Structural and Biological Characterization of Three Novel Mastoparan Peptides from the Venom of the Neotropical Social Wasp Protopolybia Exigua (Saussure)
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Toxicon 45 (2005) 101–106 www.elsevier.com/locate/toxicon Structural and biological characterization of three novel mastoparan peptides from the venom of the neotropical social wasp Protopolybia exigua (Saussure) Maria Anita Mendes, Bibiana Monson de Souza, Mario Sergio Palma* CEIS-Department of Biology, IBRC-UNESP (CAT-CEPID/FAPESP), Institute of Immunological Investigations (Millennium Institute-MCT/CNPq), Rio Claro, SP 13506-900, Brazil Available online 11 November 2004 Abstract The venom of the Neotropical social wasp Protopolybia exigua(Saussure) was fractionated by RP-HPLC resulting in the elution of 20 fractions. The homogeneity of the preparations were checked out by using ESI-MS analysis and the fractions 15, 17 and 19 (eluted at the most hydrophobic conditions) were enough pure to be sequenced by Edman degradation chemistry, resulting in the following sequences: Protopolybia MPI I-N-W-L-K-L-G-K-K-V-S-A-I-L-NH2 Protopolybia-MP II I-N-W-K-A-I-I-E-A-A-K-Q-A-L-NH2 Protopolybia-MP III I-N-W-L-K-L-G-K-A-V-I-D-A-L-NH2 All the peptides were manually synthesized on-solid phase and functionally characterized. Protopolybia-MP I is a hemolytic mastoparan, probably acting on mast cells by assembling in plasma membrane, resulting in pore formation; meanwhile, the peptides Protopolybia-MP II and -MP III were characterized as a non-hemolytic mast cell degranulator toxins, which apparently act by virtue of their binding to G-protein receptor, activating the mast cell degranulation. q 2004 Elsevier Ltd. All rights reserved. Keywords: Social wasp; Mastoparan; Hemolysis; Antimicrobial peptide; G-protein receptor 1. Introduction serotonin, norepinephrine, hyaluronidase, histidine decar- boxylase, phospholipase A2 and several polycationic Stinging accidents caused by social wasps and bees, peptides and proteins acting together to produce the generally produce severe pain, local damage and occasion- biological effects (Argiolas and Pisano, 1985). The poly- ally death in large vertebrates including man, caused by cationic peptides are involved with the occurrence of action of their venoms (Nakajima, 1984, 1986). inflammation, mainly due to mast cell degranulation, The chemical constituents of these venoms have been leading to the release of histamine from basophilic well documented for the most of social wasps species granulocytes and/or serotonin from platelets (Hancock and endemic from temperate and cold climates: acetylcholine, Diamond, 2000). The main structural features of these peptides: amphipathic, a-helical conformation, permit to some of them to assemble in the zwiterionic membranes of * Corresponding author. Tel.: C55 19 35264163; fax: C55 19 mammalian cells, producing pores and making these 3534 8523. peptides to act as hemolysins (Gallo and Huttner, 1998; E-mail address: [email protected] (M.S. Palma). Krishnakumari and Nagaraj, 1997). 0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2004.09.015 102 M.A. Mendes et al. / Toxicon 45 (2005) 101–106 In social wasp venoms the most important cytotrophic 2.2. Materials and instruments principles are the mast cell degranulator peptides, known as mastoparans (Nakajima, 1986), which are tetradecapeptides Acetonitrile (HPLC grade) was obtained from presenting from seven to ten hydrophobic amino acid ALDRICH, and trifluoroacetic acid (TFA) analytical- residues and from two to four lysine residues in their reagent grade, was from CARLO ERBA. For preparation primary sequences. They constitute the most abundant of the eluents, high-purity water (Nanopure Barnstead) was group of peptides in the venoms of social wasps (Hirai et al., used. The purification was carried out in a HPLC system 1979). Among the biological activities of mastoparans, also (SHIMADZU), model. CBM-10A, equipped with a diode may be included the activation of phospholipase-A2, array detector (SHIMADZU), model SPD-M 10A. Mass phospholipase-C, G-proteins and guanylate cyclase spectra, were acquired on an ESI-Triple Quadrupole mass (Higashijima et al., 1990; Song et al., 1993). spectrometer instrument (MICROMASS, UK), model. Even though the large number of species of social wasps Quatro II. The amino acid sequence was performed in an occur in the tropical/subtropical regions of the planet, very automatic peptide sequencer (SHIMADZU) model. PPSQ- few is known about the venom composition of these insects, 21 A. specially those from the Neotropics (Dohtsu et al., 1992, To perform the biological activities, NaCl, KCl and 1993). Social wasps cause very frequent stinging accidents CaCl2 (MERCK), NaH2PO4,KH2PO4 and glucose in the men, followed by a series of pharmacological, (SYNTH), Liquemine (heparin, ROCHE), BSA and p- inflammatory and immunopathological manifestations of nitrophenyl-N-acetyl-b-d-glucosaminidine (SIGMA), Tri- the stung victims (Nakajima, 1986). However, the venoms ton X-100 (ALDRICH) and triphenyltetrazolium chloride from the social wasps of the Neotropical regions have been (MALLINCKRODT) were used. poorly investigated, limiting our toxinological knowledge The peptides were synthesized using Fmoc-aa-OH, and creating many difficulties to handle the proper care of Novasyn TGR resin, which were acquired from NOVA- the patients after envenomation accidents with these wasps. BIOCHEM; trifluoroacetic acid, 1,2- ethanedithiol, anisole, Protopolybia exigua is an aggressive wasp and causes phenol and ethyl ether were purchased from ALDRICH. frequent stingings in the people living in the regions where this insect is endemic; thus, the biochemical and pharma- 2.3. Sample preparation and purification cological characterization of the most abundant peptide components of this venom will contribute both to a better The biological material from the dried extract was understanding about the composition and to the knowledge solubilized in 5%(v/v) MeCN in a concentration of of the envenomation mechanisms of this venom. 100 mg/ml and chromatographed under RP-HPLC with a ! The present work is reporting the structural and functional SHISEIDO Nucleosil C-18 (ODS) column (250 10.0 mm; characterization of three biologically active peptides ident- 5 mm), at a flow rate of 2 ml/min, by using a gradient from 5 ified in the venom of the wasp P. exigua. They were purified, to 60% (v/v) MeCN (containing 0.1% TFA), at 30 8C, had their molecular masses determined by ESI-MS, were during 45 min. The elution was monitored at 215 nm with a sequenced by Edman degradation chemistry and functionally UV-DAD detector (SHIMADZU, mod. SPD-M10A) and characterized. One of the peptides was a hemolytic each peak eluted was manually collected into plastic vials of mastoparan; meanwhile, the two other ones interact with 2 ml. The peaks of interest were resubmitted to chromatog- and activate Pertussis toxin-sensitive G-proteins in vitro in a raphy by using RP-HPLC with a SHISEIDO Nucleosil C-18 ! m similar manner to that of G-protein coupled receptors, (ODS) column (250 4.6 mm; 5 m), under isocratic causing an activation of a cascade of molecular events, elution with 40% (v/v) MeCN (containing 0.1% TFA) at a flow rate of 700 ml/min, during 30 min. at 30 8C. The elution which, result in mast cell degranulation. was monitored at 215 nm and each fraction was manually collected into plastic vials of 2 ml. The homogeneity of the preparation was checked through ESI-MS analysis. 2. Material and methods 2.4. Mass spectrometry 2.1. Biological material Mass spectra were acquired on a triple quadrupole (Quatro II) mass spectrometer instrument (Micromass, UK), The wasps Protopolybia exı´gua (Saussure)were col- equipped with a standard electrospray probe, adjusted to ca. lected in Rio Claro-SP, southeast Brazil. The collected 5 ml minK1. During all experiments the source temperature wasps were immediately frozen and stored at K20 8C. The was maintained at 80 8C and the needle voltage at 3.6 kV, venom was obtained by wasps dissection with surgical applying a drying gas flow (nitrogen) of 200 l h-1 and a microscissors. The venom reservoirs were removed and the nebulizer gas flow (nitrogen) of 20 l hK1.Themass venom extracted with 1:1 acetonitrile / ultra pure water. The spectrometer was calibrated with intact horse heart extract was lyophilized and kept at K20 8C. myoglobin and its typical cone-voltage induced fragments. M.A. Mendes et al. / Toxicon 45 (2005) 101–106 103 The cone sample to skimmer lens voltage, controlling the KCl (MERCK), 0.043 g NaH2PO4 (SYNTH), 0.048 g ion transfer to the mass analyzer, was maintained at 30 V. KH2PO4 (SYNTH), 0.10 g glucose (SYNTH), 0.10 g BSA About 50 pmol of each sample was injected into electro- (SIGMA), 90 mL CaCl2 (MERCK) 2 M solution, 50 ml spray transport solvent. The ESI mass spectra were obtained Liquemine (heparin, ROCHE) in a 100 ml water. Mast cells in the continuous acquisition mode, scanning from m/z 100 were incubated in the presence of peptides during 15 min at to 2000 with a scan time of 7s. 37 8C. After centrifugation, the supernatants were sampled for b-D-glucosaminidase assay. Briefly, 50 mL of the mast 2.5. Peptide sequencing cell suspensions were added to 50 mL of the substrate [3 mg of p-nitrophenyl-N-acetyl-b-D-glucosaminidine (SIGMA) The amino acid sequence was performed by using a gas- dissolved in 10 ml of 200 mM sodium citrate, pH 4.5 phase sequencer PPSQ-21 A (Shimadzu) based on auto- solution] and incubated during 6 hours at 37 8C. The mated Edman degradation chemistry. reaction was interrupted by addition of 150 ml of 0.2 M TRIS solution and the absorbance of colored product was 2.6. Peptide synthesis assessed at 405 nm in a microtitre plate reader (Biotrack, AMERSHAM BIOSCIENCE). In case of Pertussis Toxin The peptides were prepared by step-wise manual solid- [Islets Activating Protein (IAP)] treatment, the mast cells phase synthesis using N-9-fluorophenylmethoxy-carbonyl were previously incubated with 1 ug/ml IAP at 37 8C during (Fmoc) chemistry with Novasyn TGS resin (NovaBio- 60 min.