From the
Tetanus toxin and clostridial neurotoxins type B, D, F, and G
inhibit intracellular Ca
Neurotransmitter release occurs via a
Ca
Among the three
synaptic proteins cleaved by clostridial neurotoxins, VAMP appears to
be of special interest because it is the target of five of these
neurotoxins (3, 4) and is the only putative vesicular
SNAP receptor so far identified(10) . VAMP homologs are highly
conserved in eucaryotes, from yeast to human(14, 15) .
In all species, the cytoplasmic domain of VAMP consists of a N-terminal
proline-rich region followed by a large and highly conserved
hydrophilic core (Fig. 1). VAMP is anchored in the synaptic
vesicle membrane by a short lipophilic domain. Its C-terminal region is
intravesicular and of variable size. The first Met residue is probably
removed during maturation, and, most likely, the N-terminal end is
capped by an acetyl group(11, 16) . VAMP is hydrolyzed
at distinct sites by TeNT and BoNT/B, /D, /F, and /G (see Fig. 1and, for review, see Refs. 3 and 4). The enzymatic cleavage
of VAMP by these neurotoxins results in the release of a large
N-terminal cytosolic fragment whose size varies with the neurotoxin
type. Because they are released in the cytoplasm, these soluble
fragments may compete with endogenous VAMP in its function, thus
leading to an alteration of neurotransmission. Our aims were (i) to
test whether a synthetic fragment corresponding to that released by one
of these toxins (for instance TeNT) can perturb neurotransmission when
intracellularly applied and (ii) if so, to identify the putative VAMP
domains underlying this action. The best model for this purpose is
dissected ganglia from the central nervous system of Aplysia
californica as some of their largest neurons make identified
synapses (17) and afford an easy access to intracellular
spaces(5, 18, 19) . In addition,
neurotransmitter release can be easily quantified using conventional
electrophysiological techniques (for details, see Ref. 18).
The complete blockade of neurotransmission that follows the
proteolytic removal of VAMP from the surface of synaptic vesicles by
several clostridial neurotoxins and the fact that this protein was
found in a fusogenic protein complex comprising the two t-SNAP
receptors, syntaxin and SNAP-25, led to the implication of VAMP in the
exocytotic process of small synaptic
vesicles(3, 4, 10, 11, 16, 24) .
Nevertheless, until now, the mechanism of action of VAMP-synaptobrevin
in the neurotransmitter release process has been unclear. In addition,
several attempts to characterize in vivo the role of VAMP
using anti-VAMP antibodies have been
unsuccessful(5, 25, 26) . Our in vivo experiments show clearly that the potent inhibition of
acetylcholine release induced by the intraneuronal injection in
presynaptic neurons of A. californica of N-terminal
proline-rich fragments of VAMP involves the N-terminal region of this
small protein in the regulated secretion of neurotransmitters.
Comparison of the minimum active domain of Aplysia VAMP with
the N terminus of the human VAMP II isoform suggests that the
functional sequence could comprise a
PGGPXGX
The neuronal effects observed with the
N-terminal domain of VAMP suggest a competition of VAMP N-terminal
fragments with endogenous VAMP in its interaction with proteins of the
release machinery. However, the long lasting inhibition of
neurotransmitter release induced by VAMP or its N-terminal fragments
reached a maximum of 50%, contrasting with the complete blockade
ensured by intraneuronal injection of TeNT alone (Fig. 2a) or in the presence of inhibitory peptides (Fig. 5d). This suggests that the mechanism of action of
TeNT and by inference of BoNT/B, /D, /F, and /G is probably not related
to toxin-induced release of inhibitory peptides but rather to a
dissociation of the N-terminal part of synaptobrevin from the synaptic
vesicle. Moreover, these results could be interpreted in the light of
recent in vitro results. Thus, in neurosecretory cells,
synaptobrevin is distributed in two main pools. One corresponds to the
20 S complex, which include the SNAPs, syntaxin, SNAP-25, and the
vesicular protein synaptotagmin(10, 24) . In this
complex, synaptobrevin was shown to strongly interact with syntaxin and
SNAP-25 by its well conserved domain (sequence
27-96)(29, 30, 31) . In the second pool,
about 40-50% of VAMP/synaptobrevin was also recently found to be
associated in vitro with
synaptophysin(32, 33, 34) , a glycosylated
protein with four transmembrane helices and a long intracytoplasmic
domain enriched in tyrosine residues, which shows the propensity to
form channels in membranes(35) . This in vitro interaction was shown to be blocked to 60-65% at
concentration of 0.18 mM by the 1-32 N-terminal fragment
of rat VAMP II but not rat VAMP I (sequence 1-34), showing that
the interaction synaptophysin-VAMP II is mediated by the N-terminal
domain of VAMP II(33, 34) . The interaction between VAMP
and synaptophysin was proposed to inhibit the oligomerization of this
latter protein to form a pore or to interact with its synaptic targets
with subsequent formation of a fusion pore(32) . Indeed
synaptophysin was not found in the 20 S complex, suggesting that the
secretion process could be ensured by a disruption of the cytoplasmic
synaptophysin-synaptobrevin 56-kDa complex allowing reconstitution of
the temporary assembly of the 20 S complex and the oligomerization of
synaptophysin to transiently form transmembrane channels ensuring
neurosecretion (Fig. 6A)(32) .
In conclusion, in this study
the N-terminal proline-rich domain of VAMP II isoforms has been
identified as playing a physiological role in neurotransmitter release
certainly due to its interaction with synaptophysin. Since this domain
is clearly distinct from the region involved in the interaction with
SNAP receptors (domain 27-96), this confirms that in vivo synaptobrevin has more than one role in neuroexocytosis.
An
important observation is the potent antagonist properties of peptides
as short as 16 residues (in the case of Aplysia) toward the
Ca
We thank D. Ficheux and L. Martin for helpful
technical assistance and Dr. A. Beaumont for stylistic revision of the
manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-dependent neurotransmitter
release via the specific proteolytic cleavage of vesicle-associated
membrane protein (VAMP)/synaptobrevin, a highly conserved 19-kDa
integral protein of the small synaptic vesicle membrane. This results
in the release of the larger part of the cytosolic domain of this
synaptic protein into the cytoplasm. Microinjection of synthetic
peptides corresponding to this fragment into identified presynaptic
neurons of Aplysia californica led to a potent, long
lasting, and dose-dependent inhibition (
50% at 10 µM)
of acetylcholine release, probably by hindering endogenous
VAMP/synaptobrevin from interacting with synaptic proteins involved in
exocytosis. Structure activity studies showed that this effect is
confined to the N-terminal domain of VAMP/synaptobrevin isoform II and
is related to the presence of a proline-rich motif
(PGGPXGX
PP or
PAAPXGX
PP). At higher concentrations, the
inhibitory effect was lower and only transient, suggesting that the
N-terminal proline-rich domain of VAMP/synaptobrevin plays opposing
roles in neurotransmitter release very likely by interacting with
different synaptic proteins. This probably occurs by disruption of the
recently reported in vitro VAMP-synaptophysin interaction
that involves the N-terminal domain of VAMP II and was proposed to
hinder synaptophysin-related formation of a fusion pore. The observed
recovery of neurotransmitter release following injection of high
concentration of N-terminal fragments of VAMP II brings a strong in vivo support to this hypothesis. The minimum active
peptide GPGGPQGGMQPPREQS could be used for rationally designing potent
synthetic blockers of neurotransmission.
-regulated exocytosis of the content of synaptic
vesicles. Over the past year, a remarkable convergence of data has
given insights into the exocytotic machinery, and several proteins
involved in vesicle docking and/or membrane fusion processes have been
discovered(1, 2) . Among them, three proteins are
specific intracellular substrates of proteolytic neurotoxins, namely
tetanus and botulinum neurotoxins(3, 4) . More
precisely, tetanus toxin (TeNT)
(
)and botulinum
neurotoxins (BoNT)/B, /D, /F, and /G cleave vesicle-associated membrane
protein (VAMP)/synaptobrevin, an integral protein of the small synaptic
vesicle membrane, whereas the target of BoNT/A and /E is 25-kDa
synaptosomal-associated protein (SNAP-25), a protein associated with
the plasma membrane. For its part BoNT/C cleaves syntaxin, an integral
plasma membrane protein (for review, see Refs. 3 and 4). Because the
cleavage of these three synaptic proteins, in vitro (see
references quoted above), correlates with the inhibition of
neurotransmitter release induced by TeNT and BoNTs, in
vivo(5, 6, 7, 8) , it has been
inferred that the cleavage of either VAMP, SNAP-25, or syntaxin
prevents synaptic transmission. Collectively, these findings point out
a key role for these synaptic proteins in neurotransmitter release, but
their respective roles remain unclear. The three proteins associate
tightly together and with synaptotagmin (9, 10) and N-ethylmaleimide-sensitive fusion protein to form a 20 S
complex thought to be involved in the fusion of vesicles with membranes
(11). Therefore VAMP, SNAP-25, and syntaxin have been postulated to be
involved in either the docking or the fusion of synaptic vesicles with
the plasma membrane at active zones of nerve
terminals(2, 12, 13) .
Figure 1:
A, alignment of the isoform II of the human VAMP (41) and of Aplysia VAMP (21). The sequence of the C-terminal
intravesicular tail of the Aplysia isoform is not reported. B, schematic structure of VAMP. a and b, cytosolic domain comprising a variable N-terminal portion (a) and a highly conserved region (b); c membrane-anchoring domain; and d, C-terminal tail
protruding in the lumen of synaptic vesicles. C, the
sites of cleavage of Aplysia VAMP by clostridial neurotoxins
are indicated according to Yamasaki et al. (21, 40) and
Niemann et al. (4). N-terminal sequences of various VAMPS are
as follows: 1, Di Antonio et al. (41); 2,
Südhof et al. (14); 3, Yamasaki et al. (21); 4, Hunt et al. (27); 5, Trimble et al. (42); 6, Elferink et al. (43); 7, Archer et al. (39); and 8, Mac Mahon et al. (28).
Peptide Syntheses
Assembly of the protected
peptide chain was carried out using the stepwise solid-phase method of
Merrifield (22) on an Applied Biosystems 431A peptide synthesizer, with
dicyclohexylcarbodiimide/hydroxybenzotriazole as coupling reagents and N-methylpyrrolidone as solvent. Fluoren-9-ylmethoxycarbonyl
was employed as temporary protecting groups for amino acid
amines, and deprotection was achieved by treatment with piperidine. The
syntheses were run on a 0.1-mmol scale (114 mg of Wang resin loaded at
0.88 mmol of hydroxyl group/g of dry resin) except for the large
peptides A-syb 1-83, ac-A-syb 2-66, A-syb 20-83,
H-syb II 1-93, and H-syb II 24-93 for which successive
removal of peptidyl-resin at different steps of the synthesis led us to
finish on a 25-µmol scale. Peptides with proline in the last or
penultimate position were synthesized with dichlorotrityl resin, which
is known to prevent diketopiperazine formation during synthesis. The
peptide resin was treated for 2 h with 40 ml of trifluoroacetic acid in
the presence of phenol (3 g), 1,2-ethanedithiol (1 ml), thioanisole (2
ml), and H
O (2 ml) in order to cleave the peptide from the
resin and to remove the protecting groups of the amino acid side
chains. Purifications of the peptides were carried out by reverse phase
HPLC on a Vydac C4 column (250
10 mm) with a linear gradient of
10-60% B in 90 min (A, 0.1% trifluoroacetic acid in
H
O; B, 0.09% trifluoroacetic acid, 70% acetonitrile in
H
O; flow 1.5 ml/min; detection wavelength, 214 nm). The
purified peptides were then lyophilized and kept as a powder at
-80 °C. Their purity was checked by amino acid analysis,
capillary electrophoresis, and electrospray mass spectrometry.
Electrophysiological Measurements of Acetylcholine
Release in A. californica
Experiments were performed at 22
°C on identified cholinergic synapses in the buccal ganglion of Aplysia. In this ganglion, two cholinergic presynaptic
neurons, B4 and B5, make well known chloride-dependent inhibitory
synapses with the same postsynaptic cells as B3, B6, and several
others(17) . Both presynaptic cells (125-180 µM in diameter) were impaled with two glass microelectrodes (3 M KCl Ag/AgCl, 4-10 megaohms). Alternately,
once a minute, a presynaptic neuron was depolarized to elicit an action
potential. The ensuing postsynaptic response was recorded in the
postsynaptic neuron as a current change using the conventional
two-electrode voltage-clamp technique. Subsequently, by taking into
account its reversal potential, i.e. the null potential for
Cl
, the postsynaptic response was expressed as
membrane conductance, a value that is directly proportional to the
amount of acetylcholine released per impulse (for details, see Refs. 18
and 19).
Injection of Peptides
Buccal ganglia were
continuously superfused (30 ml/h, experimental chamber volume of 1 ml)
with a physiological medium (artificial sea water) containing 460
mM NaCl, 10 mM KCl, 11 mM CaCl,
25 mM MgCl
, 28 mM MgSO
, and
10 mM Tris-HCl, pH 7.8. Before intracellular administration,
peptides dissolved in 50 mM Tris-HCl, pH 7.8, 100 mM NaCl were mixed together with a solution of dye (10% by volume;
fast green FCF, Sigma). Peptides were air pressure-injected (1% by
volume of the cell body) into one of the two presynaptic neurons under
visual and electrophysiological monitoring. For this purpose, a third
electrode was implanted into the cell body and was removed after
injection.
The VAMP Fragment Released by TeNT Inhibits
Neurotransmission
Intracellular injection of TeNT (Fig. 2a) into identified presynaptic neurons of the
buccal ganglion of Aplysia produces a fast and complete
blockade of neurotransmitter release(20) . This results from the
cleavage of Aplysia VAMP at the Gln-Phe
bond(21, 22) . Given that the N-terminal fragment
is released into the cytosol, an Aplysia VAMP acetyl
2-66 peptide (ac-A-syb 2-66) was synthesized by solid-phase
peptide synthesis (23) using the Fmoc
(Fluoren-9-ylmethoxycarbonyl)/t-butyl strategy(22) .
All of the peptides used in this study were synthesized by this method.
In all cases, the peptidyl-resin was cleaved using trifluoroacetic acid
in the presence of scavengers, and the crude peptide was purified by
reverse phase HPLC. The purity of each peptide was checked by amino
acid analysis, capillary electrophoresis, and electrospray mass
spectrometry and was estimated to be higher than 95%. The synthetic
ac-A-syb 2-66 peptide was microinjected as reported previously (18, 19) into presynaptic neurons of Aplysia at
a final intrasomatic concentration of 10 µM. This resulted
in an almost 50% inhibition of acetylcholine release as shown by the
depression of the amplitude of the postsynaptic responses evoked by
presynaptic action potentials (Fig. 2b). The inhibition
of acetylcholine release was maximal within 1 h following injection,
and it remained for several hours (>6 h, not shown). Importantly, no
alteration of the presynaptic action potential was detected. This
suggested that the inhibition of neurotransmission was not the
consequence of a change in nerve conduction properties. In addition,
neither the time to peak nor the decay time of the postsynaptic
responses were modified (not shown). This indicated that the kinetics
of the release process remained unchanged.
Figure 2:
The fragment of VAMP released by TeNT
induces a 50% inhibition of neurotransmitter release in Aplysia. Neurotransmitter release was evoked at identified
cholinergic synapses in the buccal ganglion of Aplysia and
monitored (percent) following the microinjection of TeNT (A)
or of various fragments of VAMP into the cell body of a presynaptic
(either B4 or B5) neuron (B-D). The release of
neurotransmitter was quantified via the measurement of the amplitude of
postsynaptic responses recorded as described elsewhere (18). The
peptides were synthesized according to Cornille et al. (23);
they span various domains of either Aplysia of human isoform
II VAMPs (A-syb and H-syb II, respectively). The first and last amino
acids are indicated as numbers according to the published sequences
(21, 42). Note that the ac-A-syb 2-66 peptide corresponds to the
fragment of VAMP released in Aplysia neurons by either TeNT or
BoNT/B. The final intracellular concentration of each peptide is
indicated. Each of the panels reports a typical experiment from a
series of 3-5. The average inhibition value obtained for an
intraneuronal injection of 10 µM of the following peptides
is given: A-syb 2-66 (45 ± 3%), A-syb 1-83 (39
± 6%), H-syb-II 1-93(44 ±
4%).
The injection of a
C-terminally extended peptide encompassing the whole cytoplasmic domain
of Aplysia VAMP (A-syb 1-83) at the same intracellular
concentration as ac-A-syb 2-66 did not lead to a greater
inhibition (Fig. 2c). Because instead of an acetyl
group, the first Met was present in this peptide; this result suggested
that, most likely, the processing of the Met and the capping of
Ser are not essential for the intracellular activity of the
peptide. These data suggested also that TeNT might induce inhibition of
neurotransmitter release, at least partially, by generating an
inhibitory peptide. However, since a total inhibition of release was
observed upon intraneuronal injection of this toxin (Fig. 2a), it was important to test whether a total
blockade of transmission could be obtained following microinjection of
higher concentrations of the released cytosolic fragment of VAMP.
However, the intracellular concentration of the ac-A-syb 2-66 and
A-syb 1-83 peptides could not be increased due to solubility
problems in the injection micropipette (note that to reach an
intracellular concentration of
100 µM requires an
intrapipette concentration of 10 mM). This problem was solved
later by the identification of a smaller domain with similar inhibitory
properties but with increased solubility.
The Inhibitory Domain of VAMP Is Confined in its
N-terminal Portion
The cytosolic region of VAMP has a variable
N-terminal domain and a central region that is conserved among various
species (Fig. 1C). Thus, it was postulated that a
comparison of the abilities of various VAMPs to interfere with
neurotransmission might help in the identification of the inhibitory
domain. A peptide corresponding to the whole cytosolic domain of the
human II isoform was synthesized (H-syb II 1-93); it differed
mainly from the A-syb 1-83 peptide by its first 26 amino acids
and several punctual changes in the conserved domain (Fig. 1).
Intraneuronal injection of H-syb II 1-93 (10 µM final) induced a 45% inhibition of acetylcholine release (Fig. 2d) similar, on a molar ratio, to that obtained
with either A-syb 2-66 or A-syb 1-83 (Fig. 2b). Since Aplysia and human II VAMPs
differ mainly in their N-terminal portion, the highly conserved region
(H-syb II 24-93 and A-syb 20-83) was suspected to contain
the protein domain responsible for this inhibition. Therefore, the
corresponding peptides were synthesized and intraneuronally applied,
each at 10 µM; no alteration in the neurotransmission was
detected (Fig. 3c). Contrastingly, intracellular
application of peptides corresponding to the variable N-terminal
proline-rich part of the two isoforms tested A-syb 1-19 and H-syb
II 1-26 () blocked neurotransmitter release to the
same extent and over a similar time scale (Fig. 3b) as the longer
peptides (A-syb 2-66, A-syb 1-83, and H-syb II 1-93)
( Fig. 2and 4).
Figure 3:
The
proline-rich domain of VAMP is responsible for the inhibition of
neurotransmitter release in Aplysia neurons. a,
schematic representation of synaptobrevin. PPPP represents the proline-rich domain of synaptobrevin. b and c show the same kind of experiments as in Fig. 2. The
final intracellular concentration of each peptide is indicated. Each of
the panels reports a typical experiment from a series of 3-5. The
average inhibition value obtained for an intraneuronal injection of 10
µM of the peptide A-syb 1-19 is 45 ±
6%.
Dual Action of the N-terminal Proline-rich Domain of
VAMP
Because the more soluble A-syb 1-19 and H-syb II
1-26 fragments retained entirely the inhibitory properties of the
ac-A-syb 2-66 peptide or H-Syb II 1-93, it was possible to
perform dose-dependent experiments in order to test whether a complete
inhibition could be reached (Fig. 4). Upon intraneuronal
injection of either A-syb 1-19 or H-syb II 1-26 peptides at
1 µM, the observed inhibition was around 20% and reached
about 50% at 10 µM. However at a final concentration of
100 µM, no gain in inhibition was observed, but, instead,
the extent of inhibition was reduced, and the depression of
neurotransmitter release was only transient (Fig. 4, a-c and d-f). This
suggested that the N-terminal domain of VAMP altered the release
process via two counteracting effects. In the 1-10 µM range, the inhibitory effect predominated, whereas in the
10-100 µM range, an opposite so-called facilitatory
effect superimposed and allowed neurotransmission to recover to near
control levels. This was confirmed in a series of experiments where a
presynaptic neuron was submitted to two injections. When the A-syb
1-19 peptide was first injected at a final concentration of 5
µM, the second injection of 5 µM (2 h later)
produced a further inhibition (Fig. 5a). Contrastingly
when the neuron was injected twice with 10 µM of A-syb
1-19 peptide, the second application produced an increase in
neurotransmission (Fig. 5b). Because the amino acid
sequence of the A-syb 1-19 and H-syb II 1-26 peptides are
different (), it was possible that they produced similar
effects via interactions with different intraneuronal targets. However
this appeared unlikely because coinjection of 10 µM of
each peptide (Fig. 5c) led to the same weak and
transient effect observed with 20 µM of either peptide
(not shown).
Figure 4:
The
proline-rich domain of VAMP from Aplysia (1-19) and
human isoform II (1-26) have the same dual effect on
neurotransmitter release. Inhibition in the 1-10 µM range and this inhibition is lowered and transient at the
10-100 µM range. The same kind of experiments as in
Figs. 2 and 3 are shown. Each of the panels reports a typical
experiment from a series of 3-5. The average inhibition values
obtained for an intraneuronal injection of the following peptides are
as follows: at 1 µM, A-syb 1-19 (20 ± 12%),
H-syb-II 1-26 (18 ± 12%); at 10 µM, A-syb
1-19 (45 ± 6%), H-syb-II 1-26 (44 ±
4%).
Figure 5:
Double injection of A-syb
1-19 at 5 and 10 µM concentrations (a and b). Coinjection of 10 µM A-syb 1-19 and
A-syb 1-26 is shown in c. Injection of 20 nM TeNT following a 50% inhibition of acetylcholine release induced
by injection of 10 µM A-syb 1-19 is shown in d. a-c, the 50% inhibition reached by
injection of 10 µM of A-syb 1-19 peptide can be
reversed by a second injection of 10 µM of the same
peptide or can be total by the addition of 20 nM of tetanus
toxin. d, the A-syb 1-19 and H-syb II 1-26
peptides are acting on the same receptor targets. The coinjection of 10
µM of each of these peptides is similar to the injection
of 20 µM of one of them (not shown). The same kind of
experiments as in Figs. 2-4 are shown.
In addition, an intraneuronal injection of TeNT
(20 nM), 60 min after injection of 10 µM of
A-syb 1-19 peptide giving a half-inhibition of neurotransmitter
release led to a complete inhibition of neurotransmission within 2 h (Fig. 5d). This demonstrated that endogenous vesicular
synaptobrevin molecules remained accessible to the toxin and able to
interact with protein targets involved in the neurotransmitter release
process.
A Motif PX
In order to examine whether the ability of the VAMP
N-terminal fragments to alter neurotransmission was due to the presence
of a common motif, the minimum active domain of the A-syb 1-19
peptide was first determined. Various analogues of this peptide,
shortened at either their N and/or C terminus, were synthesized and
injected into presynaptic neurons of Aplysia. The minimal
active peptide corresponded to the GlyPXGX
PP Is
Responsible for the Dual Effect Observed in Aplysia
Neurons
-Ser
sequence of Aplysia synaptobrevin (A-syb 4-19) (). When intracellularly applied at 10 µM, it
exhibited a 36% inhibitory activity (), and at 100
µM, it induced the typical transient action observed with
A-syb 1-19 or H-syb II 1-26 (not shown). Furthermore, both Aplysia and human VAMP N-terminal domains share the striking
feature of being mainly composed of amino acids with no, or short,
lateral chains (Gly or Ala) and of several proline residues. However
these N-terminal domains differ by their length and by the positions of
these residues in the amino acid sequences. In order to test the
specificity of the responses observed, the synthetic 1-28
fragment of human synaptobrevin isoform I (H-syb I 1-28) () was used. Indeed this peptide shares about the same
amino acid content as H-syb II 1-26 but differs from it by the
relative positions of the proline moieties. Injection of H-syb I
1-28 (at either 10 or 100 µM) into presynaptic
neurons did not modify neurotransmission (). These
observations suggested the existence of a common motif in A-syb
4-19 and H-syb II 1-26, but not in H-syb I 1-28, is
responsible for the intracellular activity of the two former peptides.
This motif is suggested to be
PX
PXGX
PP by
comparison of the sequences of the two active peptides ().
PP or a
PAAPXGX
PP motif. The squid VAMP
8-91 fragment contains also two similar motifs
(PGGPX
PP and PGGPXGPP) in its N-terminal
sequence and, as expected, this 8-91 fragment inhibits partially
neurotransmitter release when injected inside the giant nerve terminal
of squid(27) . This biologically active motif is not found in
vertebrate VAMP I isoforms (for example the inactive H-syb I 1-28
contains a PGX
PXGXPP sequence),
in rat cellubrevin (28) (Fig. 1) or in a Drosophila VAMP(14) .
Figure 6:
Proposed target sites of N-terminal
proline-rich fragments of VAMP II injected into Aplysia neurons. A, synaptobrevin is associated by its central
part with syntaxin and SNAP-25 to form the 20 S complex while
synaptophysin forms oligomers, a step putatively required for a fusion
pore formation and a subsequent neurotransmitter release (32). The
intraneuronal injection of the VAMP peptide 1-19 may prevent the
formation of the 20 S complex (target 1, whitearrow in B) at low concentration (<10 µM)
inhibiting neurotransmitter release. B, synaptophysin
interacts with synaptobrevin to form a 56-kDa complex, and this
interaction prevents oligomerization of synaptophysin (32-34). At
concentration higher than 100 µM, the intraneuronally
injected N-terminal fragments of the VAMP inhibit this protein-protein
interaction (target 2, whitearrow) this facilitating
neurosecretion (see Fig. 4d).
Our in vivo results bring strong physiological support to these proposals,
deduced from in vitro biochemical experiments. Thus, at the
membrane level, the N-terminal peptides of VAMP/synaptobrevin probably
inhibit the binding of endogenous synaptobrevin simply by hindering the
highly conserved domain of the native peptide from reaching its site of
binding to SNAP-25 and syntaxin (Fig. 6B). This
proposition is relevant for the inhibition observed after injection of
N-terminal fragments of VAMP II at concentration below 10
µM. It is interesting to observe that peptides
corresponding to the conserved region of VAMP such as Syb 20-83
are unable to reduce neurotransmitter release (Fig. 3c),
suggesting that these peptides have no accessibility to the complex
formed between VAMP and SNAP-25 and syntaxin. Furthermore, in our in vivo conditions, the intraneuronally injected N-terminal
fragments of synaptobrevin, disjoined from the linked vesicles, behave
as pseudoagonists devoid of intrinsic activities leading to 50%
inhibition of neurotransmitter release. The U-shaped dose-response
curves (Fig. 4, c and f, and Fig. 5, b and c) often observed in pharmacological
experiments, could be due to the interaction of the N-terminal
fragments at higher concentrations (10-100 µM) with
another site resulting in activation of the neurosecretory process.
This interaction could occur at the level of the 56-kDa complex (Fig. 6B) with the N-terminal fragments of synaptobrevin
disrupting the interaction between endogenous synaptobrevin and
synaptophysin, leading to the formation of pores in the plasma membrane
and neurosecretion (Fig. 6A). In vitro, the
inhibition of synaptobrevin-synaptophysin by the 1-32 fragment of
VAMP II occurs also in the concentration range of 0.18
mM(32) , like it was observed in our in vivo conditions. This counteracting process would account for the
reversion of the inhibition of acetylcholine release (Fig. 4, c and f, and Fig. 5, b and c). It is interesting to observe that, in contrast to the
present findings, full and reversible inhibition of transmitter release
was observed following injections of peptide fragments of synaptotagmin
or SNAPs to squid giant presynaptic terminals(36, 37) .
This seems to indicate that, in this case, only one target is
critically involved in the neurosecretory function of these proteins.
Moreover, the reversibility of the inhibition was interpreted by a
dilution of the inhibitory peptides in the axon of squid giant
neuron(36, 37) . Interestingly, injection of 8-91
domain of squid synaptobrevin in the squid giant synapse was reported
to cause a slow and irreversible inhibition of about 60% of
neurotransmitter release(27) , a result in complete agreement
with our observations, supporting a difference in the mechanism of
action of these different proteins in neurosecretion and the occurrence
of two binding sites for synaptobrevin.
-dependent neurotransmitter release. This could
facilitate the development of synthetic blockers of neurotransmission,
which could be used as surrogates for BoNT/A in the therapeutic
treatment of dystonia induced by motoneuronal hyperactivity (strabism,
hemifacial spasm, blepharospasm, etc.)(38) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.