(Received for publication, April 30, 1996, and in revised form, November 21, 1996)
From the Département de Pharmacochimie Moléculaire et Structurale, U266 INSERM, URA D1500 CNRS, Université René Descartes, UFR des Sciences Pharmaceutiques et Biologiques, 75270 Paris Cedex 06, France
The light chain (L chain) of tetanus neurotoxin (TeNT) has been shown to have been endowed with zinc endopeptidase activity, selectively directed toward the Gln76-Phe77 bond of synaptobrevin, a vesicle-associated membrane protein (VAMP) critically involved in neuroexocytosis. In previous reports, truncations at the NH2 and COOH terminus of synaptobrevin have shown that the sequence 39-88 of synaptobrevin is the minimum substrate of TeNT, suggesting either the requirement of a well defined three-dimensional structure of synaptobrevin or a role in the mechanism of substrate hydrolysis for residues distal from the cleavage site. In this study, the addition of NH2- and COOH-terminal peptides of synaptobrevin, S 27-55 (S1) and S 82-93 (S2), to the synaptobrevin fragment S 56-81 allowed the cleavage of this latter peptide by TeNT to occur. This appears to result from an activation process mediated by the simultaneous binding of S1 and S2 with complementary sites present on TeNT as shown by surface plasmon resonance experiments and the determination of kinetic constants. All these results favor an exosite-controlled hydrolysis of synaptobrevin by TeNT, probably involving a conformational change of the toxin. This could account for the high degree of substrate specificity of TeNT and, probably, botulinum neurotoxins.
Tetanus toxin (TeNT)1 and the seven serotypes of botulinum neurotoxins (BoNT/A, /B, /C, /D, /E, /F, and /G), which are produced by several anaerobic bacilli of the genus Clostridium, cause the paralytic syndromes of tetanus and botulism by blocking neurotransmitter release at central and peripheral synapses, respectively (1, 2). They are formed by two disulfide-linked polypeptides, the heavy (H) chain (100 kDa) being responsible for specific binding to neurons and cell penetration, and the light (L) chain (50 kDa) for neuroexocytosis blockade, the reduction of the disulfide bond being required for this process (1, 2). The L chain of these neurotoxins contains the typical His-Glu-Xaa-Xaa-His sequence of zinc endopeptidases (3-5). Accordingly, each toxin has been shown to contain one atom of zinc (6-8), except BoNT/C for which two zinc atoms have been found (9). These L chains, but not their apo-forms, cleave specifically three proteins of the neuroexocytosis apparatus: synaptobrevin, which is hydrolyzed by TeNT, BoNT/B, /D, /F, and /G, SNAP 25, which is cleaved by BoNT/A, /C1, and /E, and syntaxin by BoNT/C1 (10-12). Moreover, as suspected from previous studies on zinc metallopeptidases (13), site-directed mutagenesis of the recombinant light chains of TeNT and BoNT/A showed that the two histidines of the motif His-Glu-Xaa-Xaa-His are zinc ligands and the glutamate plays a critical role in the catalytic process (14-17).
Among the group of zinc metallopeptidases, a particularity of
clostridial neurotoxins is a very high substrate selectivity, contrasting with the wide specificity of zinc proteases belonging to
the thermolysin and metzincin families (13). Indeed, except for
BoNT/C1 (11, 12), the clostridial neurotoxins appear to cleave a single protein substrate at a single peptide bond (10). The
target of five clostridial neurotoxins, synaptobrevin, also known as
VAMP (for vesicle-associated membrane protein), is a protein highly
conserved in eucaryotes (18-20), which play a central role in
neuroexocytosis (21-27). Human synaptobrevin isoform II contains 116 residues with a short COOH-terminal tail anchoring the molecule into
the membrane of small synaptic vesicles. Its cytosolic region is
composed of a NH2-terminal proline-rich domain of 24 residues followed by a hydrophilic domain of 72 amino acids (Fig.
1). TeNT cleaves synaptobrevin selectively at one single peptide bond Gln76-Phe77 (28). Successive
truncations of synaptobrevin at its NH2 and COOH terminus
have shown that the removal of both the proline-rich NH2-terminal 1-25 domain and the 93-116 transmembrane
anchor does not reduce the rate of hydrolysis by TeNT (29, 30) (Fig.
1). Conversely, truncations of the 27-49 acidic domain (30-32) as
well as truncations of the 82-93 basic domain (32) results in a
dramatic decrease (100 times) in the rate of cleavage (Fig. 1), the
minimum substrate of TeNT being the 50-residue peptide S 39-88 (32). Furthermore, several copies of a common acidic motif are present in the
sequence of the three targets of clostridial neurotoxins (33).
Antibodies raised against this motif cross-react with synaptobrevin,
SNAP-25, and syntaxin and inhibit the cleavage of synaptobrevin by
either TeNT or BoNT/B or /G (34). Two copies of this acidic motif are
present in the sequence of synaptobrevin and correspond to the 38-47
and 62-71 regions designated V1 and V2, respectively. Substitutions of
the acidic residues of V1 by serines in synaptobrevin abolish almost
completely its cleavage by TeNT (34, 35). Altogether these findings
suggested that domains of synaptobrevin far from the cleavage site
could play an important role in the hydrolysis of this peptide either
by inducing a cleavable conformation of synaptobrevin at the
Gln76-Phe77 bond or by promoting a proteolytic
conformation of the TeNT L chain. With the aim of answering these
questions, various peptides corresponding to the synaptobrevin (S)
acidic domain S1 (for S 27-55) and basic domain
S2 (for S 82-93) (Fig. 1) have been prepared by
solid-phase synthesis, and their influence on the cleavage of different
synthetic synaptobrevin fragments by purified TeNT L chain was studied.
We show here that the cleavage of synaptobrevin fragment S 50-93 or S
32-81, too short to be efficiently cleaved by TeNT, occurred following
addition of 1 mM S1 or 1 mM
S2, respectively, while the same concentrations of
S1 or S2 do not affect the high cleavage rate
of the complete substrate S 32-93. Moreover, the addition of both 1 mM S1 and 1 mM S2
allowed a cleavage of the short S 56-81 fragment by TeNT L chain to be
observed. Surface plasmon resonance experiments demonstrated that the
binding of TeNT L chain to S1 is dependent on the presence
of S2. Altogether these results show clearly that synaptobrevin
cleavage is dependent on the interactions of both well defined
NH2- and COOH-terminal domains S1 and
S2, with complementary exosites present in TeNT. The
influence of S1 and S2 on the kinetic constants
of the enzymatic reaction favors an allosteric mechanism (36-38),
probably through a conformational change of the toxin.
Hepes and all other buffer components were purchased from Sigma. High performance liquid chromatography (HPLC) grade solvents (acetonitrile and methanol) were obtained from SDS (Peypin, France). Solvents, reagents, and protected amino acids for peptide synthesis were obtained from Perkin-Elmer (Roissy, France). All the chemicals for surface plasmon resonance experiments were provided by Pharmacia Biosensor (Uppsala, Sweden).
Synthesis and Purification of PeptidesAssembly of the
protected peptide chains was carried out using the stepwise solid phase
method of Merrifield (39) on an Applied Biosystems (ABI) 431A automated
peptide synthesizer with ABI small scale Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry. The peptides (S
27-55, S 82-93, S 32-93, S 32-81, S 50-93, S 56-81, S 77-93, and
S 77-81) were synthesized as outlined in Cornille et al.
(29) with the exception of the fluorescent substrate [Pya88]S 39-88 whose synthesis has been previously
described (32). Peptides were cleaved from the resin, deprotected,
diethyl ether-precipitated, and washed in accordance with ABI
guidelines. All the peptides were purified by reverse phase (RP) HPLC
on a Vydac C18 column (250 × 10 mm) using
acetonitrile (CH3CN) gradients in 0.1% trifluoroacetic acid. Ion electrospray mass spectrometry allowed the molecular weight
of synaptobrevin peptides and metabolites to be verified. The
HPLC-purified peptides were lyophilized and stored at 20 °C. Peptide concentration was determined by absorbance measurements at 280 nm, on the basis of the extinction coefficient calculated from sequence
data (40).
TeNT was kindly provided by
Pasteur-Mérieux (France). The double chain was reduced by using
dithiothreitol and TeNT L chain was separated from the H chain and
purified by ion exchange chromatography as described previously in
Soleilhac et al. (32). The TeNT L chain was stored in 20 mM Hepes, pH 7.4, at 20 °C in siliconized Eppendorf
tubes. No change in peptidase activity was observed after 6 months.
For substrate peptides S 32-93, S 32-81,
S 50-93, and S 56-81, the enzymatic assays were carried out in a
typical volume of 100 µl in siliconized Eppendorf tubes. In this
case, 100 µM substrate (1 mM for S 56-81)
were incubated with 1 µg of TeNT L chain in the presence of 1 mM S1 or/and 1 mM S2 in
20 mM Hepes, pH 7.4, at 37 °C. The cleavage was stopped
by adding 50 µl of 0.2 M HCl, and this solution was
analyzed by HPLC on a Capcell C8 column (300 Å, 7 µm,
250 × 4.6 mm) under isocratic conditions (24% B for the
metabolite S 77-93 and 8% B for S 77-81. Eluent A was trifluoroacetic acid 0.05% in H2O; eluent B was
CH3CN 90%, trifluoroacetic acid 0.038% in
H2O; ultraviolet detection = 214 nm). After elution of
the cleavage product, the substrate and the activating peptide were
removed by washing the column with 100% B. The amount of cleavage
product released was determined by HPLC from a standard curve obtained
by injection of known concentrations of synthetic cleavage products (S
77-93 and S 77-81).
Real time surface
plasmon resonance sensorgrams were recorded on a Pharmacia Biosensor
BIAcore 2000 apparatus. Experiments were run with a CM5 sensor chip
(Pharmacia Biosensor, Uppsala, Sweden) on which 0.2 pmol/mm2 peptide S1 (relative response, 688 RU)
was covalently bound to the dextran matrix via free amino groups by the
N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide-N-hydroxysuccinimide activation method followed by a deactivation by ethanolamine. Another
channel was activated by the same method and deactivated in the same
way to serve as a control. The maximal response allowed for a 1/1
stoichiometry binding of TeNT to S1 was calculated to be
9800 RU given the molecular mass of TeNT L chain (50 kDa). Binding
experiments were performed at 25 °C with a flow rate of 10 µl/min
in 20 mM Hepes, pH 7.4. After each binding experiment, the
sensor chip was regenerated by a 6 M guanidinium chloride pulse of 30 s.
The enzymatic assay has been described in detail in Soleilhac et al. (32). Briefly, 20 µM substrate [Pya88]S 39-88 (50 amino acids) and different concentrations of S1 and S2 peptides were incubated in siliconized Eppendorf tubes in a total volume of 100 µl of 20 mM Hepes, pH 7.4, containing 250 ng of TeNT L chain (50 nM) for 20 min at 37 °C in the dark. The reaction was stopped by the addition of 900 µl of 72% MeOH, 0.1% trifluoroacetic acid in H2O, and the reaction mixture was then loaded on a Sep-Pak C18 cartridge (Waters) in order to separate the fluorescent cleavage product [Pya88]S 77-88 from the substrate. This metabolite was eluted with 3 ml of 65% MeOH, 0.1% trifluoroacetic acid in H2O, and the fluorescence of the eluate was read at 377 nm after excitation of the fluorophore at 343 nm using a Perkin-Elmer LS-50B fluorometer. The amount of released cleavage product was calculated from the fluorescence of known concentrations of the synthetic cleavage product [Pya88]S 77-88. The same assay was used for dose-dependent experiments with S1 and S2 peptides and to determine the kinetic constants for enzymatic cleavage in the presence of 100 µM S1 and S2 peptides. In some cases, enzymatic reactions were stopped by addition of 1 µl of trifluoroacetic acid, and the mixture was analyzed by RP-HPLC on a Capcell C8 (300 Å, 5 µm, 250 × 4.6 mm) column by using a 25-45% B gradient (A, trifluoroacetic acid 0.05% in H2O; B, CH3CN 90%, trifluoroacetic acid 0.038% in H2O) with an ultraviolet detection at 343 nm, in order to verify that the fluorescence increase was exclusively related to the formation of the cleavage product [Pya88]S 77-88.
To determine whether S1 or S2 could
promote the cleavage of synaptobrevin fragments S 50-93, S 32-81, and
S 56-81, which are poorly or not at all cleaved by TeNT L chain, the
latter peptides were incubated in the presence of 1 mM
S1 or S2 before addition of TeNT L chain. After
20 min at 37 °C, the reaction mixtures were analyzed by RP-HPLC in
order to quantify the level of cleavage products
generated. No effect on the cleavage rate of the optimal substrate S 32-93 was observed after addition of the same
concentrations of S1 (Fig. 2, C and
D) or S2 (Fig. 3A). In
contrast, the cleavage rate of S 50-93 in the presence of 1 mM S1 increased 34-fold (52 pmol·min1·µg
1) reaching a level
comparable to that of S 32-93 (102 pmol·min
1·µg
1) (Fig. 2, A
and B) but was not enhanced by the addition of 1 mM S2 (Fig. 3B). An even greater
potentiating effect was observed following addition of 1 mM
S2 to S 32-81 (Fig. 3C). This effect is
difficult to quantify because, in the absence of S2, the
cleavage of S 32-81 was undetectable; however, taking 0.1 pmol·min
1·µg
1 as the limit of
cleavage product detection, it can be assumed to be greater than
150-fold. The addition of 1 mM S1 to this
peptide did not improve its cleavage (Fig. 3C). In view of
these results the influence of the addition of 1 mM
S1 and/or 1 mM S2 was tested on the
synaptobrevin fragment S 56-81, lacking both S1 (S 27-55) and S2 (S 82-93) domains. In this case, as expected, the
simultaneous presence of the two peptides S1 and
S2 allowed a significant TeNT enzyme activity (12 pmol·min
1·µg
1) (Fig. 3D)
to be observed. Interestingly, no effects could be obtained under the
same conditions with a peptidic sequence shorter than S1, S
37-47 (data not shown), which corresponds to the acidic motif
conserved in SNAP-25 and syntaxin (33).
The Presence of S2 Is Required to Observe TeNT L Chain Binding to S1
A convenient method to observe
interactions between small soluble peptides and proteins is to use the
surface plasmon resonance technique (41). When a 6 µM
solution of TeNT L chain was injected into a flow cell containing
immobilized S1, no binding was observed (Fig.
4). Contrastingly, injection of a mixture of 6 µM TeNT L chain with 50 µM S2
showed a specific binding of about 4000 RU (Fig. 4). In a control
experiment, injection of 50 µM peptide S2
alone was shown to produce only a small response of 330 RU (Fig. 4).
The S2-promoted interaction between TeNT and S1
was dose-dependently reversed by preincubating the
TeNT-S2 mixture with a large excess of S1. With
1 mM S1, no detectable binding of TeNT was
observed (Fig. 4, inset).
Determination of the Influence of S1 and S2 on the Kinetic Constants of the Enzymatic Activity of TeNT L Chain
The minimum fluorescent substrate [Pya88]S
39-88, which contains the non-natural fluorescent amino acid
pyrenylalanine (Pya) in position 88 (32) was chosen to study the
influence of S1 and S2 on the kinetic constants
of the enzymatic reaction. Increasing concentrations of peptides
S1 and S2 induced a dose-dependent increase in [Pya88]S 39-88 cleavage, with maximal
substrate degradation obtained at 100 µM either
S1 or S2 (170 and 600%, respectively) (Fig.
5), which was therefore the concentration used to
measure the influence of the peptides on the kinetic constants of the
enzymatic reaction (Table I). The addition of peptide
S2 produced a shift of the apparent Michaelis constant
(Km) value of the substrate from 576 to 51 µM, while the maximal velocity
(Vmax) was only slightly affected (166 instead
of 111 pmol·min1·µg
1). As expected
from a partial overlap of the sequences of S1 and the
[Pya88]S 39-88 substrate, the potentiating effect
induced by the peptide S1 was lower, the
Km shifting from 576 to 479 µM and the
Vmax shifting from 111 to 169 pmol·min
1·µg
1) (Table I). In all
these studies, control experiments by RP-HPLC indicated that the
enhanced fluorescence corresponded to an increase in the formation of
the cleavage product [Pya88]S 77-88 (data not shown). No
other cleavage was observed.
|
The purpose of this study was to investigate the mechanism involved in the highly specific and selective cleavage of synaptobrevin at the Gln76-Phe77 bond (28). Previously reported data have shown that the acidic NH2-terminal sequence 27-55 and the basic COOH-terminal sequence 82-93 of synaptobrevin are essential for an efficient cleavage of this peptide (29-32). Different possibilities could account for these data. (i) The hydrolysis of synaptobrevin could require a well defined tertiary structure of the peptide substrate induced by residues far from the cleavage site. (ii) Additional interactions with TeNT could be necessary to generate the Michaelis complex. (iii) Interactions of well defined sequences of the substrate with specific exosites present on tetanus toxin could be necessary to promote synaptobrevin cleavage, as it is the case for allosteric enzymes.
In order to answer these questions, synthetic peptides S1 and S2 corresponding, respectively, to the acidic and basic motif of synaptobrevin were tested for their ability to enhance the cleavage rate of the complementary synaptobrevin sequences by TeNT. Interestingly, the cleavage of the synaptobrevin peptide S 50-93, which lacks almost completely the S1 sequence and which is very poorly cleaved by TeNT, was greatly enhanced (34-fold) by the addition of 1 mM S1. Similarly, the cleavage of the synaptobrevin peptide S 32-81, in which the full sequence of S2 is lacking and which is not cleaved by TeNT, was triggered (150-fold) by the addition of 1 mM S2. The decisive demonstration of the role of the S1 and S2 motifs came from the cleavage of S 56-81, a synaptobrevin peptide lacking both S1 and S2 domains, which was induced by the simultaneous addition of 1 mM S1 and 1 mM S2.
In all these experiments the cleavage rate measured for the mixtures of peptides was 2-7 times lower than that measured with the optimum substrate S 32-93 (Fig. 3). This was not unexpected, since the affinity for TeNT of the isolated fragments and their ability to fit the complementary sites within the protein cannot be as high as when the binding motifs are included in the substrate sequence.
These results do not support the hypothesis of a role for the NH2- and COOH-terminal peptides S1 and S2 in inducing a TeNT-cleavable conformation of synaptobrevin. Indeed, the formation of a unique tertiary structure, comparable to that of S 27-93, by mixing the three short peptides S 27-55 (S1), S 56-81, and S 82-93 (S2) is highly improbable. This is supported by the lack of a well defined tertiary structure for synaptobrevin in solution as previously observed by 1H NMR (29). A mechanism which could account for the present results is the promotion of an active conformation of TeNT induced by the binding of the S1 and S2 domains of synaptobrevin to exosites present on the toxin surface, as in the case of allosteric enzymes (36-38). Surface plasmon resonance analysis clearly showed the binding of the S1 domain of synaptobrevin to TeNT. This interaction occurs only in the presence of the S2 fragment and is not due to a direct interaction between S1 and S2 as shown by 1H NMR analysis (data not shown). This finding strongly suggests a cooperative interaction of both exosites. Conversely, when S2 was immobilized on the sensor chip, the ionic interaction of the highly basic S2 fragment with the multiple negative charges of the dextran matrix probably hampered the correct orientation of the S2 side chains, accounting for the absence of binding observed when either TeNT or a TeNT-S1 mixture was injected through a flow cell coated with S2 (data not shown). However, this interaction has been clearly demonstrated by the 10-fold decrease in the Km value of the fluorescent substrate following addition of the lacking S2 fragment (Table I).
Altogether these findings suggest that peptides S1 and
S2 favor the cleavage of synaptobrevin fragments by
interaction with corresponding exosites present at the toxin surface
and are in good agreement with an allosteric model of enzyme
functioning (36-38). According to this hypothesis, the predominant
conformational state of TeNT L chain in solution could correspond to
nonproteolytic form(s) in equilibrium with a very low percentage of the
proteolytic form. The higher affinity of the S1 and
S2 domains for the latter form would increase its
population, their interaction with the corresponding exosites on TeNT
triggering the proteolytic cleavage of the vesicle-associated membrane
protein (Fig. 6). The increased apparent affinity of the
substrate [Pya88]S 39-88, reflected by a reduction in
Km values in the presence of S1 and
especially S2 while the maximal velocity is not
significantly affected, also favors this assumption. Nevertheless, further physicochemical experiments will be necessary to physically observe the proposed change of TeNT conformation. As far as we know, a
cooperative mechanism has never been described for zinc proteases and
for proteases in general only for thrombin, a trypsin-like serine
protease, which makes use of exosites for recognition of its substrate
(42). TeNT L chain selectivity is dependent on this type of mechanism.
Given the similar behavior of BoNT/B and /G toward synaptobrevin in
terms of structure-activity relationships (34, 35), a similar mechanism
could also be involved in the selectivity of other clostridial
neurotoxins.
We thank Dr. J. R. Cartier (Pasteur-Mérieux) for kindly providing purified TeNT dichain and Dr. J. P. Changeux for critical reading of the manuscript.