heparitin-sulfate and Mast-Cell-Sarcoma

The interaction of heparan sulfate with different ligand proteins depends on the precise location of O-sulfate groups in the polysaccharide chain. We have previously shown that overexpression in human kidney 293 cells of a mouse mastocytoma 2-O-sulfotransferase (2-OST), previously thought to catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to C2 of L-iduronyl residues, preferentially increases the level of 2-O-sulfation of D-glucuronyl units [Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M. (2000) Biochem. J. 346, 463-468]. In the study presented here, we further investigated the substrate specificity of the mouse mastocytoma 2-OST. Different polysaccharide acceptor substrates were incubated with cell extracts from 2-OST-transfected 293 cells together with the sulfate donor 3'-phosphoadenosine 5'-phospho[(35)S]sulfate. Incubations with O-desulfated heparin, predominantly composed of [(4)alphaIdoA(1)-(4)alphaGlcNSO(3)(1)-](n)(), resulted in 2-O-sulfation of iduronic acid. When, on the other hand, an N-sulfated capsular polysaccharide from Escherichia coli K5, with the structure [(4)betaGlcA(1)-(4)alphaGlcNSO(3)(1)-](n)(), was used as an acceptor, sulfate was transferred almost exclusively to C2 of glucuronic acid. Substrates containing both iduronic and glucuronic acid residues in about equal proportions strongly favored sulfation of iduronic acid. In agreement with these results, the 2-OST was found to have a approximately 5-fold higher affinity for iduronic acid-containing substrate disaccharide units (K(m) approximately 3.7 microM) than for glucuronic acid-containing substrate disaccharide units (K(m) approximately 19.3 microM).

Topics: Animals; Brain; Cell Line; Genetic Vectors; Glucuronic Acid; Heparitin Sulfate; Hexuronic Acids; Humans; Lung; Mast-Cell Sarcoma; Mice; Organ Specificity; RNA, Messenger; Substrate Specificity; Sulfotransferases; Tumor Cells, Cultured

The D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate was investigated with focus on its substrate specificity, its kinetic properties, and a comparison of epimerase preparations from the Furth mastocytoma and bovine liver, which synthesize heparin and heparan sulfate, respectively. New substrates for the epimerase were prepared from the capsular polysaccharide of Escherichia coli K5, which had been labeled at C5 of its D-glucuronic and N-acetyl-D-glucosamine moieties by growing the bacteria in the presence of D-[5-(3)H]glucose. Following complete or partial ( approximately 50%) N-deacetylation of the polysaccharide by hydrazinolysis, the free amino groups were sulfated by treatment with trimethylamine.SO(3)complex, which yielded products that were recognized as substrates by the epimerase and released tritium from C5 of the D-glucuronyl residues upon incubation with the enzyme. Comparison of the kinetic properties of the two substrates showed that the fully N-sulfated derivative was the best substrate in terms of its K(m)value, which was significantly lower than that of its partially N-acetylated counterpart. The V(max)values for the E.coli polysaccharide derivatives were essentially the same but were both lower than that of the O-desulfated [(3)H]heparin used in our previous studies. Surprisingly, the apparent K(m)values for all three substrates increased with increasing enzyme concentration. The reason for this phenomenon is not entirely clear at present. Partially purified C5-epimerase preparations from the Furth mastocytoma and bovine liver, respectively, behaved similarly in terms of their reactivity towards the various substrates, but the variation in apparent K(m)values with enzyme concentration precluded a detailed comparison of their kinetic properties.

Topics: Animals; Carbohydrate Epimerases; Carbon Radioisotopes; Cattle; Escherichia coli; Glucose; Heparin; Heparitin Sulfate; Kinetics; Liver; Mast-Cell Sarcoma; Mice; Muscle Neoplasms; Polysaccharides; Sulfuric Acids; Tritium

Heparan sulfate proteoglycans, attached to cell surfaces or in the extracellular matrix, interact with a multitude of proteins via their heparan sulfate side chains. Degradation of these chains by limited (endoglycosidic) heparanase cleavage is believed to affect a variety of biological processes. Although the occurrence of heparanase activity in mammalian tissues has been recognized for many years, the molecular characteristics and substrate recognition properties of the enzyme(s) have remained elusive. In the present study, the substrate specificity and cleavage site of heparanase from human hepatoma and platelets were investigated. Both enzyme preparations were found to cleave the single beta-D-glucuronidic linkage of a heparin octasaccharide. A capsular polysaccharide from Escherichia coli K5, with the same (-GlcUAbeta1,4-GlcNAcalpha1,4-)n structure as the unmodified backbone of heparan sulfate, resisted heparanase degradation in its native state as well as after chemical N-deacetylation/N-sulfation or partial enzymatic C-5 epimerization of beta-D-GlcUA to alpha-L-IdceA. By contrast, a chemically O-sulfated (but still N-acetylated) K5 derivative was susceptible to heparanase cleavage. O-Sulfate groups, but not N-sulfate or IdceA residues, thus are essential for substrate recognition by the heparanase(s). In particular, selective O-desulfation of the heparin octasaccharide implicated a 2-O-sulfate group on a hexuronic acid residue located two monosaccharide units from the cleavage site, toward the reducing end.

Topics: Animals; Antithrombin III; Blood Platelets; Carbohydrate Sequence; Carcinoma, Hepatocellular; Escherichia coli; Glucuronidase; Glycoside Hydrolases; Heparin; Heparitin Sulfate; Humans; Isoenzymes; Liver Neoplasms; Mast-Cell Sarcoma; Mice; Microsomes; Molecular Sequence Data; Substrate Specificity; Tumor Cells, Cultured

The biosynthesis of heparin and heparan sulfate involves a series of polymer-modification reactions that is initiated by N-deacetylation and subsequent N-sulfation of N-acetylglucosamine residues. These reactions are catalysed by a combined N-deacetylase/N-sulfotransferase. Proteins expressing both activities have previously been purified from mouse mastocytoma, which generates heparin, and from rat liver, which produces heparan sulfate. In the present study, the mouse mastocytoma enzyme has been expressed in the human kidney cell line, 293, to investigate whether it could promote modification of the endogenous heparan sulfate precursor polysaccharide into a heparan-like molecule. The N-deacetylase activity of the stably transfected cell clones as approximately 8-fold higher, on a cell-protein basis, than that of control cells, while the N-sulfotransferase activity was increased approximately 2.5 fold. The amounts of glycosaminoglycans synthesized were the same in control and transfected cells, measured as incorporation of [3H]-glucosamine, whereas 35S-labeled glycosaminoglycans were approximately 50% increased in transfected cells, with an increased relative content of heparin sulfate. Structural analysis demonstrated the the glucosamine units of the "heparan sulfate" from transfected cells were almost exclusively N-sulfated, as expected for heparin, whereas more than half of the glucosamine units of the control polysaccharide remained N-acetylated. Notably, the increased N-sulfation was not accompanied by increased O-sulfation, not by C-5 epimerization of D-glucuronic to L-iduronic acid units. The implications of these findings are discussed with regard to the regulation of the biosynthetic process.

Topics: Amidohydrolases; Animals; Carbohydrate Sequence; Cells, Cultured; Heparitin Sulfate; Humans; Kidney; Mast-Cell Sarcoma; Mice; Molecular Sequence Data; Recombinant Proteins; Sulfotransferases; Transfection

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.

Topics: Animals; Carbohydrate Conformation; Carbohydrate Sequence; Chromatography, Affinity; Chromatography, High Pressure Liquid; Heparin; Heparitin Sulfate; Intestinal Mucosa; Kinetics; Mast-Cell Sarcoma; Mice; Microsomes; Molecular Sequence Data; Oligosaccharides; Polysaccharides; Sulfotransferases; Swine

Topics: Animals; Carbohydrate Epimerases; Cell Line; Heparin; Heparitin Sulfate; Kinetics; Mast-Cell Sarcoma; Mice; Microsomes; Uronic Acids

Topics: Animals; Glycosaminoglycans; Heparin; Heparitin Sulfate; Mast-Cell Sarcoma; Mice; Molecular Conformation; Polysaccharides; Racemases and Epimerases; Sulfurtransferases; Uronic Acids

Topics: Acetamides; Animals; Cell-Free System; Glucosamine; Glucuronates; Glycosaminoglycans; Heparin; Heparitin Sulfate; Mast-Cell Sarcoma; Mice; Microsomes; Polysaccharides; Sulfurtransferases; Time Factors; Uridine Diphosphate Sugars; Uronic Acids

1. A method was developed for determination of the uronic acid composition of heparin-like glycosaminoglycans. Polymers or oligosaccharides are degraded to monosaccharides by a combination of acid hydrolysis and deamination with HNO(2). The resulting uronic acid monosaccharides (accounting for about 70% of the uronic acid contents of the starting materials) are isolated and converted into the corresponding aldono-1,4-lactones, which are separated by g.l.c. The calculated ratios of glucuronic acid/iduronic acid are reproducible within 5%. 2. Samples of heparin from pig intestinal mucosa (molar ratio of sulphate/disaccharide unit, 2.40) and heparan sulphate from human aorta (sulphate/disaccharide ratio, 0.46) were subjected to uronic acid analysis. l-Iduronic acid constituted 77% and 19% respectively of the total uronic acid contents. 3. The correlation between the contents of sulphate and iduronic acid indicated by this finding also applied to the fractionated deamination products of the two polymers. The sulphated fragments varied in size from disaccharide to octasaccharide (or larger) and showed sulphate/disaccharide molar ratios in the range of 0.05-2.0. The proportion of iduronic acid increased with increasing ester sulphate contents of the oligosaccharides. 4. Previous studies on the biosynthesis of heparin in a cell-free system have shown that l-iduronic acid residues are formed by C-5 epimerization of d-glucuronic acid units at the polymer level; the process requires concomitant sulphation of the polymer. The results obtained in the present structural study conform to these findings, and suggest further that similar mechanisms may operate in the biosynthesis of heparan sulphate. The epimerization reaction appears to be linked to the sulphation of hydroxyl groups but does not seem to require sulphation of the target uronic acid residues. The significance of sulphamino groups in relation to the formation of iduronic acid is unknown.

Topics: Animals; Aorta; Carbon Radioisotopes; Cellulose; Chromatography, Gas; Chromatography, Ion Exchange; Chromatography, Paper; Electrophoresis; Electrophoresis, Paper; Heparin; Heparitin Sulfate; Hexoses; Humans; Intestinal Mucosa; Kinetics; Mast-Cell Sarcoma; Nitrites; Oligosaccharides; Spectrophotometry; Sulfates; Sulfuric Acids; Swine; Time Factors; Uronic Acids