RESUMO
Spiders are one of the most successful venomous animals, with more than 48,000 described species. Most spider venoms are dominated by cysteine-rich peptides with a diverse range of pharmacological activities. Some spider venoms contain thousands of unique peptides, but little is known about the mechanisms used to generate such complex chemical arsenals. We used an integrated transcriptomic, proteomic, and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins. Twenty-six of the 33 superfamilies are disulfide-rich peptides, and we show that 15 of these are knottins that contribute >90% of the venom proteome. NMR analyses revealed that most of these disulfide-rich peptides are structurally related and range in complexity from simple to highly elaborated knottin domains, as well as double-knot toxins, that likely evolved from a single ancestral toxin gene.
Assuntos
Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Venenos de Aranha/química , Animais , Proteínas de Artrópodes/análise , Austrália , Dípteros/efeitos dos fármacos , Dissulfetos , Evolução Molecular , Feminino , Perfilação da Expressão Gênica , Espectrometria de Massas , Peptídeos/análise , Peptídeos/química , Peptídeos/genética , Filogenia , Conformação Proteica , Proteômica/métodos , Venenos de Aranha/genética , Venenos de Aranha/toxicidade , Aranhas/genéticaRESUMO
A critical hurdle in ant venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of venom proteins and peptides. To resolve this, we used venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Paraponera clavata venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides.
Assuntos
Venenos de Formiga/química , Formigas/metabolismo , Perfilação da Expressão Gênica , Proteínas de Insetos/análise , Neurotoxinas/análise , Proteoma , Proteômica , Transcriptoma , Animais , Venenos de Formiga/genética , Venenos de Formiga/toxicidade , Formigas/genética , Cálcio/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Bases de Dados Genéticas , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/toxicidade , Camundongos Endogâmicos C57BL , Neurotoxinas/genética , Neurotoxinas/toxicidade , Espectrometria de Massas em TandemRESUMO
Using an integrated transcriptomic and proteomic approach, we characterized the venom peptidome of the European red ant, Manica rubida. We identified 13 "myrmicitoxins" that share sequence similarities with previously identified ant venom peptides, one of them being identified as an EGF-like toxin likely resulting from a threonine residue modified by O-fucosylation. Furthermore, we conducted insecticidal assays of reversed-phase HPLC venom fractions on the blowfly Lucilia caesar, permitting us to identify six myrmicitoxins (i.e., U3-, U10-, U13-, U20-MYRTX-Mri1a, U10-MYRTX-Mri1b, and U10-MYRTX-Mri1c) with an insecticidal activity. Chemically synthesized U10-MYRTX-Mri1a, -Mri1b, -Mri1c, and U20-MYRTX-Mri1a irreversibly paralyzed blowflies at the highest doses tested (30-125 nmol·g-1). U13-MYRTX-Mri1a, the most potent neurotoxic peptide at 1 h, had reversible effects after 24 h (150 nmol·g-1). Finally, U3-MYRTX-Mri1a has no insecticidal activity, even at up to 55 nmol·g-1. Thus, M. rubida employs a paralytic venom rich in linear insecticidal peptides, which likely act by disrupting cell membranes.
Assuntos
Venenos de Formiga , Formigas , Animais , Peptídeos , Proteômica , PeçonhasRESUMO
Many chemical insecticides are becoming less efficacious due to rising resistance in pest species, which has created much interest in the development of new, eco-friendly bioinsecticides. Since insects are the primary prey of most spiders, their venoms are a rich source of insect-active peptides that can be used as leads for new bioinsecticides or as tools to study molecular receptors that are insecticidal targets. In the present study, we isolated two insecticidal peptides, µ/ω-TRTX-Mb1a and -Mb1b, from venom of the African tarantula Monocentropus balfouri. Recombinant µ/ω-TRTX-Mb1a and -Mb1b paralyzed both Lucilia cuprina (Australian sheep blowfly) and Musca domestica (housefly), but neither peptide affected larvae of Helicoverpa armigera (cotton bollworms). Both peptides inhibited currents mediated by voltage-gated sodium (NaV) and calcium channels in Periplaneta americana (American cockroach) dorsal unpaired median neurons, and they also inhibited the cloned Blattella germanica (German cockroach) NaV channel (BgNaV1). An additional effect seen only with Mb1a on BgNaV1 was a delay in fast inactivation. Comparison of the NaV channel sequences of the tested insect species revealed that variations in the S1-S2 loops in the voltage sensor domains might underlie the differences in activity between different phyla.
Assuntos
Agentes de Controle Biológico/farmacologia , Peptídeos/farmacologia , Venenos de Aranha/química , Aranhas , Sequência de Aminoácidos , Animais , Baratas/efeitos dos fármacos , Dípteros/efeitos dos fármacos , Feminino , Larva/efeitos dos fármacos , Lepidópteros/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Peptídeos/genética , Proteínas Recombinantes/farmacologia , Venenos de Aranha/genéticaRESUMO
κ-Hexatoxins (κ-HXTXs) are a family of excitotoxic insect-selective neurotoxins from Australian funnel-web spiders that are lethal to a wide range of insects, but display no toxicity towards vertebrates. The prototypic κ-HXTX-Hv1c selectively blocks native and expressed cockroach large-conductance calcium-activated potassium (BKCa or KCa1.1) channels, but not their mammalian orthologs. Despite this potent and selective action on insect KCa1.1 channels, we found that the classical KCa1.1 blockers paxilline, charybdotoxin and iberiotoxin, which all block insect KCa1.1 channels, are not lethal in crickets. We therefore used whole-cell patch-clamp analysis of cockroach dorsal unpaired median (DUM) neurons to study the effects of κ-HXTX-Hv1c on sodium-activated (KNa), delayed-rectifier (KDR) and 'A-type' transient (KA) K+ channels. 1 µM κ-HXTX-Hv1c failed to significantly inhibit cockroach KNa and KDR channels, but did cause a 30 ± 7% saturating inhibition of KA channel currents, possibly via a Kv4 (Shal-like) action. However, this modest action at such a high concentration of κ-HXTX-Hv1c would indicate a different lethal target. Accordingly, we assessed the actions of κ-HXTX-Hv1c on neurotransmitter-gated ion channels in cockroach DUM neurons. We found that κ-HXTX-Hv1c failed to produce any major effects on GABAA or glutamate-Cl receptors but dramatically slowed nicotine-evoked ACh receptor (nAChR) current decay and reversed nAChR desensitization. These actions occurred without any alterations to nAChR current amplitude or the nicotine concentration-response curve, and are consistent with a positive allosteric modulation of nAChRs. κ-HXTX-Hv1c therefore represents the first venom peptide that selectively modulates insect nAChRs with a mode of action similar to the excitotoxic insecticide spinosyn A. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'
Assuntos
Neurotoxinas/toxicidade , Receptores Nicotínicos/efeitos dos fármacos , Venenos de Aranha/toxicidade , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Análise de Variância , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Estimulação Elétrica , Gryllidae , Humanos , Indóis/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Receptores Nicotínicos/química , Canais de Ânion Dependentes de Voltagem/fisiologiaRESUMO
Ants have evolved venoms rich in peptides and proteins used for predation, defense, and communication. However, they remain extremely understudied due to the minimal amount of venom secreted by each ant. The present study investigated the differences in the proteome and peptidome of the venom from the bullet ant, Paraponera clavata. Venom samples were collected from a single colony either by manual venom gland dissection or by electrical stimulation and were compared using proteomic methods. Venom proteins were separated by 2D-PAGE and identified by nanoLC-ESI-QTOF MS/MS. Venom peptides were initially separated using C18 reversed-phase high-performance liquid chromatography, then analyzed by MALDI-TOF MS. The proteomic analysis revealed numerous proteins that could be assigned a biological function (total 94), mainly as toxins, or roles in cell regulation and transport. This investigation found that ca. 73% of the proteins were common to venoms collected by the two methods. The peptidomic analysis revealed a large number of peptides (total 309) but with <20% shared by the two collection methods. There was also a marked difference between venoms obtained by venom gland dissection from different ant colonies. These findings demonstrate the rich composition and variability of P. clavata venom.
Assuntos
Venenos de Formiga/análise , Peptídeos/análise , Proteômica/métodos , Animais , Formigas/química , Formigas/patogenicidade , Eletroforese em Gel Bidimensional , Proteínas de Insetos/análise , Espectrometria de Massas em TandemRESUMO
Sheep flystrike is caused by parasitic flies laying eggs on soiled wool or open wounds, after which the hatched maggots feed on the sheep flesh and often cause large lesions. It is a significant economic problem for the livestock industry as infestations are difficult to control due to ongoing cycles of larval development into flies followed by further egg laying. We therefore screened venom fractions from the Australian theraphosid spider Coremiocnemis tropix to identify toxins active against the sheep blowfly Lucilia cuprina, which is the primary cause of flystrike in Australia. This screen led to isolation of two insecticidal peptides, Ct1a and Ct1b, that are lethal to blowflies within 24 h of injection. The primary structure of these peptides was determined using a combination of Edman degradation and sequencing of a C. tropix venom-gland transcriptome. Ct1a and Ct1b contain 39 and 38 amino acid residues, respectively, including six cysteine residues that form three disulfide bonds. Recombinant production in bacteria (Escherichia coli) resulted in low yields of Ct1a whereas solid-phase peptide synthesis using native chemical ligation produced sufficient quantities of Ct1a for functional analyses. Synthetic Ct1a had no effect on voltage-gated sodium channels from the American cockroach Periplanata americana or the German cockroach Blattella germanica, but it was lethal to sheep blowflies with an LD50 of 1687 pmol/g.
Assuntos
Proteínas de Artrópodes/isolamento & purificação , Dípteros , Inseticidas/isolamento & purificação , Venenos de Aranha/química , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/toxicidade , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Alinhamento de Sequência , Análise de Sequência de Proteína , Ovinos/parasitologia , Testes de Toxicidade , TranscriptomaRESUMO
The inexorable decline in the armament of registered chemical insecticides has stimulated research into environmentally-friendly alternatives. Insecticidal spider-venom peptides are promising candidates for bioinsecticide development but it is challenging to find peptides that are specific for targeted pests. In the present study, we isolated an insecticidal peptide (Ae1a) from venom of the African spider Augacephalus ezendami (family Theraphosidae). Injection of Ae1a into sheep blowflies (Lucilia cuprina) induced rapid but reversible paralysis. In striking contrast, Ae1a was lethal to closely related fruit flies (Drosophila melanogaster) but induced no adverse effects in the recalcitrant lepidopteran pest Helicoverpa armigera. Electrophysiological experiments revealed that Ae1a potently inhibits the voltage-gated sodium channel BgNaV1 from the German cockroach Blattella germanica by shifting the threshold for channel activation to more depolarized potentials. In contrast, Ae1a failed to significantly affect sodium currents in dorsal unpaired median neurons from the American cockroach Periplaneta americana. We show that Ae1a interacts with the domain II voltage sensor and that sensitivity to the toxin is conferred by natural sequence variations in the S1-S2 loop of domain II. The phyletic specificity of Ae1a provides crucial information for development of sodium channel insecticides that target key insect pests without harming beneficial species.
Assuntos
Inseticidas/farmacologia , Peptídeos/farmacologia , Venenos de Aranha/química , Aranhas/fisiologia , Canais de Sódio Disparados por Voltagem/química , Animais , Blattellidae/efeitos dos fármacos , Dípteros/efeitos dos fármacos , Drosophila melanogaster/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/metabolismo , Inseticidas/química , Lepidópteros/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Peptídeos/genética , Peptídeos/isolamento & purificação , Periplaneta/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Aranhas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Animal venom peptides are currently being developed as novel drugs and bioinsecticides. Because ants use venoms for defense and predation, venomous ants represent an untapped source of potential bioactive toxins. This study compared the protein and peptide components of the poneroid ants Neoponera commutata, Neoponera apicalis, and Odontomachus hastatus and the formicoid ants Ectatomma tuberculatum, Ectatomma brunneum, and Myrmecia gulosa. 1D and 2D PAGE revealed venom proteins in the mass range <10 to >250 kDa. NanoLC-ESI-QTOF MS/MS analysis of tryptic peptides revealed the presence of common venom proteins and also many undescribed proteins. RP-HPLC separation followed by MALDI-TOF MS of the venom peptides also revealed considerable heterogeneity. It was found that the venoms contained between 144 and 1032 peptides with 5-95% of peptides in the ranges 1-4 and 1-8 kDa for poneroid and formicoid ants, respectively. By employing the reducing MALDI matrix 1,5-diaminonapthalene, up to 28 disulfide-bonded peptides were also identified in each of the venoms. In particular, the mass range of peptides from poneroid ants is lower than peptides from other venoms, indicating possible novel structures and pharmacologies. These results indicate that ant venoms represent an enormous, untapped source of novel therapeutic and bioinsecticide leads.
Assuntos
Venenos de Formiga/química , Peptídeos/análise , Proteínas/análise , Animais , Formigas , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Heterogeneidade Genética , Peso Molecular , Especificidade da Espécie , Espectrometria de Massas em TandemRESUMO
Ants (Formicidae) represent a taxonomically diverse group of hymenopterans with over 13,000 extant species, the majority of which inject or spray secretions from a venom gland. The evolutionary success of ants is mostly due to their unique eusociality that has permitted them to develop complex collaborative strategies, partly involving their venom secretions, to defend their nest against predators, microbial pathogens, ant competitors, and to hunt prey. Activities of ant venom include paralytic, cytolytic, haemolytic, allergenic, pro-inflammatory, insecticidal, antimicrobial, and pain-producing pharmacologic activities, while non-toxic functions include roles in chemical communication involving trail and sex pheromones, deterrents, and aggregators. While these diverse activities in ant venoms have until now been largely understudied due to the small venom yield from ants, modern analytical and venomic techniques are beginning to reveal the diversity of toxin structure and function. As such, ant venoms are distinct from other venomous animals, not only rich in linear, dimeric and disulfide-bonded peptides and bioactive proteins, but also other volatile and non-volatile compounds such as alkaloids and hydrocarbons. The present review details the unique structures and pharmacologies of known ant venom proteinaceous and alkaloidal toxins and their potential as a source of novel bioinsecticides and therapeutic agents.
Assuntos
Venenos de Formiga/química , Alcaloides/análise , Alcaloides/química , Animais , Formigas , Humanos , Proteínas de Insetos/análise , Proteínas de Insetos/química , Peptídeos/análise , Peptídeos/químicaRESUMO
RATIONALE: Compared with other animal venoms, ant venoms remain little explored. Ants have evolved complex venoms to rapidly immobilize arthropod prey and to protect their colonies from predators and pathogens. Many ants have retained peptide-rich venoms that are similar to those of other arthropod groups. METHODS: With the goal of conducting a broad and comprehensive survey of ant venom peptide diversity, we investigated the peptide composition of venoms from 82 stinging ant species from nine subfamilies using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). We also conducted an in-depth investigation of eight venoms using reversed-phase high-performance liquid chromatography (RP-HPLC) separation coupled with offline MALDI-TOFMS. RESULTS: Our results reveal that the peptide compositions of ant venom peptidomes from both poneroid and formicoid ant clades comprise hundreds of small peptides (<4 kDa), while large peptides (>4 kDa) are also present in the venom of formicoids. Chemical reduction revealed the presence of disulfide-linked peptides in most ant subfamilies, including peptides structured by one, two or three disulfide bonds as well as dimeric peptides reticulated by three disulfide bonds. CONCLUSIONS: The biochemical complexity of ant venoms, associated with an enormous ecological and taxonomic diversity, suggests that stinging ant venoms constitute a promising source of bioactive molecules that could be exploited in the search for novel drug and biopesticide leads.
Assuntos
Venenos de Formiga/análise , Peptídeos/análise , Proteoma/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Venenos de Formiga/química , Formigas , Dissulfetos , Peptídeos/química , Proteoma/químicaRESUMO
Ants (Hymenoptera: Formicidae) represent a taxonomically diverse group of arthropods comprising nearly 13,000 extant species. Sixteen ant subfamilies have individuals that possess a stinger and use their venom for purposes such as a defence against predators, competitors and microbial pathogens, for predation, as well as for social communication. They exhibit a range of activities including antimicrobial, haemolytic, cytolytic, paralytic, insecticidal and pain-producing pharmacologies. While ant venoms are known to be rich in alkaloids and hydrocarbons, ant venoms rich in peptides are becoming more common, yet remain understudied. Recent advances in mass spectrometry techniques have begun to reveal the true complexity of ant venom peptide composition. In the few venoms explored thus far, most peptide toxins appear to occur as small polycationic linear toxins, with antibacterial properties and insecticidal activity. Unlike other venomous animals, a number of ant venoms also contain a range of homodimeric and heterodimeric peptides with one or two interchain disulfide bonds possessing pore-forming, allergenic and paralytic actions. However, ant venoms seem to have only a small number of monomeric disulfide-linked peptides. The present review details the structure and pharmacology of known ant venom peptide toxins and their potential as a source of novel bioinsecticides and therapeutic agents.
Assuntos
Venenos de Formiga/análise , Formigas/química , Biodiversidade , Evolução Biológica , Modelos Moleculares , Peptídeos/genética , Peptídeos/toxicidade , Sequência de Aminoácidos , Animais , Venenos de Formiga/classificação , Formigas/genética , Sequência de Bases , Dimerização , Descoberta de Drogas/métodos , Espectrometria de Massas , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
ß-Diguetoxin-Dc1a (Dc1a) is a toxin from the desert bush spider Diguetia canities that incapacitates insects at concentrations that are non-toxic to mammals. Dc1a promotes opening of German cockroach voltage-gated sodium (Nav) channels (BgNav1), whereas human Nav channels are insensitive. Here, by transplanting commonly targeted S3b-S4 paddle motifs within BgNav1 voltage sensors into Kv2.1, we find that Dc1a interacts with the domain II voltage sensor. In contrast, Dc1a has little effect on sodium currents mediated by PaNav1 channels from the American cockroach even though their domain II paddle motifs are identical. When exploring regions responsible for PaNav1 resistance to Dc1a, we identified two residues within the BgNav1 domain II S1-S2 loop that when mutated to their PaNav1 counterparts drastically reduce toxin susceptibility. Overall, our results reveal a distinct region within insect Nav channels that helps determine Dc1a sensitivity, a concept that will be valuable for the design of insect-selective insecticides.
Assuntos
Baratas/fisiologia , Venenos de Aranha/farmacologia , Aranhas/fisiologia , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Inseticidas , Dados de Sequência Molecular , Especificidade da Espécie , Venenos de Aranha/química , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/fisiologiaRESUMO
The rise of integrative taxonomy, a multi-criteria approach used in characterizing species, fosters the development of new tools facilitating species delimitation. Mass spectrometric (MS) analysis of venom peptides from venomous animals has previously been demonstrated to be a valid method for identifying species. Here we aimed to develop a rapid chemotaxonomic tool for identifying ants based on venom peptide mass fingerprinting. The study focused on the biodiversity of ponerine ants (Hymenoptera: Formicidae: Ponerinae) in French Guiana. Initial experiments optimized the use of automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to determine variations in the mass profiles of ant venoms using several MALDI matrices and additives. Data were then analyzed via a hierarchical cluster analysis to classify the venoms of 17 ant species. In addition, phylogenetic relationships were assessed and were highly correlated with methods using DNA sequencing of the mitochondrial gene cytochrome c oxidase subunit 1. By combining a molecular genetics approach with this chemotaxonomic approach, we were able to improve the accuracy of the taxonomic findings to reveal cryptic ant species within species complexes. This chemotaxonomic tool can therefore contribute to more rapid species identification and more accurate taxonomies. BIOLOGICAL SIGNIFICANCE: This is the first extensive study concerning the peptide analysis of the venom of both Pachycondyla and Odontomachus ants. We studied the venoms of 17 ant species from French Guiana that permitted us to fine-tune the venom analysis of ponerine ants via MALDI-TOF mass spectrometry. We explored the peptidomes of crude ant venom and demonstrated that venom peptides can be used in the identification of ant species. In addition, the application of this novel chemotaxonomic method combined with a parallel genetic approach using COI sequencing permitted us to reveal the presence of cryptic ants within both the Pachycondyla apicalis and Pachycondyla stigma species complexes. This adds a new dimension to the search for means of exploiting the enormous biodiversity of venomous ants as a source for novel therapeutic drugs or biopesticides. This article is part of a Special Issue entitled: Proteomics of non-model organisms.
Assuntos
Venenos de Formiga/metabolismo , Formigas , Proteínas de Insetos , Mapeamento de Peptídeos/métodos , Peptídeos , Filogenia , Animais , Venenos de Formiga/química , Venenos de Formiga/genética , Formigas/química , Formigas/classificação , Formigas/genética , Formigas/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Vicinal disulfide bridges, in which a disulfide bond is formed between adjacent cysteine residues, constitute an unusual but expanding class of potential allosteric disulfides. Although vicinal disulfide rings (VDRs) are relatively uncommon, they have proven to be functionally critical in almost all proteins in which they have been discovered. However, it has proved difficult to test whether these sterically constrained disulfides participate in functionally important redox transformations. We demonstrate that chemical replacement of VDRs with dicarba or diselenide bridges can be used to assess whether VDRs function as allosteric disulfides. Our approach leads to the hypothesis that not all VDRs participate in functionally important redox reactions.
Assuntos
Dissulfetos/metabolismo , Proteínas/química , Proteínas/metabolismo , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Modelos Moleculares , Oxirredução , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical knottin peptide; three of the disulfide bridges form a classical inhibitor cystine knot motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large ß hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel.
Assuntos
Miniproteínas Nó de Cistina/química , Dípteros/efeitos dos fármacos , Proteínas de Insetos/química , Neurotoxinas/química , Periplaneta/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/química , Venenos de Aranha/química , Canais de Sódio Disparados por Voltagem/química , Sequência de Aminoácidos , Animais , Miniproteínas Nó de Cistina/metabolismo , Miniproteínas Nó de Cistina/farmacologia , Dípteros/metabolismo , Dissulfetos/química , Escherichia coli/genética , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/metabolismo , Cinética , Potenciais da Membrana/efeitos dos fármacos , Modelos Moleculares , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotoxinas/metabolismo , Neurotoxinas/farmacologia , Técnicas de Patch-Clamp , Periplaneta/metabolismo , Cultura Primária de Células , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Bloqueadores dos Canais de Sódio/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Venenos de Aranha/metabolismo , Venenos de Aranha/farmacologia , Aranhas/química , Aranhas/fisiologia , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Spider venoms represent vast sources of bioactive molecules whose diversity remains largely unknown. Indeed, only a small subset of species have been studied out of the ~43,000 extant spider species. The present study investigated inter- and intra-species venom complexity in 18 samples collected from a variety of lethal Australian funnel-web spiders (Mygalomorphae: Hexathelidae: Atracinae) using C4 reversed-phase separation coupled to offline MALDI-TOF mass spectrometry (LC-MALDI-TOF MS). An in-depth investigation focusing on four atracine venoms (male Illawarra wisharti, male and female Hadronyche cerberea, and female Hadronyche infensa Toowoomba) revealed, on average, ~800 peptides in female venoms while male venoms contained ~400 peptides, distributed across most HPLC fractions. This is significantly higher than previous estimates of peptide expression in mygalomorph venoms. These venoms also showed distinct intersexual as well as intra- and inter-species variation in peptide masses. Construction of both 3D and 2D contour plots revealed that peptide mass distributions in all 18 venoms were centered around the 3200-5400m/z range and to a lesser extent the 6600-8200m/z range, consistent with previously described hexatoxins. These findings highlight the extensive diversity of peptide toxins in Australian funnel-web spider venoms that that can be exploited as novel therapeutic and biopesticide lead molecules. BIOLOGICAL SIGNIFICANCE: In the present study we describe the complexity of 18 venoms from lethal Australian funnel-web spiders using LC-MALDI-TOF MS. The study includes an in-depth investigation, focusing on four venoms, that revealed the presence of ~800 peptides in female venoms and ~400 peptides in male venoms. This is significantly higher than previous estimates of peptide expression in spider venoms. By constructing both 3D and 2D contour plots we were also able to reveal the distinct intersexual as well as intra- and inter-species variation in venom peptide masses. We show that peptide mass distributions in all 18 venoms were centered around the 3200-5400 m/z range and to a lesser extent the 6600-8200 m/z range, consistent with the small number of previously described hexatoxins from these spiders. These findings highlight the extensive diversity of peptide toxins in Australian funnel-web spider venoms that that can be exploited as novel therapeutic and biopesticide lead molecules. The present study has greatly expanded our understanding of peptide variety and complexity in these lethal mygalomorph spiders. Specifically it highlights both the utility of LC-MALDI-TOF in spider taxonomy and the massive combinatorial peptide libraries that spider venoms offer the pharmaceutical and agrochemical industry.
Assuntos
Venenos de Aranha/química , Aranhas/classificação , Animais , Austrália , Feminino , Interações Hidrofóbicas e Hidrofílicas , Masculino , Peso Molecular , Peptídeos/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Picada de Aranha , Venenos de Aranha/classificação , Venenos de Aranha/isolamento & purificaçãoRESUMO
Despite the in vivo lethality of venom, neurotoxicity has not previously been considered a significant complication of envenoming by the Australian pygmy copperhead (Austrelaps labialis). However, recent evidence has emerged demonstrating that this venom contains potent presynaptic and postsynaptic neurotoxicity. The present study describes the isolation and pharmacological characterization of the first postsynaptic neurotoxin, α-EPTX-Al2a, from the venom of A. labialis. α-EPTX-Al2a (8072.77 Da) caused a concentration-dependent block of twitch contractions and a complete block of responses to cholinergic agonists in the chick biventer cervicis nerve-muscle preparation. This action is consistent with postjunctional neurotoxicity. Monovalent tiger snake antivenom prevented the onset of neurotoxicity if applied prior to toxin administration, but was only able to partially reverse neurotoxicity once muscle paralysis had developed. α-EPTX-Al2a produced a potent pseudo-irreversible antagonism of chick muscle nicotinic acetylcholine receptors (nAChRs), with an estimated pA(2) value of 7.902 (K(B) = 12.5 nM). Interestingly, the toxin only produced a modest block of neuronal α7 nAChRs, with an IC(50) of 1.2 µM, and failed to inhibit ganglionic α3ß2/α3ß4 nAChRs in a fluorescence-based FLIPR assay using SH-SY5Y cells. α-EPTX-Al2a contained 75 amino acid residues with five disulfide bonds that had significant homology to classical long-chain α-neurotoxins. While α-EPTX-Al2a retains most pharmacophore residues critical for binding to muscle-type (α1)(2)ßγδ nAChRs it lacks the key Ala(28) and Arg(36) residues important for α7 nAChR affinity. Given that A. labialis venom contains both irreversible presynaptic and postsynaptic neurotoxins, clinicians need to be aware of potential neurotoxic complications associated with pygmy copperhead envenomation.
Assuntos
Venenos de Crotalídeos/toxicidade , Neurotoxinas/toxicidade , Receptores Nicotínicos/metabolismo , Sequência de Aminoácidos , Animais , Antivenenos/farmacologia , Carbacol/farmacologia , Linhagem Celular Tumoral , Galinhas , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Técnicas In Vitro , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/isolamento & purificação , Antagonistas Nicotínicos/toxicidade , Fosfolipases A2 Secretórias/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transmissão Sináptica , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Over 10,000 arthropod species are currently considered to be pest organisms. They are estimated to contribute to the destruction of ~14% of the world's annual crop production and transmit many pathogens. Presently, arthropod pests of agricultural and health significance are controlled predominantly through the use of chemical insecticides. Unfortunately, the widespread use of these agrochemicals has resulted in genetic selection pressure that has led to the development of insecticide-resistant arthropods, as well as concerns over human health and the environment. Bioinsecticides represent a new generation of insecticides that utilise organisms or their derivatives (e.g., transgenic plants, recombinant baculoviruses, toxin-fusion proteins and peptidomimetics) and show promise as environmentally-friendly alternatives to conventional agrochemicals. Spider-venom peptides are now being investigated as potential sources of bioinsecticides. With an estimated 100,000 species, spiders are one of the most successful arthropod predators. Their venom has proven to be a rich source of hyperstable insecticidal mini-proteins that cause insect paralysis or lethality through the modulation of ion channels, receptors and enzymes. Many newly characterized insecticidal spider toxins target novel sites in insects. Here we review the structure and pharmacology of these toxins and discuss the potential of this vast peptide library for the discovery of novel bioinsecticides.
Assuntos
Inseticidas/farmacologia , Peptídeos/farmacologia , Venenos de Aranha/farmacologia , Animais , Humanos , Inseticidas/química , Peptídeos/química , Conformação Proteica , Venenos de Aranha/químicaRESUMO
Voltage-gated sodium (Na(V)) channels play a central role in the propagation of action potentials in excitable cells in both humans and insects. Many venomous animals have therefore evolved toxins that modulate the activity of Na(V) channels in order to subdue their prey and deter predators. Spider venoms in particular are rich in Na(V) channel modulators, with one-third of all known ion channel toxins from spider venoms acting on Na(V) channels. Here we review the landscape of spider-venom peptides that have so far been described to target vertebrate or invertebrate Na(V) channels. These peptides fall into 12 distinct families based on their primary structure and cysteine scaffold. Some of these peptides have become useful pharmacological tools, while others have potential as therapeutic leads because they target specific Na(V) channel subtypes that are considered to be important analgesic targets. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides and so far only 0.01% of this diversity been characterised. Thus, it is likely that future research will reveal additional structural classes of spider-venom peptides that target Na(V) channels.