Characterisation by mass spectrometry and ¹H‐NMR of novel hexasaccharides among the acidic O‐linked carbohydrate chains of bovine submaxillary mucin

Abstract

The acidic oligosaccharide alditols released from bovine submaxillary-gland mucin by Carlson degradation were investigated by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 'H-NMR. Among the largest structures identified were four branched hexasaccharides, three of them novel, comprising two separate pairs of structures. One pair contained the sequence Fuc(al-2)Gal(~1-4)[Fuc(al-3)]GlcNAc(~l-) (Fuc, L-fucose), at C3 of N-acetylgalactosaminitol and differed only by substitution at C6 by N-acetylneuraminic or N-glycolylneuraminic acid. The other pair also differed in substitution of the sialic acid linked at C6 and contained the GalNAc- Recent reports [l, 21 on the neutral 0-linked oligosaccharides in bovine submaxillary-gland mucin (BSM) have revealed a greater diversity of structure than previously recognised [3 -61. Seventeen sugar chains ranging from disaccharides to heptasaccharides have been identified [l, 21, including oligosaccharides having blood-group-A, blood-group-H and LewisY determinants.In addition to these, there are also acidic oligosaccharides which represent about 85% of the carbohydrate in BSM and a total of 12 monosialylated oligosaccharides have been isolated so far [6 -101. The present study describes the characterisation of an additional number of mainly larger, acidic structures released from BSM by Carlson degradation. These have been characterised by a combination of liquid secondary-ion mass spectrometry (LSIMS), methylation analysis and 'H-NMR after chromatography on Bio-Gel P-4 and HPLC separation and include four hexasaccharides containing N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc) which extend the range of known structures on BSM. MATERIALS AND METHODS Isolation of BSM acidic oligosaccharide alditolsBSM, prepared by a procedure essentially the same as described by Tettamanti and Pigman [l I], was purchased from Sigma Chemical Co., Dorset, England (Type I-S).Oligosaccharides were released as alditols as previously described [2]. After removal of salt and borate, the oligosaccharide mixture was dissolved in 2 mM pyridinel2 mM acetic acid, pH 5.0, ( 2 ml) and applied to a Bio-Rad AG 1-X2 (acetate form, 10-ml bed volume) anion-exchange column equilibrated with the same buffer. The column was washed with 50 ml equilibration buffer to elute the neutral oligosaccharide alditol fraction (23 mg, dry mass from 450 mg BSM) investigated previously [2]. The column was then eluted with 50 ml of 0.3 M and 50 m10.5 M formic acid. After lyophilization of the combined formic acid eluent, 138 mg acidic oligosaccharide alditols were obtained.The acidic oligosaccharides were fractionated on a BioGel P-4 (200 -400 mesh, 1.6 cm x 90 cm) column at room temperature with elution by 50 mM pyridinel25 mM acetic acid buffer (pH 5.3) to give A1 -7 (Fig. 1) in the following amounts: Al, 16.7 mg; A2,73 mg; A3, 14.3 mg; A4, 19.1 mg; AS, 3.6 mg; A6, 1.5 mg; A7, 7.3 mg. HPLC purificationFractions AS and A6 were chromatographed on a APSHypersil-2 column (NH2...

The αGal Epitope of the Histo-Blood Group Antigen Family Is a Ligand for Bovine Norovirus Newbury2 Expected to Prevent Cross-Species Transmission

“…Besides the species-specific expression of the αGal antigen on bovine digestive epithelial cells, another species-specific bovine characteristic was evidenced with regard to HBGAs expression. Indeed, we failed to detect HBGAs based on type 1 precursor (Galβ3GlcNAc) in bovine tissues or saliva, consistent with the results from earlier structural analyses of O-linked oligosaccharides from bovine salivary mucins which described the presence of type 2-based structures only [34],[35]. Those studies also failed to detect the αGal epitope.…”

Matrix‐assisted laser desorption/ionization mass spectrometry compatible β‐elimination of O‐linked oligosaccharides

“…Several groups have studied the O ‐glycans cleaved from bovine submaxillary mucin using various analytical techniques 25–32. Interestingly, all of the studied oligosaccharide pools were obtained by the traditional β‐elimination procedure.…”

Purification, Characterization, and Preliminary X-Ray Diffraction Analysis of a Lactose-Specific Lectin from Cymbosema roseum Seeds

“…The neutral Olinked glycans of bovine submaxillary mucin include GalNAcα1-3[Fuc α1-2]Gal, Nacetyllactosamine branches terminated with fucose α1-2-Gal and the core 3 determinants [25,26]. In addition, Tn antigen accounts for >75% of the carbohydrate chains of the desialylated ovine submaxillary mucin.…”

“…Bovine submaxillary mucin is a glycoprotein bearing at least 16 different carbohydrate moieties [25,26], 85% are acidic O-linked oligosaccharide chains, including a high density of sialyl Tn antigens and sialyl core 3 saccharide sequences. The neutral Olinked glycans of bovine submaxillary mucin include GalNAcα1-3[Fuc α1-2]Gal, Nacetyllactosamine branches terminated with fucose α1-2-Gal and the core 3 determinants [25,26].…”