We report a structural and functional venomics characterization of the black-tailed horned pitviper, Mixcoatlus melanurus. The venom phenotype of this small and elusive pitviper endemic to México comprise peptides and proteins of 16 toxin families whose relative abundance mirror those of neurotoxic (type II) venoms described for some species within genera distributed in Central Asia (Gloydius) and the Americas (Sistrurus, Crotalus, Ophryacus, and Bothriechis). A novel β-neurotoxic heterodimeric PLA₂, termed Melanurutoxin was characterized. With a relative abundance of 14.8% of the total M. melanurus venom proteome and a median lethal dose of 0.31 μg/g mouse body weight, Melanurutoxin accounted for 37.8% of the lethality of the whole venom (0.82 μg/g). The low percentage (1.1%) of snake venom metalloproteinases (PIII-SVMPs) and the high content of Melanurutoxin and bradykinin-potentiating peptides (BPP, 16%) found in the type-II venom proteome of M. melanurus correlate with the severe hypotension and neurotoxicity leading to neuromuscular blockade, flaccid paralysis and respiratory arrest observed in ex vivo neuromuscular junction experiments and in vivo experimental murine envenoming. Mexican antivenoms manufactured by Birmex and Bioclon showed low neutralization potency per vial (95 LD₅₀s, Birmex; 114 LD₅₀s, Antivipmyn®), and failed to reverse completely the paralysis and the hypotensive effect induced by the black-tailed horned pitviper, Mixcoatlus melanurus. We suggest that the impaired ability of these antivenoms to neutralize the neurotoxicity of M. melanurus venom may be attributed to the use of immunization mixtures that include venom of taxa, C. basiliscus (Birmex) and C. simus (Antivipmyn®), that contain only small amounts of Melanurutoxin-like β-neurotoxic heterodimeric PLA₂s.

Biological significance

This study represents the first proteomics and funcional investigations conducted on the venom of the black-tailed horned, Mixcoatlus melanurus, a pitviper species endemic to México. The venom's features unveiled through combination of bottom-up venomics and ex vivo and in vivo functional assays provided complementary evidence pointing to severe hypotension and neurotoxicity leading to neuromuscular blockade, flaccid paralysis and respiratory arrest as the predominant mechanism of murine prey immobilization and death caused by M. melanurus. A novel β-neurotoxic heterodimeric PLA₂, coined Melanurutoxin, was identified as a major contributor to the lethality of the whole venom. Our study also showed the inefficacy of two commercial Mexican antivenoms to reverse competely the paralytic and hypotensive effects induced by M. melanurus venom in the murine model. We hypothesize that the impaired ability of these antivenoms to neutralize the neurotoxicity of M. melanurus venom should be ascribed to the use as immunogens of venoms that contain only small amounts of Melanurutoxin-like β-neurotoxic heterodimeric PLA₂s.

中文翻译：

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黑尾角（Pilotperus melanurus）的经济学和生化分析，以及一种新的crotoxin同源物Melanurutoxin的表征。

我们报道了黑尾角pitviper，的结构和功能venomics表征Mixcoatlus橙线。墨西哥特有的这种小型且难以捉摸的蛇的毒液表型包含16个毒素家族的肽和蛋白质，它们的相对丰度反映了针对中亚（Gloydius）和美洲（Sistrurus）属内某些物种描述的神经毒性（II型）毒液的相对丰度。，响尾蛇，墨西哥角蝮属，和棕榈蝮属）。表征了一种新型的β-神经毒性异二聚体PLA ₂，称为黑素毒素。相对丰富度为黑腹果蝇总数的14.8％毒液蛋白质组和小鼠的平均致死剂量为0.31μg/ g小鼠体重，黑色素毒素占整个毒液致死率（0.82μg/ g）的37.8％。在M. melanurus的II型毒液蛋白质组中发现的蛇毒金属蛋白酶（PIII-SVMPs）的百分率低（1.1％），黑色素毒素和缓激肽增强肽的高含量（BPP，16％）与低血压相关在体外神经肌肉连接实验和体内实验鼠毒液中观察到的神经毒性导致神经肌肉阻滞，松弛性麻痹和呼吸停止。Birmex和Bioclon生产的墨西哥抗蛇毒药显示每个小瓶的中和效力低（95 LD ₅₀ s，Birmex； 114 LD ₅₀s，Antivipmyn®），并且无法完全逆转由黑尾角（Mixcoatlus melanurus）引起的瘫痪和降压作用。我们认为，这些抗蛇毒血清的中和神经毒性的能力受损M.橙线毒液可以归因于使用免疫的混合物，其包括类群的毒液，的C.蛇怪（Birmex）和C. simus（Antivipmyn®），含有仅少量黑色素毒素样β-神经毒性异二聚PLA ₂ s。

生物学意义

这项研究是对墨西哥特有的黑尾（Mixcoatlus melanurus）的毒液进行的首次蛋白质组学和功能研究。通过自下而上的病毒学和离体和体内功能测定相结合揭示的毒液特征提供了补充证据，表明严重的低血压和神经毒性导致神经肌肉阻滞，松弛性麻痹和呼吸停止，这是由引起的鼠类猎物固定和死亡的主要机制M.橙线。新型β-神经毒性异二聚体PLA ₂，被称为黑色素毒素，被认为是整个毒液致死性的主要贡献者。我们的研究还表明，两种商业墨西哥抗蛇毒草素不能有效地逆转小鼠模型中由M. melanurus毒液诱导的麻痹和降压作用。我们假设这些抗蛇毒血清中和神经毒性的能力受损M.橙线毒液应归因于用作只含有少量Melanurutoxin样β神经毒性异二聚体PLA的毒液免疫₂秒。