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Aug 5, 1996 - Thus, B. arenicola IT2 represents an interesting tool to study the ...... the insect selective neurotoxin (AalT) from scorpion venom to locust.
Eur. J. Biochem. 243, 93-99 (1997) 0 FEBS 1997

Biochemical and pharmacological characterization of a depressant insect toxin from the venom of the scorpion Buthacus arenicola Sandrine CESTELE, Charles KOPEYAN, Razika OUGHIDENI, Pascal MANSUELLE, Claude GRANIER and HervC ROCHAT Laboratoire d’Ingtnierie des prottines, CNRS URA 1455, I.F.R. Jean Roche, Facult6 de mtdecine Nord, Marseille, France (Received 5 August 1996)

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EJB 96 1161/3

A depressant toxin active on insects, Buthacus arenicola IT2, was isolated from the venom of the North African scorpion B. arenicola and its structural and pharmacological properties were investigated. B. arenicola IT2 is a single polypeptide of 61 amino acid residues, including 8 half-cystines but no methionine and histidine, with a molecular mass of 6835 Da. Its amino acid sequence is 79-95% identical to other depressant toxins from scorpions. When injected into the cockroach Blutella germanica, B. arenicola IT2 induced a slow depressant flaccid paralysis with a LD,, of 175 ng. B. arenicola IT2 has two non-interacting binding sites in cockroach neuronal membranes: one of high affinity (Kd, = 0.1 1 ? 0.04 nM) and low capacity (BmaX, = 2.2 2 0.6 pmol/mg), and one of low affinity (Kd2= 24 2 7 nM) and high capacity (BmdxP = 226 2 92 pmol/mg). Its binding to these two sites was completely inhibited by Leiurus quinquestriatus quinquestriatus IT2, a depressant toxin from L. quinquestriatus quinquestriatus. Reciprocal-binding experiments between B. urenicola IT2 and the excitatory insect-toxin A. australis Hector IT revealed competition between the two toxins for the high-affinity sites of B. arenicola IT2. B. arenicola IT2 has a higher affinity than L. quinquestriatus hebraeus IT2, a depressant toxin from L. quinquestriatus hebraeus. Thus, B. arenicola IT2 represents an interesting tool to study the receptor site for depressant toxins on insect sodium channels.

Keywords: scorpion venom; neurotoxin; primary structure; insect sodium channel.

Voltage-sensitive sodium channels are the molecular structures responsible for the increase in sodium permeability during the initial phase of the action potential in most excitable cells (Hille, 1984). Sodium channels from various excitable tissues and animal phylla contain a major a-subunit with an apparent molecular mass of 260 kDa (Beneski and Catterall, 1980; Hartshorne et al., 1982; Barhanin et al., 1983; Darbon et al., 1983; Gordon et al., 1988; Jover et al., 1988). Analysis of the amino acid sequence of the sodium channels deduced from cDNA has enabled the construction of topological models of the channel within the plasma membrane. Amino-acid-sequence analysis indicates four similar domains containing six transmembrane segments/domain (Greenblatt et al., 1985; Noda et al., 1986; Guy and Conti, 1990), connected by internal and external polypeptide loops. The extracellular loops may participate in the formation of binding sites of polypeptide neurotoxins (Tejedor and Catterall, 1988; Thomsen and Catterall, 1989; Gordon et al., 1992). Vertebrate and insect sodium channels have similar primary structures (Loughney et al., 1989), topological characteristics (Gordon et al., 1992; Moskowitz et al., 1994) and basic biochemical (Gordon et al., 1988, 1990; Moskowitz et al., 1991, 1994) and pharmacological (Pelhate and Satelle, 1982; Cestkle et al., 1995) properties. Sodium channels are specific targets for various neurotoxins (Catterall, 1992). Ligand-binding studies show that these toxins occupy at least six receptor sites on voltage-sensitive sodium channels (Catterall, 1980; Couraud et al., 1982; Poli et al., 1986; Fainzilber et al., 1994) and such toxins have been used as tools Correspondence to S. Cestble, Laboratoire d’IngCnierie des prottines, CNRS URA 1455, I.F.R. Jean Roche, Facultt de mCdecine Nord, Bd Pierre Dramard, F-13916 Marseille Cedex 20, France Abbreviations. LD,,, median lethal dose; IT, insect toxin.

for functional mapping and characterization of the channel (Catterall, 1986, 1992). Scorpion venoms contain, among others, two groups of neurotoxins, the excitatory and the depressant insect toxins, named according to the symptomatology developed by injected animals. These toxins are only active on insects, as demonstrated by toxicity, electrophysiological and binding assays on in vivo and in vitro preparations derived from various animals (Zlotkin, 1991). This selectivity may suggest characteristic structural or functional features of the insect sodium channels compared with those of vertebrates. The excitatory and the depressant insect toxins are single-chain polypeptides of 60-70 amino acid residues cross-linked by 4 disulfide bridges (Darbon et al., 1982; Zlotkin et al., 1991). The excitatory toxins cause a fast excitatory paralysis on the animals and induce repetitive firing in insect nerves. The depressant toxins cause a slow depressory flaccid paralysis due to depolarization of the nerve membrane and blockage of the sodium conductance in axons (Pelhate and Zlotkin, 1982; Zlotkin et al., 1985; Zlotkin, 1991). The excitatory and depressant toxins are used as tools for the study of the pharmacology of the insect sodium channels and the design of insect-selective biopesticides (Stewart et al., 1991 ; Tomalski and Miller, 1991). We describe the purification from Buthacus arenicola venom and biochemical characterization of a depressant toxin, called B. arenicola IT2. We examined the pharmacological properties of this toxin on neuronal membranes from Periplaneta americana.

MATERIALS AND METHODS Scorpion venom. The venom of B. arenicola was obtained in the laboratory by electrical stimulation of scorpions collected in the oasis of Tozeur (Tunisia).

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Purification of B. arenicola IT2 from B. arenicola venom. The lyophilized venom (1 g) was extracted with water and dialyzed as described by Miranda et al. (1970). The dialysed and lyophilized water extract of the venom was subjected to gelfiltration chromatography on Sephadex G-50 Fine (Pharmacia), on four columns (3.2 c m x 100 cm) in series, equilibrated and developed with 0.1 M ammonium acetate, pH 8.5, flow rate 40 ml/h, at 25°C. The last step of purification was semipreparative HPLC on a Beckman RP-8 column (10 mmX250 mm) and a Kratos system. The sainple was eluted with a linear gradient from 15% to 40% acetonitrile (Carlo Erba) in 0.15 M ammonium formate, pH 2.7, over 40 min, at a flow rate of 4 ml/min (Kopeyan et al., 1990). The homogeneity of the toxin was assessed by analytical HPLC on a Merck Lichrospher 100 RP-8 column ( 5 pin, 4 mmX125 mm) with a linear gradient from 25 % to 33 % acetonitrile in 0.1 5 M ammonium formate, pH 2.7, over 40 min, at a flow rate of 1 ml/min. The detection wavelength was 280 nm. Polyacrylamide-gel electrophoresis. Electrophoresis was performed in polyacrylamide gels, with a p-alanine acetate buffer, pH 4.4, on a Phast system according to the manufacturer's instructions (Pharmacia). Proteins were precipitated with 20 96 trichloracetic acid and silver stained. Amino acid analysis. Protein samples were hydrolyzed under vacuum in a Pico-Tag work station (MilliporeNaters Associates) with 6 M HCl and 1 D/c phenol at 110°C for 20 h or 70 h. The amino acid composition was calculated from analysis by means of a Beckman 6300 apparatus. Amino-acid-sequence determination. Reduced protein was prepared by reduction of the disulfide bridges with dithioerythreitol (60-fold molar excess over disulfide bridges, in 250 mM Tris/HCI, 4 mM EDTA, 6 M guanidine, pH 8.5, under nitrogen at 40°C and for 20 h in the dark). Two alkylations were carried out: S-pyridylethylation (8 nmol) was performed by adding 2 pl 4-vinylpyridine and incubating for 20 min at room temperature, and S-carboxymethylation (20 nmol) was carried out with iodoacetic acid sodium salt (Crestfield et al., 1963). Alkylated protein was desalted on a Beckman HPLC system equipped with a C, column (4 mmX125 mm, 5 pm) from Merck. Alkylated proteins and peptides were subjected to Edman degradation. S-carboxymethylated peptides were sequenced in a Beckman 890 MM sequencer. S-pyridylethylated protein (200 pmol) and derived peptides were sequenced in an Applied Biosystems 476A sequencer. Enzymatic cleavages. S-pyridylethylated B. arenicola IT2 (500 pmol) was digested with 5 % (by mass) trypsin in 0.125 M NH,HCO,, pH 8.5, at 37"C, for 20 h. S-pyridylethylated B. arenicola IT2 (1 nmol) was digested with 2% (by mass) a-chymotrypsin in 0.1 M NH,HCO,, pH 7.8, at 37"C, for 20 h. Scarboxymethylated B. arenicola IT2 ( 5 nmol) was subjected to Lys C protease digestion 15% (by mass)] 2.5 mM Tris/HCI 0.4 mM, EDTA, pH 8.5, at 37"C, for 18 h. All peptides were purified by analytical HPLC and eluted with a linear gradient from 2% to 45% solvent B [solvent A, 0.1 ?hCF,CO,H (trifluoroacetic acid); solvent B, acetonitrile in 0.1 % CF,CO,HJ at a flow rate of 1 ml/min on a reverse phase C,, column. Electrospray mass spectrometry of the native protein was performed on a VG-Bio-Q (Bio-Tech) in the positive mode (Van Dorsselaer et al., 1990). Cockroaches-lethality assay. Median lethal doses (LD,,,) were determined according to Behrens and Karber (1935) using cockroaches (Blatellu gcrnzanica, 50 t 2 mg) and an automatic syringe from the Burkard Manufacturing Company. Immunization protocols. Three rabbits were immunized with L. qiiinquestrintus quinquestriatus IT2, a depressant toxin purified from the venom of L. quinquestriatus quinquestricztus

(Kopeyan et al., 1990) as follows. 200 pg of L. yuinquestriat~is quinquestricitus IT2 in 1 ml, NaCl/P, (6 mM Na,HPO,, 2 mM Na,PO,, 2 mM K,HPO,, 133 mM NaCl, pH 7.2), and an equal volume of complete Freund's adjuvant were injected intradermically into each rabbit on day 0. This procedure was repeated subcutaneously with incomplete Freund's adjuvant on days 30, 60, and 90 with 2 x 3 0 0 pg and 500 pg Lqq IT2, respectively. Animals were bled for serum one week after each injection. Enzyme-linked immunoabsorbent assay. 96-well microtiter plates (Nunc) were coated by incubation for 3 h at 37 "C with 100 pl of 0.5 pg/ml Lqq IT2 in NaCl/P, containing 0.05% Tweedwell. After washing with 0.15 M NaCI, 0.05% Tween, the blocking solution (NaCI/P, containing 2 % BSA), was added and incubated for 1 h at 37°C. After 6 washes, dilutions of rabbit anti-(Lqq IT2) serum (10,- 10"-fold in NaCI/P, containing 0.25 o/c BSA and 0.05 96 Tween) were added and incubated for 1 h at 37°C. The wells were washed again and incubated with 100 pl/well of a 1:1000 dilution of peroxidase-conjugated goat anti-rabbit IgG (Diagnostics Pasteur). The wells were washed and peroxidase substrate solution (TMB, Kirekegraard and Pery Labs) was added (100 pl/well). After 10 min at room temperature, the reaction was stopped by addition of 1 M phosphoric acid (50 pUwe11). The absorbance at 450 nm was measured by means of a Titertek multiscan photometer (Flow Labs). For the competition ELISA, the same protocol was used, except that the serum dilution giving half-maximum signal (1 :500000) was incubated for 1 h at 37 "C with each of a series of concentrations of the toxic fraction before transfer into the well. Radioiodination of B. arenicola IT2 and purification of the derivatives. 1 nmol B. arenicola IT2, 1 nmol Na'*'I, 20 p1 20 mM sodium phosphate, pH 7.4, and 8 nmol iodogen (Pierce Chem. Co.) were mixed in 50 pl, and incubated at room temperature for 1 h. The mixture was injected into a HPLC RP8 column and eluted at 20°C with a linear gradient from 5 96 to 35 % solvent B in solvent A for 10 min, then from 35% to 75 % solvent B in solvent A for 70 min. The flow rate was 1 ml/min. Radioactivity was measured on a gamma counter (Crystal 11, Packard). Enzymatic digestion of '"1-toxin derivatives. The relative proportions of Tyr, 'z51-Tyrand (L2'I)2-Tyrin "'I-toxin derivates were determined after enzymatic hydrolysis (20 h, 37°C) of samples from different fractions. Samples were hydrolyzed with trypsin, pronase, carboxypeptidase Y, microsomal leucine aminopeptidase and prolidase in a 1:2:1 : 1 :1 (by mass) ratio; the carboxypeptidase Y:toxin ratio was 1:35 (by mass). Reverse-phase HPLC analysis was performed as described (Marchot et al., 1988). Neuronal-membrane preparation. Insect synaptosomes (P,L preparation) were prepared from the central nervous system of adult cockroach ( P arnericana) according to established methods (Gordon et al., 1990, 1992; Moskowitz et al., 1994). All buffers contained phenylmethylsulfonyl fluoride (50 pg/ml), pepstatin A (1 mM), iodoacetamide (1 mM) and l,10-phenantroline (1 mM). Membrane protein concentrations were determinated using a Bio-Rad protein assay with BSA as standard. Binding assay. Equilibrium-saturation assays were performed with a series of concentrations of the unlabeled toxin in the presence of a fixed concentration of the labeled toxin. To obtain saturation curves, the specific radioactivity and the amount of bound toxin were calculated and determined for each toxin concentration. The binding medium was 140 mM choline chloride, 1.8 mM CaCI,, 5.4 mM KCI, 0.8 mM MgS04, 25 mM Hepes, 10 mM glucose, pH 7.4 (buffer A) containing 2 mg/ml BSA. After incubation, the reaction mixture was diluted with 0.2 ml ice-cold buffer A containing 5 mg/ml BSA, and filtered through GF/C fillers (Whatman) under vacuum, and the filters

Cestkle et al. (ELK J. Biochem. 243)

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tA

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Time (hours)

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Time (minutes) Fig. 1. Purification of depressant anti-insect toxin of B. arenicolu LT2. (A) Gel filtration on Sephadex G-50. Four columns (400 cmX3.2 cm) connected in series were equilibrated and developed with 0.1 M ammonium acetate. pH 8.5, at a flow rate of 40 mllmin. (B) Reverse-phase HPLC of fraction I1 on a semipreparative reverse-phase C, column. Linear gradient from IS cic to 40 % solvent B over 80 min ; flow rate, 4 ml/min. Peak 1 eluted at 59 min and peak 2 at 64 min. The inset shows reverse-phase HPLC of B. arenicola IT2 on an analytical RP-8 column. Linear gradient from 25% to 33% solvent B in solvent A over 40 min; flow rate, 1 ml/min. B. arenicola IT2 eluted at 22 min. FI-FIV, fractions I-IV.

washed twice. Non-specific binding was determined in the presence of 1 pM unlabeled toxin and corresponded to 30-50% of the total binding. These experiments were carried out at 22°C. Equilibrium-saturation or competition experiments were analyzed by the iterative computer program LIGAND (Elsevier Biosoft). Each data point represents the mean C SE of three experiments.

RESULTS Purification of B. arenicola IT2. B. arenicola IT2 was purified from venom extract of scorpions collected in the area of Tozeur (Tunisia) in two steps : gel-filtration and semi-preparative HPLC. Four Sephadex G-50 columns in series were used for gel filtration. The elution pattern is shown in Fig. 1A. Four fractions were obtained and the toxicity of each was tested on B. germanica cockroaches. Only fractions I and I1 were active, inducing a fast excitatory paralysis. Only the fast excitatory paralysis is generally observed in toxicity tests when the two kinds of insect toxins are present in the same fraction, because excitatory toxins are 1-200-fold more active than depressant toxins (Kopeyan et al., 1990). To detect depressant toxins during the purification, a competition ELISA was used with the depressant toxin Lqq IT2 isolated from the venom of L. quinyuestriatus quinquestriLitus and polyclonal rabbit anti-(Lqq IT2) Ig. Depressant insect toxins are similar and constitute a defined group of scorpion toxins

(Kopeyan et al., 1990). We therefore supposed that anti-(Lqq 1T2) Ig might recognize depressant toxins in B. arenicola venom. In competition ELISA, free Lqq IT2 inhibited binding in a dose-dependent manner (Cestkle, S., Granier, C. and Kopeyan, C., unpublished results), as did fraction I1 obtained by gel filtration. No significant inhibition was observed with fraction I. This immunoreactivity indicated that fraction I1 may contain antigenic analogues of Lqq IT2. In the second purification step, fraction I1 was subjected to a semipreparative HPLC on a C, column (Fig. 1B). Fraction F1 induced a slow depressory flaccid paralysis in B. germanica, whereas fraction F2 induced a fast excitatory paralysis. F1 gave a single peak in analytical HPLC on a C, column (Fig. 1 B) and a single band on polyacrylamide-gel electrophoresis (data not shown) : this fraction was therefore presumed to be homogeneous and was called B. arenicola IT2. The yield of toxin from the crude venom was 0.4% (by mass), consistent with results previously described for purifications of other scorpion toxins (Zlotkin et al., 1985; Martin and Rochat, 1986). Biochemical characterization of B. arenicola IT2. The amino acid composition of B. arenicolu IT2 is reported in Table 1. B. arenicola IT2 is composed of 61 amino acid residues. The N-terminal 54 amino acids of the S-pyridylethylated protein, except positions 52 and 53, were assigned by sequencing 200 pmol. Two tryptic peptides, T1 and T2, were particularly useful for the sequence determination. The amino acid sequence

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Table 1. Amino acid composition of B. arenicola IT2. Values calculated from 20-h and 70-h hydrolyzes. Values in parentheses are the expected values based on sequencing. ~~

~

Number of residues

Amino acid

6.9 (7) 3.4 (4) 3.2 (3) 4.2 (4) 1.3 (1) 8.8 (10) 2.1 (2) 5.5 (8) 1.1 (1) 0 (0) 0.9 (1) 3.0 (3) 3.7 (4) 1.0 (1) 4.7 (5) 0 (0) 2.7 (3) 4.0 (4)

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan' Total

E, 0

1

x

z 0

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40

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Fig. 3. Purification of the derivatives of B. arenicola '251-IT2.B. arenicola IT2 (1 nmol) was reacted with Na1251(1 nmol) as indicated in Materials and Methods. The mixture was injected into the HPLC RP8 column. The first peak of radioactivity represents free '"I-; peaks A and B represent, the monoiodide and the diiodide derivatives of B. arenicola IT2, respectively.

' Spectrophotometrically determinated.

of peptide T3 confirmed the previously established sequence 27-51 (Fig. 2). Moreover, the established sequence was confirmed by the amino acid compositions and sequencing of peptides obtained by digestion with Lys-C protease or chymotrypsin (data not shown). The molecular mass of the native protein (6834.80 2 0.40 Da), as measured by electrospray mass spectrometry, is in close agreement with the calculated mass (6834.62 Da). Biological activity of B. arenicola IT2. Toxicity. Injected into B. germanica, B. arenicola IT2 caused a slow, progressive depressant flaccid paralysis. The LD,, was 175 ng, similar to that of the depressant insect toxin Lqq IT2 (Kopeyan et al., 1990). Binding assays. Reverse-phase-HPLC was used to purify B. arenicola lZ5I-IT2derivatives (Fig. 3). Two peaks were obtained. To test the binding activity of the eluted components, samples of equal radioactivity (about 2X 1O6 c p d m l ) were incubated for 1 h with cockroach neuronal membranes in the absence (total binding) or in the presence (non-specific binding) of the native 1 0

I

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toxin (1 pM). Specific binding was obtained only with peak A. Moreover, peaks A and B, obtained by reacting 2 nmol B. arenicola IT2 with KI (toxidiodide ratio = 1) in the presence of Na'"1 as tracer, were treated with a mixture of proteases and the iodotyrosines were separated by reverse-phase-HPLC. This analysis revealed that peak A contained 87 % lZ5I-Tyrand 13% (lzsI),-Tyr, whereas peak B contained 85% (1Z51),-Tyrand 15% ('"I-Tyr). For binding experiments, only the top tube of fraction A was used. Scatchard analysis of a saturation-binding curve of B. arenicola IT2 (Fig. 4A) suggests two distinct binding sites for B. arenicola IT2 in cockroach neuronal membranes: a highaffinity (&, = 0.1 1 ? 0.04 nM) and low-capacity (Bnldx,= 2.2 2 0.6 pmol/mg) class of binding sites and a low-affinity (Kd2= 24 2 7 nM) and high-capacity (BmuxZ = 226 2 92 pmol/ mg) class. It appears that the low-affinity sites are 100-fold more numerous than high-affinity sites. Cockroach neuronal membranes were used for competitivebinding assays with Lqq IT2 and Androctonus australis Hector IT. It was previously shown that A. australis Hector IT binds to a single class of non-interacting binding sites of high affinity in various insect neuronal membranes (Gordon et al., 1984, 1985; De Lima et al., 1989). The binding of radiolabeled AaH IT is 3

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(a) DGYIRRRDGCKVSCLFGNEGCDKECKAYGGSYGGSYGYCWTWGLACWCEGLPDDK--K------(b)

AYGGSYGYCWTWGLACWCEGLPDDK

TWK SETNTCG ( e ) DGYIRRRDGCKVSCLFGNEGCDKECKAYGGSYGYCWTWGLACWCEGLPDDKTWKSETNTCG (C)

(d)

Fig. 2. Amino acid sequence of B. arenicola IT2 and comparison with amino acid sequences of other depressant insect toxins. Results from Edman degradation of S-pyridylethylated B. arenicola IT2 (a); tryptic peptide T3 (b); tryptic peptide T2 (c); tryptic peptide T1 (d). Complete amino acid sequence of B. arenicola IT2 (e). -, unidentified residue. Ba, B. urenicola; Lqh, L. quinquestriatus hebraeus; Lqq, L. quinquestriutus quinquestriatus; Bj, B. judaicus.

Cestkle et al. ( E m J. Bicichern. 243) 0.10

al., 1992; Moskowitz et al., 1994). B. arenicola IT2 possesses the pharmacological properties of a depressant toxin.

A

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y o o o o0

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[Bound] (nM)

DISCUSSION Using toxicity tests in insects and competition ELISA, we purified a depressant insect toxin, B. arenicola IT2, from the venom of Buthacus arenicola. B. arenicola IT2 is a single-chain polypeptide composed of 61 amino acid residues with a-molecular mass of 6835 Da. The molecular size and the presence of eight half-cystines are typical of Buthinae scorpion toxins. The amino acid sequence shows a high degree of similaritv with the sequences of three known depressant insect toxins from scorpions (79 - 95 % of the positions of B. arenicola IT2 are identical to those of other toxins). In particular, the position of the eight half-cystines residues fits with the highly conserved pattern, typical of mammal toxins (Kopeyan et al., 1974; Fontecilla-Camps et al., 1981 ; GrCgoire and Rochat, 1983) and thus are presumed to form four disulfide bonds as they do in other scorpion toxins. In contrast, excitatory toxins possess only three disulfide bridges in positions identical with those found in depressant and mammal toxins. Amino acid analyses of B. arenicola IT2 were in full agreement with the determined sequence, and they confirm the absence of histidine and methionine residues and the abundance of tryptophan and glycine, conforming to the composition of other depressant insect toxins such as Lqq IT2 and LqhIT2. Thus, depressant toxins form a group of similar proteins (Zlotkin et al., 1991 ; Kopeyan et al., 1990). There were two binding sites for B. arenicola IT2 on the cockroach neuronal membranes: the equilibrium-dissociation constant ( K J and the capacity (Bmdx)of the high-affinity binding sites were two orders of magnitude lower than those of the lowaffinity sites. The comparison between the affinity of two depressant toxins, B. arenicola IT2 and L. quinquestriatus hebraeus IT2, on cockroach neuronal membranes, indicates that the affinities of B. arenicola IT2 are 40-fold and 25-fold higher for the high-affinity and low-affinity sites, respectively, than that of L. quinquestriutus hebraeus IT2 (Moskowitz et al., 1994). There are only three amino-acid-sequence differences between B. arenicola IT2 and L. quinquestriatus hebraeus IT2: position 5, Arg-+Lys; position 13, Ser-+Ala; position 16, Phe--+Ile. These three mutations may explain the different affinities observed between B. arenicola "'I-IT2 and L. quinquestriatus hebraeus IT2. In competition experiments between B. arenicola Iz5I-IT2 and Lqq IT2, the two toxins displayed similar affinities for the depressant-receptor sites, in spite of the three amino acid differences between the two sequences, indicating that modifications at these positions do not influence the binding. In view of its high affinity, B. arenicolu IT2 may be a useful tool to characterize the depressant-receptor sites. In competitive-binding experiments, only Lqq IT2 was able to compete with B. arenicola ''TIT2 for both its binding sites: AaH IT competed with B. arenicola t251-IT2only for its highaffinity sites. The high-affinity binding sites for the depressant toxins are presumably located on the insect sodium channels, because of the mutual competitive binding of the depressant and excitatory toxins, and the binding inhibition by site-directed antibodies against specific external segments of the sodium channel polypeptide (Gordon et al., 1992). The nature and the function of the low-affinity, high-capacity B. arenicola IT2-binding sites is unknown: no competitive binding was observed except with depressant toxins. B. arenicola IT2 has a higher affinity than AaH IT for the high-affinity binding sites (Kd ninefold lower to that of AaH IT) but AaH IT is more toxic (175-fold more active than B. arenicola IT2). Similar discrepencies be-

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.-c: X

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-6

log ([ToxinIiM) Fig.4. Interaction of B. arenicola IT2 with insect neuronal membranes. (A) Scatchard analysis of specific binding of B. arenicola IT2 to insect neuronal membranes. Cockroach neuronal membranes (1 pg protein) were incubated with 66.6 pM B. nrenicola '2i1-IT2 and increasing concentrations of unlabeled B. arenicola 1T2 for 60 min at 22°C in 0.3 ml buffer A containing 2 mg/ml BSA, and the amount of specifically bound B. arenicola '"I-IT2 was determined after rapid filtration (see Materials and Methods). Non-specific binding (30-50% of total binding) was determinated in the presence of 1 pM B. arenicola IT2 and was always subtracted. The specific radioactivity and the amount of toxin bound were calculated for each toxin concentration, and the Scatchard plot was drawn by means of the computer program LIGAND (cold saturation). Kdl = 0.11 ? 0.4 nM, B,, = 2.2? 0.6 pmol/mg protein, Kdz = 24 L 7 nM, B,,,,, = 226?92 pmol/mg protein. (B) Competition experiments for binding to neuronal membranes between B. arenicola Iz5I-IT2, B. arenicola IT2, Lqq IT2 and AaH IT. Cockroach neuronal membranes were incubated for 60min at 22°C in the presence of 66.6 pM B. arenicolu "TIT2 and increasing concentration of Lqq IT2 (A), B. arenicola IT2 (circ) or AaH IT (0).Non-specific binding of B. arenicola lZ5I-IT2,determined in the presence of 1 pM B. arenicola IT2 was subtracted.

completely inhibited by various depressant toxins (Zlotkin et al., 1985, 1991). We found that B. arenicola IT2 qimilarly inhibited AaH IT binding (data not shown). B. arenicolu l2Y-IT2 competed for Lqq IT2 binding to cockroach neuronal membranes (Fig. 4B). AaH IT toxin inhibited only 40% of the specifically bound B. arenicola 12'I-IT2 (Fig. 4B). Under the experimental conditions used, 40 % of the labeled toxin B. arenicola IT2 occupied 100% of the high-affinity binding sites and 60% of the labeled toxin occupied low-affinity binding sites. Thus, AaH IT competes only for binding to the high-affinity B. arenicola IT2 sites. Moreover, there is a good correlation between the Kd of AaH IT obtained by Scatchard plot (0.9 2 0.2 nM; Cestkle, S., unpublished results) and the Kd (1.6 2 0.6 nM) calculated using the relationship: IC,, = Kd (1 [1251-IT2](Kdl "'I-IT2). Therefore, B. arenicola IT2 and AaH IT competed for the high-affinity sites, as previouly shown for other depressant toxins (Gordon et

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(1985) The binding of an insect-selective neurotoxin and saxitoxin to insect neuronal membranes, Biochenz. Biophys. Acia 821, 130-136. Gordon, D., Merrick, D., Wollner, D. A. & Catterall, W. A. (1988) Biochemical properties of sodium channels in a wide range of exitable tissues studied with site-directed antibodies, Biochemistry 27, 7032The authors thank Drs D. Gordon and C. Devaux, for helpful discus7038. sion, Dr R. Romi, for the gift of the enzymes, and M. Fleury, for fine Gordon, D., Moskowitz, H. & Zlotkin, E. (1990) Sodium channel polysecretarial work. We are grateful to Procida company, Marseille, France, peptides in central nervous system of various insects identified with for the generous gift of the Prriplanrta unzericana and Blatella g e r m site directed antibodies, Bioclzim. Biophgs. Acfu 1026, 80- 86. nicu. Sandrine Cestkle is the recipient of a fellowship from the Miriisr6re Gordon, D., Moskowikowitz, H., Warmer, C., Catterall, W. 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tween LD,,, and K , have been observed for toxin VII from Tityus serrulatiis (De Lima et a]., 1986, 1989) and toxin IV from L. quinquestriatus quinquestrii~rus(Gordon et al., 1996). There are several possible explanations : the nervous system of insects may be largely impermeable to crude venoms and purified toxins (Parnas et al., 1970; D’Ajello et al., 1972); B. arenicoln IT2 does not reach all its binding sites when injected into cockroaches for pharmacokinetic reasons ; or because the majority of the toxin binds to low-affinity binding sites, or because of the hydrophobic properties of the toxin; binding of B. urenicoln IT2 to the high-affinity binding sites with high affinity may induce only a low toxicity in insects; or the toxicity of B. arenicoln IT2 is due to the binding on low-affinity binding sites. The insect selectivity of the depressant toxins suggests structural differences between mammal and insect sodium channels. Further analysis of insect selectivity and particularly the structures of the toxins and of the sodium channels may allow the design of insect-selective biopesticides.

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