Halldórsdóttir Anna Margrét, Zhang Lijuan, Tollefsen Douglas M
Division of Laboratory Medicine, Department of Pathology and Immunology, Campus Box 8118, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
Glycobiology. 2006 Aug;16(8):693-701. doi: 10.1093/glycob/cwj117. Epub 2006 Apr 19.
Dermatan sulfate (DS) accelerates the inhibition of thrombin by heparin cofactor II (HCII). A hexasaccharide consisting of three l-iduronic acid 2-O-sulfate (IdoA2SO3)-->N-acetyl-D-galactosamine 4-O-sulfate (GalNAc4SO3) subunits was previously isolated from porcine skin DS and shown to bind HCII with high affinity. DS from porcine intestinal mucosa has a much lower content of this disaccharide but activates HCII with potency similar to that of porcine skin DS. Therefore, we sought to characterize oligosaccharides from porcine mucosal DS that interact with HCII. DS was partially depolymerized with chondroitinase ABC, and oligosaccharides containing 2-12 monosaccharide units were isolated. The oligosaccharides were then fractionated by anion-exchange and affinity chromatography on HCII-Sepharose, and the disaccharide compositions of selected fractions were determined. We found that the smallest oligosaccharides able to bind HCII were hexasaccharides. Oligosaccharides 6-12 units long that lacked uronic acid (UA)2SO3 but contained one or two GalNAc4,6SO3 residues bound, and binding was proportional to both oligosaccharide size and number of GalNAc4,6SO3 residues. Intact DS and bound dodecasaccharides contained predominantly IdoA but little D-glucuronic acid. Decasaccharides and dodecasaccharides containing one or two GalNAc4,6SO3 residues stimulated thrombin inhibition by HCII and prolonged the clotting time of normal but not HCII-depleted human plasma. These data support the hypothesis that modification of IdoA-->GalNAc4SO3 subunits in the DS polymer by either 2-O-sulfation of IdoA or 6-O-sulfation of GalNAc can generate molecules with HCII-binding sites and anticoagulant activity.
硫酸皮肤素（DS）可加速肝素辅因子II（HCII）对凝血酶的抑制作用。先前从猪皮DS中分离出一种由三个L-艾杜糖醛酸2-O-硫酸酯（IdoA2SO3）→N-乙酰-D-半乳糖胺4-O-硫酸酯（GalNAc4SO3）亚基组成的六糖，该六糖显示出与HCII具有高亲和力。来自猪肠黏膜的DS中这种二糖的含量要低得多，但激活HCII的效力与猪皮DS相似。因此，我们试图对猪黏膜DS中与HCII相互作用的寡糖进行表征。用软骨素酶ABC将DS部分解聚，分离出含有2-12个单糖单元的寡糖。然后通过阴离子交换和在HCII-琼脂糖上的亲和色谱对寡糖进行分级分离，并测定所选馏分的二糖组成。我们发现能够结合HCII的最小寡糖是六糖。长度为6-12个单元且缺乏糖醛酸（UA）2SO3但含有一个或两个GalNAc4,6SO3残基的寡糖能够结合，且结合与寡糖大小和GalNAc4,6SO3残基数量均成正比。完整的DS和结合的十二糖主要含有艾杜糖醛酸，但几乎不含D-葡萄糖醛酸。含有一个或两个GalNAc4,6SO3残基的十糖和十二糖可刺激HCII对凝血酶的抑制作用，并延长正常人血浆而非HCII缺陷型人血浆的凝血时间。这些数据支持这样的假设，即通过IdoA的2-O-硫酸化或GalNAc的6-O-硫酸化对DS聚合物中的IdoA→GalNAc4SO3亚基进行修饰可产生具有HCII结合位点和抗凝活性的分子。
