Novel Product Formation and Substrate Specificity of the Phospholipase D Toxins in the Venom of the Sicariidae Spider Family

Persistent Link:
http://hdl.handle.net/10150/333346
Title:
Novel Product Formation and Substrate Specificity of the Phospholipase D Toxins in the Venom of the Sicariidae Spider Family
Author:
Lajoie, Daniel M.
Issue Date:
2014
Publisher:
The University of Arizona.
Rights:
Copyright © 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.
Embargo:
Release 13-Aug-2015
Abstract:
Venoms 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.
Type:
text; Electronic Dissertation
Keywords:
Phospholipase D; Spider; Toxin; Toxinology; Venom; Biochemistry; Loxoscelism
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Biochemistry
Degree Grantor:
University of Arizona
Advisor:
Cordes, Matthew, H. J.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen
dc.titleNovel Product Formation and Substrate Specificity of the Phospholipase D Toxins in the Venom of the Sicariidae Spider Familyen_US
dc.creatorLajoie, Daniel M.en_US
dc.contributor.authorLajoie, Daniel M.en_US
dc.date.issued2014-
dc.publisherThe University of Arizona.en_US
dc.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.en_US
dc.description.releaseRelease 13-Aug-2015en_US
dc.description.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.en_US
dc.typetexten
dc.typeElectronic Dissertationen
dc.subjectPhospholipase Den_US
dc.subjectSpideren_US
dc.subjectToxinen_US
dc.subjectToxinologyen_US
dc.subjectVenomen_US
dc.subjectBiochemistryen_US
dc.subjectLoxoscelismen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorCordes, Matthew, H. J.en_US
dc.contributor.committeememberCordes, Matthew, H. J.en_US
dc.contributor.committeememberBandarian, Vaheen_US
dc.contributor.committeememberHorton, Nancyen_US
dc.contributor.committeememberMontfort, Williamen_US
dc.contributor.committeememberWhiteman, Noahen_US
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