Sugahara K, Tsuda H, Yoshida K, Yamada S, de Beer T, Vliegenthart J F
Department of Biochemistry, Kobe Pharmaceutical University, Japan.
J Biol Chem. 1995 Sep 29;270(39):22914-23. doi: 10.1074/jbc.270.39.22914.
Previously we isolated a tetrasaccharide-serine and a hexasaccharide-serine from the carbohydrate-protein linkage region of porcine intestinal heparin after digestion with a mixture of Flavobacterium heparinase and heparitinases I and II (Sugahara, K., Yamada, S., Yoshida, K., de Waard, P., and Vliegenthart, J.F.G. (1992) J. Biol. Chem. 267, 1528-1533). In this study four longer carbohydrate sequences (I-IV) attached to Ser or a dipeptide (Ser-Gly or Gly-Ser), which accounted for at least 18.2% of the total linkage region, were isolated from the same heparin preparation after digestion with heparinase only. IV was successfully isolated only after subsequent digestion with glycuronate-2-sulfatase. Their structures were determined by chemical and enzymatic analyses and 1H NMR spectroscopy and found to be the following octa- and decasaccharide sequences attached to Ser in a molar ratio of 1.1:2.3:1.0:1.3: delta HexA(2S)alpha 1-4GlcN(NS,6S)alpha 1-4GlcA beta 1-4GlcNAc alpha 1-4- GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (I), delta HexA(2S)alpha 1- 4GlcN(NS,6S)alpha 1-4IdoA alpha 1-4GlcNAc alpha 1-4GlcA beta 1- 3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (II), delta HexA(2S)alpha 1- 4GlcN(NS,6S)alpha 1- 4IdoA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-4GlcNAc-alpha 1- 4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (III), delta HexA alpha 1-4GlcN(NS,6S)alpha 1-4IdoA alpha 1-4GlcNAc(6S)alpha 1- 4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser (IV) (delta HexA, GlcA, IdoA, and GlcN represent 4,5-unsaturated hexuronic acid, D-glucuronic acid, L-iduronic acid, and D-glucosamine, whereas 2S, 6S, and NS stand for 2-sulfate, 6-sulfate, and N-sulfate, respectively). I and II contained 1 mol of Gly in addition to Ser. The four structures indicate that sulfation in heparin chains takes place on the monosaccharide residues located in closer vicinity to the core protein than found for heparan sulfate chains and that there exist at least several heparin subclass chains with different linkage region structures. The significance of the isolated structures is discussed in relation to the biological functions and the biosynthetic mechanisms of heparin.
此前，我们在用肝素黄杆菌酶与艾杜糖醛酸酶I和II的混合物消化猪肠肝素的碳水化合物 - 蛋白质连接区域后，分离出了一种四糖 - 丝氨酸和一种六糖 - 丝氨酸（菅原，K.，山田，S.，吉田，K.，德瓦尔德，P.，和弗利根哈特，J.F.G.（1992年）《生物化学杂志》267卷，第1528 - 1533页）。在本研究中，在用肝素酶单独消化同一肝素制剂后，分离出了四个与丝氨酸或二肽（丝氨酸 - 甘氨酸或甘氨酸 - 丝氨酸）相连的更长碳水化合物序列（I - IV），其占总连接区域的至少18.2%。IV仅在随后用2 - 硫酸艾杜糖醛酸酶消化后才成功分离出来。通过化学和酶促分析以及¹H NMR光谱确定了它们的结构，发现是以下八糖和十糖序列以1.1:2.3:1.0:1.3的摩尔比连接到丝氨酸上：ΔHexA(2S)α1 - 4GlcN(NS,6S)α1 - 4GlcAβ1 - 4GlcNAcα1 - 4 - GlcAβ1 - 3Galβ1 - 3Galβ1 - 4Xylβ1 - O - Ser（I），ΔHexA(2S)α1 - 4GlcN(NS,6S)α1 - 4IdoAα1 - 4GlcNAcα1 - 4GlcAβ1 - 3Galβ1 - 3Galβ1 - 4Xylβ1 - O - Ser（II），ΔHexA(2S)α1 - 4GlcN(NS,6S)α1 - 4IdoAα1 - 4GlcNAcα1 - 4GlcAβ1 - 4GlcNAc - α1 - 4GlcAβ1 - 3Galβ1 - 3Galβ1 - 4Xylβ1 - O - Ser（III），ΔHexAα1 - 4GlcN(NS,6S)α1 - 4IdoAα1 - 4GlcNAc(6S)α1 - 4GlcAβ1 - 3Galβ1 - 3Galβ1 - 4Xylβ1 - O - Ser（IV）（ΔHexA、GlcA、IdoA和GlcN分别代表4,5 - 不饱和己糖醛酸、D - 葡糖醛酸、L - 艾杜糖醛酸和D - 葡糖胺，而2S、6S和NS分别代表2 - 硫酸酯、6 - 硫酸酯和N - 硫酸酯）。I和II除了丝氨酸外还含有1摩尔甘氨酸。这四种结构表明，肝素链中的硫酸化发生在比硫酸乙酰肝素链中更靠近核心蛋白的单糖残基上，并且存在至少几种具有不同连接区域结构的肝素亚类链。结合肝素的生物学功能和生物合成机制讨论了所分离结构的意义。
