A novel type of endotoxin structure present in Bordetella pertussis. Isolation of two different polysaccharides bound to lipid A.

The endotoxin of Bordetella pertussis was cleaved by mild acidic hydrolysis to yield a polysaccharide (polysaccharide I, 15%), a glycolipid (63%) and lipid X (2%). Further treatment of the glycolipid with stronger acid released a second polysaccharide (polysaccharide II, 9%) and material similar to lipid A present in enterobacterial endotoxins. Both polysaccharides possess a single molecule of 3-deoxy-2-octulosonic acid as the reducing, terminal sugar. In polysaccharide II the octulosonic acid is phosphorylated in position 5 and presumably substituted in position 4; in polysaccharide I the octulosonic acid is not phosphorylated, but is substituted in position 5. Following treatment of the endotoxin with strong base, a fragment was isolated that contained bound, non-phosphorylated 3-deoxy-2-octulosonic acid, glucosamine phosphate and fatty acids. This indicated that polysaccharide I, like polysaccharide II, was bound to the lipid region of the endotoxin. The endotoxin structure thus defined is different from that proposed for the lipopolysaccharides of enterobacteria.     

The cell wall polysaccharide of Streptococcus gordonii 38: structure and immunochemical comparison with the receptor polysaccharides of Streptococcus oralis 34 and Streptococcus mitis J22

As part of our ongoing investigations involving lectinmediatedadhesion among oral bacteria, the receptor polysaccharide fromStreptococcus gordonii 38 was isolated and characterized. Carbohydrateanalysis of the hydrolysed S.gordonii 38 polysaccharide by high-performanceanionexchange chromatography with pulsed amperometric detection(HPAEC-PAD) showed galactose (Gal) (2 mol), N-acetylgalactosamine(GalNAc) (1 mol), rhamnose (Rha) (2 mol), glucose (Glc) (1 mol)and galactosamine-6-phosphate (1 mol). Mild acid hydrolysisof the polysaccharide yielded a heptasaccharide repeating unit.The structure of the heptasaccharide repeating unit was determinedby high-resolution NMR spectroscopy which includes various homonuclear(DOF—COSY, TQF-COSY, NOESY and HOHAHA) and heteronuclearexperiments (HMQC), including linkage assignments by ¹H-¹³Clong-range correlation (HMBC). Complete ¹H and ₁₃C NMR assignmentsfor the intact polysaccharide yielded the covalent structureof a heptasaccharide repeating unit:     

Isolation of a trisaccharide containing 2-amino-2-deoxy-D-galacturonic acid from the Bordetella pertussis endotoxin

4-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl-6-O-(2-amino-2-deoxy-alpha-D-galactopyranuronyl)-D-glucopyranose, a branched-chain trisaccharide, was isolated after hydrolysis of Bordetella pertussis endotoxin with 4 M HCl for 1 h at 100 degrees C. The trisaccharide was present in both polysaccharide moieties of the two constituent lipopolysaccharides of this endotoxin. Its structure was established by analysis of the 400-MHz nuclear magnetic resonance spectrum and by chemical and enzymatic degradation.     

Structure of the capsular polysaccharide of Klebsiella serotype K79

The structure of the capsular polysaccharide from Klebsiella K79 was determined by the techniques of methylation, periodate oxidation, beta-elimination, chromic acid oxidation, and partial hydrolysis. N.m.r. spectroscopy (1H and 13C) was used extensively to establish the nature of the anomeric linkages of the polysaccharide and of oligosaccharides derived through degradative procedures. The polysaccharide was found to have the heptasaccharide, "5 + 2" repeating unit: (Formula: see text).     

Characterization of a structurally complex heptasaccharide isolated from the pectic polysaccharide rhamnogalacturonan II

A heptasaccharide was released from the plant cell-wall, pectic polysaccharide rhamnogalacturonan II by selective acid hydrolysis of the glycosidic linkages of apiosyl residues. The heptasaccharide was purified to homogeneity by gel filtration and anion-exchange chromatography. Some of the heptasaccharide molecules were found to be mono- and some di-O-acetylated, but the location of the acetic ester groups was not determined. The heptasaccharide was found to have the following structure, where AceA = an aceryl (3-C-carboxy-5-deoxy-l-xylosyl) residue, and Api = an apiose residue.

     

Structural characterisation of the exopolysaccharide produced by Streptococcus thermophilus EU20

Streptococcus thermophilus EU20 when grown on skimmed milk secretes a high-molecular-weight exopolysaccharide that is composed of glucose, galactose and rhamnose in a molar ratio of 2:3:2. Using chemical techniques and 1D and 2D-NMR spectroscopy (1H and 13C) the polysaccharide has been shown to possess a heptasaccharide repeating unit having the following structure: [chemical structure: see text]. Treatment of the polysaccharide with mild acid (0.5 M TFA, 100 degrees C for 1 h) liberates two oligosaccharides; the components correspond to the repeating unit and a hexasaccharide equivalent to the repeating unit minus the terminal alpha-L-Rhap.     

Structural studies of the Rhizobium trifolii extracellular polysaccharide

The structure of the extracellular polysaccharide of Rhizobium trifolii has been investigated. Methylation analysis, sequential degradations by oxidation and elimination of oxidized residues, uronic acid degradation, and degradation by oxidation of the acetylated polysaccharide with chromium trioxide in acetic acid were the main methods used. It is proposed that the polysaccharide is composed of heptasaccharide repeating-units having the following structure:

An unusual feature is that some of the repeating units are incomplete and lack the terminal β-d-galactopyranosyl group. The polysaccharide contains O-acetyl groups (somewhat more than 1 mol. per unit), linked to O-2 and O-3 of 4-O-substituted d-glucopyranosyl chain-residues. A previous finding that the polysaccharide contains 2-deoxy-d-arabino-hexose (2-deoxy-d-glucose) residues is erroneous.     

Structural studies of the capsular polysaccharide of Acinetobacter calcoaceticus BD4

Compositional analysis of the intact and carboxyl-reduced capsular polysaccharide of Acinetobacter calcoaceticus BD4 (PS-4) showed it to consist of L-rhamnose, D-glucose, D-glucuronic and D-mannose in molar ratios of 4:1:1:1. 13C-nuclear magnetic resonance spectroscopy, methylation analysis, oligosaccharide analysis and base-catalyzed beta-elimination were used to elucidate the primary structure. Oligosaccharides were obtained by enzymatic depolymerization with a specific bacteriophage-induced depolymerase and by partial acid hydrolysis. Form the results it is concluded that PS-4 consists of repeating units of the heptasaccharide (Formula: see text). The bacteriophage-induced depolymerase was found to be an endo-beta-D-glucosidase that hydrolyzed the bond beta-D-Glc-(1----3)-L-Rha to generate a heptasaccharide in 40% yield.     

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)     

Application of high-resolution n.m.r. spectroscopy to the elucidation of the structure of the specific capsular polysaccharide of Streptococcus pneumoniae type 7F

The specific capsular polysaccharide of Streptococcus pneumoniae type 7F (American type 51) is a high-molecular-weight neutral polymer composed of 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, D-glucose, D-galactose, L-rhamnose, and 2-O-acetyl-L-rhamnose residues. N.m.r. spectroscopy (1H and 13C), in conjunction with composition and methylation analyses, and periodate oxidation data, showed the polysaccharide to be a branched polymer with a repeating heptasaccharide unit having the following structure. (formula; see text)     

The fatty acid content of the Bordetella pertussis endotoxin

The fatty acid content of Bordetella pertussis endotoxin has been estimated by several methods. Expressed as 3-hydroxytetradecanoic acid, it was 0.74 mumol (mg lyophilized material)-1, 0.38 mumol being ester-bound, and 0.32 mumol in amide linkage. Reported molar ratios of ester-bound to amide-bound fatty acids in endotoxins of various bacterial species range from 2.4 to 2 in B. pertussis, to 5 to 2 in Salmonella minnesota; according to these figures large differences must exist in the degree of substitution, and the substitution pattern of the glucosaminyl-beta-1,6-glucosamine unit present in the hydrophobic region of endotoxins. When fatty acids, released by acid and alkaline hydrolyses of the B. pertussis endotoxin, were extracted into chloroform, unidentified chromogenic substances appearing in the extract interfered with their colorimetric estimation; no interference was observed when hexane was used instead of chloroform.     

Lipopolysaccharide receptor on rabbit peritoneal macrophages. I. Binding characteristics

The Bordetella pertussis endotoxin, labeled with tritium ((3H)-LPS), bound irreversibly and nonspecifically to rabbit lung macrophages, but bound reversibly and specifically to both resident and elicited rabbit peritoneal macrophages. The specific binding capacity of the macrophages was saturated with about 3 X 10(4) LPS molecules per cell. The binding was inhibited with the homologous unlabeled endotoxin, but not at all with endotoxin from Proteus mirabilis, thus assessing ligand specificity. Endotoxins from other bacteria gave intermediate inhibition value. Binding of tritium-labeled pertussis endotoxin was significantly inhibited by one of the two polysaccharides (PS-1) present in this endotoxin, but neither the other polysaccharide (PS-2) nor the Lipid A fragment exhibited such activity. These results strongly suggest the presence of a lectin-like receptor for LPS on the membrane of rabbit peritoneal macrophages.     

Isolation of two protein-free and chemically different lipopolysaccharides from Bordetella pertussis phenol-extracted endotoxin.

Endotoxin prepared from several Bordetella pertussis strains in both immunological phases I and IV gave two lipopolysaccharide peaks (LPS-I and LPS-II) when analyzed on hydroxylapatite columns in a phosphate buffer containing 0.1% sodium dodecyl sulfate; these lipopolysaccharides, present in the ratio of 2:3, are true endotoxins by both chemical and biological criteria. Endotoxins isolated from Escherichia coli, Salmonella typhimurium, and Shigella flexneri gave single lipopolysaccharide peaks when analyzed by the same procedure. Upon hydrolysis with acetic acid (pH 3.4) at 100 degrees C for 1 h, LPS-I released a polysaccharide (PS-I); the linkage broken was that of the glycosidic bond of a non-phosphorylated 3-deoxy-oct-2-ulosonic acid. Treatment with 0.25 M mineral acid at 100 degrees C for 30 min was required to free the polysaccharide moiety (PS-II) of LPS-II, the linkage broken being the glycosidic bond of a phosphorylated 3-deoxy-oct-2-ulosonic acid. Chemical and physical differences of the polysaccharide moieties PS-I and PS-II present in LPS-I and LPS-II have been described previously (25). By using the technique of 125I labeling, it was shown that the totality of labeled proteins present in the endotoxin extracted from Bordetella pertussis by the phenol-water procedure could be separated from the lipopolysaccharide by column chromatography on hydroxylapatite; it follows that these proteins are not linked by covalent bonds to the lipopolysaccharide.     

Structure of the Bordetella pertussis 1414 endotoxin

The endotoxin (lipopolysaccharide) of Bordetella pertussis, the agent of whooping cough, consists of a lipid A linked to a highly branched dodecasaccharide containing several acid and amino sugars. The elucidation of the polysaccharide structure was accomplished by first analyzing the structures of fragments obtained by hydrolysis and nitrous deamination and then piecing the fragments together. The fine structure of the antigenic distal pentasaccharide, presented here, was determined by chemical analyses as well as by high-resolution nuclear magnetic resonance and mass spectrometry. The complete structure was reconstituted and confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The following structure was derived from the combined experimental data:The detailed structure combined with previously reported serological data now allows the synthesis of its epitopes for potential vaccines.     

Altered responses to modulators of guanine nucleotide binding protein activity in endotoxin tolerance

The effects of cholera toxin or pertussis toxin and nonhydrolyzable GTP analogs on Salmonella enteritidis endotoxin stimulation of iTxB2 and i6-keto-PGF1 alpha synthesis in control and endotoxin tolerant rat peritoneal macrophages were determined. Pretreatment with pertussis toxin alone had no effect on basal macrophage iTxB2 or i6-keto-PGF1 alpha production, but pertussis toxin (0.1, 1.0 and 10 ng/ml) significantly inhibited endotoxin-stimulated iTxB2 and i6-keto-PGF1 alpha synthesis. Pretreatment with cholera toxin, which did not affect basal iTxB2 or i6-keto-PGF1 alpha synthesis, significantly enhanced endotoxin-induced synthesis of iTxB2 and i6-keto-PGF1 alpha. The effects of pertussis and cholera toxin with or without endotoxin were significantly (P less than 0.05) less in macrophages from endotoxin tolerant rats compared to control macrophages. GTP[gamma-S] (100 microM) significantly increased iTxB2 synthesis and significantly augmented endotoxin-stimulated iTxB2 synthesis in control macrophages (P less than 0.05). However, in macrophages from endotoxin tolerant rats the effect of GTP[gamma-S] on iTxB2 synthesis was significantly less (P less than 0.05) compared to control macrophages. Collectively, these data suggest that: (1) guanine nucleotide binding regulatory proteins mediate endotoxin-stimulated arachidonic acid metabolism in rat peritoneal macrophages; and (2) endotoxin tolerance induces alterations in guanine nucleotide binding protein activity.     

Primary structure of the 2-O-methyl-alpha-L-fucose-containing side chain of the pectic polysaccharide,rhamnogalacturonan II

A 2-O-methylfucosyl-containing heptasaccharide was released from red wine rhamnogalacturonan II (RG-II) by acid hydrolysis of the glycosidic linkage of the aceryl acid residue (AceA) and purified to homogeneity by size-exclusion and high-performance anion-exchange chromatographies. The primary structure of the heptasaccharide was determined by glycosyl-residue and glycosyl-linkage composition analyses, ESIMS, and by 1H and 13C NMR spectroscopy. The NMR data indicated that the pyranose ring of the 2,3-linked L-arabinosyl residue is conformationally flexible. The L-Arap residue was confirmed to be alpha-linked by NMR analysis of a tetraglycosyl-glycerol fragment, [alpha-L-Arap-(1-->4)-beta-D-Galp-(1-->2)-alpha-L-AcefA-(1-->3)-beta-L-Rhap-(1-->3)-Gro], generated by Smith degradation of RG-II. Our data together with the results of a previous study,(1) establish that the 2-O-Me Fuc-containing nonasaccharide side chain of wine RG-II has the structure (Api [triple bond] apiose): [see structure]. Data are presented to show that in Arabidopsis RG-II the predominant 2-O-MeFuc-containing side chain is a mono-O-acetylated heptasaccharide that lacks the non-reducing terminal beta-L-Araf and the alpha-L-Rhap residue attached to the O-3 of Arap, both of which are present on the wine nonasaccharide.     

Synthesis of an Fmoc-Asn-heptasaccharide building block and its application to chemoenzymatic glycopeptide synthesis

The Fmoc-protected heptasaccharide asparagine building block β-GlcNAc-(1→2)-α-Man-(1→3)-[β-GlcNAc-(1→2)-α-Man-(1→6)]β-Man-(1→4)-β-GlcNAc-(1→4)-β-GlcNAc-(Fmoc)Asn was obtained by chemical synthesis. Two flexible strategies were developed with optimized conditions for the simultaneous debenzylation of the sugar and the amino acid part. The heptasaccharide asparagine building block is a partial structure of many glycoproteins and can be used for glycopeptide synthesis in solution and on the solid phase. In this work the heptasaccharide asparagine was elongated in solution to an Fmoc-glycopentapeptide methylester. After chemical cleavage of the Fmoc group the methylester was removed enzymatically by chymotrypsin. The use of β-(1→4)-galactosyltransferase and α-(2→6)-sialyltransferase in the presence of alkaline phosphatase allowed the efficient transfer of four sugar units to the acceptor resulting in an undecasaccharide glycopentapeptide.     

Detergent-accelerated hydrolysis of bacterial endotoxins and determination of the anomeric configuration of the glycosyl phosphate present in the "isolated lipid A" fragment of the Bordetella pertussis endotoxin

Due to the formation of micelles, severance of the hydrophilic (poly- or oligosaccharide) and hydrophobic ("Lipid A") domains of bacterial lipopolysaccharides at pH 3.4 or 4.5 and 100 degrees is slow and sometimes does not proceed at all; partially degraded fragments are usually formed. At pH 3.4 (100 degrees) in aqueous 1% sodium dodecylsulphate (SDS), both lipopolysaccharides of the Bordetella pertussis endotoxin are cleaved within 20-30 min, but 80% of the glycosidically bound phosphate present in the hydrophobic domain is lost. Other endotoxins behave similarly. At pH 4.5 (100 degrees) and in the absence of detergent, hydrolysis of the glycosidic bonds of 3-deoxy-D-manno-2-octulosonic acid residues of the B. pertussis endotoxin is negligible but, in aqueous 1% SDS, severance of the two regions of LPS 1 is complete within 1 h (that of LPS-2 requires 3-4 h), and the glycosidically bound phosphate of the isolated hydrophobic region is preserved. Comparison of the rate of acid-catalysed hydrolysis of the glycosidically bound phosphate present in this "isolated Lipid A" preparation with that of 2-deoxy-2-[(3R)-3-hydroxytetradecanamido]-alpha- and -beta-D-glucopyranose 1-phosphates established that the former 1-phosphate was the alpha anomer.     

Structural characterisation and enzymic modification of the exopolysaccharide produced by Lactococcus lactis subsp. cremoris B39

Lactococcus lactis subsp. cremoris B39 grown on whey permeate produced an exopolysaccharide containing L-Rha, D-Gal and D-Glc in a molar ratio of 2:3:2. The polysaccharide was modified using an enzyme preparation from Aspergillus aculeatus, resulting in the release of Gal and a polymer with approximately the same hydrodynamic volume as the native polysaccharide. Linkage analysis and 1H NMR studies of both the native and modified exopolysaccharides elucidated that terminally linked Gal was released during modification and that the chemical structure of the branches within the repeating units is: beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. 2D NMR experiments (both 1H-1H and 1H-13C) revealed that exopolysaccharide B39 consists of a branched heptasaccharide repeating unit with the following structure: [structure: see text].     

3-Deoxy-2-octulosonic acid 5-phosphate: a component of the endotoxin of Bordetella pertussis.

Upon hydrolysis with 2 N hydrochloric acid for 2 h, a 3-deoxy-octulosonic acid 5-phosphate was released from the endotoxin of Bordetella pertussis. The structure of the compound was established through chemical degradation. By periodate treatment of the intact endotoxin it was shown that positions 7 and 8 of the bound octulosonic acid phosphate were free, which, if present in a cyclic form, must be a pyranoside.