RESUMO
Background: Conotoxins exhibit great potential as neuropharmacology tools and therapeutic candidates due to their high affinity and specificity for ion channels, neurotransmitter receptors or transporters. The traditional methods to discover new conotoxins are peptide purification from the crude venom or gene amplification from the venom duct. Methods: In this study, a novel O1 superfamily conotoxin Tx6.7 was directly cloned from the genomic DNA of Conus textile using primers corresponding to the conserved intronic sequence and 3' UTR elements. The mature peptide of Tx6.7 (DCHERWDW CPASLLGVIYCCEGLICFIAFCI) was synthesized by solid-phase chemical synthesis and confirmed by mass spectrometry. Results: Patch clamp experiments on rat DRG neurons showed that Tx6.7 inhibited peak calcium currents by 59.29 ± 2.34% and peak potassium currents by 22.33 ± 7.81%. In addition, patch clamp on the ion channel subtypes showed that 10 µM Tx6.7 inhibited 56.61 ± 3.20% of the hCaV1.2 currents, 24.67 ± 0.91% of the hCaV2.2 currents and 7.30 ± 3.38% of the hNaV1.8 currents. Tx6.7 had no significant toxicity to ND7/23 cells and increased the pain threshold from 0.5 to 4 hours in the mouse hot plate assay. Conclusion: Our results suggested that direct cloning of conotoxin sequences from the genomic DNA of cone snails would be an alternative approach to obtaining novel conotoxins. Tx6.7 could be used as a probe tool for ion channel research or a therapeutic candidate for novel drug development.(AU)
Assuntos
Animais , Cálcio/isolamento & purificação , Conotoxinas/genética , Caramujo Conus/químicaRESUMO
Californiconus californicus, previously named Conus californicus, has always been considered a unique species within cone snails, because of its molecular, toxicological and morphological singularities; including the wide range of its diet, since it is capable of preying indifferently on fish, snails, octopus, shrimps, and worms. We report here a new cysteine pattern conotoxin assigned to the O1-superfamily capable of inhibiting the growth of Mycobacterium tuberculosis (Mtb). The conotoxin was tested on a pathogen reference strain (H37Rv) and multidrug-resistant strains, having an inhibition effect on growth with a minimal inhibitory concentration (MIC) range of 3.52â»0.22 µM, similar concentrations to drugs used in clinics. The peptide was purified from the venom using reverse phase high-performance liquid chromatography (RP-HPLC), a partial sequence was constructed by Edman degradation, completed by RACE and confirmed with venom gland transcriptome. The 32-mer peptide containing eight cysteine residues was named O1_cal29b, according to the current nomenclature for this type of molecule. Moreover, transcriptomic analysis of O-superfamily toxins present in the venom gland of the snail allowed us to assign several signal peptides to O2 and O3 superfamilies not described before in C. californicus, with new conotoxins frameworks.
Assuntos
Antibacterianos/farmacologia , Conotoxinas/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Conotoxinas/genética , Caramujo Conus , Farmacorresistência Bacteriana/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Peptídeos/genética , Tuberculose Resistente a Múltiplos MedicamentosRESUMO
Marine snails of the genus Conus (â¼500 species) are tropical predators that produce venoms for capturing prey, defense and competitive interactions. These venoms contain 50-200 different peptides ("conotoxins") that generally comprise 7-40 amino acid residues (including 0-5 disulfide bridges), and that frequently contain diverse posttranslational modifications, some of which have been demonstrated to be important for folding, stability, and biological activity. Most conotoxins affect voltage- and ligand-gated ion channels, G protein-coupled receptors, and neurotransmitter transporters, generally with high affinity and specificity. Due to these features, several conotoxins are used as molecular tools, diagnostic agents, medicines, and models for drug design. Based on the signal sequence of their precursors, conotoxins have been classified into genetic superfamilies, whereas their molecular targets allow them to be classified into pharmacological families. The objective of this work was to identify and analyze partial cDNAs encoding precursors of conotoxins belonging to I superfamily from three vermivorous species of the Mexican Pacific coast: C. brunneus, C. nux and C. princeps. The precursors identified contain diverse numbers of amino acid residues (C. brunneus, 65 or 71; C. nux, 70; C. princeps, 72 or 73), and all include a highly conserved signal peptide, a C-terminal propeptide, and a mature toxin. All the latter have one of the typical Cys frameworks of the I-conotoxins (C-C-CC-CC-C-C). The prepropeptides belong to the I2-superfamily, and encode eight different hydrophilic and acidic mature toxins, rather similar among them, and some of which have similarity with I2-conotoxins targeting voltage- and voltage-and-calcium-gated potassium channels.
Assuntos
Conotoxinas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Caramujos/genética , Sequência de Aminoácidos , Animais , Conotoxinas/química , Caramujo Conus/genética , DNA Complementar , México , Dados de Sequência Molecular , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Homologia de Sequência de AminoácidosRESUMO
Peptide de13a was previously purified from the venom of the worm-hunting cone snail Conus delessertii from the Yucatán Channel, México. This peptide has eight cysteine (Cys) residues in the unique arrangement C-C-C-CC-C-C-C, which defines the cysteine framework XIII ("-" represents one or more non-Cys residues). Remarkably, δ-hydroxy-lysine residues have been found only in conotoxin de13a, which also contains an unusually high proportion of hydroxylated amino acid residues. Here, we report the cDNA cloning of the complete precursor De13.1 of a related peptide, de13b, which has the same Cys framework and inter-Cys spacings as peptide de13a, and shares high protein/nucleic acid sequence identity (87%/90%) with de13a, suggesting that both peptides belong to the same conotoxin gene superfamily. Analysis of the signal peptide of precursor De13.1 reveals that this precursor belongs to a novel conotoxin gene superfamily that we chose to name gene superfamily G. Thus far superfamily G only includes two peptides, each of which contains the same, distinctive Cys framework and a high proportion of amino acid residues with hydroxylated side chains.
Assuntos
Conotoxinas/genética , Caramujo Conus/genética , Precursores de Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Conotoxinas/química , Dados de Sequência Molecular , Fases de Leitura Aberta , Precursores de Proteínas/química , Análise de Sequência de DNARESUMO
cDNA was prepared from the venom duct of a single Conus spurius specimen collected near the coast of Campeche, México. From it, PCR products were generated aiming to clone I-conotoxin precursors. Thirty clones were sequenced and predicted to encode ten distinct precursors: seven of I(2)-conotoxins and three of I(2)-like-conotoxins. These precursors contain three different, mature toxins, sr11a, sr11b and sr11c, of which two are novel and one (sr11a) has been previously purified and characterized from the venom of this species. The precursors include a 26- (I(2)) or 23- residue signal peptide (I(2)-like), a 31-residue "pro" region (I(2)-like), and a 32-residue mature toxin region (I(2) and I(2)-like). In addition, all the precursors have a 13-residue "post" region which contains a gamma-carboxylation recognition sequence that directs the gamma-carboxylation of Glu-9 and Glu-10 of toxin sr11a and, possibly, Glu-13 of toxin sr11b and Glu-9 of toxin sr11c. This is the first time that a "post" region has been found in precursors of I-conotoxins that also contain a "pro" region. The "post" peptide is enzymatically processed to yield the amidated mature toxin sr11a, which implies that gamma-carboxylation occurs before amidation. Phylogenetic analysis at the whole precursor level indicates that the I(2)-like-conotoxins of C. spurius are more related to I(2)-conotoxins than to I(1)- and I(3)-conotoxins from other species, and that they might represent a new subgroup of the I(2)-superfamily. The three I-conotoxins from C. spurius have charge differences at seven to nine positions, suggesting that they might have different molecular target types or subtypes.
Assuntos
Conotoxinas/química , Conotoxinas/metabolismo , Caramujo Conus/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Conotoxinas/classificação , Conotoxinas/genética , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Alinhamento de SequênciaRESUMO
cDNA was prepared from the venom duct of a single Conus spurius specimen collected near the coast of Campeche, Mexico. From it, PCR products were generated, sequenced, and predicted to encode eight distinct precursors of T-1-conotoxins. These precursors contain five different mature toxins, of which four are novel and one (sr5a) has been previously purified and characterized from the venom of this species. Three of the novel toxins are very similar to sr5a: two have one amino acid substitution at position 8, whereas the other is predicted to have one additional residue at the C-terminus; the fourth toxin has five amino acid substitutions and is predicted to have two additional residues at the C-terminus. In general, the precursors include a 22-residue signal peptide, a 24-residue "pro" region, and a 13- to 16-residue mature toxin region; however, the C-termini of two mature toxin regions are predicted to be altered by post-translational processing. Three precursors lack, in the same positions, 15 amino acid residues included in the "pre" (one residue) and "pro" (14 residues) regions, which suggests the existence of an exon encoding the last signal peptide residue and the first 14 residues of the "pro" region. Phylogenetic analysis indicates that the T-1-conotoxin precursors and mature toxins of C. spurius are more similar to certain precursors and toxins from molluscivorous Conus species than to any precursors and toxins from vermivorous cones. The results reported here will be useful for synthesizing the novel toxins in order to identify their molecular targets.
Assuntos
Conotoxinas/química , Caramujo Conus/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Conotoxinas/genética , Conotoxinas/isolamento & purificação , DNA Complementar/genética , México , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
A novel peptide, de7b, was isolated from the venom of Conus delessertii, a worm-hunting species collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and confirmed by mass spectrometry: it contains 28 amino acids, including six Cys residues. Peptide de7b is the second, O-conotoxin-like peptide isolated from the venom of this species, and it exists in different post-translationally modified isomorphs, some of which contain gamma-carboxy-glutamate (gamma) and/or 4-hydroxy-proline (O) at positions 4, 7, and/or 14. Its primary structure is DCI(P/O)GG(E/gamma)NCDVFR(O/P)YRCCSGYCILLLCA, with molecular masses varying from 3078.6 to 3154.6Da, depending on the number and kind of modified amino acid residues. Peptide de7b shows significant sequence identity with several O-conotoxins purified and biologically characterized from molluscivorous and piscivorous cone snails of the Indo-Pacific region, the tropical Atlantic and Eastern Pacific Oceans, especially with the delta-conotoxins but also with the omega-conotoxins from molluscivorous species, which suggests that it might affect voltage-gated Na(+) or Ca(2+)channels. Peptide de7b has 32% sequence identity with putative gamma-conotoxin de7a, previously characterized from the same species; both peptides contain the same number of amino acid residues and of non-Cys residues between the pairs of consecutive Cys residues. However, these peptides have charge differences at seven positions within the N-terminal half indicating that they might have distinct molecular targets that remain to be identified.
Assuntos
Conotoxinas/química , Caramujo Conus/genética , Sequência de Aminoácidos , Animais , Conotoxinas/genética , Evolução Molecular , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Processamento de Proteína Pós-Traducional , 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
Differential expression of gene-family members is typically associated with the specific development of certain tissues and organs, but its importance in the ecological adaptation of organisms has rarely been investigated. Several specialized feeding modes have evolved within the predatory marine gastropod genus Conus, including molluscivory and piscivory. Based on phylogenetic investigations of Conus species, it has been concluded that piscivory arose at least twice in this genus. Moreover, molecular analyses of conotoxin mRNA transcripts reveal that piscivores from independent evolutionary lineages express the same subset of four-loop conotoxins, contrary to phylogenetic expectations. These results demonstrate that differential expression of gene-family members can play a key role in adaptive evolution, particularly during shifts to new ecological niches.