RESUMO
The cysteine-free acyclic peptides present in marine cone snail venom have been much less investigated than their disulfide bonded counterparts. Precursor protein sequences derived from transcriptomic data, together with mass spectrometric fragmentation patterns for peptides present in venom duct tissue extracts, permit the identification of mature peptides. Twelve distinct gene superfamiles have been identified with precursor lengths between 64 and 158 residues. In the case of Conus monile, three distinct mature peptides have been identified, arising from two distinct protein precursors. Mature acyclic peptides are often post-translationally modified, with C-terminus amidation, a feature characteristic of neuropeptides. In the present study, 20 acyclic peptides from Conus monile and Conus betulinus were identified. The common modifications of C-terminus amidation, gamma carboxylation of glutamic acid (E to Ï), N-terminus conversion of Gln (Q) to a pyroglutamyl residue (Z), and hydroxylation of Pro (P) to Hyp (O) are observed in one or more peptides identified in this study. Proteolytic trimming of sequences by cleavage at the C-terminus of Asn (N) residues is established. The presence of an asparagine endopeptidase is strengthened by the identification of legumain-like sequences in the transcriptome assemblies from diverse Conus species. Such sequences may be expected to have a cleavage specificity at Asn-Xxx peptide bonds.
Assuntos
Conotoxinas , Caramujo Conus , Animais , Venenos de Moluscos/química , Venenos de Moluscos/genética , Venenos de Moluscos/metabolismo , Conotoxinas/química , Peptídeos/química , Caramujo Conus/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismoRESUMO
Distinct differences have been observed between L-tryptophan and D-tryptophan containing contryphan-Ar1131 in oxidative folding, trypsin binding, and photostabilization activity on avobenzone. [W5] contryphan-Ar1131 and [w5] contryphan-Ar1131 were chemically synthesized and characterized using RP-HPLC and mass spectrometry. Structural differences due to the change of configuration of tryptophan were evident from the optimized structures of contryphan-Ar1131 using density functional theory (DFT). The comparison of early events of oxidative folding has revealed the role of D-tryptophan in accelerating the formation of a disulfide bond. The optimized structures of the reduced form of peptides revealed the occurrence of aromatic-aromatic and aromatic-proline interactions in [w5] contryphan-Ar1131 which may be critical in aiding the oxidative folding reaction. The presence of the Lys6-Pro7 peptide bond indicates that contryphan-Ar1131 is resistant but may bind to trypsin allowing to assign the binding affinity of peptides to the protein surface. Competitive binding studies and molecular docking along with molecular dynamic (MD) simulations have revealed that [w5] contryphan-Ar1131 has more affinity for the active site of trypsin. Given tryptophan is a photostabilizer of FDA-approved chemical UV-A filter avobenzone, the report has compared the photostabilization activity of [W5]/ [w5] contryphan-Ar1131 on avobenzone under natural sunlight. [w5] contryphan-Ar1131 has better photostabilization activity than that of [W5] contryphan-Ar1131 and also individual D-tryptophan and L-tryptophan amino acids. These biochemical studies have highlighted the significance of D-tryptophan in contryphan-Ar1131 and its photostabilization activity on avobenzone may find applications in cosmetics.
Assuntos
Caramujo Conus , Animais , Caramujo Conus/metabolismo , Dissulfetos , Simulação de Acoplamento Molecular , Venenos de Moluscos/química , Venenos de Moluscos/metabolismo , Estresse Oxidativo , Peptídeos/química , Peptídeos Cíclicos , Prolina , Propiofenonas , Tripsina , Triptofano/químicaRESUMO
Cone snail venom biodiversity reflects dietary preference and predatory and defensive envenomation strategies across the ≈900 species of Conidae. To better understand the mechanisms of adaptive radiations in closely related species, we investigated the venom of two phylogenetically and spatially related species, C. flavidus and C. frigidus of the Virgiconus clade. Transcriptomic analysis revealed that the major superfamily profiles were conserved between the two species, including 68 shared conotoxin transcripts. These shared transcripts contributed 90% of the conotoxin expression in C. frigidus and only 49% in C. flavidus, which showed greater toxin diversification in the dominant O1, I2, A, O2, O3, and M superfamilies compared to C. frigidus. On the basis of morphology, two additional sub-groups closely resembling C. flavidus were also identified from One Tree Island Reef. Despite the morphological resemblance, the venom duct proteomes of these cryptic sub-groups were distinct from C. flavidus. We suggest rapid conotoxin sequence divergence may have facilitated adaptive radiation and the establishment of new species and the regulatory mechanisms facilitating species-specific venom evolution.
Assuntos
Caramujo Conus , Venenos de Moluscos , Animais , Caramujo Conus/genética , Caramujo Conus/metabolismo , Perfilação da Expressão Gênica , Venenos de Moluscos/genética , Venenos de Moluscos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Filogenia , Proteoma/genética , Proteoma/metabolismoRESUMO
Cone snail venoms contain a wide variety of bioactive peptides, including insulin-like molecules with distinct structural features, binding modes and biochemical properties. Here, we report an active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy (cryo-EM) and protein engineering to elucidate its interactions with the human insulin receptor (IR) ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of human insulin but also compromise therapeutic utility by delaying dissolution from the site of subcutaneous injection. This finding suggests approaches to developing improved therapeutic insulins. Curiously, the receptor displays a continuum of conformations from the symmetric state to a highly asymmetric low-abundance structure that displays coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.
Assuntos
Insulina , Venenos de Moluscos , Microscopia Crioeletrônica , Humanos , Insulina/metabolismo , Venenos de Moluscos/química , Venenos de Moluscos/metabolismo , Peptídeos , Conformação ProteicaRESUMO
Conus venoms comprise a large variety of biologically active peptides (conopeptides or conotoxins) that are employed for prey capture and other biological functions. Throughout the course of evolution of the cone snails, they have developed an envenomation scheme that necessitates a potent mixture of peptides, most of which are highly post-translationally modified, that can cause rapid paralysis of their prey. The great diversity of these peptides defines the ecological interactions and evolutionary strategy of cone snails. Such scheme has led to some pharmacological applications for pain, epilepsy, and myocardial infarction, that could be further explored to ultimately find unique peptide-based therapies. This review focuses on â¼ 60 representative post-translationally modified conopeptides that were isolated from Conus venoms. Various conopeptides reveal post-translational modifications of specific amino acids, such as hydroxylation of proline and lysine, gamma-carboxylation of glutamate, formation of N-terminal pyroglutamate, isomerization of l- to d-amino acid, bromination of tryptophan, O-glycosylation of threonine or serine, sulfation of tyrosine, and cysteinylation of cysteine, other than the more common disulfide crosslinking and C-terminal amidation. Many of the post-translationally modified peptides paved the way for the characterization, by alternative analytical methods, of other pharmacologically important peptides that are classified under 27 conopeptide families denoting pharmacological classes.
Assuntos
Conotoxinas/metabolismo , Conotoxinas/farmacologia , Venenos de Moluscos/metabolismo , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Venenos de Moluscos/química , Peptídeos/químicaRESUMO
Human insulin and its current therapeutic analogs all show propensity, albeit varyingly, to self-associate into dimers and hexamers, which delays their onset of action and makes blood glucose management difficult for people with diabetes. Recently, we described a monomeric, insulin-like peptide in cone-snail venom with moderate human insulin-like bioactivity. Here, with insights from structural biology studies, we report the development of mini-Ins-a human des-octapeptide insulin analog-as a structurally minimal, full-potency insulin. Mini-Ins is monomeric and, despite the lack of the canonical B-chain C-terminal octapeptide, has similar receptor binding affinity to human insulin. Four mutations compensate for the lack of contacts normally made by the octapeptide. Mini-Ins also has similar in vitro insulin signaling and in vivo bioactivities to human insulin. The full bioactivity of mini-Ins demonstrates the dispensability of the PheB24-PheB25-TyrB26 aromatic triplet and opens a new direction for therapeutic insulin development.
Assuntos
Antígenos CD/química , Insulina/química , Venenos de Moluscos/química , Venenos de Moluscos/metabolismo , Receptor de Insulina/química , Substituição de Aminoácidos , Animais , Antígenos CD/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Insulina/análogos & derivados , Insulina/metabolismo , Insulina/farmacologia , Camundongos Endogâmicos C57BL , Modelos Moleculares , Simulação de Dinâmica Molecular , Venenos de Moluscos/genética , Venenos de Moluscos/farmacologia , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Conformação Proteica , Ratos Sprague-Dawley , Receptor de Insulina/metabolismo , Relação Estrutura-Atividade , TirosinaRESUMO
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.
Assuntos
Conotoxinas/farmacologia , Venenos de Moluscos , Receptores Nicotínicos/efeitos dos fármacos , Animais , Humanos , Venenos de Moluscos/química , Venenos de Moluscos/isolamento & purificação , Venenos de Moluscos/metabolismo , Venenos de Moluscos/toxicidade , Oócitos , Proteínas Recombinantes/farmacologia , Caramujos/metabolismo , Xenopus laevisRESUMO
Venomous marine cone snails produce peptide toxins (conotoxins) that bind ion channels and receptors with high specificity and therefore are important pharmacological tools. Conotoxins contain conserved cysteine residues that form disulfide bonds that stabilize their structures. To gain structural insight into the large, yet poorly characterized conotoxin H-superfamily, we used NMR and CD spectroscopy along with MS-based analyses to investigate H-Vc7.2 from Conus victoriae, a peptide with a VI/VII cysteine framework. This framework has CysI-CysIV/CysII-CysV/CysIII-CysVI connectivities, which have invariably been associated with the inhibitor cystine knot (ICK) fold. However, the solution structure of recombinantly expressed and purified H-Vc7.2 revealed that although it displays the expected cysteine connectivities, H-Vc7.2 adopts a different fold consisting of two stacked ß-hairpins with opposing ß-strands connected by two parallel disulfide bonds, a structure homologous to the N-terminal region of the human granulin protein. Using structural comparisons, we subsequently identified several toxins and nontoxin proteins with this "mini-granulin" fold. These findings raise fundamental questions concerning sequence-structure relationships within peptides and proteins and the key determinants that specify a given fold.
Assuntos
Conotoxinas/química , Caramujo Conus/metabolismo , Cisteína/química , Granulinas/química , Sequência de Aminoácidos , Animais , Conotoxinas/genética , Conotoxinas/metabolismo , Dissulfetos/química , Granulinas/metabolismo , Espectroscopia de Ressonância Magnética , Venenos de Moluscos/metabolismo , Conformação Proteica em Folha beta , Dobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The salivary apparatus of the common octopus ( Octopus vulgaris) has been the subject of biochemical study for over a century. A combination of bioassays, behavioral studies and molecular analysis on O. vulgaris and related species suggests that its proteome should contain a mixture of highly potent neurotoxins and degradative proteins. However, a lack of genomic and transcriptomic data has meant that the amino acid sequences of these proteins remain almost entirely unknown. To address this, we assembled the posterior salivary gland transcriptome of O. vulgaris and combined it with high resolution mass spectrometry data from the posterior and anterior salivary glands of two adults, the posterior salivary glands of six paralarvae and the saliva from a single adult. We identified a total of 2810 protein groups from across this range of salivary tissues and age classes, including 84 with homology to known venom protein families. Additionally, we found 21 short secreted cysteine rich protein groups of which 12 were specific to cephalopods. By combining protein expression data with phylogenetic analysis we demonstrate that serine proteases expanded dramatically within the cephalopod lineage and that cephalopod specific proteins are strongly associated with the salivary apparatus.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Venenos de Moluscos/genética , Octopodiformes/genética , Proteogenômica/métodos , Saliva/metabolismo , Transcriptoma , Animais , Feminino , Ontologia Genética , Larva/química , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Masculino , Anotação de Sequência Molecular , Venenos de Moluscos/classificação , Venenos de Moluscos/metabolismo , Neurotoxinas/classificação , Neurotoxinas/genética , Neurotoxinas/metabolismo , Octopodiformes/química , Octopodiformes/crescimento & desenvolvimento , Octopodiformes/metabolismo , Filogenia , Proteoma/genética , Proteoma/metabolismo , Saliva/química , Glândulas Salivares/química , Glândulas Salivares/crescimento & desenvolvimento , Glândulas Salivares/metabolismo , Serina Proteases/classificação , Serina Proteases/genética , Serina Proteases/metabolismoRESUMO
Cone snail venoms have separately evolved for predation and defense. Despite remarkable inter- and intra-species variability, defined sets of synergistic venom peptides (cabals) are considered essential for prey capture by cone snails. To better understand the role of predatory cabals in cone snails, we used a high-throughput proteomic data mining and visualisation approach. Using this approach, the relationship between the predatory venom peptides from nine C. purpurascens was systematically analysed. Surprisingly, potentially synergistic levels of κ-PVIIA and δ-PVIA were only identified in five of nine specimens. In contrast, the remaining four specimens lacked significant levels of these known excitotoxins and instead contained high levels of the muscle nAChR blockers ψ-PIIIE and αA-PIVA. Interestingly, one of nine specimens expressed both cabals, suggesting that these sub-groups might represent inter-breeding sub-species of C. purpurascens. High throughput cluster analysis also revealed these two cabals clustered with distinct groups of venom peptides that are presently uncharacterised. This is the first report showing that the cone snails of the same species can deploy two separate and distinct predatory cabals for prey capture and shows that the cabals deployed by this species can be more complex than presently realized. Our semi-automated proteomic analysis facilitates the deconvolution of complex venoms to identify co-evolved families of peptides and help unravel their evolutionary relationships in complex venoms.
Assuntos
Caramujo Conus/fisiologia , Venenos de Moluscos/metabolismo , Peptídeos/metabolismo , Comportamento Predatório , Proteômica , Animais , Cromatografia Líquida , Conotoxinas/genética , Conotoxinas/metabolismo , Expressão Gênica , Venenos de Moluscos/genética , Peptídeos/genética , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Cone snails are biomedically important sources of peptide drugs, but it is not known whether snail-associated bacteria affect venom chemistry. To begin to answer this question, we performed 16S rRNA gene amplicon sequencing of eight cone snail species, comparing their microbiomes with each other and with those from a variety of other marine invertebrates. We show that the cone snail microbiome is distinct from those in other marine invertebrates and conserved in specimens from around the world, including the Philippines, Guam, California, and Florida. We found that all venom ducts examined contain diverse 16S rRNA gene sequences bearing closest similarity to Stenotrophomonas bacteria. These sequences represent specific symbionts that live in the lumen of the venom duct, where bioactive venom peptides are synthesized.IMPORTANCE In animals, symbiotic bacteria contribute critically to metabolism. Cone snails are renowned for the production of venoms that are used as medicines and as probes for biological study. In principle, symbiotic bacterial metabolism could either degrade or synthesize active venom components, and previous publications show that bacteria do indeed contribute small molecules to some venoms. Therefore, understanding symbiosis in cone snails will contribute to further drug discovery efforts. Here, we describe an unexpected, specific symbiosis between bacteria and cone snails from around the world.
Assuntos
Venenos de Moluscos/química , Caramujos/microbiologia , Stenotrophomonas/isolamento & purificação , Stenotrophomonas/fisiologia , Simbiose , Animais , DNA Bacteriano/genética , Microbiota , Venenos de Moluscos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Filogenia , RNA Ribossômico 16S/genética , Caramujos/classificação , Caramujos/fisiologia , Stenotrophomonas/genéticaRESUMO
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.
Assuntos
Caramujo Conus/genética , Caramujo Conus/metabolismo , Venenos de Moluscos/isolamento & purificação , Neuropeptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , Células Cultivadas , Conotoxinas/genética , Conotoxinas/isolamento & purificação , Conotoxinas/metabolismo , Conotoxinas/farmacologia , Caramujo Conus/química , Estudos de Associação Genética/métodos , Genômica , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Venenos de Moluscos/genética , Venenos de Moluscos/metabolismo , Venenos de Moluscos/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologia , ProteômicaRESUMO
Peptide neurotoxins from cone snails called conotoxins are renowned for their therapeutic potential to treat pain and several neurodegenerative diseases. Inefficient assay-guided discovery methods have been replaced by high-throughput bioassays integrated with advanced MS and next-generation sequencing, ushering in the era of 'venomics'. In this review, we focus on the impact of venomics on the understanding of cone snail biology as well as the application of venomics to accelerate the discovery of new conotoxins. We also discuss the continued importance of medicinal chemistry approaches to optimize conotoxins for clinical use, with a descriptive case study of MrIA featured.
Assuntos
Venenos de Moluscos/química , Caramujos/metabolismo , Animais , Conotoxinas/química , Conotoxinas/metabolismo , Conotoxinas/uso terapêutico , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Venenos de Moluscos/metabolismo , Venenos de Moluscos/uso terapêutico , Doenças do Sistema Nervoso/tratamento farmacológico , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Peptidomiméticos , Relação Estrutura-AtividadeRESUMO
Observations of the mollusc-hunting cone snail Conus textile during feeding reveal that prey are often stung multiple times in succession. While studies on the venom peptides injected by fish-hunting cone snails have become common, these approaches have not been widely applied to the analysis of the injected venoms from mollusc-hunters. We have successfully obtained multiple injected venom samples from C. textile individuals, allowing us to investigate venom compositional variation during prey capture. Our studies indicate that C. textile individuals alter the composition of prey-injected venom peptides during single feeding events. The qualitative results obtained by MALDI-ToF mass spectrometry are mirrored by quantitative changes in venom composition observed by reverse-phase high performance liquid chromatography. While it is unclear why mollusc-hunting cone snails inject prey multiple times prior to engulfment, our study establishes for the first time a link between this behavior and compositional changes of the venom during prey capture. Changes in venom composition during hunting may represent a multi-step strategy utilized by these venomous animals to slow and incapacitate prey prior to engulfment.
Assuntos
Mordeduras e Picadas , Caramujo Conus/metabolismo , Venenos de Moluscos/metabolismo , Animais , Venenos de Moluscos/isolamento & purificação , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Comportamento Predatório , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Cone snail venoms provide a largely untapped source of novel peptide drug leads. To enhance the discovery phase, a detailed comparative proteomic analysis was undertaken on milked venom from the mollusk-hunting cone snail, Conus textile, from three different geographic locations (Hawai'i, American Samoa and Australia's Great Barrier Reef). A novel milked venom conopeptide rich in post-translational modifications was discovered, characterized and named α-conotoxin TxIC. We assign this conopeptide to the 4/7 α-conotoxin family based on the peptide's sequence homology and cDNA pre-propeptide alignment. Pharmacologically, α-conotoxin TxIC demonstrates minimal activity on human acetylcholine receptor models (100 µM, <5% inhibition), compared to its high paralytic potency in invertebrates, PD50 = 34.2 nMol kg(-1). The non-post-translationally modified form, [Pro](2,8)[Glu](16)α-conotoxin TxIC, demonstrates differential selectivity for the α3ß2 isoform of the nicotinic acetylcholine receptor with maximal inhibition of 96% and an observed IC50 of 5.4 ± 0.5 µM. Interestingly its comparative PD50 (3.6 µMol kg(-1)) in invertebrates was ~100 fold more than that of the native peptide. Differentiating α-conotoxin TxIC from other α-conotoxins is the high degree of post-translational modification (44% of residues). This includes the incorporation of γ-carboxyglutamic acid, two moieties of 4-trans hydroxyproline, two disulfide bond linkages, and C-terminal amidation. These findings expand upon the known chemical diversity of α-conotoxins and illustrate a potential driver of toxin phyla-selectivity within Conus.
Assuntos
Caramujo Conus/metabolismo , Venenos de Moluscos/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Cromatografia Líquida de Alta Pressão , Concentração Inibidora 50 , Venenos de Moluscos/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Predatory marine snails of the genus Conus use venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable venom from Conus consors, an indo-pacific fish-hunting cone snail. By matching MS/MS data against an extensive set of venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7 kDa) and two proteins in the 25 and 50 kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone snail venom to date, and delineate the major protein families present in the delivered venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery.
Assuntos
Conotoxinas/isolamento & purificação , Caramujo Conus/química , Descoberta de Drogas/métodos , Perfilação da Expressão Gênica/métodos , Venenos de Moluscos/química , Proteômica/métodos , Sequência de Aminoácidos , Animais , Técnicas de Química Combinatória , Conotoxinas/administração & dosagem , Conotoxinas/química , Conotoxinas/genética , Caramujo Conus/genética , Caramujo Conus/metabolismo , Caramujo Conus/patogenicidade , Ensaios de Triagem em Larga Escala , Injeções , Dados de Sequência Molecular , Venenos de Moluscos/análise , Venenos de Moluscos/genética , Venenos de Moluscos/metabolismo , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Transcriptoma/fisiologiaRESUMO
A multidisciplinary strategy for discovery of new Conus venom peptides combines molecular genetics and phylogenetics with peptide chemistry and neuropharmacology. Here we describe application of this approach to the conantokin family of conopeptides targeting NMDA receptors. A new conantokin from Conus rolani, ConRl-A, was identified using molecular phylogeny and subsequently synthesized and functionally characterized. ConRl-A is a 24-residue peptide containing three gamma-carboxyglutamic acid residues with a number of unique sequence features compared to conantokins previously characterized. The HPLC elution of ConRl-A suggested that this peptide exists as two distinct, slowly exchanging conformers. ConRl-A is predominantly helical (estimated helicity of 50%), both in the presence and absence of Ca(++). The order of potency for blocking the four NMDA receptor subtypes by ConRl-A was NR2B > NR2D > NR2A > NR2C. This peptide has a greater discrimination between NR2B and NR2C than any other ligand reported so far. In summary, ConRl-A is a new member of the conantokin family that expands our understanding of structure/function of this group of peptidic ligands targeted to NMDA receptors. Thus, incorporating phylogeny in the discovery of novel ligands for the given family of ion channels or receptors is an efficient means of exploring the megadiverse group of peptides from the genus Conus.
Assuntos
Conotoxinas/metabolismo , Caramujo Conus/metabolismo , Peptídeos/metabolismo , Filogenia , Animais , Dicroísmo Circular , Conotoxinas/genética , Caramujo Conus/classificação , DNA Complementar , Eletrofisiologia , Venenos de Moluscos/metabolismo , Oócitos , Peptídeos/genética , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , XenopusRESUMO
Cone snails are carnivorous marine gastropods that have evolved potent venoms to capture their prey. These venoms comprise a rich and diverse cocktail of peptide toxins, or conopeptides, whose high diversity has arisen from an efficient hypermutation mechanism, combined with a high frequency of post-translational modifications. Conopeptides bind with high specificity to distinct membrane receptors, ion channels, and transporters of the central and muscular nervous system. As well as serving their natural function in prey capture, conopeptides have been utilized as versatile tools in neuroscience and have proven valuable as drug leads that target the nervous system in humans. This paper examines current knowledge on conopeptide sequences based on an analysis of gene and peptide sequences in ConoServer (http://www.conoserver.org), a specialized database of conopeptide sequences and three-dimensional structures. We describe updates to the content and organization of ConoServer and discuss correlations between gene superfamilies, cysteine frameworks, pharmacological families targeted by conopeptides, and the phylogeny, habitat, and diet of cone snails. The study identifies gaps in current knowledge of conopeptides and points to potential directions for future research.
Assuntos
Conotoxinas/química , Caramujo Conus/fisiologia , Venenos de Moluscos/química , Venenos/química , Análise de Sequência de Proteína/métodos , Caramujos/genética , Sequência de Aminoácidos , Animais , Conotoxinas/genética , Conotoxinas/metabolismo , Bases de Dados de Proteínas , Dados de Sequência Molecular , Venenos de Moluscos/genética , Venenos de Moluscos/metabolismo , Venenos/classificação , Venenos/metabolismo , Ligação Proteica , Conformação Proteica , Caramujos/classificaçãoRESUMO
NR2B-selective inhibitors show lower side-effects in preclinical pain models than non-selective NMDA receptor (NMDAR) antagonists, but it is unclear whether the improved safety of NR2B-selective inhibitors is due to their subtype selectivity or to a unique mode of inhibition of the receptor. In this study, the analgesic effects of intracerebral bolus injections of conantokin peptides with different NMDAR subunit selectivity were determined in mice by the standard hot-plate test, and following stimuli with acetic acid, formalin and complete Freund's adjuvant (CFA). In the standard hot-plate model, con-G[S16Y], a NR2B-selective inhibitor, showed the highest analgesic activity among conantokin peptides tested. In the acetic acid- and CFA-induced pain models, con-G[S16Y] and, to a lesser extent, con-G exhibited higher analgesic activity compared to non-selective inhibitors, such as con-R[1-17]. In the formalin test, while all conantokin peptides could partially suppress the first phase response, only con-G[S16Y] and con-G significantly inhibited the second phase response and suppressed paw edema. Our results suggest that the antinociceptive action of the conantokins may be related to their NR2B-selectivity and that these peptides may be useful as both neurobiological tools for probing mechanisms of nociception and as therapeutic agents for pain relief.
Assuntos
Analgésicos/metabolismo , Conotoxinas/metabolismo , Antagonistas de Aminoácidos Excitatórios/metabolismo , Venenos de Moluscos/metabolismo , Peptídeos/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Conotoxinas/genética , Feminino , Inflamação/induzido quimicamente , Inflamação/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Dados de Sequência Molecular , Venenos de Moluscos/genética , Atividade Motora/fisiologia , Medição da Dor , Peptídeos/genética , Tempo de Reação , Receptores de N-Metil-D-Aspartato/metabolismo , Teste de Desempenho do Rota-RodRESUMO
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.