451
Biochimica et Biophysica Acta, 585 (1979) 451--461 © Elsevier/North-Holland Biomedical Press
BBA 28919
A CYTOLYTIC PROTEIN FROM THE EDIBLE MUSHROOM, PLEUROTUS OSTREATUS
ALAN W. BERNHEIMER and LOIS S. AVIGAD
Department of Microbiology, New York University School of Medicine, New York, N Y 10016 (U.S.A.) (Received October 23rd, 1978)
Key words: Cytolysin; Hemolysin; Sphingomyelin; (Pleurotus ostreatus)
Summary Aqueous extracts of the edible mushroom, Pleurotus ostreatus, contain a substance that is lyric in vitro for mammalian erythrocytes. The hemolytic agent, pleurotolysin, was purified to homogeneity and found to be a protein lacking seven of the amino acids commonly found in proteins. In the presence of sodium dodecyl sulfate it exists as monomers of molecular weight 12 050 whereas under non-dissociating conditions it appears to exist as dimers. It is isoelectric at about pH 6.4. The sensitivity of erythrocytes from different animals correlates with sphingomyelin content of the erythrocyte membranes. Sheep erythrocyte membranes inhibit pleurotolysin-induced hemolysis and the inhibition is time and temperature dependent. Ability of membranes to inhibit hemolysis is abolished by prior treatment of membranes with specific phospholipases. Pleurotolysin-induced hemolysis is inhibited by liposomes prepared from cholesterol, dicetyl phosphate and sphingomyelin derived from sheep erythrocytes whereas a variety of other lipid preparations fail to inhibit. It is concluded that sphingomyelin plays a key role in the hemolytic reaction.
Introduction Aqueous extracts of edible mushrooms belonging to the genus Pleurotus are capable of lysing washed mammalian erythrocytes [1,2]. The potential commercial importance of Pleurotus and the development of methods for its cultivation [3--5] led us to investigate the nature and properties of the lytic agent here designated pleurotolysin. The results show that the substance responsible for cytotoxicity is a protein of unusual amino acid composition and that it differs in this and in other ways from cytolytic proteins that have been isolated from the basidiocarps of other kinds of gilled fungi.
452 Materials and Methods Pleurotus basidiocarps. Source A. Mycelium designated Pleurotus ostreatus and cultured in what appeared to be a mixture of hardwood sawdust and cereal grain [5] was purchased from Kinoko International {P.O. Box 6425, Oakland, CA 94621). Fruiting was induced by rehydrating and humidifying according to directions provided by the supplier. Source B. P. ostreatus strain Cloquet 3A, was aseptically cultivated at 20--23°C on p o t a t o dextrose agar (Difco Laboratories, Detroit, MI), 600 ml of m e d i u m / 2 8 0 0 ml Fernbach :]ask. Extracts from both sources contained approximately the same hemolytic activity, and the yield of purified p r o d u c t was approximately the same regardless of source. For the isolation o f pleurotolysin it was found necessary to use fresh material. Basidiocarps stored at --20°C for several months yielded no activity. Estimation o f hemolytic activity. Test solutions were diluted in 0.145 M NaC1/0.01 M Tris (pH 7.2) (buffer I). Volumes of toxin dilutions decreasing by a b o u t 25% were delivered into tubes (12 × 75 mm), and the volume in all tubes was brought to 1 ml by addition of the diluent. To each tube was added 1 ml washed sheep erythrocytes suspended in buffer 2. The density of the e r y t h r o c y t e suspension was adjusted to given an absorbance of 0.8 at 545 nm when complete lysis occurred. After mixing, the tubes were incubated at 37 ° C for 30 min and then briefly centrifuged. The percentage of hemolysis was estimated from the color of the hemoglobin in the supernatants as compared with that of standards. One hemolytic unit is that amount of test material needed to release the hemoglobin from 50% of the cells. Unless otherwise specified, sheep erythrocytes were used. Experiments were repeated at least once in order to test the reproducibility of the measurements. Inclusion in the system of either 5 mM CaC12 or 5 mM MgC12 did not affect the hemolytic activity. Amino acid analysis. Pleurotolysin (0.021 absorbance units) was hydrolyzed at l l 0 ° C for 22, 36 and 48 and 72 h in 6 N HC1 and approximately 0.05 mM phenol. Analysis was carried out in an automatic amino acid analyzer (Model D500, Durrum Instrument, Sunnyvale, CA), with the program set at 2.5 nm. Tryptophan was determined spectrophotometrically [6]. Erythrocyte membranes. Membranes from erythrocytes of sheep and rabbit were prepared osmotically according to method B of Ref. 7. They were stored at --20°C until used. Reagents. Staphylococcal sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) was prepared as in Ref. 8. Phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) from Corynebacterium ovis [9] was purified as described [10]. Sphingomyelin from beef liver, phosphatidylethanolamine and gangliosides were purchased from Sigma Chemical Co. (St. Louis, MO), sphingomyelin from sheep erythrocytes from Supelco Inc. (Bellefonte, PA), phosphatidylcholine and diphosphatidylglycerol from Sylvana Co. (Milburn, NJ) and phosphatidylserine from Applied Science Laboratories (State College, PA). Gangliosides and sphingomyelin were used as dispersions in buffer I prepared with the aid of a teflon bead and a cyclo-mixer (Clay-Adams, Inc., Parsippany, NJ). Dispersions of phospholipids other than sphingomyelin were prepared by drying in vacuo ethanol or benzene solutions as thin films, and suspending the films at a con-
453
centration of 1 mg/ml buffer I with the aid of a cyclo-mixer. Liposomes were prepared as described [10]. Results
Isolation of pleurotolysin Fractionations were done at a b o u t 4 ° C. (A) 64 g of basidiocarps (source A) were cut into small pieces and homogenized 60 s with 100 ml 0.9% NaC1 in a blender (Cuisinarts Inc., P.O. Box 352, Greenwich, CT 06830). The mixture was centrifuged at 15 000 rev./min for 10 min, and the sediment was re-homogenized with a second 100 ml 0.9% NaC1. After centrifugation the t w o supernatant fluids were pooled. (B) 105 g of ammonium sulfate were dissolved in the crude extract to make it approximately 70% saturated. After 30 min the mixture was centrifuged at 14 500 X g for 10 min. The precipitate was extracted with t w o 50 ml portions of 43% saturated ammonium sulfate (Table I). (C) The combined extract was brought to 70% saturation b y dissolving in it 19.9 g ammonium sulfate. After 15 min the mixture was centrifuged at 12 000 × g for 10 min, and the supernate was discarded. The precipitate was dissolved in 10 ml 50% glycerol. (D) The glycerol solution was dialyzed against 20 volumes of 50% glycerol for 3 h followed by electrofocusing for 40 h. The latter was done with 1% (v/v) ampholine, pH 5--7, in a 440 ml column (LKB Produkter, Sweden), a 5--50% sucrose gradient and a final potential difference of 750 V with the cathode at the t o p of the column. Fractions (Fig. 1) of 16 ml each were collected. The four most active fractions (pH 6.2--6.5) were pooled. (E) The pool was dialyzed for 72 h against 600 ml saturated ammonium sulfate. The precipitate was collected b y centrifugation and suspended in 1 ml saturated ammonium sulfate for storage. Portions were freed of ammonium sulfate b y dialysis against distilled water as required. Amounts of pleurotolysin
TABLE I PURIFICATION SCHEME FOR PLEUROTOLYSIN Stage
Volume (ml)
A. C r u d e e x t r a c t B. 43% s a t u r a t e d ammonium sulfate extract C. 70% s a t u r a t e d ammonium sulfate e x t r a c t D. E l e c t r o f o c u s e d activity (fraction 19--22) E. A m m o n i u m sulfate precipitate
200 100
Total hemolytic units
Activity recovered (%)
A280/ ml
A 280 ] A260
250 225
50 0 0 0 22 5 0 0
100 45
17 6.1
0.49 0.66
14.7 36.9
11
2000
22 0 0 0
44
32.5
0.76
61.5
64
171
10 9 0 0
22
0.60
0.51
285
5800
6 670
13
4.94
1.50
1174
1.15
Hemolytic units/ml
Hemolytic units/ A 280
454 1i ~° DD
}8
c o (n
~,' II
I'I '~ /'D~
o E c 0
"
I,
oD
oJ 4
6
8
I0
12
14
FrCIctlon
16
18 2 0
22 2 4
26
28 50
number
Fig. 1. I s o e l e c t r i c f o c u s i n g o f p l e u r o t o l y s i n . l y t i c a c t i v i t y ( o ) a n d p H (a).
Fractions were examined
for 2 8 0 n m a b s o r b a n c e
(o), h e m o -
are expressed as either absorbance units (280 nm absorbance using 10 mm light path) or hemolytic units.
Analysis by gel electrophoresis Samples of pleurotolysin were subjected to electrophoresis in polyacrylamide gels. Under both non
Biochimica et Biophysica Acta, 585 (1979) 451--461 © Elsevier/North-Holland Biomedical Press
BBA 28919
A CYTOLYTIC PROTEIN FROM THE EDIBLE MUSHROOM, PLEUROTUS OSTREATUS
ALAN W. BERNHEIMER and LOIS S. AVIGAD
Department of Microbiology, New York University School of Medicine, New York, N Y 10016 (U.S.A.) (Received October 23rd, 1978)
Key words: Cytolysin; Hemolysin; Sphingomyelin; (Pleurotus ostreatus)
Summary Aqueous extracts of the edible mushroom, Pleurotus ostreatus, contain a substance that is lyric in vitro for mammalian erythrocytes. The hemolytic agent, pleurotolysin, was purified to homogeneity and found to be a protein lacking seven of the amino acids commonly found in proteins. In the presence of sodium dodecyl sulfate it exists as monomers of molecular weight 12 050 whereas under non-dissociating conditions it appears to exist as dimers. It is isoelectric at about pH 6.4. The sensitivity of erythrocytes from different animals correlates with sphingomyelin content of the erythrocyte membranes. Sheep erythrocyte membranes inhibit pleurotolysin-induced hemolysis and the inhibition is time and temperature dependent. Ability of membranes to inhibit hemolysis is abolished by prior treatment of membranes with specific phospholipases. Pleurotolysin-induced hemolysis is inhibited by liposomes prepared from cholesterol, dicetyl phosphate and sphingomyelin derived from sheep erythrocytes whereas a variety of other lipid preparations fail to inhibit. It is concluded that sphingomyelin plays a key role in the hemolytic reaction.
Introduction Aqueous extracts of edible mushrooms belonging to the genus Pleurotus are capable of lysing washed mammalian erythrocytes [1,2]. The potential commercial importance of Pleurotus and the development of methods for its cultivation [3--5] led us to investigate the nature and properties of the lytic agent here designated pleurotolysin. The results show that the substance responsible for cytotoxicity is a protein of unusual amino acid composition and that it differs in this and in other ways from cytolytic proteins that have been isolated from the basidiocarps of other kinds of gilled fungi.
452 Materials and Methods Pleurotus basidiocarps. Source A. Mycelium designated Pleurotus ostreatus and cultured in what appeared to be a mixture of hardwood sawdust and cereal grain [5] was purchased from Kinoko International {P.O. Box 6425, Oakland, CA 94621). Fruiting was induced by rehydrating and humidifying according to directions provided by the supplier. Source B. P. ostreatus strain Cloquet 3A, was aseptically cultivated at 20--23°C on p o t a t o dextrose agar (Difco Laboratories, Detroit, MI), 600 ml of m e d i u m / 2 8 0 0 ml Fernbach :]ask. Extracts from both sources contained approximately the same hemolytic activity, and the yield of purified p r o d u c t was approximately the same regardless of source. For the isolation o f pleurotolysin it was found necessary to use fresh material. Basidiocarps stored at --20°C for several months yielded no activity. Estimation o f hemolytic activity. Test solutions were diluted in 0.145 M NaC1/0.01 M Tris (pH 7.2) (buffer I). Volumes of toxin dilutions decreasing by a b o u t 25% were delivered into tubes (12 × 75 mm), and the volume in all tubes was brought to 1 ml by addition of the diluent. To each tube was added 1 ml washed sheep erythrocytes suspended in buffer 2. The density of the e r y t h r o c y t e suspension was adjusted to given an absorbance of 0.8 at 545 nm when complete lysis occurred. After mixing, the tubes were incubated at 37 ° C for 30 min and then briefly centrifuged. The percentage of hemolysis was estimated from the color of the hemoglobin in the supernatants as compared with that of standards. One hemolytic unit is that amount of test material needed to release the hemoglobin from 50% of the cells. Unless otherwise specified, sheep erythrocytes were used. Experiments were repeated at least once in order to test the reproducibility of the measurements. Inclusion in the system of either 5 mM CaC12 or 5 mM MgC12 did not affect the hemolytic activity. Amino acid analysis. Pleurotolysin (0.021 absorbance units) was hydrolyzed at l l 0 ° C for 22, 36 and 48 and 72 h in 6 N HC1 and approximately 0.05 mM phenol. Analysis was carried out in an automatic amino acid analyzer (Model D500, Durrum Instrument, Sunnyvale, CA), with the program set at 2.5 nm. Tryptophan was determined spectrophotometrically [6]. Erythrocyte membranes. Membranes from erythrocytes of sheep and rabbit were prepared osmotically according to method B of Ref. 7. They were stored at --20°C until used. Reagents. Staphylococcal sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) was prepared as in Ref. 8. Phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) from Corynebacterium ovis [9] was purified as described [10]. Sphingomyelin from beef liver, phosphatidylethanolamine and gangliosides were purchased from Sigma Chemical Co. (St. Louis, MO), sphingomyelin from sheep erythrocytes from Supelco Inc. (Bellefonte, PA), phosphatidylcholine and diphosphatidylglycerol from Sylvana Co. (Milburn, NJ) and phosphatidylserine from Applied Science Laboratories (State College, PA). Gangliosides and sphingomyelin were used as dispersions in buffer I prepared with the aid of a teflon bead and a cyclo-mixer (Clay-Adams, Inc., Parsippany, NJ). Dispersions of phospholipids other than sphingomyelin were prepared by drying in vacuo ethanol or benzene solutions as thin films, and suspending the films at a con-
453
centration of 1 mg/ml buffer I with the aid of a cyclo-mixer. Liposomes were prepared as described [10]. Results
Isolation of pleurotolysin Fractionations were done at a b o u t 4 ° C. (A) 64 g of basidiocarps (source A) were cut into small pieces and homogenized 60 s with 100 ml 0.9% NaC1 in a blender (Cuisinarts Inc., P.O. Box 352, Greenwich, CT 06830). The mixture was centrifuged at 15 000 rev./min for 10 min, and the sediment was re-homogenized with a second 100 ml 0.9% NaC1. After centrifugation the t w o supernatant fluids were pooled. (B) 105 g of ammonium sulfate were dissolved in the crude extract to make it approximately 70% saturated. After 30 min the mixture was centrifuged at 14 500 X g for 10 min. The precipitate was extracted with t w o 50 ml portions of 43% saturated ammonium sulfate (Table I). (C) The combined extract was brought to 70% saturation b y dissolving in it 19.9 g ammonium sulfate. After 15 min the mixture was centrifuged at 12 000 × g for 10 min, and the supernate was discarded. The precipitate was dissolved in 10 ml 50% glycerol. (D) The glycerol solution was dialyzed against 20 volumes of 50% glycerol for 3 h followed by electrofocusing for 40 h. The latter was done with 1% (v/v) ampholine, pH 5--7, in a 440 ml column (LKB Produkter, Sweden), a 5--50% sucrose gradient and a final potential difference of 750 V with the cathode at the t o p of the column. Fractions (Fig. 1) of 16 ml each were collected. The four most active fractions (pH 6.2--6.5) were pooled. (E) The pool was dialyzed for 72 h against 600 ml saturated ammonium sulfate. The precipitate was collected b y centrifugation and suspended in 1 ml saturated ammonium sulfate for storage. Portions were freed of ammonium sulfate b y dialysis against distilled water as required. Amounts of pleurotolysin
TABLE I PURIFICATION SCHEME FOR PLEUROTOLYSIN Stage
Volume (ml)
A. C r u d e e x t r a c t B. 43% s a t u r a t e d ammonium sulfate extract C. 70% s a t u r a t e d ammonium sulfate e x t r a c t D. E l e c t r o f o c u s e d activity (fraction 19--22) E. A m m o n i u m sulfate precipitate
200 100
Total hemolytic units
Activity recovered (%)
A280/ ml
A 280 ] A260
250 225
50 0 0 0 22 5 0 0
100 45
17 6.1
0.49 0.66
14.7 36.9
11
2000
22 0 0 0
44
32.5
0.76
61.5
64
171
10 9 0 0
22
0.60
0.51
285
5800
6 670
13
4.94
1.50
1174
1.15
Hemolytic units/ml
Hemolytic units/ A 280
454 1i ~° DD
}8
c o (n
~,' II
I'I '~ /'D~
o E c 0
"
I,
oD
oJ 4
6
8
I0
12
14
FrCIctlon
16
18 2 0
22 2 4
26
28 50
number
Fig. 1. I s o e l e c t r i c f o c u s i n g o f p l e u r o t o l y s i n . l y t i c a c t i v i t y ( o ) a n d p H (a).
Fractions were examined
for 2 8 0 n m a b s o r b a n c e
(o), h e m o -
are expressed as either absorbance units (280 nm absorbance using 10 mm light path) or hemolytic units.
Analysis by gel electrophoresis Samples of pleurotolysin were subjected to electrophoresis in polyacrylamide gels. Under both non
