Compared chemical properties of dermonecrotic and lethal toxins from spiders of the genusLoxosceles (Araneae)

Loxosceles spider venom usually causes a typical dermonecrotic lesion in bitten patients, but it may also cause systemic effects that may be lethal. Gel filtration on Sephadex G-100 ofLoxosceles gaucho, L. laeta, orL. intermedia spider venoms resulted in three fractions (A, containing higher molecular mass components, B containing intermediate molecular mass components, and C with lower molecular mass components). The dermonecrotic and lethal activities were detected exclusively in fraction A of all three species. Analysis by SDS-PAGE showed that the major protein contained in fraction A has molecular weight approximately 35 kDa inL. gaucho andL. intermedia, but 32 kDa inL. laeta venom. These toxins were isolated from venoms ofL. gaucho, L. laeta, andL. intermedia by SDS-PAGE followed by blotting to PVDF membrane and sequencing. A database search showed a high level of identity between each toxin and a fragment of theL. reclusa (North American spider) toxin. A multiple sequence alignment of theLoxosceles toxins showed many common identical residues in their N-terminal sequences. Identities ranged from 50.0% (L. gaucho andL. reclusa) to 61.1% (L. intermedia andL. reclusa). The purified toxins were also submitted to capillary electrophoresis peptide mapping afterin situ partial hydrolysis of the blotted samples. The results obtained suggest thatL. intermedia protein is more similar toL. laeta toxin thanL. gaucho toxin and revealed a smaller homology betweenL. intermedia andL gaucho. Altogether these findings suggest that the toxins responsible for most important activities of venoms ofLoxosceles species have a molecular mass of 32–35 kDa and are probably homologous proteins.     

Purification and characterization of loxnecrogin,a dermonecrotic toxin from Loxosceles gaucho brown spider venom

The most common manifestation of Loxosceles spider envenoming is a dermonecrotic lesion at the bite site. Dermonecrotic toxins from Loxosceles gaucho venom were purified and characterized by mass spectrometry (capillary liquid chromatography followed by mass spectrometry detection). Two components were purified: a major one of 31,444 Da, called loxnecrogin A, and a minor one of 31,626 Da, called loxnecrogin B, being probably two isoforms of the toxin. The N-terminal sequence of loxnecrogin A showed similarity with N termini of other sphingomyelinolytic dermonecrotic toxins isolated from venoms of different Loxosceles species. The internal sequences did not present any statistically significant hits in sequence databases searches. However, loxnecrogin A partial sequence showed high similarity to regions of L. intermedia LiD1 recombinant protein sequence, recently described in the literature but not yet deposited in databanks.     

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.     

Proteome analysis of brown spider venom: identification of loxnecrogin isoforms in Loxosceles gaucho venom

Brown spiders of the Loxosceles genus are distributed worldwide. In Brazil, eight species are found in Southern states, where the envenomation by Loxosceles venom (loxoscelism) is a health problem. The mechanism of the dermonecrotic action of Loxosceles venom is not totally understood. Two isoforms of dermonecrotic toxins (loxnecrogins) from L. gaucho venom have been previously purified, and showed sequence similarities to sphingomyelinase. Herein we employed a proteomic approach to obtain a global view of the venom proteome, with a particular interest in the loxnecrogin isoforms' pattern. Proteomic two-dimensional gel electrophoresis maps for L. gaucho, L. intermedia, and L. laeta venoms showed a major protein region (30-35 kDa, pI 3-10), where at least eight loxnecrogin isoforms could be separated and identified. Their characterization used a combined approach composed of Edman chemical sequencing, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and electrospray ionization-quadropole-time of flight tandem mass spectrometry leading to the identification of sphingomyelinases D. The venom was also pre-fractionated by gel filtration on a Superose 12 fast protein liqiud chromatography column, followed by capillary liquid chromatography-mass spectrometry. Eleven possible loxnecrogin isoforms around 30-32 kDa were detected. The identification of dermonecrotic toxin isoforms in L. gaucho venom is an important step towards understanding the physiopathology of the envenomation, leading to improvements in the immunotherapy of loxoscelism.     

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.     

Sphingomyelinase D from Loxosceles laeta Venom Induces the Expression of MMP7 in Human Keratinocytes: Contribution to Dermonecrosis

Envenomation by Loxosceles spider is characterized by the development of dermonecrosis. In previous studies, we have demonstrated that increased expression/secretion of matrix metalloproteinases 2 and 9, induced by Loxosceles intermedia venom Class 2 SMases D (the main toxin in the spider venom), contribute to the development of cutaneous loxoscelism. In the present study we show that the more potent venom containing the Class 1 SMase D from Loxosceles laeta, in addition to increasing the expression/secretion of MMP2 and MMP9, also stimulates the expression of MMP7 (Matrilysin-1), which was associated with keratinocyte cell death. Tetracycline, a matrix metalloproteinase inhibitor, prevented cell death and reduced MMPs expression. Considering that L. laeta venom is more potent at inducing dermonecrosis than L. intermedia venom, our results suggest that MMP7 may play an important role in the severity of dermonecrosis induced by L. laeta spider venom SMase D. In addition, the inhibition of MMPs by e.g. tetracyclines may be considered for the treatment of the cutaneous loxoscelism.     

Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (brown spider) venom gland

Brown spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown spider venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone may produce necrotic lesions, inflammatory response and platelet aggregation. Biochemically, dermonecrotic toxin belongs to a family of toxins with 30-35 kDa characterized as sphingomyelinase-D. Here, employing a cDNA library of Loxosceles intermedia venom gland, we cloned and expressed two recombinant isoforms of the dermonecrotic toxin LiRecDT2 (1062 bp cDNA) and LiRecDT3 (1007 bp cDNA) that encode for signal peptides and complete mature proteins. Phylogenetic tree analysis revealed a structural relationship for these toxins compared to other members of family. Recombinant molecules were expressed as N-terminal His-tag fusion proteins in Escherichia coli and were purified to homogeneity from cell lysates by Ni(2+) chelating chromatography, resulting in proteins of 33.8 kDa for LiRecDT2 and 34.0 kDa for LiRecDT3. Additional evidence for related toxins containing sequence/epitopes identity comes from antigenic cross-reactivity using antibodies against crude venom toxins and antibodies raised with a purified dermonecrotic toxin. Recombinant toxins showed differential functionality in rabbits: LiRecDT2 caused a macroscopic lesion with gravitational spreading upon intradermal injection, while LiRecDT3 evoked transient swelling and erythema upon injection site. Light microscopic analysis of skin biopsies revealed edema, a collection of inflammatory cells in and around blood vessels and a proteinaceous network at the dermis. Moreover, differential functionality for recombinant toxins was also demonstrated by a high sphingomyelinase activity for LiRecDT2 and low activity for LiRecDT3 as well as greater in vitro platelet aggregation and blood vessel permeability induced by LiRecDT2 and residual activity for LiRecDT3. Cloning and expression of two recombinant dermonecrotic toxins demonstrate an intraspecific family of homologous toxins that act in synergism for deleterious activities of the venom and open possibilities for biotechnological applications for recombinant toxins as research tools for understanding the inflammatory response, vascular integrity and platelet aggregation modulators.     

Identification,cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Biotechnological applications of brown spider (Loxosceles genus) venom toxins

Loxoscelism (the term used to define accidents by the bite of brown spiders) has been reported worldwide. Clinical manifestations following brown spider bites are frequently associated with skin degeneration, a massive inflammatory response at the injured region, intravascular hemolysis, platelet aggregation causing thrombocytopenia and renal disturbances. The mechanisms by which the venom exerts its noxious effects are currently under investigation. The whole venom is a complex mixture of toxins enriched with low molecular mass proteins in the range of 5–40 kDa. Toxins including alkaline phosphatase, hyaluronidase, metalloproteases (astacin-like proteases), low molecular mass (5.6–7.9 kDa) insecticidal peptides and phospholipases-D (dermonecrotic toxins) have been identified in the venom. The purpose of the present review is to describe biotechnological applications of whole venom or some toxins, with especial emphasis upon molecular biology findings obtained in the last years.     

Venom of the Brazilian Spider Sicarius ornatus (Araneae,Sicariidae) Contains Active Sphingomyelinase D: Potential for Toxicity after Envenomation

Background

The spider family Sicariidae includes two genera, Sicarius and Loxosceles. Bites by Sicarius are uncommon in humans and, in Brazil, a single report is known of a 17-year old man bitten by a Sicarius species that developed a necrotic lesion similar to that caused by Loxosceles. Envenomation by Loxosceles spiders can result in dermonecrosis and severe ulceration. Sicarius and Loxosceles spider venoms share a common characteristic, i.e., the presence of Sphingomyelinases D (SMase D). We have previously shown that Loxosceles SMase D is the enzyme responsible for the main pathological effects of the venom. Recently, it was demonstrated that Sicarius species from Africa, like Loxosceles spiders from the Americas, present high venom SMase D activity. However, despite the presence of SMase D like proteins in venoms of several New World Sicarius species, they had reduced or no detectable SMase D activity. In order to contribute to a better understanding about the toxicity of New World Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from the Brazilian Sicarius ornatus spider and compare these with venoms from Loxosceles species of medical importance in Brazil.

Methodology/Principal Findings

SDS-PAGE analysis showed variations in the composition of Loxosceles spp. and Sicarius ornatus venoms. Differences in the electrophoretic profiles of male and female venoms were also observed, indicating a possible intraspecific variation in the composition of the venom of Sicarius spider. The major component in all tested venoms had a Mr of 32–35 kDa, which was recognized by antiserum raised against Loxosceles SMases D. Moreover, male and female Sicarius ornatus spiders'' venoms were able to hydrolyze sphingomyelin, thus showing an enzymatic activity similar to that determined for Loxosceles venoms. Sicarius ornatus venoms, as well as Loxosceles venoms, were able to render erythrocytes susceptible to lysis by autologous serum and to induce a significant loss of human keratinocyte cell viability; the female Sicarius ornatus venom was more efficient than male.

Conclusion

We show here, for the first time, that the Brazilian Sicarius ornatus spider contains active Sphingomyelinase D and is able to cause haemolysis and keratinocyte cell death similar to the South American Loxosceles species, harmful effects that are associated with the presence of active SMases D. These results may suggest that envenomation by this Sicarius spider has the potential to cause similar pathological events as that caused by Loxosceles envenomation. Our results also suggest that, in addition to the interspecific differences, intraspecific variations in the venoms composition may play a role in the toxic potential of the New World Sicarius venoms species.     

Extraction and protein component analysis of venom from the dissected venom glands of Latrodectus tredecimguttatus

Black widow spiders (genus Latrodectus) have attracted increasing attention due to frequently reported human injuries caused by them and the potential applications of biologically active components in their venoms. Although a number of studies have described the biological properties and structures of several venomous proteins such as latrotoxins, a comprehensive analysis of protein component of the venom from the spider is not available. We used combinative proteomic strategies to assess the protein components of the crude venom collected from Latrodectus tredecimguttatus by extracting the dissected venom glands. The experiments demonstrated that the crude venom of L. tredecimguttatus has a high abundance of acidic proteins with molecular masses greater than 15 kDa, and the content of proteins and peptides of below 15 kDa is low. 86 unique proteins were identified, part of which were contaminations of cellular components during the extraction, determined in comparison with venom obtained by electrostimulation. Except for members of latrotoxin family that were commonly considered as the primary toxic components of the venom, several other special enzymes and proteins were detected such as protease, phosphatase, lysozyme, inhibitory protein, and so on. These protein components, particularly the proteases, were speculated to play important roles in the action of L. tredecimguttatus venom.     

Spider Venom Toxin Protects Plants from Insect Attack

Many of the toxin proteins, that have been heterogeneously expressed in agricultural crops to provide resistance to insect pests, are too specific or are only mildly effective against the major insect pests. Spider venoms are a complex cocktail of toxins that have evolved specifically to kill insects. Here we show that the ω-ACTX-Hv1a toxin (Hvt), a component of the venom of the Australian funnel web spider (Hadronyche versuta) that is a calcium channel antagonist, retains its biological activity when expressed in a heterologous system. Expressed as a fusion protein in E. coli, the purified toxin fusion immobilized and killed Helicoverpa armigera and Spodoptera littoralis caterpillars when applied topically. Transgenic expression of Hvt in tobacco effectively protected the plants from H. armigera and S. littoralis larvae, with 100% mortality within 48 h. We conclude that the Hvt is an attractive and effective molecule for the transgenic protection of plants from herbivorous insects which should be evaluated further for possible application in agriculture. The authors Sher Afzal Khan and Zahid Mukhtar contributed equally to this work.     

Molecular cloning, heterologous expression and functional characterization of a novel translationally-controlled tumor protein (TCTP) family member from Loxosceles intermedia (brown spider) venom

Envenoming with brown spiders (Loxosceles genus) is common throughout the world. Cutaneous symptoms following spider bite accidents include dermonecrosis, erythema, itching and pain. In some cases, accidents can cause hypersensibility or even allergic reactions. These responses could be associated with histaminergic events, such as an increase in vascular permeability and vasodilatation. A protein that may be related to the effects of spider venom was identified from a previously obtained cDNA library of the L. intermedia venom gland. The amino acid sequence of this protein is homologous to proteins from the TCTP (translationally-controlled tumor protein) family, which are extracellular histamine-releasing factors (HRF) that are associated with the allergic reactions to parasites. Herein, we described the cloning, heterologous expression, purification and functional characterization of a novel member of the TCTP family from the Loxosceles intermedia venom gland. This recombinant protein, named LiRecTCTP, causes edema, enhances vascular permeability and is likely related to the inflammatory activity of the venom. Moreover, LiRecTCTP presents an immunological relationship with mammalian TCTPs.     

The phylogenetic distribution of sphingomyelinase D activity in venoms of Haplogyne spiders

The venoms of Loxosceles spiders cause severe dermonecrotic lesions in human tissues. The venom component sphingomyelinase D (SMD) is a contributor to lesion formation and is unknown elsewhere in the animal kingdom. This study reports comparative analyses of SMD activity and venom composition of select Loxosceles species and representatives of closely related Haplogyne genera. The goal was to identify the phylogenetic group of spiders with SMD and infer the timing of evolutionary origin of this toxin. We also preliminarily characterized variation in molecular masses of venom components in the size range of SMD. SMD activity was detected in all (10) Loxosceles species sampled and two species representing their sister taxon, Sicarius, but not in any other venoms or tissues surveyed. Mass spectrometry analyses indicated that all Loxosceles and Sicarius species surveyed had multiple (at least four to six) molecules in the size range corresponding to known SMD proteins (31-35 kDa), whereas other Haplogynes analyzed had no molecules in this mass range in their venom. This suggests SMD originated in the ancestors of the Loxosceles/Sicarius lineage. These groups of proteins varied in molecular mass across species with North American Loxosceles having 31-32 kDa, African Loxosceles having 32-33.5 kDa and Sicarius having 32-33 kDa molecules.     

Structure and pharmacology of spider venom neurotoxins

Spider venoms are complex mixtures of neurotoxic peptides, proteins and low molecular mass organic molecules. Their neurotoxic activity is due to the interaction of the venom components with cellular receptors, in particular ion channels. Spider venoms have proven to be a rich source of highly specific peptide ligands for selected subtypes of potassium, sodium and calcium channels, and these toxins have been used to elucidate the structure and physiological roles of the channels in excitable and non-excitable cells. Spider peptides show great variability in their pharmacological activity and primary structure but relative homogeneity in their secondary structure. Following diverse molecular evolution mechanisms, and in particular selective hypermutation, short spider peptides appear to have functionally diversified while retaining a conserved molecular scaffold. This paper reviews the composition and pharmacology of spider venoms with emphasis on polypeptide toxin structure, mode of action and molecular evolution.     

Successful refolding and NMR structure of rMagi3: A disulfide‐rich insecticidal spider toxin

The need for molecules with high specificity against noxious insects leads the search towards spider venoms that have evolved highly selective toxins for insect preys. In this respect, spiders as a highly diversified group of almost exclusive insect predators appear to possess infinite potential for the discovery of novel insect‐selective toxins. In 2003, a group of toxins was isolated from the spider Macrothele gigas and the amino acid sequence was reported. We obtained, by molecular biology techniques in a heterologous system, one of these toxins. Purification process was optimized by chromatographic methods to determine the three‐dimensional structure by nuclear magnetic resonance in solution, and, finally, their biological activity was tested. rMagi3 resulted to be a specific insect toxin with no effect on mice.     

Asymmetry of Leg Use During Prey Handling in the Spider Scytodes globula (Scytodidae)

In spitting spiders Scytodes globula collected in the field, anterior leg loss was more frequent on the left than on the right side, possibly as an outcome of predatory episodes. A laboratory study was performed in which intact adult females of S. globula were placed with individuals from three species of recluse spiders, Loxosceles intermedia, L. gaucho, and L. laeta. The frequency of probing touches by spitting spiders to their prey was significantly higher with left leg I than with right leg I. Left leg II and right leg II also differed in touching frequency, but in a less marked way. The results, which may represent the first evidence of behavioral laterality in a spider species, suggest that behavioral asymmetry may be an ancient evolutionary trait.     

Phospholipase D Toxins of Brown Spider Venom Convert Lysophosphatidylcholine and Sphingomyelin to Cyclic Phosphates

Venoms of brown spiders in the genus Loxosceles contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These toxins cleave the substrates sphingomyelin and lysophosphatidylcholine in mammalian tissues, releasing the choline head group. The other products of substrate cleavage have previously been reported to be monoester phospholipids, which would result from substrate hydrolysis. Using ³¹P NMR and mass spectrometry we demonstrate that recombinant toxins, as well as whole venoms from diverse Loxosceles species, exclusively catalyze transphosphatidylation rather than hydrolysis, forming cyclic phosphate products from both major substrates. Cyclic phosphates have vastly different biological properties from their monoester counterparts, and they may be relevant to the pathology of brown spider envenomation.     

Tetanus and Botulinum Toxins Inhibit, and Black Widow Spider Venom Stimulates the Release of Methionine-Enkephalin-Like Material In Vitro

The actions of tetanus toxin, botulinum A toxin, and black widow spider venom on the release of methionine-enkephalin-like immunoreactivity have been studied; a particulate fraction prepared from rat striata was used. Depending on the duration of preincubation, tetanus toxin diminished the release evoked by veratridine (50 microM final concentration), and abolished it at final concentrations between 0.1 and 1 micrograms/ml. Botulinum A toxin was about 10 to 20 times less potent. Heating or pretreatment with antitoxin inactivated the clostridial toxins. The particulate fraction pretreated with V. cholerae neuraminidase retained its toxin sensitivity. Tetanus toxin also depressed the release due to sea anemone toxin II and high K+. Spider venom stimulated the release in a concentration-dependent manner and required the presence of Ca2+; its effects were depressed by tetanus toxin. These results support the view that both clostridial toxins and spider venom act as broad-range presynaptic neurotoxins on peptidergic transmitter systems.