Yamada S, Murakami T, Tsuda H, Yoshida K, Sugahara K
Department of Biochemistry, Kobe Pharmaceutical University, Japan.
J Biol Chem. 1995 Apr 14;270(15):8696-705.
Eleven tetrasaccharides were isolated from the repeating disaccharide region of porcine intestinal heparin after strong digestion with Flavobacterium heparinase. Their structures were determined by composition analysis, enzymatic analysis, and 1H NMR spectroscopy. Nine of them have the common tetrasaccharide backbone, delta HexA alpha 1-4GlcN alpha 1-4IdoA alpha 1-4GlcN, where delta HexA and IdoA represent 4,5-unsaturated hexuronic acid and L-iduronic acid, respectively, and their structural variations are based upon the positions of sulfate groups. The nine compounds include one hexasulfated, three pentasulfated and five tetrasulfated compounds, and four of them have not been isolated previously as discrete structures. The other two of the 11 tetrasaccharides have the following hitherto unreported structures with novel glucuronate 2-O-sulfate at the internal position: delta HexA(2-sulfate) alpha 1- 4GlcN(N,6-disulfate) alpha 1-4GlcA(2-sulfate) beta 1-4GlcN(N-sulfate) and delta HexA(2-sulfate) alpha 1-4GlcN(N,6-disulfate) alpha 1-4GlcA(2-sulfate) beta 1-4GlcN(N,6-disulfate). Thus, 2-O-sulfated glucuronate in the highly sulfated tetrasaccharide structures typical of heparin has been demonstrated. The former and the latter tetrasaccharides account for 0.31 and 0.32% (w/w) of the starting heparin, respectively. Their yield, however, is an underestimation, since these tetrasaccharide structures in longer sequences will be degraded by heparinase. Although the latter tetrasaccharide described above was unexpectedly cleaved by heparinase into two disaccharide units, the former was not degraded by the enzyme most likely due to the lack of the 6-O-sulfate group on the GlcN residue at the reducing terminus. The results indicate its capability of catalyzing both anti and syn elimination, a property shared by heparitinases I and II and chondroitinase ABC. Both tetrasaccharides were degraded into disaccharides by heparitinase II. Therefore, it is necessary to reevaluate the disaccharide composition of heparin/heparan sulfate or oligosaccharide structures, which were previously determined after heparinase or heparitinase II digestion. It is no longer possible to conclude that the 2-O-sulfated unsaturated uronic acid residues obtained from heparin/heparan sulfate by lyase digestions are always derived from iduronate 2-O-sulfate residues in the original polymer. It is quite possible that the novel glucuronate 2-O-sulfate structure in the highly sulfated region of heparin is involved in some of the biological activities of heparin.
用肝素黄杆菌进行强烈消化后，从猪肠道肝素的重复二糖区域分离出11种四糖。通过组成分析、酶分析和1H核磁共振光谱确定了它们的结构。其中9种具有共同的四糖主链，δHexAα1-4GlcNα1-4IdoAα1-4GlcN，其中δHexA和IdoA分别代表4,5-不饱和己糖醛酸和L-艾杜糖醛酸，它们的结构变化基于硫酸酯基团的位置。这9种化合物包括1种六硫酸化、3种五硫酸化和5种四硫酸化化合物，其中4种以前未作为离散结构分离出来。11种四糖中的另外2种具有以下迄今未报道的结构，内部位置有新型葡萄糖醛酸2-O-硫酸酯：δHexA(2-硫酸酯)α1-4GlcN(N,6-二硫酸酯)α1-4GlcA(2-硫酸酯)β1-4GlcN(N-硫酸酯)和δHexA(2-硫酸酯)α1-4GlcN(N,6-二硫酸酯)α1-4GlcA(2-硫酸酯)β1-4GlcN(N,6-二硫酸酯)。因此，已证明在肝素典型的高度硫酸化四糖结构中存在2-O-硫酸化葡萄糖醛酸。前一种和后一种四糖分别占起始肝素的0.31%和0.32%（w/w）。然而，它们的产率被低估了，因为较长序列中的这些四糖结构会被肝素酶降解。尽管上述后一种四糖意外地被肝素酶切割成两个二糖单元，但前一种四糖很可能由于还原端GlcN残基上缺乏6-O-硫酸酯基团而未被该酶降解。结果表明它具有催化反式和顺式消除的能力，这是肝素酶I和II以及软骨素酶ABC共有的特性。两种四糖都被肝素酶II降解为二糖。因此，有必要重新评估肝素/硫酸乙酰肝素或寡糖结构的二糖组成，这些组成以前是在肝素酶或肝素酶II消化后确定的。不再能够得出这样的结论，即通过裂解酶消化从肝素/硫酸乙酰肝素获得的2-O-硫酸化不饱和糖醛酸残基总是来自原始聚合物中的艾杜糖醛酸2-O-硫酸酯残基。肝素高度硫酸化区域中的新型葡萄糖醛酸2-O-硫酸酯结构很可能参与了肝素的某些生物学活性。
