Active Sites of Dermatan Sulfate for Heparin Cofactor II. Isolation of a Nonasaccharide Fragment Containing Four Disaccharide Sequences [α-l-Iduronic Acid 2-O-Sulfate (1,3)-β-d-N-Acetylgalactosamine 4-Sulfate]

Abstract

The active site of dermatan sulfate (DS) for heparin cofactor II (HCII) was isolated in a fragment obtained by periodate oxidation, borohydride reduction, mild acid hydrolysis, and SE- and SAX-chromatography of beef mucosal and pig skin DS preparations. Characterization by mass spectrometry, one- and two-dimensional NMR spectroscopy, and HPLC analysis of disaccharides, obtained by exhaustive digestion with chondroitinase-ABC, indicates that the fragment has the prevalent structure 1, GalNAc-4SO₃-[IdoA-2SO₃-GalNAc-4SO₃]₄-R, where R is CH(CH₂OH)CH(COO⁻)-OH. 1, is the largest DS fragment thus far isolated containing IdoA2SO₃ as the only uronic acid. Its lower activity (30%) with respect to the parent polymeric DS is explainable by Tollefsen model, requiring longer polyanionic chains for formation of ternary complex with thrombin.

Notes

Abbreviations: IdoA2SO₃-GalNAc-4SO₃ = 2-acetamido-2-deoxy-3-O-(4-deoxy-2-O-sulfo-α-l-threo-pyranosyl uronic acid)-4-O-sulfo-d-galactose. GlcA = glucuronic acid. ΔDi-2,4-diS = 2-acetamido-2-deoxy-3-O-(4-deoxy-2-O-sulfo-α-l-threo-hex-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose; the other ΔDi-abbreviations are related to the other 6 disaccharide-type obtainable by chondroitinase ABC digestion from DS. SE-HPLC = size exclusion high performance liquid chromatography. SAX-chromatography = strong anion exchange chromatography. RO-DS = reduced (with NaBH₄) oxidized (with IO₄ ⁻)-DS. SD-DS= Smith-degraded DS.