Abstract
Incubation of a microsomal fraction from murine mastocytoma, with UDP-[1-3H]GlcA, UDP-GlcNAc, and adenosine 3'-phosphate 5'-phosphosulfate (PAPS), yielded labeled, N-sulfated polysaccharides, in which most of the incorporated O-sulfate groups were located at C2 of L-iduronic acid and at C6 of D-glucosamine units. Analysis by anion-exchange high pressure liquid chromatography of disaccharides, generated by deaminative cleavage of these polysaccharides, revealed that, in addition, an appreciable portion of the -GlcNSO3-HexA-GlcNSO3- sequences in the intact polymers contained O-sulfated (at C2 or C3) D-glucuronic acid units. Calculations based on such compositional analysis of the N- and O-sulfated biosynthetic product, isolated by chromatography on DEAE-cellulose, showed that glucuronosyl 2/3-O-sulfate accounted for approximately 12% of the total incorporated O-sulfate groups. With [35S]PAPS (at a low total PAPS concentration) as an alternative source of label, the sulfated glucuronic acid residues were again detectable, albeit in much smaller amounts (1.8% of the total O-sulfate groups). Incorporation of label from UDP-[5-3H]GlcA was retained by the O-sulfated glucuronic acid units, thus demonstrating that these components had in fact been formed by sulfation of glucuronic acid residues and not by "back epimerization" of sulfated iduronic acid units. Structural analysis of polysaccharide intermediates at various stages of biosynthetic polymer modification, separated by ion-exchange chromatography, showed O-sulfation of glucuronic and iduronic acid units to appear simultaneously and before the 6-O-sulfation of glucosamine residues.
Highlights
From the Department of Veterinan, Medical Chemistry, The Swedish University of Agricultural Sciences, The Biomedical Center,.S-751 23 l$psala, Sweden
Calculations based on such compositional analysis of the N- and O-sulfated biosynthetic product, isolated by chromatography on DEAE
Was retained by the O-sulfated glucuronic acid units, demonstrating that these components had been formed by sulfation of glucuronic acid residues and not by “back epimerization”
Summary
Calculations based on such compositional analysis of the N- and O-sulfated biosynthetic product, isolated by chromatography on DEAE-. With [3SS]PAPS (at a low total PAPS concentration) as an alternative source of label, the sulfated glucuronic acid residues were again detectable, albeit in much smaller amounts (1.8% of the total O-sulfate groups). Incorporation of label from UDP-[5-3H]GlcA was retained by the O-sulfated glucuronic acid units, demonstrating that these components had been formed by sulfation of glucuronic acid residues and not by “back epimerization”. Structural analysis of polysaccharide intermediates at various stages of biosynthetic polymer modification, separated by ion-exchange chromatography, showed 0-sulfation of glucuronic and iduronic acid units to appear simultaneously and before the 6-
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