The C-terminal domain of perfringolysin O is an essential
cholesterol-binding unit targeting to cholesterol-rich microdomains
Yukiko Shimada
1
, Mikako Maruya
2
, Shintaro Iwashita
3
and Yoshiko Ohno-Iwashita
1
1
Biomembrane Research Group, Tokyo Metropolitan Institute of Gerontology;
2
Department of Cell Biology, Tokyo Metropolitan
Institute of Medical Science;
3
Mitsubishi Kagaku Institute of Life Sciences (MITILS), Machida, Tokyo, Japan
There is much evidence to indicate that cholesterol forms
lateral membrane microdomains (rafts), and to suggest their
important role in cellular signaling. However, no probe has
been produced to analyze cholesterol behavior, especially
cholesterol movement in rafts, in real time. To obtain a
potent tool for analyzing cholesterol dynamics in rafts, we
prepared and characterized several truncated fragments of
h-toxin (perfringolysin O), a cholesterol-binding cytolysin,
whose chemically modified form has been recently shown to
bind selectively to rafts. BIAcore and structural analyses
demonstrate that the C-terminal domain (domain 4) of the
toxin is the smallest functional unit that has the same cho-
lesterol-binding activity as the full-size toxin with structural
stability. Cell membrane-bound recombinant domain 4 was

there are almost no probes that have been used to detect
and monitor cholesterol in rafts. Filipin is a reagent
currently used for the cytochemical staining of cholesterol
in fixed cells [6]. However, filipin permeabilizes the cell
membrane and binds to cell cholesterol indiscriminately
[6,7].
We have examined the cytolytic mechanism of perfringo-
lysin O (h-toxin) secreted by Clostridium perfringens,which
binds to membrane cholesterol and causes cell disruption.
Cholesterol-mediated binding to a membrane is a trigger for
forming toxin oligomers, leading to the formation of large
pores. This pore formation directly causes cell membrane
damage resulting in cell disruption. We prepared several
h-toxin derivatives that retain specific binding activity to
cholesterol but lack cytolytic activity. Ch [8] is a protease-
nicked derivative and loses the capacity to oligomerize
below 20 °C. MCh [9] and BCh [10,11] are methylated and
biotinylated derivatives of Ch, respectively, and both have
the same binding specificity and affinity for membrane
cholesterol as intact h-toxin, but cause no damage to
membranes at 37 °Corbelow.
The h-toxin derivatives bind to liposomes with high
cholesterol content but not to liposomes that contain less
than 20 mol% of cholesterol [12], which strongly suggests
their selective binding to cholesterol-enriched membrane
domains. Recently we demonstrated that BCh selectively
binds to cholesterol in cholesterol-rich microdomains of
intact cells, domains that fulfill the criteria of lipid rafts
[7]. The BCh bound to various types of cells was found to
be TX-100 insoluble at 4 °C[7].WhenBCh-bound

Rafts are abundant at the plasma membrane surface, and
are also found in intracellular compartments in the endo-
cytic pathway [14]. In a further study on lipid rafts, analysis
of the dynamic movement of intracellular rafts, for instance
raft assembly and raft trafficking, is necessary as well as that
on the membrane surface. However, as staining with BCh
requires fluorescent avidin, it is not suitable for real-time
imaging of the dynamic movement of lipid rafts in living
cells. Especially, such movement inside the cell is hard to
trace by the indirect fluorescence method. To establish a
system for real-time imaging of rafts, we have attempted to
isolate the cholesterol-binding domain of the toxin. Based
on the 3D crystal structure [15], h-toxin comprises four
b-sheet-rich domains, and only domain 4, located at the
C-terminus, is structurally autonomous [15]. There is
evidence to suggest that a cholesterol-binding site is located
within domain 4. For example, a C-terminal fragment
obtained by trypsin digestion (T2), including predominantly
domain 4, binds to cholesterol and to cholesterol-containing
membranes [16]. Furthermore, experiments with many
toxins mutated in the tryptophan-rich motif at the C-
terminus have revealed a significant reduction in the
membrane-binding activity [17]. However, the cholesterol-
binding site of h-toxin has not been clearly defined as yet.
We have characterized cholesterol binding activity in
relation to toxin stability and identified the smallest region
necessary for its activity. In this paper, we show that
domain 4 of h-toxin is an essential cholesterol-binding unit
targeting to cholesterol in lipid rafts. Furthermore, we
demonstrate that enhanced green fluorescent protein

merase chain reaction and ligated into pET-28b digested
with NheIandXhoI. The NheI restriction sites of the
polymerase chain reaction-amplified products were created
as noncomplementary ends of the amplification primers.
For DC-D4 (K363-T470), a pNSP10-derived plasmid enco-
ding D471 (K1-T470) was used as a PCR template. The poly-
merase chain reaction primers used were 5¢-CTCAGGC
TAGCAAGGGAAAAATAAACTTAGATC-3¢ (for D4
and DC-D4), 5¢-TCAGAGCTAGCAGTGGAGCCTATG
TTGCACAG-3¢ (for DN-D4) and 5¢-TGGTGGTGG
TGCTCGAGTGC-3¢. For the construction of a plasmid
encoding a His-tag-EGFP-D4 fusion protein, a DNA frag-
ment containing the EGFP-encoding region was amplified
from pEGFP-N3 (Clontech) by the polymerase chain
reaction with forward primer A (5¢-CGTTCTAGAGT
GAGCAAGGGCGAGGAGCTG-3¢) and reverse primer
B(5¢-ATCTACGTCGGCTAGCCTTGTACAGCTCGT
CCATGCCGAG-3¢). The fragment was then ligated into
the NheI site of the plasmid encoding His-tag-D4. The
DNA sequences in the resulting plasmids were confirmed
by the dideoxynucleotide chain-termination method [18].
Plasmids were introduced into E. coli strain BL21 (DE3)
[19] (Novagen, Madison, WI, USA) by transformation of
competent cells.
Protein production and purification
E. coli strain BL21(DE3) was used for the overexpression of
His-tag-T2¢, His-tag-D4, His-tag-DN-D4 and His-tag-DC-
D4 fusion proteins. After induction with IPTG, E. coli cells
were harvested by centrifugation and lysed in native lysis
buffer (50 m

phenylmethanesulfonyl fluoride. After protease treat-
ment, the pooled fraction was applied to a butyl-agarose
column equilibrated with 20 m
M
Tris/HCl, pH 7.5, con-
taining 0.8
M
(NH
4
)
2
SO
4
. The toxin fragments were eluted
with 0.2
M
(NH
4
)
2
SO
4
and dialyzed against Hepes-buffered
saline, pH 7.0, at 4 °C. The purity of the toxin fragments
was checked by SDS/PAGE [20]. The sequence GSHMAS
remains attached to the N termini of purified fragments
after thrombin cleavage. Toxin derivatives MCh and BCh,
and the T2 fragment were prepared as described previously
[9,10,16].
6196 Y. Shimada et al. (Eur. J. Biochem. 269) Ó FEBS 2002

For measurement of binding to cultured cells, MOLT-4
(10
5
cells) [21] were washed twice with phosphate-buffered
saline (NaCl/P
i
), and then incubated with or without 5 m
M
2-hydroxypropyl-b-cyclodextrin (2OHpbCD) in serum-free
RPMI 1640 for 15 min at 37 °C. The cells were then washed
twice with NaCl/P
i
, and then incubated with toxin frag-
ments (0.3 lg) in NaCl/P
i
containing 1 mgÆmL
)1
bovine
serum albumin for 30 min at 37 °C. Toxin fragments bound
to cells were obtained in the pellet by centrifugation.
Measurement of Trp fluorescence
A 0.3 nmol sample of each purified toxin fragment was
mixed with either phospholipid-liposomes [toxin fragment/
phospholipids 1 : 30 (mol/mol)] or cholesterol/phospho-
lipid-liposomes [toxin fragment/cholesterol 1 : 30 (mol/mol)]
in 1.5 mL of Hepes-buffered saline. After incubation for
10 min at room temperature, emission spectra were recor-
ded in the range of 300–400 nm at an excitation wavelength
of 295 nm with a Shimadzu spectrofluorophotometer
RF-5000.

above. Subtilisin BPN¢ was mixed with toxin fragment or
liposome-bound toxin fragment preparations in 50 m
M
phosphate buffer, pH 7.0. The mixture was incubated for
30 min at 27 °C and the cleavage reaction was stopped by
the addition of phenylmethanesulfonyl fluoride at a final
concentration of 1 m
M
.
TX-100 treatment and sucrose density gradient
fractionation
In order to isolate TX-100-insoluble membranes, MOLT-4
cells were extracted on ice for 20 min with 1% TX-100 in
TNE buffer (25 m
M
Tris/HCl, pH 7.5, 150 m
M
NaCl,
5m
M
EDTA) containing 2 m
M
phenylmethanesulfonyl
fluoride, 1 m
M
leupeptin, 25 lgÆmL
)1
aprotinin and
20 lgÆmL
)1

SO
4
. EGFP-D4 was eluted with 20 m
M
Tris/
HCl, pH 7.5. Cells were incubated with EGFP-D4 in
serum-free RPMI-1640 for 5 min at 37 °C. After washing
with RPMI 1640, fluorescence images of living cells were
observed using an Olympus fluorescent microscope. No
significant difference in cell viability was found before and
after EGFP-D4 addition by checking with trypan blue
Ó FEBS 2002 A probe for raft cholesterol in living cells (Eur. J. Biochem. 269) 6197
exclusion. More than 95% of the cells were viable after
being labeled with EGFP-D4, washed and incubated for
one hour at room temperature.
Others
Tricine-SDS/PAGE was performed by the method of
Schagger [23]. N-terminal sequences of toxin fragments
were analyzed with a precise cLC protein sequencer
(Applied Biosystems) according to the manufacturer’s
recommendations.
RESULTS
Isolation of cholesterol-binding fragments of h-toxin
As the C-terminal portion of h-toxin might retain choles-
terol binding activity, several N-terminal truncated frag-
ments were constructed and expressed in E. coli.Toxin
fragments T2¢,D4,DN-D4, and DC-D4 [Fig. 1(A)] were
purified from the cytoplasmic fraction of E. coli by
Ni
+

specific binding for cholesterol in membranes. These results
show that cholesterol-binding activity resides in domain 4,
and the binding specificity is the same as that of h-toxin.
They also indicate that the amino acid sequence of whole
domain 4 is required for folding into a stable structure
for cholesterol binding. As expected, neither T2¢ nor D4
showed hemolytic activity (data not shown) despite of their
ability to bind to cholesterol-containing membranes.
We next examined the cholesterol-binding kinetics of
these toxin fragments by surface plasmon resonance using a
sensor chip on which cholesterol-containing liposomes were
immobilized (Table 1). Association and dissociation rate
constants for T2¢ and D4 binding to cholesterol-containing
liposomes were almost the same as corresponding constants
for MCh binding (Table 1), indicating that the deletion of
domains 1–3 from the toxin did not influence the binding
kinetics. As a result, the dissociation constants also exhibit
similar values. These experiments with liposomal mem-
branes show that domain 4 retains the same binding
specificity and binding affinity for membrane cholesterol
as h-toxin.
Protease susceptibility of membrane-bound toxin
fragments
To investigate the state of the toxin fragments during
membrane binding, we analyzed the susceptibilities of the
T2¢ and D4 fragments to protease in the presence and
absence of membranes (Fig. 3). In the absence of liposomal
Fig. 1. Isolation of toxin fragments. (A) Schematic drawings of h-toxin
and its derivatives. Recombinant toxin fragments T2¢,D4,DN-D4 and
DC-D4 were produced in E. coli with an N-terminal His-tag for

cleavage occurred. On the other hand, the N-terminal
amino acid sequence of the digested product of the
liposome-bound T2¢ fragment was determined to be STE-
YSKGKIN, indicating that 27 amino acid residues were
cleaved from the N terminus of T2¢. The cleaved position is
shown in the 3D structure of the T2¢ fragment (Fig. 3),
demonstrating that the entire domain 4 region is protected
from protease digestion. This finding is consistent with the
Fig. 2. Binding of h-toxin fragments to cholesterol. (A) Specific binding
of h-toxin fragments to cholesterol on TLC plates. Lipid mixtures
containing 2 lg each of standard neutral lipids were applied to TLC
plates and the plates were developed. The plates were then incubated
with toxin fragments or derivatives and bound proteins were detected
by immunostaining with anti-(whole h-toxin) Ig. Lipids were detected
with 3% cupric acetate/8% phosphoric acid by heating at 140 °C
(ÔlipidsÕ lane). PC, phosphatidylcholine; SM, sphingomyelin. (B) Toxin
binding to liposomal membranes. h-Toxin, MCh and toxin fragments
were incubated with DOPC liposomes or DOPC/cholesterol liposomes
for 20 min at room temperature. After centrifugation, the total frac-
tion (T), and the resulting supernatant (S) and pellet (P) fractions were
separated and analyzed by SDS/PAGE followed by immunoblotting
with an antibody against h-toxin C-terminal peptide. Lane M shows
molecular size marker proteins.
Table 1. Kinetic analysis of toxin fragment binding to cholesterol by surface plasmon resonance. Kinetic analysis of toxin fragment binding to
immobilized cholesterol-containing liposomes was performed as described in ÔExperimental proceduresÕ. The binding kinetics were analyzed by the
software
BIAEVALUATION
2.1. Each value is given as mean ± SE, n ¼ 6.
Toxin fragment k
on

(1.5 ± 0.81) · 10
)7
Fig. 3. Susceptibility of T2¢ and D4 to protease. T2¢ and D4 fragments
were digested with subtilisin BPN¢ in the presence or absence of cho-
lesterol-containing liposomes. After protease treatment, the resultant
fragments were separated by Tricine-SDS/PAGE and analyzed by
Western blotting with an antibody against h-toxin C-terminal peptide.
In the lower panel the 3D structures of T2¢ and D4 are shown in black
against a gray background of the whole h-toxin structure. The arrow
indicates the position of cleavage by the protease in the presence of
cholesterol-containing liposomes. The N-terminal sequences of T2¢
and D4 are also shown in the lower panel.
Ó FEBS 2002 A probe for raft cholesterol in living cells (Eur. J. Biochem. 269) 6199
observation that the resultant fragment was nearly the same
size as the D4 fragment on Tricine-SDS/PAGE. As
liposomes not containing cholesterol do not protect the
fragments against protease digestion (data not shown),
cholesterol-dependent membrane binding is required for
protection.
Tryptophan fluorescence and circular dichroism spectra
of the fragments
To examine the conformation of the toxin fragments that
bind to liposomes, intrinsic tryptophan fluorescence was
measured (Fig. 4). Among seven tryptophan residues in
h-toxin, domain 4 contains six, while only one (Trp137) is
located in the N-terminal region (domain 1). To eliminate
the spectral contribution of Trp137, we used W137F, a
mutant h-toxin in which Trp137 in the N-terminal region is
replaced by Phe [17]. Some tryptophan residues in domain 4,
especially those within the 11-amino acid consensus

dichroism measurement. The D4 fragment is enriched in
b-sheets (data not shown), which is consistent with the
structure predicted from X-ray crystallography, supporting
the idea that biosynthesized domain 4 automatically folds
into the secondary structure of the native toxin. On the
other hand, the spectra of the T2 and T2¢ fragments exhibit
more disordered structures than that of D4. These data
imply that their extra N-terminal sequences other than
domain 4 might be a disordered structure.
Selective binding of D4 to lipid rafts
The binding characteristics of the D4 fragment to intact
cell membranes was examined. The D4 fragment was
detected in the cell fraction after incubation with MOLT-
4 cells (Fig. 5A, )2OHpbCD, pellet fraction). Treatment
with 5 m
M
2OHpbCD for 15 min at 37 °C, which
depletes cholesterol by 30%, caused significant reduction
in the number of D4 fragments bound to MOLT-4 cells
(Fig. 5A, 5 m
M
2OHpbCD), demonstrating cholesterol-
dependent binding. We have previously shown that BCh
binds selectively to lipid rafts in intact platelets [7]. To
analyze the selectivity of binding, we first examined the
detergent-insolubility of the D4 fragment bound to
MOLT-4 cells. After extraction with 1% TX-100 on
ice, the membrane-bound D4 fragment was recovered in
the Triton-insoluble membrane fraction (Fig. 5A, TX).
Next, we examined the distribution of the cell membrane-

with a fluorescent dye in live cells. As domain 4 has no
membrane-damaging activity by itself, it is a good
candidate for the construction of cholesterol-specific
probes. To prepare the fluorescent toxin fragment, the
T2¢ and D4 fragments were labeled with Alexa 546. Alexa-
labeled toxin fragments stained cell surfaces and
2OHpbCD treatment abolished this staining, indicating
cholesterol-specific binding (data not shown). We tried
another approach in which EGFP was fused to the N
terminus of D4. The EGFP-D4 fusion protein was
overproduced in E. coli and purified as described in
ÔExperimental proceduresÕ. Following incubation with
EGFP-D4 for 5 min at room temperature, clear fluores-
cent labeling was observed on the surface of live MOLT-4
cells (Fig. 6A). EGFP-D4 stains cells in a cholesterol-
dependent manner as no staining was observed in
2OHpbCD-treated cells (Fig. 6B). Together with the
finding that the D4 fragment binds selectively to lipid
rafts, EGFP-D4 allows us to visualize membrane choles-
terol in lipid rafts of live cells.
DISCUSSION
To clarify the physiological significance of lipid rafts, many
experimental tools have been used. Bacterial toxins that
target components in rafts are often used as raft markers
[29]. Cholera toxin is used to detect ganglioside GM1, which
is enriched in lipid rafts [30]. Sphingomyelin, a major
component of rafts, is a target for lysenin, which is secreted
by earthworms [31]. However, no tool has been reported for
the detection of cholesterol in lipid rafts in living cells. In this
paper, to obtain a probe for targeting raft cholesterol, we

6
cellsÆmL
)1
) treated with (5 m
M
2OHpbCD) or without () 2OHpbCD) 2OHpbCD were incubated
with D4 fragment for 15 min at 37 °C. After centrifugation, the total
fraction (T) and the resultant supernatant (S) and pellet (P) fractions
were analyzed by SDS/PAGE followed by Western blotting with an
anti C-terminal peptide antibody. A portion of the resultant pellet
obtained from the 2OHpbCD untreated sample was extracted with 1%
TX-100 for 20 min on ice and the soluble (TX, S) and insoluble (TX, P)
fractions were separated by centrifugation at 15 000 g.(B)D4frag-
ment-bound MOLT-4 cells were treated with 1% TX-100, homogen-
ized and subjected to a sucrose density gradient centrifugation. Eleven
fractions (starting at the top) and the pellet (P) were collected. The
distributions of D4, Lck and cholesterol were measured.
Fig. 6. Staining of cell surface cholesterol in living cells with EGFP-D4.
(A) MOLT-4 cells were incubated with EGFP-D4 for 5 min at room
temperature. Labeled cells were observed under a fluorescent micro-
scope. (B) MOLT-4 cells were treated with 5 m
M
2OHpbCD to deplete
cholesterol and then stained with EGFP-D4. Left panel, phase con-
trast; right panel, fluorescence.
Ó FEBS 2002 A probe for raft cholesterol in living cells (Eur. J. Biochem. 269) 6201
truncated mutants, in which a similar red shift of tryptophan
fluorescence is accompanied by the complete loss of
cholesterol-binding activity [32]. It is noteworthy that even
a small truncation of domain 4 at either the N or C terminus

that of the full-size toxin as revealed by surface plasmon
resonance measurement (Table 1). We previously showed
that binding of the toxin to membrane cholesterol triggers
its conformational change around tryptophan residues in
domain 4 [24,25]. Such conformational change occurs
without oligomerization, as a similar change was observed
for isolated T2 fragment (Fig. 4). Recently a model was
proposed that toxin oligomerization triggers the insertion of
a portion of domain 3 as b-hairpins, which contribute to the
formation of a transmembrane pore [34,35]. Although
domain 3 might play a role for pore formation, our results
clearly demonstrated that the insertion of domain 3 is not
required for maintaining the high affinity of the toxin for the
membrane, as revealed by BIAcore analysis (Table 1).
The D4 fragment has selective binding affinity for
cholesterol in lipid rafts (Fig. 5B) as demonstrated with
BCh [7], a protease-nicked and biotinylated full-size h-toxin.
Therefore the selectivity of binding to lipid rafts is ascribed
to domain 4. We also show that EGFP-D4 is a potent
probe for the detection of cell surface cholesterol in live cells.
As most membrane-bound EGFP-D4 was recovered in
FLDF (data not shown), the staining should display the
distribution of cholesterol in membrane rafts. The cell-
staining profile with EGFP-D4 is quite similar to that with
Alexa-labeled D4, suggesting the fusion with EGFP does
not influence the cholesterol-binding activity of D4. Previ-
ously we reported that BCh is a specific probe for the
detection of cholesterol by fluorescent microscopy [10,11].
Cholesterol on the outer surface of the plasma membrane
can be stained with BCh coupled with FITC-conjugated

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