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

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

partement d’ Inge
´
nierie et d’Etudes
des Prote
´
ines, CEA, Saclay, 91191 Gif-sur-Yvette Cedex, France.
E-mail: [email protected]
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 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

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

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

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

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

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

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-

. 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

)
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

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

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

[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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