RESUMEN
The venom of cone snails has been proven to be a rich source of bioactive peptides that target a variety of ion channels and receptors. α-Conotoxins (αCtx) interact with nicotinic acetylcholine receptors (nAChRs) and are powerful tools for investigating the structure and function of the various nAChR subtypes. By studying how conotoxins interact with nAChRs, we can improve our understanding of these receptors, leading to new insights into neurological diseases associated with nAChRs. Here, we describe the discovery and characterization of a novel conotoxin from Conus ateralbus, αCtx-AtIA, which has an amino acid sequence homologous to the well-described αCtx-PeIA, but with a different selectivity profile towards nAChRs. We tested the synthetic αCtx-AtIA using the calcium imaging-based Constellation Pharmacology assay on mouse DRG neurons and found that αCtx-AtIA significantly inhibited ACh-induced calcium influx in the presence of an α7 positive allosteric modulator, PNU-120596 (PNU). However, αCtx-AtIA did not display any activity in the absence of PNU. These findings were further validated using two-electrode voltage clamp electrophysiology performed on oocytes overexpressing mouse α3ß4, α6/α3ß4 and α7 nAChRs subtypes. We observed that αCtx-AtIA displayed no or low potency in blocking α3ß4 and α6/α3ß4 receptors, respectively, but improved potency and selectivity to block α7 nAChRs when compared with αCtx-PeIA. Through the synthesis of two additional analogs of αCtx-AtIA and subsequent characterization using Constellation Pharmacology, we were able to identify residue Trp18 as a major contributor to the activity of the peptide.
Asunto(s)
Conotoxinas , Caracol Conus , Receptores Nicotínicos , Animales , Ratones , Calcio , Secuencia de Aminoácidos , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Conus ateralbus is a cone snail endemic to the west side of the island of Sal, in the Cabo Verde Archipelago off West Africa. We describe the isolation and characterization of the first bioactive peptide from the venom of this species. This 30AA venom peptide is named conotoxin AtVIA (δ-conotoxin-like). An excitatory activity was manifested by the peptide on a majority of mouse lumbar dorsal root ganglion neurons. An analog of AtVIA with conservative changes on three amino acid residues at the C-terminal region was synthesized and this analog produced an identical effect on the mouse neurons. AtVIA has homology with δ-conotoxins from other worm-hunters, which include conserved sequence elements that are shared with δ-conotoxins from fish-hunting Conus. In contrast, there is no comparable sequence similarity with δ-conotoxins from the venoms of molluscivorous Conus species. A rationale for the potential presence of δ-conotoxins, that are potent in vertebrate systems in two different lineages of worm-hunting cone snails, is discussed.
Asunto(s)
Conotoxinas/química , Caracol Conus/química , Aminoácidos/genética , Animales , Cabo Verde , Conotoxinas/farmacocinética , Secuencia Conservada/genética , Femenino , Ganglios Espinales/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Péptidos/química , Péptidos/genética , Péptidos/farmacocinética , FilogeniaRESUMEN
Prey shifts in carnivorous predators are events that can initiate the accelerated generation of new biodiversity. However, it is seldom possible to reconstruct how the change in prey preference occurred. Here we describe an evolutionary "smoking gun" that illuminates the transition from worm hunting to fish hunting among marine cone snails, resulting in the adaptive radiation of fish-hunting lineages comprising â¼100 piscivorous Conus species. This smoking gun is δ-conotoxin TsVIA, a peptide from the venom of Conus tessulatus that delays inactivation of vertebrate voltage-gated sodium channels. C. tessulatus is a species in a worm-hunting clade, which is phylogenetically closely related to the fish-hunting cone snail specialists. The discovery of a δ-conotoxin that potently acts on vertebrate sodium channels in the venom of a worm-hunting cone snail suggests that a closely related ancestral toxin enabled the transition from worm hunting to fish hunting, as δ-conotoxins are highly conserved among fish hunters and critical to their mechanism of prey capture; this peptide, δ-conotoxin TsVIA, has striking sequence similarity to these δ-conotoxins from piscivorous cone snail venoms. Calcium-imaging studies on dissociated dorsal root ganglion (DRG) neurons revealed the peptide's putative molecular target (voltage-gated sodium channels) and mechanism of action (inhibition of channel inactivation). The results were confirmed by electrophysiology. This work demonstrates how elucidating the specific interactions between toxins and receptors from phylogenetically well-defined lineages can uncover molecular mechanisms that underlie significant evolutionary transitions.
Asunto(s)
Caracol Conus/fisiología , Peces/fisiología , Conducta Predatoria/fisiología , Secuencia de Aminoácidos , Animales , Bioensayo , Conotoxinas/química , Conotoxinas/toxicidad , Caracol Conus/anatomía & histología , Datos de Secuencia Molecular , Péptidos/metabolismo , FilogeniaRESUMEN
More than 100 species of venomous cone snails (genus Conus) are highly effective predators of fish. The vast majority of venom components identified and functionally characterized to date are neurotoxins specifically targeted to receptors, ion channels, and transporters in the nervous system of prey, predators, or competitors. Here we describe a venom component targeting energy metabolism, a radically different mechanism. Two fish-hunting cone snails, Conus geographus and Conus tulipa, have evolved specialized insulins that are expressed as major components of their venoms. These insulins are distinctive in having much greater similarity to fish insulins than to the molluscan hormone and are unique in that posttranslational modifications characteristic of conotoxins (hydroxyproline, γ-carboxyglutamate) are present. When injected into fish, the venom insulin elicits hypoglycemic shock, a condition characterized by dangerously low blood glucose. Our evidence suggests that insulin is specifically used as a weapon for prey capture by a subset of fish-hunting cone snails that use a net strategy to capture prey. Insulin appears to be a component of the nirvana cabal, a toxin combination in these venoms that is released into the water to disorient schools of small fish, making them easier to engulf with the snail's distended false mouth, which functions as a net. If an entire school of fish simultaneously experiences hypoglycemic shock, this should directly facilitate capture by the predatory snail.
Asunto(s)
Caracol Conus/química , Caracol Conus/fisiología , Insulina/genética , Toxinas Marinas/química , Conducta Predatoria/fisiología , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Insulina/análisis , Insulina/síntesis química , Insulina/metabolismo , Toxinas Marinas/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
The turripeptide ubi3a was isolated from the venom of the marine gastropod Unedogemmula bisaya, family Turridae, by bioassay-guided purification; both native and synthetic ubi3a elicited prolonged tremors when injected intracranially into mice. The sequence of the peptide, DCCOCOAGAVRCRFACC-NH2 (O = 4-hydroxyproline) follows the framework III pattern for cysteines (CC-C-C-CC) in the M-superfamily of conopeptides. The three-dimensional structure determined by NMR spectroscopy indicated a disulfide connectivity that is not found in conopeptides with the cysteine framework III: C1-C4, C2-C6, C3-C5. The peptide inhibited the activity of the α9α10 nicotinic acetylcholine receptor with relatively low affinity (IC50, 10.2 µM). Initial Constellation Pharmacology data revealed an excitatory activity of ubi3a on a specific subset of mouse dorsal root ganglion neurons.
Asunto(s)
Conotoxinas/química , Conotoxinas/farmacología , Caracol Conus/química , Animales , Calcio/metabolismo , Células Cultivadas , Conotoxinas/aislamiento & purificación , Caracol Conus/efectos de los fármacos , Caracol Conus/genética , Caracol Conus/crecimiento & desarrollo , Femenino , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Modelos Moleculares , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Receptores Nicotínicos/metabolismo , Xenopus laevisRESUMEN
Somatostatin (SS) is a peptide hormone with diverse physiological roles. By investigating a deep-water clade of fish-hunting cone snails, we show that predator-prey evolution has generated a diverse set of SS analogs, each optimized to elicit specific systemic physiological effects in prey. The increased metabolic stability, distinct SS receptor activation profiles, and chemical diversity of the venom analogs make them suitable leads for therapeutic application, including pain, cancer, and endocrine disorders. Our findings not only establish the existence of SS-like peptides in animal venoms but also serve as a model for the synergy gained from combining molecular phylogenetics and behavioral observations to optimize the discovery of natural products with biomedical potential.
Asunto(s)
Caracol Conus , Somatostatina , Ponzoñas , Animales , Caracol Conus/química , Filogenia , Conducta Predatoria , Somatostatina/química , Ponzoñas/químicaRESUMEN
Cannabinoid (CB) receptor agonists show robust antinociceptive effects in various pain models. However, most of the clinically potent CB1 receptor-active drugs derived from cannabis are considered concerning due to psychotomimetic side effects. Selective CB receptor ligands that do not induce CNS side effects are of clinical interest. The venoms of marine snail Conus are a natural source of various potent analgesic peptides, some of which are already FDA approved. In this study we evaluated the ability of several Conus venom extracts to interact with CB1 receptor. HEK293 cells expressing CB1 receptors were treated with venom extracts and CB1 receptor internalization was analyzed by immunofluorescence. Results showed C. textile (C. Tex) and C. miles (C. Mil) samples as the most potent. These were serially subfractionated by HPLC for subsequent analysis by internalization assays and for analgesic potency evaluated in the formalin test and after peripheral nerve injury. Intrathecal injection of C. Tex and C. Mil subfractions reduced flinching/licking behavior during the second phase of formalin test and attenuated thermal and mechanical allodynia in nerve injury model. Treatment with proteolytic enzymes reduced CB1 internalization of subfractions, indicating the peptidergic nature of CB1 active component. Further HPLC purification revealed two potent antinociceptive subfractions within C. Tex with CB1 and possible CB2 activity, with mild to no side effects in the CB tetrad assessment. CB conopeptides can be isolated from these active Conus venom-derived samples and further developed as novel analgesic agents for the treatment of chronic pain using cell based or gene therapy approaches.
Asunto(s)
Agonistas de Receptores de Cannabinoides/farmacología , Dolor Crónico/tratamiento farmacológico , Venenos de Moluscos/farmacología , Analgésicos/farmacología , Animales , Conducta Animal/efectos de los fármacos , Agonistas de Receptores de Cannabinoides/administración & dosificación , Cannabinoides/farmacología , Dolor Crónico/metabolismo , Caracol Conus/química , Terapia Genética/métodos , Células HEK293 , Humanos , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Inyecciones Espinales , Venenos de Moluscos/administración & dosificación , Dimensión del Dolor/efectos de los fármacos , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/metabolismo , Ratas , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismoRESUMEN
Venomous molluscs (Superfamily Conoidea) comprise a substantial fraction of tropical marine biodiversity (>15,000 species). Prior characterization of cone snail venoms established that bioactive venom components used to capture prey, defend against predators and for competitive interactions were relatively small, structured peptides (10-35 amino acids), most with multiple disulfide crosslinks. These venom components ("conotoxins, conopeptides") have been widely studied in many laboratories, leading to pharmaceutical agents and probes. In this review, we describe how it has recently become clear that to varying degrees, cone snail venoms also contain bioactive non-peptidic small molecule components. Since the initial discovery of genuanine as the first bioactive venom small molecule with an unprecedented structure, a broad set of cone snail venoms have been examined for non-peptidic bioactive components. In particular, a basal clade of cone snails (Stephanoconus) that prey on polychaetes produce genuanine and many other small molecules in their venoms, suggesting that this lineage may be a rich source of non-peptidic cone snail venom natural products. In contrast to standing dogma in the field that peptide and proteins are predominantly used for prey capture in cone snails, these small molecules also contribute to prey capture and push the molecular diversity of cone snails beyond peptides. The compounds so far characterized are active on neurons and thus may potentially serve as leads for neuronal diseases. Thus, in analogy to the incredible pharmacopeia resulting from studying venom peptides, these small molecules may provide a new resource of pharmacological agents.
RESUMEN
The cone snails (family Conidae) are the best known and most intensively studied venomous marine gastropods. However, of the total biodiversity of venomous marine mollusks (superfamily Conoidea, >20,000 species), cone snails comprise a minor fraction. The venoms of the family Drilliidae, a highly diversified family in Conoidea, have not previously been investigated. In this report, we provide the first biochemical characterization of a component in a Drilliidae venom and define a gene superfamily of venom peptides. A bioactive peptide, cdg14a, was purified from the venom of Clavus davidgilmouri Fedosov and Puillandre, 2020. The peptide is small (23 amino acids), disulfide-rich (4 cysteine residues) and belongs to the J-like drillipeptide gene superfamily. Other members of this superfamily share a conserved signal sequence and the same arrangement of cysteine residues in their predicted mature peptide sequences. The cdg14a peptide was chemically synthesized in its bioactive form. It elicited scratching and hyperactivity, followed by a paw-thumping phenotype in mice. Using the Constellation Pharmacology platform, the cdg14a drillipeptide was shown to cause increased excitability in a majority of non-peptidergic nociceptors, but did not affect other subclasses of dorsal root ganglion (DRG) neurons. This suggests that the cdg14a drillipeptide may be blocking a specific molecular isoform of potassium channels. The potency and selectivity of this biochemically characterized drillipeptide suggest that the venoms of the Drilliidae are a rich source of novel and selective ligands for ion channels and other important signaling molecules in the nervous system.
Asunto(s)
Caracol Conus , Venenos de Moluscos/química , Péptidos , Secuencia de Aminoácidos , Animales , Conducta Animal/efectos de los fármacos , Ganglios Espinales/citología , Ratones , Neuronas/efectos de los fármacos , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/toxicidadRESUMEN
The considerable diversity of Conus peptides in the I(1)-superfamily provides a rare opportunity to define parameters important for the post-translational l- to d-isomerization of amino acids. This subtlest of post-translational modifications is not readily detectable by most techniques, and it would be a considerable advance if one could predict its potential occurrence purely from gene sequences. We previously described three I(1)-conotoxins, iota-RXIA (formerly designated r11a), r11b and r11c, each containing a d-amino acid at the third position from the C-terminus. In this work, we investigated two novel I(1)-superfamily members, r11d and ar11a, which we show have only l-amino acids. Based on these observations and an analysis of cDNA sequences of other group members, we suggest that there is a rule to predict d-amino acids in I(1)-superfamily peptides. Two factors are important: the residue to be modified should be three amino acids from the C-terminus of the precursor sequence, and it should be in a suitable sequence context. We apply the rule to other members of the I(1)-superfamily, to determine a priori which are probably modified.
Asunto(s)
Aminoácidos/química , Conotoxinas/química , Secuencia de Aminoácidos , Animales , Clonación Molecular , Conotoxinas/farmacología , Caracol Conus , ADN Complementario , Electrofisiología , Espectrometría de Masas , Datos de Secuencia Molecular , Músculo Esquelético/efectos de los fármacos , Rana pipiens , Homología de Secuencia de AminoácidoRESUMEN
Cone snails in the Virgiconus clade prey on marine worms. Here, we identify six related conotoxins in the O1-superfamily from three species in this clade, Conus virgo, Conus terebra and Conus kintoki. One of these peptides, vi6a, was directly purified from the venom of C. virgo by following its activity using calcium imaging of dissociated mouse dorsal root ganglion (DRG) neurons. The purified peptide was biochemically characterized, synthesized and tested for activity in mice. Hyperactivity was observed upon both intraperitoneal and intracranial injection of the peptide. The effect of the synthetic peptide on DRG neurons was identical to that of the native peptide. Using the vi6a sequence, five other homologs were identified. These peptides define a glycine-rich subgroup of the O1-superfamily from the Virgiconus clade, with biological activity in mice.
Asunto(s)
Conotoxinas/química , Ganglios Espinales/efectos de los fármacos , Glicina/química , Moluscos/fisiología , Venenos de Moluscos/química , Secuencia de Aminoácidos , Animales , Conotoxinas/toxicidad , Ratones , Venenos de Moluscos/toxicidad , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
Peptide de7a was purified from the venom of Conus delessertii, a vermivorous cone snail collected in the Yucatan Channel, Mexico. Its amino acid sequence was determined by automatic Edman degradation after reduction and alkylation. The sequence shows six Cys residues arranged in the pattern that defines the O-superfamily of conotoxins, and several post-translationally modified residues. The determination of its molecular mass by means of laser desorption ionization time-of-flight mass spectrometry (average mass, 3170.0 Da) confirmed the chemical data and suggested amidation of the C-terminus. The primary structure (ACKOKNNLCAITgammaMAgammaCCSGFCLIYRCS*; O, hydroxyproline; gamma, gamma-carboxyglutamate; *, amidated C-terminus; calculated average mass, 3169.66 Da) of de7a contains a motif (gammaCCS) that has previously only been found in two other toxins, both from molluscivorous cone snails: TxVIIA from Conus textile and gamma-PnVIIA from Conus pennaceus. These toxins cause depolarization and increased firing of action potentials in molluscan neuronal systems, and toxin gamma-PnVIIA has been shown to act as an agonist of neuronal pacemaker cation currents. The similarities to toxins TxVIIA and gamma-PnVIIA suggest that peptide de7a might also affect voltage-gated nonspecific cation pacemaker channels.
Asunto(s)
Conotoxinas/química , Moluscos/metabolismo , Venenos de Moluscos/química , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Conotoxinas/aislamiento & purificación , Datos de Secuencia Molecular , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
In this study, a proteogenomic annotation strategy was used to identify a novel bioactive peptide from the venom of the predatory marine snail Conus victoriae. The peptide, conorfamide-Vc1 (CNF-Vc1), defines a new gene family. The encoded mature peptide was unusual for conotoxins in that it was cysteine-free and, despite low overall sequence similarity, contained two short motifs common to known neuropeptides/hormones. One of these was the C-terminal RF-amide motif, commonly observed in neuropeptides from a range of organisms, including humans. The mature venom peptide was synthesized and characterized structurally and functionally. The peptide was bioactive upon injection into mice, and calcium imaging of mouse dorsal root ganglion (DRG) cells revealed that the peptide elicits an increase in intracellular calcium levels in a subset of DRG neurons. Unusually for most Conus venom peptides, it also elicited an increase in intracellular calcium levels in a subset of non-neuronal cells. BIOLOGICAL SIGNIFICANCE: Our findings illustrate the utility of proteogenomics for the discovery of novel, functionally relevant genes and their products. CNF-Vc1 should be useful for understanding the physiological role of RF-amide peptides in the molluscan and mammalian nervous systems.
Asunto(s)
Caracol Conus/genética , Caracol Conus/metabolismo , Venenos de Moluscos/aislamiento & purificación , Neuropéptidos/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Células Cultivadas , Conotoxinas/genética , Conotoxinas/aislamiento & purificación , Conotoxinas/metabolismo , Conotoxinas/farmacología , Caracol Conus/química , Estudios de Asociación Genética/métodos , Genómica , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Venenos de Moluscos/genética , Venenos de Moluscos/metabolismo , Venenos de Moluscos/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Neuropéptidos/genética , Neuropéptidos/metabolismo , Neuropéptidos/farmacología , ProteómicaRESUMEN
Cone snails are renowned for producing peptide-based venom, containing conopeptides and conotoxins, to capture their prey. A novel small-molecule guanine derivative with unprecedented features, genuanine, was isolated from the venom of two cone snail species. Genuanine causes paralysis in mice, indicating that small molecules and not just polypeptides may contribute to the activity of cone snail venom.
Asunto(s)
Caracol Conus/química , Guanina/química , Guanina/aislamiento & purificación , Animales , Guanina/análogos & derivados , Guanina/farmacología , Isomerismo , Ratones , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Parálisis/inducido químicamenteRESUMEN
We describe the purification and biochemical characterization of three components from the venom of the toxoglossate gastropod Terebra subulata. The three polypeptide venom components, augertoxins s6a, s7a and s11a, are 40-41AA in length with 3-4 disulfide linkages. The arrangement of Cys residues is reminiscent of certain conopeptide superfamilies, but molecular cloning failed to show the highly conserved sequence features diagnostic of the conopeptide gene superfamily with a similar arrangement of Cys residues. One of the purified peptides, s7a, elicited an uncoordinated twisting syndrome when injected into the nematode Caenorhabditis elegans, but had no effect on mice. T. subulata belongs to the family Terebridae, one of four major groups of toxoglossate gastropods in the superfamily Conacea. The results reveal that some features of the augertoxins and conotoxins are generally similar, such as the organization of prepropeptide precursors and their proteolytic processing into mature toxins; however, Terebra may have evolved generally larger venom components that are less highly post-translationally modified. The results suggest that Conus peptide gene superfamilies probably do not extend to the Terebridae, suggesting that distinctive venom gene superfamilies may be expressed in each major division of Conacean gastropods.
Asunto(s)
Venenos de Moluscos/química , Venenos de Moluscos/genética , Caracoles/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Conotoxinas/genética , ADN Complementario/análisis , Datos de Secuencia Molecular , Venenos de Moluscos/aislamiento & purificación , Conformación Proteica , ARN/análisis , Análisis de Secuencia de Proteína , Homología de SecuenciaRESUMEN
The toxinology of the crassispirine snails, a major group of venomous marine gastropods within the superfamily Conoidea, is largely unknown. Here we define the first venom peptide superfamily, the P-like crassipeptides, and show that the organization of their gene sequences is similar to conotoxin precursors. We provide evidence that one peptide family within the P-like crassipeptide superfamily includes potassium-channel (K-channel) blockers, the κP-crassipeptides. Three of these peptides were chemically synthesized (cce9a, cce9b and iqi9a). Using conventional electrophysiology, cce9b was shown to be an antagonist of both a human Kv1.1 channel isoform (Shaker subfamily of voltage-gated K channels) and a Drosophila K-channel isoform. We assessed the bioactivity of these peptides in native mammalian dorsal root ganglion neurons in culture. We demonstrate that two of these crassipeptides, cce9a and cce9b, elicited an excitatory phenotype in a subset of small-diameter capsaicin-sensitive mouse DRG neurons that were also affected by κJ-conotoxin PlXIVA (pl14a), a blocker of Kv1.6 channels. Given the vast complexity of heteromeric K-channel isoforms, this study demonstrates that the crassispirine venoms are a potentially rich source for discovering novel peptides that can help to identify and characterize the diversity of K-channel subtypes expressed in native neurons and other cell types.
Asunto(s)
Venenos de Moluscos/química , Péptidos/química , Caracoles/química , Animales , Clonación Molecular , Drosophila , Humanos , Ratones , Ratones Endogámicos C57BL , Venenos de Moluscos/aislamiento & purificación , Venenos de Moluscos/toxicidad , Péptidos/aislamiento & purificación , Péptidos/toxicidad , Filogenia , Canales de Potasio/química , Caracoles/genética , XenopusRESUMEN
An impressive biodiversity (>10,000 species) of marine snails (suborder Toxoglossa or superfamily Conoidea) have complex venoms, each containing approximately 100 biologically active, disulfide-rich peptides. In the genus Conus, the most intensively investigated toxoglossan lineage (â¼500 species), a small set of venom gene superfamilies undergo rapid sequence hyperdiversification within their mature toxin regions. Each major lineage of Toxoglossa has its own distinct set of venom gene superfamilies. Two recently identified venom gene superfamilies are expressed in the large Turridae clade, but not in Conus. Thus, as major venomous molluscan clades expand, a small set of lineage-specific venom gene superfamilies undergo accelerated evolution. The juxtaposition of extremely conserved signal sequences with hypervariable mature peptide regions is unprecedented and raises the possibility that in these gene superfamilies, the signal sequences are conserved as a result of an essential role they play in enabling rapid sequence evolution of the region of the gene that encodes the active toxin.
Asunto(s)
Conotoxinas/genética , Caracol Conus/genética , Evolución Molecular , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia Conservada , Frecuencia de los Genes , Modelos Genéticos , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/genética , Selección Genética , Homología de Secuencia de AminoácidoRESUMEN
The crassispirids are a large branch of venomous marine gastropods whose venoms have not been investigated previously. We demonstrate that crassispirids comprise a major group of toxoglossate snails in a clade distinct from all turrids whose venoms have been analyzed. The isolation and biochemical definition of the first venom component from any crassispirid is described. Crassipeptide cce9a from Crassispira cerithina (Anton, 1838) was purified from crude venom by following biological activity elicited in young mice, lethargy and a lack of responsiveness to external stimuli. Using Edman sequencing and mass spectrometry, the purified peptide was shown to be 29 amino acid residues long, with the sequence: GSCGLPCHENRRCGWACYCDDGICKPLRV. The sequence assignment was verified through the analysis of a cDNA clone encoding the peptide. The peptide was chemically synthesized and folded; the synthetic peptide was biologically active and coelution with the native venom peptide was demonstrated. When injected into mice of various ages, the peptide elicited a striking shift in behavioral phenotype between 14 and 16 days, from lethargy to hyperactivity.
Asunto(s)
Conotoxinas/química , Venenos de Moluscos/química , Péptidos/análisis , Caracoles/metabolismo , Factores de Edad , Secuencia de Aminoácidos , Animales , Conducta Animal/efectos de los fármacos , Conotoxinas/toxicidad , ADN/aislamiento & purificación , Genoma , Hipercinesia/inducido químicamente , Ratones , Datos de Secuencia Molecular , Venenos de Moluscos/toxicidad , Péptidos/síntesis química , Péptidos/toxicidad , Análisis de Secuencia de Proteína , Caracoles/químicaRESUMEN
BACKGROUND: Ionotropic glutamate receptors (iGluRs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the central nervous system. Based on both molecular and pharmacological criteria, iGluRs have been divided into two major classes, the non-NMDA class, which includes both AMPA and kainate subtypes of receptors, and the NMDA class. One evolutionarily conserved feature of iGluRs is their desensitization in the continued presence of glutamate. Thus, when in a desensitized state, iGluRs can be bound to glutamate, yet the channel remains closed. However, the relevance of desensitization to nervous system function has remained enigmatic. RESULTS: Here, we report the identification and characterization of a novel polypeptide (con-ikot-ikot) from the venom of a predatory marine snail Conus striatus that specifically disrupts the desensitization of AMPA receptors (AMPARs). The stoichiometry of con-ikot-ikot appears reminiscent of the proposed subunit organization of AMPARs, i.e., a dimer of dimers, suggesting that it acts as a molecular four-legged clamp that holds the AMPAR channel open. Application of con-ikot-ikot to hippocampal slices caused a large and rapid increase in resting AMPAR-mediated current leading to neuronal death. CONCLUSIONS: Our findings provide insight into the mechanisms that regulate receptor desensitization and demonstrate that in the arms race between prey and predators, evolution has selected for a toxin that blocks AMPAR desensitization, thus revealing the fundamental importance of desensitization for regulating neural function.
Asunto(s)
Caracol Conus/metabolismo , Venenos de Moluscos/química , Neurotoxinas/farmacología , Péptidos/farmacología , Receptores AMPA/metabolismo , Animales , Benzotiadiazinas/farmacología , Sitios de Unión , Fraccionamiento Químico , Cromatografía Líquida de Alta Presión , Caracol Conus/química , Dimerización , Conductividad Eléctrica , Hipocampo/efectos de los fármacos , Neurotoxinas/química , Neurotoxinas/aislamiento & purificación , Técnicas de Placa-Clamp , Péptidos/química , Péptidos/aislamiento & purificación , Ratas , Receptores AMPA/química , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , XenopusRESUMEN
Two venom peptides, CPY-Pl1 (EU000528) and CPY-Fe1 (EU000529), characterized from the vermivorous marine snails Conus planorbis and Conus ferrugineus, define a new class of conopeptides, the conopeptide Y (CPY) family. The peptides have no disulfide cross-links and are 30 amino acids long; the high content of tyrosine is unprecedented for any native gene product. The CPY peptides were chemically synthesized and shown to be biologically active upon injection into both mice and Caenorhabditis elegans; activity on mammalian Kv1 channel isoforms was demonstrated using an oocyte heterologous expression system, and selectivity for Kv1.6 was found. NMR spectroscopy revealed that the peptides were unstructured in aqueous solution; however, a helical region including residues 12-18 for one peptide, CPY-Pl1, formed in trifluoroethanol buffer. Clones obtained from cDNA of both species encoded prepropeptide precursors that shared a unique signal sequence, indicating that these peptides are encoded by a novel gene family. This is the first report of tyrosine-rich bioactive peptides in Conus venom.