From the Shemyakin-Ovchinnikov Institute of
Bioorganic Chemistry, Russian Academy of Sciences, 16/10
Miklukho-Maklaya Str., V-437 Moscow GSP-7, 117997 Russia, the
§ Department of Physiology, Centre Medical Universitaire, 1 Rue Michel Servet, 1211 Geneva 4, Switzerland, and ¶ Central
Research Biophysics, Bayer AG, D-51368 Leverkusen, Germany
Received for publication, January 29, 2001
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ABSTRACT |
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A novel "weak toxin" (WTX) from Naja
kaouthia snake venom competes with
[125I] The first representative of "weak toxins," characterized by
low toxicity, was isolated from Naja melanoleuca snake venom
and sequenced in 1975 (1). Toxins of this type were later referred to
as melanoleuca (2) or miscellaneous-type (3) toxins. In protein data
banks (e.g. Swiss-Prot) they are classified together with
some other toxins in the group of weak toxins. At present, around 15 amino acid sequences of such toxins are known. They consist of 62-68
amino acid residues, contain five disulfide bridges, and belong to the
group of snake venom "three-fingered" toxins, whose characteristic
feature is the presence of three disulfide-confined loops
("fingers") (4, 5). However, most other three-fingered toxins, including cardiotoxins, muscarinic toxins, acetylcholinesterase inhibitors, and the so-called short chain In the present work, we decided to check whether a weak toxin isolated
from N. kaouthia venom (designated WTX) acts on AChRs. WTX
was found to bind to the membrane-bound Torpedo californica AChR, although much less efficiently than a short chain neurotoxin, NT-II, or a long one, CTX. However, WTX and CTX were virtually equipotent in binding to the soluble N-terminal domain of the rat WTX and Other Toxins
WTX was purified from N. kaouthia as described
previously (17). The molecular mass of WTX was determined by
matrix-assisted laser desorption ionization time of flight on a Vision
2000 (Thermo Bioanalysis Corp.) mass spectrometer. This preparation
(See Fig. 1) was used for primary structure determination (17) and for preliminary testing of biological activity. Further experiments were
done on the WTX preparations obtained after additional purification steps by ion exchange chromatography on a TSK SP-5PW column (see Fig. 4).
CTX was obtained from N. kaouthia venom as described
previously (18). NT-II was purified from the N. oxiana venom
by gel filtration (under the same conditions as those used for N. kaouthia venom) followed by ion exchange chromatography on Bio Rex
70 in a gradient of ammonium acetate from 0.05 to 0.3 M (pH
7.5). AChR Preparations
Membrane-bound nicotinic acetylcholine receptor from T. californica electric organ was kindly provided by Prof. F. Hucho. A soluble fusion protein consisting of the N-terminal domain
1-208 of rat Binding Experiments
Membrane-bound AChR from T. californica--
Varying
concentrations of competitors were added to 50 µl of membrane
suspension (25 µg of protein/ml, 50 mM Tris-HCl buffer, pH 8.0), and the mixture was incubated for 1 h at room temperature in a total volume of 195 µl. Then 5 µl of 0.4 µM
[125I] Fusion Protein--
A solution of GST- Immunological Assays
CTX was reduced under denaturing conditions, carboxymethylated,
and used to prepare a keyhole limpet hemocyanin conjugate, which
was employed to immunize rabbits, and to obtain polyclonal antibodies by a standard procedure (22). An immunoglobulin
fraction, obtained from the antiserum by fractional precipitation with
ammonium sulfate, was dissolved in phosphate-buffered saline, dialyzed against this buffer, lyophilized, and stored at Reconstitution in Xenopus Oocytes
Xenopus oocytes were isolated and prepared as
previously described (24). A volume of 10 nl containing 2 ng of
expression vector cDNA was injected into the nucleus the day
following the oocyte isolation. To allow good protein expression
levels, oocytes were kept for 2-3 days prior to recording. Each oocyte
was placed in a separate well of a 96-well microtiter plate (Nunc) at
18 °C. Oocytes were incubated in Barth's solution, a standard
medium for maintaining Xenopus oocytes in vitro,
which consists of the following (in mM): NaCl 88, KCl 1, NaHCO3 2.4, HEPES 10, MgSO4 0.82, Ca(NO3)2 0.33, CaCl2 0.41. pH was
adjusted to 7.4 with NaOH. To minimize contamination, the medium was
filtered at 0.2 µm and supplemented with antibiotics (20 µg/ml
kanamycin, 100 units/ml penicillin, and 100 µg/ml streptomycin or 50 µg/ml gentamicin).
Electrophysiology Experiments
Electrophysiological recordings were made using a dual electrode
voltage clamp (TEC 00, NPI Electronic GmbH, Tamm, Germany) as
described previously (25, 26). Cells were placed in a plexiglass chamber, voltage-clamped at The WTX isolated by sequential gel filtration, ion exchange, and
reverse-phase chromatography is homogeneous by the criteria of the
latter and has a molecular mass of 7613 Da (Fig.
1A). However, this mass is
larger than that (7483 Da) calculated from the published sequence of
the weak toxin CM-9a from N. kaouthia venom (2). There are
no tryptophan residues in CM-9a, whereas determination of the primary
structure of WTX (17) detected a Trp residue in position 36 and also
revealed two other differences from the CM-9a sequence: Lys-50 instead
of Tyr and Tyr-52 instead of Lys (17). The molecular mass calculated
for this sequence practically coincides with that found experimentally.
Therefore, WTX, whose sequence is depicted in Fig. 1B, can
be considered as a new homologue of CM-9a. When tested for a
capacity to compete with [125I]-bungarotoxin for binding to the
membrane-bound Torpedo californica acetylcholine receptor
(AChR), with an IC50 of ~2.2 µM. In this respect, it is ~300 times less potent than neurotoxin II from Naja oxiana and
-cobratoxin from N. kaouthia, representing short-type and long-type
-neurotoxins,
respectively. WTX and
-cobratoxin displaced
[125I]
-bungarotoxin from the Escherichia
coli-expressed fusion protein containing the rat
7 AChR
N-terminal domain 1-208 preceded by glutathione
S-transferase with IC50 values of 4.3 and 9.1 µM, respectively, whereas for neurotoxin II the
IC50 value was >100 µM. Micromolar
concentrations of WTX inhibited acetylcholine-activated currents in
Xenopus oocyte-expressed rat muscle AChR and human and rat
7 AChRs, inhibiting the latter most efficiently
(IC50 of ~8.3 µM). Thus, a virtually
nontoxic "three-fingered" protein WTX, although differing from
-neurotoxins by an additional disulfide in the N-terminal loop, can
be classified as a weak
-neurotoxin. It differs from the
short chain
-neurotoxins, which potently block the muscle-type but
not the
7 AChRs, and is closer to the long
-neurotoxins, which
have comparable potency against the above-mentioned AChR types.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-neurotoxins, contain four
disulfide bridges. A fifth disulfide bridge is present in the long
chain
-neurotoxins, such as
-bungarotoxin
(
Bgt)1 from Bungarus
multicinctus or
-cobratoxin from Naja
kaouthia (CTX) and
-bungarotoxin (
Bgt), which together with
short
-neurotoxins are potent antagonists of different classes of
nicotinic acetylcholine receptors (AChRs) (for reviews see Refs. 6-8).
In the long chain
- and
-neurotoxins the fifth disulfide bridge
is located in the central loop II, whereas in weak toxins the
additional disulfide is in the N-terminal loop I (see Fig.
1B). The available data show that residues of loops I, II,
and III of
-neurotoxins participate in binding to AChRs (for reviews
see Refs. 4, 5, and 7). However, depending on the type of
-neurotoxin (short or long) and AChR (muscle-type or neuronal
7), the role of particular loops may vary. For example, there
are numerous data demonstrating the involvement of identified residues
in the central loop II of short chain and long chain neurotoxins in
binding both to muscle-type (9-12) and
7 AChRs (13). Interestingly,
a fifth disulfide in loop II of the long
-neurotoxins is essential
for binding to
7 AChR but not to the Torpedo AChR (14).
The residues of loop I in short
-neurotoxins were shown to be
involved in binding to the Torpedo receptor (15), whereas
loop I in long
-neurotoxins appears to be less important for the
interaction with Torpedo and
7 AChRs (11, 13).
7
AChR expressed in Escherichia coli as a fusion protein with
glutathione S-transferase (GST). Subsequent
electrophysiological experiments revealed that in the micromolar range
WTX blocked human and rat
7 AChRs as well as rat muscle AChR
(16).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Bgt was purchased from Sigma. Radioactive
[125I]
Bgt was prepared and purified as described
previously (19).
7 AChR preceded by GST, whose denatured form was first mentioned in Ref. 20, was isolated after expression of the
respective cDNA in E. coli (data to be published
elsewhere).2 Briefly,
a gene fragment corresponding to the amino acids 1-208 was cloned into
the pGEX-KG vector (21) at the BamHI/HinIII sites, and the recombinant plasmids were used to transform the E. coli strain JM107. The inclusion bodies containing the target protein were washed extensively with 1% Triton X-100 in the presence of 0.5 M NaCl, then dissolved in 8 M
urea, 1 mM dithiothreitol, subsequently refolded at
10 °C in 20 mM Tris-HCl, pH 9.5, 0.1% CHAPS,
concentrated by ultrafiltration, and then dialyzed against 10 mM Tris-HCl, pH 8.0, 0.1% CHAPS.
Bgt (specific activity 45 Ci/mmol) were added,
and the samples were incubated for 1 h more. The mixture was
quickly filtered through Whatman GF/F filters (preincubated in
0.25% polyethylenimine for at least 2 h), the filters were washed
four times with 1 ml of 50 mM Tris-HCl buffer, pH 8.0, and
the radioactivity was determined in an Ultragamma
-counter (Amersham
Pharmacia Biotech).
7-(1-208)
protein (17 µg/ml, pH 8.0, 0.1% CHAPS) was incubated with different
concentrations of competitors in a final volume of 190 µl for 1 h at room temperature. Then 10 µl of 0.4 µM
[125I]
Bgt were added, and the mixture was incubated
for 1 h more. The unbound [125I]
Bgt was removed
by fast filtration through DE-81 filters (Whatman), and the
filters were washed four times with 1 ml of 50 mM Tris-HCl buffer, pH 8.0, containing 0.1% Triton X-100 and counted.
80 °C.
Enzyme-linked immunosorbent assay was performed in 96-well Linbro
E.I.A. microtitration plates (Flow Laboratories) as in Ref. 23,
using immunoglobulins of nonimmunized rabbits as a control. It was
found that antibodies reacted with comparable efficiency with the
reduced, carboxymethylated CTX and native CTX; the titers were 1:64,000
and 1:50,000, respectively.
80 mV, and superfused with normal frog
Ringer's solution (in mM: NaCl 115, KCl 2.5, CaCl2 1.8, HEPES 10, pH 7.2, adjusted with NaOH). ACh stock
solution (0.1 M) was kept frozen and added to the test
solutions on the day of the experiment. Cells were treated with WTX by
incubating in the measuring chamber with a WTX-containing normal frog
Ringer's solution for 20 min with perfusion stopped.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Bgt for binding to the
membrane-bound T. californica AChR, WTX was found to be
about 300 times less potent than CTX and NT-II, the long chain and
short chain
-neurotoxins (Fig.
2A and Table I).
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Fig. 1.
Analytical characteristics and primary
structure of weak toxin WTX. A, reverse-phase HPLC of
WTX on a Vydac C18 column (4.6 × 250 mm) in an acetonitrile
concentration gradient (from 15 to 45% in 30 min) in water in the
presence of 0.1% trifluoroacetic acid. The flow rate was 1 ml/min.
Inset, matrix-assisted laser desorption ionization mass
spectrum of WTX. B, amino acid sequence of WTX. Shown
against a black background is Trp-36, absent in weak toxin CM-9a. The
residue numbers are given for the fragment 50-52, wherein the Lys-50
and Tyr-52 are interchanged in CM-9a, as well as for Cys-6 and Cys-11,
characteristic for weak toxins. The polypeptide backbone of WTX is
depicted in a typical three-fingered pattern, with the respective loops
(fingers) marked I-III.
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Fig. 2.
Inhibition of
[125I] Bgt binding to the
AChR-enriched membrane preparations of the T. californica
electric organ (A) and to the solubilized fusion
protein GST-
-(1-208) (B) by
the snake venom toxins WTX (
), CTX (
), and NT-II (
) and
by
-conotoxin ImI (
). Symbols
and
* refer to the experiments done using WTX subjected to one run
and three runs of ion exchange HPLC (see Fig. 4), respectively.
[C], toxin concentration (M).
Binding of polypeptide and peptide toxins to the membrane-bound Torpedo
AChR and GST-7-(1-208) fusion protein
In addition to the muscle-type AChRs, the long neurotoxins Bgt and
CTX also bind to neuronal
7 AChR. To assess this possibility for WTX
as well, we used a bacterially expressed protein containing the
N-terminal domain (amino acid residues 1-208) of the rat brain
7
receptor preceded by GST. This chimera faithfully reproduces a number
of
7 AChR features (Fig. 2B and Table I). It interacts with
Bgt and CTX as well as with the
7-targeting
-conotoxin ImI but practically does not bind either NT-II, a short-chain
-neurotoxin, or
-conotoxin G1 acting on the Torpedo
and other muscle-type AChRs. In competition with
[125I]
Bgt for binding to the chimerical protein, WTX
was as active as CTX (Table I).
To test whether WTX binding can also lead to a functional block of the
native 7 AChRs, electrophysiological investigations on the
7 AChR
expressed in Xenopus oocytes were performed. Incubation for
20 min in the presence of 10 µM WTX caused an almost
complete block of the subsequent ACh-evoked current (Fig.
3A). Such an inhibitory
potency is much weaker than that of CTX or
Bgt, which block
7
AChRs in the nanomolar range (13, 27, 28), but is of the same order of
magnitude as the IC50 of
-conotoxin ImI in blocking the
rat
7 AChR (26, 29). We decided to check whether WTX is closer to
Bgt/CTX or to
-conotoxin ImI in the reversibility of its blocking
effect on the rat
7 AChR.
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Superfusion with control medium following WTX blockade showed very slow
recovery, taking more than 1 h (Fig. 3A). It is similar to the slow dissociation of Bgt (29) or CTX (see Ref. 30 and Fig.
6), whereas
-conotoxin ImI could be washed out almost completely in
10 min.
In view of the facts that WTX is washed out from oocytes as slowly as
CTX but in terms of acting concentrations is about 1000-fold less
potent, it was necessary to address the possibility that the effects on
7 AChRs in oocytes ascribed to WTX in fact might have arisen if
traces of CTX, even as low as 0.1%, were present in the WTX
preparations. Therefore, additional experiments were done to check and,
if necessary, to increase the degree of purity of WTX.
Fig. 1A shows only one peak in the mass spectrum of WTX
purified by reverse-phase HPLC. No sequences other than that of WTX were detected on Edman degradation (17). However, matrix-assisted laser
desorption ionization analysis of a model 1:1000 mixture of CTX:WTX
failed to detect the peak of CTX. Fig. 4
shows chromatography of ~4 µg of CTX (trace 3), which
would correspond to the 0.1% content in 4 mg of WTX. It is clear that
such a small amount can be reliably detected under the chosen
conditions. Fig. 4, trace 1 corresponds to purification of
4.2 mg of WTX from the batch used for structure determination and in
the biological activity experiments described above. In addition to the
huge peak of WTX itself, several minor peaks (each 0.1% or less) can
be distinguished, including one coinciding with the position of
CTX.
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When the pooled fractions of the repurified WTX (eluting between 45 and 65 min) were lyophilized and applied again to the same column (Fig. 4, trace 2), no peak was detected at the position of CTX. Some other minor contaminating peaks were still present, which was not surprising in view of the relatively large amount of WTX applied onto the column and the extremely high sensitivity of UV detection.
We checked for the presence of CTX traces in the repurified WTX with
the aid of polyclonal antibodies raised against reduced and
carboxymethylated CTX. As seen from Fig.
5, these antibodies allow the detection
of very low concentrations of CTX. Although at high concentrations
(>0.01 mg/ml) WTX also reacts with these antibodies, the difference in
the affinities makes possible the estimation of the upper limit of
putative CTX admixture in the repurified WTX. A virtually complete
inhibition of the 7 AChR expressed in oocytes was observed at 20 µM (0.15 mg/ml) repurified WTX (see below, Figs.
3B and 7). As seen from Fig. 5, to this concentration of WTX
corresponds an OD of ~0.16. If we assume that it is only the CTX
admixture in WTX that interacts with antibodies, this amount should be
as low as ~8.8 × 10
6 mg/ml (See Fig. 5),
i.e. not more than 0.006% of the WTX. Moreover, if there is
a slight cross-reactivity of antibodies with WTX, the admixture should
be even lower, if present at all.
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Thus, immunological data confirmed that after additional purification
by ion exchange HPLC, there are no measurable traces of CTX in WTX, at
least in the range that would force one to ascribe the biological
activity of WTX to CTX admixtures. Some of the binding
experiments (Fig. 2) and more detailed studies in oocytes (Figs. 3,
6, and 7) were done with the
repurified samples of WTX.
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Additional purification had no effect on the WTX binding either to the
Torpedo membranes or to 7 AChR fusion protein. In Fig. 2
the experimental points for the repurified WTX (marked as
diamonds and asterisks) fit the displacement
curves obtained with the preparation that did not undergo such a treatment.
Electrophysiological experiments on the additionally purified WTX gave
similar results as those described above; the WTX concentration required to achieve a virtually complete block was 20 µM
(cf. Fig. 3, A and B). It is also seen
(Fig. 3C) that at 20 µM WTX reduces by about
60% the ACh-induced current in the human 7 AChR. In the same
concentration range it efficiently blocks the rat muscle AChR expressed
in Xenopus oocytes (Fig. 3D). As mentioned above,
long chain
-neurotoxins like
Bgt or CTX block the
7 and muscle
AChR in the nanomolar range, as illustrated in Fig. 3E for
CTX action on the human
7 AChR.
From the washout experiments shown in Fig. 3, A and
B, and, in more detail, in Fig. 6, it is clear that
additional purification did not affect the virtual irreversibility of
the WTX action. In this respect, it is practically indistinguishable
from Bgt or CTX and differs greatly from
-conotoxin ImI, which
can be washed out in minutes (Fig. 6).
Fig. 7 shows the dose-response data illustrating the WTX action on the
rat and human 7 AChRs. Accurate quantitative measurements of these
blocking effects on oocytes are difficult, especially at low toxin
concentrations, because of the long incubation times required for the
WTX to manifest its activity. We were able to estimate the
IC50 value for the WTX action on the rat
7 AChR as 8.3 µM, whereas for the qualitatively less potent action on human
7 AChR we can give a very rough estimation of the
IC50 value as ~15 µM.
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DISCUSSION |
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Our initial purpose was to isolate from the N. kaouthia
snake venom a weak toxin, CM-9a, the only weak toxin previously found in this venom (2), and try to find its biological target. However, because the determined molecular mass of the corresponding fraction, designated by us as WTX, differed by 130 Da from that calculated from
the published amino acid sequence of CM-9a (2), we had to determine the
complete primary structure of WTX (17). Whereas CM-9a, like all
previously described weak toxins from cobra venoms, contains no
tryptophans, WTX has a Trp residue at position 36 and differs from
CM-9a in two other positions: Lys-50/Tyr-50 and Tyr-52/Lys-52 (see Fig.
1). Therefore, WTX appears to be a new homologue of CM-9a. These
minor alterations in the primary structure may simply be due to
collecting the venoms from different snake populations. This structural
difference can, however, influence the biological activity of the
toxins. It is known that the toxicity (LD50) of weak toxins
can vary from about 5 to 80 mg/kg (2, 31). We did not determine the
LD50 for WTX, but in doses up to 2 mg/kg (intravenous
injection) it was nontoxic to rats. Interestingly, a recently
isolated -bungarotoxin, structurally related to weak toxins (32),
has an LD50 of 0.15 mg/kg, which is comparable with those
of
-neurotoxins, but its molecular target has not been yet characterized.
As an attempt to define the biological targets for weak toxins,
we examined the activity of WTX against two types of nicotinic acetylcholine receptors. As seen from Fig. 2A and Table I,
WTX binds specifically to the membrane-bound Torpedo AChR,
but about 300 times less effectively than CTX or NT-II. A comparison of WTX with these two -neurotoxins was done also with an
7
AChR-related system using a fusion protein, GST-
7-(1-208). Although
this protein binds CTX with only a moderate affinity (Fig.
2B and Table I), the following data allow us to consider it
to be an appropriate model of the rat
7 AChR ligand-binding domain.
It does discriminate, similarly to the intact
7 AChR, between the
long and short
-neurotoxins (no inhibition of
[125I]
Bgt binding by short neurotoxin NT-II at 100 µM) as well as between the muscle-type and neuronal
-conotoxins (no inhibition by
-conotoxin G1 at 100 µM and an IC50 of 42.6 µM for
neuronal
-conotoxin ImI). Under these circumstances, the comparable
inhibitory potencies of WTX and CTX (IC50 of 4.3 and 9.1 µM, respectively) allowed us to anticipate that WTX would
bind specifically to the native
7 AChRs.
Indeed, subsequent electrophysiological recordings demonstrated the
capability of WTX to inhibit functional responses of 7 AChRs
expressed in Xenopus oocytes (Figs. 3, 6, and 7). Although WTX and CTX are virtually indistinguishable in their binding to the
fusion protein GST-
7-(1-208) (Fig. 2B), WTX is about
1000-fold less potent than CTX in blocking the ACh current on the
native
7 receptors heterologously expressed in Xenopus
oocytes. As described under "Results," additional
purification (Fig. 4) and immunological control experiments (Fig. 5)
allowed us to rule out the possibility that the observed WTX effects on
the ACh currents in oocytes might be caused by traces of CTX, if it
were present even in as small amounts as 0.1%.
During the revision of this paper, a weak toxin from the Naja
naja atra snake venom was shown to block cholinergic transmission in frog muscle preparations in the micromolar range (33), and a similar
activity on a chick muscle was reported for a weak toxin from
Bungarus candidus (34). However, these two weak toxins were
not tested against 7 AChRs, and the authors did not set a task to
rule out the presence of trace amounts of the respective
-neurotoxins.
WTX, despite acting on the 7 AChR at micromolar rather than
nanomolar concentrations, as do
Bgt or CTX, is very similar to these
two long chain
-neurotoxins in the persistency of the produced block
(Fig. 6). In this respect it differs from
-conotoxin ImI and its
analogs, which are also active in the micromolar range but can be
washed out within minutes (26, 29) (see Fig. 6).
A number of amino acid residues and certain conformational
motifs essential for binding to respective AChRs have been
identified both in -neurotoxins and
-conotoxins (4, 5, 11, 13, 35). The additional disulfide bridge in the CTX and in
-bungarotoxin is a prerequisite for their binding to neuronal AChRs, that is to
7
and
3
2, respectively (14, 36). This
disulfide fixes a helix-like scaffold in the
-neurotoxin central
loop II (37). A similar scaffold could be distinguished in
-conotoxin ImI acting on the
7 AChR as well as in the other
-conotoxins targeting the neuronal AChRs, but not in
-conotoxin
G1 blocking the Torpedo and muscle AChRs (38).
Interestingly, there are no
-helices or helix-like segments in the
N-terminal loop I of the three-fingered toxins. A helix was found in
the CD59 protein (39), which, like other members of the Ly-6 family of
immune system proteins (40, 41), has a fifth disulfide in the
N-terminal loop I, rather than in the loop II as do the long-type
-neurotoxins. The "endogenous toxin" lynx-1 recently found in
the nervous system (42) was supposed to have a helix turn in loop I,
but the conclusion was based only on modeling studies. However,
although confirming by x-ray analysis the
-structure-dominated
three-fingered motif in bucandin, a presynaptic neurotoxin, no
helical turns were found in its loop I, containing the additional
disulfide (43). Only
-structure was detected by 1H-NMR
studies of WTX (44). It seems that specificity and selectivity of a
particular three-fingered toxin toward various AChRs or other targets
depends not only on the chemical nature and distribution of functional
groups (charged, hydrophobic, etc.) along the three-fingered scaffold,
but also on the differences in the conformation and conformational
mobility of those loops. In particular, long times required to restore
the sensitivity of the
7 AChR to Ach after applying and washing out
WTX might be associated with the conformational heterogeneity of WTX
(44) and conformational changes that WTX might undergo itself on
binding to the receptor or induce in the latter. Noteworthy allosteric
effects of antagonists and agonists, some of them influencing the
channel moiety, were described for the Torpedo AChR (45,
46).
In summary, the results obtained suggest that the biological target of
WTX, a member of the family of weak toxins, may be nicotinic AChRs.
Interacting with the Torpedo, muscle, and 7 AChRs at
similar concentrations, WTX resembles the long chain
-neurotoxins
more than the short ones in this respect. Although acting only at
micromolar concentrations, WTX has an advantage of being practically
nontoxic while producing a long lasting effect. In view of the
pharmacological importance of neuronal AChRs (for review see Ref. 47),
a search for selective and efficient markers and blockers of those
receptors is still an important task. Our work shows that the
three-fingered proteins of the "weak-toxin structural subtype" are
promising in this respect.
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ACKNOWLEDGEMENTS |
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We are grateful to F. Hucho for helpful
discussions and for providing the Torpedo membranes and to
V. Witzemann, J. Patrick, and B. Weingärtner for kindly providing
the cloned rat muscle AChR cDNA, rat 7 cDNA, and
human
7 cDNA, respectively. We are also indebted to V. Starkov
for breeding snakes and supplying their venoms and to N. Dergousova
and E. Shibanova for preparing the
7 fusion protein. We thank S. Bertrand for help with electrophysiological experiments. We are
grateful to an anonymous referee, whose remarks stimulated additional
experiments on WTX.
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FOOTNOTES |
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* This work was supported by the Russian Foundation for Basic Research (Grants 99-04-48798 and 00-04-48889) and by the Swiss National Science Foundation and the "Office Federal de l'Education et des Sciences." A preliminary account was presented at the XIIIth World Congress of the International Society on Toxinology, September 18-22, 2000, Paris, France.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel. & Fax: 7 095 335 57 33; E-mail: vits@ibch.ru.
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M100788200
2 N. I. Dergousova, E. D. Shibanova, I. E. Kasheverov, C. Methfessel, and V. I. Tsetlin, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
Bgt, bungarotoxin;
CTX, -cobratoxin;
ACh, acetylcholine;
AChR, nicotinic acetylcholine
receptor;
WTX, weak toxin from N. kaouthia;
NT-II, neurotoxin II from Naja oxiana;
GST, glutathione
S-transferase;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
HPLC, high performance liquid chromatography.
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REFERENCES |
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1. | Carlsson, F. H. H. (1975) Biochim. Biophys. Acta 400, 310-321[Medline] [Order article via Infotrieve] |
2. | Joubert, F. J., and Taljaard, N. (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 425-436[Medline] [Order article via Infotrieve] |
3. | Shafqat, J., Siddiqi, A. R., Zaidi, Z. H., and Jornvall, H. (1991) FEBS Lett. 284, 70-72[CrossRef][Medline] [Order article via Infotrieve] |
4. | Menez, A. (1998) Toxicon 36, 1557-1572[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Tsetlin, V. I.
(1999)
Eur. J. Biochem.
264,
281-286 |
6. | Changeux, J.-P., and Edelstein, S. J. (1998) Neuron 21, 959-980[Medline] [Order article via Infotrieve] |
7. | Hucho, F., Tsetlin, V. I., and Machold, J. (1996) Eur. J. Biochem. 239, 539-557[Abstract] |
8. | Buisson, B., and Bertrand, D. (1998) J. Physiol. Paris 92, 89-100[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Ackermann, E. J.,
Ang, E. T.,
Kanter, J. R.,
Tsigelny, I.,
and Taylor, P.
(1998)
J. Biol. Chem.
273,
10958-10964 |
10. |
Osaka, H.,
Malany, S.,
Molles, B. E.,
Sine, S. M.,
and Taylor, P.
(2000)
J. Biol. Chem.
275,
5478-5484 |
11. |
Antil, S.,
Servent, D.,
and Menez, A.
(1999)
J. Biol. Chem.
274,
34851-34858 |
12. | Utkin, Y. N., Krivoshein, A. V., Davydov, V. L., Kasheverov, I. E., Franke, P., Maslennikov, I. V., Arseniev, A. S., Hucho, F., and Tsetlin, V. I. (1998) Eur. J. Biochem. 253, 229-235[Abstract] |
13. |
Antil-Delbeke, S.,
Gaillard, C.,
Tamiya, T.,
Corringer, P. J.,
Changeux, J. P.,
Servent, D.,
and Menez, A.
(2000)
J. Biol. Chem.
275,
29594-29601 |
14. |
Servent, D.,
Winckler-Dietrich, V.,
Hu, H. Y.,
Kessler, P.,
Drevet, P.,
Bertrand, D.,
and Menez, A.
(1997)
J. Biol. Chem.
272,
24279-24286 |
15. |
Tremeau, O.,
Lemaire, C.,
Drevet, P.,
Pinkasfeld, S.,
Ducancel, F.,
Boulain, J. C.,
and Menez, A.
(1995)
J. Biol. Chem.
270,
9362-9369 |
16. | Utkin, Y., Kukhtina, V., Chiodini, F., Bertrand, D., Methfessel, C., and Tsetlin, V. (2000) Abstracts of the XIIIth World Congress of the International Society on Toxinology, Paris, September 18-22, 2000, P162, International Society on Toxinology, Paris |
17. | Utkin, Y. N., Kukhtina, V. V., Maslennikov, I. V., Eletsky, A. V., Starkov, V. G., Weise, C., Franke, P., Hucho, F., and Tsetlin, V. I. (2001) Toxicon 39, 921-927[CrossRef][Medline] [Order article via Infotrieve] |
18. | Kukhtina, V. V., Weise, C., Osipov, A. V., Starkov, V. G., Titov, M. I., Esipov, S. E., Ovchinnikova, T. V., Tsetlin, V. I., and Utkin, Y. N. (2000) Bioorg. Khim. 26, 803-807[Medline] [Order article via Infotrieve] |
19. | Klukas, O., Peshenko, I. A., Rodionov, I. L., Telyakova, O. V., Utkin, Y. N., and Tsetlin, V. I. (1995) Bioorg. Khim. 21, 152-155[Medline] [Order article via Infotrieve] |
20. |
Ariel, S.,
Asher, O.,
Barchan, D.,
Ovadia, M.,
and Fuchs, S.
(1998)
Ann. N. Y. Acad. Sci.
841,
93-96 |
21. | Guan, K. L., and Dixon, J. E. (1991) Anal. Biochem. 192, 262-267[Medline] [Order article via Infotrieve] |
22. | Gullik, W. J. (1994) in Methods in Molecular Biology (Walker, J. M., ed), Vol. 32 , pp. 389-399, Humana Press, Totowa, NJ |
23. | Richards, D. (1988) in ELISA and Other Solid Phase Immunoassays (Kemeny, D. M. , and Challacombe, S. J., eds) , pp. 345-346, John Wiley & Sons, Inc., New York |
24. | Bertrand, D., Cooper, E., Valera, S., Rungger, D., and Ballivet, M. (1991) in Methods in Neuroscience (Conn, M., ed), Vol. 4 , pp. 174-193, Academic Press, New York |
25. | Bertrand, D., Bertrand, S., and Ballivet, M. (1992) Neurosci. Lett. 146, 87-90[CrossRef][Medline] [Order article via Infotrieve] |
26. | Utkin, Y. N., Zhmak, M. N., Methfessel, C., and Tsetlin, V. I. (1999) Toxicon 37, 1683-1695[CrossRef][Medline] [Order article via Infotrieve] |
27. | Gerzanich, V., Anand, R., and Lindstrom, J. (1994) Mol. Pharmacol. 45, 212-220[Abstract] |
28. | Couturier, S., Bertrand, D., Matter, J. M., Hernandez, M. C., Bertrand, S., Millar, N., Valera, S., Barkas, T., and Ballivet, M. (1990) Neuron 5, 847-856[Medline] [Order article via Infotrieve] |
29. | Johnson, D. S., Martinez, J., Elgoyhen, A. B., Heinemann, S. F., and McIntosh, J. M. (1995) Mol. Pharmacol. 48, 194-199[Abstract] |
30. | Wonnacott, S., Albuquerque, E. X., and Bertrand, D. (1993) Methods Neurosci. 12, 263-275 |
31. | Joubert, F. J. (1975) Hoppe-Seyler's Z. Physiol. Chem. 356, 1901-1908[Medline] [Order article via Infotrieve] |
32. | Aird, S. D., Womble, G. C., Yates, J. R., and Grigffin, P. R. (1999) Toxicon 37, 609-625[CrossRef][Medline] [Order article via Infotrieve] |
33. | Chang, L., Lin, S., Wang, J., Hu, W., Wu, B., and Huang, H. (2000) Biochim. Biophys. Acta 1480, 293-301[Medline] [Order article via Infotrieve] |
34. | Nirthanan, S., Gopalakrishnakone, P., Gwee, M. C. E., Khoo, H. E., Cheah, L. S., and Kini, M. R. (2000) Abstracts of the XIIIth World Congress of the International Society on Toxinology, Paris, September 18-22, 2000, L108, International Society on Toxinology, Paris |
35. |
Quiram, P. A.,
and Sine, S. M.
(1998)
J. Biol. Chem.
273,
11007-11011 |
36. | Grant, G. A., Luetje, C. W., Summers, R., and Xu, X. I. (1998) Biochemistry 37, 12166-12171[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Betzel, C.,
Lange, G.,
Pal, G. P.,
Wilson, K. S.,
Maelicke, A.,
and Saenger, W.
(1991)
J. Biol. Chem.
266,
21530-21536 |
38. | Maslennikov, I. V., Shenkarev, Z. O., Zhmak, M. N., Ivanov, V. T., Methfessel, C., Tsetlin, V. I., and Arseniev, A. S. (1999) FEBS Lett. 444, 275-280[CrossRef][Medline] [Order article via Infotrieve] |
39. | Kieffer, B., Driscoll, P. C., Campbell, I. D., Willis, A. C., Van der Merwe, P. A., and Davis, S. J. (1994) Biochemistry 33, 4471-4482[Medline] [Order article via Infotrieve] |
40. | Ploug, M., and Ellis, V. (1994) FEBS Lett. 349, 163-168[CrossRef][Medline] [Order article via Infotrieve] |
41. | Gumley, T. P., McKenzie, I. F. C., and Sandrin, M. S. (1995) Immunol. Cell Biol. 73, 277-296[Medline] [Order article via Infotrieve] |
42. | Miwa, J. M., Ibanez-Tallon, I., Crabtree, G. W., Sanchez, R., Sali, A., Role, L. W., and Heintz, N. (1999) Neuron 23, 105-114[Medline] [Order article via Infotrieve] |
43. | Kuhn, P., Deakon, A. M., Comoso, S., Rajaseger, G., Kini, R. M., Uson, I., and Kolatkar, P. R. (2000) Acta Crystallogr. Sect. D Biol. Crystallogr. 56, 1401-1407[CrossRef][Medline] [Order article via Infotrieve] |
44. | Eletsky, A. V., Maslennikov, I. V., Kukhtina, V. V., Utkin, Y. N., Tsetlin, V. I., and Arseniev, A. S. (2001) Bioorg. Khim. 27, 89-101[Medline] [Order article via Infotrieve] |
45. |
White, B. H.,
and Cohen, J. B.
(1992)
J. Biol. Chem.
267,
15770-15783 |
46. | Moore, M. A., and McCarthy, M. P. (1995) Biochim. Biophys. Acta 1235, 336-342[Medline] [Order article via Infotrieve] |
47. | Cordero-Erausquin, M., Marubio, L. M., Klink, R., and Changeux, J.-P. (2000) Trends Pharmacol. Sci. 21, 211-217[CrossRef][Medline] [Order article via Infotrieve] |