Antithrombin-binding octasaccharides and role of extensions of the active pentasaccharide sequence in the specificity and strength of interaction -: Evidence for very high affinity induced by an unusual Glucuronic acid residue
Antithrombin-binding octasaccharides and role of extensions of the active pentasaccharide sequence in the specificity and strength of interaction -: Evidence for very high affinity induced by an unusual Glucuronic acid residue
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DOI：
10.1074/jbc.m801102200
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发表时间：
2008-09-26
影响因子：
4.8
通讯作者：
Viskov, Christian
中科院分区：
文献类型：
--
作者：
Guerrini, Marco;Guglieri, Sara;Viskov, Christian
低分子量肝素（LMWH）的抗血栓形成活性在很大程度上与抗凝血酶（AT）结合五糖序列阿加 *IA（GlcN（NAc/NS），（6S）-GlcA-GlcN（NS），（3，6S）-IdoUA（2S）-GlcN（NS，6S））相关。预期阿加 *IA序列沿着LMWH链的位置也会影响与AT的结合。本研究旨在研究阿加 *IA序列两侧不同二糖延伸的结构和分子构象在调节AT亲和力中的作用。本研究选择了四种高纯度的八糖，分别通过分子排阻层析、高压液相层析和AT-亲和层析从低分子肝素依诺肝素中分离得到。所有四种八糖在其非还原性末端终止于4，5-不饱和糖醛酸残基（Delta U）。在两种八糖中，阿加 *IA在还原端延长了单位IdoUA（2S）-GlcN（NS，6S）（OCTA- 1）或IdoUA-GlcN（NAc），（6S）（OCTA-2）。在另外两种八糖（OCTA-3和OCTA-4）中，阿加 *IA在非还原侧分别被GlcN（NS），（6S）-IdoUA和GlcN（NS），（6S）-GlcA延长。延伸增加了八糖相对于五糖阿加 *IA对AT的亲和力，这也通过荧光滴定证实。二维NMR和对接研究清楚地表明，虽然阿加 *IA序列的延长基本上不改变阿加 *IA片段的结合构象，但延长促进了与蛋白质的额外接触.应该注意的是，如以前未报道的，OCTA-4中位于阿加 *IA序列之前的不寻常的GlcA残基相对于其含IdoUA的同源物OCTA-3诱导了对AT的亲和力意外增加1个数量级.发现这样的残基将其两个羟基定位在蛋白质残基的近距离处。因此，除了已确立的离子相互作用外，非离子相互作用可能有助于增强寡糖AT复合物。
The antithrombotic activity of low molecular weight heparins (LMWHs) is largely associated with the antithrombin ( AT)binding pentasaccharide sequence AGA*IA (GlcN(NAc/NS),(6S)-GlcA-GlcN(NS),(3,6S)-IdoUA(2S)-GlcN(NS,6S)). The location of the AGA*IA sequences along the LMWH chains is also expected to influence binding to AT. This study was aimed at investigating the role of the structure and molecular conformation of different disaccharide extensions on both sides of the AGA*IA sequence in modulating the affinity for AT. Four high purity octasaccharides isolated by size exclusion chromatography, high pressure liquid chromatography, and AT-affinity chromatography from the LMWH enoxaparin were selected for the study. All the four octasaccharides terminate at their nonreducing end with 4,5-unsaturated uronic acid residues (Delta U). In two octasaccharides, AGA*IA was elongated at the reducing end by units IdoUA(2S)-GlcN(NS,6S) (OCTA- 1) or IdoUA-GlcN(NAc),(6S) (OCTA-2). In the other two octasaccharides (OCTA-3 and OCTA-4), AGA*IA was elongated at the nonreducing side by units GlcN(NS),(6S)-IdoUA and GlcN(NS),(6S)-GlcA, respectively. Extensions increased the affinity for AT of octasaccharides with respect to pentasaccharide AGA*IA, as also confirmed by fluorescence titration. Two- dimensional NMR and docking studies clearly indicated that, although elongation of the AGA*IA sequence does not substantially modify the bound conformation of the AGA*IA segment, extensions promote additional contacts with the protein. It should be noted that, as not previously reported, the unusual GlcA residue that precedes the AGA*IA sequence in OCTA-4 induced an unexpected 1 order of magnitude increase in the affinity to AT with respect to its IdoUA- containing homolog OCTA-3. Such a residue was found to orientate its two hydroxyl groups at close distance to residues of the protein. Besides the well established ionic interactions, nonionic interactions may thus contribute to strengthen oligosaccharideAT complexes.