Vergara Irene, Pedraza-Escalona Martha, Paniagua Dayanira, Restano-Cassulini Rita, Zamudio Fernando, Batista Cesar V F, Possani Lourival D, Alagón Alejandro
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad # 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, México.
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad # 2001, Colonia Chamilpa, Cuernavaca, Morelos 62210, México.
J Proteomics. 2014 Jun 13;105:295-306. doi: 10.1016/j.jprot.2014.02.027. Epub 2014 Mar 5.
Here we show for the first time that the venom from an elapid (Micrurus fulvius) contains three finger toxin (3FTxs) peptides with low toxicity but high content of lethal phospholipases A2 (PLA2). The intravenous venom LD50 in mice was 0.3μg/g. Fractionation on a C18 column yielded 22 fractions; in terms of abundance, 58.3% of them were components of 13-14kDa and 24.9% were molecules of 6-7kDa. Two fractions with PLA2 activity represented 33.4% of the whole venom and were the most lethal fractions. Fractions with low molecular mass (<7000Da) partially and reversibly blocked the nicotinic acetylcholine receptor (nAChR), with the exception of one that blocked it completely. The fraction that blocked 100% contained two protein species whose dose-response was determined; the IC50s were 13±1 and 9.5±0.3nM. Despite the apparent effect on nAChR none of the low molecular mass fractions were lethal in mice, at concentrations of 1μg/g. From 2D-PAGE and LC-MS/MS, we identified fourteen species of PLA2, four protein species of C-type lectin, three zinc metalloproteinases, one phosphodiesterase and one 3FTx. The N-terminal amino acid sequence of fractions with biological interest was obtained.
In contrast with coral snake venoms from South America, M. fulvius has minor amounts of low molecular mass components, but high content of PLA2, which is responsible for the venom lethality of this species. The results reported here contribute to better understanding of envenomation development and to improve antivenom design and production. These findings break from the paradigm that neurotoxicity caused by Micrurus venoms is mainly attributable to 3FTx neurotoxins and encourage future studies on Micrurus evolution and venom specialization. This article is part of a Special Issue entitled Non-model organisms.
我们首次表明，一种眼镜蛇科蛇（珊瑚蛇）的毒液含有三种指状毒素（3FTxs）肽，毒性低但致死性磷脂酶A2（PLA2）含量高。小鼠静脉注射毒液的半数致死量为0.3μg/g。在C18柱上进行分级分离得到22个级分；就丰度而言，其中58.3%是13 - 14kDa的组分，24.9%是6 - 7kDa的分子。具有PLA2活性的两个级分占整个毒液的33.4%，是最具致死性的级分。低分子量（<7000Da）的级分部分且可逆地阻断烟碱型乙酰胆碱受体（nAChR），但有一个级分能完全阻断。完全阻断nAChR的级分含有两种蛋白质，测定了它们的剂量反应；半数抑制浓度（IC50s）分别为13±1和9.5±0.3nM。尽管对nAChR有明显影响，但在1μg/g的浓度下，低分子量级分在小鼠中均无致死性。通过二维聚丙烯酰胺凝胶电泳（2D - PAGE）和液相色谱 - 串联质谱（LC - MS/MS），我们鉴定出14种PLA2、4种C型凝集素蛋白、3种锌金属蛋白酶、1种磷酸二酯酶和1种3FTx。获得了具有生物学意义的级分的N端氨基酸序列。
与南美洲的珊瑚蛇毒液不同，珊瑚蛇的低分子量成分含量较少，但PLA2含量高，这是该物种毒液致死性的原因。本文报道的结果有助于更好地理解蛇伤的发展，并改进抗蛇毒血清的设计和生产。这些发现打破了珊瑚蛇毒液引起的神经毒性主要归因于3FTx神经毒素的范式，并鼓励未来对珊瑚蛇进化和毒液特化的研究。本文是名为“非模式生物”的特刊的一部分。
