Tan Choo Hock, Tan Kae Yi, Tan Nget Hong
Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
J Proteomics. 2016 Jul 20;144:33-8. doi: 10.1016/j.jprot.2016.06.004. Epub 2016 Jun 6.
Recent advances in proteomics enable deep profiling of the compositional details of snake venoms for improved understanding on envenomation pathophysiology and immunological neutralization. In this study, the venom of Australian tiger snake (Notechis scutatus) was trypsin-digested in solution and subjected to nano-ESI-LCMS/MS. Applying a relative quantitative proteomic approach, the findings revealed a proteome comprising 42 toxin subtypes clustered into 12 protein families. Phospholipases A2 constitute the most abundant toxins (74.5% of total venom proteins) followed by Kunitz serine protease inhibitors (6.9%), snake venom serine proteases (5.9%), alpha-neurotoxins (5.6%) and several toxins of lower abundance. The proteome correlates with N. scutatus envenoming effects including pre-synaptic and post-synaptic neurotoxicity and consumptive coagulopathy. The venom is highly lethal in mice (intravenous median lethal dose=0.09μg/g). BioCSL Sea Snake Antivenom, raised against the venoms of beaked sea snake (Hydrophis schistosus) and N. scutatus (added for enhanced immunogenicity), neutralized the lethal effect of N. scutatus venom (potency=2.95mg/ml) much more effectively than the targeted H.schistosus venom (potency=0.48mg/ml). The combined venom immunogen may have improved the neutralization against phospholipases A2 which are abundant in both venoms, but not short-neurotoxins which are predominant only in H. schistosus venom.
A shotgun proteomic approach adopted in this study revealed the compositional details of the venom of common tiger snake from Australia, Notechis scutatus. The proteomic findings provided additional information on the relative abundances of toxins and the detection of proteins of minor expression unreported previously. The potent lethal effect of the venom was neutralized by bioCSL Sea Snake Antivenom, an anticipated finding due to the fact that the Sea Snake Antivenom is actually bivalent in nature, being raised against a mix of venoms of the beaked sea snake (Hydrophis schistosus) and N. scutatus. However, it is surprising to note that bioCSL Sea Snake Antivenom neutralized N. scutatus venom much more effectively compared to the targeted sea snake venom by a marked difference in potency of approximately 6-fold. This phenomenon may be explained by the main difference in the proteomes of the two venoms, where H. schistosus venom is dominated by short-neurotoxins in high abundance - this is a poorly immunogenic toxin group that has been increasingly recognized in the venoms of a few cobras. Further investigations should be directed toward strategies to improve the neutralization of short-neurotoxins, in line with the envisioned production of an effective pan-regional elapid antivenom.
蛋白质组学的最新进展使得能够对蛇毒的组成细节进行深度分析，以更好地理解蛇毒中毒的病理生理学和免疫中和作用。在本研究中，澳大利亚虎蛇（Notechis scutatus）的毒液在溶液中经胰蛋白酶消化后进行纳米电喷雾电离液相色谱-串联质谱分析（nano-ESI-LCMS/MS）。采用相对定量蛋白质组学方法，研究结果揭示了一个由42种毒素亚型组成的蛋白质组，这些亚型分为12个蛋白家族。磷脂酶A2是最丰富的毒素（占总毒液蛋白的74.5%），其次是库尼茨丝氨酸蛋白酶抑制剂（6.9%）、蛇毒丝氨酸蛋白酶（5.9%）、α-神经毒素（5.6%）以及几种丰度较低的毒素。该蛋白质组与虎蛇的蛇毒中毒效应相关，包括突触前和突触后神经毒性以及消耗性凝血病。该毒液对小鼠具有高度致死性（静脉注射半数致死剂量=0.09μg/g）。BioCSL海蛇抗蛇毒血清是针对喙吻海蛇（Hydrophis schistosus）和虎蛇（添加以增强免疫原性）的毒液制备而成，它中和虎蛇毒液的致死效应（效价=2.95mg/ml）比针对的喙吻海蛇毒液（效价=0.48mg/ml）更有效。联合毒液免疫原可能提高了对两种毒液中都丰富的磷脂酶A2的中和作用，但对仅在喙吻海蛇毒液中占主导的短链神经毒素则没有效果。
本研究采用的鸟枪法蛋白质组学方法揭示了澳大利亚普通虎蛇（Notechis scutatus）毒液的组成细节。蛋白质组学研究结果提供了关于毒素相对丰度的额外信息，并检测到了先前未报道的低表达蛋白质。BioCSL海蛇抗蛇毒血清中和了毒液的强效致死效应，鉴于海蛇抗蛇毒血清实际上是双价的，是针对喙吻海蛇（Hydrophis schistosus）和虎蛇的混合毒液制备而成，这是一个预期的发现。然而，令人惊讶的是，BioCSL海蛇抗蛇毒血清中和虎蛇毒液的效果比针对的海蛇毒液有效得多，效价差异显著，约为6倍。这种现象可能是由于两种毒液蛋白质组的主要差异所致，其中喙吻海蛇毒液以高丰度的短链神经毒素为主——这是一类免疫原性较差的毒素组，在一些眼镜蛇的毒液中越来越受到关注。应进一步开展研究，以制定提高对短链神经毒素中和作用策略，这与设想生产一种有效的泛区域眼镜蛇科抗蛇毒血清相一致。
