M I N I R E V I E W

Polypeptide neurotoxins from spider venoms

Eugene Grishin

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia

Spider venoms contain a variety of toxic components. The polypeptide toxins are divided into low and high

molecular mass types.

Small polypeptide toxins interacting with cation channels display spatial structure homology. They can affect

the functioning of calcium, sodium, or potassium channels.

A family of high molecular mass toxic proteins was found in the venom of the spider genus Latrodectus. These

neurotoxins, latrotoxins, cause a massive transmitter release from a diversity of nerve endings. The latrotoxins are

proteins of about 1000 amino acid residues and share a high level of structure identity.

The structural and functional properties of spider polypeptide toxins are reviewed in this paper.

Keywords: spider venom toxins; polypeptide toxins; amino acid sequence; spatial structure; cation channels;

latrotoxins; neurotransmitter release; gene cloning.

About 40 000 different kinds of spiders are known at present.However, the venom of only several dozens of species is wellstudied. It comprises many components, and neurotoxins are ofthe most interest. They can be chemically divided into twomajor groups, viz. polyamine and polypeptide toxins. Thepolypeptide toxins in turn are also subdivided by theirfunctional and molecular characteristics. These toxins arerepresented by two main types. The first type embracesrelatively small polypeptides, which can interact with ionchannels of the excitable membrane. The second one covershigh molecular mass neurotoxins bound to receptor componentsof the presynaptic membrane and intensifying neuromediatorsecretion. The present review deals with major structure-functional properties of both the spider venom neurotoxintypes.

The venoms of various spiders contain polypeptide toxins ofmolecular mass ranging from 4 to 10 kDa, with a high numberof cysteine residues, which form intramolecular disulfidebridges. Their number in these toxins is varying from 6 to 14.As a rule, the toxins containing the same number of disulfidebridges display distinct structural homology, but differ in theirfunctional characteristics. Many spider neurotoxins affect thefunctioning of different calcium channels. They serve as perfectresearch tools in electrophysiological and/or biochemicalexperiments. Many fewer toxins are known to affect thesodium or potassium channels.

C A L C I U M C H A N N E L A N T A G O N I S T S

An extensive class of polypeptide neurotoxins has beenidentified in various spider venoms inhibiting the function ofvoltage-dependent calcium channels, initially referred to asv-toxins. A family of peptide antagonists of voltage-activatedcalcium channels, v-agatoxins, is purified from the venom of

the funnel web spider, Agelenopsis aperta. v-Agatoxins are aheterogeneous group of polypeptides varying in their molecularmass from 5 to 10 kDa [1,2]. The given toxins are classifiedinto four groups by their structural and functional charac-teristics (Fig. 1).v-Agatoxins of group I (v-Aga-I) block nerve muscular

transmission in insects, but do not influence the binding tosynaptosomes of v-conotoxin GVIA, the blocker of the N-typecalcium channel from sea-snail venom. v-Aga-IA is struc-turally a heterodimeric peptide; the main chain is made up of 66amino acids and is bound to a tripeptide (Ser-Pro-Cys) by adisulfide bridge [3]. It contains nine cysteine residues formingdisulfide bridges (four intramolecular and one intermolecular).v-Aga-I block presynaptic Ca++ channels in motorneurons andneurosecretory cells of insects (but not of birds and mammals),and also affect the Ca++ currents in rat sensory neurons.v-Agatoxins of group II (v-Aga-II) block nerve musculartransmission in insects and inhibit binding of v-conotoxinGVIA. These are peptides by their chemical compositionconsisting of 90±95 amino acid residues [1]. v-Aga-II blockthe Ca++ channels in insect motoneurons and in the synapto-somes of birds and rats. v-Agatoxins of group III (v-Aga-III)inhibit binding of v-conotoxin GVIA, but do not affect thenerve muscular transmission in insects [4]. They resemble atruncated v-Aga-II, i.e. these are peptides of 76 amino acidslong and are highly homologous to the toxins of group II.v-Aga-III block mainly the Ca++ currents of N- and L-typesin diverse rat and mouse preparations [5]. Of much interest arethe v-agatoxins of group IV (v-Aga-IV) which show selectiveblocking of Ca++ channels of P-type [6]. All the toxins of thisgroup are eight cysteine residue peptides made up of 48 aminoacid residues containing four disulfide bridges. The homologybetween the sequences of the two mostly widespread toxins,v-Aga-IVA and v-Aga-IVB, reaches 71%. The disulfidebridge configuration of these two toxins appears to beidentical: Cys4±Cys20, Cys12±Cys25, Cys19±Cys36, andCys27±Cys34. The toxins of this group have rather unusualmodifications of the C-terminal amino acid residue. Forexample, a peptide toxin PLTX-II from the spiderPlectreurys tristes venom has O-palmitoyl threonine amideresidue at its C-terminus. Such fatty acylation seems to be

Eur. J. Biochem. 264, 276±280 (1999) q FEBS 1999

Correspondence to E. V. Grishin, Shemyakin-Ovchinnikov Institute of

Bioorganic Chemistry, Moscow, Russian Academy of Sciences, Ul.

Miklukho-Maklaya, 16/10, 117871, GSP-7, Moscow, Russia,

Fax: + 95 310 7007, Tel.: + 95 330 5892, E-mail: grev@ibch.siobc.ras.ru

(Received 17 February 1999, revised 26 March 1999, accepted

29 March 1999)

q FEBS 1999 Polypeptide neurotoxins from spider venoms (Eur. J. Biochem. 264) 277

typical of all similar calcium channel blockers in the P. tristesvenom [7].

A peptide toxin, v-grammotoxin SIA, was isolated fromthe venom of the tarantula spider Grammostola spatula. Thetoxin consists of 36 amino acid residues with three disulfidebridges and an amidated C-terminal valine residue (Fig. 1).v-Grammotoxin SIA blocks N-, P-, and Q-types but not L-typevoltage-gated calcium channels [8]. A novel peptide toxin of 74amino acids (DW13.3) was isolated from the venom of thespider Filistata hibernalis. This toxin potently blocks allcalcium channel currents studied, with the exception of T-type currents. Such an inhibition of different subtypes ofcalcium channels by this toxin reflects a common binding sitepresent on all calcium channels except for T-type [9].

There is a group of relatively selective spider toxins affectingcalcium channels. A calcium channel antagonist peptide,SNX-325, was purified from the spider Segestria florentinavenom. SNX-325 toxin consists of 49 amino acid residues withfour intramolecular disulfide bridges (Fig. 1). At nanomolarconcentrations it is a selective blocker of the N-type channel,but not of the other calcium channels [10]. A potent andselective peptide antagonist of the E-class calcium channelswas isolated from the African tarantula, Hysterocrates gigas.This peptide toxin of 41 amino acids named SNX-482 affectspharmacological properties of native R-type calcium currentswithout effecting N-, L-, and P/Q-type currents [11].

S O D I U M C H A N N E L E F F E C T O R S

A family of peptide toxins affecting the sodium channels andcalled m-agatoxins (m-Aga-I to m-Aga-VI) has been found inthe venom of the spider Agelenopsis aperta [12]. There arecurrently six toxins characterized that consist of 36±38 aminoacids and contain eight cysteine residues, which form fourdisulfide bridges (Fig. 1). At least four toxins of this familyhave an amidated C-terminal residue. m-Agatoxins, by theiraction, are like scorpion excitatory insectotoxins and induce ashift of the curve of the insect Na+ channel activation to morenegative potentials. By their structure and action, the toxins

resemble curtatoxins (Ct-I and Ct-II) from the venom of thespider Hololena curta [13], or toxic polypeptides (Tx1 andTx2-9) from the venom of the spider Phoneutria nigriventer[14] and many other polypeptide toxins from various spidervenoms (Fig. 1). The mechanism of their action is still obscure.However, blocking of their effects by tetrodotoxin as well as ahigh structural homology to m-agatoxins provides evidenceabout the identity of their mechanisms of action [14].

Fig. 1. Amino acid sequences of polypeptide toxins from spider venoms. Alignment of the sequences was carried out by cysteine residue positioning. The

right column denotes the number of amino acid residues in the polypeptide chain of the corresponding toxin (N-terminal sequence is given for v-Aga-

IIA). v-Aga-IA±v-Aga-IVA, v-agatoxins from the funnel web spider Agelenopsis aperta [1±6]; PLTX-II, toxin from the spider Plectreurys tristes [7];

SIA, v-grammotoxin SIA from the tarantula spider Grammostola spatula [8]; DW13.3, toxin from the spider Filistata hibernalis [9]; SNX-325, toxin

from the spider Segestria florentina [10]; SNX-482, toxin from the African tarantula Hysterocrates gigas [11]; m-Aga-I and m-Aga-VI, m-agatoxins

from the spider Agelenopsis aperta [12]; Ct-I and Ct-II, curtatoxins from the spider Hololena curta [13]; Tx1 and Tx2-9, toxins from the spider

Phoneutria nigriventer [14]; HaTxl and HaTx2, hanatoxins from the Chilean tarantula Grammostola spatulata [15]; HpTx1, heteropodatoxin from the

spider Heteropoda venatoria [17]; HWTX-I, huwentotoxin-I from the Chinese bird spider Selenocosmia huwena [18].

Fig. 2. Schematic diagram of the tertiary fold of the v-Aga-IVA with

the triple-stranded antiparallel b-sheet. The four disulfide bridges

between numbered Cys residues are shown. The N- and C-termini are

labelled N and C. The diagram was generated using PDB entry 1IVA.

278 E. Grishin (Eur. J. Biochem. 264) q FEBS 1999

P O T A S S I U M C H A N N E L I N H I B I T O R S

The components influencing potassium channel function areless abundant in spider venom. Two highly homologous toxins(hanatoxin 1, HaTxl, and hanatoxin 2, HaTx2) have beenisolated from the venom of the Chilean tarantula, Grammostolaspatulata, which block the Kv2.1 potassium channel atnanomolar concentrations. The toxins contain 35 amino acidresidues with three disulfide bridges (Fig. 1). Hanatoxins do notdisplay any pronounced structural homology relative to theknown inhibitors of potassium channels but their primarystructure is similar (over 40% amino acid sequence identity) tothe calcium channel blocker v-grammotoxin SIA isolated fromthe same spider venom [15]. Recent studies have indicated thathanatoxin and grammotoxins can interact both with a potassiumchannel and with a calcium channel. This conclusion providedevidence for the toxin binding to voltage-gated calcium andpotassium channel regions, which actually possess an identicalstructure [16]. Another polypeptide group, also inhibitingpotassium channels, was isolated from the venom of the spiderHeteropoda venatoria. The three heteropodatoxins (HpTx1±3)consist of 30±33 amino acid residues with three disulfidebridges and display amidated C-terminal amino acid residues(Fig. 1). They all possess a high structural homology (about40%) with hanatoxins. The heteropodatoxins prolong theaction-potential duration of isolated rat ventricular myocytes,suggesting that these toxins block potassium currents. Thetoxins were shown to block the Kv4.2 current in a voltage-dependent manner with less blocking at more positivepotentials [17].

V A R I E T Y O F S P I D E R T O X I N S

Many publications are available on isolation of other poly-peptide toxins from spider venoms. As a rule, these toxins canbe classified by their biological properties and structuralcharacteristics into one of the above groups. In addition, thereis a series of publications about other quite differentmechanisms of action of spider toxins. For example, aneurotoxic polypeptide, huwentotoxin-I (HWTX-I), was iso-lated from the venom of the Chinese bird spider Selenocosmiahuwena. This toxin consists of 33 amino acid residues,including six Cys residues (Fig. 1). The action site ofHWTX-I was demonstrated to be a nicotinic acetylcholinereceptor [18].

T H R E E - D I M E N S I O N A L S O L U T I O NS T R U C T U R E O F T H E S P I D E R T O X I N S

Studies on the spatial structure of polypeptide toxins are ofmuch importance for elucidating their molecular mechanism ofaction. Recently, using NMR spectroscopy, the solutionstructures of certain spider toxins, which affect cation channelsand the choline receptor, have been determined. First, theattention has been focused on the antagonist structure ofcalcium channels of v-Aga-IVB and v-Aga-IVA [19,20]. Thespatial structure of v-IVA is composed of a short triple-stranded antiparallel b-sheet, three loops, and disordered N-and C-terminal fragments (Fig. 2). The analysis of the spatialarrangement of v-agatoxins and v-conotoxin GVIA hasrevealed their common structural features that is a unique

Fig. 3. Schematic representation and

alignment of latrotoxin structures. The figures

indicate the domain localizations. The first

N-terminal domain contains 459±470 amino acid

residues and includes two potential membrane

regions. The second domain consists of 532±717

amino acid residues with a series of ankyrin-like

repeats. The last two figures on the right

designate the domain removed upon maturation.

q FEBS 1999 Polypeptide neurotoxins from spider venoms (Eur. J. Biochem. 264) 279

example of the toxin convergent evolution [21]. The overallstructure topology of the two toxins is the same; but thehydrophobic C-terminal segment of v-Aga-IVA, lacking inv-conotoxin, plays a crucial role in the blocking action ofv-agatoxin on the P-type calcium channel. Similar structuralmotives underlay the arrangement of the spider toxins, viz.three antiparallel b-sheets forming the b-layer, flexible peptideloops, the molecular nucleus containing the cystine knot andfairly disordered N- and C-termini. The conservative residuesof basic amino acids form a positively charged surface locatedclose to the hydrophobic C-terminus which actively interactswith the channel. The v-ACTX-HV1, a potent insecticidaltoxin from the venom of an Australian spider Hadronycheversuta, displays significant spatial structure homology withthe v-agatoxins and v-conotoxins. However, this toxin inhibitsinsect, but not mammalian voltage-gated calcium channelcurrents [22].

The spider toxins affecting the sodium channel functionpossess rather a similar spatial structure. The solution structureof m-agatoxin (m-Aga-I) obtained by NMR also consists of awell-defined triple-stranded b-sheet involving residues 7±9,20±24, and 30±34 as well as four tight turns. Another similartoxin, m-Aga-IV, exhibited two distinct conformations insolution . These conformers arose from cis and trans peptidebonds involving a proline at position 15 [23].

The spider toxins with three disulfide bridges also havetypical features of the spatial arrangement. The molecule ofHWTX-I affecting the acetylcholine receptor adopts acompact structure consisting of a small triple-strandedantiparallel b-sheet and five b-turns. The three disulfidebridges are buried within the molecule [24]. On the whole,the polypeptide toxins from the spider venoms share commonspatial structure with various peptide toxins targeting a varietyof ionic channel types as well as with some protease inhibitorsand plant proteins. The key features of this spatial structure area triple-stranded, antiparallel b-sheet and a cystine knot [25].

B L A C K W I D O W S P I D E R T O X I N S

A family of high molecular mass neurotoxins, latrotoxins,(about 120 kDa) has been revealed in venoms from the spidergenus Latrodectus. The toxins provoke a massive anddestructive transmitter release of neuromediators from pre-synaptic endings of various animals [26]. The a-latrotoxin(a-LTX) is mostly studied and known as affecting thevertebrates. a-LTX induces exocytosis of small synapticvesicles without acting on large vesicles [27]. The toxininitiates secretion of all known neurotransmitters, evidentlyinfluencing the universal components of the neurosecretionsystem. The a-LTX molecule is shown to exist as a tetramer[28]. Its action is accompanied by the membrane depolarizationand Ca++ influx into the presynaptic ending [29]. At this stagea-LTX is strongly bound to the membrane receptors neurexinor latrophilin [30,31]. The black widow spider venomencourages neurosecretion in different insect and crustaceanpreparations [32]. Its action on these preparations occurs due tothe presence in the venom of insectotoxic proteins (latroin-sectotoxins, LITs) and a latrocrustotoxin (a-LCT) affectingcrustacean nerve endings [33]. Some of these toxins have ahigh-affinity receptor on the presynaptic membrane and arecapable of forming channels in lipid bilayers [34].

Cloning of the genes encoding latrotoxins revealed generalprinciples of their molecular arrangement [35±37]. Alignmentof the sequences of four latrotoxins (Fig. 3) shows that theycontain conserved regions extending over the entire proteins.

The latrotoxin molecule can be divided into four structuraldomains. The first domain, made up of 14±38 amino acids, isremoved upon the protein maturation. It differs structurallyfrom the classic signal peptide of eukaryotic proteins, butprobably performs a similar function, i.e. serves as a signal oftoxin secretion. The N-terminal domain consists of 450±480amino acid residues. Two conservative hydrophobic regionsforming transmembrane segments are found in its structure.The central domain of latrotoxins is entirely formed by ankyrinrepeats also found in the proteins, which are connected withcytoskeletal proteins, and are involved in differentiation andtranscription processes [38]. Repeats 15±17 in a-LTX and a-LIT have an unusual six cysteine residue cluster absent in the d-LIT structure. The fourth latrotoxin C-terminal domainconsisting of about 160 amino acid residues is removed uponthe toxin maturation.

Data available on the structure±functional properties oflatrotoxins provide two models of their action [39]. The firststage is the toxin tetramer binding to the presynaptic receptor.Because ankyrin repeats, according to many studies, areinvolved in interprotein interaction, this suggests that inlatrotoxins they promote the formation of their tetramers. Thenext stage envisages the insertion of the fragments of severaltoxin molecules into the membrane with the formation of thecation channel, through which Ca++ ions penetrate into thepresynaptic ending. Of much importance here are twoconservative hydrophobic regions found in the N-terminaldomain of the latrotoxins. According to the other model thetoxin binding to the receptor, which interacts with theneurosecretion proteins, activates the receptor and initiatessecretion, in the absence of Ca++ ions. Note that the two modelsdo not exclude each other; the combined mechanisms can takeplace simultaneously.

C O N C L U S I O N

The polypeptide components of the spider venoms displayspecific action on the ionic channels and receptor systems ofthe nerve cell membrane. When studying functionally impor-tant systems of excitable membranes, a variety of spider toxinsare widely employed as tools in electrophysiological andneurochemical experiments. The selectivity of some spidertoxin actions only in insects allows consideration of thesemolecules as the basis for designing novel insecticides. Furtherstudies of the spider venoms is rather promising for enlargingthe class of unique substances possessing highly specific actionon the most significant components of the nerve cell membrane.

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