Maimone M M, Tollefsen D M
Department of Medicine and Biochemistry, Washington University, St. Louis, Missouri 63110.
J Biol Chem. 1990 Oct 25;265(30):18263-71.
Dermatan sulfate increases the rate of inhibition of thrombin by heparin cofactor II (HCII) approximately 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 (ATalR), were size-fractionated and then chromatographed on an HCII-Sepharose column. The smallest HCII-binding fragments were hexasaccharides, of which approximately 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 approximately 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)----ATalR(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)----ATalR(4-SO4). Since IdoA(2-SO4)----GalNAc(4-SO4) comprises only approximately 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.
硫酸皮肤素通过提供一种催化模板，使抑制剂和蛋白酶都能与之结合，从而将肝素辅因子II（HCII）对凝血酶的抑制速率提高约1000倍。硫酸皮肤素是由D-葡萄糖醛酸（GlcA）或L-艾杜糖醛酸（IdoA）与N-乙酰-D-半乳糖胺（GalNAc）残基交替组成的线性聚合物。硫酸皮肤素的异质性源于不同程度的O-硫酸化以及两种类型糖醛酸残基的存在。为了表征硫酸皮肤素中HCII结合位点，我们分离出了与HCII具有高亲和力的硫酸皮肤素最小片段。硫酸皮肤素通过肼解进行部分N-脱乙酰化，在pH 4下用亚硝酸裂解，并用[3H]NaBH4还原。所得片段含有偶数个单糖单元，还原端的GalNAc转化为[3H]2,5-脱水-D-木糖醇（ATalR），进行尺寸分级，然后在HCII-琼脂糖柱上进行色谱分析。最小的HCII结合片段是六糖，其中约6%结合。基于离子交换色谱，结合的物质似乎是一种异质混合物，每个六糖含有四个、五个或六个硫酸基团。随后，通过使HCII-琼脂糖柱超载，分离出了对HCII具有最高亲和力的六糖。高亲和力六糖通过强阴离子交换色谱进行分级，分离出一个主要峰，约占起始六糖的2%。高亲和力六糖被裂解为二糖，通过阴离子交换色谱、纸电泳和纸色谱进行分析。观察到一种单二硫酸化二糖，即IdoA(2-SO4)----ATalR(4-SO4)，表明该六糖具有以下结构：IdoA(2-SO4)----GalNAc(4-SO4)----IdoA(2-SO4)---- GalNAc(4-SO4)----IdoA(2-SO4)----ATalR(4-SO4)。由于IdoA(2-SO4)----GalNAc(4-SO4)仅占完整硫酸皮肤素中二糖的约5%左右，这些二糖在生物合成过程中必定会聚集形成HCII的高亲和力结合位点。
