BIOSYNTHESIS OF HEPARIN HEPARAN-SULFATE - THE D-GLUCOSAMINYL 3-O-SULFOTRANSFERASE REACTIONS - TARGET AND INHIBITOR SACCHARIDES

O-Sulfation at C-3 of N-sulfated GlcN units concludes polymer modifica tion and the formation of antithrombin binding regions in the biosynth esis of heparin/heparan sulfate, The resulting GlcNSO(3)(3-OSO3) units are largely restricted to heparin chains with high affinity for antit hrombin (H-A heparin). Low affinity (L(A)) heparin fails to serve as a substrate in the 3-O-sulfotransferase reaction yet contains potential 3-O-sulfate acceptor sites (Kusche, M., Torri, G., Casu, B., and Lind ahl, U. (1990) J. Biol, Chem, 265, 7292-7300), as verified in the pres ent study using a novel sequencing procedure, O-Desulfated, re-N-sulfa ted L(A) heparin, as well as an octasaccharide fraction isolated after heparinase I digestion of L(A) heparin, both yielded labeled H-A comp onents following incubation with solubilized mouse mastocytoma microso mal enzymes and [S-35]adenosine 3'-phosphate 5'phosphosulfate (PAPS), suggesting that the 3-O-sulfotransferase may be inhibited by sulfated saccharide sequences outside the 3-O-sulfate acceptor region, Indeed, the addition of L(A) heparin precluded enzymatic 3-O-sulfation of a sy nthetic pentasaccharide substrate, The K-m for the pentasaccharide was determined to similar to be 6 mu M. Incubations of mixed pentasacchar ide substrate and saccharide inhibitors revealed K-i values for intact L(A) heparin and for a heparin octasaccharide fraction of similar to 1.3 and similar to 0.7 mu M, respectively, Inhibition experiments with selectively desulfated heparin indicated that both IdoA 2-O-sulfate a nd GlcN 6-O-sulfate groups contributed to the inhibition of the 3-O-su lfotransferase. By contrast, chondroitin sulfate or dermatan sulfate s howed no significant inhibitory activity, It is proposed that the regu lation of GlcN 3-O-sulfation during biosynthesis of heparin/heparan su lfate depends on the topological organization of the membrane-bound en zyme machinery in the intact cell.