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Abstract：
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 t90 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 t90 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 pA2 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.
摘要：
中国眼镜蛇（Najaatra）是一种在中国南方具有重要医学意义的蛇。尽管先前的研究表明突触后神经毒素占N.atra毒液的11-23％，静脉阻塞的患者没有明显的神经毒性迹象。我们先前已经表明，某些具有“神经毒性”毒液的物种在蛇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患病率有关。在这项研究中,我们描述了α-Elapitoxin-Na1a（α-EPTX-Na1a;6949Da）的分离和表征，一种短链突触后神经毒素,约占N.atra粗毒液的9%。α-EPTX-Na1a（30-300nM）产生浓度依赖性的间接抽搐抑制作用，在300nM时t90值为17±2分钟，并消除了对外源性乙酰胆碱和卡巴胆碱的收缩反应，在小鸡宫颈神经肌肉准备中。预先添加中国N.atra单价抗蛇毒血清（0.3U/ml）或澳大利亚多价蛇毒血清（2.4U/ml），预防了α-EPTX-Na1a（30nM）的体外神经毒性作用。在t90时间点添加每种抗蛇毒血清部分逆转了由α-EPTX-Na1a（30nM）引起的体外神经毒性。通过反复洗涤组织，α-EPTX-Na1a（30nM）对间接抽搐的抑制作用并未逆转。α-EPTX-Na1a对卡巴胆碱的浓度-反应曲线表现出假性不可逆拮抗作用，pA2值为8.21。α-EPTX-Na1a的从头蛋白质测序揭示了62个氨基酸的典型短链突触后神经毒素谱，与其他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。与中国从眼镜蛇中分离出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(即天冬酰胺至苏氨酸)和G49A(即甘氨酸至丙氨酸)。总之,α-EPTX-Na1a是一种有效的，伪不可逆,短链神经毒素.α-EPTX-Na1a在中国N.atra毒液中的高患病率可能解释了毒液中毒患者的轻度神经毒性。
