Isolation and characterization of FMRFamide-like peptides in the venoms of solitary sphecid wasps.

Purification of small peptide components in the venoms of the solitary sphecid wasps, Sphex argentatus argentatus and Isodontia harmandi, led to the isolation of several major peptides. Analysis of MS/MS spectra by MALDI-TOF/TOF revealed the sequence of a new peptide Sa112 (EDVDHVFLRF-NH2), which is structurally very similar to leucomyosupressin (pQDVDHVFLRF-NH2) and SchistoFLRFamide (PDVDHVFLRF-NH2), the FMRFamide-like peptides from cockroach and locust, respectively. Indeed, this new peptide, like SchistoFLRFamide, inhibited the frequency and amplitude of spontaneous contractions of the locust oviduct in a dose-dependent manner. A non-amidated peptide Sa12b (EDVDHVFLRF) was also isolated, but this peptide had no effect on spontaneous locust oviduct contraction. This is the first example of a FMRF-like peptide to be found in solitary wasp venom. Additionally, a truncated form of the myosuppressins, which has previously been synthesized and tested for biological activity, DVDHVFLRF-NH2 (Sh5b), was found for the first time as a natural product. Four other novel peptides were isolated and characterized as Sa81 (EDDLEDFNPTVS), Sa10 (EDDLEDFNPTIA), Sh41 (DDLSDFNPKV), and Sh42 (EDDLSDFNPKV). They are structurally related to each other, having a high content of acidic amino acids, but no structural similarity to any known peptides. Ion channel associated activities of Sh41 and Sh42 were tested, but did not show any activity for Na+, K+, Ca2+ channels.

Asian Hornet, Vespa velutina Lepeletier 1836 (Hym.: Vespidae), Venom Obtention Based on an Electric Stimulation Protocol.

The yellow-legged Asian hornet (Vespa velutina Lepeletier 1836 (Hymenoptera: Vespidae)) is naturally distributed in China, Southeast Asia, and India; however, recently it has been detected outside of its native area, confirmed as being established in South Korea, Europe, and Japan. Health risks and deaths caused by the invasive Vespa velutina stings have become a public health concern, being the most common cause of anaphylaxis due to hymenopterans in some European regions. This in turn has led to increased demand from medical practitioners and researchers for Vespa velutina venom for diagnostic and therapeutic purposes. In this study, a straightforward, quick, and inexpensive method for obtaining Vespa velutina venom by electric stimulation is described. The venom extracts were analyzed by nuclear magnetic resonance spectroscopy (1H-NMR). The availability of Vespa velutina venom will lead to improved diagnostic and therapeutic methods, mainly by venom immunotherapy (VIT), in patients allergic to this invasive species.

Sweet as honey, bitter as bile: Mitochondriotoxic peptides and other therapeutic proteins isolated from animal tissues, for dealing with mitochondrial apoptosis.

Mitochondria are subcellular organelles involved in cell metabolism and cell life-cycle. Their role in apoptosis regulation makes them an interesting target of new drugs for dealing with cancer or rare diseases. Several peptides and proteins isolated from animal and plant sources are known for their therapeutic properties and have been tested on cancer cell-lines and xenograft murine models, highlighting their ability in inducing cell-death by triggering mitochondrial apoptosis. Some of those molecules have been even approved as drugs. Conversely, many other bioactive compounds are still under investigation for their proapoptotic properties. In this review we report about a group of peptides, isolated from animal venoms, with potential therapeutic properties related to their ability in triggering mitochondrial apoptosis. This class of compounds is known with different names, such as mitochondriotoxins or mitocans.

Purification and molecular characterization of phospholipase, antigen 5 and hyaluronidases from the venom of the Asian hornet (Vespa velutina).

The aim of this study was to purify potential allergenic components of Vespa velutina venom, the yellow legged Asian Hornet, and perform a preliminary characterization of the purified proteins. Starting from the whole venom of V.velutina, several chromatographic steps allowed to purify the phospholipase (named Vesp v 1), as well as the antigen 5 (Vesp v 5, the only allergenic component described as such so far). The two hyaluronidase isoforms found (Vesp v 2A and Vesp v 2B) cannot be separated from each other, but they are partially purified and characterized. Purity of the isolated proteins in shown by SDSPAGE, as well as by the results of the N-terminal sequencing. This characterization and nLC-MS/MS data provide most of the sequence for Vesp v 1 and Vesp v 5 (72 and 84% coverage, respectively), confirming that the whole sequences of the isolated natural components match with the data available in public transcriptomic databases. It is of particular interest that Vesp v 1 is a glycosylated phospholipase, a fact that had only described so far for the corresponding allergen components of Dolichovespula maculata and Solenopsis invicta. The availability of the complete sequences of Vespa velutina components permits comparison with homologous sequences from other Hymenoptera. These data demonstrate the higher similarity among the species of the genera Vespa and Vespula, in comparison to Polistes species, as it is especially observed with the hyaluronidases isoforms: the isoform Vesp v 2A only exists in the former genera, and not in Polistes; in addition, the most abundant isoform (Vesp v 2B) exhibits 93% sequence identity with the Ves v 2 isoform of Vespula vulgaris. Finally, the isolated components might be useful for improving the diagnosis of patients that could be allergic to stings of this invasive Asian hornet, as it has been the case of an improved diagnosis and treatment of other Hymenoptera-sensitized patients.

Antibacterial effects of gold nanoparticles functionalized with the extracted peptide from Vespa orientalis wasp venom.

The development of novel antimicrobial strategies is necessary because of the escalation of multidrug-resistant pathogens. Recently, antimicrobial peptides and their combination with nanoparticles were regarded as a promising tool to target drug-resistant pathogens. Herein, we evaluated antimicrobial efficacy of a peptide extracted from Vespa orientalis wasp venom and also its conjugation with gold nanoparticles. Nanoparticle conjugation measurement was done by evaluating the absorbance changes of the surface plasmon resonance band of gold nanoparticles at 555 nm. A significant increase in the antibacterial activity against gram negative and positive bacteria was obtained when the extracted peptide conjugated with gold nanoparticles. Finally, the results show that this new peptide-AuNps has the high practical potential for antibacterial activity and may provide an alternative therapy for bacterial infection.

Antimicrobial activity and stability of stapled helices of polybia-MP1.

Polybia-MP1 is a well-known natural antimicrobial peptide isolated from the venom of the social wasp Polybia paulista. A recent study showed that this peptide displays a broad antibacterial spectrum as well as low toxicity to human red blood cells and normal fibroblasts. However, its moderate antimicrobial activity and high susceptibility to protease have been a major hurdle for clinical use. This study examined the possibility of developing biologically more potent, yet metabolically more stable, analogues of MP1 using an emerging technology termed "all-hydrocarbon stapling." The stapled analogues of MP1 showed more than a threefold increase in helicity as well as an approximately 70-fold enhancement in proteolytic stability. These stapled analogues also exhibited a significant increase in inhibition against some Gram-positive bacteria while displaying a modest enhancement in hemolytic activity. Overall, the current study demonstrated that the all-hydrocarbon stapling system is a highly useful tool for the development of biologically more potent and metabolically more stable analogues of natural antimicrobial peptides.

Heterologous Expression, Purification and Immunoreactivity of the Antigen 5 from Polybia paulista Wasp Venom.

Polybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy.

Isolation and chemical characterization of agelaiatoxin8 (AvTx8) from Agelaia vicina wasp venom and its biological effects on GABA neurotransmission.

Arthropod venoms are sources of molecules that may be useful tools to investigate molecular mechanisms of putative new medicines and laboratory drugs. Here we show the effects of the compound agelaiatoxin-8 (AVTx8), isolated from Agelaia vicina venom, on γ-aminobutyric acid (GABA) neurotransmission in rat brain synaptosomes. Analysis reveals that AvTx8 is composed by 14 amino acid residues with a molecular weight (MW) of 1567 Da. AvTx8 increased GABA release and inhibited GABA uptake in synaptosomes from rat cerebral cortex. AvTx8 inhibited GABA uptake and increased GABA release in the presence of Ca+ , Na+ , and K+ channel blockers, suggesting that it acts directly on GABA transporters. In addition, AvTx8 significantly decreases GABA binding in synaptic membranes from rat brain cortex, suggesting that it also modulates the activity of GABA receptors. Moreover, AvTx8 decreased GAT-1- and GAT-3-mediated GABA uptake in transfected COS-7 cells. Accordingly, we suggest that AvTx8 modulates GABA neurotransmission and might provide a novel entry point for identifying a new class of GABA-modulating neuroprotective drugs.

Beneficial effect of adipokinetic hormone on neuromuscular paralysis in insect body elicited by braconid wasp venom.

The effect of Habrobracon hebetor venom and the role of the adipokinetic hormone (AKH) in poisoned adult females of the firebug Pyrrhocoris apterus were studied 24 and 48h after treatments. Venom application elicited total neuromuscular paralysis in firebugs, but the co-application of venom and Pyrap-AKH significantly reduced paralysis (up to 3.2 times) compared to the application of venom only. Although the mechanisms of their action are unknown, both agents might affect neuromuscular junctions. Venom application significantly increased the expression of both P. apterus Akh genes (Pyrap-Akh 5.4 times and Peram-Cah-II 3.6 times), as well as the level of AKHs in the central nervous system (2.5 times) and haemolymph (3.0 times). In the haemolymph, increased AKH levels might have led to the mobilization of stored lipids, which increased 1.9 times, while the level of free carbohydrates remained unchanged. Total metabolism, monitored by carbon dioxide production, significantly declined in paralysed P. apterus individuals (1.4 times and 1.9 times, 24 and 48h after the treatment, respectively), probably because of a malfunction of the muscular system. The results suggest an active role of AKH in the defence mechanism against the stress elicited by neuromuscular paralysis, and the possible involvement of this hormone in neuronal/neuromuscular signalling.

Evaluation of the antimicrobial activity of the mastoparan Polybia-MPII isolated from venom of the social wasp Pseudopolybia vespiceps testacea (Vespidae, Hymenoptera).

Mastoparans, a class of peptides found in wasp venom, have significant effects following a sting as well as useful applications in clinical practice. Among these is their potential use in the control of micro-organisms that cause infectious diseases with a significant impact on society. Thus, the present study describes the isolation and identification of a mastoparan peptide from the venom of the social wasp Pseudopolybia vespiceps and evaluated its antimicrobial profile against bacteria (Staphylococcus aureus and Mycobacterium abscessus subsp. massiliense), fungi (Candida albicans and Cryptococcus neoformans) and in vivo S. aureus infection. The membrane pore-forming ability was also assessed. The mastoparan reduced in vitro and ex vivo mycobacterial growth by 80% at 12.5 µM in infected peritoneal macrophages but did not affect the shape of bacterial cells at the dose tested (6.25 µM). The peptide also showed potent action against S. aureus in vitro (EC50 and EC90 values of 1.83 µM and 2.90 µM, respectively) and reduced the in vivo bacterial load after 6 days of topical treatment (5 mg/kg). Antifungal activity was significant, with EC50 and EC90 values of 12.9 µM and 15.3 µM, respectively, for C. albicans, and 11 µM and 22.70 µM, respectively, for C. neoformans. Peptides are currently attracting interest for their potential in the design of antimicrobial drugs, particularly due to the difficulty of micro-organisms in developing resistance to them. In this respect, Polybia-MPII proved to be highly effective, with a lower haemolysis rate compared with peptides of the same family.

Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide anoplin.

Anoplin is a short natural cationic antimicrobial peptide which is derived from the venom sac of the solitary wasp, Anoplius samariensis. Due to its short sequence G1 LLKR5 IKT8 LL-NH2 , it is ideal for research tests. In this study, novel analogs of anoplin were prepared and examined for their antimicrobial, hemolytic activity, and proteolytic stability. Specific substitutions were introduced in amino acids Gly1 , Arg5 , and Thr8 and lipophilic groups with different lengths in the N-terminus in order to investigate how these modifications affect their antimicrobial activity. These cationic analogs exhibited higher antimicrobial activity than the native peptide; they are also nontoxic at their minimum inhibitory concentration (MIC) values and resistant to enzymatic degradation. The substituted peptide GLLKF5 IKK8 LL-NH2 exhibited high activity against Gram-negative bacterium Zymomonas mobilis (MIC = 7 µg/ml), and the insertion of octanoic, decanoic, and dodecanoic acid residues in its N-terminus increased the antimicrobial activity against Gram-positive and Gram-negative bacteria (MIC = 5 µg/ml). The conformational characteristics of the peptide analogs were studied by circular dichroism. Structure activity studies revealed that the substitution of specific amino acids and the incorporation of lipophilic groups enhanced the amphipathic α-helical conformation inducing better antimicrobial effects. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Evaluation of Thr6-bradykinin purified from Polybia occidentalis wasp venom in the choline uptake of mammal cortices.

CONTEXT: Thr6-bradykinin is a peptide found in the venom of social and solitary wasps. This kinin, along with other bradykinin-like peptides, is known to cause irreversible paralysis in insects by presynaptic blockade of cholinergic transmission. However, this activity has never been tested in mammals. OBJECTIVE: As such, the objective of this study was to evaluate the effect of Thr6-bradykinin on the cholinergic system of rats. MATERIALS AND METHODS: The peptide was isolated from the venom of the Neotropical social wasp Polybia occidentalis Olivier (Vespidae). After correct identification and quantification by ESI-MS and MS/MS, the peptide was tested in [14C]-choline uptake using rat cortical synaptosomes. Each uptake assay was accompanied by lactic acid dehydrogenase (LDH) activity measurement to evaluate synaptosome integrity in the presence of six increasing concentrations of BK or Thr6-BK (0.039, 0.156, 0.625, 2.500, 10.000 and 40.000 µM). RESULTS: Data revealed that neither BK nor Thr6-BK at any of the six concentrations tested (from 0.039 to 40.000 µM) affected [14C]-choline uptake in synaptosomes. Moreover, there was no increase in LDH in the supernatants, indicating that BK and Thr6-BK did not disrupt the synaptosomes. DISCUSSION AND CONCLUSION: In contrast to previous reports for the insect central nervous system (CNS), Thr6-BK had no effect on mammalian cholinergic transmission. Nevertheless, this selectivity for the insect CNS, combined with its irreversible mode of action may be relevant to the discovery of new sources of insecticides and could contribute to understanding the role of kinins in the mammalian CNS.

MP-V1 from the Venom of Social Wasp Vespula vulgaris Is a de Novo Type of Mastoparan that Displays Superior Antimicrobial Activities.

Mastoparans from the venom of social wasps have attracted considerable attention as effective antibiotic candidates. In this study, mastoparan V1 (MP-V1) from Vespula vulgaris was first disclosed to have a peptide amino acid sequence distinct from typical mastoparans and its biochemical properties and antimicrobial effects were compared with those of typical mastoparans MP-L, -X(V) and -B. Circular dichroism (CD) spectroscopy revealed that MP-V1 and -X(V) form more stable α-helical conformations in lipid membrane-like environments than MP-L and -B. In parallel, these two also showed more effective antimicrobial activities against the pathogens than did MP-L and -B. Although MP-V1 had a less stable α-helical conformation than MP-X(V), it showed stronger antimicrobial effects against Streptococcus mutans and Salmonella enterica than MP-X(V). In the meantime, analysis of hemolytic activity revealed a range of doses (~50 µM) that exhibited little potent cytotoxicity on human erythrocytes. Finally, the atypical MP-V1 peptide amino acid sequence provided important clues to understanding its antimicrobial mechanism from a structural perspective. Therefore, it has been concluded that MP-V1 is a de novo type of mastoparan with superior antimicrobial activities against both pathogenic bacteria and fungi, which may be useful in developing multipurpose antimicrobial drugs against infectious diseases.

Bradykinin in Hemipepsis ustulata: A novel method for safely milking wasps.

Wasp venom characterization is of interest across multiple disciplines such as medicinal chemistry and evolutionary biology. A simple method is described herein to milk wasp venom without undue risks to the researcher. The wasps were immobilized by cooling for safe handling, restrained, and their venom was collected on parafilm. Bradykinin from Hemipepsis ustulata was identified by LC-MS/MS during method verification.

Anxiolytic activity and evaluation of potentially adverse effects of a bradykinin-related peptide isolated from a social wasp venom.

Anxiety disorders are major health problems in terms of costs stemming from sick leave, disabilities, healthcare and premature mortality. Despite the availability of classic anxiolytics, some anxiety disorders are still resistant to treatment, with higher rates of adverse effects. In this respect, several toxins isolated from arthropod venoms are useful in identifying new compounds to treat neurological disorders, particularly pathological anxiety. Thus, the aims of this study were to identify and characterize an anxiolytic peptide isolated from the venom of the social wasp Polybia paulista. The peptide was identified as Polisteskinin R, with nominal molecular mass [M+H](+)=1301Da and primary structure consisting of Ala-Arg-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH. The anxiolytic effect was tested using the elevated plus maze test. Moreover, adverse effects on the spontaneous behavior and motor coordination of animals were assessed using the open field and rotarod tests. Polisteskinin R induced a dose-dependent anxiolytic effect. Animals treated with the peptide and diazepam spent significantly more time into the open arms when compared to the groups treated with the vehicle and pentylenetetrazole. No significant differences in spontaneous behavior or motor coordination were observed between the groups, showing that the peptide was well tolerated. The interaction by agonists in both known BK receptors induces a variability of physiological effects; Polisteskinin R can act on these receptors, inducing modulatory activity and thus, attenuating anxiety behaviors. The results of this study demonstrated that the compound Polisteskinin R exerted potent anxiolytic effects and its analogues are promising candidates for experimental pharmacology.

A simple non-invasive technique for venom milking from a solitary wasp Delta conoideum Gmelin (Hymenoptera: Vespidae).

Prospecting wasp, ant and bee venom for active bio-molecules has gained considerable interest among researchers in recent years. Collecting sufficient quantity of venom from solitary wasps without sacrificing them is often difficult. Here we describe a non-invasive technique for collecting venom from a solitary wasp Delta conoideum Gmelin (Red-backed potter wasp). Venom was milked by presenting an agar block to a single female wasp for stinging. The venom was extracted from the agar block using ACN: water solvent system. The total protein in venom was estimated quantitatively and the presence of peptides in the venom was confirmed by MALDI-TOF analysis. The proposed technique is non-invasive and pure venom can be repeatedly 'milked' using this method from other wasps and also bees without the need for sacrificing a large number of individuals.

Biomarker genes highlight intraspecific and interspecific variations in the responses of Pinus taeda L. and Pinus radiata D. Don to Sirex noctilio F. acid gland secretions.

Sirex noctilio F., a Eurasian horntail woodwasp recently introduced into North America, oviposits in pines and other conifers and in the process spreads a phytopathogenic fungus that serves as a food source for its larvae. During oviposition the woodwasp also deposits mucus produced in its acid (venom) gland that alters pine defense responses and facilitates infection by the fungus. A 26,496-feature loblolly pine cDNA microarray was used to survey gene expression of pine tissue responding to S. noctilio venom. Six genes were selected for further assessment by quantitative real-time polymerase chain reaction (qRT-PCR), including one that encoded an apparent PR-4 protein and another that encoded a thaumatin-like protein. Expression of both was strongly induced in response to venom, while expression of an apparent actin gene (ACT1) was stable in response to the venom. The pattern of gene response was similar in Pinus taeda L. and Pinus radiata D. Don, but the magnitude of response in P. radiata was significantly stronger for each of the induced genes. The magnitude of the biomarker gene response to venom also varied according to genotype within these two species. The qRT-PCR assay was used to demonstrate that the primary bioactive component in S. noctilio venom is a polypeptide.

Expression of enzymatically inactive wasp venom phospholipase A1 in Pichia pastoris.

Wasp venom allergy is the most common insect venom allergy in Europe. It is manifested by large local reaction or anaphylactic shock occurring after a wasp sting. The allergy can be treated by specific immunotherapy with whole venom extracts. Wasp venom is difficult and costly to obtain and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form in yeast Pichia pastoris, which can give high yields of correctly folded protein on defined minimal medium and secretes relatively few native proteins simplifying purification.Residual amounts of enzymatically active phospholipase A1 could be expressed, but the venom protein had a deleterious effect on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A). All the three mutated forms could be expressed in P. pastoris. The H229A mutant did not have any detectable phospholipase A1 activity and was secreted at the level of several mg/L in shake flask culture. The protein was purified by nickel-affinity chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. The protein could bind IgE antibodies from wasp venom allergic patients and could inhibit the binding of wasp venom to IgE antibodies specific for phospholipase A1 as shown by Enzyme Allergo-Sorbent Test (EAST). Moreover, the recombinant protein was allergenic in a biological assay as demonstrated by its capability to induce histamine release of wasp venom-sensitive basophils.The recombinant phospholipase A1 presents a good candidate for wasp venom immunotherapy.

Hyperalgesic and edematogenic effects of peptides isolated from the venoms of honeybee (Apis mellifera) and neotropical social wasps (Polybia paulista and Protonectarina sylveirae).

Stings by bees and wasps, including Brazilian species, are a severe public health problem. The local reactions observed after the envenoming includes typical inflammatory response and pain. Several studies have been performed to identify the substances, including peptides that are responsible for such phenomena. The aim of the present study is to characterize the possible nociceptive (hyperalgesic) and edematogenic effects of some peptides isolated from the venoms of the honeybee (Apis mellifera) and the social wasps Polybia paulista and Protonectarina sylveirae, in addition to characterize some of the mechanisms involved in these phenomena. For this purpose, different doses of the peptides mellitin (Apis mellifera), Polybia-MP-I, N-2-Polybia-MP-I (Polybia paulista), Protonectarina-MP-NH2 and Protonectarina-MP-OH (Protonectarina sylveirae) were injected into the hind paw of mice. Hyperalgesia and edema were determined after peptide application, by using an electronic von Frey apparatus and a paquimeter. Carrageenin and saline were used as controls. Results showed that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH(2) and Protonectarina-MP-OH peptides produced a dose- and time-related hyperalgesic and edematogenic responses. Both phenomena are detected 2 h after melittin, Polybia-MP-I, N-2-Polybia-MP-I injection; their effects lasted until 8 h. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that zileuton blocked peptide-induced hyperalgesia and induced a decrease of the edematogenic response. On the other hand, indomethacin did not interfere with these phenomena. These results indicate that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH(2), and Protonectarina-MP-OH peptides could contribute to inflammation and pain induced by insect venoms.

Bradykinin-related peptides in the venom of the solitary wasp Cyphononyx fulvognathus.

Bradykinin (BK) and its related peptides are widely distributed in venomous animals, including wasps. In fact, we have previously purified a novel BK-related peptide (BRP) named Cd-146 and the threonine(6)-bradykinin (Thr(6)-BK) from the venom of the solitary wasp Cyphononyx fulvognathus. Further survey of this same wasp venom extract allowed the structural characterization of two other novel BRPs, named here as fulvonin and cyphokinin. Biochemical characterization performed here showed that although the high primary structure similarity observed with BK, these wasp peptides are not good substrates for angiotensin I-converting enzyme (ACE) acting more likely as inhibitors of this enzyme. In pharmacological assays, only those more structurally similar to BK, namely cyphokinin and Thr(6)-BK, were able to promote the contraction of guinea-pig ileum smooth muscle preparations, which was completely blocked by the B(2) receptors antagonist HOE-140 in the same way as observed for BK. Only fulvonin was shown to potentiate BK-elicited smooth muscle contraction. Moreover, the 2 new wasp BRPs, namely fulvonin and cyphokinin, as well as Cd-146 and Thr(6)-BK, showed hyperalgesic effect in the rat paw pressure test after intraplantar injection. This effect was shown here to be due to the action of these peptides on BK receptors, since the hyperalgesia induced by both Cd-146 and fulvonin was blocked by B(1) receptor antagonist, while the effect of both cyphokinin and Thr(6)-BK was reversed by B(2) antagonist. This data give support to a better understanding of the function and targets of the kinin-related peptides widely found in several insect venoms.