Fang Lei, Zheng Fang, Zhan Chang-Guo
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, USA.
Mol Biosyst. 2014 Feb;10(2):348-54. doi: 10.1039/c3mb70313d.
Human butyrylcholinesterase (BChE) and its mutants have shown great potential in treating cocaine overdose and addiction. In order to effectively suppress cocaine reward in the brain for a long period of time after an exogenous cocaine hydrolase administration, the therapeutic enzyme should have not only a high catalytic efficiency against cocaine, but also a sufficiently long circulation time. It has been known that PEGylation (covalent attachment of polyethylene glycol) modification of a therapeutic protein can prolong the biological half-life of the protein without affecting its biological function. However, the asparagine-linked glycans on the surface of glycosylated BChE may interfere with the PEGylation modification. In this study, we built a three-dimensional (3D) model of glycosylated human BChE to investigate the influence of glycans on the PEGylation modification. Glycans did not change the overall stability of the BChE structure, but could increase the flexibility of some local structures. For further evaluating the accessibility of the PEGylation reaction sites, particularly lysine residues, on the protein surface, we calculated the Solvent Accessible Surface Areas (SASAs) of these residues. The results indicate that some lysine residues show a significant decrease in SASA due to the direct or indirect influence of their surrounding glycans. The results also indicate that PEGylation reaction agents with smaller functional groups could have a better chance to react with lysine residues. This investigation provides a structural basis for rational engineering of human BChE and its mutants as therapeutic candidates.
人丁酰胆碱酯酶（BChE）及其突变体在治疗可卡因过量和成瘾方面显示出巨大潜力。为了在外源性可卡因水解酶给药后长时间有效抑制大脑中的可卡因奖赏效应，治疗性酶不仅应具有对可卡因的高催化效率，还应具有足够长的循环时间。已知对治疗性蛋白质进行聚乙二醇化（共价连接聚乙二醇）修饰可以延长蛋白质的生物半衰期而不影响其生物学功能。然而，糖基化BChE表面的天冬酰胺连接聚糖可能会干扰聚乙二醇化修饰。在本研究中，我们构建了糖基化人BChE的三维（3D）模型，以研究聚糖对聚乙二醇化修饰的影响。聚糖并未改变BChE结构的整体稳定性，但可增加一些局部结构的灵活性。为了进一步评估蛋白质表面聚乙二醇化反应位点（特别是赖氨酸残基）的可及性，我们计算了这些残基的溶剂可及表面积（SASA）。结果表明，一些赖氨酸残基由于其周围聚糖的直接或间接影响，其SASA显著降低。结果还表明，具有较小官能团的聚乙二醇化反应剂与赖氨酸残基反应的机会可能更好。本研究为合理设计人BChE及其突变体作为治疗候选物提供了结构基础。
