THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 279, No. 33, Issue of August 13, pp. 34209 –34216, 2004
Printed in U.S.A.
Lipid Phase Coexistence Favors Membrane Insertion of
Equinatoxin-II, a Pore-forming Toxin from Actinia equina*
Received for publication, December 17, 2003, and in revised form, May 10, 2004
Published, JBC Papers in Press, June 2, 2004, DOI 10.1074/jbc.M313817200
Ariana Barli臧¶, Ion Gutiérrez-Aguirre‡¶储, José M. M. Caaveiro‡储**, Antonio Cruz‡‡,
Maria-Begoña Ruiz-Argüello‡§§¶¶, Jesús Pérez-Gil‡‡, and Juan M. González-Mañas‡储储
From the ‡Unidad de Biofı́sica (Consejo Superior de Investigaciones Cientı́ficas-Universidad del Paı́s Vasco/Euskal
Herriko Unibertsitatea) and Departamento de Bioquı́mica y Biologı́a Molecular, Universidad del Paı́s Vasco, Apdo. 644,
48080 Bilbao, Spain and ‡‡Departamento de Bioquı́mica y Biologı́a Molecular I, Facultad de Biologı́a,
Universidad Complutense, 28040 Madrid, Spain
Equinatoxin-II is a eukaryotic pore-forming toxin belonging to the family of actinoporins. Its interaction with
model membranes is largely modulated by the presence of
sphingomyelin. We have used large unilamellar vesicles
and lipid monolayers to gain further information about
this interaction. The coexistence of gel and liquid-crystal
lipid phases in sphingomyelin/phosphatidylcholine mixtures and the coexistence of liquid-ordered and liquiddisordered lipid phases in phosphatidylcholine/cholesterol or sphingomyelin/phosphatidylcholine/cholesterol
mixtures favor membrane insertion of equinatoxin-II.
Phosphatidylcholine vesicles are not permeabilized by
equinatoxin-II. However, the localized accumulation of
phospholipase C-generated diacylglycerol creates conditions for toxin activity. By using epifluorescence microscopy of transferred monolayers, it seems that lipid packing defects arising at the interfaces between coexisting
lipid phases may function as preferential binding sites for
the toxin. The possible implications of such a mechanism
in the assembly of a toroidal pore are discussed.
Equinatoxin II (Eqt-II)1 is a member of the actinoporins, a
group of sea anemone cytolysins (1). It is a 179-amino acid
residue protein with a molecular mass of 19.8 kDa and an
isoelectric point of 10.5 (2). Its three-dimensional structure has
* This work was supported by University of the Basque Country
Grant 042.310-13552/2001 and Dirección General de Educación Superior e Investigación Cientı́fica Grant BIO2003-09056. The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a postdoctoral fellowship associated to the “Programa
Movilizador” of the Basque Government.
¶ These authors contributed equally to this work.
储 Recipients of predoctoral fellowships from the Basque Government.
** Present address: Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South St., Waltham, MA 02454.
§§ Present address: Dept. of Medicine, University of Cambridge, Addenbrooke’s Hospital, Hills Rd., CB2 2QQ Cambridge, United Kingdom.
¶¶ Recipient of a predoctoral fellowship from Ministerio de Ciencia y
Tecnologı́a.
储储 To whom correspondence should be addressed. Tel.: 3494-6015379;
Fax: 3494-6013500; E-mail: [email protected].
1
The abbreviations used are: Eqt-II, equinatoxin-II; ANS, 1-anilinonaphtalene-8-sulfonic acid; ANTS, 8-aminonaphthalene-1,3,6-trisulfonic
acid; Chol, cholesterol; DAG, diacylglycerol; DPX, p-xylene-bis-pyridinium
bromide; Ld, liquid-disordered phase; Lo, liquid-ordered phase; LUV, large
unilamellar vesicle(s); NBD, 2-nitrobenzo-2-oxa-1,3-diazole; NBD-PC,
1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadizole-1-yl)amino]dodecanoyl]
sn-glycero-3-phosphocholine; PC, egg phosphatidylcholine; PLC, phospholipase C; SM, bovine brain sphingomyelin; TR, Texas RedTM; mN,
millinewton.
This paper is available on line at http://www.jbc.org
been solved by x-ray crystallography and NMR (3, 4). Eqt-II
forms cation-selective pores with a diameter of ⬃2 nm in cell
and model membranes (5–7). The mechanism of pore formation
is a multistep process consisting of (i) membrane binding of the
water-soluble monomer, (ii) oligomerization on the membrane
surface, and (iii) pore formation (1, 5–11). This mechanism is
common to other actinoporins like sticholysin-II from Stichodactyla helianthus (12, 13). Membrane insertion of Eqt-II and
sticholysins is favored by the presence of sphingomyelin within
the target membrane (6, 8, 14 –16). The recent finding of a
phosphocholine binding site in the three-dimensional structure
of sticholysin-II (13) supports the role of sphingomyelin as a
specific receptor for actinoporins, as other authors have suggested (17, 18). However, the presence of sphingomyelin is not
strictly necessary for the lytic activity of these toxins, which are
also active in phosphatidylcholine/cholesterol mixtures (14,
16). Therefore, other factors are likely to govern their mechanism of action.
Mixtures of sphingomyelin, phosphatidylcholine, and cholesterol are characteristic of the so-called rafts, microdomains in
which the concentration of membrane components (lipids or
proteins) and their physicochemical properties are different
from the surrounding environment. The increasing amount of
information pointing to the existence of lipid domains in cell
and model membranes and their implication in many crucial
biological processes has been extensively reviewed (19 –26).
One important characteristic of rafts is their resistance to
detergent solubilization (27–30). This property is associated
with the fact that lipids in rafts exist in the liquid-ordered (Lo)
phase, where their acyl chains are extended and ordered as in
the gel phase but possess lateral and rotational mobilities
characteristic of the liquid-disordered (Ld) phase (31, 32). In
monolayers, bilayers, and animal cell membranes, Lo and Ld
fluid phases are immiscible (33). Other examples of lipid phase
coexistence are known, the most common one being probably
the coexistence of gel and fluid phases in certain lipid mixtures
or in pure lipid bilayers near the gel-fluid transition temperature (34).
In the present work, we have analyzed a variety of parameters that determine the formation of distinct lipid phases (lipid
composition, temperature, presence of different sterols, and
enzymatic activity of phospholipase C). A strong correlation
was found between the coexistence of lipid phases and the
pore-forming activity of Eqt-II. Epifluorescence microscopy imaging of supported lipid monolayers revealed the preferential
localization of this eukaryotic toxin at the interface between
lipid phases.
34209
34210
Interaction of Equinatoxin-II with Model Membranes
EXPERIMENTAL PROCEDURES
Materials—Egg phosphatidylcholine (PC), bovine brain sphingomyelin (SM), and cholesterol (Chol) were from Avanti Polar Lipids
(Alabaster, AL). Ergosterol, cholestenone (4-cholesten-3-one), and Triton X-100 were from Sigma. 8-Aminonaphthalene-1,3,6-trisulfonic acid
(ANTS), 1-anilinonaphtalene-8-sulfonic acid (ANS), p-xylene-bispyridinium bromide (DPX), 1-palmitoyl-2-[12-[(7-nitro-2–1,3-benzoxadizole-1-yl)amino]dodecanoyl]sn-glycero-3-phosphocholine (NBD-PC),
and the FluoReporter® Texas RedTM-X (TR) protein labeling kit were
obtained from Molecular Probes, Inc. (Eugene, OR). Phospholipase C
from Bacillus cereus (PLC) (EC 3.1.4.3) was supplied by Roche Applied
Science, and o-phenantroline was from Merck.
Eqt-II Purification—Eqt-II was purified from the liquid exuded by
Actinia equina specimens freshly collected in the Bay of Biscay. We
followed the purification protocol described in Ref. 2. The purified
protein was concentrated to ⬃10 mg/ml with an Amicon 8050 concentrator (Danvers, MA) ultrafiltration unit equipped with a regenerated
nitrocellulose filter (Millipore Corp., Bedford, MA) with a molecular
mass cut-off of 10 kDa. Aliquots were stored at ⫺20 °C, and once
thawed they were not refrozen. Protein concentration was estimated
spectrophotometrically using a molar extinction coefficient at 280 nm of
3.61 ⫻ 104 M⫺1 cm⫺1 (35).
Labeling of Equinatoxin II with Texas RedTM—To a 190 ␮M solution
of Eqt-II in distilled water, 130 ␮l of 1 M NaHCO3 were added to raise
the pH to 8.3. The labeling reaction was started by the addition of 200
␮l of TR stock solution (5 mg/ml) (final Eqt-II/TR molar ratio of 5:1). The
mixture was incubated for 60 min at room temperature with constant
stirring and protected from light. To inactivate any remaining free dye,
47 ␮l of hydroxylamine were added to the mixture, and the solution was
stirred for an additional 30 min at room temperature. To purify the
labeled protein, the mixture was loaded on a Sephadex G-15 column
and eluted with 10 mM Hepes, 200 mM NaCl, pH 7.5. 500-␮l fractions
were collected, and absorption spectra from 250 to 650 nm were measured. Protein concentration and the degree of labeling were determined
as indicated by the manufacturer of the protein labeling kit.
Leakage of Liposomal Contents—The appropriate lipids were mixed
in organic solvent, evaporated thoroughly, and resuspended in 10 mM
Hepes 200 mM NaCl, pH 7.5, containing 25 mM ANTS and 90 mM DPX.
Large unilamellar vesicles (LUV) were prepared by the extrusion
method (36), using polycarbonate filters with a pore size of 0.1 ␮m
(Nucleopore, Pleasanton, CA). Nonencapsulated fluorescent probes
were separated from the vesicle suspension through a Sephadex G-75
gel filtration column (Amersham Biosciences). Solution osmolarities
were checked with an Osmomat 030 instrument (Gonotec, Berlin, Germany). Phospholipid concentration was measured according to Bartlett
(37).
The leakage of encapsulated solutes was assayed as described by
Ellens et al. (38). The probe-loaded liposomes (final lipid concentration ⫽ 0.1 mM) were treated with the appropriate amounts of Eqt-II in
a fluorometer cuvette at 25 °C with constant stirring. Changes in fluorescence intensity were recorded in a PerkinElmer Life Sciences LS-50
spectrofluorometer (Beaconsfield, UK) with excitation and emission
wavelengths set at 350 and 510 nm, respectively. An interference filter
with a nominal cut-off value of 470 nm was placed in the emission light
path to minimize the contribution of the light scattered by the vesicles
to the fluorescence signal. The percentage of leakage was calculated
after the complete release of the fluorescent probe by the addition of the
nonionic detergent Triton X-100 (final concentration ⫽ 0.1% w/v). When
PLC was used, the assay was carried out under optimal conditions for
its activity; buffer was 10 mM Hepes, 200 mM NaCl, 10 mM CaCl2, pH
7.5, and the experiment was carried out at 37.6 °C with constant stirring. Concentrations were 0.1 mM, 0.3 ␮M, and 1.5 units/ml for lipid,
Eqt-II, and PLC, respectively. To stop the enzyme reaction, o-phenantroline was added at a final concentration of 6 mM.
Surface Pressure Measurements—Surface pressure measurements
were carried out with a MicroTrough-S system from Kibron (Helsinki,
Finland) at 25 °C with constant stirring. The aqueous phase consisted
of 1.1 ml of 10 mM Hepes, 200 mM NaCl, pH 7.5. The lipid, dissolved in
chloroform/methanol (2:1), was gently spread over the surface. The
desired initial surface pressure was attained by changing the amount of
lipid applied to the air-water interface. After 10 min to allow for solvent
evaporation, the protein was injected through a hole connected to the
subphase. The final protein concentration in the Langmuir trough was
1 ␮M. The increment in surface pressure versus time was recorded until
a stable signal was obtained.
Supported Phospholipid Monolayers—Monolayers were formed by
spreading chloroform/methanol (3:1, v/v) solutions (1 mM) of the phos-
pholipid mixture on top of a buffered subphase (10 mM Hepes, 200 mM
NaCl, pH 7.5) in a thermostated Langmuir-Blodgett ribbon trough
(NIMA Technologies, Coventry, UK). To allow the observation by epifluorescence microscopy, 1% (mol/mol) of NBD-PC was included. After
10 min to allow for solvent evaporation, monolayers were compressed at
25 cm2/min to an initial surface pressure of 20 mN/m. After 10 min for
equilibration, TR-labeled Eqt-II from a 45 ␮M buffered stock solution
was injected into the subphase. Insertion was followed by monitoring
the increase in surface pressure. The surface pressure stabilized at 25
mN/m, and at this point, the monolayer was transferred onto glass
coverslips at a velocity of 5 mm/min. The ribbon trough was provided
with a feedback mechanism that kept the surface pressure constant by
compressing the monolayer, thereby compensating the loss of material
that took place during the transfer.
Epifluorescence microscopy observation of the planar supported
monolayers was carried out with a Zeiss Axioplan II fluorescence microscope (Carl Zeiss, Jena, Germany). Images from NBD-labeled phospholipid and TR-labeled protein were recorded separately from the
same sample by switching fluorescence filters to select the proper
emission wavelength range. The experiment was carried out at 25 °C.
RESULTS
Interaction of Eqt-II with SM-PC Mixtures—In most cases,
the interaction of Eqt-II and other actinoporins with model
membranes requires the presence of SM in the target membrane (6, 15). To gain more information on the interaction of
Eqt-II with model membranes, we prepared LUV composed of
SM and PC in different proportions. Protein-vesicle interaction
was monitored through the release of fluorescent dyes that had
been entrapped in the vesicles. In Fig. 1A, we observe a strong
dependence of the release of encapsulated ANTS/DPX on the
SM content of the vesicles. For SM molar fractions between 0.3
and 0.7, the percentages of leakage ranged from 44 to 54%, and
maximum leakage was obtained when the mixture was approximately equimolar. When one of the two phospholipids predominated, the release was reduced to ⬃25%, and for LUV made of
100% PC or 100% SM, the release was close to 8%.
Next, we prepared lipid monolayers with PC/SM mixtures to
determine whether this behavior could be observed in other
model membranes. The initial surface pressure (␲0) was set at 20
mN/m, and we measured the increase in surface pressure (⌬␲)
after injection of 1 ␮M Eqt-II into the aqueous subphase (Fig. 1A).
The insertion followed the same pattern observed in LUV: 1)
maximum values for ⌬␲ (between 12 and 14 mN/m) were observed when the molar fractions of PC and SM were similar; 2)
when one of the lipids predominated, ⌬␲ was reduced to 8 –9
mN/m, and 3) for 100% PC and 100% SM monolayers, ⌬␲ was
practically the same (5.3 and 5.5 mN/m, respectively).
We also measured the “critical pressure” (␲c) for different
PC/SM mixtures (i.e. the initial surface pressure (␲0) above
which no ⌬␲ is observed after injection of Eqt-II into the subphase). In all of the lipid compositions tested, the higher the ␲0,
the smaller the ⌬␲, because tighter lipid packing prevented
protein insertion (Fig. 1B). The critical pressures were calculated by linear fitting of the experimental ⌬␲ versus ␲0 (initial
surface pressure) values and extrapolation to ⌬␲ ⫽ 0. Fig. 1C
shows the ␲c values obtained for different SM/PC mixtures.
Again, the highest ␲c values (around 36 mN/m) were observed
in mixtures approximately equimolar and decreased when one
of the lipids was predominant. This means that at intermediate
SM molar fractions there are more binding sites available for
Eqt-II, and more protein molecules are able to penetrate the
monolayer. The mixtures producing maximum penetration
were also associated with the maximum values of proteininduced leakage from LUV (Fig. 1A). The gel to liquid-crystal
phase transition temperatures (Tm) for PC and SM are ⫺5 °C
(39) and 38 °C (40), respectively. A detailed phase diagram of
egg PC and bovine brain SM has been published (39). According
to those data, at our experimental temperature (25 °C), pure
egg PC exists in the fluid lamellar phase. The addition of SM
Interaction of Equinatoxin-II with Model Membranes
34211
FIG. 2. Effect of temperature on the lytic activity of Eqt-II.
Percentage of ANTS/DPX released from LUV composed of SM/PC (1:1)
as a function of temperature. The lipid concentration was 0.05 mM and
the lipid/protein ratio was 200. Buffer was 10 mM Hepes, 200 mM NaCl,
pH 7.5, and the experiments were carried out with constant stirring.
FIG. 1. Interaction of Eqt-II with SM/PC mixtures. A, effect of
SM molar fraction on the Eqt-II-induced increase of surface pressure
(⌬␲) in SM/PC monolayers (●) and release of encapsulated solutes from
SM/PC LUV (Œ). Initial surface pressure (␲0) in the monolayer was 20
mN/m, and the concentration of Eqt-II in the subphase was 1 ␮M. The
percentage of leakage was measured 15 min after the addition of Eqt-II
to the vesicles. Final lipid concentration was 0.1 mM, and the lipid/
protein (L/P) molar ratio was 200. The continuous and dotted lines
correspond to the fit of the experimental data to a second grade equation. Experiments were carried out at room temperature with constant
stirring. B, ⌬␲ versus ␲0 data obtained after the addition of Eqt-II
underneath phospholipid monolayers. ⌬␲ is the difference between the
final surface pressure value and ␲0. Experimental data from two independent experiments were fitted to straight lines. Intercepts with the
abscissa represent the critical pressure (␲c). Lipid compositions were as
follows: PC (●), PC/SM (80:20) (f), PC/SM (60:40) (Œ), PC/SM (40:60)
(), PC/SM (20:80) (⽧), and SM (filled hexagons). C, variation of ␲c
values obtained from B with the SM content of the monolayer. The
subphase was 10 mM Hepes, 200 mM NaCl, pH 7.5. Eqt-II concentration
in the subphase was 1 ␮M. Experiments were carried out at 25 °C with
constant stirring.
gives rise to a (PC ⫹ SM) fluid lamellar phase plus a pure SM
gel phase. Phase separation is clear above 33 mol % SM, and
above 90 mol % SM only the gel phase occurs. Our data (Fig. 1)
show maximum protein insertion and maximum bilayer permeabilization at SM mol % between 30 and 70 (i.e. in the phase
diagram region where phase separation predominates). In addition, binary phase diagrams at 23 °C of palmitoyloleoyl phosphatidylcholine and palmitoyl sphingomyelin mixtures also
showed gel/fluid coexistence at palmitoyl sphingomyelin proportions between 30 and 70 mol % (41). Thus, it seems that the
coexistence of gel and fluid lipid phases favors the degree of
protein insertion and the extent of vesicle permeabilization.
The Effect of Temperature—We studied the effect of temperature on the EqT-II-induced release of fluorescent solutes en-
capsulated in PC/SM (1:1) LUV (Fig. 2). The highest percentages of leakage were observed between 11 and 25 °C. At
temperatures higher than 25 °C, the toxin activity started to
decrease, a trend that was even more pronounced above 30 °C.
Above 40 °C, the leakage was reduced to 13%. One possible
explanation for this behavior would be a potential thermal
destabilization of the protein. In a control experiment, we
measured the fluorescence of 7.9 ␮M ANS in the presence of 2.6
␮M Eqt-II as a function of temperature (data not shown). Between 16 and 54 °C, the ANS fluorescence remained low and
constant, an indication that in this temperature interval the
folding of the protein was compact (42). Therefore, the decrease
in EqT-II activity shown in Fig. 2 was not due to protein
denaturation. However, a partial phase diagram for mixtures
of SM and PC of natural origin (43) reveals that, above 32 °C,
only the fluid phase exists. The egg PC/bovine brain SM phase
diagram (39) also suggests that at 37 °C complete miscibility of
these two lipids occurs. In addition, the phase diagram for the
equimolar mixture of palmitoyl sphingomyelin/palmitoyloleoyl
phosphatidylcholine shows that above 35 °C only the liquiddisordered phase exists (41). Thus, in our opinion, the temperature-dependent decrease in protein activity results from
changes in the membrane structure associated with an increased lipid miscibility and the disappearance of coexisting
lipid phases.
Interaction of Eqt-II with PC/Cholesterol Mixtures—Actinoporins can also permeabilize PC-cholesterol membranes
(14, 16). In Fig. 3A, we represent the kinetics of Eqt-IIinduced leakage of ANTS/DPX encapsulated in LUV made of
PC-cholesterol (70:30). At mammalian physiological temperatures, there was almost no leakage. However, at lower temperatures, the release increased, and at 4 °C it was 22%.
Fixing the temperature at 4 °C, the extent of leakage increased with the cholesterol content of the model membrane
(Fig. 3B). Thus, the effect of cholesterol is temperature- and
concentration-dependent.
In lipid monolayers formed at an initial pressure of 20 mN/m,
the increase in surface pressure after injecting 1 ␮M Eqt-II into
the subphase depends linearly on the amount of cholesterol
(Fig. 4A). We also observed a small increment in the critical
pressures (Fig. 4B).
The effects of cholesterol are not as conspicuous as those of
SM but might be related with a similar feature (i.e. the formation of different lipid phases within the model membrane). At
low temperatures, cholesterol is likely to interact with the
phospholipid to form an Lo lipid phase in coexistence with the
bulk fluid phase (44 – 46), thereby favoring the lytic action of
Eqt-II.
Interaction of Eqt-II with SM-PC/Cholesterol Mixtures—The Lo
phase has intermediate physical properties between the gel and
the liquid-crystalline phases, and it has been related to the sphin-
34212
Interaction of Equinatoxin-II with Model Membranes
FIG. 3. Permeabilization of LUV composed of PC/Chol. A, effect
of temperature on the Eqt-II-induced permeabilization of LUV composed of PC/Chol (70:30). Lipid concentration was 0.1 mM, and the
lipid/protein ratio was 150. B, effect of cholesterol on the Eqt-II-induced
permeabilization of LUV composed of PC/Chol (mixtures). Lipid concentration was 0.1 mM, lipid/protein ratio was 75, and the experiment
was performed at 4 °C with constant stirring. The excitation wavelength was set at 350 nm, and fluorescence emission was measured at
510 nm. After 25 min, Triton X-100 was added at 0.1% (w/v) final
concentration to obtain 100% permeabilization. Buffer was 10 mM
Hepes, 200 mM NaCl, pH 7.5.
FIG. 4. Insertion of Eqt-II into monolayers made of PC/Chol
mixtures. A, increment of surface pressure (⌬␲) after insertion of
Eqt-II into lipid monolayers. Initial surface pressure (␲0) was 20 mN/m.
Average values and S.D. of two independent measurements are shown.
B, ⌬␲ versus ␲0 values for different PC/Chol mixtures. Lipid compositions were as follows: PC (●), PC/Chol (90:10) (f), PC/Chol (85:15) (Œ),
PC/Chol (80:20) (), PC/Chol (70:30) (⽧), and PC/Chol (60:40) (filled
hexagon). In all of the experiments, the final toxin concentration was 1
␮M. Subphase consisted of 10 mM Hepes, 200 mM NaCl, pH 7.5. Assays
were performed at 25 °C with constant stirring.
FIG. 5. Permeabilization of LUV composed of SM/PC (50:50),
SM/PC/Chol (50:35:15), and SM/PC/Chol (50:15:35). A, kinetics of
Eqt-II-induced release of ANTS/DPX encapsulated in LUV. Lipid concentration was 0.1 mM, and the lipid/protein ratio was 1200. Triton
X-100 was added at 0.1% (w/v) final concentration to obtain 100%
permeabilization. Assays were performed at 25 °C with constant stirring. B, effect of Eqt-II concentration on the permeabilization of LUV
composed of SM/PC (50:50) (●), SM/PC/Chol (50:35:15) (f), and SM/PC/
Chol (50:15:35) (Œ). Maximal release was obtained from two independent measurements, and the average values and S.D. are shown.
golipid- and cholesterol-enriched lipidic domains known as rafts
(19, 23, 25, 45). Molecular interactions between SM and cholesterol induce Lo-Ld phase coexistence in SM/PC/cholesterol mixtures (47). We have tested the effect of Eqt-II on model membranes having two different lipid compositions, namely SM-PCcholesterol (50:35:15) and SM-PC-cholesterol (50:15:35). The
results obtained were compared with those observed on the
SM/PC (50:50) mixture.
Fig. 5A shows the kinetics of Eqt-II-induced release of ANTS/
DPX encapsulated in LUV. In the lipid mixtures containing
cholesterol, the leakage was larger than in the SM/PC (50:50)
control mixture. Moreover, release in the cholesterol-containing vesicles was less dependent on protein concentration (Fig.
5B). Therefore, the presence of cholesterol within the SMcontaining model membrane renders it much more susceptible
to the lytic activity of Eqt-II.
Eqt-II inserted to a similar extent in SM-PC-cholesterol (50:
35:15) and SM-PC (50:50) monolayers, but the rate of insertion
was faster in the cholesterol-containing mixture. In monolayers
containing SM-PC-cholesterol (50:15:35), both the degree of
protein penetration and the rate of the process increased as
compared with the cholesterol-free monolayer (Fig. 6A). The
critical pressure values also changed in the presence of cholesterol (Fig. 6B). Whereas the ␲c values for SM-PC-cholesterol
(50:35:15) and SM-PC (50:50) were nearly the same (36.8 and
37.2 mN/m, respectively), the ␲c for SM-PC-cholesterol (50:15:
35) was 46.8 mN/m. At any given initial surface pressure value,
more protein was able to insert into the SM-PC-cholesterol
(50:15:35) monolayer than in the cholesterol-free films. This
fact was particularly evident when the initial pressure approached 30 mN/m, a value that is thought to be close to the
lateral packing of phospholipids in membranes (48).
The Effect of Other Sterols—Lipid domain formation is dependent on sterol structure. Some sterols or their derivatives
Interaction of Equinatoxin-II with Model Membranes
FIG. 6. Insertion of Eqt-II into monolayers composed of SM/PC
(50:50), SM/PC/Chol (50:35:15), and SM/PC/Chol (50:15:35). A, insertion kinetics of Eqt-II into monolayers with the indicated compositions. In all cases, ␲0 was 20 mN/m. B, determination of the critical
pressures (␲c) for the insertion of Eqt-II into monolayers composed of
SM/PC (50:50) (f), SM/PC/Chol (50:35:15) (●), and SM/PC/Chol (50:15:
35) (Œ). Critical pressures were obtained from linear regression of data
from two independent experiments. Measurements were performed at
25 °C with constant stirring. Eqt-II concentration was 1 ␮M. Buffer was
10 mM Hepes, 200 mM NaCl, pH 7.5.
promote domain formation, whereas others do not (30, 49 –51).
To investigate whether this variable affects the insertion of
Eqt-II into model membranes, we prepared different SM/PC/
sterol (50:15:35) mixtures. We used cholesterol as a domainpromoting sterol, ergosterol as an even stronger promoter, and
cholestenone as an inhibitor of domain formation.
The Eqt-II-induced release of ANTS-DPX encapsulated in
LUV composed of SM/PC/sterol (50:15:35) is shown in Fig. 7A.
Final release values were 75, 59, and 45% for the liposomes
containing ergosterol, cholesterol, and cholestenone, respectively. Therefore, the permeabilizing activity of Eqt-II seems to
correlate with the ability of the different sterols to create lipid
domains within the model membrane. This dependence was
observed over a wide range of lipid/protein ratios for the three
lipid compositions (data not shown).
The insertion of Eqt-II into lipid monolayers was also dependent on the ability of the lipid mixture to form domains (Fig. 7B).
Critical pressure values were 52.3, 48.1, and 45.9 mN/m for the
SM/PC/ergosterol (50:15:35), SM/PC/cholesterol (50:15:35), and
SM/PC/cholestenone (50:15:35) monolayers, respectively. Thus,
the ergosterol-containing mixture is the one that accommodates
more protein molecules and exhibits the highest percentages of
Eqt-II-induced permeabilization of LUV.
Combined Action of Phospholipase C and Equinatoxin-II—The
lack of a measurable permeabilizing activity does not necessarily
mean that the protein is not interacting with the membrane. For
instance, although PC LUV are refractory to Eqt-II-induced permeabilization, the toxin partitions into PC LUV (14). Toxin binding to the membrane is probably just one step in the permeabilizing process. Moreover, the interaction of Eqt-II with PC
monolayers at initial surface pressures below 25 mN/m gives rise
to an increase in the surface pressure (Fig. 4B).
In order to explore conditions that could render PC vesicles
34213
FIG. 7. Effect of sterol structure on the interaction of Eqt-II
with model membranes made of SM/PC/sterol (50:15:35) mixtures. A, Eqt-II was added to ANTS-DPX-loaded LUV composed of
SM/PC/sterol (50:15:35). Sterols were cholesterol, ergosterol, or cholestenone, as indicated. Lipid concentration was 0.15 mM, and the lipid/
protein ratio was 900. Excitation and emission wavelengths were set at
350 and 510 nm, respectively. Triton X-100 was added at 0.1% (w/v)
final concentration to obtain 100% permeabilization. The experiments
were carried out in 10 mM Hepes, 200 mM NaCl, pH 7.5, at 25 °C with
constant stirring. B, determination of ␲c for the insertion of Eqt-II into
SM/PC/sterol (50:15:35) monolayers. Critical pressure values (␲c) were
obtained from the linear regression of experimental data from two
independent experiments. Sterols used were cholesterol (●), ergosterol
(f), or cholestenone (Œ). Eqt-II concentration was 1 ␮M. The buffer in
the subphase was 10 mM Hepes, 200 mM NaCl, pH 7.5. Assays were
performed at 25 °C with constant stirring.
susceptible to Eqt-II-induced permeabilization, we studied its
activity in the presence of PLC from Bacillus cereus. PLC is a
phosphodiesterase that hydrolyzes PC and generates diacylglycerol (DAG) and phosphorylcholine. When PLC or Eqt-II
was added to PC LUV, there was no protein-induced leakage of
ANTS/DPX (Fig. 8A, lower traces). However, when the vesicles
were preincubated during 5 min with Eqt-II, the subsequent
addition of PLC gave rise to the release of the fluorophore after
a lag period of 2 min (Fig. 8A, upper trace). This leakage was
dependent on PLC activity, since the addition of o-phenantroline, a specific inhibitor of PLC (52), abolished this effect (Fig.
8B). In a control experiment, we observed that LUV of PC-DAG
(9:1) were not permeabilized by Eqt-II (data not shown), thus
indicating that the homogeneously distributed DAG does not
render the vesicles susceptible to the lytic activity of the toxin.
It has been proposed that it is the localized generation of
DAG-rich domains within the outer leaflet of the membrane
what promotes vesicle aggregation and fusion without leakage
of encapsulated solutes (53–55). The time interval comprised
between the addition of o-phenantroline and the arrest of leakage is probably due to the slow access of the inhibitor (56).2 The
time length for o-phenantroline to produce maximum inhibition (i.e. tens of seconds) is several orders of magnitude longer
than the time required for DAG to diffuse out of the asymmet2
J. L. Nieva, unpublished results.
34214
Interaction of Equinatoxin-II with Model Membranes
which the fluorescent probe is excluded, and the bright area
corresponds to a disordered phase where NBD-PC accumulates.
Fig. 9, D–F, also shows the topological distribution of the fluorescently labeled Eqt-II. Although a fraction of the protein fluorescence can be observed in the form of dispersed bright spots
located in both lipid phases, equinatoxin-II binds preferentially
at their interface.
DISCUSSION
FIG. 8. Combined action of PLC and Eqt-II. A, permeabilization
of LUV composed of PC after the addition of PLC, Eqt-II, or both. When
both proteins were present, the first arrow indicates the addition of PLC
to PC LUV preincubated with Eqt-II for 5 min. The second arrow
indicates the addition of Triton X-100 to a final concentration of 0.1%
(w/v) to obtain 100% release. B, effect of o-phenantroline (specific inhibitor of PLC) on the permeabilization resulting from the combined
action of Eqt-II and PLC. The first arrow indicates the addition of PLC
to PC LUV preincubated with Eqt-II for 5 min. The second arrow
indicates the addition of o-phenantroline (final concentration ⫽ 6 mM),
and the third arrow corresponds to the addition of Triton X-100. Excitation wavelength was set at 350 nm, and fluorescence emission was
measured at 510 nm. Concentrations were 0.1 mM, 0.3 ␮M, and 1.5
units/ml for lipid, Eqt-II, and PLC, respectively. Buffer was 10 mM
Hepes, 200 mM NaCl, 10 mM CaCl2, pH 7.5. Experiments were carried
out at 37.6 °C with constant stirring.
ric domains. Transbilayer (flip-flop) movements of DAG have a
t value on the order of tens of milliseconds (57, 58). Lateral
diffusion is even faster, with “hopping” or “jumping” events
occurring with a frequency of about 10⫺7 s (59). Therefore, the
inhibition of PLC activity results in an arrest of Eqt-II insertion, because DAG diffusion overcomes DAG generation and
interdomain interfaces are blurred out.
Fluorescence Microscopy of Transferred Monolayers—Finally,
we tried to visualize the localization of Eqt-II after insertion into
monolayers known to contain coexisting domains. For this purpose, we labeled the toxin with the fluorescent marker TR. Labeling of the toxin did not affect its hemolytic activity or its ability
to insert into monolayers (data not shown). We built a monolayer
composed of SM/PC/cholesterol (50:15:35), which also included
1% NBD-PC, a fluorescence-labeled lipid that is excluded from
ordered phases and accumulates into disordered regions of the
film. The initial surface pressure was 20 mN/m, and the toxin
was injected directly into the subphase (10 mM Hepes, 200 mM
NaCl, pH 7.5). Protein insertion originated an increase in the
surface pressure, which stabilized at 25 mN/m. At this point, the
monolayer was transferred onto a glass support while keeping
constant the surface pressure. The transferred monolayer was
placed under the fluorescence microscope, and the selection of the
fluorescence filter permitted the visualization of either the
NBD-PC or the TR-labeled toxin in the same preparation. Fig. 9,
A–C, shows the distribution of NBD-PC in the film. Two phases
coexist; the dark area corresponds to an ordered phase from
At 25 °C, when the gel and liquid-crystal phases coexist in
the SM/PC mixtures (39, 41), the lytic activity of Eqt-II shows
a marked dependence on the SM/PC ratio in the model membrane; it is maximum when the mixture is approximately
equimolar and decreases when one of the two components
predominates (Fig. 1, A and B). On the other hand, in LUV
made of SM/PC (1:1), the Eqt-II-induced release of ANTS/DPX
shows a strong dependence on temperature. The extent of
permeabilization is markedly reduced above 25 °C (Fig. 2). This
is not the result of protein denaturation, because its hydrophobic core remains inaccessible to ANS in the temperature interval where changes in activity are recorded.3 Above 40 °C, when
the two lipids are totally miscible (39, 43), the percentage of
leakage is practically the same as that obtained with either
100% PC or 100% SM. Although specific interactions between
actinoporins and SM (6, 15) cannot be excluded, it is the coexistence of lipid phases that seems to modulate the interaction of
the toxin with the membrane.
The addition of cholesterol to PC model membranes enhances the lytic activity of Eqt-II (Figs. 3 and 4). The same
effect has been described for sticholysin-II, a related actinoporin from S. helianthus (16). The phase diagrams of mixtures of
different phosphatidylcholines with cholesterol obtained by a
variety of experimental approaches indicate the existence of
two immiscible fluid phases (33, 41, 44, 46, 60 – 62). At 37 °C,
Eqt-II is unable to permeabilize LUV made of PC/cholesterol
(70:30), probably because the lipids are organized in one uniform Ld phase. At 4 °C, which is close to the transition temperature of PC (39), the mixtures containing 15–30% cholesterol
fall into the phase diagram region where two immiscible lipid
phases coexist (44), and the toxin resumes its lytic activity.
The incorporation of cholesterol to PC/SM mixtures gives
rise to lipid compositions typically associated with membrane
rafts (for a review, see Ref. 63). A ternary phase diagram has
recently been published for palmitoyloleoyl phosphatidylcholine/palmitoyl sphingomyelin/cholesterol mixtures at different
temperatures (41). In our experimental conditions, the SM/PC/
cholesterol (50:15:35) mixture clearly lies in the liquid-ordered/
liquid-disordered coexistence region mixture and is particularly sensitive to the action of the toxin as evidenced by the
large increment observed in the critical pressure permitting
insertion of the protein, which increased from 36.8 mN/m (in
the PC/SM equimolar mixture) to 46.8 mN/m. The specific
interactions of cholesterol with saturated phospholipids might
give rise to condensed complexes (47, 64, 65) in which lipids
undergo an area contraction that permits the accommodation
of more protein molecules into the monolayer. A close relationship between condensed complexes, liquid-ordered phases, and
rafts has been established (66). The use of sterols distinct from
cholesterol also highlights the correlation between phase coexistence and lytic activity of Eqt-II (Fig. 7). Whereas the presence of ergosterol increases the extent of permeabilization in
LUV and the critical pressure in monolayers, cholestenone
induces opposite effects. Ergosterol is a fungal sterol that promotes tight packing of saturated phospholipids and domain
formation (49, 50), whereas cholestenone does not interact with
3
I. Gutiérrez-Aguirre, unpublished observation.
Interaction of Equinatoxin-II with Model Membranes
34215
FIG. 9. Epifluorescence microscopy images of Eqt-II inserted into a SM/PC/Chol monolayer. The initial surface pressure of the
SM/PC/Chol/NBD-PC (50:14:35:1) monolayer was initially set at 20 mN/m. After the injection of the TR-labeled protein into the subphase, the
surface pressure stabilized at 25 mN/m. At this point, the monolayer was transferred onto a glass support at 5 mm/min while keeping the surface
pressure constant. The experiment was carried out at 25 °C. The subphase was 10 mM Hepes, 200 mM NaCl, pH 7.5. A–C, different frames of the
monolayer viewed through an NBD filter (emission at 520 nm). D–F, the same frames viewed through a TR filter (emission at 590 nm). Scale bars,
50 ␮m.
sphingomyelin, does not induce domain formation (49, 51), and
restores detergent solubilization (30).
The combined action of PLC and Eqt-II further illustrates
the relevance of lipid phase coexistence for Eqt-II activity (Fig.
8). PC LUV are not permeabilized by Eqt-II or PLC alone.
However, the PLC-induced local accumulation of DAG creates
conditions for the permeabilization of the otherwise insensitive
PC LUV. Hønger et al. (67) have found a direct correlation
between the total area occupied by the interfaces between gel
and fluid lipid domains and the activity of phospholipase A2.
Moreover, Nielsen et al. (68), using atomic force microscopy,
have shown that the activity of phospholipase A2 on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine monolayers originates
3–5-Å deep depressions in the membrane that are interpreted
as areas where the product of its catalytic activity (i.e. lyso-PC)
concentrates. The edges between the intact bilayer and the
product-enriched domains must facilitate the accessibility of
phospholipase A2 (and presumably also of PLC) to the region of
the lipid molecule that undergoes its catalytic activity and,
therefore, may play a role in triggering the activity burst (54).
The strong correlation between Eqt-II activity and the coex-
istence of lipid phases could be the result of the accumulation of
the protein at the interface between immiscible lipid phases. In
monolayers, this specific localization is favored when the protein does not show a defined preference for any of the different
phases (69). The free energy per length associated to the boundaries between liquid phases is referred to as line tension, and it
has been proposed that if such an interface exists in cell membranes, it is likely to be decorated with specific proteins and/or
lipids (33). Our epifluorescence experiment with TR-labeled
Eqt-II in monolayers confirms that the protein shows a certain
preference to bind at the boundaries between ordered and
disordered regions (Fig. 9). If coexistence of immiscible phases
is comparable in monolayer and bilayer systems, as has been
recently suggested (62), it seems that the accumulation of the
protein at the boundaries between gel-fluid or liquid-ordered/
liquid-disordered lipid phases could precede membrane permeabilization. Lipid packing defects and differences in membrane
thickness occurring at these interfaces (70 –73) might facilitate
the interaction with the protein. Most likely, this interaction is
governed by a structural motif that binds phosphocholine and
is conserved among actinoporins (13).
34216
Interaction of Equinatoxin-II with Model Membranes
A number of receptors for pore-forming toxins are components of lipid rafts and therefore permit the accumulation of
toxins in two dimensions and provide a mechanism that facilitates the oligomerization of the toxin prior to pore formation
(for a review, see Ref. 74). Association with the interfaces
between domains is an even more efficient concentration strategy because it confines the toxin to a linear space where oligomerization and pore formation can take place at very low
protein bulk concentrations. Moreover, lipid molecules at interfaces might be intrinsically more disordered, perhaps offering less resistance to protein insertion. In the case of equinatoxin-II, the insertion is limited to its N-terminal ␣ helix (9, 10).
This process can be compared with the insertion of a wedge into
a fracture line and would expose momentarily the hydrophobic
cores of immiscible lipid phases. However, protein insertion
gives rise to a concomitant increase in the surface pressure of
the outer monolayer of the membrane (9), which may push
adjacent lipid molecules to fill the open gap. Therefore, the
insertion of Eqt-II could be associated to the redistribution of
lipid molecules around it and might result in the formation of
a lipidic pore whose walls might be delimited by the hydrophilic
face of its amphipathic N-terminal ␣-helix and the polar head
groups of the phospholipids (10). Such a toroidal pore structure
has been postulated for actinoporins (10 –13), antimicrobial
peptides (75, 76), and apoptotic proteins (77, 78).
Acknowledgments—We thank Prof. F. M. Goñi and Dr. G. Basañez
for critical reading of the manuscript.
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