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Introduction
Botulinum neurotoxins (BoNT) are potent toxins and are also therapeutic
agents[1]. These botulism-causing protein
toxins are produced by the anaerobic eubacterium, Clostri-dium botulinum. There are seven serologically and genetically
different BoNT, named BoNT/A-G, produced by serotypes A, B, C, D, E, F, and G Clostridium botulinum strains[2]. BoNT/A is synthesized by serotype A Clostridium botulinum strain as a single-chain protein with a molecular mass of approximately
150 kDa[3,4]. This protein is post-translationally proteolyzed to form a di-chain molecule in which the two chains, a ~50 kDa
light chain and a ~100 kDa heavy chain, remain linked by a disulfide bond and non-covalent
bonds[5,6]. The crystal structure of BoNT/A shows a linear arrangement of three functional domains named the receptor binding domain, translocation
domain, and catalytic domain[7]. These structural characters correspond to the intoxication process of BoNT/A and the
mechanism of BoNT/A intoxication consisting of 4-steps: receptor binding, internalization, translocation, and cleavage of the
synaptosomal associated protein of 25 kDa (SNAP-25). The receptor binding domain specifically binds to its neuronal
receptor, ganglioside GT1b or
GD1a, and unidentified protein
receptors[8]. After receptor binding and receptor-mediated
endocytosis of the neuro-toxins, they enter acidic neuronal organelles, synaptic vesicles, or
endosomes[8,9]. It is believed that the
translocation domain undergoes a conformational change and forms a protein-conducting channel on the membrane of the
endosome to translocate the catalytic domain into the cytoplasm of neuronal
cells[10]. The catalytic domain of BoNT/A is a
zinc protease[11,12] and is highly specific for the C-terminus of SNAP-25, a soluble NSF accessory protein receptor
(SNARE) protein complex component[13]. Cleavage of the C-terminus of SNAP-25 by the catalytic domain
ofBoNT/A inhibits SNARE complex formation as well as neurotransmitter
release[13,14]. Inhibition of the neurotransmitter, eg, acetylcholine, released in
the neuromuscular junction ultimately leads to paralysis and causes
botulism[1]. This specific action at the neuromuscular
junction of BoNT is increasingly being used to treat various neuromuscular
disorders such as strabismus, torticollis, and
blepharospasm[15,16].
Among the intoxication processes of BoNT/A and other types of BoNT, the translocation process is the least
understood[1,7]. Previous results show that BoNT can form ion channels in artificial planar lipid bilayers under acidic
conditions[10,17,18] and PC12 cell
membrane[19]. But, how can such a big soluble protein dramatically change to a hydrophobic membrane
protein? And how BoNT responds to acidic conditions to form an ion channel and how the light chain translocates across the
membrane barrier is not clear. Furthermore, the quaternary structure of BoNT/A in aqueous solution is not fully resolved.
Native gel electrophoresis had showed that the possible quaternary structure of BoNT/A is trimer or
tetramer[20,21]. However, the crystal structure of BoNT/A meant that it was initially recognized as a dimeric
species[22], but it is now recognized as a
monomeric species[23]. It is believed that the quaternary structure derived from X-ray crystallography is not reliable, because
under crystallization conditions the quaternary structure may
change[24, 25]. Thus, a clear solution to the structure of
BoNT/A in solution is critical, not only because of these conflicting reports but also because it can clarify the translocation process
of BoNT/A. In this study, sucrose density gradient centrifugation and chemical crosslinking experiments illustrated that
only after disulfide bond reduction did BoNT/A undergo dimerization in acidic conditions. And Triton-X 114
phase-separation experiments showed that acidic conditions and disulfide reduction are factors mediating the change in hydrophobicity
of BoNT/A. These results thus imply that disulfide reduction is the structural factor that corresponds to BoNT/A
translocation in synaptic vesicles or endosomes.
Materials and methods
Preparation of BoNT/A neurotoxin and toxoid We purified BoNT/A neurotoxin from cultures of C botulinum type A (ATCC 7948) according to the method developed by DasGupta and
Sathyamoorthy[3]. The purity was examined by 8%
SDS-PAGE and Coomassie blue staining (Figure 1A). The different batch preparations contained the most nicked BoNT/A and
traceable un-nicked BoNT/A. To generate antibodies against the BoNT/A toxin, we prepared the BoNT/A toxoid following
the method developed by Kozaki and
Sakaguchi[26]. BoNT/A (0.2 mg/mL) was toxoided by dialysis against 0.4% formalin in
0.1 mol/L phosphate buffer (pH 7.0) for 6 d at room temperature.
Production of antibodies The toxoid was mixed with an equal volume of Freund's complete adjuvant. Three 0.5-mL doses
of the toxoid emulsion were injected into two rabbits (2.7 and 3.2 kg) at 7-day intervals. Six weeks after the third injection, two
booster injections were given. Two weeks after the booster, the rabbits were exsanguinated from the cervical artery. Polyclonal
antibodies were purified from the rabbit's serum by protein A Sepharose 4 fast-flow affinity chromatography for IgG
antibodies[27]. To establish hybridoma cell lines that produce BoNT/A monoclonal antibodies, BALB/c mice were immunized
intraperitoneally with 250 µg toxoid in Freund's complete adjuvant and were boosted twice with 100 µg toxoid in
Freund's incomplete adjuvant at intervals of 2 weeks. Mice were boosted intraperitoneally with 50 µg toxoid in PBS 1 month later, and spleen
cells were fused with murine plasmacytoma FO cells for 4 d by the method of Galfre and
Milstein[28]. An enzyme-linked immunosorbent assay (ELISA) was used to screen culture supernatants showing positive binding to BoNT/A proteins.
Hybridomas from positive-binding wells were cloned by the limiting dilution method. To obtain monoclonal antibodies,
approximately
5×106 hybridoma cells were inoculated into the peritoneal cavity of Pristance primed BALB/c mice, and ascitic fluid was
collected on d 7_14. Monoclonal antibodies were then purified from the ascitic fluid by 40% saturated ammonium sulfate
precipitation and protein A Sepharose 4 fast-flow affinity chromatography for IgG antibodies.
ELISA assay of BoNT/A The modified ELISA method developed by Engvall and
Perlman[29] was used to analyze the antibodies against BoNT/A. Microtiter plates (PolySorp surface,Nalge Nunc International, Naperville, IL, USA) were coated
with 100 µL per well of BoNT/A protein (0.1 mg/mL) in 0.01 mol/L PBS, pH 7.0, overnight. After blocking with 1% BSA (bovine
serum albumin, Sigma Chemical Co, St Louis, MO. USA) in phosphate-buffered saline (PBS) for 1 h, plates were washed, and
diluted polyclonal antibodies or hybridoma supernatant (100 µL) were added to the wells and then incubated for 1 h. Plates
were next washed and incubated with alkaline phosphate (AP)-conjugated secondary antibodies for 1 h. Finally, the alkaline
phosphate enzyme activity was developed with the addition of substrate, and readings were taken at an optical density (OD)
of 405 nm with a Dynatech MR700 microplate reader. For the BoNT/A immunoassay, a sandwich ELISA was developed. The
ELISA plate wells were coated with 0.1 µg (1 µg/mL) monoclonal antibodies overnight and then incubated with 100 µL of the
BoNT/A protein or BoNT/E (0.007 to 1 µg/mL) or samples from sucrose density gradient centrifugation fractions for
1 h. The bound BoNT/A proteins were then labeled with 100 µL polyclonal antibodies (1
µg/mL), probed with AP-conjugated goat anti-rabbit IgG, and quantified using the colorimetric assay. Absorbance at 405 nm was measured with an ELISA plate
reader.
SDS-PAGE and
immunoblotting Proteins were separated by SDS-PAGE according to the procedure of
Laemmli[30] or the method of Weber and
Osborn[31] on a mini ProteinII system (Bio-Rad, Hercules, CA, USA). After SDS-PAGE frac-tionation,
the proteins were electrotransferred to a polyvinyli-dene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The
resulting membrane was blocked with PBS containing 0.05% Tween-20 and 5% BSA (Sigma) at room temperature. Subsequently,
the membrane was incubated with primary antibody in PBS at 4 oC overnight and then with AP- or horseradish peroxidase
(HRP)-conjugated secondary antibodies (Jackson ImmuneResearch, West Grove, PA, USA). The AP on the membrane was
detected using a Western-blue kit (Promega, Madison, WI, USA), and an enhanced chemiluminescence kit (Pierce, Rockford,
USA) was used to detect the HRP on the membrane.
Sucrose density gradient centrifugation An SW-41 rotor (Beckman, Palo Alto. CA, USA) was used to perform the
sucrose density gradient centrifugation experiments. Sucrose density gradients in 0.1 mol/L HEPES buffer (Sigma) at
different pH values (pH 7.5 and 4.5) were prepared with a sucrose gradient maker from two sucrose stock solutions containing 5%
and 20% (w/v) sucrose (Merck-Shuchardt Chemical Co, Germany). Gradients were maintained at 4 oC cold room for 4_6 h before use. Samples were then applied on top of the gradients, and were centrifuged at 5 oC for 19 h. Each sucrose density gradient centrifugation experiment consisted of a calibration control containing the marker proteins (Sigma), thyroglobulin
(19 S, 669 kDa), catalase (11.3 S, 232 kDa, purchased from Pharmacia Upjohn, Peapack, NJ, USA),
alcohol dehydrogenase (7.0 S, 150 kDa), and BSA (4.3 S, 66
kDa). Following centrifugation, sample fractions (0.65 mL) were removed from the bottom of the
centrifuge tube for the ELISA of BoNT/A.
Chemical crosslinking A homobifunctional crosslinking reagent, Bis (sulfosuccinimidyl) suberate
(BS3, Pierce), was used in this study. The functional group of
BS3 is a sulfo-NHS ester, which forms covalent linkages with the neighboring
primary amines. An 11.4-Å-long linker separates the functional groups of
BS3.
Prior to performing the crosslinking reactions, purified BoNT/A proteins (200
µg/mL) were dialyzed against the buffer solution containing 50 mmol/L HEPES and 100 mmol/L NaCl at pH 7.5, 6.1, and 4.5, respectively. For the reduced BoNT/A, the
toxins were treated with 20 mmol/L DTT at 37 oC for 30 min before the crosslinking reactions. For crosslinking BoNT/A with
BS3, 4 µL of freshly prepared 7.5 mmol/L
BS3 was added to 20 µL of purified reduced or non-reduced toxin (2 µg) and incubated
at room temperature. Aliquots of freshly prepared 7.5
mmol/L BS3 (3 µL) were added to the reaction mixtures 20 and 60 min
later. Reactions were stopped 1 h after the final addition of
BS3 by adding 50 mmol/L glycine. After the crosslinking reactions,
these samples were subjected to SDS-PAGE fractionation and assayed by Western blotting with BoNT/A polyclonal antibodies.
Triton-X 114 phase partition Purified BoNT/A proteins (200 µg/mL) were dialyzed against the buffer solution containing
50 mmol/L HEPES and 100 mmol/L NaCl at pH 7.5, 6.1, and 4.5, respectively. For the reduced BoNT/A, the toxins were treated
with 20 mmol/L DTT at 37 oC for 30 min before the phase-separation experiment.
The Triton X-114 phase-separation experiment was carried out according to the method of Bordier
(1981)[32]. A Triton X-114 (10%, 20 µL) solution was added to the BoNT/A protein toxins (180 µL) and incubated on ice for 5 min and then incubated
at 30 oC for 5 min. After centrifuging for 5 min in an Eppendorf centrifuge, 10% Triton X-114 (10 µL) was added to the water
phase, and 0.1 mol/L HEPES buffer was added to 200 µL, producing a final Triton X-114 concentration of 0.5%. Triton X-114
phase separation was repeated again, and the detergent phase was added together. Triton X-114 was again added to the
water phase to obtain a final concentration of 2%. After phase separation, the water phase was removed to a new Eppendorf
tube, and the detergent phase was discarded. Then 0.1 mol/L HEPES buffer was added to the detergent phase and water
phase solution to 500 µL, respectively. Aliquots of the detergent phase and water phase samples (40 µL) were added to 15 µL
of the SDS-PAGE sample solution. These samples were analyzed by Western blotting.
1-Anilino-8-naphthalene sulfonate (ANS) binding ANS fluorescence measurements were made on a Perkin-Elmer
spectrofluorometer model LS-50B. ANS (Molecular Probes, Eugene, OR, USA) was dissolved in absolute ethanol, and its
concentration was determined by spectrophotometer
[e372(ANS) = 8000
mol-1cm-1]. Purified BoNT/A proteins (20
µg/mL) were dialyzed
against the buffer solution containing 50
mmol/L HEPES and 100 mmol/L NaCl at pH 4.5. For the
reduced BoNT/A, the toxins were treated with 20 mmol/L DTT at 37 oC for 30 min before the ANS binding experiment. One
microliter aliquots of 5 mmol/L ANS were added into a
1-cm path length cuvette containing 1 mL of 0.125 µmol/L proteins. BoNT/A or reduced BoNT/A with ANS was then excited
at 360 nm, and the emission was recorded between 400 and 580 nm. The excitation and emission slit widths were both set to
15 nm. All measurements were made at 25 oC.
Results
Development of the immunoassay of
BoNT/A Because BoNT/A is a zinc protease[11,
12], its biochemical characterization is not convenient. To facilitate the biochemical analysis of BoNT/A, we tried to establish an immunoassay for BoNT/A. We
identified two monoclonal antibodies, named H-4F and H-3, that specifically recognize the heavy chain of BoNT/A, and one
monoclonal antibody, L-1H, that specifically recognizes the light chain of BoNT/A (Figure 1A). These three monoclonal
antibodies then served as capture antibodies for the ELISA of BoNT/A. We found that L-1H failed to act as a capture
antibody for the ELISA of BoNT/A when combined with the polyclonal antibodies generated from the rabbit. However, the
H-4F and H-3 monoclonal antibodies could serve as capture antibodies and detected BoNT/A at as low as 10 ng/mL
BoNT/A by sandwich ELISA. Further-more, this assay can distinguish BoNT/E and BoNT/A, even though the presence of
BoNT/E was as high as 100 ng/mL (data not shown). Thus, we performed the ELISA to characterize the crude extract from
culturesof C botulinum type A after sucrose density gradient centrifugation. It is well
established that in addition to a 150-kDa toxic protein com-ponent, a progenitor toxin with a molecular mass of 900 kDa,
composed of hemagglutinin proteins and a nontoxic non-hemagglutinin protein, also exists in the crude extract of cultures
of C botulinum type A[8]. Figure 1B shows that the crude extract existed as two forms of BoNT/A, 7S and 19S, which correspond
to the pure BoNT/A toxin protein and the 900-kDa BoNT/A complex protein. These results indicated that ELISA can serve as
a convenient tool to assay the biochemical properties of BoNT/A.
Sucrose density gradient centrifugation demonstrated that disulfide reduction is necessary for the dimer formation of
BoNT/A under acidic conditions Clostridium
botulinum neurotoxin, like tetanus toxin and diphtheria toxin, forms ion
channels in planar lipid bilayers under acidic
conditions[10,16,17]. The ion channel activity had been proposed as the
translocation mechanism of BoNT/A in neuronal
cells[8]. Thus, we hypothesized that type A C botulinum neurotoxin proteins can form oligomeric proteins in acidic conditions, like the anthrax protective
antigen[33]. Sucrose density gradient centrifugation
combined with the ELISA was used to analyze the oligomer states of BoNT/A. Figure 2A shows that the purified BoNT/A
contained the toxin component only, without nontoxic protein contamination. These nontoxic protein contaminations may
be associated with BoNT/A and may interfere with judgment concerning the oligomeric state of BoNT/A. After dialysis of the
purified BoNT/A in pH 4.5 and 7.5 HEPES buffer, 0.2 mg/mL BoNT/A was loaded on a 5%_20% sucrose density gradient and
centrifuged at 151 200×g for 19 h at 5 oC. We found that the BoNT/A protein migrated as a 7S, monomeric toxin with a
molecular mass of about 150 kDa, in both neutral and acidic conditions (Figure 2B). These results suggest that factors other
than an acidic condition are necessary for the oligomerization of BoNT/A.
Previous studies showed that the heavy chain of BoNT/B, but not the entire toxin, can form ion channels under acidic
conditions[10]. Recent results also indicated, after DTT reduction, that the BoNT/A structure is more flexible than the
non-reduced form[34]. Thus, we assumed that reduction of the disulfide bond between the heavy chain and the light chain may
facilitate oligomeric channel formation of BoNT/A. To test this assumption, BoNT/A proteins were treated with DTT before
being subjected to sucrose density gradient centrifugation at pH 4.5. We found that treatment of BoNT/A with 20 mmol/L
DTT at 37 oC for 30 min efficiently reduced the disulfide bond between the heavy and light chains (compare lanes 7 of Figure
4A and 4B). Figure 2B shows that the DTT-reduced BoNT/A proteins migrated as a major peak at the 12S position with an
approximate molecular weight of 300 kDa. This result suggests that BoNT/A proteins can form dimers under acidic
conditions only after disulfide bond reduction.
Chemical crosslinking studies of type A Clostridium botulinum neurotoxin The channel stoichiometry formed by the BoNT has not been fully understood, but a dimer and tetramer structure has been
proposed[10,17,18,21,35]. To further clarify the
oligomeric state of BoNT/A, we used BS3 (0.5
mmol/L) to perform chemical crosslinking on BoNT/A after extensive dialysis in pH 4.5 and 7.5 HEPES buffer, respec-tively.
Although the sucrose density gradient centrifugation experiment indicated that BoNT/A proteins exist as monomers under
these conditions, the crosslinking pattern of BoNT/A showed a ladder form, from monomer to tetramer (Figure 3, lanes 2 and
3). In contrast, the crosslinking pattern of 20 mmol/L DTT-reduced BoNT/A showed that, under neutral conditions, the final
product was a monomer (Figure 3, lane 4) and that in an acidic conditions the final products were dimers of BoNT/A (Figure
3, lane 5). From this result, combined with the sucrose density gradient centrifugation, we suspected that BoNT/A can form
dimers after reduction, and the tetramer observed in the non-reduced BoNT/A crosslinking study may have arisen from
intermolecular crosslinking artifacts.
Disulfide bond reduction and acidic pH are responsible for the hydrophobic change of type
A C botulinum neurotoxin Previous studies have shown that botulinum neurotoxins can form ion channels on planar lipid
bilayers[10,17,18]. This suggests that aqueous BoNT/A possibly becomes a hydrophobic-like protein in order to cross the hydrophobic barrier.
Interestingly, Figure 2B shows that, in addition to the dimeric BoNT/A protein, there are large BoNT/A aggregations after
disulfide bond reduction in an acidic condition. This result may imply that the hydrophobicity of BoNT/A undergoes a
dramatic change in acidic conditions after
disulfide bond reduction. It has been shown that the Triton X-114 phase separation experiment can distinguish membrane
proteins from water-soluble
proteins[32]. So we first prepared the BoNT/A proteins under different pH conditions, either
reduced with 20 mmol/L DTT or not, and then performed the Triton X-114 phase separation experiment. As shown in Figure
4A, most non-reduced BoNT/A proteins were still hydrophilic-like proteins. Densitometric scans indicated that less than
20% of the BoNT/A proteins were partitioned into the detergent phase (Figure 4A, lanes 1, 3, and 5). These results indicate
that factors other than the acidic condition are necessary for the hydrophobicity change of BoNT/A. Indeed, under an acidic
condition, reduced BoNT/A dramatically changed to hydrophobic proteins (Figure 4B, compare lanes 1 and 2 and lanes 3 and
4). A densitometric scan indicated that more than 90% of the heavy chains and light chains of BoNT/A were partitioned into
the Triton-X114 phase. However, the disulfide bond-reduced BoNT/A was still a hydrophilic-like protein under a neutral
condition, and less than 40% of the heavy chains and light chains of BoNT/A were partitioned into the Triton-X114 phase
(Figure 4B, compare lanes 5 and 6). To further confirm the results of Triton X-114 phase separation, we probed the
hydrophobic change in BoNT/A with a hydrophobic-sensitive dye, ANS. The ANS anion is an often-utilized hydrophobic probe for
proteins[36]. When excited at 360 nm, the binding of nonpolar ANS to the hydrophobic region of a protein is associated with
enhanced ANS fluorescence emission spectra at 480 nm. We found that no obvious ANS fluorescence was
observed when non-reduced BoNT/A at pH 7.5 (Figure 5, line 1). However, there was nearly a two-fold increase in the
fluorescence intensity when ANS was bound to the reduced
BoNT/A than to the non-reduced BoNT/A at pH 4.5 (Figure
5, compare lines 2 and 3). Combining the Triton X-114 phase separation and ANS binding experiments, our observations imply
that an acidic condition and disulfide reduction are
both necessary for the change in hydrophobicity of BoNT/A.
Discussion
As to their intracellular actions, many plant and bacterial toxins do not directly penetrate the plasma membrane but go
through a low-pH compartment (ie, synaptic vesicle or
endosome)[37]. These toxins have evolved sophisticated mechanisms
to translocate their active portion through the hydrophobic barrier of these acidic organelles. For example, some studies have
shown that the diphtheria toxin undergoes a conformational change and exposes hydrophobic sites within the acidic endosomal
lumen[37]. The exposure of hydrophobic sites then induces membrane insertion and channel formation, and results in
translocation of the catalytic domain. These results indicate that an acidic pH is the main factor triggering membrane
insertion of diphtheria toxin, but disulfide bond reduction and nicking of the diphtheria toxin are not necessary for membrane
insertion[38]. Similarly, there are experiments which show that an acidic pH is necessary for membrane insertion and ion
channel formation of
BoNT[8,10]. However, circular dichroism and fluorescence spectroscopic studies revealed that the conformation of BoNT/A
does not undergo a drastic change over a range of pH 6_9 [39], and near-UV circular dichroism spectroscopy indicated that the
structural features of BoNT/A change considerably upon disulfide
reduction[34]. So, disulfide reduction might influence the
membrane insertion of BoNT. Consistent with these spectroscopic studies, Triton X-114 phase separation showed that
disulfide bond reduction and an acidic condition (pH 4.5 and 6) were both required for the increase in hydrophobicity of
BoNT/A protein molecules (Figure 4). In addition, the large aggregate found in sucrose density gradient centrifugation may
reflect the hydrophobic change of the disulfide bond-reduced BoNT/A protein in an acidic condition (Figure 2B), like the
diphtheria toxin and protective antigen of anthrax toxin found in acidic
conditions[38,40]. Interestingly, the traceable un-nicked
BoNT/A did not change its hydrophobicity even after disulfide reduction (Figure 4B, lanes 2, 4, and 6). This is similar to the
anthrax toxin protective antigen in which low pH-induced proteolytically nicked
PA63 hydrophobicity increases but does not
induce a change in hydrophobicity in the intact protective
antigen[40]. Consistent with these observations, we observed that
the enhancement of fluorescence of ANS by the disulfide-reduced BoNT/A was nearly two-fold stronger than
thatof the non-reduced form in an acidic condition (Figure 5) and
Kamata et al also recently showed that botulinum type B-nicked
neurotoxin becomes hydrophobic more quickly and extensively than does the un-nicked toxin under acidic pH
conditions[41]. These results suggest that the nick between the heavy chain and the light chain may also involve membrane insertion of
botulinum neurotoxins in acidic pH conditions.
The channel stoichiometry of BoNT/A was studied by sucrose density gradient centrifugation and chemical crosslinking
experiments. Sucrose density gradient centrifugation showed that the non-reduced BoNT/A did not form oligomers even in
an acidic pH (Figure 2B). This result is consistent with the light-scattering experiment, without the hydraulic pressure in the
sucrose density gradient centri-fugation, which revealed that only the monomer of BoNT/A exists in solution over a broad pH
range[42]. But, in the chemical crosslinking study, tetramers emerged as the final product of
BS3 crosslinked, non-reduced BoNT/A (Figure 3, lanes 2 and 3). There are two possible reasons to explain these contrasting results. First, the tetramer
observed in BS3 crosslinked BoNT/A is intermolecular crosslinking artifacts and not intramolecular crosslinking under the
crosslinking condition. Second, the interaction between the tetramer of BoNT/A is weak, and the hydraulic pressure during
the sucrose density gradient centrifugation is large enough to dissociate the tetramer into monomers.
Furthermore, we also found that reduced BoNT/A proteins can form dimers under acidic conditions, either by sucrose
density gradient centrifugation (Figure 2B) or chemical crosslinking (Figure 3, lanes 5). Recently, Cai and Singh had reported
that BoNT/A, by native gel electrophoresis, chemical crosslinking, gel filtration and fluorescence anisotropy but not by
sucrose density gradient centrifugation, exists as a dimer in both reduced and non-reduced
conditions[43]. Interestingly, using fluorescence anisotropy they also found that a high concentration (above 200 nmol/L) of BoNT/A existed as a dimer,
while at a low concentration (20 nmol/L) it existed as a monomer. Comparing with the results of Cai and Singh, we also found
that BoNT/A (approximately 440 nmol/L) can exist as a dimer, trimer or tetramer by
BS3 crosslinking experiments (Figure 3,
lanes 2 and 3). This result may imply that there are weak interaction between the non-reduced BoNT/A and this may change
the equilibrium between oligomer states at neutral pH condition. But the hydraulic pressure during centrifugation may
affect the weak interactions and only monomer was observed in the sucrose density gradient centrifugation experiment
(Figure 2B). From these observations, we concluded that BoNT/A forms a dimer structure after disulfide reduction in acidic
conditions, implying that the BoNT/A channel stoichiometry is a dimer. Although, molecular modeling of the pore structure
of BoNT/A and electric density mapping of the BoNT/A protein both suggest a tetramer structure for
BoNT/A[18,34]. This result is consistent with reports by Donovan and Middlebrook, who measured the dependence of conductance on BoNT/C
concentration and also indicated that the stoichiometry of the channel is a
dimer[17].
Recently, a triterpenoid derivative, toosendanin, extracted from the bark of Melia toosendan Sieb et Zucc had been demonstrated with antibotulismic effect via interference with toxin
translocation[44,45]. In this study, we show that disulfide
reduction is responsible for the change in hydrophobicity and dimer formation of BoNT/A in an acidic condition. This
finding implies that compounds that block this disulfide bond reduction may serve as potential therapeutic agents for
botulism. Furthermore, mutations on disulfide reduction sites might reduce the toxicity of BoNT by preventing the
translocation process. These translocation-deficient mutated BoNT may serve as good vaccine candidates for botulism.
Acknowledgement
We greatly thank Mrs Rey-fun SHEU for technical assistance .
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