Bacterial Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
Chem Biol Interact. 2013 Mar 25;203(1):24-9. doi: 10.1016/j.cbi.2012.09.018. Epub 2012 Oct 6.
Human serum butyrylcholinesterase (HuBChE) is currently the most suitable bioscavenger for the prophylaxis of highly toxic organophosphate (OP) nerve agents. A dose of 200mg of HuBChE is envisioned as a prophylactic treatment that can protect humans from an exposure of up to 2 × LD50 of soman. The limited availability and administration of multiple doses of this stoichiometric bioscavenger make this pretreatment difficult. Thus, the goal of this study was to produce a smaller enzymatically active HuBChE polypeptide (HBP) that could bind to nerve agents with high affinity thereby reducing the dose of enzyme. Studies have indicated that the three-dimensional structure and the domains of HuBChE (acyl pocket, lip of the active center gorge, and the anionic substrate-binding domain) that are critical for the binding of substrate are also essential for the selectivity and binding of inhibitors including OPs. Therefore, we designed three HBPs by deleting some N- and C-terminal residues of HuBChE by maintaining the folds of the active site core that includes the three active site residues (S198, E325, and H438). HBP-4 that lacks 45 residues from C-terminus but known to have BChE activity was used as a control. The cDNAs for the HBPs containing signal sequences were synthesized, cloned into different mammalian expression vectors, and recombinant polypeptides were transiently expressed in different cell lines. No BChE activity was detected in the culture media of cells transfected with any of the newly designed HBPs, and the inactive polypeptides remained inside the cells. Only enzymatically active HBP-4 was secreted into the culture medium. These results suggest that residues at the N- and C-termini are required for the folding and/or maintenance of HBP into an active stable, conformation.
人血清丁酰胆碱酯酶（HuBChE）是目前最适合用于预防高毒性有机磷（OP）神经毒剂的生物清除剂。预计 200mg 的 HuBChE 剂量可作为预防治疗，能保护人类免受高达 2×沙林 LD50 的暴露。这种化学计量型生物清除剂的供应有限且需要多次给药，使得这种预处理变得困难。因此，本研究的目的是产生更小的具有酶活性的 HuBChE 多肽（HBP），它可以与神经毒剂高亲和力结合，从而减少酶的剂量。研究表明，HuBChE 的三维结构和结构域（酰基口袋、活性中心峡谷的唇缘和阴离子底物结合域）对于结合底物至关重要，对于抑制剂（包括 OP）的选择性和结合也是必不可少的。因此，我们通过删除 HuBChE 的一些 N-和 C-末端残基设计了三种 HBP，同时保持包括三个活性位点残基（S198、E325 和 H438）在内的活性位点核心的折叠。缺乏 45 个 C-末端残基但已知具有 BChE 活性的 HBP-4 被用作对照。含有信号序列的 HBPs 的 cDNA 被合成，克隆到不同的哺乳动物表达载体中，重组多肽在不同的细胞系中瞬时表达。用任何新设计的 HBPs 转染的细胞的培养基中均未检测到 BChE 活性，无活性的多肽仍留在细胞内。只有具有酶活性的 HBP-4 分泌到培养基中。这些结果表明，N-和 C-末端的残基对于 HBP 的折叠和/或维持为一种活性稳定构象是必需的。
