Modulation of membrane phospholipids, the cytosolic calcium influx and cell proliferation following treatment of B16-F10 cells with recombinant phospholipase-D from Loxosceles intermedia (brown spider) venom

Abstract

The mechanism through which brown spiders (Loxosceles genus) cause dermonecrosis, dysregulated inflammatory responses, hemolysis and platelet aggregation, which are effects reported following spider bites, is currently attributed to the presence of phospholipase-D in the venom. In the present investigation, through two-dimensional immunoblotting, we observed immunological cross-reactivity for at least 25 spots in crude Loxosceles intermedia venom, indicating high expression levels for different isoforms of phospholipase-D. Using a recombinant phospholipase-D from the venom gland of L. intermedia (LiRecDT1) in phospholipid-degrading kinetic experiments, we determined that this phospholipase-D mainly hydrolyzes synthetic sphingomyelin in a time-dependent manner, generating ceramide 1-phosphate plus choline, as well as lysophosphatidylcholine, generating lysophosphatidic acid plus choline, but exhibits little activity against phosphatidylcholine. Through immunofluorescence assays with antibodies against LiRecDT1 and using a recombinant GFP-LiRecDT1 fusion protein, we observed direct binding of LiRecDT1 to the membrane of B16-F10 cells. We determined that LiRecDT1 hydrolyzes phospholipids in detergent extracts and from ghosts of B16-F10 cells, generating choline, indicating that the enzyme can access and modulate and has activity against membrane phospholipids. Additionally, using Fluo-4, a calcium-sensitive fluorophore, it was shown that treatment of cells with phospholipase-D induced an increase in the calcium concentration in the cytoplasm, but without altering viability or causing damage to cells. Finally, based on the known endogenous activity of phospholipase-D as an inducer of cell proliferation and the fact that LiRecDT1 binds to the cell surface, hydrolyzing phospholipids to generate bioactive lipids, we employed LiRecDT1 as an exogenous source of phospholipase-D in B16-F10 cells. Treatment of the cells was effective in increasing their proliferation in a time- and concentration-dependent manner, especially in the presence of synthetic sphingomyelin in the medium. The results described herein indicate the ability of brown spider phospholipase-D to induce the generation of bioactive phospholipids, calcium influx into the cytoplasm and cell proliferation, suggesting that this molecule can be used as a bioactive tool for experimental protocols in cell biology.

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders

“…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).…”

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

“…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]. The most common activity assay for these enzymes detects PLD activity by monitoring choline release from substrate.…”

Whole venom of Loxosceles similis activates caspases-3, -6, -7, and -9 in human primary skin fibroblasts

“…Because this cascade can be activated by interactions outside the cell, we speculate that the venom of L. similis may interact with receptors on the cell surface. Many studies have shown the binding of Loxosceles venom toxins upon the membrane of diverse cell types, showing direct induction of cytotoxicity and supporting the hypothesis of “planted antigens” to different components of cell structures (Chaim et al, 2006; Chaves-Moreira et al, 2009; Dias-Lopes et al, 2010; Wille et al, 2013). Further research should be done to investigate which venom compounds can activate the caspases cascade investigated.…”