary-zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and multianalyte resonant mirror are used to evaluate the heterogeneity of samples of ricin toxins extracted from five horticultural varieties of Ricinus communis seeds: R. communis zanzibariensis, carmencita, impala, sanguineus, and gibsonii. The investigation is also extended to the geographical provenance of the beans. Combining mass spectrometry, CE techniques, and resonant mirror results in a powerful analytical tool capable to characterize and differentiate between different varieties of ricin toxins. Each technique complements the others, adding another level of information. This study reveals a large extent of heterogeneity for each cultivar, demonstrating that ricin toxins consist of a series of glycosylated proteins most likely originating from a multigene family. By combining these techniques, it is possible to differentiate between zanzibariensis and the other four varieties, and that variations in the functional characteristics may be observed between the different cultivars. This study demonstrates that knowledge of the variety of R. communis beans used and their geographical provenance is essential before any type of investigation of ricin toxins is carried out. Consequently, any unusual behavior observed can only be attributed to that particular cultivar studied and not automatically extended to include all R. communis varieties. 相似文献
12.
Studies on the structure of lipopolysaccharides of Salmonella minnesotta and Salmonella typhimurium R strains 总被引:7,自引:1,他引:6
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1. The composition of the lipopolysaccharides and the corresponding lipid-free polysaccharides from four R-mutants of Salmonella has been studied. All the lipopolysaccharides, from RI and RII serotypes contained d-glucose, d-galactose, heptose, N-acetylglucosamine and 3-deoxy-2-oxo-octonate. The polysaccharide obtained from the RII lipopolysaccharides also contained all these sugars. The polysaccharides from RI lipopolysaccharides lacked N-acetylglucosamine. 2. From partial hydrolysates of the lipopolysaccharides, a number of oligosaccharides have been isolated and partially characterized. Oligosaccharides containing N-acetylglucosamine or glucosamine were obtained only from RII lipopolysaccharides. Several oligosaccharides composed of glucose and galactose were common to RI and RII preparations. 3. A structural unit, based on the oligosaccharides found, is proposed for the RII lipopolysaccharide. It contains the sequence: alpha-N-acetylglucosaminyl- alpha-glucosyl-alpha-galactosyl-glucosyl.... A second alpha-galactosyl residue is bound to position 6 of the last glucosyl group. The complete unit is believed to to be attached to a polyheptose phosphate backbone in the RII antigen. 4. The RI lipopolysaccharide of Salmonella minnesota contains an analogous structure lacking the terminal N-acetylglucosamine residue. 5. A basal structure common to the lipopolysaccharides of several Salmonella species is proposed. 相似文献
13.
Nontypeable Haemophilus influenzae (NTHi) is an important pathogen responsible for otitis media in children and of pneumonitis in adults with depressed resistance. NTHi is acapsular and, therefore, capsular polysaccharide-based vaccines are ineffective for preventing infections by this pathogen. Recently it was found that a detoxified lipooligo-saccharide (LOS) conjugate from NTHi 9274 induced bactericidal antibodies effective against a large number of NTHi isolates, and conferred protection against NTHi otitis media in chinchillas (X.-X.Gu et al., 1996, Infect. Immun.,64, 4047-4053; X. -X.Gu et al., 1997., Infect. Immun.,65, 4488-4493). In this paper we report the chemical character-ization of the LOS from NTHi 9274 LOS. NTHi is capable of expressing a heterogenous population of LOS exhibited by multiple oligosaccharide (OS) epitopes. OSs released from the LOS of NTHi 9274 by mild acid hydrolysis were purified using Bio-Gel P4 gel permeation chromatography. The OSs were characterized by glycosyl composition analysis, glycosyl linkage analysis, nuclear magnetic resonance spectroscopy (NMR), fast atom bombardment mass spectro-metry (FAB-MS), matrix-assisted laser desorption time of flight mass spectro-metry (MALDITOF-MS), and tandem MS/MS. At least 17 different OS molecules were observed. These contained variable glycosyl residues, phosphate (P), and phospho-ethanolamine (PEA) substituents. These molecules contained either three, four, or five hexoses, and all contained four heptosyl residues. The four heptosyl residues consisted of one D,D-Hep and three L,D-Hep. Dephosphorylation of the OSs with aqueous 48% hydrofluoric acid (HF) reduced the number of molecules to about to seven; Hex(1)-(7)Hep(4)Kdo(1). Of these seven, Hex(2)Hep(4)Kdo(1), Hex(3)Hep(4)Kdo(1), and Hex(4)Hep(4)Kdo(1)were the major constituents. Thus, this NTHi LOS preparation is very heterogeneous, and contains structures different from those previously published for Haemophilus influenzae. The tandem MS/MS analysis and glycosyl linkage data suggest that the LOS oligosaccharides have the following structures where Hex is either a Glc or Gal residue. 相似文献
14.
The preferred conformation of the hexose and heptose regions of core saccharides from Enterobacteriaceae lipopolysaccharides was calculated. The Hard Sphere Exo Anomeric (HSEA) approach was used and the minimum energy conformation of the Salmonella typhimurium and Escherichia coli R1, R2, R3, R4 and K12 cores calculated. The results indicate that most of the cores are sterically crowded, with small degrees of freedom, and that the hexose and heptose parts form two separate regions. The core structures exhibit a 'front'-side and a 'back'-side, the former being similar for all the structures and the latter being characteristic for each core type. 相似文献
15.
Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. 总被引:104,自引:27,他引:77
Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria. It is now well established that within a single organism, size heterogeneity of this molecule can exist. We have developed a LPS isolation procedure which is effective in extracting both smooth and rough LPS in high yields (51 to 81% of the LPS present in whole cells as quantitated by using hydroxy fatty acid, heptose, and 2-keto-3-deoxyoctonate yields) and with a high degree of purity. The contamination by protein (0.1% by weight of LPS), nucleic acids (1%), lipids (2 to 5%), and other bacterial products was low. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the LPS demonstrated the presence of a high degree of size heterogeneity in the isolated smooth LPS as well as the presence of significant amounts of rough-type LPS. The Pseudomonas aeruginosa LPS interacted well with a monoclonal antibody in a variety of immunochemical analyses. The usefulness of the procedure was demonstrated by comparing LPS preparations obtained from wild-type and mutant strains of P. aeruginosa and Salmonella typhimurium. For example, it was shown that the LPS of an antibiotic supersusceptible mutant Z61 of P. aeruginosa, which was previously characterized as identical to wild type with respect to the ratio of smooth to rough LPS molecules isolated by the phenol-water procedure, actually contained only a small proportion of O-antigenic side chains. 相似文献
16.
Chain length heterogeneity of lipopolysaccharide released from Salmonella typhimurium by ethylenediaminetetraacetic acid or polycations 总被引:7,自引:0,他引:7
Cells of two smooth Salmonella typhimurium strains (SL696 and SH4247) were treated with ethylenediaminetetraacetic acid (EDTA) and the polycations poly(L-lysine) and protamine to monitor both quantitatively and qualitatively the release of [14C] galactose-labelled lipopolysaccharide into the medium to find out whether these effector substances caused selective release of certain fractions from the initially heterogenous lipopolysaccharide population. Each one of the substances released considerable amounts of lipopolysaccharide into the medium. Analysis by sodium dodecyl sulphate/polyacrylamide gel electrophoresis followed by autoradiography showed that the total lipopolysaccharide (from isolated membranes) and the released materials produced coincident banding patterns, each with a high degree of O side-chain length heterogeneity. Densitometric scans of the autoradiograms were analyzed for possible differences in the distribution and relative abundance of lipopolysaccharide molecules with different O chain lengths. It was found that in SL696 the released materials were identical to the total lipopolysaccharide; in SH4247 subtle deviations from the total lipopolysaccharide were seen. We conclude from these results that lipopolysaccharide molecules with short and long O side chains are linked to and stabilized in the outer membrane by similar mechanisms equally susceptible to the effects of EDTA and polycations. 相似文献
17.
Complete structure of lipid A obtained from the lipopolysaccharides of the heptoseless mutant of Salmonella typhimurium 总被引:17,自引:0,他引:17
A highly purified monophosphoryl lipid A, TLC-3 fraction obtained from the lipopolysaccharides of the heptoseless mutant Salmonella typhimurium G30/C21 was converted to the dimethyl pentatrimethylsilyl derivative and analyzed by proton NMR spectroscopy at 400 MHz. Substantial downfield shifts of the resonances for protons at the 3- and 3'-carbons of the glucosamine disaccharide to 5.06 and 5.15 ppm, respectively, occurred from the normal range of 3.5-4.1 ppm, indicating that these two positions on the sugar rings were acylated. Significant downfield shift of the resonances for protons at the 4- and 6'-carbons did not occur, indicating the absence of acyl groups at these two positions. Since positive ion fast atom bombardment mass spectrometry previously established the presence of hydroxymyristoyl and myristoxymyristoyl esters at the reducing end and distal subunits, respectively, these acyl groups must be attached to the oxygen of the corresponding 3- and 3'-carbons of lipid A. With these results, we can now describe the complete structure of the monophosphoryl lipid A, TLC-3 from S. typhimurium. 相似文献
18.
The development of electrospray and matrix-assisted laser desorption mass spectrometry has provided protein chemists with tools for peptide and protein structure analysis with unprecedented sensitivity and molecular weight range. The two technologies can be viewed as competitive with respect to their molecular mass determinations, but complementary with respect to their differences in instrumentation, sample preparation methods, and nature of spectra produced. 相似文献
19.
20.
Maoyin Li Ethan Baughman Mary R. Roth Xianlin Han Ruth Welti Xuemin Wang 《The Plant journal : for cell and molecular biology》2014,77(1):160-172
Plant triacylglycerols (TAGs), or vegetable oils, provide approximately 25% of dietary calories to humans and are becoming an increasingly important source of renewable bioenergy and industrial feedstocks. TAGs are assembled by multiple enzymes in the endoplasmic reticulum from building blocks that include an invariable glycerol backbone and variable fatty acyl chains. It remains a challenge to elucidate the mechanism of synthesis of hundreds of different TAG species in planta. One reason is the lack of an efficient analytical approach quantifying individual molecular species. Here we report a rapid and quantitative TAG profiling approach for Arabidopsis seeds based on electrospray ionization tandem mass spectrometry with direct infusion and multiple neutral loss scans. The levels of 93 TAG molecular species, identified by their acyl components, were determined in Arabidopsis seeds. Quantitative TAG pattern analyses revealed that the TAG assembly machinery preferentially produces TAGs with one elongated fatty acid. The importance of the selectivity in oil synthesis was consistent with an observation that an Arabidopsis mutant overexpressing a patatin‐like phospholipase had enhanced seed oil content with elongated fatty acids. This quantitative TAG profiling approach should facilitate investigations aimed at understanding the biochemical mechanisms of TAG metabolism in plants. 相似文献