Toxicon 56 (2010) 1172–1180
Contents lists available at ScienceDirect
Toxicon
journal homepage: www.elsevier.com/locate/toxicon
Functional expression of a recombinant toxin – rPnTx2-6 – active
in erectile function in rat
F.S. Torres b,1, C.N. Silva a,1, L.F. Lanza b, Agenor V. Santos b, A.M.C. Pimenta b, M.E. De Lima b, *,1,
M.R.V. Diniz a,1
a
Divisão de Ciências Biomédicas, Centro de Pesquisa Carlos Ribeiro Diniz, Fundação Ezequiel Dias, Belo Horizonte, Minas Gerais, Brazil
Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Laboratório de Venenos e Toxinas Animais, Universidade Federal de Minas Gerais,
Av. Antônio Carlos, 6627, 31.270-901 Belo Horizonte, Minas Gerais, Brazil
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 November 2009
Received in revised form 24 March 2010
Accepted 19 April 2010
Available online 24 April 2010
In the current study, the putative cDNA for PnTx2-6 toxin of the Phoneutria nigriventer
spider venom was cloned and expressed as tioredoxin fusion protein in the cytoplasm of
Escherichia coli. The fusion protein was purified from the bacterial extracts by combination
of immobilized Ni–ion affinity and gel filtration chromatographies. Then, it was cleaved by
enterokinase and the generated recombinant PnTx2-6 (rPnTx2-6) was further purified by
reverse-phase HPLC. Likewise the native toxin purified from the spider venom, rPnTx2-6
potentiates the erectile function when injected in rats. This result indicates that the
production of functional recombinant PnTx2-6 might be an alternative to provide this
basic and valuable tool for study, as well as for further understanding such complex
physiological system, including its correlation with the central nervous system and local
tissue factors.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Phoneutria nigriventer
Recombinant toxin PnTx2-6
Nitric oxide
Erectile function
1. Introduction
Penile erection is a neurovascular event modulated by
psychological and hormonal factors, which involves
a refined interaction of central nervous system with local
tissue factors. The penis is innervated by both autonomic
and somatic nerve system (Andersson and Wagner, 1995;
Giuliano and Rampin, 2000; Priviero et al., 2007). Sympathetic and parasympathetic nerves from the pelvis emerge
to form the cavernous nerves, which enter the corpus
cavernosum to regulate blood flow to the penis during
penile erection (Lue, 2000). The nerves, endothelium of
sinusoids and blood vessels and smooth muscle cells in the
penis produce transmitters and modulators that control the
erect versus flaccid state of the penis (Andersson, 2001;
* Corresponding author. Tel.: þ553134092659; fax þ553134092614.
E-mail address: [email protected] (M.E. De Lima).
1
The authors have equally contributed to this work.
0041-0101/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2010.04.010
Musicki and Burnett, 2006; Priviero et al., 2007). Upon
sexual stimulation, acetylcholine and nitric oxide (NO) are
released from the cavernous nerve terminals and also
vasoactive relaxing factors from the penile endothelial
cells, such as vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP), which relax arteries
and arterioles supplying the erectile tissue and making the
penile blood flow to increase The increased corporal arterial inflow in turn results in an increased intracavernosal
pressure and volume and penile erection as well
(Bivalacqua et al., 2000).
Nitric oxide (NO) is the principal mediator of smooth
muscle relaxation and erectile function of corpus cavernosum (Burnett et al., 1992; Burnett, 1997; Cerqueira and
Yoshida, 2002; Rajfer et al., 1992). It is synthesized from Larginine by the NO synthase (NOS) action and it spreads
locally into adjacent smooth muscle cells of the corpus
cavernosum, activating guanylate cyclase to increase
intracellular levels of cGMP (Burnett, 1997). This process
reduces calcium concentration in muscle, which trigger
F.S. Torres et al. / Toxicon 56 (2010) 1172–1180
membrane polarization, resulting in cavernosal smooth
muscle relaxation and in penile erection. Activity is
terminated by the breakdown of cGMP by the cGMPspecific type phosphodiesterase-5 (Bivalacqua et al., 2000;
Gupta et al., 1995; Lue, 2000; Nathan and Xie, 1994).
However, organic and psychogenic factors may cause
alterations in the NO/cGMP pathway and other pathways
and impair smooth muscle relaxation and/or increase
smooth muscle contraction, thereby resulting in erectile
dysfunction (ED). ED is defined as a persistent inability to
achieve or maintain erection for satisfactory sexual
performance (Bivalacqua et al., 2003; NIH Consensus
Conference, 1993). The incidence of ED can be aggravated
by diabetes mellitus, hypercholesterolemia, cardiovascular
disease, cigarette smoking and obesity (Bivalacqua et al.,
2003; Priviero et al., 2007). ED has been treated with
procedures from surgery to intracavernosal and intraurethral administered agents as well as effective oral
therapy by using phosphodiesterase-5 inhibitors, such as
apomorphine, phentolamine, sildenafil (ViagraÒ), taladafil
(CialisÒ) and vardenafil (LevitraÒ) (Leite et al., 2007;
Priviero et al., 2007).
Priapism is defined as a pathologic condition of a penile
erection that persists beyond or is unrelated to sexual
stimulation (Berger et al., 2001; Gomes et al., 2003). Peptide
toxins have shown to act mainly on sodium, potassium and
calcium channels (Borges et al., 2009; Catterall et al., 2007;
De Lima et al., 2007; Escoubas et al., 2000), and some of
them are able to induce corpus cavernosum relaxation
(Andrade et al., 2008; Nunes et al., 2008, 2009; Teixeira
et al., 2001, 2003, 2004) and priapism in experimental
animals (Brazil and Vellard, 1925; Cordeiro et al., 1992;
Rezende et al., 1991; Schenberg and Lima, 1966; reviewed
by Lucas, 1988; Vital Brazil et al., 1987).
The Phoneutria nigriventer spider is responsible for
severe human accidents (Bucaretchi et al., 2001; Vital Brazil
et al., 1987) characterized by simptoms such as intense
pain, sudoresis, agitation, salivation, tachycardia, cardiac
arrhythmia and priapism (Cordeiro et al., 1992; Vital Brazil
et al., 1987; Schenberg and Lima, 1966). Studies using P.
nigriventer spider venom have reported incidence of
priapism in mice and dogs (Bucaretchi et al., 2001). This
venom is a cocktail of toxins, containing peptides, enzymes,
free amino acids, histamine and serotonin (To reviews see:
Borges et al., 2009; Cordeiro et al., 1995; Gomez et al.,
2002). In describing the partial proteome of the P. nigriventer venom, Richardson et al. (2006) demonstrated that
the biologically active compounds comprise more than 100
different polypeptides with molecular mass ranging
between 3500 and 9000 Da. Among these, the PhTx2
family consists of four neurotoxic polypeptides (PnTx2-1,
PnTx2-5, PnTx2-6 and PnTx2-9; Cordeiro et al., 1992). The
symptoms of PnTx2-1, PnTx2-5 and PnTx2-6 are related.These toxins produce excitatory signs in mice, after
intracerebroventricular (i.c.v.) injection, which include
salivation, lachrymation, priapism, convulsions and spastic
paralysis of the anterior and posterior extremities (Rezende
et al., 1991). They have also proved to trigger inhibition of
the Naþ channel fast inactivation (Araujo et al., 1993).
Moreover, they have been shown to increase the entry
of Naþ in cortical synaptosomes, inducing membrane
1173
depolarization, calcium influx, releasing acetylcholine and
glutamate in a TTX-sensitive way (Romano-Silva et al.,
1993; Moura et al., 1998).
Despite PnTx2-5 has strong functional and sequence
homology with PnTx2-6, it is less toxic to mice and shows
lower affinity in GH3 cells, compared to PnTx2-6. Both
toxins cause priapism and they differ from each of other in
five amino acid residues (Cordeiro et al., 1992; Matavel
et al., 2009). Yonamine et al. (2004) showed that penile
erection (i.p. injection of 10 mg/mice) and others effects (as
salivation, respiratory distress and death) caused by PnTx25 were partially suppressed (w50% of animals) by pretreatment with NOS non-selective inhibitor L-NAME and
totally prevented by using nNOS-selective inhibitor (7-NI).
In the other hand, PnTx2-6 toxin has complex effects on
Naþ channel kinetics, inhibiting its activation and shifting
the activation voltage dependence toward negative
potentials (Araujo et al., 1993; Matavel et al., 2002, 2009).
Its complete amino acid sequence comprises a single-chain
basic polypeptide (48 residues) with 10 cysteine residues
(Cordeiro et al., 1992). As the other Phoneutria toxin
precursors, PnTx2-6 precursor has shown the same structural pattern, i.e the presence of a hydrophobic signal
peptide, a glutamate-rich intervening propeptide,
preceding the mature toxin sequencing, which is in some
cases followed by additional amino acid residues at the Cterminus (C terminal peptide), implying post-translational
modifications of the synthesized functional peptide (Diniz
et al., 1993). Thus the PnTx2-6 precursor toxin is synthesized as a prepropeptide (Kalapothakis et al., 1998a,b).
When injected subcutaneously (1.5 mg/mouse), PnTx2-6
causes priapism within 25–30 min, followed by death after
50-80 min (Richardson and Cordeiro, personal communication). Intracavernously injection (0.006 mg/kg) PnTx2-6
induces penile erection and it seems to have no systemic
effects, but when the toxin was intraperitoneally injected, it
produced erection and others toxic effects (Andrade et al.,
2008). Nunes et al. (2008) observed that the erectile
response of normatensive rat was potentiated after
subcutaneous or intravenous injection of PnTx2-6 and that
the pre-treatment of the animals with the non-selective
NOS inhibitor (L-NAME) inhibited erectile response caused
by toxin inoculation. In addition, these authors observed
that toxin administration increases nitric oxide levels in
cavernous corpus cells and that it was able to restore the
erectile function in hypertensive rats (DOCA-sal). Preliminary pharmacokinetic study (Yonamine et al., 2004)
employing iodinated toxin, showed that PnTx2-6 could
penetrate the blood-brain barrier. These studies suggested
that PnTx2-6 could be a possible tool for developing new
pharmacological agents for treating erectile dysfunction.
However, due to the small yields of crude spider venoms
and their complexity, as they contain so many components,
purifying and using such toxins are quite restricted.
Producing recombinant toxins in a heterologous organism
would be an alternative to overcome this problem. In this
study, the region encoding the functional polypeptide of
PnTx2-6 DNA was amplified by PCR and cloned into pET32c
(þ) vector (Novagen). A soluble thioredoxin fusion product
was expressed in the cytoplasm of Escherichia coli. The
recombinant PnTx2-6 displayed similar structural and
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F.S. Torres et al. / Toxicon 56 (2010) 1172–1180
pharmacological properties to the native toxin. It acted on
penile erection of normotensive rats following subcutaneous injection and caused hypersalivation, arrhythmia and
death following intracerebroventricular injection in rats and
mice. The expression of a soluble active toxin allows the
preparation of high quantities of the recombinant toxin and
its mutants, which can be specially designed for the study of
its action on penile erection and ion channels.
2. Materials and methods
Oligonucleotides used as primers for polymerase chain
reactions (PCR) and dideoxy-nucleotide sequencing were
synthesized by Severn Biotech Ltd., UK. Big Dye Terminator
v3.1 Cycle (Applied Biosystem, USA), restriction endonucleases, DNA-modifying enzymes, CNBr-activated Sepharose 4B, HisTrap kit, Histrap Desalting Kit and Sephasil 5 mST
4.6/250 Protein C18 were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). pET32c(þ) plasmid DNA,
NM522, BL21(DE3)pLysS competent cells, and Enterokinase
cleavage kit were supplied by Novagen Inc. (Madison, USA).
Trifuoroacetic acid and acetonitrile were purchased from
Merck (Schuchardt, Germany).
2.1. Expression construct and protein expression
Total RNA was isolated from P. nigriventer venom
glands with TRIzolÒ (Invitrogen), and the first strand cDNA
was synthesized from 4.0 mg of the total RNA, using the
RT-PCR kit with SuperScriptÔ First-Strand Synthesis
System (Invitrogen). The 147-bp DNA sequence encoding
the mature PnTx2-6 (Fig. 1) was amplified by PCR, using
specific primers – sense 50 AGAGAGAGAGCCATGGCCACATG
CGCTGGCCAA30 and antisense 50 AGAGAGAGACTCGAGTCAT
TTTTTACAGTTAGC30 - that included 50 Nco1 and 30 Xho1
restriction enzyme sites, respectively. The amplified
product was cleaved with Nco1 and Xho1, bound into
similarly digested pET32c(þ) and used to transform E. coli
NM 522 cells and cultured into Luria–Bertani (LB)
medium, containing 100 mg/ml of ampicillin at 37 C. The
PCR selected clones were certified by DNA sequencing,
using an automated sequencer ABI 3130 Genetic Analyzer
(Applied Biosystems) and following the manufacturer’s
own protocol and reagents. Expression host E. coli BL21
(DE3)pLysS was transformed with the isolated recombinant plasmid. The individual clones were grown at 37 C in
LB broth, containing 100 mg/mL ampicillin and 34 mg/mL
chloramphenicol. The recombinant protein expression was
induced by adding isopropyl-b-D-thiogalactoside (IPTG) at
a final concentration of 1 mM when cells reached the
logarithmic phase (OD600 ¼ 0.5 or a cell density of 5 108
cells/mL). After growing for an additional 3 h, the cells
were harvested by centrifugation and the resulting pellets
were then frozen at 70 C. The expression profiles of
recombinant protein were examined by SDS-PAGE on
12.5% (w/v) polyacrilamide gels and detected with Coomassie blue staining.
2.2. Purification and characterization of rPnTx2-6
Induced frozen cells were thawed and resuspended in
30 mM imidazole, 0.5 M NaCl and 20 mM NaHPO4, pH 7.4
(binding buffer) and the cells were further lysed by sonication. After centrifugation, the supernatant containing
soluble recombinant fusion protein was loaded onto
HistrapÔ HP Chelating affinity column (Amersham Biosciences, HPLC) charged with 500 mM NiSO4 and equilibrated
with binding buffer. Unbound proteins were eluted from
the chelating column with binding buffer. Fusion proteins
were eluted with linear gradient of 500 mM imidazole,
0.5 M NaCl and 20 mM NaHPO4, pH 7.4 (elution buffer) and
the fractions containing thioredoxin-PnTx2-6 fusion
protein were detected by PAGE-SDS 12.5% (w/v).
The fraction containing the thioredoxin-PnTx2-6 fusion
protein was pooled, further purified and desalted by
filtration gel chromatography (HitrapÔ HP Desalting,
Amersham Biosciences, HPLC). After cleavage with
enterokinase (following the Novagen protocol), the products were subjected to reverse-phase chromatography
(Sephasil Peptide C18 5 m ST 4.6/250; HPLC). The recombinant protein was detected by mass spectrometry MALDITOF analysis and used for biological tests.
A
PnTx2-6 coding region
Signal sequence
Glu rich reg.
Mature toxin
MKVAILFLSILVLAVASESIEESRDDFAVEELGRATCAGQDQPCKETCDCCGERGECVCGGPCICRQGYFWIAWYKLANCKK
PnTx2-6 coding region amplified for PCR
EK
B
Insertion of PnTx2-6 DNA into the pET32c(+)
DDDDK AMATCAGQDQPCKETCDCCGERGECVCGGPCICRQGYFWIAWYKLANCKK
pET32c(+)
NcoI
pET32c(+)
XhoI
Fig. 1. Diagrammatic representation of PnTx2-6 preprotoxin coding regions and the cloning of the PCR product coding the PnTx2-6 toxin. (A) The functional
domains, including the signal sequence ( ), the Glu-rich sequence (,), the mature toxin (-), which are cleaved at the indicated points (:) to form the mature
toxin. The DNA coding the mature toxin PnTx2-6 was amplified by PCR. (B) The NcoI-XhoI PCR cleaved product the mature toxin PnTx2-6 was inserted into
pEt32c(þ) expression vector. The residues DDDDK correspond the cleavage site of enterokinase (EK), which are cleaved at the point shown with a (:), and the
residues 2AM1 correspond to the expression of pET32c(þ).
F.S. Torres et al. / Toxicon 56 (2010) 1172–1180
2.3. Mass spectrometry analysis of rPnTx2-6
Mass spectrometry analyzes of rPnTx2-6 were carried
out using a MALDI-TOF/TOF MS AutoFlex III (Bruker Daltonics, Billerica, USA) instrument in positive/reflector mode
controlled by Flex-Control software. Instrument calibration
was achieved by using Peptide Calibration Standard II
(Bruker Daltonics, USA) as reference and a-cyano-4hydroxycinnamic acid was used as matrix. Samples were
spotted to MTPAnchorChip-400/384 (Bruker Daltonics,
USA) targets using standard protocols for the dried droplet
method. MS data analysis was performed by using the FlexAnalysis software (Bruker Daltonics, USA).
2.4. In vivo measurements of ICP/MAP
All experimental protocols were approved by the Ethics
Committee of the Universidade Federal de Minas Gerais
(Process no. CETEA 22/1996 and 102/2009). Male wistar
rats (240–270 g) were provided by the Centro de Bioterismo –
CEBIO of the Instituto de Ciências Biológicas (the animal
breeding center of the Institute of Biological Sciences,
Universidade Federal de Minas Gerais) and maintained with
free access to standard food and drinking water, and kept
on a 12 h light/dark cycle.
Rats were anesthetized with urethane (1400 mg/kg; i.p.)
and placed on a heating pad. The left femoral artery was
exposed and cannulated (using a 30 G needle connected to
PE-10 tubing filled with heparinized saline) for continuous
monitoring of mean arterial pressure (MAP). The shaft of
the penis was freed of skin and fascia, and the right corpus
cavernosum was cannulated by insertion of a 30 G needle
connected to a pressure transducer, permitting continuous
monitoring of corpus cavernosum pressure (ICP) as
described elsewhere (Mills et al., 1994 adapted for Nunes
et al., 2008). The abdominal cavity was opened, the right
major pelvic ganglion (MPG) exposed, and silver bipolar
electrodes were positioned on it for electrical stimulation.
Voltage–response curves were performed with continuous
stimulation of the MPG (30 s duration for each voltage)
using a crescent range (0.5, 0.75, 1.0, 1.2, 1.5, 2.0, 2.5 and
3.0 V) in a 5 ms pulse duration and a 12 Hz frequency.
Results were expressed as mean SEM. Statistical
analyses were performed using two-way analyses of variance (ANOVA) followed by Bonferroni post-hoc test for
multiple comparisons. A value of p < 0.05 was considered
statistically significant.
1175
peptide), implying post-translational modifications of the
synthesized functional peptide (Diniz et al., 1993;
Kalapothakis et al., 1998a,b). The conversion of the
precursor into mature polypeptide would seem to require
at least two different proteolytic activities (Fig 1A). As
bacteria do not seem to have the necessary enzymes for the
proteolytic cleavage of the precursor in order to generate
the final functional toxin, only the region encoding the
mature polypeptide PnTx2-6 (GenBank: AY054746) was
cloned into the vector (Fig. 1B).
The PCR positive clones from ampicillin-resistant colonies were sequenced to confirm in-frame insertion of DNA
encoding the mature toxin. These clones were used to
transform BL21(DE3)pLysS E. coli. The clones were then
grown in 250 mL medium and induced with IPTG as
described previously. Aliquots of cell extracts were
analyzed under denaturing conditions by polyacrilamide
gel electrophoresis with SDS. Extracts from induced
cultures showed the presence of a protein band of Mr of
approximately 22.5 kDa in the cell lysate, in a relatively
large amount compared with that observed in extracts
from uninduced cultures as shown in Fig. 2.
3.2. Purification and structural features of recombinant
PnTx2-6
The E. coli cell lysate containing PnTx2-6 fusion protein
was centrifuged and the supernatant was subjected to
chromatography on a metal chelate affinity column (Fig 3).
Presence of rPnTx2-6 in the fractions during the chromatographic purification step was detected by SDS-PAGE
(Fig. 3) and Western blot technique using antibodies
against the P. nigriventer spider venom (data not shown).
The affinity purified products were pooled and then
desalted by gel filtration chromatography (not shown) and
3. Results
3.1. Construction and expression of the rPnTx2-6
The characterization of cDNA clones has established that
Phoneutria toxins peptides are first translated as prepropeptide precursors. The primary structure of the Phoneutria toxins polypeptide precursors have indicated four
distinctive segments: (i) a well defined signal sequence (the
“pre” region) followed by (ii) a short glutamate-rich
segment (the “pro” region), (iii) the sequence encoding the
mature protein and, in some cases, followed by (iv) additional amino acid residues at the C-terminus (C terminal
Fig. 2. SDS/PAGE [15% (w/v) gel] analyses of expression of PnTx2-6 protein
fusion in Escherichia coli cells transformed with the PnTx2-6-pET32c(þ)
constructs. Lane MW, standard proteins (molecular weighs indicated at the
left) Bench MarkÔ Protein Ladder (7 mL); lane 1, cell extract before IPTG
(25mL); lane 2, cell extract after IPTG (25 mL). Arrow indicates PnTx2-6
protein fusion. Conditions: The electrophoresis was developed in Tris–
glycine buffer, pH 8.3, at 20mA. The samples were under reduced conditions.
The gel was stained with Coomassie blue R-250 (0,2% w/v).
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F.S. Torres et al. / Toxicon 56 (2010) 1172–1180
Fig. 3. Purification of PnTx2-6 protein fusion. Affinity chromatography on HistrapÔ Chelating HP (1.6 2.5 cm/5 mL). The fraction contained PnTx2-6 protein
fusion (indicated by an asterisk) was pooled. The splited lines indicate the cut point of pools. Binding buffer: 30 mM imidazole, 0.5 M NaCl, 20 mM NaHPO4, pH
7.4. Charge buffer: 500 mM NiSO4. Elution buffer: 500 mM imidazole, 0.5 M NaCl and 20 mM NaHPO4, pH 7.4. Fractions of 1 mL were collected at a flow rate of
1 mL/min. Inset, SDS/PAGE [15% (w/v) gel] analyses of purification steps on affinity and filtration gel chromatographies of PnTx2-6 protein fusion. Lane 1, standard
proteins (molecular weights indicated at the left) Bench MarkÔ Protein Ladder (7 mL); lane 2, cell extract after IPTG induction; lane 3, affinity chromatography
(25 mL); lane 4, gel filtration chromatography (25 mL). Conditions: The electrophoresis was developed as indicated in Fig. 2.
the resultant fraction containing fusion protein was digested with enterokinase. The recombinant toxin was cleaved
at -2 position, resulting in rPnTx2-6 with a N-terminal
extension of two amino acid residues (Ala2–Met1) (Fig
1B). Enterokinase-treated samples were applied to
a reverse phase Sephasil Peptide C18 5 ST4.6/250 column
(Fig. 4A). Mass spectrometric analysis, of the rPnTx2-6,
confirmed the presence of the two additional amino acid
residues when compared to native toxin (Fig. 4B).
The observed masses by mass spectrometry analyses for
the native and recombinant toxins were 5290.03 Da and
5486.29 Da, respectively. They were in accordance with
those calculated for the toxin native 5288.11 Da and
5490.39 Da for the recombinant toxin (data not shown).
Mass spectrometry analysis showed that all the Cys residues
present in rPnTx2-6 were found to be oxidized (Fig. 4B).
3.3. Recombinant PnTx2-6 potentiates rat erectile function
after subcutaneous injection
The effect of rPnTx2-6 (12 mg/kg s.c.) on ganglionic
stimulation induced elevation of ICP/MAP was evaluated in
normotensive control rats. Based on preliminary tests with
the native toxin PnTx2-6, by using a range of doses (3–48 mg/
kg), the best effect was that verified with 12 mg/kg, s.c.
(Nunes et al., 2008). In light of these data, all in vivo experiments were performed using this dose. The voltage–
response curves (0.5–3.0 V, 12 Hz, 0.1 ms, 30 s each step)
were performed before (control) and 15 min after injection
of the recombinant toxin. The erectile response, represented
as the ICP/MAP ratio, was significantly potentiated after
subcutaneous injection of rPnTx2-6 (Fig. 5).
4. Discussion
Penile erection is a complex neurovascular process
involving relaxation of the cavernosal smooth muscle
combined with increased arterial flow into the penis and
restricted venous outflow from the organ. The nitric oxide
pathway is of critical importance in the physiologic induction and maintenance of erection (Andersson, 2001; Burnett
et al., 1992; Drewes et al., 2003; Ignarro et al., 1990).
It is well known that following accidents caused by
venomous animals such as scorpions (Teixeira et al., 2004)
and spiders (Cordeiro et al., 1992), victims show to suffer
from priapism. These venoms have been postulated as
potential source of drug models to treat erectile dysfunction
(Nunes et al., 2008, 2009, in press). The crude venom of
P. nigriventer spider induced cavernosal relaxation (LopesMartins et al., 1994) and two a-toxins, PnTx2-6 and PnTx25, isolated from this venom were able to cause penile
erection (Andrade et al., 2008; Nunes et al., 2008, Yonamine
et al., 2004). Nunes et al. (2008, 2009); Villanova et al. (2009)
suggested that the PnTx2-6 toxin is a potential tool for
developing new therapeutics for treating ED and that
priapism induced by this toxin is a consequence of a highly
specific interference with the NO pathway.
Pharmacological studies need to be carried out for
a better understanding on how such toxins act, their
possible secondary sites, collateral effects, which frequently
F.S. Torres et al. / Toxicon 56 (2010) 1172–1180
1177
Fig. 4. (A) Analytical reverse phase HPLC purification of rPnTx2-6 after digested with enterokinase enzyme. The analyses were performed on a column of Sephasil
Peptide C18 5m ST 4.6/250; the samples were applied in 0.1% trifluoroacetc acid and the column eluted with gradient of acetonitrile in 0.1% trifluoroacetc acid.
Fractions of 1 mL were collected at a flow rate of 1 mL/min. (B) Mass spectrometry analyses of rPnTx2-6. The molecular mass of rPnTx2-6 was further determined
by matrix-assisted laser desorption ionization-time-of-fight MS.
result in toxicity, among others. These studies require
substantial quantity of toxins that are usually poorly represented in venoms. The expression of these molecules is
a convenient alternative to obtain the necessary material.
Purification of minor toxin constituents or closely related
molecular species, whose pharmacological properties can
be very distinct, is not an easy task. Protein engineering
techniques, including cloning and expression of gene coding
for toxins, can overcome these problems, and be an
exploratory tool for analyses of protein structure/function
relationships. However, purifying recombinant proteins,
while maintaining their intact native conformation, is
a major hurdle for using this strategy. It is known that
expression level can be improved by fusing not only bacterial gene, but also bacteriophage, mammalian and synthetic
genes. To date, various effective affinity and solubility tags
such as GST, His6-tag, FLAG-tag, and MBP-tag have been
developed (Arnau et al., 2006; Hunt, 2005; Wang, 2005).
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F.S. Torres et al. / Toxicon 56 (2010) 1172–1180
Fig. 5. Penile erection induced by ganglionic stimulation in normotensive
control rats after subcutaneous injection of rPnTx2-6. Ganglionic stimulation
(0.5–3.0 V) induces an increase in ICP/MAP ratio, which was significantly
potentiated by subcutaneous (n ¼ 3) injection of rPnTx2-6 (12 mg/ Kg).
**P < 0.01 (two-way ANOVA followed by Bonferroni test), ***P < 0.001 (twoway ANOVA followed by Bonferroni test).
Here, Tioredoxin (Trx) and His6-tag were fused to the
target toxin PnTx2-6 as affinity and solubility tags. The
PnTx2-6-Trx fusion protein was cloned and expressed in
the E. coli cytoplasm. Subsequently, it was cleaved and the
recombinant toxin purified, as described above. Other study
has also used the pET32 expression system to express and
purify P. nigriventer toxin and the recombinant PnTx1
neurotoxin has been expressed as a soluble and active form
(Diniz et al., 2006). These recombinant toxins were similarly
active in a dose-dependent manner, as their native equivalents, suggesting that both toxins were correctly folded
maintaining a biologically active conformation. Such activities were evaluated throughout biological assays as toxicity
symptoms (for both toxins), binding affinity (to PnTx1) and
the property to potentiate the erectile function, as observed
for PnTx2-6. PnTx2-6 possesses 10 Cys residues, having
clear implications regarding the toxin structure and, ultimately, its function. Disulphide bonds are rarely found in
cytoplasmic proteins (Hwang et al., 1995). The absence of
protein with stable disulphide bonds and the presence of
such bonds in exported proteins are generally attributed to
different reducing environments of subcellular compartments (Gilbert, 1998; Hwang et al., 1995). Solubility may be
manipulated by choosing the vector. Vectors designed to
produce a thioredoxin fusion protein can improve the yield
of soluble products (Derman et al., 1993; La Vallie et al.,
2000; Riggs et al., 2001). The above considerations may
explain the expression of this toxin with so many cysteine
residues in the cytoplasm of E. coli.
The vast majority of spider toxins identified to date have
been characterized as cysteine-rich polypeptides, and they
seem to rely on a common structural scaffold, the inhibitor
cystine knot motif (ICK). These are highly constrained
structures, due to their high density of disulphide bonds,
and they are known to occur in a wide variety of peptides
and protein toxins from plants and animals (Craik et al.,
2001). The disulfide-rich nature of PnTx2-6 and related
peptides has clear implications for the structure and function of these toxins. Small modifications during the
formation of disulphide bridges can alter the bioactive
three-dimensional structure of the toxins that carry this
kind of structural motif (Escoubas et al., 2000). On the other
hand, the excellent stability of the molecules containing
this structural motif and the great variability of sequences
that can be modified and accommodate in their framework,
combined with their bioactivity, can make them attractive
models for drug design purposes.
Erection induced by rPnTx2-6 could be observed 15 min
after a subcutaneous injection. Probably, this is the necessary time span for the toxin to reach its target. Recent
results showed that PnTx2-6 labeled with technetium is
found in cavernosal tissue, among others (Nunes, 2008;
Nunes et al., in press). This observation gives support to
a possible existence of target sites for this molecule in the
cavernosal tissue. Our results, concerning the corpus cavenosum experiments, demonstrated that rPnTx2-6 can
induce erection in a similar fashion as the native toxin (see
Nunes et al., 2008). Additional studies are required to reveal
the precise mechanism by which rPnTx2-6 causes penile
erection. We are currently engaged in studies involving
site-directed mutagenesis coupled with expression of DNA
encoding this molecule in order to elucidate this structure–
function relationship. A series of short-term studies
involving expression of DNA coding for rPnTx2-6 mutants
are planned to try to define the key amino acid residues
implicated in toxin interaction with receptors on sodium
channels, and its relationship with erectile response.
Acknowledgments
This work was partially supported by Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais
(FAPEMIG), Instituto Nacional de Ciência e Tecnologia em
Toxinas (INCTTOX-FAPESP) and Rede Proteoma Nacional
(MCT/FUNDEP). F.S. Torres and L.F. Lanza have been granted
research scholarships from CNPq, C.N. Silva, from CAPESINCTTOX-FAPESP, and A.V. Santos, M.E. De Lima and A.M.C.
Pimenta from CNPq. M.R.V. Diniz has been fellowship
recipient from FAPEMIG. The authors are grateful to Mrs.
Ana Luiza Bittencourt Paiva, Ana do Carmo Valentim, Raoni
Almeida de Souza and Júlio César dos Reis for their valuable
technical assistance. Authors also thank Dr. Kenia Pedrosa
Nunes for support.
Ethical statement
All experimental protocols were approved by the Ethics
Committee of the Federal University of Minas Gerais
(Process no. CETEA 22/1996 and 102/2009). No pain was
inflicted to the animals.
The nucleotide(s) sequence of cDNA PnTx2-6 was
deposited with DDBJ/EMBL/GenBank. RefSeq accession
number AY054746, and reported previously (Matavel
et al., 2002).
Conflict of interest
The authors declare that they have no conflicts of interest.
F.S. Torres et al. / Toxicon 56 (2010) 1172–1180
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