Peptides 26 (2005) 2157–2164

Structural and functional characterization of two novel peptide toxins isolated from the venom of the social paulista Bibiana M. Souza a, Maria A. Mendes a, Lucilene D. Santos a, Maur´ıcio R. Marques a, Lilian M.M. Cesar´ a, Roberta N.A. Almeida a, Fernando C. Pagnocca a,b, Katsuhiro Konno c, Mario S. Palma a,∗

a CEIS, Department of Biology/IBRC-UNESP (CAT-CEPID/FAPESP), Institute of Immunological Investigations (Millennium Institute-MCT/CNPq), Rio Claro, SP 13506-900, b Department of Biochemistry and Microbiology, IBRC-UNESP, Rio Claro, SP, Brazil c Institute Butantan (CAT-CEPID/FAPESP), S˜ao Paulo, SP, Brazil

Received 14 February 2005; received in revised form 19 April 2005; accepted 20 April 2005 Available online 29 August 2005

Abstract

Two novel inflammatory peptides were isolated from the venom of the social wasp . They had their molecular masses determined by ESI-MS and their primary sequences were elucidated by Edman degradation chemistry as:

• Polybia-MPI:IDWKKLLDAAKQIL-NH2 (1654.09 Da), • Polybia-CP:ILGTILGLLKSL-NH2 (1239.73 Da). Both peptides were functionally characterized by using Wistar rat cells. Polybia-MPI is a mast cell lytic peptide, which causes no hemolysis to rat erythrocytes and presents chemotaxis for polymorphonucleated leukocytes (PMNL) and with potent antimicrobial action both against Gram-positive and Gram-negative bacteria. Polybia-CP was characterized as a chemotactic peptide for PMNL cells, presenting antimicrobial action against Gram-positive bacteria, but causing no hemolysis to rat erythrocytes and no mast cell degranulation activity at physiological concentrations. © 2005 Elsevier Inc. All rights reserved.

Keywords: Polybia paulista; ; Polycationic peptide; Wasp venom; Mastoparans; Chemotactic peptides

1. Introduction the ; mellitin, MCD-peptide and apamine among the bees [1,15]. Stings by Hymenoptera such as hornets, yel- Mast cell degranulating peptides and chemotactic peptides low jackets and honeybees are manifested by symptoms of are among the major components of vespid venoms [13]. pain, local edema and cardiovascular disturbances [5]. The The mastoparans are amidated tetradecapeptides responsible Hymenoptera venoms are constituted of biogenic amines for histamine release from the mast cells, of serotonine (histamine, serotonin, dopamine, and norepinephrine), some from platelets, of catecholamines and adenylic acids from proteins (phospholipases, hyaluronidase, antigen 5) and a adrenal chromafin cells [24] and even causes exocytosis in series of biologically active polycationic peptides such as: rat pituitary cells [26] and pancreatic ␤-cells [34]. mastoparans, chemotactic peptides and waspkinins among Mastoparans are thought to cause the formation of ion channels in lipid membranes leading to cell lysis [17], ∗ Corresponding author. Tel.: +55 193526 4163; fax: +55 193534 8523. and they are also known to increase permeability of ions E-mail address: [email protected] (M.S. Palma). and small molecules through the biological membranes by

0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.04.026 2158 B.M. Souza et al. / Peptides 26 (2005) 2157–2164 forming pores at high peptide concentrations [25]. These 2. Materials and methods peptides also promote an increase of intracellular Ca2+ concentration in neutrophils [27]. 2.1. Sample preparation Investigations directed toward elucidation of the regu- latory mechanism of mastoparans have shown that these The wasps collected in Rio Claro-SP, southeast Brazil, peptides are involved with the modulation of the activities were immediately frozen and stored at −20 ◦C. The venom of proteins as diverse as GTP-binding proteins (G proteins), reservoirs of 3000 worker wasps were removed by dissection phospholipase A2 and phospholipase C [10,32]. with surgical microscissors and washed with 1:1 acetoni- The second most important group of biologically active trile (MeCN, Aldrich): water containing 0.1% (v/v) triflu- peptides from wasp venoms are the chemotactic peptides, oroacetic acid (TFA, Aldrich) to solubilize the peptides. The which are tridecapeptides presenting many hydrophobic extract was then centrifuged at 8000 × g during 15 min at amino acid residues and generally a single basic residue; 4 ◦C; the supernatant was collected and used to purify the they generally attract macrophages and polymorphonuclear peptides. leukocytes to the region around the site of stinging [33]. Depending on their primary sequences, these peptides can 2.2. Peptide purification cause hemolysis and exhibit a reduced activity of mast cell degranulation [11,23,12]. The supernatant described above was chromatographed in Polycationic peptides usually contain from 12 to 50 amino a CAPCELL PACK C-18 UG120 column (10 mm × 250 mm, acids residues, and a net positive charge from +2 to +7 due 5 ␮m, Shisheido) under a linear gradient from 5 to 60% to the excess of basic amino acid residues; the hydropho- (v/v) MeCN [containing 0.1% (v/v) trifluoroacetic acid], at bic residues represent more than 50% of their amino acid a flow rate of 2.0 mL/min over 60 min by monitoring at sequences [8]. These structural features contribute to the UV 214 nm. The extracts were manually collected and dried formation of amphipathic, ␣-helical conformations, making by using a lyophilizer (MLW LGA-05, Heto). The peaks them able to interact with the anionic components of the of interest (fractions 11 and 12) were re-chromatographed bacterial membranes, providing their assembly in these mem- under reversed phase chromatography with the same column branes with consequent pore formation [7]. Because of this, described above, by using isocratic elution with 41% (v/v) some of these peptides also can present antimicrobial activ- MeCN for the fraction 11 and 45% (v/v) MeCN for the frac- ities [15,19]. The amphipathic, ␣-helical conformations also tion 12 (containing 0.1% TFA in both situations) at a flow may permit the assembling of some of these cationic pep- rate of 2.0 mL/min during 20 min at 30 ◦C. The elution was tides with the zwitterionic membranes of mammalian cells, monitored at 215 nm and fractions were manually collected making some of these peptides act as hemolysins of this cells in 5 mL glass vials. [15]. Recently, novel pharmacologically active peptides from 2.3. ESI mass spectrometry analysis tropical wasp venoms have been reported: three novel mastoparan peptides were described in the venom of All the mass spectrometric analysis were performed in a Protopolybia exigua, with two of them characterized as triple quadrupole mass spectrometer (MICROMASS, mod. modulators of mast cell degranulation by virtue of their Quattro II). The experimental protocol was based in details interaction with G-protein receptors [21]. Micro-scale described in a previous publication [19] and adapted for the bioassay guided low abundant inflammatory peptides, present investigation. The mass spectrometer was outfitted naturally occurring in the venom of the wasp Agelaia with a standard probe electrospray (ESI—Micromass, Altrin- pallipes pallipes were sequenced by using tandem mass chan). The samples were injected into electrospray transport spectrometry protocols, permitting the identification of solvent by using a micro syringe (500 ␮L) coupled to a micro novel mastoparan and chemotactic peptides [20]. Two novel infusion pump (KD Scientific) at a flow rate of 4 ␮L/min. inflammatory peptides were described in the venom of the The mass spectrometer was calibrated with intact horse social wasp Polybia paulista, presenting acetylation at the heart myoglobin and its typical cone-voltage induced frag- N-terminus, responsible by the modulation of the PMNL ments to operate at resolution 4000. The samples were cells chemotaxys and histamine release from mast cells dissolved in 50% (v/v) acetonitrile [containing 0.1% (v/v) [28]. formic acid] to be analyzed by positive electrospray ion- In the present work we report two novel inflammatory pep- ization (ESI+) using typical conditions: a capillary volt- tides isolated from the venom of the social wasp P. paulista. age of 3.5 kV, a cone voltage of 30 V, a dessolvation gas They were purified by HPLC under reversed phase condi- temperature of 80 ◦C and flow of nebulizer gas (nitrogen) tions, had their molecular masses determined by ESI-MS and about 15 L/h and drying gas (nitrogen) 200 L/h. The spectra their sequences were determined by automated Edman dedra- were obtained in the continuous acquisition mode, scanning dation chemistry. Their functional characterization revealed from m/z 100 to 2500 at a scan time of 5 s. The acquisi- that both peptides exhibit inflammatory and antimicrobial tion and treatment of data were performed with MassLynx activities. software. B.M. Souza et al. / Peptides 26 (2005) 2157–2164 2159

2.4. Amino acid sequencing of NADH min−1 at 25 ◦C and pH 7.4) and then converted into relative activity by using the total LDH activity of rat The amino acid sequence was performed by using a gas- mast cells lysed in presence of 0.1% (v/v) Triton X-100 phase sequencer PPSQ-21 A (Shimadzu) based on automated (considered as 100% reference). Results are expressed as Edman Degradation Chemistry. mean ± S.D. of five experiments.

2.5. Biological activities 2.5.3. Hemolytic activity Five hundred microliters of washed rat red blood cells 2.5.1. Mast cell degranulation activity (WRRBC) were washed three times with physiological saline Degranulation was determined by measuring the release solution and suspended in 50 mL of physiological saline of the granule marker, ␤-d-glucosaminidase, which co- solution [0.9% (m/v) NaCl]. Ninety microliters of this cell localizes with histamine, as previously described [9]. Mast solution were incubated with 10 ␮L of peptide solution in cells was obtained by peritoneal washing of female adult ◦ different concentrations, at 37 C for 2 h. The samples were Wistars rats. The mast cells were washed three times by then centrifuged and the absorbance of the supernatants was re-suspension and centrifugation in a mast cell medium measured at 540 nm. The absorbance measured from lysed (150 Mm NaCl (MERCK), 4 mM KCl (MERCK), 4 mM WRRBC in presence of 1% (v/v) Triton X-100 was consid- NaH PO (SYNTH), 3 mM KH PO (SYNTH), 5 mM 2 4 2 4 ered to be 100% and physiological saline solution activity glucose (SYNTH), 15 ␮M BSA (SIGMA), 2 mM CaCl 2 as being 0%. Results are expressed as mean ± S.D. of five (MERCK), 50 ␮L Liquemine (5000 UI/0.250 mL, ROCHE)). experiments. The cells were incubated with various peptide con- centrations for 15 min at 37 ◦C and after centrifugation the supernatants were sampled for ␤-d-glucosaminidase. 2.5.4. Chemotaxis assays Briefly, 50 ␮L substrate, 5 mM p-nitrophenyl-N-acetyl-␤-d- Chemotaxis was assayed in a specific multi-chamber appa- glucosaminide in 0.2 M citrate, pH 4.5, and 50 ␮L of the ratus (NEURO PROBE) [6] by using polymorphonucleated samples of the medium were incubated in 96-well plates leukocytes (PMNL), obtained from subcutaneous inflamma- for 6 h at 37 ◦C to yield the chromophore, p-nitrophenol. tory induction in Wistar rats. The upper chambers were filled ␮ ∼ × 5 After incubation, 50 ␮L of the previous solution were added with 200 L of a PMNL suspension ( 2.7 10 cells/mL to 150 ␮L of 0.2 M Tris and absorbance was measured at in 0.9% NaCl solution–physiological saline solution) and ␮ 405 nm. The values were expressed as the percentage of total the lower chambers were filled with 400 L of physiolog- ␤-d-glucosaminidase activity from rat mast cell suspensions, ical saline solution containing various concentrations of the −7 −4 determined in lysed mast cells in presence of 0.1% (v/v) peptides (10 to 10 M). A polycarbonate membrane con- ␮ Triton X-100 (considered as 100% reference). Results are taining pores of 10 m of diameter (NEURO PROBE) was expressed as mean ± S.D. of five experiments. placed between both chambers. The chemotaxis chamber was incubated at 37 ◦C for 1 h. After incubation, cells in the lower 2.5.2. Mast cell lysis chamber were counted by using a Neubauer¨ chamber under a This method measures the leakage of lactate dehydroge- microscope after violet crystal staining. Results are expressed ± nase (LDH, EC 1.1.1 27) from the cytoplasm of rat mast cells as mean S.D. of five experiments. into the surrounding medium as an indicator of mast cell lysis by the peptide toxins. 2.5.5. Antimicrobial activity LDH catalyzes the reversible reduction of pyruvate to The minimal inhibitory concentrations (MIC) of the lactate with NADH as coenzyme. The activity of lactate dehy- peptides were determined based on methods previously drogenase was assayed with the supernatants of rat peritoneal described elsewhere [2,18]. The microorganisms used were: mast cells incubated with the toxin peptides also used for Bacilus subtilis (CCT 2576), Staphylococcus aureus (ATCC mast cell degranulation assay as described above (Section 6538), Escherichia coli (ATCC 25922) and Pseudomonas 2.5.1). The LDH activity was assayed by using the UV-LDH aeruginosa (ATCC 15422). Assay Kit from Biobras Diagnostics; 20 ␮L each supernatant The assays were performed in 96-well plates. The inoc- was pre-incubated with 800 ␮L of LDH buffer (50 mM Tris ula were prepared in saline solution [0.9% (v/v) NaCl] by pH 7.4, containing 1.2 mM pyruvate, 5 mM EDTA) during suspending a 18 h old culture in Muller–Hinton¨ broth to the 5 min, at 25 ◦C. The standard spectrophotometric LDH assay 0.5 value of a McFarland scale, a concentration equivalent to measured the decreasing of absorbance at 340 nm due to the 1 × 104 cells/mL and 50 ␮L were inoculated into the wells, consumption of NADH in this reaction. containing 50 ␮L of solution of each peptide solubilized in The reaction was initiated by the addition of 200 ␮Lof Muller–Hinton¨ broth medium. The concentration range of LDH substrate (0.15 mM NADH); the kinetics of NADH con- the peptides was from 0.5 to 1000 ␮g/mL. sumption was monitored by acquiring the decreasing of the The plates were incubated at 37 ◦C for 24 h and after 20 ␮L ◦ absorbance at 340 nm during 12 min (A340)at25 C. The of a triphenyltetrazolium chloride solution (TTC) 0.5% (w/v) results were initially calculated as catalytic units (micromoles was added. The plates were then incubated for an additional 2160 B.M. Souza et al. / Peptides 26 (2005) 2157–2164 period of 2 h at 37 ◦C. The minimal inhibitory concentration was that where the dye was not reduced and tetracycline in a concentration range from 0.2 to 600 ␮g/mL was used as control. Results are expressed as mean ± S.D. of five exper- iments.

3. Results

3.1. Peptides purification and sequencing

The crude venom of P. paulista was initially fractionated under reversed phase-HPLC by using a linear gradient from 5 to 60% (v/v) acetonitrile [containing 0.1% (v/v) trifluoracetic acid]. The chromatographic profile (Fig. 1) shows the exis- tence of 13 peaks, which were submitted to preliminary bio- logical assays. Fractions 1 and 2 were constituted by complex mixtures of endogenous biogenic amines and neurotrans- mitters, while fractions 3–12 contained unidentified venom components; the fraction 13 consisted of a mixture of two acetylated inflammatory peptides already characterized both structurally and functionally [28] and unknown venom com- Fig. 2. (a) Chromatogram profile of re-fractionation of fraction 11 under ponents. The fraction 11 presented some mast cell degranula- reverse-phase HPLC with a C-18 (ODS) CAPCELL PACK UG120 column (250 mm × 10 mm, 5 ␮m), under isocratic condition (41% (v/v) MeCN (con- tion, chemotaxis for PMNL cells and potent inhibitory activ- taining 0.1% TFA)), at a flow rate of 2.0 mL/min over 20 min by monitoring at ity against both Gram-positive and Gram-negative bacteria, UV 214 nm. (b) Chromatogram profile of re-purification of fraction 12 under whereas the fraction 12 presented some mast cell degran- reverse-phase HPLC with a C-18 (ODS) CAPCELL PACK UG120 column ulation, chemotaxis for PMNL cells and potent inhibitory (250 mm × 10 mm, 5 ␮m), under isocratic condition (45% (v/v) MeCN (con- activity against Gram-positive bacteria. Afterward, the purity taining 0.1% TFA)), at a flow rate of 2.0 mL/min over 20 min by monitoring at UV 214 nm. of fractions were tested by ESI-MS. These observations lead to further purification of the peptide components of these fractions by reverse-phase HPLC under isocratic conditions The primary sequences determined for both peptides were: (Fig. 2a and b). The biological activities described above were • Fraction 11C: IDWKKLLDAAKQIL-NH2 found associated to the peaks 11C (Fig. 2a) and 12A (Fig. 2b); (1654.09 Da) ESI-MS spectra revealed the high purity of the isolated pep- • Fraction 12A:ILGTILGLLKSL-NH2 (1239.73 Da) tides with molecular ion peaks at m/z 1654.09 and 1239.73 Da for fractions 11C and 12A, respectively (not shown results). The sequences above just fit to the experimental values Therefore, both peptides were considered pure enough to be of the respective molecular masses if the C-terminal residues sequenced by Edman Degradation Chemistry and to be bio- were considered in the amidated form, as with most of the logically characterized. peptide toxins from the venoms of Hymenoptera [14,30]. The primary sequence of the peptide present in the frac- tion 11C was compared to other similar peptides from the venom of social wasps as shown in Table 1, revealing some

Table 1 Amino acid sequences of Polybia-MPI compared to other mastoparan pep- tides from social wasp venoms Peptide Primary sequence Wasp species Polybia-MPI IDWKKLLDAAKQIL P. paulista Mastoparan-A IKWKAILDAVKKVL Vespa analis Mastoparan-M INLKAIAALAKKLL Vespa mandarina -MP INWKALLDAAKKVL P. sylveirae Mastoparan-C INWKALLAVAKKIL Vespa crabro Protopolybia-MPI INWLKLGKKVSAIL P. exigua Fig. 1. Chromatogram profile of fractionation of venom extract of the Parapolybia-MP INWKKMAATALKMI P. indica Polybia paulista venom under reverse-phase HPLC with a C-18 (ODS) Protopolybia-MPII INWKAIIEAAKQAL P. exigua CAPCELL PACK UG120 column (250 mm × 10 mm, 5 ␮m), under linear Agelaia-MP INWLKLGKAIIDAL A. p. pallipes gradient from 5 to 60% (v/v) MeCN (containing 0.1% TFA), at a flow rate Protopolybia-MPIII INWLKLGKAVIDAL P. exigua of 2.0 mL/min over 60 min by monitoring at UV 214 nm. B.M. Souza et al. / Peptides 26 (2005) 2157–2164 2161

Table 2 Primary sequence of Polybia-CP compared to other chemotactic peptides from social wasps venoms Peptide Primary sequence Wasp species Paulista-CP ILGTILGLLKSL P. paulista Protonectin ILGTILGLLKGL P. sylveirae/A. p. pallipes Ves-CP-T FLPILGKILGGLL V. tropica Ves-CP-M FLPIIGKLLSGLL V. mandarina Ves-CP-X FLPIIAKLLGGLL V. xanthoptera Ves-CP-P FLPIIAKLVSGLL Paravespula lewisi conservation in relation to the sequences of the mastoparan peptides, particularly in relation to Protonectarina-MP iso- Fig. 4. LDH activity in rat peritoneal mast cell. The activity was deter- mined by measuring the presence of the lactate dehydrogenase activity to lated from the venom of the tropical social wasp Protonec- the medium; the results were shown as relative activity by using the total tarina sylveirae, which has 71% of similarity in relation LDH activity contents of rat mast cells lysed in presence of 0.1% (v/v) Triton to the peptide component of fraction 11C. Thus, this pep- X-100 (considered as 100%). Results are expressed as mean ± S.D. (n = 5). tide was classified as a mastoparan (MP) and was named Polybia-MPI. When the sequence of peptide component and Polybia-CP, respectively); in fact, this concentration is from fraction 12A is compared to the sequences avail- too high to speculate about any important action under phys- able in the literature, as shown in Table 2, it is possible iological conditions. to observe 90% of similarity in relation to the chemotac- Fig. 4 shows the results of rat peritoneal mast cell lysis by tic peptide previously reported as Protonectin in the ven- LDH measurement in the medium. Polybia-MPI presented oms of two different species of neotropical social wasps, about 50% of LDH activity at 4.5 × 10−5 M and Polybia- Protonectarina sylveirae [4] and A. p. pallipes [20]. Thus, CP only reach this percentage at concentration higher than the peptide isolated from fraction 12A will be referred as 10−4 M. Polybia-CP. Hemolysis of rat erythrocytes was also examined for both peptides and the results are represented in Fig. 5. Polybia-MPI 3.2. Biological activities presented no hemolytic activity, while Polybia-CP presented a very reduced hemolysis at 10−5 M, but caused 78% hemol- Biological activities of Polybia-MPI and Polybia-CP were − ysis at the concentration of 10 4 M. investigated by assaying mast cell degranulation, release of Polybia-MPI and Polybia-CP presented a high chemo- LDH activity, hemolysis, chemotaxis and antimicrobial activ- taxys of PMNL cells (2.4 × 104 and 1.7 × 104 cells/ml, ities. − respectively) at the concentration of 10 5 M(Fig. 6). Fig. 3 shows the results of rat peritonial mast cell degran- The antimicrobial activity was examined and the results ulation for Polybia-MPI and Polybia-CP, revealing that both summarized in Table 3. Polybia-MPI presented a potent peptides presented a reduced activity at 10−5 M, while a rea- antimicrobial activity against both Gram-positive and sonable activity was observed when the peptides concentra- Gram-negative bacteria, while Polybia-CP demonstrated a tion was 10−4 M (62 and 52% degranulation for Polybia-MPI significant antimicrobial activity only against Gram-positive bacteria.

Fig. 3. Degranulation activity in rat peritoneal mast cell. The activity was determined by measuring the release of the granule marker, ␤-d- glucosaminidase, which co-localizes with histamine and the values for Fig. 5. Hemolytic activity in washed rat red blood cells (WRRBC). The ␤-d-glucosaminidase released in the medium were expressed in the per- absorbance measured at 540 nm from lysed WRRBC in presence of 1% centage of total ␤-d-glucosaminidase. Values are mean ± S.D. (n = 5). (v/v) Triton X-100 was considered as 100%. Values are mean ± S.D. (n = 5). 2162 B.M. Souza et al. / Peptides 26 (2005) 2157–2164

• A central cationic Lys residue is observed at the sev- enth position in the chemotactic peptides from cold cli- mate species, while Polybia-CP and Protonectin contain a cationic Lys residue at the 10th position. • The only structural difference between Protonectin and Polybia-CP is the replacement of the Gly residue at the 11th position in the sequence of Protonectin by a Ser in Polybia-CP.

Delivery of stored compounds from mast cell granules may occur either due to the cytolytic effect of the peptides or due to exocytosis activated by the binding of the peptides to Fig. 6. Chemotaxis of PMNL cells for Polybia-MPI and Polybia-CP. Values G-protein coupled receptors, which in turn activate a cascade are mean ± S.D. (n = 5). of molecular events, resulting in mast cell degranulation [10]. Table 3 Some mastoparan peptides seem to be involved in guanylate Antimicrobial activities of the peptides Polybia-CP and Polybia-MPI cyclase activation, either interacting directly with the enzyme Peptides Minimal inhibitory concentration (␮g/mL) or through other proteins [32]. E. coli P. aeruginosa B. subtilis S. aureus Despite classification as a mastoparan peptide and to present a high similarity of primary sequence when com- Polybia-MPI 8 8 4 15 Polybia-CP 250 250 15 15 pared to Protonectarina-MP, the rat mast cell degranulation −5 Tetracycline 2 18 18 0.5 activity of Polybia-MPI is reduced (EC50 = 4.5 × 10 M) −6 (reference) when compared to Protonectarina-MP (EC50 =4× 10 M) [3]. Polybia-MPI caused the release of 62% of the contents × −5 4. Discussion from rat mast cells at 4.5 10 M(Fig. 3), however it also promoted the delivering of 50% of LDH activity from these The results above reveal two novel peptide toxins from cells at the same concentration range. Therefore, taking into the venom of the social wasp P. paulista, presenting a series account both results together and the standard deviation of of different biological activities and the following primary both experiments, it may be concluded that the peptide caused sequences: the lysis of 50% of rat mast cells, which in turn led to the delivering the granules contents of these cells. • Polybia-MPI:IDWKKLLDAAKQIL-NH2 Polybia-MPI induced no hemolytic suggesting that it • Polybia-CP:ILGTILGLLKSL-NH2 demonstrates a poor interaction with the zwitterionic mem- One of the characteristic structural features of the branes of rat erythrocytes (Fig. 5), despite application of mastoparan peptides is the existence of some basic amino increasingly high doses in the assays. Thus, Polybia-MPI acid residues (generally lysine), which are responsible for the seems to be present a much higher affinity to interact with formation of positive charges, generally at the positions 4, 5, the membranes of mast cells than of erythrocytes. 11 and 12. In most mastoparan peptides from two to four Polybia-CP presented both a reduced mast cell degranu- −5 Lys residues are located in the positions mentioned above. lation activity at 10 M(Fig. 3) and mast cell lysis (Fig. 4) Thus, Polybia-MPI presents the Lys residues at the positions as expected for the most of chemotactic peptides from wasp 4, 5 and 11, while its similar natural analogue Protonectarina- venoms [22]; this peptide also has a poor hemolytic activity MP presents the basic residues at the positions 4, 11 and 12 in rat erythrocytes in the same concentration range (Fig. 5), (Table 1). as well as its natural analogue Protonectin [3,20]. The activ- −4 The primary sequences of Polybia-CP and other chemo- ities observed at the concentration of 10 M is too high to tactic peptides isolated from venoms of other species of social have any physiological meaning. Thus, the peptide Polybia- wasps [22,24] are compared in Table 2. This comparison CP does not seems to interact with the zwiterionic membranes reveals some interesting structural aspects: of rat erythrocytes and rat mast cells. Chemotaxis is the phenomenon in which bacteria or sin- • Most chemotactic peptides of the wasps endemic to cold gle cells of multicellular organisms direct their movements regions of the planet (Ves-CP-T, Ves-CP-M,Ves-CP-Xand according to the presence of specific chemical signals in Ves-CP-P) present a sequence of 13 amino acid residues, their environment. The recruitment of leukocytes to a site while the chemotactic peptides from the venom of tropical of tissue injury caused by a wasp sting, constitutes a cause species (Polybia-CP and Protonectin) present 12 residues. for inflammatory responses. Mechanistically, it involves a • Most chemotactic peptides of wasp species endemic to cascade of cellular events precisely regulated by temporal cold regions of the planet present a characteristic FLP and spatial presentation of a repertoire of molecules in the tripeptide at the amino terminal side, which is missing in migrating leukocytes and their surroundings (microenviron- Polybia-CP and Protonectin. ments) [16]. Eukaryotic cells in general sense the presence of B.M. Souza et al. / Peptides 26 (2005) 2157–2164 2163 chemotactic stimuli through stereospecific 7-transmembrane bacteria, while the peptide Polybia-CP was effective only heterotrimeric G-protein coupled receptors; the activity of against Gram-positive bacteria. The difference between the the chemotactic peptides generally is dependent on specific sequences of both peptides is the replacement of Gly residue signals mediated by G-proteins located on the plasma mem- at the 11th of Protonectin, by a Ser residue in Polybia-CP. brane of the chemoattracted cells, making the cell/peptide The side chain for the Gly residue is hydrogen, while for the interaction e relatively selective [22]. Although the mecha- Ser residue is a hydroxy methyl group. Thus, the C-terminal nism of induction of PMNL cells attraction by the peptides region of Polybia-CP is more hydrophylic than the equiva- Polybia-MPI and Polybia-CP is not known, these peptides lent region of Protonectin, which could at first, explains the probably elicit their effect by interacting with G-proteins at better interaction of Protonectin with the more hydrophobic the level of the PMNL cell membrane, as previously reported characteristics of the outer membrane of the Gram-negative for other chemotatic peptides from the venom of the tropical bacteria. social wasp P. exigua [21]. Thus, the present investigation describes the isolation, Generally, the peptide wasp toxins are polycationic pep- purification, sequencing and biological characterization of tides which may adopt a ␣-helix conformation and present two novel biologically active peptide toxins from the venom amphiphylic properties, which are essential to exhibit their of the tropical social wasp P.paulista. These toxins constitute biological activities [30]. Thus, they can interact with the members of polycationic, linear peptides deprived of cys- anionic components of the bacterial membranes in different teine residues, and demonstrating multifunctional activities ways, sometimes resulting in irreversible damage to the cell. such as: mast cell degranulation, chemotaxys of PMNL cells, Among the known toxins from wasps venoms the peptides cytolysis of erythrocytes and antimicrobial activity. Crabrolin [15], Anoplin [14] and Protonectin [20] were also reported to present antimicrobial activity. Polybia-MPI presented antimicrobial activity against both Acknowledgements Gram-positive and Gram-negative bacteria, while Polybia- CP presented antimicrobial action only against the Gram- This work was supported by a grant from the Sao˜ Paulo positive bacteria. In order to explain these effects based State Research Foundation (FAPESP); M.A.M. is Postdoc- on the possible interactions of each peptide with the bac- toral fellow from FAPESP (Proc. 01/05060-4), B.M.S. and terial membranes, the secondary structures of Polybia- M.R.M are Doctoral students fellows from FAPESP; L.D.S. MPI and Polybia-CP were predicted by using the software and L.M.M.C are Doctoral student fellow from CAPES. “Consensus Secondary Structure Prediction”, from the Net- Mario Sergio Palma (Proc. 300377/2003-5) and Fernando work Protein Sequence Analysis (http://npsa-pbil.ibcp.fr/cgi- Carlos Pagnocca are researching for the Brazilian Council bin/npsa automat.pl?page=/NPSA/npsa seccons.html). The for Scientific and Technological Development (CNPq). algorithm predicted that Polybia-MPI has about 71.43% of ␣-helix conformation and 28.57% of coil structure, while Polybia-CP was predicted to present 50% of random coil References and 50% of ambiguous conformations. Thus, it seems clear that the high content of ␣-helix of Polybia-MPI must aid its [1] Argiolas A, Pisano J. Facilitation of phospholipase A2 activity by assembly in the bacterial membrane, reflecting the lower MIC mastoparans, a new class of mast cell degranulating peptides from values observed for this peptide, when compared to Polybia- wasp venom. J Biol Chem 1983;258:13697–702. [2] Coyle B, Kavanagh K, McCann M, Devereux M, Geraghty M. Mode CP. of anti-fungal activity of 1,10-phenanthroline and its Cu(II), Mn(II) The cell wall is a complex structure, fundamentally dif- and Ag(I) complexes. BioMetals 2003;16:321–9. ferent in Gram-positive and Gram-negative bacteria. The [3] Dohtsu K, Hagiwara K, Palma MS, Nakajima T. Isolation and cell wall of bacteria consists of a polymer of disaccharides sequence analysis of peptides from the venom of Protonectarina cross-linked by short chain peptides, forming a type of pep- sylveirae (Hymenoptera ). Nat Toxins 1993;1:271–6. [4] Dohtsu K, Hagiwara K, Palma MS, Nakajima T. Determination of tidoglycan called murein. In the Gram-positive bacteria, the the structure and mechanism of action of novel peptides from the cell wall is thick (15–80 nm), consisting of several layers of venom of Protonectarina sylveirae (II). Peptide chemistry. Holland: peptidoglycan complexed with molecules of teichoic acids. ESCON Printing Co; 1992. In the Gram-negative bacteria, the cell wall is relatively thin [5] Eno AE. Pharmacological Investigation of Oedema Induced by (10 nm) and composed of a single layer of peptidoglycan venom from the Wasp Polistes fuscatus. Toxicon 1997;35:1691–8. [6] Falk W, Goodwin Jr RH, Leonard EJ. Chemotaxis assay. J Immunol surrounded by a membranous structure, the outer membrane, Meth 1980;33:239–47. which invariably also contains lipopolysaccharides. Thus, the [7] Gallo RL, Huttner KM. Antimicrobial peptides: an emerging concept outer membrane is more hydrophobic in the Gram-negative in cutaneous biology. J Investig Dermatol 1998;111:739–43. than in the Gram-positive bacteria and constitutes the target [8] Hancock REW, Chapple DS. Peptide antibiotics. Antimicrob Agents for attack by hydrophobic agents and other antibiotic agents Chemother 1999;43:1317–23. [9] Hide I, Bennett JP, Pizzey A, Boonem GM, Bar-Sagi D, Gomperts [29,31]. BD, Tatham PER. Degranulation of individual mast cells in response According to Mendes et al. [19] Protonectin has antimi- to Ca2+ and guanine nucleosides: all-or-nothing event. J Cell Biol crobial action against both Gram-positive and Gram-negative 1993;123:585–93. 2164 B.M. Souza et al. / Peptides 26 (2005) 2157–2164

[10] Higashijima T, Burnier J, Ross EM. Regulation of Gi and G0 by [22] Nagashima K, Masamichi K, Saito K, Yasuhara T, Tsukamoto T, mastoparan related peptides and hydrophilic amines. J Biol Chem Mori M, Fujino M, Nakajima T. The role of lysine at seventh posi- 1990;265:14176–86. tion of wasp chemotactic peptides. Biochem Biophys Res Commun [11] Hirai Y, Yasuhara T, Yoshida H, Nakajima T, Fujino M, Kitada C. 1990;168:844–9. A new mast cell degranulating peptide “mastoparan” in the venom [23] Nakajima T. Biochemistry of vespid venoms. In: Tu AT, editor. of Vespula lewisii. Chem Pharm Bull 1979;27:1942–4. Handbook of natural toxins, vol. 2. New York and Basel: Marcel [12] Ho CL, Hwang LL. Structure and biological activities of a new Dekker; 1984. mastoparan isolated from the venom of the hornet (Vespa basalis). [24] Nakajima T, Uzu S, Wakamatsu K, Saito K, Miyazawa T, Yasuhara Biochem J 1991;274:453–6. T, Tsukamoto Y, Fujino M. Amphiphilic peptides in wasp venom. [13] Ho CL, Shih YP, Wang KT, Yu HM. Enhancing the hypotensive Biopolymers 1986;25:115–21. effect and diminishing the cytolytic activity of hornet mastoparan B [25] Nakajima T, Wakamatsu K, Mukai H. Mastoparan as a G protein by d-amino acid substitution. Toxicon 2001;39:1561–6. activator. In: Rochat H, Martin-Eauclaire MF, editors. toxins. [14] Konno K, Hisada M, Fontana R, Lorenzi CCB, Naoki H, Itagaki Basel, Switzerland: Birkhauser¨ Verlag; 2000. p. 388–98. Y, Miwa A, Kawai N, Nakata Y, Yasuhara T, Ruggiero J, Azevedo [26] Ozaki Y, Matsumoto Y, Yatomi Y, Higashihara M, Kariya T, Kume WF, Palma MS, Nakajima T. Anoplin a novel antimicrobial peptide S. Mastoparan, wasp venom activates platelets via pertussis toxin- from the venom of the solitary wasp Anoplius samariensis. Biochim sensitive GTP-binding proteins. Biochem Biophys Res Commun Biophys Acta 2001;1550:70–80. 1990;170:779–85. [15] Krishnakumari V, Nagaraj P. Antimicrobial and hemolytic activities [27] Perianin A, Snyderman R, Mastoparan. A wasp venom peptide, iden- of crabolin, a13-residues peptide from the venom of the european tifies two discrete mechanism for elevating cytosolic calcium and Hornet, Vespa crabro, and its analogs. J Peptide Res 1997;50:88–93. inositol trisphosphates in human polymorphonuclear leukocytes. J [16] Le Y, Gong W, Li B, Dunlop NM, Shen W, Su SB, Ye RD, Wang JM. Immunol 1989;143:1669–73. Utilization of two seven-transmembrane, G-protein coupled recep- [28] Ribeiro SP, Mendes MA, Santos LD, Souza BM, Marques MR, tors, formyl peptide receptor-like 1 and formyl peptide receptor, by Azevedo Jr WF, Palma MS. Structural and functional characterization the synthetic hexapeptide WKYMVM for human phagocyte activa- of N-terminally blocked peptides isolated from the venom of the tion. J Immunol 1999;163:6777–84. social wasp Polybia paulista. Peptides 2004;25:2069–78. [17] Li ML, Lao RW, Qiu JW, Wang ZJ, Wu TM. Antimicrobial activ- [29] Schwarz G, Reiter R. Negative cooperativity and aggregation in ity of synthetic all-D masoparan M. Int J Antimicrobial Agent biphasic binding of mastoparan × peptide to membranes with acidic 2000;13:203–8. lipids. Biophys Chem 2001;90:269–77. [18] Meletiadis J, Meis´ JGM, Mouton JW, Donnelly Verweij PE. Com- [30] Sforza ML, Oyama Jr S, Canduri F, Lorenzi CCB, Pertinhez T, parison of NCCLS and 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H- Konno K, Souza BM, Palma MS, Ruggiero-Neto J, Azevedo Jr WF, tetrazolium bromide (MTT) methods of in vitro susceptibility testing Spisni A. How C-terminal carboxyamidation alters the biological of filamentous fungi and development of a new simplified method. activity of peptides from the venom of the Eumenine solitary wasp. J Clin Microbiol 2000;38:2949–54. Biochemistry 2004;43:5608–17. [19] Mendes MA, Souza BM, Santos LD, Palma MS. Structural char- [31] Singleton P. Bacteria in biology. In: Biotechnology and medicine. acterization of novel chemotactic and mastoparan peptides from John Wiley & Sons Inc; 2004. the venom of the social wasp Agelaia pallipes pallipes by high- [32] Song DL, Chang GD, Ho CL, Chang CH. Structural requirements performance liquid chromatography/electropray ionization tandem of mastoparan for activation of membrane-bound guanylate cyclase. mass spectrometry. Rap Commun Mass Spectrom 2004;18:636–42. Eur J Pharmacol 1993;247:283–8. [20] Mendes MA, de Souza BM, Marques MR, Palma MS. Structural and [33] Yasuhara T, Nakajima T, Fukuda K, Tsukamoto Y, Mori M, Kitada biological characterization of two novel peptides from the venom C, Fujino M. Structure and activity of chemotactic peptide from the of the neotropical social wasp Agelaia pallipes pallipes. Toxicon venom sac of vespinae. In: Munekata E, editor. Peptide chemistry. 2004;44:67–74. Osaka: Protein Research Foundation; 1983. p. 185–90. [21] Mendes MA, de Souza BM, Palma MS. Structural and biological [34] Yokokawa N, Komatsu M, Takeda T, Aizawa T, Yamada T, characterization of three novel mastoparan peptides from the venom Mastoparan. Mastoparan, a wasp venom stimulates insulin release of the neotropical social wasp Protopolybia exigua (Saussure). Tox- by pancreatic islets through pertussis toxin sensitive GTP-binding icon 2005;45:101–6. protein. Biochem Biophys Res Commun 1989;158:712–6.