Structural studies of S-7, another exocellular polysaccharide containing 2-deoxy-arabino-hexuronic acid

The exocellular polysaccharide S-7, a heteropolysaccharide from Azotobacter indicus var. myxogenes has been studied using methylation analysis, Smith degradation, partial acid hydrolysis, NMR spectroscopy and mass spectrometry as the principal methods. It is concluded that the repeating unit has the following structure: [structure: see text] The absolute configuration of the deoxyhexuronic acid was deduced from 1H NMR chemical shifts and is most likely D. Approximately two O-acetyl groups per repeating unit are present, one of which is presumably on the Rha residue. The structure bears great resemblance to another polysaccharide, recently studied, produced by Sphingomonas paucimobilis I-886.     

Structure of acidic o-specific polysaccharide from the marine bacterium <Emphasis Type="Italic">Cellulophaga baltica</Emphasis>

The structure of an acidic O-specific polysaccharide from the marine bacterium Cellulophaga baltica was established by chemical methods and NMR spectroscopy. The polysaccharide was shown to consist of repeating tetrasaccharide units containing two mannose residues, one N-acetyl-D-glucosamine residue, and one D-glucuronic acid residue. An O-acetyl group was also found in the polysaccharide in nonstoichiometric amount. The polysaccharide had the following structure:

Open image in new window

     

Structural studies of the extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain H10b

The extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain H10b, when grown under strictly anaerobic conditions with glucose as carbohydrate source, has been studied by chemical and spectroscopic techniques. The results demonstrate that the polysaccharide consists of hexasaccharide repeating units with the following structure: [structure: see text] The isolated polysaccharide was found to be approximately 65% acetylated at O-2 of the 3-O-[(S)-1-carboxyethyl]-beta-D-Glcp residue. The absolute configuration of the 1-carboxyethyl groups was determined by circular dichroism.     

A structural investigation of the capsular polysaccharide of Klebsiella K14

The structure of the capsular polysaccharide isolated from Klebsiella serotype K14 has been investigated employing a combination of chemical and spectroscopic methods. The repeating structure is shown to be of the “4 + 1 + 1” type, and it carries a 1-carboxyethylidene acetal substituent at positions 4 and 6 of a terminal glucose residue. The polysaccharide is one of a group of only three Klebsiella polysaccharides that have been found to contain a galactofuranose residue in the repeating unit. The repeating unit has the following structure.     

The structure of the polysaccharide part of the LPS from Serratia marcescens serotype O19, including linkage region to the core and the residue at the non-reducing end

The structure of the LPS from Serratia marcescens serotype O19 was investigated. Deamination of the LPS released the O-chain polysaccharide together with a fragment of the core oligosaccharide. The following structure of the product was determined by NMR spectroscopy, mass spectrometry, and chemical methods: [carbohydrate structure: see text] The main polymer consists of a repeating disaccharide V-U and is present on average of 18 units per chain as estimated by integration of signals in the NMR spectra. The residue O corresponds to the primer, which initiates biosynthesis of the O-chain, and an oligomer of a disaccharide R-S is an insert between the primer and the main polymer. The polysaccharide has a beta-Kdo residue at the non-reducing end, a feature similar to that observed previously in the LPS from Klebsiella O12.     

Structure of an acylated exopolysaccharide synthesized by Acinetobacter sp

The complex preparation ethapolan synthesized by Acinetobacter sp. consists of neutral (minor component) and two acidic exopolysaccharides (EPS) one of which is acylated. On the basis of chemical modification of EPS, solvolysis with anhydrous hydrogen fluoride resulting in a penta- and octasaccharide fragments, Smith degradation, 1H- and 13C NMR analysis the following structure of the acylated polysaccharide repeating unit has been established (scheme): It is suggested that in the acylated EPS at least one glucose residue and the galactose residue are O-acylated.     

Purification and determination of the structure of capsular polysaccharide of Vibrio vulnificus M06-24.

Virulence of Vibrio vulnificus has been strongly associated with encapsulation and an opaque colony morphology. Capsular polysaccharide was purified from a whole-cell, phosphate-buffered saline-extracted preparation of the opaque, virulent phase of V. vulnificus M06-24 (M06-24/O) by dialysis, centrifugation, enzymatic digestion, and phenol-chloroform extraction. Nuclear magnetic resonance spectroscopic analysis of the purified polysaccharide showed that the polymer was composed of a repeating structure with four sugar residues per repeating subunit: three residues of 2-acetamido-2,6-dideoxyhexopyranose in the alpha-gluco configuration (QuiNAc) and an additional residue of 2-acetamido hexouronate in the alpha-galactopyranose configuration (GalNAcA). The complete carbohydrate structure of the polysaccharide was determined by heteronuclear nuclear magnetic resonance spectroscopy and by high-performance anion-exchange chromatography. The 1H and 13C nuclear magnetic resonance spectra were completely assigned, and vicinal coupling relationships were used to establish the stereochemistry of each sugar residue, its anomeric configuration, and the positions of the glycosidic linkages. The complete structure is: [----3) QuipNAc alpha-(1----3)-GalpNAcA alpha-(1----3)-QuipNAc alpha-(1----]n QuipNAc alpha-(1----4)-increases The polysaccharide was produced by a translucent phase variant of M06-24 (M06-24/T) but not by a translucent, acapsular transposon mutant (CVD752). Antibodies to the polysaccharide were demonstrable in serum from rabbits inoculated with M06-24/O.     

Structural determination of the capsular polysaccharide of Neisseria meningitidis group I: a two-dimensional NMR analysis

The capsular polysaccharide antigen of Neisseria meningitidis group I was isolated by Cetavlon precipitation and purified by ion-exchange chromatography. The structure of the I polysaccharide was determined largely by comprehensive proton and carbon-13 nuclear magnetic resonance studies in which both one-dimensional and two-dimensional experiments were carried out directly on the I polysaccharide. The I polysaccharide is composed of the repeating unit----4)alpha-L-GulpNAcA(1----3)[4-OAc]beta-D-ManpNA-cA(-- --in which the former residue adopts the 4C1 (L) conformation and the latter residue adopts the 4C1 (D) conformation. The one-bond coupling between the anomeric carbon and proton (1J13C,H) of the 2-acetamido-2-deoxy-beta-D-mannuronopyranosyl residue is not consistent with its beta-D configuration. This anomalous value of 1J13C,H for this residue is due to through-space anisotropy effects on its anomeric proton, generated by the proximity of the carboxyl group of the neighboring 2-acetamido-2-deoxy-alpha-L-guluronopyranosyl residue. The O-acetyl substituents of the I polysaccharide are essential for its antigenicity to group I polysaccharide-specific antibodies.     

Preliminary structure determination of the capsular polysaccharide of Vibrio cholerae O139 Bengal Al1837.

Vibrio cholerae O139 Bengal has recently been identified as a cause of epidemic cholera in Asia. In contrast to V. cholerae O1, V. cholerae O139 Bengal has a polysaccharide capsule. As determined by high-performance anion-exchange chromatography and 1H nuclear magnetic resonance analysis, the capsular polysaccharide of V. cholerae O139 Bengal strain Al1837 has six residues in the repeating subunit; this includes one residue each of N-acetylglucosamine, N-acetylquinovosamine (QuiNAc), galacturonic acid (GalA), and galactose and two residues of 3,6-dideoxyxylohexose (Xylhex). The proposed structure is [formula: see text]     

Structure of the capsular polysaccharide of Klebsiella serotype K40

The capsular polysaccharide from Klebsiella Serotype K40 contains D-galactose, D-mannose, L-rhamnose, and D-glucuronic acid in the ratios of 4:1:1:1. Methylation analysis of the native and carboxyl-reduced polysaccharide provided information about the glycosidic linkages in the repeating unit. Degradation of the permethylated polymer with base established the identity of the sugar unit preceding the glycosyluronic acid residue. The modes of linkages of different sugar residues were further confirmed by Smith degradation and partial hydrolysis of the K40 polysaccharide. The anomeric configurations of the different sugar residues were determined by oxidation of the peracetylated native and carboxyl-reduced polysaccharide with chromium trioxide. Based on all of these results, the heptasaccharide structure 1 was assigned to the repeating unit of the K40 polysaccharide. (Formula: see text)     

Structural characterization of the outer core and the O-chain linkage region of lipopolysaccharide from Pseudomonas aeruginosa serotype O5.

The point of attachment of the O-chain in the outer core region of Pseudomonas aeruginosa serotype O5 lipopolysaccharide (LPS) was determined following a detailed analysis of the extended core oligosaccharide, containing one trisaccharide O-chain repeating unit, present in both the wild-type strain PAO1 and O-chain deficient mutant strains AK1401 and PAO-rfc. The structure of the extended core oligosaccharide was determined by various mass spectrometric methods as well as one-dimensional and two-dimensional NMR spectroscopy. Furthermore, the one-dimensional analogues of NOESY and TOCSY experiments were applied to confirm the structure of the outer core region in the O-chain polysaccharide. In both the extended core oligosaccharide and the core of the smooth LPS, a loss of one of the beta-glucosyl residues and the translocation of the alpha-rhamnosyl residue, followed by the attachment of the first O-chain repeating unit was observed. This process is complicated and could involve two distinct rhamnosyltransferases, one with alpha-1, 6-linkage specificity and another with alpha-1,3-linkage specificity. It is also plausible that an alpha-1,3 rhamnosyltransferase facilitates the addition of the 'new' alpha-rhamnosyl residue that will act as a receptor for the attachment of the single O-antigen repeating unit in the LPS of the semi-rough mutant. The 2-amino-2-deoxy-fucosyl residue of the first O-chain repeating unit directly attached to the core was found to have a beta-anomeric configuration instead of an alpha configuration, characteristic for this residue as a component of the O-chain polysaccharide. The results of this study provide the first example of the mechanistic implications of the structure of the outer core region in a fully assembled O-chain containing LPS, differing from the O-chain deficient rough LPS.     

Structure of a colitose-containing O-specific polysaccharide of the marine bacterium Pseudoalteromonas tetraodonis IAM 14160(T).

O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Pseudoalteromonas tetraodonis type strain IAM 14160(T) and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, 1H,(13)C HMQC and HMBC experiments. The polysaccharide was found to consist of hexasaccharide repeating units containing one residue each of D-Gal, D-GlcA, D-GalNAc and D-GlcNAc and two residues of 3,6-dideoxy-L-xylo-hexose (colitose, Col) and having the following structure:In common with the polysaccharides of some other bacteria, the polysaccharide studied contains a tetrasaccharide fragment alpha-Colp-(1-->2)-beta-D-Galp-(1-->3)-[alpha-Colp-(1-->4)]-beta-D-GlcpNAc, which is a colitose ('3-deoxy-L-fucose') analogue of the Lewis(b) blood group antigenic determinant.     

Structure of a new 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose-containing O-specific polysaccharide from the lipopolysaccharide of Citrobacter gillenii PCM 1542

The O-specific polysaccharide of Citrobacter gillenii PCM 1542 from serotype O-12a,12 b is composed of one residue each of D-glucose, D-GlcNAc, 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose (D-GlcNAcyl) and two GalNAc residues. On the basis of sugar and methylation analyses of the intact and Smith degraded polysaccharides, along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the branched pentasaccharide repeating unit of the O-specific polysaccharide was established:This structure differs significantly from that of the O-specific polysaccharide of C. gillenii PCM 1544 from the same serotype O-12a,12 b, which has been established earlier (Kübler-Kielz.shtsls;b, J. et al. Carbohydr. Res. 2001, 331, 331-336). Serological studies confirmed that the two O-antigens are not related and suggested that strains PCM 1542 and 1544 should be classified into different O-serogroups.     

Structural studies of the Escherichia coli O-149 O-antigen polysaccharide

The structure of the O-antigen polysaccharide from Escherichia coli O-149 has been investigated; methylation analysis, partial hydrolysis with acid, and n.m.r. spectroscopy were the principal methods used. It is concluded that the polysaccharide is composed of trisaccharide repeating-units having the following structure. (Formula: see text). The absolute configuration at the acetalic carbon atom of the pyruvic acid residue is S.     

Complete structure of the adhesin receptor polysaccharide of Streptococcus oralis ATCC 55229 (Streptococcus sanguis H1).

This report describes the determination of the complete primary structure of the adhesin receptor polysaccharide of Streptococcus oralis ATCC 55229 (previously characterized as Streptococcus sanguis H1), a Gram-positive bacteria implicated in dental plaque formation. The polysaccharide was isolated from S. oralis ATCC 55229 cells after deproteination, enzymatic hydrolysis, and ion exchange chromatography. It was shown to consist of rhamnose, galactose, glucose, glycerol, and phosphate, in molar ratios of 2:3:1:1:1. Sequence and linkage assignments of the glycosyl residues were obtained by methylation analysis followed by gas-liquid chromatography and electron-impact mass spectrometry. 31P NMR spectroscopy revealed that phosphate was present in a diester, connecting glycerol to one of the galactosyl residues. High-performance liquid chromatography of a partial acid hydrolysate of the polysaccharide confirmed this finding by showing galactose 6-phosphate and glycerol 1-phosphate. The structural determination was completed by the combination of two-dimensional homonuclear Hartmann-Hahn and NOE experiments and heteronuclear [1H,13C] and [1H,31P] multiple-quantum coherence experiments. Thus, the adhesin receptor polysaccharide of S. oralis ATCC 55229 was found to be a polymer composed of hexasaccharide repeating units that contain glycerol linked through a phosphodiester to C6 of the alpha-galactopyranosyl residue and are joined end-to-end through galactofuranosyl-beta(1-->3)-rhamnopyranosyl linkages: [formula: see text] This structure is novel among bacterial cell surface polysaccharides in general and specifically among those implicated in dental plaque formation.     

On the Steric Configuration of the Urushi Polysaccharide

This paper presents the steric configuration of a polysaccharide obtained from the latex of the Japanese lacquer tree. Thus, the polysaccharide consisted of galactose and galacturonic acid, and the main chain of polysaccharide was proved to be linked with 1 → 3 linkage of galactose residue of 6-O-galacturonosyl galactose unit by the periodate oxidation method. The molecular weight was calculated to be 6 × 10⁴ by Archibald’s ultracentrifugal method. The configuration of the polysaccharide was discussed to be α helical structure of which one turn of the helices consists of 6 to 7 6-O-galacturonosyl galactose units and the diameter of the helix is 21 Å and one pitch is 9.5 Å on the basis of viscosity measurements, color reaction with iodine, crystallization with capronic acid and consideration of molecular models.     

The structure of the O-polysaccharide of the Pseudoalteromonas rubra ATCC 29570T lipopolysaccharide containing a keto sugar

The structure of the phenol-soluble polysaccharide from Pseudoalteromonas rubra type strain ATCC 29570T has been elucidated using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, gNOESY, ROESY, 1H,13C gHMQC and gHMBC experiments. It is concluded that the trisaccharide repeating unit of the polysaccharide has the following structure: [carbohydrate structure: see text] where Sug is 2-acetamido-2,6-dideoxy-D-xylo-hexos-4-ulose, Am is acetimidoyl and Acyl is a malic acid residue, which is O-acetylated in approximately 70% of the units.     

The structure of the O-specific polysaccharide chain of Proteus penneri strain 16 lipopolysaccharide

O-Specific polysaccharide was obtained by mild acid degradation of Proteus penneri strain 16 lipopolysaccharide and found to contain D-glucose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, and 3,6-dideoxy-3-[(R)-3-hydroxybutyramido]- D-galactose in the ratio of 2:1:1:1 as well as a small proportion of O-acetyl groups. On the basis of one-dimensional 1H-NMR13C-NMR and NOE spectroscopy, two-dimensional homonuclear-shift-correlated spectroscopy with one-step and two-step relayed coherence transfer and heteronuclear 1H/13C NMR shift-correlated spectroscopy, it was concluded that the O-specific polysaccharide of P. penneri strain 16 has the following structure: (formula; see text) This structure was confirmed by methylation analysis and structural analysis of a linear tetrasaccharide fragment prepared by cleavage of the polysaccharide with anhydrous hydrogen fluoride followed by conversion of the alpha-tetrosyl fluoride obtained in to the corresponding free oligosaccharide and alditol. O-Acetyl groups were tentatively located at position 3 of the glucuronic acid residue and at position 4 of the 6-substituted glucose residue, the degree of acetylation being less than 20% of the total. Cross-reactions of P. penneri strain 16 anti-(O-specific polysaccharide) antiserum with lipopolysaccharides from several other Proteus strains and the role of 3,6-dideoxy-3-(R)-3-hydroxybutyramido-D-galactose in the serological specificity of P. penneri strain 16 are discussed.     

Somatic antigens of Shigella and Escherichia coli. Determination of the structure of O-specific polysaccharide from Shigella boydii type 12 and its serological properties compared with the O-specific polysaccharide from Escherichia coli

The structure of the O-specific polysaccharide of the somatic antigen (lipopolysaccharide) of Shigella boydii, type 12, was established by 1H- and 13C-NMR, methylation analysis and partial acid hydrolysis methods. The polysaccharide consists of pentasaccharide repeating units of the following structure: (formula; see text) The amount of O-acetyl groups was far less than stoichiometric, only about 2 for 3-4 repeating units. Nevertheless, the results of serological studies revealed 3-O-acetyl-alpha-L-rhamnose residue to be the major immunodominant group. In spite of the presence of similar trisaccharide fragments, the lipopolysaccharide and polysaccharide from Shigella boydii type 12 gave no crossreaction with lipopolysaccharide and polysaccharide from Escherichia coli 07. The possible reasons of the absence of serological relatedness between the Sh. boydii, type 12, and E. coli 07 cells were discussed.     

Macromolecular properties and end-group analysis of heparin isolated from bovine liver capsule.

Glycosaminoglycans were extracted from bovine liver capsule with 4 M-guanidinium chloride, resulting in solubilization of approx. 90% of the total uronic acid-containing polysaccharide of the tissue. The extracted polysaccharide was purified and fractionated by anion-exchange chromatography on DEAE-cellulose, density-gradient ultracentrifugation in CsCl and finally gel chromatography on Sepharose 4B. By using these procedures, the two major polysaccharide components, dermatan sulphate and heparin, which constituted 55 and 30% respectively of the total glycosaminoglycan content of the tissue, were separated from each other. Analysis of the macromolecular properties of the two polysaccharides showed that heparin existed exclusively as single polysaccharide chains, whereas dermatan sulphate occurred largely as a proteoglycan (protein content, 74% dry wt.). The purified heparin preparation was subjected to sedimentation-equilibrium ultracentrifugation, indicating a molecular weight of 8800. Analysis for neutral sugars (by g.l.c.) showed 0.1 residue of xylose and 0.2 residue of galactose/polysaccharide chain; serine amounted to 0.3 residue/polysaccharide chain. Reduction of the heparin with NaB3H4 resulted in incorporation of 3H, approximately corresponding to one reducible group/polysaccharide chain. The 3H-labelled sugar residue was liberated by a combination of acid hydrolysis and deaminative cleavage of the polysaccharide with HNO2; it was subsequently identified as an aldonic acid by paper electrophoresis. Most of the heparin chains thus contained a uronic acid residue in reducing position. It is suggested that heparin isolated from bovine liver capsule is a degradation product released from larger molecules by an endo-glycuronidase.