Venomic study on cone snails (Conus spp.) from South Africa

From six Conus species (Conus coronatus, Conus lividus, Conus mozambicus f. lautus, Conus pictus, Conus sazanka, Conus tinianus) collected off the eastern coast of South Africa the venoms were analyzed using MALDI-TOF mass spectrometry. Between 56 and 151 molecular masses most in a range of 1000 to 2500 Da, were identified. Among the six venoms, between 0 and 27% (C. coronatus versus C. sazanka) of the peptide masses were found to be similar. In a study on venoms from 6 Conus species collected in the Philippines, the percentage of identical masses was between none and 9% only. The venoms from the South African Conus species antagonized the rat neuronal nicotinic acetylcholine receptors (nAChRs) α3β2, α4β2, and α7, except for C. coronatus venom that blocked the α4β2 and α7 nAChRs only. HPLC-fractionation of C. tinianus venom led to the isolation of a peptide that is active on all three receptor subtypes. It consists of 16 amino acid residues cross-linked by two disulfide bridges as revealed by de novo sequencing using tandem mass spectrometry: GGCCSHPACQNNPDYC. Posttranslational modifications include C-terminal amidation and tyrosine sulfation. The new peptide is a member of the α-conotoxin family that are competitive antagonists of nAChRs. Phylogenetic analysis of the 16S RNA from numerous Conus species has clarified the evolutionary position of endemic South African Conus species and provided the first evidence for their close genetic relationships.     

The effects of crude venoms of Conus magus and Conus striatus on the contractile response and electrical activity of guinea-pig cardiac musculature.

R. Endean, P. Gyr and J. Surridge. The effects of crude venoms of Conus magus and Conus striatus on the contractile response and electrical activity of guinea-pig cardiac musculature. Toxicon17, 381–395, 1979.—Positive inotropic activity of long duration elicited in atrial or ventricular musculature by the venom of Conus striatus or that of C. magus can be antagonized by manganese, lanthanum, verapamil or tetrodotoxin. The block of ventricular contractility by each of these four blocking agents can be overcome by either venom as can the block of atrial contractility by tetrodotoxin or lanthanum. Positive inotropic activity similar to that in non-reserpinized guinea-pigs was produced in ventricular musculature from reserpinized guinea-pigs by standard doses of either venom but atrial musculature from reserpinized guinea-pigs required increased amounts of the venoms before such activity was exhibited. Although the crude venoms did not affect resting potentials they caused marked increases in overshoot and overall duration of action potentials in both atrial and ventricular musculature. The abolishing, by manganese and lanthanum, of the plateau of the action potentials in atrial musculature was reversed by C. striatus venom but not C. magus. The venom of C. magus, but not C. striatus, antagonizes the depressant effect of tetrodotoxin on the rate of rise and overshoot of the action potential of atrial or ventricular musculature.     

The contryphans,a d-trvptophan-containing family of Conus peptides: interconversion between conformers

We previously characterized contryphan-R, a d -tryptophan-containing octapeptide from the venom of Conus radiatus. In this study, we present evidence that the contryphan family of peptides is widely distributed in venoms of the fish-hunting cone snails. We purified, synthesized and characterized contryphan-Sm from Conus stercusmuscarum venom, and obtained molecular evidence for the existence of a third peptide, contryphan-P from Conus purpurascens venom ducts. The sequences of these three contryphans showed identity in seven of eight amino acids and a conserved pattern of post-translational modification. We also demonstrate that contryphan-Sm equilibrates between two distinct conformational states.     

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.     

Isolation and characterization of three hemorrhagic factors from the venom of Vipera palaestinae

Three hemorrhagic factors were purified from the venom of Vipera palaestinae by gel filtration on Sephadex G-75, ammonium sulfate precipitation and DEAE-cellulose chromatography. One hemorrhagic factor is basic (HR1), one is weakly acidic and one is strongly acidic. All these factors have a similar mol. wt of about 60,000 and each of them shows one precipitin line in immunoelectrophoresis. HR1 and HR2 show both gelatinase and caseinase activity; HR3 has no detectable proteolytic activity. The basic hemorrhagic principle is a glycoprotein (positive PAS staining). Together with the weekly acidic factor it accounts for most of the hemorrhagic activity; the specific activity of each of these two components is 10 times higher than that of the crude venom.     

Purification and characterization of a naturally occurring antihemorrhagic factor in the serum of the hispid cotton rat (Sigmodon hispidus)

The serum of the cotton rat contains a factor which neutralizes the hemorrhagic activity of Crotalus atrox venom. The antihemorrhagic factor was purified from S. hispidus by DEAE-Sephadex and flat-bed isoelectrofocusing. In the purified fraction there was one band as determined by disc electrophoresis and one symmetrical peak by ultracentrifugation. The purified antihemorrhagic factor had no gelatinase or caseinase activity and was stable at pH 3–10. The neutralization capacity of the purified preparation was approximately 20 times that of crude serum. The molecular weight was near 90,000 and the isoelectric point was 5·4. Neither the purified factor nor crude serum formed a precipitin line with C. atrox venom. The results suggest that the antihemorrhagic factor of S. hispidus is probably α-globulin, which has similar characteristics to the antihemorrhagic factors isolated in snake serum.     

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。     

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.     

海南产桶形芋螺毒管cDNA文库构建

目的构建海南产桶形芋螺毒管cDNA文库,为桶形芋螺毒素基因资源的永续保存和新型药物的研发提供依据。方法以总RNA抽提试剂盒,从桶形芋螺毒管中提取总RNA,采用SMART技术,LD-PCR扩增获得双链cDNA,经SfiⅠ酶切和CHROMA SPIN-400柱分级分离后,将500bp以上的片段与载体pDNR-LIB连接,通过电穿孔转入E.coli JM109,获得原始文库;随机挑取单菌落,经菌液PCR鉴定重组率及插入片段大小,最后扩增文库。结果经鉴定,所得文库的容量约为5.0×106个克隆,原始文库的平均滴度为5.01×108,插入片段平均长度为1.0kb,文库的重组率达到92%。结论所构建的文库是合格的,为新型芋螺毒素的发现和研究利用提供了依据。     

Some effects on muscle and nerve of crude venom from the gastropod Conus striatus

The venom of Conus striatus elicits spontaneous activity in nerves and in skeletal muscles. In frog sartorius muscle the venom lowers resting potentials of muscle fibres and triggers action potentials. The action potentials are of smaller amplitude and much longer duration than normal. Skeletal musculature and nerve ultimately become refractory to electrical stimulation if exposed to adequate concentrations of venom. Spontaneous twitches with amplitudes much greater than normal occur in isolated guinea-pig atria exposed to the venom. The venom has a spasmogenic action on the musculature of rat ilea which can be blocked by the 5-hydroxytryptamine antagonist 2-brom-d-lysergic acid diethylamide bitartrate. Active material in the venom is dialysable. The activity of the venom is modified by heating at 100°C. The pharmacological activity of the venom of C. striatus is similar to but not identical with that of the venom of the piscivorous species C. magus.     

A toxin from the venom of the marine snail Conus geographus which acts on the vertebrate central nervous system

A toxin from the venom of the marine snail, Conus geographus Linne, has been purified to homogeneity and characterized. The toxin is a heat stable acidic protein with an apparent monomer mol. wt of 13,000. It has no detectable toxicity to mice on i.p. injection, but is a potent convulsant following intracerebral injection. Thus, it is distinct from other toxins in the venom which act on the peripheral neuromuscular system.     

Recombinant expression and insecticidal properties of a Conus ventricosus conotoxin-GST fusion protein

A novel conotoxin, conotoxin Vn2, was recently isolated from the venom of Conus ventricosus, a worm-hunting cone snail species living in the Mediterranean Sea. Analysis of conotoxin Vn2 amino acid sequence suggested that it is a member of the O1 superfamily of conotoxins. Conotoxin Vn2 displays quite a high degree of sequence similarity with bioactive peptides targeting calcium channels and in particular with the ω conotoxin PnVIB, extracted from the venom of the molluscivorous cone snail Conus pennaceus. In this work we describe the development of a heterologous expression system to obtain a glutathione-S-transferase (GST) fusion product of conotoxin Vn2 in a pure form and in a sufficient amount to characterize its bioactivity. The fusion product has been expressed in recombinant form in Escherichia coli cells, purified, and its neurotoxic activity has been assayed on the larvae of the moth Galleria mellonella, a simple experimental model to test the toxicity of compounds in insects. Moreover the conotoxin Vn2 Asp2His mutant has been produced to analyse the role of this aspartic acid residue in the toxin bioactivity, as an acidic amino acid is conserved in this position in all the O1 superfamily C. ventricosus conotoxins. Results obtained indicate that indeed conotoxin Vn2 has strong insecticidal properties at a dose of only 100 pmol/g of body weight. Surprisingly, mutation of Asp2 to His leads to enhanced toxicity in the larvae model system opening up interesting possibilities for the use of conotoxin Vn2 variants in environmental friendly crop protection applications.     

Some effects of crude venom from the cones Conus striatus and Conus magus on isolated guinea-pig atria.

Long-lasting positive inotropic activity is shown by the venoms of Conus striatus and C. magus. The activity persists after repeated and prolonged washout but is blocked by prior β-adrenoceptor blockade with propranolol and also by the 5-hydroxytryptamine antagonist BOL-148. The activity is not exerted on atria from reserpinized animals. It is suggested that the activity stems from an ability of each venom to release monoamines from cardiac tissues. In addition to their positive inotropic activity the venoms also induce spontaneous contractions in isolated atria. These contractions are not blocked by propranolol or BOL-148.     

Determination of sphingomyelinase-D activity of Loxosceles venoms in sphingomyelin/cholesterol liposomes containing horseradish peroxidase

Based on degradation of sphingomyelin/cholesterol liposomes containing entrapped horseradish peroxidase, we evaluated the Sphingomyelinase-D (SMase-D) activity of scorpion, spider and snake venoms by monitoring spectrophotometrically the product of oxidation of HRP released. The results indicate that Loxosceles crude venoms (Loxosceles intermedia, Loxosceles laeta, Loxosceles gaucho and Loxosceles similis) displayed SMase-D activity in a concentration-dependent manner. Furthermore, this activity was blocked by the anti-loxoscelic antivenom. However, Tityus serrulatus scorpion venom, Phoneutria nigriventer spider venom and Bothrops jararaca, Crotalus durissus, Lachesis muta and Micrurus frontalis snake venoms did not show measurable SMase-D activity.     

The role of the fibrinolytic enzyme system in the haemostatic defects following snake envenomation

The effect of five Egyptian snake venoms on fibrinolysis both in vitro and in vivo were studied. The viper venoms C. cerastes, C. vipera and E. carinatus lysed fibrin in vitro in both unheated and heated fibrin-agar plates, although this effect was markedly reduced on heated plates. Insignificant fibrin lysis was produced by the cobra venom N. nigricollis and N. haje. The three viper venoms produced lysis of fibrinogen on incubation with the purified material, whereas the cobra venoms produced fibrinogenolysis only after 30–90 min incubation periods.Rabbits treated with lethal doses of N. nigricollis venom displayed blood incoagulability due to an antithromboplastic action; no in vivo fibrinogenolysis could be observed. The venom of N. haje produced insignificant changes in blood clotting and fibrinolysis when injected in rabbits. Rabbits injected with C. vipera venom displayed blood hypocoagulability associated with prolonged thrombin clotting times, indicating fibrinolysis. Likewise, injection of C. cerastes venom produced a weak fibrinolytic state associated with blood hypercoagulability. E. carinatus venom caused blood incoagulability and severe hypofibrinogenemia in rabbits as a result of activation of the fibrinolytic system as well as a direct lytic action of the venom on fibrinogen.     

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.     

General biochemical and immunological characteristics of the venom from Peruvian scorpion Hadruroides lunatus

This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD₅₀ determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector.     

Venom of marine snail Conus californicus: biochemical studies of a cholinomimetic component.

An extract from the venom of the marine snail Conus californicus, which has cholinomimetic effects in Mercenaria heart and Aplysia central neurons, was partially purified by gel filtration and preparative silica gel and cellulose thin layer chromatography. Cholinomimetic activity was retained after heating to 100°C for one hour, exposure to pH 2 or pH 10, and incubation with proteases, nucleases, and cholinesterases, and was destroyed by boiling in 1-0 M NaOH or 1-0 M HCl for one hour. Gel filtration, thin layer electrophoresis and analytical cellulose thin layer chromatography using indicator spray reagents indicated that the cholinomimetic is a low molecular weight, positively charged alkaloid compound.