Yamada S, Yoshida K, Sugiura M, Sugahara K, Khoo K H, Morris H R, Dell A
Department of Physiological Chemistry, Kobe Women's College of Pharmacy, Japan.
J Biol Chem. 1993 Mar 5;268(7):4780-7.
Three discrete tetrasaccharide structures which are resistant to Flavobacterium heparinase and heparitinases I and II were isolated from porcine intestinal heparin after exhaustive digestion with a mixture of all the above enzymes, and the tri-, tetra-, and penta-sulfated structures were determined by negative ion mode fast atom bombardment mass spectrometry and 500-MHz 1H NMR analysis as delta 4,5GlcA beta 1-4GlcNAc (6-sulfate)alpha 1-4GlcA beta 1-4GlcN(N,3-disulfate), delta 4,5 GlcA beta 1-4GlcNAc(6-sulfate)alpha 1-4GlcA beta 1-4GlcN (N,3,6-trisulfate), and delta 4,5GlcA beta 1-4GlcN (N,6-disulfate)alpha 1-4GlcA beta 1-4GlcN(N,3,6-trisulfate). The three components share the 3-O-sulfated reducing GlcN and the 6-O-sulfated internal GlcN, indicating that they are structural variants derived from the nonreducing portion of the minimal pentasaccharide sequence required for binding to antithrombin III. Isolation of the pentasulfated component has never been reported. Their unexpected resistance to heparitinases I and II indicates that 3-O-sulfation of the reducing GlcN contributes to the resistant nature of these tetrasaccharides to the enzymes. The present study demonstrates that the nonreducing trisaccharide portion of the structural variants of the antithrombin III-binding pentasaccharide sequence can be isolated in tetrasaccharides resistant to heparinase/heparitinases I and II, while the rest of the repeating region is degraded into disaccharide units. The lyase treatment is applicable to evaluation of heparin/heparan sulfate preparations in terms of the presence or absence of the specific structure containing the 3-O-sulfated GlcN representing biosynthetic precursors, intermediates or final products of the binding site.
在用上述所有酶的混合物进行彻底消化后，从猪肠道肝素中分离出三种对黄杆菌肝素酶、肝素酶I和肝素酶II具有抗性的离散四糖结构，并通过负离子模式快原子轰击质谱和500兆赫1H核磁共振分析确定了三硫酸化、四硫酸化和五硫酸化结构，分别为δ4,5GlcAβ1-4GlcNAc(6-硫酸酯)α1-4GlcAβ1-4GlcN(N,3-二硫酸酯)、δ4,5GlcAβ1-4GlcNAc(6-硫酸酯)α1-4GlcAβ1-4GlcN(N,3,6-三硫酸酯)和δ4,5GlcAβ1-4GlcN(N,6-二硫酸酯)α1-4GlcAβ1-4GlcN(N,3,6-三硫酸酯)。这三种成分都具有3-O-硫酸化的还原型GlcN和6-O-硫酸化的内部GlcN，表明它们是源自与抗凝血酶III结合所需的最小五糖序列非还原部分的结构变体。从未有过五硫酸化成分分离的报道。它们对肝素酶I和肝素酶II意外的抗性表明，还原型GlcN的3-O-硫酸化有助于这些四糖对酶的抗性。本研究表明，抗凝血酶III结合五糖序列结构变体的非还原三糖部分可以在对肝素酶/肝素酶I和II具有抗性的四糖中分离出来，而重复区域的其余部分则降解为二糖单元。裂解酶处理可用于评估肝素/硫酸乙酰肝素制剂中是否存在含有代表结合位点生物合成前体、中间体或最终产物的3-O-硫酸化GlcN的特定结构。
