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Infection and Immunity, October 1998, p. 4811-4816, Vol. 66, No. 10
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Characterization of Clostridium
botulinum Type B Neurotoxin Associated with Infant Botulism
in Japan
Shunji
Kozaki,1,*
Yoichi
Kamata,1
Tei-ichi
Nishiki,2
Hiroaki
Kakinuma,3
Hiromi
Maruyama,3
Hiroaki
Takahashi,3
Tadahiro
Karasawa,4
Kiyotaka
Yamakawa,4 and
Shinichi
Nakamura4
Department of Veterinary Science, College of
Agriculture, Osaka Prefecture University, Sakai,
Osaka,1
Mitsubishi Kasei Institute of
Life Science, Machida, Tokyo,2
Department of Pediatrics, Kanazawa Medical University,
Uchinada,3 and
Department of
Bacteriology, School of Medicine, Kanazawa University,
Kanazawa,4 Ishikawa, Japan
Received 1 June 1998/Returned for modification 7 July 1998/Accepted 29 July 1998
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ABSTRACT |
The neurotoxin of strain 111 (111/NT) associated with type B infant
botulism showed antigenic and biological properties different from that
(Okra/NT) produced by a food-borne botulism-related strain, Okra. The
specific toxicity of 111/NT was found to be about 10 times lower than
that of Okra/NT. The monoclonal antibodies recognizing the light chain
cross-reacted with both neurotoxins, whereas most of the antibodies
recognizing the carboxyl-terminal half of the heavy chain of Okra/NT
did not react to 111/NT. Binding experiments with rat brain
synaptosomes revealed that 125I-labeled 111/NT bound to a
single binding site with a dissociation constant
(Kd) of 2.5 nM; the value was rather lower than
that (0.42 nM) of 125I-Okra/NT for the high-affinity
binding site. In the lipid vesicles reconstituted with ganglioside
GT1b, 125I-Okra/NT interacted with the amino-terminal
domain of synaptotagmin 1 (Stg1N) or synaptotagmin 2 (Stg2N), fused
with the maltose-binding protein, in the same manner as the respective
full-length synaptotagmins, and the Kd values
accorded with those of the low- and high-affinity binding sites in
synaptosomes. However, 125I-111/NT only exhibited a low
capacity for binding to the lipid vesicles containing Stg2N, but not
Stg1N, in the presence of ganglioside GT1b. Moreover, synaptobrevin-2,
an intracellular target protein, was digested to the same extent by the
light chains of both neurotoxins in a concentration-dependent manner.
These findings indicate that the 111/NT molecule possesses the
receptor-recognition site structurally different from Okra/NT, probably
causing a decreased specific toxicity.
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INTRODUCTION |
Since the first case of infant
botulism was diagnosed in the United States in 1976 (21,
31), more than 1,000 cases have been reported from around the
world. Infant botulism is represented by neuromuscular paralysis due to
the toxin produced in the intestines after germination and outgrowth of
ingested spores of Clostridium botulinum (1).
This disease, which affects children up to 6 months old, with rare
exceptions, is characterized by constipation, generalized weakness, and
various neurological disorders, although cases represent a spectrum of
disease ranging from subclinical infection to the most fulminant form
of the disease, which presents unexpected sudden death (5).
Most cases have been caused by C. botulinum type A or B
(2), although there have been a few exceptions caused by
type C, E, or F. The toxigenic organisms causing type E and F infant
botulism were not C. botulinum, but were culturally and
biochemically identical to Clostridium butyricum and
Clostridium baratii, respectively (7, 20). Since
the first Japanese case of type A infant botulism reported in 1986 (29), there have been 14 cases, 9 due to type A and 1 due to type C (30). The types of toxin in the other four cases were not described. In 1995, there was a 6-month-old patient with infant botulism identified in Ishikawa Prefecture; this was found to be the
first reported case of type B infant botulism in Japan (9).
The properties of the neurotoxin produced by the organism associated
with infant botulism have extensively been examined physicochemically, immunochemically, and genetically. The neurotoxin from type A isolates
associated with infant botulism in Japan was antigenically similar but
not identical to that produced by food-borne botulism-related type A
strains (33). In addition, such antigenic difference was
restricted to the heavy chain (~100 kDa), which is comprised in the
neurotoxin together with the light chain (~50 kDa) (15). The antigenic dissimilarity was also found between the neurotoxins produced by type E organisms and those produced by toxigenic C. butyricum (17). These observations were supported by
the notion that the nucleotide sequence of the infant neurotoxin gene
was clearly related but not identical to that of the authentic
neurotoxin gene (22). These findings evoke the question of
whether type B neurotoxin associated with infant botulism in Japan
possesses properties different from those of the authentic neurotoxin.
In the present study, we found that this infant botulism-related neurotoxin shows toxicity lower than that of the previously known neurotoxin, which is probably due to a low capacity for binding to the
toxin receptor.
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MATERIALS AND METHODS |
Strains and neurotoxins.
Strain 111 was isolated from the
feces of a patient with type B infant botulism (9). Strain
Okra, associated with food-borne botulism, was used for preparation of
the authentic type B neurotoxin. The progenitor toxins of both strains
111 and Okra were purified according to a method previously described
(18). After dialysis against 10 mM phosphate buffer (pH
7.5), the progenitor toxin was loaded onto a DEAE-Sepharose Fast Flow
column (Pharmacia, Uppsala, Sweden) equilibrated with the same buffer.
The neurotoxin was eluted with an NaCl linear gradient from 0 to 0.3 M,
concentrated with YM-10 membrane (Amicon, Inc., Beverly, Mass.), and
then converted to the nicked form by treatment with
N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin (Sigma Chemical Co., St. Louis, Mo.) at a
toxin/enzyme ratio of 100:1 for 15 min at pH 7.5 and 37°C. The purified neurotoxins were referred to as 111/NT and Okra/NT,
respectively. Toxicity was assayed by the time-to-death method by
intravenous injection into mice (10). Samples were also
titrated by intraperitoneal injection into mice with serial twofold
dilutions to obtain a mean 50% lethal dose (LD50) by the
Reed and Muench calculation (32).
MAbs and ELISA.
Monoclonal antibodies (MAbs) against 111/NT
were obtained by a method described elsewhere (16). Five
cell lines were established in this study. MAbs were purified from
ascitic fluid by Affi-Gel-protein A (Bio-Rad Laboratories, Richmond,
Calif.) chromatography. The subclass and light chain of each MAb were
determined by the method described previously (12). In
addition, 13 MAbs against Okra/NT prepared by the procedures described
previously (16) were used to determine their reactivities to
111/NT: 4 MAbs reacted with the amino-terminal half of the heavy chain,
another 5 reacted with the carboxyl-terminal half of the heavy chain,
and the other 4 reacted with the light chain. The reactivities of MAbs
to neurotoxin were examined by enzyme-linked immunosorbent assay
(ELISA) according to the previously described method (16)
and immunoblotting as described below.
Binding of 125I-labeled neurotoxin to
synaptosomes.
The neurotoxin was radioiodinated with Na
125I (Dupont, NEN Research Products, Boston, Mass.) by the
chloramine-T method as described previously (11). The
specific activities of 125I-111/NT and
125I-Okra/NT were 5.1 to 7.5 mCi/mg of protein (28 to 41 Mbq/nmol), and the residual toxicities were higher than 80% of that of
the unlabeled toxin. Synaptosomes were prepared from rat brain
(26) and suspended in 3 mM
2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES)-NaOH
buffer (pH 7.0) containing 120 mM NaCl, 2.5 mM KCl, 2 mM
MgCl2, 2 mM CaCl2, and 0.1% bovine serum
albumin (HBS-BSA). Synaptosomes were incubated for 30 min at 37°C
with 125I-labeled neurotoxin in 0.2 ml of HBS-BSA. To test
the inhibition of binding of the 125I-labeled neurotoxin,
synaptosomes were preincubated for 10 min at 37°C with unlabeled
neurotoxin at various concentrations. The 125I-labeled
neurotoxin having bound to synaptosomes was separated by filtration
through a Millititer-HA plate well (Millipore Corp., Bedford, Mass.).
The filters were washed five times each with 0.25 ml of chilled
HBS-BSA. The radioactivity retained on the filter was determined in a
gamma counter. Specific binding was determined as the difference
between means of triplicate assays in the presence or absence of
200-fold excess unlabeled toxin. Scatchard plot analysis was performed
with the computer program SP123 (8).
Expression of recombinant amino-terminal domains of
synaptotagmins 1 and 2 and their toxin-binding activities.
The PCR
products of rat synaptotagmins 1 and 2 were cloned into pET-3a vector
(Novagen Inc., Madison, Wis.) as described before (26, 28)
and used as templates to generate the synaptotagmin-truncation mutants
denoted Stg1N (amino acids 1 to 78) and Stg2N (amino acids 1 to 87),
respectively. The cDNA fragments encoding Stg1N and Stg2N were
amplified by PCR with the following primers: forward, 5'-AGGCGCCATGGTGAGTGCCAGTCATCCTGAGGCCCTG-3' (Stg1N) and
5'-AGGCGCCATGAGAAACATCTTCAAGAGG-3' (Stg2N); reverse,
5'-GCTCTAGATTAACAAAAGCAGCAGGTTACGAC-3' (Stg1N) and
5'-GCTCTAGACTATTAACAGATGCAGAAGCAGCAGGTGAG-3' (Stg2N), where EheI and XbaI sites were included at the ends of
the forward and reverse primers, respectively. After digestion with
EheI and XbaI, the PCR fragment was ligated into
XmnI- and XbaI-digested pMAL-c2 vector (New
England Biolabs, Beverly, Mass.) and verified by DNA sequencing.
Expression of recombinant synaptotagmin mutants with the pMAL-c2 fusion
protein expression system (New England Biolabs) was performed by the
manufacturer's protocol. Overnight cultures of Escherichia
coli DH5 or TB1 cells containing the recombinant plasmid were
grown in rich medium (tryptone, 10 g; yeast extract, 5 g; NaCl, 5 g; glucose, 2 g per liter) containing 100 µg of
ampicillin per ml at 37°C, with shaking, to an
A600 of 0.5. Isopropyl-
-D-thiogalactopyranoside (IPTG; Wako Pure
Chemicals, Osaka, Japan) was added to 0.3 mM (final concentration), and
incubation was continued for additional 2 h. The cells were
harvested by centrifugation at 4°C and suspended in buffer A (20 mM
Tris, 0.2 M NaCl, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride
[PMSF; Sigma], 60 µM leupeptin [Peptide Institute, Inc., Osaka,
Japan], and 40 µM pepstatin A [Peptide Institute, Inc.] [pH
7.4]) at 0.1 volume of the original culture and lysed on ice by
sonication with two pulses of 15 s each. Lysates were centrifuged at 8,000 × g for 20 min at 4°C. The clear
supernatant was loaded onto a column of amylose resin, equilibrated
with buffer A. After the column had been washed with buffer A, bound
proteins were eluted with buffer A containing 10 mM maltose.
The purified recombinant truncation mutants were incorporated into
phosphatidylcholine lipid vesicles together with ganglioside GT1b
(Wako) by the acetone-precipitation method as described previously (27). The binding of 125I-111/NT or
125I-Okra/NT to the reconstituted lipid vesicles was
measured by the filtration assay as described above.
Expression of rat VAMP-2 (synaptobrevin-2).
A cDNA fragment
encoding vesicle-associated membrane protein 2 (VAMP-2
[synaptobrevin]) was amplified by PCR with rat brain cDNA
(6) and the following primers: forward,
5'-AGACATAATGTCGGCTACCGCTGCCACCGTC-3'; reverse,
5'-CTTGGATCCTATTAAGTGCTGAAGTAAACGATGAT-3'. NdeI
and BamHI sites were included at the ends of the forward and
reverse primers, respectively. The amplified product was cleaved with
NdeI and BamHI and ligated with NdeI-
and BamHI-digested pET-16b vector (Novagen). After
confirmation of the integrity of the coding region by sequencing, the
recombinant plasmid was introduced into E. coli
BL21(DE3)pLysS.
Expression of recombinant VAMP-2 was performed according to the pET
System manual (Novagen). Cultures were grown at 37°C in
Luria-Bertani
broth containing 50 µg of carbenicillin per ml and
34 µg of
chloramphenicol per ml, with shaking, to an
A600
of 0.5
at 600 nm. After addition of 30 µM IPTG (final concentration),
incubation was continued for an additional 2 h. The cells were
collected by centrifugation and suspended in buffer B (20 mM Tris,
0.5 M NaCl, 5 mM imidazole, 0.1 mM PMSF, 60 µM leupeptin, and
40 µM
pepstatin A [pH 7.9]) at 0.02 volume of the original culture.
After
treatment with 0.1 mg of lysozyme per ml for 15 min at 30°C,
the
cells were lysed on ice by sonication with three pulses of
10 s
each. The lysates were centrifuged at 80,000 ×
g for
1 h
at 4°C. The supernatant was discarded, and the pellet was
dissolved
in buffer B containing 1%
n-octyl-
-glucoside
(Dojindo Laboratories,
Kumamoto, Japan). After centrifugation at
80,000 ×
g for 1 h at
4°C, the clear
supernatant was loaded onto a column of His · Bond
resin
(Novagen), equilibrated with buffer B. After the column
had been
washed, bound proteins were eluted with buffer B containing
1 M
imidazole. The eluate was concentrated with YM-10 membrane
(Amicon) and
dialyzed against 5 mM 3-morpholinopropanesulfonic
acid (MOPS
[Dojindo])-0.3 M glycine buffer (pH 6.5).
In vitro cleavage of VAMP-2.
The neurotoxin (150 nM) was
reduced for 30 min at 37°C in 5 mM MOPS-0.3 M glycine buffer (pH
6.5) containing 10 mM dithiothreitol (DTT). The reduced neurotoxin was
then incubated with the recombinant VAMP-2 at a final concentration of
5 µM. After 1 h at 37°C, the reaction was terminated by
boiling for 3 min with 1% sodium dodecyl sulfate (SDS), and the
mixture underwent SDS-polyacrylamide gel electrophoresis (PAGE) as
described below. After electrophoresis, the gel was stained with
Coomassie brilliant blue, and the intensity of stained band was
quantified by scanning with a dual-wavelength densitometer (CS-9000
[Shimadzu, Kyoto, Japan]).
Other methods.
SDS-PAGE was performed in a 10 or 15% gel by
the method of Laemmli (19). For immunoblotting, the protein
was transferred to nitrocellulose (TM-2 [Tokyo Roshi, Tokyo, Japan]).
The nitrocellulose paper was then incubated for 30 min with the
respective MAbs. The immunoreactive bands were visualized with the
ProtBlot Western AP system (Promega Corp., Madison, Wis.). Polyclonal
rabbit antiserum against Okra/NT was prepared as described previously
(18). Protein contents were determined by the method of
Bradford with bovine gamma globulin as a standard (3). The
quantity of ganglioside was defined as the amount of
N-acetylneuraminic acid (NeuAc) determined by the method of
Svennerholm (37).
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RESULTS |
Properties of 111/NT.
111/NT was purified from the culture in
the same way as for Okra/NT. In SDS-PAGE under reducing conditions,
111/NT was separated into heavy and light chains with molecular masses
of 94 and 49 kDa, which were, respectively, 5 and 3 kDa smaller than
the heavy (99 kDa) and light (51 kDa) chains of Okra/NT (Fig.
1). The toxicity of 111/NT, titrated by
intraperitoneal injections to obtain a mean LD50, was
5.4 × 106 LD50/mg of protein, which was
about 1/10 that of Okra/NT (6.0 × 107
LD50/mg of protein). These results suggest that 111/NT
possesses some characteristics distinguishable, on a molecular basis,
from those of Okra/NT.
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FIG. 1.
SDS-PAGE of 111/NT and Okra/NT in the presence of DTT. A
sample (2 µg of each neurotoxin per lane) was applied to a 10%
polyacrylamide gel. The two minor bands in Okra/NT were derivatives of
the heavy chain (14). The positions of molecular mass
standards are shown on the left. H, heavy chain; L, light chain.
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Antigenicity of 111/NT.
In order to explore the issues
discussed above, we first examined the antigenic similarity and
dissimilarity between 111/NT and Okra/NT. In the agar gel diffusion
test with polyclonal rabbit anti-Okra/NT, each of the two toxins formed
a single precipitate line which joined in a line of identity, except
for a spur formed by Okra/NT (data not shown), suggesting that the
antigenicity of 111/NT was similar but not identical to that of
Okra/NT. We then examined the cross-reactivities between 111/NT and
Okra/NT by using ELISA and immunoblotting with MAbs against the two
neurotoxins (Table 1 and Fig.
2). Of the 13 MAbs against Okra/NT, all
recognizing the amino-terminal half of the heavy chain (B-8, B-10,
B-12, and B-13) and the light chain (B-56, B-90, B-96, and B-101) were
found to cross-react with 111/NT, whereas those recognizing the
carboxyl-terminal half of the heavy chain (B-14, B-15, B-16, and B-17),
except one (B-18), showed no reactivity. Of the five MAbs against
111/NT, two (BI-4 and BI-7) reacting to the light chain bound to
Okra/NT, but one (BI-2) recognizing the heavy chain did not. These
results suggest that 111/NT possesses antigenic sites different from
that of Okra/NT, which may be located on the carboxyl-terminal half of
the heavy chain. Moreover, the remaining two MAbs (BI-3 and BI-6)
reacted specifically to 111/NT in ELISA but not to any band in
immunoblotting, which may indicate the existence of particular epitopes
derived from the conformation of the 111/NT molecule.
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FIG. 2.
Immunoblotting analyses of 111/NT (I) and Okra/NT (F)
with MAbs. The MAb used is shown on top of the lane. The data presented
are representative of MAbs reacting to the same fragment.
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Binding of 125I-labeled 111/NT to synaptosomes and
synaptotagmin truncation mutants.
125I-111/NT bound to
synaptosomes in a saturable manner, and Scatchard analysis indicated a
single class of binding site with a dissociation constant
(Kd) of 2.5 nM and a maximum binding activity (Bmax) of 0.76 pmol/mg of protein. On the other
hand, the binding experiments with 125I-Okra/NT indicated
that there were two classes of binding sites with
Kd values of 0.42 nM (high affinity) and 11.7 nM
(low affinity). The Bmaxs for high- and
low-affinity sites were 1.12 and 3.21 pmol/mg of protein, respectively
(Fig. 3). Okra/NT completely inhibited
the binding of 125I-111/NT to synaptosomes (data not
shown), indicating the possibility that both neurotoxins share the same
binding site(s) on synaptosomal membranes.
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FIG. 3.
Scatchard analyses of 125I-111/NT (A) and
125I-Okra/NT (B) binding to rat brain synaptosomes.
Synaptosomes (10 µg of protein) were incubated at 37°C for 30 min
with increasing concentrations of 125I-labeled toxin in the
absence or presence of excess unlabeled toxin. Specific binding was
plotted after correction for nonspecific binding. The data presented
are from one experiment and are representative of three experiments
with similar results. A binding saturation curve is shown in an inset
in each panel.
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Our previous data suggested that Okra/NT recognizes the amino-terminal
domain of synaptotagmins 1 and 2 as the binding site
in the presence of
ganglioside GT1b or GD1a (
28). In order to
investigate the
role of synaptotagmin as a receptor protein of
111/NT, we examined the
binding activities of Stg1N and Stg2N
incorporated into lipid vesicles
together with ganglioside GT1b
(Stg1N/GT1b and Stg2N/GT1b,
respectively).
125I-111/NT bound to Stg2N/GT1b lipid
vesicles in a concentration-dependent
and saturable manner, while it
did not interact with Stg1N/GT1b
lipid vesicles (Fig.
4A). Scatchard analysis indicated a
single
class of binding site with a
Kd of 2.4 nM, which was comparable
to that obtained with synaptosomes.
125I-Okra/NT bound not only to Stg2N/GT1b lipid vesicles
but also
to Stg1N/GT1b lipid vesicles, and their
Kds were 0.52 and 3.7
nM for Stg2N and Stg1N,
respectively (Fig.
4B). These values resembled
those obtained with
full-length synaptotagmins and synaptosomes
(
26,
28). The
Bmax of
125I-Okra/NT for Stg2N/GT1b
lipid vesicles was higher than that of
125I-111/NT.
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FIG. 4.
Binding of 125I-labeled toxins to
recombinant amino-terminal domain of synaptotagmins 1 (Stg1N) and 2 (Stg2N) associated with ganglioside GT1b. (A) Dose dependence of
125I-labeled 111/NT (solid symbols) and
125I-labeled Okra/NT (open symbols) to Stg1N (triangles)
and Stg2N (circles) incorporated into lipid vesicles with ganglioside
GT1b. The recombinant synaptotagmin (10 ng of protein) reconstituted
into lipid vesicles with ganglioside GT1b (2 ng of NeuAc) was incubated
at 37°C for 30 min with increasing concentrations of
125I-labeled toxin. Values are the means ± standard
errors from three experiments. Error bars smaller than the symbols were
omitted. (B) Scatchard plot of the binding data shown in panel A,
except for 125I-labeled 111/NT binding to the lipid
vesicles containing Stg1N and ganglioside GT1b, for which the values
were too low to show in the panel.
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Cleavage of VAMP-2 by 111/NT.
The light chain of type B
neurotoxin was found to exhibit a zinc-dependent endopeptidase in
response to the synaptic vesicle protein, VAMP-2 (34). After
reduction of 111/NT with 10 mM DTT, we examined the proteolytic
activity with recombinant VAMP-2 and compared it with that of Okra/NT.
As shown in Fig. 5, VAMP-2 was digested
to the same extent by reduced 111/NT and Okra/NT in a concentration-dependent manner.
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FIG. 5.
Dose dependence of proteolysis of recombinant VAMP-2 by
111/NT and Okra/NT. (A) Samples were treated at 37°C for 1 h
with reduced 111/NT (solid circles) or reduced Okra/NT (open circles)
at different concentrations and electrophoresed on a 15%
polyacrylamide gel. The gel was stained with Coomassie brilliant blue
and quantified by densitometry. Results are expressed as a percentage
of the initial VAMP-2 content. The data presented are the means of
three independent experiments. (B) SDS-PAGE profile of VAMP-2 after
incubation with or without reduced 111/NT (I) and reduced Okra/NT (F).
The positions of VAMP-2 and the toxin-induced fragment are indicated by
solid and open arrowheads, respectively.
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DISCUSSION |
No attempt has been made to investigate dissimilarity among the
neurotoxins produced by proteolytic type B strains, although there has
been a report only on the antigenic difference between the neurotoxins
produced by proteolytic and nonproteolytic type B strains (14,
23). The present data indicate that 111/NT of the isolate
implicated in infant botulism possesses some properties, including
toxicity, distinguishable on molecular basis from those of Okra/NT
produced by a food-borne botulism-related strain. SDS-PAGE revealed
that the heavy and light chains derived from 111/NT have slightly
smaller molecular sizes than those of Okra/NT. The ELISA results showed
that 111/NT has multiple antigenic sites different from those of
Okra/NT. Such differences in antigenicity appeared to be conserved
mainly in the carboxyl-terminal half of the heavy chain, but not in the
light chain. Since the carboxyl-terminal half of the heavy chain is
responsible for the toxin binding to the receptor on the presynaptic
membrane (13, 24), these results led us to ask to what
extent the antigenic diversity between 111/NT and Okra/NT reflects upon
their receptor recognition on the plasma membrane. In fact, binding
experiments with synaptosomes showed that 125I-111/NT bound
to a single site with a Kd value about one-sixth that of 125I-Okra/NT for the high-affinity binding site,
although both neurotoxins appeared to share the same binding site.
Furthermore, the light chains of both neurotoxins showed in vitro
cleavage of VAMP-2 to the same extent, suggesting that there is no
difference in the intracellular action between the two light chains.
Perhaps 111/NT shows low toxicity because of its lower capability of
binding to the receptor.
We have previously identified synaptotagmins 1 and 2 as the low- and
high-affinity protein receptors, respectively, for Okra/NT (28). However, their toxin-binding activities were only
observed in the presence of ganglioside GT1b or GD1a, suggesting that
synaptotagmins from the toxin-binding site by associating with these
gangliosides. Synaptotagmin is an integral membrane protein present on
synaptic vesicles and is considered to be involved in their
Ca2+-dependent exocytosis at the nerve terminal
(36). Synaptotagmin has a single transmembrane region, a
short amino-terminal intravesicular domain, and a large cytoplasmic
domain (4). After synaptic vesicle exocytosis, the
amino-terminal domain is exposed outside the nerve terminal. Therefore,
it is likely that the amino-terminal domain consists of the
toxin-binding site in association with the specific ganglioside. The
present data demonstrated that Stg1N and Stg2N, which represent the
amino-terminal domain of synaptotagmin fused with maltose-binding
protein, exhibit a 125I-Okra/NT-binding activities
equivalent to those of full-length synaptotagmins 1 and 2 (28). These results indicate that the carboxyl-terminal
cytoplasmic domain does not contribute to form the toxin recognition
site. On the other hand, 125I-111/NT appeared to lose the
capacity to bind to Stg1N and had a lower Kd and
Bmax for Stg2N, compared with
125I-Okra/NT binding to Stg1N and Stg2N. The
Kd value of 125I-111/NT for Stg2N
accorded with that of the single binding site on synaptosomes. Since
the action of neurotoxin after binding to the receptor involves
subsequent internalization and translocation into cytosol, where the
light chain reaches a specific target protein (35),
functional differences at such subsequent steps between the two
neurotoxins still remain to be clarified. The present findings,
however, strongly indicate that the receptor recognition site on the
111/NT molecule differs structurally from that of Okra/NT, and this may
be supported partly by the results showing that 111/NT contains
specific epitopes that recognize the steric conformation. Moreover,
synaptotagmin 1 does not seem to play a critical role as the protein
receptor for type B neurotoxin.
The genes of botulinum neurotoxins have already been cloned, and their
nucleotide sequences have been determined (22). These data
revealed the nature of the light chain which elicits the proteolytic
activity in the cell cytosol to specifically cleave proteins of the
exocytotic apparatus, thereby blocking neurotransmitter release
(24, 25). In contrast to the function of the light chain,
the mechanism of receptor recognition by the neurotoxin has been
obscure, being probably due not only to serotype specificity of the
toxin receptor but also to heterogeneity of the receptor recognition
site on the neurotoxins. Therefore, it is of great value to know the
primary structure of the carboxyl-terminal regions of 111/NT and
Okra/NT in order to define the receptor-binding site. Studies are now
in progress to determine the amino acid sequences of these regions in
both neurotoxins.
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ACKNOWLEDGMENTS |
This work was partly supported by a grant for Scientific Research
from the Ministry of Education, Science, Sports, and Culture and by a
grant for Health Science Research of Emerging and Reemerging Infectious
Diseases from the Ministry of Health and Welfare, Japan.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Veterinary Science, College of Agriculture, Osaka Prefecture
University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan. Phone:
81-722-54-9486. Fax: 81-722-54-9918. E-mail:
kozaki{at}center.osakafu-u.ac.jp.
Editor:
J. T. Barbieri
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|
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Infection and Immunity, October 1998, p. 4811-4816, Vol. 66, No. 10
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
