A novel structural class of toxins: the methionine-rich peptides from the venoms of turrid marine snails (Mollusca, Conoidea).

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.     

疣缟芋螺毒素的二维液相色谱分离方法

目的建立一种新的基于二维液相色谱技术的疣缟芋螺毒素分离方法,了解其毒素组构成特点。方法从疣缟芋螺毒管中提取芋螺毒素总肽,在传统的凝胶色谱和反相色谱方法的基础上,根据芋螺毒素的等电点和疏水性,利用目标蛋白快速分离系统(Proteome Lab TMPF2D),对其进行二维分离。结果经过传统分离方法,能够检测得到40个左右的芋螺毒素条带,且分离效果不明显。而通过二维液相色谱分离方法,pI/UV图谱显示共检测到约200个芋螺毒素条带。结论同传统分离方法相比,采用Proteome LabTMPF2D系统对毒素分离更快速、分辨率更高,因此更利于鉴定芋螺毒素肽组分,也为下游序列结构特征与生物活性研究奠定了良好的基础。     

Conotoxins - new vistas for peptide therapeutics

There are approximately 500 species of predatory cone snails within the genus Conus. They comprise what is arguably the largest single genus of marine animals alive today. It has been estimated that the venom of each Conus species has between 50 and 200 components. These highly constrained sulfur rich components or conotoxins represent a unique arsenal of neuropharmacologically active peptides that have been evolutionarily tailored to afford unprecedented and exquisite selectivity for a wide variety of ion-channel subtypes. Remarkable divergence occurs when cone snails speciate. Consequently, the complement of venom peptides in any one Conus species is distinct from that of any other species. Hence many thousands of peptides that modulate ion channel function are present within Conus venoms. Evolutionary pressures have afforded a "pre-optimized," structurally sophisticated library that has been "fine tuned" over 50 million years. The statistics associated with sampling such libraries bear testimony to the validity and feasibility of this strategy. Although approximately 100 conotoxin sequences have been published in the scientific literature, representing a mere 0.2 % of the estimated library size, this sample has already afforded a peptide of proven clinical utility and several pre-clinical leads for CNS disorders. Conus libraries represent a rich pharmacopoeia and the potential to "therapeutically mine" such a resource appears limitless. The paucity of synthetic methodologies necessary to achieve the regioisomeric folding patterns present in these native peptides precludes access to synthetic conotoxin libraries, further validating the overall "mining" strategy. In this article, we will present a pragmatic overview of the molecular diversity as well as the neurobiological mechanisms that define each major class of conotoxin.     

Drugs from the sea: conopeptides as potential therapeutics

Marine cone snails from the genus Conus are estimated to consist of up to 700 species. These predatory molluscs have devised an efficient venom apparatus that allows them to successfully capture polychaete worms, other molluscs or in some cases fish as their primary food sources. The toxic venom used by the cone shells contains up to 50 different peptides that selectively inhibit the function of ion channels involved in the transmission of nerve signals in animals. Each of the 700 Conus species contains a unique set of peptides in their venom. Across the genus Conus, the conotoxins represent an extensive array of ion channel blockers each showing a high degree of selectivity for particular types of channels. We have undertaken a study of the conotoxins from Australian species of Conus that have the capacity to inhibit specifically the nicotinic acetylcholine receptors in higher animals. These conotoxins have been identified by mass spectroscopy and their peptide sequences in some cases deduced by the application of modern molecular biology to the RNA extracted from venom ducts. The molecular biological approach has proven more powerful than earlier protein/peptide based technique tor the detection of novel conotoxins [1,2]. Novel conotoxins detected in this way have been further screened for their abilities to modify the responses of tissues to pain stimuli as a first step in describing their potential as lead compounds for novel drugs. This review describes the progress made by several research groups to characterise the properties of conopeptides and to use them as drug leads for the development of novel therapeutics for the treatment of a range of neurological conditions.     

Analysis of a cone snail's killer cocktail - The milked venom of Conus geographus

“Snails can kill” is a statement that receives much disbelief. Yet the venom from Conus geographus, as delivered by a disposable hypodermic-like needle, has indeed killed many unsuspecting human victims. Our understanding of their milked venom the essence of these fatalities, is in itself non-existent. Here, we present the molecular mass analysis of the milked venom of C. geographus, providing the first insight into the composition of its deadly cocktail.     

Novel conopeptides of the I-superfamily occur in several clades of cone snails.

The I-superfamily of conotoxins represents a new class of peptides in the venom of some Conus species. These toxins are characterized by four disulfide bridges and inhibit or modify ion channels of nerve cells. When testing venoms from 11 Conus species for a functional characterization, blocking activity on potassium channels (like Kv1.1 and Kv1.3 channels, but not Kv1.2 channels) was detected in the venom of Conus capitaneus, Conus miles, Conus vexillum and Conus virgo. Analysis at the cDNA level of these venoms using primers designed according to the amino acid sequence of a potassium channel blocking toxin (ViTx) from C. virgo confirmed the presence of structurally homologous peptides in these venoms. Moreover, peptides belonging to the I-superfamily, but with divergent amino acid sequences, were found in Conus striatus and Conus imperialis. In all cases, the sequences of the precursors' prepro-regions exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between species. We then performed phylogenetic analyses of new and published mitochondrial 16S rDNA sequences representing 104 haplotypes from these and numerous other Conus species, using Bayesian, maximum-likelihood, maximum-parsimony and neighbor-joining methods of inference. Cone snails known to possess I-superfamily toxins were assigned to five different major clades in all of the resulting gene trees. Moreover, I-superfamily conopeptides were detected both in vermivorous and piscivorous species of Conus, thus demonstrating the widespread presence of such toxins in this speciose genus beyond evolutionary and ecological groups.     

Conotoxins down under.

In the four decades since toxinologists in Australia and elsewhere started to investigate the active constituents of venomous cone snails, a wealth of information has emerged on the various classes of peptides and proteins that make their venoms such potent bioactive cocktails. This article provides an overview of the current state of knowledge of these venom constituents, several of which are of interest as potential human therapeutics as a consequence of their high potency and exquisite target specificity. With the promise of as many as 50,000 venom components across the entire Conus genus, many more interesting peptides can be anticipated.     

New conopeptides of the D-superfamily selectively inhibiting neuronal nicotinic acetylcholine receptors

The venom of cone snails (Conus spp.) is a rich source of peptides exhibiting a wide variety of biological activities. Several of these conopeptides are neuronal nicotinic acetylcholine receptor (nAChR) antagonists and belong to the A-, M-, S-, C and the recently described D-superfamily (αD-conopeptides). Here we describe the discovery and characterization of two αD-conopeptides isolated from the venom of Conus mustelinus and Conus capitaneus. Their primary structure was determined by Edman degradation, MS/MS analysis and by a PCR based approach. These peptides show close structural homology to the αD-VxXIIA, -B and -C conopeptides from the venom of Conus vexillum and are dimers (about 11 kDa) of similar or identical peptides with 49 amino acid residues and a characteristic arrangement of ten conserved cysteine residues. These novel types of conopeptides specifically block neuronal nAChRs of the α7, α3β2 and α4β2 subtypes in nanomolar concentrations. Due to their high affinity, these new ligands may provide a tool to decipher the localisation and function of the various neuronal nAChRs.     

Conorfamide, a Conus venom peptide belonging to the RFamide family of neuropeptides.

A novel Conus peptide, conorfamide-Sr1, has been characterized. The sequence of the natural peptide was determined using standard Edman sequencing methods and mass spectrometry, and confirmed by chemical synthesis. The peptide has 12 amino acids and no cysteine residues. The following sequence was obtained: GPMGWVPVFYRF-NH(2).No other peptide from a vermivorous Atlantic Conus species has previously been characterized. Conorfamide-Sr1 belongs to the RFamide neuropeptide family, and is the first RFamide peptide to be found in any venom. The presence of conorfamide-Sr1 as a major peptide in Conus spurius venom suggests that Conus lineages in the Atlantic may have evolved novel Conus venom peptide families.     

Novel non-enzymatic toxic peptide of Daboia russelii (Eastern region) venom renders commercial polyvalent antivenom ineffective.

The snake venoms are typically complex mixtures of enzymes and non-enzymatic peptides. Regional variation in the non-enzymatic fraction of Russell's viper venom from three regions of India studied. The eastern, western and southern regional venom upon gel permeation chromatography on sephadex-G-75 column resolved into three peaks. All the three overlapping peaks differ in their lethality and enzymatic potency. Peak III of all the regional venom found to be non-enzymatic, Western and southern regional venom has trypsin inhibitory activity with varying potencies. Interestingly, the peak III of eastern region is devoid of trypsin inhibitory activity. But it is highly lethal with a LD50 0.7 mg/kg body weight and also it exhibited post-synaptic neurotoxicity. On the other hand southern and western regional venom's non-enzymatic peak is non-lethal and did not induce neurotoxic symptoms in experimental model. The antibodies developed against the eastern regional venom cross-reacted with the peaks I and II of other regional venom, but failed to cross-react with the peak III of western and southern regional Russell's viper venom. Commercial anti-venom prepared to neutralize the toxic effects of common poisonous snakes of India, showed positive cross-reaction against peaks I, II and III of all three regional venom tested, except peak III of eastern regional venom. Commercial anti-venom neutralized the lethal toxicity of both western and southern regional Russell's viper venom, and failed to neutralize the lethal effects of eastern regional Russell's viper venom.     

A biochemical characterization of the major peptides from the Venom of the giant Neotropical hunting ant Dinoponera australis

Venom from the “false tocandira” Dinoponera australis, a giant Neotropical hunting ant, paralyzes small invertebrate prey and induces a myriad of systemic effects in large vertebrates. HPLC/DAD/MS analyses revealed that the venom has over 75 unique proteinaceous components with a large diversity of properties ranging in size, hydrophobicity, and overall abundance. The six most abundant peptides, demonstrative of this diversity and hereafter referred to as Dinoponeratoxins, were de novo sequenced by exact mass precursor ion selection and Edman degradation. The smallest peptide characterized, Da-1039, is hydrophilic and has similarities to vasoactive peptides like kinin and bombesin. The two largest and most abundant peptides, Da-3105 and Da-3177, have a 92.9% identity in a 28 residue overlap and share ∼50 of their sequence with ponericin G2 (an antimicrobial from another ponerine ant Pachycondyla goeldii). One peptide, Da-1585, is a hydrophilic cleavage product of an amphipathic peptide, Da-2501. The most hydrophobic peptide, Da-1837, is amidated (a PTM observed in one half of the major peptides) and shares homology with poneratoxin, a sodium channel modifier found in the bullet ant Paraponera clavata. This study is the first examination of potential pharmacophores from venom of the genus Dinoponera (Order: Hymenoptera).     

Alterations in sodium channel gating produced by the venom of the marine mollusc Conus striatus.

The action of the venom from the marine mollusc Conus striatus was studied using the voltage-clamp technique on myelinated nerve. Conus venom applied to an isolated node of Ranvier at 1.1 micrograms protein/ml produced repetitive firing of action potentials when the node was depolarized under current-clamp conditions. Venom application produced a leftword (depolarizing) shift in both the peak sodium current-voltage and the permeability-voltage relationships. A concomitant decrease in maximum peak current and permeability also occurred. The time course of sodium current decline (inactivation) was slowed at all voltages by the presence of venom. Venom treatment caused only a slight depolarizing shift (5 mV) in the voltage-dependence of steady-state Na inactivation. The closing to the resting state of previously activated Na channels, "deactivation", was judged from Na "tail" currents following membrane repolarization, and was slowed more than four-fold by venom treatment. The changes in Na channel gating produced by Conus striatus venom can best be described as a stabilization of the open state of the Na channel and a shift in the voltage dependence of the opening of Na channels. The slowing of both inactivation and deactivation of Na channels can be simulated by alterations in the rate constants of a five state Markov model.     

Intersexual variations in Northern (Missulena pruinosa) and Eastern (M. bradleyi) mouse spider venom.

Venoms of both sexes of Australian Northern (Missulena pruinosa) and Eastern (Missulena bradleyi) mouse spiders were studied in order to determine intersexual variations in venom yield, composition and bioactivity. Females of both species yielded more venom than males. High-performance liquid chromatography (HPLC) and mass spectrometry data further indicate a substantial degree of intersexual variation in the venom composition of both species. In a cricket (Acheta domestica) acute toxicity assay, only small intersexual differences were observed, but M. bradleyi venom was found to be considerably more potent than M. pruinosa venom. In the chick biventer cervicis nerve-muscle preparation, male but not female M. bradleyi venom induced large and sustained muscle contractions with fasciculation and decreased twitch height that could be reversed by CSL funnel-web spider antivenom. In contrast, venoms of both sexes of M. pruinosa did not induce significant effects in the chick biventer cervicis nerve-muscle preparation. We therefore conclude that female M. bradleyi venom and venoms from male and female M. pruinosa appear to contain few, if any, orthologs of delta-missulenatoxin-Mb1a, the toxin responsible for the effects of male M. bradleyi venom in vertebrates. These findings are consistent with clinical reports that mouse spiders, particularly species other than male M. bradleyi, do not appear to be a major medical problem in humans.     

Screening for post-translational modifications in conotoxins using liquid chromatography/mass spectrometry: an important component of conotoxin discovery.

Mass spectrometry has emerged as an important technique for conotoxin analysis due to its capacity for selective, sensitive, information-rich analyses. Using liquid chromatography/mass spectrometry, Conus venom can be fractionated and the peptides surveyed for specific post-translational modifications, indicating those toxin components likely to have an important biological function. With Conus striatus and Conus victoriae venom as models, bromination, carboxylation and glycosylation modifications are identified through characteristics such as isotopic distribution and labile losses observed during mass spectrometric analysis. This modification screening approach enables the identification of a C. victoriae bromo-carboxy-conotoxin, designated vc5c, as a candidate for detailed mass spectrometric analysis. Using a cDNA sequence coupled with liquid chromatography/mass spectrometry and nanoelectrospray ionization-ion trap-mass spectrometry, the sequence of vc5c is determined to be ICCYPNXWCCD, where W is 6-bromotryptophan, X is gamma-carboxy glutamate and C is disulfide-linked cysteine. This represents the ninth T-superfamily (-CC-CC- scaffold) toxin that has been isolated from venom and characterized.     

Effects of venom from Conus striatus on the delayed rectifier potassium current of molluscan neurons

The effect of crude venom extracted from venom ducts of Conus striatus on the delayed rectifier potassium current (IK) of the marine mollusc Aplysia californica was studied using voltage clamp techniques. Initial experiments indicated that the venom had phospholipase activity which destroyed the cells. The use of phospholipase inhibitors prevented destruction of the cell and permitted long-term electrophysiological measurements to be made. Application of the venom to unclamped cells caused a dramatic increase in the frequency of action potentials associated with a depolarization of the membrane potential. A broadening of the action potential was also observed. Three separate effects of the venom were observed on IK in voltage clamped cells: an increase in peak current (effect I), a slowing of both the activation and inactivation kinetics (effect K) and a decrease in the peak current (effect D). All three effects were dose dependent and both effects on peak current were greater at more depolarized membrane potentials. The data suggest that the three effects on IK are caused by different components of the venom. Effect D appears to be caused by a heat-labile compound of molecular weight greater than 50,000, effect I by a heat-stable compound of less than 50,000 and effect K by a heat-stable compound of intermediate molecular size.     

Electrophysiological characterization of a novel small peptide from the venom of Conus californicus that targets voltage-gated neuronal Ca channels

Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. californicus has been shown to be distantly related to all Conus species, but showing unusual biological features. We report a novel peptide isolated from C. californicus with a significant inhibitory action over neuronal voltage-gated calcium channels. The new toxin is formed by 13-amino acid residues with two disulfide bonds, whose sequence (NCPAGCRSQGCCM) is strikingly different from regular ω-conotoxins. In the HPLC purification procedure, the venom fraction eluted in the first 10-15 min produced a significant decrease (54% ± 3%) of the Ca²⁺ current in Xenopus laevis oocytes transfected with purified rat-brain mRNA. A specific peptide obtained from the elution at 13 min decreased the Ca²⁺ current in the adult rat dorsal-root ganglion neurons in a primary culture by 34% ± 2%. The cysteine pattern of this peptide corresponds to the framework XVI described for the M-superfamily of conopeptides and is unprecedented among Conus peptides acting on Ca²⁺ channels.     

Characterization of voltage-dependent calcium channel blocking peptides from the venom of the tarantula Grammostola rosea

Voltage-dependent calcium channel blocking peptides were purified and sequenced from the venom of the tarantula, Grammostola rosea. cDNAs encoding the peptide sequences were cloned from the venom gland cDNA library. The electrophysiological effects of the peptides on several types of voltage-dependent calcium channels were evaluated using a Xenopus laevis oocyte expression system. A peptide contained in one of the HPLC peak fractions inhibited P/Q type voltage-dependent calcium channels (Ca_v2.1). The amino acid sequence of this peptide is identical to that of ω-grammotoxin SIA. A peptide from another discrete peak, which is identical to GsAFII except for one tryptophan residue in the C-terminus, inhibited L-type voltage-dependent calcium channels (Ca_v1.2). A novel peptide, named GTx1-15 (Accession number, AB201016), shows 76.5% sequence homology with the sodium channel blocker phrixotoxin 3, however, GTx1-15 preferentially inhibited T-type voltage-dependent calcium channels (Ca_v3.1). In silico secondary and tertiary structure prediction revealed that GTx1-15 and sodium channel blockers such as hainantoxin-IV, phrixotoxin 3, and ceratotoxin 2 show very similar β-strand composition, distribution of Optimal Docking Areas (continuous surface patches likely to be involved in protein-protein interactions), and surface electrostatic potential. These findings suggest that these peptide toxins evolved from common ancestors by gene duplication to maintain surface atmospheres appropriate for interaction with low-voltage-dependent ion channels.     

The neutralizing effect of a polyclonal antibody raised against the N-terminal eighteen-aminoacid residues of birtoxin towards the whole venom of Parabuthus transvaalicus.

Scorpion venom is composed among other things of a large number of neurotoxic peptides affecting all major types of ion channels. The majority of the toxicity of the venom is attributed to the presence of these peptides. In our previous studies using a combination of HPLC and mass spectrometry, we showed that birtoxin like peptides are the major peptidic components of the venom of Parabuthus transvaalicus. These peptides are quite similar to each other differing by only few amino acid residues. In addition they all share a common N-terminus of eighteen amino acid residues. We hypothesize that neutralization of this domain will decrease the toxicity of the whole venom of P. transvaalicus. Polyclonal antibodies against the common N-terminal region of the peptides are generated. Here we show by bioassays that the polyclonal antibodies neutralize the venom of P. transvaalicus in a dose dependent manner and by mass spectrometry and western blotting that these peptides indeed react with the polyclonal antibodies. Previously antibodies generated against a single major toxic component of venom have proven to be an effective strategy for antivenin production. In the case of P. transvaalicus the generated antibody is against the majority of the peptidic fraction due to the presence of several highly similar and highly toxic components in this venom. We show that using the knowledge obtained through biochemical characterization studies it is possible to design very specific antibodies that will be useful for clinical applications against Parabuthus envenomation.