Structure of the major exopolysaccharide produced by Azotobacter beijerinckii B-1615.

A. beijerinckii strain B-1615 produced two acidic exopolysaccharides in the ratio approximately 9:1. The minor polysaccharide contained mannuronic and guluronic acids in the ratio 2.3:1 and is a bacterial alginate. The major polysaccharide consisted of D-galactose, L-rhamnose, and pyruvic acid in the ratios 2:1:1 and was acetylated. On the basis of methylation analysis, and 1H- and 13C-n.m.r. spectroscopy of the polysaccharide before and after removal of the pyruvic acid residues and O-deacetylation, it was concluded that the major polysaccharide had the structure [formula; see text] with up to 1.5 OAc groups per repeating unit.

A selective method for sequential splitting of O- and N-linked glycans from N,O-glycoproteins.

O-Linked oligosaccharides from N,O-glycoproteins were selectively split off by treatment with alkaline sodium borohydride in the presence of cadmium salt. The side reaction of reductive cleavage of N-glycosylamide and peptide bonds, observed under standard conditions of splitting of O-linked chains (M NaBH4 and 50mM NaOH, 16 h, 50 degrees), was inhibited by addition of 50-10 mM cadium acetate and 5-10mM EDTA.Na4, as shown by treatment of model compounds and several glycoproteins (ovomucoid, group-specific glycoproteins H and B, fetuin, and asialofetuin). This treatment, in combination with the previously developed procedure for the release of the N-linked oligosaccharide chains by lithium borohydride, allows a sequential, selective cleavage of O-, and then N-linked oligosaccharides from N,O-glycoproteins by chemical methods.

The carbohydrate chains of influenza virus hemagglutinin.

The major surface antigen of influenza virus A/Leningrad/385/80 (H3N2), H3 hemagglutinin, as well as its heavy and light subunits were obtained by bromelain treatment, followed by gel chromatography. Carbohydrate chains were split off from both subunits by lithium borohydride-lithium hydroxide in aqueous 2-methyl-2-propanol, and individual oligosaccharides isolated. The main oligosaccharides, whose structure was determined by 1H-n.m.r. spectroscopy and chemical methods, are of the ordinary oligomannoside and complex types. It was found that, in spite of the great difference in number of glycosylation sites in heavy and light subunits, the amount and even relative abundance of variants of carbohydrate chains in both subunits are very similar.

Specific method for the fragmentation of the polypeptide chain of glycoproteins. Distribution of carbohydrate chains on the peptide core of blood-group-specific glycoprotein.

A method for specific fragmentation of the polypeptide backbone of glycoproteins at the glycosylated serine and threonine residues has been developed. The fragmentation includes beta-elimination of the carbohydrate chains, followed by bromination of the resulting enamine groups, and cleavage of the brominated amino acid residues by alkaline sodium borohydride. The method was employed for fragmentation of the peptide core of pig blood-group substance H. Essentially all the serine and threonine residues were shown to be O-glycosylated, and rather frequently either adjacent or separated by a single amino acid (mainly alanine). When they were separated by two or three amino acid residues, proline was preponderant.

Characterization of the primary structure and the microheterogeneity of the carbohydrate chains of porcine blood-group H substance by 500-MHz 1H-NMR spectroscopy.

In blood-group H substance from pig stomach cell linings, carbohydrate structures are known to occur which are O-glycosidically linked via GalNAc to Ser or Thr of the polypeptide backbone. They have in common the Gal(beta 1 leads to 3)GalNAc core unit, which bears one or more N-acetyllactosamine branches. The latter are terminated either by Fuc in (alpha 1 leads to 2) linkage or by GlcNAc in (alpha 1 leads to 4) linkage to Gal. Both terminal sequences are considered to be porcine blood-group H antigenic determinants. Employing 500-MHz 1H-NMR spectroscopy, the spectral features of the oligosaccharide-alditols corresponding to these chains were established. Insight could be gained into the microheterogeneity displayed by some of the alditol fractions as to the distribution of the terminating Fuc and GlcNAc residues over the various branches. Such information can hardly be obtained using other approaches.

The structure of carbohydrate chains of blood-group substance. Isolation and elucidation of the structure of higher oligosaccharides from blood-group substance H.

Twenty individual higher reduced oligosaccharides, having from seven to eleven monosaccharide units, were isolated after sodium borohydride degradation of blood-group substance H from pig stomach linings. Anion-exchange high-pressure liquid chromatography appears to be a very convenient and effective method for this kind of higher oligosaccharide mixtures separation. The oligosaccharide structures were determined by means of periodate oxidation, methylation analysis, partial acid and enzymic hydrolysis. It has been found that all the oligosaccharides investigated can be divided into four series. The oligosaccharides belonging to each series have the common oligosaccharide fragment to which terminal L-fucose and/or N-acetyl-D-glucosamine residues are attached. Comparison of all the oligosaccharide structures, including tri, penta and hexasaccharides described earlier, shows that the lower oligosaccharides represent the structural element of the higher oligosaccharides.

The structure of pentasaccharides and hexasaccharides from blood group substance H.

Six individual reduced pentasaccharides and hexasaccharides were isolated after sodium borohydride degradation of blood group substance H from pig stomach linings. Their structure was determined by means of periodate oxidation, methylation analysis, mass spectrometry, partial acid and enzymic hydrolysis and chromic anhydride oxidation. It has been found that all the oligosaccharides investigated contain the common tetrasaccharide core to which terminal alpha-L-fucose and N-acetyl-alpha-D-glucosamine residues are attached. Some problems of carbohydrate chain heterogeneity in blood group substances are discussed.