Novel Product Formation and Substrate Specificity of the Phospholipase D Toxins in the Venom of the Sicariidae Spider Family
AuthorLajoie, Daniel M.
AdvisorCordes, Matthew, H. J.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractVenoms of the Sicariidae spider family contain phospholipase D (PLD) enzyme toxins that can cause severe dermonecrosis and even death in humans. PLD toxins are known to cleave the substrates sphingomyelin (SM) and lysophosphatidylcholine (LPC) in mammalian tissues, releasing a choline headgroup and a reported monoester phospholipid formed via a hydrolytic reaction. However, some PLD toxins have demonstrated the ability to utilize substrates besides SM and LPC and other PLD toxins have demonstrated no activity against either SM or LPC. Given that the etiology of the disease state following envenomation is not well understood, we postulated that PLD toxins could be utilizing other phospholipid substrates in vivo. To determine the level of promiscuity among the PLD toxins, we developed a novel ³¹P-NMR assay to measure phospholipase activity against a panel of potential phospholipid substrates. While developing the assay, we made the surprising discovery that recombinant PLD toxins, as well as whole venoms from diverse Sicariidae species, exclusively generates cyclic phosphate rather than hydrolytic products. We also found that a distantly related PLD toxin from a pathogenic bacterium, with low sequence identity to the spider PLDs, exclusively generates cyclic phosphate products. We then established that St_βIB1i, a PLD with extremely diminished activity toward SM and LPC, actually demonstrates large preferential specificity towards ethanolamine phospholipid substrates. We solved the crystal structure of St_βIB1i to compare to PLD toxins of known structure, toward an understanding of the molecular basis of substrate specificity. The cyclic phosphate products generated by the PLD toxins have extremely different biochemical properties than their monoester counterparts and may be relevant to the pathology following envenomation or bacterial infection. In addition the specificity St_βIB1i has for ethanolamine substrates may have biological implications, as insects have high concentrations of ethanolamine-containing phospholipids.
Degree ProgramGraduate College