New insights into the proteomic characterization of the coral snake Micrurus pyrrhocryptus venom
New insights into the proteomic characterization of the coral snake Micrurus pyrrhocryptus venom
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DOI：
10.1016/j.toxicon.2018.08.003
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发表时间：
2018-10-01
期刊：
影响因子：
2.8
通讯作者：
Possani, Lourival D.
中科院分区：
文献类型：
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作者：
Olamendi-Portugal, Timoteo;Batista, Cesar V. F.;Possani, Lourival D.
本文报道了珊瑚蛇毒的蛋白质组学分析。采用反相高效液相色谱法分离了全可溶性毒液，质谱法测定了各组分的分子量（≥100）。鉴定出三组主要成分，分别为分子质量为5 ~ 8 kDa的肽、12 ~ 16 kDa的蛋白和20 ~ 30 kDa的蛋白。两种成分被完全测序：一种是7210 Da的a-神经毒性肽，对烟碱乙酰胆碱受体（nAChR）有轻微的阻断活性，另一种是分子量为13517 Da的磷脂酶a (2) (PLA(2))，对nAChR没有影响。采用反相高效液相色谱法测定各组分的PLA(2)活性。此外，利用Edman降解得到了11个组分的n端序列。其中，3种与已知的PLA(2)相似，6种与三指毒素（3FTx）相似，1种与kunitz型丝氨酸蛋白酶抑制剂相似。毒液的双向凝胶电泳可以分离出大约30个点，分子量从25到70 kDa不等。从凝胶中提取了17个斑点，经胰蛋白酶酶切，并对85个斑点进行了质谱/质谱分析，鉴定出与蛇毒金属蛋白酶（SVMP）、PLA(2) s、l -氨基酸氧化酶（LAAO）、乙酰胆碱酯酶（AChE）和丝氨酸蛋白酶（SP）相似的氨基酸序列。此外，从整个可溶性毒液的胰蛋白酶消化中获得的肽进行LC-MS分析，可以鉴定出695条肽，其氨基酸序列与其他蛇毒中发现的至少355种成分相对应，其中发现了c型凝集素，vespryns，锌指蛋白和waprins等。这些结果显示了该蛇毒的复杂性，并为今后对该蛇毒成分的鉴定和活性测定工作提供了重要的知识。
A proteomic analysis of the soluble venom of the coral snake Micrurus pyrrhocryptus is reported in this work. The whole soluble venom was separated by RP-HPLC and the molecular weights of its components (over 100) were determined by mass spectrometry. Three main sets of components were identified, corresponding to peptides with molecular masses from 5 to 8 kDa, proteins from 12 to 16 kDa and proteins from 20 to 30 kDa. Two components were fully sequenced: one a-neurotoxic peptide of 7210 Da with slight blocking activity of the nicotinic acetylcholine receptor (nAChR) and a phospholipase A(2) (PLA(2)) with molecular weight 13517 Da and no effect on the nAChR. PLA(2) activity was evaluated for all RP-HPLC components. In addition, N-terminal sequence was obtained for eleven components using Edman degradation. Among these, three were similar to known PLA(2)'s, six to three-finger toxins (3FTx) and one to Kunitz-type serine protease inhibitors. Two-dimensional gel electrophoresis of the venom allowed the separation of about thirty spots with components of molecular weights from 25 to 70 kDa. Seventeen spots were recovered from the gel, digested with trypsin and the corresponding peptides (85) were sequenced by MS/MS allowing identification of amino acid sequences with similarities to snake venom metalloproteases (SVMP), PLA(2)'s, L-amino acid oxidases (LAAO), acetylcholinesterases (AChE) and serine proteases (SP). In addition, LC-MS analysis of peptides obtained from tryptic digestion of whole soluble venom allowed the identification of 695 peptides, whose amino acid sequence could correspond to at least 355 components found in other snake venoms, where C-type lectins, vespryns, zinc finger proteins, and waprins were found, among others. These results show the complexity of the venom and provide important knowledge for future work on identification and activity determination of venom components from this coral snake.