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

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.

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- GloTM luminescent cell viability Assay

kit(Promega).

B. Software programs for macromolecular

crystallography: DM density modification package

release 2.1, CCP4 (comprehensive computing suit

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 150

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 superdex TM

-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

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

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 Plo5 ostreolysin)

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 250

C

.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 370 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 370 C in presence of 10% Tergitol- type NP-

40.Tryptic peptide mixture was denatured with 1X

Glycoprotein denaturing buffer at 1000C 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 370

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

SUPELCOSILTM 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 ZitexR -

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

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 10 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

No. of crystals

Resolution(0A)

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 ith

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.

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 3.50A). 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 900 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 31P 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.

Fig 5: NMR spectrum of native ostreolysin

(by Bruker AVANCETM 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 <5oA 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).

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 570A. 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 510A . 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 0A b=182.0

0A c=175.5

0A. There is one monomer in asymmetric unit that corresponds to solvent content of 66% ,

Rfactor= 0.59, Rfree=0.60, Resolution= 2.7 0A )

b.Ribbon model of modified ostreolysin

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 0A b=182.0 0A c=175.5 0A. There is one monomer in asymmetric unit that

corresponds to solvent content of 66% , Rfactor= 0.59, Rfree=0.60, Resolution= 2.7 0A )

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 0A b=182.0

0A c=175.5

0A. There is one monomer in asymmetric unit that

corresponds to solvent content of 66% , Rfactor= 0.59, Rfree=0.60, Resolution= 2.7 0A )

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(0A)

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)

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.

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

370C for 24 hours. Cell were plated in 96-well plates

separately at density of 2X104 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 96TM 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 –GloTM 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

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 0.4X104 0.4X104

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 0.43X104

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 0.43X104

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

2X104 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

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 10A 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-

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 2.70A 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

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.

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

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