Ernst S, Rhomberg A J, Biemann K, Sasisekharan R
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4182-7. doi: 10.1073/pnas.95.8.4182.
Heparinase I from Flavobacterium heparinum has important uses for elucidating the complex sequence heterogeneity of heparin-like glycosaminoglycans (HLGAGs). Understanding the biological function of HLGAGs has been impaired by the limited methods for analysis of pure or mixed oligosaccharide fragments. Here, we use methodologies involving MS and capillary electrophoresis to investigate the sequence of events during heparinase I depolymerization of HLGAGs. In an initial step, heparinase I preferentially cleaves exolytically at the nonreducing terminal linkage of the HLGAG chain, although it also cleaves internal linkages at a detectable rate. In a second step, heparinase I has a strong preference for cleaving the same substrate molecule processively, i.e., to cleave the next site toward the reducing end of the HLGAG chain. Computer simulation showed that the experimental results presented here from analysis of oligosaccharide degradation were consistent with literature data for degradation of polymeric HLGAG by heparinase I. This study presents direct evidence for a predominantly exolytic and processive mechanism of depolymerization of HLGAG by heparinase I.
来自肝素黄杆菌的肝素酶I在阐明类肝素糖胺聚糖（HLGAGs）复杂的序列异质性方面具有重要用途。由于用于分析纯或混合寡糖片段的方法有限，对HLGAGs生物学功能的理解受到了阻碍。在此，我们使用涉及质谱和毛细管电泳的方法来研究HLGAGs被肝素酶I解聚过程中的一系列事件。在第一步中，肝素酶I优先从HLGAG链的非还原末端连接处进行外切裂解，不过它也能以可检测的速率裂解内部连接。在第二步中，肝素酶I强烈倾向于对同一底物分子进行连续性裂解，即朝着HLGAG链的还原端裂解下一个位点。计算机模拟表明，这里通过寡糖降解分析得出的实验结果与文献中关于肝素酶I对聚合HLGAG降解的数据一致。本研究为肝素酶I对HLGAG解聚主要是外切和连续性机制提供了直接证据。
