Search

The potent neurotoxic venom of the black widow spider contains a cocktail of seven phylum-specific latrotoxins (LTXs), but only one, α-LTX, targets vertebrates. This 130 kDa toxin binds to receptors at presynaptic nerve terminals and triggers a massive release of neurotransmitters. It is widely accepted that LTXs tetramerize and insert into the presynaptic membrane, thereby forming Ca 2+ -conductive pores, but the underlying mechanism remains poorly understood. LTXs are homologous and consist of an N-terminal region with three distinct domains, along with a C-terminal domain containing up to 22 consecutive ankyrin repeats. Here we report cryoEM structures of the vertebrate-specific α-LTX tetramer in its prepore and pore state. Our structures, in combination with AlphaFold2-based structural modeling and molecular dynamics simulations, reveal dramatic conformational changes in the N-terminal region of the complex. Four distinct helical bundles rearrange and together form a highly stable, 15 nm long, cation-impermeable coiled-coil stalk. This stalk, in turn, positions an N-terminal pair of helices within the membrane, thereby enabling the assembly of a cation-permeable channel. Taken together, these data give insight into a unique mechanism for membrane insertion and channel formation, characteristic of the LTX family, and provide the necessary framework for advancing novel therapeutics and biotechnological applications., (© 2024. The Author(s).)

Selective one-dimensional 13 C- 13 C spin-diffusion solid-state nuclear magnetic resonance (SSNMR) provides evidence for CH/π ring packing interactions between Pro and Tyr residues in 13 C-enriched Latrodectus hesperus dragline silk. The secondary structure of Pro-containing motifs in dragline spider silks consistently points to an elastin-like type II β-turn conformation based on 13 C chemical shift analysis. 13 C- 13 C spin diffusion measurements as a function of mixing times allow for the measurement of spatial proximity between the Pro and Tyr rings to be ∼0.5-1 nm, supporting strong Pro-Tyr ring interactions that likely occur through a CH/π mechanism. These results are supported by molecular dynamics (MD) simulations and analysis and reveals new insights into the secondary structure and Pro-Tyr ring stacking interactions for one of nature's toughest biomaterials.

Widow spiders are widely known for their potent venom toxins that make them among the few spiders of medical concern. The latrotoxins are the most well-studied widow toxins and include both the vertebrate-specific latrotoxins and the insect-specific latroinsectotoxins (LITs). Previous studies have shown that toxins are not limited to expression in the venom glands of adult spiders; however, gaps exist in latrotoxin screening across all life stages for brown widows, Latrodectus geometricus and southern black widows, Latrodectus mactans. In this study, we screened male and female venom gland, cephalothorax, and abdomen tissues, spiderling cephalothorax and abdomen tissues, and eggs of both L. geometricus and L. mactans, for the presence of three latrotoxins: α-latrotoxin (α-LTX), and α- and δ-latroinsectotoxins (α/δ-LITs). Widows were locally collected. Extracted RNA was used to prepare cDNA that was analyzed by PCR for the presence or absence of latrotoxin expression. Results show that expression profiles between the two species are very similar but not identical. Expression of α-LTX was found in all life stages in all tissues examined for both species. For both species, no LIT expression was detected in eggs and variable patterns of α-LIT expression were detected in spiderlings and adults. Notably, δ-LIT could only be detected in females for both species. Our results show that latrotoxin expression profiles differ within and between widow species. Data on their expression distribution provide further insight into the specific latrotoxins that contribute to toxicity profiles for each life stage in each species and their specific role in widow biology., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Latrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α-latrotoxin (α-LTX)), five specialized on insects (α, β, γ, δ, ε- latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca 2+ -conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the α-LCT monomer and the δ-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small β-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca 2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family., (© 2021. The Author(s).)

Spider silk has outstanding mechanical properties, rivaling some of the best materials on the planet. Biochemical analyses of tubuliform silk have led to the identification of TuSp1, egg case protein 1, and egg case protein 2. TuSp1 belongs to the spidroin superfamily, containing a non-repetitive N - and C -terminal domain and internal block repeats. ECP1 and ECP2, which lack internal block repeats and sequence similarities to the highly conserved N - and C -terminal domains of spidroins, have cysteine-rich N -terminal domains. In this study, we performed an in-depth proteomic analysis of tubuliform glands, spinning dope, and egg sacs, which led to the identification of a novel molecular constituent of black widow tubuliform silk, referred to as egg case protein 3 or ECP3. Analysis of the translated ECP3 cDNA predicts a low molecular weight protein of 11.8 kDa. Real-time reverse transcription-quantitative PCR analysis performed with different silk-producing glands revealed ECP3 mRNA is predominantly expressed within tubuliform glands of spiders. Taken together, these findings reveal a novel protein that is secreted into black widow spider tubuliform silk.

This paper explores the structures of exogenous protein molecules that can effectively improve the mechanical properties of silkworm silk. Several transgenic vectors fused with the silkworm fibroin light chain and type 3 repeats in different multiples of the ampullate dragline silk protein 1 (MaSp1) from black widow spider with different lengths of the polyalanine motifs were constructed for this study. Transgenic silkworms were successfully obtained by piggy Bac-mediated microinjection. Molecular detection showed that foreign proteins were successfully secreted and contained within the cocoon shells. According to the prediction of PONDR ® VSL2 and PONDR ® VL-XT, the type 3 repeats and the polyalanine motif of the MaSp1 protein were amorphous. The results of FTIR analysis showed that the content of β-sheets in the silk of transgenic silkworms engineered with transgenic vectors with additional polyalanine was significantly higher than that of wild-type silkworm silk. Additionally, silk with a higher β-sheet content had better fracture strength and Young's modulus. The mechanical properties of silk with longer chains of exogenous proteins were improved. In general, our results provide theoretical guidance and technical support for the large-scale production of excellent bionic silk.

Spider dragline silk represents a biomaterial with outstanding mechanical properties, possessing high-tensile strength and toughness. In black widows at least eight different proteins have been identified as constituents of dragline silk. These represent major ampullate spidroins MaSp1, MaSp2, MaSp', and several low-molecular weight cysteine-rich protein (CRP) family members, including CRP1, CRP2, and CRP4. Molecular modeling predicts that CRPs contain a cystine slipknot motif, but experimental evidence to support this assertion remains to be reported. To advance scientific knowledge regarding CRP function, we recombinantly expressed and purified CRP1 and CRP4 from bacteria and investigated their secondary structure using circular dichroism (CD) under different chemical and physical conditions. We demonstrate by far-UV CD spectroscopy that these proteins contain similar secondary structure, having substantial amounts of random coil conformation, followed by lower levels of beta sheet, alpha helical and beta turn structures. CRPs are thermally and pH stable; however, treatment with reagents that disrupt disulfide bonds impact their structural conformations. Cross-linking mass spectrometry (XL-MS) data also support computational models of CRP1. Taken together, the chemical and thermal stability of CRPs, the cross-linking data, coupled with the structural sensitivity to reducing agents, are experimentally consistent with the supposition CRPs are cystine slipknot proteins.

Latrodectus species are among the most venomous of spiders, with abundant toxic proteinaceous components in their venomous glands and other tissues, as well as their eggs. To date, several proteinaceous toxins with insecticidal potential, including α-insectotoxin and δ-insectotoxin, two of the most potent known insecticidal toxins, have been purified and characterized by comprehensively utilizing conventional biochemical techniques. This has greatly enhanced our knowledge of the molecular basis and mechanism of action of their toxicity. Application of proteomic and transcriptomic techniques further revealed the synergistic action of multiple Latrodectus proteinaceous toxins and toxin-like components. Insecticidal toxins from Latrodectus spiders have great potential in insect pest control; however, more studies are needed to further reveal their mechanisms of action and understand their structures and properties before any practical application, for example, the insecticidal toxin-containing fusion proteins with oral activity. Here, we review current knowledge of the molecular basis and mechanism of action underlying the insecticidal activity of venoms and toxins from Latrodectus spiders, and examine their potential application in insect pest control. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Many natural silks produced by spiders and insects are unique materials in their exceptional toughness and tensile strength, while being lightweight and biodegradable-properties that are currently unparalleled in synthetic materials. Myriad approaches have been attempted to prepare artificial silks from recombinant spider silk spidroins but have each failed to achieve the advantageous properties of the natural material. This is because of an incomplete understanding of the in vivo spidroin-to-fiber spinning process and, particularly, because of a lack of knowledge of the true morphological nature of spidroin nanostructures in the precursor dope solution and the mechanisms by which these nanostructures transform into micrometer-scale silk fibers. Herein we determine the physical form of the natural spidroin precursor nanostructures stored within spider glands that seed the formation of their silks and reveal the fundamental structural transformations that occur during the initial stages of extrusion en route to fiber formation. Using a combination of solution phase diffusion NMR and cryogenic transmission electron microscopy (cryo-TEM), we reveal direct evidence that the concentrated spidroin proteins are stored in the silk glands of black widow spiders as complex, hierarchical nanoassemblies (∼300 nm diameter) that are composed of micellar subdomains, substructures that themselves are engaged in the initial nanoscale transformations that occur in response to shear. We find that the established micelle theory of silk fiber precursor storage is incomplete and that the first steps toward liquid crystalline organization during silk spinning involve the fibrillization of nanoscale hierarchical micelle subdomains., Competing Interests: The authors declare no conflict of interest.

The outstanding material properties of spider dragline silk fibers have been attributed to two spidroins, major ampullate spidroins 1 and 2 (MaSp1 and MaSp2). Although dragline silk fibers have been treated with different chemical solvents to elucidate the relationship between protein structure and fiber mechanics, there has not been a comprehensive proteomic analysis of the major ampullate (MA) gland, its spinning dope, and dragline silk using a wide range of chaotropic agents, inorganic salts, and fluorinated alcohols to elucidate their complete molecular constituents. In these studies, we perform in-solution tryptic digestions of solubilized MA glands, spinning dope and dragline silk fibers using five different solvents, followed by nano liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis with an Orbitrap Fusion™ Tribrid™. To improve protein identification, we employed three different tryptic peptide fragmentation modes, which included collision-induced dissociation (CID), electron transfer dissociation (ETD), and high energy collision dissociation (HCD) to discover proteins involved in the silk assembly pathway and silk fiber. In addition to MaSp1 and MaSp2, we confirmed the presence of a third spidroin, aciniform spidroin 1 (AcSp1), widely recognized as the major constituent of wrapping silk, as a product of dragline silk. Our findings also reveal that MA glands, spinning dope, and dragline silk contain at least seven common proteins: three members of the Cysteine-Rich Protein Family (CRP1, CRP2 and CRP4), cysteine-rich secretory protein 3 (CRISP3), fasciclin and two uncharacterized proteins. In summary, this study provides a proteomic blueprint to construct synthetic silk fibers that most closely mimic natural fibers., Competing Interests: The authors declare no conflict of interest.

The eggs of oviparous animals are storehouses of maternal proteins required for embryonic development. Identification and molecular characterization of such proteins will provide much insight into the regulation of embryonic development. We previously analyzed soluble proteins in the eggs of the black widow spider (Latrodectus tredecimguttatus), and report here on the extraction and mass spectrometric identification of the egg membrane proteins. Comparison of different lysis solutions indicated that the highest extraction of the membrane proteins was achieved with 3%-4% sodium laurate in 40 mmol/L Tris-HCl buffer containing 4% CHAPS and 2% DTT (pH 7.4). SDS-PAGE combined with nLC-MS/MS identified 39 proteins with membrane-localization annotation, including those with structural, catalytic, and regulatory activities. Nearly half of the identified membrane proteins were metabolic enzymes involved in various cellular processes, particularly energy metabolism and biosynthesis, suggesting that relevant metabolic processes were active during the embryonic development of the eggs. Several identified cell membrane proteins were involved in the special structure formation and function of the egg cell membranes. The present proteomic analysis of the egg membrane proteins provides new insight into the molecular mechanisms of spider embryonic development.

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

Latrodectus tredecimguttatus (commonly known as black widow spiders) have toxins not only in their venom glands, but also in other parts of their body, in their eggs and even in the newborn spiderlings. The study on the toxins in venom and materials outside the venom glands of the spiders to elucidate their differences and similarities, evolutional relationship and biological functions is of important theoretical and applicable significance. The development of modern protein chemistry and proteomics techniques has provided efficient means for the study of protein and peptide toxins of L. tredecimguttatus. By using such techniques, the molecular base and action mechanism of the toxins can be revealed at the levels of both single purified proteins and omics. Up to now, although protein chemistry and proteomics study on L. tredecimguttatus toxins have achieved a certain progress, the relevant work particularly that on the toxins in the materials outside the venom glands has to be further deepened.

Background: Black widow spider (L. tredecimguttatus) has toxic components not only in the venomous glands, but also in other parts of the body and its eggs. It is biologically important to investigate the molecular basis of the egg toxicity., Results: In the present work, an aqueous extract was prepared from the eggs of the spider and characterized using multiple physiological and biochemical strategies. Gel electrophoresis and mass spectrometry demonstrated that the eggs are rich in high-molecular-mass proteins and the peptides below 5 kDa. The lyophilized extract of the eggs had a protein content of 34.22% and was shown to have a strong toxicity towards mammals and insects. When applied at a concentration of 0.25 mg/mL, the extract could completely block the neuromuscular transmission in mouse isolated phrenic nerve-hemidiaphragm preparations within 12.0 ± 1.5 min. Using whole-cell patch-clamp technique, the egg extract was demonstrated to be able to inhibit the voltage-activated Na+, K+ and Ca2+ currents in rat DRG neurons. In addition, the extract displayed activities of multiple hydrolases. Finally, the molecular basis of the egg toxicity was discussed., Conclusions: The eggs of black widow spiders are rich in proteinous compounds particularly the high-molecular-mass proteins with different types of biological activity The neurotoxic and other active compounds in the eggs are believed to play important roles in the eggs' toxic actions.

The early research found that the spiderlings of black widow spider (Latrodectus tredecimguttatus) exhibited obvious toxicity to animals. The present work performed a systematical analysis of the aqueous extract of newborn black widow spiderlings. The extract was shown to contain 69.42% of proteins varying in molecular weights and isoelectric points. Abdominal injection of the extract into mice and cockroaches caused obvious poisoning symptoms as well as death, with LD50 being 5.30 mg/kg in mice and 16.74 µg/g in Periplaneta americana. Electrophysiological experiments indicated that the extract at a concentration of 10 µg/mL could completely block the neuromuscular transmission in isolated mouse nerve-hemidiaphragm preparations within 21 ± 1.5 min, and 100 µg/mL extract could inhibit a certain percentage of voltage-activated Na⁺, K⁺, and Ca²⁺ channel currents in rat dorsal root ganglion neurons. These results demonstrate that the spiderlings are rich in neurotoxic components, which play important roles in the spiderling toxicity., (© 2014 Wiley Periodicals, Inc.)

The eggs of black widow spider (L. tredecimguttatus) have been demonstrated to be rich in biologically active components that exhibit great research value and application foreground. In the present study, a protein toxin, named Latroeggtoxin-II, was isolated from the eggs using the combination of gel filtration, ion exchange chromatography and reversed-phase high performance liquid chromatography. Electrospray mass spectrometric analysis indicated that the molecular weight of the protein was 28.69 kDa, and Edman degradation revealed that its N-terminal sequence was ESIQT STYVP NTPNQ KFDYE VGKDY-. After being abdominally injected into mice and P. americana, the protein could make the animals especially P. americana display a series of poisoning symptoms. Electrophysiological experiments demonstrated that the protein could selectively inhibit tetrodotoxin-resistant Na(+) channel currents in rat dorsal root ganglion neurons, without significant effect on the tetrodotoxin-sensitive Na(+) channel currents. Using multiple proteomic strategies, the purified protein was shown to have only a few similarities to the existing proteins in the databases, suggesting that it was a novel protein isolated from the eggs of black widow spiders., (Copyright © 2014. Published by Elsevier B.V.)

This study provides a detailed secondary structural characterization of major ampullate dragline silk from Latrodectus hesperus (black widow) spiders. X-ray diffraction results show that the structure of black widow major ampullate silk fibers is comprised of stacked β-sheet nanocrystallites oriented parallel to the fiber axis and an amorphous region with oriented (anisotropic) and isotropic components. The combination of two-dimensional (2D) (13)C-(13)C through-space and through-bond solid-state NMR experiments provide chemical shifts that are used to determine detailed information about the amino acid motif secondary structure in black widow spider dragline silk. Individual amino acids are incorporated into different repetitive motifs that make up the majority of this protein-based biopolymer. From the solid-state NMR measurements, we assign distinct secondary conformations to each repetitive amino acid motif and, hence, to the amino acids that make up the motifs. Specifically, alanine is incorporated in β-sheet (poly(Alan) and poly(Gly-Ala)), 3(1)-helix (poly(Gly-Gly-Xaa), and α-helix (poly(Gln-Gln-Ala-Tyr)) components. Glycine is determined to be in β-sheet (poly(Gly-Ala)) and 3(1)-helical (poly(Gly-Gly-X(aa))) regions, while serine is present in β-sheet (poly(Gly-Ala-Ser)), 3(1)-helix (poly(Gly-Gly-Ser)), and β-turn (poly(Gly-Pro-Ser)) structures. These various motif-specific secondary structural elements are quantitatively correlated to the primary amino acid sequence of major ampullate spidroin 1 and 2 (MaSp1 and MaSp2) and are shown to form a self-consistent model for black widow dragline silk.

Up to now, there have been a few reports on the toxic components purified from black widow spider (Latrodectus tredecimguttatus) eggs. In the present study, a novel neurotoxic protein was purified from the eggs by gel filtration combined with ion-exchange chromatography. Its molecular weight was 23.752 kDa determined by electrospray mass spectrometry. The protein could block the neuromuscular transmission in mouse-isolated phrenic nerve-hemidiaphragm preparations completely in a reversible manner and activate tetrodotoxin-sensitive sodium current in rat dorsal root ganglion cells. The N-terminal sequence of the protein was identified by the Edman degradation to be N-S-I-A-D-D-R-Y-R-W-P-G-Y-P-G-A-G-L-I-P-Y-I-I-D-S-. When the sequence was used to search against protein database with a sequence query in Mascot engine there was no matched sequence or protein whereas the Basic Local Alignment Search Tool (BLAST) analysis indicated that no significant similarity was found. These results demonstrated that the protein (named Latroeggtoxin-I) is a novel neurotoxic protein purified from the eggs of black widow spiders., (© 2013 Wiley Periodicals, Inc.)

Adhesive spider glues are required to perform a variety of tasks, including web construction, prey capture, and locomotion. To date, little is known regarding the molecular and structural features of spider glue proteins, in particular bioadhesives that interconnect dragline or scaffolding silks during three-dimensional web construction. Here we use biochemical and structural approaches to identify and characterize two aggregate gland specific gene products, AgSF1 and AgSF2, and demonstrate that these proteins co-localize to the connection joints of both webs and wrapping silks spun from the black widow spider, Latrodectus hesperus. Protein architectures are markedly divergent between AgSF1 and AgSF2, as well as traditional spider silk fibroin family members, suggesting connection joints consist of a complex proteinaceous network. AgSF2 represents a nonglycosylated 40-kDa protein that has novel internal amino acid block repeats with the consensus sequence NVNVN embedded in a glycine-rich matrix. Analysis of the amino acid sequence of AgSF1 reveals pentameric QPGSG iterations that are similar to conserved modular elements within mammalian elastin, a rubber-like elastomeric protein that interfaces with collagen. Wet-spinning methodology using purified recombinant proteins show AgSF1 has the potential to self-assemble into fibers. X-ray fiber diffraction studies performed on these synthetic fibers reveal the presence of noncrystalline domains that resemble classical rubber networks. Collectively, these data support that the aggregate gland serves to extrude a protein mixture that contains substances that allow for the self-assembly of fiber-like structures that interface with dragline silks to mediate prey capture.

The investigation was undertaken to study the biology and ecology of Latrodectus, the possibilities of its importation to Russia from other countries, to breed Latrodectus in the laboratory setting, and to design the first homeopathic matrix of Latrodectus to manufacture homeopathic remedies. The authors were the first to devise a method for Latrodectus breeding in the laboratory setting of Moscow and its vicinities. The Latrodectus bred in the laboratory is suitable to manufacture drugs and in captivity they do not lose its biological activity. The authors were the first to prepare a homeopathic Latrodectus matrix for homeopathic medicines, by using the new Russian extragent petroleum. Chromatography mass spectrometry was used to identify more than a hundred chemical compounds in the Russian petroleum. The biological activity of the petroleum Latrodectus matrix for the manufacture of homeopathic remedies was highly competitive with that of the traditional Latrodectus venom matrix made using ethyl alcohol. The homeopathic Latrodectus matrix made using glycerol lost its biological activity because of glycerol. The biological activity of homeopathic matrixes made from Latrodectus inhabiting the USA, Uzbekistan, and the south of Russia and from that bred in the laboratory was studied. The homeopathic matrix made from the Latrodectus living in the Samarkand Region, Republic of Uzbekistan, has the highest biological activity.

The extragent used to prepare a Latrodectus mactans hydrocarbon extract is a multicomponent system composed of alkanes, alkenes, and arenes. More than 100 compounds were identified in the hydrocarbon extract (petroleum). The petroleum matrix of Latrodectus mactans was first obtained to manufacture homeopathic remedies. The authors could prepare the first Russian homeopathic medicine from Latrodectus mactans, which proved to be effective in treating canine eclampsia. Canine experiments provide a rationale for the authors' choice as the only homeopathic remedy among thousand known drugs to treat female eclampsia. It is Latrodectus mactans that is in the list of homeopathic medicines permitted for use in accordance with Order No. 335 (Supplement 2) of the Ministry of Health and Medical Industry of Russia, issued on November 29, 1995. It is manufactured from Latrodectus mactans living in the USA.

The authors have detected atypical paecilomycosis-associated myocarditis with impaired amino acid exchange and pain syndrome for the first time. At first, pain occurs in the chest and radiates into the axilla, to the left arm to the finger tips, by paralyzing the arm. In some patients, pain manifests itself in both arms with radiation to the belly, by accompanying by fainting. The skin is wet, cold; the pulse is frequent and of poor volume and difficult-to-count. Heart pain spreads into the armpit and down the arm, by making the fingers numb. Attempts to use current analgesics (movalis, sirdalud, nimesil, morphine) in combination with fungicides (diflucan, mycosist, orungal) have failed to yield positive results. The homeopathic drug Latrodectus mactans, prepared from caracurt venom, in combination with the authors' designed diet and other homeopathic agents have relieved pain syndrome and normalized amino acid exchange, which offered possibilities for successful surgical treatment for echinococcosis with later recovery.

α-Latrotoxin (α-LTX) is known to cause massive exocytosis from presynaptic nerve terminals. We investigated the effects of α-LTX on exocytotic release from mast cells, typical non-neuronal secretory cells. When we transfected mast cells with latrophilin, a specific receptor for α-LTX, α-LTX caused intracellular Ca(2+) to increase and led to exocytosis in the presence of extracellular Ca(2+). On the other hand, neither Ca(2+) increase nor exocytosis was observed in the absence of extracellular Ca(2+). These results indicate that α-LTX, together with latrophilin, works as a Ca(2+) ionophore. However, α-LTX had additional effects on signal transduction in mast cells. We found that inhibitors of protein kinase C (PKC) partially suppressed exocytosis. Furthermore, several soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including SNAP-23, were phosphorylated by α-LTX. These results suggest that exocytosis induced by α-LTX can be explained by (1) elevation of intracellular Ca(2+), (2) phosphorylation of SNARE proteins including SNAP-23, syntaxin-4 and VAMP-8 through PKC-dependent and -independent pathways. Our study may provide a new system to investigate the action of α-LTX and the mechanism of exocytosis in mast cells., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Alpha-latrotoxin induces neurotransmitter release by stimulating synaptic vesicle exocytosis via two mechanisms: (1) A Ca(2+)-dependent mechanism with neurexins as receptors, in which alpha-latrotoxin acts like a Ca(2+) ionophore, and (2) a Ca(2+)-independent mechanism with CIRL/latrophilins as receptors, in which alpha-latrotoxin directly stimulates the transmitter release machinery. Here, we show that the Ca(2+)-independent release mechanism by alpha-latrotoxin requires the synaptic SNARE-proteins synaptobrevin/VAMP and SNAP-25, and, at least partly, the synaptic active-zone protein Munc13-1. In contrast, the Ca(2+)-dependent release mechanism induced by alpha-latrotoxin does not require any of these components of the classical synaptic release machinery. Nevertheless, this type of exocytotic neurotransmitter release appears to fully operate at synapses, and to stimulate exocytosis of the same synaptic vesicles that participate in physiological action potential-triggered release. Thus, synapses contain two parallel and independent pathways of Ca(2+)-triggered exocytosis, a classical, physiological pathway that operates at the active zone, and a novel reserve pathway that is recruited only when Ca(2+) floods the synaptic terminal.

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.

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 silk proteins are well-known for their extraordinary mechanical properties, displaying remarkable strength and toughness. In this study, matrix-assisted laser desorption ionization (MALDI) tandem time-of-flight (TOF) mass spectrometry (MS/MS) and reverse genetics were used to isolate a new cDNA sequence that encodes for a protein assembled into egg case silk from the black widow spider, Latrodectus hesperus. Analysis of the primary sequence of this protein reveals approximately 52% identity to the egg case protein 1 (ECP-1) fibroin-like family member. On the basis of the similarity in the primary sequence and expression pattern, we have named this factor egg case protein 2 (ECP-2). Alignments of ECP-1 and ECP-2 demonstrate highly conserved N termini, with 16 Cys residues found within the first 153 amino acids. Traditional ensemble repeats found within reported fibroins were poorly represented in the primary sequence of ECP-2, but scattered blocks of polyalanine were present, along with a C terminus rich in GA repeats. Reverse transcription quantitative PCR analysis showed that ECP-2 is predominantly expressed in the tubuliform gland. Relative to ECP-1, ECP-2 mRNA levels were determined to be >2-fold higher. MALDI MS/MS analysis of peptide fragments generated from the large-diameter core fiber after enzymatic digestion and acid hydrolysis demonstrated the presence of a fiber that is trimeric in nature, containing tubuliform spidroin 1 (TuSp1), ECP-1, and ECP-2. We also report an additional primary sequence for TuSp1, demonstrating that TuSp1 contains two Cys residues within a nonrepetitive N-terminal region. In combination with the distinctive protein architectures of ECP-1 and ECP-2, along with their co-localization with TuSp1 in the core fiber, our findings suggest that ECP-1 and ECP-2 play important structural roles in the egg case silk fiber.

Spider silks are among the strongest and toughest known materials, but investigation of these remarkable properties has been confined largely to orb-weaving spiders. We investigated the mechanical performance of silk from the cobweb-weaving spider Latrodectus hesperus. Both silk from the scaffolding region of the web and sticky gumfooted capture lines had material properties similar to the major ampullate silk that orb weavers use as the framework for their orb webs. Major ampullate fibers obtained from anaesthetized Latrodectus spiders were similar, but exhibited increased stiffness and reduced extensibility. Novel continuous dynamic analysis of the silks revealed that the loss tangent (tandelta) increased rapidly during the first 2-3% of extension and reached a maximum near the yield point of fibers. The loss tangent then rapidly declined at an ever-decreasing rate until failure. We suggest that these data support molecular models for the mechanics of spider silk. We also demonstrate that the addition of sticky aggregate glue to the ends of the gumfooted lines modulates their mechanical performance--reducing stiffness and increasing extensibility. The storage modulus of viscid regions of the gumfooted lines was much lower than dry regions. This may be explained by disruption of hydrogen bonding within the amorphous regions of the fibers due to hydration from the glue.

Molecular and material properties of major ampullate silk were studied for the cobweb-building black widow spider Latrodectus hesperus. Material properties were measured by stretching the silk to breaking. The strength was 1.0 +/- 0.2 GPa, and the extensibility was 34 +/- 8%. The secondary structure of the major ampullate silk protein was studied using carbon-13 NMR spectroscopy. Alanine undergoes a transition from a coiled structure in pre-spun silk to a beta sheet structure in post-spun silk. We have also isolated two distinct cDNAs (both about 500 bp) which encode proteins similar to major ampullate spidroin 1 and 2 (MaSp1 and MaSp2). The MaSp1-like silk contains polyalanine runs of 5-10 residues as well as GA and GGX motifs. The MaSp2-like silk contains polyalanine runs of varying length as well as GPG(X)(n) motifs. L. hesperus major ampullate silk is more like major ampullate silk from other species than other L. hesperus silks.

The black widow spider venom contains several large protein toxins--latrotoxins--that are selectively targeted against different classes of animals: vertebrates, insects, and crustaceans. These toxins are synthesised as large precursors that undergo proteolytic processing and activation in the lumen of the venom gland. The mature latrotoxins demonstrate strong functional structure conservation and contain multiple ankyrin repeats, which mediate toxin oligomerisation. The three-dimensional structure has been determined for alpha-latrotoxin (alphaLTX), a representative venom component toxic to vertebrates. This reconstruction explains the mechanism of alphaLTX pore formation by showing that it forms tetrameric complexes, harbouring a central channel, and that it is able to insert into lipid membranes. All latrotoxins cause massive release of neurotransmitters from nerve terminals of respective animals after binding to specific neuronal receptors. A G protein-coupled receptor latrophilin and a single-transmembrane receptor neurexin have been identified as major high-affinity receptors for alphaLTX. Latrotoxins act by several Ca(2+)-dependent and -independent mechanisms based on pore formation and activation of receptors. Mutant recombinant alphaLTX that does not form pores has been used to dissect the multiple actions of this toxin. As a result, important insights have been gained into the receptor signalling and the role of intracellular Ca(2+) stores in the effect of alphaLTX.

We report here the first three-dimensional structure of alpha-latrotoxin, a black widow spider neurotoxin, which forms membrane pores and stimulates secretion in the presence of divalent cations. We discovered that alpha-latrotoxin exists in two oligomeric forms: it is dimeric in EDTA but forms tetramers in the presence of Ca2+ or Mg2+. The dimer and tetramer structures were determined independently at 18 A and 14 A resolution, respectively, using cryo-electron microscopy and angular reconstitution. The alpha-latrotoxin monomer consists of three domains. The N- and C-terminal domains have been identified using antibodies and atomic fitting. The C4-symmetric tetramers represent the active form of alpha-latrotoxin; they have an axial channel and can insert into lipid bilayers with their hydrophobic base, providing the first model of alpha-latrotoxin pore formation.

We present the material analysis of scaffolding silk from the cobweb of the black widow spider Latrodectus hesperus. 30 strands were tested from the webs of nine spiders. Strands were stretched at 0.211 mm/s as force and extension were recorded. Cross-sectional area was measured under 1000 x oil-immersion light microscopy. The stress strain curve shows that cobweb silk is a distinct material from other known spider silks. The average breaking point for this cobweb silk is 1.1 +/- 0.5 GPa at 0.22 +/- 0.05 strain. All samples increased stiffness as they were stretched, but to different extents. Variation in stiffness might be due to differential crystallization or alignment of the silk proteins during stretching.

alpha-Latrotoxin is a presynaptic neurotoxin isolated from the venom of the black widow spider Latrodectus tredecimguttatus. It exerts toxic effects in the vertebrate central nervous system by depolarizing neurons, by increasing [Ca2+]i and by stimulating uncontrolled exocytosis of neurotransmitters from nerve terminals. The actions of alpha-latrotoxin are mediated, in part, by a GTP-binding protein-coupled receptor referred to as CIRL or latrophilin. Exendin-4 is also a venom toxin, and it is derived from the salivary gland of the Gila monster Heloderma suspectum. It acts as an agonist at the receptor for glucagon-like peptide-1(7-36)-amide (GLP-1), thereby stimulating secretion of insulin from pancreatic beta-cells of the islets of Langerhans. Here is reported a surprising structural homology between alpha-latrotoxin and exendin-4 that is also apparent amongst all members of the GLP-1-like family of secretagogic hormones (GLP-1, glucagon, vasoactive intestinal polypeptide, secretin, pituitary adenylyl cyclase activating polypeptide). On the basis of this homology, we report the synthesis and initial characterization of a chimeric peptide (Black Widow GLP-1) that stimulates Ca2+ signaling and insulin secretion in human beta-cells and MIN6 insulinoma cells. It is also reported here that the GTP-binding protein-coupled receptors for alpha-latrotoxin and exendin-4 share highly significant structural similarity in their extracellularly-oriented amino-termini. We propose that molecular mimicry has generated conserved structural motifs in secretagogic toxins and their receptors, thereby explaining the evolution of defense or predatory strategies that are shared in common amongst distantly related species including spiders, lizards, and snakes. Evidently, the toxic effects of alpha-latrotoxin and exendin-4 are explained by their ability to interact with GTP-binding protein-coupled receptors that normally mediate the actions of endogenous hormones or neuropeptides.

Toxic components of the Latrodectus pallidus spider venom were isolated and characterized. The venom was shown to contain a toxin specific for mammals and at least one insectospecific toxin. Partial amino acid sequences of both toxins were determined, and their high structural homology with previously studied alpha-latrotoxin and alpha-latroinsectotoxin from L. mactans tredecimguttatus was found.

A cDNA encoding a polypeptide of 88 amino acids was cloned following the rapid amplification of cDNA ends (RACE) procedure using mRNA isolated from the venom glands of the Mediterranean black widow spider (Latrodectus tredecimguttatus) and oligonucleotides based on the sequence of a tryptic fragment putatively from alpha-latrotoxin. Apart from a potential signal peptide, the rest of this small protein, named latrodectin, was highly hydrophilic, having a calculated molecular mass of 7945 Da and a pI of 4.3. Northern-blot analysis showed that the mRNA was specifically expressed in the venom gland of L. tredecimguttatus and that it was well conserved between two geographically remote species (L. geometricus and L. indistinctus). A polyclonal serum raised in rabbits against the C-terminal sequence of latrodectin detected cross-reactive proteins in the venom fluid, venom gland extracts, and in purified alpha-latrotoxin, suggesting that latrodectin is intimately associated with alpha-latrotoxin. Finally, we produced a recombinant protein in a cell system infected with baculovirus and developed an immunoaffinity purification procedure for latrodectin to facilitate further structural and functional analyses of the molecule.

We have studied the action of an alpha-latrotoxin (alpha-LTX) complex of two polypeptides (LTX 130 kDa and low molecular weight protein (LMWP) 8 kDa) and the action of a venom fraction containing LTX with excess LMWP on calcium influx into synaptosomes and PC12 cells as well as on [14C]GABA release from synaptosomes. Both preparations considerably activate calcium influx and stimulate [14C]GABA release from synaptosomes. Preincubation of both preparations with antibodies against a 14 amino acid residue C-terminal peptide of LMWP differentially modulates these effects. Antibodies inhibit induced calcium influx and enhance induced GABA release.