An insecticidal peptide from the theraposid Brachypelma smithi spider venom reveals common molecular features among spider species from different genera

peptides 29 (2008) 1901–1908

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An insecticidal peptide from the theraposid Brachypelma smithi spider venom reveals common molecular features among spider species from different genera§ Gerardo Corzo a,*, Elia Diego-Garcı´a a,b, Herlinda Clement a, Steve Peigneur b, George Odell a,1, Jan Tytgat b, Lourival D. Possani a, Alejandro Alago´n a a

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologı´a, Universidad Nacional Auto´noma de Me´xico, UNAM. Apartado Postal 510-3, Cuernavaca, Morelos 61500, Mexico b Laboratory of Toxicology, University of Leuven, Campus Gasthuisberg, O&N2, PO Box 922, Herestraat 49, 3000 Leuven, Belgium

article info

abstract

Article history:

The soluble venom of the Mexican theraposid spider Brachypelma smithi was screened for

Received 9 April 2008

insecticidal peptides based on toxicity to house crickets. An insecticidal peptide, named Bs1

Received in revised form

(which stands for Brachypelma smithi toxin 1) was obtained in homogeneous form after the

7 July 2008

soluble venom was fractionated using reverse-phase and cation-exchange chromatography.

Accepted 7 July 2008

It contains 41 amino acids cross-linked by three disulfide bridges. Its sequence is similar to an

Published on line 17 July 2008

insecticidal peptide isolated from the theraposid spider Ornithoctonus huwena from China, and another from the hexathelid spider Macrothele gigas from Japan, indicating that they are

Keywords:

phylogenetically related. A cDNA library was prepared from the venomous glands of B. smithi

Insecticidal

and the gene that code for Bs1 was cloned. Sequence analysis of the nucleotides of Bs1 showed

Peptide

similarities to that of the hexathelid spider from Japan proving additional evidence for close

cDNA

genetic relationship between these spider peptides. The mRNAs of these toxins code for signal

Spider toxin

peptides that are processed at the segment rich in acidic and basic residues. Their C-terminal

Theraposid

amino acids are amidated. However, they contain only a glycine residue at the most Cterminal position, without the presence of additional basic amino acid residues, normally required for post-translation processing of other toxins reported in the literature. The possible mechanism of action of Bs1 was investigated using several ion channels as putative receptors. Bs1 had minor, but significant effects on the Para/tipE insect ion channel, which could indirectly correlate with the observed lethal activity to crickets. # 2008 Elsevier Inc. All rights reserved.

1.

Introduction

Spiders have evolved highly selective toxins for insects [12]. They are a very diversified group of insect predators (ca. 37,000

described species) that appear to possess a high potential for the discovery of novel insect-selective toxins [3,12]. Comparative studies showed that, spider as well as scorpion venoms have been demonstrated to contain very lethal components

§

The gene sequence reported in this paper has been submitted to the GenBank and has accession number EU196048 for Bs1cDNA. * Corresponding author at: Instituto de Biotecnologı´a, Universidad Nacional Auto´noma de Me´xico, UNAM, Av. Universidad 2001, Cuernavaca, Morelos 62210, Mexico Tel.: +52 777 317 1209; fax: +52 777 317 2388. E-mail address: [email protected] (G. Corzo). 1

Sadly we lost Prof. George Odell during the time this manuscript was been prepared. Abbreviations: TFA, trifluoroacetic acid; ESI, electro spray ionization; LD50, dose that kills 50% of the animals; Bs1, toxin1 from the spider Brachypelma smithi. 0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2008.07.003

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against pest insects [4,5]. Therefore, venoms of spiders are attractive because they represent a diversified pool of active peptides with a great potential for drug discovery [15]. Molecular mass fingerprints of crude spider venoms have shown that spider venoms may contain more than 200 different components, toxic to both: vertebrates and insects [11,14,21]. The venomous glands of theraposid spiders produce abundant amount of venom, compared to other spiders that might contain a more diverse type of different components, but in smaller quantities [3,15]. Since the size of theraposids are generally larger than that of other spider families, it seems that their prey-capture system is based on the advantages given by their body sizes rather than by their venom components toxicity. It has been described that the venom from species of the Araneae family is richest in peptide diversity than the venom from species of theraposids [13]. Even so, the venom of theraposids contains insecticidal peptides targeted at ion channel receptors, and thus they represent an interesting material for the discovery of new insect receptors. Pharmacological data on their venom components are still scarce because of the lack of proper systems for testing insect receptors compared to that of the huge amount of vertebrate receptors already available. The precise targeting of insect receptors by spider toxins may be useful for the understanding of the molecular basis of toxin selectivity at the receptor level, and may eventually lead to the design of more effective and safer pesticides. In the present work, we report both the isolation of the insecticidal peptide Bs1 and the cDNA sequence of the same peptide from the venomous glands of the Mexican theraposid Brachypelma smithi, thereafter abbreviated B. smithi. A comparative study with peptides from other genera of spiders is described, showing similarities and particularities, especially related to post-translational modifications. Additionally, the mechanism of action of Bs1 was investigated conducting experiments on several voltage-gated ion channels.

monitored at 230 nm. Fractions with insecticidal activity were further fractionated by cation-exchange HPLC on a TSK-gel sulfopropyl column (SP-5PW; 7.5 mm  75 mm, Tosoh, Japan), equilibrated in 0.5 M acetic acid (pH 2.9). A linear gradient of 0.5 M acetic acid in 2 M ammonium acetate (pH 5.9) was applied (0–20% in 20 min) at a flow rate of 1.5 ml/min. The active fractions were finally purified on a reverse-phase C18 column (4.6 mm  250 mm, Nacalai Tesque, Japan) using the same gradient system as above, with a flow rate of 1 ml/min.

2.3.

Insecticidal toxins were reduced and alkylated prior to Edman sequencing, following techniques described by our group [7]. Peptides were also subjected to enzymatic hydrolysis. Tryptic hydrolysis was carried out in 0.1 M sodium bicarbonate buffer (pH 8.1) at 37 8C for 3 h, using a 1:50 (w/w) enzyme to substrate ratio. The tryptic digests were fractionated by reverse-phase HPLC using a C18 column (4.6 mm  250 mm, Nacalai Tesque, Japan) and a linear gradient of acetonitrile in 0.1% aqueous TFA. All fractions were analyzed by electro-spray ionization mass spectrometry using a Finnigan LCQDUO ion trap mass spectrometer (San Jose, CA). All tryptic fragments were sequenced by automated Edman degradation, using a LF 3000 Protein Sequencer from Beckman (San Diego, CA) as earlier described [7].

2.4.

Experimental

2.1.

Biological materials

B. smithi venom was obtained by electrical stimulation of fieldcollected spiders from Mexico. The crude venom was frozen and stored at 20 8C until use. It is necessary to clarify that our group has an official permit (SEMARNAT FAUT-0184 and MORIN-166-0704) to collect and maintain a colony of these spiders alive. They are in good conditions (well fed, healthy and are now living in the laboratory for many years).

2.2.

Peptide purification

B. smithi soluble venom (100 ml) was dissolved in 0.1% aqueous TFA containing 5% acetonitrile, and the insoluble material was removed by centrifugation at 14,000  g for 5 min. Diluted venom was first fractionated using a reverse-phase semipreparative C18 column (5C18MS, 10 mm  250 mm, Nacalai Tesque, Japan) equilibrated in 0.1% TFA, and eluted with a linear gradient of acetonitrile/0.1% TFA (0–60% in 60 min), at a flow rate of 2 ml/min. The absorbance of the effluent was

cDNA library construction

A cDNA library was constructed with mRNA extracted from a pair of venomous glands from one specimen of B. smithi following a protocol already reported [27]. For RNA isolation, the ‘‘Total RNA Isolation System’’ from Promega (Madison, WI) was used. With this material a full-length cDNA plasmid library was prepared using the SMART cDNA Library Construction Kit (CLONTECH Lab., Palo Alto, CA).

2.5.

2.

Sequence determination

Gene cloning and DNA sequencing

Based on the information obtained from direct peptide sequencing, several oligonucleotides were synthesized using a model 391 DNA Synthesizer (Applied Biosystems, Foster City, CA): Bs1CCR5 50 -TGYTGYMGNGARTAYGARTGY-30 ; Bs2CCY5 50 -TGYTGYTAYGARTAYGARTGY-30 ; and BsCDKC5 50 -TGYGAYAARGAYGAYCC-30 (M, N, R and Y stands for degenerated nucleotides). For the polymerase chain reaction (PCR), each one of these primers was used in conjunction with the pair CD3/30 primer [8]. The plasmid library was used as template for the PCR reaction. The PCR products were purified using a Centricon 100 column (Amicon, USA) following the manufacturer’s instructions, and then, they were ligated into a pGEMplasmid (Promega, Madison, WI). The ligation reaction was used to transform competent E. coli DH5-a cells. Positive clones were sequenced from both ends using a model 3100 DNA sequencer (Applied Biosystems, Foster City, CA). The positive colonies (blue/white color) were analyzed, with the following protocol: hot start 4 min denaturalization at 94 8C, 1 min at 94 8C, 1 min at 50 8C and 1 min at 72 8C, for 32 cycles and final step of 5 min at 72 8C. The plasmid DNA of these colonies was sequencing from both ends.

peptides 29 (2008) 1901–1908

2.6.

Biological activity

Crickets (Achaeta spp.) were weighed and groups of six individuals with the same weight were placed together. Crickets were injected intra-thoracically between the second and third pair of legs, with 5 ml of five different doses of spider toxin previously dissolved in distillated water according to their weight (0, 3.7, 7.5, 15, 30 and 60 mg/g body weight). The LD50 value for the toxins is given in micrograms of toxin per gram of cricket, which represents the lethal dose to kill 50% of treated crickets at 15 min after injection. The LD50 value was obtained by Probit analysis of data from two independent groups of 30 crickets injected with the spider peptide and two independent groups of 6 crickets injected with distilled water.

2.7.

Electrophysiological experiments

The physiological effect of Bs1 was analyzed by heterologously expression of rNav1.2, rNav1.4, hNav1.5, mNav1.6, Nav1/tipE, hKv1.3 and Shaker IR channels in oocytes from Xenopus laevis. cRNAs transcripts were synthesized from the linearized plasmids using the large-scale T7 or SP6 mMESSAGE mMACHINE transcription kit (Ambion, Foster City, CA). Stage-V and -VI X. laevis oocytes were harvested by partial ovariectomy under anaesthesia (3-aminobenzoic acid ethyl ester methanesulfonate salt, 0.5 g/l from Sigma (Bornem, Belgium)). Anaesthetized animals were kept on ice during dissection. The oocytes were defolliculated by treatment with 2 mg/ml collagenase (Sigma, Bornem, Belgium) in Ca2+-free ND-96 solution (in mM: NaCl 96, KCl 2, MgCl2 1, HEPES 5 adjusted to pH 7.5). Between 1 and 24 h after defolliculation, oocytes were injected with 50 nl of 50–100 ng/ml cRNA using the microinjector (Drummond Scientific, USA). The oocytes were then incubated in ND-96 solution (supplemented with 50 mg/l gentamycin sulphate) at 16 8C for 1–5 days. Electrophysiological measurements were performed by the two-electrode voltage-clamp technique, at room temperature (18–22 8C). The recordings were processed by GeneClamp 500 amplifier (Axon Instruments, USA) controlled by a pClamp data acquisition system (Axon Instruments, USA). Whole-cell currents from oocytes were recorded 3–5 days after injection. Bath solution composition was (in mM): NaCl 96; KCl 2; CaCl2 1.8; MgCl2 2 and HEPES 5, pH 7.4. Voltage and current electrodes were filled with 3 M KCl. Resistances of both electrodes were kept low (<0.5 MV). Currents were sampled at 2 kHz and filtered at 10 kHz using a four-pole low-pass Bessel filter. Leak subtraction was performed using a P/4 protocol. Current traces were evoked in oocytes expressing the cloned sodium channels by 5 mV depolarization steps from a holding potential of 90 mV. Recordings of potassium channels were registered by depolarization from a holding potential of 90 to 0 mV. The pClamp program was used for data acquisition and data files (Molecular devices, Sunnyvale, CA) were directly imported, analyzed and visualized with Origin software. Averaged data are indicated as mean  S.E.M. Statistical analysis of differences between groups was carried out with Student’s t-test and a probability of 0.05 was taken as the level of statistical significance.

3.

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Results

The whole soluble venom of B. smithi was fractionated using reverse-phase HPLC (Fig. 1A). Sixty fractions were manually collected and vacuum-dried. Dry HPLC fractions were resuspended in distilled water and assayed for insecticidal activity toward crickets. Only fraction 41 showed a clear insecticidal activity at the tested doses, and it was further fractionated by cation-exchange chromatography. A pure insecticidal peptide was obtained and named Bs1 (Fig. 1B). This peptide was assayed in vivo using crickets and a LD50 of 27  11 mg/g (body weight) was experimentally estimated. Edman degradation of both the native Bs1 and a sample of the reduced and alkylated peptide allowed the identification of the first 39 amino acid residues (Table 1A). These were further confirmed by sequencing two sub-peptides obtained by tryptic digestion, which gave unequivocal identification of amino acids from position C1 to R14 and C15 to K39 (data not shown). Based on the molecular mass experimentally determined for the native Bs1 of 4916.57 Da, it was assumed that about two amino acid residues were still missing at the C-terminal region. In order to solve this uncertainty a cDNA library was prepared with the aim to clone the gene responsible for the synthesis of Bs1. A pair of venomous glands from a healthy B. smithi male was dissected, and a cDNA library was prepared. Since most of the amino acid sequence of Bs1 was known by Edman degradation, three degenerate oligonucleotides were designed and used to amplify the nucleotide sequence of Bs1. Several sequences with the expected size were obtained from PCR reactions using such cDNA library. The amino acid sequence deduced from the cDNA that corresponds to the Bs1 peptide was obtained; nevertheless, the complete sequence of the precursor was obtained using the information from the amino acid sequence of the mature peptides and a SMART adaptor primer using a new random screening of the cDNA library. The Bs1 gene was found in several bacteria colonies. After proper nucleotide sequence, it was found that the messenger coding for Bs1 codifies for 99 amino acid residues from which 57 residues are part of the signal peptide, and 41 residues correspond to the mature protein. The last residue is the donor of the amino group for the last C-terminal situated residue of Bs1 (Fig. 2). Table 1A shows the complete 41 amino acid sequence of the mature insecticidal peptide Bs1 thus, confirming the last two residues K40 and S41. The molecular weight theoretically expected for the first 41 amino acids of the mature peptide is 4917.58 Da, in close agreement with the experimental value found, which was 4916.57 Da. Thus, the molecular mass found for the pure peptide has 1.0 unit of mass less than expected, supporting the idea of a C-terminal amidated serine. It is worth recalling that the amino acid before the stop codon is glycine, known to be a donor of amides for amidation of peptides at the C-terminal region. The molecular mass found also is consistent with the idea that Bs1 is folded by three disulfide bridges, as seen in any other active multiple cysteines-containing peptide toxins [3,12]. Sequence analysis of the primary structure of Bs1 showed that it is quite similar to the insecticidal peptides found in the venom of spiders from the Asian-Pacific area, such as that found from the theraposid Ornithoctonus huwena from China [30] and the hexathelid spider Machrotele gigas from Japan [5](Table 1B).

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Fig. 1 – Reverse-phase HPLC profile from the whole venom of B. smithi. (A) The soluble venom (5 mg) was fractionated using a reverse-phase semi-preparative C18 column (5C18MS, 10 mm T 250 mm) equilibrated in 0.1% TFA, and eluted with a linear gradient of acetonitrile from 0 to 60% in 0.1%TFA, run for 60 min at a flow rate of 2 ml/min. (B) Cation-exchange HPLC of fraction 41 from the previous HPLC separation. Five hundred milligrams of this fraction was applied to the column. The major component, labeled Bs1 correspond to the pure peptide.

Recently, the sequence of LTx4 from the spider Lasidora sp. from Brazil has been deposited in the Gene Bank with an accession number EF219061. LTx4 has 76% identity with the amino acid sequence of the Bs1 precursor that includes the signal peptide and pro-peptide sequence of Bs1 (Tables 1 and 2). Since B. smithi is a theraposid from the Southern Pacific Coast of Mexico, it is assumed that these peptides might have been originated from a parent peptide from an organism located in the land before the separation of America and East Asia. This supposition is supported in part by analysis of the similarities of the mRNA organization of spider species located in both continents (Table 2). For example, it is anticipated that Bs1 undergoes post-translational modification at both the Nterminal and the C-terminal regions of the molecule. At the Nterminal section of the mature peptide, the gene of Bs1 embraces an amino acid sequence that is also found in the mRNAs of the similar insecticidal toxins Tox6 and Magi2 from M. gigas [27](Table 2). Moreover, the cDNA of Bs1, Tox6 and Magi2 contain a C-terminal glycine alone, which is cleaved to yield a C-terminal amidated serine. All three peptides: Bs1, Tox6 and Magi2 have similar post-translational modifications at both N- and the C-terminal segments. Therefore, these amino acid regions are common endoproteolytic sites for Nterminal and C-terminal processing in these two spider genera. These similarities between Bs1, Tox6 and Magi2 indicate a phylogeny relationship despite of the differences in genus and geographical localization. Bs1 toxin was tested on several voltage-gated sodium ion channels (Nav) such rNav1.2, rNav1.4, hNav1.5, mNav1.6 and Para/tipE. Moreover, Bs1 was tested on two voltage-gated potassium ion channels, hKv1.3 and Shaker IR. The results of the electrophysiological assays on these ion channels showed that Bs1 has no significant effects on rNav1.2, rNav1.4, hNav1.5, mNav1.6, hKv1.3 and Shaker IR (see Supplemental Material). However, Bs1 at concentration of 2 mM showed an effect in the insect Para/tipE channel (Fig. 3A and 3B). A small change of sodium currents was observed in the presence of Bs1 at 2 mM (Fig. 3A). Fig. 3B show the activation and the steady-state inactivation curves of sodium currents. Both figures represent the normalized conductance–voltage relationship of Para/tipE in the absence and in the presence of

Table 1 – Amino acid sequence of Bs1 (A) and alignment to similar spider toxins (B)

a Exp. and Theo.; experimental and theoretical molecular weights; bIdentity to Bs1; cAccs, accession number. *Means amidation at the Cterminus.

peptides 29 (2008) 1901–1908

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Fig. 2 – Nucleotide and amino acid sequence of Bs1. The abbreviations corresponding to nt_c and aa_c are the cDNA nucleotide and predicted amino acid sequences, respectively. Putative signal peptide and pro-peptide sequence are in italics and underlined; amino acid +1 correspond to the first amino acid of toxin peptide. Lower cases are untranslated 30 region; lower cases and italics showed the putative poly-A signal sequence. Numbers at the right indicate the corresponding size in the sequences. Lower case ‘g’, stands for the glycine residue at the C-terminal region expected to be processed for amidation of the last residue Ser.

2 mM toxin. The differences observed by effect to Bs1 in Para/ tipE are statistically significant (the mean  S.E.M. from six experiments).

4.

Discussion

In general, undiscriminated random screening of cDNAs derived from tissues or organs can generate an enormous number of possible genes that might correspond to any possible protein present in the source of the biological material used for preparation of the cDNA library. In order to direct the search for specific genes it is better to start with known amino acid sequences of the peptide/protein possibly coded by the gene under analysis. For the present case, it was essential to have first determined the amino acid sequence of Bs1. Oligonucleotides were designed as mentioned in the Section 2.5, that permitted to obtain the appropriate gene. Since cDNAs encoding spider peptide toxins with multiple cysteines have been found to be shorter than 0.6 kilobase pairs (Kb) (see [2,5,9,20,22,27], for this work, clones having only 0.4– 0.6 Kb were selected, after direct colony PCR procedures. Based on this presumption, the sequence analysis of 10 cDNA clones obtained from the venom gland of the theraposid B. smithi was performed. They all have the expected length of 0.4–0.6 Kb. In addition, several nucleotide sequences for Bs1 from several independent colonies were obtained, confirming that none of the cDNA sequences were generated by artifacts or PCR errors. The amino acid sequence encoding Bs1 was shown to consist

of a hydrophobic signal peptide moiety, a relatively hydrophilic spacer sequence with various lengths, and a cysteinerich region reminiscent of typical multi-cysteine peptide toxins specific for ion channels. This structural organization is typical for precursors of known spider multi-cysteine peptide toxins [2,5,9,20,22,27] supporting the expectation that the designed oligonucleotides were useful for obtaining the Bs1 cDNA clone. Sequences of the mature forms and precursors of Bs1 as well as other spider peptide toxins indicated that their Nterminal region were generated by cleavage at Asn/Ser/Arg/X/ Lys-Arg/Arg (shaded in gray in Table 2) of the pro-peptide, which precedes the signal peptide. Similarly, the N-terminal segment of spider peptide toxins are generated at Glu/Gln/Lys/ Arg-Arg endoproteolytic sites in precursor polypeptide toxins [2,5,9,20,22,27], where the common feature is the presence of a dibasic peptide (see last two residues above). Proteolytic processing of the precursor to a mature toxin is predicted. It is assumed that the Bs1 peptide is obtained after an endoproteolytic cleavage by a protease acting on the dibasic amino acid motif, KR (K56–R57), similar to the endoproteolytic cleavage of Magi2, Tox6 and LTx4 peptides (AB121202, Q75WH1, A3F7X1 respectively, see Table 2). The endoproteolytic motif RXRR has been observed in other precursors of spider toxins such Magi4 [5]. The sequence RXKR seems to be conserved in the cDNAs of Bs1, Magi2, Tox6 and LTx4 and also in the precursors of the peptide members of the superfamily HwTx-XVI [19]. The C-terminal amino acid of these spider peptide toxins is anticipated to undergo a post-translational

Table 2 – Amino acid sequences of toxin precursors

a

GenBank accession numbers: Bs1, Brachipelma smithi (EU196048); LTx4, Lasiodora sp. (EF219061); Magi2, Macrothele gigas (AB121202); Magi4, M. gigas (AB105149); Tox6, M. gigas (AB121200). The shadow in Bs1 and LTx4 represents high identity in their precursors. Last row: a star means that the residues in that column are identical in all sequences in the alignment; a colon means that conserved substitutions have been observed; a dot means that semi-conserved substitutions are observed.

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Fig. 3 – Electrophysiological effects of Bs1 on Para/tipE voltage gated ion channels expressed in Xenopus oocytes. (A) The inset on the left shows an averaged trace before and after addition of 2 mM Bs1. The arrow indicates the selected trace after application of the peptide. Traces were evoked by depolarizations to S20 mV and +20 mV from a holding potential of S90 mV. On the right, current–voltage relationship of a representative experiment in control conditions and in the presence of 2 mM peptide. Each point represents the mean W S.E.M. from six experiments. Current traces were evoked by 5 mV depolarization steps from a holding potential of S90 mV. B) Activation and steady-state inactivation curves. Both figures represent the normalized conductance–voltage relationship of Para/tipE in the absence and in the presence of 2 mM toxin. Data are presented as a Boltzmann sigmoidal fit. Each point represents the mean W S.E.M. from six experiments. Activation curves were derived from the currents used for the current–voltage relationships. Steady state inactivation current traces were evoked by 100 ms depolarizations of 5 mV from S120 mV to S10 mV followed by a 100-ms pulse to 0 mV. In all panels the close circles represent control conditions and the open circles represent the effect of 2 mM Bs1 as indicated. All data are represented as mean W standard error of the mean.

modification to become amidated. Normally, the last residues contain a glycine followed by a single or double basic amino acid residues, which is the endoproteolytic site for amidation signal [1,10]. The peptide Bs1 was found to have a glycine alone at its C-terminus, exactly as earlier shown for Magi2 and Tox6 [27]. The insecticidal toxin Bs1 possesses a cysteine framework represented by – CX6CX6CCX4CX14C – (X is any amino acid residue). Such positional patterns of cysteine residues are in good agreement with that expected for an ‘inhibitory cysteine knot (ICK) motif’, – CX3-7CX3-6CX0-5CX1-4CX 3-13C –, which is commonly conserved in diverse animal peptide toxins [23,25,26].

The peptide Bs1 was tested on several ion channels (rNav1.2, rNav1.4, hNav1.5, mNav1.6 and Para/tipE, hKv1.3 and Shaker IR) channels. The rationale for testing on Nav was based on previous work performed with spider toxic peptides that have insecticidal activity, similar to Bs1, but show no mammalian toxicity. In this way, it would be possible to verify whether Bs1 was specific for insects. Several typical mammalian Nav channels were not sensitive to Bs1 applications (see supporting material). However, the insect specific sodium channel Para/tipE was affected by addition of Bs1 (at 2 mM concentration). This could eventually explain the lethal activity of Bs1 found in the in vivo experiments with crickets. The shift of the voltage-activation curve to more negative

peptides 29 (2008) 1901–1908

potentials in the presence of Bs1 (Fig. 3) is an electrophysiological characteristic of spider peptide toxins that bind to the site 4 of insect sodium channels, which have been linked to its insecticidal activity [6,16]. The spider peptides m-agatoxins and palutoxins are toxic to insects (LD50 9–48 mg/g in Lepidoptera) and they also modify sodium currents (Para/tipE) affecting the shift voltage activation [4,24,29]. Although, the Para/tipE was isolated from Drosophila melanogaster, homologous receptors to Para/tipE have been found in other insect species such cockroach, housefly, crickets and tobacco budworm [18,28]. Therefore, the insect sodium channel could be the principal target of Bs1, but a possible interaction of Bs1 with other insect receptors is not ruled out at this stage, mainly due to the relatively high concentration needed for the visual effect (2 mM). No significant sequence similarity between other peptide sequences, and any known peptide toxins was found except those represented in Table 1B. Furthermore Bs1 contains a cationic amino acid cluster at the C-terminal region, similar to other spider peptide toxins (underlined in Table 1B), which is presumed to play an important role in the interactions with ion channels [17,31]. Taken together, these results indicate the possibility that the Bs1 and the other spider peptide toxins constitute novel ion channel blockers or modulators that could possess unique pharmacological properties in insects.

Acknowledgments This work was supported in part by a grant from Laboratorios Silanes S.A. de C.V. and Institute Bioclo´n S.A. de C.V., and by a grant from the Direccio´n General de Asuntos del Personal Acade´mico (DGAPA-UNAM) IN226006 and CONACyT 49773/ 24968. The confirmation of the amino acid sequence of Bs1 by Dr. Fernando Zamudio and the mass spectrometry determination conducted by Dr. Cesar Batista is greatly acknowledged. This work was also supported in part by grants OT/05-064, G.330.06 from the Fund for Scientific Research (FWO)-Flanders, P6/31 (Interuniversity attraction Poles Program- Belgian StateBelgian Science Policy.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.peptides. 2008.07.003.

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