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Coastal taipan (Oxyuranus scutellatus) envenoming causes life-threatening neuromuscular paralysis in humans. We studied the time period during which antivenom remains effective in preventing and arresting in vitro neuromuscular block caused by taipan venom and taipoxin. Venom showed predominant pre-synaptic neurotoxicity at 3 &micro, g/mL and post-synaptic neurotoxicity at 10 &micro, g/mL. Pre-synaptic neurotoxicity was prevented by addition of Australian polyvalent antivenom before the venom and taipoxin and, reversed when antivenom was added 5 min after venom and taipoxin. Antivenom only partially reversed the neurotoxicity when added 15 min after venom and had no significant effect when added 30 min after venom. In contrast, post-synaptic activity was fully reversed when antivenom was added 30 min after venom. The effect of antivenom on pre-synaptic neuromuscular block was reproduced by washing the bath at similar time intervals for 3 &micro, g/mL, but not for 10 &micro, g/mL. We found an approximate 10&ndash, 15 min time window in which antivenom can prevent pre-synaptic neuromuscular block. This time window is likely to be longer in envenomed patients due to the delay in venom absorption. Similar effectiveness of antivenom and washing with 3 &micro, g/mL venom suggests that antivenom most likely acts by neutralizing pre-synaptic toxins before they interfere with neurotransmission inside the motor nerve terminals.

Intra-specific venom variation has the potential to provide important insights into the evolution of snake venom, but remains a relatively neglected aspect of snake venom studies. We investigated the venom from 13 individual coastal taipans Oxyuranus scutellatus from four localities on the north-east coast of Australia, spanning a distance of 2000km. The intra-specific variation in taipan venom was considerably less than the inter-specific variation between it and the other Australian elapids to which it was compared. The electrophoretic venom profile of O. scutellatus was visually different to six other genera of Australian elapids, but not to its congener inland taipan O. microlepidotus. There was minimal geographical variation in taipan venom, as the intra-population variation exceeded the inter-population variation for enzymatic activity, procoagulant activity, and the abundance of neurotoxins. The pre-synaptic neurotoxin (taipoxin) was more abundant than the post-synaptic neurotoxins (3FTx), with a median of 11.0% (interquartile range (IQR): 9.7% to 18.3%, range: 6.7% to 23.6%) vs. a median of 3.4% (IQR: 0.4% to 6.7%, range: 0% to 8.1%). Three taipan individuals almost completely lacked post-synaptic neurotoxins, which was not associated with geography and occurred within two populations. We found no evidence of sexual dimorphism in taipan venom. Our study provides a basis for evaluating the significance of intra-specific venom variation within a phylogenetic context by comparing it to the inter-specific and inter-generic variation. The considerable intra-population variation we observed supports the use of several unpooled individuals from each population when making inter-specific comparisons.

Envenomation by snakes is a major neglected human disease. Hospitalization and use of animal-derived antivenom are the primary therapeutic supports currently available. There is consensus that additional, not expensive, treatments that can be delivered even long after the snake bite are needed. We recently showed that the drug dubbed NUCC-390 shortens the time of recovery from the neuroparalysis caused by traumatic or toxic degeneration of peripheral motor neurons. These syndromes are characterized by the activation of a pro-regenerative molecular axis, consisting of the CXCR4 receptor expressed at the damaged site in neuronal axons and by the release of its ligand CXCL12α, produced by surrounding Schwann cells. This intercellular signaling axis promotes axonal growth and functional recovery from paralysis. NUCC-390 is an agonist of CXCR4 acting similarly to CXCL12α. Here, we have tested its efficacy in a murine model of neuroparalytic envenoming by a Papuan Taipan (Oxyuranus scutellatus) where a degeneration of the motor axon terminals caused by the presynaptic PLA2 toxin Taipoxin, contained in the venom, occurs. Using imaging of the neuromuscular junction and electrophysiological analysis, we found that NUCC-390 administration after injection of either the purified neuroparalytic Taipoxin or the whole Taipan venom, significantly accelerates the recovery from paralysis. These results indicate that NUCC-390, which is non-toxic in mice, should be considered for trials in humans to test its efficacy in accelerating the recovery from the peripheral neuroparalysis induced by Taipans. NUCC-390 should be tested as well in the envenomation by other snakes that cause neuroparalytic syndromes in humans. NUCC-390 could become an additional treatment, common to many snake envenomings, that can be delivered after the bite to reduce death by respiratory deficits and to shorten and improve functional recovery., Author summary We have taken here a different angle in the search for novel treatments of snake envenomings by focusing of the phase of recovery from the peripheral neuroparalysis caused by neurotoxic snakes. This is a critical period that may require mechanical ventilation and prolonged hospitalization. We have found a drug, dubbed NUCC-390, which accelerates the functional recovery from peripheral neuroparalysis caused by the Taipan venom in mice, by acting on the CXCR4 receptor. In fact, we found here that this receptor is expressed by the motor neuron at the site of damage that, in the case of many snake venoms containing presynaptic PLA2 neurotoxins, consists in the degeneration of the axon terminal. NUCC-390 binds to CXCR4 and acts similarly to its natural agonist the chemokine CXCL12α. The compound is not toxic and can be administered after the snakebite and therefore it has the potential of becoming a non-expensive addition to the currently available antisera treatment whose efficacy is limited by the need of delivery shortly after snakebite.

Publisher Summary This chapter covers natural agents that prolong the existence of acetylcholine after it is released from cholinergic nerve terminals. Natural toxins are chemical agents of biological origin including chemical agents and proteins, and can be produced by all types of organisms; such is the case of onchidal and fasciculins. Although a chemical and a protein, respectively, onchidal and fasciculins share the same toxic effect of inhibiting AChE, which is concentrated in synaptic regions and is responsible for the rapid catalysis of the hydrolysis of acetylcholine. As such, the natural toxins onchidal and fasciculins can produce disorders of neuromuscular transmission clinically categorized as either pre- or postsynaptic; some toxins simultaneously affect both sites. Chemical agents associated with neuromuscular transmission syndromes include the fasciculins, crotoxin, taipoxin, tubocurarine, organophosphorus compounds, and other antidotal therapy of the toxic effects of cholinesterase inhibitors such as onchidal and fasciculins used as potential chemical warfare agents is directed to blocking the effects of excessive acetylcholine stimulation and reactivating the inhibited enzyme.

A chicken-derived antivenom (ChDAv) towards taipan snake (Oxyuranus scutellatus) venom was produced by purifying anti-taipan IgY from egg yolks of hens immunized with taipan venom. The productivity, antivenomic profile, neutralization ability, pharmacokinetic properties and immunogenicity of the ChDAv were compared with those of an antivenom produced in horses (EDAv). We found that 382 eggs are required to produce the mass of anti-taipan antibodies contained in one liter of equine hyperimmune plasma, and that 63 chickens would be needed to generate the amount of anti-taipan antibodies annually produced by one horse. It was estimated that, in Costa Rica, the production of antitaipan antibodies could be 40% cheaper if chickens were used as immunoglobulin source, instead of horses. During antivenomic assessment, ChDAv showed lower ability to immunocapture the a subunit of taipoxin, the most important neurotoxin in the venom. ChDAv showed a lower ability to neutralize the coagulant and lethal activities of taipan venom. ChDAv was more immunogenic in rabbits than EDAv, probably due to the fact that chickens are phylogenetically more distant to rabbits than horses. This finding may explain why clearance from rabbit bloodstream was faster for chicken-IgY than for equineIgG in a pharmacokinetic study. In conclusion, the production of anti-taipan antivenom was less effective when chicken egg yolks were used as source of immunoglobulins instead of horses. Universidad de Costa Rica/[741-B3-017]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Envenomation Envenoming by snake bite is recorded unsystematically. The World Health Organization reports that more than 5 million people are bitten by snakes annually. They estimate that up to 138,000 die of complications, and many victims are left disabled. Stazi et al . developed a mouse model of envenoming by the Papuan taipan ( Oxyuranus scutellatus ). This snake's poison, taipoxin, is a phospholipase A2 neurotoxin akin to tetanus and botulinum toxins. Taipoxin causes the degeneration of motor axon synaptic transport but does not kill the neurons, and a slow recovery occurs if the victim is given respiratory support. Signaling by the G protein–coupled receptor CXCR4 is implicated in this synaptic pathophysiology. The authors studied the effects of a previously identified agonist of CXCR4, NUCC-390, a nontoxic compound in mice that accelerates regeneration of their synapses, and found that it significantly accelerated recovery from paralysis. NUCC-390 thus has the potential to enhance the recovery of patients hospitalized with neuroparalytic snake envenomation. PLOS Negl. Trop. Dis. 14 , e0008547 (2020).

BackgroundTaipans of the genus Oxyuranus are predominately mammal-eating specialists and a majority of Australian brown snakes of the sister genus Pseudonaja are generalist predators, feeding on mammals, lizards and frogs. In this paper, venom composition of several related mammal-eating species was compared using shotgun proteomics.ResultsVenom of Oxyuranus temporalis consisted predominately of α-neurotoxins (three-finger toxin family) and was deficient in phospholipase A2 neurotoxins. In contrast, PLA2 neurotoxins (taipoxin and paradoxin) were abundant in the venoms of other mammal-eating taipan species – Oxyuranus scutellatus and O. microlepidotus. Variation in neurotoxic PLA2 expression was also recorded in mammal-eating brown snakes, some species having high venom levels of textilotoxin or related homologues, for example Pseudonaja textilis and P. nuchalis, and others, such as P. ingrami, lacking them. Venom prothrombinase proteins (fX and fV) were expressed in most mammalivorous lineages, being particularly abundant in some Pseudonaja species. Notably, Oxyuranus temporalis venom was deficient in venom prothrombinase despite a mammal-based diet. Expression of an α-neurotoxin that is lethal to rodents (pseudonajatoxin b) was profoundly down-regulated in Pseudonaja textilis venom sample from Queensland and highly up-regulated in the sample from South Australia despite a report that the snake feeds on rodents in both regions.ConclusionRelated species of taipans and brown snakes that feed on small mammals express different sets of venom proteins toxic to this vertebrate group. This suggests an involvement of factors other than prey type selection in shaping venom proteome composition.

Antivenoms manufactured by bioCSL Limited (Australia) and Instituto Clodomiro Picado (Costa Rica) against the venom of the taipan snakes (Oxyuranus scutellatus) from Australia and Papua New Guinea (PNG), respectively, were compared using antivenomics, an analytical approach that combines proteomics with immunoaffinity chromatography. Both antivenoms recognized all venom proteins present in venom from PNG O. scutellatus, although a pattern of partial recognition was observed for some components. In the case of the Australian O. scutellatus venom, both antivenoms immunorecognized the majority of the components, but the CSL antivenom showed a stronger pattern of immunoreactivity, which was revealed by the percentage of retained proteins in the immunoaffinity column. Antivenoms interacted with taipoxin in surface plasmon resonance. These observations on antivenomics agree with previous neutralization studies. Fondos del Sistema Consejo Nacional de Rectores/[741-B2-652]/FEES-CONARE/Costa Rica Universidad de Costa Rica/[741- B3-017]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Despite the unparalleled diversity of venomous snakes in Australia, research has concentrated on a handful of medically significant species and even of these very few toxins have been fully sequenced. In this study, venom gland transcriptomes were sequenced from eleven species of small Australian elapid snakes, from eleven genera, spanning a broad phylogenetic range. The particularly large number of sequences obtained for three-finger toxin (3FTx) peptides allowed for robust reconstructions of their dynamic molecular evolutionary histories. We demonstrated that each species preferentially favoured different types of α-neurotoxic 3FTx, probably as a result of differing feeding ecologies. The three forms of α-neurotoxin [Type I (also known as (aka): short-chain), Type II (aka: long-chain) and Type III] not only adopted differential rates of evolution, but have also conserved a diversity of residues, presumably to potentiate prey-specific toxicity. Despite these differences, the different α-neurotoxin types were shown to accumulate mutations in similar regions of the protein, largely in the loops and structurally unimportant regions, highlighting the significant role of focal mutagenesis. We theorize that this phenomenon not only affects toxin potency or specificity, but also generates necessary variation for preventing/delaying prey animals from acquiring venom-resistance. This study also recovered the first full-length sequences for multimeric phospholipase A2 (PLA2) ‘taipoxin/paradoxin’ subunits from non-Oxyuranus species, confirming the early recruitment of this extremely potent neurotoxin complex to the venom arsenal of Australian elapid snakes. We also recovered the first natriuretic peptides from an elapid that lack the derived C-terminal tail and resemble the plesiotypic form (ancestral character state) found in viper venoms. This provides supporting evidence for a single early recruitment of natriuretic peptides into snake venoms. Novel forms of kunitz and waprin peptides were recovered, including dual domain kunitz-kunitz precursors and the first kunitz-waprin hybrid precursors from elapid snakes. The novel sequences recovered in this study reveal that the huge diversity of unstudied venomous Australian snakes are of considerable interest not only for the investigation of venom and whole organism evolution but also represent an untapped bioresource in the search for novel compounds for use in drug design and development.

Pre-synaptic PLA(2) neurotoxins are important components of many Australasian elapid snake venoms. These toxins disrupt neurotransmitter release. Taipoxin, a pre-synaptic neurotoxin isolated from the venom of the coastal taipan (Oxyuranus scutellatus), causes necrosis and muscle degeneration. The present study examined the myotoxic and cytotoxic activities of venoms from the Papuan taipan (O. scutellatus) and Irian Jayan death adder (Acanthophis rugosus), and also tested their pre-synaptic neurotoxins: cannitoxin and P-EPTX-Ar1a. Based on size-exclusion chromatography analysis, cannitoxin represents 16% of O. scutellatus venom, while P-EPTX-Ar1a represents 6% of A. rugosus venom. In the chick biventer cervicis nerve-muscle preparation, A. rugosus venom displayed significantly higher myotoxic activity than O. scutellatus venom as indicated by inhibition of direct twitches, and an increase in baseline tension. Both cannitoxin and P-EPTX-Ar1a displayed marked myotoxic activity. A. rugosus venom (50-300 μg/ml) produced concentration-dependent inhibition of cell proliferation in a rat skeletal muscle cell line (L6), while 300 μg/ml of O. scutellatus venom was required to inhibit cell proliferation, following 24-hr incubation. P-EPTX-Ar1a had greater cytotoxicity than cannitoxin, inhibiting cell proliferation after 24-hr incubation in L6 cells. Lactate dehydrogenase levels were increased after 1-hr incubation with A. rugosus venom (100-250 μg/ml), O. scutellatus venom (200-250 μg/ml) and P-EPTX-Ar1a (1-2 μM), but not cannitoxin (1-2 μM), suggesting venoms/toxin generated cell necrosis. Thus, A. rugosus and O. scutellatus venoms possess different myotoxic and cytotoxic activities. The proportion of pre-synaptic neurotoxin in the venoms and PLA(2) activity of the whole venoms are unlikely to be responsible for these activities.

There is limited information on the cross-neutralisation of neurotoxic venoms with antivenoms. Cross-neutralisation of the in vitro neurotoxicity of four Asian and four Australian snake venoms, four post-synaptic neurotoxins (α-bungarotoxin, α-elapitoxin-Nk2a, α-elapitoxin-Ppr1 and α-scutoxin; 100 nM) and one pre-synaptic neurotoxin (taipoxin; 100 nM) was studied with five antivenoms: Thai cobra antivenom (TCAV), death adder antivenom (DAAV), Thai neuro polyvalent antivenom (TNPAV), Indian Polyvalent antivenom (IPAV) and Australian polyvalent antivenom (APAV). The chick biventer cervicis nerve-muscle preparation was used for this study. Antivenom was added to the organ bath 20 min prior to venom. Pre- and post-synaptic neurotoxicity of Bungarus caeruleus and Bungarus fasciatus venoms was neutralised by all antivenoms except TCAV, which did not neutralise pre-synaptic activity. Post-synaptic neurotoxicity of Ophiophagus hannah was neutralised by all antivenoms, and Naja kaouthia by all antivenoms except IPAV. Pre- and post-synaptic neurotoxicity of Notechis scutatus was neutralised by all antivenoms, except TCAV, which only partially neutralised pre-synaptic activity. Pre- and post-synaptic neurotoxicity of Oxyuranus scutellatus was neutralised by TNPAV and APAV, but TCAV and IPAV only neutralised post-synaptic neurotoxicity. Post-synaptic neurotoxicity of Acanthophis antarcticus was neutralised by all antivenoms except IPAV. Pseudonaja textillis post-synaptic neurotoxicity was only neutralised by APAV. The α-neurotoxins were neutralised by TNPAV and APAV, and taipoxin by all antivenoms except IPAV. Antivenoms raised against venoms with post-synaptic neurotoxic activity (TCAV) cross-neutralised the post-synaptic activity of multiple snake venoms. Antivenoms raised against pre- and post-synaptic neurotoxic venoms (TNPAV, IPAV, APAV) cross-neutralised both activities of Asian and Australian venoms. While acknowledging the limitations of adding antivenom prior to venom in an in vitro preparation, cross-neutralization of neurotoxicity means that antivenoms from one region may be effective in other regions which do not have effective antivenoms. TCAV only neutralized post-synaptic neurotoxicity and is potentially useful in distinguishing pre-synaptic and post-synaptic effects in the chick biventer cervicis preparation.

Snake pre-synaptic neurotoxins endowed with phospholipase A(2) activity (SPANs) are potent inducers of paralysis through specific disruption of the neuromuscular junction pre-synaptic membrane and represent a valuable tool for investigating neuronal degeneration and recovery. They have different structural complexity and a wide range of lethal potency and enzymatic activity, but share similar mechanism of action. Whilst no correlation has been reported between neurotoxicity and enzymatic activity, toxicity increases with structural complexity and phospholipase A(2) oligomers show 10-fold lower LD(50) values when compared to their monomeric counterparts. Up to date, no structural study has been done on multimeric SPANs to shed light on the correlation between structural complexity and neurotoxicity. In this study we investigated the structure of taipoxin, a trimeric phospholipase A(2) neurotoxin, and of its subunits by X-ray crystallography and small angle X-ray scattering analysis. We present here the high-resolution structure of two isoforms of taipoxin β subunit, which show no neurotoxic activity but enhance the activity of the other subunits in the complex. One isoform shows no structural change that could justify the lack of activity. The other displays three point mutations in critical positions for the catalytic activity. Moreover, we designed a model for the quaternary structure of taipoxin under physiological conditions, in which the three subunits are organized into a flat holotoxin with the substrate binding sockets exposed on the same side of the complex, suggesting a role for this interface in the toxin-membrane interaction. Language: en

Neuroendocrine chromaffin cells were used to study the mechanism of the snake phospholipase A2 (PLA2) neurotoxin enhancement of exocytosis. Notexin, beta-bungarotoxin, taipoxin or textilotoxin enhanced the fast release of catecholamines elicited by flash photolysis of cytosolic caged calcium. Such an increase correlates with the capacity of these neurotoxins to cause fragmentation of the F-actin cortical barrier with subsequent accumulation of vesicles in the proximity of the plasma membrane. These PLA2 neurotoxins do not act via protein kinase C activation, which is known to promote F-actin fragmentation. Lithium, RO31-8220 and SB216763, three inhibitors of the glycogen synthase kinase 3, prevent both the alteration of the F-actin peripheral cortex and the enhancement of fast release elicited by these neurotoxins. In addition, glycogen synthase kinase 3 has been detected by immunolocalization in a membranous compartment of the chromaffin cell endoplasmic reticulum (ER). These results suggest that the activation of this enzyme plays a major role in the enhancement of exocytosis of the readily releasable granules caused by PLA2 neurotoxins in neuroendocrine chromaffin cells.

The neuromuscular junction activity of Oxyuranus scutellatus venom and its presynaptic neurotoxin, taipoxin, and their neutralization by two antivenoms were examined in mouse phrenic nerve-diaphragm preparations. The action of taipoxin was also studied at 21°C. The efficacy of the antivenoms was also assessed in an in vivo mouse model. Both antivenoms were effective in neutralizing the neuromuscular blocking activity in preincubation-type experiments. In experiments involving independent addition of venom and antivenoms, neutralization depended on the time interval between venom addition and antivenom application. When taipoxin was incubated for 5, 10 or 20min at 21°C, and antivenom added and temperature increased to 37°C, neutralization was achieved only when the toxin was incubated for 5 or 10min. The neutralization by the two antivenoms in an in vivo model showed that both whole IgG and F(ab')2 antivenoms were effective in neutralizing lethality. Our findings highlight the very rapid action of taipan venom at the nerve terminal, and the poor capacity of antivenoms to revert neurotoxicity as the time interval between venom or taipoxin application and antivenom addition increased. Additionally the disparity between molecular masses of the active substances of the two antivenoms did not result in differences in neutralization.

Paradoxin and taipoxin are neurotoxic phospholipases from the inland and coastal species of Australian taipan. Despite their relatively high sequence homology of 70% and 84% for the acidic and basic chains respectively, they differ substantially in reported assays of neurotoxicity. This study provides the first characterisation of paradoxin, which like taipoxin, is a trimer at physiological pH. More broadly, these toxins were found to be composed of a more diverse range of subunits than previously recognised, including newly discovered γTPx isoforms, which give rise to an additional, major conformation of TPx.

Motor neurons transmit impulses to muscle fibers through synapses called neuromuscular junctions (NMJs). Motor neuron degeneration is a characteristic of some peripheral neuropathies. Neurotoxins cause degeneration of the motor axon terminal, and recovery is assisted by local Schwann cells. Duregotti et al . found that injured motor neurons release danger signals that activate Schwann cells. Exposure to the black widow spider venom α-latrotoxin (α-LTX) or snake venom–derived taipoxin (TPX) induced the production of mitochondrial H 2 O 2 in cultured primary rat motor neurons, as well as the release of mitochondrial DNA (mtDNA) and cytochrome c (Cyt c) into the culture medium. The phosphorylation of ERK (extracellular signal–regulated kinase) was increased in Schwann cells either cocultured with toxin-treated motor neurons or treated with H 2 O 2 , mtDNA, or Cyt c or a combination thereof. Phosphorylated ERK was also increased in NMJ-associated Schwann cells in mice injected subcutaneously with sublethal doses of the toxins. Pretreating these cocultures or mice with catalase, which converts H 2 O 2 to water and O 2 , inhibited the increase in phosphorylated ERK. Pretreating mice with a MEK (ERK kinase) inhibitor delayed muscle recovery from α-LTX–induced paralysis. Isolated soleus muscles pretreated with catalase had decreased markers of synaptic regeneration after exposure to α-LTX. Exosomes secreted from toxin-treated motor neurons contained mtDNA, and phagocytic markers increased in NMJ-associated Schwann cells in isolated toxin-treated muscles. The findings suggest that injured motor neurons activate nearby Schwann cells to prevent the degeneration of neuromuscular junctions. E. Duregotti, S. Negro, M. Scorzeto, I. Zornetta, B. C. Dickinson, C. J. Chang, C. Montecucco, M. Rigoni, Mitochondrial alarmins released by degenerating axon terminals activate perisynaptic Schwann cells. Proc. Natl. Acad. Sci. U.S.A. 112 , E497–E505 (2015). [Abstract][Full Text]

The mechanisms of action of four snake presynaptic phospholipase A2 neurotoxins were investigated in cultured neurons isolated from various parts of the rat brain. Strikingly, physiological concentrations of notexin, β-bungarotoxin, taipoxin or textilotoxin induced a dose-dependent formation of discrete bulges at various sites of neuronal projections. Neuronal bulging was paralleled by the redistribution of the two synaptic vesicle markers synaptophysin I (SypI) and vesicle-attached membrane protein 2 (VAMP2) to the bulges, and by the exposure of the luminal domain of synaptotagmin on the cell surface. These neurotoxins induced glutamate release from cultured neurons similarly to the known evoked release of acetylcholine from neuromuscular junctions. In addition, partial fragmentation of F-actin and neurofilaments was observed in neurons, but not in astrocytes. These findings indicate that these snake presynaptic neurotoxins act with by same mechanism and that the observed phenotype results from the fusion of synaptic vesicles with the plasma membrane not balanced by an adequate membrane retrieval. These changes closely resemble those occurring at neuromuscular junctions of intoxicated animals and fully qualify these primary neuronal cultures as pertinent models for studying the molecular mode of action of these neurotoxins.

We identified, by affinity chromatography, two putative binding proteins for the presynaptic snake venom toxin taipoxin. We have previously characterized one of these proteins [neuronal pentraxin (NP)] as a neuronally secreted protein with homology to acute-phase proteins. Here we report the identification of the second protein as a 49-kDa lumenal calcium binding protein that we have named taipoxin-associated calcium binding protein 49 (TCBP-49). This protein contains six EF-hand putative calcium binding domains and the carboxyl-terminal sequence His-Asp-Glu-Leu (HDEL), identical to the yeast endoplasmic reticulum retention signal. Message for this protein is present in brain, liver, muscle, heart, kidney, and testis. Antibodies to this protein label reticular organelles of neurons and glia. This localization and the specific enrichment of native and recombinant TCBP-49 on columns of immobilized taipoxin raise the possibility that this protein interacts with internalized taipoxin, perhaps mediating its activation. The availability of pure TCBP-49 will allow direct tests of whether TCBP-49 alters the integrity of the oligomeric structure, phospholipase activity, or toxicity of taipoxin.

Neuronal pentraxin 1 (NP1), neuronal pentraxin 2 (NP2), and neuronal pentraxin receptor (NPR) are members of a new family of proteins identified through interaction with a presynaptic snake venom toxin taipoxin. We have proposed that these three neuronal pentraxins represent a novel neuronal uptake pathway that may function during synapse formation and remodeling. We have investigated the mutual interactions of these proteins by characterizing their enrichment on taipoxin affinity columns; by expressing NP1, NP2, and NPR singly and together in Chinese hamster ovary cells; and by generating mice that fail to express NP1. NP1 and NP2 are secreted, exist as higher order multimers (probably pentamers), and interact with taipoxin and taipoxin-associated calcium-binding protein 49 (TCBP49). NPR is expressed on the cell membrane and does not bind taipoxin or TCBP49 by itself, but it can form heteropentamers with NP1 and NP2 that can be released from cell membranes. This is the first demonstration of heteromultimerization of pentraxins and release of a pentraxin complex by proteolysis. These processes are likely to directly effect the localization and function of neuronal pentraxins in neuronal uptake or synapse formation and remodeling.

The presynaptically active, toxic phospholipases known as notexin and taipoxin are principal components of the venom of the Australian tiger snake and the Australian taipan respectively. The inoculation of the toxins into one hind limb of rats caused, within 1 h, the depletion of transmitter from the motor nerve terminals of the soleus muscle. This was followed by the degeneration of the motor nerve terminals and of the axonal cytoskeleton. By 24 h 70% of muscle fibers were completely denervated. Regeneration and functional reinnervation were almost fully restored by 5 days, but collateral innervation was common in the regenerated muscles, and this abnormality persisted for at least 9 months. The data provide an explanation for both the severity of neuromuscular paralysis that can accompany envenoming bites by tiger snakes and taipans and the difficulty experienced by physicians in managing the envenomed subjects.

After intracerebral injection, some toxic secreted phospholipases A2 (sPLA2) can induce epileptic seizures which bases are currently ill known. We undertook the detailed study of the central neurotoxicity of paradoxin (PDX), an analog of taipoxin, in rodents. Since literature strongly suggests a high variability in the sPLA2 epileptogenic properties, we compared, in an acute model, PDX with crotoxin (CTX), known to induce seizures and that may bind to similar neuronal receptors. Related toxic enzymes (ammodytoxin A, ATX A, and CTX subunit CB) and the non neurotoxic sPLA2 from pancreas and PLA2 analog ammodytin L (AML) were also tested. Despite being highly neurotoxic, PDX did not induce either convulsions or long-lasting seizure fits. The results obtained with the other enzymes showed that toxic sPLA2s can effectively be differentiated based on two criteria: the presence of cortically recorded epileptic paroxysmal discharges (E) and convulsions (C). We thus propose to classify the toxic sPLA2s into different groups depending on their epileptogenic properties: E-C- (PDX), E+C+ (CTX, CB), and E-C+ (ATX A). The non toxic AML and pancreatic enzyme were E-C-. Moreover, the results obtained with AML, and preliminarily with chemically inhibited CB, suggested that phospholipid hydrolysis is important to trigger seizures and convulsions. However, PDX and CTX that possess highly different epileptogenic properties exerted comparable, although slightly different, catalytic activities. Similarly, histological evaluations of the brain of PDX and CTX-treated rats (H&E staining, GFAP immunodetection, hsp70 and c-fos mRNA detection) did not provide satisfactory clues to explain these large differences. Further studies are strongly required. J. Neurosci. Res. 54:848–862, 1998. © 1998 Wiley-Liss, Inc.

We have identified the first putative integral membrane pentraxin and named it neuronal pentraxin receptor (NPR). NPR is enriched by affinity chromatography on columns of a snake venom toxin, taipoxin, and columns of the taipoxin-binding proteins neuronal pentraxin 1 (NP1), neuronal pentraxin 2 (NP2), and taipoxin-associated calcium-binding protein 49 (TCBP49). The predominant form of NPR contains an putative NH2-terminal transmembrane domain and all forms of NPR are glycosylated. NPR has 49 and 48% amino acid identity to NP1 and NP2, respectively, and NPR message is expressed in neuronal regions that express NP1 and NP2. We suggest that NPR, NP1, NP2, and TCBP49 are involved in a pathway responsible for the transport of taipoxin into synapses and that this may represent a novel neuronal uptake pathway involved in the clearance of synaptic debris.

Taipans are amongst the most venomous snakes in the world, and neurotoxicity is a major life-threatening symptom of envenoming by these snakes. Three species of taipans exist, and the venom from each species contains a presynaptic neurotoxin which accounts for much of the neurotoxicity observed following human envenoming. The high cost of antivenom used to treat neurotoxicity has resulted in the need to develop alternative but effective therapies. Therefore, in this study, we examined the ability of the P2Y receptor antagonist suramin to prevent the in vitro neurotoxic effects of the three presynaptic neurotoxins in taipan venoms: taipoxin, paradoxin and cannitoxin. Toxins were purchased from commercial sources or purified in house, using multiple steps of gel filtration chromatography. All three toxins (11 nM) inhibited nerve-mediated twitches in the chick biventer cervicis nerve–muscle preparation within 300 min. The presence of suramin (0.3 mM) completely blocked the taipoxin and cannitoxin-mediated inhibition of nerve-mediated twitches within the course of the experiment (P

Envenomation by Australian copperheads results mainly in muscle paralysis largely attributed to the presence of postsynaptic α-neurotoxins. However, poorly reversible neurotoxic effects suggest that these venoms may contain snake presynaptic phospholipase A2 neurotoxins (SPANs) that irreversibly inhibit neurotransmitter release. Using size-exclusion liquid chromatography, the present study isolated the first multimeric SPAN complex from the venom of the Australian common copperhead, Austrelaps superbus. The multimeric SPAN P-elapitoxin-As1a (P-EPTX-As1a) along with two novel monomeric SPANs and a new postsynaptic α-neurotoxin were then pharmacologically characterized using the chick biventer cervicis nerve-muscle preparation. All SPANs inhibited nerve-evoked twitch contractions at the neuromuscular junction without inhibiting contractile responses to cholinergic agonists or KCl. These actions are consistent with a prejunctional action to inhibit neurotransmitter release, without direct myotoxicity. Furthermore, the multimeric P-EPTX-As1a caused tetanic ‘fade’ in muscle tension under high frequency nerve stimulation, and produced a triphasic alteration to neurotransmitter release. These actions have been previously noted with other multimeric SPAN complexes such as taipoxin. Moreover, the neurotoxic α-subunit of P-EPTX-As1a shows high homology to taipoxin α-chain. Several other coagulopathic and myotoxic high mass proteins including a class PIII snake venom metalloproteinase, C-type lectin, l-amino acid oxidase, acetylcholinesterase and phospholipase B were also identified that may contribute to the overall toxicity of A. superbus venom. In conclusion, clinicians should be aware that early antivenom intervention might be necessary to prevent the onset of irreversible presynaptic neurotoxicity caused by multimeric and monomeric SPANs and that A. superbus venom is potentially capable of producing coagulopathic and myotoxic effects.

Crotoxin and taipoxin are both neurotoxic phospholipases A 2 capable of affecting the presynaptic activity to bring about ultimate blockade of synaptic transmission. The enzymatic activity has generally been considered to be necessary but not sufficient for the blockade. Since many phospholipases A 2 with comparable or even higher enzymatic activity are not toxic, it has been postulated that the difference lies in the affinity of binding to the presynaptic membrane. In confirmation of this proposition, we and others have previously shown that iodinated crotoxin and taipoxin bind specifically with high affinity to the isolated synaptic membrane fraction from guinea-pig brain, whereas specific binding is not detected with the nontoxic pancreatic phospholipase A 2 . Experiments based on photoaffinity labeling and simple chemical cross-linking techniques have led to the identification of three polypeptides preferentially present in neuronal membranes as (subunits of) the binding protein(s) for crotoxin and/or taipoxin. Some, but not all, other toxic phospholipases A 2 also appear to be ligands for the three polypeptides. We now report studies on partial purification of these polypeptides using affinity chromatography and other techniques. In order to learn the normal physiological roles played by the toxin-binding proteins, the phospholipase-independent effects of the toxins on the synaptosomes have been sought. We have found that under Ca 2+ -free condition, taipoxin or crotoxin inhibits with ic 50 of 20–1000 nM the Na + -dependent uptake of norepinephrine, dopamine and serotonin by the synaptosomes. In contrast, choline uptake is not affected. Furthermore, the high-affinity site for [ 3 H]desipramine binding, known to be the norepinephrine transporter, is inhibited with an ic 50 of 14nM by taipoxin independent of phospholipase activity. These results are strong indications that certain synaptosomal biogenic amine transporters are part of the binding proteins for taipoxin and crotoxin. Chemical modification at Tyr-21 of the phospholipase subunit of crotoxin greatly reduces the neurotoxicity and the binding affinity with little effect on the enzymatic activity of the toxin. Hence, Tyr-21 may be an important residue for the binding of crotoxin and perhaps other phospholipase A 2 neurotoxins. Even stronger evidence comes from the finding that replacement of the corresponding Phe residue of bovine pancreatic phospholipase A 2 with Tyr by site-directed mutagenesis enables it to compete with an ic 50 of 1 μM for the binding of [ 125 I]crotoxin, contrasting sharply with the complete lack of such ability with the wild type even at 50 μM. In addition, rat phospholipase A 2 , which has a corresponding Tyr residue, can inhibit the binding of [ 125 I]crotoxin.

Venom from the common tiger snake, Notechis scutatus scutatus , contains several toxic acidic proteins which promote hypotension and hemorrhage in mice. One of these toxins, HT e , has a phospholipase A 2 (PLA 2 ) amino acid sequence. It contains 125 amino acids rather than the 119/120 found in other N. s. scutatus PLA 2 s, because it also has the loop of residues (62-66) found in helix D of pancreatic PLA 2 s, the γ-subunit of taipoxin, and the D-subunit of textilotoxin. High sequence identity is found between the first 57 and the last 25 amino acids of HT e and other N. s. scutatus PLA 2 s. In the central section containing the pancreatic loop and the β-wing, sequence similarity with other N. s. scutatus PLA 2 s is low. The β-wing amino acids are highly homologous to taipoxin-γ. HT g , an isoform of HT e , has a sequence almost identical to that of HT e in the central section. Neuropharmacological and neurophysiological studies show that HT e blocks neuromuscular transmission, but it does not produce blockade by virtue of a selective action on nerve endings. Instead, the toxin acts both on nerve and on muscle. Unlike taipoxin-γ and textilotoxin-D, HT e and HT g are not glycosylated and are not otherwise modified. HT e , HT g , and the other acidic proteins hydrolyze the synthetic PLA 2 substrate, 3-octanoyloxy-4-nitrobenzoic acid, as well as L-α-phosphatidylcholine.

2082-04 Embargo por política editorial The venom proteomes of populations of the highly venomous taipan snake, Oxyuranus scutellatus, from Australia and Papua New Guinea (PNG), were characterized by reverse-phase HPLC fractionation, followed by analysis of chromatographic fractions by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. Proteins belonging to the following seven protein families were identified in the two venoms: phospholipase A2 (PLA2), Kunitz-type inhibitor, metalloproteinase (SVMP), three-finger toxin (3FTx), serine proteinase, cysteine-rich secretory proteins (CRISP), and coagulation factor V-like protein. In addition, C-type lectin/lectin-like protein and venom natriuretic peptide were identified in the venom of specimens from PNG. PLA2s comprised more than 65% of the venoms of these two populations. Antivenoms generated against the venoms of these populations showed a pattern of cross-neutralization, corroborating the immunological kinship of these venoms. Toxicity experiments performed in mice suggest that, at low venom doses, neurotoxicity leading to respiratory paralysis represents the predominant mechanism of prey immobilization and death. However, at high doses, such as those injected in natural bites, intravascular thrombosis due to the action of the prothrombin activator may constitute a potent and very rapid mechanism for killing prey Universidad de Costa Rica/[741-A7-611]/UCR/Costa Rica Universidad de Costa Rica/[741-A9-506]/UCR/Costa Rica Ministerio de Ciencia e Innovación/[BFU2010-17373]//España Generalitat Valenciana/[PROMETEO/2010/005]//España Consejo Superior de Investigaciones Científicas/2009CR0021/CRUSA-CSIC/España UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Several phospholipase A(2) (PLA(2)) neurotoxins from snake venoms can affect acetylcholine release at the neuromuscular junction. In isolated nerve-muscle preparations three distinct phases have been described for this phenomenon: An initial transient decrease in twitch tension; a second facilitatory phase during which twitch height is greater than control twitch height; and the last phase which causes a reduction in twitch height that finally results in paralysis. Suramin has been reported to inhibit the toxic effects of β-bungarotoxin and another PLA(2) neurotoxin, crotoxin in vitro and in vivo. We have further examined the effects of suramin on the three phases of the effects of the presynaptic PLA(2) neurotoxins β-bungarotoxin, taipoxin and ammodytoxin on mouse phrenic nerve-hemidiaphragm preparations. When preparations were pre-treated with suramin (0.3mM), the early biphasic effects (depression followed by facilitation) were abolished, and the time taken for final blockade induced by β-bungarotoxin, taipoxin and ammodytoxin A was significantly prolonged. In contrast, suramin did not significantly affect the facilitation induced by the potassium channel blocking toxin dendrotoxin I when applied under the same conditions. In addition, application of 0.3mM suramin did not prevent the facilitatory actions of 3,4-diaminopyridine (3,4-DAP) and tetraethylammonium chloride (TEA). Overall, the mechanism whereby suramin reduces the effects of PLA(2) neurotoxins remains elusive. Since suramin reduces both enzyme-dependent and enzyme-independent effects of the toxins, suramin is not acting as a simple enzyme inhibitor. Furthermore, the observation that suramin does not affect actions of standard K(+) channel blockers suggests that suramin does not stabilise nerve terminals.

Certain phospholipases A2 isolated from snake venoms are highly potent neurotoxins acting primarily at the presynatic sites of some peripheral and central synapses Among the most extensively studied are β-bungarotoxin. crotoxin and taipoxin. At the neuromuscular junction. the early effects vary with the toxins and experimental conditions. an initial decrease in neurotransmission followed by an increase being common to most of the presynaptically toxic phospholipases A2. However the ultimate blockade of transmission is the principal cause of neurotoxicity for these toxins. The phospholipase activity has been generally regarded to be related to, but not by itself sufficient for. the neuromuscular blockade by these toxins in physiological solution. Direct evidence for binding to a receptor is provided by reversible radioligand binding assays coupled with chemical cross-linking and photoaffinity labeling techniques. Specific binding of the presynaptic toxin with high to very high affinity to neuron-sp...

Experiments were conducted on mouse hemidiaphragm preparations using five phospholipase A2 neurotoxins of differing chain structures and antigenicities [notexin (one chain); crotoxin (two chains not covalently bound), beta-bungarotoxin (two chains covalently bound); taipoxin (three chains), and textilotoxin (five chains; one copy each of three chains and two copies of a fourth chain)]. Three clostridial neurotoxins (botulinum neurotoxin types A and B, and tetanus toxin) were used in comparison experiments. Phospholipase A2 neurotoxins produced concentration-dependent blockade of neuromuscular transmission. There was no obvious relationship between chain structure and potency, but there was an indication of a relationship between chain structure and binding. The binding of notexin was substantially reversible, the binding of crotoxin was slightly reversible, and the binding of beta-bungarotoxin, taipoxin and textilotoxin was poorly reversible. Experiments with neutralizing antibodies indicated that phospholipase A2 neurotoxins became associated with binding sites on or near the cell surface. This binding did not produce neuromuscular blockade. When exposed to physiological temperatures and nerve stimulation, bound toxin disappeared from accessibility to neutralizing antibody. This finding suggests that there was some form of molecular rearrangement. The two most likely possibilities are: (1) there was a change in the conformation of the toxin molecule, or (2) there was a change in the relationship between the toxin and the membrane. The molecular rearrangement step did not produce neuromuscular blockade. At a later time there was onset of paralysis; the amount of time necessary for onset of blockade was a function of toxin concentration. Phospholipase A2 neurotoxins were not antagonized by drugs that inhibit receptor-mediated endocytosis. In addition, phospholipase A2 neurotoxins did not display the pH-induced conformational changes that are typical of other endocytosed proteins, such as clostridial neurotoxins. However, phospholipase A2 neurotoxins were antagonized by strontium, and this antagonism was expressed against toxins that were free in solution and toxins that were bound to the cell surface. Limited antagonism was expressed after toxins had undergone molecular rearrangement, and no antagonism was expressed after toxin-induced neuromuscular blockade. The cumulative data suggest that phospholipase A2 neurotoxins are not internalized to produce their poisoning effects. These toxins appear to act on the plasma membrane, and this is the site at which they initiate the events that culminate in neuromuscular blockade.

Snake pre-synaptic phospholipase A(2) neurotoxins paralyse the neuromuscular junction by releasing phospholipid hydrolysis products that alter curvature and permeability of the pre-synaptic membrane. Here, we report results deriving from the first chemical analysis of the action of these neurotoxic phospholipases in neurons, made possible by the use of high sensitivity mass spectrometry. The time-course of the phospholipase A(2) activity (PLA(2)) hydrolysis of notexin, beta-bungarotoxin, taipoxin and textilotoxin acting in cultured neurons was determined. At variance from their enzymatic activities in vitro, these neurotoxins display comparable kinetics of lysophospholipid release in neurons, reconciling the large discrepancy between their in vivo toxicities and their in vitro enzymatic activities. The ratios of the lyso derivatives of phosphatidyl choline, ethanolamine and serine obtained here together with the known distribution of these phospholipids among cell membranes, suggest that most PLA(2) hydrolysis takes place on the cell surface. Although these toxins were recently shown to enter neurons, their intracellular hydrolytic action and the activation of intracellular PLA(2)s appear to contribute little, if any, to the phospholipid hydrolysis measured here.

Snake presynaptic neurotoxins with phospholipase A2 (PLA2) activity cause degeneration of the neuromuscular junction. They induce depletion of synaptic vesicles and increase the membrane permeability to Ca(2+) which fluxes from the outside into the nerve terminal. Moreover, several toxins were shown to enter the nerve terminals of cultured neurons, where they may display their PLA2 activity on internal membranes. The relative contribution of these different actions in nerve terminal degeneration remains to be established. To gather information on this point, we have compared the effects of beta-bungarotoxin, taipoxin, notexin and textilotoxin with those of alpha-latrotoxin on the basis of the notion that this latter toxin is well known to cause massive Ca(2+) influx and exocytosis of synaptic vesicles. All the parameters analysed here, including calcium imaging, are very similar for the two classes of neurotoxins. This indicates that Ca(2+) overloading plays a major role in the degeneration of nerve terminals induced by the snake presynaptic neurotoxins.

Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes. Toxicon 29, 973–988 1991.—Crotoxin, the presynaptic neurotoxin from Crotalus durissus terrificus , was iodinated and used to demonstrate high affinity, specific binding to guineapig ( Cavia porcellus ) brain synaptosomes and synaptosomal membrane fragments. 125 I-crotoxin binding to the membrane fragments displays two binding plateaus, ( K d1 = 4 nM and K d2 = 87 nM, B max1 = 2 and B max2 = 4 pmoles/mg membrane protein), but binding to whole synaptosomes revealed only one plateau ( K d = 2 nM and B max = 5 pmoles/mg membrane protein). Rosenthal analyses of Scatchard plots yielded similar binding constants in the presence or absence of 0.025% Triton X-100. In addition to equilibrium analyses, kinetic analyses of 125 I-crotoxin binding to synaptosomal membrane fragments gave a K d -value of 3 nM. The K d value was not significantly changed by the exclusion of added calcium, but the binding site number was lowered. Crotoxin binding was inhibited by the acidic subunit of crotoxin and several presynaptic neurotoxins, which were classified according to their inhibitory properties as, strong (acidic subunit of crotoxin, Mojave toxin, concolor toxin, taipoxin and pseudexin), moderate (ammodytoxin A and textilotoxin), weak (notexin and scutoxin A), very weak (notechis II-5) and non-inhibitory (basic subunit of crotoxin, β-bungarotoxin, Crotalus atrox and porcine pancreatic phospholipases A 2 , dendrotoxin, and notechis III-4). Purified acidic subunit of crotoxin, the most potent competitor of crotoxin binding, was somewhat more competitive than intact crotoxin and the other strong inhibitors on a molar basis. Strong, moderate and weak inhibitor groups each differed from the preceding group by requiring about a ten fold increase in concentration to effect a 50% inhibition of crotoxin binding. The weak group was therefore at least two-orders of magnitude less effective than the strong inhibition shown by the acidic subunit of crotoxin. Treatment of synaptosomal membranes with protease K lowered 125 I-crotoxin binding, whereas treatment with trypsin did not. Iodinated, phospholipase A 2 from C. atrox venom showed no specific binding to whole synaptosomes. Our results demonstrate the presence and describe some of the properties of high affinity, specific binding sites in brain tissue for crotoxin and related presynaptic neurotoxins.

R. D. Crosland . Effect of drugs on the lethality in mice of the venoms and neurotoxins from sundry snakes. Toxicon 29, 613–631, 1991.—I investigated the efficacy of 10 drugs with respect to reducing the lethality in mice of the following venoms and their respective neurotoxins: Bungarus caeruleus venom; Bungarus multicinctus venom, α-bungarotoxin, β-bungarotoxin; Crotalus durissus terrificus venom, crotoxin; Notechis scutatus scutatus venom; Oxyuranus scutellatus venom, taipoxin. The drugs diltiazem, nicergoline, primaquine, verapamil and vesamicol protected mice from the lethality of B. caeruleus venom, B. multicinctus venom, and/or β-bungarotoxin. Dexamethasone provided protection from B. multicinctus venom, β-bungarotoxin, crotoxin, O. scutellatus venom and taipoxin. Protective activity resided in amphiphilic drugs and correlated with the charge on the drug at physiological pH. Protection from lethality was maximal when the drugs were administered immediately after injection of the venom or toxin. Nifedipine, piracetam and reserpine provided no protection from any of the venoms or toxins tested.

Snake presynaptic neurotoxins with phospholipase A2 activity are potent inducers of paralysis through inhibition of the neuromuscular junction. These neurotoxins were recently shown to induce exocytosis of synaptic vesicles following the production of lysophospholipids and fatty acids and a sustained influx of Ca2+ from the medium. Here, we show that these toxins are able to penetrate spinal cord motor neurons and cerebellar granule neurons and selectively bind to mitochondria. As a result of this interaction, mitochondria depolarize and undergo a profound shape change from elongated and spaghetti-like to round and swollen. We show that snake presynaptic phospholipase A2 neurotoxins facilitate opening of the mitochondrial permeability transition pore, an inner membrane high-conductance channel. The relative potency of the snake neurotoxins was similar for the permeability transition pore opening and for the phospholipid hydrolysis activities, suggesting a causal relationship, which is also supported by the effect of phospholipid hydrolysis products, lysophospholipids and fatty acids, on mitochondrial pore opening. These findings contribute to define the cellular events that lead to intoxication of nerve terminals by these snake neurotoxins and suggest that mitochondrial impairment is an important determinant of their toxicity.

Crotoxin is a neurotoxic phospholipase A2 capable of blocking synaptic transmission by inhibiting the release of neurotransmitters. The photoaffinity labeling technique was used to identify the neural membrane molecules involved in the binding of crotoxin. A photoactivatable, radioactive derivative of crotoxin was synthesized by reacting crotoxin with N-hydroxysuccinimidyl-4-azidobenzoate and with Na[125I]. Photoirradiation of synaptosomes from guinea pig brains in the presence of the crotoxin derivative resulted in the formation of a major radioactive conjugate of 100,000 daltons as revealed by autoradiography of a sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern. Pretreatment of the synaptosomes with trypsin, Staphylococcus aureus protease, or papain prevented the formation of this conjugate. The conjugate was not detected when plasma membranes from several nonneural tissues replaced the brain synaptosomes. Unmodified crotoxin inhibited the formation of this adduct with an IC50 of about 10(-8)M. Mojave toxin, caudoxin, notexin, Naja naja PLA, and taipoxin also inhibited adduct formation with different potencies, while beta-bungarotoxin and pancreatic PLA were ineffective. We concluded that an 85,000-dalton protein is the major component responsible for the binding of crotoxin to synaptosomal membranes.

Several protein neurotoxins from a variety of animals are known to increase the release of acetylcholine at the neuromuscular junction. Some toxins stimulate release in an uncontrolled fashion by inducing depolarization of the nerve terminal. Such toxins include those that alter the activation and inactivation characteristics of sodium channels, and those that create transmembrane pores. Toxins that facilitate evoked neurotransmitter release are more interesting because they can be used to explore the normal control mechanisms that modulate physiologically relevant release. Several snake toxins with phospholipase activity can block release, although they initially cause a stimulation. These include β-bungarotoxin, crotoxin, notexin, and taipoxin. The facilitation of transmitter release in mammalian nerve-muscle preparations is associated with a blockade of some of the potassium channels of the nerve terminal. This will slow the repolarization after an action potential, and thus allow calcium ions ...

Snake presynaptic phospholipase A2 neurotoxins (SPANs) paralyze the neuromuscular junction (NMJ). Upon intoxication, the NMJ enlarges and has a reduced content of synaptic vesicles, and primary neuronal cultures show synaptic swelling with surface exposure of the lumenal domain of the synaptic vesicle protein synaptotagmin I. Concomitantly, these neurotoxins induce exocytosis of neurotransmitters. We found that an equimolar mixture of lysophospholipids and fatty acids closely mimics all of the biological effects of SPANs. These results draw attention to the possible role of local lipid changes in synaptic vesicle release and provide new tools for the study of exocytosis.

The Papuan taipan (Oxyuranus scutellatus canni) is widely distributed throughout much of Papua New Guinea. Although neurotoxicity is a major symptom of envenomation, no neurotoxins have been isolated from this venom. Using a series of size exclusion chromatography steps, we report the isolation of cannitoxin, a presynaptic neurotoxin (44,848 Da) that represents approximately 16% of the whole venom. The toxin displayed high phospholipase A2 (PLA2 activity (330 +/- 5 micromol/min/mg) and caused concentration-dependent (11-66 nM) inhibition of indirect (0.2 ms; 0.1 Hz; supramaximal V) twitches of the chick biventer cervicis nerve-muscle preparation without effecting nicotinic receptor agonists. Prior addition of CSL Taipan antivenom (5 U/ml) or inhibition of phospholipase A2 activity by incubation with 4-bromophenacyl bromide prevented the inhibition of twitches. Cannitoxin is composed of three different subunits, alpha, beta, and gamma, with the possibility of two beta isomers. However, only the alpha subunit displayed in vitro neurotoxic activity of its own. Thus, cannitoxin is similar in structure and pharmacology to taipoxin, which has been isolated from the closely related Australian species O. scutellatus scutellatus (coastal taipan).

The application of the snake neurotoxin taipoxin to hippocampal neurons in culture induced Ca(2+)-dependent synaptic vesicle (SV) exocytosis, with swelling of nerve terminals and redistribution of SV proteins to the axolemma. Using digital imaging videomicroscopy to measure fluorescence resonance energy transfer in live neurons, we also found that taipoxin modulates the machinery for neurosecretion by causing dissociation of the SV proteins synaptobrevin 2 and synaptophysin I at a stage preceding taipoxin-induced facilitation of SV fusion. These early effects of the toxin are followed by severe impairment of SV exo-endocytosis, which might underlie the prevention of neurotransmitter release reported after intoxication by taipoxin.