RESUMO
Latrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α-latrotoxin (α-LTX)), five specialized on insects (α, ß, γ, δ, ε- latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca2+-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the α-LCT monomer and the δ-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small ß-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.
Assuntos
Viúva Negra/química , Cálcio/química , Neurotoxinas/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Venenos de Aranha/química , Animais , Sítios de Ligação , Viúva Negra/patogenicidade , Cálcio/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Transporte de Íons , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Potenciais da Membrana/fisiologia , Modelos Moleculares , Neurotoxinas/genética , Neurotoxinas/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Venenos de Aranha/genética , Venenos de Aranha/metabolismoRESUMO
CsTx-1, the main neurotoxic acting peptide in the venom of the spider Cupiennius salei, is composed of 74 amino acid residues, exhibits an inhibitory cysteine knot motif, and is further characterized by its highly cationic charged C terminus. Venom gland cDNA library analysis predicted a prepropeptide structure for CsTx-1 precursor. In the presence of trifluoroethanol, CsTx-1 and the long C-terminal part alone (CT1-long; Gly-45-Lys-74) exhibit an α-helical structure, as determined by CD measurements. CsTx-1 and CT1-long are insecticidal toward Drosophila flies and destroys Escherichia coli SBS 363 cells. CsTx-1 causes a stable and irreversible depolarization of insect larvae muscle cells and frog neuromuscular preparations, which seem to be receptor-independent. Furthermore, this membranolytic activity could be measured for Xenopus oocytes, in which CsTx-1 and CT1-long increase ion permeability non-specifically. These results support our assumption that the membranolytic activities of CsTx-1 are caused by its C-terminal tail, CT1-long. Together, CsTx-1 exhibits two different functions; as a neurotoxin it inhibits L-type Ca(2+) channels, and as a membranolytic peptide it destroys a variety of prokaryotic and eukaryotic cell membranes. Such a dualism is discussed as an important new mechanism for the evolution of spider venomous peptides.
Assuntos
Evolução Molecular , Neurotoxinas/química , Venenos de Aranha/química , Aranhas/química , Animais , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , DNA Complementar/genética , Drosophila melanogaster , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Feminino , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Neurotoxinas/genética , Estrutura Terciária de Proteína , Rana temporaria , Venenos de Aranha/genética , Aranhas/genética , Xenopus laevisRESUMO
When 21 species of sea anemones were screened for Kv1 potassium channel toxins by competitive inhibition of the binding of (125)I-α-dendrotoxin to rat synaptosomal membranes, 11 species (two species of Actiniidae, one species of Hormathiidae, five species of Stichodactylidae and three species of Thalassianthidae) were found to be positive. Furthermore, full-length cDNAs encoding type 1 potassium channel toxins from three species of Stichodactylidae and three species of Thalassianthidae were cloned by a combination of RT-PCR, 3'RACE and 5'RACE. The precursors of these six toxins are commonly composed of signal peptide, propart and mature peptide portions. As for the mature peptide (35 amino acid residues), the six toxins share more than 90% sequence identities with one another and with κ(1.3)-SHTX-She1a (Shk) from Stichodactyla helianthus but only 34-63% identities with the other type 1 potassium channel toxins.
Assuntos
Toxinas Marinhas/farmacologia , Neurotoxinas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Anêmonas-do-Mar/química , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Superfamília Shaker de Canais de Potássio/metabolismo , Sinaptossomos/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar , Avaliação Pré-Clínica de Medicamentos , Venenos Elapídicos/metabolismo , Toxinas Marinhas/química , Toxinas Marinhas/genética , Toxinas Marinhas/metabolismo , Dados de Sequência Molecular , Neurotoxinas/química , Neurotoxinas/genética , Neurotoxinas/metabolismo , Oceanos e Mares , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/metabolismo , Ratos , Anêmonas-do-Mar/genética , Sinaptossomos/efeitos dos fármacosRESUMO
We have developed a model genetic system for analyzing the function of peptide toxins from animal venoms. We engineered and propagated strains of Drosophila melanogaster expressing heat-inducible transgenes encoding either kappa-ACTX-Hv1c or omega-ACTX-Hv1a, two insect-specific neurotoxic peptides found in the venom of the Australian funnel-web spider Hadronyche versuta. Heat induction of transgene expression for 20 min was sufficient to kill all transgenic flies, indicating that the ion channels targeted by these toxins are viable insecticide targets. The unusual phenotype of flies induced to express omega-ACTX-Hv1a recapitulates that of a hypomorphic allele of the high-voltage-activated calcium channel Dmca1D, suggesting that this is likely to be the target of omega-ACTX-Hv1a.
Assuntos
Peptídeos/fisiologia , Venenos de Aranha/genética , Toxinas Biológicas/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Viúva Negra/genética , Viúva Negra/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Expressão Gênica , Vetores Genéticos/genética , Modelos Biológicos , Dados de Sequência Molecular , Neurotoxinas/genética , Neurotoxinas/metabolismo , Peptídeos/genética , Alinhamento de Sequência , Venenos de Aranha/metabolismo , Toxinas Biológicas/genética , Transformação GenéticaRESUMO
JZTX-XI is a peptide toxin isolated from the venom of the Chinese spider Chilobrachys jingzhao. It contains 34 residues including six cysteine residues with disulfide bridges linked in the pattern of I-IV, II-V, and III-VI. Using 3'- and 5'-RACE methods, the full-length cDNA was identified as encoding an 86-residue precursor of JZTX-XI. In the electrophysiological assay, JZTX-XI shows activity toward the Kv2.1 channel in a way similar to hanatoxin1 and SGTx1 that both the activation and the deactivation processes are affected, which is in accordance with the high sequence homology among them (over 60% identity). On the other hand, JZTX-XI also exhibits specific interaction against the Nav channels of rat cardiac myocytes with a significant reduction in the peak current and slowing of channel inactivation. The solution structure of native JZTX-XI was determined by 1H NMR methods to identify the structural basis of these specific activities. Structural comparison of JZTX-XI with other gating modifier toxins shows that they all adopt a similar surface profile, a hydrophobic patch surrounded by charged residues such as Arg or Lys, which might be a common structural factor responsible for toxin-channel interaction. JZTX-XI might be an ideal tool to further investigate how spider toxins recognize various ion channels as their targets.
Assuntos
Neuropeptídeos/química , Neurotoxinas/química , Peptídeos/química , Peptídeos/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Sódio/metabolismo , Venenos de Aranha/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Cristalografia por Raios X , DNA Complementar/isolamento & purificação , Feminino , Ativação do Canal Iônico/genética , Ativação do Canal Iônico/fisiologia , Masculino , Camundongos , Dados de Sequência Molecular , Neuropeptídeos/genética , Neuropeptídeos/isolamento & purificação , Neuropeptídeos/fisiologia , Neurotoxinas/genética , Neurotoxinas/isolamento & purificação , Neurotoxinas/toxicidade , Oócitos/química , Oócitos/metabolismo , Peptídeos/genética , Peptídeos/isolamento & purificação , Periplaneta , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/metabolismo , Bloqueadores dos Canais de Sódio/toxicidade , Venenos de Aranha/genética , Venenos de Aranha/isolamento & purificação , Venenos de Aranha/toxicidade , Xenopus laevisRESUMO
Scorpion venoms are a particularly rich source of neurotoxic proteins/peptides that interact in a highly specific fashion with discrete subtypes of ion channels in excitable and non-excitable cells. Here we have employed a recently developed technique to effect molecular cloning and structural characterization of a novel putative potassium channel-blocking toxin from the same sample of venom from the North African scorpion, Androctonus amoreuxi. The deduced precursor open-reading frame is composed of 59 amino acid residues that consists of a signal peptide of approximately 22 amino acid residues followed by a mature toxin of 37 amino acid residues. The mature toxin contains two functionally important residues (Lys27 and Tyr36), constituting a functional dyad motif that may be critical for potassium channel-blocking activity that can be affirmed from structural homologs as occurring in the venoms from other species of Androctonus scorpions. Parallel proteomic/transcriptomic studies can thus be performed on the same scorpion venom sample without sacrifice of the donor animal.
Assuntos
Neurotoxinas/genética , Peptídeos/genética , Bloqueadores dos Canais de Potássio/química , Venenos de Escorpião/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Proteínas de Insetos , Dados de Sequência Molecular , Neurotoxinas/química , Peptídeos/química , Venenos de Escorpião/química , Escorpiões/metabolismo , Análise de Sequência de DNA , Análise de Sequência de ProteínaRESUMO
A novel toxin, named Cll9, was isolated from the venom of the scorpion Centruroides limpidus limpidus Karsch. It is composed of 63 amino acid residues closely packed by four disulfide bridges. It showed no apparent effect when injected to insects, crustaceans and i.p. to mice. However, when i.c.v. injected in the rat it immediately induced sleep, suggesting that it has a neurodepressant effect. We confirmed this by showing that it has a strong antiepileptic action, as assessed with the penicillin focus model. Its effectiveness in inhibiting Na(+) permeability in (cultured) rat peripheral ganglia further supports its neurodepressant actions. However, this peptide did not affect other Na(+) channels such as those from cerebellum granular cells in culture or the rSkM1 Na(+) channels expressed in HEK293. The cDNA and genomic regions encoding this peptide were cloned and sequenced. This peptide is synthesized as a precursor of 84 amino acid residues and processed by removing 19 amino acids (signal peptide) from the amino terminal region and a couple of lysine residues from the carboxyl end. The presence of an intron of 777 bases interrupting the region encoding the signal peptide was also revealed. A comparison of its primary sequence, with more than 100 scorpion toxins known, showed that together with toxin CsE9 they constitute a new subfamily of peptides considered to be one of the most divergent groups of scorpion toxin-like peptides discovered.
Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Neurotoxinas/farmacologia , Peptídeos/farmacologia , Sistema Nervoso Periférico/efeitos dos fármacos , Venenos de Escorpião/química , Venenos de Escorpião/genética , Venenos de Escorpião/farmacologia , Sequência de Aminoácidos , Animais , Anticonvulsivantes/farmacologia , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Avaliação Pré-Clínica de Medicamentos/métodos , Gânglios/citologia , Gânglios/efeitos dos fármacos , Gryllidae/efeitos dos fármacos , Íntrons , Masculino , Camundongos , Dados de Sequência Molecular , Neurotoxinas/genética , Neurotoxinas/isolamento & purificação , Peptídeos/genética , Peptídeos/isolamento & purificação , Ratos , Ratos Wistar , Venenos de Escorpião/isolamento & purificação , Homologia de Sequência de Aminoácidos , Sono/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/metabolismoRESUMO
The aim of the present study was the molecular cloning of toxins active on calcium channels expressed by the spider Phoneutria nigriventer. Clones encoding the toxins Pn3-3A, Pn3-4A, Tx3-5, Pn3-5A, Tx3-6, Pn3-6A and Pn3-6B were identified from a cDNA library derived from the venom gland of this spider, revealing toxins of 49, 76, 45, 39, 55 and 58 amino acids residues, respectively, with polypeptide precursors being composed of three major portions: a signal peptide, a propeptide and finally, the mature toxin. A high degree of homology with the amino acid sequence was found between Pn3-3A and the neurotoxin Tx3-3 (identity of 79%), and between Pn3-4A and the neurotoxin Tx3-4 (identity of 95%). The deduced amino acid sequence for the mature polypeptides Tx3-5 and Tx3-6 confirms the polypeptide sequence previously published for these neurotoxins. In addition, the toxin Pn3-5A showed 58% identity to the Tx3-5 amino acid sequence, and the toxins Pn3-6A and Pn3-6B showed 85 and 33% identity, respectively, to the Tx3-6 amino acid sequence.
Assuntos
Neurotoxinas/genética , Venenos de Aranha/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Canais de Cálcio/efeitos dos fármacos , Clonagem Molecular , DNA Complementar/genética , Biblioteca Gênica , Dados de Sequência Molecular , Neurotoxinas/toxicidade , Peptídeos/genética , Alinhamento de Sequência , Homologia de Sequência , Venenos de Aranha/toxicidade , AranhasRESUMO
1. The spiders of medical importance in the Asia-Pacific region include widow (family Theridiidae) and Australian funnel-web spiders (subfamily Atracinae). In addition, cupboard (family Theridiidae) and Australian mouse spiders (family Actinopodidae) may contain neurotoxins responsible for serious systemic envenomation. Fortunately, there appears to be extensive cross-reactivity of species-specific widow spider antivenom within the family Theridiidae. Moreover, Sydney funnel-web antivenom has been shown to be effective in the treatment of mouse spider envenomation. 2. alpha-Latrotoxin (alpha-LTx) appears to be the main neurotoxin responsible for the envenomation syndrome known as "latrodectism" following bites from widow spiders. This 120 kDa protein binds to distinct receptors (latrophilin 1 and neurexins) to induce neurotransmitter vesicle exocytosis via both Ca2+-dependent and -independent mechanisms, resulting in vesicle depletion. This appears to involve disruption to a process that normally inhibits vesicle fusion in the absence of Ca2+. Precise elucidation of the mechanism of action of alpha-LTx will lead to a major advancement in our understanding of vesicle exocytosis. 3. delta-Atracotoxins (delta-ACTX) are responsible for the primate-specific envenomation syndrome seen following funnel-web spider envenomation. These peptides induce spontaneous repetitive firing and prolongation of action potentials in excitable cells. This results from a hyperpolarizing shift of the voltage-dependence of activation and a slowing of voltage-gated Na+ channel inactivation. This action is due to voltage-dependent binding to neurotoxin receptor site-3 on insect and mammalian voltage-gated Na+ channels in a manner similar, but not identical, to scorpion alpha-toxins and sea anemone toxins. delta-Atracotoxins provide us with highly specific tools to study Na+ channel structure and function 4. omega- and Janus-faced ACTX, from funnel-web spider venom, are novel neurotoxins that show selective toxicity to insects. In particular omega-ACTX define a new insecticide target due to a specific action to block insect voltage-gated Ca2+ channels. Both these ACTX show promise for the development of baculoviral recombinant biopesticides expressing these toxins for the control of insecticide-resistant agricultural pests. In addition, they should provide valuable tools for the pharmacological and structural characterization of insecticide targets.
Assuntos
Neurotoxinas/efeitos adversos , Picada de Aranha/etiologia , Venenos de Aranha/efeitos adversos , Aranhas , Sequência de Aminoácidos , Animais , Antivenenos/uso terapêutico , Ásia , Austrália , Viúva Negra , Humanos , Dados de Sequência Molecular , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/genética , Neurotoxinas/farmacologia , Ilhas do Pacífico , Homologia de Sequência de Aminoácidos , Picada de Aranha/tratamento farmacológico , Venenos de Aranha/antagonistas & inibidores , Venenos de Aranha/genética , Venenos de Aranha/farmacologia , Aranhas/genéticaRESUMO
BnSP-7, a Lys49 myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis venom, was structurally and functionally characterized. Several biological activities were assayed and compared with those of the chemically modified toxin involving specific amino acid residues. The cDNA produced from the total RNA by RT-PCR contained approximately 400 bp which codified its 121 amino acid residues with a calculated pI and molecular weight of 8.9 and 13,727, respectively. Its amino acid sequence showed strong similarities with several Lys49 phospholipase A(2) homologues from other Bothrops sp. venoms. By affinity chromatography and gel diffusion, it was demonstrated that heparin formed a complex with BnSP-7, held at least in part by electrostatic interactions. BnSP-7 displayed bactericidal activity and promoted the blockage of the neuromuscular contraction of the chick biventer cervicis muscle. In addition to its in vivo myotoxic and edema-inducing activity, it disrupted artificial membranes. Both BnSP-7 and the crude venom released creatine kinase from the mouse gastrocnemius muscle and induced the development of a dose-dependent edema. His, Tyr, and Lys residues of the toxin were chemically modified by 4-bromophenacyl bromide (BPB), 2-nitrobenzenesulfonyl fluoride (NBSF), and acetic anhydride (AA), respectively. Cleavage of its N-terminal octapeptide was achieved with cyanogen bromide (CNBr). The bactericidal action of BnSP-7 on Escherichia coli was almost completely abolished by acetylation or cleavage of the N-terminal octapeptide. The neuromuscular effect induced by BnSP-7 was completely inhibited by heparin, BPB, acetylation, and CNBr treatment. The creatine kinase releasing and edema-inducing effects were partially inhibited by heparin or modification by BPB and almost completely abolished by acetylation or cleavage of the N-terminal octapeptide. The rupture of liposomes by BnSP-7 and crude venom was dose and temperature dependent. Incubation of BnSP-7 with EDTA did not change this effect, suggesting a Ca(2+)-independent membrane lytic activity. BnSP-7 cross-reacted with antibodies raised against B. moojeni (MjTX-II), B. jararacussu (BthTX-I), and B. asper (Basp-II) myotoxins as well as against the C-terminal peptide (residues 115-129) from Basp-II.
Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/química , Lisina/química , Neurotoxinas/química , Neurotoxinas/toxicidade , Fosfolipases A/química , Fosfolipases A/toxicidade , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Edema/metabolismo , Escherichia coli/metabolismo , Fosfolipases A2 do Grupo II , Heparina/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/efeitos dos fármacos , Neurotoxinas/genética , Peroxidase/metabolismo , Fosfolipases A/genética , Proteínas de Répteis , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Temperatura , Fatores de Tempo , Difração de Raios XRESUMO
The structural specificity of alpha-PMTX, a novel peptide toxin derived from wasp venom has been studied on the neuromuscular synapse in the walking leg of the lobster. alpha-PMTX is known to induce repetitive action potentials in the presynaptic axon due to sodium channel inactivation. We synthesized 29 analogs of alpha-PMTX by substituting one or two amino acids and compared threshold concentrations of these mutant toxins for inducing repetitive action potentials. In 13 amino acid residues of alpha-PMTX, Arg-1, Lys-3 and Lys-12 regulate the toxic activity because substitution of these basic amino acid residues with other amino acid residues greatly changed the potency. Determining the structure-activity relationships of PMTXs will help clarifying the molecular mechanism of sodium channel inactivation.
Assuntos
Junção Neuromuscular/fisiologia , Neurotoxinas/metabolismo , Canais de Sódio/metabolismo , Transmissão Sináptica/fisiologia , Venenos de Vespas/metabolismo , Substituição de Aminoácidos/genética , Animais , Proteínas de Insetos , Nephropidae , Junção Neuromuscular/efeitos dos fármacos , Neurotoxinas/genética , Neurotoxinas/toxicidade , Ligação Proteica , Relação Estrutura-Atividade , Transmissão Sináptica/efeitos dos fármacos , Venenos de Vespas/genética , Venenos de Vespas/toxicidadeRESUMO
The primary structure of the crusta gene encoding alpha-latrocrustoxin (alpha-LCT), a high molecular mass neurotoxin specific to crustaceans, was determined in the black widow spider Latrodectus mactans tredicimguttatus genome. The total length of the sequenced DNA was 4693 bp. The structural part of the black widow spider chromosome gene encoding alpha-LCT does not contain introns. The sequenced DNA contains a single extended open reading frame (4185 bp) and encodes a protein precursor of alpha-LCT, comprising 1395 aa. We assume the Met residue at position -10 relative to the N-terminal residue of Glu1 of the mature toxin to be the first one in the protein precursor. The calculated molecular mass of the precursor (156147 Da) exceeds that of the mature toxin by approximately 30 kDa. These data are in agreement with the notion that over the course of maturation the protein precursor undergoes double processing--cleavage of a decapeptide from the N-terminal part and of a approximately 200-aa fragment from the C-terminal part. alpha-LCT displayed a number of imperfect ankyrin-like repeats and areas of structural homology with earlier studied latrotoxins; the highest homology degree (62%) was revealed with alpha-latroinsectotoxin (alpha-LIT).
Assuntos
Viúva Negra/genética , Venenos de Aranha/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Genes de Insetos , Dados de Sequência Molecular , Neurotoxinas/genética , Alinhamento de Sequência , Análise de SequênciaRESUMO
According to the known primary sequences of three neurotoxins active on small conductance Ca2+-activated potassium channels from the scorpion Buthus martensi Karsch, their corresponding cDNAs were cloned and sequenced using 3'- and 5'-RACE. All of them encoded a signal peptide composed of 28 residues and a mature toxin of 29, 28 and 33 residues, respectively. Their cDNA deduced sequences were totally consistent with those determined, and the C-terminal amidation of one neurotoxin was confirmed. The genomic DNAs of these three toxins were also amplified by PCR, cloned and sequenced. They all consisted of two exons disrupted by a small single intron. All of these introns were inserted within the signal peptide at the same -10 position upstream from the mature toxin, consisting of 94, 78 and 87 bp, respectively.
Assuntos
Mapeamento Cromossômico , Neurotoxinas/genética , Canais de Potássio/efeitos adversos , Venenos de Escorpião/genética , Escorpiões/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Éxons , Íntrons , Dados de Sequência Molecular , Neurotoxinas/química , Neurotoxinas/toxicidade , Reação em Cadeia da Polimerase , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/toxicidade , Venenos de Escorpião/química , Venenos de Escorpião/toxicidadeRESUMO
Scorpions have survived successfully over millions of years without detectable changes in their morphology. Instead, they have developed an efficient alomonal machinery and a stinging device supporting their needs for prey and defense. They produce a large variety of polypeptidic toxins that bind and modulate ion channel conductance in excitable tissues. The binding site, mode of action, and chemical properties of many toxins have been studied extensively, but little is known about their genomic organization and diversity. Genes representing each of the major classes of Buthidae scorpion toxins, namely, "long" toxins, affecting sodium channels (alpha, depressant, and excitatory), and "short" toxins, affecting potassium and chloride channels, were isolated from a single scorpion segment and analyzed. Each toxin type was found to be encoded by a gene family. Regardless of toxin length, 3-D structure, and site of action, all genes contain A+T-rich introns that split, at a conserved location, an amino acid codon of the signal sequence. The introns vary in length and sequence but display identical boundaries, agree with the GT/AG splice junctions, and contain T-runs downstream of a putative branch point, 5'-TAAT-3'. Despite little sequence similarity among all toxin classes, the conserved gene organization, intron features, and common cysteine-stabilized alpha-helical (CSH) core connecting an alpha-helix to a three-stranded beta-sheet suggest, that they all evolved from an ancestral common progenitor. Furthermore, the vast diversity found among genomic copies, cDNAs, and their protein products for each toxin suggests an extensive evolutionary process of the scorpion "pharmaceutical factory," whose success is due, most likely, to the inherent permissiveness of the toxin exterior to structural alterations.
Assuntos
Canais de Cloreto/efeitos dos fármacos , Neurotoxinas/genética , Canais de Potássio/efeitos dos fármacos , Venenos de Escorpião/química , Canais de Sódio/efeitos dos fármacos , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar , Íntrons , Dados de Sequência Molecular , Neurotoxinas/farmacologia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
alpha-Latrotoxin is a presynaptic neurotoxin isolated from the venom of the black widow spider Latrodectus tredecimguttatus. It exerts toxic effects in the vertebrate central nervous system by depolarizing neurons, by increasing [Ca2+]i and by stimulating uncontrolled exocytosis of neurotransmitters from nerve terminals. The actions of alpha-latrotoxin are mediated, in part, by a GTP-binding protein-coupled receptor referred to as CIRL or latrophilin. Exendin-4 is also a venom toxin, and it is derived from the salivary gland of the Gila monster Heloderma suspectum. It acts as an agonist at the receptor for glucagon-like peptide-1(7-36)-amide (GLP-1), thereby stimulating secretion of insulin from pancreatic beta-cells of the islets of Langerhans. Here is reported a surprising structural homology between alpha-latrotoxin and exendin-4 that is also apparent amongst all members of the GLP-1-like family of secretagogic hormones (GLP-1, glucagon, vasoactive intestinal polypeptide, secretin, pituitary adenylyl cyclase activating polypeptide). On the basis of this homology, we report the synthesis and initial characterization of a chimeric peptide (Black Widow GLP-1) that stimulates Ca2+ signaling and insulin secretion in human beta-cells and MIN6 insulinoma cells. It is also reported here that the GTP-binding protein-coupled receptors for alpha-latrotoxin and exendin-4 share highly significant structural similarity in their extracellularly-oriented amino-termini. We propose that molecular mimicry has generated conserved structural motifs in secretagogic toxins and their receptors, thereby explaining the evolution of defense or predatory strategies that are shared in common amongst distantly related species including spiders, lizards, and snakes. Evidently, the toxic effects of alpha-latrotoxin and exendin-4 are explained by their ability to interact with GTP-binding protein-coupled receptors that normally mediate the actions of endogenous hormones or neuropeptides.
Assuntos
Viúva Negra/química , Glucagon/química , Glucagon/genética , Neurotoxinas/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Precursores de Proteínas/química , Precursores de Proteínas/genética , Venenos de Aranha/química , Peçonhas , Sequência de Aminoácidos , Animais , Viúva Negra/genética , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Exenatida , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Mimetismo Molecular , Dados de Sequência Molecular , Neurotoxinas/genética , Neurotoxinas/toxicidade , Fragmentos de Peptídeos/metabolismo , Peptídeos/química , Peptídeos/genética , Precursores de Proteínas/metabolismo , Receptores de Glucagon/metabolismo , Homologia de Sequência de Aminoácidos , Venenos de Aranha/genética , Venenos de Aranha/toxicidadeRESUMO
A new potassium channel toxin, HmK, has been isolated from the sea anemone Heteractis magnifica. It inhibits the binding of [125I]-alpha-dendrotoxin (a ligand for voltage-gated K channels) to rat brain synaptosomal membranes with a Ki of about 1 nM, blocks K+ currents through Kv 1.2 channels expressed in a mammalian cell line, and facilitates acetylcholine release at the avian neuromuscular junction. HmK comprises of 35 amino acids (Mr 4055) with the sequence R1TCKDLIPVS10ECTDIRCRTS20MKYRLNLCRK30TCGSC35. A full assignment of the disulfide linkages was made by using partial reduction with tri(2-carboxyethyl)phosphine (TCEP) at acid pH and rapid alkylation with iodoacetamide. The disulfide bridges were identified as Cys3-Cys35, Cys12-Cys28, and Cys17-Cys32. A cDNA clone encoding HmK was isolated using RT-PCR from the total RNA obtained from sea anemone tentacles, while the 5'- and 3'-flanking regions of the cDNA were amplified by RACE. The full-length cDNA was 563 bp long and contained a sequence encoding a signal peptide of 39 amino acids. The coding region for matured HmK toxin was cloned and expressed as a glutathione S-transferase (GST) fusion product in the cytoplasm of Escherichia coli. After affinity purification and cleavage, the recombinant toxin was shown to be identical to native HmK in its N-terminal sequence, chromatographic behavior, and binding to dendrotoxin binding sites on rat brain membranes.
Assuntos
Venenos de Cnidários/genética , Neurotoxinas/genética , Bloqueadores dos Canais de Potássio , Anêmonas-do-Mar/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Encéfalo/metabolismo , Clonagem Molecular , Venenos de Cnidários/metabolismo , Venenos de Cnidários/farmacologia , DNA Complementar/genética , Venenos Elapídicos/metabolismo , Escherichia coli/genética , Membranas/metabolismo , Dados de Sequência Molecular , Junção Neuromuscular/efeitos dos fármacos , Neurotoxinas/metabolismo , Neurotoxinas/farmacologia , Ligação Proteica , Proteínas Recombinantes de Fusão/farmacologia , Análise de Sequência , Homologia de Sequência de AminoácidosRESUMO
A cDNA clone (ACLPREMT1) for a K49 phospholipase A2 (PLA2) myotoxin from Agkistrodon contortrix laticinctus snake venom was isolated from a venom gland library and sequenced. The ACLPREMT1 cDNA is 734 bp in length and has an open reading frame of 414 bp. It codes for a K49 phospholipase A2 with 121 amino acid residues. The sequence of the first 20 amino acid residues of the predicted mature protein matches exactly with the N-terminal sequence of the purified myotoxin. Comparison of the ACLPREMT1 cDNA sequence with PLA2 cDNAs from Viperidae snakes shows that it has a similar organization: highly conserved 5' and 3' untranslated regions, a sequence encoding a 16-amino acid signal peptide, and the mature protein coding region. Comparison of the predicted sequence of ACL myotoxin and other K49 and D49 PLA2 myotoxins shows that, despite the homology (85-97%) at the nucleotide level, K49 PLA2 myotoxins are distinct from the D49 PLA2s and form a highly conserved protein family. In addition to the substitution of D49K, K49 myotoxins have several invariant residues not found in the D49 group, including K7, K78, K80, K115, and K116. There are also some conserved residues (E12, T13, K16, and N17) in all myotoxic proteins, including some neurotoxic and myotoxic PLA2s. Molecular modeling of ACL myotoxin shows that these residues are close together on the surface of one side of the molecule which suggests a potential site for binding to membranes and/or induction of toxicity.