The five hexa- or heptasulfated hexasaccharides share the common core pentasulfated structure ΔHexA(2S)α1-4GlcN(NS,6S)α1-4IdoAα/GlcAβ1-4GlcN(6S)α1-4GlcAβ1- 4GlcN(NS) with one or two additional sulfate groups (ΔHexA, GlcN, IdoA, and GlcA represent 4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, whereas 2S, 6S, and NS stand for 2-O-, 6-O-, and 2-N-sulfate, respectively). The tetrasaccharide has the hexasulfated structure typical of heparin. In this study, the structures of one tetra- and five hexasaccharides from the repeat region were determined by chemical and enzymatic analyses as well as 500-MHz 1H NMR spectroscopy. The latter structures were reported recently (Sugahara, K., Tsuda, H., Yoshida, K., Yamada, S., de Beer, T., and Vliegenthart, J. Six contained oligosaccharides derived from the repeating disaccharide region, whereas four contained glycoserines from the glycosaminoglycan-protein linkage region. Subfractionation of the hexasaccharide fraction by anion exchange high pressure liquid chromatography yielded 10 fractions. D-Glucuronosyl 5-epimerization not accompanied by 2-O-sulfation occurs at a still earlier stage of polymer modification the resulting L-iduronic acid units appear to remain nonsulfated throughout the subsequent modification reactions.Ībstract Porcine intestinal heparin was extensively digested with Flavobacterium heparinase and size-fractionated by gel chromatography. The results suggest that 2-O-sulfation of L-iduronic acid units is tightly coupled to the formation of these units (by 5-epimerization of D-glucuronic acid residues) and, furthermore, that both processes are completed before 6-O-sulfation of the polysaccharide molecule is initiated. Furthermore, structural analysis of microsomal heparin-precursor polysaccharides showed a distinct intermediate species that contained 2-O-sulfated L-iduronic acid units but essentially no 6-O-sulfate groups on the (N-sulfated) D-glucosamine residues. Characterization of the labeled products showed that O-sulfation occurs preferentially in the vicinity of N-sulfate groups that 2-O-sulfation of L-iduronic acid residues occurs preferentially or exclusively in the absence of a 6-O-sulfate group on adjacent D-glucosamine units and that 6-O-sulfation of D-glucosamine residues occurs readily in the presence of 2-O-sulfate groups on adjacent L-iduronic acid units. The substrate specificity of O-sulfotransferases involved in the biosynthesis of heparin was studied by incubating exogenous polysaccharide acceptors with mouse mastocytoma microsomal fraction in the presence of phosphoadenylylsulfate. The unexpected finding that oligosaccharides associated with later stages in HS biosynthesis inhibit HS2ST indicates that the enzyme must be separated temporally and/or spatially from downstream products during biosynthesis in vivo, and highlights a challenge for the enzymatic synthesis of lengthy HS chains in vitro. ![]() Structural analysis predicts a mode of inhibition in which 6-O-sulfate groups located on glucosamine residues present in highly-sulfated oligosaccharides occupy the canonical binding site of the nucleotide cofactor. Inhibition assays also indicate that the IC50 values correlate simply with degree of oligosaccharide sulfation. Surprisingly, heavily sulfated oligosaccharides formed by later-acting sulfotransferases bind more tightly to HS2ST than those corresponding to its natural substrate or product. Here, the binding preferences of the uronyl 2-O-sulfotransferase (HS2ST) are examined with variably-sulfated hexasaccharides. Heparan Sulfate (HS) is a cell signaling molecule linked to pathological processes ranging from cancer to viral entry, yet fundamental aspects of its biosynthesis remain incompletely understood.
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