Identification of N-sulphated disaccharide units in heparin-like polysaccharides

Abstract

Preparations of heparin and heparan sulfate [from pig mucosa, mouse mastocytoma, whale and bovine lung] were degraded with HNO2. The resulting disaccharides were isolated by gel chromatography, reduced with either NaBH4 or NaB3H4 and then fractionated into non-sulfated, monosulfated and disulfated species by ion-exchange chromatography or by paper electrophoresis. The non-sulfated disaccharides were separated into 2, and the monosulfated disaccharides into 3 components by paper chromatography. The uronic acid moieties of the various non- and mono-sulfated disaccharides were identified by means of radioactive labels selectively introduced into uronic acid residues (3H and 14C in D-glucuronic acid, 14C only in L-iduronic acid units) during biosynthesis of the polysaccharide starting material. Labeled uronic acids were also identified by paper chromatography after liberation from disaccharides by acid hydrolysis or by glucuronidase digestion. Similar procedures applied to disaccharides treated with NaB3H4 indicated 2,5-anhydro-D-mannitol as the reducing terminal unit. Based on these results and the known positions and configurations of the glycosidic linkages in heparin, the 2 non-sulfated disaccharides were identified as 4-O-(.beta.-D-glucopyranosyluronic acid)-2,5-anhydro-D-mannitol and 4-O-(.alpha.-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol. The 3 monosulfated [1-3H]anhydromannitol-labeled disacharrides were subjected to Smith degradation or to digestion with homogenates of human skin fibroblasts, and the products were analyzed by paper electrophoresis. The results, along with the 1H NMR spectra of the corresponding unlabeled disaccharides, permitted the allocation of O-sulfate groups to various positions in the disaccharides. These were thus identified as 4-O-(.beta.-D-glucopyranosyluronic acid)-2,5-anhydro-D-mannitol 6-sulfate, 4-O-(.alpha.-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol 6-sulfate and 4-O-(.alpha.-L-idopyranosyluronic acid 2-sulfate)-2,5-anhydro-D-mannitol. The last-mentioned disaccharide was a poor substrate for the iduronate sulfatase of human skin fibroblasts compared with the disulfated species 4-O-(.alpha.-L-idopyranosyluronic acid 2-sulfate)-2,5-anhydro-D-mannitol 6-sulfate. The identified [1-3H]anhydromannitol-labeled disaccharides were used as reference standards in a study of the disaccharide composition of heparins and heparan sulfates. Low N-sulfate contents, most pronounced in the heparan sulfates, were associated with high ratios of mono-O-sulfated/di-O-sulfated (n-sulfated) disaccharide units and with relatively large amounts of 2-sulfated L-iduronic acid residues bound to C-4 of N-sulfo-D-glucosamine units lacking O-sulfate substituents.