Structural and Biological Characterization of Three Novel Mastoparan Peptides from the Venom of the Neotropical Social Wasp Protopolybia Exigua (Saussure)

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).

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. chem). Side-chain protective groups included t-butyl for The values were expressed as the mean percentage of serine and t-butoxycarbonyl for lysine. Cleavages of the total b-D-glucosaminidase activityGSD from five exper- peptides-resin complexes were performed by treatment iments with rat mast cell suspensions, determined in lysed with trifluoroacetic acid/1,2- ethanedithiol / anisole/ mast cells in presence of 0.1% (v/v) Triton X-100 phenol/ water (82.5:2.5:5:5:5 by volume), using 10 ml (considered as 100% reference). per gram of complex at room temperature during 2 h. After filtering to remove the resin, ethyl ether at 4 8C was added 2.7.2. Hemolytic activity to the soluble material causing precipitation of the crude Washed rat red blood cells (WRRBC) were used to peptides, which were collected as a pellet after a evaluate the hemolytic activity of the peptides. WRRBC centrifugation at 1000g, during 15 min at room tempera- were prepared by washing 50 mL of Wistar rats red blood ture. The crude peptides were solubilized in water and cell suspensions 3 times with physiological saline solution chromatographed under RP-HPLC using a semi-prepara- [NaCl 0.85% (w/v) and CaCl 10 mM], and suspending in ! 2 tive column (SHISEIDO C18, 250 10 mm, 5 mm), under 50 ml of the same solution. Aliquots of WRRBC were then isocratic elution with 40% (v/v) acetonitrile in water incubated at 378 C in the presence of each peptide for [containing 0.1% (v/v) trifluoroacetic] at a flow rate of 120 min, with gentle mixing. Samples were then centri- 2 ml/min. The elution was monitored at 215 nm with a fuged, and the absorbance of the supernatants was measured UV-DAD detector (SHIMADZU, mod. SPD-M10A) and at 540 nm. The absorbance measured from lysed WRRBC in each fraction eluted was manually collected into plastic presence of 1% (v/v) Triton X-100 was considered as 100%. vials of 2 ml. The homogeneity and correct sequence of the Results are expressed as meansGSD of five experiments. 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 3. Results and discussion used to check the peptides purity (considering as criteria the presence of a single molecular ion, equivalent the 3.1. Purification expected molecular mass for the amino sequence of each peptide); and finally the sequence of the synthetic material The venom extracts of P. exı´gua were subjected to was confirmed by automated sequencing based on Edman reverse-phase HPLC fractionation, resulting in the elution of degradation chemistry. 20 fractions (Fig. 1), which were collected and submitted to a series of biological assays. Fractions 1 and 2 were 2.7. Biological assays constituted of complex mixtures of endogenous biogenic amines and neurotransmitters; fractions 3, 4, 9, 10, 11, 12, 2.7.1. Mast cell degranulation 16 and 18 presented low amount of biological material to Mast cell degranulation was determined by measuring permit their biochemical identification. The fraction 5 was the release of b-D-glucosaminidase, which co-localizes with identified as serotonin, while the fractions 6, 7, 8 13, 15, 17, histamine, as proposed by Hide et al. (1993). Mast cells 19 and 20 were constituted of unidentified peptide were obtained by peritoneal washing of adult Wistar rats components. The bioassay of the fraction 15, 17 and 19 with a solution containing 0.877 g NaCl (MERCK), 0.028 g (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-K- Protolybia

K-V-S-A-I-L -NH2 exigua Protonectarina-MP I-N-W-K-A-L-L-D- Protonectarina

A-A-K-K-V-L-NH2 syleirae Mastoparan-X I-N-W-K-G-I-A-A- Vespa

M-A-K-K-L-L-NH2 xanthoptera Parapolybia-MP I-N-W-K-K-M-A-A- Parapolybia

T-A-L-K-M-I-NH2 indica Protopolybia-MP-II I-N-W-K-A-I-I-E-A- Protopolybia

A-K-Q-A-L-NH2 exı´gua Protopolybia-MP-III I-N-W-L-K-L-G-K- Protopolybia

A-V-I-D-A-L-NH2 exigua Fig. 1. Chromatogram profile of fractionation of venom extract of Ropalidia-MP I-N-W-A-K-L-G-K- Ropalidia sp. the Protopolybia exigua under reverse-phase HPLC with a L-A-L-Q-A-L-NH2 Nucleosil C-18 (ODS) SHISEIDO column (250!10 mm), under Polistes-MP V-D-W-K-K-I-G-Q- Polistes linear gradient from 5% to 60% (v/v) MeCN (containing 0.1% H-I-L-S-V-L-NH2 jadwigae TFA), at a flow rate of 2.0 mL/min. over 45 min. at 30 8Cby Nakajima (1986); Dohtsu et al. (1993) monitoring at UV 215 nm. peptides usually present lysine residues at the positions 4 cell degranulation activity, which stimulated the further or 5 and 11/12 (or even 4/5 and 11 or 12), such as some characterization of these fractions. examples shown in the Table 1 (Protonectarina-MP, The ESI-MS spectra revealed that the fractions 15, 17 Mastoparan-X and Parapolybia-MP); however the peptide and 19 were constituted of peptide components presenting of fraction 15 presents the lysine residues at the positions molecular masses 1581.02, 1566.90, and 1551.93 Da, 5, 8 and 9. Therefore, the peptides of fractions 17 and 19 respectively (not disclosed results). The mass spectra seem to belong another sub-group of mastoparan peptides, shown that those peptides were pure enough to be submitted which present only two lysine residues in their sequences, to amino acid sequencing by automated Edman degradation generally at different positions from the 4th to 12th chemistry protocols. residues, as the examples showed in the Table 1 (Polistes- MP and Ropalidia-MP). Thus, the three novel peptides 3.2. Structural analysis identified in the venom of P. exigua,seemtobe mastoparans bearing the tripeptide INW at the amino The primary sequences of both peptides assigned by terminal side of all toxins, also observed in the Edman degradation chemistry were: mastoparans of other social wasps, endemic both from the temperate zones (Vespa xanthoptera and Parapolybia Fr. 15 I-N-W-L-K-L-G-K-K-V-S-A-I-L-NH2, indica) and tropical/subtropical regions (Protopolybia (1581.02 Da) exigua and Ropalidia sp.). Fr. 17: I-N-W-K-A-I-I-E-A-A-K-Q-A-L-NH2, The peptide component of fraction 15 was named (1566.90 Da) Protopolybia-MP-I; while those of fractions 17 and 19 Fr. 19 I-N-W-L-K-L-G-K-A-V-I-D-A-L-NH2, were named Protopolybia-MP-II and Protopolybia-MP-III, (1551.93 Da) respectively. The sequences above just fit to the experimental values of the respective molecular masses if the C-terminal 3.3. Biological activities residues were considered in the amidated form, as the most of peptide toxins from Hymenopteran venoms The biological activities of the peptides Protopolybia- (Nakajima, 1986; Konno et al., 2000, 2001). MP-I, -II and -III were investigated by using synthetic A search in the literature shows that the sequence of the peptides. Mast cell degranulation (in absence and presence peptides of fractions 15, 17 and 19 from the venom of of IAP) and hemolytic activities were assayed for both P. exigua(Saussure) are relatively conserved when com- peptides. pared to the mastoparan peptides from other social wasps Fig. 2 shows the results of rat peritoneal mast species, as shown in the Table 1. The peptide from fraction cell degranulation activities for the mastoparan (MP) 15 belongs a sub-group of mastoparans presenting three as a reference compound, and also for Protopolybia-MP-I, lysine residues in their sequences; the classical mastoparan -II and -III; at 10 mM Mastoparan (MP) caused M.A. Mendes et al. / Toxicon 45 (2005) 101–106 105

Fig. 2. Degranulation activity in rat peritoneal mast cells for Mastoparan (MP) peptide and also for the peptides Protopolybia- Fig. 3. Comparative degranulation activity in rat peritoneal mast MP-I, -II and -III. The activity was determined by measuring the cells for the peptides Protopolybia-MP-I, -II and -III, both in presence ( ) and in absence (L) of IAP. Bars represent the mean release of the granule marker, b-D-glucosaminidase, which co- G SD (nZ5). localizes with histamine and the values for b-D-glucosaminidase released in the medium were expressed in the percentage of total Protopolybia-MP-I is the same for both in absence and in the ezyme activity. Values are meanGSD (nZ5). presence of mast cells previously treated with IAP. In spite the degranulation of 57% of mast cells present in the assay, the activity of Protoplybia-MP-II and -III are reduced when while Protopolybia-MP-I, -II and -III presented 43, 30 and compared to Protopolybia-MP-I, the degranulating activity 20% of mast cell degranulation, respectively. of former peptides decreased signiﬁcantly when the mast In fact the delivering of stored compounds from mast cells were previously IAP-treated. cells granules may occur either due to the cytolytic effect of These results suggest that Protopolybia-MP-I probably the peptides or due to exocytosis activated by the binding of acts on mast cells due to effects caused on membrane the peptides to G-protein coupled receptors, which in turn perturbation, such as pore formation; however, the peptides activate a cascade of molecular events, resulting in mast cell Protopolybia-MP-II and -III seem to induce mast cell degranulation (Higashijima et al., 1990). degranulation due to their binding to the Gi component, Some mastoparan peptides seem to be involved on which in turn activates the cascade of molecular events guanylate cyclase activation, either interacting directly with cAMP-regulated, resulting in mast cell degranulation. the enzyme or through other proteins (Song et al., 1993). Hemolytic activity was also examined (Fig. 4). At 10 mM Mastoparans are known to activate various G-proteins, such the peptides mastoparan (MP) and Protopolybia-MP-I must as Gi,Go,Gs and transducin (Higashijima et al., 1990). act causing about 40% hemolysis in rat erythrocytes, while 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, Fig. 4. Hemolytic activity in washed rat red blood cells (WRRBC) may contribute to the better understanding of the mechan- for Mastoparan (MP) and also for the peptides Protopolybia-MP I, ism of action of these peptides. The Fig. 3 shows the -II and - III. The absorbance measured at 540 nm from lysed results of mast cell degranulation caused by the WRRBC in presence of 1% (v/v) Triton X-100 was considered as peptides Protopolybia-MP-I, -II and -III. The activity of 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 Bokoch, G.M., Gilman, A.G., 1984. Inhibition of receptor- 100 mM. Thus, these results suggest that Protopolybia-MP-I mediated release of arachidonic acid by pertussis toxin. Cell causes cytolysis, while Protopolybia-MP-II and -III appar- 39 (2), 301–308. ently must not cause membrane perturbation. Thus, the Dohtsu, K., Okumura, K., Hagiwara, K., Palma, M.S., Nakajima, T., results of hemolysis assays for P. exiguamastoparans are 1992. Isolation and sequence analysis of peptides from the venom sac of Protonectarina sylveirae, in: Ynaihara, N. (Ed.), consistent with the mechanism proposed above, in which Peptide Chemistry. ESCON, Amsterdan, pp. 586–588. Protopolybia-MP-I is a cytolytic peptide, while Protopoly- Dohtsu, K., Okumura, K., Hagiwara, K., Palma, M.S., Nakajima, T., bia-MP-II and -III seem to act by binding to Gi component 1993. Isolation and sequence analysis of peptides from the of adenylate cyclase regulating system, resulting in mast cell venom of Protonectarina sylveirae (Hymenoptera-Vespidae). exocytosis. Nat. Toxins 1, 272–276. It is interesting to emphasize that the peptides Proto- Gallo, R.L., Huttner, K.M., 1998. Antimicrobial peptides: an polybia-MP-I and -III present about 64% of sequence emerging concept in cutaneous biology. J. Investig. Dermatol. similarity to each other, in which the main difference is the 111, 739–743. presence of three lysine residues in Protopolybia-MP-I and Hancock, R.E.W., Diamond, G., 2000. The role of cationic two lysine residues in Protopolybia-MP-III; despite the antimicrobial peptides in innate host defenses. TIMS 8, 402– sequence similarity between these peptides, they seem to act 410. Hide, I., Bennett, J.P., Pizzey, A., Boonen, G., Sagi, D.B., on mast cells through different mechanisms. Gomperts, B.D., Tatham, P.E.R., 1993. Degranulation of It was previously reported that the presence of positive individual mast cell in response to Ca2C and guarine charges, the occurrence of C-terminal residue in the nucleotides: an all-or-none event. J. Cell Biol. 123, 585–593. amidated form and a-helical conformation are essential Higashijima, T., Burnier, J., Ross, E.M., 1990. Regulation of Gi and requisites for the stimulatory effect of mastoparans on G0 by mastoparan related peptides and hydrophilic amines. membrane-bound guanylate cyclase (Song et al., 1993). J. Biol. Chem. 265, 14176–14186. Analyzing the sequences of the three peptides it may be Hirai, Y., Yasuhara, T., Yoshida, H., Nakajima, T., Fujino, M., observed that Protopolybia-MP-I presents a net charge of Kitada, C., 1979. A new mast cell degranulating peptide 3C, while Protopolybia-MP-II and -III present net charge of mastoparan in the venom of Vespula lewisii. Chem. Pharm. 1C. By using the bioinformatic tool PSIPRED Protein Bull. 27, 1942–1944. Structure Prediction Server (http://bioinf.cs.ucl.ac.uk/ Konno, K., Hisada, M., Naoki, H., Itagaki, Y., Kawai, N., Miwa, A., Yasuhara, T., Motimoto, Y., Nakai, Y., 2000. Structure and psipred/) to predict the secondary structure of these peptides biological activitiesof eumenine mastoparan-AF (EMP-AF), a it was estimated that Protopolybia-MP-I, -II and -III, must new mast cell degranulating peptide in venom of the solitary present about 71, 71 and 42% of their secondary structure in wasp (Anterhynchium flavomarginatum micado). Toxicon 38, a-helical conformation, respectively. Thus, the three pep- 1505–1515. tides fill the basic structural requirements suggested by Song Konno, K., Hisada, M., Fontana, R., Lorenzi, C.C.B., Naoki, H., et al. (1993) to activate the membrane-bound guanylate Itagaki, Y., Miwa, A., Kawai, N., Nakata, Y., Yasuhara, T., cyclase; however, only the peptides Protopolybia-MP-II and Ruggiero, J., Azevedo, W.F., Palma, M.S., Nakajima, T., 2001. -III apparently bind to the Gi subunity of guanylate cyclase, Anoplin, a novel antimicrobial peptide from the venom of the suggesting that this interaction requires an ideal net charge solitary wasp Anoplius samariensis. Biochim. Biophys. Acta of 1Cto promote this activation. 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. Acknowledgements 50, 88–93. Manning, D.R., Gilman, A.G., 1983. The regulatory components of This work was supported by a grant from the Saõ Paulo adenylate cyclase and transducin. A family of structurally State Research Foundation (FAPESP). 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