Brown Spencer, Kuberan Balagurunathan
Department of Medicinal Chemistry, University of Utah, 30 South 2000 East, Skaggs #307, Salt Lake City, UT, 84112, USA.
Methods Mol Biol. 2015;1229:21-9. doi: 10.1007/978-1-4939-1714-3_3.
Glycosaminoglycans (GAG) are most commonly isolated as large polymers from various animal origins, the functional units of which are oligosaccharides, which bind their target proteins to induce conformational changes, compete with other ligands, or facilitate the formation of signaling complexes. One example, the extensively studied heparin pentasaccharide sequence-which binds antithrombin-III, inducing a conformational change that increases its serpin protease activity by 1,000-fold-is unique in that no other specific GAG-protein structure-function relations have been described to the same degree. Thus, production of heparan sulfate (HS) oligosaccharides is critical for obtaining specific structural information regarding the binding interactions of GAG and their ligands (typically proteins). Purely synthetic methods of oligosaccharide synthesis are possible, but the cost, time requirement, and difficulty of their preparation prohibit library synthesis in significant amounts. Herein, the use of bacterial heparin lyases for the production of HS oligosaccharides via enzymatic depolymerization of HS polymers is discussed. The separation and purification of these oligosaccharides by liquid chromatography are also described.
糖胺聚糖（GAG）最常作为来自各种动物来源的大聚合物分离出来，其功能单元是寡糖，寡糖与它们的靶蛋白结合以诱导构象变化、与其他配体竞争或促进信号复合物的形成。一个例子是经过广泛研究的肝素五糖序列，它与抗凝血酶III结合，诱导构象变化，使其丝氨酸蛋白酶抑制剂活性增加1000倍，其独特之处在于尚未有其他特定的GAG-蛋白质结构-功能关系被如此详细地描述。因此，硫酸乙酰肝素（HS）寡糖的生产对于获得有关GAG及其配体（通常是蛋白质）结合相互作用的特定结构信息至关重要。寡糖合成的纯化学合成方法是可行的，但其成本、时间需求以及制备难度阻碍了大量文库的合成。本文讨论了使用细菌肝素裂解酶通过HS聚合物的酶促解聚来生产HS寡糖。还描述了通过液相色谱对这些寡糖进行分离和纯化的方法。
