Li He, Schopfer Lawrence M, Masson Patrick, Lockridge Oksana
Eppley Institute and Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA.
Biochem J. 2008 Apr 15;411(2):425-32. doi: 10.1042/BJ20071551.
BChE (butyrylcholinesterase) protects the cholinergic nervous system from organophosphorus nerve agents by scavenging these toxins. Recombinant human BChE produced from transgenic goat to treat nerve agent intoxication is currently under development. The therapeutic potential of BChE relies on its ability to stay in the circulation for a prolonged period, which in turn depends on maintaining tetrameric quaternary configuration. Native human plasma BChE consists of 98% tetramers and has a half-life (t((1/2))) of 11-14 days. BChE in the neuromuscular junctions and the central nervous system is anchored to membranes through interactions with ColQ (AChE-associated collagen tail protein) and PRiMA (proline-rich membrane anchor) proteins containing proline-rich domains. BChE prepared in cell culture is primarily monomeric, unless expressed in the presence of proline-rich peptides. We hypothesized that a poly-proline peptide is an intrinsic component of soluble plasma BChE tetramers, just as it is for membrane-bound BChE. We found that a series of proline-rich peptides was released from denatured human and horse plasma BChE. Eight peptides, with masses from 2072 to 2878 Da, were purified by HPLC and sequenced by electrospray ionization tandem MS and Edman degradation. All peptides derived from the same proline-rich core sequence PSPPLPPPPPPPPPPPPPPPPPPPPLP (mass 2663 Da) but varied in length at their N- and C-termini. The source of these peptides was identified through database searching as RAPH1 [Ras-associated and PH domains (pleckstrin homology domains)-containing protein 1; lamellipodin, gi:82581557]. A proline-rich peptide of 17 amino acids derived from lamellipodin drove the assembly of human BChE secreted from CHO (Chinese-hamster ovary) cells into tetramers. We propose that the proline-rich peptides organize the 4 subunits of BChE into a 340 kDa tetramer, by interacting with the C-terminal BChE tetramerization domain.
丁酰胆碱酯酶（BChE）通过清除这些毒素来保护胆碱能神经系统免受有机磷神经毒剂的侵害。目前正在研发由转基因山羊生产的重组人BChE，用于治疗神经毒剂中毒。BChE的治疗潜力依赖于其在循环系统中长时间停留的能力，而这又取决于维持四聚体四级结构。天然人血浆BChE由98%的四聚体组成，半衰期（t(1/2)）为11 - 14天。神经肌肉接头和中枢神经系统中的BChE通过与含有富含脯氨酸结构域的ColQ（乙酰胆碱酯酶相关胶原尾蛋白）和PRiMA（富含脯氨酸的膜锚定蛋白）蛋白相互作用而锚定在膜上。在细胞培养中制备的BChE主要是单体，除非在富含脯氨酸的肽存在下表达。我们推测富含脯氨酸的肽是可溶性血浆BChE四聚体的固有组成部分，就像它对于膜结合型BChE一样。我们发现一系列富含脯氨酸的肽从变性的人及马血浆BChE中释放出来。通过高效液相色谱法纯化了8种质量在2072至2878 Da之间的肽，并通过电喷雾电离串联质谱和埃德曼降解法进行测序。所有肽均源自相同的富含脯氨酸的核心序列PSPPLPPPPPPPPPPPPPPPPPPPPLP（质量2663 Da），但在其N端和C端长度不同。通过数据库搜索确定这些肽的来源为RAPH1 [含Ras相关和PH结构域（普列克底物蛋白同源结构域）的蛋白1；片状伪足蛋白，gi:82581557]。源自片状伪足蛋白的17个氨基酸的富含脯氨酸的肽促使从中国仓鼠卵巢（CHO）细胞分泌的人BChE组装成四聚体。我们提出富含脯氨酸的肽通过与BChE C端四聚化结构域相互作用，将BChE的4个亚基组织成340 kDa的四聚体。
