Biosynthesis of heparin/heparan sulfate. The D-glucosaminyl 3-O-sulfotransferase reaction: target and inhibitor saccharides.
O-Sulfation at C-3 of N-sulfated GlcN units concludes polymer modification and the formation of antithrombin binding regions in the biosynthesis of heparin/heparan sulfate. The resulting GlcNSO3(3-OSO3) units are largely restricted to heparin chains with high affinity for antithrombin (HA heparin). Low affinity (LA) 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 Lindahl, U. (1990) J. Biol. Chem. 265, 7292-7300), as verified in the present study using a novel sequencing procedure. O-Desulfated, re-N-sulfated LA heparin, as well as an octasaccharide fraction isolated after heparinase I digestion of LA heparin, both yielded labeled HA components following incubation with solubilized mouse mastocytoma microsomal enzymes and [35S]adenosine 3'-phosphate 5'phosphosulfate (PAPS), suggesting that the 3-O-sulfo-transferase may be inhibited by sulfated saccharide sequences outside the 3-O-sulfate acceptor region. Indeed, the addition of LA heparin precluded enzymatic 3-O-sulfation of a synthetic pentasaccharide substrate. The Km for the pentasaccharide was determined to approximately be 6 microM. Incubations of mixed pentasaccharide substrate and saccharide inhibitors revealed Ki values for intact LA heparin and for a heparin octasaccharide fraction of approximately 1.3 and approximately 0.7 microM, respectively. Inhibition experiments with selectively desulfated heparin indicated that both IdoA 2-O-sulfate and GlcN 6-O-sulfate groups contributed to the inhibition of the 3-O-sulfotransferase. By contrast, chondroitin sulfate or dermatan sulfate showed no significant inhibitory activity. It is proposed that the regulation of GlcN 3-O-sulfation during biosynthesis of heparin/heparan sulfate depends on the topological organization of the membrane-bound enzyme machinery in the intact cell.
Razi
N
N
Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.
Lindahl
U
U
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006493:Heparin / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D012515:Mast-Cell Sarcoma / Q000201:enzymology*; D051379:Mice; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D011134:Polysaccharides / Q000494:pharmacology*; D015238:Sulfotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013552:Swine
D006493:Heparin; D006497:Heparan Sulfate; D009844:Oligosaccharides; D011134:Polysaccharides; D015238:Sulfotransferases; C093554:heparin-glucosamine 3-O-sulfotransferase