Molecular Toxinology Laboratory, Department of Molecular Biology and Biotechnology, Tezpur University, Assam 784028, India.
Department of Neurological Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
J Proteomics. 2019 Sep 15;207:103463. doi: 10.1016/j.jprot.2019.103463. Epub 2019 Jul 22.
In the present study, venom composition, toxic effects, and immunological characteristics of Naja kaouthia venom from North East India has been studied. Using RP-HPLC, venom components were separated and proteins in the fractions were identified using ESI-LC MS/MS. Proteins identified belong to 9 different snake venom protein families. Three finger toxins and PLA were the most abundant protein families detected by mass spectrometry analysis. The other minor proteins families identified in the venom were kunitz-type serine inhibitors, waprin, L-amino acid oxidase, CRISP, vespyrn, nerve growth factor and metalloproteinase. This proteome composition correlated with the tested enzymatic and toxic activities of the venom. Western blot and third generation antivenomics analysis using Vins polyvalent antivenom revealed immunoreactivity towards Naja kaouthia venom of North East India. Concentration-dependent immunocapturing profile carried out using RP-HPLC displayed immunerecognition of majority of venom proteins of Naja kaouthia except few three-finger toxins. Presence of such non-immunodepleted toxins apparently may affect the performance of Vins polyvalent antivenom. Thus, inclusion of antibodies of most relevant non-immunorecognized toxins in antivenom might help to improve the quality of antivenom. BIOLOGICAL SIGNIFICANCE: Envenomings by genus Naja, represent a serious medical problem in Asian countries including North east India. In North East India, Naja kaouthia is most prevalent cobra species causing a large number of fatalities. To gain deeper insight into the spectrum of medically relevant toxins, we applied proteomics approach to unveil the proteome profile of Naja kaouthia venom. The proteomic analysis divulged the presence of two major protein families: three finger toxins and phospholipases A. In general, polyvalent antivenom is administered for Naja kaouthia envenomings, however, this venom is not included in the immunization mixtures (only Indian Big Four venoms) for production of these polyvalent antivenoms. For the first time, third generation antivenomics approach was used to decipher maximal binding capacity of Indian polyvalent antivenom against Naja kaouthia venom. Although Vins polyvalent antivenom was effective in immunocapturing majority of venom components, however, large amount of antivenom was required to immunocapture the venom proteins. Moreover, the study revealed poor immunorecognition capacity of Vins antivenom towards four three finger toxin subtypes. This may have significant impact on antivenom efficacy in treating Naja kaouthia envenomings.
在本研究中，研究了来自印度东北部的圆斑蝰蛇毒液的组成、毒性作用和免疫学特性。使用 RP-HPLC 分离毒液成分，并使用 ESI-LC MS/MS 鉴定馏分中的蛋白质。鉴定出的蛋白质属于 9 种不同的蛇毒蛋白家族。通过质谱分析，三指毒素和 PLA 是检测到的最丰富的蛋白家族。毒液中鉴定出的其他较小的蛋白家族包括 Kunitz 型丝氨酸抑制剂、Waprin、L-氨基酸氧化酶、CRISP、Vespyrn、神经生长因子和金属蛋白酶。这种蛋白质组组成与毒液的酶学和毒性活性测试结果相关。Western blot 和使用 Vins 多价抗蛇毒血清进行的第三代抗蛇毒血清分析显示，该多价抗蛇毒血清对来自印度东北部的圆斑蝰蛇毒液具有免疫反应性。使用 RP-HPLC 进行的浓度依赖性免疫捕获谱显示，除了少数三种毒素外，大多数圆斑蝰蛇毒液蛋白都能被免疫识别。显然，存在这种未免疫耗竭的毒素会影响 Vins 多价抗蛇毒血清的性能。因此，在抗蛇毒血清中加入大多数相关的非免疫识别毒素的抗体可能有助于提高抗蛇毒血清的质量。生物学意义：属 Naja 的蛇类毒液在包括印度东北部在内的亚洲国家是一个严重的医疗问题。在印度东北部，圆斑蝰是最常见的引起大量死亡的眼镜蛇物种。为了更深入地了解具有医学相关性的毒素谱，我们应用蛋白质组学方法揭示了圆斑蝰蛇毒液的蛋白质组图谱。蛋白质组学分析揭示了两种主要蛋白质家族的存在：三指毒素和磷脂酶 A。一般来说，多价抗蛇毒血清用于治疗圆斑蝰蛇咬伤，但这种毒液不包括在这些多价抗蛇毒血清的免疫混合物（仅包括印度四大毒液）中用于生产这些多价抗蛇毒血清。这是首次使用第三代抗蛇毒血清组学方法来破译印度多价抗蛇毒血清对圆斑蝰蛇毒液的最大结合能力。尽管 Vins 多价抗蛇毒血清能有效免疫捕获大多数毒液成分，但需要大量抗蛇毒血清才能免疫捕获毒液蛋白。此外，该研究还揭示了 Vins 抗蛇毒血清对四种三指毒素亚型的识别能力较差。这可能对抗蛇毒血清在治疗圆斑蝰蛇咬伤方面的疗效产生重大影响。
