The tetrasaccharide beta-D-glucopyranosyl-(1,3)-beta-D-glucopyranuronyl-(1, 2)-L-glycero-alpha-D-manno-heptopyranosyl-(1,5)-3-deoxy-D-manno-2- octulosonic acid was isolated after treatment of polysaccharide 1 of Bordetella pertussis endotoxin with nitrous acid. Taking into account previously identified di- and trisaccharide fragments and analytical data obtained for the intact polysaccharide 1, we present the structure of a heptasaccharide that is thought to represent the region immediately adjacent to the hydrophobic (lipid A) moiety of lipopolysaccharide 1 of the B. pertussis endotoxin. This heptasaccharide represents 50 to 60% of the complete polysaccharide structure

Chaby, R

Moreau, M

Szabo, L

PubMed

The tetrasaccharide P-D-glucopyranosyl-(1,3)-4-D-glucopyranuronyl-(1,2)-L-glycero-a-D-manno-heptopyranosyl-(1,5)-3-deoxy-D-manno-2-octulosonic acid was isolated after treatment of polysaccharide 1 of Bordetella pertussis endotoxin with nitrous acid. Taking into account previously identified di-and trisaccharide fragments and analytical data obtained for the intact polysaccharide 1, we present the structure of a heptasaccharide that is thought to represent the region immediately adjacent to the hydrophobic (lipid A) moiety of lipopolysaccharide 1 of the B. pertussis endotoxin. This heptasaccharide represents 50 to 60% of the complete polysaccharide structure. Polysaccharides isolated from endotoxins of smooth-type, gram-negative bacteria usually contain two regions, one composed of repeating oligosaccharide units and the other composed of nonrepeating monosaccharides. In the case of endotoxins isolated from enterobacteria, the first region is referred to as the 0-specific chain and the second as the core. While exact structures are known for many repeating units present in endotoxins (17), core regions have been less frequently investigated. For the latter studies, R forms of enteric bacteria have been used primarily  In previous studies we have established that the endotoxin isolated from vaccinal, phase I strains of Bordetella pertussis is made up of two major lipopolysaccharides, lipopolysaccharides 1 and 2 (which account for almost all of the mass of the endotoxin), and that the isolated polysaccharide chains (polysaccharides 1 [PS-1] and 2) of these are, in fact, oligosaccharides; their estimated molecular masses of about 3,000 daltons indicate that they contain approximately 12 to 16 monosaccharide units. As their 13C nuclear magnetic resonance (NMR) spectra do not suggest the presence of repeating units, they could represent core-type oligosaccharides. In agreement with this, the rate of migration of B. pertussis endotoxin was found to be similar to that of endotoxins isolated from R mutants of enteric bacteria  Furthermore, it has been demonstrated that the binding of the B. pertussis endotoxin to rabbit peritoneal macrophages is mediated by the polysaccharide region (9) and that in human monocytes, secretion of interleukin 1 is stimulated by the isolated PS-1 of this endotoxin (7a). Knowledge of the exact structure of this polysaccharide is therefore clearly of interest. In the present work the structure of the tetrasaccharide that contains the terminal reducing 3-deoxy-Dmanno-2-octulosonic acid (KDO) unit of PS-1 is described. * Corresponding author. MATERIALS AND METHODS Endotoxin. B. pertussis 1414 (phase I, vaccinal; Institut Mdrieux, Lyon, France) was grown in the liquid medium of Cohen and Wheeler, and the endotoxin was isolated by the phenol-water method as previously described  General methods. For descending chromatography on Whatman no. 1 paper, the solvents butan-2-one-methanolformic acid-water (8:4:4:1, vol/vol) (Si) and butan-i-olpyridine-water (6:4:3, vol/vol) (S2) were used. High-voltage paper electrophoresis was carried out on Whatman 3MM paper in a flat-plate electrophoresis apparatus with a pyridine-acetic acid-water (16:11:973, vol/vol; pH 5) buffer. Compounds containing vicinal diols were detected by treatment with periodate-alkaline silver nitrate (24). On paper chromatograms and electropherograms radioactive material was located with a Berthold LB 280 scanner; in solutions it was measured by scintillation with 2,5-di-(5-tert-butyl-2-benzoxazolyl)thiophene (4 g/liter in toluene), using an Intertechnique SL 30 counter. Sugars and polyols were peracetylated with sodium acetate-acetic anhydride at 100°C for 1 h, solvents were removed, the products were extracted from the residue with chloroform, which was then evaporated, and the peracetates, dissolved in ethyl acetate, were used for analysis. For combined gas-liquid chromatography and mass spectrometry (GLC-MS), a stainless steel column (2,000 by 3.2 mm, charged with 3% SE-30 on GasChrom Q, 100-120 mesh) was used with a temperature gradient of 2°C/min from 120 to 2000C. The analytical methods used to estimate hexoses, heptoses, hexosamines, hexuronic acids, and nitrogen were those employed previousl

Monique Moreau

Richard Chaby

Ladislas Szabo

CiteSeerX

Structure of the terminal reducing heptasaccharide of polysaccharide 1 isolated from the Bordetella pertussis endotoxin

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=215687

Structure of the terminal reducing heptasaccharide of polysaccharide 1 isolated from the Bordetella pertussis endotoxin.

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