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(Received for publication, June 1,
1995; and in revised form, January 6, 1996) From the
Clostridial neurotoxins are zinc endopeptidases that block
neurotransmission and have been shown to cleave, in vitro,
specific proteins involved in synaptic vesicle docking and/or fusion.
We have used immunohistochemistry and immunoblotting to demonstrate
alterations in toxin substrates in intact neurons under conditions of
toxin-induced blockade of neurotransmitter release. Vesicle-associated
membrane protein, which co-localizes with synaptophysin, is not
detectable in tetanus toxin-blocked cultures. Syntaxin, also
concentrated in synaptic sites, is cleaved by botulinum neurotoxin C.
Similarly, the carboxyl terminus of the synaptosomal-associated protein
of 25 kDa (SNAP-25) is not detectable in botulinum neurotoxin A-treated
cultures. Unexpectedly, tetanus toxin exposure causes an increase in
SNAP-25 immunofluorescence, reflecting increased accessibility of
antibodies to antigenic sites rather than increased expression of the
protein. Furthermore, botulinum neurotoxin C causes a marked loss of
the carboxyl terminus of SNAP-25 when the toxin is added to living
cultures, whereas it has no action on SNAP-25 in in vitro preparations. This study is the first to demonstrate in
functioning neurons that the physiologic response to these toxins is
correlated with the proteolysis of their respective substrates.
Furthermore, the data demonstrate that botulinum neurotoxin C, in
addition to cleaving syntaxin, exerts a secondary effect on SNAP-25.
Clostridial neurotoxins (CNTs) ( VAMP,
SNAP-25, and syntaxin interact with N-ethylmaleimide-sensitive
fusion protein and soluble N-ethylmaleimide-sensitive fusion
protein attachment proteins (SNAPs), cytosolic elements essential for
intracellular membrane fusion(15) . Since SNAPs must bind to
membrane receptors prior to N-ethylmaleimide-sensitive fusion
protein attachment, the SNAP receptors have been designated as SNAREs,
with the vesicular protein (VAMP/synaptobrevin) as the v-SNARE and the
target membrane proteins (SNAP-25 and syntaxin) as the
t-SNAREs(15) . Furthermore, it has been demonstrated that VAMP,
SNAP-25, and syntaxin themselves form a stable
complex(16, 17, 18) . Two isoforms of VAMP
in neuronal tissue and a nonneuronal homologue cellubrevin have been
identified in a number of animal species (19, 20, 21, 22) . VAMP is comprised
of three major domains: the NH Syntaxin has
two isoforms that are anchored to membranes by a single COOH-terminal
transmembrane domain(26, 27) . Syntaxin binds to
presynaptic calcium channels and to synaptotagmin located in the
synaptic vesicle membrane(26, 28, 29) .
Botulinum neurotoxin C cleaves syntaxin at a site near the
transmembrane domain(13, 14, 30) . SNAP-25
is a membrane-associated cytoplasmic protein implicated in the fusion
of synaptic vesicles with the presynaptic membrane (15) and in
membrane addition leading to constitutive axonal growth(31) .
SNAP-25 is a hydrophilic protein that is palmitylated at one to four of
its closely spaced cysteine residues and acts as an integral membrane
protein(32, 33) . Botulinum neurotoxin A cleaves SNAP
25 at a site nine amino acids from the COOH terminus between residues
Gln Tetanus toxin, BoNT A, and BoNT C act in vivo to block
neurotransmitter release. Both BoNT A and TeNT have been shown
previously to block synaptic transmission in spinal cord cell cultures (34, 35) . TeNT-induced disappearance of inhibitory
and excitatory postsynaptic potentials coincides in the same system
with the blockade of inhibitory and excitatory neurotransmitter
release(36) . In the present study, we have characterized the
effect of TeNT, BoNT A, and BoNT C on the vSNARE, VAMP/synaptobrevin,
and the tSNARES, SNAP-25 and syntaxin, in cells in which we have
demonstrated the arrest of neurotransmitter release. This is the first
study to examine VAMP, SNAP-25, and syntaxin in intact functioning
neurons, where it has been possible to observe the action of BoNT C on
two of the three SNARE proteins.
Spinal cord cell cultures contain a heterogeneous population
of neurons growing on a monolayer of nonneuronal cells (Fig. 1A). To confirm that TeNT, BoNT A, and BoNT C
have blocked synaptic neurotransmission, spinal cord cultures are
assayed for inhibitory and excitatory neurotransmitter release.
Cultures exposed to these toxins are radiolabeled with
[
Figure 1:
Neurotransmitter release in spinal cord
cell cultures. A, interference contrast photomicrograph shows
that spinal cord cell cultures are a heterogeneous mixture of neurons. Magnification bar, 25 µm. B and C,
control and toxin-exposed cultures are radiolabeled and assayed for
potassium-stimulated calcium-dependent release of glycine and
glutamate. Tetanus toxin and botulinum neurotoxins A and C (0.06 nM for 20 h) completely block release of the inhibitory
neurotransmitter glycine (B) and the excitatory
neurotransmitter glutamate (C).
Tetanus toxin abolishes synaptic immunostaining in mouse spinal cord
neurons of both VAMP-1 (Fig. 2) and VAMP-2 (not shown). Loss of
VAMP from neuronal terminals after TeNT proteolysis is demonstrated
clearly by double-labeling experiments using mouse anti-synaptophysin,
a marker for synaptic terminals, detected with fluorescein (Fig. 2, A, C, and E) and rabbit
anti-VAMP-1 detected with rhodamine (Fig. 2, B, D, and F). In control cultures, VAMP-1 immunostaining
of synaptic terminals (Fig. 2B) co-localizes with
synaptophysin immunoreactivity (Fig. 2A). In
TeNT-exposed cultures, although synaptic terminals are stained with
anti-synaptophysin (Fig. 2C), there is no synaptic
labeling with anti-VAMP-1 (Fig. 2D). An additional
control for the specificity of TeNT action on VAMP was obtained using
BoNTs A and C, which also are zinc endopeptidases, but which cleave
other components of the vesicle docking-fusion complex(15) .
VAMP immunoreactivity (Fig. 2F) is unaffected by BoNT A
exposure at a time when neurotransmitter release is completely blocked.
Similar results were observed in BoNT C-exposed cultures (data not
shown). Consistent with the immunohistochemistry, VAMP is absent from
immunoblots of cultures exposed to TeNT (Fig. 3).
Figure 2:
Cell cultures double-labeled with
antibodies against VAMP and against synaptophysin. Control cultures
show synaptophysin immunostaining (A) in synaptic terminals;
VAMP immunoreactivity (B) has an almost identical
distribution. In cultures synaptically blocked by TeNT (0.06 nM for 20 h), synapses are identified clearly with anti-synaptophysin (C), although VAMP immunostaining (D) is totally
abolished. In contrast, in BoNT A-blocked cultures (0.06 nM for 20 h), synapses are stained with both anti-synaptophysin (E) and with anti-VAMP (F) antibodies. Magnification bar, 25 µm.
Figure 3:
Immunoblot analysis for VAMP and syntaxin
in toxin-treated cultures. Spinal cord cell cultures were incubated
with BoNT A, BoNT C, or TeNT (0.06 nM) for 16 h. Homogenates
were prepared and analyzed for VAMP and syntaxin as described under
``Experimental Procedures.'' VAMP is lost completely from
cultures exposed to TeNT. Syntaxin is cleaved to a lower molecular
weight by BoNT C.
In control
cultures, syntaxin is localized at the neuronal surface, particularly
along axonal membranes, but also appears concentrated at synaptic
membrane sites marked by synaptophysin immunoreactivity (Fig. 4, A and B). However, syntaxin staining persists in
cultures known to be intoxicated by BoNT C (Fig. 4, C and D). This is consistent with the persistence of
syntaxin on immunoblots (Fig. 3). Botulinum neurotoxin C cleaves
syntaxin near the transmembrane domain producing a soluble fragment of
syntaxin that is not degraded further(13) . Immunoblots of
homogenates prepared from BoNT C-exposed spinal cord cell cultures show
syntaxin cleaved to a lower molecular weight band. None of the other
toxins have any effect on syntaxin (Fig. 3).
Figure 4:
Cell cultures double-labeled with
antibodies against syntaxin and against synaptophysin. A, synaptophysin immunostaining defines synaptic terminals in control
cultures. B, syntaxin is associated with the neuronal membrane
including that of axonal branches, although intense immunofluorescence
tends to coincide with synaptic sites. In BoNT C-blocked cultures,
syntaxin immunoreactivity (D) persists, both at
synaptophysin-positive sites (C) and along axonal membranes. Magnification bar, 25 µm.
The localization
of SNAP-25 and the effects of BoNT A, TeNT, or BoNT C on its
distribution were analyzed by double-label immunohistochemistry using
antibodies against synaptophysin (Fig. 5, A, C, E, and G) and against the COOH terminus
of SNAP-25 (Fig. 5, B, D, F, and H). The pattern of immunostaining for SNAP-25 in control
cultures is similar to that of syntaxin (Fig. 5, A and B); i.e. presence along axonal and synaptic
membranes. Botulinum neurotoxin A cleaves the last nine amino acids
from the COOH terminus of
SNAP-25(6, 10, 11, 12) . Synaptic
terminals identified by synaptophysin immunostaining in BoNT A-exposed
cultures (Fig. 5C) do not stain with antibodies against
the COOH terminus of SNAP-25. (Fig. 5D). SNAP-25 is
lost not only from the synaptic membranes but also from the other
neuronal surface membranes including those of axons and cell bodies.
Unexpectedly, alterations in SNAP-25 are seen also when cultures are
exposed to TeNT or BoNT C. In TeNT-exposed cultures, SNAP-25
immunofluorescence clearly is more intense than in control cultures (Fig. 5F). In contrast, the immunoreactivity of the
SNAP-25 COOH terminus is markedly reduced in BoNT C-exposed neurons (Fig. 5H).
Figure 5:
Cell cultures double-labeled with
antibodies against the carboxyl terminus of SNAP-25 and against
synaptophysin. In control cultures, SNAP-25 immunostaining (B)
shows a distribution pattern similar to that for syntaxin (compare with Fig. 4B); the most intense fluorescence co-localizes
with synaptic sites marked by synaptophysin immunoreactivity (A). Synapses identified by synaptophysin immunoreactivity in
BoNT A-blocked cultures (C) show no staining for the COOH
terminus of SNAP-25 (D). Additionally, SNAP-25 immunostaining
is lost from all neuronal surface membranes in BoNT A-treated cultures (D). In TeNT-blocked cultures, SNAP-25 immunoreactivity (F) over axonal membranes is more intense than in control
cultures (compare with panel B; reacted, photographed, and
printed under the same conditions), whereas synaptophysin staining (E) is similar to controls. In BoNT C-blocked cultures (0.06
nM for 20 h), staining for the COOH terminus of SNAP-25 (H) is almost totally eliminated from structures that stain
with anti-synaptophysin antibodies (G). Magnification
bar, 25 µm.
SNAP-25 in control and toxin-treated cell
cultures was analyzed further by immunoblotting (Fig. 6).
Treatment of cultures with BoNT A for 24 h results in the loss of the
COOH terminus of SNAP-25 (Fig. 6A). Botulinum
neurotoxin C exposure causes a similar loss of the COOH terminus of
SNAP-25, consonant with the immunohistochemistry of intact neurons. In
contrast, TeNT has no effect on SNAP-25 when analyzed by immunoblot (Fig. 6A). Immunoblots prepared from another set of
BoNT A or BoNT C-blocked cultures show two bands detected with a
monoclonal antibody against the NH
Figure 6:
Immunoblot analysis of SNAP-25 in
toxin-treated spinal cord cultures. Cultures are incubated with BoNT A,
BoNT C, or TeNT (0.06 nM) for 24 h (A) or 20 h (B). Homogenates are prepared and analyzed for SNAP-25
immunoreactivity using antibodies against the COOH or NH
Cleavage of SNAP-25 was examined after
4, 8, and 16 h of toxin exposure to compare BoNT A and BoNT C effects (Fig. 7). Proteolysis of SNAP-25 by BoNT A is more rapid and
more complete than by BoNT C as evidenced with both SNAP-25 antibodies.
Some COOH terminus immunoreactivity persists after 16 h in BoNT C,
whereas there is none left with BoNT A treatment. Similarly, antibodies
against the NH
Figure 7:
Time course analysis of SNAP-25 cleavage
in BoNT A and BoNT C-treated cultures. Spinal cord cultures were
incubated in BoNT A or BoNT C (0.3 nM) for 4, 8 and 16 h.
Homogenates were prepared and analyzed for SNAP-25 and syntaxin. BoNT A
cleaves SNAP-25 more rapidly than BoNT C. Whereas there is total
cleavage of SNAP-25 in BoNT A-treated cultures, some uncleaved SNAP-25
remains after 16 h with BoNT C. The proteolysis of SNAP-25 in BoNT
C-treated cultures appears to follow the more complete cleavage of
syntaxin.
To determine if cleavage of
SNAP-25 in BoNT C-exposed cultures were due to contamination with BoNT
A, toxins used in these studies were immunoblotted with antibodies
against BoNT A. Preparations of BoNT C were not recognized by
antibodies against BoNT A, providing evidence against the possibility
of contamination by BoNT A (data not shown). Additionally,
immunohistochemistry and immunoblots of BoNT A and BoNT C-treated
cultures were repeated using a mixture of toxin with an excess of
antibodies against BoNT A. When cultures are exposed to the BoNT A
preparation premixed with antibodies against BoNT A, immunoreactivity
for SNAP-25 persists, and no cleavage of SNAP-25 is detected by
immunoblot, i.e. BoNT A is rendered ineffective. However, BoNT
C premixed with anti-BoNT A is equally as effective as BoNT C alone in
altering the staining patterns of both SNAP-25 and syntaxin (data not
shown). These data demonstrate that the effect of BoNT C on SNAP-25
cannot be explained by the presence of contaminating amounts of BoNT A. BoNT C action on SNAP-25 has not been described before, although the
previous studies were carried out using subcellular preparations. We
investigated the action of BoNT C in vitro on postnuclear
supernatants prepared from spinal cord cell cultures. For in vitro studies, BoNT A and BoNT C (150 nM final concentration)
are activated (3, 4, 5, 11) prior to
addition to the postnuclear supernatants for 90 min at 37 °C. Under
these conditions, immunoblots using antibodies against either the
NH This study is the first to demonstrate in physiologically
relevant cells, i.e. in intact functioning neurons, a direct
correlation between the clostridial neurotoxin-induced block in
neurotransmitter release and the cleavage of toxin-specific protein
substrates, VAMP, SNAP-25, or syntaxin. The cleavage of synaptic
proteins may not be the only mechanism whereby these toxins induce
their prolonged neuroparalysis(41, 42, 43) .
Nonetheless, our data clearly demonstrate by immunohistochemistry and
immunoblot analysis that VAMP, SNAP-25, and syntaxin are cleaved by
TeNT, BoNT A, or BoNT C, respectively, in the same neurons in which
neurotransmitter release is shown to be blocked. These findings confirm
that the principal mechanism of action of clostridial neurotoxins is
proteolytic cleavage of specific synaptic proteins necessary for
neurotransmitter release. Additionally, this study provides insight
into the interaction of these proteins preceding synaptic vesicle
exocytosis in intact neurons. Treatment of cultures with TeNT results
in the cleavage of VAMP and an increase in the intensity of SNAP-25
immunoreactivity as detected by immunohistochemistry with the COOH
terminus antibody. This suggests that VAMP/synaptobrevin binds to the
COOH terminus of SNAP-25. In contrast, immunohistochemistry of the
NH When BoNT
C is added to intact spinal cord neurons in culture, not only is
syntaxin cleaved, as expected, but there is a concomitant loss of
immunostaining for the COOH terminus of SNAP-25. The reduction in
SNAP-25 immunoreactivity in intact neurons is seen both by
immunohistochemistry and by immunoblotting. Furthermore, in support of
this result, an antibody against the NH Recent findings demonstrate the
presence of syntaxin (proposed as a t-SNARE) in purified synaptic
vesicle fractions(37) . Although the majority of syntaxin
appeared in a plasma membrane fraction, BoNT C preferentially cleaved
vesicular syntaxin leaving the plasma membrane syntaxin largely
unaffected. In the spinal cord cultures, however, much lower
concentrations of BoNT C cleaved almost all syntaxin. Differences
between in vivo and in vitro preparations as well as
differences in the conditions of BoNT C incubation might account for
this discrepancy. In summary, the findings reported here demonstrate
the actions of the clostridial neurotoxins in vivo, seen
together as the blockade of neurotransmitter release with the
proteolytic cleavage of the respective toxin substrates. The data also
provide evidence for the first time that, in addition to cleavage of
syntaxin, BoNT C has a secondary action on the COOH terminus of
SNAP-25. The finding that BoNT C is the only clostridial neurotoxin
that acts on two of the three SNARE proteins might be significant in
terms of its efficacy for the clinical treatment of muscle spasm
disorders.
Volume 271,
Number 13,
Issue of March 29, 1996 pp. 7694-7699
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
BOTULINUM NEUROTOXIN C ACTS ON SYNAPTOSOMAL-ASSOCIATED PROTEIN OF
25 kDa (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)are synthesized as
single polypeptides of 150 kDa and subsequently are cleaved to active
disulfide-linked dichain toxins. The heavy chain (100 kDa) carries the
receptor binding and transmembrane domains of the toxin, and the light
chain (50 kDa) contains the catalytic domain that blocks
neurotransmitter release(1, 2) . Clostridial
neurotoxins are zinc
endopeptidases(3, 4, 5) , which cleave
specific proteins thought to be involved in the synaptic vesicle
docking-fusion complex. Vesicle-associated membrane protein (VAMP),
also known as synaptobrevin, is cleaved by the majority of the CNTs
including tetanus toxin (TeNT) and botulinum neurotoxins (BoNTs) B, D,
F, and
G(3, 5, 6, 7, 8, 9) .
Botulinum neurotoxins A and E cleave SNAP-25 (synaptosomal-associated
protein of 25 kDa)(6, 10, 11, 12) ,
and BoNT C cleaves syntaxin(13, 14) .
terminus is a variable
domain, the middle or B domain is highly conserved, and the COOH
terminus contains the transmembrane spanning region with a short
projection into the synaptic vesicle lumen. Tetanus toxin cleaves the
peptide bond Gln-Phe
within the
conserved domain of VAMP(3) . Recent in vitro studies
have shown that VAMP can bind to syntaxin(17) , to SNAP-25 (17) , and to
synaptophysin(23, 24, 25) .
and
Arg
(6, 10, 11, 12) .
Materials
Purified tetanus toxin (2 
10
mouse lethal doses/mg of protein) was provided by Dr.
William Habig (Food and Drug Administration, Bethesda, MD). Purified
BoNT A was from List Biological Laboratories, Inc., Campbell, CA, and
BoNT C was from the Centre for Applied Microbiology and Research,
Porton Down, UK (5.2 10 and 1.0 10 mouse lethal doses/mg of protein, respectively).
[
H]Glutamine (specific activity, 52 Ci/mmol) and
[H]glycine (specific activity, 12.2 Ci/mmol) were
from Amersham Corp., Arlington Heights, IL. 5-Fluoro-2` -deoxyuridine
was a gift from Hoffman-LaRoche Inc., Nutley, NJ. Horse antibodies
against BoNT A and against BoNT C were from the United States Army
Medical Research Institute of Infectious Diseases, Ft. Detrick, MD.
Mouse monoclonal antibody against synaptophysin was from Boehringer
Mannheim. Anti-VAMP antibodies were produced in rabbits to synthetic
peptides corresponding to amino acids 1-32 of the VAMP variable
domain from each of the two isoforms found in rat brain and affinity
purified using the synthetic peptides. ()Monoclonal antibody
against the NH terminus of SNAP-25 was from Chemicon,
Temecula, CA. Polyclonal antibodies were raised in rabbits against a
peptide consisting of the COOH-terminal 12 amino acids of SNAP-25
conjugated to keyhole limpet hemocyanin and affinity purified from
columns containing the peptide coupled to Sepharose. Antibodies
specific for syntaxin were obtained by immunizing rabbits with
recombinant syntaxin 1A and purifying the IgG fraction.Spinal Cord Cell Cultures
Spinal cords from 13-day
fetal mice were dissociated and plated in 35-mm Vitrogen- (Collagen
Corp., Palo Alto, CA) coated culture dishes as described
previously(38, 39) . Cultures were grown for 3 weeks
in a humidified 10% CO atmosphere at 35 °C. On the 5th
day after plating, 5-fluoro-2` deoxyuridine was added to cultures for
96 h to inhibit nonneuronal cell growth.Evoked Neurotransmitter Release
Spinal cord
cultures were rinsed once in minimum essential medium and incubated for
20 h in 0.06 nM TeNT, BoNT A, or BoNT C in serum-free culture
medium at 35 °C. Cell cultures were rinsed and radiolabeled with 2
µCi/ml [H]glycine for 30 min or 5 µCi/ml
[H]glutamine for 60 min in isosmotic
HEPES-buffered salts solution (HBSS) containing 136 mM NaCl, 3
mM KCl, 2 mM CaCl, 1 mM MgCl, 10 mM HEPES, 10 mM glucose,
and 0.1% (w/v) bovine serum albumin. The pH was adjusted to 7.25, and
the osmolality was adjusted with sucrose to 325 ± 5 mmol/kg. To
evoke neurotransmitter release, cultures were exposed sequentially to
1.25 ml of the following solutions for 4.5-min intervals at 35 °C
as described previously(36) : HBSS without calcium and with 0.5
mM EGTA; HBSS containing 56 mM KCl, 83 mM NaCl and no CaCl; HBSS containing 56 mM KCl,
83 mM NaCl, and 2 mM CaCl; and finally
HBSS containing 0.5 mM EGTA and no calcium. In a previous
study, we demonstrated by thin-layer chromatography that virtually all
of the radioactivity, [H]glycine or
[H]glutamate, released under these conditions
co-migrates with free glycine or free glutamate,
respectively(36) . Cell-associated radioactivity was assayed
after dissolving the cells in 0.2 N NaOH and neutralizing with
HCl. Calcium-dependent release was calculated as the amount of
radiolabel released in the presence of calcium minus the amount
released in the absence of calcium and normalized to the total
radioactivity in the culture at the start of the release assay.Immunohistochemistry
Control and toxin-treated
(0.06 nM for 20 h) cultures were fixed in 2% paraformaldehyde
in HBSS without calcium, magnesium, or bovine serum albumin for 30 min
at room temperature. After rinsing, 0.1 M glycine in
phosphate-buffered saline (PBS) was added for 30 min to block free
aldehyde groups, after which cells were permeabilized with 0.05%
saponin in PBS for 30 min at room temperature. The cultures were
incubated overnight at 4 °C in a primary antibody solution
containing a mixture of mouse anti-synaptophysin (1:70) with either
affinity-purified rabbit anti-VAMP-1 (1:400), anti-VAMP-2 (1:400),
anti-SNAP-25 (1:400), or anti-syntaxin (1:500) in PBS containing 5%
normal goat serum (NGS). After rinsing 3 times (10 min each) in
PBS/NGS, cultures were incubated in a mixture of goat anti-rabbit
IgG-rhodamine and goat anti-mouse IgG-fluorescein (each 1:50) in
PBS/NGS for 60 min at 35 °C. Cultures were rinsed twice (10 min
each) in PBS/NGS, twice in PBS, and stored at 4 °C in n-propyl gallate in glycerol to prevent fluorescence
photobleaching(40) .Electrophoresis and Immunoblot Analysis
After
incubation with toxins (0.06 nM or 0.3 nM; see
Figs.), spinal cord neurons were detached from tissue culture dishes by
trypsinization and washed once with PBS. Cells were dissolved by
boiling for 5 min in electrophoresis sample buffer containing 2% SDS
and dithiothreitol (DTT). Protein samples were run on 10-20%
SDS-polyacrylamide gels (Bio-Rad, Hercules, CA) and transferred to
nitrocellulose membranes (Bio-Rad). Membranes were blocked for 1 h in
Tris buffered saline (20 mM Tris, 500 mM NaCl, pH
7.5) containing 5% nonfat dry milk and 0.05% Tween 20 (TTBS), incubated
sequentially with primary antibodies (dilutions were the same used for
immunohistochemistry) in TTBS and the appropriate alkaline phosphatase
conjugated secondary antibody (Bio-Rad), and developed using alkaline
phosphatase color development reagents.
H]glycine or [H]glutamine.
Neurotransmitter release is evoked by potassium-induced depolarization
in the presence of calcium. With potassium stimulation, control
cultures release 25-30% of the total
[H]glycine (Fig. 1B) and
4-5% of the total [H]glutamate (Fig. 1C) taken up by cultures. Tetanus toxin, BoNT A,
and BoNT C completely block potassium-evoked release of both
neurotransmitters (Fig. 1, B and C).
terminus of SNAP-25 (Fig. 6B); the predominant band is the cleaved lower
molecular weight form of SNAP-25 and the other band corresponds to the
remaining uncleaved SNAP-25.
termini. In BoNT A- or BoNT C-treated cultures, SNAP-25 (COOH
terminus) immunoreactivity is lost completely (A). SNAP-25
immunoreactivity in TeNT-blocked cultures is similar to controls (A). In another set of BoNT A- or BoNT C-treated cultures,
staining of the NH terminus confirms SNAP-25 proteolysis (B).
terminus indicate that some uncleaved
SNAP-25 remains in BoNT C-treated cultures, although the BoNT A-exposed
cultures show a clear progression with time to the total cleavage of
SNAP-25. The time course of syntaxin cleavage demonstrates that
virtually all of syntaxin is cleaved by BoNT C in 16 h, whereas more
SNAP-25 remains intact. Thus, BoNT C action on SNAP-25 appears to
follow its proteolysis of syntaxin.
or COOH terminus demonstrate proteolysis of SNAP-25 by
BoNT A but not by BoNT C (data not shown). Thus, the action of BoNT C
on SNAP-25 is observed only when the toxin is added to intact neurons
and gains access to synaptic proteins under physiologic conditions.
terminus of SNAP-25 in intact neurons remains unchanged
(data not shown). Furthermore, immunoblot analysis of SNAP-25 (COOH
terminus) did not show any difference between control and TeNT-exposed
cultures. Thus, the increased immunoreactivity of the COOH terminus of
SNAP-25 in TeNT-treated cultures is not due to increased levels of
SNAP-25 but rather to increased accessibility to the antibody. Direct
binding of SNAP-25 to VAMP and to syntaxin was demonstrated in an in vitro system using purified fusion proteins and deletion
analysis (17, 18) . Furthermore, Chapman et al.(17) reported that deletion of nine amino acids from the
COOH terminus of SNAP-25 reduced VAMP binding by 73%. Our observations
in spinal cord neurons are consistent with these findings. terminus of SNAP-25
recognizes two bands due to SNAP-25 cleavage in BoNT C-treated
cultures. Possible explanations for the effect include 1) that syntaxin
proteolysis by BoNT C changes the conformation or accessibility of
SNAP-25, increasing its susceptibility to endogenous proteases or 2)
that BoNT C itself proteolytically cleaves SNAP-25 albeit at a slower
rate than it cleaves syntaxin. The action of BoNT C on SNAP-25
apparently occurs only when living neurons are exposed to this toxin.
SNAP-25 is not affected when BoNT C is added to spinal cord cell
culture homogenates, although BoNT A cleaves SNAP-25 in the same
homogenates. Furthermore, BoNT C does not cleave recombinant SNAP-25. ()Thus, the action of BoNT C on SNAP-25 in vivo might occur only when SNAP-25 is complexed with another protein
and/or plasma membranes. Alternatively, SNAP-25 may be protected from
proteolysis by endogenous proteases when it is complexed to syntaxin,
and the cleavage of syntaxin by BoNT C increases SNAP-25's
susceptibility to proteolysis.
)))
We thank Dr. Ornella Rossetto, University of Padova,
Padova, Italy for affinity-purified antibodies against VAMP, SNAP-25,
and syntaxin; Sandra Fitzgerald for preparation of cell cultures; and
Linda Bowers and Annette Vertino-Bell for preparation of final figures.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 S. Kalandakanond and J. A. Coffield Cleavage of Intracellular Substrates of Botulinum Toxins A, C, and D in a Mammalian Target Tissue J. Pharmacol. Exp. Ther., March 1, 2001; 296(3): 749 - 755. [Abstract] [Full Text]
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S. Kalandakanond and J. A. Coffield Cleavage of SNAP-25 by Botulinum Toxin Type A Requires Receptor-Mediated Endocytosis, pH-Dependent Translocation, and Zinc J. Pharmacol. Exp. Ther., March 1, 2001; 296(3): 980 - 986. [Abstract] [Full Text]
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 J. B. Bergsman and R. W. Tsien Syntaxin Modulation of Calcium Channels in Cortical Synaptosomes As Revealed by Botulinum Toxin C1 J. Neurosci., June 15, 2000; 20(12): 4368 - 4378. [Abstract] [Full Text] [PDF]
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 J. A. Chaddock, J. R. Purkiss, L. M. Friis, J. D. Broadbridge, M. J. Duggan, S. J. Fooks, C. C. Shone, C. P. Quinn, and K. A. Foster Inhibition of Vesicular Secretion in Both Neuronal and Nonneuronal Cells by a Retargeted Endopeptidase Derivative of Clostridium botulinum Neurotoxin Type A Infect. Immun., May 1, 2000; 68(5): 2587 - 2593. [Abstract] [Full Text] [PDF]
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 G. Schiavo, M. Matteoli, and C. Montecucco Neurotoxins Affecting Neuroexocytosis Physiol Rev, April 1, 2000; 80(2): 717 - 766. [Abstract] [Full Text] [PDF]
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L.-S. Chin, R. D. Nugent, M. C. Raynor, J. P. Vavalle, and L. Li SNIP, a Novel SNAP-25-interacting Protein Implicated in Regulated Exocytosis J. Biol. Chem., January 14, 2000; 275(2): 1191 - 1200. [Abstract] [Full Text] [PDF]
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G. A. O'Sullivan, N. Mohammed, P. G. Foran, G. W. Lawrence, and J. O. Dolly Rescue of Exocytosis in Botulinum Toxin A-poisoned Chromaffin Cells by Expression of Cleavage-resistant SNAP-25. IDENTIFICATION OF THE MINIMAL ESSENTIAL C-TERMINAL RESIDUES J. Biol. Chem., December 24, 1999; 274(52): 36897 - 36904. [Abstract] [Full Text] [PDF]
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 E. A. Neale, L. M. Bowers, M. Jia, K. E. Bateman, and L. C. Williamson Botulinum Neurotoxin A Blocks Synaptic Vesicle Exocytosis but Not Endocytosis at the Nerve Terminal J. Cell Biol., December 13, 1999; 147(6): 1249 - 1260. [Abstract] [Full Text] [PDF]
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P. G. P. Foran, L. M. Fletcher, P. B. Oatey, N. Mohammed, J. O. Dolly, and J. M. Tavare Protein Kinase B Stimulates the Translocation of GLUT4 but Not GLUT1 or Transferrin Receptors in 3T3-L1 Adipocytes by a Pathway Involving SNAP-23, Synaptobrevin-2, and/or Cellubrevin J. Biol. Chem., October 1, 1999; 274(40): 28087 - 28095. [Abstract] [Full Text] [PDF]
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C. Leoni, A. Menegon, F. Benfenati, D. Toniolo, M. Pennuto, and F. Valtorta Neurite Extension Occurs in the Absence of Regulated Exocytosis in PC12 Subclones Mol. Biol. Cell, September 1, 1999; 10(9): 2919 - 2931. [Abstract] [Full Text]
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S. D. Conner and G. M. Wessel Syntaxin Is Required for Cell Division Mol. Biol. Cell, August 1, 1999; 10(8): 2735 - 2743. [Abstract] [Full Text]
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 G Lalli, J Herreros, S. Osborne, C Montecucco, O Rossetto, and G Schiavo Functional characterisation of tetanus and botulinum neurotoxins binding domains J. Cell Sci., January 8, 1999; 112(16): 2715 - 2724. [Abstract] [PDF]
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J. M. Canaves and M. Montal Assembly of a Ternary Complex by the Predicted Minimal Coiled-coil-forming Domains of Syntaxin, SNAP-25, and Synaptobrevin. A CIRCULAR DICHROISM STUDY J. Biol. Chem., December 18, 1998; 273(51): 34214 - 34221. [Abstract] [Full Text] [PDF]
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 S. Wu, K.-C. Lim, J. Huang, R. F. Saidi, and C. L. Sears Bacteroides fragilis enterotoxin cleaves the zonula adherens protein, E-cadherin PNAS, December 8, 1998; 95(25): 14979 - 14984. [Abstract] [Full Text] [PDF]
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 S.-Y. Hua, D. A. Raciborska, W. S. Trimble, and M. P. Charlton Different VAMP/Synaptobrevin Complexes for Spontaneous and Evoked Transmitter Release at the Crayfish Neuromuscular Junction J Neurophysiol, December 1, 1998; 80(6): 3233 - 3246. [Abstract] [Full Text] [PDF]
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 R. D. Fields Clostridial Neurotoxins in Synaptic Research Neuroscientist, September 1, 1998; 4(5): 324 - 328. [Abstract] [PDF]
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K. Sadoul, A. Berger, H. Niemann, U. Weller, P. A. Roche, A. Klip, W. S. Trimble, R. Regazzi, S. Catsicas, and P. A. Halban SNAP-23 Is Not Cleaved by Botulinum Neurotoxin E and Can Replace SNAP-25 in the Process of Insulin Secretion J. Biol. Chem., December 26, 1997; 272(52): 33023 - 33027. [Abstract] [Full Text] [PDF]
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J. Marsal, B. Ruiz-Montasell, J. Blasi, J. E. Moreira, D. Contreras, M. Sugimori, and R. Llinas Block of transmitter release by botulinum C1 action on syntaxin at the squid giant synapse PNAS, December 23, 1997; 94(26): 14871 - 14876. [Abstract] [Full Text] [PDF]
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 B. Hohne-Zell, A. Galler, W. Schepp, M. Gratzl, and C. Prinz Functional Importance of Synaptobrevin and SNAP-25 during Exocytosis of Histamine by Rat Gastric Enterochromaffin-Like Cells Endocrinology, December 1, 1997; 138(12): 5518 - 5526. [Abstract] |
