New weak toxins from the cobra venom

A protein with M 7485 Da containing five disulfide bonds was isolated from the venom of cobra Naja oxiana using various types of liquid chromatography. The complete amino acid sequence of the protein was determined by protein chemistry methods, which permitted us to assign it to the group of weak toxins. This is the first weak toxin isolated from the venom of N. oxiana. In a similar way, two new toxins with M 7628 and 7559 Da, which fall into the range of weak toxin masses, were isolated from the venom of the cobra N. kaouthia. The characterization of these proteins using Edman degradation and MALDI mass spectrometry has shown that one of these proteins is a novel weak toxin, and the other is the known weak toxin WTX with an oxidized methionine residue in position 9. Such a modification was detected in weak toxins for the first time. A study of the biological activity of the toxin from N. oxiana showed that, like other weak toxins, it can be bound by α7 and muscle-type nicotinic acetylcholine receptors.     

Snake Venom Cardiotoxins-Structure,Dynamics, Function and Folding

Abstract

Snake cardiotoxins are highly basic (pI>10) small molecular weight (～6.5 kDa), all β-sheet proteins. They exhibit a broad spectrum of interesting biological activities. The secondary structural elements in these toxins include antiparallel double and triple stranded β-sheets. The three dimensional structures of these toxins reveal an unique asymmetric distribution of the hydrophobic and hydrophilic amino acids. The 3D structures of closely related snake venom toxins such as neurotoxins and cardiotoxin-like basic proteins (CLBP) fail to show similar pattern(s) in the distribution of polar and nonpolar residues. Recently, many novel biological activities have been reported for cardiotoxins. However, to-date, there is no clear structure-function correlation(s) available for snake venom cardiotoxins. The aim of this comprehensive review is to summarize and critically evaluate the progress in research on the structure, dynamics, function and folding aspects of snake venom cardiotoxins.     

Proteins toxic to arthropods in the venom of elapid snakes.

It has been found that the lethal action of elapid snake venoms to arthropods (fly larvae and isopods) is due to proteic factors differing from the toxins which are strongly and specifically active on mammals.This conclusion was based on the following: (1) Lack of any correlation between the toxic activity on larvae, isopods, and mice of ten elapid snake venoms. (2) Absence of any toxicity to arthropods in pure toxins isolated and purified from several elapid snake venoms according to their lethality. (3) Electrophoretical separation of the venom of the snake Naja mossambica mossambica (= N. nigricollis mossambica) resulted in fractions active either to arthropods and/or to mice. (4) Separation of the above venom by gel filtration on Sephadex G-50 enabled the isolation of fractions highly toxic to arthropods. (5) The above fractions demonstrated a high phospholipase activity corresponding to about 80 per cent of the total activity of the whole venom. The link between phospholipase and toxicity to arthropods will serve as a target for further investigation.It appears that the phenomenon of diversity in toxic activities of different proteins to different groups of organism, as previously demonstrated in scorpion venoms, is equally shared by elapid snake venoms.     

Evaluation of the physiological activity of venom from the Eurasian water shrew <Emphasis Type="Italic">Neomys fodiens</Emphasis>

Background

Animal toxins can have medical and therapeutic applications. Principally, toxins produced by insects, arachnids, snakes and frogs have been characterized. Venomous mammals are rare, and their venoms have not been comprehensively investigated. Among shrews, only the venom of Blarina brevicauda has been analysed so far, and blarina toxin has been proven to be its main toxic component. It is assumed that Neomys fodiens employs its venom to hunt larger prey. However, the toxic profile, properties and mode of action of its venom are largely unknown. Therefore, we analysed the cardio-, myo- and neurotropic properties of N. fodiens venom and saliva of non-venomous Sorex araneus (control tests) in vitro in physiological bioassays carried out on two model organisms: beetles and frogs. For the first time, we fractionated N. fodiens venom and S. araneus saliva by performing chromatographic separation. Next, the properties of selected compounds were analysed in cardiotropic bioassays in the Tenebrio molitor heart.

Results

The venom of N. fodiens caused a high decrease in the conduction velocity of the frog sciatic nerve, as well as a significant decrease in the force of frog calf muscle contraction. We also recorded a significant decrease in the frog heart contractile activity. Most of the selected compounds from N. fodiens venom displayed a positive chronotropic effect on the beetle heart. However, one fraction caused a strong decrease in the T. molitor heart contractile activity coupled with a reversible cardiac arrest. We did not observe any responses of the insect heart and frog organs to the saliva of S. araneus. Preliminary mass spectrometry analysis revealed that calmodulin-like protein, thymosin β-10, hyaluronidase, lysozyme C and phospholipase A2 are present in the venom of N. fodiens, whereas thymosin β4, lysozyme C and β-defensin are present in S. araneus saliva.

Conclusion

Our results showed that N. fodiens venom has stronger paralytic properties and lower cardioinhibitory activity. Therefore, it is highly probable that N. fodiens might use its venom as a prey immobilizing agent. We also confirmed that S. araneus is not a venomous mammal because its saliva did not exhibit any toxic effects.

     

Triacontyl p-coumarate: An inhibitor of snake venom metalloproteinases

Snake venom metalloproteinases (SVMPs) participate in a number of important biological, physiological and pathophysiological processes and are primarily responsible for the local tissue damage characteristic of viperid snake envenomations. The use of medicinal plant extracts as antidotes against animal venoms is an old practice, especially against snake envenomations. Such plants are sources of many pharmacologically active compounds and have been shown to antagonize the effects of some venoms and toxins. The present study explores the activity of triacontyl p-coumarate (PCT), an active compound isolated from root bark of Bombacopsis glabra vegetal extract (Bg), against harmful effects of Bothropoides pauloensis snake venom and isolated toxins (SVMPs or phospholipase A₂). Before inhibition assays, Bg or PCT was incubated with venom or toxins at ratios of 1:1 and 1:5 (w/w; venom or isolated toxins/PCT) for 30 min at 37 °C. Treatment conditions were also assayed to simulate snakebite with PCT inoculated at either the same venom or toxin site. PCT neutralized fibrinogenolytic activity and plasmatic fibrinogen depletion induced by B. pauloensis venom or isolated toxin. PCT also efficiently inhibited the hemorrhagic (3MDH – minimum hemorrhagic dose injected i.d into mice) and myotoxic activities induced by Jararhagin, a metalloproteinase from B. jararaca at 1:5 ratio (toxin: inhibitor, w/w) when it was previously incubated with PCT and injected into mice or when PCT was administered after toxin injection. Docking simulations using data on a metalloproteinase (Neuwiedase) structure suggest that the binding between the protein and the inhibitor occurs mainly in the active site region causing blockade of the enzymatic reaction by displacement of catalytic water. Steric hindrance may also play a role in the mechanism since the PCT hydrophobic tail was found to interact with the loop associated with substrate anchorage. Thus, PCT may provide a alternative to complement ophidian envenomation treatments.     

Astacin-like metalloproteases are a gene family of toxins present in the venom of different species of the brown spider (genus Loxosceles)

Brown spiders have a worldwide distribution, and their venom has a complex composition containing many different molecules. Herein, we report the existence of a family of astacin-like metalloprotease toxins in Loxosceles intermedia venom, as well as in the venom of different species of Loxosceles. Using a cDNA library from the L. intermedia venom gland, we cloned two novel cDNAs encoding astacin-like metalloprotease toxins, LALP2 and LALP3. Using an anti-serum against the previously described astacin-like toxin in L. intermedia venom (LALP1), we detected the presence of immunologically-related toxins in the venoms of L. intermedia, Loxosceles laeta, and Loxosceles gaucho. Zymographic experiments showed gelatinolytic activity of crude venoms of L. intermedia, L. laeta, and L. gaucho (which could be inhibited by the divalent metal chelator 1,10-phenanthroline) at electrophoretic mobilities identical to those reported for immunological cross-reactivity. Moreover, mRNAs extracted from L. laeta and L. gaucho venom glands were screened for astacin-like metalloproteases, and cDNAs obtained using LALP1-specific primers were sequenced, and their deduced amino acid sequences confirmed they were members of the astacin family with the family signatures (HEXXHXXGXXHE and MXY), LALP4 and LALP5, respectively. Sequence comparison of deduced amino acid sequences revealed that LALP2, LALP3, LALP4, and LALP5 are related to the astacin family. This study identified the existence of gene family of astacin-like toxins in the venoms of brown spiders and raises the possibility that these molecules are involved in the deleterious effects triggered by the venom.     

Pharmacokinetics of Naja sumatrana (Equatorial Spitting Cobra) Venom and Its Major Toxins in Experimentally Envenomed Rabbits

Background

The optimization of snakebite management and the use of antivenom depend greatly on the knowledge of the venom''s composition as well as its pharmacokinetics. To date, however, pharmacokinetic reports on cobra venoms and their toxins are still relatively limited. In the present study, we investigated the pharmacokinetics of Naja sumatrana (Equatorial spitting cobra) venom and its major toxins (phospholipase A₂, neurotoxin and cardiotoxin), following intravenous and intramuscular administration into rabbits.

Principal findings

The serum antigen concentration-time profile of the N. sumatrana venom and its major toxins injected intravenously fitted a two-compartment model of pharmacokinetics. The systemic clearance (91.3 ml/h), terminal phase half-life (13.6 h) and systemic bioavailability (41.9%) of N. sumatrana venom injected intramuscularly were similar to those of N. sputatrix venom determined in an earlier study. The venom neurotoxin and cardiotoxin reached their peak concentrations within 30 min following intramuscular injection, relatively faster than the phospholipase A₂ and whole venom (T_max = 2 h and 1 h, respectively). Rapid absorption of the neurotoxin and cardiotoxin from the injection site into systemic circulation indicates fast onsets of action of these principal toxins that are responsible for the early systemic manifestation of envenoming. The more prominent role of the neurotoxin in N. sumatrana systemic envenoming is further supported by its significantly higher intramuscular bioavailability (F_i.m. = 81.5%) compared to that of the phospholipase A₂ (F_i.m. = 68.6%) or cardiotoxin (F_i.m. = 45.6%). The incomplete absorption of the phospholipase A₂ and cardiotoxin may infer the toxins'' affinities for tissues at the injection site and their pathological roles in local tissue damages through synergistic interactions.

Conclusion/Significance

Our results suggest that the venom neurotoxin is absorbed very rapidly and has the highest bioavailability following intramuscular injection, supporting its role as the principal toxin in systemic envenoming.     

Purification and properties of mammalian toxins from the venom of the Brazilian scorpion Tityus serrulatus Lutz and Mello

The water-soluble part of the dried venom from the scorpion, Tityus serrulatus Lutz and Mello (range, Southeastern Brazil), showed 16 polypeptide bands on polyacrylamide gel electrophoresis. This material exhibited toxic and hyaluronidase activity but no phospholipase, phosphodiesterase, protease, or fibrinolytic activity. Fractionation on glycinamide-treated Sephadex G-50 afforded three protein fractions, which were non-toxic, equitoxic, and three times more toxic than the water-soluble venom. Subsequent separation of the toxic fractions on carboxymethyl-cellulose with phosphate buffers furnished five toxic components, which were further purified on carboxymethyl-cellulose with a salt gradient in acetate buffer. Toxin γ, the major and most basic toxin, is a 62-residue protein that, unlike other scorpion toxins, contains methionine. Automated Edman degradation showed the amino-terminal sequence to be H-Lys-Glu-Gly-Tyr-Leu-Met-Asp-His-Glu-Gly-Cys-Lys-Leu-Ser-Cys-Phe-Ile-Arg-Pro-Ser-Gly-Tyr-Cys-Gly-Arg-Glu-Cys-Gly-Ile-. Toxin γ is the first example of a fifth structural type of mammalian toxin from scorpion venom. Its amino-terminal sequence shows greater homology with toxins similar to Centruroides suffusus suffusus toxin III and Androctonus australis toxin II than with toxins similar to A. australis toxin I or Bhutus occitanus tunetanus toxin I.     

Cloning,expression and characterization of a phospholipase D from Loxosceles gaucho venom gland

Loxosceles venom comprises a mixture of diverse toxins that induces intense local inflammatory reaction, dermonecrotic injury, platelet aggregation, hemolytic anemia and acute renal failure. Among several toxins in the venom, phospholipases D (PLDs), also called dermonecrotic toxins, are the most important and best studied, since they account for the main effects observed in loxoscelism. Despite their importance, biological analysis of PLDs is hampered by the minute amounts normally purified from the venom, and therefore many efforts have been made to clone those toxins. However, to date, no PLD from Loxosceles gaucho has been obtained in a heterologous system. Thus, in this work we show the cloning of a PLD from L. gaucho venom gland, named LgRec1, which was successfully expressed in a bacterial system. LgRec1 evoked local reaction (edema, erythema, ecchymosis, and paleness), dermonecrosis and hemolysis. It was also able to hydrolyze sphingomyelin and promote platelet aggregation. ELISA and Western blot analysis showed that LgRec1 was recognized by an anti-L. gaucho venom serum, a commercial arachnidic antivenom as well as a monoclonal antibody raised against the dermonecrotic fraction of L. gaucho venom. In addition, LgRec1 demonstrated to be highly immunogenic and antibodies raised against this recombinant toxin inhibited local reaction (∼65%) and dermonecrosis (∼100%) elicited by L. gaucho whole venom. Since PLDs are considered the major components accounting for the local and systemic envenomation effects caused by spiders from genus Loxosceles, the information provided here may help to understand the mechanisms behind clinical symptomatology.     

Chironex fleckeri (Box Jellyfish) Venom Proteins: EXPANSION OF A CNIDARIAN TOXIN FAMILY THAT ELICITS VARIABLE CYTOLYTIC AND CARDIOVASCULAR EFFECTS*

The box jellyfish Chironex fleckeri produces extremely potent and rapid-acting venom that is harmful to humans and lethal to prey. Here, we describe the characterization of two C. fleckeri venom proteins, CfTX-A (∼40 kDa) and CfTX-B (∼42 kDa), which were isolated from C. fleckeri venom using size exclusion chromatography and cation exchange chromatography. Full-length cDNA sequences encoding CfTX-A and -B and a third putative toxin, CfTX-Bt, were subsequently retrieved from a C. fleckeri tentacle cDNA library. Bioinformatic analyses revealed that the new toxins belong to a small family of potent cnidarian pore-forming toxins that includes two other C. fleckeri toxins, CfTX-1 and CfTX-2. Phylogenetic inferences from amino acid sequences of the toxin family grouped CfTX-A, -B, and -Bt in a separate clade from CfTX-1 and -2, suggesting that the C. fleckeri toxins have diversified structurally and functionally during evolution. Comparative bioactivity assays revealed that CfTX-1/2 (25 μg kg⁻¹) caused profound effects on the cardiovascular system of anesthetized rats, whereas CfTX-A/B elicited only minor effects at the same dose. Conversely, the hemolytic activity of CfTX-A/B (HU₅₀ = 5 ng ml⁻¹) was at least 30 times greater than that of CfTX-1/2. Structural homology between the cubozoan toxins and insecticidal three-domain Cry toxins (δ-endotoxins) suggests that the toxins have a similar pore-forming mechanism of action involving α-helices of the N-terminal domain, whereas structural diversification among toxin members may modulate target specificity. Expansion of the cnidarian toxin family therefore provides new insights into the evolutionary diversification of box jellyfish toxins from a structural and functional perspective.     

The First Recombinant Viper Three-Finger Toxins: Inhibition of Muscle and Neuronal Nicotinic Acetylcholine Receptors

Genes encoding two three-finger toxins TFT-AF and TFT-VN, nucleotide sequences of which were earlier determined by cloning cDNA from venom glands of vipers Azemiops feae and Vipera nikolskii, respectively, were expressed for the first time in E. coli cells. The biological activity of these toxins was studied by electrophysiological techniques, calcium imaging, and radioligand analysis. It was shown for the first time that viper three-finger toxins are antagonists of nicotinic acetylcholine receptors of neuronal and muscle type.     

Action of different toxins from the scorpion Androctonus australis on a locust nerve-muscle preparation

The neuromuscular effects of four purified toxins and crude venom from the scorpion Androctonus australis were investigated in the extensor tibiae nerve-muscle preparation of the locust Locusta migratoria. Insect and crustacean toxin and the mammal toxins I and II which have previously been shown to act on fly larvae, isopods, and mice all paralyse locust larvae. The paralytic potencies decrease in the following order: insect toxin → mammal toxin I → crustacean toxin → mammal toxin II.The toxins and crude venom cause repetitive activity of the motor axons. This leads to long spontaneous trains of junction potentials in the case of crude venom and insect toxin. The other toxins chiefly cause short bursts of action and junction potentials following single stimuli.The ‘slow’ excitatory motor axon invariably is affected sooner than the inhibitory or the ‘fast’ excitatory one. The minimal doses of toxins required to affect the ‘slow’ motor axon decrease in an order somewhat different from that established for their paralytic potencies: insect toxin → crustacean toxin → mammal toxin I → mammal toxin II.Crude venom depolarises and destabilises the muscle membrane potential at low doses. At high doses it decreases the membrane resistance, whereas insect toxin leads to an increase.Crude venom and insect toxin enhance the frequency of mejps, whereas mammal toxin I leads to the occurrence of ‘giant’ mejps.The pattern of axonal activities indicates that the various peripheral branches of the motor nerve are the primary target of the toxins.The time course of nerve action potentials is affected by mammal toxin I and crustacean toxin which cause anomalous shapes and prolongations not caused by insect toxin.The results with other animals suggest that only the insect toxin is selective in its activity. The way it affects the axon might be quite different from that previously reported for scorpion venoms or toxins.     

Protein toxins of the Echis coloratus viper venom directly activate TRPV1

BackgroundPeptide and protein toxins are essential tools to dissect and probe the biology of their target receptors. Venoms target vital physiological processes to evoke pain. Snake venoms contain various factors with the ability to evoke, enhance and sustain pain sensation. While a number of venom-derived toxins were shown to directly target TRPV1 channels expressed on somatosensory nerve terminals to evoke pain response, such toxins were yet to be identified in snake venoms.MethodsWe screened Echis coloratus saw-scaled viper venom's protein fractions isolated by reversed phase HPLC for their ability to activate TRPV1 channels. To this end, we employed heterologous systems to analyze TRPV1 and NGF pathways by imaging and electrophysiology, combined with molecular biology, biochemical, and pharmacological tools.ResultsWe identified TRPV1 activating proteins in the venom of Echis coloratus that produce a channel-dependent increase in intracellular calcium and outwardly rectifying currents in neurons and heterologous systems. Interestingly, channel activation was not mediated by any of its known toxin binding sites. Moreover, although NGF neurotropic activity was detected in this venom, TRPV1 activation was independent of NGF receptors.ConclusionsEchis coloratus venom contains proteins with the ability to directly activate TRPV1. This activity is independent of the NGF pathway and is not mediated by known TRPV1 toxins' binding sites.General significanceOur results could facilitate the discovery of new toxins targeting TRPV1 to enhance current understanding of this receptor activation mechanism. Furthermore, the findings of this study provide insight into the mechanism through which snakes' venom elicit pain.     

Characterization of Kbot21 Reveals Novel Side Chain Interactions of Scorpion Toxins Inhibiting Voltage-Gated Potassium Channels

Scorpion toxins are important pharmacological tools for probing the physiological roles of ion channels which are involved in many physiological processes and as such have significant therapeutic potential. The discovery of new scorpion toxins with different specificities and affinities is needed to further characterize the physiology of ion channels. In this regard, a new short polypeptide called Kbot21 has been purified to homogeneity from the venom of Buthus occitanus tunetanus scorpion. Kbot21 is structurally related to BmBKTx1 from the venom of the Asian scorpion Buthus martensii Karsch. These two toxins differ by only two residues at position 13 (R /V) and 24 (D/N).Despite their very similar sequences, Kbot21 and BmBKTx1 differ in their electrophysiological activities. Kbot21 targets K_V channel subtypes whereas BmBKTx1 is active on both big conductance (BK) and small conductance (SK) Ca²⁺-activated K⁺ channel subtypes, but has no effects on Kv channel subtypes. The docking model of Kbot21 with the Kv1.2 channel shows that the D24 and R13 side-chain of Kbot21 are critical for its interaction with K_V channels.     

Hemorrhagic principles in the venom of Bitis arietans,a viperous snake. I. Purification and characterization

Two hemorrhagic principles (Bitis arietans hemorrhagin a and b: abbreviated as BHRa and BHRb) were purified from the venom of the viperous snake Bitis arietans (puff adder) by gel filtration, ion-exchange and absorption chromatography. A 10-fold purification was achieved for BHRa and 7-fold for BHRb with an overall yield of 6.4% of hemorrhagic activity. The hemorrhagins were homogeneous according to disc- and SDS-polyacrylamide gel electrophoresis and immunodiffusion. BHRa and BHRb consist of 623 and 685 amino-acid residues and their apparent molecular weights were 68 000 and 75 000, respectively. They were also immunologically distinct. The purified hemorrhagins express proteolytic activity with heat-denatured casein and hide powder azure. The proteolytic activity with heat-denatured casein was almost the same as that of the crude venom, but that with hide powder azure was less than one-tenth of that of the crude venom. The purified hemorrhagins were free of arginine esterase and phospholipase A₂ activities and they are acid labile hemorrhagic toxins. Their hemorrhagic activity was inhibited by EDTA, cysteine and by polyvalent anti-snake serum, but not by phenylmethanesulfonyl fluoride or soybean trypsin inhibitor.     

Comparison of Phylogeny,Venom Composition and Neutralization by Antivenom in Diverse Species of Bothrops Complex

In Latin America, Bothrops snakes account for most snake bites in humans, and the recommended treatment is administration of multispecific Bothrops antivenom (SAB – soro antibotrópico). However, Bothrops snakes are very diverse with regard to their venom composition, which raises the issue of which venoms should be used as immunizing antigens for the production of pan-specific Bothrops antivenoms. In this study, we simultaneously compared the composition and reactivity with SAB of venoms collected from six species of snakes, distributed in pairs from three distinct phylogenetic clades: Bothrops, Bothropoides and Rhinocerophis. We also evaluated the neutralization of Bothrops atrox venom, which is the species responsible for most snake bites in the Amazon region, but not included in the immunization antigen mixture used to produce SAB. Using mass spectrometric and chromatographic approaches, we observed a lack of similarity in protein composition between the venoms from closely related snakes and a high similarity between the venoms of phylogenetically more distant snakes, suggesting little connection between taxonomic position and venom composition. P-III snake venom metalloproteinases (SVMPs) are the most antigenic toxins in the venoms of snakes from the Bothrops complex, whereas class P-I SVMPs, snake venom serine proteinases and phospholipases A₂ reacted with antibodies in lower levels. Low molecular size toxins, such as disintegrins and bradykinin-potentiating peptides, were poorly antigenic. Toxins from the same protein family showed antigenic cross-reactivity among venoms from different species; SAB was efficient in neutralizing the B. atrox venom major toxins. Thus, we suggest that it is possible to obtain pan-specific effective antivenoms for Bothrops envenomations through immunization with venoms from only a few species of snakes, if these venoms contain protein classes that are representative of all species to which the antivenom is targeted.     

Snake venomics and antivenomics of Bothrops atrox venoms from Colombia and the Amazon regions of Brazil,Perú and Ecuador suggest the occurrence of geographic variation of venom phenotype by a trend towards paedomorphism

The venom proteomes of Bothrops atrox from Colombia, Brazil, Ecuador, and Perú were characterized using venomic and antivenomic strategies. Our results evidence the existence of two geographically differentiated venom phenotypes. The venom from Colombia comprises at least 26 different proteins belonging to 9 different groups of toxins. PI-metalloproteinases and K49-PLA₂ molecules represent the most abundant toxins. On the other hand, the venoms from Brazilian, Ecuadorian, and Peruvian B. atrox contain predominantly PIII-metalloproteinases. These toxin profiles correlate with the venom phenotypes of adult and juvenile B. asper from Costa Rica, respectively, suggesting that paedomorphism represented a selective trend during the trans-Amazonian southward expansion of B. atrox through the Andean Corridor. The high degree of crossreactivity of a Costa Rican polyvalent (Bothrops asper, Lachesis stenophrys, Crotalus simus) antivenom against B. atrox venoms further evidenced the close evolutionary kinship between B. asper and B. atrox. This antivenom was more efficient immunodepleting proteins from the venoms of B. atrox from Brazil, Ecuador, and Perú than from Colombia. Such behaviour may be rationalized taking into account the lower content of poorly immunogenic toxins, such as PLA₂ molecules and PI-SVMPs in the paedomorphic venoms. The immunological profile of the Costa Rican antivenom strongly suggests the possibility of using this antivenom for the management of snakebites by B. atrox in Colombia and the Amazon regions of Ecuador, Perú and Brazil.     

Cysteine-rich toxins from Lachesana tarabaevi spider venom with amphiphilic C-terminal segments

Venom of Lachesana tarabaevi (Zodariidae, “ant spiders”) exhibits high insect toxicity and serves a rich source of potential insecticides. Five new peptide toxins active against insects were isolated from the venom by means of liquid chromatography and named latartoxins (LtTx). Complete amino acid sequences of LtTx (60-71 residues) were established by a combination of Edman degradation, mass spectrometry and selective proteolysis. Three toxins have eight cysteine residues that form four intramolecular disulfide bridges, and two other molecules contain an additional cystine; three LtTx are C-terminally amidated. Latartoxins can be allocated to two groups with members similar to CSTX and LSTX toxins from Cupiennius salei (Ctenidae) and Lycosa singoriensis (Lycosidae). The interesting feature of the new toxins is their modular organization: they contain an N-terminal cysteine-rich (knottin or ICK) region as in many neurotoxins from spider venoms and a C-terminal linear part alike some cytolytic peptides. The C-terminal fragment of one of the most abundant toxins LtTx-1a was synthesized and shown to possess membrane-binding activity. It was found to assume amphipathic α-helical conformation in membrane-mimicking environment and exert antimicrobial activity at micromolar concentrations. The tails endow latartoxins with the ability to bind and damage membranes; LtTx show cytolytic activity in fly larvae neuromuscular preparations. We suggest a membrane-dependent mode of action for latartoxins with their C-terminal linear modules acting as anchoring devices.