Structures of Five Sulfated Hexasaccharides Prepared from Porcine Intestinal Heparin Using Bacterial Heparinase
Structures of Five Sulfated Hexasaccharides Prepared from Porcine Intestinal Heparin Using Bacterial Heparinase
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细菌肝素酶从猪肠肝素制备五种硫酸六糖的结构
DOI：
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发表时间：
1996
影响因子：
4.8
通讯作者：
K. Sugahara
中科院分区：
文献类型：
--
作者：
Hiromi Tsuda;S. Yamada;Yukari Yamane;Keiichi Yoshida;J. Hopwood;K. Sugahara
用黄杆菌肝素酶对猪肠型肝素进行广泛消化，并用凝胶层析法进行分级。用阴离子交换高效液相色谱对六糖组分进行精馏，得到10个组分。六个含有来自重复二糖区域的低聚糖，而四个含有来自糖胺聚糖-蛋白质连接区的糖丝氨酸。后者是最近报道的(Sugahara，K.，Tsuda，H.，Yoshida，K.，Yamada，S.，De Beer，T.，和Vliegenthart，J.F.G.(1995)J.Biol)。化学。22914-22923)。在本研究中，通过化学分析、酶分析和500 MHz~1H核磁共振波谱确定了来自重复区域的一个四糖和五个六糖的结构。四糖具有典型的肝素的六硫酸盐结构。这五个六酸或七酸六糖具有共同的核心五硫酸盐结构ΔHexA(2S)α1-4GlcN(NS，6S)α1-4IdoAα/GlcAβ1-4GlcN(6S)α1-4GlcAβ1-4GlcN(NS)，并带有一个或两个额外的硫酸盐基团(Δ六糖、GlcN、IOA和GLCA分别代表4-脱氧-α-L-苏六-4-吡喃葡萄糖糖醛酸、D-氨基葡萄糖、L-艾杜糖酸和D-葡萄糖醛酸，而2S、6S和NS分别代表2-O-、6-O-和2-N-硫酸盐)。三个组分具有以下迄今未报道的结构：ΔHexA(2S)α1-4GlcN(NS，6S)α1-4GlcAβ1-4GlcN(NS，6S)α1-4GlcAβ1-4GlcN(NS，6S)，ΔHexA(2S)α1-4GlcN(NS，6S)α1-4IdoAα1-4GlcNAc(6S)α1-4GlcAβ1-4GlcN(NS，3S)，和ΔHexA(2S)α1-4GlcN(NS，6S)α1-4IdoA(2S)α1-4GlcNAc(6S)α1-4GlcAβ1-4GlcN(NS，6S)。五种六糖中有两种是从抗凝血酶III结合部位衍生的结构变体，还原末端含有3-O-磺化的GlcN，在还原的N，3-二硫化的GlcN残基上有或没有6-O-硫酸基团。另一个含有与上述七硫酸化抗凝血酶III结合部位片段相同的结构，但缺少3-O-硫酸盐基团，因此是结合部位的前体。另一种在这种亲形式的内部idoA残基上有额外的硫酸盐基团，因此可以认为已经偏离了生物合成途径中的结合位置。因此，本研究中分离的六糖包括三对重叠的结构变体，具有明显的生物合成前体-产物关系，这可能反映了结合部位的生物合成调节机制。
Porcine intestinal heparin was extensively digested with Flavobacterium heparinase and size-fractionated by gel chromatography. Subfractionation of the hexasaccharide fraction by anion exchange high pressure liquid chromatography yielded 10 fractions. Six contained oligosaccharides derived from the repeating disaccharide region, whereas four contained glycoserines from the glycosaminoglycan-protein linkage region. The latter structures were reported recently (Sugahara, K., Tsuda, H., Yoshida, K., Yamada, S., de Beer, T., and Vliegenthart, J. F. G.(1995) J. Biol. Chem. 270, 22914-22923). In this study, the structures of one tetra- and five hexasaccharides from the repeat region were determined by chemical and enzymatic analyses as well as 500-MHz 1H NMR spectroscopy. The tetrasaccharide has the hexasulfated structure typical of heparin. The five hexa- or heptasulfated hexasaccharides share the common core pentasulfated structure ΔHexA(2S)α1-4GlcN(NS,6S)α1-4IdoAα/GlcAβ1-4GlcN(6S)α1-4GlcAβ1- 4GlcN(NS) with one or two additional sulfate groups (ΔHexA, GlcN, IdoA, and GlcA represent 4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, whereas 2S, 6S, and NS stand for 2-O-, 6-O-, and 2-N-sulfate, respectively). Three components have the following hitherto unreported structures: ΔHexA(2S)α1-4GlcN(NS,6S)α1-4GlcAβ1-4GlcN(NS,6S)α1-4GlcAβ1-4GlcN(NS,6S), ΔHexA(2S)α1-4GlcN(NS,6S)α1-4IdoAα1-4GlcNAc(6S)α1-4GlcAβ1-4GlcN(NS,3S), and ΔHexA(2S)α1-4GlcN(NS,6S)α1-4IdoA(2S)α1-4GlcNAc(6S)α1-4GlcAβ1- 4GlcN(NS,6S). Two of the five hexasaccharides are structural variants derived from the antithrombin III-binding sites containing 3-O-sulfated GlcN at the reducing termini with or without a 6-O-sulfate group on the reducing N,3-disulfated GlcN residue. Another contains the structure identical to that of the above heptasulfated antithrombin III-binding site fragment but lacks the 3-O-sulfate group and therefore is a pro-form for the binding site. Another has an extra sulfate group on the internal IdoA residue of this pro-form and therefore can be considered to have diverged from the binding site in the biosynthetic pathway. Thus, the isolated hexasaccharides in this study include the three overlapping pairs of structural variants with an apparent biosynthetic precursor-product relationship, which may reflect biosynthetic regulatory mechanisms of the binding site.
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DOI：
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发表时间：
1992
期刊：
The Journal of biological chemistry
影响因子：
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DOI：
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发表时间：
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期刊：
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影响因子：
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影响因子：
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KLAGSBRUN, M