Brown recluse spider (Loxosceles reclusa) venom phospholipase D (PLD) generates lysophosphatidic acid (LPA)

Abstract

Envenomation by the brown recluse spider (Loxosceles reclusa) may cause local dermonecrosis and, rarely, coagulopathies, kidney failure and death. A venom phospholipase, SMaseD (sphingomyelinase D), is responsible for the pathological manifestations of envenomation. Recently, the recombinant SMaseD from Loxosceles laeta was demonstrated to hydrolyse LPC (lysophosphatidylcholine) to produce LPA (lysophosphatidic acid) and choline. Therefore activation of LPA signalling pathways may be involved in some manifestations of Loxosceles envenomation. To begin investigating this idea, we cloned a full-length cDNA encoding L. reclusa SMaseD. The 305 amino acid sequence of the L. reclusa enzyme is 87, 85 and 60% identical with those of L. arizonica, L. intermedia and L. laeta respectively. The recombinant enzyme expressed in bacteria had broad substrate specificity. The lysophospholipids LPC, LPI (18:1-1-oleyol lysophosphatidylinositol), LPS, LPG (18:1-1-oleoyl-lysophosphatidylglycerol), LBPA (18:1-1-oleoyl-lysobisphosphatidic acid) (all with various acyl chains), lyso-platelet-activating factor (C16:0), cyclic phosphatidic acid and sphingomyelin were hydrolysed, whereas sphingosylphosphorylcholine, PC (phosphatidylcholine; C22:6, C20:4 and C6:0), oxidized PCs and PAF (platelet-activating factor; C16:0) were not hydrolysed. The PAF analogue, edelfosine, inhibited enzyme activity. Recombinant enzyme plus LPC (C18:1) induced the migration of A2058 melanoma cells, and this activity was blocked by the LPA receptor antagonist, VPC32183. The recombinant spider enzyme was haemolytic, but this activity was absent from catalytically inactive H37N (His37-->Asn) and H73N mutants. Our results demonstrate that Loxosceles phospholipase D hydrolyses a wider range of lysophospholipids than previously supposed, and thus the term 'SMaseD' is too limited in describing this enzyme.

Figure 1. Complete cDNA and translated peptide sequence of L. reclusa SMaseD

Total RNA was extracted from the venom glands of L. reclusa (SpiderPharm). The degenerate primers designed from the sequence of SMaseD from related species, L. arizonica, were used for amplification of the initial fragment of SMaseD (580–963 bp of full ORF). The full cDNA sequence was identified by 3′- and 5′-RACE. Underlined, putative signal peptide; boxed, catalytic histidine residues, potential N-glycosylation site.

Figure 2. Peptide sequence alignment of SMaseDs

Intermedia, L. intermedia SMaseD; Reclusa, L. reclusa SMaseD; Arizonica, L. arizonica SMaseD; Laeta, L. laeta SMaseD; CP–PLD, C. pseudotuberculosis PLD. The GCG program, PRETTY, was used for the analysis. Solid line box, putative signal sequences; ●, histidine residues essential for enzyme activity. The 305 amino acid peptide sequence of L. reclusa enzyme is 87, 85 and 60% identical with those of L. arizonica, L. intermedia and L. laeta respectively.

Figure 3. The expression of recombinant SMaseD in a bacterial expression system

(A) Schematic diagram of recombinant SMaseD construct expressed in bacteria. EK, enterokinase cleavage site. (B) Coomassie Blue staining (lane 1, 2.4 μg of SMaseD loaded) and Western blotting with S-protein–HRP conjugate (lane 2, 0.1 μg of SMaseD loaded) of the SMaseD purified from the bacterial cell lysates by sequential affinity purification using Ni-Sepharose (Amersham Biosciences). Lane M, molecular mass standards.

Figure 4. Substrate specificity of the purified recombinant SMaseD

(A) LysoPLD activity was assayed by measuring hydrolysis of various lysophospholipids with choline head groups. Briefly, the substrate in Hepes-buffered saline was incubated with SMaseD (125 ng) for 1 h at 37 °C in a total volume of 0.1 ml. The amount of choline released from the reactions was measured as described in the Experimental section. K_m and V_max values were calculated using the Michaelis–Menten equation. Results shown are from a representative experiment of at least three independent assays. (B) Hydrolysis of non-choline lysophospholipids and cyclic phosphatidic acid by SMaseD was measured. The LPA produced from the reaction of 1 mM substrate with SMaseD (1 ng) was measured by direct LPA assay described in the Experimental section. LPA18:1 was used as a positive control, whereas lysoPAF was used as negative control for the assay. (Although SMaseD hydrolyses lysoPAF, the product, ether LPA, cannot be detected because lysophospholipase in LPA assay enzyme cocktail does not cleave ether-linkage.) The results shown are the means±S.E.M. for triplicate experiments.

Figure 5. His³⁷ and His⁷³ are required for enzyme activity of the recombinant L. reclusa SMaseD

(A) The mutated amino acids are indicated by large bold letters. Potential signal peptide is underlined. (B) LysoPLD activity of the wild-type and mutant SMaseD proteins (2 ng/reaction) expressed in bacteria. Briefly, 1 mM LPC18:1 in Hepes-buffered saline was incubated with SMaseD for 1.5 h at 37 °C in a total volume of 0.1 ml. The amount of choline released from the reactions was measured as described in the Experimental section. The presence of the wild-type and mutant proteins (100 ng/lane) was confirmed by Western blotting using S-protein–HRP conjugate. The results shown are the means±S.E.M. for triplicate experiments.

Figure 6. Recombinant L. reclusa SMaseD induced the migration of human melanoma A2058 cells

Purified wild-type, H37N and H73N recombinant SMaseDs were used for the induction of A2058 cell migration as described in the Experimental section. Lipid concentrations were: LPA18:1, 50 nM; LPC18:1, 2.5 μM; and VPC32183, 10 μM. WT and mutant SMaseDs (5 ng) and LPC18:1 (1 mM) were pre-incubated for 2 h at 37 °C in a total volume of 20 μl after which 5 μl of the final reaction mixture was added to the 2 ml of assay buffer in the lower chamber of transwell. The results shown are the means±S.E.M. for duplicate experiments.

Figure 7. The purified recombinant L. reclusa SMaseD showed dose-dependent erythrocyte lysis

Human erythrocytes (freshly donated) were washed three times with VBS²⁺ and resuspended in the same buffer at 2%. The cells were incubated with the purified recombinant SMaseDs [indicated amounts of wild-type SMaseD (A) and 2 ng of wild-type and mutant SMaseD (B)] for 30 min at 37 °C. Control samples were incubated with VBS²⁺ buffer alone. After washing with VBS²⁺, the cells were resuspended to the original volume of VBS²⁺ (0.1 ml) and mixed with 0.1 ml of autologous plasma. After incubation for 1 h at 37 °C, non-lysed cells were discarded by centrifugation (1000 g and 1 min); the light absorbance of the released haemoglobin was measured at 405 nm. Background or total cell lysis was evaluated by incubation of erythrocytes with VBS²⁺ (no SMaseD) or distilled water (dH₂O) respectively. The results shown are the means±S.E.M. for duplicate experiments.

Figure 8. Edelfosine inhibits lysoPLD activity of the recombinant L. reclusa SMaseD

Wild-type SMaseD (1 ng) was incubated with 1 mM LPC18:1 and various concentrations of edelfosine for 1 h at 37 °C in a total volume of 0.1 ml of Hepes-buffered saline. The amount of choline released from the reactions was measured by the enzymatic choline detection assay as described in the Experimental section. The results shown are the means±S.E.M. for triplicate experiments.