Guerrini Marco, Guglieri Sara, Casu Benito, Torri Giangiacomo, Mourier Pierre, Boudier Christian, Viskov Christian
G. Ronzoni Institute for Chemical and Biochemical Research, via G. Colombo 81, 20133 Milan, Italy.
J Biol Chem. 2008 Sep 26;283(39):26662-75. doi: 10.1074/jbc.M801102200. Epub 2008 Jul 17.
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 (DeltaU). 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 oligosaccharide-AT complexes.
低分子量肝素（LMWHs）的抗血栓活性很大程度上与抗凝血酶（AT）结合五糖序列AGA()IA（GlcN(NAc/NS,6S)-GlcA-GlcN(NS,3,6S)-IdoUA(2S)-GlcN(NS,6S)）相关。AGA()IA序列在LMWH链上的位置预计也会影响与AT的结合。本研究旨在调查AGA()IA序列两侧不同二糖延伸结构和分子构象在调节对AT亲和力方面的作用。通过尺寸排阻色谱、高压液相色谱和AT亲和色谱从低分子量肝素依诺肝素中分离出四种高纯度八糖用于该研究。所有四种八糖在其非还原端均以4,5-不饱和糖醛酸残基（ΔU）终止。在两种八糖中，AGA()IA在还原端通过IdoUA(2S)-GlcN(NS,6S)（OCTA-1）或IdoUA-GlcN(NAc,6S)（OCTA-2）单元延长。在另外两种八糖（OCTA-3和OCTA-4）中，AGA()IA分别在非还原侧通过GlcN(NS,6S)-IdoUA和GlcN(NS,6S)-GlcA单元延长。荧光滴定也证实，与五糖AGA()IA相比，延伸增加了八糖对AT的亲和力。二维核磁共振和对接研究清楚地表明，虽然AGA()IA序列的延长并没有实质性改变AGA()IA片段的结合构象，但延伸促进了与蛋白质的额外接触。应当指出，如之前未报道的那样，OCTA-4中AGA(*)IA序列之前的异常GlcA残基相对于其含IdoUA的同系物OCTA-3，使对AT的亲和力意外增加了1个数量级。发现该残基将其两个羟基定位在与蛋白质残基近距离处。除了已确立的离子相互作用外，非离子相互作用可能因此有助于加强寡糖-AT复合物。
