Beijing Institute of Biotechnology, Beijing 100071, China.
Pharmaceutical College, Henan University, Kaifeng 475001, China.
Toxins (Basel). 2023 Mar 6;15(3):200. doi: 10.3390/toxins15030200.
Botulinum neurotoxins (BoNTs) can cause nerve paralysis syndrome in mammals and other vertebrates. BoNTs are the most toxic biotoxins known and are classified as Class A biological warfare agents. BoNTs are mainly divided into seven serotypes A-G and new neurotoxins BoNT/H and BoNT/X, which have similar functions. BoNT proteins are 150 kDa polypeptide consisting of two chains and three domains: the light chain (L, catalytic domain, 50 kDa) and the heavy chain (H, 100 kDa), which can be divided into an N-terminal membrane translocation domain (HN, 50 kDa) and a C-terminal receptor binding domain (Hc, 50 kDa). In current study, we explored the immunoprotective efficacy of each functional molecule of BoNT/F and the biological characteristics of the light chain-heavy N-terminal domain (FL-HN). The two structure forms of FL-HN (i.e., FL-HN-SC: single chain FL-HN and FL-HN-DC: di-chain FL-HN) were developed and identified. FL-HN-SC could cleave the vesicle associated membrane protein 2 (VAMP2) substrate protein in vitro as FL-HN-DC or FL. While only FL-HN-DC had neurotoxicity and could enter neuro-2a cells to cleave VAMP2. Our results showed that the FL-HN-SC had a better immune protection effect than the Hc of BoNT/F (FHc), which indicated that L-HN-SC, as an antigen, provided the strongest protective effects against BoNT/F among all the tested functional molecules. Further in-depth research on the different molecular forms of FL-HN suggested that there were some important antibody epitopes at the L-HN junction of BoNT/F. Thus, FL-HN-SC could be used as a subunit vaccine to replace the FHc subunit vaccine and/or toxoid vaccine, and to develop antibody immune molecules targeting L and HN domains rather than the FHc domain. FL-HN-DC could be used as a new functional molecule to evaluate and explore the structure and activity of toxin molecules. Further exploration of the biological activity and molecular mechanism of the functional FL-HN or BoNT/F is warranted.
肉毒神经毒素（BoNTs）可引起哺乳动物和其他脊椎动物的神经麻痹综合征。BoNTs 是已知最毒的生物毒素，被归类为 A 类生物战剂。BoNTs 主要分为 7 种血清型 A-G 和新型神经毒素 BoNT/H 和 BoNT/X，它们具有相似的功能。BoNT 蛋白是由两条链和三个结构域组成的 150 kDa 多肽：轻链（L，催化结构域，50 kDa）和重链（H，100 kDa），可分为 N 端膜转位结构域（HN，50 kDa）和 C 端受体结合结构域（Hc，50 kDa）。在本研究中，我们探索了 BoNT/F 每种功能分子的免疫保护效力和轻链-重链 N 端结构域（FL-HN）的生物学特性。开发并鉴定了 FL-HN 的两种结构形式（即 FL-HN-SC：单链 FL-HN 和 FL-HN-DC：双链 FL-HN）。FL-HN-SC 可在体外切割囊泡相关膜蛋白 2（VAMP2）底物蛋白，与 FL-HN-DC 或 FL 相同。然而，只有 FL-HN-DC 具有神经毒性，可进入神经-2a 细胞切割 VAMP2。结果表明，FL-HN-SC 的免疫保护效果优于 BoNT/F 的 Hc（FHc），这表明 L-HN-SC 作为抗原，在所有测试的功能分子中，对 BoNT/F 提供了最强的保护作用。对 FL-HN 不同分子形式的进一步深入研究表明，BoNT/F 的 L-HN 连接处存在一些重要的抗体表位。因此，FL-HN-SC 可作为亚单位疫苗替代 FHc 亚单位疫苗和/或类毒素疫苗，并开发针对 L 和 HN 结构域而不是 FHc 结构域的抗体免疫分子。FL-HN-DC 可作为一种新的功能分子，用于评估和探索毒素分子的结构和活性。进一步探索功能 FL-HN 或 BoNT/F 的生物学活性和分子机制是必要的。
