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 31P 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.
Highlights
Envenomation by brown spiders in the genus Loxosceles can induce a disease state called loxoscelism in mammalian tissue
Cutaneous loxoscelism can result in ulcer formation, edema, and dermonecrosis at the site of envenomation
Phospholipase D (PLD) toxins from Loxosceles bind to mammalian cell surfaces [9] and have enzymatic activity against common phospholipids in mammalian tissue, including lysophosphatidylcholine (LPC) and sphingomyelin (SM) [2], [4], [10]
Summary
Envenomation by brown spiders in the genus Loxosceles can induce a disease state called loxoscelism in mammalian tissue. PLD toxins from Loxosceles bind to mammalian cell surfaces [9] and have enzymatic activity against common phospholipids in mammalian tissue, including lysophosphatidylcholine (LPC) and sphingomyelin (SM) [2], [4], [10]. Liberation of choline from SM or LPC is commonly assumed to result from substrate hydrolysis, giving either ceramide-1-phosphate (C1P) or lysophosphatidic acid (LPA), respectively, as a second product [10,11,12,13,14]. We use 31P NMR spectroscopy and mass spectrometry to directly observe formation of the phosphate-containing products from the action of a recombinant Loxosceles PLD toxin, as well as diverse whole venom samples, on the substrates SM and LPC. Instead of the hydrolytic products C1P and LPA, respectively, we observe exclusive formation of cyclic phosphate products resulting from intramolecular transphosphatidylation
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