RESUMEN
Almost all marine snails within superfamily Conoidea produce venoms containing numerous neuroactive peptides. Most toxins characterized from members of this superfamily are produced by species belonging to family Conidae. These toxins (conotoxins) affect diverse membrane proteins, such as voltage- and ligand-gated ion channels, including nicotinic acetylcholine receptors (nAChRs). Family Turridae has been considerably less studied than their Conidae counterpart and, therefore, turrid toxins (turritoxins) have just been barely described. Consequently, in this work the most prominent chromatographic (RP-HPLC) fractions from the East Pacific species Polystira nobilis venom duct extract were isolated. The biological activity of six selected fractions was assayed on human (h) α7 AChRs expressed in Xenopus laevis oocytes. One of these fractions, F21, inhibited the acetylcholine-elicited response by 62 ± 12%. Therefore, this fraction was further purified and the F21-2 peptide was obtained. This peptide (at 5.6 µM) strongly and irreversibly inhibited the acetylcholine-induced response on hα7 and hα3ß2 nAChRs, by 55 ± 4 and 91 ± 1%, respectively. Electrospray mass spectrometry indicates that the average molecular mass of this toxin is 12 358.80 Da. The affinity for hα3ß2 nAChRs is high (IC50 of 566.2 nM). A partial sequence without cysteines was obtained by automated Edman degradation: WFRSFKSYYGHHGSVYRPNEPNFRSFAS ; blastp search revealed that this sequence has low similarity to some non-Cys-containing turripeptides. This is the first report of a turritoxin from a species of the American Pacific and the second description of a turripeptide inhibiting nAChRs.
Asunto(s)
Conotoxinas/farmacología , Venenos de Moluscos , Receptores Nicotínicos/efectos de los fármacos , Animales , Humanos , Venenos de Moluscos/química , Venenos de Moluscos/aislamiento & purificación , Venenos de Moluscos/metabolismo , Venenos de Moluscos/toxicidad , Oocitos , Proteínas Recombinantes/farmacología , Caracoles/metabolismo , Xenopus laevisRESUMEN
Background: Conopeptides are neuropharmacological peptides derived from the venomous salivary glands of cone snails. Among 29 superfamilies based on conserved signal sequences, T-superfamily conotoxins, which belong to the smallest group, include four different frameworks that contain four cysteines denominated I, V, X and XVI. In this work, the primary structure and the cysteine connectivity of novel conotoxin of Conus bandanus were determined by tandem mass spectrometry using collision-induced dissociation. Methods: The venom glands of C. bandanus snails were dissected, pooled, and extracted with 0.1% trifluoroacetic acid in three steps and lyophilized. The venom was fractionated and purified in an HPLC system with an analytical reversed-phase C18 column. The primary peptide structure was analyzed by MALDI TOF MS/MS using collision-induced dissociation and confirmed by Edman's degradation. The peptides cysteine connectivity was determined by rapid partial reduction-alkylation technique. Results: The novel conotoxin, NGC1C2(I/L)VREC3C4, was firstly derived from de novo sequencing by MS/MS. The presence of isoleucine residues in this conotoxin was confirmed by the Edman degradation method. The conotoxin, denominated Bn5a, belongs to the T1-subfamily of conotoxins. However, the disulfide bonds (C1-C4/C2-C3) of Bn5a were not the same as found in other T1-subfamily conopeptides but shared common connectivities with T2-subfamily conotoxins. The T1-conotoxin of C. bandanus proved the complexity of the disulfide bond pattern of conopeptides. The homological analysis revealed that the novel conotoxin could serve as a valuable probe compound for the human-nervous-system norepinephrine transporter. Conclusion: We identified the first T1-conotoxin, denominated Bn5a, isolated from C. bandanus venom. However, Bn5a...(AU)
Asunto(s)
Animales , Venenos de Moluscos/análisis , Venenos de Moluscos/química , Disulfuros/análisis , Conotoxinas/aislamiento & purificación , Caracol Conus/patogenicidadRESUMEN
Conorfamides (CNFs) are toxins initially characterized from the venom duct of the venomous marine snail Conus spurius from the Gulf of Mexico; at their C-termini, these toxins are amidated and have high sequence similarity with the molluskan cardioexcitatory tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide or FMRFa) and other FMRFa-related peptides (FaRPs) found in the five molluskan classes, and in other invertebrate and vertebrate phyla. These peptides were the first FaRPs found to be present in any venom, and they are biologically active in mice, limpets, and/or freshwater snails. However, the molecular targets of the known CNFs (CNF-Sr1 and CNF-Sr2 from C. spurius, and CNF-Vc1 from C. victoriae) remain unidentified. Very recently, three FaRPs from C. textile have been found to potentiate the currents of acid-sensing ion channels. In this work, we characterized a novel conorfamide, CNF-Sr3 (ATSGPMGWLPVFYRF-NH2), comprised of 15 amino acid residues, and with a specific blocking activity for the Shaker subtype of the voltage-gated potassium channels, without significant effect on the Shab, Shaw, Shal and Eag channels. This peptide is the third type of disulfide-free conotoxins that has been discovered to target K+ channels.
Asunto(s)
Caracol Conus/química , Venenos de Moluscos/química , Neuropéptidos/farmacología , Péptidos/farmacología , Canales de Potasio de la Superfamilia Shaker/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Baculoviridae , Células HEK293 , Humanos , Venenos de Moluscos/síntesis química , Venenos de Moluscos/farmacología , Neuropéptidos/síntesis química , Neuropéptidos/química , Péptidos/química , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Células Sf9/virologíaRESUMEN
The Terebridae are a diverse family of tropical and subtropical marine gastropods that use a complex and modular venom apparatus to produce toxins that capture polychaete and enteropneust preys. The complexity of the terebrid venom apparatus suggests that venom apparatus development in the Terebridae could be linked to the diversification of the group and can be analyzed within a molecular phylogenetic scaffold to better understand terebrid evolution. Presented here is a molecular phylogeny of 89 terebrid species belonging to 12 of the 15 currently accepted genera, based on Bayesian inference and Maximum Likelihood analyses of amplicons of 3 mitochondrial (COI, 16S and 12S) and one nuclear (28S) genes. The evolution of the anatomy of the terebrid venom apparatus was assessed by mapping traits of six related characters: proboscis, venom gland, odontophore, accessory proboscis structure, radula, and salivary glands. A novel result concerning terebrid phylogeny was the discovery of a previously unrecognized lineage, which includes species of Euterebra and Duplicaria. The non-monophyly of most terebrid genera analyzed indicates that the current genus-level classification of the group is plagued with homoplasy and requires further taxonomic investigations. Foregut anatomy in the family Terebridae reveals an inordinate diversity of features that covers the range of variability within the entire superfamily Conoidea, and that hypodermic radulae have likely evolved independently on at least three occasions. These findings illustrate that terebrid venom apparatus evolution is not perfunctory, and involves independent and numerous changes of central features in the foregut anatomy. The multiple emergence of hypodermic marginal radular teeth in terebrids are presumably associated with variable functionalities, suggesting that terebrids have adapted to dietary changes that may have resulted from predator-prey relationships. The anatomical and phylogenetic results presented serve as a starting point to advance investigations about the role of predator-prey interactions in the diversification of the Terebridae and the impact on their peptide toxins, which are promising bioactive compounds for biomedical research and therapeutic drug development.
Asunto(s)
Estructuras Animales/anatomía & histología , Evolución Biológica , Filogenia , Caracoles/anatomía & histología , Caracoles/genética , Estructuras Animales/fisiología , Animales , Secuencia de Bases , Teorema de Bayes , Cartilla de ADN/genética , ADN Ribosómico/genética , Tracto Gastrointestinal/anatomía & histología , Funciones de Verosimilitud , Madagascar , Modelos Genéticos , Datos de Secuencia Molecular , Venenos de Moluscos/química , Venenos de Moluscos/fisiología , Mozambique , Oceanía , Panamá , Análisis de Secuencia de ADN , Caracoles/clasificación , Especificidad de la EspecieRESUMEN
Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. californicus has been shown to be distantly related to all Conus species, but showing unusual biological features. We report a novel peptide isolated from C. californicus with a significant inhibitory action over neuronal voltage-gated calcium channels. The new toxin is formed by 13-amino acid residues with two disulfide bonds, whose sequence (NCPAGCRSQGCCM) is strikingly different from regular ω-conotoxins. In the HPLC purification procedure, the venom fraction eluted in the first 10-15 min produced a significant decrease (54% ± 3%) of the Ca(2+) current in Xenopus laevis oocytes transfected with purified rat-brain mRNA. A specific peptide obtained from the elution at 13 min decreased the Ca(2+) current in the adult rat dorsal-root ganglion neurons in a primary culture by 34% ± 2%. The cysteine pattern of this peptide corresponds to the framework XVI described for the M-superfamily of conopeptides and is unprecedented among Conus peptides acting on Ca(2+) channels.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/metabolismo , Conotoxinas/farmacología , Caracol Conus , Venenos de Moluscos/fisiología , Neuronas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Bloqueadores de los Canales de Calcio/química , Canales de Calcio Tipo N/fisiología , Células Cultivadas , Fraccionamiento Químico , Cromatografía Líquida de Alta Presión , Conotoxinas/química , Fenómenos Electrofisiológicos/efectos de los fármacos , Fenómenos Electrofisiológicos/fisiología , Femenino , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Datos de Secuencia Molecular , Venenos de Moluscos/química , Neuronas/metabolismo , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Ratas , Transfección , Xenopus laevis/fisiologíaRESUMEN
There is little information on the egg proteins of gastropod mollusks. Here we focus on PV2, a novel neurotoxin from snail eggs, studying its size, shape, structure, and stability, using small angle X-ray scattering (SAXS), absorption and fluorescence spectroscopy, circular dichroism, electron microscopy and partial proteolysis. Results indicate that PV2 is a compact and well folded oligomer of 130x44 A. It is an octamer of four 98 kDa heterodimers composed of 67 and 31 kDa subunits. Subunits are held together by disulfide bonds. Dimers are assembled into native PV2 by non-covalent forces. The larger subunit is more susceptible to proteolysis, indicating it is less compactly folded and/or more exposed. Quenching of tryptophan fluorescence showed a single class of tryptophyl side chains occluded in hydrophobic regions. Native structure shows loss of secondary structure (alpha+beta) at 6 M urea or 60-70 degrees C; the effects on the quaternary structure suggest an unfolding without disassembling of the protein. The 3D model of PV2 presented here is the first for an egg proteinaceous neurotoxin in animals.
Asunto(s)
Venenos de Moluscos/química , Neurotoxinas/química , Animales , Dicroismo Circular , Disulfuros/química , Endopeptidasa K/química , Moluscos , Conformación Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Dispersión de Radiación , Caracoles , Espectrometría de Fluorescencia/métodos , Triptófano/química , Rayos XRESUMEN
A novel peptide, pal9a, was purified from the venom duct extract of the turrid snail, Polystira albida (superfamily Conoidea, family Turridae), collected in the Gulf of Mexico. Its primary structure was determined by automated Edman degradation and confirmed by mass spectrometry. Turritoxin pal9a contains 34 amino acid residues, including 6 Cys residues arranged in the pattern C-C-C-C-C-C (framework IX, where "-" represents one or more non-Cys amino acids), which characterizes the P-conotoxins. Peptide pal9a is the first P-conotoxin-like turritoxin characterized from a member of family Turridae of the Western Atlantic. The primary structure of turritoxin pal9a, NVCDGDACPDGVCRSGCTCDFNVAQRKDTCFYPQ-nh(2) (-nh(2), amidated C-terminus; calculated monoisotopic mass, 3679.48Da; experimental monoisotopic mass, 3678.84Da), shows variable degrees of low sequence similarity with framework IX-toxins from turrid (three species of Lophiotoma, and four species of Gemmula), terebrid (Hastula hectica), and Conus species of the Indo-Pacific (C. textile, C. gloriamaris, C. amadis, and C. litteratus) and of the Western Atlantic (C. regius). During the comparison of peptide pal9a with the other framework IX-toxins known to date, we realized that, in general, these peptides are hydrophilic, acidic compounds that have not been found in the fish-hunting Conus species studied thus far; we also found support for the notion that they may belong to several distinct gene superfamilies, even those from the same species. Given the broad distribution of framework IX-toxins within superfamily Conoidea, it will be interesting to identify the still-unknown molecular targets of P-conotoxins, P-conotoxin-like turritoxins, and P-conotoxin-like augertoxins.
Asunto(s)
Venenos de Moluscos/química , Secuencia de Aminoácidos , Animales , Conotoxinas/química , Evolución Molecular , Gastrópodos/genética , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
As part of continuing studies of the venom components present in Conus austini (syn.: Conus cancellatus), a vermivorous cone snail collected in the western Gulf of Mexico, Mexico, two major peptides, as14a and as14b, were purified and characterized. Their amino acid sequences were determined by automatic Edman sequencing after reduction and alkylation. Their molecular masses, established by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, confirmed the chemical analyses and indicated that as14a and as14b have free C-termini. Each peptide contains 4-Cys residues arranged in a pattern (C-C-C-C, framework 14). The primary structure of as14a is GGVGRCIYNCMNSGGGLNFIQCKTMCY (experimental monoisotopic mass 2883.92Da; calculated monoisotopic mass 2884.20Da), whereas that of as14b is RWDVDQCIYYCLNGVVGYSYTECQTMCT (experimental monoisotopic mass 3308.63Da; calculated monoisotopic mass 3308.34Da). Both purified peptides elicited scratching and grooming activity in mice, and as14b also caused body and rear limb extension and tail curling immediately upon injection. The high sequence similarity of peptide as14a with peptide vil14a from the vermivorous C. villepinii suggests that the former might block K+ channels.
Asunto(s)
Sistema Nervioso Central/efectos de los fármacos , Conotoxinas/química , Conotoxinas/farmacología , Venenos de Moluscos/química , Secuencia de Aminoácidos , Animales , Conducta Animal/efectos de los fármacos , Fármacos del Sistema Nervioso Central/química , Fármacos del Sistema Nervioso Central/farmacología , Conotoxinas/genética , Caracol Conus/química , Caracol Conus/genética , Masculino , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
A novel peptide, conorfamide-Sr2 (CNF-Sr2), was purified from the venom extract of Conus spurius, collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and amino acid analysis, and confirmed by electrospray ionization mass spectrometry. Conorfamide-Sr2 contains 12 amino acids and no Cys residues, and it is only the second FMRFamide-related peptide isolated from a venom. Its primary structure GPM gammaDPLgammaIIRI-nh2, (gamma, gamma-carboxyglutamate; -nh2, amidated C-terminus; calculated monoisotopic mass, 1468.72Da; experimental monoisotopic mass, 1468.70Da) shows two features that are unusual among FMRFamide-related peptides (FaRPs, also known as RFamide peptides), namely the novel presence of gamma-carboxyglutamate, and a rather uncommon C-terminal residue, Ile. CNF-Sr2 exhibits paralytic activity in the limpet Patella opea and causes hyperactivity in the freshwater snail Pomacea paludosa and in the mouse. The sequence similarities of CNF-Sr2 with FaRPs from marine and freshwater mollusks and mice might explain its biological effects in these organisms. It also resembles FaRPs from polychaetes (the prey of C. spurius), which suggests a natural biological role. Based on these similarities, CNF-Sr2 might interact with receptors of these three distinct types of FaRPs, G-protein-coupled receptors, Na+ channels activated by FMRFamide (FaNaCs), and acid-sensing ion channels (ASICs). The biological activities of CNF-Sr2 in mollusks and mice make it a potential tool to study molecular targets in these and other organisms.
Asunto(s)
Ácido 1-Carboxiglutámico/química , Caracol Conus/química , FMRFamida/química , Venenos de Moluscos/química , Neuropéptidos/química , Péptidos/química , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Conducta Animal/efectos de los fármacos , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/fisiología , Masculino , Ratones , Ratones Endogámicos , Peso Molecular , Moluscos , Venenos de Moluscos/aislamiento & purificación , Venenos de Moluscos/farmacología , Actividad Motora/efectos de los fármacos , Músculos/efectos de los fármacos , Músculos/fisiología , Neuropéptidos/aislamiento & purificación , Neuropéptidos/farmacología , Péptidos/aislamiento & purificación , Péptidos/farmacología , Poecilia , Análisis de Secuencia de Proteína , CaracolesRESUMEN
A novel 31-residue toxin, named as7a, was isolated and characterized from the venom of Conus austini, a vermivorous cone snail collected in the western Gulf of Mexico. The complete amino acid sequence, TCKQKGEGCSLDVgammaCCSSSCKPGGPLFDFDC, was determined by automatic Edman sequencing after reduction and alkylation. The sequence shows six Cys residues arranged in the pattern that defines the O-superfamily of conotoxins, and the sequence motif -gammaCCS-, which has only been found in the gamma-conotoxin family. The molecular mass of the native peptide was determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, which confirmed the chemical analyses and suggested a free C-terminus. The purified peptide elicited toxic effects in the freshwater snail Pomacea paludosa after intramuscular injection, but it had no effect when injected intracerebrally into mice. The structural similarity of peptide as7a to other gamma-conotoxins suggests that modulation of pacemaker channels could be responsible for its biological activity.
Asunto(s)
Conotoxinas/química , Conotoxinas/farmacología , Secuencia de Aminoácidos , Animales , Conducta Animal/efectos de los fármacos , Caracol Conus/química , Caracol Conus/fisiología , Conducta Alimentaria , Ratones , Datos de Secuencia Molecular , Venenos de Moluscos/química , Poliquetos , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The objective of this investigation was to purify and characterize polypeptides from the venom ducts of the turrid snails Polystira albida and Gemmula periscelida (superfamily: Conoidea, family: Turridae), collected in Mexican waters. Venoms of other groups in the superfamily (family: Conidae, genus: Conus) have peptide toxins ('conotoxins'), but no venom components have been characterized from any turrid species. Crude venoms were fractionated using reversed-phase high performance liquid chromatography, and one major component from each venom was characterized. In contrast to most conotoxins, the polypeptides characterized contain a high proportion of Met, Tyr and Arg residues, and few, if any, Cys residues. The two peptides had some regions of homology, but were not significantly similar to other peptides. Both peptides are predicted to contain alpha-helical structures, and the peptide from P. albida is predicted to form a coiled-coil motif. This structural motif could provide conformational stability for these turrid venom components ("turritoxins"), which in the case of conotoxins is primarily achieved by disulfide bonds. Thus, the first turritoxins characterized are strikingly different from the conotoxins, suggesting divergent biochemical strategies in the venoms of different major groups included in the superfamily Conoidea.
Asunto(s)
Venenos de Moluscos/química , Péptidos/química , Caracoles/química , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Metionina/química , México , Datos de Secuencia Molecular , Venenos de Moluscos/genética , Venenos de Moluscos/aislamiento & purificación , Péptidos/genética , Péptidos/aislamiento & purificación , Estructura Secundaria de Proteína , Agua de Mar , Análisis de Secuencia de ProteínaRESUMEN
A novel Conus peptide, conorfamide-Sr1, has been characterized. The sequence of the natural peptide was determined using standard Edman sequencing methods and mass spectrometry, and confirmed by chemical synthesis. The peptide has 12 amino acids and no cysteine residues. The following sequence was obtained: GPMGWVPVFYRF-NH(2). No other peptide from a vermivorous Atlantic Conus species has previously been characterized. Conorfamide-Sr1 belongs to the RFamide neuropeptide family, and is the first RFamide peptide to be found in any venom. The presence of conorfamide-Sr1 as a major peptide in Conus spurius venom suggests that Conus lineages in the Atlantic may have evolved novel Conus venom peptide families.