Spider Neurotoxins, Short Linear Cationic Peptides and Venom Protein Classification Improved by an Automated Competition between Exhaustive Profile HMM Classifiers.

Spider venoms are rich cocktails of bioactive peptides, proteins, and enzymes that are being intensively investigated over the years. In order to provide a better comprehension of that richness, we propose a three-level family classification system for spider venom components. This classification is supported by an exhaustive set of 219 new profile hidden Markov models (HMMs) able to attribute a given peptide to its precise peptide type, family, and group. The proposed classification has the advantages of being totally independent from variable spider taxonomic names and can easily evolve. In addition to the new classifiers, we introduce and demonstrate the efficiency of hmmcompete, a new standalone tool that monitors HMM-based family classification and, after post-processing the result, reports the best classifier when multiple models produce significant scores towards given peptide queries. The combined used of hmmcompete and the new spider venom component-specific classifiers demonstrated 96% sensitivity to properly classify all known spider toxins from the UniProtKB database. These tools are timely regarding the important classification needs caused by the increasing number of peptides and proteins generated by transcriptomic projects.

Manual classification strategies in the ECOD database.

ECOD (Evolutionary Classification Of protein Domains) is a comprehensive and up-to-date protein structure classification database. The majority of new structures released from the PDB (Protein Data Bank) each week already have close homologs in the ECOD hierarchy and thus can be reliably partitioned into domains and classified by software without manual intervention. However, those proteins that lack confidently detectable homologs require careful analysis by experts. Although many bioinformatics resources rely on expert curation to some degree, specific examples of how this curation occurs and in what cases it is necessary are not always described. Here, we illustrate the manual classification strategy in ECOD by example, focusing on two major issues in protein classification: domain partitioning and the relationship between homology and similarity scores. Most examples show recently released and manually classified PDB structures. We discuss multi-domain proteins, discordance between sequence and structural similarities, difficulties with assessing homology with scores, and integral membrane proteins homologous to soluble proteins. By timely assimilation of newly available structures into its hierarchy, ECOD strives to provide a most accurate and updated view of the protein structure world as a result of combined computational and expert-driven analysis.

Structure of membrane-active toxin from crab spider Heriaeus melloteei suggests parallel evolution of sodium channel gating modifiers in Araneomorphae and Mygalomorphae.

We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called "inhibitor cystine knot" or "knottin" fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 µm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a "membrane access" mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders.

First report of in vitro selection of RNA aptamers targeted to recombinant Loxosceles laeta spider toxins.

BACKGROUND: Loxoscelism is the envenomation caused by the bite of Loxosceles spp. spiders. It entails severe necrotizing skin lesions, sometimes accompanied by systemic reactions and even death. There are no diagnostic means and treatment is mostly palliative. The main toxin, found in several isoforms in the venom, is sphingomyelinase D (SMD), a phospholipase that has been used to generate antibodies intended for medical applications. Nucleic acid aptamers are a promising alternative to antibodies. Aptamers may be isolated from a combinatorial mixture of oligonucleotides by iterative selection of those that bind to the target. In this work, two Loxosceles laeta SMD isoforms, Ll1 and Ll2, were produced in bacteria and used as targets with the aim of identifying RNA aptamers that inhibit sphingomyelinase activity. RESULTS: Six RNA aptamers capable of eliciting partial but statistically significant inhibitions of the sphingomyelinase activity of recombinant SMD-Ll1 and SMD-Ll2 were obtained: four aptamers exert ~17% inhibition of SMD-Ll1, while two aptamers result in ~25% inhibition of SMD-Ll2 and ~18% cross inhibition of SMD-Ll1. CONCLUSIONS: This work is the first attempt to obtain aptamers with therapeutic and diagnostic potential for loxoscelism and provides an initial platform to undertake the development of novel anti Loxosceles venom agents.

Spit and venom from scytodes spiders: a diverse and distinct cocktail.

Spiders from the family Scytodidae have a unique prey capturing technique: they spit a zig-zagged silken glue to tether prey to a surface. Effectiveness of this sticky mixture is based on a combination of contraction and adhesion, trapping prey until the spider immobilizes it by envenomation and then feeds. We identify components expressed in Scytodes thoracica venom glands using combined transcriptomic and proteomic analyses. These include homologues of toxic proteins astacin metalloproteases and potentially toxic proteins including venom allergen, longistatin, and translationally controlled tumor protein (TCTP). We classify 19 distinct groups of candidate peptide toxins; 13 of these were detected in the venom, making up 35% of the proteome. Six have significant similarity to toxins from spider species spanning mygalomorph and nonhaplogyne araneomorph lineages, suggesting their expression in venom is phylogenetically widespread. Twelve peptide toxin groups have homologues in venom gland transcriptomes of other haplogynes. Of the transcripts, approximately 50% encode glycine-rich peptides that may contribute to sticky fibers in Scytodes spit. Fifty-one percent of the identified venom proteome is a family of proteins that is homologous to sequences from Drosophila sp. and Latrodectus hesperus with uncharacterized function. Characterization of these components holds promise for discovering new functional activity.

First report of in vitro selection of RNA aptamers targeted to recombinant Loxosceles laeta spider toxins

BACKGROUND: Loxoscelism is the envenomation caused by the bite of Loxosceles spp. spiders. It entails severe necrotizing skin lesions, sometimes accompanied by systemic reactions and even death. There are no diagnostic means and treatment is mostly palliative. The main toxin, found in several isoforms in the venom, is sphingomyelinase D (SMD), a phospholipase that has been used to generate antibodies intended for medical applications. Nucleic acid aptamers are a promising alternative to antibodies. Aptamers may be isolated from a combinatorial mixture of oligonucleotides by iterative selection of those that bind to the target. In this work, two Loxosceles laeta SMD isoforms, Ll1 and Ll2, were produced in bacteria and used as targets with the aim of identifying RNA aptamers that inhibit sphingomyelinase activity. RESULTS: Six RNA aptamers capable of eliciting partial but statistically significant inhibitions of the sphingomyelinase activity of recombinant SMD-Ll1 and SMD-Ll2 were obtained: four aptamers exert ~17% inhibition of SMD-Ll1, while two aptamers result in ~25% inhibition of SMD-Ll2 and ~18% cross inhibition of SMD-Ll1. CONCLUSIONS: This work is the first attempt to obtain aptamers with therapeutic and diagnostic potential for loxoscelism and provides an initial platform to undertake the development of novel anti Loxoscelesvenom agents.

Unravelling the complex venom landscapes of lethal Australian funnel-web spiders (Hexathelidae: Atracinae) using LC-MALDI-TOF mass spectrometry.

Spider venoms represent vast sources of bioactive molecules whose diversity remains largely unknown. Indeed, only a small subset of species have been studied out of the ~43,000 extant spider species. The present study investigated inter- and intra-species venom complexity in 18 samples collected from a variety of lethal Australian funnel-web spiders (Mygalomorphae: Hexathelidae: Atracinae) using C4 reversed-phase separation coupled to offline MALDI-TOF mass spectrometry (LC-MALDI-TOF MS). An in-depth investigation focusing on four atracine venoms (male Illawarra wisharti, male and female Hadronyche cerberea, and female Hadronyche infensa Toowoomba) revealed, on average, ~800 peptides in female venoms while male venoms contained ~400 peptides, distributed across most HPLC fractions. This is significantly higher than previous estimates of peptide expression in mygalomorph venoms. These venoms also showed distinct intersexual as well as intra- and inter-species variation in peptide masses. Construction of both 3D and 2D contour plots revealed that peptide mass distributions in all 18 venoms were centered around the 3200-5400m/z range and to a lesser extent the 6600-8200m/z range, consistent with previously described hexatoxins. These findings highlight the extensive diversity of peptide toxins in Australian funnel-web spider venoms that that can be exploited as novel therapeutic and biopesticide lead molecules. BIOLOGICAL SIGNIFICANCE: In the present study we describe the complexity of 18 venoms from lethal Australian funnel-web spiders using LC-MALDI-TOF MS. The study includes an in-depth investigation, focusing on four venoms, that revealed the presence of ~800 peptides in female venoms and ~400 peptides in male venoms. This is significantly higher than previous estimates of peptide expression in spider venoms. By constructing both 3D and 2D contour plots we were also able to reveal the distinct intersexual as well as intra- and inter-species variation in venom peptide masses. We show that peptide mass distributions in all 18 venoms were centered around the 3200-5400 m/z range and to a lesser extent the 6600-8200 m/z range, consistent with the small number of previously described hexatoxins from these spiders. These findings highlight the extensive diversity of peptide toxins in Australian funnel-web spider venoms that that can be exploited as novel therapeutic and biopesticide lead molecules. The present study has greatly expanded our understanding of peptide variety and complexity in these lethal mygalomorph spiders. Specifically it highlights both the utility of LC-MALDI-TOF in spider taxonomy and the massive combinatorial peptide libraries that spider venoms offer the pharmaceutical and agrochemical industry.

Molecular evolution of α-latrotoxin, the exceptionally potent vertebrate neurotoxin in black widow spider venom.

Black widow spiders (members of the genus Latrodectus) are widely feared because of their potent neurotoxic venom. α-Latrotoxin is the vertebrate-specific toxin responsible for the dramatic effects of black widow envenomation. The evolution of this toxin is enigmatic because only two α-latrotoxin sequences are known. In this study, ~4 kb α-latrotoxin sequences and their homologs were characterized from a diversity of Latrodectus species, and representatives of Steatoda and Parasteatoda, establishing the wide distribution of latrotoxins across the mega-diverse spider family Theridiidae. Across black widow species, α-latrotoxin shows ≥ 94% nucleotide identity and variability consistent with purifying selection. Multiple codon and branch-specific estimates of the nonsynonymous/synonymous substitution rate ratio also suggest a long history of purifying selection has acted on α-latrotoxin across Latrodectus and Steatoda. However, α-latrotoxin is highly divergent in amino acid sequence between these genera, with 68.7% of protein differences involving non-conservative substitutions, evidence for positive selection on its physiochemical properties and particular codons, and an elevated rate of nonsynonymous substitutions along α-latrotoxin's Latrodectus branch. Such variation likely explains the efficacy of red-back spider, L. hasselti, antivenom in treating bites from other Latrodectus species, and the weaker neurotoxic symptoms associated with Steatoda and Parasteatoda bites. Long-term purifying selection on α-latrotoxin indicates its functional importance in black widow venom, even though vertebrates are a small fraction of their diet. The greater differences between Latrodectus and Steatoda α-latrotoxin, and their relationships to invertebrate-specific latrotoxins, suggest a shift in α-latrotoxin toward increased vertebrate toxicity coincident with the evolution of widow spiders.

Novel class of spider toxin: active principle from the yellow sac spider Cheiracanthium punctorium venom is a unique two-domain polypeptide.

Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a-c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe.

A rational nomenclature for naming peptide toxins from spiders and other venomous animals.

Molecular toxinology research was initially driven by an interest in the small subset of animal toxins that are lethal to humans. However, the realization that many venomous creatures possess a complex repertoire of bioactive peptide toxins with potential pharmaceutical and agrochemical applications has led to an explosion in the number of new peptide toxins being discovered and characterized. Unfortunately, this increased awareness of peptide-toxin diversity has not been matched by the development of a generic nomenclature that enables these toxins to be rationally classified, catalogued, and compared. In this article, we introduce a rational nomenclature that can be applied to the naming of peptide toxins from spiders and other venomous animals.

Cyto-insectotoxins, a novel class of cytolytic and insecticidal peptides from spider venom.

Eight linear cationic peptides with cytolytic and insecticidal activity, designated cyto-insectotoxins (CITs), were identified in Lachesana tarabaevi spider venom. The peptides showed antibiotic activity towards Gram-positive and Gram-negative bacteria at micromolar concentrations as well as toxicity to insects. The primary structures of the toxins were established by direct Edman sequencing in combination with enzymatic and chemical polypeptide degradation and MS. CITs represent a novel class of cytolytic molecules and spider venom toxins. They are the first example of molecules showing equally potent antimicrobial and insecticidal effects. Analysis of L. tarabaevi venom gland expressed sequence tag database revealed the primary structures of the protein precursors; eight peptides homologous with the purified toxins were additionally predicted. CIT precursors share a conventional prepropeptide structure with an acidic prosequence and a processing motif common to most spider toxin precursors. The most abundant peptide, CIT 1a, was chemically synthesized, and its lytic activity on different bacterial strains, human erythrocytes and lymphocytes, insect cells, planar lipid bilayers and lipid vesicles was characterized. The spider L. tarabaevi is suggested to have evolved to rely on a unique set of linear cytolytic toxins, as opposed to the more common disulfide-containing spider neurotoxins.

Insect-selective spider toxins targeting voltage-gated sodium channels.

The voltage-gated sodium (Na(v)) channel is a target for a number of drugs, insecticides and neurotoxins. These bind to at least seven identified neurotoxin binding sites and either block conductance or modulate Na(v) channel gating. A number of peptide neurotoxins from the venoms of araneomorph and mygalomorph spiders have been isolated and characterized and determined to interact with several of these sites. These all conform to an 'inhibitor cystine-knot' motif with structural, but not sequence homology, to a variety of other spider and marine snail toxins. Of these, spider toxins several show phyla-specificity and are being considered as lead compounds for the development of biopesticides. Hainantoxin-I appears to target site-1 to block Na(v) channel conductance. Magi 2 and Tx4(6-1) slow Na(v) channel inactivation via an interaction with site-3. The delta-palutoxins, and most likely mu-agatoxins and curtatoxins, target site-4. However, their action is complex with the mu-agatoxins causing a hyperpolarizing shift in the voltage-dependence of activation, an action analogous to scorpion beta-toxins, but with both delta-palutoxins and mu-agatoxins slowing Na(v) channel inactivation, a site-3-like action. In addition, several other spider neurotoxins, such as delta-atracotoxins, are known to target both insect and vertebrate Na(v) channels most likely as a result of the conserved structures within domains of voltage-gated ion channels across phyla. These toxins may provide tools to establish the molecular determinants of invertebrate selectivity. These studies are being greatly assisted by the determination of the pharmacophore of these toxins, but without precise identification of their binding site and mode of action their potential in the above areas remains underdeveloped.

Role of IgG(T) and IgGa isotypes obtained from arachnidic antivenom to neutralize toxic activities of Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus venoms

The ability of IgG(T) and IgGa subclasses—isolated by liquid chromatography from equine arachnidic antivenom (AAV)—to neutralize toxic activities of Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus venoms as well as to remove venom toxins from circulation was investigated. These subclasses showed similar antibody titers against L. gaucho, P. nigriventer and T. serrulatus venoms, and by immunoblotting few differences were observed in the recognition pattern of venom antigens. IgG(T) and IgGa neutralized 100% lethality induced by L. gaucho and 50% of P. nigriventer venom, but IgGa failed to neutralize T. serrulatus venom, in contrast to IgG(T). Both subclasses neutralized local reactions and dermonecrosis induced by L. gaucho venom in rabbits. In mice, IgG(T) and IgGa partially neutralized the edematogenic activity induced by P. nigriventer and T. serrulatus venoms, but only IgG(T) neutralized (ca. 81%) the nociceptive activity induced by T. serrulatus venom. Both subclasses failed to neutralize nociceptive activity induced by P. nigriventer venom. IgG(T) reduced the serum venom levels of animals injected with L. gaucho, P. nigriventer or T. serrulatus venoms, while IgGa solely reduced L. gaucho and P. nigriventer venoms levels. Our results demostrate that IgG(T) and IgGa subclasses neutralize toxic activities induced by P. nigriventer, T. serrulatus and L. gaucho venoms with different efficacies, as well as depurate these venoms from circulation.

Classification of spider neurotoxins using structural motifs by primary structure features. Single residue distribution analysis and pattern analysis techniques.

In recent years the data on the novel structures of spider toxins have been greatly increasing. The sequence data should be classified. We introduced two primary structure analysis techniques-single residue distribution analysis (SRDA) and pattern analysis for classifying spider polypeptide toxins with molecular weight less than 10kDa. For multiple sequence alignment, we also introduced three novel sequence representation formats named as a simple record, motif record and a pattern record, which can be useful for large-scale analysis of structures. About 300 sequences of spider toxins were analyzed and nine primary structure motifs were identified. New classification of spider toxins was proposed on the basis of previously described principal structural motif (PSM) and extra structural motif (ESM) [Kozlov, S.A., Malyavka, A.A., McCutchen, B., Lu, A., Schepers, E., Herrmann, R., Grishin, E.V., 2005. A novel strategy for the identification of toxin-like structures in spider venom. Proteins 59 (1), 131-140]. Five main structural classes were revealed, and for putative ion channel inhibitors from the most numerous classes 1, 2, and 3, five-digital personal ID numbers were introduced. A reference table with simple, motif and pattern representation sequence formats was created for all analyzed structures.

[Spider bites: araneidism of medical importance]. / Morsures d'araignées: les aranéismes d'importance médicale.

LIMITED RISKS: Although most species of spiders are venomous, only ten or so are able to induce human envenomations. From a systematic point of view, it is possible to distinguish the araneomorph spiders - or "true" spiders - from the mygalomorph spiders. Dangerous species for humans can be found in both groups. Regarding "true' spiders, two kinds of envenomation are frequent, ubiquitous and potentially severe: latrodectism (neurotoxic symptomatology) due to the Widow spiders of the Latrodectus species,and loxoscelism (viscero-cutaneous symptomatology). Regarding the mygalomorph spiders, the Australian species responsible for atraxism (neurotoxic symptomatology) are considered as the most dangerous. Most of the other mygalomorph spiders, when they bite, only provoke benign loco regional problems. A supplementary defensive weapon exists in certain South-American species: urticating hairs which may induce severe ocular damage.

Agatoxins: ion channel specific toxins from the American funnel web spider, Agelenopsis aperta.

Agatoxins from Agelenopsis aperta venom target three classes of ion channels, including transmitter-activated cation channels, voltage-activated sodium channels, and voltage-activated calcium channels. The alpha-agatoxins are non-competitive, use-dependent antagonists of glutamate receptor channels, and produce rapid but reversible paralysis in insect prey. Their actions are facilitated by the micro-agatoxins, which shift voltage-dependent activation of neuronal sodium channels to more negative potentials, causing spontaneous transmitter release and repetitive action potentials. The omega-agatoxins target neuronal calcium channels, modifying their properties in distinct ways, either through gating modification (omega-Aga-IVA) or by reduction of unitary current (omega-Aga-IIIA). The alpha-agatoxins and omega-agatoxins modify both insect and vertebrate ion channels, while the micro-agatoxins are selective for insect channels. Agatoxins have been used as selective pharmacological probes for characterization of ion channels in the brain and heart, and have been evaluated as candidate biopesticides.

Structure determination of an organometallic 1-(diazenylaryl)ethanol: a novel toxin subclass from the web of the spider Nephila clavipes.

A novel chemical subclass of toxin, [1-(3-diazenylphenyl)ethanol]iron, was identified among the compounds present in the web of the spider Nephila clavipes. This type of compound is not common among natural products, mainly in spider-venom toxins; it was shown to be a potent paralytic and/or lethal toxin applied by the spider over its web to ensure prey capture only by topical application. The structure was elucidated by means of ESI mass spectrometry, 1H-NMR spectroscopy, high-resolution (HR) mass spectrometry, and ICP spectrometry. The structure of [1-(3-diazenylphenyl)ethanol]iron and the study of its insecticidal action may be used as a starting point for the development of new drugs for pest control in agriculture.

Pharmacologically active spider peptide toxins.

Advances in mass spectrometry and peptide biochemistry coupled to modern methods in electrophysiology have permitted the isolation and identification of numerous novel peptide toxins from animal venoms in recent years. These advances have also opened up the field of spider venom research, previously unexplored due to methodological limitations. Many peptide toxins from spider venoms share structural features, amino acid composition and consensus sequences that allow them to interact with related classes of cellular receptors. They have become increasingly useful agents for the study of voltage-sensitive and ligand-gated ion channels and the discrimination of their cellular subtypes. Spider peptide toxins have also been recognized as useful agents for their antimicrobial properties and the study of pore formation in cell membranes. Spider peptide toxins with nanomolar affinities for their receptors are thus promising pharmacological tools for understanding the physiological role of ion channels and as leads for the development of novel therapeutic agents and strategies for ion channel-related diseases. Their high insecticidal potency can also make them useful probes for the discovery of novel insecticide targets in the insect nervous system or for the development of genetically engineered microbial pesticides.

PhTx4, a new class of toxins from Phoneutria nigriventer spider venom, inhibits the glutamate uptake in rat brain synaptosomes.

We report the characterization of a new class of glutamate uptake inhibitors isolated from Phoneutria nigriventer venom. Glutamate transport activity was assayed in rat cerebrocortical synaptosomes by using [(3)H]-L-glutamate. PhTx4 inhibited glutamate uptake in a dose dependent manner. The IC(50) value obtained was 2.35+/-0.9 microg/ml which is in the observed range reported for glutamate uptake blockers. Tx4-7, one of PhTx4 toxins, showed the strongest inhibitory activity (50.3+/-0.69%, n=3).