Structure of an acidic exopolysaccharide of Burkholderia pseudomallei

A recently described water-soluble exopolysaccharide of Burkholderia pseudomallei recognized by the IgG 1 monoclonal antibody 3015 [Steinmetz, I., Rohde, M. & Brenneke, B. (1995) Infect. Immun. 63, 3959-3965] was isolated by repetitive ethanol-precipitation steps and by anion-exchange chromatography. The structure of the polysaccharide was determined by a combination of chemical-derivatization and mass-spectrometric techniques (compositional and methylation analysis, GC/MS, and electrospray-ionization-MS/MS of reduced and permethylated hydrolytic fragments), and two-dimensional 1H-NMR methods (COSY, TOCSY and NOESY) and confirmed by isolation and structural characterization of the depolymerized repeating unit of the polysaccharide. The combined structural data established a linear tetrasaccharide repeating unit consisting of three galactose residues, one bearing a 2-linked O-acetyl group, and a 3-deoxy-D-manno-2-octulosonic acid residue. [-->3)-beta-D-Galp2Ac-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-Galp-(1- ->5)-beta-Kdo-(2-->]n     

Structure of the repeating unit of acid polysaccharide from Alteromonas sp. 4MS17

An acidic polysaccharide from Alteromonas sp. 4MC17 is built up of trisaccharide repeating units containing D-glucose, D-mannose and D-galacturonic acid residues. On the basis of methylation studies, 1H and 13C NMR-spectroscopy data, including two-dimensional homonuclear correlation spectroscopy and nuclear Overhauser effects, the following structure was suggested for the polysaccharide repeating unit: -->4)-beta-D-Glcp-(1-->4)-beta-D-GalpA-(1-->4)-beta-D-Manp-( 1-->.     

Structural determination of the O-antigenic polysaccharide from the enterotoxigenic Escherichia coli O147

The structure of the O-antigenic polysaccharide from enterotoxigenic Escherichia coli O147 has been determined by NMR spectroscopy, and component and methylation analyses. The sequence of the sugar residues could be determined by NOESY and heteronuclear-multiple-bond-connectivity NMR experiments. It is concluded that the polysaccharide is composed of tetrasaccharide repeating units with the following structure: -->4)-beta-D-GalpA-(1-->3)-beta-D-GalpNAc-(1-->2)-alpha-L-Rhap+ ++-(1-->2)-alpha-L-Rhap-(1-->, where Rha represents 6-deoxymannose. The O-antigen of E. coli O147 is identical to the repeating unit of Shigella flexneri serotype 6 lipopolysaccharide, except that the latter contains an O-acetyl group at C3 of the rhamnosyl residue substituted by the N-acetylgalactosamine residue. Immunochemical analyses using a monoclonal antibody specific for the S. flexneri serotype 6 O-antigen showed an identical reactivity with both lipopolysaccharides.     

Structural elucidation of the lipopolysaccharide core regions of the wild-type strain PAO1 and O-chain-deficient mutant strains AK1401 and AK1012 from Pseudomonas aeruginosa serotype O5

Lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype O5 wild-type strain PAO1 and derived rough-type mutant strains AK1401 and AK1012 was isolated by a modified phenol/chloroform/petroleum-ether extraction method. Deoxycholate/PAGE of the LPS from the rough mutant AK1401 indicated two bands near the dye front with mobilities similar to those of the parent strain, indicating that both LPS contain a complete core and a species comprising a core and one repeating unit. Composition analysis of the LPS from strains PAO1 and AK1401 indicated that the complete core oligosaccharide was composed of D-glucose (four units), L-rhamnose (one unit), 2-amino-2-deoxy-D-galactose (one unit), L-glycero-D-manno-heptose (Hep; two units), 3-deoxy-D-manno-octulosonic acid (Kdo; two units), L-alanine (one unit) and phosphate (three units). The glycan structure of the LPS was determined by one-dimensional and two-dimensional (2D) NMR techniques in combination with MS-based methods on oligosaccharide samples obtained from the LPS by delipidation procedures. The locations of three phosphomonoester groups on the first heptose residue were established by a two-dimensional 31P (omega1)-half-filtered COSY experiment on the reduced core oligosaccharide sample of the LPS from the wild-type strain. The presence of a 7-O-carbamoyl substituent was observed on the second heptose. The structure of the core region of the O-chain-deficient LPS from P. aeruginosa serotype 05 is as follows: [structure: see text] where R1 is beta-D-Glcp-(1-->2)-alpha-L-Rhap-(1-->6)-alpha-D-Glcp-(1--> and R2 is alpha-D-Glcp-(1-->6)-beta-D-Glcp-(1->. A structural model is presented that is also representative of that for P. aeruginosa serotype O6 LPS. A revised structure for the serotype O6 mutant strain A28 is presented.     

The O-specific polysaccharide chain of Campylobacter fetus serotype B lipopolysaccharide is a D-rhamnan terminated with 3-O-methyl-D-rhamnose (D-acofriose)

An O-specific polysaccharide was liberated from Campylobacter fetus subsp. fetus serotype B lipopolysaccharide by mild acid hydrolysis followed by gel chromatography. This polysaccharide was found to contain D-rhamnose and 3-O-methyl-D-rhamnose (D-Rha3Me, D-acofriose) in a ratio of approximately 24:1, as well as lipopolysaccharide core constituents. The structure of the polysaccharide was studied by 1H-NMR and 13C-NMR spectroscopy, which included two-dimensional COSY, rotating-frame NOE spectroscopy (ROESY), and computer-assisted analysis of the 13C-NMR spectrum. Methylation analysis using [2H3]methyl iodide and Smith degradation followed by GLC/MS of the derived acetylated oligosaccharide-alditols was used to determine the location of D-acofriose. The O-specific polysaccharide is linear, consists on average of 12 disaccharide repeating units, and is terminated by a residue of D-acofriose. The following structure of the D-rhamnan chain was established: [sequence: see text]     

Lack of association between the alpha-adducin locus and essential hypertension in the Japanese population

A neutral O-specific polysaccharide obtained from the lipopolysaccharide of Proteus penneri strain 26 was studied using sugar analysis and 1H and 13C NMR spectroscopy, including two-dimensional NMR techniques. The following structure of the trisaccharide repeating unit was established: -->6)-alpha-D-GlcpNAc-(1-->3)-alpha-L-QuipNAc-(1-->3)-alpha-D-Glcp NAc-(1--> where L-QuiNAc is 2-acetamido-2,6-dideoxy-L-glucose (N-acetyl-L-quinovosamine). Cross-reactivity of the Proteus penneri 26 anti-O serum with other strains of P. penneri isolated in Poland and USA and one strain of P. vulgaris is discussed.     

Structural investigation of two cell-wall polysaccharides of Penicillium expansum strains

The structure of two cell-wall polysaccharides isolated from three different strains of Penicillium expansum, the type species of the genus, have been established by 1D and 2D NMR spectroscopy, and also by methylation analyses. The water-soluble polysaccharide F1S-B consisted of a linear tetrasaccharide repeating unit with the following structure: [-->6)-beta-D-Galf-(1-->5)-beta-D-Galf-(1-->5)-beta-D-Gal f-(1-->5)-beta-D- Galf-(1-->]n The alkali-soluble polysaccharide F1I is a (1-->3)-alpha-D-glucan.     

Somatic antigens of pseudomonads: structure of the O-specific polysaccharide of Pseudomonas fluorescens biovar B, strain IMV 247

The O-specific polysaccharide of Pseudomonas fluorescens biovar B, strain IMV 247, was studied by acid hydrolysis, GLC-MS and 1H and 13C NMR spectroscopy, including 1D and 2D NOE, 2D hybrid TOCSY and ROESY (TORO), and 2D H-detected heteronuclear multiple-bond correlation (HMBC) experiments. The polysaccharide was found to contain L-rhamnose, 3.6-dideoxy-3-[(S)-3-hydroxybutyramido]-D-glucose (D-Qui3NHb), 2-acetamido- 2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido-D-glucose (D-QuiNAc4NHb) and 2-acetamido-2- deoxy-D-galacturonic acid (D-GalNAcA). Partial acid hydrolysis of the polysaccharide resulted in a non-reducing GalNAcA-->QuiNAc4NHb disaccharide with the 3-hydroxybutyryl group glycosylated intramolecularly by the QuiN4N residue. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established:-->4) -alpha-D-GalpNAcA-(1-->3)- alpha-D-QuipNAc4NHb-(1-->2)-beta-D-Quip3NHb-(1-->2)-alpha-L- Rhap(1-->.     

Molecular cloning of a novel human receptor gene with homology to the rat adrenomedullin receptor and high expression in heart and immune system

Lipopolysaccharide was isolated from strain LMG 6999 of Burkholderia vietnamiensis. Degradative and NMR spectroscopic studies established the presence of two polymeric fractions based on the following trisaccharide repeating units: I:-->3)-alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc- (1-->; II:-->3)-alpha-D-GalpNAc-(1-->3)-beta-D-GalpNAc-(1-->4)- alpha-L-Rhap-(1-->. The same polymers have previously been found together in lipopolysaccharide from the reference strain for Burkholderia cepacia serogroup O4 and, individually, in those from B. cepacia serogroups C (I) and A (II).     

Synthesis of four spacer-containing 'tetrasaccharides' that represent four possible repeating units of the capsular polysaccharide of Streptococcus pneumoniae type 6B

In the framework of studies towards oligosaccharide-conjugate based vaccines against Streptococcus pneumoniae, the synthesis is reported of four spacer-containing tetrasaccharides that each can be conceived as representing a repeating unit of the capsular polysaccharide of S. pneumoniae serotype 6B, namely, 3-aminopropyl D-ribityl-(5-->hydrogen phosphate-->2)-alpha-D-galactopyranosyl-(1-->3) -alpha-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranoside, 3-aminopropyl alpha-L-rhamnopyranosyl-(1-->4)-D-ribityl-5(-->hydrogen phosphate-->2)-alpha-D-galactopyranosyl-(1-->3)-alpha-D-glucopyranoside, 3-aminopropyl alpha-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl-(1-->4) -D-ribityl-(5-->hydrogen phosphate-->2) -alpha-D-galactopyranoside, and alpha-D-galactopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->3)-alpha-L -rhamnopyranosyl-(1-->4)-5-O-(3-aminopropyl hydrogen phosphate)-D-ribitol. Phosphorylations were carried out using the H-phosphonate method.     

Structure of a highly acidic O-specific polysaccharide of lipopolysaccharide of Pseudoalteromonas haloplanktis KMM 223 (44-1) containing L-iduronic acid and D-QuiNHb4NHb

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide isolated by phenol-water extraction of Pseudoalteromonas haloplanktis strain KMM 223 (44-1). L-Iduronic acid (IdoA) was found to be a component of the polysaccharide and identified by NMR spectroscopy and after carboxyl-reduction followed by acid hydrolysis and acetylation, by GLC-MS as 2,3,4-tri-O-acetyl-1,6-anhydroidose. On the basis of 1H and 13C NMR spectroscopic studies, including 1D NOE, 2D NOESY, HSQC and HMBC experiments, the following structure of the branched pentasaccharide repeating unit of the polysaccharide was established: -->4)-beta-D-GlcpAI-(1-->4)-beta-D-GlcpAII-(1-->3)-beta-D-++ +QuipNHb4NHbII- (1-->2)-alpha-L-IdopA-(-->4 increases 1 alpha-D-QuipNAc4NAcI where QuiNAc4NAc and QuiNHb4NHb are 2,4-diacetamido-2,4,6-trideoxyglucose and 2,4,6-tri-deoxy-2,4- di[(S)-3-hydroxybutyramido]glucose, respectively. This is the first report of L-iduronic acid in a lipopolysaccharide and of D-QuiNHb4NHb in nature.     

Fine chemical structure analysis of oligosaccharides produced by an ulvan-lyase degradation of the water-soluble cell-wall polysaccharides from Ulva sp, (Ulvales, Chlorophyta)

A marine bacterium degrading the water-soluble cell wall polysaccharides from Ulva sp. (ulvan) has been isolated. The good correlation between ulvan degradation monitored by reducing-power, UV absorbance and viscosimetry, indicated that the crude enzymatic extract contains essentially an endo-ulvan lyase activity. This activity was rapidly inhibited by the reaction products which consisted of a series of ulvanobiouronic acid A 3-sulfate [-->4)-beta-D-GlcpA-(1-->4)-alpha-L-Rhap 3-sulfate-(1-->]n with 4-deoxy-L-threo-hex-4-enopyranosiduronic acid at the non-reducing end. Other deviant repeating structures with beta-D-Xylp or alpha-IdopA replacing beta-D-GlcpA in the repeating ulvanobiouronic acid disaccharide and the presence of two consecutive (1-->4) linked beta-D-Glc pA demonstrated the great variability and complexity of ulvan chemical structure.     

Structural studies of the O-specific polysaccharide of Hafnia alvei strain PCM 1206 lipopolysaccharide containing D-allothreonine

The structure of the O-specific side-chain of the Hafnia alvei strain PCM 1206 lipopolysaccharide has been investigated. Methylation analysis, partial acid hydrolysis, FAB-MS/MS and 1H-NMR and 13C-NMR spectroscopy were the principal methods used. D-Allothreonine (D-aThr), amide-linked to the D-galacturonic acid, was identified as a constituent in the polysaccharide and the following structure of a pentasaccharide repeating unit was established: [structure: see text].     

NMR spectroscopic elucidation of the structure of a glycerol teichoic acid-like O-specific polysaccharide of the bacterium Hafnia alvei strain PCM 1199

The structure of the acidic O-specific polysaccharide of a Gram-negative bacterium, H. alvei strain PCM 1199, was studied by NMR spectroscopy including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), 1H, 13C heteronuclear single-quantum coherence (HSQC), 1H, 13C heteronuclear multiple-bond correlation (HMBC), and one-dimensional 1H, 31P heteronuclear multiple-quantum coherence (HMQC) experiments. It was found that the polysaccharide contains D-galactose, 2-acetamido-2-deoxy-D-glucose, 4-acetamido-4,6-dideoxy-D-glucose, glycerol, and phosphate in the ratios 1:2:1:1:1, as well as O-acetyl groups in non-stoichiometric amounts. The polysaccharide is similar in structure to teichoic acids of Gram-positive bacteria and has the following structure of the repeating unit: 3)-beta-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4NAc-(1- ->1)-Gro- 3-P-(O--> [formula: see text] beta-D-GlcpNAc [formula: see text] The O-specific polysaccharide of H. alvei PCM 1199 is structurally related to another teichoic acid-like O-specific polysaccharide of H. alvei PCM 1205 studied by us earlier.     

Structural study on a sulfated polysaccharide-peptidoglycan complex produced by Arthrobacter sp

The structure of a sulfated polysaccharide-peptidoglycan complex (SP-PG) produced by Arthrobacter sp. was analyzed by NMR spectroscopy. In addition, oligosaccharide fragments of the SP-PG-L obtained by HF degradation were analyzed by NMR spectroscopy. These findings indicated that the sulfated polysaccharide (SP) contains a repeating unit composed of two galactofuranosides and a glucopyranoside. The main chain of the trisaccharide is [-->6) beta-D-Galf(1-->6)-beta-D-Galf(1-->ln, with beta-D-Glcp linked to one of the Galfs through a (1-->2) linkage. The sulfated positions of the trisaccharide were identified as C-3 and C-5 of the beta-glucosylated Galf residues, and C-2 or C-3 of the other Galf residue.     

Monosialogangliosides of human myelogenous leukemia HL60 cells and normal human leukocytes. 2. Characterization of E-selectin binding fractions, and structural requirements for physiological binding to E-selectin

E-selectin binding gangliosides were isolated from myelogenous leukemia HL60 cells, and the E-selectin binding pattern was compared with that of human neutrophils as described in the preceding paper in this issue. The binding fractions were identified as monosialogangliosides having a series of unbranched polylactosamine cores. Structures of fractions 12-3, 13-1, 13-2, and 14, which showed clear binding to E-selectin under the conditions described in the preceding paper, were characterized by functional group analysis by application of monoclonal antibodies, 1H-NMR, FAB-MS, and electrospray mass spectrometry with collision-induced dissociation of permethylated fractions. Fractions 12-3, 13-1, and 13-2 were characterized by the presence of a major ganglioside with the following structure: NeuAc alpha 2-->3Gal beta 1-->4 GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3)-GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->4 Glc beta Cer. Fractions 12-3 and 13-2 contained, in addition, small quantities (10-15%) of extended SLex with internally fucosylated structures: NeuAc alpha 2-->3 Gal beta 1-->4-(Fuc alpha 1-->3) GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->3 Gal beta 1-->4 (+/- Fuc alpha 1-->3)GlcNA c beta 1-->3 Gal beta beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->Glc Beta Cer. Fraction 13-1, showing stronger E-selectin binding activity than 12-3 and 13-2, contained only a trace quantity (< 1%) of SLex. Fraction 14, which also showed clear binding to E-selectin, was characterized by the presence of the following structures, in addition to two internally monofucosylated structures (XX and XXI, Table 2, text): NeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3Gal beta 1-->4 GlcNAc beta 1-->3 Gal beta 1-->4 GlcNAc beta 1-->3 Gal beta 1-->4 Glc beta Cer; andNeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4 (Fuc alpha 1--3)-GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1--4Glc beta Cer. SLex determinant was completely absent. Thus, the E-selectin binding epitope in HL60 cells is carried by unbranched terminally alpha 2-->3 sialylated polylactosamine having at least 10 monosaccharide units (4 N-acetyllactosamine units) with internal multiple fucosylation at GlcNAc. These structures are hereby collectively called "myeloglycan". Monosialogangliosides from normal human neutrophils showed an essentially identical pattern of gangliosides with selectin binding property. Myeloglycan, rather than SLex, provides a major physiological epitope in E-selectin-dependent binding of leukocytes and HL60 cells.     

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p -2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.     

Structure of the O-specific polysaccharide of Citrobacter freundii O3a,3b,1c

The following structure of the O-specific polysaccharide of Citrobacter freundii O3a,3b,1c containing D-mannose and D-rhamnose was established using sugar analysis and NMR spectroscopy, including computer-assisted analysis of the 13C NMR spectrum, 2D COSY, H,H-relayed COSY, heteronuclear 13C, 1H correlation (HETCOR), and rotating-frame NOE spectroscopy (ROESY):-->4)-alpha-D-Manp-(1-->3)-beta-D-Rhap-(1-->4) -beta-D-Rhap-(1-->.     

Degeneration of Purkinje cells in parasagittal zones of the cerebellar vermis after treatment with ibogaine or harmaline

The exopolysaccharide acetan, elaborated by Acetobacter xylinum, has been investigated. The polysaccharide and a heptasaccharide, obtained on enzymic hydrolysis, corresponding to the repeating unit were characterised by sugar and methylation analysis and by NMR spectroscopy and MS. It is concluded that the polysaccharide is composed of repeating units with the following structure. [formula: see text] The polysaccharide further contains approximately two O-acetyl groups per repeating unit, which have not been assigned, but it appears that they are on primary locations.