Bittenbinder Mátyás A, Wachtel Eric, Pereira Daniel Da Costa, Slagboom Julien, Casewell Nicholas R, Jennings Paul, Kool Jeroen, Vonk Freek J
Naturalis Biodiversity Center, Leiden, the Netherlands.
AIMMS Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands.
Toxicon X. 2024 Apr 7;22:100197. doi: 10.1016/j.toxcx.2024.100197. eCollection 2024 Jun.
Snakebite envenoming is a global health issue that affects millions of people worldwide, and that causes morbidity rates surpassing 450,000 individuals annually. Patients suffering from snakebite morbidities may experience permanent disabilities such as pain, blindness and amputations. The (local) tissue damage that causes these life-long morbidities is the result of cell- and tissue-damaging toxins present in the venoms. These compounds belong to a variety of toxin classes and may affect cells in various ways, for example, by affecting the cell membrane. In this study, we have developed a high-throughput assay that can be used to study membrane disruption caused by snake venoms using phospholipid vesicles from egg yolk as a substrate. Resuspended chicken egg yolk was used to form these vesicles, which were fluorescently stained to allow monitoring of the degradation of egg yolk vesicles on a plate reader. The assay proved to be suitable for studying phospholipid vesicle degradation of crude venoms and was also tested for its applicability for neutralisation studies of varespladib, which is a PLA inhibitor. We additionally made an effort to identify the responsible toxins using liquid chromatography, followed by post-column bioassaying and protein identification using high-throughput venomics. We successfully identified various toxins in the venoms of and which are likely to be involved in the observed vesicle-degrading effect. This indicates that the assay can be used for screening the membrane degrading activity of both crude and fractionated venoms as well as for neutralisation studies.
蛇咬伤中毒是一个全球性的健康问题，影响着全球数百万人，每年导致超过45万人发病。患有蛇咬伤疾病的患者可能会经历永久性残疾，如疼痛、失明和截肢。导致这些终身疾病的（局部）组织损伤是毒液中存在的细胞和组织损伤毒素的结果。这些化合物属于多种毒素类别，可能以多种方式影响细胞，例如，通过影响细胞膜。在本研究中，我们开发了一种高通量检测方法，可用于研究蛇毒引起的膜破坏，该方法以蛋黄中的磷脂囊泡为底物。重悬的鸡卵黄用于形成这些囊泡，对其进行荧光染色，以便在酶标仪上监测卵黄囊泡的降解。该检测方法被证明适用于研究粗毒液的磷脂囊泡降解，并且还测试了其在瓦雷普拉德（一种PLA抑制剂）中和研究中的适用性。我们还努力使用液相色谱法鉴定相关毒素，随后进行柱后生物测定，并使用高通量毒液组学进行蛋白质鉴定。我们成功鉴定了 和 的毒液中的各种毒素，这些毒素可能与观察到的囊泡降解效应有关。这表明该检测方法可用于筛选粗毒液和分级毒液的膜降解活性以及进行中和研究。
