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 Stinging accidents caused by social wasps and bees, generally produce severe pain, local damage and occasionally death in large vertebrates including man, caused by action of their venoms (Nakajima, 1984, 1986). The chemical constituents of these venoms have been well documented for the most of social wasps species endemic from temperate and cold climates: acetylcholine,

* Corresponding author. Tel.: C55 19 35264163; fax: C55 19 3534 8523. E-mail address: [email protected] (M.S. Palma). 0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2004.09.015

serotonin, norepinephrine, hyaluronidase, histidine decarboxylase, phospholipase A2 and several polycationic peptides and proteins acting together to produce the biological effects (Argiolas and Pisano, 1985). The polycationic peptides are involved with the occurrence of inflammation, mainly due to mast cell degranulation, leading to the release of histamine from basophilic granulocytes and/or serotonin from platelets (Hancock and 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 mammalian cells, producing pores and making these peptides to act as hemolysins (Gallo and Huttner, 1998; Krishnakumari and Nagaraj, 1997).

102

M.A. Mendes et al. / Toxicon 45 (2005) 101–106

In social wasp venoms the most important cytotrophic principles are the mast cell degranulator peptides, known as mastoparans (Nakajima, 1986), which are tetradecapeptides presenting from seven to ten hydrophobic amino acid residues and from two to four lysine residues in their primary sequences. They constitute the most abundant group of peptides in the venoms of social wasps (Hirai et al., 1979). Among the biological activities of mastoparans, also may be included the activation of phospholipase-A2, phospholipase-C, G-proteins and guanylate cyclase (Higashijima et al., 1990; Song et al., 1993). Even though the large number of species of social wasps occur in the tropical/subtropical regions of the planet, very few is known about the venom composition of these insects, specially those from the Neotropics (Dohtsu et al., 1992, 1993). Social wasps cause very frequent stinging accidents in the men, followed by a series of pharmacological, inflammatory and immunopathological manifestations of the stung victims (Nakajima, 1986). However, the venoms from the social wasps of the Neotropical regions have been poorly investigated, limiting our toxinological knowledge and creating many difficulties to handle the proper care of the patients after envenomation accidents with these wasps. Protopolybia exigua is an aggressive wasp and causes frequent stingings in the people living in the regions where this insect is endemic; thus, the biochemical and pharmacological characterization of the most abundant peptide components of this venom will contribute both to a better understanding about the composition and to the knowledge of the envenomation mechanisms of this venom. The present work is reporting the structural and functional characterization of three biologically active peptides identified in the venom of the wasp P. exigua. They were purified, had their molecular masses determined by ESI-MS, were sequenced by Edman degradation chemistry and functionally characterized. One of the peptides was a hemolytic mastoparan; meanwhile, the two other ones interact with and activate Pertussis toxin-sensitive G-proteins in vitro in a similar manner to that of G-protein coupled receptors, causing an activation of a cascade of molecular events, which, result in mast cell degranulation.

2. Material and methods 2.1. Biological material The wasps Protopolybia exı´gua (Saussure)were collected in Rio Claro-SP, southeast Brazil. The collected wasps were immediately frozen and stored at K20 8C. The venom was obtained by wasps dissection with surgical microscissors. The venom reservoirs were removed and the venom extracted with 1:1 acetonitrile / ultra pure water. The extract was lyophilized and kept at K20 8C.

2.2. Materials and instruments Acetonitrile (HPLC grade) was obtained from ALDRICH, and trifluoroacetic acid (TFA) analyticalreagent grade, was from CARLO ERBA. For preparation of the eluents, high-purity water (Nanopure Barnstead) was used. The purification was carried out in a HPLC system (SHIMADZU), model. CBM-10A, equipped with a diode array detector (SHIMADZU), model SPD-M 10A. Mass spectra, were acquired on an ESI-Triple Quadrupole mass spectrometer instrument (MICROMASS, UK), model. Quatro II. The amino acid sequence was performed in an automatic peptide sequencer (SHIMADZU) model. PPSQ21 A. To perform the biological activities, NaCl, KCl and CaCl 2 (MERCK), NaH 2PO4, KH2 PO4 and glucose (SYNTH), Liquemine (heparin, ROCHE), BSA and pnitrophenyl-N-acetyl-b-d-glucosaminidine (SIGMA), Triton X-100 (ALDRICH) and triphenyltetrazolium chloride (MALLINCKRODT) were used. The peptides were synthesized using Fmoc-aa-OH, Novasyn TGR resin, which were acquired from NOVABIOCHEM; trifluoroacetic acid, 1,2- ethanedithiol, anisole, phenol and ethyl ether were purchased from ALDRICH. 2.3. Sample preparation and purification The biological material from the dried extract was solubilized in 5%(v/v) MeCN in a concentration of 100 mg/ml and chromatographed under RP-HPLC with a SHISEIDO Nucleosil C-18 (ODS) column (250!10.0 mm; 5 mm), at a flow rate of 2 ml/min, by using a gradient from 5 to 60% (v/v) MeCN (containing 0.1% TFA), at 30 8C, during 45 min. The elution was monitored at 215 nm with a UV-DAD detector (SHIMADZU, mod. SPD-M10A) and each peak eluted was manually collected into plastic vials of 2 ml. The peaks of interest were resubmitted to chromatography by using RP-HPLC with a SHISEIDO Nucleosil C-18 (ODS) column (250!4.6 mm; 5 mm), under isocratic 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 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. 2.4. Mass spectrometry Mass spectra were acquired on a triple quadrupole (Quatro II) mass spectrometer instrument (Micromass, UK), equipped with a standard electrospray probe, adjusted to ca. 5 ml minK1. During all experiments the source temperature was maintained at 80 8C and the needle voltage at 3.6 kV, applying a drying gas flow (nitrogen) of 200 l h-1 and a nebulizer gas flow (nitrogen) of 20 l hK1. The mass spectrometer was calibrated with intact horse heart myoglobin and its typical cone-voltage induced fragments.

M.A. Mendes et al. / Toxicon 45 (2005) 101–106

The cone sample to skimmer lens voltage, controlling the ion transfer to the mass analyzer, was maintained at 30 V. About 50 pmol of each sample was injected into electrospray transport solvent. The ESI mass spectra were obtained in the continuous acquisition mode, scanning from m/z 100 to 2000 with a scan time of 7s. 2.5. Peptide sequencing The amino acid sequence was performed by using a gasphase sequencer PPSQ-21 A (Shimadzu) based on automated Edman degradation chemistry. 2.6. Peptide synthesis The peptides were prepared by step-wise manual solidphase synthesis using N-9-fluorophenylmethoxy-carbonyl (Fmoc) chemistry with Novasyn TGS resin (NovaBiochem). Side-chain protective groups included t-butyl for serine and t-butoxycarbonyl for lysine. Cleavages of the peptides-resin complexes were performed by treatment with trifluoroacetic acid/1,2- ethanedithiol / anisole/ phenol/ water (82.5:2.5:5:5:5 by volume), using 10 ml per gram of complex at room temperature during 2 h. After filtering to remove the resin, ethyl ether at 4 8C was added to the soluble material causing precipitation of the crude peptides, which were collected as a pellet after a centrifugation at 1000g, during 15 min at room temperature. The crude peptides were solubilized in water and chromatographed under RP-HPLC using a semi-preparative column (SHISEIDO C18, 250!10 mm, 5 mm), under isocratic elution with 40% (v/v) acetonitrile in water [containing 0.1% (v/v) trifluoroacetic] at a flow rate of 2 ml/min. The elution was monitored at 215 nm with a UV-DAD detector (SHIMADZU, mod. SPD-M10A) and each fraction eluted was manually collected into plastic vials of 2 ml. The homogeneity and correct sequence of the synthetic peptides were evaluated by comparing their retention times in the RP-HPLC under isocratic conditions with 40% (v/v) MeCN [containing 0.1% (v/v) TFA] against the natural peptides; ESI-MS analysis was also used to check the peptides purity (considering as criteria the presence of a single molecular ion, equivalent the expected molecular mass for the amino sequence of each peptide); and finally the sequence of the synthetic material was confirmed by automated sequencing based on Edman degradation chemistry. 2.7. Biological assays 2.7.1. Mast cell degranulation Mast cell degranulation was determined by measuring the release of b-D-glucosaminidase, which co-localizes with histamine, as proposed by Hide et al. (1993). Mast cells were obtained by peritoneal washing of adult Wistar rats with a solution containing 0.877 g NaCl (MERCK), 0.028 g

103

KCl (MERCK), 0.043 g NaH2PO4 (SYNTH), 0.048 g KH2PO4 (SYNTH), 0.10 g glucose (SYNTH), 0.10 g BSA (SIGMA), 90 mL CaCl2 (MERCK) 2 M solution, 50 ml Liquemine (heparin, ROCHE) in a 100 ml water. Mast cells were incubated in the presence of peptides during 15 min at 37 8C. After centrifugation, the supernatants were sampled for b-D-glucosaminidase assay. Briefly, 50 mL of the mast cell suspensions were added to 50 mL of the substrate [3 mg of p-nitrophenyl-N-acetyl-b-D-glucosaminidine (SIGMA) dissolved in 10 ml of 200 mM sodium citrate, pH 4.5 solution] and incubated during 6 hours at 37 8C. The reaction was interrupted by addition of 150 ml of 0.2 M TRIS solution and the absorbance of colored product was assessed at 405 nm in a microtitre plate reader (Biotrack, AMERSHAM BIOSCIENCE). In case of Pertussis Toxin [Islets Activating Protein (IAP)] treatment, the mast cells were previously incubated with 1 ug/ml IAP at 37 8C during 60 min. The values were expressed as the mean percentage of total b-D-glucosaminidase activityGSD from five experiments with rat mast cell suspensions, determined in lysed mast cells in presence of 0.1% (v/v) Triton X-100 (considered as 100% reference). 2.7.2. Hemolytic activity Washed rat red blood cells (WRRBC) were used to evaluate the hemolytic activity of the peptides. WRRBC were prepared by washing 50 mL of Wistar rats red blood cell suspensions 3 times with physiological saline solution [NaCl 0.85% (w/v) and CaCl2 10 mM], and suspending in 50 ml of the same solution. Aliquots of WRRBC were then incubated at 378 C in the presence of each peptide for 120 min, with gentle mixing. Samples were then centrifuged, and the absorbance of the supernatants was measured at 540 nm. The absorbance measured from lysed WRRBC in presence of 1% (v/v) Triton X-100 was considered as 100%. Results are expressed as meansGSD of five experiments.

3. Results and discussion 3.1. Purification The venom extracts of P. exı´gua were subjected to reverse-phase HPLC fractionation, resulting in the elution of 20 fractions (Fig. 1), which were collected and submitted to a series of biological assays. Fractions 1 and 2 were constituted of complex mixtures of endogenous biogenic amines and neurotransmitters; fractions 3, 4, 9, 10, 11, 12, 16 and 18 presented low amount of biological material to permit their biochemical identification. The fraction 5 was identified as serotonin, while the fractions 6, 7, 8 13, 15, 17, 19 and 20 were constituted of unidentified peptide components. The bioassay of the fraction 15, 17 and 19 (assigned with asterisks in Fig. 1) revealed pronounced mast

104

M.A. Mendes et al. / Toxicon 45 (2005) 101–106 Table 1 Amino acid sequences of mastoparan peptides from different species of social waspsa,b Peptides

Sequences

Wasp species

Protopolybia-MP-I

I-N-W-L-K-L-G-KK-V-S-A-I-L -NH2 I-N-W-K-A-L-L-DA-A-K-K-V-L-NH2 I-N-W-K-G-I-A-AM-A-K-K-L-L-NH2 I-N-W-K-K-M-A-AT-A-L-K-M-I-NH2 I-N-W-K-A-I-I-E-AA-K-Q-A-L-NH2 I-N-W-L-K-L-G-KA-V-I-D-A-L-NH2 I-N-W-A-K-L-G-KL-A-L-Q-A-L-NH2 V-D-W-K-K-I-G-QH-I-L-S-V-L-NH2

Protolybia exigua Protonectarina syleirae Vespa xanthoptera Parapolybia indica Protopolybia exı´gua Protopolybia exigua Ropalidia sp.

Protonectarina-MP Mastoparan-X Parapolybia-MP Protopolybia-MP-II Protopolybia-MP-III Fig. 1. Chromatogram profile of fractionation of venom extract of the Protopolybia exigua under reverse-phase HPLC with a Nucleosil C-18 (ODS) SHISEIDO column (250!10 mm), under linear gradient from 5% to 60% (v/v) MeCN (containing 0.1% TFA), at a flow rate of 2.0 mL/min. over 45 min. at 30 8C by monitoring at UV 215 nm.

cell degranulation activity, which stimulated the further characterization of these fractions. The ESI-MS spectra revealed that the fractions 15, 17 and 19 were constituted of peptide components presenting molecular masses 1581.02, 1566.90, and 1551.93 Da, respectively (not disclosed results). The mass spectra shown that those peptides were pure enough to be submitted to amino acid sequencing by automated Edman degradation chemistry protocols. 3.2. Structural analysis The primary sequences of both peptides assigned by Edman degradation chemistry were: Fr. 15 I-N-W-L-K-L-G-K-K-V-S-A-I-L-NH 2, (1581.02 Da) Fr. 17: I-N-W-K-A-I-I-E-A-A-K-Q-A-L-NH2 , (1566.90 Da) Fr. 19 I-N-W-L-K-L-G-K-A-V-I-D-A-L-NH2, (1551.93 Da) The sequences above just fit to the experimental values of the respective molecular masses if the C-terminal residues were considered in the amidated form, as the most of peptide toxins from Hymenopteran venoms (Nakajima, 1986; Konno et al., 2000, 2001). A search in the literature shows that the sequence of the peptides of fractions 15, 17 and 19 from the venom of P. exigua(Saussure) are relatively conserved when compared to the mastoparan peptides from other social wasps species, as shown in the Table 1. The peptide from fraction 15 belongs a sub-group of mastoparans presenting three lysine residues in their sequences; the classical mastoparan

Ropalidia-MP Polistes-MP

Polistes jadwigae

Nakajima (1986); Dohtsu et al. (1993)

peptides usually present lysine residues at the positions 4 or 5 and 11/12 (or even 4/5 and 11 or 12), such as some examples shown in the Table 1 (Protonectarina-MP, Mastoparan-X and Parapolybia-MP); however the peptide of fraction 15 presents the lysine residues at the positions 5, 8 and 9. Therefore, the peptides of fractions 17 and 19 seem to belong another sub-group of mastoparan peptides, which present only two lysine residues in their sequences, generally at different positions from the 4th to 12th residues, as the examples showed in the Table 1 (PolistesMP and Ropalidia-MP). Thus, the three novel peptides identified in the venom of P. exigua, seem to be mastoparans bearing the tripeptide INW at the amino terminal side of all toxins, also observed in the mastoparans of other social wasps, endemic both from the temperate zones (Vespa xanthoptera and Parapolybia indica) and tropical/subtropical regions (Protopolybia exigua and Ropalidia sp.). The peptide component of fraction 15 was named Protopolybia-MP-I; while those of fractions 17 and 19 were named Protopolybia-MP-II and Protopolybia-MP-III, respectively. 3.3. Biological activities The biological activities of the peptides ProtopolybiaMP-I, -II and -III were investigated by using synthetic peptides. Mast cell degranulation (in absence and presence of IAP) and hemolytic activities were assayed for both peptides. Fig. 2 shows the results of rat peritoneal mast cell degranulation activities for the mastoparan (MP) as a reference compound, and also for Protopolybia-MP-I, -II and -III; at 10 mM Mastoparan (MP) caused

M.A. Mendes et al. / Toxicon 45 (2005) 101–106

Fig. 2. Degranulation activity in rat peritoneal mast cells for Mastoparan (MP) peptide and also for the peptides ProtopolybiaMP-I, -II and -III. The activity was determined by measuring the release of the granule marker, b-D-glucosaminidase, which colocalizes with histamine and the values for b-D-glucosaminidase released in the medium were expressed in the percentage of total ezyme activity. Values are meanGSD (nZ5).

the degranulation of 57% of mast cells present in the assay, while Protopolybia-MP-I, -II and -III presented 43, 30 and 20% of mast cell degranulation, respectively. In fact the delivering of stored compounds from mast cells granules may occur either due to the cytolytic effect of the peptides or due to exocytosis activated by the binding of the peptides to G-protein coupled receptors, which in turn activate a cascade of molecular events, resulting in mast cell degranulation (Higashijima et al., 1990). Some mastoparan peptides seem to be involved on guanylate cyclase activation, either interacting directly with the enzyme or through other proteins (Song et al., 1993). Mastoparans are known to activate various G-proteins, such as Gi, Go, Gs and transducin (Higashijima et al., 1990). It has been found that the action of IAP on the Gi regulatory component of adenylate cyclase is believed to be responsible for the various physiological and cellular effects of the toxin. Cells treated with the toxin fail to respond to agents that normally block cAMP accumulation. (Sato et al, 1981). Thus, IAP became a valuable tool in the study of the regulation of adenylate cyclase. The action of IAP on the Gi component of adenylate cyclase has also been found to inhibit various metabolic responses; it has been found that IAP catalyzes the ADP-ribosylation of transducin, a guanine nucleotide-binding regulatory protein (Manning and Gilman, 1983; Bokoch and Gilman, 1984), preventing the binding of some mastoparan peptides to the Gi regulatory component of adenylate cyclase (Higashijima et al., 1990). Thus, the study of mast cell degranulation by mastoparan peptides, both in absence and in presence of IAP, may contribute to the better understanding of the mechanism of action of these peptides. The Fig. 3 shows the results of mast cell degranulation caused by the peptides Protopolybia-MP-I, -II and -III. The activity of

105

Fig. 3. Comparative degranulation activity in rat peritoneal mast cells for the peptides Protopolybia-MP-I, -II and -III, both in presence ( ) and in absence (L) of IAP. Bars represent the mean G SD (nZ5).

Protopolybia-MP-I is the same for both in absence and in the presence of mast cells previously treated with IAP. In spite the activity of Protoplybia-MP-II and -III are reduced when compared to Protopolybia-MP-I, the degranulating activity of former peptides decreased significantly when the mast cells were previously IAP-treated. These results suggest that Protopolybia-MP-I probably acts on mast cells due to effects caused on membrane perturbation, such as pore formation; however, the peptides Protopolybia-MP-II and -III seem to induce mast cell degranulation due to their binding to the Gi component, which in turn activates the cascade of molecular events cAMP-regulated, resulting in mast cell degranulation. Hemolytic activity was also examined (Fig. 4). At 10 mM the peptides mastoparan (MP) and Protopolybia-MP-I must act causing about 40% hemolysis in rat erythrocytes, while

Fig. 4. Hemolytic activity in washed rat red blood cells (WRRBC) for Mastoparan (MP) and also for the peptides Protopolybia-MP I, -II and - III. The absorbance measured at 540 nm from lysed WRRBC in presence of 1% (v/v) Triton X-100 was considered as 100%. Values are meanGSD (nZ5).

106

M.A. Mendes et al. / Toxicon 45 (2005) 101–106

Protopolybia-MP-II and -III were not hemolytic, even at 100 mM. Thus, these results suggest that Protopolybia-MP-I causes cytolysis, while Protopolybia-MP-II and -III apparently must not cause membrane perturbation. Thus, the results of hemolysis assays for P. exiguamastoparans are consistent with the mechanism proposed above, in which Protopolybia-MP-I is a cytolytic peptide, while Protopolybia-MP-II and -III seem to act by binding to Gi component of adenylate cyclase regulating system, resulting in mast cell exocytosis. It is interesting to emphasize that the peptides Protopolybia-MP-I and -III present about 64% of sequence similarity to each other, in which the main difference is the presence of three lysine residues in Protopolybia-MP-I and two lysine residues in Protopolybia-MP-III; despite the sequence similarity between these peptides, they seem to act on mast cells through different mechanisms. It was previously reported that the presence of positive charges, the occurrence of C-terminal residue in the amidated form and a-helical conformation are essential requisites for the stimulatory effect of mastoparans on membrane-bound guanylate cyclase (Song et al., 1993). Analyzing the sequences of the three peptides it may be observed that Protopolybia-MP-I presents a net charge of 3C, while Protopolybia-MP-II and -III present net charge of 1C. By using the bioinformatic tool PSIPRED Protein Structure Prediction Server (http://bioinf.cs.ucl.ac.uk/ psipred/) to predict the secondary structure of these peptides it was estimated that Protopolybia-MP-I, -II and -III, must present about 71, 71 and 42% of their secondary structure in a-helical conformation, respectively. Thus, the three peptides fill the basic structural requirements suggested by Song et al. (1993) to activate the membrane-bound guanylate cyclase; however, only the peptides Protopolybia-MP-II and -III apparently bind to the Gi subunity of guanylate cyclase, suggesting that this interaction requires an ideal net charge of 1Cto promote this activation.

Acknowledgements This work was supported by a grant from the Sa˜o Paulo State Research Foundation (FAPESP). Maria Anita Mendes is Postdoctoral fellows from FAPESP (Proc. 01/05060-4), Bibiana Monson de Souza is Doctoral student fellow from FAPESP (Proc. 03/00985-5). Mario Sergio Palma is researching for the Brazilian Council for Scientific a Technological Development (CNPq, 300377/2003-5).

References Argiolas, A., Pisano, J.J., 1985. Bombolitins, a new class of mast cells degranulating peptides from the venom of the Bumblebee Megabombus pennsylvanicus. J. Biol. Chem. 260 (3), 1437– 1444.

Bokoch, G.M., Gilman, A.G., 1984. Inhibition of receptormediated release of arachidonic acid by pertussis toxin. Cell 39 (2), 301–308. Dohtsu, K., Okumura, K., Hagiwara, K., Palma, M.S., Nakajima, T., 1992. Isolation and sequence analysis of peptides from the venom sac of Protonectarina sylveirae, in: Ynaihara, N. (Ed.), Peptide Chemistry. ESCON, Amsterdan, pp. 586–588. Dohtsu, K., Okumura, K., Hagiwara, K., Palma, M.S., Nakajima, T., 1993. Isolation and sequence analysis of peptides from the venom of Protonectarina sylveirae (Hymenoptera-Vespidae). Nat. Toxins 1, 272–276. Gallo, R.L., Huttner, K.M., 1998. Antimicrobial peptides: an emerging concept in cutaneous biology. J. Investig. Dermatol. 111, 739–743. Hancock, R.E.W., Diamond, G., 2000. The role of cationic antimicrobial peptides in innate host defenses. TIMS 8, 402– 410. Hide, I., Bennett, J.P., Pizzey, A., Boonen, G., Sagi, D.B., Gomperts, B.D., Tatham, P.E.R., 1993. Degranulation of individual mast cell in response to Ca2C and guarine nucleotides: an all-or-none event. J. Cell Biol. 123, 585–593. Higashijima, T., Burnier, J., Ross, E.M., 1990. Regulation of Gi and G0 by mastoparan related peptides and hydrophilic amines. J. Biol. Chem. 265, 14176–14186. Hirai, Y., Yasuhara, T., Yoshida, H., Nakajima, T., Fujino, M., Kitada, C., 1979. A new mast cell degranulating peptide mastoparan in the venom of Vespula lewisii. Chem. Pharm. Bull. 27, 1942–1944. Konno, K., Hisada, M., Naoki, H., Itagaki, Y., Kawai, N., Miwa, A., Yasuhara, T., Motimoto, Y., Nakai, Y., 2000. Structure and biological activitiesof eumenine mastoparan-AF (EMP-AF), a new mast cell degranulating peptide in venom of the solitary wasp (Anterhynchium flavomarginatum micado). Toxicon 38, 1505–1515. Konno, K., Hisada, M., Fontana, R., Lorenzi, C.C.B., Naoki, H., Itagaki, Y., Miwa, A., Kawai, N., Nakata, Y., Yasuhara, T., Ruggiero, J., Azevedo, W.F., Palma, M.S., Nakajima, T., 2001. Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis. Biochim. Biophys. Acta 1550, 70–80. Krishnakumari, V., Nagaraj, R., 1997. Antimicrobial and hemolytic activities of crabrolin, a 13-residue from the venom of the European hornet, Vespa crabro, and its analogs. J. Peptide Res. 50, 88–93. Manning, D.R., Gilman, A.G., 1983. The regulatory components of adenylate cyclase and transducin. A family of structurally homologous guanine nucleotide-binding proteins. J. Biol. Chem. 258, 7059–7063. Nakajima, T., 1984. Biochemistry of vespid venoms, in: Tu, A.T. (Ed.), Handbook of Natural Toxins, vol. 2. Marcel Dekker, New York, pp. 109–133. Nakajima, T., 1986. Pharmacological biochemistry of vespid venoms, in: Piek, T. (Ed.), Venom of Hymenoptera. Academic Press, London, pp. 309–327. Sato, Y., Izumiya, K., Sato, H., Cowell, J.L., Manclark, C.R., 1981. Role of antibody to leukocytosis-promoting factor hemagglutinin and to filamentous hemagglutinin in immunity to pertussis. Infect. Immun. 31 (3), 1223–1231. Song, D.L., Chang, G.D., Ho, C.L., Chang, C.H., 1993. Structural requirements of mastoparan for activation of membrane-bound guanylate cyclase. Eur. J. Pharmacol. 247, 283–288.