Spider Venoms and Spider Toxins

Abstract

Spider venoms and toxins are useful tools for the study of ion channels and synaptic functions of neurons in vertebrates and invertebrates. The components of spider venom, such as proteins, peptides, polyamines and bioamines, are species-specific. The various functions of these toxins are reviewed in this paper.     

Recent Advances in Research on Widow Spider Venoms and Toxins

Widow spiders have received much attention due to the frequently reported human and animal injures caused by them. Elucidation of the molecular composition and action mechanism of the venoms and toxins has vast implications in the treatment of latrodectism and in the neurobiology and pharmaceutical research. In recent years, the studies of the widow spider venoms and the venom toxins, particularly the α-latrotoxin, have achieved many new advances; however, the mechanism of action of the venom toxins has not been completely clear. The widow spider is different from many other venomous animals in that it has toxic components not only in the venom glands but also in other parts of the adult spider body, newborn spiderlings, and even the eggs. More recently, the molecular basis for the toxicity outside the venom glands has been systematically investigated, with four proteinaceous toxic components being purified and preliminarily characterized, which has expanded our understanding of the widow spider toxins. This review presents a glance at the recent advances in the study on the venoms and toxins from the Latrodectus species.     

Hemorrhagic Toxins from Snake Venoms

Abstract

One of the more dramatic consequences of envenomation by crotalid and viperid snakes is the occurrence of hemorrage. In cases where the envenomation is less severe, the hemorrhagic is generally observed to be localized at the site of the bite. However, hemorrhage can be found disseminated through a substantial area of the involved extremity. In cases where the envenomation is severe, bleeding in organs such as heart, lungs, kidneys and brain may also occur. From the biochemical investigations on these toxins over the past 30 years, the nature of the venom toxins and their mechanism of activity are now becoming clear. Virtually all of the hemorrhagic toxins isolated and characterized thus far have been determined to be metalloproteinases. In this review we discuss the history of the isolation and characterization of these toxins in an attempt to clarify some of the confusion surrounding these toxins and their biochemical activities. We also survey the data available on the natural and synthetic inhibitors against the toxins. Finally, based upon the literature, we propose possible biochemical mechanisms which may give rise to the hemorrhagic pathology associated with crotalid/viperid envenomation.     

A Short Review of the Venoms and Toxins of Spider Wasps (Hymenoptera: Pompilidae)

Parasitoid wasps represent the plurality of venomous animals, but have received extremely little research in proportion to this taxonomic diversity. The lion’s share of investigation into insect venoms has focused on eusocial hymenopterans, but even this small sampling shows great promise for the development of new active substances. The family Pompilidae is known as the spider wasps because of their reproductive habits which include hunting for spiders, delivering a paralyzing sting, and entombing them in burrows with one of the wasp’s eggs to serve as food for the developing larva. The largest members of this family, especially the tarantula hawks of the genus Pepsis, have attained notoriety for their large size, dramatic coloration, long-term paralysis of their prey, and incredibly painful defensive stings. In this paper we review the existing research regarding the composition and function of pompilid venoms, discuss parallels from other venom literatures, identify possible avenues for the adaptation of pompilid toxins towards human purposes, and future directions of inquiry for the field.     

Conus Venoms: A Rich Source of Neuroactive Peptides

Abstract

Purification of toxins from the venoms of two fish-hunting gastropod cone snails, Conus geographus and Conus magus has revealed the presence of three classes of paralytic peptide toxins. These are: 1) the w-conotoxins, which block voltage activated calcium channels at the presynaptic terminus; 2) the α-conotoxins, which block the acetylcholine receptor and 3) the ω-conotoxins, which inhibit muscle sodium channels, and therefore prevent propagation of the muscle action potential. These toxins are basic peptides from 13–27 amino acids long, rich in cystine residues which are present as disulfides. A number of α-conotoxins and one a-conotoxin have been chemically synthesized.

In addition to the paralytic conotoxins, the venoms of Conus have other toxins which have not yet been completely characterized. A large number of neuroactive peptides and proteins have also been found. Since there are approximately 300 species of Conus, all of which produce venoms, the cone snails promise to be a rich source of neuroactive peptides in the years ahead.     

Toxins from Mamba Venoms that Facilitate Neuroiluscular Transmission

Abstract

Venoms from the four species of African mambas have many neurotoxins that are different from other snake neurotoxins. Postjunctional α-neurotoxins, which bind to nicotinic cholinoceptors, appear to be the only type of toxin that mambas have in common with other snakes with neurotoxic venoms. Typical mamba neurotoxins are prejunctional facilitatory toxins and anticholinesterase toxins or fasciculins.

The prejunctional facilitatory toxins enhance the amount of transmitter that is released in response to nerve stimulation. This activity was first demonstrated with dendrotoxin (from Dendroaspis anqusticeps) at the neuromuscular junction, but it can also be observed in both the sympathetic and parasympathetic branches of the autonomic nervous system and in the central nervous system. These facilitatory neurotoxins have 57-60 amino acids in a single polypeptide chain cross-linked by three disulphide bonds. They are structurally homologous to the Kunitz type protease inhibitors such as bovine pancreatic trypsin inhibitor.

The anticholinesterase toxins are specific and powerful in inhibitors (K_i of about 10^?10 M) of acetylcholinesterase from several sources (human erythrocytes, rat brain and muscle, eel electroplax) but they have no effect on acetylcholinesterase from cobra venom or chick brain and muscle. The first two of these inhibitors (from Dendroaspis angusticeps) were called fasciculins because of the long-lasting muscle fasciculations that they produced in mice. Fasciculins have 61 amino acids and four disulphides and show sequence homology with the postjunctional short neurotoxins.

Synergistic interactions between components are characteristic of mamba venoms. One of these components is always a member of the so-called “angusticeps-type” toxins (short neurotoxin homologues of 58-60 amino acids and four disulphides). These toxins are divided into four subgroups, the anticholinesterases constituting subgroup I. Angusticeps-type toxins interact with facilitatory toxins or with so-called “synergistic-type” proteins (toxins of two subunits, each with 62-63 amino acids and joined together by disulphide bonds). The pharmacology of these interactions is not known, only data on the increased lethality of the synergistic mixtures is available.     

Development of a Broad-Spectrum Antiserum against Cobra Venoms Using Recombinant Three-Finger Toxins

Three-finger toxins (3FTXs) are the most clinically relevant components in cobra (genus Naja) venoms. Administration of the antivenom is the recommended treatment for the snakebite envenomings, while the efficacy to cross-neutralize the different cobra species is typically limited, which is presumably due to intra-specific variation of the 3FTXs composition in cobra venoms. Targeting the clinically relevant venom components has been considered as an important factor for novel antivenom design. Here, we used the recombinant type of long-chain α-neurotoxins ({"type":"entrez-protein","attrs":{"text":"P01391","term_id":"128930"}}P01391), short-chain α-neurotoxins ({"type":"entrez-protein","attrs":{"text":"P60770","term_id":"46397632"}}P60770), and cardiotoxin A3 ({"type":"entrez-protein","attrs":{"text":"P60301","term_id":"41688806"}}P60301) to generate a new immunogen formulation and investigated the potency of the resulting antiserum against the venom lethality of three medially important cobras in Asia, including the Thai monocled cobra (Naja kaouthia), the Taiwan cobra (Naja atra), and the Thai spitting cobra (Naja Siamensis) snake species. With the fusion of protein disulfide isomerase and the low-temperature settings, the correct disulfide bonds were built on these recombinant 3FTXs (r3FTXs), which were confirmed by the circular dichroism spectra and tandem mass spectrometry. Immunization with r3FTX was able to induce the specific antibody response to the native 3FTXs in cobra venoms. Furthermore, the horse and rabbit antiserum raised by the r3FTX mixture is able to neutralize the venom lethality of the selected three medically important cobras. Thus, the study demonstrated that the r3FTXs are potential immunogens in the development of novel antivenom with broad neutralization activity for the therapeutic treatment of victims involving cobra snakes in countries.     

Precious Natural Peptides from Spider Venoms: New Tools for Studying Potassium Channels

Among the large variety of animal toxins that target potassium channels, spider peptides constitute an unique class of voltage-dependent K⁺ (Kv) current inhibitors according to their structure and pharmacological properties. Spider toxins that block Kv currents are small basic peptides the include three disuflide bridges and belong to the family of inhibitor cystine knot (ICK) molecules. Unlike snake, bee, scorpion, or sea anemone toxins that block Kv1 or Kv3 channels, ICK spider toxins target Kv2 and Kv4 channels, which are expressed in the central nervous system (CNS) and in the cardiovascular system. Their selective affinities for Kv2 and/or Kv4 subfamilies are very useful for dissecting these currents in neuronal and cardiac cells and for the determination of their contribution in physiological processes. Their mode of action is also original, since they induce a shift of channel opening to more depolarized potentials that alter the voltage-dependent properties of K currents. Then they are called gating modifiers. Structure-function studies of these gating modifiers were recently facilitated by solving their tridimensional structure together with the crystallization of prokaryotic K⁺ channels. Spider toxins present an active molecular surface, including a hydrophobic patch surrounded by charged residues, which are important for their binding on Kv channels. Gating modifiers interact with important residues in the S3C-S4 external loop via both hydrophobic and electrostatic interactions. Several dynamic interaction models were proposed, but all of them remain putative.     

An Overview of The Three Dimensional Structure of Short Spider Toxins

Arthropods are one of the most diverse animal groups on the Earth. Spiders belong to this phylum and they are ancient animals with a history going back some three hundred million years. They are abundant, widespread, and natural controllers of insect populations. They use their venom to capture prey or to fight against predators. This venom is constituted of various peptides and enzymes with different activities. Among these proteins, toxic peptides are responsible for the macroscopic effect of the venom.

Most of the toxins are known to interact with ion channels (mainly potassium channels, sodium channels, and calcium channels). These transmembrane molecules are ubiquitous in the cells. They underlie a broad range of the most basic biological processes, from excitation and signaling to secretion and absorption. Like enzymes they are diverse and ubiquitous macromolecular catalysts with high substrate specificity and subject to strong regulation. Animal toxins and, more specifically, spider toxins are effectors of these channels. Depending on the peptide, they have ability to block the channel by plugging into its pore of conduction, or by modifying the opening and closing capacity of the channels, binding on a few specific sites along the structure of the channel.

Most of these peptides fold according to the overall same pattern, the inhibitor cystine knot (ICK) scaffold. Basically, it consists of a ring formed by a part of the backbone of the peptide and two disulfide bridges, penetrated by a third disulfide bridge. An additional disulfide bridge might be found in some toxins. Another fold has been found in a few toxins and has been described as the DDH scaffold. This motif lacks the knot and comprises an antiparallel β -hairpin stabilized by two conserved disulfide bridges.

This paper will try to summarize the structural characteristics of the spider toxins for which the fold has been described in the literature.     

A Note on the Evolution of Spider Toxins Containing the Ick-Motif

Most spider neurotoxins comprise a large group of structurally related polypeptides adopting the inhibitor cystine knot motif (ICK). The plethora of interesting biological functions they perform makes them worthy to study the evolutionary patterns that have been shaping their functional diversification. However, such a task remains elusive due to the lack of significant sequence similarities among the full set of spider neurotoxins. In this communication the phylogenetic relationships of a comprehensive set of spider neurotoxins with the ICK motif is analyzed by Bayesian inference. This procedure satisfactorily resolves terminal clusters, though high-order relationships remain poorly defined. Both paralogous expansion and orthologous diversification are well represented during the evolution of these ICK motif–containing peptides. Phylogenetic relationships are discussed in the light of what is known about spider neurotoxin functionalization.     

Patent Evaluation: The Use of Spider Toxins for the Treatment of Neurodegenerative Disease

Summary

Novelty: A series of 3-hydroxyindole-polyamines are claimed as calcium channel antagonists. The isolation of naturally occurring polyamines from spider venom is described along with their use for the treatment of stroke, brain trauma, Alzheimer's disease, seizures and hypertension.

Biology: A number of graphs are given, showing the specified polyamine is capable of reversibly blocking electrical current through calcium channels of GH₄C₁ clonal pituitary cells, and can block up to 45% of the high threshold current remaining during nimodipine treatment of NIE-115 cells. A graph is also given showing a concentration dependence of the block of calcium currents by the specified compound.

Chemistry: A scheme is presented showing how the synthetic polyamines were prepared and the use of this method to synthesize a sample of the natural polyamine and experimental details were given for both the isolation of the spider venom and the synthesis of the polyamine, 3-(aminopropyl-aminopropylaminopropylaminopentylaminocarbonylmethyl)-4-hydroxyindole (DOC1). This indole polyamine is one of nine specificallyl claimed compounds.

Structure:      

Snake and Spider Toxins Induce a Rapid Recovery of Function of Botulinum Neurotoxin Paralysed Neuromuscular Junction

Botulinum neurotoxins (BoNTs) and some animal neurotoxins (β-Bungarotoxin, β-Btx, from elapid snakes and α-Latrotoxin, α-Ltx, from black widow spiders) are pre-synaptic neurotoxins that paralyse motor axon terminals with similar clinical outcomes in patients. However, their mechanism of action is different, leading to a largely-different duration of neuromuscular junction (NMJ) blockade. BoNTs induce a long-lasting paralysis without nerve terminal degeneration acting via proteolytic cleavage of SNARE proteins, whereas animal neurotoxins cause an acute and complete degeneration of motor axon terminals, followed by a rapid recovery. In this study, the injection of animal neurotoxins in mice muscles previously paralyzed by BoNT/A or /B accelerates the recovery of neurotransmission, as assessed by electrophysiology and morphological analysis. This result provides a proof of principle that, by causing the complete degeneration, reabsorption, and regeneration of a paralysed nerve terminal, one could favour the recovery of function of a biochemically- or genetically-altered motor axon terminal. These observations might be relevant to dying-back neuropathies, where pathological changes first occur at the neuromuscular junction and then progress proximally toward the cell body.     

Oral Tolerance Induction by Bothrops jararaca Venom in a Murine Model and Cross-Reactivity with Toxins of Other Snake Venoms

Oral tolerance is defined as a specific suppression of cellular and humoral immune responses to a particular antigen through prior oral administration of an antigen. It has unique immunological importance since it is a natural and continuous event driven by external antigens. It is characterized by low levels of IgG in the serum of animals after immunization with the antigen. There is no report of induction of oral tolerance to Bothrops jararaca venom. Here, we induced oral tolerance to B. jararaca venom in BALB/c mice and evaluated the specific tolerance and cross-reactivity with the toxins of other Bothrops species after immunization with the snake venoms adsorbed to/encapsulated in nanostructured SBA-15 silica. Animals that received a high dose of B. jararaca venom (1.8 mg) orally responded by showing antibody titers similar to those of immunized animals. On the other hand, mice tolerized orally with three doses of 1 µg of B. jararaca venom showed low antibody titers. In animals that received a low dose of B. jararaca venom and were immunized with B. atrox or B. jararacussu venom, tolerance was null or only partial. Immunoblot analysis against the venom of different Bothrops species provided details about the main tolerogenic epitopes and clearly showed a difference compared to antiserum of immunized animals.     

A Combined Bioassay and Nanofractionation Approach to Investigate the Anticoagulant Toxins of Mamba and Cobra Venoms and Their Inhibition by Varespladib

Envenomation by elapid snakes primarily results in neurotoxic symptoms and, consequently, are the primary focus of therapeutic research concerning such venoms. However, mounting evidence suggests these venoms can additionally cause coagulopathic symptoms, as demonstrated by some Asian elapids and African spitting cobras. This study sought to investigate the coagulopathic potential of venoms from medically important elapids of the genera Naja (true cobras), Hemachatus (rinkhals), and Dendroaspis (mambas). Crude venoms were bioassayed for coagulant effects using a plasma coagulation assay before RPLC/MS was used to separate and identify venom toxins in parallel with a nanofractionation module. Subsequently, coagulation bioassays were performed on the nanofractionated toxins, along with in-solution tryptic digestion and proteomics analysis. These experiments were then repeated on both crude venoms and on the nanofractionated venom toxins with the addition of either the phospholipase A₂ (PLA₂) inhibitor varespladib or the snake venom metalloproteinase (SVMP) inhibitor marimastat. Our results demonstrate that various African elapid venoms have an anticoagulant effect, and that this activity is significantly reduced for cobra venoms by the addition of varespladib, though this inhibitor had no effect against anticoagulation caused by mamba venoms. Marimastat showed limited capacity to reduce anticoagulation in elapids, affecting only N. haje and H. haemachatus venom at higher doses. Proteomic analysis of nanofractionated toxins revealed that the anticoagulant toxins in cobra venoms were both acidic and basic PLA₂s, while the causative toxins in mamba venoms remain uncertain. This implies that while PLA₂ inhibitors such as varespladib and metalloproteinase inhibitors such as marimastat are viable candidates for novel snakebite treatments, they are not likely to be effective against mamba envenomings.     

Bioactive Components in Fish Venoms

Animal venoms are widely recognized excellent resources for the discovery of novel drug leads and physiological tools. Most are comprised of a large number of components, of which the enzymes, small peptides, and proteins are studied for their important bioactivities. However, in spite of there being over 2000 venomous fish species, piscine venoms have been relatively underrepresented in the literature thus far. Most studies have explored whole or partially fractioned venom, revealing broad pharmacology, which includes cardiovascular, neuromuscular, cytotoxic, inflammatory, and nociceptive activities. Several large proteinaceous toxins, such as stonustoxin, verrucotoxin, and Sp-CTx, have been isolated from scorpaenoid fish. These form pores in cell membranes, resulting in cell death and creating a cascade of reactions that result in many, but not all, of the physiological symptoms observed from envenomation. Additionally, Natterins, a novel family of toxins possessing kininogenase activity have been found in toadfish venom. A variety of smaller protein toxins, as well as a small number of peptides, enzymes, and non-proteinaceous molecules have also been isolated from a range of fish venoms, but most remain poorly characterized. Many other bioactive fish venom components remain to be discovered and investigated. These represent an untapped treasure of potentially useful molecules.     

Effects of Snake Venoms on Hemostasis

Abstract

Proteins found in venoms, especially of theViperidae snake family, exert, often with a narrow specificity, activating, inactivating, or other converting effects on different components of the hemostatic and fibrinolytic systems, respectively. Some purified snake venom proteins have become valuable tools in basic research and in diagnostic procedures in hemostaseology. “Procoagulant” as well as “anticoagulant” venom components have been identified inin vitro test systems. “Procoagulant” snake venom components may causein vivo, upon massive application as in the case of snake-bite of small prey animals, intravascular coagulation leading to circulatory arrest and rapid death. Smaller doses of procoagulant venom components applied to large organisms as in the case of snake-bite accidents in humans, may cause a consumption coagulopathy with localized or generalized bleeding. Highly purified, specific fibrinogen coagulant venom proteinases are used in human medicine to produce therapeutic defibrinogenation. These practically nontoxic venom enzymes may act synergistically with other components aggravating their toxic effects.     

Complement Activation by Animal Venoms

Abstract

During activation of complement (C) system, C4b and C3b attach covalently to targets such as cell membranes. Deposited C4b serve both as the focus for the assembly of the C3 convertases and as ligands for C3b receptors or inactivators. C4a, C3a and C5a, mediators of the early events of inflammation, are released into the fluid phase while C5b serves to organize the cytolytical complex C5b-C9. Among the Arthropoda the venom from some spiders (Loxosceles) contain activators of the mammalian C system, although the exact mechanism and point of the activation cascade in where they act were not yet elucidated. In the insect venoms as honeybee, yellow jacket, yellow hornet, white-facet hornet, caterpillars and wasp there are some components capable of reducing total haemolytic and serum levels. Interestingly, mellitin, a soluble haemolytic peptide present in bee venom shares structural homology with human C9. Scorpion venom apparently are not active on the C system. Snakes, belonging to ELAPIDAE, CROTALIDAE and VIPERIDAE families produce venoms containing components with a vast range of action on mammalian C system, some acting by cleaving directly a particular component, while others interact with C components, the resulting complex being able as an amplificator, to activate part of the C cascade. Soluble mediators of inflammation, cell bound fragments of C components recognizable by specific receptors disposed on immune or inflammatory cells, or the formation of multimolecular complex potentially capable of damage host cells are the presumable consequences of C activation by animal venoms.