![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 289, Issue 3, 1509-1516, June 1999
Department of Physiology and Pharmacology (J.A.C.), College of Veterinary Medicine, University of Georgia, Athens, Georgia and Department of Medicine; and Department of Biochemistry and Molecular Pharmacology (N.M.B., A.B.M., L.L.S.), Jefferson Medical College, Philadelphia, Pennsylvania
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Botulinum toxin blocks transmitter release by proceeding through a
series of four steps: binding to cell surface receptors, penetration of
the cell membrane by receptor-mediated endocytosis, penetration of the
endosome membrane by pH-induced translocation, and intracellular
proteolysis of substrates that govern exocytosis. Each of these steps
is essential for toxin action on intact cells. Therefore, alterations
in cell structure or cell function that impede any of these steps
should confer resistance to toxin. In the present study, screening for
susceptibility to four serotypes of botulinum toxin revealed that the
cutaneous-pectoris nerve-muscle preparation of Rana
pipiens is resistant to type B botulinum toxin. Resistance was
demonstrated both by electrophysiologic techniques and by dye-staining
techniques. In addition, resistance to serotype B was demonstrated at
toxin concentrations that were 2 orders of magnitude higher than those
associated with blockade produced by other serotypes. In experiments on
broken cell preparations, type B toxin cleaved synaptobrevin from frog
brain synaptosomes. However, the toxin did not bind to frog nerve
membranes. These findings suggest that resistance is due to an absence
of cell surface receptors for botulinum toxin type B. The fact that
cutaneous-pectoris preparations were sensitive to other botulinum toxin
serotypes (A, C, and D), as well as other neuromuscular blocking agents (
-latrotoxin, 
-bungarotoxin), indicates that botulinum toxin type
B receptors are distinct.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Botulinum
toxin acts in the cytosol of vulnerable cells to block spontaneous and
evoked transmitter release. Although the toxin exerts this effect on
most nerve cells, it acts preferentially on cholinergic nerve endings.
Dose-response experiments indicate that the vertebrate neuromuscular
junction is the site that is most sensitive to toxin action (for
reviews, see Simpson 1989
; Montecucco, 1994).
To produce blockade of neuromuscular transmission, botulinum toxin
proceeds through a series of steps (Simpson, 1980, 1981). This sequence
involves binding to the plasma membrane, internalization by
receptor-mediated endocytosis, escape to the cytosol by pH-induced translocation, and eventual expression of zinc-dependent
metalloendoprotease activity. The substrates for the protease activity
of botulinum toxin are synaptobrevin, synaptosomal-associated protein
of 25 kDa (SNAP-25), and syntaxin, each of which is essential for
transmitter release (Schiavo et al., 1994). Of the seven toxin
serotypes, four act on synaptobrevin (types B, D, F, and G), two cleave
SNAP-25 (types A and E), and one cleaves both syntaxin and SNAP-25
(type C).
There is a substantial literature showing that almost all cholinergic neuromuscular junctions are sensitive to botulinum toxin. In fact, every junction that has been tested is poisoned by at least one of the seven toxin serotypes. Because of the near universality of toxin action, relatively little attention has been devoted to identification of cholinergic neuromuscular junctions that are resistant. This is unfortunate, because a comparison of sensitive and resistant cell lines could shed light on the complex sequence of events that underlies toxin action. In addition, a comparison of sensitive and resistant lines could provide novel insights into fundamental aspects of cell biology.
At least in theory, there are four alterations in cell structure or function that could produce resistance to botulinum toxin: 1) absence of cell surface receptors; 2) loss of receptor-mediated endocytosis; 3) loss of the endosomal proton pump; and 4) absence of substrate. Each of these corresponds to the four steps that underlie toxin action.
To date, no cholinergic neuromuscular junctions have been identified
that are resistant to toxin action because of alterations in
receptor-mediated endocytosis or pH-induced translocation. However, the
discovery of such cells is conceptually possible because drugs that
neutralize endosomal pH (Simpson, 1982, 1983) or block the endosomal
proton pump (Simpson et al., 1994) do inhibit toxin action. A small
number of cholinergic neuromuscular junctions have been identified that
are resistant to toxin action because of an absence of substrate. For
example, Burgen et al. (1949) reported that the rat phrenic
nerve-hemidiaphragm preparation is relatively resistant to serotype B. Interestingly, rat synaptobrevin has a mutation (Gln 
Val) at the
site of toxin-induced proteolysis, suggesting that the rat
neuromuscular junction is relatively resistant to botulinum toxin type
B due to an absence of vulnerable substrate (Patarnello et al., 1993).
No one has yet identified a cholinergic neuromuscular junction that is
resistant to botulinum toxin because of an absence of cell surface
receptors. Therefore, a concerted effort has been made in the present
study to find a tissue that is relatively or absolutely resistant to at
least one, but not all, of the seven toxin serotypes. This search has
resulted in the discovery that the cutaneous-pectoris nerve-muscle
preparation of Rana pipiens is resistant to botulinum toxin
type B, and resistance is related to an absence of binding sites on the
cell surface. This discovery will make it possible to address three
related aspects of botulinum toxin action, as follows: 1) the role of
synaptotagmin as a receptor for serotype B; 2) the competence of
high-affinity and low-affinity toxin-binding sites in evoking
neuromuscular blockade; and 3) the relationship between the receptor
for serotype B and receptors for other botulinum toxin serotypes (i.e.,
A, C, and D) and other neuromuscular blocking agents (i.e.,
-latrotoxin and -bungarotoxin).
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Toxins.
Botulinum toxin types A and B were isolated and
tested for potency as described previously (Simpson et al., 1988);
serotypes C and D were purchased from WAKO Fine Chemicals (Dallas, TX). Serotypes A, C, and D were in the nicked and activated form. Serotype B
was activated by adding it to N-tosyl-phenylalanine
chloromethylketone-treated trypsin that was coupled to agarose
beads [trypsin-toxin, 1:40 (w/w)]. The mixture was incubated at
37°C for 15 min in 0.02 M sodium phosphate buffer, pH 7.0. The
reaction was terminated by centrifugation and aspiration of activated
toxin. The homogeneity and molecular structure of the toxins were
confirmed by polyacrylamide gel electrophoresis in the presence of SDS,
and biological activity of the toxins was measured on mouse phrenic
nerve-hemidiaphragm preparations as described previously (Simpson and
DasGupta, 1983).
Neuromuscular Preparations.
Frog cutaneous-pectoris
preparations were exposed to botulinum neurotoxin under two conditions.
In the initial experiments, various serotypes of botulinum toxin were
added to tissues at 30°C, nerve stumps were stimulated, and twitch
responses were recorded. The purpose of the experiments was to
determine which serotypes block frog neuromuscular transmission. In the
second set of experiments, toxin was added to tissues, and spontaneous miniature endplate potentials (MEPPs) were monitored. Standard intracellular recordings were obtained using glass microelectrodes filled with 3 M KCl (tip resistance, 20-40 M
). The purpose of these
experiments was to determine the rate at which various serotypes could
paralyze tissues in which vesicles had previously been stained with
FM1-43 (see below). Nerve stimulation was not applied during development of paralysis so that vesicles would not destain.
Visualization of Exocytosis and Toxin Action.
Procedures for
staining vesicles with the styryl dye FM1-43 and for monitoring
stimulation-evoked exocytosis have been described in detail (Betz et
al., 1992; Betz and Bewick, 1993). Briefly, cutaneous-pectoris
nerve-muscle preparations were dissected from R. pipiens and
pinned in Sylgard-lined dishes. Unless otherwise noted, tissues were
maintained at room temperature (about 23°C) in Ringer solution
consisting of 120 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, and 2.4 mM NaHCO3.
Vesicles were stained by adding 2 µM FM1-43 to the incubation medium
and stimulating nerves continuously (10 Hz) for 4.5 to 5 min. Tissues
were then washed for 30 to 90 min at 4°C to remove dye associated
with surface membranes.
Iodination of Botulinum Toxin and Ligand-Binding Experiments. Botulinum toxin type B was radioiodinated with Bolton-Hunter reagent. Purified toxin (100 µg) was mixed with 1 mCi of 125I-Bolton-Hunter reagent in 100 mM borate buffer, pH 8.0, for 30 min at room temperature. Iodinated toxin was separated from free iodine by fractionation on Sephadex G-50 columns. Preparations of labeled material typically had a specific activity of 600 to 900 Ci/mmol and a residual toxicity of 70 to 90%.
A crude membrane preparation was obtained by homogenizing frog brain in iced Tris·HCl buffer (50 mM, pH 7.4). The homogenate was centrifuged for 10 min at 1000g, and the resulting homogenate was resuspended in fresh buffer and recentrifuged for 45 min at 40,000g. The final pellet was resuspended in Tris·HCl buffer (as above). The binding of iodinated botulinum toxin type B to membrane preparations was measured by a centrifugation assay as described previously (Bakry et al., 1991b; Coffield et al., 1997).
The ligand was mixed with a specified concentration of membrane (see
Results) in 100 µl of buffer containing 50 mM Tris·HCl,
pH 7.4, 100 mM NaCl, and 1 mg/ml BSA. The binding reaction was allowed
to proceed at room temperature (about 23°C) for the amount of time
necessary to reach equilibrium (see Results). The reaction
was terminated by centrifugation (15,000g, 2 min), after
which the pellet was washed with fresh buffer and recentrifuged.
Membrane-associated ligand was collected and quantified, and the
results were corrected for nonspecific binding. The data were evaluated
by using the equilibrium binding analysis program of McPherson (1982).
Vesicle Isolation and Incubation with Toxin. Frog or mouse brain vesicles were isolated according to standard procedures. All isolation steps were performed at 3°C (on ice). Briefly, frog or mouse brains were excised and minced in homogenization buffer (255 mM sucrose, 1 mM EDTA, 20 mM HEPES, pH 7.4). The resulting suspensions were homogenized in a glass-Teflon homogenizer (2 × 20 strokes). The homogenate was fractionated using an SS-34 rotor in a Sorvall RC5B centrifuge (Sorvall, Inc., Newtown, CT). Homogenates were spun at 1000g for 5 min. The resulting supernatant (S-1) was recentrifuged at 10,000g for 10 min. The second supernatant (S-2) was recentrifuged at 250,000g for 1 h, and the resulting pellet (P-3; brain vesicle fraction) was resuspended in homogenization buffer and used for digestion experiments.
Nicked, reduced (10 mM dithiothreitol; 2 h) toxin was preincubated with 200 µM tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) for 2 h at room temperature. A sample of toxin without TPEN was processed in parallel. Subsequently, these toxin preparations were added to aliquots of frog or mouse brain vesicles. Vesicles were incubated for 4 h with 1 × 10
8 M
nicked toxin in the presence or absence of a final concentration of 30 µM TPEN. Aliquots processed identically but without toxin served as controls.
After incubation, reducing sample buffer was added to each tube, and
the digests were separated using polyacrylamide gel electrophoresis.
Peptide Antibodies and Western Blot Analysis. Monoclonal antibodies against VAMP-1 and VAMP-2 were generously provided by Dr. Reinhard Jahn (Boyer Center for Molecular Medicine and Howard Hughes Medical Institute, Yale University). Antibodies against syntaxin and SNAP-25 were purchased from Sigma (St. Louis, MO) and Sternberger Monoclonals Inc. (Baltimore, MD), respectively.
Samples for Western blot analysis were separated in 12% Tris-tricine gels (Schägger and von Jagow, 1987). Subsequent to separation, proteins were transferred to NitroPure membranes (Micron Separations Inc., Westboro, MA) in Tris-glycine transfer buffer at 50 V for 30 to
45 min. Blotted membranes were rinsed in distilled water and stained
for 1 min with 0.2% Ponceau S in 1% acetic acid. Following a brief
rinse with distilled water, molecular weight markers and transferred
proteins were identified. Membranes were destained in PBS-Tween (pH
7.5; 0.1% Tween 20), blocked with 5% nonfat powdered milk in
PBS-Tween overnight at 3°C. For identification of membrane or vesicle
proteins, membranes were washed again (3×) and incubated with primary
antibodies at the appropriate dilution in 0.05 to 1.0% milk for 2 to
5 h at room temperature.
For visualization of synaptobrevin in toxin digestion experiments,
membranes were incubated in 5% milk with a 1:2000 dilution of Cl 10.1 anti-synaptobrevin monoclonal antibody for 6 h at room temperature.
Membranes were washed again (3×) and visualized using enhanced
chemiluminescence (Amersham-Pharmacia Biotech, Piscataway, NJ.)
according to the manufacturer's instructions. Membranes were exposed
to film (Hyperfilm-ECL) for times adequate to visualize chemiluminescent bands. Peptides were identified by comparison with
known standards.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Botulinum Toxin Blocks Frog Neuromuscular Transmission. A number of elegant studies have been published showing that botulinum toxin blocks frog neuromuscular transmission. However, most of these studies were published more than two decades ago. This means that most of the work was done 1) before the discovery that there is a multistep sequence of events that underlies toxin action and 2) before the discovery that all serotypes of botulinum neurotoxin are metalloendoproteases. A further limitation on these studies is that, with few exceptions, attention has been limited to serotype A.
In the present work, three toxin serotypes were selected for study based on substrate specificity as follows: serotype A, SNAP-25; serotype B, synaptobrevin; and serotype C, syntaxin. Each serotype was added to tissues (n = 5) at a concentration of 3 × 109 M, and nerve-stimulus-evoked muscle
twitch was monitored for at least 180 min.
Botulinum toxin types A and C produced irreversible blockade of
transmission. The respective paralysis times were serotype A, 165 ± 12 min; serotype C, 116 ± 9 min. In contrast, serotype B had
no observable effect on evoked twitch, even when monitored for 180 min.
A similar set of experiments was done by monitoring spontaneous MEPP
frequency, except that serotype D
which cleaves synaptobrevinwas added to the study. The results demonstrated that serotypes A, C, and D
blocked transmission (Fig. 1), but
serotype B had no effect.
|
7 M). Interestingly, there was
still no evidence that this serotype had an effect on neuromuscular transmission.
For those serotypes that did act on frog neuromuscular junctions, the
characteristics of toxin-induced blockade were the same as those
routinely observed at mammalian neuromuscular preparations. For
example, lowering temperature had a profound effect on toxin-induced paralysis. When serotypes A, C, or D (3 × 109 M) were added to tissues at 4°C, there
was no onset of paralysis within a 12-h observation period. The marked
effect of temperature has been interpreted to mean that toxin-induced
paralysis is an energy-dependent process (viz., receptor-mediated
endocytosis; see Discussion).
Another similarity between toxin action on mammalian and frog
neuromuscular junctions is the existence of a lag time. As shown in
Fig. 1, there was an interval between addition of toxin to tissues and
the initial signs of neuromuscular blockade. This interval has been
interpreted to mean that there is a lag time necessary for the toxin to
be productively internalized and reach its intracellular substrate (see
Discussion).
Toxin Action at the Frog Neuromuscular Junction Can Be Demonstrated
by Visual as Well as Electrophysiologic Techniques.
FM1-43 is a
fluorescent dye that can be used to stain the membranes of recycling
synaptic vesicles. The dye has been previously used to study normal
exocytotic activity at neuromuscular junctions (Betz and Bewick, 1992),
as well as abnormal activity in the presence of neuromuscular blocking
agents such as -latrotoxin (Henkel and Betz, 1995) and botulinum
toxin (Henkel et al., 1996). In the present study, nerve terminals were
loaded with FM1-43, and nerve terminals were photographed to obtain
baseline values for intensity and distribution of vesicle staining.
Botulinum toxin types A, B, C, or D were subsequently added to tissues
(108 M), which were allowed to incubate for at
least 2 h at 23°C. Single nerve pulses were applied at 120 min,
and at approximately 20-min intervals thereafter, until transmission
was blocked.
8 M)
produced little (<10%) destaining (Fig. 2, panel 2), and the same
result was obtained in tissues poisoned with serotypes C and D. By
contrast, tissues treated with botulinum toxin type B were
indistinguishable from control tissues (Fig. 2, panel 1, C and D).
|
|
Serotype B Binds to Mouse But Not to Frog Membrane
Preparations.
Studies that successfully quantify the rate
constants for toxin association and dissociation at cholinergic
neuromuscular junctions have not been reported. The absence of such
work is due to the small amount of nerve ending membrane at
neuromuscular junctions, the small number of receptors in these
membranes, and the apparent high affinity of toxin for these receptors
(i.e., low toxin concentrations needed to achieve binding, as deduced by toxicity assays). Therefore, as an alternative, investigators have
studied toxin binding to central nervous system receptors. This
approach is based on the observation that the toxin blocks transmitter
release from central nerve endings by the same mechanism that accounts
for blockade of transmitter release at peripheral nerve endings
(Ahnert-Hilger and Bigalke, 1995; Schiavo et al., 1995).
) and human membrane
preparations (Coffield et al., 1997). Binding was dependent on time,
with a T1/2 to equilibrium of approximately 30 min. Binding was also dependent on protein concentration, with half-maximal binding at approximately 40 to 50 µg/ml. The apparent Kd value for association with
high-affinity sites was 4.1 nm, which is similar to that observed in
rat tissues (Bakry et al., 1997). The
Bmax value was 8.4 pmol/mg protein,
which is also similar to that previously reported.
When mouse membrane preparations were incubated in a 50-fold molar
excess of unlabeled homologous toxin, the binding of labeled toxin was
diminished an average of 87% (three experiments done in triplicate).
When membranes were incubated in a 50-fold molar excess of heterologous
toxin (serotype C), binding was never reduced by more than 12% (three
experiments done in triplicate).
Identical experiments were done with frog membranes and iodinated
serotype B, but the results were strikingly different. The amount of
specific binding was so low as to be indistinguishable from background.
Regardless of incubation time or membrane protein concentration,
high-affinity binding sites for serotype B could not be detected.
Triticum Vulgaris Lectin Antagonizes Toxin Binding
and Activity.
The receptor for botulinum toxin either possesses a
sialic acid residue or is in close proximity to a sialic acid residue. One piece of evidence to support this concept is the finding that lectins with affinity for sialic acid compete with botulinum toxin for
binding sites on rodent brain membranes and antagonize toxin action at
rodent neuromuscular junctions (Bakry et al., 1991a).
4). As shown in Fig.
4, the lectin produced a significant
reduction in toxin binding. For both serotypes, the amount of binding
in the presence of lectin was less than half of that in the absence of
lectin. In companion experiments, the effect of botulinum toxin type C
on spontaneous MEPPs was monitored in the absence and presence of
lectin (106 M). The
lectin by itself did not affect the rate of spontaneous MEPPs. However,
the presence of lectin significantly delayed the onset of poisoning by
botulinum toxin (Table 1).
|
|
Methylamine Hydrochloride Antagonizes Toxin Activity. Work on other vertebrate neuromuscular junctions has shown that botulinum toxin must proceed through an acid-dependent step to block transmission. For example, drugs that block acidification of the endosomal lumen (e.g., bafilomycin) or neutralize acidification of the lumen (e.g., methylamine hydrochloride, chloroquine) substantially antagonize toxin action (see Introduction).
In the present work, methylamine hydrochloride was selected as a prototype agent to determine whether neutralization of frog endosomes would delay onset of toxin action. Tissues (n = 5) were pretreated with the drug (1 mM) for 30 min, after which they were exposed to botulinum toxin type C (1 × 108 M), and MEPPS were monitored. In the
absence of the drug, the toxin blocked transmission in 53 ± 3 min; in the presence of the drug, the toxin blocked transmission in
114 ± 14 min. The difference in paralysis times is highly
significant (p < 0.01).
Frog Nerve Endings Have the Three Principal Substrates for
Botulinum Toxin.
Frog and mouse brain synaptosomes were isolated
as described in Materials and Methods and then lysed and
submitted to polyacrylamide gel electrophoresis. The gels were
subsequently used in immunoblots for SNAP-25, synaptobrevin 1, synaptobrevin 2, and syntaxin. As shown in Fig.
5, all three substrates were present in
both frog and mouse. However, frog brain synaptobrevin 1 was present to the near exclusion of synaptobrevin 2.
|
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Mechanism of Toxin Action.
There is substantial
electrophysiologic evidence that botulinum toxin type A acts on frog
neuromuscular junctions to block transmission (e.g., Miledi and
Spitzer, 1974). Thus, serotype A has been shown to reduce the
magnitudes of nerve stimulus-induced endplate potentials and twitch,
resulting in complete paralysis of transmission, and to reduce the
frequency of spontaneous MEPPs to levels that are barely 1% of normal.
These observations are suggestive evidence that botulinum toxin action
at the frog neuromuscular junction is the same as its action at other
vertebrate neuromuscular junctions. However, the marked differences
between toxin action on certain mammalian nerve endings (e.g., mouse
phrenic-nerve hemidiaphragm) and on certain nonmammalian preparations
(e.g., Aplysia buccal ganglion) argue strongly that one must be
cautious about the notion that a single scheme can account for all
aspects of the cellular and subcellular effects of the toxin (see, for example, Poulain et al., 1989). Therefore, experiments were done to
demonstrate that botulinum toxin action at the frog neuromuscular junction involves binding and internalization, pH-induced translocation to the cytosol, and zinc-dependent metalloprotease action. In addition,
work was done to show that the pharmacological characteristics of these
steps are similar to those at mammalian neuromuscular junctions.
Resistance to Serotype B. In contrast to serotypes A, C, and D, botulinum toxin type B did not block frog neuromuscular transmission. This result was obtained even when using toxin concentrations that were orders of magnitude higher than those used with other serotypes. By combining deduction and experimental findings, one can reasonably conclude that the absence of effect by type B was due to an absence of cell surface receptors.
Resistance to this one serotype could not have been due to an absence of receptor-mediated endocytosis. Endocytosis is a process upon which all active serotypes depend, and thus paralysis induced by any one serotype is good presumptive evidence for the existence of a mechanism for receptor-mediated endocytosis. In addition, resistance to serotype B could not have been due to an absence of endosomal acidification, as indicated by two observations. First, all active serotypes must proceed through pH-induced translocation to escape the endosome and reach the cytosol. The fact that several serotypes act on frog tissues is highly suggestive evidence that endosomes are acidified. Second, methylamine hydrochloride, which acts to neutralize endosomal lumens, was an antagonist of the toxin serotypes that act on frog tissues. Finally, resistance to serotype B could not have been due to an absence of susceptible substrate. Western blot analysis with appropriate antibodies showed that substrate was present and, furthermore, that this substrate was susceptible to zinc-dependent proteolysis by serotype B. By process of elimination, the most plausible explanation for resistance to serotype B is an absence ofor alteration ofthe cell
surface receptor. The observation that iodinated serotype B did not
bind to frog nerve membranes strongly reinforces the concept that
resistance is linked to the receptor.
The fact that a specific type of nerve ending lacks receptors for a
specific type of toxin creates the opportunity to address several
issues that pertain to toxin action and nerve function. Some of the
more important issues are described below.
1. Synaptotagmin may be a receptor for botulinum toxin type B. Several studies have been published suggesting that one or more of
the synaptotagmins could be part of the receptor for serotype B
(Nishiki et al., 1993, 1994, 1996). Synaptotagmin is enriched in
membranes of synaptic vesicles, and ordinarily it would not be
accessible to toxin on the cell surface. However, the lumenal domain of
synaptotagmin does become accessible during exocytosis. Indeed, de
Camilli and colleagues (Kraszewski et al., 1995) have prepared a
fluorescent derivative of an antibody against the lumenal domain of
synaptotagmin and used it to label synaptic vesicles.
The discovery that frog nerve terminals are resistant to serotype B by
virtue of an absence of receptors presents a unique opportunity. It may
be possible to prepare a family of antibodies directed individually
against the family of synaptotagmin homologues and then use
cytohistochemical techniques to determine whether any of the homologues
is missing.
2. If a low-affinity receptor and a high-affinity receptor play a
role in type B toxin binding, these receptors are 1) missing from frog
membranes; 2) not capable of acting individually as fully competent
receptors; or 3) both of the above. Monteccuco (1986) has advanced
the interesting idea that toxin binding may involve two separate
receptors. A low-affinity receptor would play the role of collecting
toxin molecules on the nerve surface, and a high-affinity receptor
would play the role of initiating events that lead to toxin action
(viz., receptor-mediated endocytosis). In the present study, frog nerve
endings were resistant to high concentrations of, and lengthy exposure
to, serotype B. If the dual receptor hypothesis is correct, it means
that one or both of the following two premises must be correct. First,
the frog is resistant because it has simultaneously lost both classes
of receptor, or second, the frog is resistant because it has lost one
receptor, and the remaining receptoreven when exposed to high toxin
concentrationsis not competent to initiate subsequent steps in toxin action.
3. Resistance to type B cannot be due to the loss of a sialic
acid residue that all toxin serotypes share to produce paralysis. Incubation of botulinum toxin with sialic acid-containing molecules such as gangliosides causes loss of toxicity (Simpson and Rapport, 1971; Kitamura et al., 1980; Kamata et al., 1993). Similarly, incubation of nerve membranes with lectins that have affinity for
sialic acid blocks toxin binding and action (Bakry et al., 1991a). These observations suggest that there is a sialic
acid residue in or close to receptors for each serotype. The fact that frog nerve endings are sensitive to types A, C, and D, but not to type
B, means that resistance to any individual serotype cannot be explained
on the basis that the membrane has lost a common sialic acid binding
site. This conclusion is reinforced by the finding that Limax
flavus lectin continued to act as an antagonist of type C toxin in
the cutaneous-pectoris preparation. This means, for example, that the
various serotypes cannot share a common low-affinity receptor (viz., a
ganglioside) whose loss accounts for resistance to serotype B. There
must be some additional deficit to explain resistance (see above).
4. The binding site whose loss accounts for resistance to type B
is not essential for binding of other clostridial neurotoxins, -bungarotoxin or -latrotoxin. Ligand-binding studies
reported by other investigators have demonstrated that, with the
possible exception of serotypes C and D, the various toxin serotypes do not share a common receptor. The findings reported here strongly reinforce that point. Even in the absence of binding sites for serotype
B, several other serotypes apparently bind quite well. This line of
reasoning can be expanded to include other neuromuscular blocking
agents. Both -bungarotoxin (Abe et al., 1977; Caratsch et al., 1985)
and -latrotoxin (Pumplin and Reese, 1977) act on frog nerve endings
that are resistant to botulinum toxin type B, which likely reflects an
absence of overlap in binding sites.
Nerve endings that lacked receptors for type B toxin were otherwise
normal in their ability to store and release transmitter. Clearly, the
toxin receptor is not essential for cell viability in general or to the
process of exocytosis in particular. This is in stark contrast to the
toxin substrate. In the absence of synaptobrevin, individual nerves
would be unable to maintain excitation-secretion coupling, and intact
organisms would be unable to survive. The challenge now is to determine
what role type B toxin receptors play in the structure or function of
nerve membranes. The answer to this question may be facilitated by the
identification of other nerves that lack receptors for single serotypes
of botulinum toxin. Work to identify such nerves is underway.
| |
Acknowledgments |
|---|
We are grateful to Dr. W. J. Betz, in whose laboratory some of these experiments were performed, and to Steve Fadul, who provided expert technical assistance.
| |
Footnotes |
|---|
Accepted for publication January 25, 1999.
Received for publication August 25, 1999.
1 This work was supported in part by National Institutes of Health Grant NINDS22153 (to L.L.S.).
Send reprint requests to: Lance L. Simpson, Ph.D., Room 314-JAH, Jefferson Medical College, 1020 Locust Street, Philadelphia, PA. E-mail: Lance.Simspon{at}mail.tju.edu
| |
Abbreviations |
|---|
MEPP, miniature endplate potential; SNAP, synaptosomal-associated protein of 25 kDa; TPEN, tetrakis (2-pyridylmethyl) ethylenediamine.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  S. Kalandakanond and J. A. Coffield Cleavage of Intracellular Substrates of Botulinum Toxins A, C, and D in a Mammalian Target Tissue J. Pharmacol. Exp. Ther., March 1, 2001; 296(3): 749 - 755. [Abstract] [Full Text]
|
|
|
|
|
|
S. Kalandakanond and J. A. Coffield Cleavage of SNAP-25 by Botulinum Toxin Type A Requires Receptor-Mediated Endocytosis, pH-Dependent Translocation, and Zinc J. Pharmacol. Exp. Ther., March 1, 2001; 296(3): 980 - 986. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
, solid line; type B: [10
, dashed-dotted line; type C: [10
,
dotted line; type D: [10
, dashed line.
