Monash Venom Group, Department of Pharmacology, Biomedical Discovery Institute, Monash University, Clayton, VIC 3800, Australia; Department of Emergency Medicine, The First Affiliated Hospital of Guangzhou Medical University, 151 Yanjiang Rd, Guangzhou 510120, Guangdong, PR China.
Monash Venom Group, Department of Pharmacology, Biomedical Discovery Institute, Monash University, Clayton, VIC 3800, Australia.
Biochem Pharmacol. 2020 Nov;181:114059. doi: 10.1016/j.bcp.2020.114059. Epub 2020 May 28.
The Chinese Cobra (Naja atra) is an elapid snake of major medical importance in southern China. Although previous studies have shown that postsynaptic neurotoxins account for 11-23% of N. atra venom, envenomed patients do not display marked signs of neurotoxicity. We have previously shown that the lack of clinical neurotoxicity following snake envenoming by some species with 'neurotoxic' venoms may be related to the high prevalence of short-chain postsynaptic neurotoxins in these venoms. In this study, we describe the isolation and characterization of α-Elapitoxin-Na1a (α-EPTX-Na1a; 6949 Da), a short-chain postsynaptic neurotoxin, which accounts for approximately 9% of N. atra crude venom. α-EPTX-Na1a (30-300 nM) produced concentration-dependent inhibition of indirect-twitches, with a t value of 17 ± 2 min at 300 nM, and abolished contractile responses to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The prior addition of either Chinese N. atra monovalent antivenom (0.3 U/ml) or Australian polyvalent snake antivenom (2.4 U/ml), prevented the in vitro neurotoxic effects of α-EPTX-Na1a (30 nM). Addition of each of these antivenoms at the t time point partially reversed the in vitro neurotoxicity caused by α-EPTX-Na1a (30 nM). The inhibition of indirect twitches by α-EPTX-Na1a (30 nM) was not reversed by repeatedly washing the tissue. α-EPTX-Na1a displayed pseudo-irreversible antagonism of concentration-response curves to carbachol with a pA value of 8.21. De novo protein sequencing of α-EPTX-Na1a revealed a typical short-chain postsynaptic neurotoxin profile of 62 amino acids which shared >98% amino acid sequence similarity with short-chain postsynaptic neurotoxins from other Naja species. When compared to short-chain neurotoxins isolated from cobras in China, α-EPTX-Na1a contained novel residues K47Q (i.e. lysine to glutamine), N48T (i.e. asparagine to threonine) and G49A (i.e. glycine to alanine). In conclusion, α-EPTX-Na1a is a potent, pseudo-irreversible, short-chain neurotoxin. The high prevalence of α-EPTX-Na1a in Chinese N. atra venom is likely to explain the mild neurotoxicity experienced by envenomed patients.
中国眼镜蛇（Naja atra）是中国南方具有重要医学意义的一种眼镜蛇。尽管先前的研究表明，突触后神经毒素占 N. atra 毒液的 11-23%，但中毒患者并没有明显的神经毒性迹象。我们之前曾表明，某些具有“神经毒性”毒液的蛇类中毒后缺乏临床神经毒性，可能与这些毒液中短链突触后神经毒素的高流行率有关。在这项研究中，我们描述了α-Elapitoxin-Na1a（α-EPTX-Na1a；6949 Da）的分离和特性，α-EPTX-Na1a 是一种短链突触后神经毒素，约占 N. atra 粗毒液的 9%。α-EPTX-Na1a（30-300 nM）产生浓度依赖性抑制间接抽搐，在 300 nM 时 t 值为 17±2 min，并消除了鸡双颈椎神经-肌肉标本对外源性乙酰胆碱和卡巴胆碱的收缩反应。在体外，预先加入中国 N. atra 单价抗蛇毒血清（0.3 U/ml）或澳大利亚多价蛇抗蛇毒血清（2.4 U/ml）可预防α-EPTX-Na1a（30 nM）的神经毒性作用。在 t 时间点加入这两种抗蛇毒血清均可部分逆转由α-EPTX-Na1a（30 nM）引起的体外神经毒性。α-EPTX-Na1a（30 nM）对间接抽搐的抑制作用不能通过反复洗涤组织来逆转。α-EPTX-Na1a 对卡巴胆碱的浓度反应曲线表现出假性不可逆的拮抗作用，pA 值为 8.21。α-EPTX-Na1a 的从头蛋白质测序显示了 62 个氨基酸的典型短链突触后神经毒素特征，与来自其他 Naja 物种的短链突触后神经毒素具有>98%的氨基酸序列相似性。与中国眼镜蛇分离的短链神经毒素相比，α-EPTX-Na1a 含有新的残基 K47Q（即赖氨酸到谷氨酰胺）、N48T（即天冬酰胺到苏氨酸）和 G49A（即甘氨酸到丙氨酸）。总之，α-EPTX-Na1a 是一种强效、假性不可逆的短链神经毒素。α-EPTX-Na1a 在中华眼镜蛇毒液中的高流行率可能解释了中毒患者所经历的轻度神经毒性。
