NeuNAc Alpha Research Articles

Relationship between cell surface asparagine-linked glycoproteins and myoblast differentiation. Analysis of wheat germ agglutinin-resistant mutants.

Alterations in the oligosaccharides of complex surface glycoproteins have been studied in wild-type rat skeletal myoblasts (L6-9/1) and two wheat germ agglutinin resistant mutants, selected for either high level of resistance (WGARI) or low level of resistance (WGARII). All three lines possessed high mannose oligosaccharides of the structure Man(n)GlcNAc, where n = 6, 7, or 8. L6-9/1 had complex oligosaccharides of the type (+/- NeuNAc alpha 2-3Gal beta GlcNAc beta)n-Man3GlcNAc(+/- Fuc)GlcNAc where n = 3 or 4, in addition to small amounts of biantennary and higher order structures. WGARII, the mutant with low resistance to wheat germ agglutinin, possessed similar complex oligosaccharides except that they had undergone some base-sensitive modification which rendered them resistant to Clostridium perfringens sialidase. WGARI, mutant of high resistance to wheat germ agglutinin, possessed complex oligosaccharides of the type (GlcNAc beta)nMan3GlcNAc(+/- Fuc)GlcNAc, with n = 2, 3, or 4. Since the WGARI mutants were unaltered in differentiation, it is concluded that the terminal sialic acid and galactose residues on the protein-bound oligosaccharides are not required for differentiation.

Chemical and carbon-13 nuclear magnetic resonance studies of the blood group M and N active sialoglycopeptides from human glycophorin A.

The NH2-terminal sialoglycopeptides from human erythrocyte glycophorin A have been obtained by specific proteolytic cleavage and gel filtration chromatography. By cyanogen bromide cleavage, a glycosylated octapeptide was obtained from blood group M donors having an amino acid composition and 13C NMR spectrum consistent with the structure (formula: see text) was demonstrated. By Staphylococcus aureus protease cleavage, a glycosylated pentapeptide was obtained from N donors having the same structure as II, without the carboxyl-terminal sequence Val . Ala . Hse. Methanolysis/gas chromatographic analysis and 13C NMR spectroscopy of I and II and their asialo derivatives reveal that the M- and N-active sialoglyco-octapeptides both have identical oligosaccharide structures, each containing three O-linked tetrasaccharides with the structure NeuNAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr). The demonstration of the anomeric form of GalNAc-peptide linkages revealed by 13C NMR has previously been inaccessible by chemical analysis. Conformationally, I and II appear identical and both manifest several unusual resonance shifts suggestive of a glycopeptide secondary structure involving four specific hydrogen bonds. Calcium ion titration was also found to induce shifts in the NeuNAc 13C resonances that may be of functional significance. Serological studies reveal that both the M and N glyco-octapeptides and the N glyco-pentapeptide retain all of the M and N activity of the parent structure. Deamination and/or desialylation completely destroys this activity. These data are consistent with a model in which the M or N determinant is the NH2-terminal amino acid and a NeuNAc residue(s). From these data it is concluded that there is no chemical basis for assertions in the literature that M and N antigens differ in their oligosaccharide structure or that the N antigen is biosynthetically transformed to the M antigen by sialylation.

Antibodies against sialyloligosaccharides coupled to protein.

The beta-(p-aminophenyl)ethylamine derivatives of sialyloligosaccharides can be coupled to proteins via their phenylisothiocyanate intermediates under conditions that preserve labile sugar linkages. Bovine serum albumin containing 10 to 40 mol of oligosaccharides/mol of protein and keyhole limpet hemocyanin containing 1,100 mol of oligosaccharide/mol of protein have been prepared with the following oligosaccharides: Neu-NAc alpha 2-3Gal beta 1-4Glc, NeuNAc alpha 2-6Gal beta 1-4Glc, Neu-NAc alpha 2-6Gal beta 1-4GlcNAc beta 1-4Glc, Gal beta 1-3[Neu-NAc alpha 2-6]GlcNAc beta 1-4Glc, and NeuNAc alpha 2-3Gal- beta 1-3[NeuNAc alpha 2-6]GlcNAc beta 1-4Glc. Rabbits immunized with these synthetic glycoproteins produce antibodies directed against the oligosaccharides. The specificities of these antibodies are determined by comparing inhibitory activities of structurally related oligosaccharides in radioimmunoassay and by double diffusion analysis in agarose gels using oligosaccharide-protein conjugates as precipitating antigens. The antibodies distinguish positional isomers of sialic acid.

Gangliosides of human erythrocytes. A novel ganglioside with a unique N-acetylneuraminosyl-(2 leads to 3)-N-acetylgalactosamine structure.

A novel ganglioside having both N-acetylgalactosamine and N-acetylglucosamine was isolated from human erythrocyte membranes. Its structure was characterized by successive exoglycosidase treatment, methylation analysis, and direct-probe mass spectrometry of permethylated intact and desialylated glycolipid. The core structure of the ganglioside was found to be N-acetylgalactosaminyl paragloboside with sialosyl substitution by 2 leads to 3 linkage at the terminal GalNAc residue: NeuNAc alpha 2 leads to 3 GalNAc beta 1 leads to -3Gal beta 1 leads to 4 GlcNAc beta 1 leads to 3 Gal beta 1 leads to 4Glc leads to Cer. The ganglioside was characterized by the presence of a ceramide with myristic acid as the major component.

The carbohydrate portions of milk glycoproteins.

kappa-Casein is the main glycoprotein of cow's milk. Its polysaccharide part is O-glycosidically linked to threonine residue 133. It contains only 3 different sugars (Gal, GalNAc, NeuNAc), but a microheterogeneity has been detected at the sugar level. Two main polysaccharides have so far been characterized. The structure of the trisaccharide is NeuNAc alpha 2 leads to 3 Gal beta 1 leads to GalNAc; the tetrasaccharide contains one additional sialic acid. The polysaccharide part of ovine kappa-casein resembles that of bovine kappa-casein, but contains also N-glycolyl neuraminic acid. Human kappa-casein contains 3 times more carbohydrate than bovine kappa-casein with 2 additional sugars, GlcNAc and Fuc. The various polysaccharide parts isolated from bovine colostrum kappa-caseinoglycopeptide are much more complex than those obtained from the normal glycopeptide, indicating an evolution of the sugar part as a function of time after parturition. Some aspects of the secondary structure of kappa-casein and the role of the sugar part are discussed. The carbohydrate moiety of another milk protein, human lactotransferrin, is also discussed briefly. it is comprised of 2 identical glycan groups, N-glycosidically linked to the protein, and quite different from the kappa-casein carbohydrate moiety.