Brown Spider Venom Phospholipase-D Activity upon Different Lipid Substrates

Abstract

Brown spider envenomation results in dermonecrosis, characterized by an intense inflammatory reaction. The principal toxins of brown spider venoms are phospholipase-D isoforms, which interact with different cellular membrane components, degrade phospholipids, and generate bioactive mediators leading to harmful effects. The Loxosceles intermedia phospholipase D, LiRecDT1, possesses a loop that modulates the accessibility to the active site and plays a crucial role in substrate. In vitro and in silico analyses were performed to determine aspects of this enzyme’s substrate preference. Sphingomyelin d18:1/6:0 was the preferred substrate of LiRecDT1 compared to other Sphingomyelins. Lysophosphatidylcholine 16:0/0:0 was preferred among other lysophosphatidylcholines, but much less than Sphingomyelin d18:1/6:0. In contrast, phosphatidylcholine d18:1/16:0 was not cleaved. Thus, the number of carbon atoms in the substrate plays a vital role in determining the optimal activity of this phospholipase-D. The presence of an amide group at C2 plays a key role in recognition and activity. In silico analyses indicated that a subsite containing the aromatic residues Y228 and W230 appears essential for choline recognition by cation-π interactions. These findings may help to explain why different cells, with different phospholipid fatty acid compositions exhibit distinct susceptibilities to brown spider venoms.

A Brief Overview of the Toxic Sphingomyelinase Ds of Brown Recluse Spider Venom and Other Organisms and Simple Methods To Detect Production of Its Signature Cyclic Ceramide Phosphate

“…Most of the subsequent studies used easier assays that followed release of the product choline using commercially available kits, such as the Amplex TM Red Phospholipase D Assay Kit, (Molecular Probes/Invitrogen, Eugene, OR) (Binford et al, 2009;Chaves-Moreira et al, 2023;Mariutti et al, 2017;Zobel-Thropp et al, 2012). Another assay (Felicori et al, 2006;Young and Pincus, 2001) utilized TNPAL-SM (trinitrophenylaminolauryl-SM), which is cleaved to release TNPAL-Cer that is extracted into a heptane-rich phase for detection of its absorbance (Gatt et al, 1981).…”

“…Comparison of LPC and LPE with the α-and β-clade enzymes of the preceding paragraph found the same headgroup selectivities--i.e., choline was preferred by αclade PLD whereas the β-clade enzymes utilize both, or only ethanolamine. One of the factors that accounts for the preference for choline headgroups in the α-clade is a "cage" of three tyrosines that establish cation-π interactions with the choline (Chaves-Moreira et al, 2023;Moutoussamy et al, 2022). This interaction was seen in both substrate selection and in the binding of the enzymes to liposomes of different composition and molecular dynamics simulations.…”

“…In addition to headgroup preferences, SMase D/PLD also display differences in activity depending on the lipid backbones (Chaves-Moreira et al, 2023). Assays of the recombinant SMase D/PLD from L. intermedia venom, LiRecDT1, using SM and LPC with different fatty acyl chains found highest activity with d18:1/C6:0 SM (i.e., an 18-carbon sphingosine with an N-acyl fatty acid of 6 carbons) and decreasing activities as the N-acyl chain increased in length.…”