Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 221 Maryland Hall, 3400 N. Charles St. Baltimore, Maryland, 21218, USA.
Department of Materials Science and Engineering, Johns Hopkins University, 204C Shaffer Hall, 3400 N. Charles St. Baltimore, Maryland, 21218, USA.
Biotechnol J. 2018 Mar;13(3):e1700479. doi: 10.1002/biot.201700479. Epub 2017 Nov 17.
Human butyrylcholinesterase (BChE), predominantly tetramers with a residence time of days, offers the potential to scavenge organophosphorus pesticides and chemical warfare agents. Efficient assembly of human BChE into tetramers requires an association with proline-rich peptide chaperones. In this study, the incorporation of different proline-rich peptide chaperones into BChE is investigated computationally and experimentally. First, the authors applied molecular dynamic (MD) simulations to interpret the interactions between proline-rich chaperones with human BChE tetramer domains. The P24 chaperone which contains 24 prolines, promoted the association of BChE tetramer with a 74% simulated helicity of BChE subunits, whereas the control without chaperone and BChE with an 8-proline chaperone (P8) complex exhibited 55.8 and 60.6% predicted helicity, respectively. The interaction of proline-rich chaperones with BChE subunits (B-P) provides a conduit to facilitate the interactions between BChE subunits (B-B) of the complex, which is mainly attributed to hydrophobic interactions and hydrogen-bond binding. Experimental assessment of these two proline-rich chaperones plus a 14-proline chaperone (P14) was performed and confirmed that P24 has superior capability to facilitate recombinant BChE (rBChE) tetramerization with >60% rBChE tetramer in P24-transfected rBChE cells, whereas P14- and P8-transfected rBChE cells had 44 and 33% rBChE tetramer, respectively. The rBChE control had 14% tetramer. Finally, we developed a stable rBChE tetramer expression system in CHO cells by enriching P24 expression in rBChE expressing cells. Overall, our simulations provided a design concept for identifying proline-rich peptides that promote the rBChE tetramerization in CHO cells.
人丁酰胆碱酯酶（BChE）主要以具有数天停留时间的四聚体形式存在，具有清除有机磷农药和化学战剂的潜力。BChE 高效组装成四聚体需要与富含脯氨酸的肽伴侣结合。在这项研究中，通过计算和实验研究了不同富含脯氨酸的肽伴侣在 BChE 中的掺入。首先，作者应用分子动力学（MD）模拟来解释富含脯氨酸的伴侣与人类 BChE 四聚体结构域之间的相互作用。含有 24 个脯氨酸的 P24 伴侣促进了 BChE 四聚体与 BChE 亚基模拟 74%螺旋度的结合，而没有伴侣的对照物和含有 8 个脯氨酸伴侣（P8）的复合物分别表现出 55.8%和 60.6%的预测螺旋度。富含脯氨酸的伴侣与 BChE 亚基（B-P）的相互作用提供了一个通道，以促进复合物中 BChE 亚基（B-B）之间的相互作用，这主要归因于疏水相互作用和氢键结合。对这两种富含脯氨酸的伴侣以及 14 个脯氨酸伴侣（P14）进行了实验评估，并证实 P24 具有促进重组 BChE（rBChE）四聚化的卓越能力，在 P24 转染的 rBChE 细胞中超过 60%的 rBChE 为四聚体，而 P14 和 P8 转染的 rBChE 细胞中 rBChE 四聚体分别为 44%和 33%。rBChE 对照物的四聚体为 14%。最后，我们通过在表达 rBChE 的细胞中富集 P24 表达，开发了 CHO 细胞中稳定的 rBChE 四聚体表达系统。总的来说，我们的模拟为识别在 CHO 细胞中促进 rBChE 四聚化的富含脯氨酸的肽提供了一个设计概念。
