The type-specific substance from Pneumococcus type 29

1. A pentasaccharide, corresponding to the dephosphorylated repeating unit of the specific substance, S.29, from Pneumococcus type 29, was obtained by hydrolysis with alkali followed by enzymic dephosphorylation. 2. The pentasaccharide was shown to be O-2-acetamido-2-deoxy-beta-d-galactopyranosyl-(1-->6)-O-beta-d-galactofuranosyl-(1-->3)-O-beta-d-galactopyranosyl-(1-->6)-O-beta-d-galactofuranosyl-(1-->1)-ribitol. 3. The phosphodiester linkages in S.29 join the hydroxyl group at position 5 of ribitol and the hydroxyl group at position 3 or 4 of a 2-acetamido-2-deoxy-d-galactose residue in the next repeating unit. 4. A partial structure for S.29 was deduced from these experiments.     

The type-specific substance from Pneumococcus type 13

1. The type-specific substance, S.13, from Pneumococcus type 13 was subjected to hydrolysis with alkali, followed by enzymic dephosphorylation, to yield a pentasaccharide. 2. The pentasaccharide, corresponding to the dephosphorylated repeating unit of S.13, was shown to be O-beta-d-galactopyranosyl-(1-->4)-O-beta- d-glucopyranosyl-(1-->3)-O-beta-d- galactofuranosyl-(1-->4)-O-2-acetamido-2-deoxy-beta-d- glucopyranosyl-(1-->2)-ribitol. 3. The phosphodiester linkages in S.13 join the hydroxyl group at position 1 of ribitol and the hydroxyl group at position 4 of a galactopyranosyl residue in the next repeating unit. 4. Ester groups, presumably O-acetyl, are located on positions 2 or 3 of most glucopyranosyl residues in S.13. 5. A partial structure for S.13 is proposed.     

The immunochemistry of Salmonella chemotype VI O-antigens. The structure of oligosaccharides from Salmonella group U (o 43) lipopolysaccharides

1. A series of oligosaccharides was isolated from Salmonella milwaukee lipopolysaccharide by partial acid hydrolysis. 2. Structural studies on these oligosaccharides indicated that the O-specific side chain of this lipopolysaccharide has a repeating pentasaccharide unit that is probably alpha-d-galactosyl-(1-->3)-beta-d-galactosyl- (1-->3)-N-acetylgalactosaminyl-(1-->3)-N-acetyl- d-glucosaminyl-(1-->4)-l-fucose. 3. Another oligosaccharide, which is not structurally related to the repeating pentasaccharide unit, has also been isolated and it is indistinguishable from an oligosaccharide obtained from Salmonella ;rough' (R) lipopolysaccharides. The isolation of this and similar core oligosaccharides from all chemotype VI lipopolysaccharides supports the view that Salmonella S-lipopolysaccharides have a common core that is probably identical with RII lipopolysaccharide.     

Complete lipopolysaccharide of Plesiomonas shigelloides O74:H5 (strain CNCTC 144/92). 1. Structural analysis of the highly hydrophobic lipopolysaccharide, including the O-antigen, its biological repeating unit, the core oligosaccharide, and the linkage between them

The lipopolysaccharide of Plesiomonas shigelloides serotype O74:H5 (strain CNCTC 144/92) was obtained with the hot phenol/water method, but unlike most of the S-type enterobacterial lipopolysaccharides, the O-antigens were preferentially extracted into the phenol phase. The poly- and oligosaccharides released by mild acidic hydrolysis of the lipopolysaccharide from both phenol and water phases were separated and investigated by (1)H and (13)C NMR spectroscopy, MALDI-TOF mass spectrometry, and sugar and methylation analysis. The O-specific polysaccharide and oligosaccharides consisting of the core, the core with one repeating unit, and the core with two repeating units were isolated. It was concluded that the O-specific polysaccharide is composed of a trisaccharide repeating unit with the [-->2)-beta-d-Quip3NAcyl-(1-->3)-alpha-l-Rhap2OAc-(1-->3)-alpha-d-FucpNAc-(1-->] structure, in which d-Qui3NAcyl is 3-amino-3,6-dideoxy-d-glucose acylated with 3-hydroxy-2,3-dimethyl-5-oxopyrrolidine-2-carboxylic acid. The major oligosaccharide consisted of a single repeating unit and a core oligosaccharide. This undecasaccharide contains information about the biological repeating unit and the type and position of the linkage between the O-specific chain and core. The presence of a terminal beta-d-Quip3NAcyl-(1--> residue and the -->3)-beta-d-FucpNAc-(1-->4)-alpha-d-GalpA element showed the structure of the biological repeating unit of the O-antigen and the substitution position to the core. The -->3)-beta-d-FucpNAc-(1--> residue has the anomeric configuration inverted compared to the same residue in the repeating unit. The core oligosaccharide was composed of a nonphosphorylated octasaccharide, which represents a novel core type of P. shigelloides LPS characteristic of serotype O74. The similarity between the isolated O-specific polysaccharide and that found on intact bacterial cells and lipopolysaccharide was confirmed by HR-MAS NMR experiments.     

The structure of the O-specific polysaccharide chain of the Shewanella algae BrY lipopolysaccharide

An acidic O-specific polysaccharide was obtained by mild acid degradation of the Shewanella algae strain BrY lipopolysaccharide and was found to contain L-rhamnose, 2-acetamido-4-[D-3-hydroxybutyramido)]-2,4,6-trideoxy-D-glucose (D-BacNAc4NHbu), and 2-amino-2,6-dideoxy-L-galactose, N-acylated by the 4-carboxyl group of L-malic acid (L-malyl-(4-->2)-alpha-L-FucN) in the ratio 2:1:1. 1H and 13C NMR spectroscopy was applied to the intact polysaccharide, and the following structure of the repeating unit was established:-3)-alpha-D-BacNAc4NHbu-(1-->3)-alpha-L-Rha-(1-->2)-alpha-L-Rha-(1-->2)-L-malyl-(4-->2)-alpha-L-FucN-(1-. The repeating unit includes linkage via the residue of malic acid, reported here for the first time as a component of bacterial polysaccharides.     

The O-chain structure from the LPS of the bacterium Naxibacter alkalitolerans YIM 31775T

The O-chain polysaccharide of the lipopolysaccharide from the bacterium Naxibacter alkalitolerans strain YIM 31775(T) was characterized. The structure was studied by means of chemical analysis and 2D NMR spectroscopy and shown to be built up by the following tetrasaccharide repeating unit: -->3)-alpha-D-FucpNAc-(1-->2)-beta-D-Quip3NHBu-(1-->2)-alpha-D-Rhap-(1-->)-beta-D-Galp-(1--> where HBu is hydroxy-butanoyl.     

The structure of the O-specific polysaccharide from Salmonella cerro (serogroup K, O:6,14,18)

The following structure of the Salmonella cerro LPS O-chain repeating unit has been determined using NMR and chemical methods: -->4)-alpha-D-Man(1-->2)-alpha-D-Man(1-->2)-beta-D-Man(1-->3)-alpha-D-GalNAc-(1-->.     

Enzymatic synthesis of fucose-containing disaccharides employing the partially purified alpha-L-fucosidase from Penicillium multicolor

The alpha-L-Fucp-(1 --> 3)-D-GlcpNAc disaccharide structure is a vital core unit of the oligosaccharide components of glycoconjugates isolated from human milk and blood group substances. Alpha-L-Fucosidase from Penicillium multicolor catalyses the transfer of L-fucose from donor structures such as alpha-L-FucpOpNP and alpha-L-FucpF to various GlcpNAc derivatives and Glcp, forming alpha-(1 --> 3) linkages. The synthesis of several biologically relevant disaccharides including alpha-L-Fucp-(1 --> 3)-alpha-D-GlcpNAcOMe, alpha-L-Fucp-(1 --> 3)-alpha-D-GlcpNAcOAll, alpha-L-Fucp-(1 --> 3)-beta-D-GlcpNAcOAll, alpha-L-Fucp-(1 --> 3)-D-GlcpNAc and alpha-L-Fucp-(1 --> 3)-D-Glcp has been achieved in up to 34% yields by application of this enzyme.     

Structural studies of the O-specific polysaccharide of Vibrio cholerae O8 using solvolysis with triflic acid

The O-specific polysaccharide (OPS) of Vibrio cholerae 08 was isolated by mild acid degradation of the lipopolysaccharide and studied by two-dimensional NMR spectroscopy, including NOESY and heteronuclear multiple-bond correlation (HMBC) experiments. The OPS was found to have a tetrasaccharide repeating unit with the following structure: --> 4)-beta-D-Glcp NAc3NAcylAN-(1 --> 4)-beta-D-Manp NAc3NAcAN-(1 --> 4)-alpha-L-Gulp NAc3NAcA-(1 --> 3) -beta-D-QuipNAc4NAc-(1 --> where QuiNAc4NAc is 2,4-diacetamido-2,4,6-trideoxyglucose, GlcNAc3NAcylAN is 2-acetamido-3-(N-formyl-L-alanyl)amino-2,3-dideoxyglucuronamide, ManNAc3NAcAN is 2,3-diacetamido-2,3-dideoxymannuronamide, and GulNAc3NAcA is 2,3-diacetamido-2,3-dideoxyguluronic acid. The OPS was stable towards acid hydrolysis and solvolysis with anhydrous hydrogen fluoride, but could be cleaved selectively with trifluoromethanesulfonic (triflic) acid by the glycosidic linkages of beta-QuiNAc4NAc and alpha-GulNAc3NAcA. The structures of the oligosaccharides obtained that were elucidated by electrospray ionization (ESI) MS and NMR spectroscopy, confirmed the OPS structure.     

The surface layer (S-layer) glycoprotein of Geobacillus stearothermophilus NRS 2004/3a. Analysis of its glycosylation.

Geobacillus stearothermophilus NRS 2004/3a possesses an oblique surface layer (S-layer) composed of glycoprotein subunits as the outermost component of its cell wall. In addition to the elucidation of the complete S-layer glycan primary structure and the determination of the glycosylation sites, the structural gene sgsE encoding the S-layer protein was isolated by polymerase chain reaction-based techniques. The open reading frame codes for a protein of 903 amino acids, including a leader sequence of 30 amino acids. The mature S-layer protein has a calculated molecular mass of 93,684 Da and an isoelectric point of 6.1. Glycosylation of SgsE was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->](n = 13-18), with a 2-O-methyl group capping the terminal trisaccharide repeating unit at the non-reducing end of the glycan chains. The glycan chains are bound via the disaccharide core -->3)-alpha-l-Rhap-(1-->3)-alpha-L-Rhap-(L--> and the linkage glycose beta-D-Galp in O-glycosidic linkages to the S-layer protein SgsE at positions threonine 620 and serine 794. This S-layer glycoprotein contains novel linkage regions and is the first one among eubacteria whose glycosylation sites have been characterized.     

Structural analysis of the O-antigen polysaccharide from the Shiga toxin-producing Escherichia coli O172.

The structure of the O-antigen polysaccharide from Escherichia coli O172 has been determined. In combination with sugar analysis, NMR spectroscopy shows that the polysaccharide is composed of pentasaccharide repeating units. Sequential information was obtained by mass spectrometry and two-dimensional NMR techniques. An O-acetyl group was present as 0.7 equivalent per repeating unit. Treatment of the O-deacetylated polysaccharide with aqueous 48% hydrofluoric acid rendered cleavage of the phosphodiester in the backbone of the polymer and the pentasaccharide isolated after gel permeation chromatography was structurally characterized. Subsequent NMR experiments on polymeric materials revealed the structure of the repeating unit of the O-polysaccharide from E. coli O172 as:-->P-4)-alpha-D-Glcp-(1-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D- GlcpNAc-(1-->3)-alpha-L-FucpNAc-(1-->4)-alpha-D-Glcp6Ac-(1-->     

Structure of the O-specific polysaccharide of the lipopolysaccharide of Azospirillum brasilense Sp245

An O-specific polysaccharide was isolated from the lipopolysaccharide of a plant-growth-promoting bacterium Azospirillum brasilense Sp245 and studied by sugar analyses along with one- and two-dimensional 1H and 13C NMR spectroscopy, including NOESY. The polysaccharide was found to be a new rhamnan with a pentasaccharide repeating unit having the following structure:-->2)-beta-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->     

Methylobacterium sp. isolated from a Finnish paper machine produces highly pyruvated galactan exopolysaccharide

The slime-forming bacterium Methylobacterium sp. was isolated from a Finnish paper machine and its exopolysaccharide (EPS) was produced on laboratory scale. Sugar compositional analysis revealed a 100% galactan (EPS). However, FT-IR showed a very strong peak at 1611 cm(-1) showing the presence of pyruvate. Analysis of the pyruvate content revealed that, based on the sugar composition, the EPS consists of a trisaccharide repeating unit consisting of D-galactopyranose and [4,6-O-(1-carboxyethylidene)]-D-galactopyranose with a molar ratio of 1:2, respectively. Both linkage analysis and 2D homo- and heteronuclear 1H and 13C NMR spectroscopy revealed the following repeating unit: -->3)-[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)-alpha-D-Galp-(1-->. By enrichment cultures from various ground and compost heap samples a polysaccharide-degrading culture was obtained that produced an endo acting enzyme able to degrade the EPS described. The enzyme hydrolysed the EPS to a large extent, releasing oligomers that mainly consisted out of two repeating units.     

Conformation of the O-specific polysaccharide of Shigella dysenteriae type 1: molecular modeling shows a helical structure with efficient exposure of the antigenic determinant alpha-L-Rhap-(1-->2)-alpha-D-Galp.

The O-specific polysaccharide of Shigella dysenteriae type 1, which has the repeating tetrasaccharide unit -->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->3)-alpha-D-GlcNAcp-(1--> (A-B-C-D), is a major virulence factor, and it is believed that antibodies against this polysaccharide confer protection to the host. The conformational properties of fragments of this O-antigen were explored using systematic search with a modified HSEA method (GLYCAN) and with molecular mechanics MM3(96). The results show that the alpha-D-Gal-(1-->3)-alpha-D-GlcNAc linkage adopts two favored conformations, phi/psi approximately equal to -40 degrees /-30 degrees (I) and approximately 15 degrees /30 degrees (II), whereas the other glycosidic linkages only have a single favored phi/psi conformational range. MM3 indicates that the trisaccharide B-C-D and tetrasaccharides containing the B-C-D moiety exist as two different conformers, distinguished by the conformations I and II of the C-D linkage. For the pentasaccharide A-B-C-D-A' and longer fragments, the calculations show preference for the C-D conformation II. These results can explain previously reported nuclear magnetic resonance data. The pentasaccharide in its favored conformation II is sharply bent, with the galactose residue exposed at the vertex. This hairpin conformation of the pentasaccharide was successfully docked with the binding site of a monoclonal IgM antibody (E3707 E9) that had been homology modeled from known crystal structures. For fragments made of repetitive tetrasaccharide units, the hairpin conformation leads to a left-handed helical structure with the galactose residues protruding radially at the helix surface. This arrangement results in a pronounced exposure of the galactose and also the adjacent rhamnose in each repeating unit, which is consistent with the known role of the as alpha-L-Rhap-(1-->2)-alpha-D-Galp moiety as a major antigenic epitope of this O-specific polysaccharide.     

Structure of the O-polysaccharide of Pseudomonas putida FERM P-18867

The O-polysaccharide of the lipopolysaccharide of Pseudomonas putida FERM P-18867 was found to contain D-mannose and D-rhamnose and have the following structure of the trisaccharide repeating unit:-->2)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-beta-D-Manp-(1-->     

Structural investigation of the capsular polysaccharide from Klebsiella serotype K-34 and its characterization by N.M.R. spectroscopy

The repeating unit of the capsular polysaccharide from Klebsiella type K-34 has been established by methylation, partial hydrolysis, and Smith degradation to consist of a hexasaccharide repeating-unit built up of four l-rhamnose, one d-glucose, and one d-galacturonic acid residues. The anomeric configurations of the linkages was determined by proton and ¹³C-n.m.r. spectroscopy at each step of the degradation procedures. Further evidence for the configurations of the glycosidic linkages involved the use of proton T₁ relaxation-times and oxidation by chromium trioxide. The data allowed assignment of the following structure for the repeating unit:     

Structural differences between the alkali-extracted water-soluble cell wall polysaccharides from mycelial and yeast phases of the pathogenic dimorphic fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a pathogenic dimorphic fungus causing paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. We have studied the structure of the alkali-extracted water-soluble cell wall polysaccharides (F1SS) from both mycelial and yeast phases of this fungus by using chemical analysis and NMR spectroscopic techniques. The F1SS polysaccharide from the mycelial phase consists of a trisaccharidic repeating unit of -->6)-[alpha-Galf -(1-->6)-alpha-Manp-(1-->2)]-alpha-Manp-(1-->. The F1SS polysaccharide of the yeast phase maintains 10% of the structure of the mycelium phase, but the main structure contain a disaccharide repeating unit of -->6)-[-alpha-Manp-(1-->2)]-alpha-Manp-(1-->, alternating with a trisaccharide repeating block of -->6)-[beta-Galf -(1-->6)-alpha-Manp-(1-->2)]-alpha-Manp-(1-->.     

Structure and heterogeneity of the O-antigen chain of Salmonella Agona lipopolysaccharide

Lipopolysaccharide of Salmonella Agona smooth-type cells was obtained from bacteria by a hot phenol-water extraction procedure. Mild acid hydrolysis of lipopolysaccharide, followed by gel filtration, yielded the pure O-polysaccharide. Abequose, rhamnose, mannose, galactose and glucose in the molar ratio 0.8 : 1.0 : 1.0 : 1.1 : 0.5 were detected, and their linkages were established. Sugar configurations were determined by gas chromatography. Two repeating units, namely -->2)-[alpha-Abep-(1-->3)-]-alpha-d-Manp-(1-->4)-alpha-l-Rhap-(1-->3)-alpha-d-Galp-(1-->and -->2)-[alpha-Abep-(1-->3)-]-alpha-d-Manp-(1-->4)-alpha-l-Rhap-(1-->3)-[alpha-d-Glcp-(1-->4)-]-alpha-d-Galp-(1-->, were deduced from nuclear magnetic resonance studies. The effort to separate them was unsuccessful. An immunochemical test performed by means of Western blotting with anti O12 serum demonstrated that glucose was present in the longer lipopolysaccharide chains, at some distance from the core region.     

Structure of the O-specific polysaccharide of Hafnia alvei PCM 1196

An acidic O-specific polysaccharide was isolated from Hafnia alvei PCM 1196 lipopolysaccharide and studied by sugar and methylation analyses along with one- and two-dimensional 1H and 13C NMR spectroscopy, including NOESY and HMBC experiments. The following structure of the pentasaccharide repeating unit was established: -->4)-alpha-D-GalpA-(1-->3)-beta-D-GlcpNAc-(1-->2)-beta-D-Galp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-GlcpNAc-(1-->.     

Structure of the sialic acid-containing O-specific polysaccharide from Salmonella enterica serovar Toucra O48 lipopolysaccharide.

Lipopolysaccharide was extracted from cells of Salmonella enterica serovar Toucra O48 and, after mild acid hydrolysis (1% AcOH, 1 h, 100 degrees C or 0.1 M NaOH-AcOH, pH 4.5, 5 h, 100 degrees C), the O-specific polysaccharide was isolated and characterized. The core and an oligosaccharide containing a fragment of the repeating unit linked to the core region were also obtained, depending on hydrolysis conditions. On the basis of sugar and methylation analyses and NMR spectroscopy of the hydrolysis products, the biological repeating unit of the O-specific polysaccharide was shown to be the following trisaccharide: -->4)-alpha-Neup5Ac(2-->3)-L-alpha-FucpNAc(1-->3)-D-beta-Glc pNAc(1--> The polysaccharide O-chain was substituted with a single molar equivalent of O-acetyl group, distributed between the Neu5Ac O-9 and O-7 positions, in an approximate ratio of 7 : 3.