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

Abstract

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 (...

Role of Deacetylase Activity of N-Deacetylase/N-Sulfotransferase 1 in Forming N-Sulfated Domain in Heparan Sulfate

“…In a control experiment, the two compounds exhibited comparable reactivity to 6-OST-1 modification consistent with this modification not being influenced by the level of N-sulfation (35)(36)(37). The data suggest that change in the N-S domain structures impacts the biosynthesis of HS.…”

Overexpression of Heparan Sulfate 6-O-Sulfotransferases in Human Embryonic Kidney 293 Cells Results in Increased N-Acetylglucosaminyl 6-O-Sulfation

“…The insert was excised with EcoRI and subcloned into the corresponding site of the pcDNA3 plasmid vector (Invitrogen). The 6-OST2 and 6-OST3 expression constructs have been described previously (25). The different expression plasmids were transfected into HEK 293 cells using Lipofectamine (Invitrogen).…”

Genetic Analysis of the Heparan Modification Network in Caenorhabditis elegans

“…Whereas we observe HST-6 clearly exhibiting a strong preference for an adjacent 2-O-sulfate in vivo (as there is almost no D0S6 detected from HS purified from worms), it is completely capable of sulfating GlcNS in the hst-2(ok595) null mutant. Human HS6st-2 and -3 were shown by Jemth et al (36) to have a strong preference for 2-O-sulfated substrates in vitro. One possibility is that the 6-O-sulfotransferase reaction may be coupled to the 2-O-sulfotransferase reaction, for example, due to physical interactions between the enzymes.…”