RESUMO
The present study details the purification, the amino acid sequence determination, and a preliminary characterization of the biological effects in mice of a new conotoxin from the venom of Conus cancellatus (jr. syn.: Conus austini), a worm-hunting cone snail collected in the western Gulf of Mexico (Mexico). The 23-amino acid peptide, called as25a, is characterized by the sequence pattern CX1CX2CX8CX1CCX5, which is, for conotoxins, a new arrangement of six cysteines (framework XXV) that form three disulfide bridges. The primary structure (CKCPSCNFNDVTENCKCCIFRQP*; *, amidated C-terminus; calculated monoisotopic mass, 2644.09Da) was established by automated Edman degradation after reduction and alkylation, and MALDI-TOF and ESI mass spectrometry (monoisotopic mass, 2644.12/2644.08Da). Upon intracranial injection in mice, the purified peptide provokes paralysis of the hind limbs and death with a dose of 240 pmol (~0.635 µg, ~24.9 ng/g). In addition, a post-translational variant of this peptide (as25b) was identified and determined to contain two hydroxyproline residues. These peptides may represent a novel conotoxin gene superfamily.
Assuntos
Conotoxinas/química , Caramujo Conus , Cisteína/química , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Conotoxinas/isolamento & purificação , Conotoxinas/toxicidade , Masculino , Camundongos , Dados de Sequência Molecular , Neuropeptídeos/química , Neuropeptídeos/toxicidade , Paraplegia/induzido quimicamente , Análise de Sequência de Proteína , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Venenos de Moluscos/química , Sequência de Aminoácidos , Animais , Conotoxinas/química , Evolução Molecular , Gastrópodes/genética , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
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.
Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Conotoxinas/química , Conotoxinas/farmacologia , Venenos de Moluscos/química , Sequência de Aminoácidos , Animais , Comportamento Animal/efeitos dos fármacos , Fármacos do Sistema Nervoso Central/química , Fármacos do Sistema Nervoso Central/farmacologia , Conotoxinas/genética , Caramujo Conus/química , Caramujo Conus/genética , Masculino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Ácido 1-Carboxiglutâmico/química , Caramujo Conus/química , FMRFamida/química , Venenos de Moluscos/química , Neuropeptídeos/química , Peptídeos/química , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Comportamento Animal/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos , Peso Molecular , Moluscos , Venenos de Moluscos/isolamento & purificação , Venenos de Moluscos/farmacologia , Atividade Motora/efeitos dos fármacos , Músculos/efeitos dos fármacos , Músculos/fisiologia , Neuropeptídeos/isolamento & purificação , Neuropeptídeos/farmacologia , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Poecilia , Análise de Sequência de Proteína , CaramujosRESUMO
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.
Assuntos
Conotoxinas/química , Conotoxinas/farmacologia , Sequência de Aminoácidos , Animais , Comportamento Animal/efeitos dos fármacos , Caramujo Conus/química , Caramujo Conus/fisiologia , Comportamento Alimentar , Camundongos , Dados de Sequência Molecular , Venenos de Moluscos/química , Poliquetos , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A major peptide, de13a from the crude venom of Conus delessertii collected in the Yucatan Channel, Mexico, was purified. The peptide had a high content of posttranslationally modified amino acids, including 6-bromotryptophan and a nonstandard amino acid that proved to be 5-hydroxylysine. This is the first report of 5-hydroxylysine residues in conotoxins. The sequence analysis, together with cDNA cloning and a mass determination (monoisotopic mass of 3486.76 Da), established that the mature toxin has the sequence DCOTSCOTTCANGWECCKGYOCVNKACSGCTH, where O is 4-hydroxyproline, W 6-bromotryptophan, and K 5-hydroxylysine, the asterisk represents the amidated C-terminus, and the calculated monoisotopic mass is 3487.09 Da. The eight Cys residues are arranged in a pattern (C-C-C-CC-C-C-C) not described previously in conotoxins. This arrangement, for which we propose the designation of framework #13 or XIII, differs from the ones (C-C-CC-CC-C-C and C-C-C-C-CC-C-C) present in other conotoxins which also contain eight Cys residues. This peptide thus defines a novel class of conotoxins, with a new posttranslational modification not previously found in other Conus peptide families.
Assuntos
Conotoxinas/química , Peptídeos/química , Processamento de Proteína Pós-Traducional , Caramujos , Sequência de Aminoácidos , Aminoácidos/química , Animais , Conotoxinas/isolamento & purificação , Conotoxinas/metabolismo , Dados de Sequência Molecular , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Caramujos/metabolismoRESUMO
Carnivorous mollusks belonging to the genus Conus paralyze their prey by injecting a rich mixture of biologically active peptides. Conus regius is a vermivorous member of this genus that inhabits Brazilian tropical waters. Inter-, intra-species and individual variations of cone snail venom have been previously reported. In order to investigate intra-specific differences in C. regius venom, its feeding behavior and the correlation between these two factors, animals were pooled according to gender, size and season of collection, and their venom composition was compared by high performance liquid chromatography (HPLC). Both the whole venom and one specific peak were monitored by HPLC. Chromatographic profiles revealed no significant differences in their peak areas, indicating that the venom composition, based solely in the presence or absence of the major peaks, is stable regardless of season, gender and size. Therefore, analysis of one given toxin, eluting in one of the major peaks, is representative among the population. Moreover, this work presents the identification of one novel conotoxin (rg11a), which amino acid sequence was deduced by mass spectrometry.
Assuntos
Conotoxinas/isolamento & purificação , Comportamento Alimentar/fisiologia , Caramujos/química , Sequência de Aminoácidos , Animais , Tamanho Corporal , Cromatografia Líquida de Alta Pressão , Conotoxinas/química , Feminino , Masculino , Espectrometria de Massas , Dados de Sequência Molecular , Caramujos/fisiologiaRESUMO
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.