Here we des-cribe both chemically synthesized poneratoxin and pon-eratoxin obtained by expression in insect cells.. For the first PCR step, an upstream primer containing the 5¢ signal pep
Trang 1Poneratoxin, a neurotoxin from ant venom
Structure and expression in insect cells and construction of a bio-insecticide
Ewa Szolajska1, Jaroslaw Poznanski1, Miguel Lo´pez Ferber2, Joanna Michalik1, Evelyne Gout3,
Pascal Fender3, Isabelle Bailly3, Bernard Dublet3and Jadwiga Chroboczek3
1
Institute of Biochemistry and Biophysics (IBB), Polish Academy of Sciences, Warsaw, Poland;2Laboratoire de Pathologie Compare´e, UMR 5087, INRA-CNRS-Universite´ de Montpellier II, St Christol les Ales;3Institute 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)
Trang 2an 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 andP clavata venom
The peptide with the sequence FLPLLILGSLLMTPPVI
QAIHDAQR-NH2was 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
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¢-GAATTCATGCTACTAGTAAAT
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
GAAAGCATGCAAAGGCAGA-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 andits purification (A) Total cell extract of poneratoxin-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) are shown on the left of both gels.
Trang 3Technology) 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· l06per 35-mm dish) were infected at MOI 10 For
time course of expression, harvested cells were resuspended
in 50 mM Tris/1 mM EDTA/100 mM 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 mM
Tris/1 mM EDTA/100 mM NaCl, pH 8 The cells were
suspended in lysis buffer [50 mM Tris/1% (v/v) Nonidet
P-40/200 mM NaCl/1 mM EDTA, pH 8.5] containing
Complete protease inhibitors (Boehringer) and sonicated
The extract was centrifuged at 10 000 g
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 mM Tris/150 mM
NaCl/1 mM EGTA/2 mM MgCl2/0.4% (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 frugiperdalarvae were obtained from a laboratory colony
reared on semiartificial diet [16] at 22.5 ± 0.5C, 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 ± 0.5 lL each
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 105pfu was sufficient to kill all the larvae, and so the three baculoviruses were diluted with NaCl/Pito reach this dose
in 8 lL The mock-infected group was injected with 8 lL NaCl/Pi To evaluate the LT50, 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 mMacetic 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 25C 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 lMstock 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 lM) 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 Æcm2Ædmol)1
Nuclear magnetic resonance (NMR) experiments NMR measurements were performed on the Bruker AMX
600 MHz spectrometer at 298 K Peptide solution (3 mM) in the 25 : 65 : 10 (v/v/v) mixture of2H-enriched-TFE, H2O and 2H2O was adjusted to pH 5.5 The standard COSY, TOCSY as well as 100 and 200 ms mixing time NOESY spectra were accumulated, processed byNMRPIPE[21] and analyzed byX-EASYprogram [22] Structure determination was obtained withDYANAsoftware in the REDAC strategy mode [23] Final refinement was carried out by simulated annealing procedure with help ofX-PLOR[24] The ponera-toxin structure was deposited in the Protein Data Bank (http://www.rcsb.org), accession code PDB1G92 Chemical
Trang 4shift 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 clavatais 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-NH2 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
For the tests on S 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.
Trang 5by signal peptide (SPx) They were obtained with titers of
2· 107, 2.5· 107and 1.5· 107pfuÆmL)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 P¼ 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
(Q¼ 3.22, P < 0.005) Thus, the expression of
poneratox-in gives virus with improved killponeratox-ing properties Surprisponeratox-ingly,
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 mMacetic 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, injectedwith 105pfu of SPx, Px andcontrol 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 ponpon-eratoxin (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
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) andof 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).
Trang 6which 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.
Trang 7the 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 [TMPRED 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-giperdaor 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
Trang 8the 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 californicabaculovirus 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 australisor the mite Pyemotes tritici
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 Also, the amounts of toxin released from
ingested larvae may or may not be high enough to have an effect on the predators Clearly, more work is required to understand how this improvement in virus killing rate occurs and what are its implications for the development of safe baculovirus recombinant bio-insecticides
Acknowledgements
This work was supported in part by NATO Linkage Grant no 940881.
ES was supported in part by a poste rouge of CNRS We are indebted
to J O Schmidt (South-west Venoms, Tucson, AZ, USA) for a sample
of P clavata venom The help of the Laboratory of Magnetic Resonance (IBS) in the structural part of this work, G Goch (IBB)
in CD spectroscopy, H Kozlowska (IBB) in HPLC and of M Jerka-Dziadosz (Nencki Institute, Warsaw) in immunofluorescence technique
is acknowledged We are grateful to A Wyslouch (IBB) and to
M Jaquinod and J.-P Andreini (IBS) for discussions and to R Wade for reading our manuscript.
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Trang 10Supplementary 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 3JHaHN 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