RESUMEN
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.
Asunto(s)
Alérgenos , Venenos de Avispas/química , Alérgenos/química , Alérgenos/genética , Alérgenos/inmunología , Alérgenos/aislamiento & purificación , Humanos , Hipersensibilidad/diagnóstico , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Modelos Moleculares , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Venenos de Avispas/genética , Venenos de Avispas/inmunología , Venenos de Avispas/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Membranas Sinápticas/metabolismo , Transmisión Sináptica/efectos de los fármacos , Sinaptosomas/metabolismo , Venenos de Avispas , Avispas/química , Ácido gamma-Aminobutírico/metabolismo , Animales , Células COS , Chlorocebus aethiops , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/metabolismo , Ratas , Ratas Wistar , Membranas Sinápticas/patología , Sinaptosomas/patología , Venenos de Avispas/química , Venenos de Avispas/aislamiento & purificación , Venenos de Avispas/toxicidadRESUMEN
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.
Asunto(s)
Antiinfecciosos/farmacología , Bacterias/efectos de los fármacos , Hongos/efectos de los fármacos , Péptidos/farmacología , Infecciones Estafilocócicas/tratamiento farmacológico , Venenos de Avispas/farmacología , Avispas/química , Administración Tópica , Animales , Antiinfecciosos/aislamiento & purificación , Modelos Animales de Enfermedad , Femenino , Voluntarios Sanos , Humanos , Péptidos y Proteínas de Señalización Intercelular , Macrófagos Peritoneales/microbiología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Péptidos/aislamiento & purificación , Resultado del Tratamiento , Venenos de Avispas/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Bradiquinina/metabolismo , Corteza Cerebral/metabolismo , Colina/metabolismo , Venenos de Avispas/metabolismo , Animales , Bradiquinina/aislamiento & purificación , Bradiquinina/farmacología , Radioisótopos de Carbono/metabolismo , Corteza Cerebral/efectos de los fármacos , Colina/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Masculino , Ratas , Ratas Wistar , Venenos de Avispas/aislamiento & purificación , Venenos de Avispas/farmacología , AvispasRESUMEN
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.
Asunto(s)
Ansiolíticos/uso terapéutico , Ansiedad/tratamiento farmacológico , Bradiquinina/uso terapéutico , Fragmentos de Péptidos/uso terapéutico , Venenos de Avispas/uso terapéutico , Animales , Ansiolíticos/efectos adversos , Ansiolíticos/aislamiento & purificación , Ansiedad/psicología , Bradiquinina/efectos adversos , Bradiquinina/aislamiento & purificación , Evaluación Preclínica de Medicamentos/métodos , Masculino , Fragmentos de Péptidos/efectos adversos , Fragmentos de Péptidos/aislamiento & purificación , Ratas , Ratas Wistar , Venenos de Avispas/efectos adversos , Venenos de Avispas/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Venenos de Abeja/efectos adversos , Abejas/química , Mordeduras y Picaduras/inducido químicamente , Edema/inducido químicamente , Hiperalgesia/inducido químicamente , Venenos de Avispas/efectos adversos , Animales , Venenos de Abeja/inmunología , Venenos de Abeja/aislamiento & purificación , Mordeduras y Picaduras/inmunología , Edema/inmunología , Humanos , Hiperalgesia/inmunología , Masculino , Ratones , Dolor/inducido químicamente , Dolor/inmunología , Venenos de Avispas/inmunología , Venenos de Avispas/aislamiento & purificación , Avispas/químicaRESUMEN
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.
Asunto(s)
Bradiquinina/análogos & derivados , Bradiquinina/fisiología , Péptidos/fisiología , Venenos de Avispas/farmacología , Secuencia de Aminoácidos , Animales , Bradiquinina/aislamiento & purificación , Femenino , Motilidad Gastrointestinal/efectos de los fármacos , Cobayas , Íleon/efectos de los fármacos , Íleon/fisiología , Masculino , Datos de Secuencia Molecular , Dimensión del Dolor/métodos , Péptidos/aislamiento & purificación , Conejos , Ratas , Ratas Wistar , Venenos de Avispas/aislamiento & purificación , AvispasRESUMEN
Hymenoptera venoms are complex mixtures of biochemically and pharmacologically active components such as biogenic amines, peptides and proteins. Polycationic peptides generally constitute the largest group of Hymenoptera venom toxins, and the mastoparans constitute the most abundant and important class of peptides in the venom of social wasps. These toxins are responsible for histamine release from mast cells, serotonin from platelets, and catecholamines and adenylic acids from adrenal chromafin cells. The present work reports the structural and functional characterization of two novel mastoparan peptides identified from the venom of the neotropical social wasp Polybia paulista. The mastoparans Polybia-MP-II and -III were purified, sequenced and synthesized on solid phase using Fmoc chemistry and the synthetic peptides used for structural and functional characterizations. Polybia-MP-II and -III are tetradecapeptides, amidated at their C-termini, and form amphipathic alpha-helical conformations under membrane-mimetic conditions. Both peptides were polyfunctional, causing pronounced cell lysis of rat mast cells and erythrocytes, in addition to having antimicrobial activity against both Gram-positive and Gram-negative bacteria.
Asunto(s)
Péptidos/farmacología , Venenos de Avispas/química , Avispas/metabolismo , Animales , Degranulación de la Célula/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Bacterias Aerobias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Hemólisis/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular , L-Lactato Deshidrogenasa/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Pruebas de Sensibilidad Microbiana , Neutrófilos/citología , Neutrófilos/fisiología , Péptidos/síntesis química , Péptidos/química , Péptidos/aislamiento & purificación , Ratas , Ratas Wistar , Espectrometría de Masa por Ionización de Electrospray , Venenos de Avispas/aislamiento & purificación , Venenos de Avispas/farmacologíaRESUMEN
A novel antimicrobial peptide, eumenitin, was isolated from the venom of the solitary eumenine wasp Eumenes rubronotatus. The sequence of eumenitin, Leu-Asn-Leu-Lys-Gly-Ile-Phe-Lys-Lys-Val-Ala-Ser-Leu-Leu-Thr, was mostly analyzed by mass spectrometry together with Edman degradation, and corroborated by solid-phase synthesis. This peptide has characteristic features of cationic linear alpha-helical antimicrobial peptides, and therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the CD spectra of eumenitin in the presence of TFE or SDS showed a high content of alpha-helical conformation. Eumenitin exhibited inhibitory activity against both Gram-positive and Gram-negative bacteria, and moderately stimulated degranulation from the rat peritoneal mast cells and the RBL-2H3 cells, but showed no hemolytic activity against human erythrocytes. This antimicrobial peptide in the eumenine wasp venom may play a role in preventing potential infection by microorganisms during prey consumption by their larvae.
Asunto(s)
Antibacterianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/genética , Venenos de Avispas/química , Venenos de Avispas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Dicroismo Circular , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Venenos de Avispas/genética , Venenos de Avispas/farmacología , AvispasRESUMEN
Two novel peptides were isolated from the crude venom of the social wasp Polybia paulista, by using RP-HPLC under a gradient of MeCN from 5 to 60% (v/v) and named Polybine-I and -II. Further purification of these peptides under normal phase chromatography, rendered pure enough preparations to be sequenced by Edman degradation chemistry. However, both peptides did not interact with phenylisothiocyanate reagent, suggesting the existence of a chemically blocked N-terminus. Therefore, the sequences of both peptides were assigned by ESI-MS/MS under CID conditions, as follows: Polybine-I Ac-SADLVKKIWDNPAL-NH2 (Mr 1610 Da) and Polybine-II Ac-SVDMVMKGLKIWPL-NH2 (Mr 1657 Da). During the tandem mass spectrometry experiments, a loss of 43 a.m.u. was observed from the N-terminal residue of each peptide, suggesting the acetylation of the N-terminus. Subsequently, the peptides with and without acetylation were synthesized on solid phase and submitted to functional characterizations; the biological activities investigated were: hemolysis, chemotaxis of polymorphonucleated leukocytes (PMNL), mast cell degranulation and antibiosis. The results revealed that the acetylated peptides exhibited more pronounced chemotaxis of PMNL cells and mast cell degranulation than the respective non-acetylated congeners; no hemolytic and antibiotic activities were observed, irrespective to the blockage or not of the alpha-amino groups of the N-terminal residues of each peptide. Therefore, the N-terminal acetylation may be related to the increase of the inflammatory activity of both peptides.
Asunto(s)
Péptidos/aislamiento & purificación , Venenos de Avispas/química , Venenos de Avispas/aislamiento & purificación , Acetilación , Secuencia de Aminoácidos , Animales , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Proteínas Hemolisinas/aislamiento & purificación , Proteínas Hemolisinas/farmacología , Mastocitos/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Ratas , Espectrometría de Masa por Ionización de Electrospray , Venenos de Avispas/farmacología , AvispasRESUMEN
It has previously been shown that the denatured crude extract of Agelaia vicina wasp venom inhibits glutamate and GABA uptake in rat cerebral cortex synaptosomes. To identify the components responsible for these effects, the neurotoxin AvTx 7 (molecular weight of 1210 Da) was isolated from A. vicina venom and its effects on glutamate neurotransmission investigated. AvTx 7 inhibits glutamate uptake in a dose-dependent and uncompetitive manner. AvTx 7 was found to stimulate the glutamate release in the presence of calcium and sodium channel blockers, suggesting that its action is not mediated through these channels. AvTx 7 potentiates glutamate release in the presence of K(+) channel blockers tetraethylammonium and 4-aminopyridine, indicating that the toxin may act through these drugs-sensible K(+) channels. We suggest that AvTx 7 can be a valuable tool to enhance our understanding of K(+) channels' involvement in the release of glutamate.
Asunto(s)
Ácido Glutámico/metabolismo , Neurotoxinas/toxicidad , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Venenos de Avispas/toxicidad , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Cinética , Espectrometría de Masas , Neurotoxinas/química , Neurotoxinas/aislamiento & purificación , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Ratas , Ratas Wistar , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Venenos de Avispas/química , Venenos de Avispas/aislamiento & purificación , Avispas/químicaRESUMEN
A novel antimicrobial peptide, anoplin, was purified from the venom of the solitary wasp Anoplius samariensis. The sequence was mostly analyzed by mass spectrometry, which was corroborated by solid-phase synthesis. Anoplin, composed of 10 amino acid residues, Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2, has a high homology to crabrolin and mastoparan-X, the mast cell degranulating peptides from social wasp venoms, and, therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the circular dichroism (CD) spectra of anoplin in the presence of trifluoroethanol or sodium dodecyl sulfate showed a high content, up to 55%, of the alpha-helical conformation. A modeling study of anoplin based on its homology to mastoparan-X supported the CD results. Biological evaluation using the synthetic peptide revealed that this peptide exhibited potent activity in stimulating degranulation from rat peritoneal mast cells and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, this is the first antimicrobial component to be found in the solitary wasp venom and it may play a key role in preventing potential infection by microorganisms during prey consumption by their larvae. Moreover, this peptide is the smallest among the linear alpha-helical antimicrobial peptides hitherto found in nature, which is advantageous for chemical manipulation and medical application.
Asunto(s)
Antibacterianos/aislamiento & purificación , Oligopéptidos/química , Oligopéptidos/aislamiento & purificación , Venenos de Avispas/química , Venenos de Avispas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos , Degranulación de la Célula , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Femenino , Mastocitos/efectos de los fármacos , Mastocitos/fisiología , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Oligopéptidos/farmacología , Ratas , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Venenos de Avispas/farmacología , AvispasRESUMEN
Mastoparans are tetradecapeptides found to be the major component of vespid venoms. These peptides present a wide spectrum of biological activities, such as mast cell degranulation, hemolytic activity and also reveals antimicrobial activity. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized. At room temperature these crystals diffracted to 2.8 A resolution. However, upon cooling to cryogenic temperature around 85 K, the original resolution limit could be improved to 2.0 A. Crystals were determined to belong to the space group P3(1) (P3(2)). This is the first mastoparan to be crystallized and it will provide further insights in the conformational significance of mastoparan toxins, with respect to their potency and activity in G protein regulation.
Asunto(s)
Cristalografía por Rayos X/métodos , Proteínas de Insectos/química , Venenos de Avispas/química , Avispas/metabolismo , Animales , Frío , Cristalización , Proteínas de Insectos/aislamiento & purificación , Venenos de Avispas/aislamiento & purificaciónRESUMEN
Micro-scale (sub-pmol) isolation and sequence determination of three peptides from the venom of the solitary spider wasp Cyphononyx dorsalis is described. We isolated two novel peptides Cd-125 and Cd-146 and a known peptide Thr(6)-bradykinin from only two venom sacs of solitary spider wasp Cyphononyx dorsalis without bioassay-guided fractionation, but instead guided by MALDI-TOF MS. The MALDI-TOF MS analysis of each fraction showed the purity and molecular weight of the components, which led to the isolation of the peptides virtually without loss of sample amount. The sequences of the novel peptides Cd-125 (Asp-Thr-Ala-Arg-Leu-Lys-Trp-His) and Cd-146 (Ser-Glu-Thr-Gly-Asn-Thr-Val-Thr-Val-Lys-Gly-Phe-Ser-Pro-Leu-Arg) were determined by Edman degradation together with mass spectrometry, and finally corroborated by solid-phase synthesis. The known peptide Thr(6)-bradykinin (Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg) was identified by comparison with the synthetic authentic specimen. This is the first example for any kinins to be found in Pompilidae wasp venoms. The procedure reported here can be applicable to studies on many other components of solitary wasp venoms with limited sample availability.
Asunto(s)
Venenos de Avispas/química , Secuencia de Aminoácidos , Animales , Femenino , Espectrometría de Masas , Datos de Secuencia Molecular , Venenos de Avispas/aislamiento & purificaciónRESUMEN
A new mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF), was isolated from the venom of the solitary wasp Anterhynchium flavomarginatum micado, the most common eumenine wasp found in Japan. The structure was analyzed by FAB-MS/MS together with Edman degradation, which was corroborated by solid-phase synthesis. The sequence of EMP-AF, Ile-Asn-Leu-Leu-Lys-Ile-Ala-Lys-Gly-Ile-Ile-Lys-Ser-Leu-NH(2), was similar to that of mastoparan, a mast cell degranulating peptide from a hornet venom; tetradecapeptide with C-terminus amidated and rich in hydrophobic and basic amino acids. In fact, EMP-AF exhibited similar activity to mastoparan in stimulating degranulation from rat peritoneal mast cells and RBL-2H3 cells. It also showed significant hemolytic activity in human erythrocytes. Therefore, this is the first example that a mast cell degranulating peptide is found in the solitary wasp venom. Besides the degranulation and hemolytic activity, EMP-AF also affects on neuromuscular transmission in the lobster walking leg preparation. Three analogs EMP-AF-1 approximately 3 were snythesized and biologically tested together with EMP-AF, resulting in the importance of the C-terminal amide structure for biological activities.