Title:
Development of an Assay for Sphingomyelinase D Products in Venom from Loxosceles reclusa and Other Spiders
Development of an Assay for Sphingomyelinase D Products in Venom from Loxosceles reclusa and Other Spiders
Author(s)
Lachmayr, Hannah Lowndes
Advisor(s)
Merrill, Alfred H.
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Abstract
The venoms of Loxosceles reclusa, the brown recluse spider, and some other Loxosceles species cause dermonecrotic lesions—a dying of the flesh—and in severe cases, systemic injury and death. Since this type of injury results from a bite, diagnosis can be cryptic unless the culprit spider is available to be identified by an expert. The distribution of Loxosceles reclusa is also not well known, which further complicates diagnosis. These spiders are of additional interest because one of the agents in the venom is a sphingomyelinase D (SMase D) that cleaves the head group of sphingomyelin (SM) and causes intramolecular transphosphatidylation to produce ceramide 1,3-cyclic phosphate (Cer(1,3)P), a lipid that is not known to be present in humans otherwise. Therefore, a simple assay to analyze the presence of SMase D, by either activity assays or detection of its product Cer(1,3)P, could aid in identification of spider bites and possibly facilitate development of a treatment. A key reagent for such studies is Cer(1,3)P which is currently not commercially available, so we have developed a method for its synthesis and identified conditions for distinguishing Cer(1,3)P from the substrate SM and alternative hydrolysis products (ceramide-1-phosphate and ceramide) by thin-layer chromatography. The availability of this chemical and its synthesis procedure enables an assay of SMase D for better characterization of this important component of brown recluse spider venom (and possibly other organisms). Moreover, the assay could be used to search for enzymes that hydrolyze Cer(1,3)P, and such enzymes might prove useful in the development of a treatment for these wounds.
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Date Issued
2020-05
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Text
Resource Subtype
Undergraduate Thesis