Black Widow Spider Venom Research Articles

Preying dangerously: black widow spider venom resistance in sympatric lizards.

Lizards and spiders are natural adversaries, yet little is known of adaptations that lizards might possess for dealing with the venomous defences of spider prey. In the Western USA, two lizard species (Elgaria multicarinata and Sceloporus occidentalis) are sympatric with and predate western black widow spiders (Latrodectus hesperus). The consequences of black widow spider venom (BWSV) can be severe, and are well understood for mammals but unknown for reptiles. We evaluated potential resistance to BWSV in the lizards that consume black widows, and a potentially susceptible species (Uta stansburiana) known as prey of widows. We investigated BWSV effects on whole-animal performance (sprint) and muscle tissue at two venom doses compared with control injections. Sprint speed was not significantly decreased in E. multicarinata or S. occidentalis in any treatment, while U. stansburiana suffered significant performance reductions in response to BWSV. Furthermore, E. multicarinata showed minimal tissue damage and immune response, while S. occidentalis and U. stansburiana exhibited increased muscle damage and immune system infiltration in response to BWSV. Our data suggest predator–prey relationships between lizards and spiders are complex, possibly leading to physiological and molecular adaptations that allow some lizards to tolerate or overcome the dangerous defences of their arachnid prey.

Neutralization of black widow spider (Latrodectus mactans) venom with rabbit polyclonal serum hyperimmunized with recombinant alpha-latrotoxin fragments

Alpha-latrotoxin (ɑLTx) is the component responsible for causing the pathophysiology in patients bitten by spiders from the genus Latrodectus, commonly known as black widow spiders. The current antivenom used to treat these envenomations in Mexico is produced using the venom of thousands of spiders, obtained through electrical stimulation. This work aimed to produce this protein as well as two of its fragments in a bacterial model, to evaluate their use as immunogens to produce neutralizing hyperimmune sera, in rabbits.ɑLTx is a 130 kDa protein which has not yet been obtained in a soluble active form using bacterial models. In the present work, ɑLTx and two of its fragments, ankyrin domain and amino terminal domain (LTxAnk and LTxNT) were produced in bacteria and solubilized from inclusion bodies using N-lauroyl sarcosine. These three proteins were used for hyperimmunization in order to evaluate their potential as immunogens for the production of neutralizing hyperimmune sera against the complete venom of Latrodectus mactans.The hyperimmune sera obtained using the complete ɑLTx as well as the LTxNT, was capable of preventing death of mice envenomated with 3 LD50s of venom, both in preincubation and rescue experiments. Conversely, the serum obtained using the LTxAnk fragment, generated only partial protection and a delay in the time of death, even with a maximum dose of 450 μL. We therefore conclude that the produced proteins show great potential for their use as immunogens and should be further tested in large animals, such as horses.

Latrotoxin-Induced Neuromuscular Junction Degeneration Reveals Urocortin 2 as a Critical Contributor to Motor Axon Terminal Regeneration.

We used α-Latrotoxin (α-LTx), the main neurotoxic component of the black widow spider venom, which causes degeneration of the neuromuscular junction (NMJ) followed by a rapid and complete regeneration, as a molecular tool to identify by RNA transcriptomics factors contributing to the structural and functional recovery of the NMJ. We found that Urocortin 2 (UCN2), a neuropeptide involved in the stress response, is rapidly expressed at the NMJ after acute damage and that inhibition of CRHR2, the specific receptor of UCN2, delays neuromuscular transmission rescue. Experiments in neuronal cultures show that CRHR2 localises at the axonal tips of growing spinal motor neurons and that its expression inversely correlates with synaptic maturation. Moreover, exogenous UCN2 enhances the growth of axonal sprouts in cultured neurons in a CRHR2-dependent manner, pointing to a role of the UCN2-CRHR2 axis in the regulation of axonal growth and synaptogenesis. Consistently, exogenous administration of UCN2 strongly accelerates the regrowth of motor axon terminals degenerated by α-LTx, thereby contributing to the functional recovery of neuromuscular transmission after damage. Taken together, our results posit a novel role for UCN2 and CRHR2 as a signalling axis involved in NMJ regeneration.

Catching Latrophilin With Lasso: A Universal Mechanism for Axonal Attraction and Synapse Formation.

Latrophilin-1 (LPHN1) was isolated as the main high-affinity receptor for α-latrotoxin from black widow spider venom, a powerful presynaptic secretagogue. As an adhesion G-protein-coupled receptor, LPHN1 is cleaved into two fragments, which can behave independently on the cell surface, but re-associate upon binding the toxin. This triggers intracellular signaling that involves the Gαq/phospholipase C/inositol 1,4,5-trisphosphate cascade and an increase in cytosolic Ca2+, leading to vesicular exocytosis. Using affinity chromatography on LPHN1, we isolated its endogenous ligand, teneurin-2/Lasso. Both LPHN1 and Ten2/Lasso are expressed early in development and are enriched in neurons. LPHN1 primarily resides in axons, growth cones and presynaptic terminals, while Lasso largely localizes on dendrites. LPHN1 and Ten2/Lasso form a trans-synaptic receptor pair that has both structural and signaling functions. However, Lasso is proteolytically cleaved at multiple sites and its extracellular domain is partially released into the intercellular space, especially during neuronal development, suggesting that soluble Lasso has additional functions. We discovered that the soluble fragment of Lasso can diffuse away and bind to LPHN1 on axonal growth cones, triggering its redistribution on the cell surface and intracellular signaling which leads to local exocytosis. This causes axons to turn in the direction of spatio-temporal Lasso gradients, while LPHN1 knockout blocks this effect. These results suggest that the LPHN1-Ten2/Lasso pair can participate in long- and short-distance axonal guidance and synapse formation.

Latrophilins updated.

Latrophilins (LPHN) are part of a yet unexplored family of receptors comprising three isoforms, LPHN1-3, and belonging to a unique branch of G protein-coupled receptors (GPCR) named adhesion GPCR (aGPCR). LPHN are considered to be prototypical models for the study of aGPCR as they are one of the most evolutionary conserved members. Previously described as the target for a potent neurotoxin from the black widow spider venom, LPHN are now being studied under a whole new perspective. Indeed, recent advances have provided a better understanding of different aspects of this prototypical family of receptors: 1) elucidation of LPHN ectodomain organization by crystallography has unveiled a new functional domain with great repercussion on all the other members of the aGPCR family, 2) proteomic approaches have opened the gate to unsuspected functional characteristics of LPHN cellular role, and 3) genetic approaches have provided hints into the physiological functions of LPHN in specific systems and organisms. Moreover, genomic linkage studies screening human patients from diverse genetic backgrounds have involved LPHN gene defects in human disorders such as attention-deficit hyperactivity disorder and cancer. In this review, we will provide a historical perspective addressing experimental research on these receptors while highlighting the new advances and discoveries concerning LPHN functions. As GPCR still represent the most studied targets for the development of pharmacological approaches aiming at alleviating human disorders, the relevance of studying LPHN retains a high pertinence to better understand these receptors for the treatment of human diseases.

Recruitment and diversification of an ecdysozoan family of neuropeptide hormones for black widow spider venom expression

Venoms have attracted enormous attention because of their potent physiological effects and dynamic evolution, including the convergent recruitment of homologous genes for venom expression. Here we provide novel evidence for the recruitment of genes from the Crustacean Hyperglycemic Hormone (CHH) and arthropod Ion Transport Peptide (ITP) superfamily for venom expression in black widow spiders. We characterized latrodectin peptides from venom gland cDNAs from the Western black widow spider (Latrodectus hesperus), the brown widow (Latrodectus geometricus) and cupboard spider (Steatoda grossa). Phylogenetic analyses of these sequences with homologs from other spider, scorpion and wasp venom cDNAs, as well as CHH/ITP neuropeptides, show latrodectins as derived members of the CHH/ITP superfamily. These analyses suggest that CHH/ITP homologs are more widespread in spider venoms, and were recruited for venom expression in two additional arthropod lineages. We also found that the latrodectin 2 gene and nearly all CHH/ITP genes include a phase 2 intron in the same position, supporting latrodectin's placement within the CHH/ITP superfamily. Evolutionary analyses of latrodectins suggest episodes of positive selection along some sequence lineages, and positive and purifying selection on specific codons, supporting its functional importance in widow venom. We consider how this improved understanding of latrodectin evolution informs functional hypotheses regarding its role in black widow venom as well as its potential convergent recruitment for venom expression across arthropods.

Methods to produce, culture and evaluate embryonic stem-cell-derived neurons as a research tool for botulinum toxin and the black widow spider venom latrotoxin

Methods to produce, culture and evaluate embryonic stem-cell-derived neurons as a research tool for botulinum toxin and the black widow spider venom latrotoxin

FLRT Proteins Are Endogenous Latrophilin Ligands and Regulate Excitatory Synapse Development

Latrophilins (LPHNs) are a small family of G protein-coupled receptors known to mediate the massive synaptic exocytosis caused by the black widow spider venom α-latrotoxin, but their endogenous ligands and function remain unclear. Mutations in LPHN3 are strongly associated with attention deficit hyperactivity disorder, suggesting a role for latrophilins in human cognitive function. Using affinity chromatography and mass spectrometry, we identify the FLRT family of leucine-rich repeat transmembrane proteins as endogenous postsynaptic ligands for latrophilins. We demonstrate that the FLRT3 and LPHN3 ectodomains interact with high affinity in trans and that interference with this interaction using soluble recombinant LPHN3, LPHN3 shRNA, or FLRT3 shRNA reduces excitatory synapse density in cultured neurons. In addition, reducing FLRT3 levels with shRNA in vivo decreases afferent input strength and dendritic spine number in dentate granule cells. These observations indicate that LPHN3 and its ligand FLRT3 play an important role in glutamatergic synapse development.

High Affinity Neurexin Binding to Cell Adhesion G-protein-coupled Receptor CIRL1/Latrophilin-1 Produces an Intercellular Adhesion Complex

The G-protein-coupled receptor CIRL1/latrophilin-1 (CL1) and the type-1 membrane proteins neurexins represent distinct neuronal cell adhesion molecules that exhibit no similarities except for one common function: both proteins are receptors for α-latrotoxin, a component of black widow spider venom that induces massive neurotransmitter release at synapses. Unexpectedly, we have now identified a direct binding interaction between the extracellular domains of CL1 and neurexins that is regulated by alternative splicing of neurexins at splice site 4 (SS4). Using saturation binding assays, we showed that neurexins lacking an insert at SS4 bind to CL1 with nanomolar affinity, whereas neurexins containing an insert at SS4 are unable to bind. CL1 competed for neurexin binding with neuroligin-1, a well characterized neurexin ligand. The extracellular sequences of CL1 contain five domains (lectin, olfactomedin-like, serine/threonine-rich, hormone-binding, and G-protein-coupled receptor autoproteolysis-inducing (GAIN) domains). Of these domains, the olfactomedin-like domain mediates neurexin binding as shown by deletion mapping. Cell adhesion assays using cells expressing neurexins and CL1 revealed that their interaction produces a stable intercellular adhesion complex, indicating that their interaction can be trans-cellular. Thus, our data suggest that CL1 constitutes a novel ligand for neurexins that may be localized postsynaptically based on its well characterized interaction with intracellular SH3 and multiple ankyrin repeats adaptor proteins (SHANK) and could form a trans-synaptic complex with presynaptic neurexins.

The reserve pool of synaptic vesicles acts as a buffer for proteins involved in synaptic vesicle recycling

Presynaptic nerve terminals contain between several hundred vesicles (for example in small CNS synapses) and several tens of thousands (as in neuromuscular junctions). Although it has long been assumed that such high numbers of vesicles are required to sustain neurotransmission during conditions of high demand, we found that activity in vivo requires the recycling of only a few percent of the vesicles. However, the maintenance of large amounts of reserve vesicles in many evolutionarily distinct species suggests that they are relevant for synaptic function. We suggest here that these vesicles constitute buffers for soluble accessory proteins involved in vesicle recycling, preventing their loss into the axon. Supporting this hypothesis, we found that vesicle clusters contain a large variety of proteins needed for vesicle recycling, but without an obvious function within the clusters. Disrupting the clusters by application of black widow spider venom resulted in the diffusion of numerous soluble proteins into the axons. Prolonged stimulation and ionomycin application had a similar effect, suggesting that calcium influx causes the unbinding of soluble proteins from vesicles. Confirming this hypothesis, we found that isolated synaptic vesicles in vitro sequestered soluble proteins from the cytosol in a process that was inhibited by calcium addition. We conclude that the reserve vesicles support neurotransmission indirectly, ensuring that soluble recycling proteins are delivered upon demand during synaptic activity.

Alpha-Latrotoxin Rescues SNAP-25 from BoNT/A-Mediated Proteolysis in Embryonic Stem Cell-Derived Neurons

The botulinum neurotoxins (BoNTs) exhibit zinc-dependent proteolytic activity against members of the core synaptic membrane fusion complex, preventing neurotransmitter release and resulting in neuromuscular paralysis. No pharmacologic therapies have been identified that clinically relieve botulinum poisoning. The black widow spider venom α-latrotoxin (LTX) has the potential to attenuate the severity or duration of BoNT-induced paralysis in neurons via the induction of synaptic degeneration and remodeling. The potential for LTX to antagonize botulinum poisoning was evaluated in embryonic stem cell-derived neurons (ESNs), using a novel screening assay designed around the kinetics of BoNT/A activation. Exposure of ESNs to 400 pM LTX for 6.5 or 13 min resulted in the nearly complete restoration of uncleaved SNAP-25 within 48 h, whereas treatment with 60 mM K+ had no effect. Time-lapse imaging demonstrated that LTX treatment caused a profound increase in Ca2+ influx and evidence of excitotoxicity, though ESNs remained viable 48 h after LTX treatment. This is the first instance of a cell-based treatment that has shown the ability to eliminate BoNT activity. These data suggest that LTX treatment may provide the basis for a new class of therapeutic approach to BoNT intoxication and may contribute to an improved understanding of long-term mechanisms of BoNT intoxication and recovery. They further demonstrate that ESNs are a novel, responsive and biologically relevant model for LTX research and BoNT therapeutic drug discovery.

From the black widow spider to human behavior: Latrophilins, a relatively unknown class of G protein‐coupled receptors, are implicated in psychiatric disorders

The findings of a recent study associate LPHN3, a member of the latrophilin family, with an increased risk of developing attention deficit/hyperactivity disorder (ADHD), the most common psychiatric disorder in childhood and adolescence. Latrophilins comprise a new family of G protein-coupled receptors of unknown native physiological function that mediate the neurotoxic effects of α-latrotoxin, a potent toxin found in black widow spider venom. This receptor-toxin interaction has helped to elucidate the mechanistic aspects of neurotransmitter and hormone release in vertebrates. Such unprecedented discovery points to a new direction in the assessment of ADHD and suggest that further study of this receptor family may provide novel insights into the etiology and treatment of ADHD and other related psychiatric conditions.

Prediction of Epitopes in Closely Related Proteins Using a New Algorithm

Latrophilin 1 (presynaptic receptor) binds α-latrotoxin from black widow spider venom and regulates neurotransmitter release from nerve endings. The study of the mechanism of action of this receptor is impeded by the existence of closely related latrophilins 2 and 3. A profile of differences detecting the most differing and identical sites in several proteins was developed in order to obtain highly specific antibodies for differentiation between isoforms of related proteins. In addition, we used an algorithm for prediction of immunogenic sites of the protein, based on the basic vector method. The peptides selected using this algorithm were used for immunization of animals. The resultant sera exhibited the estimated specificity and high affinity for the corresponding receptor forms.

Latrophilin is an essential gene and mediates the toxicity of black widow spider venom in C. elegans

Latrophilin is an essential gene and mediates the toxicity of black widow spider venom in C. elegans

Synaptic effects of low molecular weight components from Chilean Black Widow spider venom

α-Latrotoxin is the principal component of the venom from the euroasiatic Black Widow spider and has been studied for its pharmacological use as a synaptic modulator. Interestingly, smaller molecular weight fractions have been found to be associated with this toxin, but their cellular actions have not been studied in detail. The venom from the Chilean Black Widow spider ( Latrodectus mactans) does not produce α-latrotoxin, however it does contain several small polypeptides. We have recently demonstrated cellular effects of these peptides at the synaptic level using whole-cell patch clamp techniques. Purified venom from the glands of L. mactans was studied in 12 DIV rat hippocampal neuronal cultures. Venom at a concentration of 10 nM was able to decrease neuronal conductance thereby increasing membrane resistance. This effect on the passive properties of the neurons induced a change in action potential kinetics simulating the action of classic potassium channel blockers. These changes produced an increase in spontaneous synaptic activity in rat hippocampal cultures in the presence of the venom in a concentration- and time-dependent manner. These results indicate that venom from Chilean spider L. mactans is capable of increasing cell membrane resistance, prolonging the action potential and generating an increase in synaptic activity demonstrating an interesting pharmacological effect of these low molecular weight fragments.

Proteomic analysis of Latrodectus tredecimguttatus venom for uncovering potential latrodectism‐related proteins

Black widow spider is one of the most poisonous spiders in the world. Up to now, there have been few systematic analyses of the spider venom components, and the mechanism of action of the venom has not been completely understood. In this work, we employed combinative proteomic strategy to analyze the venom collected from living adult spider Latrodectus tredecimguttatus by electrical stimulation. The experiments demonstrated that the venom is primarily composed of high molecular weight proteins and has high abundance proteins around 100 kDa. The content of peptides and proteins with low molecular weight is low. A total of 75 nonredundant venom proteins with distinct function were unambiguously identified. Besides the known black widow spider venom proteins including latrotoxins, a variety of hydrolases and other proteins with special activity were found in the venom, such as proteinase, phospholipase, phosphatase, nuclease, fucolectin, venom allergen antigen 5-like protein and trypsin inhibitor, and so on. Their possible biological actions and relationship with latrodectism were discussed. The results help to understand the complexity and action mechanism of L. tredecimguttatus venom.

Solution structure and sugar-binding mechanism of mouse latrophilin-1 RBL: a 7TM receptor-attached lectin-like domain.

SummaryLatrophilin-1 (Lat-1), a target receptor for α-Latrotoxin, is a putative G protein-coupled receptor implicated in synaptic function. The extracellular portion of Lat-1 contains a rhamnose binding lectin (RBL)-like domain of unknown structure. RBL domains, first isolated from the eggs of marine species, are also found in the ectodomains of other metazoan transmembrane proteins, including a recently discovered coreceptor of the neuronal axon guidance molecule SLT-1/Slit. Here, we describe a structure of this domain from the mouse Lat-1. RBL adopts a unique α/β fold with long structured loops important for monosaccharide recognition, as shown in the structure of a complex with L-rhamnose. Sequence alignments and mutagenesis show that residues important for carbohydrate binding are often absent in other receptor-attached examples of RBL, including the SLT-1/Slit coreceptor. We postulate that this domain class facilitates direct protein-protein interactions in many transmembrane receptors.

Monoclonal antibody fragment from combinatorial phage display library neutralizes alpha-latrotoxin activity and abolishes black widow spider venom lethality, in mice

Alpha-latrotoxin ( α-ltx), a component of the venom of black widow spiders (BWSV), binds to higher vertebrates presynaptic nerve terminals, stimulating massive neurotransmitter release. This neurotoxic protein is responsible for most of the symptoms elicited in men by the bite of black widow spider (BWS), i.e. a neurological syndrome named latrodectism. By reasoning that targeting this single component would abrogate most of the effect of BWS envenomation, we took advantage of the antibody phage display technology to generate monoclonal Fab fragments able to bind and neutralize the α-ltx. To this aim, we immunized Balb/c mice with purified toxin and cloned their antibody repertoire in the pCombIII phage display vector. By combining a high-stringency affinity selection with a sensitive 45Ca 2+ uptake assay, we isolated a Fab fragment (FM1) able to bind the α-ltx in the low nM range and neutralize its ionophore activity, in vitro and in vivo. After the onset of overt symptomatology, administration of FM1 to experimentally envenomed mice induced remission of symptoms and prevented lethality. Since α-ltx is the only molecule responsible for the great toxicity of BWS bites in mammals, the FM1 Fab, highly effective in neutralizing the toxin in vivo, represents a promising immunotherapy reagent for treating latrodectic patients.

Zinc release at the synaptic terminals of rod photoreceptors

The presence of reactive zinc (Zn 2+) within photoreceptor terminals, and evidence that exogenous zinc affects the electrophysiological activity of the distal retina, led to the suggestion that its co-release with glutamate could play an essential role in the modulation of information at the first synapse in the visual pathway. Although we had shown previously that zinc release could be visualized in the region of the outer synaptic layer of a retinal slice preparation, it could not be ascertained with certainty that the release sites were at the presynaptic terminal rather than from the mitochondria-rich inner segment or from zinc within the distal processes of photoreceptors and Müller cells. Using membrane permeant and membrane impermeant forms of a fluorescent zinc indicator (Newport green), we show both the intracellular distribution of Zn 2+ and its depolarization-dependent discharge from the terminals of isolated zebrafish photoreceptors in culture. Zinc release could be detected in the dark-adapted preparation, and was further enhanced by brief exposures to black widow spider venom or high K +. Synaptically released zinc may significantly influence neural processing in the vertebrate retina by modulating the activity of excitatory and/or inhibitory receptors as well as intracellular signaling proteins.

The geometry of the ionic chànnel lumen formed by α-latroinsectotoxin from black widow spider venom in the bilayer lipid membranes

The dependence of single channel conductance formed by α-latroinsectotoxin (α-LIT) from black widow spider venom in the planar phospholipid membrane on the hydrodynamic radii of different nonelectrolytes allowed to determine the geometry of α-LIT water lumen. It was found that the cis- and trans-entrances of α-LIT channel had the same effective radii of 0.55–0.58 nm. Relatively small conductance of α-LIT channel (23.5 + 3.7 pS) in a symmetrical membrane bathing solution of 100 mM KCl (pH 7.4) may result from the constriction inside the channel with apparent radius of 0.37 nm located 32.5% of channel length away from the cis-entrance.