Glycosaminoglycans as naturally occuring combinatorial libraries: Spectrometry-Based strategy for characterization of anti-thrombin interaction strategy with low molecular weight heparin and heparin oligomers
Glycosaminoglycans as naturally occuring combinatorial libraries: Spectrometry-Based strategy for characterization of anti-thrombin interaction strategy with low molecular weight heparin and heparin oligomers
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DOI：
10.1021/ac0710432
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发表时间：
2007-08-15
影响因子：
7.4
通讯作者：
Kaltashov, Igor A.
中科院分区：
文献类型：
--
作者：
Abzalimov, Rinat R.;Dubin, Paul L.;Kaltashov, Igor A.
肝素是一种带密集电荷的多糖，以其抗凝活性而闻名，尽管它也调节大量其他生物过程。与合成受到独特基因模板严格控制的生物聚合物不同，肝素分子表现出显着的结构异质性，这对肝素-蛋白质相互作用的研究提出了严峻的挑战。这种分析挑战通常是通过将肝素的庞大结构简化为小分子模型来解决的。在本文中，我们描述了一种受组合化学实验方法启发的不同方法。使用聚合度固定但化学成分不同的寡肝素分子混合物（通过用细菌肝素酶对猪肠肝素进行酶消化的尺寸排阻色谱法获得的肝素六糖）以及肝素衍生药物制剂 Tinzaparin（最多 22 聚体的肝素寡糖）研究抗凝血酶 III (AT) 与类肝素的相互作用。 AT 结合剂根据蛋白质-低聚肝素缔合形成的复合物的 ESI MS 测量结果进行鉴定。此外，在 AT 存在的情况下，溶液中游离肝素寡聚物的差异消耗可用于验证结合偏好。部分变性条件下寡肝素-AT 相互作用的 ESI MS 表征允许监测蛋白质-聚阴离子结合的构象异构体特异性。这些研究中出现的模型引用了 AT 上明确的结合位点的概念，灵活的伙伴（肝素）适应该结合位点以最大化有利的分子间静电相互作用。这项研究证明了 ESI MS 作为分析工具的巨大潜力，可以在普遍接受的还原论范式之外研究高度异质糖胺聚糖与其同源蛋白质的相互作用，通过使用结构明确的同质低分子量模拟物降低肝素的内在复杂性。
Heparin is a densely charged polysaccharide, which is best known for its anticoagulant activity, although it also modulates a plethora of other biological processes. Unlike biopolymers whose synthesis is strictly controlled by a unique genetic template, heparin molecules exhibit a remarkable degree of structural heterogeneity, which poses a serious challenge for studies of heparin-protein interactions. This analytical challenge is often dealt with by reducing the enormous structural repertoire of heparin to a model small molecule. In this paper, we describe a different approach inspired by the experimental methodologies from the arsenal of combinatorial chemistry. Interaction of anti-thrombin III (AT) with heparinoids is studied using a mixture of oligoheparin molecules of fixed degree of polymerization, but varying chemical composition (heparin hexasaccharides obtained by size exclusion chromatography of an enzymatic digest of porcine intestinal heparin with bacterial heparinase), as well as a heparin-derived pharmaceutical preparation Tinzaparin (heparin oligosaccharides up to a 22-mer). AT binders are identified based on the results of ESI MS measurements of complexes formed by protein-oligoheparin association. Additionally, differential depletion of free heparin oligomers in solution in the presence of AT is used to verify the binding preferences. ESI MS characterization of oligoheparin-AT interaction under partially denaturing conditions allowed the conformer specificity of the protein-polyanion binding to be monitored. A model emerging from these studies invokes the notion of a well-defined binding site on AT, to which a flexible partner (heparin) adapts to maximize favorable intermolecular electrostatic interactions. This study demonstrates the enormous potential of ESI MS as an analytical tool to study the interactions of highly heterogeneous glycosaminoglycans with their cognate proteins outside of the commonly accepted reductionist paradigm, which reduces the intrinsic complexity of heparin by using structurally defined homogeneous low molecular weight mimetics.