Mátyás A. Bittenbinder , Eric Wachtel , Daniel Da Costa Pereira , Julien Slagboom , Nicholas R. Casewell , Paul Jennings , Jeroen Kool , Freek J. Vonk
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引用次数: 0
Abstract
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 in vitro 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 PLA2 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 C. rhodostoma and N. mossambica, 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.
毒蛇咬伤是一个全球性的健康问题,影响着全球数百万人,每年的发病率超过 45 万人。被蛇咬伤的患者可能会终身残疾,如疼痛、失明和截肢。造成这些终身疾病的(局部)组织损伤是毒液中的细胞和组织损伤毒素造成的。这些化合物属于多种毒素类别,可通过各种方式影响细胞,例如影响细胞膜。在这项研究中,我们开发了一种高通量体外检测方法,可以利用蛋黄中的磷脂囊泡作为底物来研究蛇毒造成的细胞膜破坏。我们使用重悬的鸡卵黄来形成这些囊泡,并对其进行荧光染色,以便在平板阅读器上监测卵黄囊泡的降解情况。事实证明,这种检测方法适用于研究粗制毒液的磷脂囊泡降解,同时还测试了它是否适用于varespladib(一种PLA2抑制剂)的中和研究。此外,我们还努力利用液相色谱法鉴定毒素,然后进行柱后生物测定,并利用高通量毒液组学鉴定蛋白质。我们成功鉴定了 C. rhodostoma 和 N. mossambica 毒液中的多种毒素,这些毒素很可能参与了观察到的囊泡降解效应。这表明该检测方法可用于筛选粗制和分馏毒液的膜降解活性以及中和研究。