Báo cáo khoa học: Poneratoxin, a neurotoxin from ant venom Structure and expression in insect cells and construction of a bio-insecticide pot

Poneratoxin, a neurotoxin from ant venom Structure and expression in insect cells and construction of a bio-insecticide Ewa Szolajska 1 , Jaroslaw Poznanski 1 , Miguel Lo ´ pez Ferber 2 , Joanna Michalik 1 , Evelyne Gout 3 , Pascal Fender 3 , Isabelle Bailly 3 , Bernard Dublet 3 and Jadwiga Chroboczek 3 1 Institute of Biochemistry and Biophysics (IBB), Polish Academy of Sciences, Warsaw, Poland; 2 Laboratoire de Pathologie Compare ´ e, UMR 5087, INRA-CNRS-Universite ´ de Montpellier II, St. Christol les Ales; 3 Institute of Structural Biology (IBS), Grenoble, France Poneratoxin is a small neuropeptide found in the venom of the ant Paraponera clavata. It is stored in the venom reservoir as an inactive 25-residue peptide. Here we des- cribe both chemically synthesized poneratoxin and pon- eratoxin obtained by expression in insect cells. When expressed in insect cells, poneratoxin was observed attached to cell membranes. Both synthetic and recom- binant ponerotoxins were soluble below pH 4.5. The structure of synthetic poneratoxin was characterized by circular dichroism and solved by nuclear magnetic reso- nance. In an environment imitating a lipid bilayer, at pH within the range of insect hemolymph, synthetic ponera- toxin has a V shape, with two a-helices connected by a b-turn. Insect larvae were paralyzed by injection of either of the purified toxins, with the recombinant one acting faster. The recombinant toxin-producing baculovirus reduced the average survival time of the insect host by 25 h compared with unmodified virus. Mass spectrometry analysis showed that the recombinant toxin has an N-terminal 21-residue extension, possibly improving its stability and/or stabilizing the membrane-bound state. The potential use of poneratoxin for the construction of bio- logical insecticide is discussed. Keywords: synthetic poneratoxin; recombinant poneratoxin; baculovirus; insecticide; peptide atomic structure. Living organisms have developed natural toxins targeting key metabolic pathways of either their predators or their prey. These toxins are used in research as molecular probes, targeting with high affinity selected ion channel subtypes. As such, they are very useful for understanding the mechanism of synaptic transmission. Moreover, studies on toxin entry into cells have been important for unraveling the mechanism of cell endocytosis and the functioning of membrane receptors. Many arthropod species such as scorpions and spiders, as well as insects (bees, wasps and ants) produce venom, which is a mixture of different neurotoxins, arthropods’ natural insecticides. Some of these neurotoxins are peptides. The insect viruses, baculoviruses, have been used as insect pest control agents since the last century [1]. They have a relatively narrow host range, which might allow specific pests to be targeted. However, the baculovirus life cycle is complex and long, so it takes several days before the infected insect dies, leading to considerable damage to crops. To overcome this limitation, several attempts have been made to obtain baculoviruses with enhanced toxicity. Recombinant baculoviruses have been constructed with genes coding for regulators of insect metabolism such as hormones and enzymes [2,3], but also for natural toxins of scorpions, mites, or spiders [4–8]. When compared with the wild-type virus, some of these recombinants were able to reduce the life span of infected insects. The tropical ant Paraponera clavata is a predator of small animals such as insect larvae. Its venom contains a potent insect-specific peptide neurotoxin, poneratoxin. Ponera- toxin affects the voltage-dependent sodium channels and blocks the synaptic transmission in the insect central nervous system in a concentration-dependent manner [9–11]. It appears to be a good candidate for the construc- tion of a baculovirus insecticide apt to immobilize the infected insect. In this study, we have solved the atomic structure of poneratoxin. In addition, we have expressed the toxin in baculovirus and explored the biological properties of such recombinant virus. Experimental procedures Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis Mass spectra were obtained with a Perseptive Biosystems (Framingham, MA, USA) Voyager Elite Xl time of flight mass spectrometer with delayed extraction, operating with a pulsed nitrogen laser at 337 nm. Positive-ion mass spectra were acquired using a linear, delayed extraction mode with Correspondence to J. Chroboczek, Institute of Structural Biology (IBS), 41 J. Horowitz, 38027 Grenoble, France. Fax: + 33 4 38785494, Tel.: + 33 4 38789590, E-mail: wisia@ibs.fr Abbreviations: AcMNPV, Autographa californica nuclear polyhedrosis virus; MOI, multiplicity of infection; PC, phosphatidylcholine; pfu, plaque-forming units; Px, poneratoxin; SPx, poneratoxin preceded by signal peptide; TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroethanol. (Received 19 December 2003, revised 10 March 2004, accepted 30 March 2004) Eur. J. Biochem. 271, 2127–2136 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04128.x an accelerating potential of 20 kV, a 94% grid potential, a 0.15% guide wire voltage, and a delay time of 100 ns. Each spectrum is the results of 100 averaged laser pulses. Aliquots of 1 lL samples and of 1 lL of a saturated solution of a-cyano-4-hydroxycinnamic acid prepared in 50% aqueous acetonitrile/0.3% trifluoroacetic acid (TFA; v/v/v) were mixed on the stainless steel sample plate and dried in air prior to analysis. External calibration was performed with standards using the averaged m/z values of 1297.51, 2094.46, 2466.72, 3660.19 and 5734.59. Synthetic poneratoxin, antibody and P. clavata venom The peptide with the sequence FLPLLILGSLLMTPPVI QAIHDAQR-NH 2 was synthesized by solid phase synthe- sis using t-boc chemistry. It was purified by reverse phase HPLC on Vydac C 18 column with an acetonitrile gradient of 35 to 90% in 0.1% (v/v) TFA and it eluted at 73% (v/v) acetonitrile (Fig. 1B). Peptide identity, integrity and purity were analyzed by MALDI-TOF mass spectrometry. This analysis revealed a peak with molecular mass of 2756. The peptide was coupled to ovalbumin by the benzidine method at a ratio 17/1 and used to immunize rabbits. P. clavata dried venom was a kind gift of J. O. Schmidt [12]. 1 Genes, plasmids, viruses and cells Synthetic oligonucleotides made with the codon choice suitable for AcMNPV [13] were used for the construction of the poneratoxin gene [11]. Two oligonucleotides: forward 5¢- 2 GATCCATGTTTCTTCCGCTTCTGATCCTTGGCT CTCTTCTGATGAC-3¢ and reverse 5¢-CGGCGTCATCA GAAGAGAGCCAAGGATCAGAAGCGGAAGAAA CATG-3¢, were used to synthesize the N-terminal fragment of the poneratoxin gene. Two others: forward 5¢-GCC GCCCGTGATACAGGCGATCCACGATGCGCAGA GGTAGTAATGAG-3¢ and reverse 5¢-AATTCTCATTA CTACCTCTGCGCATCGTGGATCGCCTGTATCAC GGG-3¢ were used to construct the C-terminal fragment. After phosphorylation and annealing, both fragments were ligated. The full-length gene contained 75 nucleotides with an ATG codon in front of the gene and three stop codons at the end of it, and with flanking regions containing the restriction sites for BamHI and EcoRI. The gene was inserted into the pFastBac transfer vector (Life Technology) giving a recombinant plasmid pFastBacPx. The second construct containing the poneratoxin gene with the up- stream signal sequence of AcMNPV glycoprotein gp67 [14] was made by a three-step PCR amplification. For the first PCR step, an upstream primer containing the 5¢ signal peptide sequence: 5¢-GAATTC ATGCTACTAGTAAAT CAG-3¢ (number 1) and downstream primer with a sequence complementary to the 5¢ end of poneratoxin gene and 3¢ end of a signal peptide: 5¢-CAGAAGCGGAA GAAA GCATGCAAAGGCAGA-3¢ (number 2), were used. In the second PCR the plasmid pFastBacPx was used as a template with upstream and downstream primers containing, respectively, the first 15 nucleotides of the 3¢ end of signal peptide sequence and 15 nucleotides of the 5¢ end of the poneratoxin gene (number 3) and 12 nucleotides complementary to the 3¢ end of poneratoxin gene (number 4): 5¢- TCTGCCTTTGCATGCTTTCTTCCGCTTCTG-3¢ and 5¢-GAATTCTCATTACTACCT-3¢. Finally, the mix- ture of these two PCR reactions was used as a template with primers numbers 1 and 4 in a 30-cycles run. In all of these oligonucleotides the sequence of the poneratoxin gene is in bold letters and that of signal peptide is underlined. The final DNA products were digested with BamHI and EcoRI and inserted into the vector pFastBac, yielding, respectively, pFastBacSPx and pFastBacPx. All constructs were confirmed by DNA sequencing. The recombinant baculoviruses with signal peptide (SPx) or without it (Px) were generated in the Bac-to-Bac Expression System (Life Fig. 1. Expression of recombinant poneratoxin in the baculovirus system and its purification. (A)Totalcellextractofponeratoxin-expressing Sf21 cells (5 · 10 5 ) was subjected to 20% SDS/PAGE and Western blot. Lane 1, crude cell lysate; lane 2, synthetic poneratoxin (0.5 lg); lane 3, P. clavata venom (62.5 lg). (B) Fractionation on reverse-phase HPLC C18 column of the poneratoxin pool after Superdex column. Fractions containing the recombinant poneratoxin are indicated with the arrow. (C) Purified recombinant poneratoxin was analyzed on 20% SDS/PAGE and revealed with silver stain. Lane 1, fractions no. 49–51; lane 2, synthetic poneratoxin. Molecular mass markers (in kDa) areshownontheleftofbothgels. 2128 E. Szolajska et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Technology). They were propagated in Sf21 cells (IPBL from Spodoptera frugiperda), as well as in HF (High Five or BTI-TN-5B1-4 from Trichoplusia ni)(Invitrogen),main- tained in TC 100 medium supplemented with 5% (v/v) fetal bovine serum [15]. Protein expression Virus stocks were prepared by infection of Sf21 monolayers at the multiplicity of infection (MOI) 0.1 with the supernatants obtained after cell transfection with recom- binant baculovirus DNA. For protein expression, insect cells (l.5 · l0 6 per 35-mm dish) were infected at MOI 10. For time course of expression, harvested cells were resuspended in 50 m M Tris/1 m M EDTA/100 m M NaCl, pH 8.0, and lysed by three cycles of freezing and thawing. The total cell extract was analyzed for the presence of poneratoxin by 20% SDS/PAGE followed by Western blot with the antiponeratoxin antibody (500-fold dilution). Electrotrans- fer onto poly(vinylidene difluoride) membrane was carried out in the presence of 10% (v/v) methanol. Production and purification of recombinant poneratoxin Three days after infection with the recombinant baculovirus the insect cells were collected and washed twice with 50 m M Tris/1 m M EDTA/100 m M NaCl,pH8.Thecellswere suspended in lysis buffer [50 m M Tris/1% (v/v) Nonidet P-40/200 m M NaCl/1 m M EDTA, pH 8.5] containing Complete protease inhibitors (Boehringer) and sonicated. The extract was centrifuged at 10 000 g 3 for 20 min and the resulting pellet was dissolved in 70% formic acid. It was fractionated on the Superdex Peptide HR 10/30 column (Pharmacia) using 70% (v/v) formic acid for elution. Fractions containing the recombinant peptide were applied onto C18-218 TP54 column (4.6 · 250 mm, Vydac) and eluted with acetonitrile/water gradient (35–100%) contain- ing 0.1% TFA (v/v/v). Recombinant poneratoxin eluted between 87% and 91% acetonitrile (v/v) (Fig. 1B). Western blot with antiponeratoxin serum was used to identify fractions containing poneratoxin. Confocal microscopy Recombinant baculovirus-infected cells were collected by low-speed centrifugation, transferred onto cover slips and fixed for 6 min in 50% ethanol/0.2% Triton X-100 (v/v/v). They were washed twice with TBST [20 m M Tris/150 m M NaCl/1 m M EGTA/2 m M MgCl 2 /0.4% (v/v) Tween 20, pH 7.2] and incubated for 1 h with the antiponeratoxin serum diluted 1/400 in TBST. After TBST wash, the samples were incubated with FITC-conjugated goat antiserum against rabbit IgG (Pasteur Institute, Paris) for 1 h at room temperature, washed three times with TBS, mounted with Citifluor (Citifluor Ltd, UK) and photo- graphed with Leitz–Wetzlar confocal microscope. Toxicity assays S. frugiperda larvae were obtained from a laboratory colony reared on semiartificial diet [16] at 22.5 ± 0.5 °C, 70% humidity, with 16 h photoperiod. Groups of 12 fourth instar larvae aged 11 days, with average weight 148.6 ± 3.7 mg, were injected with 8 lL±0.5lLeach of infectious supernatant of either unmodified parental baculovirus bMON14272 (Bac-to-Bac Expression System), or virus containing poneratoxin gene (Px) and virus with poneratoxin gene preceded by signal peptide (SPx). Preliminary experiments indicated that a dose of 10 5 pfu was sufficient to kill all the larvae, and so the three baculoviruses were diluted with NaCl/P i to reach this dose in 8 lL. The mock-infected group was injected with 8 lL NaCl/P i . To evaluate the LT 50 , a total of 132 larvae were injected each with SPx or Px virus supernatants, and 84 with the unmodified control virus. Survival was scored every 8 h. The data were analyzed using the Kruskal–Wallis non- parametric test with the correction for tied ranks [17]. Individual comparisons were carried out using the Dunn test [18]. Synthetic and recombinant poneratoxins were dissolved in 50 m M acetic acid/NaOH, pH 4.5. Groups of 12 larvae (approximate weight 158 mg) were injected with 10 ng of each peptide and with the P. clavata venom equivalent to 10 ng of poneratoxin [11] or with 8 lL solvent as a control. The time needed to paralyze larvae as well as paralysis duration was monitored. Circular dichroism (CD) measurements The spectra were collected at 25 °C in 185–270 nm wavelength range with a 0.2 nm spectral step size on an AVIV 202 spectropolarimeter, using 1 cm path-length cell. Each spectrum was recorded as an average of three scans and then corrected for the buffer background. For all CD measurements the same 360 l M stock solution of ponera- toxin prepared in 1% (v/v) aqueous 2,2,2-trifluoroethanol (TFE) was used. The presence of TFE permitted studies at pH 5.5. Aqueous solutions of SDS and PC were used at 10 and 1% (v/v), respectively. CD measurements were carried out with samples obtained by mixing the adequate amounts of 1% (v/v) aqueous TFE, peptide stock, and TFE or SDS or PC solutions. Before each experiment the exact peptide concentration (set initially at  5 l M )was determined from the absorption at 280 nm using the extinction coefficient calculated according to the peptide sequence [19]. Estimations of secondary structure elements were carried out using deconvolution by back-propagation of neural networks implemented by Bo ¨ hm et al.[20].All the CD data were expressed as mean residue ellipticity given in °Æcm 2 Ædmol )1 . Nuclear magnetic resonance (NMR) experiments NMR measurements were performed on the Bruker AMX 600 MHz spectrometer at 298 K. Peptide solution (3 m M )in the 25 : 65 : 10 (v/v/v) mixture of 2 H-enriched-TFE, H 2 O and 2 H 2 O was adjusted to pH 5.5. The standard COSY, TOCSY as well as 100 and 200 ms mixing time NOESY spectra were accumulated, processed by NMRPIPE [21] and analyzed by X - EASY program [22]. Structure determination was obtained with DYANA software in the REDAC strategy mode [23]. Final refinement was carried out by simulated annealing procedure with help of X - PLOR [24]. The ponera- toxin structure was deposited in the Protein Data Bank (http://www.rcsb.org), accession code PDB1G92. Chemical Ó FEBS 2004 Structure and expression of an ant neurotoxin (Eur. J. Biochem. 271) 2129 shift and coupling constants have been deposited in BioMagResBank (http://www.bmrb.wisc.edu), accession code BMRB-4921. Results One of the major components of the venom of tropical ant P. clavata is poneratoxin, a peptide largely responsible for the venom’s neurotoxic activity [9,10,12]. We were interested in the structure of this peptide and its anti-insect neuro- toxicity with the idea of using it for engineering a baculovirus capable of serving as a bio-insecticide. Peptide synthesis and expression of recombinant poneratoxin Using the published poneratoxin sequence [12], the peptide FLPLLILGSLLMTPPVIQAIHDAQR-NH 2 was chemic- ally synthesized and purified by HPLC. Mass spectrometry analysis of the product gave the correct molecular mass of 2756 (theoretical 2757). This peptide was used for structural studies and to raise the polyclonal poneratoxin-specific antibody. For the poneratoxin expression, two recombinant bacu- loviruses with the poneratoxin gene were engineered, one containing a signal peptide and another without it. As the signal for poneratoxin secretion is not known, the 39 amino acid signal sequence of the major envelope glycoprotein gp67 (MLLVNQSHQGFNKEHTSKMVSAIVLYVLLA AAAHSAFA) was used. The same sequence was success- fully employed in the baculovirus system for expression of the insect-specific neurotoxin of the scorpion Androctonus australis [6]. The sequence includes all the nucleotides from the first ATG of the open reading frame. During the cloning procedure, a cysteine residue was introduced between this signal and the mature poneratoxin. Surprisingly, ponera- toxin expression was detected only in cells infected with baculovirus containing the poneratoxin gene with the signal peptide. The maximum level of expression was seen at three days post infection, in good agreement with the usual activity of the polyhedrin promoter. A similar expression level was observed in both, Sf21 and HF, cell lines. Therefore Sf21 cells were used for the rest of the studies as they are easier to grow in suspension. Toxin expression was analyzed on 20% (w/v) polyacryl- amide denaturing gel followed by immunoblot. No toxin was detected in the extracellular medium (tested using reverse-phase C18 SepPac and Western blot, not shown). Analysis of fractions derived from the crude lysate revealed that poneratoxin is expressed in an insoluble form (not shown). The recombinant toxin was retarded on denaturing gels in comparison with the synthetic peptide (Fig. 1A) but this mobility difference cannot be explained by the disulfide bridge as electrophoresis was run in the presence of reducing agent. The mass spectroscopy data for the 25-amino acid synthetic peptide confirmed its integrity (molecular mass 2756). However, the mass spectroscopy of the recombinant poneratoxin contained in the formic acid extract showed that it has molecular mass of 4861, compatible with a longer peptide starting with the methionine in the middle of the signal peptide (theoretical mass 4861). It is relevant that when gp64 (called also gp67) is expressed during AcMNPV infection, the second ATG in the open reading frame is used as the translation initiation codon and that downstream sequences encode a functional signal peptide [25]. In addition, the preparation showed the presence of a second species with mass of 4888, suggesting the postranslational modification by formylation of the initiator methionine (theoretical mass 4889), which explained the difficulties encountered in the N-terminal sequencing. The possible dimerization of the recombinant peptide mediated by the N-terminal cysteine (added during the cloning steps) was excluded by repeating the mass spectrometry analysis under reductive conditions, with unchanged results. The pH of P. clavata venom is very low, due to the high concentration of formic acid. Accordingly, the synthetic peptide is soluble below pH 4.5 and such conditions were used for the extraction and purification of recombinant poneratoxin. On reverse-phase C18 column the recombin- ant poneratoxin eluted at higher acetonitrile concentration than the synthetic peptide (Fig. 1B). It is relevant in this context that the recombinant peptide has the N-terminal extension MVSAIVLYVLLAAAAHSAFAC, which will likely reinforce its hydrophobic character. Confocal microscopy was used to determine the cellular localization of the recombinant poneratoxin in insect cells. The toxin was observed at the cell periphery (Fig. 2), and treatment with the mild detergent NP-40 did not liberate it from the insoluble fraction (data not shown). This suggests that poneratoxin synthesized in the cytoplasm becomes insoluble upon its transfer towards the cell membrane. Toxicity studies ForthetestsonS. frugiperda larvae three baculoviruses were used: the unmodified parental virus obtained after infection of insect cells with the initial unmodified shuttle vector, the recombinant virus with the poneratoxin gene (Px) and the third virus with the poneratoxin gene preceded Fig. 2. Confocal microscopy of the Sf21 cells expressing recombinant poneratoxin. Baculovirus-infected cells were collected on the cover slips, fixed, incubated with the antiponeratoxin serum and observed with Leitz–Wetzlar confocal microscope as described in the Materials and methods. Magnification ·1000. 2130 E. Szolajska et al. (Eur. J. Biochem. 271) Ó FEBS 2004 by signal peptide (SPx). They were obtained with titers of 2 · 10 7 ,2.5· 10 7 and 1.5 · 10 7 pfuÆmL )1 , respectively. Global analysis of the data (Fig. 3 and Table 1) confirms the difference in the killing rate of the three viruses (Kruskal–Wallis H-values equal 59.91 after correction for tied ranks, with a P ¼ 9.77 · 10 )14 ). The highest killing rate was observed with the baculovirus expressing recombinant poneratoxin. The killing rate of 50% was reached at about 131 h post injection 4 by SPx and at 160 h post injection by the parental virus. The difference is statistically significant (Q ¼ 3.22, P < 0.005). Thus, the expression of poneratox- in gives virus with improved killing properties. Surprisingly, the Px baculovirus containing the poneratoxin gene but unable to express the peptide, killed the larvae 35 h later than the parental baculovirus. The Px virus seems to be disabled in its multiplication due to instability; we observed titers decreasing with time for Px, with constant titers for two other viruses. Nevertheless, toxicity studies on freshly obtained viruses resulted in 100% larvae killing by all three viruses (Fig. 3). To estimate the paralyzing activity of these neurotoxins, the larvae were injected with synthetic and recombinant poneratoxins as well as the P. clavata venom containing an equivalent amount of poneratoxin (estimated according to Piek et al. [11]) or with the solvent alone (50 m M acetic acid/ NaOH, pH 4.5). The strongest paralyzing effect was exerted by venom (Table 2), which suggests that other venom components might also be neurotoxins. Recombinant poneratoxin was more toxic than the synthetic one. It should be borne in mind that the recombinant poneratoxin has the 21 amino acid extension compared with the synthetic one. Unless this difference in activity is due to some as yet uncharacterized post-translational modifica- tions of the recombinant toxin, it seems that the N-terminal extension increases its neurotoxicity. It is conceivable that the hydrophobic extension might improve toxin stability resulting in longer bioavailability. Structure characterization by CD All the CD spectra were analyzed at pH 5.5. The CD spectrum obtained for the synthetic peptide in 1% (v/v) TFE solution was dominated by a minimum located at 200 nm and exhibited no maximum below 200 nm (Fig. 4), Fig. 3. Cumulative mortality (in percentage) of S. frugiperda fourth instar larvae, injected with 10 5 pfu of SPx, Px and control virus or buffer (mock) injected. Table 1. Average survival times (in hours) of 4th instar S. frugiperda larvae. S. frugiperda larvae were injected with 10 5 pfu of the parental virus (control, shuttle vector bMON14272), the virus expressing pon- eratoxin (SPx), and the virus unable to express poneratoxin (Px). The 95% interval is the confidence interval for a type 1 error of > 0.05. It shows that the real average value obtained from our data would be between the lower and higher values in 95% of the experiments using the same population. Virus 95% interval Median Lower Higher SPx 136.17 130.95 148.85 Control 161.23 153.36 178.67 Px 196.36 189.36 203.486 Table 2. Direct paralyzing effect of poneratoxin. Groups of 12 S. fru- giperda larvae were injected with 10 ng of each peptide and with the P. clavata venom equivalent to 10 ng of poneratoxin. Larvae were scored as paralyzed if they were unable to right themselves within 30 s of being placed on their backs. As a control, 12 larvae were injected with 8 lL of sample solvent. They showed some reduction in mobility at 2 min after injection and then recuperated. Toxin Paralysis observed after Recovery after Venom 30 s 25 min Recombinant 3 min 7 min Synthetic 11 min 3 min Solvent Not observed Fig. 4. Molar ellipticity of synthetic poneratoxin as a function of SDS concentration in 1% TFE (A) and of TFE concentration (B). In both figures, curve (a) was obtained at 1% TFE. Concentrations of SDS in (A) were 0.2% (b), 1.4% (c) and 2.5% (d); and TFE in (B) 3% (b), 6% (c), 12% (d), 25% (e) and 50% (f). Ó FEBS 2004 Structure and expression of an ant neurotoxin (Eur. J. Biochem. 271) 2131 which is characteristic of a highly populated unordered random conformation [26]. However, CD experiments performed upon PC addition with the aim of imitating lipid bilayer environment demonstrated stabilization of the a-helical conformation (results not shown). Upon addition of PC the minimum moved toward longer wavelengths becoming deeper and a maximum appeared at 192 nm, which is indicative of the stabilization of a-helical structure. However, due to strong light scattering, the concentration range of PC was very limited and only qualitative analysis could be performed. In contrast, use of SDS, a detergent with micelles exhibiting no significant scattering, permitted quantitative analysis of the helix stabilization induced by the micellar phase. The SDS titration of poneratoxin in 1% (v/v) TFE demonstrated a systematic increase of the positive peak at 192 nm accompanied by the buildup of the minima at 208 nm and 223 nm, clearly indicating SDS-induced stabilization of peptide helical structure (Fig. 4A). The estimated partition of helical structure exceeded 30% for peptide in 1.4% (v/v) aqueous SDS. TFE is known to promote a stable secondary structure of the polypeptide chain in peptides [27] by strengthening the internal H-bonds of the peptide [28]. TFE-induced con- formational change is close to that observed for SDS (Fig. 4B); the CD spectra recorded in 12% TFE and 1.4% SDS are almost identical. The maximal effect of the secondary structure stabilization (63% a-helical, 12% b-turn, 18% random) was observed in 25% (v/v) TFE solution. The increase of the TFE concentration above 25% did not result in any significant change of CD spectra. Additional titration experiment showed no significant changes of peptide secondary structure in the pH range of 5.3–7.8 in 35% (v/v) TFE solution (data not presented). Therefore, in order to minimize amide proton exchange rates, the 25% (v/v) TFE aqueous solution of the synthetic poneratoxin at pH 5.5 was used for the NMR analysis. Structure of synthetic poneratoxin by NMR The solution structure of poneratoxin was determined on the basis of 428 experimentally derived distance restraints. Finally 10 structures exhibiting occasional residual viola- tions larger than 0.3 A ˚ were accepted. NMR-derived structure showed the presence of two helical regions: PLLILGS(3–9) and IQAIHDAQ(17–24), with residues LLMTPPV(10–16) forming a turn. The structure of the central LMTPPV(11–16) region of the peptide is almost identical to the open turn type III conformation of LMTDPV(151–156) fragment from the haloalkane dehalo- genase of Xanthobacter autotrophicus [29]. For both helices Fig. 5. Structural properties of the synthetic poneratoxin in solution. (A) Sausage model of the mean structure. The thickness of the tube is a measure of local backbone flexibility. Helical regions are in red. (B) Hydrophobic potential on peptide surface. Hydrophobic residues are in red, hydrophilic in blue. (C) The putative structure–function rela- tion. The apolar N-terminal helix is marked in red, the C-terminal polar helix is marked in blue. The helical regions are separated by a turn (in green) The N-terminus is dark green. (D) Stick representation of poneratoxin. Amino acids participating in the long–range hydro- phobic interactions stabilizing V-shaped conformation are space-filled. 2132 E. Szolajska et al. (Eur. J. Biochem. 271) Ó FEBS 2004 the experimentally determined pattern of short-range con- tacts is consistent with the proposed secondary structure. The overall backbone root-mean-square deviation (rmsd) is 1.32 A ˚ for 10 lowest-energy structures obtained after simulated annealing procedure, indicating the high quality of the obtained model. The average structure of the peptide is presented in the sausage model (Fig. 5A). The radius of the tube modeling the Ca backbone is proportional to the local structure deviations among the ensemble of 10 selected conformations. Detailed analysis showed that rmsd value (which is a measure of structure quality) determined separately for helical regions PLLILGS(3–9) or IQ- AIHDAQ(17–24) is significantly lower (< 0.4 A ˚ ). This clearly demonstrates the internal stability of the helical regions with their relative spatial organization weakly defined, resulting in lowered number of experimental long- range interhelical restraints. In conclusion, the solution structure of synthetic poner- atoxin can be modeled as two loosely interacting helices with a preferred V-shaped orientation. The central loop is stabilized by a small, flexible, but well defined hydrophobic core built from Ile6, Leu10, Pro15, Leu17 and Gln18 side- chains (Fig. 5D). The helix-break-helix organization of poneratoxin is similar to that found for other peptides interacting with the plasma membrane [30,31]. Taking into account the distribution of polar/apolar residues along the sequence and the sequence based prediction of peptide localization [TM PRED at http://www.ch.embnet.org/soft ware/TMPRED_form.html predicted transmembrane localization of FLPLLILGSLLMTPPVI(1–17) fragment] it is conceivable that the N-terminal apolar helix favors transmembrane localization while the C-terminal amphi- philic helix, is either solvent exposed or interacting with the membrane surface. Discussion Poneratoxin is a potent insect-specific toxin produced by the predatory ant P. clavata. The primary sequence of poner- atoxin has been obtained from the peptide mixture stored in the venom reservoir [10]. The gene has not been character- ized and nothing is known of the toxin processing during synthesis and secretion into the venom reservoir. A peptide was synthesized using the published amino acid sequence [11]. Synthetic poneratoxin is a very hydrophobic peptide of 25 amino acid residues with a rather charged C-terminal part. Our CD data show that under conditions imitating the membrane surroundings it has a propensity to acquire an ordered structure. The NMR structure shows the peptide in the form of two a-helices connected by a b-turn. The two helices have quite different characters. The first, PLLILGS(3–9), is apolar, whereas the second, IQ- AIHDAQR(17–25), contains polar and charged amino acids. This will result in different interactions with cell membranes. The extremely hydrophobic N-terminal helix will easily interact with uncharged lipid bilayers composed of phosphatidylcholine [32]. The C-terminal helix, slightly positively charged and terminating with arginine, will be able to attach to negatively charged cell surfaces similar as found for other membrane interacting peptides [30,31]. Such a toxin can thus use two different complementary modes of interaction to attain its target, cellular membranes. Moreover, the poneratoxin sequence starts with a bulky hydrophobic phenylalanine, which enhances the peptide hydrophobicity index and may be important for membrane penetration [33]. To analyze the biological activity of poneratoxin, we constructed two recombinant baculoviruses, one with the poneratoxin gene and another in which the peptide is preceded by a secretion signal from a baculovirus gene. However, no poneratoxin was detected when the gene devoid of a signal peptide was used (virus Px). It seems likely that when not exported, this peptide is either destroyed inside the cell or is toxic for the cells. Sequence analysis suggested that the part LPLLILGSLLMTPPVIQA(2–19), which looks like a transmembrane segment is similar to signal peptides of a variety of proteins [34]. If without an authentic signal peptide this fragment is seen by the expressing cell as a signal peptide, it could be degraded by the appropriate enzymatic system such as one that cleaves the signal peptide of preprolactine within its hydrophobic core, between two leucine clusters [35]. When the toxin was preceded by a signal peptide, baculovirus produced a recombinant toxin containing an N-terminal extension of 21 amino acid residues. Interest- ingly, the ATG coding for the middle methionine of the signal peptide is contained in a perfect Kozak consensus sequence AAGATGG, ensuring proper translation initi- ation [36]. Thus, the recombinant poneratoxin seems to be synthesized through the initiation from the second ATG in the open reading frame, similar to insect protein gp64 [25], a source of signal peptide. However, the signal peptide was not cleaved from the poneratoxin resulting in an uncleaved intracellular form, with no extra-cellular poneratoxin detected. It cannot be excluded that the amount of mature secreted toxin is too low for detection, but important enough to be responsible for the biological activity. Alternatively, the toxin liberated by cells dying from the infection could be responsible for the increased pathogen- icity observed in the biological assays. The results of the biological assay demonstrate that the 21 amino acid residue N-terminal extension improves the paralyzing activity of the recombinant peptide when compared with the synthetic one. The extension is quite hydrophobic in character and it is conceivable that this improves toxin stability and therefore its bioavailability. Alternatively, the N-terminal extension could stabilize the active toxin conformation. Additional experiments are needed to clarify these questions. The pH of the lepidopteran larvae hemolymph is between 6.6 and 6.8 [37,38], which is the upper pH limit of poneratoxin solubility. However, the infection of S. fru- giperda or T. ni larvae with AcMNPV by intrahaemocoelic injection starts with the progeny virus observed first in fat bodies and epithelium, with a rather slow build-up in the hemolymph [39]. It is conceivable that the conditions on the surface of the epithelium are sufficient to allow partial toxin solubility promoting its interaction with epithelial cell lipidic membranes. The poneratoxin activation is likely to be a multistep process and the poneratoxin structure studies give some insights into this process. Secretion of the native ponera- toxin to the venom reservoir is most probably triggered by the specific secretion signal. The neurotoxin stored in the ant venom reservoir should be inactive, preventing damage to Ó FEBS 2004 Structure and expression of an ant neurotoxin (Eur. J. Biochem. 271) 2133 the ant, suggesting that the acidic conditions present in the venom reservoir will forbid the structure conformation necessary for exerting poneratoxin neurotoxicity. Upon injection of the venom in the hemocoel of the prey, the membrane insertion and the pH change could trigger conformational changes, yielding the active neurotoxin. Few ant toxins are so far described [10,11,40–42] and only one three-dimensional structure of an ant venom peptide toxin is known [43]. This toxin, ectatomin, is built from two chains, each consisting of two a-helices bound by a hinge region. However, this structure seems to be much more rigid than that of poneratoxin, as each chain forms a hairpin stabilized by disulfide bridges. Furthermore, the chains are connected by a third S–S bridge resulting in a four-alpha- helical bundle structure. It should be noted that contrary to the majority of known sequences of venom peptides specific for sodium channels [44] native poneratoxin does not contain cysteine and it is conceivable that it has a distinct mode of action. Classical arthropod toxins, such as those of scorpions, form a family of small proteins of 30–70 amino acids affecting the kinetics of sodium or potassium channels. Autographa californica baculovirus recombinants expressing some of these toxins present improved insecticide activity as compared with wild type virus [45]. A well-documented example of a recombinant baculovirus is that carrying the sequence of the toxin from A. australis Hector scorpion venom [46,47]. Several recombinant baculoviruses with different toxin synthetic genes have been studied in the laboratory [6,48,49] and in controlled conditions in the field [47,50]. We thought that poneratoxin, another insect toxin, might be a good candidate for the reinforcement of the insecticide action of a baculovirus, perhaps providing an alternative insecticide activity with a mechanism of action possibly different from that of spiders and mite toxins. Toxicity studies showed that the baculovirus engineered to express poneratoxin is a better killer than the parental virus. The gain in time is considerable if we remember that the feeding period of S. frugiperda larvae extends up to 10 days. This is shortened with the parental baculovirus infection to about 7 days and to 5–6 days with recombinant SPx baculovirus. Similar results have been obtained with recombinant baculoviruses expressing neurotoxins of the scorpion A. australis or the mite Pyemotes tritici 5 [6,7,49]. We think that further improvements could be obtained by adjusting the secretion pathway to imitate the native one. Also the development of a bio-insecticide expressing in parallel two toxins targeting different pathways may significantly increase killing speed [51]. Many concerns have been raised about the risk of using genetically modified baculoviruses as insecticides in the field. One is the possible toxicity of the recombinant protein to the environment. To date, no detailed information exists on the per os toxicity of poneratoxin to other animals, birds or mammals, especially when released in nature. Predators will ingest the almost-dead larvae containing the expressed toxin. Poneratoxin appears to be soluble in acidic condi- tions, close to those existing in the stomach of mammals. However, it is not clear if the protein present in the larvae cadavers is solubilized and released, and, if so, if it will remain active. 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(2003) Further enhance- ment of baculovirus insecticidal efficacy with scorpion toxins that interact cooperatively. FEBS Lett. 537, 106–110. Ó FEBS 2004 Structure and expression of an ant neurotoxin (Eur. J. Biochem. 271) 2135 Supplementary material The following material is available from http://blackwell publishing.com/products/journals/suppmat/EJB/EJB4128/ EJB4128sm.htm Table S1. Structural statistics and restraint violations for the ensemble of 10 structures representing solution structure of poneratoxin. In parentheses is the range of estimated values. Fig. S1. NMR derived restraints analyzed in the terms of range categories (upper) and position in sequence (lower). Fig. S2. Distribution of the sequential and short range NMR constraints. The systematic pattern of i,i+3 NOEs accompanied by the lowered values of 3 J HaHN vicinal coupling constants permitted the assignment of the secon- dary structure. The helical regions in the peptide sequence (top) are marked in bold letters. Fig. S3. The CD spectra of poneratoxin recorded in 35% TFE solution at pH 5.3 and 7.8. For comparison the spectrum obtained for 25% TFE, pH 5.5 adopted from manuscript is presented. 2136 E. Szolajska et al. (Eur. J. Biochem. 271) Ó FEBS 2004 . some of these recombinants were able to reduce the life span of infected insects. The tropical ant Paraponera clavata is a predator of small animals such as insect larvae. Its venom contains a potent insect- specific. Poneratoxin, a neurotoxin from ant venom Structure and expression in insect cells and construction of a bio-insecticide Ewa Szolajska 1 , Jaroslaw Poznanski 1 , Miguel Lo ´ pez Ferber 2 , Joanna. 5¢-GAATTC ATGCTACTAGTAAAT CAG-3¢ (number 1) and downstream primer with a sequence complementary to the 5¢ end of poneratoxin gene and 3¢ end of a signal peptide: 5¢-CAGAAGCGGAA GAAA GCATGCAAAGGCAGA-3¢