Therefore, domain 4 of h-toxin is an essential choles-terol-binding unit targeting to cholesterol in membrane rafts, providing a very useful tool for further studies on lipid rafts on ce
Trang 1The C-terminal domain of perfringolysin O is an essential
cholesterol-binding unit targeting to cholesterol-rich microdomains
Yukiko Shimada1, Mikako Maruya2, Shintaro Iwashita3and Yoshiko Ohno-Iwashita1
1
Biomembrane Research Group, Tokyo Metropolitan Institute of Gerontology;2Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science;3Mitsubishi 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
detected in the floating low-density fractions and was found
to be cofractionated with the raft-associated protein Lck, indicating that recombinant domain 4 also binds selectively
to cholesterol-rich rafts Furthermore, an enhanced green fluorescent
mem-brane surfaces in a cholesterol-dependent manner in living cells Therefore, domain 4 of h-toxin is an essential choles-terol-binding unit targeting to cholesterol in membrane rafts, providing a very useful tool for further studies on lipid rafts
on cell surfaces and inside cells
Keywords: raft; microdomain; cholesterol; BIAcore; perfringolysin O
In recent years, accumulating evidence has indicated that
cholesterol forms lateral membrane microdomains (lipid
rafts) in which sphingolipids are also enriched [1,2] This
microdomain is a scaffold where specific proteins assemble
and plays a pivotal role in signal transduction and many
other cellular functions [3] Lipid rafts have been isolated
by sucrose density gradient centrifugation after treatment
of cells with Triton X-100 (TX-100) by taking advantage of
their insolubility in detergent at 4C [2] Changes in
cholesterol content, either by inhibition of its biosynthesis
or by its removal from the plasma membrane, affect the
localization of proteins associated with rafts, and thus
affect raft function [4,5], suggesting (an) essential role(s) of
cholesterol in the structural maintenance and function of
rafts Although interest in cholesterol functions and
demand for their analyses have been increasing rapidly,
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 20C 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 37C or below
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 4C [7] When BCh-bound platelets were treated with TX-100 and fractionated on a sucrose-density gradient, BCh was predominantly localized
in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin and Src family kinases are enriched [7] Depletion of one-third of the cholesterol from cells with b-cyclodextrin, which was accompanied by more than a 70% reduction in cholesterol from FLDF, almost
Correspondence to Y Shimada or Y Ohno-Iwashita, Biomembrane
Research Group, Tokyo Metropolitan Institute of Gerontology,
35–2 Sakae-cho, Itabashi-ku, Tokyo 173–0015, Japan.
Fax: +81 3 35794776, Tel: +81 3 39643241 ext 3063 or 3068,
E-mail: yshimada@tmig.or.jp or iwashita@tmig.or.jp
Abbreviations: Br-DSPC, brominated distearoylphosphatidylcholine;
DOPC, dioleoylphosphatidylcholine; DSPC,
distearoylphospha-tidylcholine; EGFP, enhanced green fluorescent protein; FLDF,
floating low-density fractions; IPTG, isopropyl thio-b- D -galactoside;
2OHpbCD, 2-hydroxypropyl-b-cyclodextrin; PE,
phosphatidyl-ethanolamine; TX-100, Triton X-100.
(Received 5 August 2002, revised 10 October 2002,
accepted 30 October 2002)
Trang 2completely abolished BCh binding to lipid rafts This
indicates that the binding of BCh to lipid rafts depends on
their cholesterol content BCh, coupled with
fluorescent-avidin or colloidal gold-fluorescent-avidin, has been used as a probe
to analyze the distribution of membrane cholesterol by
fluorescence microscopy and electron microscopy
[10,11,13]
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
(EGFP)-tagged domain 4 may be a promising tool for
analyzing raft dynamics in living cells
E X P E R I M E N T A L P R O C E D U R E S
Materials
Anti-h-toxin antibody was raised in rabbits as described
previously [16] A rabbit antibody specific to the C-terminus
of h-toxin was produced using the peptide antigen
CGTTLYPGSSITYN (amino acids C449-N472 of the
mature form of h-toxin) Cholesterol and isopropyl
thio-b-D-galactoside (IPTG) were purchased from Sigma (St Louis,
MO, USA) Hydroxylapatite was from Seikagaku (Tokyo,
Japan) Peroxidase-conjugated anti-rabbit IgG was
pur-chased from Medical & Biological Laboratories (Nagoya,
Japan) BIAcore sensor chip SA was from BIAcore
(Uppsala, Sweden) Alexa FluorTM546 was from Molecular
Probes, Inc (Eugene, OR, USA)
Plasmid construction
Plasmid pNSP10 containing the perfringolysin O gene
(pfoA) [17] was used to construct pfoA derivatives encoding
the C-terminal region of the toxin The DNA fragment
containing the T2¢ (V337-N472)-encoding region was
pre-pared by digesting pNSP10 with SpeI and XhoI and inserting it between the NheI and XhoI sites on the 3¢ side of the sequence encoding His-tag and a thrombin-cleavage site
in the expression vector pET-28b The DNA fragment containing the D4 (K363-N472)- or DN-D4 (S371-N472)-encoding region was amplified from pNSP10 by a poly-merase chain reaction and ligated into pET-28b digested with NheI and XhoI 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 colistrain 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 mM phosphate buffer, pH 8.0, 300 mM NaCl,
10 mM imidazole) by ultrasonication The overexpressed proteins were partially purified from the cytoplasmic fraction of E coli by Ni+-NTA agarose column chroma-tography His-tagged toxin fragments bound to Ni+-NTA agarose were eluted with 250 mM imidazole For further purification, the fractions containing His-tagged toxin fragments were loaded onto a hydroxylapatite column equilibrated with 20 mMphosphate buffer, pH 8.0, and the flow through fraction was collected Active fragments were recovered in the flow through fraction, while inactive ones were adsorbed to hydroxylapatite The pooled fraction was incubated with thrombin at an enzyme to substrate ratio of
1 : 100 for 5 h at room temperature to cleave the His-tag and the cleavage reaction was stopped by the addition of
1 mMphenylmethanesulfonyl fluoride After protease treat-ment, the pooled fraction was applied to a butyl-agarose column equilibrated with 20 mM Tris/HCl, pH 7.5, con-taining 0.8M(NH4)2SO4 The toxin fragments were eluted with 0.2M(NH4)2SO4and dialyzed against Hepes-buffered saline, pH 7.0, at 4C 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]
Trang 3Binding to cholesterol on TLC plates
The cholesterol-binding activity of each toxin fragment was
examined on TLC plates as described previously [16] Toxin
fragments bound to the TLC plates were detected with
anti-whole h-toxin antibody
Preparation of lipid vesicles
Phospholipids [dioleoylphosphatidylcholine (DOPC) or
brominated distearoylphosphatidylcholine (Br-DSPC)]
alone or 1 : 1 (mol/mol) mixtures of cholesterol and
phospholipids were evaporated to make lipid films
Hepes-buffered saline was added to the lipid films and
mixed vigorously The lipid dispersion was sonicated with a
Branson Sonifier and centrifuged at 9000 g for 5 min to
remove undispersed lipids For kinetic analysis by the
BIAcore system, 0.1 mol% of
biotin-phosphatidylethanol-amine (PE) was added to the lipid mixture before
evapor-ation The lipid dispersion containing biotin-PE was frozen
and thawed 10 times, and the resultant multilamellar
vesicles were extruded through 100 nm polycarbonate
membranes in a Liposofast apparatus (Avestin Inc.,
Ottawa, Canada)
Binding of toxin fragments to liposomes and MOLT-4
cells
The toxin fragments were incubated individually with
liposomes in Hepes-buffered saline containing 1 mgÆmL)1
bovine serum albumin for 30 min at room temperature The
mixtures were centrifuged at 350 000 g for 30 min at 4C
Both the pellets and the supernatants were analyzed by
Western blotting using anti-C-terminal peptide antibody
For measurement of binding to cultured cells, MOLT-4
(105cells) [21] were washed twice with phosphate-buffered
saline (NaCl/Pi), and then incubated with or without 5 mM
2-hydroxypropyl-b-cyclodextrin (2OHpbCD) in serum-free
RPMI 1640 for 15 min at 37C The cells were then washed
twice with NaCl/Pi, and then incubated with toxin
frag-ments (0.3 lg) in NaCl/Pi containing 1 mgÆmL)1 bovine
serum albumin for 30 min at 37C 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
Circular dichroism spectra
Circular dichroism spectra were recorded on a JASCO
J-720 spectropolarimeter with 5 mm pathlength cells Toxin
fragments in NaCl/Piwere scanned from 250 to 200 nm
Molecular ellipticity ([h]) was calculated based on the mean
residue weight of each fragment
Kinetic analysis of toxin fragments in cholesterol binding
The cholesterol-binding kinetics of toxin fragments were determined by surface plasmon resonance [22] using BIA-core 1000TM All experiments were carried out at 25C in degassed Hepes-buffered saline DOPC/cholesterol lipo-somes containing biotin-PE were injected and immobilized
on a sensor chip SA that has dextran matrix-attached streptavidin After immobilization, the final signal increase was 1000 response units (RU) MCh, T2¢ and D4 were dialyzed against the same buffer and applied to the liposome-immobilized sensor chip Analyses were per-formed at a flow rate of 20 lLÆmin)1 Another sensor chip
SA bearing immobilized DOPC/biotin-PE liposomes was used as a control
Susceptibility of toxin fragments to a protease Liposome-bound toxin fragments were obtained by ultra-centrifugation after incubation with liposomes as described above Subtilisin BPN¢ was mixed with toxin fragment or liposome-bound toxin fragment preparations in 50 mM phosphate buffer, pH 7.0 The mixture was incubated for
30 min at 27C and the cleavage reaction was stopped by the addition of phenylmethanesulfonyl fluoride at a final concentration of 1 mM
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 mM Tris/HCl, pH 7.5, 150 mM NaCl,
5 mM EDTA) containing 2 mM phenylmethanesulfonyl fluoride, 1 mM leupeptin, 25 lgÆmL)1 aprotinin and
20 lgÆmL)1soybean trypsin inhibitor Then the TX-100-soluble and -inTX-100-soluble fractions were separated by centri-fugation at 15 000 g for 15 min and analyzed by Western blotting For sucrose density gradient fractionation, TX-100-treated cells were homogenized with a Potter– Elvehjem homogenizer and mixed with an equal volume of 80% sucrose, overlaid with 2.4 mL of 35% sucrose and 1.3 mL of 5% sucrose in TNE buffer After centrifugation
at 250 000 g for 18 h at 4C in a SW55 rotor, 11 fractions
of 0.4 mL each were collected from the top and the pellet was suspended in 0.4 mL of TNE buffer
Fluorescence microscopy For fluorescence microscopic observation, EGFP-D4 was overexpressed in E coli and purified with Ni+-NTA agarose as described for the purification of the T2¢ and D4 fragments The fractions eluted from the Ni+-NTA agarose column were applied to a butyl-agarose column equilibrated with 20 mM Tris/HCl, pH 7.5, containing 0.8M(NH4)2SO4 EGFP-D4 was eluted with 20 mMTris/ HCl, pH 7.5 Cells were incubated with EGFP-D4 in serum-free RPMI-1640 for 5 min at 37C 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
Trang 4exclusion 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
R E S U L T S
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+-NTA agarose, hydroxylapatite and butyl agarose
column chromatographies We also prepared the T2
fragment [16] and a toxin derivative, MCh [9], from
recombinant h-toxin protein by biochemical modification
as described before Three fragments (T2, T2¢ and D4) were
obtained as single proteins [Fig 1(B)] On the other hand,
DN-D4, which is truncated by eight-amino acids from the N
terminus of domain 4, and DC-D4, which has only two
amino acids deleted from the C terminus of domain 4, were
not stable during the purification process Therefore small
amounts of the DN-D4 and DC-D4 fragments were
recovered N-terminal sequence analysis of the T2¢ and
D4 fragments revealed that they have the expected
N-terminal sequence
Binding specificity and affinity of toxin fragments
for cholesterol
We first examined the binding specificity of the toxin
fragments to cholesterol by immunostaining with
anti-h-toxin antibody on lipid-developed TLC plates The T2¢ and
D4 fragments, as well as T2 and MCh, bound only to free
cholesterol among lipids [Fig 2(A)], indicating specific
recognition of free cholesterol by these toxin fragments To
investigate the binding specificity to cholesterol as a
mem-brane component further, liposomes containing cholesterol
were prepared After incubation with DOPC/cholesterol
liposomes, the T2¢ and D4 fragments were detected in the
pellet fraction [Fig 2(B)], which shows that the fragments
have binding activity similar to those of intact h-toxin, MCh
and T2 Negligible amounts of toxin fragments were bound
to liposomes prepared without cholesterol, indicating the
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 purification After thrombin digestion, an extra of six amino acids remained at the N terminus of each fragment (dotted rectangles) T2 is
a tryptic fragment of h-toxin and the N-terminal sequence was deter-mined previously [8] Amino acid numbers shown in each toxin derivative correspond to the positions in h-toxin The black rectangles represent the tryptophan-rich motif in domain 4 The arrowhead indicates the position of a protease-nicked site located between T144 and H145 in MCh and BCh (B) SDS/PAGE of purified toxin frag-ments and derivatives Toxin fragfrag-ments (T2¢ and D4) were expressed in
E coliand purified from the cytoplasmic fraction by a series of column chromatographies as described in Experimental procedures MCh and T2 were obtained from recombinant h-toxin by biochemical modifi-cations as described before Lane M shows molecular size marker proteins.
Trang 5membranes, both fragments were digested by subtilisin
BPN¢ into undetectable pieces (Fig 3, lanes 2 and 6) When
the D4 fragment bound to DOPC/cholesterol liposomes
was treated with subtilisin BPN¢, no changes in fragment
size were observed on Tricine-SDS/PAGE (Fig 3, lane 8)
In the case of T2¢, after binding to DOPC/cholesterol
liposomes, subtilisin BPN¢ digestion produced a proteolytic fragment with a molecular size similar to that of the D4 fragment (Fig 3, lane 4) The resultant proteolytic frag-ments were recovered from the gel (lanes 4 and 8) and their N-terminal sequences were analyzed by a protein sequencer The N-terminal amino acid sequence of the liposome-bound D4 fragment after digestion was found to be GSHMASKGKI, which corresponds to the N-terminal sequence of the intact D4 fragment, indicating that no 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
(lipids 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) fracfrac-tions 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 Experimental procedures 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 ( M )1 Æs)1) k off (s)1) K D ( M )
D4 (1.1 ± 0.29) · 10 5 (6.0 ± 0.47) · 10)3 (5.2 ± 0.14) · 10)8 T2¢ (1.7 ± 0.47) · 10 5 (1.4 ± 0.17) · 10)2 (8.8 ± 0.30) · 10)8
(6.5 ± 0.11) · 10)3 (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.
Trang 6observation 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
sequence (tryptophan-rich motif), insert into the membrane
lipid layer when intact h-toxin binds to the membrane [24]
Upon binding to cholesterol-containing membranes,
tryp-tophan fluorescence in all toxin fragments was markedly
enhanced (Fig 4, dotted and dashed line), as is observed in
intact h-toxin [25] Next we examined whether the enhanced
tryptophan fluorescence of the toxin fragments is quenched
by liposomes containing Br-DSPC [24,25], in which
bro-mines were placed on the 9,10-carbon atoms of the acyl
chains It is known that bromine atoms quench intrinsic
tryptophan fluorescence in their vicinity [26,27] When T2,
T2¢ and D4 bound to Br-DSPC/cholesterol liposomes, the
tryptophan fluorescence enhanced by binding to cholesterol
was remarkably quenched by Br-DSPC (Fig 4, long dashed
line), in a similar manner to those of h-toxin and W137F Compared to W137F and MCh, the maximal emission wavelengths of the T2, T2¢ and D4 fragments are distinctly longer (336–338 vs 345–346 nm) in the absence of lipo-somes (Fig 4, solid line) These results show that the environment of the tryptophan residues in the T2, T2¢ and D4 fragments is more exposed to solvent than in the case of the full-size toxins However, after binding to cholesterol-containing liposomes, T2, T2¢ and D4 showed a blue shift in the maximal emission wavelength resembling that (336 nm)
of intact h-toxin, MCh and W137F (Fig 4, dotted and dashed line) These results suggest that the toxin fragments may change the conformation of their cholesterol-binding site to that of the full-size toxin, when they bind to membrane cholesterol
We also analyzed their secondary structures by circular 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 mM 2OHpbCD for 15 min at 37C, which depletes cholesterol by 30%, caused significant reduction
in the number of D4 fragments bound to MOLT-4 cells (Fig 5A, 5 mM 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-bound D4 fragment in sucrose gradient fractionation following solubilization with TX-100 The D4 fragment was recovered predominantly in the FLDF where lipid rafts are known to be located (Fig 5B) Cholesterol and
a tyrosine kinase, Lck [2,28], were recovered in the same fractions (Fig 5B), confirming that these fractions are enriched in lipid rafts In parallel experiments, BCh-bound cells were treated with TX-100 and fractionated
on a sucrose gradient, which showed that the distribution pattern of D4 is the same as that of BCh (data not shown) These results show that the D4 fragment binds selectively to lipid rafts, indicating that the binding characteristics of BCh targeting to lipid rafts can be ascribed to domain 4
Visualization of the probe
To understand the biological function of lipid rafts, it is necessary to examine the distribution of cell membrane
Fig 4 Fluorescence emission spectra of toxin fragments and derivatives.
The intrinsic tryptophan fluorescence of toxin fragments and
deriva-tives was examined (solid lines) as described in Experimental
proce-dures These proteins were incubated with three types of liposomes,
DOPC liposomes (dotted lines), DOPC/cholesterol liposomes (dotted
and dashed lines), and Br-DSPC/cholesterol liposomes (long dashed
lines) After 10 min at room temperature, the fluorescence emission
spectra were measured Maximal emission wavelengths in the presence
of DOPC/cholesterol liposomes and in the absence of liposomes are
displayed on the graphs in nanometers.
Trang 7cholesterol in real time Thus we attempted to visualize
membrane cholesterol using domain 4 directly labeled
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
Experimental procedures 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
D I S C U S S I O N
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 isolated and characterized the minimal toxin fragment of h-toxin that binds to cholesterol with the high specificity of native h-toxin without cytolytic activity The D4 fragment, which corresponds to domain 4, retains a stable structure and has cholesterol-binding activity with high affinity
As to the structural features of the purified D4 fragment, the circular dichroism measurements revealed that the isolated domain 4 exhibits a secondary structure enriched
in b-sheets, in good agreement with that predicted from crystal structure [15] However, the maximal emission wavelength of the intrinsic tryptophan fluorescence is red-shifted in the D4 fragment (Fig 4), which indicates that the environment of the tryptophan residues in the D4 fragment
is more exposed to solvent than in the native toxin As no tryptophan is present at the interface of domain 4 with domains 1–3, the above results imply a structural change accompanied by the removal of domains 1–3 Therefore, domain 4 is an autonomous domain, but some co-operation with domains 1–3 is necessary for folding into the native tertiary structure However, it should be noted that this structural difference does not affect the binding affinity and specificity of D4 to membrane cholesterol In addition, upon binding to cholesterol, the polarity of the local environment around the tryptophan residues in the D4 fragment becomes the same as in the intact toxin (Fig 4), suggesting that binding to membranes restores the conformation of the D4 fragment to that in the membrane-bound form of the full-size toxin This is in striking contrast with some C-terminal
Fig 5 D4 fragment is enriched in FLDF after binding to membrane
cholesterol (A) MOLT-4 cells (106 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) D4
frag-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.
Trang 8truncated 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
causes instability of the products Especially, the deletion of
only two amino acids from the C terminus might cause
product instability, resulting in little production Therefore,
we conclude that the D4 fragment, which corresponds to
domain 4, is the smallest cholesterol-binding unit that is
structurally stable Recently recombinant domain 4 (r-d4)
of streptolysin O was isolated by a combination of
TX-100-extraction, denaturation with urea and ethanol precipitation
[33] Such drastic procedures might affect the conformation
of native structure Furthermore, bovine serum albumin
was required for the renaturation of r-d4, resulting in failure
in analysis of the toxin structure On the other hand, our
procedures for the purification of recombinant domain 4
are under native conditions without the use of any
detergents or denaturants, which makes it possible to
analyze its structure precisely At present, the amino acid
residues involved in cholesterol binding remain unidentified
Recombinant domain 4 should provide a good tool for
analyzing cholesterol-binding sites, for instance, by NMR
on the toxin–cholesterol interaction
The cytolytic activity of h-toxin depends on its
oligo-merization, which leads to pore formation in the cell
membrane We have previously shown that T2 lacks
oligomerization activity due to the deletion of the
N-terminal portion, resulting in no cytolytic activity [16]
T2¢ and D4, shorter fragments than T2, also lack cytolytic
activity In this report we demonstrate that the isolated
domain 4 has the ability to recognize and bind to membrane
cholesterol, the first step in h-toxin action The isolated
domain 4 has a binding affinity for cholesterol similar to
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 avidin On the other hand EGFP-D4 can be used to visualize cell surface cholesterol in both living and fixed cells
in only one step Therefore staining with EGFP-D4 has the advantage that the distribution and movement of choles-terol in living cells can be monitored without artifacts linked
to the cross-linkage of FITC-conjugated avidin Further-more, it is possible to use EGFP-D4 to detect intracellular distribution of cholesterol We are now constructing an expression vector for intracellular EGFP-D4 to analyze the movement of cholesterol-containing microdomains within live cells Thus, the recombinant domain 4 could become a key tool for investigating the distribution and movement of not only the membrane surface, but also intracellular cholesterol in lipid rafts
A C K N O W L E D G M E N T S
We thank Dr Ichiro Yahara for providing facilities of kinetic analysis
by the BIAcore systems We thank Drs Yoshitaka Nagai and Koichi Suzuki for support and encouragement We are grateful to Drs Inomata and Hayashi for technical advice and to Dr M.M Dooley-Ohto for reading the manuscript.
This work was supported by grants from the Japan Science and Technology Corporation, the Japan Science Society (to Y.S.) and from
a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, ONO Medical Research Foundation and Life Science Foundation of Japan (to Y.O.-I.) Y.S is a Domestic Research Fellow of the Japan Science and Technology Corporation.
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