Tài liệu Báo cáo khoa học: A synthetic weak neurotoxin binds with low affinity to Torpedo and chicken a7 nicotinic acetylcholine receptors docx

A synthetic weak neurotoxin binds with low affinity to
Torpedo
and chicken a7 nicotinic acetylcholine receptors
Siew Lay Poh
1,2
, Gilles Mourier
1
, Robert Thai
1
, Arunmozhiarasi Armugam
2
, Jordi Molgo
´
3
, Denis Servent
1
,
Kandiah Jeyaseelan
2
and Andre
´
Me
´
nez
1
1
CEA, Saclay, Gif-sur-Yvette, France;
2
National University of Singapore, Singapore;
3
UPR, CNRS, Gif-sur-Yvette, France

Weak neurotoxins from snake venom are small proteins
with five disulfide bonds, which have been shown to be poor
binders of nicotinic acetylcholine receptors. We report on the
cloning and sequencing of four cDNAs encoding weak
neurotoxins from Naja sputatrix venom glands. The protein
encoded by one of them, Wntx-5, has been synthesized by
solid-phase synthesis and characterized. The physicochemi-
cal properties of the synthetic toxin (sWntx-5) agree with
those anticipated for the natural toxin. We show that this
toxin interacts with relatively low affinity (K
d
¼ 180 n
M
)
with the muscular-type acetylcholine receptor of the electric
organ of T. marmorata, and with an even weaker affinity
(90 l
M
) with the neuronal a7 receptor of chicken. Electro-
physiological recordings using isolated mouse hemidia-
phragm and frog cutaneous pectoris nerve–muscle
preparations revealed no blocking activity of sWntx-5 at l
M
concentrations. Our data confirm previous observations that
natural weak neurotoxins from cobras have poor affinity for
nicotinic acetylcholine receptors.
Keywords: snake neurotoxins; nicotinic acetylcholine
receptors.
During the past three decades, the most ÔobviousÕ venom
toxins have been uncovered either because they are present

in large amounts and/or because they have been directly
associated with the search for an important target. At
present, two additional approaches may be considered to
discover new toxin functions. One of them is a proteomic-
type approach, which aims at isolating all components of
the ÔtoxinomeÕ [1,2]. The second approach involves investi-
gation of the vast number of venom components that have
already been isolated, and sometimes chemically character-
ized, but whose biological activity still remains mysterious.
These functionally unknown components are often classi-
fied as miscellaneous types of toxins, even though they
usually belong to well-identified structural families [3]. This
is the case of the so-called Ôweak neurotoxinsÕ (Wntxs) found
in elapid snakes and isolated for the first time 26 years ago
from the venom of Naja melanoleuca [4]. Since then, more
such toxins have been isolated [5–19].
The Wntxs possess 62–68 amino acids and belong to the
structural family of Ôthree-fingeredÕ folded toxins, which
includes the cardiotoxins, muscarinic toxins, acetylcholin-
esterase inhibitors and the a-neurotoxins that block mus-
cular and/or neuronal nicotinic acetylcholine receptors
(AChRs) [20–22]. The fold adopted by all these toxins is
characterized by three adjacent loops rich in b-pleated sheet,
tethered by four conserved disulphides. A fifth loop is
sometimes observed in the second loop of the a/j-neuro-
toxins and j-neurotoxins [22], where it specifically contri-
butes to the binding of the toxins to the neuronal AChR
[23–26]. Wntxs also possess a fifth disulfide bond, but this is
located in the first loop [16,27,28].
Using Wntxs isolated from venom, it was shown that

these molecules interact with AChRs but with low affinities
[10,29]. Many efforts have been made to obtain pure Wntxs.
However, it cannot be completely ruled out that their low
activity may be due to the presence of minor but highly
potent contaminants, as was previously observed in the case
of j-bungarotoxin [30]. We identified four cDNAs enco-
ding Wntxs in venom glands of the cobra N. sputatrix
(previously known as Naja naja sputatrix [31]) and selec-
ted one of them. Then, we synthesized the correspond-
ing Wntx (Wntx-5) by chemical means, characterized its
physicochemical properties and investigated its biological
properties. We show that sWntx-5 is a weak binder of
muscular-type AChR from Torpedo marmorata’s electric
organ and an even weaker binder of the a7 neuronal-type
receptor from chicken. Our data generally agree with a
report published recently [29]. Moreover, the low AChR
binding activity of Wntx-5 can be accounted for by the
presence of a few residues that are also found in potent
three-fingered snake neurotoxins [22,32,33].
MATERIALS AND METHODS
Materials
Bacterial strains used, JM109 [34] and EpicureanÒ coli
SUREÒ cells, were from Stratagene (USA). Oligonucleo-
tides were synthesized at the National University of Singa-
pore. Molecular biology reagents were from Amersham
International Inc. (UK), Promega, New England Biolabs,
Correspondence to A. Me
´
nez, De
´

partement d’ Inge
´
nierie et d’Etudes
des Prote
´
ines, CEA, Saclay, 91191 Gif-sur-Yvette Cedex, France.
E-mail:
Abbreviations: Wntx, weak neurotoxin; AchR, nicotinic
acetylcholine receptor; TCEP, tris(2-carboxyethyl)-phosphine
hydrochloride; Bgtx, bungarotoxin; Ea, erabutoxin a.
Note: The cDNA sequences reported in this paper have the
GenBank accession numbers AF026891, AF026892, AF098923 and
AF098923.
(Received 18 February 2002, revised 17 June 2002,
accepted 12 July 2002)
Eur. J. Biochem. 269, 4247–4256 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03113.x
Novagen or Perkin Elmer (USA). Protected amino acid
derivatives, resins, dicyclohexylcarbodiimide and N-hydro-
xybenzotriazole were from Nova-Biochem (Meudon,
France). Piperidine, N-methylpyrrolidone, dichloro-
methane, methanol, trifluroacetic acid and ter-butyl-
methyloxide were from SDS (Peypin, France). TCEP [tris
(2-carboxyethyl)-phosphine hydrochloride] was from Pierce
(Rockford, Illinois, USA, or Saint-Quentin-Fallavier,
France). Oxidized and reduced glutathione (GSH and GSSH
respectively) were from Sigma (St Louis, MO). Automated
chain assembly was performed on a standard Applied
Biosystems 431 peptide synthesiser. cDNA of the chimeric
a7-V201–5HT
3

was kindly provided by J. P. Changeux
(Pasteur Institute, Paris, France.) and
125
I-labelled bungaro-
toxin (210–250 CiÆmmol
)1
) was from Amersham.
RT-PCR and subcloning
Total RNA prepared from the venom glands of N. sputatrix
[35] was used in RT-PCR. Reverse transcription reactions
were performed with 3 lgofRNAinatotalreaction
volume of 10 lLof1·RT buffer (100 m
M
Tris/HCl,
pH 8.4; 250 m
M
KCl; 12.5 m
M
MgCl
2
and 0.5 mgÆmL
)1
BSA) containing 10 U of MuMLV reverse transcriptase,
0.5 m
M
of dNTP and 40 ng of antisense primer X191;
5¢-gCggCggAATTCTTTTTTTTTTTTTTTTTT-3¢.The
reaction was carried out at 42 °C for 1 h, and terminated by
heating at 95 °C for 5 min. The entire mixture was used in a
50-lL polymerase chain reaction. The full-length cDNA

was cloned using two pairs of primers, which recognized
conserved regions of genes encoding Wntxs. The first pair
was X289 (5¢ TgTgCTACTTgCC CTggAA 3¢)andX191.
The second pair was X133 (5¢ TCC AgAAAAgATCgCAA
gATg 3¢) [35] and X300 (5¢ AgAgC CAAgCTTTTACT
ATCggTT 3¢).
The PCR products were fractionated using a low melting
point agarose gel (1.2%). The DNA band was cut out and
purified using freeze–thaw or centrifuged methods as
described previously [36,37]. The amplified products were
ligated to pT7 Blue(R) vector using procedures described by
the supplier (Novagen, USA). The ligated products were
transformed into E. coli, JM109 or SURE cells [34] by
electroporation and selected on LB-ampicillin (50 lgÆmL
)1
)
plates supplemented with 5-bromo-4-chloroindol-3-yl b-
D
-
galactoside (X-gal) and isopropyl thio-b-
D
-pyranoside
(IPTG). Putative recombinant plasmids were sequenced
on both strands with M13/pUC forward and reverse
universal primers using the dideoxy chain termination
method [38] on an automated DNA sequencer (Model 373,
Applied Biosystems, USA), using the manufacturer’s pro-
tocol and reagents.
Sequence analysis
Searches for homologous proteins on GenBank databases

(National Center for Biotechnology Information, USA)
were performed using the
BLAST
program, with deduced
amino acid sequences from the cDNAs [39].
Chemical synthesis of toxin
The peptide was assembled by a stepwise solid-phase method
with dicyclohexylcarbodiimide/hydroxybenzotriazole as
coupling reagents and N-methyl pyrrolidone as a solvent.
Fmoc-protected amino acids were used with t-butyl ester
(Glu, Asp), t-butyl ether (Ser, Thr, Tyr), trityl (Cys, Asn,
Gln), t-butylcarbonyl (Lys) and 2,2,5,7,8-pentamethyl-
chromane-6-sulfonyl (Arg) [40]. Wntx-5 was assembled
on Fmoc-Arg-(Pmc)-Wang resin (loading 0.52 mmolÆg
)1
)
[41]. The synthesis was carried out using a version derived
from the Applied Biosystem standard Fmoc 0.1 mmol
small-scale program [42]. At the end of the synthesis, the
peptide was cleaved from the resin and the protecting
groups were removed from the amino acid side chains
using a mixture of trifluoroacetic acid (90%), triisopropyl-
silan (5%) and deionized water (5%). After 2 h incubation
at room temperature with constant mixing, the mixture
was filtered into cold t-butyl methyloxide (peroxide-free)
and centrifuged at
2
960 g for 30 min. The peptide precipi-
tates were washed three times and dried, dissolved in 10%
acetic acid and lyophilized. The synthetic toxin was

reduced with molar excess of TCEP under acidic condi-
tions and purified by RP-HPLC using a Vydac C18
column (250 · 10 mm) with a gradient of 40–60% of 60%
acetonitrile mixed with 0.1% trifluoroacetic acid in water.
The flow rate was 3 mLÆmin
)1
and the detection was
monitored at 214 nm. Peptide purity was assessed using an
analytical Vydac C18 column (250 · 0.46 mm) using the
same elution conditions.
Disulfide bond formation and protein purification
The reduced synthetic peptide was oxidized in a refolding
buffer (0.1
M
sodium acetate, 1
M
4
GdnHCl and 1 m
M
EDTA, pH 7.8) containing GSH and GSSH in a molar
ratio of 10 : 1. The reduced synthetic peptide was dissolved
in 0.2 mL of 0.1% trifluoroacetic acid, and immediately
diluted into oxidation buffer to a final concentration of
0.05 mgÆmL
)1
. After incubation for up to 3 days at room
temperature, the peptide was acidified with 30% trifluoro-
acetic acid and purified on a Vydac C18 semipreparative
column using the gradient employed to purify the reduced
toxin form. The protein concentration was determined by

means of spectrometry.
Mass analysis, amino acid composition and sequence
determination
The masses of both the reduced and refolded peptides were
determined using an ion spray mass spectra system,
Micromass Platform II (Micromass, Altrincham, UK).
For amino acid composition analysis, the peptide was
hydrolysed in a sealed vial heated at 120 °C in the presence
of 6
M
HCl for 16 h. The hydrolysate was analysed using an
Applied Biosystem Model 130A automatic analyser
equipped with an online 420A derivatiser for the conversion
of the free amino acid into phenyl thiocarbamoyl deriva-
tives. The amino acid sequence of the peptide was
determined using an applied Biosystems 477A protein
sequencer.
Circular dichroism
CD spectra were recorded on a CD6 Jobin Yvon dichro-
graph (Roussel Uclaf, France). Routinely, measurements
were performed at 20 °C in 0.1 cm pathlength quartz cells
4248 S. L. Poh et al.(Eur. J. Biochem. 269) Ó FEBS 2002
(Hellma, Germany) under continuous nitrogen gas flow
with a peptide concentration around 4.5 · 10
)5
M
in
deionized water. Spectra were recorded in the 186–260 nm
wavelength range. Each spectrum represents the average of
four spectra.

Binding to acetylcholine receptors
Binding assays were performed using
125
I-labelled a-bung-
arotoxin (a-Bgtx, 210–250 CiÆmmol
)1
, Amersham) as com-
petitor. The AChR-rich membranes from the electric organ
of T. marmorata were prepared as described previously [43].
The chimeric a7 receptors were obtained by expressing the
chimeric cDNA (a7–5HT
3
) in HEK cells [23]. In compet-
itive experiments with AChR from T. marmorata
we measured, at equilibrium, the effect of toxins on the
125
I-labelled a-Bgtx binding. Varying amounts of toxins
were incubated with 3 n
M
of active sites of receptors
and 5 n
M
of
125
I-labelled a-Bgtx for at least 4 h. With a7
receptors, the toxin was incubated at different concentra-
tions with 250 lL of cells suspended in NaCl/P
i
for at least
30 min. Cell suspensions were filtered 6 min after addition

of 5 n
M
125
I-labelled a-Bgtx. The curves were fitted with the
empirical Hill equation. K
d
values for Torpedo AChR were
calculated according to Cheng and Prusoff [44]. For a7
receptors, protection constants (K
p
) calculated by fitting the
competition data using the Hill equation, correspond to K
d
values [45].
Electrophysiological recordings
Electrophysiological recordings were carried out on both
isolated mouse hemidiaphragm preparations (removed
from adult female Swiss–Webster mice killed by dislocation
of the cervical vertebrae followed by immediate exsanguin-
ation), and from isolated cutaneous pectoris nerve-muscle
preparations removed from double-pithed male frogs
(Rana temporaria), as described previously [46]. Briefly,
the motor nerve was stimulated with a suction microelec-
trode adapted to the diameter of the nerve, with pulses of
0.05–0.1 ms duration and supra-maximal voltage (typically
3–8 V) supplied by a S-44 stimulator (Glass Instruments,
West Warwick, USA) linked to a stimulus isolation unit.
Membrane potentials and synaptic potentials were recor-
ded, from endplate regions with intracellular microelec-
trodes filled with 3

M
KCl (8–18 MX resistance), using
conventional techniques and an Axoclamp-2A system
(Axon Instruments, Union City, CA, USA). Recordings
were made continuously from the same endplate before and
after application of toxins tested. Electrical signals after
amplification were collected and digitized, at a sampling rate
of 25 kHz, with the aid of a computer equipped with an
analogue-to-digital interface board (DT2821, Data Trans-
lation, Marlboro, USA). Endplate potentials and miniature
endplate potentials were analysed individually for amplitude
and time course.
RESULTS
Cloning and sequencing of cDNAs
Thirty-three putative clones were obtained from a cDNA
library prepared from venom glands of N. sputatrix,usinga
conventional RT-PCR-based approach. The ORFs of these
cDNAs encode a set of four novel proteins that were named
Wntx-5, 6, 8 and 9 (Fig. 1). The putative leader sequences
contain 21 amino residues and are typical of secreted
proteins [47]. Only the isoform Wntx-5 showed variation in
its signal peptide region due to a single first base substitution
(Fig. 1). The calculated theoretical molecular masses of
these basic Wntxs were 7504.5 Da, 7509.1 Da, 7508.1 Da
and 7535 Da. The four derived amino acid sequences
(Seq.1–4 in Fig. 2A) show high similarity. Wntx-6 possesses
an aspartic acid at position 21 whilst other sequences have
an asparagine, Wntx-5 has a lysine at position 29 whereas
other sequences have a methionine, and Wntx-9 has an
asparagine at position 65 whilst other sequences have a

serine. They all exhibit high sequence similarity with other
Wntxs (Fig. 2A) but they are clearly more similar to those
from cobras than to those found in kraits, mamba and coral
snake venom [4–19].
Comparative analysis of Wntx sequences
Figure 2A shows a comparison of amino acid sequences of
26 putative Wntxs including those derived from cDNAs
isolated from N. sputatrix. The high degree of identity of
the sequences isolated from cobras is striking, both in
terms of length and amino acid distribution. Those from
kraits, mambas and coral snakes display more deviations
and a smaller number of conserved residues (see for
example the three Wntxs at the bottom of the group).
Thus, 25 positions (indicated by open boxes in Fig. 2A)
are strictly conserved among cobra Wntxs. These include
10 half-cystines and 15 additional residues. Using Fig. 2A
numbering, these additional residues are Leu1, Pro7, Glu8,
Gly22, Glu23, Phe27, Lys28, Tyr43, Gly46, Ala48, Thr50,
Pro52, Thr66, Asp67 and Asn70. Sixteen additional
positions of cobra Wntxs are occupied by highly conserved
residues ( 80%, shaded boxes in Fig. 2). These residues
include Thr2, Leu4, Phe/Tyr10, Asn21, Lys24, Lys/Arg29,
Arg33, Arg42, Arg45, Lys55, Pro56, Arg/Lys57, Asp/
Glu58, Val61, Ser65 and Lys/Arg68. Therefore, cobras
Wntxs form a highly homogeneous group of proteins,
which share at least 56% sequence similarity (excluding
their disulfide bonds). We noted that Wntx-5 has a
particularly high degree (62–97%) of identity with other
cobra Wntx sequences, making it a potential prototype of
Wntxs from cobras. Therefore, we decided to synthesize

Wntx-5 for the investigation of biological properties of
cobra Wntxs.
Synthesis and purification of synthetic Wntx-5 (sWntx-5)
Wntx-5 was synthesized chemically using a modified version
of the Fmoc/small-scale (0.1 mmol) programme developed
by Applied Biosystems [42] using a preloaded Arg-(Pmc)-
Wang resin as solid support [41]. After treatment with the
trifluoroacetic acid cleavage mixture and lyophilization, the
crude peptide was treated in acidic conditions with TCEP, a
reducing agent, and was purified by reverse-phase HPLC
on a C18 column. Figure 3A shows that the RP-HPLC
profile of the crude peptide displayed three major peaks (a, b
and c). Electrospray mass analyses revealed that peak a
was a truncated form of sWntx-5 terminated at Pro33
(3749.6 Da), peak b contained peptides ranging from
Ó FEBS 2002 Synthetic weak neurotoxin (Eur. J. Biochem. 269) 4249
7513.5 to 7530.5 Da and peak c contained a peptide with
the calculated mass of the reduced form of sWntx-5
(7514.5 Da). This fraction corresponded to approximately
17% of the total crude mixture. The purity of the reduced
sWntx-5 toxin was assessed on an analytical C18 column
(Fig. 3B). Reduced sWntx-5 was oxidized using a redox
buffer containing a mixture of GSH and GSSH in a
peptide : GSH : GSSH molar ratio of 1 : 10 : 1 at pH 7.8.
The resulting glutathione-mediated oxidation mixture was
acidified and submitted to RP-HPLC, revealing that the
oxidized sWntx-5 (Fig. 3) eluted as a major component
(yield ¼ 10% of the reduced form), approximately 10 min
before the reduced form (Fig. 3B). Amino acid composi-
tion, N-terminal amino acid sequencing up to 75% of the

total length of the protein and electrospray mass analyses
(mass ¼ 7504.4 Da, which is virtually identical to the
calculated value) confirmed the purity and identity of the
sWntx-5.
Circular dichroic spectrum of sWntx-5
As shown in Fig. 4, the far UV spectrum of the sWntx-5
displayed a positive band at 196 nm and a broad negative
band at 222 nm, together with a slight shoulder around
210 nm. This pattern is highly reminiscent of the presence
of b-structure in proteins [48,49]. This conclusion agrees
with the previous structural studies made on the other
weak neurotoxins bucandin [16,28] and WTX [27]. We
compared this spectrum with that previously monitored
for the natural homologue NNA2/NNAM2 that is present
in Taiwan cobra venom, and which differs in sequence
from that of sWntx-5 by only three substitutions [10].
Fig. 1. Nucleotide sequences of cDNA-encoding weak neurotoxins in Naja sputatrix. The 3¢ ends of primers used in RT-PCR are in bold and
underlined. The regions coding for the putative signal peptides (CDS) and neurotoxins are shown. The encoded amino acids are indicated in capital
letters below the second base of each codon. The nucleotides that vary among isoforms are indicated (+), the stop codon is shown (*) and the
variant residues are in bold.
4250 S. L. Poh et al.(Eur. J. Biochem. 269) Ó FEBS 2002
Fig. 2. Alignment of neurotoxin sequences. (A) Sequence alignment of snake weak neurotoxins. Sequences 1–18 are cobra weak neurotoxins [4–12], while sequences 19–25 are from kraits, coral snakes and
mambas [13–19]. Sequences 1–4 were determined in this study. All other sequences are taken from the databanks, GenBank, SWISS-PROT or TrEMBL. For simplicity, the accession numbers, species names
and references are shown. The names of cobra species are those from the original papers that described the toxin sequences. From a taxonomy viewpoint, however, some species names have changed [31]. The
numbering at the top includes all insertions. Residues that are strictly conserved and 80% conserved in the respective groups of weak neurotoxins are indicated with open and shaded boxes, respectively. (B)
Optimized sequence alignment of Ea and Wntx-5. The 11 functional residues previously identified for Ea are indicated (+). Those that have a homologous residue in Wntx-5 are in bold boxes, whereas those
specific to Ea are underlined.
Ó FEBS 2002 Synthetic weak neurotoxin (Eur. J. Biochem. 269) 4251
A similar strong negative band around 220 nm is observed
for both toxins. The slightly weaker band that is observed

with NNA2 can be explained by the presence of a positive
signal at 208 nm. This band might correspond to the
shoulder observed at 210 nm for sWntx-5. Nevertheless,
the common presence of a negative band of comparable
intensity around 220 nm strongly suggests that the level of
b-sheet content is comparable in both toxins. We also
compared the spectrum of sWntx-5 with the spectra of
toxin a from N. nigricollis [50,51] a short-chain neuro-
toxin, and a-cobratoxin [52], a long-chain neurotoxin,
which both possess highly similar three-fingered structures
[53,54]. The overall pattern displayed by these two
neurotoxins clearly agrees with the presence of b-sheet
structure, with a positive band around 196–199 nm and a
negative trough centred around 212–216 nm. The CD
spectrum displayed by sWntx-5 is globally comparable,
with some differences, however. In particular, its negative
band is centred at a somewhat longer wavelength.
However, this is not so surprising, since the minimum
wavelength associated the n-p* transition of a peptide
chromophore in b-sheet structure can be shifted to 223 nm
[49]. Therefore, our data indicate that sWntx-5 adopts an
overall structure rich in b-sheet.
Probing biological activity of sWntx-5
The ability of sWntx-5 to bind to muscular-type and a7
neuronal-type AChRs was estimated from competition
experiments using, respectively, T. mamorata and a chimer-
ical version of chicken a7)5HT
3
receptor expressed in HEK
cells [23], and

125
I-labelled a-Bgtx as a tracer. The deduced
K
d
with Torpedo receptor was estimated to be 180 n
M
(Fig. 5, Table 1). With the a7 neuronal receptor, the affinity
was much lower and we have not been able to complete the
competition curve, due to a lack of material (Fig. 5).
Nevertheless, from the available data we assumed that 90%
of the binding of
125
I-labelled a-bungarotoxin should be
inhibited by 10
)2
M
sWntx-5, and we calculated a theoret-
ical curve based on the limited number of available points.
Hence, we estimated that sWntx-5 should inhibit the
binding of
125
I-labelled a-Bgtx to a7–5HT
3
receptor with
a K
d
value of approximately 10
)4
M
(Table 1). All these

binding data not only indicate that sWntx-5 is a weak binder
of AChR from electric organ of T. marmorata but also that
it is an even weaker binder of the chicken a7 neuronal-type
AChR.
It was previously shown that l
M
concentration of the
weak neurotoxin NNA2 inhibits at least 50% of the ACh-
induced contraction of nerve-muscle preparations from frog
[10]. Since, Wntx-5 and NNA2 shows high sequence identity
(three amino acid residues different, Fig. 2A), we investi-
gated the ability of sWntx-5 to block neuromuscular
transmission in both isolated frog cutaneous pectoris
nerve-muscle and mouse hemidiaphragm preparations,
using electrophysiological techniques. In the frog nerve–
muscle preparation, sWntx-5 caused no blockage of neuro-
muscular activity at concentrations up to 9 l
M
. In contrast,
the control a-cobratoxin blocked both washed out and
Fig. 3. RP-HPLC of (A) crude peptide giving 3 major peaks (a, b and c)
representing the 3 major products present in the crude mixture, (B)
purified reduced sWntx-5 and (C) refolded sWntx-5 present in the oxi-
dation medium. A Vydac C18 column (0.46 · 25 cm) was used. Elution
was performed with a profile of 40% of a solution of
6
60% acetonitrile
and 0.1% trifluoroacetic acid in H
2
O for 15 min, followed by a gra-

dient of 40–60% in 40 min, at 1 minÆmL
)1
flow rate. Protein was
monitored at 214 nm.
Fig. 4. Far UV CD spectra of snake neurotoxins. The spectrum of
sWntx-5 was monitored between 190 nm and 250 nm, in the presence
of nitrogen. The cell path-length and temperature of measurement
were 0.05 mm and 20 °C, respectively. Previously described venom-
derived spectra of a short neurotoxin, toxin a [50], a long neurotoxin,
a-cobratoxin [58], and NNA2, another weak neurotoxin from cobra
venom [10], are also shown.
4252 S. L. Poh et al.(Eur. J. Biochem. 269) Ó FEBS 2002
nonwashed out preparations at 0.2 l
M
in 2 min. Phrenic
nerve stimulation of isolated mouse hemidiaphragms,
previously treated with formamide (to uncouple excita-
tion-contraction coupling), elicited action potentials at
junctional areas without contraction triggered by endplate
potentials. Addition of 6 l
M
sWntx-5 to the standard
solution did not block neuromuscular transmission, even
after 30-min incubation. In contrast, the potent a-cobra-
toxin used as a control, on both washed out and nonwashed
out preparations, blocked neuromuscular transmission by
60% at 0.6 l
M
and completely at 1.6 l
M

. Therefore, sWntx-
5atl
M
concentrations does not seem able to block muscle
AChRs from frogs and mice.
DISCUSSION
A weak neurotoxin is currently defined as a protein isolated
from elapid venom that possesses about 65 residues
including 10 cysteines, eight of which can be readily aligned
with those of the well-known three-fingered toxins
[21,22,55]. That Wntxs also adopt this fold has been
confirmed recently with the resolution of the X-ray and
NMR structures of the Wntx called bucandin and WTX
[16,27,28]. When we started this work, 22 amino acid
sequences of Wntxs were known and it was clear to us
that this family of proteins could be divided into two
categories. The first one includes the cobra Wntxs whereas
the second category involves mostly those from kraits,
mambas (Dendroaspis jamesoni) and coral snakes (Micru-
rus corallinus). We confirmed the homogeneous character of
the subgroup of cobra Wntxs by introducing four new
sequences (Wntx-5, Wntx-6, Wntx-7 and Wntx-9) derived
from cDNAs isolated from venom glands of Naja sputatrix.
This subgroup is highly homogenous, with few insertions or
deletions and about 56% of the residues other than the half-
cystines, that are strictly or highly conserved. In view of such
a high degree of sequence similarities, we anticipate that all
toxins from this subgroup may exert a highly similar
biological function. This may be in contrast to the Wntxs
from the second subgroup, which display many deviations

and few conserved residues (besides the conserved half-
cystines).
During the past few years, a number of studies have been
attempted to identify the biological function of Wntxs.
Recent reports have shown that Wntxs from cobra venom
are low-affinity blockers of muscular and a7 neuronal
AChRs [10–12,29,56]. However, these results were deduced
from experiments done with venom-derived toxins. There-
fore, despite many efforts to obtain highly purified toxins, it
Fig. 5. Inhibition of binding of
125
I-labelled a-bungarotoxin to (A)
nicotinic acetylcholine receptor from T. marmorata and (B) chick
chimeric a7 receptor (a7-V201–5HT
3
) expressed in HEK cells by
varying amounts of toxin a (N. nigricollis), a-cobratoxin (N. kaouthia)
and sWntx-5. The continuous lines correspond to theoretical dose–
responses fitted through the data points using the nonlinear Hill
equation.
Table 1. Summary of the effects of weak neurotoxins on various types of AchRs in competitive binding experiments. Data for sWntx-5 were from this
study, while those of WTX have been previously reported [12,29]. ND, not determined.
Ligands Types of AchRs K
d
(
M
)IC
50
(
M

)
Muscular-type AChR
sWntx-5 T. marmorata 1.8 · 10
)7
1.8 · 10
)5
WTX T. californica 9.0 · 10
)8
2.2 · 10
)6
a7-neuronal AChR
sWntx-5 Chick chimeric a7-V201–5HT3 (HEKcells) ± 9.0 · 10
)5a
± 9.0 · 10
)5a
WTX GST-Rat a7 (1–208) fusion protein ND 4.3 · 10
)6
a
Estimated K
d
due to lack of points at high concentrations of ligands.
Ó FEBS 2002 Synthetic weak neurotoxin (Eur. J. Biochem. 269) 4253
could not be totally excluded that these low activities may
have resulted from contamination by a potent neurotoxin.
For example, the poorly reproducible activity of venom-
derived j-bungarotoxin toward muscular AChRs, which
was contaminated by a potent a-neurotoxin [30]. We
therefore decided to produce an artificial Wntx and to
study its activity on AChRs. In this paper, we have
described the chemical synthesis of a cobra Wntx and the

activity of this synthetic toxin on muscular and a7AChRs.
We synthesized Wntx-5 because its amino acid sequence
shares between 62% and 97% identity with other toxins
from the cobra subgroup, and so it appeared to us as a
potential prototype of this subgroup.
Chemical synthesis of proteins of the size of Wntx-5 is
now feasible, even if they possess a high density of
disulfide bonds, as shown in a previous study with long
and short neurotoxins [42]. Similarly, Wntx-5 has been
synthesized successfully using an Fmoc-based chemical
approach and the resulting synthetic toxin, named sWntx-
5, was obtained with a final yield of approximately 10%
of the reduced form. Mass spectrometry and amino acid
analyses indicated that the oxidized peptide had the
expected chemical characteristics of the natural toxin.
Also, amino acid sequencing of the first 49 residues
confirmed that the sequence of sWntx-5 was identical to
that expected. Since no native toxin was available, it was
not possible to compare the chromatographic behaviour
of sWntx-5 with that of the wild-type toxin. However,
inspection of the far-UV CD spectrum of sWntx-5
recorded between 205 nm and 250 nm strongly confirms
that it adopts a structure rich in b-sheet. We have not
identified the pairings of the cysteines of sWntx-5.
However, we assumed that they correspond to the
expected ones because it has been shown repeatedly that
the presence of the conserved disulphides of all three-
fingered toxins is indispensable for their fold to be
acquired [22].
Wntxs isolated from cobra venom have been described

as poor blockers of muscular-type AChRs [10–12,29,56,].
Thus, using preparations of AChR from Torpedo califor-
nica, a weak neurotoxin from Naja kaouthia (WTX) was
found to inhibit binding of radioactive a-bungarotoxin
with apparent K
d
values around 90 n
M
[12,29]. In close
agreement with this observation, sWntx-5 inhibits binding
of radioactive a-bungarotoxin to AChRs from T. marmo-
rata,withaK
d
value of 180 n
M
. That these results agree so
well confirms the view that a Wntx from cobra venom can
bind with moderate affinity to muscular type AChRs, at
least in vitro. Though acting as a binder of muscular-type
AChR, the Wntx from N. kaouthia was nontoxic to
rodents, even when high doses (2 mgÆkg
)1
) were adminis-
tered by intravenous injection. Due to a lack of material,
we have not tested the toxic activity in vivo of sWntx-5.
Instead, we investigated its ability to block neuromuscular
transmission in both isolated mouse hemidiaphragm and
isolated frog cutaneous pectoris muscle, using electrophys-
iological techniques. We found that 6 l
M

of sWntx-5 failed
to block neuromuscular transmission in mouse phrenic
nerve hemidiaphragm muscle. Previously, it was reported
that NNA2, a weak neurotoxin from the Formosan cobra,
inhibits ACh-induced contraction of frog muscle prepara-
tions, with IC
50
concentrations around 1–4 l
M
[10]. In
contrast, 3–9 l
M
of sWntx-5 caused no effect on a
stimulated frog cutaneous pectoris nerve muscle toxin
preparation. The toxin also had no effect on the more
sensitive miniature endplate potentials. Therefore, although
sWntx-5 and NNA2 share a high degree of sequence
identity (Fig. 2), they behave differently in the frog
cutaneous pectoris nerve–muscle experiments. This situ-
ation could be due to one or more of the three mutations
that differentiate the two toxins, or to differences in the
experimental protocols, such as, for example, the use of
different frog species that may discriminate between
neurotoxins [60]. It has also been shown that the Wntx
from N. kaouthia is an antagonist of human and rat a7
AChRs [29]. In vitro binding experiments and electrophys-
iological assays showed that this toxin has a low affinity
(IC
50
) in the range of 4–15 l

M
for these receptors. On the
basis of competition binding experiments with a chimerical
version of chicken a7–5HT
3
receptor, we found that
sWntx-5 has an affinity (IC
50
)closeto90l
M
for this
receptor. This is 6–22 times lower than that observed for
WTX from N. kaouthia. Considering that the two toxins
display 11 residue differences and that the competition
systems used (human and rat on one hand, and chicken on
the other) are not identical in the two studies, the two
toxins appear to behave as comparable weak antagonists
of neuronal a7 receptors.
Do cobra Wntxs and the potent a-neurotoxins bind to
muscular AChRs using similar determinants? To address
this question, the sequence of sWntx-5 was optimally
aligned with that of erabutoxin a (Ea), a short chain and
potent neurotoxin from sea snake that possesses 11
functionally important residues [32,33] (Fig. 2B). Five of
these amino acids (shown in bold) are observed at
homologous positions in Wntx-5. These are Lys29
(homologous to Lys27 in Ea), Phe36 (Phe32), Arg39
(Arg33), Arg42 (Ile36), and Lys52 (Lys47). Note that
mutation of Ile36 into an Arg increases the affinity of Ea
for the muscular receptor by 7-fold [33] and that an

arginine is found in Wntx-5 at this location. Therefore, if
we assume that these common residues have a comparable
binding function in both toxins, sWntx-5 appears to lack
six of the 11 functional residues of Ea, which may explain
its low potency to muscular AChRs. In agreement with our
observation that sWntx-5 binds with a very low affinity to
the neuronal a7 receptor, we found only two residues
(Phe36 and Arg39) whose positions could be aligned with
those identified to be critical for this particular binding in
a-cobratoxin.
Another intriguing question concerns the significance of a
180-n
M
affinity for a toxin isolated from a venom, which
also possesses toxins acting on the same target with much
higher affinities (with K
d
s varying from n
M
to p
M
). It has
been shown that despite their low affinities, some weak
neurotoxins can be slow-dissociating proteins [17,56]. This
might also be the case for sWntx-5. What is the role of the
disulfide bond that is uniquely present in the first loop of the
weak neurotoxins? Previously, it was demonstrated that
the additional disulfide that is present in the second loop of
the long neurotoxins is specifically involved in the capacity
of these toxins to interact with a7 neuronal receptors

[23,24,26,57]. We suggest therefore that the disulfide bond
that is found in the first loop of Wntxs may be associated
with a binding to a specific tissue target, which however,
remains to be identified.
4254 S. L. Poh et al.(Eur. J. Biochem. 269) Ó FEBS 2002
ACKNOWLEDGEMENTS
This work was supported by research grants from CEA and National
University of Singapore (RP 960324). S. L. Poh is a research scholar
of NUS and received scholarships from NUS (Singapore), ARET
(France) and EGIDE (France).
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