Yamada S, Yamane Y, Tsuda H, Yoshida K, Sugahara K
Department of Biochemistry, Kobe Pharmaceutical University, Japan.
J Biol Chem. 1998 Jan 23;273(4):1863-71. doi: 10.1074/jbc.273.4.1863.
The major structure of the low sulfated irregular region of porcine intestinal heparin was investigated by characterizing the hexasaccharide fraction prepared by extensive digestion of the highly sulfated region with Flavobacterium heparinase and subsequent size fractionation by gel chromatography. Structures of a tetrasaccharide, a pentasaccharide, and eight hexasaccharide components in this fraction, which accounted for approximately 19% (w/w) of the starting heparin representing the major oligosaccharide fraction derived from the irregular region, were determined by chemical and enzymatic analyses as well as 1H NMR spectroscopy. Five compounds including one penta- and four hexasaccharides had hitherto unreported structures. The structure of the pentasaccharide with a glucuronic acid at the reducing terminus was assumed to be derived from the reducing terminus of a heparin glycosaminoglycan chain and may represent the reducing terminus exposed by a tissue endo-beta-glucuronidase involved in the intracellular post-synthetic fragmentation of macromolecular heparin. Eight out of the 10 isolated oligosaccharides shared the trisaccharide sequence, -4IdceA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-, and its reverse sequence, -4GlcA beta 1-4GlcNAc alpha 1-4IdceA alpha 1-, was not found. The latter has not been reported to date for heparin/heparan sulfate, indicating the substrate specificity of the D-glucuronyl C-5 epimerase. Furthermore, seven hexasaccharides shared the common trisulfated hexasaccharide core sequence delta HexA(2-sulfate)alpha 1-4GlcN(N-sulfate)alpha 1-4IdceA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-4GlcN(N-sulfate) which contained the above trisaccharide sequence (delta HexA, IdceA, GlcN, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, L-iduronic acid, D-glucosamine, and D-glucuronic acid, respectively) and additional sulfate groups. The specificity of the heparinase used for preparation of the oligosaccharides indicates the occurrence of the common pentasulfated octasaccharide core sequence, -4GlcN(N-sulfate)alpha 1-4HexA(2-sulfate)1-4GlcN(N-sulfate) alpha 1-4IdceA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-4 GlcN(N-sulfate)alpha 1-4HexA(2-sulfate)1-, where the central hexasaccharide is flanked by GlcN(N-sulfate) and HexA(2-sulfate) on the nonreducing and reducing sides, respectively. The revealed common sequence constituted a low sulfated trisaccharide representing the irregular region sandwiched by highly sulfated regions and should reflect the control mechanism of heparin biosynthesis.
通过对用肝素黄杆菌对高硫酸化区域进行充分消化并随后通过凝胶色谱进行大小分级分离得到的六糖部分进行表征，研究了猪肠道肝素低硫酸化不规则区域的主要结构。通过化学和酶分析以及1H NMR光谱法确定了该部分中一种四糖、一种五糖和八种六糖成分的结构，这些成分约占起始肝素的19%（w/w），代表了来自不规则区域的主要寡糖部分。包括一种五糖和四种六糖在内的五种化合物具有迄今未报道的结构。推测还原端带有葡萄糖醛酸的五糖结构源自肝素糖胺聚糖链的还原端，可能代表参与大分子肝素细胞内合成后片段化的组织内切β-葡萄糖醛酸酶暴露的还原端。分离出的10种寡糖中有8种具有三糖序列-4IdceAα1-4GlcNAcα1-4GlcAβ1-，未发现其反向序列-4GlcAβ1-4GlcNAcα1-4IdceAα1-。迄今为止，肝素/硫酸乙酰肝素尚未报道过后者，这表明D-葡萄糖醛酸C-5差向异构酶的底物特异性。此外，七种六糖具有共同的三硫酸化六糖核心序列δHexA(2-硫酸酯)α1-4GlcN(N-硫酸酯)α1-4IdceAα1-4GlcNAcα1-4GlcAβ1-4GlcN(N-硫酸酯)，其中包含上述三糖序列（δHexA、IdceA、GlcN和GlcA分别代表4-脱氧-α-L-苏-己-4-烯吡喃糖醛酸、L-艾杜糖醛酸、D-葡萄糖胺和D-葡萄糖醛酸）以及额外的硫酸基团。用于制备寡糖的肝素酶的特异性表明存在常见的五硫酸化八糖核心序列-4GlcN(N-硫酸酯)α1-4HexA(2-硫酸酯)1-4GlcN(N-硫酸酯)α1-4IdceAα1-4GlcNAcα1-4GlcAβ1-4GlcN(N-硫酸酯)α1-4HexA(2-硫酸酯)1-，其中中央六糖在非还原侧和还原侧分别由GlcN(N-硫酸酯)和HexA(2-硫酸酯)侧翼。所揭示的共同序列构成了一个低硫酸化三糖，代表夹在高硫酸化区域之间的不规则区域，应该反映了肝素生物合成的控制机制。
