Heparanase as an Additional Tool for Detecting Structural Peculiarities of Heparin Oligosaccharides
Heparanase as an Additional Tool for Detecting Structural Peculiarities of Heparin Oligosaccharides
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DOI：
10.3390/molecules24234403
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发表时间：
2019-12-01
期刊：
影响因子：
4.6
通讯作者：
Naggi, Annamaria
中科院分区：
文献类型：
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作者：
Alekseeva, Anna;Urso, Elena;Naggi, Annamaria
由于肝素和低分子肝素(LMWH)的生物学特性，在阐明其微观结构和发现新的结构特性方面不断取得进展至关重要。需要制定有效的战略，以监测制造过程和评估更具限制性的规范，如目前的管理机构所强加的那样。因此，我们应用一种有效的基于肝素酶的策略来确定达特肝素的两个主要异构体八糖的结构，并研究牛粘膜肝素的抗凝血酶结合区(ATBR)产生的四糖。肝素酶，特别是当与其他样品制备方法(如大小排除、亲和层析、肝素酶解聚)相结合时，被证明是提供有关肝素结构特性的相关信息的有力工具。应用的方法提供了直接证据，证明在非还原末端含有葡萄糖糖醛酸-氨基葡萄糖-3-O-硫酸盐的低聚物代表达肝素的重要结构特征。当扩展到与ATBR相关的牛肝素四聚体时，基于肝素酶的方法允许阐明尚未报道的小序列的结构。鉴于监管机构和制造商对开发低分子肝素仿制药以及牛肝素作为替代来源的兴趣日益增长，所获得的结果具有重要意义。
Due to the biological properties of heparin and low-molecular-weight heparin (LMWH), continuous advances in elucidation of their microheterogeneous structure and discovery of novel structural peculiarities are crucial. Effective strategies for monitoring manufacturing processes and assessment of more restrictive specifications, as imposed by the current regulatory agencies, need to be developed. Hereby, we apply an efficient heparanase-based strategy to assert the structure of two major isomeric octasaccharides of dalteparin and investigate the tetrasaccharides arising from antithrombin binding region (ATBR) of bovine mucosal heparin. Heparanase, especially when combined with other sample preparation methods (e.g., size exclusion, affinity chromatography, heparinase depolymerization), was shown to be a powerful tool providing relevant information about heparin structural peculiarities. The applied approach provided direct evidence that oligomers bearing glucuronic acid-glucosamine-3-O-sulfate at their nonreducing end represent an important structural signature of dalteparin. When extended to ATBR-related tetramers of bovine heparin, the heparanase-based approach allowed for elucidation of the structure of minor sequences that have not been reported yet. The obtained results are of high importance in the view of the growing interest of regulatory agencies and manufacturers in the development of low-molecular-weight heparin generics as well as bovine heparin as alternative source.