Neutral oligosaccharides of bovine submaxillary mucin

Abstract

Twenty-two neutral 0-linked oligosaccharides ranging from monosaccharides to octasaccharides were identified in bovine submaxillary-gland-mucin glycoprotein by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 'H-NMR. Only five of these have been previously detected in bovine submaxillary-gland mucin although several have been described from other sources of mucin. The structures include short linear sequences 3-linked to N-acetylgalactosaminitol (GalNAcol) and branched structures based on either a GlcNAc(p1-6)[Gal(p1-3)IGalNAcol or GlcNAc(B1 -6)[GlcNAc(pl-3)IGalNAcol core region. Oligosaccharides not previously characterised from any source were the disaccharide GalNAcal -6GalNAcol (GalNAc, N-acetylgalactosamine and the hexasaccharide GlcNAc(p1-6){GalNAc (al-Oligosaccharides of the blood-group-A type have not been detected previously in bovine submaxillary-gland mucin although their occurrence on bovine gastric-mucosal glycoproteins has been established by classical immunochemical studies.Bovine submaxillary-gland mucin (BSM) was among the first mucin glycoproteins to be purified and studied [l -51. Early structural investigations of its 0-linked carbohydrate chains (E 70% of the mass) indicated a predominance of the acidic disaccharide NeuAc(a2 -6)GalNAc [6 -81. Later, the presence of galactose, L-fucose (Fuc) and/or N-acetylglucosamine [9] was detected in addition to N-acetylneuraminic acid and N-acetylgalactosaminitol, consistent with the occurrence of longer chains and more complex oligosaccharides in BSM. This has been confirmed by studies on acidic oligosaccharides [lo-131.Neutral oligosaccharides comprise approximately one fifth of the total oligosaccharides and five structures have been characterised previously [lo]. In the present study a more comprehensive analysis of neutral oligosaccharides released from BSM by Carlson degradation [14] has been made by a combination of liquid secondary-ion mass spectrometry (LSIMS), GC-MS and 'H-NMR, to establish the range of structures present and allow comparison with other mucins. The results show greater diversity of chains than previously reported, among which are a series of oligosaccharides with blood-group-A-rela ted sequences. MATERIALS AND METHODS Preparation and isolation of BSM neutral oligosaccharide alditolsBSM, prepared by a procedure essentially as described by Tettamanti and Pigman [3], was purchased from Sigma Chemical Co., Dorset, England (Type 1-S).Oligosaccharides were released from BSM by the digestion procedure of Carlson [14]. In brief, BSM (450 mg) was incubated with 0.05 M NaOH/1.0 M NaBH, (20 ml) for 16 h at 45°C. After acidification to pH 5 by addition of acetic acid/ H 2 0 (1 : 1, by vol.), the reaction mixture was passed through a column of ion-exchange resin (Bio-Rad) with a 15 ml lower bed containing AG 50W-X2 (H' form) and a 20 ml upper bed containing AG 50W-X8 (H' form), and washed with four column volumes of HzO. The combined effluent and washes were lyophilized and the boric acid removed by co-ev...

Ligands for the β-Glucan Receptor, Dectin-1, Assigned Using “Designer” Microarrays of Oligosaccharide Probes (Neoglycolipids) Generated from Glucan Polysaccharides

“…Methylation Analysis-Oligosaccharides were reduced with NaBD 4 before they were methylated, hydrolyzed, reduced, and acetylated as described (29). Gas chromatography-MS analysis of the products, partially methylated alditol acetates, was performed on a ThermoQuest Trace system (Thermo Electron, Hemel Hempstead, UK), using a 15-m RTX-5 capillary column (Hewlett-Packard).…”

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

“…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.…”

“…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.…”

A stereocontrolled construction of 2-azido-2-deoxy-1,2-cis-α-galactosidic linkages utilizing 2-azido-4,6-O-benzylidene-2-deoxygalactopyranosyl diphenyl phosphates: stereoselective synthesis of mucin core 5 and core 7 structures

“…39 Oligosaccharide with core 7 was detected in bovine submaxillary-gland mucin. 40 In 1995, Paulsen and co-workers reported the synthesis of core 5 and core 7 building blocks employing the Koenigs-Knorr method, in which glycosidations of disaccharide donors with a threonine acceptor gave exclusively -glycosides in 73-74% yields. 41 This protocol depends on conventional labile glycosyl bromides that must be prepared just prior to glycosidation, thus clearly diminishing synthetic flexibility.…”