[Jounal of Toxicology.Toxin reviews,17(3),405-426(1998)] [執筆は1997年の4月でしたが，発行が１年以上遅れました] ＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝ Spider Venoms and Spider Toxins Masahisa ORI* and Hiroyoshi IKEDA** *The Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki,852,Japan ** Yamakita Senior High School, Yamakita-cho, Kanagawa 258-01, Japan 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. INTRODUCTION About 40,000 species of spiders have been described throughout the world (1), most of which are harmless to humans. Of those, only 180 species bite man, and only a few are truly venomous. However, some spiders are truly venomous, examples of which are the widow spider, Sydney funnel-web spider, banana spider and recluse spider(2). 1,200 species of spiders have been reported in Japan, none of which are venomous. However, in the autumn and winter of 1995, some widow spiders, the red back spider, Latrodectus hasseltii, and the brown widow spider, Latrodectus geometricus, were found in Japan (3,4). There have been many reviews on venomous spiders and/or their toxins (5-15), especially on the neurotoxin of black widow spiders, which is a high molecular weight protein named alpha-latroroxin (16,17). The chemical properties of the toxin of the red back spider which was introduced in Japan were studied and found to be almost the same as those of the Australian red back spider toxin, which was alpha-latrotoxin (18). Recently, however, the study of harmless spider toxin has become active from a neurobiological viewpoint. Spider venoms are a rich source of potential probes for ion channel and receptor in neurones (11-14,19). 1.Spider bite and classification of spider toxin Intoxication produced by a spider bite is called arachnidism, araneism or araneidism. We are of the opinion that it would be more appropriate to use the terms araneism or araneidism, introduced in 1910 by Sommer and Grecco, rather than arachnidism, since the order Arachnida includes not only spiders but scorpions, pseudoscorpions, ticks and mites. The syndrome caused by a particular species of spider is known by terms such as latrodectism, chiracanthism, loxoscelism, ctenism, etc., particular to that species. Among these, latrodectism, caused by the bite of the black widow spider, is the most frequently found form of araneism, not only in Europe but also in the USA. Latrodectism has been extensively studied since the classical study of Baerg (20), who allowed a black widow spider to bite his finger. Like other venoms, spider venoms are quite complex and contain a variety of protein and nonprotein components. These components include ATPase, esterase, hyaluronidase, phosphodiesterase, protease, GABA, histamine, serotonine, spermine, polyamines, nucleotides, neurotoxins, insecticidal toxins, and necrotizing toxins (9). The major biological activity resides in the protein components. In Table 1 we have classified spider toxins according to their major component and the action of the toxin. Biochemical properties and the molecular structure of spider venoms were little known until 1964, because of the small size of the gland producing the venom, from which an insufficient amount of venom could be extracted for analysis. At that time, techniques for analysis were poor and not adequate for examining such small samples. Table 1. Major spider toxins and their actions ================================================================================================== Toxin Action Major toxic component Spider Species ------------------------------------------------------------------------------------------------- neurotoxin nerve terminal protein Latrodectus spp. Ca channnel opened Atrax spp. -------------------------------------------------------------------- nerve terminal peptide Phoneutria spp. Na channel opened Cupiennius salei -------------------------------------------------------------------- nerve terminal Agelenopsis aperta Ca channnel blocked Agelena opulenta Selenocosmia huwena --------------------------------------------------------------------- postsynaptically polyamine toxin Nephila spp. Araneus spp. Argiope spp. Argiope spp. Tarantula toxin -------------------------------------------------------------------------------------------------- necrotoxin necrosis protein Loxosceles spp. Lycosa godeffroyi Lycosa godeffroyi Brachypelma smithii =================================================================================================== 2.Neurotoxin 2.1. Black Widow Spider Venom Widow spiders, which belong to the genus Latrodectus, are comb-footed spiders (Family Theridiidae). There are 40 species of this genus worldwide (15). Among these, the American black widow spider, Latrodectus mactans, Mediterranean black widow spider, and Latrodectus tredecimguttatus are well known. In 1976, studies of black widow spider venom and widow spider biology were extensively reviewed by Maretic and Lebez (6). However, since that time there have been many important studies of Mediterranean black widow spider venom. Venomous effects: In an early study of latrodectism by the American black widow spider, Latrodectus mactans, Bogen reported 150 cases with 12 deaths from the USA and Canada; the majority of these cases were males from California who had been bitten on the penis or adjacent parts while sitting on the seats of outdoor privies (21). Duchen and Gomez (10) noted that well-controlled experiments with widow spider venom were first done by D'Amour et al. (22). Venom glands from American black widow spiders were macerated in physiological saline and injected intraperitoneally into young adult rats. The venom was found to be 15 times more potent in terms of dry weight than the venom of the prairie rattlesnake. The first signs of intoxication were stiffness of gait, awkwardness of movement, predominantly in the hind limbs, lacrimation, and closure of the eyes. The rats would remain immobile unless disturbed, respiration was labored or shallow, and all died in 8-36 hr. Sampayo, in a thorough account of the pharmacological actions of the venom, concluded that the venom's principal action was on the central nervous system (23). Cantore observed that the venom abolished contraction in response to indirect stimulation in the phrenic nerve-diaphragm preparation of the rat. He concluded that the venom had an irreversible blocking effect on the motor end plates (24). In 1970, the effect of the toxin of Latrodectus was demonstrated on neuromuscular transmission, using a single neuromuscular junction of the sartorius muscle of the frog. The results showed that Latrodectus venom in five to ten minutes induced an increase of the miniature end-plate potentials (MEPP) from 0.5-1.5 /s to 300- 1000/s. After reaching the peak, the MEPP frequency fell to a low value, less than 1/s. The amplitude of end- plate potential (EPP) first increased by several increments and then fell to zero. The nerve ending spikes disappeared at approximately the same time, probably due to depolarization of the nerve ending. The authors attribute these findings to the venom reacting with the nerve ending membrane and the release of transmitter substance. This release of transmitter occurred independently of the presence of calcium ions and independently of the depolarization of the ending (25,26). The venom caused neurotransmitter depletion on not only cholinergic nerve terminals but also on adrenergic nerve fibers in the rat iris (27) and terminals of several mammalian organs (28). The extract depolarized the cell body of the crayfish stretch receptor (29) and induced a discharge of impulses in the axon (30). Venomous component: The venom is multicomponent, with some of the active agents found to have characteristics of protein (31,20,22). Many workers attempted to analyze the protein toxin by electrophoresis in 1960's, but these methods were not as good as at present and the impurity of extracts of the venom glands caused inconsistent results. Among these studies, significant differences in protein composition of gland extracts were demonstrated among four species of Latrodectus: L.mactans, L.variolus, L.geometricus and L.bishopi (32). Since the mid-1960's, gel filtration and ion exchange chromatography methods have been widely used for the separation and purification of proteins. Whole extracts of venom glands have been fractionated by these techniques. These are: (1) fraction having no toxicity in houseflies and mice, (2 fraction with high toxicity in the frog, mouse and guinea pig, but nontoxic for houseflies, (3) fraction producing quick paralysis in houseflies, (4) fraction having a slow toxic action on houseflies, and (5) fraction having toxicity on the crayfish stretch receptor (33-35). Purified protein fraction B5 which was active against vertebrates was named alpha-latrotoxin. Fraction B5 was responsible for both the increase in the frequency of MEPPs and for the depletion of vesicles (35,16). Table 2. Toxic effect of various fractions of the Latrodectus venom (34-35) ============================================================================================================== Fraction Frog Mouse Housefly Crayfish 1964(33) 1972(34) whole + + + + extract A - - - - A B + + - - C + + + - LV1 C LV2 B D - - - - E - - ND + ------------------------------------------------------------------------- B5 + + - - LV3 D C3 - - + ND C5 + + + ND E2 - - ND + LV4 ------------------------------------------------------------------------------------------------------------- Fractions A-E of Latrodectus venoms were referenced from work in 1976 (35).+, active; -, inactive; ND, not determined. LV1-LV4 were fractionated in 1964 (33). A-D were fractionated in 1972 (34). ========================================================================== Study of LTX (alpha-latrotoxin): LTX interacts with lipid bilayers to make them permeable to certain ions and appears more selective for alkali cations over anions. The bilayers definitely become permeable to Ca(II), suggesting that permanent channels are formed in the lipid bilayer by LTX, and that these remain open (36). It was also found that LTX prevented the uptake of labeled gamma aminobutyrate (GABA) and norepinephrine by rat brain synaptosomes. The release of GABA that stimulated the LTX was not dependent on extracellular Ca(II). The release of norepinephrine was Ca(II) dependent (37). The LTX stimulated the release of transmitters, dopamine and norepinephrine in a neurosecretory cell line (PC12) derived from rat phenochromocytoma in culture (38). The amino acid composition of LTX, labeled LV3, was determined with a Beckman model 120 amino acid analyzer and found to consist of 1219 residues; SDS polyacrylamide gel electrophoresis revealed its the molecular weight to be 130 kDa (39). Variable results of the amino acid sequence of the LTX have been reported by Russian workers (40). Thereafter, the total amino acid sequence of the LTX was deduced from cDNA sequencing (41).The cDNA contained a 4203 base-pair open reading frame corresponding to the 156 855 Da protein composed of 1401 amino acids. Molecular weight differed from that earlier determined by means of SDS gel electrophoresis. Therefore, the toxin can be considered as a precursor, and processing takes place in the C-terminal region of the polypeptide chain during its maturation. This hypothesis seems more reasonable because all studied peptides of tryptic hydrolysate are equally distributed in the toxin fragment with coordinates 1-1170. Molecular mass of this fragment (Mr 131 kDa) is in good agreement with the apparent molecular mass of the isolated toxin. Moreover, the C-terminal fragment of over 200 amino acid residues with coordinates 1171-1381 was absent in the products of toxin tryptic hydrolysate despite many cleavable regions (41). The highly purified toxin preparations were found to contain two components: polypeptides of 1401 (alpha latrotoxin) and 70 (low molecular weight protein) amino acid residues (42). The LTX is thought to act by binding to high-affinity receptors which are found in susceptible tissues (43,44). This receptor is a member of the neurexin family of proteins (45) and is found in the membranes of presynaptic nerve terminals where it interacts with synaptotagmin (a synaptic vesicle protein) (46). This neurexin- synaptotagmin complex is thought to be important in neurotransmitter secretion (47,48). It has also been suggested that neurexins are involved in cell recognition in the nervous system (49). The low molecular weight protein named latrodectin contains 88 amino acid residues, but a fragment of 18 amino acid residues of the protein is lost after protein maturation (50-52). This protein exhibits certain structural homology with erabutoxin-a from the sea snake (42) and with the crustacean hyperglycemic hormones (53). Structural analogy of two proteins is not necessarily associated with functional similarity. The function of latrodectin is not clear. Attempts have been made to identify the other black widow spider venom fractions which are responsible for the effects observed on invertebrate tissues. A protein with a single band, alpha-latroinsectotoxin, was identified as having a molecular weight of 120 kDa. Latroinsectotoxin has an amino acid content similar to that of LTX and acts as a specific insectotoxin causing rapid transmitter release from insect nerve endings (54,55). 2.2. Studies of Sydney funnel-web spider venom The Sydney funnel-web spider, Atrax robustus, may be considered to be the world's most dangerous spider because of its strength and aggressiveness, the lethal potency of its venom, its nocturnal habits, and its penchant for invading houses. Its distribution coincides with the metropolitan area of Australia's largest city. Venomous effect: The venom produces a syndrome characterized by generalized muscle fasciculation, sweating, salivation, hypertension, and tachycardia (56). The venom, like the black widow spider venom, induces a massive release of neurotransmitter from motor end plates and nerve terminals throughout the autonomic nervous system. Unlike widow spider venom, its effects are transient and reversible. They can be blocked in vitro by gallamine, suxamethonium, lignocaine and diazepam (57). In contrast to the widow spider venom, its venom has little effect on the ultrastructure of nerve endings in isolated rat diaphragms, and there is no obvious depletion of synaptic vesicles from the terminals (58). Venomous components: The lethal neurotoxin from the venom of the male Sydney funnel-web spider, robustoxin, is a polypeptide of 42 residues which was isolated and its amino acid sequence determined (59). One of related species is Hadronyche (recently transferred from the genus Atrax) verstus, which is located in the hilly areas west of Sydney. The lethal neurotoxin from the venom of both the male and female, versutoxin, is a polypeptide of 42 residues. Eight of the 42 residues were found to differ between robustoxin and versutoxin. The two toxins were tightly folded polypeptides (60). Several other components or properties of male Atrax robustus venom have been reported. These include GABA, permine Complex, Lactic acid, Trimethylsilyl or pentafluoropropionate derivatives, citric acid, glycerol, urea, glucose, glycine, spermidine, thyamine, and octopamine. 2.3 Ctenid Spider Venom In Brazil, most cases of envenomation by the spider are caused by Phoneutria nigriventer (Family Ctenidae). Venomous effects: Neurogenic shock can be observed more frequently in children and is characterized by cold sudoresis, agitation, salivation, priapism and death. The effect of venom on the isolated auricle of guinea pigs is produced through the release of acetylcholine and noradrenalin neurotransmitters by the parasympathetic and sympathetic nerve terminals. This release occurs by the action of the venom within the sodium channels of the nerve terminal membrane (61). These effects are abolished by tetrodotoxin (62). However, there are various polypeptides in the venom which have particular functions. Venomous component: The venom of Phoneutria nigriventer contains histamine, serotonine and other polypeptides (63,64). The contraction of the rat diaphragm induced by the spider bite was due to the release of acetylcholine by a basic component that could be separated by paper electrophoresis (65). Three neurotoxic fractions (PhTx1,PhTx2,PhTx3), lethal to mice, were isolated from the venom by gel filtration and reverse phase chromatography and the complete amino acid sequences of 11 peptides were determined to be from 32 to 77 amino acid residues (62,66-68). PhTx3 blocks the neuromuscular transmission of skeletal muscles by acting prejunctionally to depress nerve-evoked transmitter release. The effect was related to a diminished Ca(II) entry into the nerve terminal associated with inhibition exocytosis (69). The venom of the neotropical wandering ctenid spider, Cupiennius salei, contained proteins and peptides, some with enzymatic activity, others with a neurotoxic mode of action, and several as neurotoxic acting low molecule mass substances. 13 peptides (CSTX-1 to CSTX-13) had toxicity of various strengths, with the most toxic peptide being CSTX-1 and the least toxic peptide, CSTX-13. CSTX-1 has a sequence of 74 amino acids. Comparisons of the amino acid sequences of this toxin with those of the closely related ctenid spider, Phoneutria nigriventer, showed no distinct similarity in nine of 17 cases, revealed some N-terminal similarity with six out of 17 amino acids, and complete identity in only two cases (70) . 2.4. Chiracanthium Spider Venom Most cases of envenomation by spiders in Japan are caused by Chiracanthium japonicum. Venomous effects and components: This venom was purified with ion exchange gel chromatography and the toxin was determined to be neurotoxic in action. The toxic responses in mice were dyspnea, prostration, flaccid paralysis and death. The minimal lethal dose of the purified venom for mice was 10fA. The relationship between lethal dose (LD) and time to death showed correlation in doses from 10fA to 155fA. The LD50 against a house fly was 0.069fA. The molecular weight of the toxin was determined as 63,000±2,000 by SDS polyacrylamide gel electrophoresis (71). The toxin has not been analyzed yet. Other components of the venom have been reported: norepinephrine, epinephrine, octopamine, serotonin, spermine and histamine (72). These catecholamines and histamine caused severe pain through the spider bite. 2.5. Orb Weaving Spider Venom Polyamine Toxin and Glutamate Receptor In 1982, a neurotoxin from the "Joro spider," Nephila clavata, was newly found in Japan. The toxin named Joro spider toxin (JSTX) is an unexpectedly low molecular weight substance (ca. 600 Da) which acts postsynaptically to block the glutamate receptors in the crustacean neuromuscular synapse (73). Similar postsynaptically-acting toxins to JSTX have subsequently been found in many other orb-web spiders, and their structures were confirmed by chemical analysis and NMR. These include the toxins of the venom from Araneus ventricosus and Neoscona nautica (74), NSTX (from Nephila maculata) (75), and Argiopin (from Argiope lobata) (76). These polyamine-toxins consist of an aromatic subunit, amino acid linker, and a polyamine side chain. Some of these toxins were synthesized (77). The JSTX analogue, 1-naphtyl acetyl spermine (Naspm), was synthesized and utilized for further study of glutamate receptors (12,78). Additional neurotoxins from venom glands of Nephila clavata were identified and synthesized (79). UV-spectra enabled the classification of the venom components into three types: argiopin, argiopinins, and pseudoargiopinins. Molecular masses of the compounds are within the 630-759 range, and only pseudoargiopinin III has the smaller mass of 373. Amino acid analysis revealed arginine and aspartic acid residues in the majority of the venom components. All arginines possess a free amino group(80). The mere immobilizing effect of the venom of the other orb-weaving spider, Argiope bruennichi, on the cockroach and the common meal beetle is far more relevant to prey capture than to causing death. This weak effect on insects is adequate, considering that the method of hunting includes wrapping up the prey before biting (81). 2.6. American funnel web spider venom and the neurotoxic peptide The American funnel-web spider, Agelenopsis aperta, possesses a remarkable array of biological activities when considering both vertebrate and invertebrate systems. The spider has several toxins, designated agatoxins, which are of interest to neuroscientists studying glutamate receptors and Ca(II) channels (82,83). The peptide omega-Aga-IVA has been identified as a potent selective blocker of P-type calcium channels (84,85). The polyamine toxin FTX is also found in the venom. FTX is believed to selectively block P-channels. Table 3. The properties and effects of omega-Agatoxin(82-86) =============================================================================================== omega-Aga-IA Structure heterodimetric peptide consisting of 66 amino acids Effect paralytc effect on insects Action blockade of presynaptic Ca(II) channels in insect motorneurone; fully blocks high threshold Ca(II) currents and partially occludes T-type currents in rat sensory neurones --------------------------------------------------------------------------------------------- omega-Aga-IIA Structure not determined Effect paralysis in insects Action blocks Ca(II) flux in both chick (homogenous N-channel) and rat (almost P-channel) synaptosomes --------------------------------------------------------------------------------------------- omega-Aga-IIIA Structure peptide of 76 amino acids Action non-selective Ca(II) channel blocker; voltage-dependent blockade of L-,N-, and P-channels in rat neurone --------------------------------------------------------------------------------------------- omega-Aga-IVA Structure peptide of 48 amino acids Action selectively blocks P-type Ca(II) channels blockade of glutamate release depolarization unblock phenomenon blocks presynaptic Ca(II) channels at mammalian neuromuscular junction -------------------------------------------------------------------------------------------------- omega-Aga-IVB Structure peptide of 48 amino acids (closely related in size to omega-Aga-IVA ) Action highly potent blocker of P-type calcium channels in rat neurones ===================================================================================================== From the Japanese funnel-web spider, Agelena opulenta, was isolated the toxin, agelenin, which had 38 residues and blocked the transmitter presynaptically at the lobster neuromuscular junction(14,87). Plectoxin from the venom of the primitive hunting spider, Plectreus tristis (Family Plectreuridae), was bioassayed and purified to 9 peptides. The amino acid sequences of 7 of the 9 peptides were determined. Most peptides were short (46-49 amino acids) and showed variable species specificity (88). The venom of the cellar spider, Segestria florentina, caused the depolarization of excitable membranes, an increase in the secretion of mediator, and a prolongation of the action potential through the inactivation of the Na channels. The complete amino acid sequences of the insectotoxin of the venom is 35 residues (89). The venom of the funnel web spider, Hololena curta, contains a mixture of compounds. One insecticidal peptide that contains 38 amino acids and 10 acylpolyamine toxins named curatoxins were purified. The acylpolyamines instantly paralyzed lepidopteran larvae following injection (90). 2.7. Tarantula Spider Venom The tarantula is a large spider which belongs the Family Theraphosidae and has as its habitat a hole under the ground. Tarantula spider venoms contain various components, such are bioamines, polyamines, adenosine nucleotides and proteins (91,92). The venom from the Mexican red knee tarantula, Brachypelma smithii, is considered not be hazardous to man. Two proteins of the toxin were isolated and sequenced. Protein-1 has 39 residues, including six cysteine residues with three disulfide bonds (93). It is identical to one of the isoforms of cockroach-toxic ESTX from the venom of another tarantula, Eurypelma californium, that contains 39 residues (94). Protein-5 has 34 residues, including six cysteine residues with three disulfide bonds, and is most similar to Tx2-9 from the Brazilian 'armed' spider, although it has only 41% sequence identity (66-68,94). The venom from the Chinese bird spider, Selenocosmia huwena, which lives in holes underground in the south of China, caused paralysis and rapid respiratory failure in mice (95). Neurotoxic components were proteins. Among the neurotoxic components, was huwentoxin-I, a peptide of 33 residues, isolated from the venom. The structure of huwentoxin-I was similar to omega-agatoxin V from the funnel web spider, Agelenopsis aperta, which had three disulfide bonds. However, the biological activities of the two toxins were quite different. Huwentoxin-I was shown to reversibly block the neuromuscular transmission in an isolated mouse phrenic nerve-diaphragm preparation (96) and the omega-agatoxins are known to be insecticidal neurotoxins, which can induce repetitive firing and massive transmitter release from presynaptic stores at the neuromuscular junction of insects (86,97). A study of the three-dimensional structure of huwentoxin-I is in progress (98). 3. Necrotoxin 3.1. Recluse Spider Venom Recluse spiders (genus Loxosceles) are cosmopolitan, found in urban environments and make irregular web structures. There are in excess of 50 species, only a few of which have been implicated in loxoscelism. Venomous effects: In 1957, the bite of Loxosceles reclusa in the USA was discovered to have caused necrotic arachnidism (99). There have been at least 126 cases of necrotic spider bite (Loxoscelism) in the USA and about 400 cases in South America (100). In most cases there was local skin necrosis; however, it is important to note the occurrence of systematic reactions, including hemolysis, renal failure, and death (101). Venomous component: Sphingomyelinase D is an important component of Loxoscheles venom, and is responsible, at least in part, for local skin necrosis, intravascular haemolysis and platelet aggregation. The role of polymorphonuclear leucocytes seems to be important in the development of local vasculitis, the putative cause of necrotic lesions. 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