Phospholipase D Toxins of Brown Spider Venom Convert Lysophosphatidylcholine and Sphingomyelin to Cyclic Phosphates

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.

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders

“…The three toxins in this study are as follows: La_␣IB2bi (gb:AY699703); La_␤ID1 from L. arizonica (gb:KM884812); St_␤IB1i from S. terrosus (gb:KM884813). La_␣IB2bi has been identified and studied previously (15,30) and is used here as a representative of the SicTox ␣ clade. St_␤IB1i from S. terrosus was chosen as a representative of the ␤ clade.…”

“…Cloning of DNA sequences, encoding the mature PLD toxins St_␤IB1i and La_␤ID1 obtained from cDNA libraries, into pHis8 expression vectors (34) was performed as described previously for La_␣IB2bi (15,30). Sequence variants of St_ ␤IB1i were generated using QuikChange site-directed mutagenesis according to the manufacturer's protocol.…”

“…Heterologous Expression and Purification of SicTox PLD Toxins-N-terminally His 8 -tagged recombinant proteins were expressed from pHis8-toxin constructs in Escherichia coli strain BL21(DE3), closely following published methods (15,35). Freshly transformed cells were grown with shaking at 250 rpm at 37°C in 1 liter of 2ϫ YT media (20 mg/ml tryptone, 10 mg/ml Bacto yeast extract, 5 mg/ml NaCl (pH 7)) containing 30 g/ml kanamycin.…”

“…In the case of ␤1A1 (SMase II) from L. laeta, SMase D activity has been observed but with a 3-fold higher K m value and a reported 2-fold lower reaction rate at saturating substrate concentrations compared with ␣III1 (SMase I) from the same species (26). References for structure and activity data are as follows: IsSMase (74); LiRecDT6 (11); LiRecDT7 (14); SMase I (8,18,27,35,59,64,75); Ll2 (8); LiRecDT4 (9); LgRec1 (76); Lr1/Lb1/Lb2 (8,25); L. arizonica ␣IB2bi (15,30); Lr2 (19,25); P1/P2 (77-79); LiRecDT1 (6,7,12,13,20,60); 3RLG (61); 3RLH (28); LiRecDT2 (7); LiRecDT5 (9); Lb3 (8,25); LiRecDT3 (7,10); and SMase II (26).…”

“…Here, we utilize 31 P NMR-based substrate screening to identify substrates and substrate preferences for two diverse ␤I clade members as follows: St_␤IB1i from Sicarius terrosus, a New World species that lacks venom SMase D activity in vitro and has no known ␣ clade toxins (24); and La_␤ID1 from Loxosceles arizonica, a species that is known to cause loxoscelism and does produce ␣ clade toxins (30 -32). We include parallel characterization of La_␣IB2bi, a previously characterized ␣ clade toxin from L. arizonica (15,30), both as a positive control for activity against SM and LPC ( Fig. 2) and to provide a direct comparison between substrate preferences of ␣ clade and ␤I clade members.…”