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Phospholipase D toxins of brown spider venom convert lysophosphatidylcholine and sphingomyelin to cyclic phosphates.

Lajoie DM, Zobel-Thropp PA, Kumirov VK, Bandarian V, Binford GJ, Cordes MH - PLoS ONE (2013)

Bottom Line: Venoms of brown spiders in the genus Loxosceles contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans.These toxins cleave the substrates sphingomyelin and lysophosphatidylcholine in mammalian tissues, releasing the choline head group.Cyclic phosphates have vastly different biological properties from their monoester counterparts, and they may be relevant to the pathology of brown spider envenomation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona, USA.

ABSTRACT
Venoms of brown spiders in the genus Loxosceles contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These toxins cleave the substrates sphingomyelin and lysophosphatidylcholine in mammalian tissues, releasing the choline head group. The other products of substrate cleavage have previously been reported to be monoester phospholipids, which would result from substrate hydrolysis. Using (31)P NMR and mass spectrometry we demonstrate that recombinant toxins, as well as whole venoms from diverse Loxosceles species, exclusively catalyze transphosphatidylation rather than hydrolysis, forming cyclic phosphate products from both major substrates. Cyclic phosphates have vastly different biological properties from their monoester counterparts, and they may be relevant to the pathology of brown spider envenomation.

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Recombinant SicTox enzyme αIB2bi generates cyclic phosphatidic acid from octanoyl lysophosphatidylcholine (8∶0 LPC).(A) Degradation of 8∶0 LPC by SicTox enzyme αIB2bi as measured by 31P-NMR. Two isomers of LPC (1 and 2) are observed (see text) and the only observed product upon enzyme addition is a far downfield chemical shift. Trimethyl phosphate (TMP) is an internal chemical shift and concentration standard. (B) LC-MS characterization of the NMR sample from (A), before (top) and after (bottom) addition of enzyme. Chromatograms represent total ion count (TIC) from reverse phase LC-MS as a function of retention time. Insets show time-averaged negative-ion mode mass spectra of substrate (red) and product peak (blue).
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pone-0072372-g002: Recombinant SicTox enzyme αIB2bi generates cyclic phosphatidic acid from octanoyl lysophosphatidylcholine (8∶0 LPC).(A) Degradation of 8∶0 LPC by SicTox enzyme αIB2bi as measured by 31P-NMR. Two isomers of LPC (1 and 2) are observed (see text) and the only observed product upon enzyme addition is a far downfield chemical shift. Trimethyl phosphate (TMP) is an internal chemical shift and concentration standard. (B) LC-MS characterization of the NMR sample from (A), before (top) and after (bottom) addition of enzyme. Chromatograms represent total ion count (TIC) from reverse phase LC-MS as a function of retention time. Insets show time-averaged negative-ion mode mass spectra of substrate (red) and product peak (blue).

Mentions: Loxosceles PLD toxins can utilize lysophospholipids of various acyl chain lengths as substrates [10]. Thus, to improve product solubility, we repeated the 31P NMR assays with octanoyl (08∶0) LPC, a more soluble substrate (Figure 2). Under the assay conditions, octanoyl LPC exists as an equilibrium mixture of two isomers [23], 1-octanoyl-sn-glycero-3-phosphorylcholine (LPC 1) and 2-octanoyl-sn-glycero-phosphorylcholine (LPC 2), with LPC 1 predominating by a factor of ∼6. Incubation of αIB2bi with octanoyl LPC led to the appearance of species with a far downfield resonance similar to that observed when enzyme was added to palmitoyl LPC substrate (Figure 2A). With octanoyl LPC, however, loss of the substrate resonance is accompanied by gain of a comparable amount of product signal (see also Figure 3 for a quantitative analysis), and no precipitation is observed. The chemical shift of the product (+17.9 ppm) is clearly inconsistent with LPA, but agrees closely with the value reported for 1-octanoyl-glycero-2,3-cyclic-phosphate (08∶0 CPA) under similar reaction conditions [19].


Phospholipase D toxins of brown spider venom convert lysophosphatidylcholine and sphingomyelin to cyclic phosphates.

Lajoie DM, Zobel-Thropp PA, Kumirov VK, Bandarian V, Binford GJ, Cordes MH - PLoS ONE (2013)

Recombinant SicTox enzyme αIB2bi generates cyclic phosphatidic acid from octanoyl lysophosphatidylcholine (8∶0 LPC).(A) Degradation of 8∶0 LPC by SicTox enzyme αIB2bi as measured by 31P-NMR. Two isomers of LPC (1 and 2) are observed (see text) and the only observed product upon enzyme addition is a far downfield chemical shift. Trimethyl phosphate (TMP) is an internal chemical shift and concentration standard. (B) LC-MS characterization of the NMR sample from (A), before (top) and after (bottom) addition of enzyme. Chromatograms represent total ion count (TIC) from reverse phase LC-MS as a function of retention time. Insets show time-averaged negative-ion mode mass spectra of substrate (red) and product peak (blue).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756997&req=5

pone-0072372-g002: Recombinant SicTox enzyme αIB2bi generates cyclic phosphatidic acid from octanoyl lysophosphatidylcholine (8∶0 LPC).(A) Degradation of 8∶0 LPC by SicTox enzyme αIB2bi as measured by 31P-NMR. Two isomers of LPC (1 and 2) are observed (see text) and the only observed product upon enzyme addition is a far downfield chemical shift. Trimethyl phosphate (TMP) is an internal chemical shift and concentration standard. (B) LC-MS characterization of the NMR sample from (A), before (top) and after (bottom) addition of enzyme. Chromatograms represent total ion count (TIC) from reverse phase LC-MS as a function of retention time. Insets show time-averaged negative-ion mode mass spectra of substrate (red) and product peak (blue).
Mentions: Loxosceles PLD toxins can utilize lysophospholipids of various acyl chain lengths as substrates [10]. Thus, to improve product solubility, we repeated the 31P NMR assays with octanoyl (08∶0) LPC, a more soluble substrate (Figure 2). Under the assay conditions, octanoyl LPC exists as an equilibrium mixture of two isomers [23], 1-octanoyl-sn-glycero-3-phosphorylcholine (LPC 1) and 2-octanoyl-sn-glycero-phosphorylcholine (LPC 2), with LPC 1 predominating by a factor of ∼6. Incubation of αIB2bi with octanoyl LPC led to the appearance of species with a far downfield resonance similar to that observed when enzyme was added to palmitoyl LPC substrate (Figure 2A). With octanoyl LPC, however, loss of the substrate resonance is accompanied by gain of a comparable amount of product signal (see also Figure 3 for a quantitative analysis), and no precipitation is observed. The chemical shift of the product (+17.9 ppm) is clearly inconsistent with LPA, but agrees closely with the value reported for 1-octanoyl-glycero-2,3-cyclic-phosphate (08∶0 CPA) under similar reaction conditions [19].

Bottom Line: Venoms of brown spiders in the genus Loxosceles contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans.These toxins cleave the substrates sphingomyelin and lysophosphatidylcholine in mammalian tissues, releasing the choline head group.Cyclic phosphates have vastly different biological properties from their monoester counterparts, and they may be relevant to the pathology of brown spider envenomation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona, USA.

ABSTRACT
Venoms of brown spiders in the genus Loxosceles contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These toxins cleave the substrates sphingomyelin and lysophosphatidylcholine in mammalian tissues, releasing the choline head group. The other products of substrate cleavage have previously been reported to be monoester phospholipids, which would result from substrate hydrolysis. Using (31)P NMR and mass spectrometry we demonstrate that recombinant toxins, as well as whole venoms from diverse Loxosceles species, exclusively catalyze transphosphatidylation rather than hydrolysis, forming cyclic phosphate products from both major substrates. Cyclic phosphates have vastly different biological properties from their monoester counterparts, and they may be relevant to the pathology of brown spider envenomation.

Show MeSH
Related in: MedlinePlus