A 16 kDa cytolytic protein called ostreolysin was purified from the fruiting body of Pleurotus ostreatus strain PLo5. The complete 50 amino acid peptide sequence of ostreolysin was determined and deposited in a public database. A modified form of ostreolysin was created by changing an antiparallel beta strand in domain 4, altering its cholesterol binding site. This modified ostreolysin was shown to selectively kill cancer cell lines at nanomolar concentrations due to their higher membrane cholesterol levels, while having no effect on normal cell lines.

1. Purification, peptide sequencing and modeling of ostreolysin
from Pleurotus ostreatus strain Plo5 : Formation of a modified
ostreolysin with cytolytic effect only on cancer cell lines
Antik K. Bose
Affilations I Corresponding author
Affiliations
Fred Hutchinson Cancer Research Center,
1100 Fairview Avenue N, Seattle,
Washington 98109, United States.
Antik K. Bose
A 16 kDa ostreolysin ,a cytolytic protein has been purified from the fruiting body of Pleurotus ostreatus
strain PLo5 using Q-sepharose, Superdex TM
-75 gel filtration, Vydac C-18 reverse phase HPLC and SDS-
PAGE. The complete peptide sequencing of the 50 amino acids ostreolysin was done and deposited in
public protein database; UniPort B. Modeling of the 4 domains of ostreolysin and quaternary structure

2. of the native ostreolysin was elucidated. A modified ostreolysin was prepared on converting an
antiparallel ß strand in domain 4 of the protein and changing its cholesterol binding site. Modified
ostreolysin could kill cancer lines at nanomolar concentrations because of their higher membrane
cholesterol levels ,and it has no effect on normal cell lines. Stability of Modified ostreolysin was shown
by Ramachandran Plot. Modeling of Modified ostreolysin was also done.
Abbreviation:
SDS-PAGE- sodium dodecyl sulphate poly acrylamide gel
electrophoresis
EDTA-ethylene diamine tetra acetic acid
Ab- Antibody
HRP- Horse raddish peroxidase
Ve- elusion volume
Vo- void volume
HPLC- High performance liquid chromatography
PTH- phenythiohydantoin
MTT-3-(4,5- dimethyl thiazol-zyl)-2,5 diphenyl tetrazolium
bromide)
ATP- adenosine triphosphate
PI-phosphatidyl inositol
LDH- lactate hehydrogenase
Introduction:
Ostreolysin is a 16KDa cytosolic protein belonging to
aerolysin family of proteins found in bacteria, fungi
and plants, but its biological role is unknown. It
appears in peripheral parts of fruiting bodies and
lamelliae during primordium formation( Rebolj Katja,
Kristina Sepcic ;2008). It forms transmembrane
pores in natural and artificial lipid membranes. The
lysis results from specific interaction of ostreolysin
with cholesterol – enriched raft- like membrane
domains; which differ from those binding caveolin or
choera toxin subunit B. Mutants of ostreolysin can
be used as specific markers for cholesterol – rich raft
like membrane domains and for studies or raft
heterogeneity. At nM concentration; the protein
lysed human , bovine and sheep erythrocytes by a
colloid –osmotic mechanism with formation of 4nm
diameter pores. Interaction with lipid vesicles and
their permeabilisation is correlated with increase in
intrinsic fluorescence and α- helical content of the
protein. (Kritina Sepeic, Sabina Berne, Christina
Potrich,Tom Tirk, Peter Macek, Gianfranco
Menestria;2003). Depletion of 40% membrane
cholesterol by methyl –ß cylodextrin dramatically
decreased ostreolysin binding. Immunostaining
showed that ostreolysin is not co-localised with
raft-binding proteins, cholera toxin ß-subunit or
caveolin suggestiong that natural membranes
display heterogeneity of cholesterol enriched raft-
like membranes (H.helena Chowdhury ,Katjo Rebolj,
Marko Kreft, Robert Zonea,Peter Macek and Kristina
Sepecic ;2008). Ostreolysin binds to mono and
bilayers containing cholesterol, ergosterol, ß-
sitosterol, stigmasterol, lonosterol, 7-
dehydrocholesterol, cholesteryl acetate and 5
cholestene 3-one,in 1/1 molar ratio .Lytic activity is
dependent on sterol 3ß-OH group and decreases by
double bond and methylation of steroid skeleton or
C17 –isooctyl chain.

3. Ostreolysin expressed in primordium and fruiting
body, is found to inhibit growth of mycelium,
induces primordial formation into fruiting bodies. It
is not directly involved in sporulation as detected in
non-sporulating strains of P. astreatus. It is induced
by polymeric 3- alkyl pyrimidine salts. (S.Berne,
J.pohleven ,I Vidie, K Rebolj, F.pohleven, T.turk,
P.Macck;2007)
Using ligand design program LUDI , it was found that
3β-OH group of cholesterol forms H-bond with Glu-
46 and Lys -48 of ostreolysin. Binding triggers
membrane insertion because loop containing Trp 45
of ostreolysin is hydrophobic ,together with aliphatic
side chains of cholesterol, could act as a dagger for
penetration. A modified ostreolysin protein was
prepared using subtilisin Carlsberg protease (Cθ)
which digests ostreolysin at Cys43 of domain 4
resulting is release of the anti parallel β-strand
carrying 43-Cys-Gln-Trp-, Glu-Lys-Ile-Ile-50 and re
introduced in the protein but in opposite orientation
; forming a parallel β strand in the modified
ostreolysin. Using LUDI design program, it was found
that 3β-OH group of cholesterol can form H-bond
with Glu-46 but not Lys-48 because its orientation
has been reversed in respect to cholesterol 3-βOH
group in modified ostreolysin. So , modified
ostreolysin required higher membrane cholesterol
concentration for binding and membrane
penetration.
The membrane cholesterol content of cancer cells is
much higher than normal cells due to upregulation
of HMG-CoA reductase and increased concentration
of mevalonate in cancer cells (Ying Chun Li, Mi Jung
Park,Sang-Kyu Ye,Chul-Woo Kim, Yong – Nyun Kim
;2006). So, modified ostreolysin can selectively kill
cancer cell lines by membrane penetration and it has
no effect on normal hepatocytes and Monkey kidney
fibroblast cell lines (COS-7)
Materials :
A. Chemicals: Pleurotus ostreatus strain Plo5
(purchased from ZIM collection of Biotechnical
Facility ,University of Ljubljana, Solvenia),ß-
mercaptoethanol , Benzamidine hydrochloride
hydrate 98% ( catalogue no. 206752-36-5 B6506,
sigma Aldrich) ,leupeptin (chemicon
,Millipore,catalogue no 18) Q-sepharose (M-grade
weak anion exchanger,fast flow column, Amershan
Biosciences), anti IgG monoclonal Ab against
Pleurotus ostreatus ostreolysin (Abbiotech LLC),
3,3’,5,5’ tetramethyl benzidine (Litton Bionetics,
Kensington), anti IgG Ab conjugated to HRP (
Abazyme), Superdex
TM
-75 (separation range 3000-
70000, matrix spherical composite of cross linked
agarose and dextrin, GE Health Care Lifesciences,
USA), Bovine Pancreatic chymotrypsin Assay kit
(Sigma – Aldrich, Ref no. FGAP03),chicken lactate
dehydrogenase Assay kit(Bioo scientific, Texas,USA)
,Ribonuclease A assay kit (Sigma-Aldrich), Horse
liver catalase (Cal biochem,Biosciences Inc; catalog
no. 219265, USA), Horse heart myoglobin (Sigma
Aldrich), PD10 desalting column( G.E healthcare ,life
sciences) trypsin (Sigma-Aldrich), N-Glycosidase F
and glycoprotein denaturating buffer (New England
Biolabs), endoprotease Lys C (Sigma Aldrich)
endoprotease Glu C (P 8100,New England biolabs),
Dulbeeco’s Modified Eagle’s Medium (GIBCO BRL,
catalogue no. 31600, Grand Island , NY), Insulin like
growth factor- ß (sigma- Aldrich) , MCF 7 cell line
(Lonza AG,USA), Hep G2 cell line (ATCC no. HB-8065,
Abcam USA), COS-7 human hepatocyte cell line,
Sawano, CACO-2, MOLT-4, HL-60,Jurkat, HeLa
(Abcam ,USA) cell lines; cell Titer 96TM
non-
radioactive cell proliferation Assay kit (Promega),
Titer- Glo
TM
luminescent cell viability Assay
kit(Promega).
B. Software programs for macromolecular
crystallography: DM density modification package
release 2.1, CCP4 (comprehensive computing suit

4. for macromolecular crystallography, SIGMAA CCP4,
HKL package for DENZO, X- Display F and Scalepack,
Maximum likelihood heavy atom refinement
(MLPHARE).
Procedure and Result:
1.Purification of ostreolysin:
Ostreolysin , a 16 kDa cytolytic protein has been
purified from fruiting body of Pluerotus Ostreatus
strain Plo5 (taken from ZIM collection of the
Biotechnical facility University of Ljubtjana,
Solvenia).The strain Plo5 was propagated on 2%
Malt extract agar after using a liquid culture media,
described by Mansur et al (1997) at pH 5.0 with 20
mM sodium 2,2 dimethyl succinate and 50 mM (2
morpholino) ethane sulfonic acid (MES) buffer and
incubated at 280
c in 500 ml Erlenmeyer flask
containing 150ml culture and agitated at 100 rpm for
16 days. The fruiting body was used as a source of
ostreolysin. 12gm fruiting body was crushed with 50
mM Tris- Hcl buffer (pH 5.0) containing 2mM EDTA,
1%(v/v) β- mercaptoethanol , 2 mM Benzamidine, 2
µg/ml Leupeptin (extraction buffer) and centrifuged
at 10,000 rpm for 15min at 15
0
c. Ostreolysin was
purified by passing the extract through Q-sepharose
(fast Flow column ,Amersham Biosciences)
equilibrated with assay buffer and eluted with
500mM NaCl prepared in assay buffer (pH 5.0) with
a single peak . 6% SDS-PAGE of 500 mM NaCl elute
showed a single band of 16kDa.
Fig:1 Fig:2
Fig 1. A 16 KDa band of ostreolysin was observed in lanes 2,3,4 and 5 (from left) lane 1 was loaded with Horse
heart myoglobin (16.9 KDa). Stained with Coomassie Brilliant Blue G-250.
Fig 2: Tube 543 elute of superdexTM
-75 column showed a single band of 16KDa in lane 2 of 6% SDS-PAGE (from
left).16.9 KDa MW marker Horse heart myoglobin was loaded in lane 1 and 3 and staining with Coomassie Brilliant
Blue G-250

5. The 16 kDa band of ostreolysin was detected by
western blotting with anti ostreolysin monoclonal
Antibody of P. ostreatus (Abbiotech LLC) and anti IgG
conjugated to HRP secondary Ab (Abazyme). 500mM
NaCl elute from Q-sepharose column was loaded in
superdex
TM
-75 column (GE Health care Life sciences
USA), equilibrated with 50 mM Tris- HCl buffer (pH
5.0) and 150 mM NaCl. Blue dextran -2000 R
(GE
Health care Life Sciences ) was used to calculate the
void volume (Vo=5.53ml). MW markers like Bovine
Pancreas Ribonuclease A (12.6 kDa),Bovine
pancreatic chymotrypsin (20.6 KDa), Chicken lactate
dehydrogenase (H) (150 k Da), Horse liver Catalase
(222kDa), Pleurotus sajor-caju urease (450 K Da) and
Squid haemocyanin (612 KDa) were used. Elution
was done with assay buffer using Gilsons prep FCTM
fraction collector.
Table 1: Determination of Ve/Vo for superdex TM
-75 column elute containing ostreolysin :
Tube No. Ve/Vo Log 10Ve/Vo Retention constant
R=Vo/Ve
543 49.124 1.975 0.606
Table 2: Determination of Ve/Vo for MW markers (Vo=5.53ml):
Name of MW
markers
Tube no. Ve/Vo Log10 Ve/Vo Retention Constant
R=Vo/Ve
1.Ribonuclease
A(Bovine Pancreas)
(12.6 KDa)
550 49.746 2.0 0.020102
2.Bovine Pancreatic
chymotrypsin (20.6
542 49 1.970 0.020408

6. K Da)
3.Chicken Lactate
dehydrogenase (H)
(150 KDa)
440 39.78 1.60 0.025138
4.Horse liver
catalase (222KDa)
311 28.18 1.45 0.0355
5.Urease (Pleurotus
sajor-caju)(450 KDa)
55 4.97 0.75 0.502513
6.Squid
Haemocyanin (612
KDa)
10 1.99 0.3 0.502513
Tube No. Volume of elution buffer required (Ve)(ml)
10 11.0 ml (Squid haemocyanin)
55 27.51 ml (P. sajor-caju urease)
311 155.8 ml (Horse liver Catalase)
440 220 ml(Chicken lactate dehydrogenase(H) )
542 271ml (Bovine pancreatic chymotrypsin)
543 271.66ml (Pleurotus Otreatus strain Plo5ostreolysin)
550 275 ml (Bovine Pancreas Ribonuclease A)
Table 3: Determination of elution volume of ostreolysin and MW markers.
Flow rate was maintained at 1.5 ml/min and 0.5 ml was collected in each tube using Gilson’s prep FC
TM
collector.
Protein concentration of tube 543 containing ostreolysin was found to be 1.64 µg/ml. Mol weight of ostreolysin
was calculated from log Ve/Vo v/s Mol mass plot and calculated to be 16 kDa .6% SDS-PAGE of tube 543 of
Superdex TM
-75 column gave a single band of 16 kDa.
The 16 KDa band was detected by Western blotting
using anti -ostreolysin monoclonal Ab of P. ostreatus
(Abbiotech LLC) and anti IgG conjugated to HRP
secondary Ab (Abazyme).
Peptide sequencing of ostreolysin:
The purified ostreolysin was incubated with 0.4 mM
Ellman’s reagent (5,5’ dithiobis (2 nitrobenzoic
acid)), 6 (M) urea, 0.1 mM Na2EDTA and 100 mM
Tris- HCl buffer (pH 8.0) for 30 min at 25
0
C

7. .Absorption at 412 nm (€=11400 Mcm-1
)was taken
and concentration of SH groups was found to be
0.4.mM and number of disulfide bonds is 2 and
number of cysteine residues is 4 (J. Kenneth ,O.
Callaghan, J.Lee Byrne,F.Mick Tulte and R.L Zerner
;1983).
The protein was reduced in 0.25 (M) Tris- Hcl buffer
(pH 8.5),1.25 mM EDTA (containing 6(M) guanidium
chloride),0.1% (v/v) dithiothreitol at 370
C for 2
hours. Free cysteine residues were alkylated using
10mM idoacetamide for 1 hour at room
temperature in dark. Protein samples were made
excess salt and reagent free by passing the reaction
mixture through a PD 10 desalting column (G.E
Health Care Lifesciences):equilibrated and eluted
with 0.4% Ammonium bicarbonate(pH 8.,5).
Trypsin, Endoproteinase Lys-C and endoproteinase
Glu-C digestions were performed on
carboxamidomethylated ostreolysin sample in 0.4%
ammonium bicarbonate (pH 8.5)at 37
0
C overnight
using protein –substrate ratio of 1:50.Tryptic peptide
mixture was deglycosylated with 0.15 units of N-
glycosidase F (PNGase F)(New England Biolabs) over
night at 37
0
C in presence of 10% Tergitol- type NP-
40.Tryptic peptide mixture was denatured with 1X
Glycoprotein denaturing buffer at 100
0
C for 10 mins.
Similarly, the protein was incubated in 0.4%
Amminium bicarbonate (pH 8.5) with
endoproteinase Lys-C (2 µg/ml) (Sigma Aldrich) and
endoproteinase Glu-C (4 µg/ml)(sigma Aldrich) at 37
0
C overnight. The HPLC fractionation of digest
(20µl,200 p mol) was performed on an HP 1090 A
HPLC fitted with Vydac C-18 Reverse phase.2.1mm X
25 cm column (Grace Vydac);separation was
achieved with a linear gradient of 5-50% acetonitrile
containing 0.1% Trifluoroacetic acid over a period of
60 mins at flowrate of 0.2ml/min. N-terminal protein
sequence analysis was performed using a Perkin –
Elmer Applied Biosystems 477A pulsed –liquid
protein sequencer equipped with model 120 A
phenyl thiohydantion analyser. PTH-amio acids from
the sequencer were separated on 2.1 mm ID
SUPELCOSIL
TM
LC-18-D8 HPLC columns (Sigma-
Aldrich catalogue no.T195867) using 10-50% Triethyl
amine and acetic acid. C-terminal degradation
products of endoproteinase Lys-C and
endoproteinase Glu-C were filtered through Zitex
R
-
G -filter membrane (Saint Gobain performance
plastic) and analysed by same sequenator.
Fig:3 HPLC elution profile of native ostreolysin(by HP1090A fitted with Vydac C-18 column)
Uniprot KB Accession No. P83467
Entry Name - OSTL- PLEOS
Sequence Length – 50AA
Compositional bias 7-10 4 poly – Ile

8. 10 20 30 40 50
A Y A Q W V I I I I
H N V G C Q D V K I
K N L K A C W G K L
H A D G D K D A E V
C A C N W E G K I I
In PBLAST it showed 98% homology with ostreolysin
from P. ostreatus strain V-184 (P83 465) and 50%
with Agrocybe aegerita Aegerolysin Aa-
Pri1(O42717), Moniliophthora perniciosa ( strain
FA553/isolate (CPO2) aegerolysin (E2LQH3); P.
eryngii aegerolysin (E2LMN6).
Model Building and phasing of ostreolysin:
Crystals of ostreolysin were prepared. All data were
collected from crystals at room temperature using
rotation method either on Beam line 6A2 using X-
rays at wavelength 1
0
A or with Cukα X-rays
generated by a Rigaku RU-200 rotating anode
generator ,Diffraction data were processed and
analysed using Denzo (otwinkski 1993),SCALEPACK
and programs in comprehensive computing suite
program for macromolecular crystallography (CCP4
program suit ;1994)
Data collection Statistics:
Data set Native :
PCMBS Hg(AC)2 PIP Uo2 Uo2(No3)2
Table 4: X-ray diffraction data of native ostreolysin:
X-Ray source Beam line Beam line RigaKu RigaKu
6A2 6A2 RU-200 RU-200
Soak time (days)
Soaking concentration (mM)
0.5 . 0. 5 0 .5 1
5 5 1 20

9. No. of crystals
Resolution(0
A)
No. of observations
No of unique reflections
Data completeness(%)
Rmerge(%)
MFID(%)
Sites
Rcullis(%)
(IFHI)/E
1 1 1 1 1
2.7 2.8 3.03 3.03 3.3 3.1
80;907, 65;751, 160; 101, 78;979, 100;880
21;854, 28;344 ,32;461, 19;803 ,22;591
89(94), 81(83), 99(98) ,88(91), 84(87)
8.1(39.2) ,6.2(40.1) ,9.2(28.7),13.8(39.9),13.2(34.0)
17.0, 14.3, 21.3, 25.3
A,DA,EB,FC,G,H
68, 70, 71, 76
1.4(1.3),1.3(1.1),1.0(.8),1.1(.9)
PCMBS-p-choloromercuribenzenesulfonate
Hg(Ac)2 -mercury acetate
PIP-(di-µ-iodobis (ethylenediamine)-diplatinum
(II)nitrate
Uo2(No3)2 –uranyl nitrate
Rmerge= ∑hkI ∑i/Ii –<I>I/I<I>I,where Ii is the intensity
or the i
th
measurement of an equivalent reflection
with indices h,k,I.
MFID=∑II FPH/ -/FPII/∑IFP I where FPH refers to
derivative data and F to native data.
Rcullis =∑FPHC/-/FPHII/∑II FPH /-/FPII
The summation is over all centric reflections. FPHC
and FPH are measured derivative structure factor
and amplitudes respectively. FP is the native
structure factor. <IFHI</E is a measure of phasing
power of the derivative;<IFHI is the rms heavy atom
derivative structure factor amplitude and E is the
lack-of-closure errors.

10. Note: a. The number of unique reflections for the
derivatives includes Bijvoet pairs separately.
b. The values in parenthesis are for highest
resolution bin ( approx .1
0
A interval)
The crystals were soaked in artificial mother liquor
containing the derivative at room temperature. One
major site was located in the isomorphous
difference Patterson of PCMBS derivative.
Subsequent sites were found by cross-difference
Fourier using phases derived from Student
instructional report data (SIR) and from solvent
flattening. Major sites are denoted (A) to (C) and
minor sites (D) and (H). Anamolous scattering data
were collected for 4 derivatives and were used to
establish unequivocally the correct handedness of
the structure. Heavy atom parameters were refined
and phases calculated using maximum likelihood
heavy atom refinement (MLPHAR-E) CCP4 program
suite ; 1994). overall figure-of-merit was 0.58 (for
resolution shell 15 to 3.5
0
A). initial MIR map was of
reasonable quality with some interpretable features.
The program package; Density modification package,
release 2.1 (DM) was used to carry out density
modification . the initial free Rfactor of 53.4% dropped
to 34.5 % after solvent flattering and histogram
matching. The starting model was built into density-
modified electron density map using program O
(Jones et al ;1991) with skeletonized maps. The
initial model crystallographic Rfactor =55.8%
(Rfree=56.3%) was comprised of 5 fragments with
majority built as either X-rays sequence or
polyalanine.
.
Fig4: CD spectrum for native (left) and modified( right) ostreolysin.[ native ostreolysin, 68.3% α helix, 4.7% random
coil, 27% β pleated sheet (10% parallel β pleated sheet and 17% anti parallel β pleated sheet), modified ostreolysin
68.3% α helix, 28.7% β pleated sheet ( 11% parallel β pleared sheet, 17.7% anti parallel β pleated sheet )]
NMR spectroscopy:
Phosphorus -31 wideline NMR measurements were
carried out on a CMX infinity 500 spectromer at a
proton frequency of 500 m,Hz.. Typically 5µmol of
lipid dispersion were used in a 4mm rotor using an
HX Apex probe. A single 90
0
pulse was used for
detection with broad band decoupling at the proton
frequency during acquisition. The 900
pulse length
was 4 µs and strength of photon decoupling field
was 20KHz. Dwell time used was 40µs and 2048
points were collected 31
P chemical shifts are
measured relative to 0 ppm for 10% v/v phosphoric
acid. All the spectra were obtained with 50 Hz line
broadening fir the wide line spectra.

11. Fig 5: NMR spectrum of native ostreolysin
(by Bruker AVANCE
TM
DRX NMR Spectrometer) Fig:6 2D –NMR of native ostreolysin
A NOESY spectrum (Fig6 )of ostreolysin presented as
a contour plot with two frequency areas w1 and w2.
The conventional 1D-NMR spectrum of the
ostreolysin ,which occurs along the diagonal of the
plot (w1=w2) is too crowded with peaks to be directly
interpretable. The off-diagonal so-called peaks ,each
arise from the interaction of the two protons that
are <5
o
A apart in space whose 1D-NMR peaks are
located near horizontal and vertical lines through the
cross peak intersect the diagonal (a Nuclear Over
hauser effect (NOE)) lines to left to the spectrum
shows the extended polypeptide chain with its N and
C with position of 4 protons a to d. the dashed
arrows indicate the diagonal NMR peaks to which
these protons give rise. Cross peaks ,such as i,j, and k
,which are located at intersections of horizontal
vertical lines through two diagonal peaks ,are
indicative of an NOE between corresponding 2
protons indicating that they are <50
A apart.
Phase Refinement:
Phases were improved gradually via a boot
strapping procedure entailing iterative cycles of
model building, refinement using the slow cool
protocol of XPLOR-NIH (Brunger;1993) , phase
combination with SIGMAA (CCP4 program suite;
1994) and further cycles of density modification with
DM (CCP4 program suite ;1994). The final model
contains 50 residues and 4 H2o molecules
,crystallographic Rfactor=59.0% (Rfree= 60%0) for
measurement between infinity and 2.70
A for bond
length and 1.60
for bond angles. The rms deviation
for dihedrals is 26.5 0
and rms on impropers is 1.4 o
,
more than 86% of residues fall in favoured region of
Ramachandran plot, none fall in disallowed regions
(Laskowski et al ;1993).

12. The molecule is composed of 4 discontinuous
domains. Domain 1 (residues 3-5,9-17,22-27,35-37)
has an α/β structure containing a 3 stranded anti
parallel β sheet .Domain 2 (residues 6-8,38-39)
consists of 4 mixed β strands with – 3X,+1,+1
topology. Domain 3 (residues 18-21,28-34) is
comprised of an α/β/α layered structure. The 2
stranded anti parallel sheet is continuation of the
sheet structure in domain 1 that has highly
pronounced curvature centered about the
domain/domain interface. The interface of domain 2
and 3 covering a surface area of 570
A. Domain 2 is
constructed from packing of a helix against the β
sheet of domain 2 and consist of predominantly
polar interactions. Domain 2 is connected to domain
4 through a glycine linker at residue 39. Domain 4
(residue 40-50) is folded into a compact β-sandwich
consisting of 2 and 3 stranded β-sheets. One is anti
parallel with topology +1, 0, -2X,-1 while the other is
of mixed topology -1,+2,+1. The interface between
domain 2 and 4 measures 510
A . Domain 3 consists
of a salt link between Lys 19 of domain 3 and Glu 39
of domain 2. A second salt link connects Lys 29 of
domain 3 ang Glu 46 of domain 4. A number of H-
bonding interactions join Trp 5 ,Trp 27 and Trp 45.
Cys 43 located near the tip of domain 4 ,sandwiched
between a β sheet and Trp 45,which is part of
elongated loop that points into the sheet and it is a
potential cholesterol binding site. Trp 45 is
surrounded by Lys 48,Gln 44 and Trp 27.
Using ligand design program LUDI ( BIOSYM
technologies Inc, SanDiego ,California),it was found
that 3 β-OH group of cholesterol forms H-bond with
Glu-46 and Lys-48. Binding triggers membrane
insertion because loop is hydrophobic together with
aliphatic side chains of cholesterol ,could act as
dagger for penetration. Cys-43 is sandwiched
between one of the β sheets in domain 4 and Trp-45
containing loop. Bulky thiol blocking reagent
methylmethanethiosulfonate (MMTS) disturb tight
packaging of Cys-43 leading to changes in
conformation in Trp-45 containing loop and
inactivation of ostreolysin.
.
Fig:7 a. Ribbon model of native ostreolysin,(the crystals belong to space group C2221 with cell dimensions a=47.8
0
A b=182.0 0
A c=175.5 0
A. There is one monomer in asymmetric unit that corresponds to solvent content of 66% ,
Rfactor= 0.59, Rfree=0.60, Resolution= 2.7 0
A )
b.Ribbon model of modified ostreolysin

13. c.Active site of native ostreolysin
d. Cholesterol binding site of native ostreolysin
Fig 8 : a. Ribbon model of native ostreolysin, b.Ribbon model of modified ostreolysin,(the crystals belong to space
group C2221 with cell dimensions a=47.8 0
A b=182.0 0
A c=175.5 0
A. There is one monomer in asymmetric unit that
corresponds to solvent content of 66% , Rfactor= 0.59, Rfree=0.60, Resolution= 2.7
0
A )
Fig 9: a, Averaged images of ostreolysin monomers obtained by classification of different conformations.
Schematic views (left), negative strain (NS; middle) and cryo-electron microscopy (cryo;right)of two conformations.
b,c, single-particle negative strain reconstructions of ostreolysin monomer( grey surface), with the crystal structure
docked in ,showing rotation (arrow) of the domain 4 relative to the head domain. ,(the crystals belong to space
group C2221 with cell dimensions a=47.8
0
A b=182.0
0
A c=175.5
0
A. There is one monomer in asymmetric unit that
corresponds to solvent content of 66% , Rfactor= 0.59, Rfree=0.60, Resolution= 2.7 0
A )

14. Formation of modified ostreolysin protein with
activity only against cancer cell lines:
Substilysin Carls berg protease (Cθ) (Sigma- Aldrich)
cleaves ostreolysin at Cys-43 of domain 4 releasing
the fragment 43-Cys-Gln-Lys-Ile-Ile-50 present on
anti parallel β strand. The fragment is re- introduced
in the protein under conditions that favour peptide
bond formation but in opposite orientation i.e N-Ile-
Ile-Lys-Glu-Trp-Gln-Cys-c forming a parallel β pleated
sheet in domain 4. The modeling ,phasing, and phase
refinement of the modified ostreolysin were done
and Ramachandran plot of the modified protein
showed 82% residues in favoured regions.
Data collection Statistics :
Data set set native:
PCMBS,Uo2(No3)2 ,PIP, Hg(Ac)2
X –Ray source Beam line Beam line RigaKu RigaKu
6A2 6A2 RU-200 RU-200
Soak time (days)
Soaking concentration (mM)
No. of crystals
Resolution(0
A)
No. of observations
No of unique reflections
Data completeness(%)
0.5 . 0.5 0 .5 1
5 5 1 20
1 1 1 1 1
2.7 2.8 3.03 3.03 3.3 3.1
81,907; 63,748; 163,98; 73,973; 98,880
21,820; 20,321; 28,428; 15,802; 19,592
3σ1(%) ,60(34),51(20),60(18),53(28),51(20)

15. Rmerge(%)
MFID(%)
Sites
Rcullis(%)
(IFHI)/E
8.0(35.6),6.4(38.9),9.0(29.8),13.9(40.4),13.2(32.4)
18.0, 14.1 ,28.3, 26.3
A,DA,EB,FC,G,H
63,75,73,74
1.4(1.2), 1.3(1.2), 1.0(.9), 1.1(.8)
Table 5: X-ray diffraction data of modified ostreolysin
Using Ligand design program LUDI (BIOSYM
technologies Inc,San diego, california ); it was found
at 3β-OH group of cholesterol in modified
ostreolysin can form H-bond with Glu-46 but not
with Lys -48 because its orientation has been
reversed in respect to cholesterol 3 β –OH. However
the loop containing Trp-45 is directed towards the β-
sheet in domain 4. So, modified ostreolysin will
require higher cholesterol concentration for
membrane binding.

16. Fig 9: Ramachandran plot of native ostreolysin( left)
and modified ostreolysin (right).Blue regions show
allowed while green regions show moderately
allowed conformations.
.
Fig:10 Fig:11
Fig 10: .High resolution atomic force micrograph of
native ostreolysin induced pore formation in
hepatocytes.(by CypherTM
atomic force
microscope,magnification 2500X, image resized 100
times)
Fig 11:Electronmicrograph of ostreolysin oligomeric
membrane pore complex showing individual
monomers and their topography a.Hep
G2,b.MCF7,c.CACO,d.MOLT-4,e.HeLa,f.HL-60.(model
H-7100; Hitachi;5000X magnification, image resized
50 times)
Determination of cell viability:
5 weeks old Hep G2 (human liver cancer cell
line),human breast cancer cell line (MCF7) ,human
endometrial adenocarcinoma cell line (Sawano),
human colon carcinoma cell line (CACO-2), human
acute lymphoblastic leukemia cell line (MOLT-4),HL-
60(promyelocytic leukemia cell line), Jurkat (human
T-cell lymphoblast –like cell line),human epithelial
carcinoma cell line (HeLa), and normal hepatocutes
were grown in RPMI1640 media containing 10% FBS
and 20 ng/ml native and Modified ostreolysin at
370
C for 24 hours. Cell were plated in 96-well plates
separately at density of 2X10
4
cells/well. The viable
cells were measured by (3-(4,5-dimethyl thiazol-2yl)-
2,5 diphenyl tetrazolium bromide) (MTT) assay using
a cell titer 96
TM
non-radioactive cell proliferation
assay kit (Promega) by reading absorbance at
490nm. Cell viability was also measured by
quantification of ATP , which indicates metabolically
active cells using a cell Titer –Glo
TM
luminescent cell
viability assay kit (Promega). A negative control was
prepared where cell lines were incubated with
buffer and a positive control was made using 10µM
Valinomycin.
Ultra thin sections of the cells were prepared and
observed using electron microscope (Model H-
7100,Hitachi):
Cell lines No. of viable cells /µl
Ostreolysin Modified ostreolysin
Cos-7 0 0

17. Hep G2 0 0
MCF 7 0 0
Sawano 0 0
CACO-2 0 0
MOLT-4 0 0
HL-60 0 0
Jurkat 0 0
HeLa 0 0
Hepatocytes 0 0.4X104
Positive control
10µM valinomycin
0 0
Negative control 0.4X10
4
0.4X10
4
Table 6: MTT assay to determine cell viability using cell titer 96TM
non-radioactive cell proliferations assay kit
(Promega) using native and Modified ostreolysin (concentration 20 ng/ml).
Cell lines No. of viable cells /µl
Ostreolysin Modified ostreolysin
Cos-7 0 0.43X10
4
Hep G2 0 0
MCF 7 0 0
Sawano 0 0
CACO-2 0 0

18. MOLT-4 0 0
HL-60 0 0
Jurkat 0 0
HeLa 0 0
Hepatocytes 0 0.43X10
4
Positive control
10µM valinomycin
0 0
Negative control 0.43X104
0.43X104
Table 7 : Identification of metabolically active cells by quantification of ATP using Titer GloTM
luminescent cell
viability assay kit (ostreolysin and modified ostreolysin ;concentration used is 20 ng/ml):
Protein Efflux and PI influx:
Cells were plated in 96-well plates at density of
2X10
4
cells/well and cultured over night. After two
washes with phosphate –buffered saline; ostreolysin
and modified ostreolysin (20ng/ml) were added to
cells in DMEM medium without FBS. For
determination of LDH efflux from the cells, the
media was centrifuged to remove floating cells. Next
the resultant supernatant was mixed with solution of
LDH cytotoxicity detection kit (Takara) and optical
densities at 490nm were measured with microplate
reader model 550(Bio-rad). To inhibit LDH efflux ,30
mM PEG (Wako) in DMEM was added to the cells
followed by treatment with both native and
modified ostreolysin for 8 hrs. The amount of leaked
LDH were represented as % of LDH activity obtained
after treatment. In negative control buffer was used
in place of ostreolysin and in the positive control
1%(w/v) Triton x-100 were used. For phosphatidyl
inositol (PI) uptake;cells were grown (2X104
cells/well) on 96 well plates over night and washed
twice with PBS, before PI(final concentration
5µg/ml) in DMEM was added with both native and
modified ostreolysin. Uptake of PI into cells was
measured by FLA-5000 phosphor –Image (Fuji film)
with excitation at 510 nm and emission at 665 nm
.100% PI entry was measured using Triton X-100.
Cell lines % of residual LDH activity obtained
After treatment
Amount of PI uptake (µg/µl)
After treatment
Ostreolysin Modified ostreolysin Ostreolysin Modified ostreolysin
Cos-7 0 100% 5 0

19. Hepatocytes 0 100% 5 0
Hep G2 0 0 5 5
MCF 7 0 0 5 5
Sawano 0 0 5 5
CACO-2 0 0 5 5
MOLT-4 0 0 5 5
HL-60 0 0 5 5
Jurkat 0 0 5 5
HeLa 0 0 5 5
Positive control 0 0 5 µg/µl 5 µg/µl
Negative control 100% 100% 0 0
Table 8:Protein efflux determination using LDH cytotoxicity detection kit
Discussion:
Ostreolysin , has been purified from the fruiting
body of Pleurotus ostreatus strain Plo5 using Q-
sepharose, Superdex
TM
-75 gel filtration, Vydac C-18
reverse- phase HPLC and SDS-PAGE. Similar reports
for purification of ostreolysin has been observed by
others ( Rebolj Katja, Kristina Sepcic ;2008, Sabina
Berne, Christina Potrich,Tom Tirk, Peter Macek,
Gianfranco Menestria;2003). The 16 KDa band
obtained was confirmed by Western blotting with
anti- ostreolysin monoclonal Ab from Pleurotus
ostreatus (Abbiotech LLC). Similar observations has
been made by M. Kreft, R. Zorec, P.Macek
,K.Sepcic;2008). Complete peptide sequence of
ostreolysin by Perkin Elmer Applied Biosystem 477 A
pulsed-liquid protein sequencer gave a 50
aminoacids polypeptide chain with a 4 poly Ile
repeat (7-10). It was deposited in protein database
Uniport KB with accession number P83467. It
showed 98% homology with ostreolysin from
Pleurotus ostreatus strain v-184 (P83465) suggesting
that ostreolysin is conserved in Pleurotus ostreatus
strain. It showed 50% homology with aegerolysin of
Agrocybe aegerita Aa-Pri1 (042717), Monoliophthera
perniciosa (strain FA 553/ isolate CP02 ) (E2LQH3)
and P. eryngii (E2LQH3).
Crystals of ostreolysin soaked in mother liquor
containing the derivative PCMBS,Uo2(No3)2 ,PIP,
Hg(Ac)2 . Diffraction data were collected using Beam
line 6A2 using x rays at wavelength 1
0
A or with Cukα
x-rays generated by a Riga Ku RU-200. Diffraction
data was processed and analysed using DENZO
(Otwinoski,1993), SCALEPACK and CCP4 program suit
;1994. Subsequent sites were found by cross
difference Fouriers using phases derived from SIR
and solvent flattening. Heavy atom parameters were
refined and phases calculated using MLPHAR-E
(CCP4 program suit;1994). Overall figure of merit
was 0.58 (for resolution shell 15 to 3.50
A).DM
release 2.1 was used for density Modification. The
initial Rfactor was 55.8% (Rfree= 56.3%). Phases were
improved gradually via boot strapping procedure
entailing interactive cycles of model building,
refinement using the slow cool protocol of XPLOR-

20. NIH ( Brunegr;1999), phase combination with
SIGMAA (CCP4 program suit,1994) and density
modifications. The final Rfactor was 59.0% (Rfree=60%)
for measurement between infinity and 2.7
0
A for
both native and Modified ostreolysin.
The native ostreolysin is composed of 4
discontinuous domains. Domain 1 ( residues 3-5,9-
17,22-27,35-37) has an α/ß structure containing 3
stranded antiparallel ß sheet. Domain 2 ( residues 6-
8, 38-39) consist of 4 mixed ß strands with -3X;+1;+1
topology (NMR studies). Domain 3 (residues 18-
21,28-34) is comprised of α/ ß/α 3 layered structure
which showed high homology with domains of
Perfringolysin (Jamie Roisjohn,Susanne. C. Feil,
William J. Mckinstry, Rodney K.Twente, Michael W.
Parker; 1997). The 2 stranded antiparallel sheet is
continuation of the sheet structure in domain 1.
Domain 2 is constructed from packing of a helix
against the ß sheet of domain 2 and consist
predominantly of polar interactions. Domain 2 is
connected to domain 3 through a glycine linker at
residue 39. Domain 4 (residues 40-50) is folded into
a compact ß-sandwich consisting of 2 and 3 stranded
sheets. One is antiparallel with topology +1,0,-2X,
(NMR studies). There is a salt link between Lys19 of
domain 3 and Glu 39 of domain 2. A second salt link
connects Lys 29 of domain 3 and Glu 46 of domain 4
. Trp 45 is part of an elongated loop that points into
the sheet. It surrounded by Lys 48, Gln 44 and Trp 27
using ligand design program LUDI( BIOSYS
technologies Inc, San Diego, California). It was found
that 3 ß-OH group of cholesterol forms H-bond with
Glu-46 and Lys-48 of native ostreolysin. A subtilisin
Carlsberg Protease (Cθ) cleaved modified ostreolysin
was prepared which cleaves after Cys 43 of domain 4
releasing the fragment 43-Cys-Gln-Trp-Glu-Lys-Ile-
Ile-50 present on the antiparallel ß-strand. The
strand was reintroduced in the protein but in
opposite orientation ; such that 3 ß-OH group of
cholesterol can form H_bond with Glu-46 but not
with Lys 48 in domain 4 because the orientation on
Lys 48 has been reversed in respect to 3- ß-OH
group. So, Modified ostreolysin required a higher
membrane cholesterol concentration for membrane
insertion. As the membrane cholesterol content of
cancer cell lines was found to be higher due to
upregulation of cholesterol biosynthetic enzyme ß-
hydroxymethyl glutaryl – CoA reductase (ß- HMG-
CoA) and higher concentration of cholesterol
precursor mevalonate. High membrane cholesterol
content activates Akt or PKB kinases by
phosphorylation at serine 473 and Thr 308 and
upregulates anti-apoptotic genes such as Bcl-XL and
FLICE inhibitory proteins (FLIP) preventing apoptosis
and causing cancer (Ying chun Li, MiJung Park, Sang
–Kyu Ye, Chul- Woo Kim, Yong –Nyun –Kim;2006).
In cell viability tests , it was found that native
ostreolysin killed both normal ( monkey kidney
fibroblast cell line, COS-7 and normal hepatocytes )
as well as cancer cell lines like Hep G2 (human liver
cancer cell line) , MCF7 (human breast cancer cell
line), Sawano (human endometrial adenocarcinoma
cell line), MOLT-4 (human acute lymphoblastic
leukemia cell line ), HL-60 ( pro-myelocytic leukemia
cell line ) and HeLa (human epithelial carcinoma cell
lines) but modified ostreolysin killed only the cancer
cell lines due to their high membrane cholesterol
content at 20ng/ml concentrations but not normal
cell lines. Cell viability was studied bt MTT assay
using a cell titer 96
TM
non radioactive cell
proliferation assay kit (Promega) (which is based on
reduction of MTT to purple formazon by reductase
present in living cells) and by Titer –Glo TM
luminescent cell viability assay kit (Promega) (which
is based on quantification of ATP in viable cells . In
positive control maximum cell death observed using
10 µM valinomycin and negative control no cell
death was observed ( Mosmann , Tim ;1983).
Protein efflux was studied by % of residual LDH
activity after treatment with ostreolysin and
modified ostreolysin. Native ostreolysin at 20 ng/ml
concentration causes membrane pore formation in
both normal and cancer cell lines showing no
residual LDH activity but modified ostreolysin
showed 100% residual LDH activity in normal cells

21. and 0% in cancer cells suggesting that it specifically
kills cancer cells. Phosphatidyl inositol (PI) influx was
measured to study the ostreolysin induced
membrane pore formation and influx of molecules
from surrounding media. Modified ostreolysin
showed maximum PI uptake in all cancer cells in
comparison with positive control (using 10 µM
Valinomycin) and no PI uptake in normal cells
suggesting that it specifically make pores in cancer
cells. Native ostreolysin showed PI uptake in all cell
lines.

22. References:
1. Berne S, PohlevenJJ, Vidic I Rebolj K, Pohleven F, Turk T, Macek P (2007), Ostreolysin enhances fruiting initiation in oyster
mushroom (Pleurotus ostreatus ) , Mycol Res ;Dec 11(pt 12): 1431-6
2. Rebolj Katja, Sepcic Kristina (2008); Ostreolysin, a cytolytic protein from culinary medicinal oyster mushroom Pleurotus
ostreatus (Jacq:Fr) P. Kumm ( Agaricomycetideae) and its potential use in medicine and Biotechnology, International journal
of medicinal Mushroom, vol-10, issue -4:121-128
3. Sepcic kristina, Berne Sabima, Potrich Christina, Turk Tom , Macck Peter, Menestrina Gianfrance(2003), Interaction of
ostreolysin ;a catalytic protein from edible mushroom Pleurotus ostreatus ,with lipid membranes and modulation by
lysophospholipids , Eur J. Biochem; 270(6): 1199-2100
4. Chowdhury Helena H, Robolj Katja, Kreft Marko, Zoreco Robert, Macck Peter ans Sepcic Kristina (2008), lysophospholipids
prevent binding of cytolytic protein ostreolysin to cholesterol-enriched membrane domains, Toxicon ,51(8): 1345-56
5. Rebolj katja, Poklar Natasa, Macek Peter, Sepcic Kristina (2006),steroid structural requirements for interaction of
ostreolysin,a lipid-reft binding cytolysin , with lipid mono and bilayers , Biochem. Biophys.Acta ,1758:1662-70
6. Rossjohn Jamie, Feil Susanne C, Mekinstry Willam J, Twenten Rodney K, Parker Michael W, (1997), structure of cholesterol
binding ,thiol activated cytolysin and a model of its membrane form ,Cell, 89(5): 685-692
7. Mosmann Tim (1983), Rapid colorimetric assay for cellular growth and survival , application to proliferation and cytotoxicity
assays, Journal of Immunological methods , 65 (1-2):55-63
8. Fischer L, Work T. S, Burdon R.H (1980) Laboratory Techniques in Biochemistry and Molecular biology, Volume 1, part-2,
Biomedical Press.