Báo cáo khoa học: Interaction of ostreolysin, a cytolytic protein from the edible mushroom Pleurotus ostreatus, with lipid membranes and modulation by lysophospholipids pptx

Interaction of ostreolysin, a cytolytic protein from the edible
mushroom
Pleurotus ostreatus
, with lipid membranes
and modulation by lysophospholipids
Kristina Sepc
ˇ
ic
´
1,2
, Sabina Berne
2
, Cristina Potrich
1
, Tom Turk
2
, Peter Mac
ˇ
ek
2
and Gianfranco Menestrina
1
1
CNR-ITC, Istituto di Biofisica – Sezione di Trento, Povo, Italy;
2
Department of Biology, Biotechnical Faculty,
University of Ljubljana, Slovenia
Ostreolysin is a 16-kDa cytolytic protein specifically
expressed in primordia and fruiting bodies of the edible
mushroom Pleurotus ostreatus. To understand its interaction
with lipid membranes, we compared its effects on mamma-

lian cells, on vesicles prepared with either pure lipids or total
lipid extracts, and on dispersions of lysophospholipids or
fatty acids. At nanomolar concentrations, the protein lysed
human, bovine and sheep erythrocytes by a colloid-osmotic
mechanism, compatible with the formation of pores of 4 nm
diameter, and was cytotoxic to mammalian tumor cells. A
search for lipid inhibitors of hemolysis revealed a strong
effect of lysophospholipids and fatty acids, occurring below
their critical micellar concentration. This effect was distinct
from the capacity of ostreolysin to bind to and permeabilize
lipid membranes. In fact, permeabilization of vesicles
occurred only when they were prepared with lipids extracted
from erythrocytes, and not with lipids extracted from
P. ostreatus or pure lipid mixtures, even if lysophospholipids
or fatty acids were included. Interaction with lipid vesicles,
and their permeabilization, correlated with an increase in the
intrinsic fluorescence and a-helical content of the protein,
and with aggregation, which were not detected with
lysophospholipids. It appears that either an unknown lipid
acceptor or a specific lipid complex is required for binding,
aggregation and pore formation. The inhibitory effect of
lysophospholipids may reflect a regulatory role for these
components on the physiological action of ostreolysin and
related proteins during fruiting.
Keywords: fungal fruiting; hemolytic protein; lysophos-
pholipid; oyster mushroom; Pleurotus ostreatus.
The oyster mushroom (or white-rot fungus) belongs to the
genus Pleurotus which comprises a group of edible, ligni-
nolytic fungi with medicinal, biotechnological, and envi-
ronmental applications [1,2]. Despite its widespread and

massive cultivation, a major lack of information remains on
the cellular processes that lead to the initiation of fruiting
body development, as is also true for other edible mush-
rooms. Several mushrooms have been examined for genes
specifically expressed during formation of primordia and
fruiting bodies [1]. Recently, expressed sequence tags (ESTs)
of P. ostreatus were compared within liquid-cultured
mycelium and fruiting body to investigate changes in the
genes expressed during fruiting [3]. Among the 1069 ESTs
identified in fruiting bodies, one set of unigene sequences,
with a redundancy number of 29, was found to be differen-
tially expressed. These sequences were highly homologous
to the Aa-Pri1 gene expressed during primordia and fruiting
body initiation by the mushroom Agrocybe aegerita [3,4].
Moreover, 13 of the ESTs, if translated, are identical with a
138-amino-acid protein (PriA) translated from P. ostreatus
cDNA (EMBL/GenBank/DDBJ databases: Q8X1M9).
The existence of the translation products was confirmed
by isolation of the corresponding proteins, ostreolysin
(TrEMBL db: P83467) and aegerolysin, specifically
expressed in primordia and fruiting bodies of P. ostreatus
and A. aegerita, respectively [5]. These homologous, ther-
molabile proteins have a molecular mass of % 16 kDa, a low
isoelectric point, and hemolytic activity at nanomolar
concentrations. Searches in the nucleotide and protein
databases revealed that the sequence of the ostreolysin
N-terminal 50 amino acids was 88% identical with the
putative PriA protein of P. ostreatus and its translated
Correspondence to G. Menestrina, CNR-ITC, Istituto di Biofisica –
Sezione di Trento, Via Sommarive 18, 38050 Povo (TN), Italy.

Fax: + 39 0461 810 628, Tel.: + 39 0461 314 256,
E-mail:
Abbreviations: BHT, butylated hydroxytoluene; EST, expressed
sequence tag; FTIR spectroscopy, Fourier-transform infrared
spectroscopy; HC
0.5
, amount of hemolysin causing 50% lysis in 2 min;
LDL, low-density lipoprotein; LUV, large unilamellar vesicle;
lyso-PtdCho, l-a-lysophosphatidylcholine; lyso-PtdCho: 16:0,
l-a-lysophosphatidylcholine, palmitoyl; lyso-PtdEtn, l-a-lysophos-
phatidylethanolamine; lyso-PtdIns, l-a-lysophosphatidylinositol;
lyso-PtdOH, l-a-lysophosphatidic acid, oleoyl; MTT, 3-(4,5-
dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide;
PamOleGroEthyl-PCho, 1,2-palmitoyloleoyl-sn-glycero-3-ethylphos-
phocholine; PamOleGroPCho, 1-palmitoyl-2-oleoyl-sn-glycero-
3-phosphocholine; PtdCho, phosphatidylcholine; PtdEtn,
phosphatidylethanolamine; PtdGro, phosphatidylglycerol;
PtdGroPtd, cardiolipin; PtdIns, phosphatidylinositol; PtdIns-3-P,
phosphatidylinositol 3-phosphate; PtdOH, phosphatidic acid; PtdSer,
phosphatidylserine; Sph1P, sphingosine 1-phosphate; SEL, sheep
erythrocyte lipids; SUV, small unilamellar vesicle; t
0.5
, time necessary
for 50% hemolysis.
(Received 28 November 2002, revised 21 January 2003,
accepted 24 January 2003)
Eur. J. Biochem. 270, 1199–1210 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03480.x
ESTs. It was also homologous with the cDNA-derived
amino-acid sequence of the putative Aa-Pri1 protein [4],
with its isoform aegerolysin [5], with Asp-hemolysin from

the mold Aspergillus fumigatus [6], with two Clostridium
bifermentans hemolysin-like proteins expressed during spor-
ulation [7], and with hypothetical proteins from Neurospora
crassa (TrEMBL db: Q8WZT0) and Pseudomonas aerugi-
nosa (TrEMBL db: Q9I710). It has been speculated that
Aa-Pri1, and similar proteins, may have important roles in
the initial phase of fungal fruiting, such as hyphae aggre-
gation [4], or in apoptosis [1]. Their exact biological role is,
however, not yet clear.
In this work, we have undertaken a functional charac-
terization of ostreolysin, with the aim of shedding some light
on its physiological role(s), and possibly on the role of the
whole group of homologous proteins, so far observed only
in fungi and bacteria. As hemolytic activity was a common
trait, we focused first on this aspect, in particular on the
interaction with the lipid membrane. We have found that
ostreolysin permeabilizes red blood cells and tumor cells, by
forming pores in their plasma membrane. We were also able
to reproduce pore formation in artificial membranes formed
of erythrocyte total lipid extracts, suggesting the presence of
a specific lipid acceptor(s). When the ability of lipids to
inhibit ostreolysin-mediated hemolysis was investigated, we
observed a strong and specific inhibition by a series of
lysophospholipids, in particular lysophophatidylinositol
and sphingosine-1-phosphate, and, to a lesser degree, by
nonesterified fatty acids. However, the membrane acceptor
for pore formation did not appear to be a lysophospholipid.
Our studies rather suggest that ostreolysin, and related
proteins, in addition to being hemolytic, may be modulated
by lysophospholipids.

Materials and methods
Materials
Proteins. Ostreolysin was purified from the fruiting bodies
of freshly collected mushrooms as described previously [5].
The protein stock solution was desalted, concentrated by
ultrafiltration, and kept in aliquots at )20 °C. Before use, the
protein was diluted in 140 m
M
NaCl/20 m
M
Tris/HCl buffer,
pH 8.0 (vesicle buffer) unless otherwise stated. Nontoxic
phospholipase A
2
, i.e. ammodytin I
2
(880 UÆmg
)1
), was a gift
from Dr Igor Krizˇ aj, J. Stefan Institute, Ljubljana, Slovenia.
Porcine trypsin in Hanks balanced salt solution, Bacillus sp.
and Serratia marcescens proteases, Saccharomyces cerevisiae
proteinase A, and Clostridium perfringens neuraminidase
were all supplied by Sigma.
Cells. Bovine, sheep, or human erythrocytes were centri-
fuged from freshly collected citrated blood and washed
twice with an excess of 0.9% saline and once with vesicle
buffer. Transformed cell lines, HT 1080 from human
fibrosarcoma and MCF 7 from human breast adenocarci-
noma, were obtained from the Istituto Zooprofilattico

Sperimentale della Lombardia e dell’Emilia, Brescia, Italy.
Lipids. A series of natural and synthetic lipids and
derivatives were used. Egg phosphatidylcholine (Ptd-
Cho), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(PamOleGroPCho), cholesterol, cardiolipin (PtdGroPtd),
egg phosphatidic acid (PtdOH), egg phosphatidylethanol-
amine (PtdEtn), egg phosphatidylglycerol (PtdGro), liver
phosphatidylinositol (PtdIns), 1,2-palmitoyl-oleoyl-sn-
glycero-3-ethylphosphocholine (PamOleGroEthyl-PCho),
egg sphingomyelin, and brain phosphatidylserine (PtdSer)
were all obtained from Avanti Polar Lipids. Bovine
brain gangliosides and cerebrosides, ceramides, l-a-lyso-
phosphatidylinositol (lyso-PtdIns), egg yolk l-a-lysophos-
phatidylethanolamine (lyso-PtdEtn), egg yolk l-a-lysophos-
phatidylcholine (lyso-PtdCho), l-a-lysophosphatidylcholine,
palmitoyl (lyso-PtdCho 16:0), l-a-lysophosphatidic acid,
oleoyl (lyso-PtdOH), sphingosine 1-phosphate (Sph1P),
myristic, palmitic and stearic acid, dilauroyl, dimyristoyl,
dipentadecanoyl, dipalmitoyl and distearoyl phosphatidyl-
choline, and human low-density lipoprotein (LDL) were all
from Sigma. Phosphatidylinositol 3-phosphate (PtdIns3P)
was obtained from Matreya. All the lipids were dissolved in
chloroform, or other organic solvents, in accordance with
manufacturers’ instructions.
Membranes of sheep erythrocytes were prepared by
hypo-osmotic lysis in vesicle buffer diluted with distilled
water (1 : 4), followed by 5 min centrifugation at 4 °Cand
18 500 g. The supernatant was discarded, and the pellet was
resuspended in vesicle buffer. The washing procedure was
repeated 5 times to remove all cytosolic proteins. Total

lipids were then extracted from pelleted membranes, essen-
tially as described by Bligh & Dyer [8]. The membranes were
combined with 3 mL chloroform/methanol (1 : 2, v/v) and
vortex-mixed for 30 s. Chloroform and water, 1 mL each,
were then added, vortex-mixed again and gently centrifuged
to separate solvent phases. The chloroform phase was
removed, dried under argon, and used as total sheep
erythrocyte lipids (SELs). To exclude the presence of small
hydrophobic peptides co-extracted with the lipid phase,
SEL extracts were dissolved in chloroform (50 lgÆmL
)1
),
applied to a standard TLC silica plate, and run with
chloroform/methanol/acetic acid/acetone/water (35 : 25 :
4 : 14 : 2, v/v). The plates were then sprayed with the
ninhydrinreagentandheatedinanovenat100°C.
Red–violet spots were observed only corresponding to the
amine-containing lipids PtdEtn and PtdSer (as confirmed
using the appropriate phospholipid standards). No other
spots, not even at the origin or the front of the plate, were
detected.
Total lipids from fruiting bodies of P. ostreatus, or fresh
sheep brain, were obtained by Folch extraction [9]: 10 g
tissue and 10 mL distilled water were homogenized on ice,
followed by centrifugation (26 300 g, 30 min, 4 °C), and
extraction of the sediment with 100 mL chloroform/
methanol (1 : 1, v/v). The extract obtained was separ-
ated from the sediment by centrifugation (110 g, 10 min,
25 °C). The sediment was re-extracted sequentially with
chloroform/methanol (1 : 1, v/v), chloroform/methanol

(1 : 2, v/v), and chloroform/methanol/water (60 : 30 : 4.5,
v/v), 20 mL each. Respective supernatants were combined,
dried by rotary evaporation, and kept at )20 °C under
argon.
All the extraction procedures were performed in dupli-
cate. To one half of all the extraction mixtures, 0.05% (w/v)
butylhydroxytoluene (BHT; Sigma) was added as antioxi-
dant; the other half was kept without antioxidant.
1200 K. Sepc
ˇ
ic
´
et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Other reagents. Poly(ethylene glycol)s were from Pharma-
cia or Fluka. Triton X-100 was from Merck. Eagle’s
minimum essential medium, Dulbecco’s phosphate buffered
saline, Mes, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetra-
zolium bromide (MTT), and calcein were obtained from
Sigma, and fetal bovine serum was from Euroclone
(Wetnerby, UK).
Hemolytic activity
Hemolytic activity was measured by a turbidimetric method
as described previously [10]. Typically, 25 lLostreolysin
solution in vesicle buffer was added to 175 lL either sheep,
human or bovine erythrocyte suspension with an apparent
D
650
of 0.1. The decrease in D
650
was recorded for 30 min

using a kinetic microplate reader (Molecular Devices) to
define the time necessary for 50% hemolysis (t
0.5
), and the
maximal rate of hemolysis, i.e. the maximal slope of the
hemolysis kinetics. All the experiments were performed at
25 °C. HC
0.5
(lgÆmL
)1
) was defined as the hemolysin
concentration causing 50% of lysis in 2 min. If not
otherwise stated, sheep erythrocytes were used.
For osmotic protection, glucose, sucrose, raffinose,
stachiose, and a series of poly(ethylene glycol)s with
molecular masses ranging from 900 to 6000 Da were used,
as previously described [10]. Sheep erythrocytes were mixed
with 30 m
M
of an osmotic protectant in vesicle buffer, then
0.5 lgÆmL
)1
ostreolysin was added and the time course of
hemolysis was followed for up to 60 min in the kinetic
microplate reader. The size of the pores produced by
ostreolysin was estimated as described by Renkin [11].
Cytotoxic activity
Cytotoxic activity of ostreolysin was assayed using HT 1080
(human fibrosarcoma cells) and MCF 7 (human breast
adenocarcinoma cells). After thawing, cells were grown for a

week in Eagle’s minimum essential medium, supplemented
with 10% fetal bovine serum, 2 m
ML
-glutamine,
0.15 mgÆmL
)1
gentamycin, and 1 m
M
sodium pyruvate in
the case of MCF 7 cells. The cell lines were grown as
monolayers in 75-cm
2
tissue culture flasks, in a humidified
CO
2
incubator (5% CO
2
, 37 °C). When cells reached 80%
confluence, they were washed using Dulbecco’s phosphate
buffered saline with 1 m
M
EDTA, and then trypsinized with
1 mL porcine trypsin (2.5 mgÆmL
)1
in Hanks balanced salt
solution). Thereafter, 2 mL Eagle’s minimum essential
medium was added to block tryptic activity, and the cells
were washed three times with Eagle’s minimum essential
medium without fetal bovine serum, by 5 min centrifuga-
tion at 200 g. Finally, they were resuspended in 1 mL of the

same medium, counted in a Burker chamber, and plated at a
similar cell density. Various dilutions of ostreolysin in
culture medium were then added for 2 h, followed by 10%
fetal bovine serum and a further 22 h of incubation (5%
CO
2
, 37 °C). When this complete, cell viability was checked
by a standard MTT test, as described [12].
Preparation of vesicles
Lipid films were formed by removing the organic solvents
from a lipid solution in a rounded flask with rotary
evaporation and final vacuum drying. Lipids, at a final
concentration of 1–10 mgÆmL
)1
, were swollen in vesicle
buffer and vortex-mixed vigorously to obtain multilamellar
vesicles. Small unilamellar vesicles (SUVs), or micelles of
lysophospholipids and fatty acids, were prepared by 30 min
pulsed sonication with a Vibracell ultrasonic disintegrator
(Sonics and Materials), using output scale 4 and 50% duty
cycle (room temperature). For large unilamellar vesicles
(LUVs), the multilamellar vesicle suspension was subjected
to eight cycles of freeze–thawing, and pressure-extruded
through 0.1-lm polycarbonate filters (Millipore).
Permeabilization of lipid membranes was studied on
LUVs loaded with fluorescent calcein. These were prepared
essentially as above, except that calcein (at the self-
quenching concentration of 80 m
M
) was included in the

vesicle buffer. Extravesicular calcein was removed by gel
filtration on a Sephadex G-50 (medium) column. Dimen-
sions and homogeneity of the vesicles were routinely
estimated by dynamic light scattering using a Malvern
Zeta-Sizer 3 apparatus (Malvern, UK) as described previ-
ously [13].
Inhibition of ostreolysin-induced hemolysis
Binding of ostreolysin to lipids, human LDL and sheep
erythrocyte membranes was estimated by measuring the
residual hemolytic activity of unbound lysin. Typically,
75 lL micelles, SUVs, LUVs, LDL, or erythrocyte mem-
branes, all at various concentrations in vesicle buffer, were
pipetted into a multiwell plate. Then 25 lL ostreolysin
(4 lgÆmL
)1
) was added to each well, and the plate was
incubated for 30 min at 37 °C to allow ostreolysin binding.
Hemolysis was started by adding 100 lLerythrocyte
suspension in vesicle buffer and recorded for 30 min. The
lysing mixture had an initial D
650
of 0.1.
To assess the effect of partial enzymatic hydrolysis, either
pure phospholipids or LDL were treated with phospho-
lipase A
2
(ammodytin I
2
). In these experiments, LDL (1 mg
proteinÆmL

)1
), or pure sonicated PtdCho, PtdOH, PtdEtn,
PtdIns, PtdSer, and PtdGro (1 lmol lipid), were incubated
for 5 min with 1 U ammodytin I
2
[14] in vesicle buffer
supplemented with 2 m
M
CaCl
2
. Thereafter, 25 lL ostreo-
lysin (4 lgÆmL
)1
) was added, and the hemolytic assay was
performed as described above. The appearance of lipid
hydrolytic products of ammodytin I
2
, i.e. lysophospholipids
and fatty acids, was confirmed by standard TLC and
electrospray ionization mass spectroscopy. Ammodytin I
2
alone did not affect ostreolysin-induced hemolysis, and
was not hemolytic by itself, or in combination with
LUVs or LDL.
Permeabilization of LUVs
Vesicle permeabilization were assayed in a fluorescence
microplate reader (SLT Fluostar, Ma
¨
nnedorf, Switzerland)
with excitation and emission set at 494 nm and 520 nm,

respectively. Ostreolysin at various concentrations in vesicle
buffer (100 lL) was dispensed into a multiwell microplate,
followed by an appropriate amount of calcein-loaded
LUVs. The release of calcein was then recorded as described
[15,16]. For pH values ranging from 6.5 to 9.5, we used the
Tris/HCl vesicle buffer supplemented with 1 m
M
EDTA,
Ó FEBS 2003 Ostreolysin interaction with lipids (Eur. J. Biochem. 270) 1201
and for values between 4.0 and 6.0 we used 140 m
M
NaCl/
20 m
M
Mes/1 m
M
EDTA. Inhibition of calcein release by
lysophospholipids was studied by either preincubating
ostreolysin with various amounts of sonicated lyso-PtdIns
for 20 min before adding the LUVs or preincubating LUVs
and then adding ostreolysin.
Fluorescence measurements
Steady-state intrinsic fluorescence of ostreolysin, either
alone or in combination with lipids, was measured at
25 °C in a Fluoromax spectrofluorimeter (Spex, Edison,
NJ, USA) equipped with a thermostatically controlled cell
holder and a magnetic stirrer. Excitation and emission slits
were set at 5 nm. Samples were excited at 295 nm.
Fluorescence emission spectra of 320 n
M

ostreolysin were
taken over the range 300–450 nm. Intrinsic tryptophan
fluorescence signals were corrected for the dilution factor,
and the background was subtracted using the appropriate
blanks. To monitor the kinetics of ostreolysin interaction
with lipids (SUVs composed of SELs or sonicated lyso-
phospholipids), tryptophan emission intensity was recorded
at 339 nm. All the fluorescence measurements were taken in
50 m
M
Tris/HCl buffer, pH 8.0.
Electrophoresis
Proteins or proteolipid complexes were dissolved in electro-
phoresis buffer containing SDS without reducing agents.
The samples were analyzed by SDS/PAGE using an 8–25%
gradient polyacrylamide gel (Phast System; Pharmacia); gels
were double stained, first with Coomassie Blue and then,
after destaining, with silver nitrate.
Fourier-transform infrared (FTIR) spectroscopy
experiments
FTIR spectroscopy was used to assess the secondary
structure of ostreolysin in solution or adsorbed to the lipid
phase by analysis of the amide I¢ band as described [17].
Ostreolysin was incubated for 30 min with LUVs composed
of pure SELs (protein/lipid, 1 : 11.3, w/w) or LUVs
composed of PamOleGroPCho/lyso-PtdIns (9 : 1, w/w)
(protein/lipid, 1 : 1000, mol/mol), all in 10 m
M
Hepes
buffer, pH 8.0. The mixtures were centrifuged, together

with controls, in an Optima TL ultracentrifuge (Beckman).
A fixed-angle rotor (TLA-100.2) was used at 400 000 g, for
1.5 h at 5 °C. After centrifugation, the supernatant and the
pellet (resuspended in a starting volume of 10 m
M
Hepes
buffer, pH 8.0) were checked for residual hemolytic activity
and analysed by SDS/PAGE. Finally, they were deposited
on germanium crystals and gently dried by nitrogen
flushing. Spectra were collected, in an ATR geometry, on
a FTS 185 spectrometer (Bio-Rad), with MCT detector,
first on hydrated, and then on deuterated films, with or
without a polariser set at either 0 ° or 90 ° (with respect to
the plane of reflections).
In the case of lipid-bound protein, the spectrum of the
protein was obtained by subtracting the contribution of the
lipid alone, with a weight that minimized the band
remaining at 1738 cm
)1
(stretching of the carbonyl groups
in the phospholipids). This was also necessary in view of the
fact that SELs contain lipids comprising the ceramide
moiety, e.g. sphingomyelin and gangliosides, that contribute
a signal in the amide I¢ region. This amounted to % 40% of
the total. The protein spectrum was then subtracted from
the original lipid–protein one to provide the lipid-alone
contribution.
Secondary structures were obtained from analysis of
the amide I¢ band. The original spectrum was deconvoluted
to obtain the component frequencies, which were assigned

as follows: bands in the regions 1696–1680 cm
)1
and 1670–
1660 cm
)1
, b-turn; band at 1672 ± 2 cm
)1
, antiparallel
b-sheet; band at 1651 ± 3 cm
)1
, a-helix; band at 1640 ±
2cm
)1
, random coil; bands in the region 1638–1616 cm
)1
,
b-sheet (parallel plus antiparallel). These were used to
curve fit the original spectrum, and the relative areas were
taken as the proportion of the related structure present.
Additional bands around 1610 cm
)1
and 1600 cm
)1
, derive
from side chain contributions, and were excluded from the
total [17].
The lipid to protein ratio (L/P) in the pellet, was
calculated from the following algorithm [18,19]:
L/P ¼ 0:208 ðn
res

À 1Þ
ð1 À S
amide I
0
Þ
ð1 þ S
L
=2Þ
R
2980
2800
A
90

ðv
L
Þdv
R
1690
1600
A
90

ðv
amide I
0
Þdv
ð1Þ
where n
res

is the number of residues of the protein
(assumedtobe140),A
90°
is the absorption with the 90 °
polarizer, and S are order parameters calculated from the
ratio of the parallel and perpendicular absorption bands. S
L
is for the lipid chains, derived from the symmetric and
asymmetric CH
2
stretching (bands centered at 2850 and
2920 cm
)1
, respectively), using h (the angle between the
direction of the dipole moment change and that of the long
axis of the molecule) set at 90 °. S
amide I¢
is the order
parameter for the amide I¢ band (between 1600 and
1700 cm
)1
, with h ¼ 0 °). The integrals were calculated
from the corrected spectra, that with suffix L from the lipid
alone, and that with suffix amide I¢ from the protein alone.
The order parameter for the a-helix, S
a
, was obtained using
the Lorentzian components at 1650 ± 3 cm
)1
with

h ¼ 30 ° [20–22].
Results
Hemolytic and cytotoxic activity of ostreolysin
Ostreolysin was able to lyse sheep, bovine or human
erythrocytes, all with an HC
0.5
of about 1 lgÆmL
)1
(or
64 n
M
). The time course of hemolysis was characterized by
an initial lag phase followed by a relatively fast lysis, both
dependent on protein concentration (Fig. 1A). Even when
delayed, hemolysis always ran to completion (not shown).
The maximal rate of hemolysis, but not the 1/t
0.5
values,
exhibited saturation with ostreolysin concentration
(Fig. 1B).
Osmotic protectants larger than 1.500 kDa markedly
decreased the rate of hemolysis, and complete protection
was observed with molecules of 6000 kDa or more
1202 K. Sepc
ˇ
ic
´
et al.(Eur. J. Biochem. 270) Ó FEBS 2003
(Fig. 1C). The inner diameter of the ostreolysin-induced
pore was estimated to be about 4 nm, by fitting the

experimental data to the Renkin equation [11].
Exposure of HT 1080 (fibrosarcoma) and MCF 7 (mam-
malian tumor) cells to ostreolysin showed cytotoxicity with
an effective concentration producing a 50% effect of
10 lgÆmL
)1
(or 640 n
M
, Fig. 2). Direct microscope obser-
vation of cell morphology confirmed that ostreolysin had a
similar effect on both cell lines, producing swelling, blebbing
and degranulation. The activity had already peaked after
2 h of incubation.
Inhibition of ostreolysin-induced hemolysis
Hemolytic activity of ostreolysin could be inhibited by
preincubation with either washed erythrocyte membranes
Fig. 1. Time course of hemolysis of sheep erythrocytes by ostreolysin.
(A) Concentration dependence. The concentrations of hemolysin
(curves 1–9) were: 17.5, 8.8, 4.4, 2.2, 1.1, 0.6, 0.3, 0.15, and
0.08 lgÆmL
)1
. (B) Dependence of 1/t
0.5
(j) and of the maximal rate of
hemolysis, V
max
(s), on ostreolysin concentration. t
0.5
is the time
necessary to induce 50% hemolysis; V

max
is the maximal slope of the
change in apparent absorbance as seen in (A). (C) Delaying effects of
osmotic protectants. Traces were obtained in the presence of 30 m
M
glucose, sucrose or poly(ethylene glycol)s of different average
molecularmassesasindicated.
Fig. 2. Cytotoxic activity of ostreolysin towards HT 1080 fibrosarcoma
(d) and MCF 7 mammalian tumor (s) cell lines. Cells that were grown
overnight in complete Eagle’s minimum essential medium and washed
with Dulbecco’s phosphate buffered saline, received the indicated
concentrations of ostreolysin. After 24 h incubation in Eagle’s mini-
mum essential medium, the first 2 h without fetal bovine serum and the
rest with 10% fetal bovine serum, cell viability was estimated by the
MTT assay (absorbance at 575 nm). The points represent mean ± SD
from duplicate experiments. Typical cell counts, before ostreolysin
addition, were 5.2 · 10
5
HT 1080 cellsÆmL
)1
or 6.7 · 10
5
MCF 7
cellsÆmL
)1
.
Fig. 3. Inhibition of ostreolysin-induced hemolysis by SEL-containing
LUVs. Ostreolysin (0.5 lgÆmL
)1
) was preincubated (37 °C, 30 min)

with LUVs composed of pure PamOleGroPCho (d), total SELs (j),
or LUVs composed of PamOleGroPCho/SELs (1 : 1) (h). The
residual hemolytic activity was measured after the addition of sheep
erythrocytes (t
0.5
is as in Fig. 1). Each result is the mean from three to
five experiments, the standard error of which did not exceed 5%. The
mean ± SD diameters of the LUVs, determined by dynamic light
scattering, were 125 ± 50 (SELs), 125 ± 60 (SELs/PamOle-
GroPCho, 1 : 1) and 100 ± 18 nm (PamOleGroPCho). Errors are
standard deviations calculated from the polydispersity, which was
0.166, 0.266, and 0.036, respectively.
Ó FEBS 2003 Ostreolysin interaction with lipids (Eur. J. Biochem. 270) 1203
or LUVs of SELs, or from mushroom fruiting bodies or
sheep brain. Notably, the extent of inhibition was strongly
dependent on the presence or absence of an antioxidant
(BHT) during the storage of the extracted lipids. Without
BHT, the LUV concentration that decreased 1/t
0.5
by a
factor of two was 3 lgÆmL
)1
for SELs (Fig. 3), and about
6 lgÆmL
)1
and 120 lgÆmL
)1
for mushroom and brain lipid
extracts (Table 1). When lipid extracts were stored with
BHT, instead, a similar inhibition was obtained only at % 50

times higher lipid concentrations.
Ostreolysin inhibition was explained by permanent
binding to SEL LUVs. In fact, after incubation with an
excess of SEL LUVs, and ultracentrifugation of the mixture,
almost all the protein was found in the sediment, as
demonstrated by SDS/PAGE (Fig. 4A) and FTIR spectro-
scopy (Fig. 7). In the mean time, its hemolytic activity was
completely abolished. As controls, LUVs alone also sedi-
mented, whereby ostreolysin alone remained, fully active, in
the supernatant. Similar experiments indicated that ostreo-
lysin did not cosediment in a tight proteolipid complex with
Table 1. Inhibition of ostreolysin-induced hemolysis by natural and synthetic lipids, treated or not with phospholipase A
2
(PLA
2
, ammodytin I
2
),
oxidizing conditions (200 l
M
CuSO
4
and 2 m
M
H
2
O
2
), or an antioxidant scavenger (BHT). Ostreolysin (0.5 lgÆmL
)1

) was preincubated for 30 min at
37 °C with various amounts of lipid. Sheep erythrocytes were added and the remaining hemolytic activity was measured. The numbers reported are
the concentration that caused 50% inhibition of ostreolysin-induced hemolysis (in lgÆmL
)1
). NI, No inhibition (up to 750 lgÆmL
)1
); –, not
determined.
Tested component
Treatment
None PLA
2
Oxidation
Single lipid components
Egg PtdCho NI – –
PamOleGroPCho NI 2.8 100
Dilauroyl PtdCho NI 23 –
Dimyristoyl PtdCho NI 0.6 –
Dipentadecanoyl PtdCho NI 0.75 –
Dipalmitoyl PtdCho NI 0.5 –
Palmitoyl myristoyl PtdCho NI – –
Dioleoyl PtdCho NI – –
Distearoyl PtdCho NI 1.0 –
PtdOH NI 5.6 –
PtdEtn NI 23 –
PtdGro NI 0.65 –
PtdSer NI 0.65 –
PtdIns NI 1.5 –
Sphingomyelin NI NI –
Ceramides, cerebrosides, gangliosides,

Chol, PtdGroPtd, PtdIns 3-P
NI – –
Fatty acids
Myristic acid 1.0 – –
Palmitic acid 0.75 – –
Stearic acid 2.0 – –
Lipoproteins and plasma proteins
Human plasma 1 : 8
a
––
Human LDL 90 15 40
Myoglobin
b
NI – –
Egg PtdCho mixtures
1 : 1 with PamOleGroEthyl-PCho, Chol, PtdGroPtd,
PtdOH, PtdEtn, PtdGro, PtdIns, SPM, PtdSer
NI – –
1 : 2 with PtdGroPtd, PtdOH, PtdGro, PtdSer NI – –
PtdCho: PtdEtn: PtdGroPtd (10 : 10 : 0.5) NI – –
PtdCho: PtdEtn: PtdSer: PtdGroPtd (10 : 10 : 2 : 0.5) NI – –
Natural lipid mixtures BHT
Eythrocyte (membrane extract)
c
15 – –
Sheep brain total lipids 120 – –
Sheep erythrocyte total lipids 3 – 150
P. ostreatus total lipids 6 – 120
a
Dilution factor in vesicle buffer.

b
The same result was obtained with the polysaccharide chitin.
c
The same values were observed when
eythrocyte membrane extracts were treated with Bacillus sp. or S. marcescens proteases, endonuclease, trypsin, and neuraminidase.
1204 K. Sepc
ˇ
ic
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et al.(Eur. J. Biochem. 270) Ó FEBS 2003
LUVs composed of PamOleGroPCho/lyso-PtdIns (9 : 1,
w/w). By FTIR spectroscopic analysis, the amount of
cosedimented protein was less than 15% of that observed
with SEL LUVs, too little to give a band in SDS/PAGE
(Fig. 4B, lane 5). Furthermore, in the case of PamOle-
GroPCho/lyso-PtdIns LUVs, both the sediment and the
supernatant were hemolytic, suggesting that the small
amount of sedimented protein was probably that entrapped
between the LUVs. Together these results suggest the
presence of ostreolysin acceptor molecule(s) in the erythro-
cyte membrane, the lipid nature of which was confirmed by
the fact that interaction was not decreased by membrane
treatment with proteases (Bacillus sp. or S. marcescens
proteases, proteinase A, trypsin) or neuraminidase. In
addition, the hypothetical presence of nonlipid components,
such as short hydrophobic peptides, in SELs was directly
excluded by TLC analysis. Notably, ostreolysin formed
aggregates of around 34, 64, and 100 kDa (probably
dimers, tetramers, and hexamers) when bound to SEL
LUVs, but much less in the absence of lipids (Fig. 4), further

evidence for a specific interaction.
We then assayed a series of pure lipids, or lipid mixtures,
for ostreolysin inhibition (Table 1). None of the fully
acylated lipids of varying length and degree of saturation,
nor cholesterol were inhibitory, unless supplemented with a
certain proportion of SELs, as shown for the case of
PamOleGroPCho (Fig. 3). Ostreolysin-induced hemolysis
was, however, markedly inhibited by pure sonicated lyso-
phospholipids at concentrations at which these compounds
were not themselves hemolytic (Fig. 5A). The most effective
inhibitor was lyso-PtdIns, causing 50% reduction of 1/t
0.5
at
0.2 lgÆmL
)1
(% 10
)7
M
), whereas egg lyso-PtdCho, lyso-
PtdCho 16:0, Sph1P, lyso-PtdEtn, and lyso-PtdOH induced
the same effect at 0.7, 0.7, 1.1, 2.3, and 50 lgÆmL
)1
(or
10
)4
M
), respectively. This suggests that their activity was
neither dependent on the charge of the polar group (the two
negatively charged, lyso-PtdIns and lyso-PtdOH, were the
most and the least effective, respectively), nor on the fatty

acid composition (lyso-PtdCho 16:0 and egg lyso-PtdCho
had the same effect). Instead, it may depend, at least in part,
on the chemical nature of the polar head, because lyso-
PtdCho and Sph1P (with the same choline head group) had
similar effects. All lysophospholipids showed a similar
sigmoidal dose-dependent inhibition, except for Sph1P,
which had a less steep dependence (Fig. 5A).
The interaction of ostreolysin with lysophospholipids was
further analysed by preparing PamOleGroPCho LUV
containing 10% of different lysophospholipids (Fig. 5B).
Apart from LUVs containing Sph1P, the inhibitory ability
of these mixtures was dramatically decreased with respect to
pure lysophospholipids, and a similar level of inhibition was
obtained only with concentrations at least 100-fold higher
(corresponding to a 10-fold higher amount of the lyso-
phospholipid present). The order of inhibitory activity
was Sph1P > lyso-PtdIns > lyso-PtdCho > lyso-PtdEtn.
When the amount of lyso-PtdIns included in PamOle-
GroPCho LUVs was varied (Fig. 5C), it was again
apparent that the loss of inhibitory activity was larger than
the corresponding decrease in lyso-PtdIns concentration.
The inhibition of ostreolysin by lysophospholipids was
further confirmed by enzymatic hydrolysis of pure phos-
pholipids. We found that even a partial hydrolysis of pure
PtdCho, PtdOH, PtdEtn, PtdIns, PtdSer, and PtdGro by
ammodytin I
2
, a phospholipase A
2
, markedly inhibited

ostreolysin-induced hemolysis (Table 1). Furthermore,
although intact LDL was not inhibitory, it became so after
10minhydrolysiswithammodytinI
2
(Table 1).
As the binding of single-chained lysophospholipids could
be promoted by their fatty acid moiety, we also assayed
myristic, palmitic and stearic acid (sonicated for 30 min) for
inhibition. We found that all of them could inhibit
ostreolysin-induced hemolysis, but only with a molar
Fig. 4. SDS/PAGE analysis of ostreolysin interacting with LUVs composed of SELs (A) or PamOleGroPCho/lyso-PtdIns (9 : 1) (B). The samples
were ultracentrifuged and the sediments and supernatants obtained were analyzed. (A) lane 1, Pharmacia low molecular mass standards; lane 2,
ostreolysin (noncentrifuged); lane 3, ostreolysin (supernatant); lane 4, ostreolysin (sediment); lane 5, LUVs (sediment); lane 6, ostreolysin + LUVs
(sediment). (B) lane 1, Pharmacia low molecular mass standards; lane 2, LUVs (sediment); lane 3, ostreolysin (noncentrifuged); lane 4, ostreolysin
(supernatant); lane 5, ostreolysin + LUVs (sediment); lane 6, ostreolysin + LUVs (supernatant). Samples were stained with 0.5% silver nitrate. As
reported [5], ostreolysin without reducing agents appeared as a doublet at about 16 kDa. The hemolytic activities of the analysed samples, expressed
as HC
0.5
, were as follows: ostreolysin + SEL LUVs, supernatant or resuspended pellet, no activity; ostreolysin + PtdCho/lyso-PtdIns (9 : 1)
LUVs, supernatant 4 lgÆmL
)1
, resuspended pellet 8 lgÆmL
)1
; ostreolysin alone, supernatant 1 lgÆmL
)1
, resuspended pellet no activity; noncen-
trifuged ostreolysin, 1 lgÆmL
)1
.
Ó FEBS 2003 Ostreolysin interaction with lipids (Eur. J. Biochem. 270) 1205

efficiency % 10-fold lower than lysophospholipids. The
inhibition was virtually independent of the fatty acid chain
length (Table 1).
Permeabilization experiments
In agreement with the absence of inhibition, we found that
calcein-loaded LUVs composed of pure phospholipids or
sphingolipids and cholesterol, in various combinations,
could not be permeabilized by ostreolysin. Even LUVs
containing up to 10% of lyso-PtdIns, or made of total lipids
from P. ostreatus (with or without BHT), were insensitive,
despite being inhibitory to various extents. Only LUVs
containing SEL extracts could be permeabilized. The extent
of calcein release was dependent on the lysin dose and the
pH of the bathing solution (Fig. 6). It was optimal in the pH
range 8.0–9.0, where 0.5 lgÆmL
)1
ostreolysin produced
50% calcein release from SEL LUVs. In contrast with
inhibition, SEL LUV permeabilization was not affected by
the presence of BHT during lipid storage. The release was,
however, abolished by the presence of sonicated lysophos-
pholipids at sublytic concentrations.
FITR spectroscopy
FTIR spectra were recorded for ostreolysin alone, or
cosedimented with either SEL LUVs or PamOleGroPCho:
lyso-PtdIns (9 : 1, w/w) LUVs. The secondary structure of
ostreolysin was estimated by FTIR spectroscopy, analysing
the amide I¢ band. Spectra were first deconvoluted to find a
suitable set of single Lorentzian components, the sum of
which was then used to curve-fit the original spectra. The

single Lorentzian bands were attributed to four secondary
structures (Fig. 7, Table 2). The resulting curves suggested
that ostreolysin was composed of % 50% b-structure
(comprising 15% b-turn and 35% b-sheet), plus 20%
a-helix and 30% random coil.
A significant association of ostreolysin with SEL LUV
pellets was observed. The lipid/protein molar ratio estima-
ted using Eqn (1) was % 300, corresponding to a w/w ratio
of 12 : 1. When compared with the precentrifugation ratio
of 11 : 1, this suggested that more than 90% of the protein
was associated. With PtdCho:lyso-PtdIns (9 : 1, w/w)
LUVs instead, the estimated lipid/protein ratio was
% 1900, indicating a much weaker association, if any.
When ostreolysin bound to SEL LUVs, we observed an
increase in its a-helical structure from 20% to 30%
(Table 2). This occurred mainly at the expense of
b-structures. Such an increase may suggest rearrangement
of a portion of the protein with insertion of a newly formed
a-helix into the lipid matrix. Interestingly, from the 0 ° and
90 ° polarized spectra of the inserted protein, it was possible
to calculate the dichroic ratio of the helix and its orientation
Fig. 5. Inhibition of ostreolysin-induced hemolysis by lysophospholipids.
Ostreolysin (0.5 lgÆmL
)1
) was preincubated for 30 min at 37 °Cwith
various amounts of lipids. Sheep erythrocytes were added and the
remaining hemolytic activity was measured (t
0.5
is as in Fig. 1).
Asterisks indicate a concentration above which the lipids alone became

lytic. Ostreolysin was incubated with: (A) different sonicated lyso-
phospholipids, as reported; (B) LUVs, composed of a 9 : 1 mixture of
PamOleGroPCho with the indicated lysophospholipids; (C) LUVs,
composed of PamOleGroPCho and the indicated percentage of lyso-
PtdIns. Each result is a mean from three to five repetitions, and the
standard error did not exceed 5%. The mean ± SD diameters (in nm)
of LUVs in (B) and (C), determined as in Fig. 3, were 88 ± 40
(PamOleGroPCho/lyso-PtdIns, 9 : 1), 99 ± 45 (PamOleGroPCho/
lyso-PtdCho, 9 : 1), 100 ± 27 (PamOleGroPCho/lyso-PtdOH, 9 : 1),
103 ± 25 (PamOleGroPCho/lyso-PtdEtn, 9 : 1), 98 ± 24 (PamOle-
GroPCho/lyso-PtdIns, 99 : 1), 97 ± 30 (PamOleGroPCho/lyso-
PtdIns, 98 : 2), and 96 ± 37 (PamOleGroPCho/lyso-PtdIns, 95 : 5).
The width of the distributions were calculated, as in Fig. 3, from the
polydispersity (0.219, 0.213, 0.072, 0.058, 0.06, 0.095 and 0.152,
respectively).
Fig. 6. pH-dependence and dose-dependence of calcein release from
LUVs composed of SELs. Calcein release from SEL LUVs was
measured as a function of ostreolysin concentration at different pH
values (as reported).
1206 K. Sepc
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et al.(Eur. J. Biochem. 270) Ó FEBS 2003
around the perpendicular to the plane of the membrane [23].
We obtained an average angle of 45 °. However, consider-
ing that the average orientation shown by the lipid chains in
the same spectra was between 42 ° and 44 °, we could
recalculate the relative orientation of the a-helix with respect
to the lipid chains [23], obtaining an angle of 20–22 °.This

suggested an a-helix orientation nearly perpendicular to the
plane of the membrane.
Fluorescence measurements
Intrinsic tryptophan fluorescence was finally used to explore
changes in the local environment of ostreolysin. As in the case
of permeabilization, only SEL vesicles affected the fluores-
cence of ostreolysin. The intensity increased and the emission
maximum shifted from 339 to 333 nm (Fig. 8A), suggesting
that at least some of the tryptophan residues of the protein
are transferred into a more hydrophobic environment. The
time course of fluorescence increase was rather fast, as shown
in Fig. 8B. Fluorescence intensity was maximal at a lipid/
protein ratio (w/w) above 16.3, corresponding to an
approximate molar ratio of 400. No changes were detected
on addition of pure lyso-PtdIns (Fig. 8C).
Discussion
Our study provides direct evidence that ostreolysin has at
least two different ways of interacting with lipids. First, it
can permeabilize cell membranes and artificial lipid bilayers
of specific composition, and secondly, it is modulated by
lysophospholipids. In fact, the latter class of physiologically
very important lipid derivatives efficiently inhibits the most
adverse effect of this protein, i.e. cell lysis.
Ostreolysin is equally lytic to human, bovine and sheep
erythrocytes, and only slightly less potent on some human
tumor cell lines (Figs 1A and 2). There are several pieces of
evidence that hemolysis is of the colloid-osmotic type,
caused by the formation of ostreolysin pores in the lipidic
portion of the cell membrane. Hemolysis could be prevented
by the presence of osmotically active solutes large enough to

exceed the pore size (Fig. 1C), as previously reported for
other pore-forming proteins [10,24]. The Renkin estimate of
the ostreolysin inner pore diameter was % 4nm,whichis
similar to that of flammutoxin, a 31-kDa cytolytic protein
from the edible mushroom Flammulina velutipes [25]. In
addition, ostreolysin was able to release the fluorescent
marker calcein (diameter % 1.1 nm) from LUVs comprised
of total erythrocyte lipids (Fig. 6). The pore may result from
Fig. 7. Infrared attenuated total reflection spectra of ostreolysin with or without lipids. (A) Deuterated films containing: ostreolysin bound to SEL
LUVs (a), ostreolysin alone (b), or SEL LUVs alone (c). Indicated bands correspond to: OH stretching, from incompletely deuterated water and
amide A vibrations; CH
2
stretching (either symmetric, s, or asymmetric, as); OD stretching of deuterated water; C¼O stretching of the phospholipid
carbonyl groups; amide I¢ and II¢ bands. (B, C) Analysis of the amide I¢ band. The original ostreolysin spectrum (thin line in B) was deconvoluted
(C) to obtain the component frequencies. These are indicated as: t (b-turn); b
1
(antiparallel b-sheet); a (a-helix); r (random coil); b
2
(parallel plus
antiparallel b-sheet). Bands below 1610 cm
)1
derive from side chain contributions [34]. Corresponding Lorentzian bands, dotted lines in (B), were
adapted in size by least-squares fitting of their sum (thick dashed line) to the original spectrum. The percentages of secondary structures, evaluated
from the relative areas of the Lorentzian bands excluding those of the side chains, are reported in Table 2.
Table 2. FTIR spectroscopic determination of the secondary structure of
ostreolysin with and without lipids. Values are mean ± SD. b
1
, Anti-
parallel b-sheet; b
2

, parallel and antiparallel b-sheet; t, b-turn; a,
a-helix; r, random coil; b
tot
, total b-structure (i.e. b
1
+ b
2
+ t).
Protein
% Secondary structure
b
1
b
2
t a r b
tot
Ostreolysin 6 ± 1 28 ± 3 14 ± 2 19 ± 2 33 ± 3 48
Ostreolysin + SELs 3 ± 1 20 ± 2 11 ± 2 31 ± 3 35 ± 3 34
Ó FEBS 2003 Ostreolysin interaction with lipids (Eur. J. Biochem. 270) 1207
aggregation of several protein molecules, as we have
observed the occurrence of SDS-resistant ostreolysin aggre-
gates of two, four, and six monomers on SEL LUVs.
Similar aggregates appeared only very faintly in the absence
of lipids (Fig. 4A). The rather long lag phase preceding fast
hemolysis (Fig. 1A) may also indicate that the formation of
a functional pore requires the growth of ostreolysin
aggregates on, or within, the erythrocyte membrane. The
observation that maximal hemolysis rate was saturated at
high ostreolysin concentrations, whereas 1/t
0.5

was not
(Fig. 1B), confirmed that lysin binding and aggregation
(which are likely to affect 1/t
0.5
) are slower processes than
diffusion of solutes through the opened pores (limiting the
maximal rate). Asp-hemolysin, a similar protein, has also
been reported to form large aggregates on erythrocytes,
which could be visualized by electron microscopy [26].
Membranes made of the total lipid extract from fruit bod-
ies of P. ostreatus were not susceptible to permeabilization,
but were nonetheless strong inhibitors of ostreolysin-
induced hemolysis. One explanation could be that ostreo-
lysin may bind to these vesicles, but not permeabilize them.
Another possibility is that they contain a diffusible compo-
nent that can be transferred from the vesicle to the protein
and inactivate it. The observation that lysophospholipids,
either alone or in combination with other lipids (Fig. 5), can
inhibit ostreolysin hemolytic and permeabilizing activity,
but not cosediment it (Fig. 4), supports the latter explan-
ation. Concentrations of various lysophospholipids neces-
sary for 50% inhibition of hemolysis were always below
their critical micellar concentrations, which is % 70 l
M
for
lyso-PtdIns [27] and 1.3 m
M
for lyso-PtdOH (Avanti Polar
Lipids, web page). This suggests that lysophospholipids may
inhibit ostreolysin pore-forming activity in their monomeric,

rather than micellar, form.
It is known that biological membranes [28], and also
LDL [29], contain various amounts of lysophospholipids
that could be diffusively exchanged between membranes [27]
and that lysophospholipid content may be increased by
oxidative processes [30] or the action of phospholipase A
2
[29]. Accordingly, we were able to modify both noninhi-
bitory vesicles and normal LDL to become inhibitors of
ostreolysin hemolysis by 2 h of oxidation with 2 m
M
H
2
O
2
in the presence of 200 l
M
CuSO
4
, as reported also for Asp-
hemolysin and LDL [31,32]. Moreover, the inhibitory
activity, but not the permeabilization, was clearly higher if
the vesicles were prepared from lipids stored without an
antioxidant scavenger (Table 1). As oxidative degradation
of phospholipids results in a variety of products, in addition
to lysophospholipids [30], we also employed partial diges-
tion of LUVs and LDL with ammodytin I
2
, a phospho-
lipase A

2
, to prove that emerging lysophospholipids were in
fact responsible for the observed inhibition (Table 1).
Fluorescence and FTIR spectroscopy confirmed that the
interaction of ostreolysin with SEL membranes and lyso-
phospholipids (or lysophospholipid-containing membranes)
was different. Whereas addition of SEL LUVs enhanced the
protein intrinsic fluorescence and blue-shifted the wave-
length of emission maximum, lysophospholipids did not
(Fig. 8). Similarly, the FTIR spectra revealed structural
changes in ostreolysin on binding to SEL SUVs, but little or
no binding with LUVs containing 10% lyso-PtdIns. The
collective results (Figs 7 and 8, and Table 2) suggest that
binding to the lipid bilayer and pore formation induced
changes in ostreolysin conformation concomitant with
Fig. 8. Kinetics of ostreolysin binding to SEL SUVs. (A) Steady-state intrinsic fluorescence spectra of 5.13 lgÆmL
)1
ostreolysin alone (dashed line)
or combined with SEL SUVs (solid line) at the lipid/protein ratio (w/w) 3.25. (B) Time course of the increase of relative fluorescence intensity F/F
0
.
SEL SUVs were added to 5.13 lgÆmL
)1
ostreolysin at the indicated lipid/protein ratio (L/P, w/w). Molar ratio is % 25 times the w/w ratio. (C)
Dependence of the steady-state intrinsic fluorescence of ostreolysin (5.13 lgÆmL
)1
) on the SEL SUV/protein (j), or sonicated lyso-PtdIns/protein
ratio (h) (w/w). F
0
, fluorescence of ostreolysin emitted at 339 nm; F, fluorescence of ostreolysin combined with lipids at 339 nm. Excitation was at

295 nm. All the experiments were carried out in 50 m
M
Tris/HCl, pH 8.0.
1208 K. Sepc
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et al.(Eur. J. Biochem. 270) Ó FEBS 2003
transfer of certain tryptophan residues to a more hydro-
phobic environment, which were not seen on interaction
with lysophospholipids. Such changes may correspond to
the movement of the tryptophan residues into the lipid
phase, or into a hydrophobic pocket created by aggregation
of the protein molecules. According to the FTIR spectro-
scopic results, the transferred protein portion was conceiv-
ably an a-helix, inserted almost perpendicular to the plane
of the membrane.
In conclusion, we have observed two different interaction
modes of ostreolysin with lipids. One occurs on mammalian
cell membranes, or on model membranes containing total
erythrocyte lipids, and consists of tight and stable binding,
followed by permeabilization via the formation of 4 nm
pores. This goes together with tryptophan fluorescence
changes indicative of protein insertion into the lipid film,
and an increase in a-helix at the expense of b-structure. Such
effects are independent of the presence of antioxidants. The
other is the effect of lysophospholipids, and some related
components such as fatty acids, which induce loss of the
pore-forming ability, without permanent association with
membranes containing these components, nor a change in

the intrinsic fluorescence. This effect also occurs in the
presence of membranes or proteins containing these com-
ponents (e.g. mushroom membranes or LDL), probably via
exchange of lysophospholipid molecules through the solu-
tion. It is enhanced by conditions that favor the production
of lysophospholipids, e.g. the absence of scavengers, direct
oxidation or phospholipase A
2
attack.
The mode of action of ostreolysin is probably also
common to other members of this protein family, so far
found only in fungi and bacteria. For example, the homol-
ogous Asp-hemolysin of A. fumigatus was reported to be a
lyso-PtdCho-binding protein [32] and to enhance infection of
mice by this organism [26]. However, the edible mushroom
P. ostreatus is not a pathogenic species and it therefore seems
unlikely that ostreolysin and similar proteins, such as
Aa-Pri1, are toxins. Specific expression of ostreolysin and
aegerolysin during initiation of fungal fruiting [4,5], expres-
sion of a hemolysin-like protein of B. bifermentans at the
stage of sporulation [7], and our present observations may
rather suggest that these proteins are involved in cell
signaling. Lysophospholipids, in particular lyso-PtdIns and
Sph1P, are in fact well-known signaling molecules which
participate in a variety of cell processes [27] including
differentiation [33]. It is tempting to speculate that the level of
lysophospholipids present in the organism may regulate the
membrane-binding and pore-forming ability of these pro-
teins, thus controlling their participation in cell differentita-
tion in an as yet undiscovered way.

Acknowledgements
The Slovenian authors were supported by a grant from the Ministry of
Science, Education and Sport of the Republic of Slovenia. The Italian
authors were supported by a grant from Consiglio Nazionale delle
ricerche (CNR), Istituto Trentino di Cultura (ITC) and, in part, also by a
grant from Provincia Autonoma di Trento (PAT) Fondo Progetti
(Project AgriBio). K. S. was the recipient of a NATO fellowship (band
219.33) during her stay in Trento. We thank Dr Gabriella Viero for
technical help and Dr Graziano Guella for some TLC and electrospray
ionization mass spectroscopy analyses.
References
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