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

Abstract

1. Preparations of heparin and heparan sulphate were degraded with HNO2. The resulting disaccharides were isolated by gel chromatography, reduced with either NaBH4 or NaB3H4 and were then fractionated into non-sulphated, monosulphated and disulphated species by ion-exchange chromatography or by paper electrophoresis. The non-sulphated disaccharides were separated into two, and the monosulphated disaccharides into three, components by paper chromatography. 2. The uronic acid moieties of the various nonand mono-sulphated 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. Labelled 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 reducing terminal unit. On the basis of these results, and the known positions and configurations of the glycosidic linkages in heparin, the two non-sulphated disaccharides were identified as 4-0-(fi-D-glucopyranosyluronic acid)-2,5-anhydro-D-mannitol and 4-0-(a-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol. 3. The three monosulphated [1-3H]anhydromannitol-labelled disaccharides were subjected to Smith degradation or to digestion with homogenates of human skin fibroblasts, and the products were analysed by paper electrophoresis. The results, along with the 1H n.m.r. spectra ofthe corresponding unlabelled disaccharides, permitted the allocation of 0-sulphate groups to various positions in the disaccharides. These were thus identified as 4-0-(fl-D-glucopyranosyluronic acid)-2,5-anhydro-D-mannitol 6-sulphate, 4-0-(a-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol 6-sulphate and 4-0-(a-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol. The last-mentioned disaccharide was found to be a poor substrate for the iduronate sulphatase of human skin fibroblasts, as compared with the disulphated species, 4-0-(a-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-Dmannitol 6-sulphate. 4. The identified [1 -3H]anhydromannitol-labelled disaccharides were used as reference standards in a study of the disaccharide composition of heparins and heparan sulphates. Low N-sulphate contents, most pronounced in the heparan sulphates, were associated with high ratios of mono-O-sulphated/di-O-sulphated (N-sulphated) disaccharide units, and in addition, with relatively large amounts of 2-sulphated L-iduronic acid residues bound to C-4 of N-sulpho-D-glucosamine units lacking 0-sulphate substituents.

Structure of the antithrombin-binding site in heparin.

“…Exhaustive digestion of antithrombinbinding fragment with a-L-iduronidase did not significantly affect its affinity for antithrombin, as shown by subsequent affinity chromatography (as in Fig. 1 6.2 * The method used to determine the disaccharide composition of heparin preparations is described in detail elsewhere (11). Briefly, polysaccharide (10- Deaminattve cleavage of heparin-like polysaccharides yields tetrasaccharides where isolated N-acetylated disaccharide units occur surrounded by N-sulfated units (10).…”

“…11 and outlined in the legend to Table 2. Periodate Oxidation. Periodate oxidation followed by alkali treatment and reduction was carried out as described (16).…”

“…The enzyme degraded iduronosyl-2,5-anhydro-[1-3H]mannitol 6-sulfate to a labeled product that migrated like anhydromannitol 6-sulfate on paper electrophoresis (11) but had no detectable effect on either glucuronosyl-*anhydro- Effluent fractions were analyzed for uronic acid (0) or for radioactivity (0). The salt gradient (starting at arrow) used for elution is indicated by the broken line.…”

“…However, a significant fraction was absorbed to the gel; on gradient elution a peak of uronic acid-containing components, corresponding to 2-3% of the starting material, §'appeared in the elution position of high-affinity heparin. A portion of the material eluting at ¢1 M NaCl was reduced with sodium boro[3H hydride (11) Determined by the British Pharmacopoeia whole-blood assay (left column), or by the colorimetric antithrombin-activation assay (right column). § The terms "high-affinity" and "low-affinity" refer to the interaction of heparin with antithrombin.…”

Biosynthesis of heparin/heparan sulphate: mechanism of epimerization of glucuronyl C-5

“…1 paper in ethyl acetate\acetic acid\water (3 : 1 : 1, by vol. ), along with GlcA-[1-$H]aMan R and IdoA-[1-$H]aMan R standards [20]. After the separation, the paper strips were dried and cut into 1 cm pieces which were transferred to 6 ml scintillation vials.…”