(Received for publication, December 27, 1994; and in revised form, July 7, 1995)
From the
In this work, a new family of Conus peptides, the
A-conotoxins, which target the nicotinic acetylcholine receptor,
is defined. The first members of this family have been characterized
from the eastern Pacific species, Conus purpurascens (the
purple cone); three peptides that cause paralysis in fish were purified
and characterized from milked venom. The sequence and disulfide bonding
pattern of one of these,
A-conotoxin PIVA, is as follows: where O
represents trans-4-hydroxyproline. The two other peptides
purified from C. purpurascens venom are the
under-hydroxylated derivatives,
A-conotoxin PIVA and
[Pro
]
A-conotoxin PIVA.
The peptides
have been chemically synthesized in a biologically active form. Both
electrophysiological experiments and competition binding with
-bungarotox- in demonstrate that
A-PIVA acts as an antagonist
of the nicotinic acetylcholine receptor at the postsynaptic membrane.
Many venomous animals paralyze their prey by inhibiting
communication between motor neurons and the skeletal muscles they
innervate. In many instances, a key molecular target of the neurotoxins
in the venom is the nicotinic acetylcholine receptor (nAChR) ()in the postsynaptic membrane at the neuromuscular
junction. The first nAChR-targeted venom components to be extensively
characterized were the
-neurotoxins from snakes, typified by
-bungarotoxin from the Formosan krait, Bungarus
multicinctus. Another large class of toxin molecules that inhibit
the nAChR at the vertebrate neuromuscular junction are the
-conotoxins, which have been characterized from three different
fish-hunting Indo-Pacific Conus species: Conusgeographus(1) , Conusmagus(2) , and Conusstriatus(3, 4) .
All of the
-conotoxins from fish-hunting Indo-Pacific cone snails show high
structural homology and a conserved sequence motif. The seven peptides
that have been purified share the consensus sequence XCC(H/N)PACGXX(Y/F)XC. All of these peptides
appear to be potent blockers of neuromuscular transmission in teleosts
but differ significantly in their potency when tested in other
vertebrates; thus,
-conotoxin GI appears to be very potent in
blocking the nicotinic acetylcholine receptor at the neuromuscular
junction of all vertebrates tested, while
-conotoxins SI and SII
are highly potent in teleosts, but not mammals(3, 4) .
In addition to the
-conotoxins from Indo-Pacific fish-hunting Conus, two
-conotoxins have been purified from
non-fish-hunting Indo-Pacific species(5, 6) . Although
the peptides purified differ significantly from the consensus sequence
given above, they do retain the same pattern of Cys residues (the
``Cys framework''), i.e.X
CCX
CX
CX
.
Because this basic motif was found in both fish-hunting and
non-fish-hunting Conus species, it seemed reasonable to expect
that all nAChR-targeted peptides in Conus venoms would share
the same conserved -conotoxin Cys framework. In this report, we
describe a novel nAChR-targeted conotoxin from C. purpurascens, the purple cone (Fig. 1). C. purpurascens is definitely a piscivorous snail, and it was
therefore a surprise to find that in this species, the group of
peptides that blocks nicotinic acetylcholine receptors does not have a
Cys framework typical of
-conotoxins.
Figure 1: A, a specimen of the purple cone, C. purpurascens, from the Cocos Islands, in the Eastern Pacific marine province. B, C. purpurascens with its proboscis extended, in response to the presence of a fish. A dose of venom (about 5 µl) has been transferred from the venom duct to the proboscis. If the snail is able to strike the fish, it will inject the venom through a disposable harpoon-like tooth, which it has already transferred from a quiver-like organ, the radula sac, to the tip of the proboscis. Photograph by Alex Kerstitch.
A unique feature of the study described below is that in contrast to all previous biochemical studies on Conus venoms, which were carried out with dissected venom ducts, the venom used here was milked from living animals. The milking procedure we have developed allows us to harvest much larger amounts of venom from relatively few cone snail specimens.
Figure 2: Milking procedure. A, a fish is placed in front of the snail until it extends its proboscis. The collection tube (surrogate fish) is quickly substituted for the fish as the snail is about to strike. B, once the snail has harpooned the collection tube, the harpoon is cut off. Rightpanel shows the collection tube assembly. The cap of a microcentrifuge tube is hollowed out and placed over a membrane with a cut up fish tail to seal the top of the tube. C. purpurascens will generally be reluctant to strike unless the tip of its proboscis actually contacts fish tissue; the fish tail suffices for this purpose. Once the venom has been collected, the microcentrifuge tube is resealed with its own cap and some parafilm, and the venom is collected by centrifugation.
There seemed to be considerable variation in how often it was possible to milk individual snails. Some specimens seemed unable or unwilling to harpoon the surrogate at all under these conditions, but the majority of the specimens of C. purpurascens could be milked regularly twice a week. Most of the specimens of C. purpurascens could be milked on this schedule for 6 months to a year. Among the other Conus species milked by this procedure are: C. striatus, Conus obscurus, Conus stercusmuscarum, Conus ermineus, Conus monachus and Conus catus.
Peptide
was removed from the resin and deprotected by treatment (2 h, 20
°C) with trifluoroacetic
acid/HO/ethanedithiol/phenol/thioanisole (36/2/1/3/2 by
volume), and the whole mixture was filtered rapidly into t-butyl methyl ether at -10 °C. The precipitate was
collected by centrifugation, dissolved in 60% CH
CN
containing 0.092% trifluoroacetic acid, and diluted 10-fold with 0.1%
trifluoroacetic acid in water. Linear peptide was purified by
reversed-phase HPLC on a C
preparative column (Vydac
218TP1022, 20 ml/min), with a gradient of CH
CN
(6-30%) in 0.1% trifluoroacetic acid. The peptide solution was
diluted to approximately 20 µM, glutathione was added to
give final concentrations of 1.0 mM reduced and 0.5 mM oxidized, and the pH was adjusted to 7.5 with NaOH.
[Pro] Conotoxin PIVA was built using
standard t-butyloxycarbonyl chemistry on a Beckman 990B
synthesizer; couplings were carried out with dicyclohexylcarbodiimide
in dimethylformamide or dichloromethane/dimethylformamide. All amino
acids were from Bachem (Torrance, CA). Side-chain protection, synthetic
methodology, cleavage, air oxidation and desalting on Bio-Rex 70 were
essentially as described for
-conotoxin GVIA(9) .
Histidine was protected as the p-toluenesulfonyl derivative
and coupled using hydroxybenzotriazole and
benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphinium
hexafluorophosphate.
Purification was carried out on a Vydac
C column (5
30 cm) using a gradient of
CH
CN in TEAP buffer at pH 5.30 (6-21%/60 min; 100
ml/min). Fractions were collected and screened for purity by analytical
HPLC on a Vydac C
column (0.46
25 cm) eluted
isocratically with 16.2% CH
CN in 0.1% trifluoroacetic acid
(2 ml/min). Samples were also analyzed by capillary electrophoresis at
20 kV, using 0.1 M sodium phosphate at pH 2.50 and a fused
silica capillary (50 cm
75 µm). Fractions of the highest
purity were pooled and repurified/desalted on the same preparative
column, using a gradient of CH
CN in 0.1% trifluoroacetic
acid (6-36%/60 min; 100 ml/min).
Figure 3:
Panel A, reversed-phase HPLC chromatogram
of C. purpurascens milked venom. 0.5 ml of milked venom was
injected into a C Vydac preparative column, and peptides
were eluted with a gradient of CH
CN (0-36%) in
trifluoroacetic acid as described under ``Materials and
Methods.'' PeaksA, B, and C with arrows correspond to three forms of
A-conotoxin
PIVA. Panel B, further purification of peakA with an analytical C
Vydac column. A small portion of peakA was purified using a gradient of
CH
CN (6
24%) in trifluoroacetic acid as described under
``Materials and Methods.'' PanelC, the
peptide purified from panelB was reloaded on the
column and eluted under the same conditions as panelB. A single homogeneous peak was
obtained.
After completion of the
synthesis of peptides A and C (below), and completion of the biological
characterization, more snails became available, allowing reisolation of
all three isoforms. LSIMS analysis was successfully carried out on
intact peptides, and the monoisotopic molecular ions MH were in agreement with those predicted from the above sequences:
A (observed, 2648.0; theoretical, 2647.93); B (observed, 2631.9;
theoretical, 2631.93); C (observed, 2615.9; theoretical, 2615.94).
Figure 4:
A,
reversed-phase HPLC chromatogram of A-conotoxin PIVA, partially
reduced by TCEP (10 mM, pH 3, 5 min at 61 °C). The
absorbance peaks labeled 1, 2, and 3 correspond to the partially reduced peptides PR1, PR2, and PR3; N and R correspond to the native and completely
reduced peptides, respectively. An analytical C
Vydac
column was used for separating individual peaks using a gradient of
CH
CN (6-30%) in trifluoroacetic acid (see
``Materials and Methods''). B, disulfide bridge
arrangements in native and partially reduced
peptides.
Peptide C was constructed using t-butyloxycarbonyl
chemistry. Crude linear peptide was air-oxidized by stirring at pH 7.0;
the reaction was judged complete after 4 days. Purification was carried
out by adsorption onto Bio-Rex 70 cation exchanger, followed by
preparative HPLC using TEAP buffers, with a final desalting and
purification by HPLC in the trifluoroacetic acid system. TEAP at pH
5.30 was found to be preferable to the usual TEAP, pH 2.25, since the
latter did not yield high purity fractions or a good yield of purified
peptide. The advantage of using TEAP at various pH values has been
recognized previously in the purification of synthetic
peptides(12) . The resulting peptide, which co-eluted with
material isolated from venom, appeared to be 99% pure as analyzed by
analytical HPLC and capillary electrophoresis. LSIMS showed the
expected molecular ion MH (observed, 2616.0;
theoretical, 2615.94).
The major refolded material for both synthetic peaks A and C proved to be identical to their native counterparts; a mixture of native and synthetic peptide in each case gave a single sharp peak upon HPLC analysis (see Fig. 5). Both synthetic peptides were found to be biologically active by the fish paralysis assay. The chemical synthesis of these two peptides therefore confirms the sequence assignments given above, including the C-terminal amidation.
Figure 5:
A,
reversed-phase HPLC chromatogram of synthetic A-conotoxin PIVA
(fully hydroxylated peptide). The peptide was eluted from a C
Vydac analytical column with a gradient of CH
CN
(6-24%) in trifluoroacetic acid (see ``Materials and
Methods''). B, co-elution experiment. Equal amounts of
the synthetic and native peptides were mixed and co-eluted using the
same column, gradient, flow rate, and buffers as in panelA.
Because these peptides, like the -conotoxins,
inhibit the nicotinic acetylcholine receptor (see below), we have
designated peak A
A-conotoxin PIVA, where the Roman numeral
indicates the new structural class. (
)Since peaks B and C
are clearly under-hydroxylated derivatives of peak A, we designate them
as [Pro
]
A-conotoxin PIVA and
[Pro
]
A-conotoxin PIVA. However, the
proportion of the under-hydroxylated forms (peaks B and C) relative to
the fully hydroxylated toxin (peak A) varies from one milked venom
preparation to the next. Although we previously detected
under-hydroxylated forms of other conotoxins (see, for example (13) ), they have never been major species in any venom
previously characterized. The under-hydroxylated forms may arise as a
consequence of artificial aquarium conditions.
Figure 6:
A, exposure to 10 µM [Pro]
A-conotoxin PIVA reversibly
blocks endplate currents. The motor nerve of a frog cutaneus pectoris
muscle whose ACh receptors were partially blocked by
-bungarotoxin
was stimulated every 30 s, and the evoked synaptic currents from a
population of endplates was recorded extracellularly (see
``Materials and Methods''). Peak amplitudes of the evoked
synaptic currents are plotted as a function of time.
[Pro
]
A-conotoxin PIVA was applied at time
0. Open circles, responses before exposure to, and during
washout of, toxin. Closed circles, responses in the presence
of toxin. The response was rapidly blocked when the peptide was
introduced and recovered relatively slowly upon peptide washout. B, exposure to 1 µM
[Pro
]
A-conotoxin PIVA reduces the
endplate current amplitude 3-fold without affecting its time course. Boldsolidtrace, control response in the
absence of toxin. Finer solid trace, response in the presence
of toxin. Dotted trace, response in the presence of toxin
normalized with respect to the control response by 3-fold expansion of
its verticalaxis. The normalized trace of the
response in toxin coincides with the control trace, indicating that the
toxin attenuates the postsynaptic response without altering its
kinetics. Each trace represents the average of eight responses obtained
under each condition. Rapid transients at t
2 ms are stimulus
artifacts.
This possibility was supported by competition binding experiments
with -[
I]bungarotoxin used as a reporter
for receptor occupancy of high affinity sites in the Torpedo electric organ, which are well-established to be on the nicotinic
acetylcholine receptor. The results of binding experiments with
A-conotoxin PIVA and the Pro
derivative are shown
in Fig. 7. Both peptides competitively inhibit
-bungarotoxin binding, indicating that these peptides target the
macrosite that
-bungarotoxin binds to on the Torpedo receptor.
Figure 7:
Competition binding of PIVA versus
-[
I]bungarotoxin. Competition binding with Torpedo electroplax membrane was performed as described under
``Materials and Methods.'' Data points are the mean of three
determinations at each concentration. B
is the
amount of
-[
I]bungarotoxin bound in the
absence of competing peptide. The opencircles and closedtriangles are points for
A- and
[Pro
]
A-conotoxin PIVA,
respectively.
A survey of the in vivo biological activity of these peptides is shown in Table 2.
The results described above establish that the major
paralytic toxin in C. purpurascens venom targeted to
nAChRs, A-conotoxin PIVA, has a strikingly different amino acid
sequence from all other nAChR-targeted peptides previously
characterized from Conus venoms. Despite the striking structural divergence, the mechanism by which this peptide
causes paralysis is nevertheless similar to the well-characterized
-conotoxins from other fish-hunting Conus species, i.e. the peptide blocks the ACh binding site of the nAChR at
the neuromuscular junction.
Comparison of the sequence of this new
toxin, A-conotoxin PIVA, with previously characterized
-conotoxins is shown in Table 3. With one exception
(
-conotoxin SII), the latter toxins have two disulfide bonds; in
contrast,
A-PIVA has three. Furthermore, all paralytic
-conotoxins from fish-hunting Conus share the following
conserved patterns of Cys and non-Cys amino acids, i.e. CCX
CX
C. The
-conotoxins from non-fish-hunting Conus species that have
been described so far(5, 6) , while having spacing
different from that for fish-hunters as indicated above, nevertheless
have the same Cys framework. In contrast, the new peptide,
A-PIVA,
not only has a different Cys framework, but the spacing between the
first pair of Cys residues and the third Cys is 7 amino acids instead
of the 3 or 4 amino acids found in the nine characterized
-conotoxins. Thus, the new peptide is the first member of a new
family of nAChR-targeted Conus peptides. Homologs of the C. purpurascens peptide will be designated
A-conotoxins
(because of the divergence in the Cys framework, peptides belonging to
this structural class will be given Roman numeral IV, as opposed to the
regular
-conotoxins, which are always numbered I or II). In
addition, Conus peptides with a Cys framework similar to that
of PIVA but which are not targeted to the nAChR (and are
therefore not
A-conotoxins) have also been characterized. (
)
Thus, A-PIVA is both the first member of a new
family of Conus peptides, the
A-conotoxins, and the first
representative of a new structural class of Conus peptides.
The availability of a new group of nAChR-targeted Conus peptides that have significantly diverged from the
-conotoxin
series provides new opportunities for probing the nAChR. Previously,
reporter groups were attached to
-conotoxins at specific
loci(14) ; such an approach can in principle be used to map the
topology of the nAChR (15) . Since Conus peptides are
extensively cross-linked by disulfide bonds and are, therefore, fairly
rigid, they provide a structurally discrete probe, which can be used to
pinpoint the locations of residues in the receptor. Some
-conotoxins have been analyzed by multidimensional NMR
techniques(16, 17) ; sufficient amounts of
A-conotoxin PIVA have been synthesized, and a structural analysis
of these peptides by NMR methods is presently being carried out. Once
the structural work is complete,
A-PIVA could be a useful probe
for the nAChR based on an entirely different structural framework from
that of the
-conotoxins.
The degree of under-hydroxylation of
proline residues in A-conotoxin PIVA deserves comment. Different
samples of milked C. purpurascens venom show
considerable variation in the degree of under-hydroxylation; in some
venom samples, the two under-hydroxylated species described above are
present at higher levels than the completely hydroxylated
A-PIVA
(in contrast to the venom sample shown in Fig. 3A).
Small differences in IC
s shown in Fig. 7are seen
reproducibly, raising the possibility that the different hydroxylated
forms have functional biological significance. We think that at least
some of the under-hydroxylation observed may be an artifact of
maintaining C. purpurascens in aquaria for extended
periods of time. We have observed that several different Conus species become increasingly susceptible to pathology both in the
periostracum and in laying down new shell if kept in aquaria in
artificial sea water. Thus, it is possible that some under-hydroxylated
forms observed in the milked venom are present at lower levels in C. purpurascens under natural conditions and that the
under-hydroxylation observed may be a biochemical manifestation of a
progressive pathology that occurs in aquaria. It is possible that some
factors necessary for proline hydroxylation may become limiting. The
pattern of under-hydroxylation suggests that the Pro residues are not
equivalent as substrates for the hydroxylation enzyme, and that the
ease of hydroxylation is in the order: Pro
> Pro
> Pro
. In any case, the results in Fig. 7were unexpected and surprising, and further studies
investigating functional effects of proline hydroxylation are clearly
desirable.
C. purpurascens is believed to be the
only fish-hunting Conus species in the eastern Pacific marine
geographic province. It has probably been isolated from fish-hunting
Indo-Pacific species for an extended period of time. Its closest
relative is thought to be C. ermineus, the major
fish-hunting Conus species in the Atlantic marine province. An
A-conotoxin that differs significantly in sequence from
A-conotoxin PIVA has recently been identified in this species. (
)The discovery of the nAChR-targeted peptides in C. purpurascens that are so divergent from those
found in other Conus species raises the intriguing possibility
that fish-hunting may have evolved more than once in the Conidae. It
will be of interest to determine which Conus species use the
A-conotoxin family as nAChR ligands instead of members of the
-conotoxin family.