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Structure and pharmacology of spider venom neurotoxins

Pierre Escoubasa,b*, Sylvie Diochotb, Gerardo CorzocaUniversit Pierre-et-Marie-Curie, 9, quai Saint-Bernard, 75252 Paris cedex 05, France

bInstitut de Pharmacologie Molculaire et Cellulaire, CNRS, Valbonne, FrancecSuntory Institute for Bioorganic Research, Osaka, Japan

(Received 29 March 2000; accepted 4 August 2000)

Abstract Spider venoms are complex mixtures of neurotoxic peptides, proteins and low molecular mass organic molecules. Theirneurotoxic activity is due to the interaction of the venom components with cellular receptors, in particular ion channels. Spider venomshave proven to be a rich source of highly specific peptide ligands for selected subtypes of potassium, sodium and calcium channels,and these toxins have been used to elucidate the structure and physiological roles of the channels in excitable and non-excitable cells.Spider peptides show great variability in their pharmacological activity and primary structure but relative homogeneity in theirsecondary structure. Following diverse molecular evolution mechanisms, and in particular selective hypermutation, short spiderpeptides appear to have functionally diversified while retaining a conserved molecular scaffold. This paper reviews the compositionand pharmacology of spider venoms with emphasis on polypeptide toxin structure, mode of action and molecular evolution. 2000Socit franaise de biochimie et biologie molculaire / ditions scientifiques et mdicales Elsevier SAS

spiders / neurotoxins / peptides / ion channels / structure / pharmacology

1. Introduction

Spiders have long held a special place in popular mythsand folklore, due to their secretive habits, physical aspect,predatory behavior, and their perceived potential lethality.Nevertheless, although their biological and ecologicaldiversity is immense (ca. 40 000 described species andprobably more than 100 000 not yet described), very few

species represent a true medical problem. Spiders aredivided into two main groups according to the position oftheir chelicera: in the more primitive orthognath spiders(mygalomorphs), the large size of many specimen doesnot correlate with high venom toxicity, with the exceptionof Australian species of the genus Hadronyche (Atrax).These spiders display a very aggressive behavior, coupledwith a venom which is highly toxic for humans, and havecaused at least 14 recorded deaths. Other dangerousmygalomorphs include species in the genus Trechona(Dipluridae) and Harpactirella (Barychelidae). In thefamily Theraphosidae, whose members have becomewildly popular as pets, the dangerous character of thevenom is mostly limited to hearsay, and cases of severeenvenomation are usually unsubstantiated. No deaths havebeen officially recorded to date, although several speciesare reported to provoke severe envenomation symptoms.

The most dangerous spider species are found in thegroup of labidognaths (araneomorphs):Latrodectus(black

widows - Theridiidae), Loxosceles (violin or gauchospiders - Loxoscelidae) and Phoneutria(banana spiders -Ctenidae) which are responsible for many severe enveno-mation cases, and recorded mortality. Several other fami-lies are reported to be dangerous, although most reports ofenvenomation are not adequately documented: Segestri-idae, Agelenidae, Salticidae, Gnaphosidae, Thomisidae,Heteropodidae, Clubionidae and Lycosidae. These reports

must be considered carefully in the light of the naturalpolymorphism of the envenomation syndrome, and theuncertainty of taxonomic identification. To date, only thethree genera Atrax, Latrodectus and Loxosceles are rec-ognized as responsible for human death. However, allspiders possess a venom apparatus (with the exception ofthe family Uloboridae) and secrete a complement ofneurotoxins designed to paralyze and kill their prey. Thesetoxins have been the subject of sustained research effortsin recent years and are becoming increasingly important inneurobiology as natural probes of cellular receptors.

Due to the fast progress of molecular biology tech-niques, discovery of novel genes coding for cellular toxin

receptors such as ion channels is progressing at anever-increasing pace. The diversity of cellular subtypes oftoxin receptors and the complexity of heteromultimericassociation of receptor subunits, which contributes to thediversity of subtypes, is only beginning to be understood.The understanding of this cellular complexity and its rolein modulating the function of excitable cells is oftenlimited by the lack of specific pharmacological tools tostudy the molecular diversity of receptors and theirrespective roles in complex physiological pathways.

* Correspondence and reprints.E-mail address:[email protected] (Pierre Escou-bas).

Biochimie 82 (2000) 893907 2000 Socit franaise de biochimie et biologie molculaire / ditions scientifiques et mdicales Elsevier SAS. All rights reserved.S0300908400011664/REV


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Evolution has equipped venomous animals with acomplex arsenal of neurotoxins acting against cellularreceptors with exquisite sensibility and selectivity. Toxinscan thus be used by the neurobiologist as molecularscalpels, and the study of animal venoms will helpdevelop new perspectives in molecular pharmacology and

physiology. Among the different groups of venomousanimals, spiders have certainly been the least explored inthis respect, although their venoms are potentially a richsource of new ligands. These venoms are complex mix-tures, comprising both low-molecular mass componentsand polypeptide toxins that are highly reticulated bydisulfide bridges. We review here the current knowledgeon spider venom toxins with emphasis on biochemistry,structure and pharmacology of polypeptide toxins.

2. Composition of spider venoms

Spider venoms are multicomponent systems of poorlyunderstood complexity. In particular, the interactions be-tween the various chemical classes present in the venomsand their respective role in the envenomation process havenot been well defined. Spider venom components can begrouped into three major chemical classes: low molecularmass organic molecules (Mr < 1 000 Da), polypeptides(Mr3 00010 000 Da) and high molecular mass proteins(Mr > 10 000 Da).

2.1. Inorganic and organic constituents

Spider venoms contain inorganic ions and salts (Ca2+,Na+, K+, Mg2+, Cl), free acids (citric, lactic, dihydrophe-

nylacetic), glucose, free amino acids, biogenic amines(spermine, spermidine, putrescine, cadaverine) and neu-rotransmitters (glutamate, aspartate, GABA, histamine,dopamine, serotonin, epinephrin, epinin) [19]. The roleof those constituents is unknown although they maypotentiate the action of the neurotoxins in some cases.They might also represent degradation products of othervenom components, as a result of the collection process.

Another chemical class, the acylpolyamines, was char-acterized for the first time in spider venoms [1014].Spider polyamines comprise a hydrophobic moiety linkedto a lateral chain which comprises one to nine aminopro-pyl, aminobutyl or aminopentyl units, sometimes methy-

lated or hydroxylated, for a total length of 7 to 43 atoms.This hydrophilic chain can be complexified by the addi-tion of internal or terminal amino acids. The hydrophobicmoiety is composed of an aromatic carboxylic acid suchas mono- or dihydroxylated benzoic or indoleacetic acid.Currently, five structural types of these polyamines areknown (A to E) [15] and represent natural combinatorialchemistry by the spider resulting in pharmacologicaldiversification of the venom [16]. All amino acid contain-ing polyamines have been described in araneomorphs

(Araneidae) [17, 18], and polyamines devoid of aminoacids were found in different families, both in mygalo-morphs and araneomorphs: Theraphosidae (Apc600,Apc728) [46, 19, 20], Dipluridae, Ctenizidae, Agelenidae(-agatoxins) [21] and Pisauridae (CNS2103) [22]. Spiderpolyamines are neurotoxic and act as antagonists of the

different subtypes of ionotropic glutamate receptors, withsome polyamines targeting more specifically NMDA orAMPA subtypes [23]. They act as voltage-dependentopen-channel blockers, and they have been suggested toact as pore blockers of the ion channel associated with theglutamate receptor [24]. Polyamines also act on voltage-sensitive calcium channels (VSCC) (Arg636, CNS2103)[22]. The toxin FTX (funnel web toxin) permitted thediscovery of the P-type calcium channel in rat cerebellumPurkinje neurons [25]. Some polyamines also act onnicotinic acetylcholine receptors. Their mode of action,chemical synthesis and pharmacological properties havebeen recently reviewed [24, 26].

Although some polyamines can be toxic to vertebrates,by direct intracranial administration, they seem to possessessentially an insecticidal activity and are responsible forthe fast insect paralysis observed during predation. Thisparalysis is mediated by the fast and reversible block ofthe insect neuromuscular junction where glutamate is themain neurotransmitter. Our own work has shown theirpresence in tarantula venoms, in a variable manner, inparticular in South-American tarantulas [27]. In somespecies they represent the major venom components, arare profile in Theraphosidae (Escoubas, unpublisheddata). Their presence, diversity and abundance in thesevenoms may reflect the relative importance of insectsversus vertebrate prey in the diet of the spider, and an

adaptation to different ecological niches and mode ofpredation in Theraphosidae.

Two other organic compounds have been isolated fromspider venoms although their biological function remainsunknown: bis(agmatine)-oxalamide from the venom ofPlectreurys tristis [28] and HF-7, a glyconucleoside di-sulfate from the venom ofHololena curta [29].

2.2. Polypeptides

The vast majority of spider toxins identified to date arepolypeptides with a molecular mass of ca. 3 000 to8 000 Da, highly reticulated by several disulfide bridges.

In combination with polyamines, peptides appear to rep-resent the main toxic arsenal of spiders. Their presence iseasily confirmed by their retention time measurement inreversed-phase chromatography. UV detection with adiode-array detector as well as retention time permits easydistinction of peptides and polyamines which constitutethe early-eluting part of the venom (figure 1). This com-mon venom elution pattern has been widely illustrated inthe literature, and we found all tarantula venoms toconform to this model [30].

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To this day, more than 60 peptide toxins have beendescribed (figure 2). These toxins appear to essentiallytarget various membrane ion channels. They can block therelease of neurotransmitters by affecting exocytosis ofpresynaptic vesicules and induce abnormal modificationsof synaptic transmission resulting in flaccid paralysis.Some peptides may also provoke excitatory paralysis

resulting from paroxystic activity induced by excessivedepolarization. Toxin activity can also be specific forsome zoological groups (vertebrates, insects). The follow-ing paragraphs describe in more detail structure andpharmacology of spider venom polypeptides.

2.3. Proteins

Venom proteins include both high-molecular massneurotoxins and enzymes. However, with the exception ofhighly necrotic venoms such as Loxosceles, in whichenzymes have been well characterized, the presence ofenzymes in spider venoms should be viewed with caution.

Proteases, hyaluronidases, sphingomyelinases, phospholi-pases and isomerases have been reported in spider venoms[7, 8]. Nevertheless, in many cases contamination bysaliva or digestive fluids may be suspected, in particularafter electrical milking. As spiders regurgitate digestivefluids of high lytic power for external digestion of prey,simultaneous stimulation of all anterior muscles duringvenom collection may explain many of the observedenzymatic activities, in particular in older reports. Lend-ing credence to this proposal, published results are con-

tradictory, and the presence of such enzymes was notdetected by some authors [31].

Dermonecrotic and hemolytic activity of Loxoscelesvenom is due to the presence of sphingomyelinases D ofca. 35 kDa [32]. Hyaluronidases in venoms may potentiate

the action of other venom components, facilitating pen-etration in various cellular compartments and tissues.However, the exact biological function of most spidervenom enzymes remains to be elucidated.

Several high molecular mass toxins have been de-scribed in spider venoms. In the genusLatrodectus(blackwidows), the high neurotoxicity of the venom to verte-brates and invertebrates is due to the presence of a familyof ca. 110 kDa proteins called latrotoxins [33]. The mosttoxic for vertebrates is-latrotoxin (LTX) which provokesmassive neurotransmitter release. Its molecular mode ofaction is complex and not completely elucidated yet [34].It comprises a calcium-dependent mechanism in which the

toxin binds to neurexin, activating a cascade of proteinsignaling involved in synaptic vesicle trafficking. LTXalso forms non-selective cationic membrane channels,allowing a flux of calcium and small molecules(glutamate, acetylcholine). In another, calcium-independent mechanism, the toxin binds to latrophilin(CIRL), a receptor of the secretin and G-protein familyand induces a positive modulation of exocytosis of smallsynaptic vesicles possibly through IP3-mediated mobili-zation of intracellular calcium. The overall effect of LTXis massive neurotransmitter release [3537]. The macro-scopic effect of latrotoxin action is a rapid contracturantparalysis, resulting from hyperstimulation of post-synapticreceptors. The structure of

-latrotoxin obtained by

single-particle cryo-electron microscopy was recentlypublished [38] showing that the toxin is active as atetramer, comprising a central pore and different receptorbinding and pore lining domains. Five insect-specific (-,-, -, - and -latroinsectotoxins) and a crustacean-specific toxin (-latrocrustatoxin) have also been de-scribed from the venom of L. mactans tredecimguttatus.All Latrodectus venoms appear to share a common modeof action and probably contain similar toxins. In otherTheridiidae such as Steatoda capensis and Steatoda

paykulliana, the venom activity on rat cardiac preparationappears to indicate the presence of related toxins [39].

There are few reports of other high molecular masstoxins in spider venoms. Two insecticidal toxins of 22850and 27704 Da were isolated from the venom ofFilistatahibernalis (Filistatidae) [40]. Other insecticidal toxins of

Mr > 100 kDa were also discovered in the venom ofPhidippus audax (Salticidae) [41], but their completestructure has not been published yet. These results offer atantalizing glimpse into the enormous and yet unexploredbiochemical and pharmacological diversity of spider ven-oms.

Figure 1. HPLC (A) and MALDI-TOF MS (B) profile of atarantula venom (Lasiodora parahybana) showing elution pat-terns of peptides and polyamines.

Structure and pharmacology of spider venom neurotoxins 895


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3. Pharmacology of peptide toxins

Purification of peptide toxins from spider venoms hasbeen of great usefulness in the electrophysiological, phar-

macological and structural study of ion channels duringthe past 20 years. Ion channels are membrane proteins,which allow ionfluxes across the membrane and regulate

Figure 2. Primary structure of selected spider toxins (references in the text). Toxins are grouped according to length and disul fidebridge number: 2 (A), 3 (B), 4 (C, D), 5 (E), 6 (F) and 7 bridges (G). Groups H (-agatoxin IA, heterodimeric) et I (toxins ofBrachypelma / Eurypelma / Lasiodora/ Selenocosmia) represent different structures as described in the text. In each group, toxins arealigned on the central Cys-Cys doublet (underlined) when present.

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membrane potential and ionic balance. They can be gatedby variation of membrane potential or specific ligandsincluding ATP and neurotransmitters. They are involved inthe control of electrical potential across the membrane andby their modulatory effect on this potential, they controldiverse cellular functions such as excitation-contraction

coupling, hormone and neurotransmitter secretion andgene expression. The study of whole-cell currents andsingle channels with the patch-clamp technique allowedthe discovery of the immense structural and functionaldiversity of ion channels. In parallel, the investigation ofvenom toxins gave to biochemists and pharmacologists anarsenal of useful molecular tools, which permitted a finedissection of cellular currents by specifically blocking ionchannels. The goal of toxin discovery efforts is to providemolecular scalpels targeting selected subtypes of ionchannels with high affinity and the highest possiblespecificity. These toxins are useful in understanding therole of ion channels in forming native currents, their

physiological significance, and they permit the mapping ofreceptor sites that are potentially of interest as drugtargets.

3.1. Spider toxins and calcium channels

Spider venoms contain a diversity of peptide toxinsacting on voltage-sensitive calcium channels (VSCCs).These channels play a fundamental role in cardiac, mus-cular and neuronal function. They are grouped into severaltypes according to their activation threshold, their kineticsof calcium current and their pharmacology [4245].T-type current is activated by small depolarizations, inac-tivates rapidly and has a modulatory activity of pace-

maker type repetitive activity in cardiac cells and neurons.Other types of VSCCs are activated by stronger depolar-izations and are essentially implicated in neurotransmitterrelease. L-type channels are well represented in cardiaccells and are the target of the dihydropyridines used for thetreatment of cardiovascular pathologies (hypertension,angor). P-type was first identified in Purkinje cells, andtogether with N-type (neuronal), Q-type and R-type, iswidespread in central and peripheral nervous system cells.Structurally, VSCCs are channels formed by the associa-tion of an 1 covalent tetrameric subunit forming thechannel pore, and several auxiliary subunits (2,, and in skeletal muscle) which modulate kinetics and elec-

trophysiological properties of the Ca

2+

current. The dif-ferent genes coding for the1 subunit have been classifiedaccording to their sequence homologies (classes A, B, C,D, E, F, G, H, I and S) and then according to the variouspharmacological types of currents.

The venom ofAgelenopsis aperta(Agelenidae) was thefirst source of calcium channel-active spider toxins. First,a polyamine (FTX) was purified [25], followed by a seriesof polypeptides (-agatoxins) [4652]. The -agatoxinsblock the high activation threshold L, P, Q and N calcium

currents, with nanomolar affinity and variable selectivity[53]. Spider toxins have allowed the characterization andclassification of different calcium currents in CNS cellsand have also permitted the discovery of the R-typecalcium current [54] that is insensitive to all known toxins.All of these toxins, with the exception of FTX, are 30 to

80 amino acid polypeptides containing three to sevendisulfide bridges.

The -agatoxins are grouped in different classes ac-cording to their pharmacological properties against N, Land P/Q calcium currents. Some -agatoxins are veryspecific such as type I, II and IV -agatoxins, whichrespectively block L, N and P currents [47, 52]. Type III-agatoxins are less selective and block both N and Lcurrents with the same potency [50, 53]. It was demon-strated that -agatoxins block P/Q currents in a voltage-dependent manner and do not bind to the channel pore butinstead modify its gating properties. They bind to closedchannels with high affinity, on an external site close to the

pore, and their binding is negatively affected by strongdepolarization [55]. In addition to their diversity of actionagainst various VSCC subtypes, -agatoxins show selec-tivity for calcium channels of various zoological groups(mammalians, birds, insects). As an example,-agatoxinsIA and IIIA are, respectively, inactive against chick andinsect Ca2+ channels [56]. Conversely, other toxins such asthe -atracotoxins from Hadronyche spp. block exclu-sively insect Ca2+ channels [57]. Toxin selectivity thusreveals the occurrence of molecular variants of the sametype of Ca2+ channels in various phyla.

Following the discovery of-agatoxins, a large varietyof toxic peptides acting on VSCCs have been purifiedfrom both mygalomorph (-grammotoxin SIA from

Grammostola spatulata [5860], PhTX toxins from Pho-neutria nigriventer [6163], -atracotoxins from Hadro-nychespp. [57]) and araneomorph spiders (PLTX-II fromPlectreurys tristis[64], SNX325 fromSegestriaflorentina[65], DW13.3 from Filistata hibernalis [66], toxins from

Hololena curta [64]). A recent and important discovery isSNX482 from the venom of the tarantula Hysterocratesgigas, the first ligand of the R-type channel [54]. Thispeptide permitted a clear characterization of the molecularidentity of the R current (associated to the 1E gene) dueto a specific pharmacology.

The biological function of all calcium toxins for thespider could be to produce muscular paralysis of prey via

a block of calcium entry and presynaptic neurotransmitterrelease, resulting in flaccid paralysis, as observed withmany venoms.

3.2. Spider toxins and sodium channels

With the use of tetrodotoxin (TTX), two main catego-ries of sodium channels have been characterized: theTTX-sensitive channels found essentially in brain andskeletal muscle (genes I, II, III, PN1, NaCh6 and SkM10),



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and the TTX-insensitive channels found in heart (gene h1)and sensory neurons in peripheral ganglia (gene SNS)[67]. Voltage-dependent Na+ channels are present mostlyin neurons and muscle cells and are responsible for thedepolarization phase of the action potential. They aremajor contributors to the genesis and propagation of the

action potential in excitable cells. Their structure is similarto calcium channels with a tetrameric 1 subunit associ-ated with auxiliary subunits which modulate theirelectrophysiological properties [42, 68, 69]. Six inhibitorbinding sites have been revealed by peptide toxins fromcone snails (-conotoxins), sea anemone and scorpions (andtoxins), and small alkaloid toxins from various plantand animal origin [70, 71]. The discovery of-agatoxinsin the venom ofAgelenopsis aperta, thefirst spider toxinsacting on sodium channels, is relatively recent [46, 72]when compared to the existing body of literature onscorpion toxins discovered since 1970. The -agatoxinsare 3637 amino acid peptides containing four disulfide

bridges. These toxins increase the sodium ionflux acrossthe membrane through the voltage-dependent Na+ chan-nels. Cells are then strongly and durably depolarized, andthis induces a presynaptic neuronal stimulation resultingin massive neurotransmitter release and paralysis. Spiderpeptides acting on voltage-dependent sodium channels allhave a common mode of action, similar to that of scorpion-toxins, or sea anemone toxins. They slow down theinactivation kinetics of sodium currents, thus preventingtotal inactivation.

The venoms of Australian tarantulas in the group ofHadronychessp. (Atrax ssp.) demonstrate significant tox-icity to vertebrates including humans. Envenomationsymptoms following bites include paralysis, hypertension,

intense dehydration and vomiting [73]. From these ven-oms, several sodium channel toxins have been identified,and their mode of action has been well characterized [74,75]. -atracotoxin Ar1 (robustoxin) from Atrax robustusand -atracotoxin Hv1 (versutoxin) from Hadronycheversutus differ only by a few amino acids and competewith nanomolar affinity with scorpion -toxins on ratbrain membranes or insect neurons expressing TTX-sensitive sodium channels [7678]. Those toxins thusshare the same binding site on sodium channels [79].Other toxins were purified from the venom ofPhoneutrianigriventer, such as PhTX2 [80], and possess 4853amino acids andfive disulfide bridges. Electrophysiologi-cal studies on frog skeletal muscle preparations havedemonstrated their effectiveness on voltage-dependentsodium channels [80].

3.3. Spider toxins and potassium channels

Potassium channels are ubiquitous and constitute themost physiologically and structurally diversified class ofion channels. This diversity is apparent from the cloningof numerous channel subtypes (more than 65 to date in

mammals). They maintain the equilibrium membranepotential of excitable cells and contribute to membranerepolarization during the action potential, thus controllingfrequency and duration of electrical signals [8183]. Themultiplicity of potassium channel subtypes results in anincreased level of potassium current complexity due to the

diversity of channel types found in a single cell as well astheir capability of forming heteromultimeric assemblies,forming channels with varied electrophysiological andpharmacological properties. The discovery of novel toxinswith high specificity towards selected channel subtypes isof increasing importance in the elucidation of the func-tional role of the different potassium channels subunitsdescribed to date. In addition the ubiquity and functionaldiversity of potassium channels make them attractivetargets for the development of novel therapeutic agents.

Three main classes of potassium channels are definedby their structure and electrophysiological properties: 1)outward-rectifying, voltage-dependent channels (Kv) and

calcium-activated potassium channels (KCa) are tet-rameric assemblies of four six-transmembrane segmentssubunits. A ca. 20-amino acid stretch forms the channelpore (P domain); 2) inward-rectifying channels (Kir) aretetramers of subunits including only 2 transmembranesegments and one P domain; and 3) two P-domainchannels which are insensitive to voltage, and are outwardor inward-rectifying, have recently been cloned [84]. Allof these channels can be modulated by -subunits orauxiliary proteins.

Potassium channels are the target of various animaltoxins, which have been used to elucidate channel struc-ture and function, in particular in the group of sixtransmembrane segment channels: Kv1 (scorpion, sea

anemone, snake, bee toxins), KCa (scorpion and beetoxins), Kv3 (sea anemone toxins) [8587]. Spider toxinshave permitted the study of new Kv channel subfamilies(Kv2 and Kv4) heterologously expressed in Xenopusoocytes or mammalian cell lines. The discovery of hana-toxins in the venom of the tarantula Grammostola spatu-lata, the first spider toxins acting on voltage-dependentpotassium channels (Kv2.1), is recent [88] and confirmsthe pharmacological diversity of spider venoms. One canassume that potassium channel toxins act in combinationwith sodium channel toxins to depolarize excitable cellsand evoke neurotransmitter release thus inducing paraly-sis. Hanatoxins permitted the pharmacological character-

ization of Kv2.1 channels as well as the mapping of theirreceptor site [8890]. They are peptides of 35 amino acidscross-linked by three disulfide bridges, that act in avoltage-dependent manner by modifying the gating prop-erties of the channels and appear to bind to four equivalentexternal sites close to the channel voltage sensor (S4segment). Contrary to scorpion toxins which act by poreocclusion, spider toxins appear to bind to sites outside thepore and modify gating properties of the channels. Thehanatoxin-Kv2.1 interaction involves hydrophobic and/or

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basic residues located on the extracellular S3-S4 stretch[89, 90]. This mode of action is also identical to the actionof-agatoxin IVA on P-type calcium channels.

Thefirst toxins blocking Kv4 channels to be identifiedwere the heteropodatoxins from Heteropoda venatoria(Heteropodidae) [91], followed by the phrixotoxins

(PaTX) fromPhrixotrichus auratus[92]. Today, a numberof arguments concerning the kinetic properties and phar-macology of Kv4.2 and Kv4.3 channels indicate that theyconstitute the molecular determinants of the Ito1 transi-tory potassium current characterized in mammalian car-diac cells. This current modulates the form and duration ofthe action potential and plays an important role in cardiacphysiology. Other potassium channels strongly expressedin heart such as Kv1.4 and Kv 1.5 were proposed aspotential candidates for the molecular identity of Ito1, butheteropodatoxins and phrixotoxins have allowed an unam-biguous determination of its molecular constituents [91,92]. In vivo studies using mouse ECGs established the

role of Ito1 in the ventricular repolarization phase afterintravenous administration of PaTX1. Physiological stud-ies show that the phrixotoxins block the Kv4 channel viaa mechanism similar to that of hanatoxins on Kv2 chan-nels. The toxins affect preferentially the closed channel, ina voltage-dependent manner [92].

Thus spider venoms appear to represent a very richsource of novel potassium channel ligands. There is nodoubt that in the coming years, we will see a rapidincrease in the discovery of novel pharmacological toolsfrom these venoms.

3.4. Toxins and ASIC channels

Extracellular acidity associated with nociception aswell as taste transduction and perception of pH fluctua-tions in the brain is mediated by a recently discoveredclass of H+-gated cationic ion channels, the ASIC (acid-sensing ion channels) family [93]. We recently describedPcTX1, a novel 40 amino acid toxin from the venom ofthe tarantula Psalmopoeus cambridgei, which potentlyblocks (IC50 = 0.9 nM) the ASIC1a subclass [94]. Thischannel subtype is highly expressed in both centralnervous system neurons and sensory neurons from dorsalroot ganglia. This new toxin is the first high-affinity andhighly selective pharmacological agent for this novel classof channels and it blocks selectively homomultimeric

assemblies of ASIC1a. Other members of the ASIC familyand heteromultimeric assemblies of ASIC1a with otherASIC subunits are insensitive to this toxin. PcTX1 istherefore an important tool for the characterization ofnative ASIC currents in central neurons, and for theexploration of the physiological role of ASIC channels.The primary structure of PcTX1 is similar to that of othershort tarantula toxins, including conservation of Cysresidues, although its pharmacology is strikingly differentfrom that of previously described toxins.

3.5. Toxin selectivity

Toxin selectivity has been described at three differentlevels: molecular (receptor subtype), cellular (differenttissues) or whole organism (vertebrate/invertebrate). Theselectivity of spider toxins towards different organisms

and in particular towards insects [95], represents a verypromising potential application in agronomy, through theuse of genetically modified insect pathogens. The insectspecificity of spider toxins has been demonstrated inseveral instances. -agatoxins I and IV (Agelenopsisaperta) [72], curtatoxins I, II and III (Hololena curta)[9698] and toxins DTX9.2, 10 and 11 (Diguetia canities)[99, 100] specifically activate insect sodium channels.Similarly, -agatoxins IA, IIA and IIIA (Agelenopsisaperta) [47, 52], plectoxins V to XI (Plectreurys tristis)[101, 102] and-atracotoxins (Hadronychespp.) [57] arespecific blockers of insect calcium channels. However,there is a gradation of effects according to channel subtype

and phyletic origin, as comparative studies with-agatoxins of type I have demonstrated [56]. Finally,several insecticidal toxins with as yet undetermined sitesof action have been isolated from the venoms ofAptosti-chus schlingeri(aptotoxins I to IX) [103], Cupiennus salei(CSTX1) [104],Phoneutria nigriventer(TX4(6, 1)) [105],Tegenaria agrestis (TaITX-1, -2, -3) [106], Segestria sp.(peptide A) [107], Calisoga sp. (peptides A, B, C) [108]andFilistata hibernalis (FIL376, 377, 501 and 502) [40].

Relationships between primary structure of the toxinsand specificity towards vertebrates or insects have notbeen elucidated yet. The molecular masses of the insecti-cidal toxins listed above vary between 3 300 and27 000 Da, and the molecular determinants of their speci-

ficity remain unknown to date.

4. Structure and molecular biology of spider toxins

4.1. Primary structure and spatial structure

Compared to other animal toxin groups, relatively littlestructural information has been gathered on polypeptidespider toxins. Several structures have been determined by2D-NMR, providing insights into the general three-dimensional structure of these toxins. The NMR structuresof-agatoxins IVA [109] and IVB [110], -agatoxin I

[111],-atracotoxin Hv1 [57], -atracotoxin Ar1 (robus-toxin) [112], -atracotoxin Hv1 (versutoxin) [113], hu-wentoxin I [114, 115] and hanatoxin 1 [116] have beendetermined (figure 3), as well as the structure of a taran-tula venom peptide of unknown function, SHLP-I [117].Based on experimental data, structural models have beenproposed for other toxins such as-agatoxin IV [111]. Allof these peptides show a common structural motif com-posed of a triple-stranded antiparallel -sheet, stabilizedby internal disulfide bridges forming a cystine knot [118].



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Four structural elements are present: a I sheet connectedto a II sheet by a short turn or a short -helix, a -turnand a third III sheet. In the toxins where the disulfidebridge arrangement has been studied, three or four disul-fide bridges are conserved according to the consensussequence CIX3-7CIIX4-6CIIIX0-5CIVX1-4CVX4-13CVI(C arecysteine residues and X any residue, with number indi-cated by the range) in which bridges are formed betweencysteines IIV, IIV and IIIVI (figure 3).

The molecular scaffold is highly stabilized by thedisulfide bridges, with the CysIIIVI bridge going throughthe ring formed by bridges CysIIVand CysIIVto form theknot. In four-bridge toxins, a supplementary bridge is

formed either in one of the loops (-agatoxin IVA) orbetween a loop and the C-terminal end (robustoxin) tofurther stabilize the structure. This knottin-type fold isthus characterized by the simultaneous occurrence of theantiparallel-sheets and the disulfide bridges and repre-sents one of the most stable and compact globular confor-mations for a small-size protein. Cysteine residues andside chains from closely located residues form a globularand hydrophobic core, and the molecular scaffold isorganized to allow a maximum presentation of the amino

acid residues in the polypeptide backbone chain to thesolvent. The majority of residues are exposed and definethe charge distribution of the peptide. Toxins of this typehave in common several surface areas bearing eitherpositive or negative charges as well as a hydrophobic area.These areas are most likely to be involved in the interac-tion with the receptor site, although at this time noinformation has been obtained regarding the exact natureof molecular interactions between a spider toxin and itsreceptor. Additionally, exposing most of the residuesoffers more combinatorial possibilities and thus ensures apotentially very high variability of the pharmacologicalproperties of the toxins.

The fold described above is also found in cone snailtoxins [119] and in numerous animal and plant peptidespossessing common inhibitory properties for enzymes orreceptors. The knottin fold appears to have been selectedat various times in the evolutionary process as an optimalconfiguration for inhibitory polypeptides [111, 118]. Thenovel insecticidal peptides J-ACTXs [120] which possesstwo cysteine pairs in their sequence and a vicinal disulfidebridge were recently discovered. Their fold conforms tothe classical spider structure, but the vicinal bridge is

Figure 3. 3-D structures of selected spider toxins and schematic diagram of the cystine knot motif, for a three-disul fide bridge toxin.Disulfide bridges are indicated by bold lines, and cysteine residues by numbered dots. Structures were prepared with the programRASMOL, using coordinates from the Protein DataBank database (http:\\molbio.info.nih.gov\cgi-bin\pdb\doc\mrus\searching.html).

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functionally important for insecticidal activity, a strikingfeature for these toxins.

Peptides of known structure have very divergent phar-macology. -agatoxins and -atracotoxins block presyn-aptic, voltage-activated calcium channels, -atracotoxinsand -agatoxins are activators of TTX-sensitive voltage-dependent sodium channels, and huwentoxin I is a blockerof postsynaptic nicotinic acetylcholine receptors. Spidertoxins showing a consensus distribution of cysteine resi-dues with toxins of known structure (3 or 4 disulfidebridges) include blockers of voltage dependent potassiumchannels in the Kv2.1 (hanatoxins) and Kv4.2/4.3 sub-types (heteropodatoxins, phrixotoxins), toxins acting oncalcium channels (-grammotoxin SIA, SNX325) andtoxins with unknown pharmacological activity (BsTX5,plectoxins). Recent studies have demonstrated that toxinsof similar spatial structure were able to recognize recep-tors of different pharmacological classes, indicating thepresence of functionally similar recognition sites on the

receptor surface [121]. Interestingly, the same structuralmotif is found in both mygalomorph and araneomorphvenom peptides, showing a high phylogenic conservationacross the group. The majority of short polypeptide spidertoxins discovered to date appear to conform to this model.The structural homogeneity due to the conserved scaffoldis combined with a very high surface variability resultingin an extremely important pharmacological diversifica-tion. At the same time, short peptide spider toxins showsequence hypervariability between the highly conservedcysteine residues. The only toxin possessing only 2disulfide bridges described to date is a small insecticidaltoxin (3 988 Da) from the venom ofSegestria florentina(SIT) [122]. A number of polypeptide toxins of larger size,

from 60 to 80 amino acids, have also been described andcomprise 8 to 14 cysteines paired into disulfide bridges(aptotoxins, CSTX1,-agatoxin IIIA, PhTX1). The pres-ence of a central Cys-Cys doublet in all of these peptidesindicates a structural homogeneity, and the presence ofsupplementary disulfide bridges could be interpreted as astabilization of longer peptide chains, based on the con-served knottin motif. However no direct experimentalevidence has been obtained to date regarding either thethree-dimensional structure or the disulfide bridge ar-rangement of such peptides. Indirect evidence of thestructure has been obtained with the discovery of peptideACTX-Hvf17 from the venom of Hadronyche versutus,

which has significant homology to the snake peptideMIT1 and the colipase protein family [123]. The overallstructure of the latter shows two symmetrical domainscontaining antiparallel -sheets.

Several other spider peptide toxins have been describedwhich deviate from this structural family. -Agatoxin IAis characterized by an heterodimeric chain with a 66-amino acid primary chain and a secondary chain com-posed of three residues (Ser-Pro-Cys), linked by a disul-fide bridge [124]. This is the only described occurrence of

such a structure in spider venoms. However, the longestchain has an odd number of Cys residues, and such chainshave been found also in other toxins such as -agatoxinIB or Hololena curta calcium channel toxins. It thusseems possible that the presence of a secondary chain andan heterodimeric structure could have been overlooked

during automated Edman degradation, and that it may befound in other toxins. Yet another structural arrangementis found in toxins from Central-American tarantulas in thegenera Brachypelma (BsTX1) [125, 126] and Eurypelma(EsTX1) [127, 128], where toxins do not show the centraldoublet of Cys residues. More recently, related toxinshave been described in venoms of the South-Asian taran-tulas Selenocosmia huwena (huwentoxin II) [129] andCoremiocnemis validus [130] and the labidognath Tege-naria agrestis [106].

The study of the disulfide arrangement of BsTX1, themajor toxin from the venom of Brachypelma smithi hasshown that the disulfide bridges are conserved according

to the motif IIV, IIV and IIIVI [125]. The three-dimensional structure remains undetermined. These toxinsdisplay low toxicity against vertebrates and a characteris-tic succession of neurotoxic symptoms after intracere-broventricular injection in mice: gyration, convulsions,seizures and death (Escoubas, unpublished data), but theirmolecular receptor remains unknown to date. Similarneurotoxic symptoms are observed for the major peptidesin the venom of Lasiodora parahybana, a Braziliantarantula (LpTX1, LpTX2) [27]. LpTX1 and LpTX2 areisoform toxins with a strong homology to Brachypelmaand Eurypelma toxins, with an internal supplementarystretch of 10 mostly positively charged amino acids (5Lys) and one additional disulfide bridge. The addition of

this supplementary loop, which appears to be functionallyrelevant due to its highly charged nature, could representa mechanism of molecular diversification in this group oftoxins.

The breadth of our current knowledge of spider toxinsdoes not allow us at this point to clearly define structure-activity relationships similarly to the work done withscorpion or cone snail toxins. For longer peptides such astype III -agatoxins, only activities against the voltage-dependent calcium channels have been demonstrated todate. Such partial results do not allow for definitiveconclusions, but it is possible that an increased complexityof the toxin structure could be correlated with a higherspecificity towards selected receptors, such as the varioussubtypes of calcium channels.

4.2. Genomic structure

Few studies have addressed the structure of genescoding for polypeptide spider toxins. Genomic sequencesof several calcium or sodium channel toxins (PhoneutriaTx2-1, Agelenopsis aperta -agatoxin IA, Plectreurystristis PlTX, Diguetia canities DTX9.2) [100, 101,



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131133] appear to be relatively similar. They code forproteins including a 17 to 20 amino acid signal peptide inN-terminal position, which is highly homologous for agiven series of toxins, followed by a glutamate-richpropeptide sequence of 13 to 44 amino acids and thesequence coding for the mature toxin. In C-terminal

position, several residues that are excised during toxinmaturation are also often found. A possible role in thepost-translational modification of the C-terminus has beensuggested for these residues. The signal sequence includesa consensus peptidase cleavage site and a possible impli-cation of the propeptide in post-translational modificationand toxin refolding has also been suggested [101].

4.3. Structural variability and molecular evolution

As venoms may contain up to 50 or 60 differentpeptides, as revealed by MALDI-TOF (matrix-assistedlaser desorption-ionization time-of-flight) mass spectro-

metry analysis, the structure of their peptide toxins hasonly been partially explored, usually following up onspecific activity bioassays. It is therefore difficult toconstruct a global picture of the pharmacological com-plexity of a single venom as well as to understand thestructural diversity of the peptides involved. The bestexplored venom remains that of Agelenopsis aperta inwhich both acylpolyamines (-agatoxins), and polypep-tide toxins (type I, II and III-agatoxins,-agatoxins) ofvariable length and disulfide bridge number have beenidentified. The other species where venom compositionhas been investigated in detail is Phoneutria nigriventer(Ctenidae) that has a complement of toxins of similarstructure [134137]. With the goal of determining thedistribution of the different types of polypeptide toxinsacross a wide variety of species, we have undertaken astudy using reversed-phase HPLC and MALDI-TOF massspectrometry peptide profiling of 55 tarantula venoms[30]. The analysis of chromatographic profiles showed theconcomitant presence of peptides and polyamines in anumber of venoms, but an overall dominance of peptidesas venom constituents(figure 2). A detailed analysis of themass profiles and the distribution of molecular massesshowed a bimodal distribution for a total of ca. 1 500peptides identified. All species included, the molecularmasses are roughly divided into two main classes center-ing on molecular masses 3 5004 500 Da and

6 5007 000 Da. Peptides of Mr 3 5004 500 Da are themain constituents of these venoms. These results arelargely compatible with our own experience of tarantulaspider toxins and the published structures of polypeptidetoxins from these venoms.Mrvalues appear to correspondto the two main groups of structures, the first oneincluding the shorter toxins with 34 disulfide bridges (ca.4 000 Da) and the second one to larger peptides possess-ing four or more disulfide bridges (6 500 Da and up). Inthe family Theraphosidae (tarantulas), the pharmacologi-

cal diversity that is starting to emerge appears to beglobally correlated with a structural homogeneity center-ing on a knottin-fold scaffold with three disulfide bridges,although the sequencing of more peptides is clearlynecessary to confirm this model. Spider toxins appear tobe produced in a semi-combinatorial model in which only

the residues which are functionally relevant are mutatedwhile residues maintaining the overall structure are totallyconserved, following the model proposed by Olivera et al.[138] for cone snail toxins. In the O-, -, - andj-conotoxin families, pharmacological diversity is ac-quired through hypermutation of residues located in asimilar molecular scaffold defined by loops highly con-strained by conserved cysteines. In the relevant genes,only the mature peptide is modified, with high conserva-tion of the signal peptides and propeptides. In spiders, asin cone snails, the diversification of molecular targets isremarkable and appears to have followed a similar evolu-tionary pathway.

As another molecular diversification mechanism,highly homologous or isoform toxins can be found in onevenom or in venoms from closely related species, differingonly by a very limited number of amino acids (Brachy-

pelma, Lasiodora, Eurypelma toxins, hanatoxins, agatox-ins, phrixotoxins). These limited mutations will permit avery fine control of the pharmacological properties of atoxin family against a selected receptor. An additionaldiversification mechanism is the insertion of chargedresidues in a conserved sequence, forming additionalloops and interaction sites as described above for

Lasiodora parahybana toxins. This mechanism does notalter the overall structure of the toxins but considerablyaffects their electrostatic properties and results in addi-

tional pharmacologicalflexibility.Another evolutionary strategy is the D/L isomerization

described for -agatoxin IVB (or -agatoxin TK). AD-amino acid alters the structure of a toxin and appears toresult from a post-translational modification via anisomerase [139]. A study on both D and L isomers of-agatoxins IVB and IVC demonstrated the crucial im-pact of isomerization on toxin selectivity and stabilizationagainst proteolysis [140]. Although this is the only de-scribed occurrence of such an isomerase, it might not beunique to spider venoms. However, D amino acids cannotbe detected by Edman degradation, and the widespreadoccurrence of this structural variation in spider venom

toxins remains to be established. Other post-translationalmodifications have been described for spider toxins.C-terminal amidation is relatively frequent, and a moreunusual modification was described for toxin PlTX-IIfrom Plectreurys tristis where the C-terminal threoninehas a O-palmitoyl group, thus changing significantly itshydrophobicity, steric hindrance and surface properties[141].

Although the registry of toxin modifications appears tobe much larger in other venomous animals such as cone

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snails, it is probable that spider toxins will reveal morestructural diversity as our knowledge of venoms increases.

5. Conclusions

Several molecular mechanisms have been discoveredthat allow spiders to produce a rich diversity of neurotox-ins resulting in venoms of high efficiency as demonstratedby biochemical and toxicological analysis. This structuraldiversity results in great pharmacological diversity thatpermits a very precise and selective targeting of multiplecellular receptors. The presence of a diversified pharma-copoeia in a single venom represents an evolutionaryadvantage for a predator, resulting in the highly efficientcapture of diverse prey.

For the biologist, spider venoms thus represent anincredibly rich and diversified source of novel moleculartools for the exploration of the physiology of excitable

cells, leading to a better understanding of ligand-receptorinteractions at the molecular level, and the development ofnovel therapeutic strategies following natures lead.

Acknowledgments

The authors are grateful to Dr. M. Gelb and C.K. Kristensen(SpiderPharm Inc.) for a critical reading of the manuscript and toDr. T. Nakajima (SUNBOR) for continued support.

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