Biosynthesis of heparan sulphate with diverse structures and functions: two alternatively spliced forms of human heparan sulphate 6-O-sulphotransferase-2 having different expression patterns and properties

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Background: Heparin regulates mast cell proteases. Results: Both HS6ST-1 and HS6ST-2 are involved in 6-O-sulfation of heparin. The contents of tryptase and CPA in mast cells depend on 6-O-sulfation of heparin but chymase does not. Conclusion:The 6-O-sulfation pattern regulates differently the storage of MC-specific proteases. Significance: The fine structure of heparin may be essential for MC homeostasis.

Section: Discussionmentioning

confidence: 69%

Heparan Sulfate 6-O-Sulfotransferase Isoform-dependent Regulatory Effects of Heparin on the Activities of Various Proteases in Mast Cells and the Biosynthesis of 6-O-Sulfated Heparin

Anower-E-Khuda

Habuchi

Nagai

et al. 2013

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“…We demonstrated previously that in mammals, heparan sulfate 6-O-sulfotransferases exist in three isoforms (HS6ST-1, -2, and -3) and one alternatively spliced form (HS6ST-2S) (30,31). Although the substrate specificities of these isoforms largely overlap, the individual isoforms exhibit a characteristic preference for the uronic acid residue neighboring the N-sulfoglucosamine.…”

Section: Heparan Sulfate (Hs)mentioning

confidence: 99%

Mice Deficient in Heparan Sulfate 6-O-Sulfotransferase-1 Exhibit Defective Heparan Sulfate Biosynthesis, Abnormal Placentation, and Late Embryonic Lethality

Habuchi

Nagai

Sugaya

et al. 2007

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116

101

Heparan sulfate (HS) plays critical roles in a variety of developmental, physiological, and pathogenic processes due to its ability to interact in a structure-dependent manner with numerous growth factors that participate in cellular signaling. The divergent structures of HS glycosaminoglycans are the result of the coordinate actions of several N-and O-sulfotransferases, C5-epimerase, and 6-O-endosulfatases. We have shown that 6-O-sulfation of the glucosamine residues in HS are catalyzed by the sulfotransferases HS6ST-1, -2, and -3. To determine the biological and physiological importance of HS6ST-1, we now describe the creation of transgenic mice that lack this sulfotransferase. Most of our HS6ST-1-null mice died between embryonic day 15.5 and the perinatal stage, and those mice that survived were considerably smaller than their wild-type littermates. Some of these HS6ST-1-null mice exhibited development abnormalities, and histochemical and molecular analyses of these mice revealed an ϳ50% reduction in the number of fetal microvessels in the labyrinthine zone of the placenta relative to that in the wild-type mice. Because we observed a modest reduction in VEGF-A mRNA and protein in the tissues of HS6ST-1-null mice, an HS-dependent defect in cytokine signaling probably contributes to increased embryonic lethality and decreased growth. Biochemical studies of the HS chains isolated from various organs of our HS6ST-1-null mice revealed a marked reduction of GlcNAc(6SO 4 ) and HexA-GlcNSO 3 (6SO 4 ) levels and a reduced ability to bind Wnt2. Thus, despite the presence of three closely related 6-O-sulfotransferase genes in the mouse genome, HS6ST-1 is the primary one used in HS biosynthesis in most tissues. Heparan sulfate (HS)2 proteoglycans on the cell surfaces interact with numerous growth factors, morphogens, receptors, and extracellular matrix proteins. These complex interactions regulate the activity, gradient formation, and stability of many receptor-ligand interactions. Thus, HS has important functions in a variety of developmental, morphogenetic, and pathogenic processes (1-3). The specificities of the interactions between HS and ligands are thought to be due, at least in part, to the fine structure of HS characterized by the specific localized patterns of sulfates and the hexuronic acid isoform residues (4 -8). The fine and divergent structures of HS are generated in the Golgi apparatus through the coordinate actions of HS modification enzymes, namely N-deacetylase/N-sulfotransferases (NDSTs), Hsepi (C5-epimerase), and 2-O-, 6-O-, and 3-O-sulfotransferases, following the biosynthesis of the HS backbone structure comprising alternating GlcA and GlcNAc residues (9, 10). Further modification of HS occurs at the cell surface by the action of 6-O-endosulfatase (11-13). The localization of some of these HS modification enzymes to the Golgi may be facilitated by their tendency to form multienzyme complexes, as shown in the case of Hsepi and HS2ST (HS 2-O-sulfotransferase) (14, 15). These modification enzymes, with...

“…28) cDNAs. No PCR product was observed using primers for 6-OST3, indicating that the 293 cells express 6-OST1 and 6-OST2S mRNA endogenously, as verified by sequencing of the purified PCR fragments (data not shown).…”

Section: Enzymatic 6-o-sulfation Of 3 H-labeled Octasaccharides-mentioning

confidence: 99%

Oligosaccharide Library-based Assessment of Heparan Sulfate 6-O-Sulfotransferase Substrate Specificity

Jemth

Smeds

et al. 2003

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Heparan sulfate mediates numerous complex biological processes. Its action critically depends on the amount and the positions of O-sulfate groups (iduronyl 2-O-sulfates, glucosaminyl 6-O-and 3-O-sulfates) that form binding sites for proteins. The structures and distribution of these protein-binding domains are influenced by the expression and substrate specificity of heparan sulfate biosynthetic enzymes. We describe a general approach to assess substrate specificities of enzymes involved in glycosaminoglycan metabolism, here applied to 6-O-sulfotransferases involved in heparan sulfate biosynthesis. To understand how 2-O-sulfation affects subsequent 6-O-sulfation reactions, the substrate specificity of 6-O-sulfotransferase 3 was probed using substrates from a heparin-based octasaccharide library. Purified 3 H-labeled N-sulfated octasaccharides from a library designed to sample 2-O-sulfated motifs were used as sulfate acceptors, 3 -phosphoadenosine 5 -phosphosulfate as sulfate donor, and cell extract from 6-Osulfotransferase 3-overexpressing 293 cells as enzyme source in the 6-O-sulfotransferase-catalyzed reactions. The first 6-O-sulfate group was preferentially incorporated at the internal glucosamine unit of the octasaccharide substrate. As the reaction proceeded, the octasaccharides acquired three 6-O-sulfate groups. The specificities toward competing octasaccharide substrates, for 6-Osulfotransferase 2 and 6-O-sulfotransferase 3, were determined using overexpressing 293 cell extracts and purified octasaccharides. Both 6-O-sulfotransferases showed a preference for 2-O-sulfated substrates. The specificity toward substrates with two to three 2-O-sulfate groups was three to five times higher as compared with octasaccharides with no or one 2-O-sulfate group.Heparan sulfate (HS) 1 is a linear polysaccharide present on virtually all cells and in the extracellular matrix (1). HS chains are heterogeneous, with a large number of complex sequences based on variable patterns of N-acetyl, N-sulfate, and O-sulfate groups (2-4). The biosynthesis of the polysaccharide chains begins with the synthesis of an oligosaccharide primer covalently attached to a serine residue in a proteoglycan protein core. HS chains are then generated by the sequential addition of N-acetyl-D-glucosamine (GlcNAc) and D-glucuronate (GlcUA). Along with polymerization the chains undergo a series of modification steps that involve five distinct enzyme families; (i) N-deacetylase/N-sulfotransferase removes the N-acetyl group from GlcNAc units and replaces it with an N-sulfate group (GlcNS); (ii) GlcUA C5 epimerase converts GlcUA to its C5 epimer L-iduronate (IdoUA); (iii) 2-O-sulfotransferase adds 2-O-sulfate groups to IdoUA and GlcUA residues; (iv) 6-O-sulfotransferase (6-OST), and (v) 3-O-sulfotransferase (3-OST) transfer O-sulfate groups to C6 and C3, respectively, of GlcNAc or GlcNS units (2-4). The N-deacetylase/N-sulfotransferase (5-8), the 6-OST (9), and the 3-OST (10 -12) families each contain several members, whereas only one GlcUA C5 epimerase (...