Biosynthesis of heparin. Enzymatic sulfation of pentasaccharides

Heparin-derived pentasaccharides with the general structures GlcN-GlcA/IdoA-GlcN-GlcA/IdoA-GlcN (where GlcA represents D-glucuronic acid and IdoA represents L-iduronic acid) and GlcNSO3-GlcA/IdoA-GlcNSO3-GlcA/IdoA- GlcNSO3 (where -NSO3 represents an N-sulfate group) were tested as exogenous sulfate acceptors in incubations with adenosine 3'-phosphate 5'-[35S]phosphosulfate and microsomal enzymes from a heparin-producing mouse mastocytoma. No transfer occurred to the N-unsubstituted pentasaccharide containing only L-iduronic acid, but the other three isomers incorporated various amounts of 35S, which was totally present in N-sulfate groups. After complete chemical N-sulfation, all four pentasaccharides served as acceptors in O-sulfotransferase reactions and incorporated from 20 to greater than 200 times as much radioactivity as did the nonsulfated parent compounds. The C-6 position of the internal glucosamine unit was labeled preferentially, irrespective of the structures of the adjacent hexuronic acid units. Significant 2-O-35S-sulfation of IdoA units occurred in both -IdoA-Glc-NSO3-GlcA- and -GlcA-GlcNSO3-IdoA- sequences, whereas no significant sulfation of GlcA residues was detected. The pentasaccharide GlcNSO3-GlcA-Glc-NSO3-GlcA-GlcNSO3 thus can be used as a selective substrate in assays for glucosaminyl-6-O-sulfotransferase activity. The antithrombin-binding region, essential for the blood anticoagulant activity of heparin, has been identified as a pentasaccharide sequence with the predominant structure GlcNR(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-++ +IdoA(2-OSO3)-GlcNSO3(6-OSO3) (where R represents either a sulfate or an acetyl group and -OSO3 represents an O-sulfate/ester sulfate group, with locations of O-sulfate groups indicated in parentheses) (Lindahl U., Thunberg, L., Bäckström, G., Riesenfeld, J., Nordling, K., and Björk, I. (1984) J. Biol. Chem. 259, 12368-12376). The products of [35S]sulfate transfer to the pentasaccharide GlcNSO3-GlcA-GlcNSO3-IdoA-GlcNSO3 contained molecules with high affinity for antithrombin, corresponding to 0.3-0.5% of the total label. Structural analysis suggested the occurrence of O-[35S]sulfate groups at both C-6 of the nonreducing terminal glucosamine unit and C-3 of the internal glucosamine unit. No products with high affinity for antithrombin were formed from the pentasaccharides that had a different monosaccharide sequence than the binding region; and moreover, these oligosaccharides appeared unable to incorporate glucosaminyl 3-O-sulfate groups. These findings point to the importance of the uronic acid sequence in the generation of the antithrombin-binding region of heparin.