Title:
Assay development for and evaluation of sphingomyelinase D and associated activities in venoms from Loxosceles reclusa and Kukulcania hibernalis and in isolated soil bacteria
Assay development for and evaluation of sphingomyelinase D and associated activities in venoms from Loxosceles reclusa and Kukulcania hibernalis and in isolated soil bacteria
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Lachmayr, Hannah L.
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Abstract
Since a sphingomyelinase D (SMase D) enzyme is presumably the major agent in the necrosis and toxicity of the brown recluse spider venom by cleaving sphingomyelin (SM) to ceramide 1,3-cyclic phosphate (Cer(1,3)P), a rigorous but simple assay would aid in detecting the presence of this sphingomyelinase activity. Several Sicariidae spiders, such as the brown recluse spider Loxosceles reclusa, and some pathogenic bacteria contain phospholipase D (PLase D) enzymes that act on phospholipid substrates. PLases D that cleave SM to release the headgroup choline are also called SMases D. Although the lipid product of the L. reclusa SMase D was initially thought to be ceramide 1-phosphate (Cer1P), a transphosphatidylation mechanism by L. reclusa SMase D has been shown to produce Cer(1,3)P. For this thesis, fluorescent nitrobenzoxadiazole (NBD) analogs of products that are known to be made by various types of SMases (i.e., ceramide, Cer1P and Cer(1,3)P) were synthesized and an elution solvent system was identified that fully resolves these compounds as well as the substrate NBD-SM using silica gel thin-layer chromatography plates. When venom from L. reclusa was assayed, the product was the expected NBD-labeled Cer(1,3)P. Venom from Kukulcania hibernalis, a spider that has been suggested to have SMase D but whose products have not yet been determined, was assayed as well and NBD-Cer(1,3)P was found to be produced. To explore additional biomedically relevant applications of these methodologies, such as to discover bacterial enzymes that can degrade sphingolipids, we attempted to isolate bacteria from the soil with enzymatic activity to degrade SM and/or cyclic Cer(1,3)P. Single isolates of soil microorganisms were selected based on their ability to grow on the restrictive carbon sources SM or Cer(1,3)P and then examined for their cleavage of NBD-SM and/or NBD-Cer(1,3)P. Of four candidate isolates, genome sequencing of two isolates was taxonomically assigned to Klebsiella variicola, a gram-negative bacterium and opportunistic human pathogen. Unfortunately, in the timeframe of this thesis, the conditions to assay the degradatory enzymes were not found. In summary, this work has developed a facile assay for SMase D, characterized for the first time Cer(1,3)P as a breakdown product of the action of K. hibernalis venom on SM, and expanded the methodologies that are available for analysis of activities that produce and degrade Cer(1,3)P.
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2021-08-02
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