©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Botulinum Neurotoxin Type C Cleaves a Single Lys-Ala Bond within the Carboxyl-terminal Region of Syntaxins (*)

Giampietro Schiavo (1), Clifford C. Shone (2), Mark K. Bennett (3), Richard H. Scheller (4), Cesare Montecucco (1)

From the (1) Centro Consiglio Nazionale delle Ricerche Biomembrane and Dipartimento di Scienze Biomediche, Universit di Padova, Via Trieste 75, 35121 Padova, Italy, the (2) Protein Toxins Section, Centre of Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom, the (3) Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, and the (4) Howard Hughes Medical Institute, Department for Molecular and Cellular Physiology, Stanford University, Stanford, California 94305

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-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.


INTRODUCTION

Botulism is characterized by a flaccid paralysis caused by botulinum neurotoxins (BoNT),() 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) .

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.


MATERIALS AND METHODS

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) .

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 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 NaHPO, 150 mM NaCl, pH 7.4.

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, 1 mM MgCl, 1.2 mM NaHPO, 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 NaHPO, 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 NaHPO, 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 NaHPO, 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).

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).

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.


RESULTS

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) ).

It is noteworthy that another Lys-Ala peptide bond (Lys-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.


DISCUSSION

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) .


FOOTNOTES

*
This work was supported by Telethon-Italy Grant 473. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: BoNT, botulinum neurotoxins; DTT, dithiothreitol; GST-syntaxin, recombinant glutathione S-methyltransferase syntaxin fusion protein; OP, ortho-phenanthroline; VAMP, vesicle-associated membrane protein; PAGE, polyacrylamide gel electrophoresis; L, light; H, heavy.


ACKNOWLEDGEMENTS

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.


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