Alkaline borohydride reductive cleavage (B-elimination) of hog submaxillary glycoproteins from three immunologically determined phenotypes, viz. A', H + and A-H-, resulted in the release of a series of neutral and acidic oligosaccharide-alditols. 360-M Hz 'H-NMR spectroscopy in combination with methylation analysis and mass spectrometry were used for reinvestigation of the structures of these oligosaccharide-alditols. All are partial structures representing the possible complete and biosynthetically incomplete stages of the chain of a pentasaccharide-N-acetylgalactosaniinitol, present in the glycoprotein with blood-group-A activity : lY.
xThree distinct phenotypes can be discerned immunologically in the aqueous extracts of mucins derived from individual hog submaxillary glands, namely: A', H', and A-H-; this was ascribed, at least in part, to differences in the oligosaccharide structures of the m u c h glycoproteins [l]. Alkaline borohydride reductive cleavage (fi-elimination reaction) is a useful method to release the oligosaccharides from their linkage to serine or threonine in the polypeptide backbone [2]. The availability of such a facile procedure for the cleavage of the carbohydrate chains from the protein core makes it possible to isolate the oligosaccharides, determine their structures, and thereby correlate structure with biological activity.The first such complete accounting of the oligosaccharides of porcine submaxillary mucin was made by Carlson [3,4] using A+ or A-porcine submaxillary glands. A subsequent study was undertaken by Aminoff and colleagues [5-71 in order to establish the role of the carbohydrate structure in determining the serological differences between A', H C and A-H-phenotypically active glycoproteins. In the studies of Carlson [3,4] the structures of the isolated oligosaccharides were established by several techniques including periodate oxidation, enzymic methods and methylation analysis. The structures proposed by Abbreviations. GalNAc, N-acetylgalactosamine; GalNAc-ol, Nacetylgalactosaminitol ; Fuc, fucose; NeuGc, N-glycoloylneuraminic acid; NeuAc, N-acetylneuraminic acid; NMR, nuclear magnetic resonance.
Aminoff et al.[5 -71 were mainly based on periodate oxidation. The results of both investigations were controversial, as the results of Aminoff implicated the possible attachment of sialic acid in an (or2+6) linkage to the galactose, as well as to the protein-linked N-acetylgalactosamine residues of the oligosaccharide chains in porcine submaxillary mucin, whereas only the latter type occurs according to Carlson.A thorough study, utilizing at least two independent methods for structural analysis, seemed to be required for bringing this controversy to an end. Since 360-MHz 'H-NMR spectroscopy has proved to be a very powerful technique in the determination of carbohydrate structures [8,9], this method in conjunction with methylation analysis and mass spectrometry was applied to shed light on this problem. A preliminary report on these findings has previously been presented [lo].
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