Linkage of carbohydrate to hydroxyamino acids in mucopolysaccharides and mucoproteins

Abstract

1. Glycosidic linkage of carbohydrate to the primary hydroxyl groups of threonine and serine has been established in human blood-group A and Le(a) substances, bovine submaxillary-gland mucin and human pseudomyxomatous mucin. 2. Treatment of these substances in 0.09n-lithium hydroxide at 100 degrees for 1hr. led to beta-elimination at these glycosidic linkages with the resultant formation of alpha-oxobutyric acid and glycine from threonine linkages, and pyruvic acid from serine linkages. Though most of the threonine was destroyed in every case, about one-third to one-half of the serine residues resisted alkaline cleavage. Such results, indicative of the presence of unbound serine residues, allow, in submaxillary mucin, for a close correlation between the remaining serine, threonine, glutamic acid and aspartic acid and the available sialyl-(2-->6)-N-acetylgalactosamine prosthetic groups. 3. The stoichiometry of the beta-eliminations has been demonstrated for pseudomyxomatous mucin. The alpha-oxo acids were separated and determined as their quinoxalinol derivatives by thin-layer chromatography on silica gel. Reaction at the threonine centres favoured alpha-oxobutyric acid formation (70%, via the intermediary dehydropeptide) over the alternative pathway to glycine (30%). 4. 100% of the hexosamine was destroyed in submaxillary-gland mucin, 85% in pseudomyxomatous mucin and about 60% in the blood-group substances. In the latter cases, the glucosamine/galactosamine ratio was increased from about 4:1 to 8-10:1, suggesting a preferential destruction of galactosamine. Evidence was obtained, however, for a further destruction of hexosamine, in addition to that which could be theoretically attached to peptide at possible known binding sites. 5. The major part of the alkali-resistant hexosamine in the blood-group substances was non-diffusible and was accompanied by the constituent carbohydrates in similar molar proportions to the native materials.