A synthetic weak neurotoxin binds with low affinity to Torpedoand chicken a7 nicotinic acetylcholine receptors 1 CEA, Saclay, Gif-sur-Yvette, France;2National University of Singapore, Si
Trang 1A synthetic weak neurotoxin binds with low affinity to Torpedo
and chicken a7 nicotinic acetylcholine receptors
1
CEA, Saclay, Gif-sur-Yvette, France;2National University of Singapore, Singapore;3UPR, 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
with the muscular-type acetylcholine receptor of the electric
organ of T marmorata, and with an even weaker affinity
Electro-physiological recordings using isolated mouse
concentrations Our data confirm previous observations that natural weak neurotoxins from cobras have poor affinity for nicotinic acetylcholine receptors
receptors
During the past three decades, the most obvious 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 toxinome [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 weak neurotoxins (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 three-fingered 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-neuroa/j-neuro-toxins [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]
M A T E R I A L S A N D M E T H O D S Materials
Bacterial strains used, JM109 [34] and Epicurean coli SURE 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: andre.menez@cea.fr
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)
Trang 2Novagen or Perkin Elmer (USA) Protected amino acid
derivatives, resins, dicyclohexylcarbodiimide and
N-hydro-xybenzotriazole were from Nova-Biochem (Meudon,
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
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 lg of RNA in a total reaction
BSA) containing 10 U of MuMLV reverse transcriptase,
5¢-gCggCggAATTCTTTTTTTTTTTTTTTTTT-3¢ The
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¢) and X191
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
(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)
amino acid sequences from the cDNAs [39]
Chemical synthesis of toxin
The peptide was assembled by a stepwise solid-phase method
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
[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
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
acetonitrile mixed with 0.1% trifluoroacetic acid in water
monitored at 214 nm Peptide purity was assessed using an
same elution conditions
Disulfide bond formation and protein purification The reduced synthetic peptide was oxidized in a refolding
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
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
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 CDspectra were recorded on a CD6 Jobin Yvon dichro-graph (Roussel Uclaf, France) Routinely, measurements
Trang 3(Hellma, Germany) under continuous nitrogen gas flow
deionized water Spectra were recorded in the 186–260 nm
wavelength range Each spectrum represents the average of
four spectra
Binding to acetylcholine receptors
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
we measured, at equilibrium, the effect of toxins on the
receptors, the toxin was incubated at different
30 min Cell suspensions were filtered 6 min after addition
calculated according to Cheng and Prusoff [44] For a7
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
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
R E S U L T S
Cloning and sequencing of cDNAs
Thirty-three putative clones were obtained from a cDNA
library prepared from venom glands of N sputatrix, using a
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
Trang 47513.5 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
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
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.
Trang 5Fig
Trang 6A 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 neuroneuro-toxin,
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
(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%
theoret-ical curve based on the limited number of available points Hence, we estimated that sWntx-5 should inhibit the
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
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
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
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)1flow 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.
Trang 7nonwashed out preparations at 0.2 lM 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
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
AChRs from frogs and mice
D I S C U S S I O N
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 125I-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 ND4.3 · 10)6
a
Estimated K due to lack of points at high concentrations of ligands.
Trang 8could 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 a7 AChRs
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 CDspectrum 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
agreement with this observation, sWntx-5 inhibits binding
of radioactive a-bungarotoxin to AChRs from T
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
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
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
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 mutsitu-ations 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
basis of competition binding experiments with a chimerical
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
also possesses toxins acting on the same target with much
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
Trang 9A C K N O W L E D G E M E N T S
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).
R E F E R E N C E S
1 Sto¨cklin, R., Mebs, D., Boulain, J.C., Panchaud, P.A., Virelizier,
H & Gillard–Factor, C (2000) Identification of snake species by
toxin mass fingerprinting of their venoms Methods Mol Biol.
(2000) 146, 317–335.
2 Sherman, N., Shannon, J., Gallagher, P., Dragulev, B., Kamiguti,
A.S., Theakston, R.D.G., Bland, L & Fox, J.W (2000) Discovery
Science in toxinology: the genomic/proteomic interface in venom
research 13th World Congress on Animal, Plant and Microbial
Toxins, Paris.
3 Dufton, M.J & Hider, R.C (1983) Conformational properties
of the neurotoxins and cytotoxins isolated from Elapid snake
venoms CRC Crit Rev Biochem 14, 113–171.
4 Carlsson, F.H.H (1975) Snake venom toxins: the primary
struc-ture of protein S 4 C 11 A neurotoxin homologue from the venom of
forest cobra (Naja melanoleuca) Biochim Biophys Acta 400, 310–
321.
5 Joubert, F.J (1975) The purification and amino acid sequence of
toxin CM-13b from Naja haje annulifera (Egyptian cobra) venom.
Hoppe Seylers Z Physiol Chem 356, 1901–1908.
6 Joubert, F.J & Talijaard, N (1978) Naja haje haje (Egyptian
cobra) venom Some properties and the complete primary
struc-ture of three toxins (CM-2, CM-11 and CM-12) Eur J Biochem.
90, 359–367.
7 Joubert, F.J & Talijaard, N (1980) Snake venoms The amino
acid sequences of two Melanoleuca-type toxins Hoppe Seylers Z.
Physiol Chem 361, 425–436.
8 Shafqat, J., Siddiqi, A.R., Zaidi, Z.H & Jornvall, H (1991)
Extensive multiplicity of the miscellaneous type of neurotoxins
from the venom of the cobra Naja naja naja and structural
char-acterization of major components FEBS Lett 284, 70–72.
9 Qian, Y.C., Fan, C.Y., Gong, Y & Yang, S.-L (1998) cDNA
sequence analysis and expression of four long neurotoxin
homo-logues from Naja naja atra Biochim Biophys Acta 1443, 233–238.
10 Chang, L., Lin, S., Wang, J., Hu, W.P., Wu, B & Huang, H.
(2000) Structure-function studies on Taiwan cobra long
neuro-toxin homolog Biochim Biophys Acta 1480, 293–301.
11 Lin, S.R., Huang, H.B., Wu, B.N & Chang, L.S (1998)
Char-acterization and cloning of long neurotoxin homolog from Naja
naja atra Biochem Mol Biol Int 46, 1211–1217.
12 Utkin, Y.N., Kukhtina, V.V., Maslennikov, I.V., Eletsky, A.V.,
Starkov, V.G., Weise, C., Franke, P., Hucho, F & Tsetlin, V.I.
(2001) First tryptophan-containing weak neurotoxin from cobra
venom Toxicon 39, 921–927.
13 Qian, Y.-C., Fan, C.-Y., Gong, Y & Yang, S.-L (1998) cDNA
cloning and sequence analysis of six neurotoxin-like proteins from
Chinese continental banded krait Biochem Mol Biol Int 46,
821–828.
14 Chang, L.-S & Lin, J (1997) cDNA sequence of a novel
neuro-toxin homolog from Taiwan banded krait Biochem Mol Biol.
Int 43, 347–354.
15 Aird, S.D., Womble, G.C., Yates, J.R & Griffin, P.R (1999)
Pri-mary structure of c-bungarotoxin, a new postsynaptic neurotoxin
from venom of Bungarus multicinctus Toxicon 37, 609–625.
16 Khun, P., D eacon, A.M., Comoso, S., Rajaseger, G., Kini, R.M.,
Uson, I & Kolatkar, P.R (2000) The atomic resolution structure
of bucandin, a novel toxin isolated from the Malayan krait,
determined by direct methods Acta Crystallogr D Biol
Crystal-logr 56, 1401–1407.
17 Nirthanan, S., Charpantier, E., Gopalakrishnakone, P., Gwee, M.C., Khoo, H.E., Cheah, L.S., Bertrand, D & Kini, R.M (2002) Candoxin, a novel toxin from Bungarus candidus is a reversible antagonist of muscle (abcd) but a poorly reversible antagonist of neuronal alpha 7 nicotinic acetylcholine receptors J Biol Chem.
277, 17811–17820.
18 Ho, P.L., Soares, M.B., Yamane, T & Raw, I (1995) Reverse biology applied to Micrurus corallinus, a South American coral snake J Toxicol Toxin Rev 14 (3), 309–326.
19 Joubert, F.J & Taljaard, N (1979) Complete primary structure of toxin S 6 C 4 from Dendroaspis jamesoni kaimosae (Jameson’s mamba) S Afr J Chem 32, 151–155.
20 Me´nez, A (1993) Les structures des toxins des animaux venimeux Pour Sci 190, 34–40.
21 Ohno, M., Me´nez, R., Ogawa, T., D anse, J.M., Shimohigashi, Y., Fromen, C., Ducancel, F., Zinn-Justin, S., Du Le, M.H., Boulain, J.-C., Tamiya, T & Me´nez, A (1998) Molecular evolution of snake toxins: is the functional diversity of snake toxins associated with a mechanism of accelerated evolution? Prog Nucleic Acid Res Mol Biol 59, 307–364.
22 Servent, D & Me´nez, A (2001) Snake toxins that interact with nicotinic acetylcholine receptors In Neurotoxicological Handbook Vol I (Massaro, E.J., ed.), Humana Press, Totowa, NJ.
23 Servent, D., Winckler-Dietrich, V., Hu, H.Y., Kessler, P., Drevet, P., Bertrand, D & Me´nez, A (1997) Only snake curaremimetic toxins with a fifth disulfide bond have high affinity for the neu-ronal a 7 nicotinic receptor J Biol Chem 272, 24279–24286.
24 Servent D., Thanh, H.L., Antil, S., Bertrand, D., Corringer, P.J., Changeux, J.P & Me´nez, A (1998) Functional determinants by which snake and cone snail toxins block the alpha 7 neuronal nicotinic acetylcholine receptors J Physiol Paris 92, 107–111.
25 Grant, G.A., Luetje, C.W., Summers, R & Xu, X.L (1998) Dif-ferential roles for disulfide bonds in the structural integrity and biological activity of j-Bungarotoxin, a neuronal nicotinic acet-ylcholine receptor antagonist Biochemistry 37, 12166–12171.
26 Antil-Delbeke, S., Gaillard, C., Tamiya, T., Corringer, P.-J., Changeux, J.P., Servent, D & Me´nez, A (2000) Molecular determinants by which a long chain toxin from snake venom interacts with the neuronal alpha 7-nicotinic acetylcholine receptor J Biol Chem 275, 29594–29601.
27 Eletskii, A.V., Maslennikov, I.V., Kukhtina, V.V., Utkin IuN., Tsetlin, V.I & Arsen’ev, A.S (2001) Structure and conformational heterogeneity of the weak toxin from the cobra Naja kaouthia venom Bioorg Khim 27, 89–101.
28 Torres, A.M., Kini, R.M., Selvanayagam, N & Kuchel, P.W (2001) NMR structure of bucandin, a neurotoxin from the venom
of the Malayan krait (Bungarus candidus) Biochem J 360, 539– 548.
29 Utkin, Y.N., Kukhtina, V.V., Kryukova, E.V., Chiodini, F., Bertrand, D , Methfessel, C & Tsetlin, V.I (2001) Weak toxin from Naja kaouthia is a nontoxic antagonist of alpha 7 and muscle-type nicotinic acetylcholine receptors J Biol Chem 276, 15810–11815.
30 Fiordalisi, J.J., Al-Rabiee, R., Chiappinelli, V.A & Grant, G.A (1994) Affinity of native j-bungarotoxin and site directed mutants for the muscle nicotinic acetylcholine receptor Biochemistry 33, 12963–12967.
31 Wu¨ster, W (1996) Taxonomic changes and toxinology: systematic revisions of the Asiatic cobras (Naja naja species complex) Toxicon 34, 399–406.
32 Pillet, L., Tre´meau, O., Ducancel, F., Drevet, P., Zinn-Justin, S., Pinkasfeld, S., Boulain, J.-C & Me´nez, A (1993) Genetic engineering of snake toxins Role of invariant residues in the structural and functional properties of a curaremimetic toxin, as probed by site-directed mutagensis J Biol Chem 268, 909–916.
33 Tre´meau, O., Lemaire, C., Drevet, P., Pinkasfeld, S., Ducancel, F., Boulain, J.-C & Me´nez, A (1995) Genetic engineering of snake
Trang 10toxins, the functional site of erabutoxin a as delineated by
site-directed mutagensis, includes variant residues J Biol Chem 268,
9362–9369.
34 Yanish-Perron, C., Vieire, J & Messing, J (1985) Improved M13
phage cloning vectors and host strains: nucleotide sequences of
M13mp18 and pUC19 vectors Gene 33, 103–199.
35 Afifiyan, F., Armugam, A., Tan, C.H., Gopalakrishnakone, P &
Jeyaseelan, K (1999) Postsynaptic alpha-neurotoxin gene of the
spitting cobra, Naja naja sputatrix: structure, organization, and
phylogenetic analysis Genome Res 9, 259–366.
36 Sambrook, J., Fritsch, E.F & Maniatis, T (1989) Molecular
cloning: A Laboratory Manual, 2nd edn Cold Spring Harbor
Laboratory Press.
37 Weichenhan, D (1991) Fast recovery of DNA from agarose gel by
centrifugation through blotting paper Trends Genet 7, 109.
38 Sanger, F., Nicklen, S & Coulson, A.R (1977) DNA sequencing
with chain-terminating inhibitors Proc Natl Acad Sci USA 74,
5463–5467.
39 Altschul, S.F., Gish, W., Miller, W., Myers, E.W & Lipman, D.J.
(1990) Basic local alignment search tool J Mol Biol 215, 403–
410.
40 Riniker, B., Flo¨rsheimer, A., Fretz, H., Sieber, P & Kamber, B.
(1993) A general strategy for the synthesis of large peptides:
combined solid-phase and solution approach Tetrahedron 49,
9307–9320.
41 Wang, S.-S (1973) p-Alkoxybenzyl alcohol resin and p-alkoxy
benzyloxycarbonylhydrazide resin for solid phase synthesis of
protected peptide fragments J Am Chem Soc 94, 1328.
42 Mourier, G., Servent, D., Zinn-Justin, S & Me´nez, A (2000)
Chemical engineering of a three-fingered toxin with anti-a7
neuronal acetylcholine receptor activity Protein Eng 13, 217–
225.
43 Saitoh, T., Oswald, R., Wennogle, L.P & Changeux, J.P (1980)
Conditions for the selective labelling of the 66 000 dalton chain of
the acetylcholine receptor by the covalent non-competitive blocker
5-azido-[ 3 H]trimethisoquin FEBS Lett 116, 30–36.
44 Cheng, Y.C & Prusoff, W.H (1973) Relationship between the
inhibition constant (K i ) and the concentration of inhibitor, which
causes 50 per cent inhibition of an enzymatic reaction Biochem.
Pharmacol 22, 3099–3108.
45 Weber, M & Changeux, J.-P (1974) Binding of Naja nigricolis
[3H]a-toxin to membrane fragments from Electrophorus and
Torpedo electric organs Mol Pharmacol 10, 15–34.
46 Favreau, P., Krimm, I., Le Gall, F., Bobenrieth, M.J., Lamthanh,
H., Bouet, F., Servent, D , Molgo, J., Me´nez, A., Letourneux, Y.
& Lancelin, J.M (1999) Biochemical characterization and
nuclear magnetic resonance structure of novel alpha-conotoxins
isolated from the venom of Conus consors Biochemistry 38, 6317–
6326.
47 Blobel, G & Dobberstein, B (1975) Transfer of proteins across membrane I-Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma J Cell Biol 67, 852–862.
48 Woody, R.W (1995) Circular Dichroism Methods Enzymol 246, 34–71.
49 Sreerama, N., Manning, M.C., Powers, M.E., Zhang, J.X., Goldenberg, D P & Woody, R.W (1999) Tyrosine, phenylala-nine, and disulfide contributions to the circular dichroism of proteins: circular dichroism spectra of wild-type and mutant bovine pancreatic trypsin inhibitor Biochemistry 38, 10814–10822.
50 Me´nez, A., Bouet, F., Tamiya, N & Fromageot, P (1976) Conformational changes in two neurotoxic proteins from snake venoms Biochim Biophys Acta 26, 121–132.
51 Me´nez, A., Langlet, G., Tamiya, N & Fromageot, P (1978) Conformation of snake toxic polypeptides studied by a method of prediction and circular dichroism Biochimie 60, 505–516.
52 Hider, R.C., D rake, A.F & Tamiya, N (1988) An analysis of the 225–230-nm CDband of elapid toxins Biopolymers 27, 113–122.
53 Zinn-Justin, S., Roumestand, C., Gilquin, B., Bontems, F., Me´nez, A & Toma, F (1992) Three-dimensional solution struc-ture of a curaremimetic toxin from Naja nigricollis venom: a proton NMR and molecular modeling study Biochemistry 31, 11335–11347.
54 Le Goas, R., LaPlante, S.R., Mikou, A., Delsuc, M.A., Guittet, E., Robin, M., Charpentier, I & Lallemand, J.Y (1992) Alpha-cobratoxin: proton NMR assignments and solution structure Biochemistry 31, 4867–4875.
55 Me´nez, A (1998) Functional architectures of animal toxins: a clue
to drug design? Toxicon 36, 1557–1572.
56 Vulfius, C.A., Krasts, I.V., Utkin, Y.N & Tsetlin, V.I (2001) Nicotinic receptors in Lymnea stagnalis neurons are blocked by alpha-neurotoxins from cobra venoms Neurosci Lett 309, 189– 192.
57 Servent, D., Antil-Delbeke, S., Gaillard, C., Corringer, P.-J., Changeux, J.P & Me´nez, A (2000) Molecular characterization of the specificity of interactions of various neurotoxins on two dis-tinct nicotinic acetylcholine receptors Eur J Pharmacol 393, 197–204.
58 Antil, S., Servent, D & Me´nez, A (1999) Variability among the sites by which curaremimetic toxins bind to torpedo acetylcholine receptor, as revealed by identification of the functional residues of alpha-cobratoxin J Biol Chem 274, 34851–34858.
59 Sato, S & Tamiya, N (1971) The amino acid sequence of erabutoxins, neurotoxic proteins of sea-snake (Laticauda semi-fasciata) venom Biochem J 122, 453–461.
60 Chang, C.C (1979) The action of snake venom on nerve and muscle In Snake Venoms, Handbook of Experimental Pharma-cology (Lee, C.Y., ed.), pp 309–376.