Development of a membrane-disruption assay using phospholipid vesicles as a proxy for the detection of cellular membrane degradation
Toxicon X, vol. 22, pp. 100197
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.
Authors 8
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Naturalis Biodiversity Center · Vrije Universiteit Amsterdam
Affiliation as printed
AIMMS Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands
Centre for Analytical Sciences Amsterdam (CASA), Amsterdam, the Netherlands
Naturalis Biodiversity Center, Leiden, the Netherlands
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Affiliation as printed
AIMMS Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands
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Affiliation as printed
AIMMS Division of Molecular and Computational Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands
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Affiliation as printed
AIMMS Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands
Centre for Analytical Sciences Amsterdam (CASA), Amsterdam, the Netherlands
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Liverpool School of Tropical Medicine
Affiliation as printed
Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
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Affiliation as printed
AIMMS Division of Molecular and Computational Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands
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Jeroen Kool corresponding
Affiliation as printed
AIMMS Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands
Centre for Analytical Sciences Amsterdam (CASA), Amsterdam, the Netherlands
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Naturalis Biodiversity Center · Vrije Universiteit Amsterdam
Affiliation as printed
AIMMS Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands
Centre for Analytical Sciences Amsterdam (CASA), Amsterdam, the Netherlands
Naturalis Biodiversity Center, Leiden, the Netherlands
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