Agelotoxin: a Phospholipase A2 from the Venom of the Neotropical Social

Toxicon 38 (2000) 1367±1379 www.elsevier.com/locate/toxicon Agelotoxin: a phospholipase A2 from the venom of the neotropical social wasp cassununga (Agelaia pallipes pallipes) (Hymenoptera-Vespidae) Helena Costa, Mario Sergio Palma* Center of Study of Social Insects (CEIS), Department of Biology, Institute of Biosciences of Rio Claro, University of SaÄo Paulo State (UNESP), CEP 13506-900 Rio Claro, SP, Brazil Received 16 April 1999; accepted 23 August 1999 Abstract The neotropical wasp Agelaia pallipes pallipes is aggressive and endemic in southeast of Brazil, where very often it causes stinging accidents in rural areas. By using gel ®ltration on Sephadex G-100, followed by high performance reversed phase chromatography in a C-18 column under acetonitrile/water gradient, the agelotoxin was puri®ed: a toxin presenting phospholipase A2 (PLA2) activity, which occurs under equilibrium of three dierent aggregation states: monomer (mol. wt 14 kDa), trimer (mol. wt 42 kDa) and pentamer (mol. wt 74 kDa). The enzyme presents high sugar contents attached to the protein chain (22% [w/w]) and a transition of the values of pH optimum for the substrate hydrolysis from 7.5 to 9.0, under aggregation from monomer to pentamer. All the aggregation states present Michaelian steady-state kinetic behavior and the monomer polymerization caused a decreasing of phospholipasic activity due a non-competitive inhibition promoted by the formation of a quaternary structure. The PLA2 catalytic activity of agelotoxin changes according to its state of aggregation (from 833 to 12533 mmol mg1 min1) and both the monomeric and oligomeric forms present lowest activities than the PLA2 from Apis mellifera venom and hornetin from Vespa basalis. Agelotoxin is also a very potent direct hemolysin; the monomer of agelotoxin presented hemolytic actions until 200 times higher than the PbTx from P. paulista, 740 times higher than the PLA2 from A. mellifera, 570 times higher than that of neutral PLA2 from N. nigricolis and about 1250 times than that of cardiotoxin from Naja naja atra venom. 7 2000 Elsevier Science Ltd. All rights reserved. * Corresponding author. Fax: +55-19-524-0227. 0041-0101/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S0041-0101(99)00199-3 1368 H. Costa, M.S. Palma / Toxicon 38 (2000) 1367±1379 1. Introduction Hymenoptera venoms are complex mixtures of biochemically and pharmacologically active components such as biogenic amines, peptides and proteins (Nakajima, 1986). The composition of vespid venoms have been subject to little investigation, since the production of venoms by the social wasps is very reduced and there is a limited avaiability of vespid venoms as raw materials. It has been shown that the Vespinae venoms contain many dierent components such as phospholipases A and B, hyaluronidases, acid phosphatases, proteases and nucleotidases (Nakajima, 1986). The compositions of the various Vespinae venoms are rather similar each other (Habermann, 1972), however, very few is known about the neotropical Polistinae venoms. It has been demonstrated that vespid venoms frequently cause allergic reactions in humans (Homan, 1985; Reisman and Osur, 1987; Castro et al., 1994) and the PLA2 are recognized as one of the major allergens from these venoms (Homan, 1978; 1985). PLA2 (E.C. 3.1.1.4) catalyzes the speci®c hydrolysis of ester bonds at the C2 position of 1,2-diacyl-3-sn-glycerophospholipids into their corresponding lyso compounds with release of free fatty acids. Thus, PLA2 is able to disrupt the phospholipid packings from several types of biological membranes, leading to pore formation and/or cell lysis (Dotimas and Hider, 1987). Recently we described the puri®cation and biochemical characterization of the polybitoxins from the venom of the South American social wasp Polybia paulista, presenting PLA2 activity and very potent hemolytic actions in washed red cells (Oliveira and Palma, 1998). The aim of present paper is to describe the puri®cation and some biochemical properties of a novel toxin from the venom of a neotropical social wasp presenting PLA2 and hemolytic activities: the agelotoxin from Agelaia pallipes pallipes, which naturally occurs in dierent states of aggregation (monomer, trimer and pentamer). This toxin is probably among the most powerful hemolysins of animal venoms already known. 2. Material and methods 2.1. Biological material and venom extraction Workers of A. pallipes pallipes were captured in the University Campus, at Rio Claro, SP, southeast of Brazil. The freshly collected wasps were immediately frozen and dissected. The venom reservoirs were removed from the sting apparatuses by pulling with forceps and cutting with microscissors, under a stereomicroscope, minimizing contamination from extraneous tissues. The reservoirs were then carefully washed in a small volume of isotonic solution, thawed, punctured, followed by several washings with distilled water to extract the venom and followed by centrifugation at 12,000g, during 15 min at 48C; the supernatant was freeze dried and kept at 808C until be used. H. Costa, M.S. Palma / Toxicon 38 (2000) 1367±1379 1369 2.2. Protein assay Protein was determined by the method of Lowry (Hartree, 1972), using BSA as standard. 2.3. Determination of phospholipase speci®city In order to determine the type of phospholipase activity the crude venom of A. pallipes pallipes and the puri®ed toxins were incubated at 378C in presence of natural and synthetic phospholipids as substrates: egg phosphatydylcholine, egg lysophosphatydylcholine, 1-stearoyl-2-oleoyl-3-sn-glycero phosphoryl choline and 1-oleoyl-2-stearoyl-3-sn-glycerophosphorylcholine (Sigma Chem. Co.). Fine suspensions of phospholipids were prepared by sonication in 1 mM Tris±HCl (pH 7.9), containing 100 mM sodium chloride, 20 mM potassium chloride, 10 mM calcium chloride and 0.5% (v/v) Triton X-100 in an ultrasonic bath. For the purpose of identi®cation of products formed by phospholipase digestion, the same buer as described above was used, but the concentration of Tris±HCl was increased to 50 mM. The digests were examined by thin-layer chromatography (TLC) on silica gel plates (Whatman LK6DF) as described by King et al. (1984): 35 ml (1.5 mg ml1 phospholipid) was applied to the pre-adsorbent zone and the plate was developed in chloroform±methanol±0.1 N HCl (60:35:5). The spots were visualized by exposure to iodine vapor for detection of monoacyl phospholipids. The digests were also examined directly for the presence of saturated fatty acids by chromatography on freshly prepared silica gel plates which had been dipped in 5% (w/v) AgNO3 and then dried at 1108C for 1 h. After developing in presence of hexane±diethyl ether±acetic acid (70:30:1), the spots were visualized under UV light after spraying with 0.2% (w/v) dichloro¯uorescein in ethanol. The procedure described above was also applied to PLA2 from honeybee venom (Sigma Chem. Co.) in order to be used as a control experiment. 2.4. Phospholipase A2 activity The assays were routinely carried out by using a spectrophotometric method based on pH change due to the liberation of fatty acids, as described by Araujo and Radvanyi (1987). The reaction medium contained 15 mmol phosphatydylcholine, 18 mmol Triton X-100, 5 mmol calcium chloride, 80 mmol phenol red and 7.5 mmol Tris in a ®nal volume of 2.5 ml, at pH 7.9. The absorbance was initially read at 558 nm against a proper reference; the reaction was initiated by addition of either crude venom or puri®ed toxins. The decrease in the absorbance of phenol red, caused by the acidi®cation of medium was measured after 5 min minutes of incubation at 378C. The DA558 was proportional to the liberation of fatty acids in the assay conditions. One unit of PLA2 activity was de®ned as the amount of enzyme necessary to hydrolyse 1 mmol phosphatidylcholine h1 in 1 ml of the reactional medium at pH 7.9 and 378C. 1370 H. Costa, M.S. Palma / Toxicon 38 (2000) 1367±1379 2.5. Hemolysis Direct hemolytic activity was assayed on washed red cells of mouse by slight modi®cations in the procedure described by Ho and Ko (1988). The toxins were dissolved in 0.14 M saline Tris (0.01 M) buered at pH 7.4 and the red cells, suspended in the same solution at the hematocrit 50%, were mixed and incubated at 378C for 60 min. The hemolysis was stopped by addition of cooled (48C) Tris buered saline to a ®nal volume of 5 ml and the degree of hemolysis was determined by measuring the released hemoglobin at 545 nm. Similarly, control samples were incubated in absence of the toxins, by hemolysing the red cells in water under similar conditions. The hemolytic potency was expressed as percent of hemolysis, assuming the lysis in water to be equal 100% in the given incubation time. The determinations above were run in triplicate at the end of ®ve independent preparations. The results are expressed as means2S.E. 2.6. Agelotoxin puri®cation All puri®cation steps were carried out at 0±48C, unless speci®ed. The freeze dried venom (11.65 mg) was solubilized in 5 mM ammonium acetate pH 6.8 and applied to a Sephadex G-100 (51.0 Â 2.5 cm) previously equilibrated with the same buer. Elution was performed with 5 mM ammonium acetate pH 6.8 at a ¯ow rate of 12 ml h1 and fractions of 3 ml were collected in presence of 100 mlof 10% (v/v) glycerol (previously added to each collection tube). Protein elution was monitored along the pro®le of elution by measuring the absorbance at 280 nm and PLA2 activity assayed as described above for each collected fraction. The fractions presenting phospholipasic activity were pooled and freeze dried.

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