Lomonte Bruno, Escolano José, Fernández Julián, Sanz Libia, Angulo Yamileth, Gutiérrez José María, Calvete Juan J
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
J Proteome Res. 2008 Jun;7(6):2445-57. doi: 10.1021/pr8000139. Epub 2008 Apr 30.
We report the comparative proteomic characterization of the venoms of two related neotropical arboreal pitvipers from Costa Rica of the genus Bothriechis, B. lateralis (side-striped palm pit viper) and B. schlegelii (eyelash pit viper). The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venom proteomes of B. lateralis and B. schlegelii comprise similar number of distinct proteins belonging, respectively, to 8 and 7 protein families. The two Bothriechis venoms contain bradykinin-potentiating peptides (BPPs), and proteins from the phospholipase A 2 (PLA 2), serine proteinase, l-amino acid oxidase (LAO), cysteine-rich secretory protein (CRISP), and Zn (2+)-dependent metalloproteinase (SVMP) families, albeit each species exhibit different relative abundances. Each venom also contains unique components, for example, snake venom vascular endothelial growth factor (svVEGF) and C-type lectin-like molecules in B. lateralis, and Kazal-type serine proteinase inhibitor-like proteins in B. schlegelii. Using a similarity coefficient, we estimate that the similarity of the venom proteins between the two Bothriechis taxa may be <10%, indicating a high divergence in their venom compositions, in spite of the fact that both species have evolved to adapt to arboreal habits. The major toxin families of B. lateralis and B. schlegelii are SVMP (55% of the total venom proteins) and PLA 2 (44%), respectively. Their different venom toxin compositions provide clues for rationalizing the distinct signs of envenomation caused by B. schlegelii and B. lateralis. An antivenomic study of the immunoreactivity of the Instituto Clodomiro Picado (ICP) polyvalent antivenom toward Bothriechis venoms revealed that l-amino acid oxidase and SVMPs represent the major antigenic protein species in both venoms. Our results provide a ground for rationalizing the reported protection of the ICP polyvalent antivenom against the hemorrhagic, coagulant, defibrinating, caseinolytic and fibrin(ogen)olytic activities of Bothriechis ( schlegelii, lateralis) venoms. However, these analyses also evidenced the limited recognition capability of the polyvalent antivenom toward a number of Bothriechis venom components, predominantly BPPs, svVEGF, Kazal-type inhibitors, some PLA 2 proteins, some serine proteinases, and CRISP molecules.
我们报告了来自哥斯达黎加的两种相关新热带树栖蝰蛇（睫角棕榈蝰属）毒液的比较蛋白质组学特征，即侧纹棕榈蝰（Bothriechis lateralis）和睫角棕榈蝰（Bothriechis schlegelii）。粗毒液通过反相高效液相色谱法进行分离，随后对每个色谱馏分进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳、N端测序、基质辅助激光解吸电离飞行时间质谱指纹分析以及胰蛋白酶肽段的碰撞诱导解离串联质谱分析。侧纹棕榈蝰和睫角棕榈蝰的毒液蛋白质组分别包含数量相似的不同蛋白质（分别属于8个和7个蛋白质家族）。两种睫角棕榈蝰属的毒液都含有缓激肽增强肽（BPPs），以及来自磷脂酶A2（PLA2）、丝氨酸蛋白酶、L - 氨基酸氧化酶（LAO）、富含半胱氨酸的分泌蛋白（CRISP）和锌（2 +）依赖性金属蛋白酶（SVMP）家族的蛋白质，尽管每个物种表现出不同的相对丰度。每种毒液还含有独特的成分，例如，侧纹棕榈蝰中的蛇毒血管内皮生长因子（svVEGF）和C型凝集素样分子，以及睫角棕榈蝰中的Kazal型丝氨酸蛋白酶抑制剂样蛋白。使用相似系数，我们估计这两种睫角棕榈蝰属分类群之间毒液蛋白质的相似度可能小于10%，这表明尽管这两个物种都已进化以适应树栖习性，但它们的毒液成分存在高度差异。侧纹棕榈蝰和睫角棕榈蝰的主要毒素家族分别是SVMP（占毒液总蛋白的55%）和PLA2（占44%）。它们不同的毒液毒素成分有助于解释由睫角棕榈蝰和侧纹棕榈蝰引起的不同中毒症状。对克洛多米罗·皮卡多研究所（ICP）多价抗蛇毒血清对睫角棕榈蝰属毒液免疫反应性的抗蛇毒血清组学研究表明，L - 氨基酸氧化酶和SVMPs是两种毒液中的主要抗原蛋白种类。我们的结果为解释所报道ICP多价抗蛇毒血清对睫角棕榈蝰属（睫角棕榈蝰、侧纹棕榈蝰）毒液的出血、凝血、去纤维蛋白、酪蛋白溶解和纤维蛋白（原）溶解活性的保护作用提供了依据。然而，这些分析也证明了多价抗蛇毒血清对许多睫角棕榈蝰属毒液成分的识别能力有限，主要是BPPs、svVEGF、Kazal型抑制剂、一些PLA2蛋白、一些丝氨酸蛋白酶和CRISP分子。
