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 SDSPAGE. 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 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.

Vo- void volume

Abbreviation:
SDS-PAGE- sodium dodecyl sulphate poly acrylamide gel electrophoresis EDTA-ethylene diamine tetra acetic acid

HPLC- High performance liquid chromatography PTH- phenythiohydantoin MTT-3-(4,5- dimethyl thiazol-zyl)-2,5 diphenyl tetrazolium bromide) ATP- adenosine triphosphate

Ab- Antibody PI-phosphatidyl inositol HRP- Horse raddish peroxidase LDH- lactate hehydrogenase Ve- elusion volume

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 - 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 cylodextrin dramatically decreased ostreolysin binding. Immunostaining showed that ostreolysin is not co-localised with raft-binding proteins, cholera toxin -subunit or caveolin suggestiong that natural membranes display heterogeneity of cholesterol enriched raftlike 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, sitosterol, stigmasterol, lonosterol, 7dehydrocholesterol, cholesteryl acetate and 5 cholestene 3-one,in 1/1 molar ratio .Lytic activity is dependent on sterol 3-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 3-OH group of cholesterol forms H-bond with Glu46 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 (C) which digests ostreolysin at Cys43 of domain 4 resulting is release of the anti parallel -strand carrying 43-Cys-Gln-Trp-, Glu-Lys-Ile-Ile-50 and re

introduced in the protein but in opposite orientation ; forming a parallel strand in the m odified ostreolysin. Using LUDI design program, it was found that 3-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-OH 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),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 ( TM Abazyme), Superdex -75 (separation range 300070000, 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), Dulbeecos Modified Eagles Medium (GIBCO BRL, catalogue no. 31600, Grand Island , NY), Insulin like growth factor- (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 nonradioactive cell proliferation Assay kit (Promega), TM Titer- Glo 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 (MLPHARE).

refinement

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) - mercaptoethanol , 2 mM Benzamidine, 2 g/ml Leupeptin (extraction buffer) and centrifuged 0 at 10,000 rpm for 15min at 15 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 TM superdex -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 0.606

543

49.124

1.975

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) 2.Bovine Pancreatic chymotrypsin (20.6

550

49.746

2.0

0.020102

542

49

1.970

0.020408

K Da) 3.Chicken Lactate dehydrogenase (H) (150 KDa) 4.Horse liver catalase (222KDa) 5.Urease (Pleurotus sajor-caju)(450 KDa) 6.Squid Haemocyanin (612 KDa) 440 39.78 1.60 0.025138

311

28.18

1.45

0.0355

55

4.97

0.75

0.502513

10

1.99

0.3

0.502513

Tube No. 10 55 311 440 542 543 550

Volume of elution buffer required (Ve)(ml) 11.0 ml (Squid haemocyanin) 27.51 ml (P. sajor-caju urease) 155.8 ml (Horse liver Catalase) 220 ml(Chicken lactate dehydrogenase(H) ) 271ml (Bovine pancreatic chymotrypsin) 271.66ml (Pleurotus Otreatus strain Plo5 ostreolysin) 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 Gilsons prep FC collector. Protein concentration of tube 543 containing ostreolysin was found to be 1.64 g/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).
TM

Peptide sequencing of ostreolysin:

The purified ostreolysin was incubated with 0.4 mM Ellmans reagent (5,5 dithiobis (2 nitrobenzoic acid)), 6 (M) urea, 0.1 mM Na2EDTA and 100 mM 0 Tris- HCl buffer (pH 8.0) for 30 min at 25 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 Nglycosidase F (PNGase F)(New England Biolabs) over 0 night at 37 C in presence of 10% Tergitol- type NP-

40.Tryptic peptide mixture was denatured with 1X 0 Glycoprotein denaturing buffer at 100 C for 10 mins. Similarly, the protein was incubated in 0.4% Amminium bicarbonate (pH 8.5) with endoproteinase Lys-C (2 g/ml) (Sigma Aldrich) and 0 endoproteinase Glu-C (4 g/ml)(sigma Aldrich) at 37 C overnight. The HPLC fractionation of digest (20l,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 (SigmaAldrich 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

Model Building and phasing of ostreolysin:

10 20 30 40 50 AYAQWV II I I HNVGCQDVKI K NLKACWGKL HADGDKDAEV CACNWEGKII In PBLAST it showed 98% homology with ostreolysin from P. ostreatus strain V-184 (P83 465) and 50% with Agrocybe aegerita Aegerolysin AaPri1(O42717), Moniliophthora perniciosa ( strain FA553/isolate (CPO2) aegerolysin (E2LQH3); P. eryngii aegerolysin (E2LMN6). Crystals of ostreolysin were prepared. All data were collected from crystals at room temperature using rotation method either on Beam line 6A2 using X0 rays at wavelength 1 A or with Cuk 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 6A2 Beam line 6A2 RigaKu RU-200 RigaKu RU-200

Soak time (days) Soaking concentration (mM)

0.5 . 5

0. 5 5

0 .5 1 20

No. of crystals Resolution(0A) No. of observations

1 2.7

1 1 2.8 3.03

1 3.03 3.3

1 3.1

80;907, 65;751, 160; 101, 78;979, 100;880

No of unique reflections

21;854, 28;344 ,32;461, 19;803 ,22;591

Data completeness(%)

89(94), 81(83), 99(98) ,88(91), 84(87)

Rmerge(%)

8.1(39.2) ,6.2(40.1) ,9.2(28.7),13.8(39.9),13.2(34.0)

MFID(%)

17.0, 14.3, 21.3, 25.3

Sites

A,DA,EB,FC,G,H

Rcullis(%)

68, 70, 71, 76

(IFHI)/E PCMBS-p-choloromercuribenzenesulfonate Hg(Ac)2 -mercury acetate

1.4(1.3),1.3(1.1),1.0(.8),1.1(.9) MFID=II FPH/ -/FPII/IFP I where FPH refers to derivative data and F to native data. Rcullis =FPHC/-/FPHII/II FPH /-/FPII

PIP-(di--iodobis (II)nitrate

(ethylenediamine)-diplatinum 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.

Uo2(No3)2 uranyl nitrate Rmerge= hkI i/Ii <I>I/I<I>I,where Ii is the intensity th or the i measurement of an equivalent reflection with indices h,k,I.

Note: a. The number of unique reflections for the derivatives includes Bijvoet pairs separately. b. The values in parenthesis are for highest 0 resolution bin ( approx .1 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 0 resolution shell 15 to 3.5 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 densitymodified 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% helix, 4.7% random coil, 27% pleated sheet (10% parallel pleated sheet and 17% anti parallel pleated sheet), modified ostreolysin 68.3% helix, 28.7% pleated sheet ( 11% parallel pleared sheet, 17.7% anti parallel 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 5mol of lipid dispersion were used in a 4mm rotor using an 0 HX Apex probe. A single 90 pulse was used for detection with broad band decoupling at the proton frequency during acquisition. The 900 pulse length was 4 s and strength of photon decoupling field was 20KHz. Dwell time used was 40s 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 / structure containing a 3 stranded anti parallel sheet .Domain 2 (residues 6-8,38-39) consists of 4 mixed strands with 3X,+1,+1 topology. Domain 3 (residues 18-21,28-34) is comprised of an // 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 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 -sandwich consisting of 2 and 3 stranded -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 Hbonding interactions join Trp 5 ,Trp 27 and Trp 45. Cys 43 located near the tip of domain 4 ,sandwiched between a 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 -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 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 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 0 corresponds to solvent content of 66% , Rfactor= 0.59, Rfree=0.60, Resolution= 2.7 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 0 0 0 group C2221 with cell dimensions a=47.8 A b=182.0 A c=175.5 A. 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:

Data collection Statistics : Data set set native:

PCMBS,Uo2(No3)2 ,PIP, Hg(Ac)2

Substilysin Carls berg protease (C) (Sigma- Aldrich) cleaves ostreolysin at Cys-43 of domain 4 releasing the fragment 43-Cys-Gln-Lys-Ile-Ile-50 present on anti parallel strand. The fragment is re- introduced in the protein under conditions that favour peptide bond formation but in opposite orientation i.e N-IleIle-Lys-Glu-Trp-Gln-Cys-c forming a parallel 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. X Ray source Beam line 6A2 Beam line 6A2 RigaKu RU-200 RigaKu RU-200

Soak time (days) Soaking concentration (mM) No. of crystals Resolution(0A) No. of observations

0.5 . 5 1 2.7

0.5 5 1 1 2.8 3.03 1 1

0 .5 20 1 3.3

3.03

3.1

81,907; 63,748; 163,98; 73,973; 98,880

No of unique reflections

21,820; 20,321; 28,428; 15,802; 19,592

Data completeness(%)

31(%) ,60(34),51(20),60(18),53(28),51(20)

Rmerge(%)

8.0(35.6),6.4(38.9),9.0(29.8),13.9(40.4),13.2(32.4)

MFID(%)

18.0, 14.1 ,28.3, 26.3

Sites

A,DA,EB,FC,G,H

Rcullis(%)

63,75,73,74

(IFHI)/E

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 3-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 OH. However the loop containing Trp-45 is directed towards the 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 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),HL60(promyelocytic leukemia cell line), Jurkat (human

Fig:11 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 10M Valinomycin. Ultra thin sections of the cells were prepared and observed using electron microscope (Model H7100,Hitachi):

Cell lines Ostreolysin Cos-7 0

No. of viable cells /l Modified ostreolysin 0

Hep G2 MCF 7 Sawano CACO-2 MOLT-4 HL-60 Jurkat HeLa Hepatocytes Positive control 10M valinomycin Negative control

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0.4X104 0

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 Ostreolysin Cos-7 Hep G2 MCF 7 Sawano CACO-2 0 0 0 0 0

No. of viable cells /l Modified ostreolysin 0.43X10 0 0 0 0

4

MOLT-4 HL-60 Jurkat HeLa Hepatocytes Positive control 10M valinomycin Negative control

0 0 0 0 0 0

0 0 0 0 0.43X10 0
4

0.43X104

0.43X104

Table 7 : Identification of metabolically active cells by quantification of ATP using Titer Glo TM 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 5g/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 Ostreolysin Modified ostreolysin 100%

Amount of PI uptake (g/l) After treatment Ostreolysin 5 Modified ostreolysin 0

Cos-7

Hepatocytes Hep G2 MCF 7 Sawano CACO-2 MOLT-4 HL-60 Jurkat HeLa Positive control Negative control

0 0 0 0 0 0 0 0 0 0 100%

100% 0 0 0 0 0 0 0 0 0 100%

5 5 5 5 5 5 5 5 5 5 g/l 0

0 5 5 5 5 5 5 5 5 5 g/l 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 QTM sepharose, Superdex -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 0 line 6A2 using x rays at wavelength 1 A or with Cuk 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%) 0 for measurement between infinity and 2.7 A for both native and Modified ostreolysin. The native ostreolysin is composed of 4 discontinuous domains. Domain 1 ( residues 3-5,917,22-27,35-37) has an / structure containing 3 stranded antiparallel sheet. Domain 2 ( residues 68, 38-39) consist of 4 mixed strands with -3X;+1;+1 topology (NMR studies). Domain 3 (residues 1821,28-34) is comprised of / / 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 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 -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 -OH group of cholesterol forms H-bond with Glu-46 and Lys-48 of native ostreolysin. A subtilisin Carlsberg Protease (C) cleaved modified ostreolysin was prepared which cleaves after Cys 43 of domain 4 releasing the fragment 43-Cys-Gln-Trp-Glu-Lys-IleIle-50 present on the antiparallel -strand. The strand was reintroduced in the protein but in opposite orientation ; such that 3 -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- -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 hydroxymethyl glutaryl CoA reductase (- HMGCoA) 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 M 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 M 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.

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