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
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
Marine cone snails are predatory gastropods characterized by a well-developed venom apparatus and highly evolved hunting strategies that utilize toxins to paralyze prey and defend against predators. The venom of each species of cone snail has a large number of pharmacologically active peptides known as conopeptides or conotoxins that are usually unique in each species. Nevertheless, venoms of only very few species have been characterized so far by transcriptomic approaches. In this study, we used transcriptome sequencing technologies and mass spectrometric methods to describe the diversity of venom components expressed by a worm-hunting species, Conus bayani. A total of 82 conotoxin sequences were retrieved from transcriptomic data that contain 54 validated conotoxin sequences clustered into 21 gene superfamilies including divergent gene family, 17 sequences clustered to 6 different conotoxin classes, and 11 conotoxins classified as unassigned gene family. Seven new conotoxin sequences showed unusual cysteine patterns. We were also able to identify 19 peptide sequences using mass spectrometry that completely overlapped with the conotoxin sequences obtained from transcriptome analysis. Importantly, herein we document the presence of 16 proteins that include five post-translational modifying enzymes obtained from transcriptomic data. Our results revealed diverse and novel conopeptides of an unexplored species that could be used extensively in biomedical research due to their therapeutic potentials.
Assuntos
Conotoxinas/genética , Caramujo Conus/genética , Enzimas/genética , Perfilação da Expressão Gênica , Venenos de Moluscos/genética , Peptídeos/genética , Proteômica , Animais , Conotoxinas/metabolismo , Caramujo Conus/enzimologia , Bases de Dados Genéticas , Enzimas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Espectrometria de Massas , Venenos de Moluscos/enzimologia , Peptídeos/metabolismo , Proteoma , TranscriptomaRESUMO
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
Predatory gastropods of the superfamily Conoidea number over 12,000 living species. The evolutionary success of this lineage can be explained by the ability of conoideans to produce complex venoms for hunting, defense, and competitive interactions. Whereas venoms of cone snails (family Conidae) have become increasingly well studied, the venoms of most other conoidean lineages remain largely uncharacterized. In the present study, we present the venom gland transcriptomes of two species of the genus Clavus that belong to the family Drilliidae. Venom gland transcriptomes of two specimens of Clavus canalicularis and two specimens of Clavus davidgilmouri were analyzed, leading to the identification of a total of 1,176 putative venom peptide toxins (drillipeptides). Based on the combined evidence of secretion signal sequence identity, entire precursor similarity search (BLAST), and the orthology inference, putative Clavus toxins were assigned to 158 different gene families. The majority of identified transcripts comprise signal, pro-, mature peptide, and post-regions, with a typically short (<50 amino acids) and cysteine-rich mature peptide region. Thus, drillipeptides are structurally similar to conotoxins. However, convincing homology with known groups of Conus toxins was only detected for very few toxin families. Among these are Clavus counterparts of Conus venom insulins (drillinsulins), porins (drilliporins), and highly diversified lectins (drillilectins). The short size of most drillipeptides and structural similarity to conotoxins were unexpected, given that most related conoidean gastropod families (Terebridae and Turridae) possess longer mature peptide regions. Our findings indicate that, similar to conotoxins, drillipeptides may represent a valuable resource for future pharmacological exploration.
Assuntos
Evolução Biológica , Caramujo Conus/genética , Variação Genética , Venenos de Moluscos/genética , Fragmentos de Peptídeos/genética , Transcriptoma , Animais , FilogeniaRESUMO
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
Animal venoms comprise a diversity of peptide toxins that manipulate molecular targets such as ion channels and receptors, making venom peptides attractive candidates for the development of therapeutics to benefit human health. However, identifying bioactive venom peptides remains a significant challenge. In this review we describe our particular venomics strategy for the discovery, characterization, and optimization of Terebridae venom peptides, teretoxins. Our strategy reflects the scientific path from mollusks to medicine in an integrative sequential approach with the following steps: (1) delimitation of venomous Terebridae lineages through taxonomic and phylogenetic analyses; (2) identification and classification of putative teretoxins through omics methodologies, including genomics, transcriptomics, and proteomics; (3) chemical and recombinant synthesis of promising peptide toxins; (4) structural characterization through experimental and computational methods; (5) determination of teretoxin bioactivity and molecular function through biological assays and computational modeling; (6) optimization of peptide toxin affinity and selectivity to molecular target; and (7) development of strategies for effective delivery of venom peptide therapeutics. While our research focuses on terebrids, the venomics approach outlined here can be applied to the discovery and characterization of peptide toxins from any venomous taxa.
Assuntos
Venenos de Moluscos , Peptídeos , Animais , Descoberta de Drogas , Estrutura Molecular , Moluscos/genética , Venenos de Moluscos/química , Venenos de Moluscos/genética , Venenos de Moluscos/uso terapêutico , Venenos de Moluscos/toxicidade , Peptídeos/química , Peptídeos/genética , Peptídeos/uso terapêutico , Peptídeos/toxicidade , FilogeniaRESUMO
BACKGROUND: Hematophagy arose independently multiple times during metazoan evolution, with several lineages of vampire animals particularly diversified in invertebrates. However, the biochemistry of hematophagy has been studied in a few species of direct medical interest and is still underdeveloped in most invertebrates, as in general is the study of venom toxins. In cone snails, leeches, arthropods and snakes, the strong target specificity of venom toxins uniquely aligns them to industrial and academic pursuits (pharmacological applications, pest control etc.) and provides a biochemical tool for studying biological activities including cell signalling and immunological response. Neogastropod snails (cones, oyster drills etc.) are carnivorous and include active predators, scavengers, grazers on sessile invertebrates and hematophagous parasites; most of them use venoms to efficiently feed. It has been hypothesized that trophic innovations were the main drivers of rapid radiation of Neogastropoda in the late Cretaceous. We present here the first molecular characterization of the alimentary secretion of a non-conoidean neogastropod, Colubraria reticulata. Colubrariids successfully feed on the blood of fishes, throughout the secretion into the host of a complex mixture of anaesthetics and anticoagulants. We used a NGS RNA-Seq approach, integrated with differential expression analyses and custom searches for putative secreted feeding-related proteins, to describe in detail the salivary and mid-oesophageal transcriptomes of this Mediterranean vampire snail, with functional and evolutionary insights on major families of bioactive molecules. RESULTS: A remarkably low level of overlap was observed between the gene expression in the two target tissues, which also contained a high percentage of putatively secreted proteins when compared to the whole body. At least 12 families of feeding-related proteins were identified, including: 1) anaesthetics, such as ShK Toxin-containing proteins and turripeptides (ion-channel blockers), Cysteine-rich secretory proteins (CRISPs), Adenosine Deaminase (ADA); 2) inhibitors of primary haemostasis, such as novel vWFA domain-containing proteins, the Ectonucleotide pyrophosphatase/phosphodiesterase family member 5 (ENPP5) and the wasp Antigen-5; 3) anticoagulants, such as TFPI-like multiple Kunitz-type protease inhibitors, Peptidases S1 (PS1), CAP/ShKT domain-containing proteins, Astacin metalloproteases and Astacin/ShKT domain-containing proteins; 4) additional proteins, such the Angiotensin-Converting Enzyme (ACE: vasopressive) and the cytolytic Porins. CONCLUSIONS: Colubraria feeding physiology seems to involve inhibitors of both primary and secondary haemostasis, anaesthetics, a vasoconstrictive enzyme to reduce feeding time and tissue-degrading proteins such as Porins and Astacins. The complexity of Colubraria venomous cocktail and the divergence from the arsenal of the few neogastropods studied to date (mostly conoideans) suggest that biochemical diversification of neogastropods might be largely underestimated and worth of extensive investigation.
Assuntos
Gastrópodes/química , Perfilação da Expressão Gênica/métodos , Venenos de Moluscos/genética , Análise de Sequência de RNA/métodos , Animais , Bases de Dados Genéticas , Esôfago/metabolismo , Gastrópodes/genética , Especificidade de Órgãos , Glândulas Salivares/metabolismoRESUMO
Venom peptides from predatory organisms are a resource for investigating evolutionary processes such as adaptive radiation or diversification, and exemplify promising targets for biomedical drug development. Terebridae are an understudied lineage of conoidean snails, which also includes cone snails and turrids. Characterization of cone snail venom peptides, conotoxins, has revealed a cocktail of bioactive compounds used to investigate physiological cellular function, predator-prey interactions, and to develop novel therapeutics. However, venom diversity of other conoidean snails remains poorly understood. The present research applies a venomics approach to characterize novel terebrid venom peptides, teretoxins, from the venom gland transcriptomes of Triplostephanus anilis and Terebra subulata. Next-generation sequencing and de novo assembly identified 139 putative teretoxins that were analyzed for the presence of canonical peptide features as identified in conotoxins. To meet the challenges of de novo assembly, multiple approaches for cross validation of findings were performed to achieve reliable assemblies of venom duct transcriptomes and to obtain a robust portrait of Terebridae venom. Phylogenetic methodology was used to identify 14 teretoxin gene superfamilies for the first time, 13 of which are unique to the Terebridae. Additionally, basic local algorithm search tool homology-based searches to venom-related genes and posttranslational modification enzymes identified a convergence of certain venom proteins, such as actinoporin, commonly found in venoms. This research provides novel insights into venom evolution and recruitment in Conoidean predatory marine snails and identifies a plethora of terebrid venom peptides that can be used to investigate fundamental questions pertaining to gene evolution.
Assuntos
Evolução Molecular , Venenos de Moluscos/genética , Caramujos/genética , Animais , Variação Genética , Família Multigênica , Peptídeos/genética , Filogenia , Processamento de Proteína Pós-Traducional , Alinhamento de Sequência , Caramujos/classificação , Caramujos/enzimologia , TranscriptomaRESUMO
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
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
BACKGROUND: The venomous marine gastropods, cone snails (genus Conus), inject prey with a lethal cocktail of conopeptides, small cysteine-rich peptides, each with a high affinity for its molecular target, generally an ion channel, receptor or transporter. Over the last decade, conopeptides have proven indispensable reagents for the study of vertebrate neurotransmission. Conus bullatus belongs to a clade of Conus species called Textilia, whose pharmacology is still poorly characterized. Thus the genomics analyses presented here provide the first step toward a better understanding the enigmatic Textilia clade. RESULTS: We have carried out a sequencing survey of the Conus bullatus genome and venom-duct transcriptome. We find that conopeptides are highly expressed within the venom-duct, and describe an in silico pipeline for their discovery and characterization using RNA-seq data. We have also carried out low-coverage shotgun sequencing of the genome, and have used these data to determine its size, genome-wide base composition, simple repeat, and mobile element densities. CONCLUSIONS: Our results provide the first global view of venom-duct transcription in any cone snail. A notable feature of Conus bullatus venoms is the breadth of A-superfamily peptides expressed in the venom duct, which are unprecedented in their structural diversity. We also find SNP rates within conopeptides are higher compared to the remainder of C. bullatus transcriptome, consistent with the hypothesis that conopeptides are under diversifying selection.
Assuntos
Caramujo Conus/genética , Genoma/genética , Venenos de Moluscos/genética , Peptídeos/genética , Animais , Composição de Bases/genética , Perfilação da Expressão Gênica , Anotação de Sequência MolecularRESUMO
Diversity among Conus toxins mirrors the high species diversity in the Indo-Pacific region, and evolution of both is thought to stem from feeding-niche specialization derived from intra-generic competition. This study focuses on Conus californicus, a phylogenetic outlier endemic to the temperate northeast Pacific. Essentially free of congeneric competitors, it preys on a wider variety of organisms than any other cone snail. Using molecular cloning of cDNAs and mass spectrometry, we examined peptides isolated from venom ducts to elucidate the sequences and post-translational modifications of two eight-cysteine toxins (cal12a and cal12b of type 12 framework) that block voltage-gated Na(+) channels. Based on homology of leader sequence and mode of action, these toxins are related to the O-superfamily, but differ significantly from other members of that group. Six of the eight cysteine residues constitute the canonical framework of O-members, but two additional cysteine residues in the N-terminal region define an O+2 classification within the O-superfamily. Fifteen putative variants of Cal12.1 toxins have been identified by mRNAs that differ primarily in two short hypervariable regions and have been grouped into three subtypes (Cal12.1.1-3). This unique modular variation has not been described for other Conus toxins and suggests recombination as a diversity-generating mechanism. We propose that these toxin isoforms show specificity for similar molecular targets (Na(+) channels) in the many species preyed on by C. californicus and that individualistic utilization of specific toxin isoforms may involve control of gene expression.
Assuntos
Caramujo Conus/química , Venenos de Moluscos/genética , Peptídeos/genética , Bloqueadores dos Canais de Sódio/toxicidade , Animais , Sequência de Bases , California , Clonagem Molecular , Primers do DNA/genética , Eletrofisiologia , Biblioteca Gênica , Espectrometria de Massas , Dados de Sequência Molecular , Venenos de Moluscos/análise , Venenos de Moluscos/classificação , Oceano Pacífico , Peptídeos/análise , Peptídeos/classificação , Análise de Sequência de DNARESUMO
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
Peroxiredoxins (Prxs) play an important role against various oxidative stresses and intra-cellular signal transduction. Peroxiredoxin 6 (PrxVI) was identified from the disk abalone Haliotis discus discus cDNA library and named HdPrxVI. The full length cDNA of HdPrxVI was 1457 bp with a 654 bp open reading frame (ORF) encoding 218 amino acids. The predicted molecular mass and estimated isoelectric point (pI) of HdPrxVI were 24 kDa and 7.3, respectively. The deduced amino acid sequence demonstrated the greatest degree (72.4%) of identity with Crassostrea gigas PrxVI. The conserved peroxidase catalytic center (42PVCTTE47) with a conserved cysteine residue (Cys44) and a catalytic center for PLA2 activity (27GGSWA31) were observed in the sequence, indicating that it is a member of 1-Cys Prx. Real time PCR results revealed that HdPrxVI mRNA is constitutively expressed in all tissues in a tissue-specific manner. During exposure to haemorrhagic septicaemia virus (VHSV), HdPrxVI mRNA transcription was down-regulated in the gill, suggesting that the abalone responded to the viral infection quickly, and HdPrxVI played a physiological role against virus-induced oxidative stress. The purified recombinant HdPrxVI, together with dithiothreitol (DTT), was shown to scavenge H2O2 in human leukemia THP-1 cells and provided protection against H2O2-induced apoptosis.
Assuntos
Antioxidantes/metabolismo , Regulação da Expressão Gênica , Venenos de Moluscos/enzimologia , Venenos de Moluscos/genética , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismo , Sequência de Aminoácidos , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Sequência de Bases , Clonagem Molecular , Fragmentação do DNA/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Estresse Oxidativo/fisiologia , Peroxirredoxina VI/farmacologia , Filogenia , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Alinhamento de SequênciaRESUMO
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
Vasopressins and oxytocins are homologous, ubiquitous and multifunctional peptides present in animals. Conopressins are vasopressin/oxytocin-related peptides that have been found in the venom of cone snails, a genus of marine predatory molluscs that envenom their prey with a complex mixture of neuroactive peptides. In the present paper, we report the purification and characterization of a unique conopressin isolated from the venom of Conus villepinii, a vermivorous cone snail species from the western Atlantic Ocean. This novel peptide, designated gamma-conopressin-vil, has the sequence CLIQDCPgammaG* (gamma is gamma-carboxyglutamate and * is C-terminal amidation). The unique feature of this vasopressin/oxytocin-like peptide is that the eighth residue is gamma-carboxyglutamate instead of a neutral or basic residue; therefore it could not be directly classified into either the vasopressin or the oxytocin peptide families. Nano-NMR spectroscopy of the peptide isolated directly from the cone snails revealed that the native gamma-conopressin-vil undergoes structural changes in the presence of calcium. This suggests that the peptide binds calcium, and the calcium-binding process is mediated by the gamma-carboxyglutamate residue. However, the negatively charged residues in the sequence of gamma-conopressin-vil may mediate calcium binding by a novel mechanism not observed in other peptides of this family.
Assuntos
Ácido 1-Carboxiglutâmico/metabolismo , Caramujo Conus/química , Venenos de Moluscos/química , Ocitocina/análogos & derivados , Peptídeos/química , Vasopressinas/química , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Dados de Sequência Molecular , Venenos de Moluscos/genética , Venenos de Moluscos/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ocitocina/química , Ocitocina/genética , Ocitocina/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Conkunitzin-S1 (Conk-S1) is a 60-residue neurotoxin from the venom of the cone snail Conus striatus that interacts with voltage-gated potassium channels. Conk-S1 shares sequence homology with Kunitz-type proteins but contains only two out of the three highly conserved cysteine bridges, which are typically found in these small, basic protein modules. In this study the three-dimensional structure of Conk-S1 has been solved by multidimensional NMR spectroscopy. The solution structure of recombinant Conk-S1 shows that a Kunitz fold is present, even though one of the highly conserved disulfide cross-links is missing. Introduction of a third, homologous disulfide bond into Conk-S1 results in a functional toxin with similar affinity for Shaker potassium channels. The affinity of Conk-S1 can be enhanced by a pore mutation within the Shaker channel pore indicating an interaction of Conk-S1 with the vestibule of potassium channels.