From the
Botulinum neurotoxin serotype C (BoNT/C) is a 150-kDa protein
produced by Clostridium botulinum, which causes animal
botulism. In contrast to the other botulinum neurotoxins that contain
one atom of zinc, highly purified preparations of BoNT/C bind two atoms
of zinc per toxin molecule. BoNT/C is a zinc-endopeptidase that cleaves
syntaxin 1A at the Lys
Botulism is characterized by a flaccid paralysis caused by
botulinum neurotoxins (BoNT),
Recently, BoNT/A, /B, /E, and /F, as well as tetanus neurotoxin,
were shown to contain one atom of zinc bound to the zinc binding motif
of zinc-endopeptidases, present in the central part of their L chains
(5, 6, 7, 8, 9) . The sequence
of BoNT/C shows the presence of the same His-Glu- Xaa-Xaa-His
motif
(10, 11) .
The specific proteolytic target of
BoNT/B, /D, /F, /G, and tetanus neurotoxin is VAMP/synaptobrevin
(VAMP), a synaptic vesicle membrane protein involved in vesicle
exocytosis at the presynaptic terminals
(5, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) .
VAMP is cleaved within the cytoplasmic portion at different sites by
the different neurotoxins
(4) . By contrast, BoNT/A and /E
cleave two different peptide bonds within SNAP-25
(13, 22, 23, 24) , a protein associated
with the cytoplasmic face of the presynaptic membrane
(25) .
Blasi et al.(26) have shown that BoNT/C selectively
recognizes and cleaves syntaxin. This synaptic terminal protein was
first identified by cloning the gene from a rat brain cDNA library
(27, 28) . It associates with synaptotagmin and the
N-type calcium channels in the active zones of the presynaptic membrane
(27, 28) . Recently, several isoforms with different
tissue and cellular distribution were isolated; in particular syntaxin
1A and 1B are specifically localized in the nervous tissue, while
syntaxin 2, 3, 4, and 5 are broadly expressed
(27, 29) .
Here we show that BoNT/C is a zinc-dependent proteinase containing
two zinc atoms and we identify the peptide bond of syntaxin
specifically hydrolyzed by this novel metalloproteinase, as well as its
specificity for the known syntaxin isoforms.
Captopril
([2 S]-1-[3-mercapto-2-methyl-propionyl]-L-proline)
was from Squibb (Italy). Dimyristoylglycerophosphocholine,
dimyristoylglycerophosphoethanolamine, dimyristoylglycerophosphoserine,
dimyristoylglycerophosphate, dimyristoylglycerophosphoinositol, and
cholesterol were from Sigma). Bovine brain mixed gangliosides were
kindly provided by Dr. G. Kirschner (FIDIA Research Laboratories, Abano
Terme, Italy). Their composition and that of soybean mixed lipids
(asolectin) were as described previously
(31) . Liposomes were
obtained by mixing chloroform/methanol (2:1) stock solutions of lipids.
After drying under N
Synaptosomes, synaptic membrane fraction (LP1) and small synaptic
vesicles were isolated from rat cerebral cortex
(13, 32) . LP1, prepared as detailed by Huttner et
al.(32) , were centrifuged in an SS34 Sorvall rotor at
16,500 rpm for 20 min and the pellet was resuspended in 140 mM
NaCl, 5 mM KCl, 5 mM NaHCO
Samples were transferred onto nitrocellulose as described elsewhere
(13) and treated with anti-syntaxin-specific antisera (1:200
dilution), anti-VAMP (1:500), anti-synaptophysin (1:2,000), or
anti-synaptotagmin (1:2,000). Primary antibodies were detected by
immunostaining with an anti-rabbit (1:10,000 dilution; Boehringer
Mannheim) or anti-mouse antibody conjugated with alkaline phosphatase
(1:1,000; Sigma)
(13) . Amount of staining was determined by
scanning the nitrocellulose paper with a dual wavelength densitometer
(Shimadzu CS-630).
It is noteworthy that another
Lys-Ala peptide bond (Lys
This paper reports on the biochemical characterization of the
zinc-endopeptidase activity of BoNT/C. This neurotoxin shows unique
features that differentiate it from the other seven clostridial
neurotoxins recently studied. ( a) It binds two atoms of zinc,
rather than one as the related toxins do. In this respect it resembles
the collagen-specific zinc-endopeptidases, whose structure has been
recently resolved
(36, 37) . On this basis, we suggest
also that in BoNT/C one zinc atom plays a catalytic role, while the
other plays a structural role. ( b) As recently shown by Blasi
et al.(26) , BoNT/C is the only clostridial neurotoxin
that cleaves syntaxin. Here, we have reported the identification of the
precise cleavage site as an unique Lys-Ala peptide bond, among the two
such bonds present in the carboxyl-terminal region of syntaxin.
( c) The peptide bond cleaved by BoNT/C differs from those
hydrolyzed by the other clostridial neurotoxins. No common pattern of
proteolytic cleavage can be identified in the peptide segments
containing the cleavage site of each neurotoxin, even within the same
protein target. This suggests that the region including the peptide
bond cleaved is not the sole determinant of the target specificity of
the clostridial neurotoxin. Additional segment(s) of the target protein
must be involved and, in fact, we have recently identified a common
neurotoxin binding motif on VAMP, SNAP-25, and syntaxin
(39) .
Hence, the basis of the remarkable specificity of the clostridial
neurotoxins is a double interaction with their substrate via a shared
motif present in VAMP, SNAP-25, and syntaxin and a region containing
the peptide bond to be hydrolyzed, unique of each target.
In
conclusion, this paper shows that the molecular pathogenesis of
botulism type C is related to a specific proteolytic activity of
BoNT/C, which penetrates into the neuronal cytosol and removes the
majority of the cytoplasmic domain of syntaxin. This will inevitably
result in the prevention of the assembly of a functional 20 S SNAREs
complex
(19) , thus accounting for the persistent inhibition of
neuroexocytosis caused by BoNT/C
(2) . The present results
provide the molecular basis for the use of BoNT/C as a tool to probe
the involvement of syntaxin isoforms in cellular processes and for its
use in the treatment of dystonias and strabismus, as it is already a
common medical practice with BoNT/A, /B, and /F
(42) .
We thank G. Milan for help in some of the experiments,
Dr. L. Lozzi (University of Siena) for peptide synthesis, F. Cattalini
and G. Rocco (University of Padua) for atomic absorption measurements,
and Dr. P. Polverino de Laureto (University of Padua) for protein
sequence.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Ala
and syntaxin 1B
at the Lys
-Ala
peptide bonds, only when
they are inserted into a lipid bilayer. The other Lys-Ala bond present
within the carboxyl-terminal region is not hydrolyzed. Syntaxin
isoforms 2 and 3 are also cleaved by BoNT/C, while syntaxin 4 is
resistant. These data suggest that BoNT/C recognizes a specific spatial
organization of syntaxin, adopted upon membrane insertion, which brings
a selected Lys-Ala peptide bond of its carboxyl-terminal region to the
active site of this novel metalloproteinase.
(
)
produced by
Clostridia, in seven different types (from A to G)
(1, 2) . BoNT/C has been associated mainly with botulism
of birds
(2) , although a case of human infant botulism was
described
(3) . BoNTs are synthesized as a single polypeptide
chain of 150 kDa, cleaved by proteases at an exposed loop with the
generation of two disulfide-linked chains. The heavy chain (H, 100 kDa)
is responsible for neurospecific binding and membrane translocation,
while the light chain (L, 50 kDa) blocks neuroexocytosis
(4) .
Proteins and Chemicals
Exponentially grown
Clostridium botulinum type C (strain NCTC 8264) bacteria were
harvested by tangential flow filtration, washed with ice-cold 50
mM sodium acetate, pH 5.0, and finally recovered by
centrifugation at 10,000 g for 20 min. Cells were
extracted with 0.2 M sodium phosphate buffer, pH 6.0, for 16 h
at 4 C and then protease-free ribonuclease A (Sigma) was added (final
concentration: 0.1 mg/ml). The suspension was further incubated for 2 h
at 37 °C and, after centrifugation at 25,000
g,
the supernatant was precipitated with 60% of
(NH
)
SO
. After stirring for 30 min
at 25 °C, the precipitated toxin was collected by centrifugation at
25,000
g for 30 min. The pellet was resuspended in 50
mM triethanolamine, pH 8.0, and dialyzed extensively against
the same buffer at 4 °C. The clarified BoNT/C solution was loaded
onto a Q Sepharose column (Pharmacia Biotech Inc.) equilibrated with 50
mM triethanolamine, pH 8.0, and eluted with the latter buffer
containing 100 mM NaCl. BoNT/C was further purified with a
Mono S column (Pharmacia), followed, when necessary, by a Mono Q
(Pharmacia) step. Immobilized metal ion affinity chromatography was
used to remove traces of contaminant proteases
(30) .
flux, lipids were resuspended in
diethyl ether, dried, and sonicated until optical clarity was achieved
at the concentration of 5 mg/ml in 10 mM
NaH
PO
, 150 mM NaCl, pH 7.4.
, 1 mM
MgCl
, 1.2 mM Na
HPO
, 10
mM glucose, 20 mM HEPES-Na, pH 7.4, at a
2.5-4.4 mg/ml protein concentration. Small synaptic vesicles were
prepared omitting the glass bead chromatography step
(32) ,
resuspended in the same buffer as LP1 at a 1.5-2.2 mg/ml protein
concentration, and used thereafter for addition of 100 µM
phenylmethylsulfonyl fluoride and 2 µg/ml pepstatin (Sigma).
Recombinant glutathione S-methyltransferase syntaxin 1A or 1B
fusion proteins (GST-Syn 1A or GST-Syn 1B) were obtained by inserting
codons 4-288 of syntaxin 1A and codons 3-288 of syntaxin 1B
cDNAs into the vector pGEX-KG
(33) and transformation into the
AB1899 strain of Escherichia coli. GST-fusion proteins were
purified by affinity chromatography on GSH-agarose matrix (Sigma) and
the recombinant portion of the fusion protein was released by thrombin
cleavage
(33) . Peptides SDTKKAVKY and KVKDASKTY were kindly
prepared by Dr. L. Lozzi (University of Siena) with a SMP 350 automatic
synthesizer (Zynsser Analytic, Frankfurt), employing a Fmoc chemistry.
The peptides were purified by reverse-phase chromatography on a C8
Ultra-Sphere preparative column (Beckman).
Determination of Metal Content
The content of
zinc, nickel, iron, copper, cobalt, and manganese was measured, after
dialysis of the toxin in metal-free buffers
(5) , by atomic
adsorption with a Perkin Elmer 4000 atomic adsorption flame
spectrophotometer with impact bed loading. Zinc was removed by 3
8 h of dialysis at 4 °C against 10 mM HEPES-Na,
150 mM NaCl, 2 mM ortho-phenantroline (OP),
pH 7.4, and subsequent extensive dialysis against the same buffer
without OP. Zinc reuptake by apo-BoNT/C was performed in 300
µM zinc chloride, 10 mM HEPES-Na, 150 mM
NaCl, pH 7.4; after 24 h at 4 °C, samples were extensively dialyzed
against the same buffer without zinc and metal content was determined
as above. Toxin samples of 0.2-0.3 mg were used for each atomic
adsorption measurement.
Proteolytic Activity of BoNT/C and Other BoNT Serotypes
on Synaptosomes, Synaptic Membrane Fractions, Small Synaptic Vesicles,
and Recombinant Syntaxin Fusion Proteins
50 µg of
synaptosomes, isolated from rat brain cortex, were incubated with the
different BoNTs (100 nM), in 140 mM NaCl, 5
mM KCl, 5 mM NaHCO, 1 mM
MgCl
, 1.2 mM Na
HPO
, 10
mM glucose, 20 mM HEPES-Na, pH 7.4, for 4 h at 37
°C. Native LP1 (50 µg) and small synaptic vesicles (25 µg),
or solubilized with 0.5%
n-octyl-
-D-glucopyranoside, were incubated with
BoNT/C (50-100 nM), previously reduced with 10
mM dithiothreitol (DTT) for 30 min at 37 C, in 4 mM
HEPES-Na, 300 mM glycine, 150 mM NaCl, 1 mM
MgCl
, 0.3 mM CaCl
, 0.02%
NaN
, pH 7.3, for 3 h at 37 °C. In some samples the
toxin was preincubated for 30 min at 37 °C with different
inhibitors (captopril, 2 mM; OP, 1 mM; EDTA, 1
mM). Samples were subjected to SDS-polyacrylamide gel
electrophoresis and stained with Coomassie Blue or silver as before
(12) . Recombinant syntaxin 1A and 1B and the correspondent
GST-syntaxin proteins (2 µg/sample) were incubated for 1 h at 37
°C mixed with 100 µg of liposomes of different lipid
composition: asolectin;
dimyristoylglycerophosphocholine:dimyristoylglycerophosphoethanolamine:cholesterol,
4:4:1 (A); A plus 10% dimyristoylglycerophosphate (w/w); A plus 10%
dimyristoylglycerophosphoserine; A plus 10%
dimyristoylglycerophosphoinositol; A plus 10% mixed brain gangliosides.
After dialysis against 10 mM NaH
PO
,
150 mM NaCl, pH 7.4, for 3 days at 4 °C, to remove the
detergent used for syntaxin purification, liposome-bound syntaxin was
recovered by centrifugation for 60 min at 200,000
g in
a Ti50 rotor (Beckman). Liposomes were resuspended by gentle
pipetteting into 1 ml of the same buffer, centrifuged, and finally
homogenized by 8 passages in a 25-gauge needle at a final protein
concentration of 0.5 mg/ml. BoNT/C reduced with DTT as above (100
nM final concentration) was added and, after 3 h at 37 °C,
samples were diluted with 600 µl of 10 mM
NaH
PO
, 150 mM NaCl, pH 7.4, and
centrifuged for 60 min at 300,000
g in a SW55 rotor
(Beckman). Supernatants were precipitated with trichloroacetic acid,
centrifuged 15 min at 15,000
g, and pellets were
dissolved in 8% SDS, 10 mM Tris acetate, 0.1 mM EDTA,
pH 8.2. Pellets derived from the SW55 centrifugation were solubilized
in the same buffer and boiled for 2 min. Samples were analyzed in a 12%
polyacrylamide SDS gels or in a high resolving SDS gel system
(35) and, after protein staining, scanned with a dual wavelength
Shimadzu CS-630 densitometer.
Antibodies and Immunoblotting
Rabbit polyclonal
antisera recognizing rat syntaxin isoforms were generated against
purified bacterially expressed proteins as reported
(27, 34) . Rabbit polyclonal antibodies against cysteine
string protein was a kind gift of Dr. A. Mastrogiacomo (University of
Rome). A mouse monoclonal antibody against rat retina synaptophysin was
purchased from Sigma, while the one specific for rat synaptotagmin was
kindly provided by Dr. M. Popoli (University of Milan, Italy).
Protein Sequencing
A 4-kDa syntaxin fragment was
electroeluted from polyacrylamide gels in an ISCO apparatus fitted with
a Spectra/Por dialysis membrane (Spectrum, CA) with a 500-Da cut-off.
Freeze-dried samples were applied to ProSpin tubes (Applied
Biosystems), and sequenced in a pulsed liquid Applied Biosystem model
477A protein sequencer.
Botulinum Neurotoxin Type C Contains Two Zinc
Atoms
BoNT/C is a proteinase that cleaves syntaxin within the
carboxyl-terminal region
(26) . The biochemical aspects of this
proteolytic activity are not characterized. On the basis of its
sequence
(10) , which shows the presence of the
His-Glu- Xaa-Xaa-His zinc-binding motif of zinc-endopeptidases,
it is expected to be a metalloendoproteinase. Fig. 1shows that
highly purified preparations of BoNT/C do contain zinc. However, at
variance with the other clostridial neurotoxins that contain one zinc
atom per toxin molecule
(5, 6, 7, 8, 9) , BoNT/C harbors
two atoms of zinc. This feature of BoNT/C is shared by neutrophil
collagenase, whose three-dimensional structure has been recently solved
(36, 37) . The active-site zinc of this
metalloproteinase is bound to two histidines of the motif and to a
third His residue. The second zinc is tetra-coordinated via three
histidines and an aspartate. At variance from the catalytic metal atom,
this latter zinc is not exchangeable and is thought to play a
structural role
(36, 37) . Fig. 1also shows that
both zinc atoms can be removed upon incubation with OP, thus generating
an apo-BoNT/C. Unfortunately, the relative Kvalues of the two zinc atoms could not be determined as we have
done before for other botulinum neurotoxins
(7) , because of the
present unavailability of BoNT/C in amounts sufficient for such
measurements. The two metal ions can be regained upon incubation in
zinc-containing buffers (Fig. 1). The content of cobalt, copper,
iron, manganese, and nickel was below detection.
Figure 1:
Zinc content, measured by atomic
absorption, of native BoNT/C and after treatment with zinc chelators.
Amount of zinc bound to BoNT/C, in atoms per protein molecule, before
( C) or after treatment with 2 mM OP or after OP
incubation, dialysis, incubation with 300 µM zinc
chloride, and further dialysis ( R). The average zinc content
of BoNT/A, /B, /E, and /F is also reported (BoNTs). Cobalt, copper,
iron, manganese, and nickel were also assayed and were found to be
below the detection limits. Data are the mean of four independent
measurements made on samples in the 0.2-0.3-mg protein range,
derived from two different batches of BoNT/C and bars represent ± S.D.
Botulinum Neutoxins Type C Cleaves Syntaxin in
Synaptosomes
Fig. 2A shows that in synaptosomes
incubated with BoNT/C a doublet of syntaxins is cleaved with the
production of two fragments of higher electrophoretic mobility. As a
control, it is also reported in the same panel A that BoNT/B,
/D, /F, and /G affect VAMP without altering the amount of syntaxin.
Fig. 2B shows that this BoNT/C activity is inhibited by
OP, EDTA, and partially by captopril, three known inhibitors of
metalloproteinases
(11, 38) . BoNT/C-induced syntaxin
proteolysis is also prevented by dissolving the synaptosomal membranes
with mild detergents, such as octylglucoside (Fig. 2 B).
This indicates that syntaxin has to be inserted in the lipid bilayer in
order to be recognized and cleaved by BoNT/C.
Figure 2:
Effect of different BoNT serotypes on the
level of synaptic terminal proteins. A, rat brain synaptosomes
were treated with BoNT/C, /B, /D, /F, and /G as detailed under
``Material and Methods,'' electrophoresed, and blotted onto
nitrocellulose membranes. Samples were incubated with a mouse
anti-synaptotagmin or with anti-synaptophysin monoclonal IgG, rabbit
anti-syntaxin, anti-cysteine string protein ( CSP), or
anti-VAMP antisera and stained with the appropriated alkaline
phosphatase-conjugated anti-IgG antibodies. In the BoNT/C-treated
sample, the filled arrowhead points to the residual intact
syntaxin doublet and the empty arrowheads to the fragments
generated by proteolysis. B, LP1 synaptosomal membranes and
small synaptic vesicles were treated with native or DTT-reduced BoNT/C
without or with n-octyl--D-glucopyranoside
( OG), OP, EDTA, and captopril, processed as in A and
then revealed with a rabbit anti-syntaxin polyclonal
antibody.
Botulinum Neurotoxin Type C Cleaves Recombinant Syntaxin
Incorporated in Lipid Bilayers
When BoNT/C was assayed with
full-length recombinant syntaxin-1A or syntaxin-1B, no proteolysis was
observed, even upon prolonged incubations with high concentrations of
BoNT/C (not shown), in agreement with the results of Blasi et al.(26) . However, both neuronal isoforms of syntaxin are
cleaved when incorporated in lipid bilayers and, in both cases, a large
fragment of estimated molecular mass of 31-32 kDa is produced
(not shown). As reported in Fig. 3also, the
NH-terminal GST-syntaxin fusion protein is efficiently
cleaved, thus suggesting that BoNT/C cleaves syntaxin within the
cytoplasmic domain at a site close to the carboxyl-terminal
transmembrane region. This result clearly indicates that no additional
synaptosomal factors are required for syntaxin proteolysis by BoNT/C.
The large proteolytic syntaxin fragment dissociates from the membrane
and is recovered in the supernatant (Fig. 3, left
panel), while the smaller carboxyl-terminal fragment remains
membrane-bound. It migrates in SDS-PAGE at the lipid front and is
barely visible, because the excess of lipids interferes with protein
staining.
Figure 3:
BoNT/C cleaves recombinant GST-syntaxin
1B. Silver-stained SDS-PAGE profile of GST-syntaxin 1B ( GST-Syn
1B) incorporated in asolectin vesicles and incubated at 37 °C
without or with DTT-reduced BoNT/C (+ BoNT/C). Half of the
sample was directly processed for SDS electrophoresis ( T),
while the other half was diluted and ultracentrifuged as detailed under
``Material and Methods,'' thus obtaining a pellet
( P) and a supernatant ( SN). The position of
GST-syntaxin and its BoNT/C-induced fragment are indicated by open and closed triangles,
respectively.
Among the lipid mixtures tested here, maximal cleavage
efficiency of BoNT/C was found with asolectin and minimal activity with
mixture A (see ``Materials and Methods''), thus suggesting
that negatively charged phospholipids affect BoNT/C activity and/or
syntaxin conformation.
Syntaxin Is Cleaved by Botulinum Neurotoxin C at a Single
Lys-Ala Peptide Bond
To determine the site(s) of BoNT/C
proteolytic cleavage of syntaxin, synaptosomes and LP1 membranes (a
plasmalemma-rich fraction) were tested, but their complex protein
composition prevented such an analysis. Hence, recombinant GST-syntaxin
1A and 1B were incorporated into asolectin liposomes and, after BoNT/C
cleavage, liposomes were recovered and loaded onto a high resolution
SDS-PAGE (Fig. 4 A). Due to their hydrophobicity and to
the unfavorable lipid:protein ratio, the small syntaxin fragments
migrated just above the lipid front, from where they were electroeluted
and sequenced. Fragments derived from syntaxin 1A and 1B gave the
identical sequence AVKYQSxA, corresponding to BoNT/C cleavage of the
peptide bond Lys-Ala
of 1A and
Lys
-Ala
of 1B (Fig. 4 B). The
1A and 1B fragments have a molecular mass of 3,779 and 3,790 daltons
and are composed of 35 and 36 residues, respectively.
Figure 4:
Sequences of syntaxin fragments generated
by BoNT/C proteolysis. A, recombinant GST-syntaxin 1A and 1B
fusion proteins, inserted is asolectin vesicles, were incubated at 37
°C alone or with DTT-reduced BoNT/C. Samples were recovered by
ultracentrifugation and the corresponding pellets were applied onto a
high resolution low molecular weight SDS-polyacrylamide gel system
(35). The 4-kDa BoNT/C-induced syntaxin 1A and 1B fragments were
electroeluted and sequenced. Both fragments provided the sequence
AVKYQSxA, shown in larger letters. B, scheme of the structure
of syntaxin; the portion of the sequences of the syntaxin family,
containing the cleavage site of BoNT/C and the transmembrane region
( black), is enlarged.
The
nonapeptide SDTKKAVKY, corresponding to the cleavage site is not
cleaved by BoNT/C, but partially inhibits the BoNT/C-mediated cleavage
of syntaxin (not shown), while a scrambled peptide is ineffective. This
peptide is not a strong inhibitor (and this can be explained in terms
of the recent finding that clostridial neurotoxins recognize their
substrates via a double interaction that involves the cleavage site as
well as a recognition site located in a different part of the substrate
protein molecule
(39) ).
-Ala
in
syntaxin-1A; Lys
-Ala
in syntaxin-1B) is
present in the vicinity, but it is not cleaved at any rate. This
finding also indicates that BoNT/C recognizes the tertiary, rather than
the primary, structure of syntaxin with a precise relative spatial
position of the binding and the cleavage sites
(39) .
Syntaxin Isoform Specificity of Botulinum Neurotoxin Type
C
Syntaxin isoform-specific antibodies are available for
isoforms 1, 2, 3, and 4, while isoform 5 is less clearly identifiable.
Immunoblotting and densitometric analysis shows that syntaxin 2 and 3
are cleaved by BoNT/C, while syntaxin isoform 4 is not (Fig. 5).
Under the conditions used here, the cleavage of syntaxins contained in
the LP1 preparation of synaptosomal membrane is never complete (Figs. 2
and 5). This result may reflect syntaxin heterogeneity in the
preparation or, as suggested by Hayashi et al.(40) ,
the presence of a multiple protein complex formed by syntaxin, SNAP 25,
and VAMP/synaptobrevin, in which syntaxin is resistant to clostridial
neurotoxin cleavage.
Figure 5:
Proteolytic cleavage of syntaxin isoforms
1, 2, 3, and 4. Synaptosomal membranes were incubated with BoNT/C or
with buffer, electrophoresed, incubated with syntaxin isoform-specific
antibodies, stained, and the spots were quantitated by densitometric
scanning. Empty columns are controls (-) that were taken in each
experiment as 100% and dotted columns are the amount of intact syntaxin
left after BoNT/C proteolysis (+). Bars are the mean
± S.D. of three different
experiments.
The susceptibility to BoNT/C proteolysis of the
various syntaxin isoforms if fully consistent with their sequence at
the cleavage site since 1A, 1B, 2, and 3 are identical, while 4 has a
Ile rather than a Lys at the P1 site. A replacement of a hydrophilic
residue with an aliphatic one at the P1 site makes rat VAMP isoform 1
resistant to the proteolytic activity of tetanus neurotoxin and BoNT/B
(12, 41) . No clear results could be obtained with
respect to isoform 5, which shows an amino acid substitution at the P1`
site. These results are also consistent with the identification of a
neurotoxin binding motif in syntaxin, additional to the cleavage site
(39) . The motif is present in two copies: X1 (segment
29-38 of rat syntaxin 1A) and X2 (segment 164-173 of rat
syntaxin 1A). X1 is present only in syntaxin isotypes 1A and 1B, while
X2 is conserved in isotypes 1A, 1B, 2, 3; in contrast, X2 shows a Gln
for Asp substitution in isoform 4 and is absent in syntaxin 5. The
present findings suggest that it is X2 that is the functional BoNT/C
binding segment of syntaxin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.