Cleavage of a C-terminal Peptide Is Essential for Heptamerization
of Clostridium perfringens
-Toxin in the
Synaptosomal Membrane*
Shigeru
Miyata
,
Osamu
Matsushita
,
Junzaburo
Minami§,
Seiichi
Katayama
,
Seiko
Shimamoto
, and
Akinobu
Okabe
¶
From the
Department of Microbiology, Faculty of
Medicine, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa
761-0793 and the § Department of Medical Technology, Kagawa
Prefectural College of Health Sciences, 281-1 Mure-cho, Kita-gun,
Kagawa 761-0123, Japan
Received for publication, December 20, 2000, and in revised form, January 23, 2001
 |
ABSTRACT |
Activation of Clostridium
perfringens
-protoxin by tryptic digestion is accompanied by
removal of the 13 N-terminal and 22 C-terminal amino acid residues. In
this study, we examined the toxicity of four constructs: an
-protoxin derivative (PD), in which a factor Xa cleavage site was
generated at the C-terminal trypsin-sensitive site; PD without the 13 N-terminal residues (
N-PD); PD without the 23 C-terminal residues
(
C-PD); and PD without either the N- or C-terminal residues
(
NC-PD). A mouse lethality test showed that
N-PD was inactive, as
is PD, whereas
C-PD and
NC-PD were equally active.
C-PD and
NC-PD, but not the other constructs formed a large SDS-resistant
complex in rat synaptosomal membranes as demonstrated by
SDS-polyacrylamide gel electrophoresis. When
NC-PD and
C-PD, both
labeled with 32P and mixed in various ratios, were
incubated with membranes, eight distinct high molecular weight bands
corresponding to six heteropolymers and two homopolymers were detected
on a SDS-polyacrylamide gel, indicating the active toxin forms a
heptameric complex. These results indicate that C-terminal processing
is responsible for activation of the toxin and that it is essential for
its heptamerization within the membrane.
 |
INTRODUCTION |
-Toxin produced by Clostridium perfringens types B
and D is the third most potent clostridial toxin after botulinum and
tetanus toxins, and is responsible for the pathogenesis of fatal
enterotoxemia in domestic animals caused by the organisms (1). This
toxin exhibits toxicity toward neuronal cells via the glutamatergic system (2, 3) or extravasation in the brain (4) after infection of
experimental animals. It has been suggested to be a pore-forming toxin
based on the following observations. (i)
-Toxin can form a large
complex in the membrane of
MDCK1 cells, and it
permeabilizes them (5, 6); (ii) the large complex formed by
-toxin
is not dissociated by SDS treatment (6), which is a common feature of
pore-forming toxins such as aerolysin (7), Clostridium
septicum
-toxin (8), and Pseudomonas aeruginosa
cytotoxin (9); and (iii) the CD spectrum of
-toxin shows it mainly
consists of
-sheets (10), as is characteristically observed for
pore-forming
-barrel toxins.
The structures of many bacterial pore-forming toxins or toxin
components such as perfringolysin O (11), Bacillus
thuringiensis
-toxin (12), aerolysin (13), staphylococcal
-toxin (14), and protective antigen of anthrax toxin (15) have been
determined. These pore-forming toxins are believed to undergo a drastic
conformational change upon interaction with a membrane. Since these
toxins are inserted into the cytoplasmic membrane without the aid of
other proteins such as chaperones or the translocation machinery,
characterization of their metamorphosis has been regarded as a novel
means for studying membrane-protein interactions (16). A characteristic feature of
-toxin is its potent neurotoxicity, which is not seen for
the structurally well defined pore-forming toxins. Thus, it could serve
as a useful tool for extending our knowledge of how proteins gain entry
into a membrane. Another characteristic feature of
-toxin is that
activation of the inactive precursor form (
-protoxin) by proteases
such as trypsin, chymotrypsin (17), and
-protease (18, 19) is
accompanied by removal of both N- and C-terminal peptides. In a
previous study, we determined the N/C termini of
-toxin activated by
trypsin, trypsin plus chymotrypsin, and
-protease to be
Lys-14/Lys-274, Lys-14/Tyr-267, and Met-11/Tyr-267, respectively (19).
This study aimed to answer the following questions. (i) Which
peptide(s), i.e. the N-, or C-, or both terminal peptides,
regulates the activity of
-toxin; ii) can the activated toxin form a
large complex in the rat synaptosomal membrane; and iii) how many toxin monomers are present in the membrane complex?
 |
EXPERIMENTAL PROCEDURES |
Construction of Expression Vectors--
Escherichia
coli NovaBlue (Novagen, Madison, WI) was used for the construction
of all recombinant plasmids. DNA fragments encoding
-protoxin and an
N-terminally truncated form of it were obtained by PCR amplification
using total DNA from C. perfringens type B NCIB 10691 (19)
as the template and the following pairs of synthetic primers:
5'-GGCCAAGGAAATATCTAATACAGTATCTAATGAA-3' (etx 1F primer) and
5'-GAATTCTTATTTTATTCCTGGTGCCTTAATAGAAAG-3' (etx 1R primer) for
-protoxin (Lys-1 to Lys-296); and
5'-GGCCAAAGCTTCTTATGATAATGTAGATACATTA-3' (etx 2F primer) and etx 1R
primer for
N-
-protoxin (Lys-14 to Lys-296). These DNA fragments
were cloned into the pT7Blue T-vector (Novagen), and then inserted into
the MscI and EcoRI sites of pET22b(+) (Novagen)
so that recombinant toxins could be directed to the periplasmic space
of E. coli by a signal peptide encoded in the vector. The
resultant plasmids, which expressed
-protoxin and
N-
-protoxin,
were designated as pEP1 and pEN1, respectively.
To construct an
-protoxin derivative (PD) and an N-terminally
truncated form of it (
N-PD), in which a factor Xa recognition sequence, IEGR, was inserted between Lys-273 and Lys-274 of
-protoxin, sequential PCR amplification was carried out using
pEP1 as the template and the following pairs of primers:
5'-TAATACGACTCACTATAGGG-3' (T7 primer) and
5'-TTTACGACCTTCGATTTTATCTACAGGTATTACATATTCTTG-3' (antisense, sequence corresponding to IEGR is underlined); and 5'-GATAAAATCGAAGGTCGTAAAGAAAAAAGTAATGATTCAAAT-3' (sense,
sequence corresponding to IEGR is underlined) and
5'-GCTAGTTATTGCTCAGCGGTGG-3' (T7ter primer). The resulting PCR
products were used as mixed templates, and T7 and T7ter were used as
primers for a second PCR amplification. The specific PCR products were
digested with AvrII and EcoRI, and then cloned
into pEP1 and pEN1. The resultant plasmids, which expressed PD and
N-PD, were named pEP4 and pEN4, respectively.
The MscI-NotI insert DNA fragments from pEP4 and
pEN4 were subcloned into the SmaI and NotI sites
of pBluescript II KS+ (Stratagene, La Jolla, CA). The EcoRI
fragments from these plasmids were inserted into pGEX-2TK (Amersham
Pharmacia Biotech, Uppsala, Sweden). The resultant plasmids, named pEP9
and pEN9, enabled the expression of GST-PD and GST-
N-PD fusion
proteins, respectively, which contained a pentapeptide recognized by
the cyclic-AMP-dependent protein kinase (20). The accuracy
of all the final DNA constructions was confirmed by DNA sequencing.
Expression and Purification of
-Protoxin
Derivatives--
Transformants of E. coli BL21(DE3)pLysS
(Novagen) carrying plasmids pEP1, pEN1, pEP4, and pEN4 were used to
prepare
-protoxin,
N-
-protoxin, PD, and
N-PD, respectively.
Each transformant was grown in 1 liter of LB broth supplemented with
ampicillin (100 µg/ml) and chloramphenicol (33.4 µg/ml) to an
optical density at 600 nm of 0.7. Isopropyl
-D-thiogalactopyranoside was added to a final
concentration of 1 mM, and then the cultures were grown for
another 3.5 h. The cells were collected and treated with polymyxin B to obtain the periplasmic fraction, as described previously (21).
Proteins in this fraction were precipitated with ammonium sulfate (60%
saturation), dialyzed against 5 mM sodium phosphate buffer
(pH 7.0), and then applied to a DEAE-Sephadex A-25 column (1.5 × 12 cm; Amersham Pharmacia Biotech), which had been equilibrated with
the buffer used for dialysis. The flow-through fraction was collected
and dialyzed against 50 mM Tris-HCl (pH 7.5) containing 1 M ammonium sulfate. This fraction was then subjected to
hydrophobic interaction high performance liquid chromatography on a
Phenyl-Superose HR 5/5 column (1 ml; Amersham Pharmacia Biotech).
Proteins were eluted with a 20-ml linear gradient of ammonium sulfate
(1-0 M) in 50 mM Tris-HCl (pH 7.5). Fractions
containing
-protoxin or its derivatives, as determined by SDS-PAGE,
were pooled and dialyzed against 20 mM Tris-HCl (pH 7.5).
Purified toxins were stored at
80 °C.
C-terminally truncated PD (
C-PD), and N- and C-terminally truncated
PD (
NC-PD) were obtained from purified PD and
N-PD, respectively,
by cleaving them at the factor Xa-sensitive site. Digestion with factor
Xa (restriction grade, Novagen) and its removal on a Xarrest-agarose
column (Novagen) were carried out according to the manufacturer's
instructions. GST-PD and GST-
N-PD were prepared from cultures of
E. coli BL21 carrying pEP9 and pEN9, respectively, which
were grown and induced as described above. The fusion proteins were
purified by affinity chromatography on a glutathione-Sepharose 4B
column (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Mouse Lethality Test and Cytotoxicy Assay--
The lethality of
-protoxin and its derivatives toward mice were determined as
described previously (19). A group of 15 male ddY mice weighing 37 g each were injected intravenously with 0.25 ml of the toxin solution,
and deaths occurring within 24 h were recorded.
MDCK cells were cultured in Eagle's minimum essential medium
containing Earle's salts, penicillin (100 units/ml), and streptomycin (100 µg/ml), supplemented with 10% heat-inactivated fetal bovine serum, in a cell culture incubator (under 5% CO2 at
37 °C). Freshly trypsinized cells were cultured in 96-well
microculture plates for 48 h to give monolayers. The medium was
exchanged for 200 µl of minimum essential medium, followed by the
addition of 50 µl of PBS or PBS containing one of the purified
-protoxin derivatives. After a 6-h incubation, the cells in each
well were washed with PBS containing Mg2+ and
Ca2+. Cell viability was determined by the 3-(4,
5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (Promega, Madison, WI) conversion assay. The absorbance, which is proportional to the number of viable cells, was read at 490 nm
using an enzyme-linked immunosorbent assay plate reader. Percentage of
cell viability was calculated as follows: the mean absorbance value of
a toxin-group/that of a control.
Preparation of Rat Brain Synaptosomal
Membranes--
Synaptosomes were prepared from rat brains as reported
previously (22) with some modifications. Briefly, rat brains (7.5 g)
were homogenized in nine volumes (w/v) of buffer A (10 mM
Tris-HCl (pH 7.0), 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine HCl, 1 µM
pepstatin A, 20 µM leupeptin) containing 0.32 M sucrose, by means of 10 passes through a glass homogenizer with a Teflon plunger. The homogenate was centrifuged at
1,000 × g for 10 min, and the supernatant was
centrifuged at 15,000 × g for 30 min. Then the pellet
was re-suspended in a small volume of buffer A containing 0.32 M sucrose, loaded onto a 0.8 M sucrose solution
layered on a 1.1 M sucrose solution in a centrifuge tube,
and finally centrifuged at 63,000 × g for 2 h.
The synaptosomal membrane fraction at the 0.8 M/1.1
M interface was isolated and collected by centrifugation at
100,000 × g for 1 h. The fraction was
re-suspended in buffer A containing 0.14 M NaCl.
Preparation of Radiolabeled
-Protoxin
Derivatives--
-Protoxin derivatives were radiolabeled with
125I as follows. The purified toxin derivatives (8 µg)
were incubated with 36.2 GBq of 125I (643.8 GBq/mg;
PerkinElmer Life Sciences) and IOAD-BEADS iodination reagent
(Pierce) in 60 µl of PBS for 15 min at room temperature. The
radioiodinated proteins were separated from free iodine by gel
filtration on a Sephadex G-25 column (0.75 × 5.5 cm; Amersham Pharmacia Biotech), equilibrated with Tris-buffered saline (TBS; 150 mM NaCl, 50 mM Tris-HCl (pH 7.4)). The specific
activities of 125I-labeled PD,
N-PD,
C-PD, and
NC-PD were 430, 630, 540, and 390 kcpm/µg of protein, respectively.
Phosphorylation of
-protoxin derivatives with 32P was
performed as follows. Approximately 300 µg of GST-PD or GST-
N-PD
were loaded onto a glutathione-Sepharose 4B column (bed volume, 100 µl; Amersham Pharmacia Biotech), and then phosphorylated using the
catalytic subunit of bovine heart protein kinase (Sigma) and [
-32P]ATP (167 TBq/mmol; ICN Biochemicals, Costa Mesa,
CA) according to the protocol recommended by the manufacturer. The
specific activities of 32P-labeled GST-PD and GST-
N-PD
were about 21 and 48 kcpm/µg of protein, respectively.
[32P]
C-PD and [32P]
NC-PD were
prepared from 32P-labeled GST-PD and GST-
N-PD,
respectively, by cleavage at the factor Xa-specific site within the
C-terminal portion and also at a nonspecific but sensitive site in a
GST linker sequence (see "Results").
Formation of an SDS-resistant Complex in Synaptosomal
Membranes--
Fifteen nanograms of each 125I-labeled
toxin derivative were added to 2.2 µg of synaptosomal membranes in 12 µl of TBS containing 0.1% BSA. After incubation at 37 °C for 90 min, the reaction mixture was centrifuged at 17,000 × g for 5 min. The membrane pellet was washed three times with
200 µl of TBS at 4 °C, and then dissolved by heating in SDS sample
buffer (62.5 mM Tris-HCl (pH 6.8), 2%
-mercaptoethanol,
1% SDS, 0.01% bromphenol blue) at 95 °C for 5 min. Samples were
electrophoresed on a SDS-PAGE gel, followed by exposure to an imaging
plate (Fuji Photo Film, Kanagawa, Japan) for autoradiography.
Heteromeric polymerization of
-protoxin derivatives was carried out
using [32P]
C-PD and [32P]
NC-PD in
various ratios. A total of 20-40 ng of each labeled toxin derivative
was added to 2.2 µg of synaptosomal membranes in 10 µl of TBS.
After incubation at 37 °C for 90 min, SDS sample buffer was added to
the reaction mixtures, followed by heating at 95 °C for 5 min. To
separate heteromeric polymers, SDS-PAGE was performed on a 5%
polyacrylamide gel prepared in a DNA sequencing gel apparatus (800 × 170 × 0.4 mm; Bio-Rad) at 15 mA and room temperature for
20 h.
Analytical Methods--
N-terminal amino acid sequencing of the
purified toxins was performed with a protein sequencer (model 492 Procise; Applied Biosystems), as described previously (23). Protein
concentrations were measured using Pierce bicinchoninic acid protein
assay reagent (Pierce) with bovine serum albumin as the standard.
SDS-PAGE was performed as described by Laemmli (24). High and low
molecular weight marker proteins were obtained from Amersham Pharmacia Biotech.
Mass determinations were carried out with a MALDI-TOF mass spectrometry
instrument (Voyager DE PRO; Applied Biosystems) in the positive linear
mode at an acceleration voltage of 15 kV with delayed extraction.
Samples were mixed with sinapinic acid (10 mg/ml in 0.1%
trifluoroacetic acid, 50% acetonitrile), which was used as the
absorbing matrix. Horse skeletal muscle myoglobin was used as the standard.
Circular dichroism (CD) spectra in the far UV region (260-205 nm) were
obtained with a J720WI spectropolarimeter (Jasco, Tokyo, Japan) using a
cell with a 1-mm light path at 25 °C. All samples were dialyzed
against PBS at 4 °C, and the dialysate was also measured as the
background. The measurements were repeated four times, and the results
were averaged.
 |
RESULTS |
Construction and Characterization of
-Protoxin
Derivatives--
Both an N-terminal peptide of 13 amino acids and a
C-terminal peptide of 22 amino acids are removed upon tryptic
activation of
-protoxin. To determine which peptide inactivates
-protoxin, we constructed recombinant
-protoxin derivatives
without the N- or C-terminal peptide using an E. coli
expression system. The recombinant
-protoxins with and without the
N-terminal peptide were inactive but activated by treatment with
trypsin plus chymotrypsin, as shown by the mouse lethality test (Table
I). The LD50 of their activated forms was 50 ng/kg of body weight, which coincided with the
value reported for
-toxin from C. perfringens cultures.
When the recombinant
-protoxin without the C-terminal peptide was expressed in E. coli, it precipitated as inclusion bodies,
which could be solubilized with 6 M urea, but it could not
be recovered in a soluble form after dialysis (data not shown). Thus,
we constructed two
-protoxin derivatives, with and without the
N-terminal peptide, respectively, in which IEGR, a coagulation factor
Xa recognition sequence, was inserted between Lys-273 and Lys-274
(trypsin cleavage site), as shown in Fig.
1A. This construction enabled
the recovery of these proteins, i.e. PD and
N-PD, from
the periplasmic space and also allowed cleavage at the relevant site
with factor Xa.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 1.
Construction and purification of
-protoxin derivatives. A, schematic
representation of recombinant -protoxin and its derivatives showing
the positions of the factor Xa-sensitive site, and the N and C termini.
The N- and C-terminal peptides, and the factor Xa recognition sequence
are shown as dotted, hatched, and
filled boxes, respectively. The molecular masses
calculated from amino acid sequences are given on the right,
and those determined by MALDI-TOF mass spectrometry are given in
parentheses. The amino acid sequences around the processing
sites are also shown, and the numbers above the
residues refer to the amino acid positions in -protoxin. The factor
Xa recognition sequence is underlined. B,
purification of -protoxin derivatives from an E. coli
expression system. The purified proteins (2 µg) were loaded into the
indicated lanes, electrophoresed on a 13% SDS-PAGE gel, and then
stained with Coomassie Brilliant Blue R. The sizes of the molecular
mass markers are indicated on the left. Lane
1, PD; lane 2, N-PD;
lane 3, C-PD; lane 4,
NC-PD.
|
|
PD and
N-PD were purified from E. coli cultures to
homogeneity, as demonstrated by SDS-PAGE (Fig. 1B). Another
two derivatives,
C-PD and
NC-PD, were obtained by treatment of
the purified PD and
N-PD, respectively, with factor Xa (Fig.
1B). The identities of all the constructs were confirmed by
nucleotide sequencing of the recombinant plasmids, N-terminal amino
acid sequencing (data not shown), and molecular mass determination by
MALDI-TOF mass spectrometry (Fig. 1A). CD analysis revealed
that all the derivatives gave a negative ellipticity band at 215 nm,
indicating a predominance of
-structure. Some minor differences were
observed in the CD spectra, which may be due to a minor change in the
conformation arising from the insertion of the factor Xa recognition
sequence or removal of the N- and/or C-terminal sequence(s). However,
there were no remarkable differences in the CD spectra, strongly
suggesting neither the insertion nor the removal(s) affects the overall
structure of
-protoxin (Fig. 2).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 2.
CD spectra of
-protoxin and its derivatives. Measurements
were carried out on protein solutions in PBS. The mean residue weight
values used were calculated from the deduced amino acid sequences.
Panel A, -protoxin; panel
B, PD; panel C, N-PD;
panel D, C-PD; panel E,
NC-PD.
|
|
Toxicity of
-Protoxin Derivatives--
The toxicity of the
-protoxin derivatives was determined by means of a mouse lethality
test (Table I). The LD50 of PD with an insertion of the
IEGR sequence was 31,000 ng/kg body weight, slightly lower than that
previously reported for
-protoxin purified from C. perfringens cultures (70,000 ng/kg of body weight). The LD50 of
N-PD was the same as that of PD, being higher
than that of
C-PD by a factor of about 60. The LD50 of
NC-PD was almost the same as that of
C-PD, although both were
slightly higher than that reported for trypsin-activated
-toxin (320 ng/kg). This might be due to the difference in the C-terminal region:
NC-PD and
C-PD have four extra amino acids after the trypsin cleavage site. To eliminate the possibility that N-terminal processing has an additional activating effect, the toxicities of the four derivatives were determined by means of an in vitro assay
method using MDCK cells, which is less sensitive but more accurate than the mouse lethality test (Fig. 3).
C-PD and
NC-PD exhibited almost the same toxicity toward MDCK
cells, and there was no significant difference in the 50% cytotoxicity
dose between the two derivatives. No cytotoxicity was detectable for
N-PD and PD at the highest concentration (600 ng/ml) used in this
study. These results clearly indicate that only cleavage at the
C-terminal region is responsible for activation of
-protoxin.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
Cytotoxicity of
-protoxin derivatives toward MDCK cells.
Various concentrations of -protoxin derivatives were added to MDCK
cells cultured on microculture plates. Viability was assayed as
described under "Experimental Procedures," and is expressed as a
percentage of the value for a control culture. Bars indicate
the standard deviation of two determinations. , PD; , NC-PD;
, N-PD; , C-PD.
|
|
Large Complex Formation in Synaptosomal Membranes--
Activated
-toxin has been shown to form an SDS-resistant 174-kDa complex in
the membranes of MDCK cells (6), and the complex has also been reported
in brain homogenates incubated with the toxin (5). Such a complex may
also be formed in synaptosomal membranes, which could be responsible
for the neuronal cell death in the hippocampus observed in
-toxin
injected mice (2). In order to assess this possibility, membranes were
incubated with 125I-labeled toxin derivatives and analyzed
by SDS-PAGE (Fig. 4A). In the
samples incubated with 125I-labeled
C-PD and
NC-PD,
large SDS-resistant complexes were detected, with apparent molecular
masses of ~200 and 180 kDa, respectively. No SDS-resistant complex
was detected in membranes incubated with 125I-labeled PD
and
N-PD. When equal amounts of 125I-labeled
C-PD and
unlabeled
NC-PD or vice versa were incubated with the membranes, a smeared band corresponding to 200-180-kDa complexes was detected on a SDS-PAGE gel (Fig. 4B),
indicating that both active forms are equally capable of forming the
complex.

View larger version (69K):
[in this window]
[in a new window]
|
Fig. 4.
Formation of a large SDS-resistant complex
by -protoxin derivatives in synaptosomal
membranes. A, complex formation through homomeric toxin
assembly. 125I-Labeled toxin derivatives (15 ng each) were
added to synaptosomal membranes (2.2 µg of protein) in 12 µl of TBS
containing 0.1% BSA. After incubation, the membranes were washed three
times with TBS and then dissolved by heating in SDS-sample buffer.
Samples were subjected to SDS-PAGE (4-20% gradient gel) and
subsequent autoradiography. Lane 1, PD;
lane 2, N-PD; lane 3,
C-PD; lane 4, NC-PD. B, complex
formation through heteromeric toxin assembly. 125I- C-PD
or 125I- NC-PD (11 ng) mixed with an equal amount of
unlabeled C-PD or NC-PD were incubated with the membranes and
then subjected to SDS-PAGE (6% gel) as described above.
Lane 1, 125I- C-PD plus unlabeled
C-PD; lane 2, 125I- C-PD plus
unlabeled NC-PD; lane 3,
125I- C-PD plus 125I- NC-PD;
lane 4, 125I- NC-PD plus unlabeled
C-PD; lane 5, 125I- NC-PD plus
unlabeled NC-PD. The sizes of the molecular mass markers are
indicated on the left.
|
|
Heptamerization of Active
-Toxin--
The molecular masses of
C-PD and
NC-PD are 31 and 29 kDa, respectively. Therefore, the
200- or 180-kDa large complex should consist of five toxin molecules
and an intrinsic membrane protein, or at maximum seven toxin molecules,
if they are unprocessed in the membrane. The result of an experiment
involving a mixture of the two active forms suggested that the smeared
band corresponds to heteromultimers consisting of the two forms in
various ratios, and that the number of toxin molecules per complex is
independent of the ratio. If this is the case, complexes of different
molecular sizes will form, which can be separated to determine the
number of toxin molecules in a complex. The heteromeric complexes
formed by 125I-labeled toxins were not well separated even
with a SDS-PAGE gel system for large polypeptides. To circumvent this
problem, we constructed GST fusion proteins containing a protein kinase recognition site (RRXSV) at the N termini of the two toxin
derivatives (Fig. 5A). These
constructs were cleaved by factor Xa not only at the C-terminal
specific site but also between the two arginine residues in the
RRXSV sequence. Thus, the fusion proteins were purified
(Fig. 5B, lanes 1 and 2),
phosphorylated, and cleaved with factor Xa (Fig. 5B,
lanes 3 and 4). The resultant
phosphorylated
C-PD and
NC-PD were designated as P-
C-PD and
P-
NC-PD, respectively. The identities of these protein derivatives
were verified by N-terminal amino acid sequencing (data not shown), and
molecular mass determination by MALDI-TOF mass spectrometry (Fig.
5A). There was no significant difference in mouse lethality
between these two constructs, although their lethalities were slightly
lower than those of
N-PD and
NC-PD (Table I). They also retained
the ability to form an SDS-resistant complex in synaptosomal membranes
(Fig. 5C). The two 32P-labeled proteins were
mixed in appropriate ratios, incubated with membranes, and then
electrophoresed on a 5% SDS-PAGE gel in a DNA sequencing gel apparatus
(Fig. 5D). Six distinct bands were observed between those of
the P-
C-PD and P-
NC-PD homopolymers, indicating that the large
complex contains seven monomers.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 5.
Construction and heptamerization of
phosphorylated N-PD and
NC-PD. A, schematic diagrams of
phosphorylated N-PD and NC-PD. The filled,
hatched, and dotted boxes are as in
Fig. 1A, and the gray box indicates
the GST-linker peptide. The protein kinase recognition sequence is
underlined, and the phosphorylated residue is indicated by
an asterisk. The factor Xa-sensitive site in the N-terminal
region is indicated by an arrow. The molecular masses
calculated from amino acid sequences are given on the right,
and those determined by MALDI-TOF mass spectrometry are given in
parentheses. B, purification of recombinant GST
fusion proteins and their cleavage by factor Xa. The purified proteins
(3 µg each) were heated in SDS-sample buffer and then electrophoresed
on a 12.5% SDS-PAGE gel, followed by staining with Coomassie Brilliant
Blue R. The sizes of the molecular mass markers are indicated on the
left. Lane 1, GST-PD; lane
2, GST- N-PD; lane 3, factor
Xa-cleaved GST-PD; lane 4, factor Xa-cleaved
GST- N-PD. C, formation of a large SDS-resistant complex
by P- N-PD and P- NC-PD. Purified GST-PD and GST- N-PD were
labeled with 32P and then cleaved with factor Xa.
32P-Labeled P- C-PD and P- NC-PD were purified as
described under "Experimental Procedures." Twenty nanograms of each
of these active toxins were incubated with and without synaptosomal
membranes (2.2 µg of protein) in 10 µl of TBS. The samples were
heated in SDS-sample buffer, electrophoresed on a 4-20% gradient
SDS-PAGE gel, and autoradiographed. Lane 1,
32P-labeled P- C-PD; lane 2,
32P-labeled P- NC-PD; lane 3,
32P-labeled P- C-PD with membranes; lane
4, 32P-labeled P- NC-PD with membranes.
D, heptameric complex formation through copolymerization of
[32P] C-PD and [32P] NC-PD.
[32P] C-PD and [32P] NC-PD were mixed
in different proportions: 1:0 (lane 1), 2:1
(lane 2), 1:1 (lane 3), 1:2
(lane 4), and 0:1 (lane 5).
Each mixture (12 ng of protein) was incubated with synaptosomal
membranes (2.2 µg of protein) in 12 µl of TBS. Samples were heated
in SDS-sample buffer and separated on a 5% SDS-PAGE gel (800 × 170 × 0.4 mm), followed by autoradiography. The predicted molar
ratios of [32P] C-PD to [32P] NC-PD are
given on the left.
|
|
Inhibition of Complex Formation by
-Protoxin--
The effect of
-protoxin on the ability of
C-PD to form an SDS-resistant complex
was examined. Complex formation was inhibited in a
dose-dependent fashion by
-prototoxin and completely
blocked by a 10-fold excess of
-protoxin (Fig.
6A). Such
dose-dependent inhibition by
-protoxin was
also observed for complex formation by
NC-PD (Fig. 6B).
The inhibition of SDS-resistant complex formation seems to be due to
competition for receptor binding, but it may also involve a subsequent
complex formation step. The labeled toxins were each detected as a
monomer in the membranes to a similar extent, independent of the amount
of
-protoxin added. If they represent monomers dissociated
from a receptor but associated with the membrane,
-protoxin would
inhibit complex formation by these monomers.

View larger version (63K):
[in this window]
[in a new window]
|
Fig. 6.
Inhibition of active
-toxin heptamerization by
-protoxin. Eleven nanograms of
125I- C-PD (A) or 125I- NC-PD
(B) alone (lane 1) or mixed with 1.1 (lane 2), 3.3 (lane 3), 11 (lane 4), 33.3 (lane 5), or
110 ng (lane 6) of unlabeled -protoxin were
incubated with synaptosomal membranes (2.2 µg of protein) in 12 µl
of TBS containing 0.1% BSA. After washing three times with TBS, the
membranes were dissolved by heating in SDS-sample buffer, subjected to
SDS-PAGE (4-20% gradient gel) and subsequent autoradiography.
|
|
 |
DISCUSSION |
This paper demonstrates that removal of the C- but not the
N-terminal peptide is responsible for the activation of
-toxin. In a
previous study, we showed that
-protoxin is cleaved not only at an
N-terminal region but also a C-terminal region by trypsin, chymotrypsin, and
-protease, and that
-protoxin activated by such
proteases differs in both its N and C termini (19). The different forms
also differ in toxicity, with the trypsin plus chymotrypsin-activated
-toxin being the most potent. Since the presence or absence of the
N-terminal peptide did not affect toxicity, the potency of
-toxin is
likely to be determined solely by the difference in the C-terminal
cleavage site. This paper also provides evidence of the ability of
active
-toxin to form a large complex in rat synaptosomal membranes.
We previously demonstrated that the toxin is neurotoxic to the brains
of mice and rats (2, 3). The finding that the toxin forms a large
complex in the synaptosomal membrane strongly suggests that the
neurotoxicity is due to large complex formation, which also is
implicated in membrane permeabilization and cell death of
-toxin-treated MDCK cells.
Aerolysin has been proposed to form heptameric oligomers, based on the
results of SDS-PAGE, scanning transmission electron microscopic mass
measurement of single oligomers, and image analysis of two-dimensional
crystals, which were obtained by reconstitution of purified aerolysin
and E. coli phospholipids (13, 25). To solve the problem
that low resolution electron crystallography gave artifactual data for
other pore-forming toxins, Moniatte et al. (26) determined
the molecular masses of oligomers formed in a solution by MALDI-TOF
mass spectrometry, providing further evidence of heptamerization.
However, analysis of the complex in biological membranes is a
prerequisite for proving heptamerization and for elucidating its
molecular mechanism, since aerolysin polymerizes at higher
concentrations (27). Taking advantage of the fact that the N-terminally
processed and unprocessed
-toxins both form the large complex, we
have demonstrated that seven monomers are present in the synaptosomal
membrane complex. This is the first demonstration of a heptameric toxin
complex in a biological membrane, and also supports the validity of the
heptermerization suggested for aerolysin and other pore-forming toxins.
The inhibition of SDS-resistant complex formation by
-protoxin shown
in this study can be explained simply by competition for receptor
binding. Alternatively,
-protoxin associated with the membrane may
exert its inhibitory effect by sequestering active
-toxin, making it
unavailable to assemble into a productive complex. Whichever of these
possibilities is true, the results unequivocally show the ability of
-protoxin to bind to a receptor and its inability to form the
productive large complex. Thus, it seems very likely that a receptor
binding site is exposed on the surface of
-protoxin, and that the
C-terminal peptide sterically hinders exposure of a site or
conformational change required for complex formation.
-Protoxin exerts no toxicity unless it encounters proteases such as
trypsin, chymotrypsin, and
-protease. This may imply that the
C-terminal peptide functions as an intramolecular chaperone. It should
be noted that C-terminally truncated forms of
-protoxin were
produced as inclusion bodies, and that several attempts to obtain
active forms through solubilization of the precipitate with urea or
guanidine HCl and subsequent dilution or dialysis were unsuccessful.
However, all
-toxin constructs containing the C-terminal peptide
were obtained in a soluble form. It has been reported that unfolding of
proaerolysin in 7 M urea was reversible upon dilution in
urea-free buffer (28). These observations suggest that these C-terminal
peptides aid proper folding of toxin molecules. Another likely
chaperonic function of the C-terminal peptide is to prevent the active
-toxin from aggregating in solution. The 5.1-kDa C-terminal
propeptide of C. septicum
-toxin has been shown to be
associated with the toxin even after proteolytic activation, preventing
the toxin from aggregating (8). Further study, of the
association/dissociation of the C-terminal peptide with the active
-toxin, and its effect on unfolding/refolding of the toxin, should
be undertaken to elucidate the roles of the C-terminal peptide in the
structure and function of
-toxin.
 |
ACKNOWLEDGEMENTS |
We thank Yuki Taniguchi for excellent
technical assistance. We also thank Drs. Hiroshi Tokumitsu, Ryoji
Kobayashi (Department of Chemistry, Kagawa Medical University, Kagawa,
Japan), Ryuichi Moriyama, Shio Makino (Department of Applied
Molecular Biosciences, Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya, Japan), and David B. Wilson (Department of
Molecular Biology and Genetics, Cornell University, Ithaca, NY) for
valuable discussions.
 |
FOOTNOTES |
*
This work was supported by Grant-in-Aid from Japan Society
for the Promotion of Science.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel./Fax:
81-87-891-2129; E-mail: microbio@kms.ac.jp.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M011527200
 |
ABBREVIATIONS |
The abbreviations used are:
MDCK, Madin-Darby
canine kidney;
PCR, polymerase chain reaction;
GST, glutathione
S-transferase;
PBS, Dulbecco's phosphate-buffered saline;
TBS, Tris-buffered saline;
PAGE, polyacrylamide gel electrophoresis;
MALDI-TOF, matrix-assisted laser desorption ionization/time of flight;
PD,
-protoxin derivative;
N-PD,
-protoxin derivative without
the 13 N-terminal residues;
C-PD,
-protoxin derivative without
the 23 C-terminal residues;
NC-PD,
-protoxin derivative without
either the N- or C-terminal residues.
 |
REFERENCES |
1.
|
Sakurai, J.
(1995)
Rev. Med. Microbiol.
6,
175-185
|
2.
|
Miyamoto, O.,
Minami, J.,
Toyoshima, T.,
Nakamura, T.,
Masada, T.,
Nagao, S.,
Negi, T.,
Itano, T.,
and Okabe, A.
(1998)
Infect. Immun.
66,
2501-2508[Abstract/Free Full Text]
|
3.
|
Miyamoto, O.,
Sumitani, K.,
Nakamura, T.,
Yamagami, S.-I.,
Miyata, S.,
Itano, T.,
Negi, T.,
and Okabe, A.
(2000)
FEMS Microbiol. Lett.
189,
109-113[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Finnie, J. W.,
Blumbergs, P. C.,
and Manavis, J.
(1999)
J. Comp. Pathol.
120,
415-420[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Nagahama, M.,
Ochi, S.,
and Sakurai, J.
(1998)
J. Nat. Toxins
7,
291-302[Medline]
[Order article via Infotrieve]
|
6.
|
Petit, L.,
Gibert, M.,
Gillet, D.,
Laurent-Winter, C.,
Boquet, P.,
and Popoff, M. R.
(1997)
J. Bacteriol.
179,
6480-6487[Abstract]
|
7.
|
Garland, W. J.,
and Buckley, J. T.
(1988)
Infect. Immun.
56,
1249-1253[Medline]
[Order article via Infotrieve]
|
8.
|
Sellman, B. R.,
and Tweten, R. K.
(1997)
Mol. Microbiol.
25,
429-440[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Ohnishi, M.,
Hayashi, T.,
and Terawaki, Y.
(1998)
J. Biol. Chem.
273,
453-458[Abstract/Free Full Text]
|
10.
|
Habeeb, A. F.,
Lee, C. L.,
and Atassi, M. Z.
(1973)
Biochim. Biophys. Acta
322,
245-250[Medline]
[Order article via Infotrieve]
|
11.
|
Rossjohn, J.,
Feil, S. C.,
McKinstry, W. J.,
Tweten, R. K.,
and Parker, M. W.
(1997)
Cell
89,
685-692[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Li, J. D.,
Carroll, J.,
and Ellar, D. J.
(1991)
Nature
353,
815-821[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Parker, M. W.,
Buckley, J. T.,
Postma, J. P.,
Tucker, A. D.,
Leonard, K.,
Pattus, F.,
and Tsernoglou, D.
(1994)
Nature
367,
292-295[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Song, L.,
Hobaugh, M. R.,
Shustak, C.,
Cheley, S.,
Bayley, H.,
and Gouaux, J. E.
(1996)
Science
274,
1859-1866[Abstract/Free Full Text]
|
15.
|
Petosa, C.,
Collier, R. J.,
Klimpel, K. R.,
Leppla, S. H.,
and Liddington, R. C.
(1997)
Nature
385,
833-838[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Lesieur, C.,
Vecsey-Semjen, B.,
Abrami, L.,
Fivaz, M.,
and Gisou van der Goot, F.
(1997)
Mol. Membr. Biol.
14,
45-64[Medline]
[Order article via Infotrieve]
|
17.
|
Hunter, S. E. C.,
Clarke, I. N.,
Kelly, D. C.,
and Titball, R. W.
(1992)
Infect. Immun.
60,
102-110[Abstract]
|
18.
|
Jin, F.,
Matsushita, O.,
Katayama, S.,
Jin, S.,
Matsushita, C.,
Minami, J.,
and Okabe, A.
(1996)
Infect. Immun.
64,
230-237[Abstract]
|
19.
|
Minami, J.,
Katayama, S.,
Matsushita, O.,
Matsushita, C.,
and Okabe, A.
(1997)
Microbiol. Immunol.
41,
527-535[Medline]
[Order article via Infotrieve]
|
20.
|
Kaelin, W. G. J.,
Krek, W.,
Sellers, W. R.,
DeCaprio, J. A.,
Ajchenbaum, F.,
Fuchs, C. S.,
Chittenden, T.,
Li, Y.,
Farnham, P. J.,
Blanar, M. A.,
Livingston, D. M.,
and Flemington, E. K.
(1992)
Cell
70,
351-364[Medline]
[Order article via Infotrieve]
|
21.
|
Okabe, A.,
Matsushita, O.,
Katayama, S.,
and Hayashi, H.
(1986)
Antimicrob. Agents Chemother.
30,
82-87[Medline]
[Order article via Infotrieve]
|
22.
|
Gray, E. G.,
and Whittaker, V. P.
(1962)
J. Anat.
96,
79-88
|
23.
|
Matsushita, O.,
Jung, C.-M.,
Minami, J.,
Katayama, S.,
Nishi, N.,
and Okabe, A.
(1998)
J. Biol. Chem.
273,
3643-3648[Abstract/Free Full Text]
|
24.
|
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
|
25.
|
Wilmsen, H. U.,
Leonard, K. R.,
Tichelaar, W.,
Buckley, J. T.,
and Pattus, F.
(1992)
EMBO J.
11,
2457-2463[Abstract]
|
26.
|
Moniatte, M.,
van der Goot, F. G.,
Buckley, J. T.,
Pattus, F.,
and van Dorsselaer, A.
(1996)
FEBS Lett.
384,
269-272[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Fivaz, M.,
Velluz, M. C.,
and van der Goot, F. G.
(1999)
J. Biol. Chem.
274,
37705-37708[Abstract/Free Full Text]
|
28.
|
Lesieur, C.,
Frutiger, S.,
Hughes, G.,
Kellner, R.,
Pattus, F.,
and van der Goot, F. G.
(1999)
J. Biol. Chem.
274,
36722-36728[Abstract/Free Full Text]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.