Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity

M. M. Maimone, D. M. Tollefsen

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Dermatan sulfate increases the rate of inhibition of thrombin by heparin cofactor II (HCII) ~1000-fold by providing a catalytic template to which both the inhibitor and the protease bind. Dermatan sulfate is a linear polymer of D-glucuronic acid (GlcA) or L-iduronic acid (IdoA) alternating with N-acetyl-D-galactosamine (GalNAc) residues. Heterogeneity in dermatan sulfate results from varying degrees of O-sulfation and from the presence of the two types of uronic acid residues. To characterize the HCII-binding site in dermatan sulfate, we isolated the smallest fragment of dermatan sulfate that bound to HCII with high affinity. Dermatan sulfate was partially N-deacetylated by hydrazinolysis, cleaved with nitrous acid at pH 4, and reduced with [3H]NaBH4. The resulting fragments, containing an even number of monosaccharide units with the reducing terminal GalNAc converted to [3H]2,5-anhydro-D-talitol (ATal(R)), were size-fractionated and then chromatographed on an HCII-Sepharose column. The smallest HCII-binding fragments were hexasaccharides, of which ~6% bound. Based on ion-exchange chromatography, the bound material appeared to comprise a heterogeneous mixture of molecules possessing four, five, or six sulfate groups per hexasaccharide. Subsequently, hexasaccharides with the highest affinity for HCII were isolated by overloading the HCII-Sepharose column. The high-affinity hexasaccharides were fractionated by strong anion-exchange chromatography, and one major peak representing ~2% of the starting hexasaccharides was isolated. The high-affinity hexasaccharide was cleaved to disaccharides that were analyzed by anion-exchange chromatography, paper electrophoresis, and paper chromatography. A single disulfated disaccharide, IdoA(2-SO4)→ATal(R)(4-SO4) was observed, indicating that the hexasaccharide has the following structure: IdoA(2-SO4)→GalNAc(4-SO4)→IdoA(2-SO4)→GalNAc(4-SO4)→IdoA(2- SO4)→ATal(R)(4-SO4). Since IdoA(2-SO4)→GalNAc(4-SO4) comprises only ~5% of the disaccharides present in intact dermatan sulfate, clustering of these disaccharides must occur during biosynthesis to form the high-affinity binding site for HCII.

Original languageEnglish
Pages (from-to)18263-18271
Number of pages9
JournalJournal of Biological Chemistry
Issue number30
StatePublished - 1990


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