JP2752684B2 - Hemolytic protein from the fruit body of Oyster mushroom - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a protein obtained from basidiomycetes, and more specifically, a novel and useful bioactive protein derived from the fruiting body of Pleurotus ostreatus, a kind of basidiomycete About.

(Problems to be solved by the prior art and the invention) When basidiomycetes are examined for extracts of their fruiting bodies, many basidiomycetes contain hemolysin in addition to hemagglutinin (lectin), and their broad distribution is A major feature of this highly developed fungus. A typical hemolysin is Phallolysin, a fruiting body of Amanita phalloides, whose presence was reported in the late 19th century. The highly toxic protein was later rediscovered by Finne et al. In 1967, but many researchers have since refined the protein preparation by examining its protein chemistry, membrane damage (Membrane damag
e) Alternatively, studies on cytotoxicity have been conducted and the results have been reported [Seeger, R. Burkhardt, M. Nannyn-Sch
miedeberg's Arch. Pharmacol. 293 , 163-170 (1976); Se
itz, j., Adler, G., Stofft, E. Eur. J. Cell Biol. 25 , 46-
53 (1981) et al.]. Amanitaceae (Amanitaceae)
Among the basidiomycetes, Amanita mushroom (A. rubescens), fly agaric (A. muscarina), and Kotamagoten mushroom (A. citrin)
It is known that hemolysin is also contained in fruiting bodies such as a) [Seeger, R., Kraus, H., Wiedmann, R. Arch. Tox
ikol. 30 , 215-226 (1973)]. Gantake haemolysin (Rube
scenslysin) was purified and examined for its membrane damage and specificity for phospholipids [Seeger, R., Burkhardt, M. Bio.
Chim. Biophys. Acta 645 , 59-62 (1981) and others]. For other basidiomycetes, see the bamboo shoots (Volvariella volvac
ea), hemolysin present in fruit bodies of enokitake (Flammulina veltipes) and oyster mushroom (Pleurotus ostreatus) have been purified. Hemolysin of syrup (Volnatoxin)
Hemolysin (Flammutoxin) is highly toxic to myocardium in addition to hemolytic activity, and the latter has an effect of causing swelling of Erich ascites tumor cells.

Hemolysin (Pleurotolysin) purified to date from the fruit body of Pleurotus ostreatus is a dimer consisting of a subunit with a molecular weight of 12 kDa, and its action on sphingomyelin has been suggested [Bernheimer, AW, Avigad, LS, Biochim.
Biophys. Acta 585 , 451-461 (1979)]. However, the yield of the hemolysin obtained to date is lower than the strong hemolytic activity of the fruiting body extract, and the discovery and use of a new protein based on a new purification method or technique is strongly expected. It has been rare.

An object of the present invention is to provide a novel and useful protein derived from Pleurotus ostreatus fruiting bodies.

(Means for Solving the Problems) The novel proteins provided by the present invention are the following two types.

1. With a molecular weight of 18 kDa and a pI value (isoelectric point) of 6.3, it binds to a sphingomyelin-containing biomembrane and acts synergistically with the following protein POH-B to show strong hemolytic activity from the fruiting body of Pleurotus ostreatus Hemolytic protein (POH-A).

2. A hemolytic protein (POH-B) derived from the fruit body of Pleurotus ostreatus, which has a molecular weight of 48 kDa and a pI value of 6.3, which acts synergistically with the protein POH-A and exhibits strong hemolysis.

These proteins POH-A and POH-B were purified by, for example, the following method, separated from each other, and their properties were examined.

I. Materials and Experimental Methods A. Materials The fruiting body of Pleurotus ostreatus was directly purchased from a farming farmer in Ogo Town, Gunma Prefecture, and was kept at -80 until immediately before use.
Stored frozen at ℃.

Sphingomyelin from yolk, sphingomyelin from bovine spinal cord, cardiolipin from bovine heart muscle, phosphatidylglycerol from yolk, phosphatidylserin from yolk, D-glucocerebroside, phosphatidylcholine from yolk, and cholesterol are from Sigma Was used. The soybean-derived phosphatidylinositol used was manufactured by Hosei Refinery Co., and the bovine brain-derived phosphatidylserine used was provided by Dr. Yasuda.
Carboxyfluorescein was obtained from Eastman Kodack, using di- [125I] -Bolton-Hunter reagent (specific activity = 4400
Ci / mmol) is from New Euglnad Nuclear, 4N-methanesulfonic acid is Pierce, Freund's complete adjuvant is Iatron, Coomassie Brilliant Blue-R-250,
Electrophoretic agarose GP-36 is from Hanoi Chemicals, bovine serum albumin is from Sigma, Coomassie-Brilliant Blue G
-250 was from Fluka. DEAE-cellulose (DE
-52) is Whatman, Sephadex G-25, G-50, G-7.
5 and G-100 were from Pharmacia, and the ampholine for isoelectric focusing was from LKB. Hydroxyapatite was prepared by the method of Tiselius (Tiselius, A., Hjerta
n, S., Levin, O. Arch. Biophys. Biochem. 65 , 132-155 (19
56)].

B. Experimental method Preparation of blood cell suspension Erythrocytes within one month from the blood collection date
14.5 mM sodium-phosphate buffer containing 0 mM NaCl, pH
Suspend in 7.2 (PBS) and centrifuge at 2,000 rpm for 5 minutes to remove the supernatant. After repeating this operation three times to wash the red blood cells, the Pached volume of the red blood cells is read, and PBS or GVB
(Veronal buffer containing 0.1% gelatin and 0.9% NaCl, pH 7.3) was added to adjust to an appropriate temperature.

Measurement of hemolytic activity Hemolytic activity was measured in the following two reaction systems. The first reaction system is as follows. Dispense an appropriate amount of sample and PBS into a test tube to make the total volume 1.4 ml. This reaction solution was kept at 37 ° C. for 5 minutes in advance, and 0.1 ml of a 7.5% blood cell suspension was added thereto (final blood cell concentration = 0.5%).
Keep warm. After 20 minutes, the reaction solution is immediately cooled with ice and stopped. After cooling on ice for 5 minutes, centrifuge at 2,000 rpm for 5 minutes,
The absorbance at 541 nm of the supernatant is measured. As a control for 0% hemolysis, 0.1 ml of a 7.5% blood cell suspension was added to 1.4 ml of PBS, and as a control for 100% hemolysis, 0.1 ml of a 7.5% blood cell suspension was mixed with 1.4 ml of distilled water to cause hemolysis. The prepared solutions are prepared, and the absorption at 541 nm of the supernatant after centrifugation is measured.

 The second reaction system was performed as follows.

96-well U-shaped microtiter plate (Nunc, Denmar
Using k), add 0.7 μL to 20 μl of the 2-fold dilution series
% Blood cell suspension (final blood cell concentration = 0.5%),
After gentle stirring, keep at 37 ° C. After 40 minutes, 2,000 rpm
And the supernatant at 541 nm is measured. For 0% hemolysis control, 20 μl of GVB plus 50 μl of 0.7% blood cell suspension, and for 100% hemolysis control, 10% (w / v) Triton X-100, 0.7 μl in 20 μl
A hemolyzed solution is prepared by adding 50 μl of a blood cell suspension, and after centrifugation, the absorption at 541 nm of the supernatant is measured.

 The degree of hemolysis of each reaction solution was determined as follows.

The unit of hemolytic activity was a reaction at 37 ° C. for 20 minutes, and the amount of hemolysin required to cause 50% hemolysis of human O-type blood cells was defined as 1 unit, and was determined as follows.

When performing a qualitative hemolysis reaction, a sample solution was prepared on a whole glass, 25 μl of a 2% blood cell suspension was added and mixed, and the presence or absence of activity was visually determined.

 Unless otherwise specified, human O-type red blood cells were used.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed according to the method of Laemmli [Laemmli, UK Nature 2
27 , 680-685 (1970)], and the separation gel was performed using a 12.5% gel. Protein staining was 7.5% (v / v) with 0.25% (w / v) Coomassie Brilliant Blue R-250.
The reaction was performed at 60 ° C. for 1 hour using a mixed solution of acetic acid and 50% (v / v) methanol. The stained gel was decolorized in a mixture of 7.5% acetic acid and 5% methanol. The following molecular weight markers were used to measure the molecular weight.

β-galactosidase (Mr = 115 kDa), phosphorylase b (Mr = 92.5 kDa), bovine serum albumin (Mr = 66.2 kDa)
Da), ovalbumin (Mr = 45kDa), carbonic
Anhydrase (Mr = 31 kDa), soybean trypsin inhibitor (Mr = 21.5 kDa), myoglobin (Mr = 17 kDa),
Lysozyme (Mr = 14.4kDa), cytochrome c (Mr = 12.5kDa)
Da).

Isoelectric Focusing Isoelectric focusing is performed by the method of Vesterberg [Vesterberg,
O. Methods Enzymol. 22 , 389-412 (1971)]
This was performed using an 8101 type isoelectric focusing apparatus. The carrier ampholine was electrophoresed using LKB 5.0-7.0 at 4 ° C. and a constant voltage of 600 V for 2 days. After the completion of the electrophoresis, 1 ml of the sample was collected and the absorption at pH 280 nm and the hemolytic activity were measured.

Analysis of amino acid composition To analyze the amino acid composition, 1 ml of 4N-methanesulfonic acid was added to the samples (about 1 mg each) dried under reduced pressure, sealed under reduced pressure, and hydrolyzed at 105 ° C for 24,48,72 hours. . The pH of the hydrolyzed sample was adjusted to pH 4 with 3.5N sodium hydroxide. For analysis of cysteine, 1 ml of 6N hydrochloric acid was added to a sample in which cysteine was formic acid oxidized to form cysteic acid, sealed under reduced pressure, and then hydrolyzed at 105 ° C. for 24 hours.

The amino acid composition of each sample was analyzed using an orthophthalaldehyde / amino acid analysis system of Hitachi High Performance Liquid Chromatography Model 655 using a citrate buffer.

Preparation of antibody P containing 2 mg of purified P. ostreatus hemolysin A (POH-A)
An emulsion is made by mixing the BS solution with an equal amount of complete incomplete adjuvant. This emulsion was subcutaneously injected into two rabbits in equal amounts. One week after the first injection, a second injection was performed, and thereafter, injections were performed every two weeks, for a total of 5 injections of a total of 10 mg of POH-A.
Three weeks after the last injection, whole blood was collected from the carotid artery. The antibody titer of the antiserum was 128. The reaction between serum and antigen at the time of test blood collection is mainly based on the overlay method and Ouchterlony
Was examined by immunodiffusion method.

Immunological methods For detection of antigens, Ouchterlony's immunodiffusion method [Ouc
hterlony, O., and Nilson, LAHandbood of Experimenta
l Immunology, 3rd Ed., Weir, DM (Ed.), vol. 1, Chapter
r 19 , ppl-44 Blackwell Scientific Pub.Co., Oxford
& Edinburgh (1978)]. First, the slide glass was degreased with acetone, and then immersed in hot distilled water at 100 ° C. for 10 minutes for washing. 0.1% (w / v) Na
Soaked in 0.2% (w / v) agarose GP-36 solution containing N _3, dried and left on a horizontal plate at room temperature, performing pre-coating. After the gel dried, it was heated and dissolved in boiling water
1% (w / v) containing 0.1 M lactose and 0.01% (w / v) NaN ₃
v) Pour agarose GP-36 PBS solution onto the pre-coated plate so that the gel surface is horizontal,
Allow the gel to solidify at room temperature. A hole was formed at an appropriate position in the gel, an antiserum was placed in the center, and a sample was placed in the periphery. The sedimentation reaction in the gel is performed at 4 ° C, 6-18 in a container keeping sufficient humidity.
After a period of time, sedimentation lines were detected under dark field. After confirming the sedimentation line, the plate was immersed in a 1% (w / v) NaCl solution at 4 ° C. for 48 hours to remove the protein in the gel, and then washed in distilled water for 4 hours.
Wash at 6 ° C for 6 hours, then immerse in ethanol at 4 ° C overnight to dehydrate. Thereafter, the plate was dried and stored.

Immunoelectrophoresis [Graber, P., williams, CAJr. Biochim
Biophys. Acta 10 , 193-205] is heated 0.1 M lactose, 0.1% (w / v) NaN ₃ , barbital buffer, pH 8.6 (I
= 0.10) containing 1.2% (w / v) agarose GP-36 solution,
Pour the gel surface horizontally onto a precoated glass plate and leave at room temperature to solidify the gel. Make this gel hole and groove, put the sample in the hole, 10 ℃, 10V
Electrophoresis at / cm for 1 hour. After the electrophoresis was completed, antiserum was put in the groove and left in a wet box at 4 ° C. for 6-18 hours, and then a sedimentation line was detected. The gel plate was stored according to the method described above.

Preparation of erythrocyte membrane (ghost) Ghost was prepared according to Marchesi and Palade Dodge et al.
eck et al. [Marchesi, VT, and Palade, GEJ Cell B
iol. 35 , 385-404 (1967); Dodge, JT, Mitchell, C., and
Hanahan, DJArch. Biochem. Biophys. 100 , 131-139 (19
63); Steck, TL, and Kant, JAMethods Enzymol. 31, 17,
2-180 (1974)]. Human O-type blood, 200ml 4
Centrifuge at 4,000 xg for 20 minutes. After centrifugation, the supernatant is removed by suction. Add PBS to the precipitate and add 4,000x at 4 ° C
g, centrifuge for 20 minutes to wash red blood cells. This operation is repeated twice. Add 20 volumes of 10 mM sodium-to the washed red blood cells
Phosphate buffer, pH 8.0 (buffer A) is added and stirred overnight at 4 ° C. to lyse erythrocytes. Add a glucose solution to a final concentration of 100 mM, and centrifuge at 4 ° C. at 21,000 × g for 50 minutes. After centrifugation, the supernatant is removed, buffer A is added, and the mixture is centrifuged at 4 ° C. at 21,000 × g for 50 minutes. This operation is repeated three times. The resulting precipitate is suspended in a small amount of buffer A,
Stored at -80 ° C. The protein concentration at this time is 6.
It was 60 mg / ml. Preparation of ¹²⁵ I-labeled protein The labeling of ¹²⁵ I of POH-A was performed according to the method of Bolton and Hunter [Bolto
n, AE, and Hunter, WM Biochem. J. 133 , 529-539 (197
3)]. Di- [dissolved in benzene
¹²⁵ I] -Bolton-Hunter reagent (BHR, specific activity = 4,400 Ci / mm
ol) Nitrogen gas is blown on 0.1 ml to completely evaporate benzene. 5 μl of dimethylformamide, 0.2 M
Add 5 μl of a borate buffer, pH 8.5, and 2.45 μg of protein, and allow to stand on ice. After standing for 15 minutes, 100 µl of 0.1 M borate buffer containing 0.1 M glycine, pH 8.5, was added to stop the reaction. This solution was washed with Sephadex G-equilibrated with 50 mM sodium phosphate buffer containing 0.25% gelatin, pH 7.2.
Gel is run using 25 columns and unreacted BHR and protein (
¹²⁵ I-POH-A) was separated.

And ¹²⁵ I-POH-A binding ¹²⁵ I-POH-A (specific activity ^{= 1.2 × 10 10 cpm / mg} ) 50μl and erythrocytes 100μl (1.09 × 10 ⁸ cells) and GVB 850Myueru against erythrocytes were mixed, at 20 ° C. Reacted. One hour later, the reaction was stopped by adding 4 ml of GVB, and a cellulose acetate membrane filter (Schleicher & Schuell OE & Membranfil) was used.
The reaction suspension was aspirated using ter0.45-μm pores). Washing of the membrane filter was performed three times using GVB containing 0.2 M sucrose, and the Pachard Auto-gammasc
γ-rays of the membrane filter were measured using an intillation spectrometer type 5260.

Preparation of liposomes Large-scale lamellar vesicles (MLV) were prepared by the method of Yasuda et al. [Y
asuda, T., Naito, Y., Tsumita, T., and Tadakuma, TJImmu
nol. Methods 44 , 153-158 (1981)].
Phospholipid and cholesterol dissolved in chloroform are mixed in an eggplant type flask to make the total lipid mass 4 μmoles. When this is dried under reduced pressure, a film-like membrane of lipid is formed. After further drying under reduced pressure for 30 minutes, 0.1 ml of 0.1 M carboxyfluorescein (CF) or PBS is added, and vigorously shaken to detach lipids adhered to the wall of the flask. The supernatant obtained by centrifuging the ribosome suspension at 23,000 × g for 20 minutes is removed. After repeating the operation of washing the liposome three times, the liposome is suspended in an appropriate amount of GVB.

Measurement of liposome sensitivity The sensitivity of liposomes to hemolysin was determined by measuring the amount of free carboxyfluorescein (CF) [Ka
ise, S., Yasuda, T., Kashukawa, R., Nishimaki, T., and Tsu
mita, T. Vox Sang. 49 , 292-300 (1985)]. Using a 96-well microtiter plate, add 50 μl of liposome to 20 μl of the prepared two-fold dilution line of hemolysin, gently stir, and incubate at 37 ° C. for 40 minutes. The fluorescence intensity of the released CF was measured using the MTP-32 Microplate Reader (Corona Electronic).
Was measured with excitation light of 490 nm and emission light of 520 nm. GV
B, 20 μl plus 50 μl liposome, 10%
Prepared by adding 50 μl of liposome to 20 μl of Triton X-100, and used as 0% and 100% controls, respectively.

Hemolysin binding to multilamellar vesicles (MLVs) Two hemolytic proteins, POH-A and
POH-B binding experiments were performed according to the method of Watanabe et al. [Watanabe, M., Tomita, T., and Yasuda, T. Biochim. Bio
phys. Acta 898 , 257-265 (1987)]. POH-A (10μ
g) or POH-B (10 μg) and liposome (50 μl)
And kept warm at 37 ° C. for 40 minutes. After reaction, 15,000rpm
The operation of removing the supernatant obtained by centrifugation at for 10 minutes was repeated three times to wash the liposomes. Add SD to the washed liposomes
20 μl of S-PAGE sample buffer was added to solubilize. After heating this solution at 100 ° C. for 5 minutes, the bound proteins were analyzed by SDS-PAGE.

Quantitative analysis Phospholipids are determined by the method of Garrach and Deuticke for the determination of phosphate [Ger
lach, E., and Deuticke, B. Biochem. Z. 337 , 477-479 (196
3)]. A sample dissolved in chloroform is blown with nitrogen gas to evaporate chloroform, and then 0.7 ml of a reagent prepared by mixing equal amounts of 70% perchloric acid and concentrated sulfuric acid is added. When the sample is heated in the fume hood and turns yellow, it is left to cool to room temperature. 0.5 ml of distilled water and 4.0 ml of 1% ammonium molybdate are added thereto and stirred. Further, 0.2 ml of FS reagent * is added and stirred, followed by heating in a boiling water bath for 10 minutes. After cooling, 820n of sample
Measure the absorption at m. Disodium hydrogen phosphate was used as a standard substance.

* FS reagent: 15 containing 0.25% (w / v) 1-amino-2-naphthol 4-sulfonic acid and 1.0% (w / v) Na ₂ SO ₃
% (W / v) Na ₂ S ₂ O ₅ solution The protein was quantified by Bradford [Bradford, MM (1978) A
nal. Biochem. 86 , 142-146; Sector, T. (1978) Anal. Bioc.
hemz. 86 , 142-146], using bovine serum albumin as a standard substance.

II. Examples Purification of novel proteins POH-A and POH-B All purification operations were performed at 4 ° C.

1) Extraction 373 g (green weight) of Pleurotus ostreatus fruit body is 2-3 times the amount of 5m
M EDTA, 2 mM 1,10-phenanthroline and 2 mM PMSF
The protein (hemolysin) was extracted by crushing with a blender together with 50 mM Tris-HCl buffer, pH 8.0 (buffer A) containing. The homogenate is centrifuged at 13,000 xg for 30 minutes,
The supernatant was collected. This supernatant was used as an extract.

2) Ammonium sulfate fractionation Powdery solid ammonium sulfate was gradually added to the extract to make it 60% saturated, and the mixture was allowed to stand at 4 ° C overnight. 13,000xg, 30
Collect by centrifugation for 10 minutes, 10 mM Tris-HCl buffer, pH
Dissolve in 8.2 (buffer B), leave for 1 hour, 28,000xg, 20
Centrifugation was performed for minutes to remove insolubles. Sephadex G-25 column (6.0 × 50 cm) in which the supernatant was equilibrated with buffer B
To make ammonium sulfate fractionation.

3) DEAE-cellulose column chromatography When passing through a DEAE-cellulose column (3.6 x 22.5 cm) previously equilibrated with buffer B, hemolysin is recovered in a non-adsorbed fraction. Fractions containing hemolytic activity were collected and designated as DEAE-cellulose fraction.

4) Hydroxyapatite column chromatography DEAE-cellulose fraction was Sephadex G equilibrated with 10 mM potassium-phosphate buffer, pH 6.8 (buffer C).
The gel was passed through a -25 column (6.0 × 50 cm) to exchange the buffer. The active fraction is passed through a hydroxyapatite column (4.0 × 14.5 cm) equilibrated with buffer C to adsorb hemolysin. When the concentration of buffer C is increased linearly from 10 mM to 400 mM, hemolysin elutes as a single activity peak, as in the elution profile shown in FIG. 1-A. The active fraction was collected and concentrated by freeze-drying to obtain a hydroxyapatite fraction.

5) Sephadex G-75 column chromatography The hydroxyapatite fraction contains highly viscous glucan and interferes with the operation of gel filtration. Therefore, β-1,3-glucanase of zymolyase 100T purified by isoelectric focusing is used. 4.3 μg) was reacted at pH 6.8, 15 ° C. for 10 minutes. As a result, the sample whose viscosity was reduced was subjected to gel filtration using a Sephadex G-75 column (2.2 × 144 cm) equilibrated with buffer C. As is apparent from the activity and protein elution profile shown in FIG. 1-B, hemolysin was separated into two protein components having different molecular weights. Both components showed only a weak hemolytic activity when used alone, but when both were mixed, a strong activity was developed. Thus, it has been clarified that two protein components having different molecular weights are involved in hemolysin, so that the low molecular weight component is POH-A and the high molecular weight component is POH-B. As described above, the hemolytic activity was determined using a reaction in which both components coexist. After the gel run, the two separated protein component fractions were collected, the POH-A fraction was freeze-dried, and the POH-B fraction was YM-
It was concentrated by ultrafiltration with 5 membranes.

6) Isoelectric focusing The POH-A fraction and the POH-B fraction were each purified by isoelectric focusing on a LKB 8101 column using a carrier ampholine having a pH range of 5.0 to 7.0. 1-C and FIG.
-D is the activity peak and pH gradient obtained by measuring the activity and pH of each fraction after electrophoresis of POH-A and POH-B, respectively. Both proteins have an isoelectric point (pI) of pH 6. 3 is shown. POH-A was subjected to gel filtration using Sephadex G-50 previously equilibrated with distilled water, the POH-A fraction was collected, lyophilized, and then subjected to a 20 Mm Tris-HCl buffer, pH
It was dissolved in 7.4 to obtain a final purified sample. About POH-B,
Gel filtration was performed using Sephadex G-100 previously equilibrated with 20 mM sodium-borate buffer, pH 9.0, and POH
The -B fraction was collected and concentrated by ultrafiltration using a YM-5 membrane to obtain a final purified sample.

Table 1 summarizes the purification process of the two protein components. From the four-stage purification method, POH-A was 3
9.0 mg and 0.59 mg of POH-B were obtained.

Analysis of POH-A and POH-B by SDS-PAGE The final purified samples of POH-A and POH-B were analyzed by SDS-PAGE. Although purified POH-A gave a single protein band, purified POH-B detected 5-6 protein bands in addition to one major protein band. Therefore,
In order to confirm the protein band of POH-B, a purified sample of POH-B (12 μg) was subjected to electrophoresis using a pH 9.5, 7.5% gel.
A 2 mm-wide gel disk was cut out from the electrophoresed gel, and the hemolytic activity of each extracted protein fraction was measured. As a result, a fraction containing POH-B was identified, and when analyzed by SDS-PAGE, a single band was detected at a position corresponding to the main band. When POH-A and POH-B were analyzed by SDS-PAGE, the apparent molecular weights of the main bands of POH-A and POH-B obtained by the standard curve prepared by the molecular weight of the migrated molecular weight marker and the moving distance were as follows: 18kDa and 42kDa respectively
It was calculated.

Using a rabbit antiserum prepared using POH-A as an antigen,
The purity of OH-A was examined, and the presence or absence of common antigenicity with POH-B was examined. The cross-reactivity of POH-A and POH-B to the antiserum was examined by Ouchterlony in-gel double diffusion method.
As a result, POH-A reacted with the antiserum to form a single sediment line. On the other hand, POH-B did not react with the antiserum, and no sedimentation line was detected. In addition, POH-A produced a single sedimentation line in the reaction with the antiserum, and together with the result of the in-gel sedimentation reaction,
The purified sample of HA was proved to be a protein of high purity. Although POH-B was reacted with antiserum after immunoelectrophoresis, no sedimentation line was formed, and POH-B is a completely different protein having no common antigenicity with POH-A. It could be confirmed.

Amino acid composition 200 μl of 4N-methanesulfonic acid was added to a purified preparation (240 μg) of POH-A, and after degassing, the mixture was added at 105 ° C. in a sealed tube at 105 ° C.
Hydrolyzed for 4,48 and 72 hours. The content of cysteine was analyzed by subjecting POH-A (240 μg) to formic acid oxidation to convert cysteine to cysteic acid, followed by analysis as a sample. The amounts of serine and threonine were extrapolated, and valine was used for 72 hours. Table 2 shows the amino acid composition of POH-A. This protein is composed of aspartic acid, threonine,
It is characterized by being rich in lysine and glycine.

Stability POH-A, POH-B and a mixture of POH-A and POH-B at 20 mM
After heating in Tris-HCl buffer, pH 8.0 for 10 minutes,
Residual hemolytic activity was measured under standard reaction conditions to determine thermal stability. POH-A is stable up to 40 ° C,
The activity gradually decreased between 0 ° C., and when heated at a temperature of 50 ° C. or more, the activity was rapidly abolished. POH-B was also relatively stable up to 40 ° C, but was completely deactivated at 50 ° C. Even when both were present, the hemolytic activity was lost at 50 ° C., and no change was observed in thermal stability. These results are in good agreement with the conditions for completely inactivating hemolysin (heating at 56 ° C. for 30 minutes).

The pH stability against POH-A and POH-B was determined by measuring the hemolytic activity under standard reaction conditions after standing at 4 ° C. for 24 hours using buffers in various pH ranges. POH-A is in the acidic region (p
H5.5-6.5) and the residual activity is reduced to 50% or less. The most stable is weakly alkaline (around pH 7.5) and stable up to pH 10 on the alkaline side. The same applies to POH-B, which is stable on the alkaline side (pH 8.0 or higher) and easily deactivated when acidic. Even when both coexist, there is no particular effect on the pH stability, and the activity is maintained at pH 8.0 or higher.

Various divalent metal ions for POH-A and POH-B (Mg
²⁺ , NI ²⁺ , CO ²⁺ , Mn ²⁺ , Cd ²⁺ , Ca ²⁺ , Sr ²⁺ , and Ba ²⁺ ) and EDTA
In order to investigate the effect of the reaction, add a metal ion or a chelating agent (final concentration 2 mM, pH 8.0) to POH-A or POH-B, and add
, And the hemolytic activity was measured. As a result, it was found that the activity of both proteins was not affected by each metal ion or EDTA.

Action on erythrocytes Action of hemolysin on human erythrocytes POH-A or POH-B was allowed to act on human O-type erythrocytes at different concentrations, and the hemolytic activity was measured. The activity curves A and B in FIG. 2 were obtained. It was found that both proteins showed hemolytic activity only at high concentrations. On the other hand, the concentration of POH-A or POH-B was kept constant, the concentration was changed to the other protein, respectively, and added to the reaction solution, and the protein was allowed to act on erythrocytes under the condition that both proteins coexist. C
And D were obtained. When POH-A and POH-B coexist, a similar hemolytic activity was observed at a much lower concentration than each alone, revealing that the two different proteins are synergistically involved. Was.

Inhibition of Hemolytic Activity by Erythrocyte Membrane (Ghost) Ghosts prepared by the method described in <Experimental method> were subjected to POH-A
(295 ng / ml) and POH-B (181 ng / ml),
Dispense by l. Next, 25% of a 1.3% blood cell suspension was added thereto, and the mixture was incubated at 37 ° C. for 40 minutes, centrifuged at 2,000 rpm for 10 minutes, and the supernatant was taken to measure the absorbance at 541 nm. FIG.
Is a curve showing the inhibitory effect on hemolysis by the erythrocyte membrane (ghost). As the membrane concentration (μg / ml) increases, the hemolytic activity decreases and is completely inhibited at a concentration of about 100 μg / ml. This phenomenon of hemolysis inhibition indicates that POH-A or POH-B, or both, bind to the erythrocyte membrane and are excluded from the reaction.

Sensitivity to hemolysis of red blood cells Sensitivity to hemolysis of erythrocytes from five mammals, the concentration of POH-B was kept constant, and the lysed serum was measured under the conditions of changing the concentration of POH-A. The erythrocyte activity curves of the animals were compared and examined. FIG. 4-A is a hemolytic activity curve obtained from sheep, human, rabbit, dog and horse erythrocytes, where the hemolytic activity increases depending on the increase in the concentration of POH-A, but the sheep erythrocyte has the lowest concentration. Hemolyses with POH-A in the order of humans and rabbits, and high concentrations of POH are found in red blood cells of dogs and horses.
-A is required. Therefore, the erythrocytes of these animals are less sensitive to hemolysis by the POH-AB system in the order sheep>human>rabbit>dog> horse. Furthermore, in the presence of a certain concentration of POH-A, the concentration of POH-B was varied to act on erythrocytes of each animal, and the hemolytic activity curves were compared. As shown in FIG. 4-B, the erythrocytes of the five animals had little dependence on the change in the concentration of POH-B as observed under the conditions of changing the concentration of POH-A, and the hemolytic activity curve There is no noticeable difference. These results indicate that POH-A and P
OH-B synergy is required, suggesting that POH-A is a factor in determining sensitivity. In addition, the degree of sensitivity in hemolysis has a clear correlation with the content of sphingomyelin (SM), which is one of the main components of the erythrocyte membrane, and erythrocytes with a higher SM content are more sensitive to hemolytic activity. Using ¹²⁵ binding ¹²⁵ I-POH-A of I-POH-A to the red blood cells were examined sheep, humans, and the binding of erythrocytes horse. The concentration of red blood cells was adjusted to be constant by completely lysing the cells with distilled water and measuring the absorption of the centrifugal supernatant at 541 nm. Bound to three types of red blood cells
^When the radioactivity (cpm) of ¹²⁵ I-POH-A was measured, POH-
The amount of A bound to sheep erythrocytes increases with increasing concentration, whereas the amount bound to erythrocytes in humans and horses is limited, consistent with sensitivity to hemolysis, suggesting a relationship with SM content. I have.

Analysis of the hemolysis phenomenon The hemolysis process by the POH-AB system
The change with time of the absorption (turbidity) at 00 nm was continuously recorded and analyzed (FIG. 5). A suspension of human O-type erythrocytes was added to a solution (1.4 ml) containing POH-A (2.06 μg) and POH-B (2.27 μg), which had been previously kept at 37 ° C. for 5 minutes, at a final concentration of 0.5%.
The reaction solution (total volume 1.5 ml) was added at 37 ° C.
When it is kept warm, the hemolysis reaction starts after about 4 minutes. From the curve A in FIG. 5, the maximum rate of this reaction is from 6 minutes after starting the reaction to 11 minutes.
Minutes, its value (-dA ₇₀₀ / dt) is 0.15. Next, when POH-B was added to a mixture of erythrocytes and POH-A kept under the same conditions as above, the reaction started within 1 minute, and the maximum hemolysis rate was obtained from 3.5 minutes to 5.5 minutes from curve B. , Its value was 0.36. On the other hand, when POH-A is added to a mixture of erythrocytes and POH-B and reacted, POH-A and POH-B are reacted.
As in the case of simultaneous action of B, hemolysis occurs after a delay of 4 minutes, and as can be seen from curve C, the maximum rate of hemolysis is between 6 and 8 minutes after the start of the reaction, with a value of 0.23
It is. According to these experiments, (1) hemolysis of erythrocytes previously reacted with POH-A starts almost without delay by the addition of POH-B. (2) In the reaction in which POH-A and POH-B act on erythrocytes simultaneously and the reaction in which POH-A acts on erythrocytes that have acted on POH-B first, hemolysis is found to start after a delay of several minutes. did. Such a phenomenon requires that POH-A acts on red blood cells,
This suggests that hemolysis occurs only when POH-B acts on the membrane to which -A is bound.

Further, in order to clarify the quantitative relationship between POH-A and POH-B in hemolysis, various quantitative ratios of POH-A and POH-B were used.
After keeping the mixture at 37 ° C. for 5 minutes, the mixture was allowed to act on erythrocytes, and the time course of hemolysis was examined. FIG. 6 shows the erythrocyte suspension at 700 nm.
2 is a hemolysis curve in which absorption was continuously recorded. The hemolysis curve A shows that when POH-A and POH-B were simultaneously acted at a molar ratio of 2.5: 1.0, almost no hemolysis was observed and the phenomenon of absorption started slightly after 20 minutes. .
2.5 times the amount of POH-A was added, and the POH-A: POH-B ratio was increased to 6.25: 1.0.
When acted on, as is apparent from the hemolysis curve B,
Although there is a delay in the reaction (10 minutes), hemolysis progresses gradually.
When the amount of POH-B was increased by a factor of 2.5 and the two proteins were added in equimolar (molar ratio, 2.5: 2.5), the hemolysis curve C showed
The delay in the onset of the hemolytic reaction was further reduced, with clear hemolysis occurring after 6.5 minutes. Furthermore, under the reaction conditions of a molar ratio of 6.25: 2.5, in which the amount of POH-A was increased by 2.5 times, it was observed that the marked hemolysis seen in the hemolysis curve D progressed after a slight delay. Thus, although excess POH-A tends to be required to cause hemolysis, there is no strict association at molecular level between the two proteins.

The data obtained from the above analysis of the hemolysis show that these two proteins independently form POH-A and then POH-B independently of red blood cells, rather than forming a complex with a certain number of molecules and participating in hemolysis. It has a unique effect on the membrane, indicating the potential for hemolysis.

C. Effects on liposomes Sensitivity of liposomes of different lipid composition To analyze the relationship between membrane damage (membrane damage) caused by hemolytic proteins and lipid components of membranes, cholesterol (Chol) is equimolar to various phospholipids. Using liposomes containing carboxyfluorescein (CF) as a component, the fluorescent substance released as a result of membrane damage was measured, and the relationship between phospholipid component sensitivity and membrane damage was examined. SM−
When a hemolytic protein is allowed to act on the liposome, POH-A and POH-B alone can be used at high concentrations even at high concentrations.
It did not act on liposomes and no CF release was detected. Therefore, as in the case of the hemolytic reaction, the amount of CF released is measured by a system in which one of the two types of hemolytic proteins is kept constant and the other concentration is changed, and the obstacle to the SM-liposome and the concentration of the CF are measured. Investigated the relationship. As shown in FIG. 7, although each concentration is different, POH-A and POH-B
(Egg white sphingomyelin) -When acted on liposomes, release of CF occurred due to membrane impairment, and essentially similar CF-release curves were obtained.

Based on the experimental results, liposomes containing various phospholipids were prepared, and the effect of the POH-AB system on the membrane was examined. In FIG. 8, the ordinate represents the amount of POH-B, and the abscissa represents POH.
Taking the amount of OH-A, the quantitative relationship of the protein required to release 50% of CF from the liposome was shown. For reference, an example of 50% hemolysis of red blood cells was recorded. As is clear from the data in FIG. 8, membrane damage due to the cooperative action of two hemolytic proteins similar to hemolysis was highly dependent on POH-A quantitatively, but was also observed on SM-liposomes. Also,
In the membrane damage of SM-liposome, the sensitivity of the liposome containing SM derived from yolk changes in accordance with the change in the concentration of both POH-A and POH-B, but in the liposome prepared using SM derived from bovine spinal cord, The concentration of POH-B decreases and POH-A
As the concentration of was increased, the tendency for sensitivity to decrease was observed.

On the other hand, the data in FIG. 8 also show that cardiolipin (CL) derived from bovine heart muscle, phosphatidylinositol (PI) derived from soybean, and phosphatidylglycerol (PG) derived from egg yolk.
Damage to liposomes prepared using phosphatidylserine (PS) derived from citrus and egg yolk, respectively
Independent of POH-A, CF release is shown to occur only by the action of POH-B. The sensitivity of the liposome also depends on the phospholipid component, and decreases in the order of CL>PI>PG> PS. In particular, the sensitivity of CL-liposomes is POH-A
Is more likely to be reduced by the presence of POH-B, and the sensitivity when POH-B alone is acted upon is doubled compared to the sensitivity in the presence of POH-A. In addition to these phospholipids,
Phosphatidylcholine (PC) from egg yolk, from bovine brain
The sensitivity of the liposomes containing PS and D-glycocerebroside (Glc-Cer) to the POH-AB system was examined, but none of them released CF.

These experimental results show that membrane damage is similar to hemolysis as POH-A
Only occur in SM-liposomes, depending on POH
-Suggests a direct effect of -A on SM. Also,
Other liposomes containing phospholipids with POH-B alone as C
Recognition of L, PI, PG, and PS was found to cause membrane damage.

Binding to liposomes It was shown that the two types of hemolytic proteins may be involved in membrane damage by different mechanisms depending on the phospholipids that make up the liposome. That is, it is necessary for the two proteins to work jointly for membrane damage of SM-liposome,
Liposomes containing L, PG and PC are damaged by the action of POH-B alone. The effects of these proteins on membranes are unknown, but are apparently selective for phospholipids,
As a means of directly proving the difference in action, the possibility of binding between membrane and protein was examined. The outline of the experiment is as follows. Mixing 10 μg of each of POH-A or POH-B with liposomes having different phospholipid components and not including CF,
After incubating at 37 ° C for 40 minutes, the plate is washed three times at 15,000 rpm.
The liposome is heat-treated with SDS as it is under standard conditions.
The presence or absence of binding of the protein to the membrane was examined by -PAGE. POH
-A alone and in the presence of POH-B gave a single, distinct protein band of the same mobility as the control. On the other hand, POH
In -B, it was not detected as a protein bound to the membrane under any of the reaction conditions. Similarly, as a result of examining the binding to CL-liposome and PC-liposome, POH-A alone and other than POH-A bound to SM-liposome as a control were used alone and in the presence of POH-B. When applied to phospholipid liposomes, no protein band was detected. POH-B alone causes damage to CL- and PC-liposomes, but no protein band is observed. Therefore, PO
HA recognizes the liposome SM independently of POH-B,
It has been shown to bind tightly to the membrane. Also, POH-B
Alone cause membrane damage to CL- and PC-liposomes,
It does not bind to the membrane and appears to act on the membrane with a different mechanism of action than POH-A.

Effect of cholesterol content on liposome sensitivity Since it was observed that the liposome's susceptibility to membrane damage by hemolytic proteins was affected by its cholesterol (Chol) content, SM from bovine spinal cord and various concentrations of Ch
A liposome consisting of ol was prepared, acted on the POH-AB system, and the release of CF was measured. When the concentration of one of the two proteins is kept constant and the other is changed, the membrane damage of liposomes having different Chol contents is examined. Release is hardly recognized, but the Chol content is reduced to 40 mol% and 50 mol%.
For the first time, the SM-liposome POH
Clear membrane damage due to -AB system was observed. This tendency was the same even when the concentration of POH-A or POH-B was changed. In particular, in the case of liposomes containing 50 mol% of Chol, membrane damage occurred rapidly when excess POH-B was allowed to act in the presence of a low concentration of POH-A. Thus, it was found that the sensitivity of the SM-liposome depends on the Chol content, and a Chol content of 40 mol% or more is required.

On the other hand, the influence of the Chol content on membrane damage of CL-liposomes by POH-B was examined by preparing six types of liposomes having different Chol contents in the range of 0 to 50 mol% and measuring CF release. As a result, from the obtained CF-
B revealed that CL-liposome membrane damage tended to increase somewhat with increasing Chol content, but occurred regardless of Chol.

The above experimental results show that the membrane damage of SM-liposomes by the POH-AB system requires the presence of a high concentration of cholesterol, whereas the action of POH-B alone results in the formation of CL-liposoda. The case indicates that Chol is not involved.

(Effects of the Invention) As described above in detail, the present invention provides a biomembrane-dissolving protein from two highly purified oyster mushrooms,
One of them was found to bind to and specifically dissolve sphingomyelin-containing biological membranes. Together, these have biomembrane solubility, so they can be combined with various affinity agents such as highly specific lectins to lyse specific cells (cancer cells, etc.) and used for cell organ preparation. It has a useful use in the treatment of cancer and the like. Further, if a dye is bound to POH-A using the specificity of POH-A for sphingomyelin, the site where sphingomyelin is present can be stained. That is, the substances provided by the present invention are new and have industrially useful uses.

[Brief description of the drawings]

FIG. 1 shows the elution pattern of the protein of the present invention in each purification step. A: Hydroxyapatite column chromatography Column size is 4.0 × 14.5 cm, flow rate is 100 ml / h, 10
ml samples were collected. −- ○: Hemolysis degree per μl of each fraction ………: Absorption at 280 nm B: Sephadex G-75 column chromatography Column size is 2.2 × 144 cm, flow rate is 18 ml / h, 3.6 m
l samples were collected. ○-○: Hemolysis of a mixture of 20 μl of each fraction and POH-A ●-●: Hemolysis of a mixture of 20 μl of each fraction and POH-B ………: Absorption at 280 nm C: Isoelectric point of POH-A fraction Electrophoresis pattern ○-○: Hemolysis of a mixture of 25 μl of each fraction and POH-B ………: Absorption at 280 nm D: Isoelectric focusing pattern of POH-B fraction ○-○: Degree of hemolysis of a mixture of 25 μl of each fraction and POH-A ………: Absorption at 280 nm FIG. 2 is a hemolytic activity curve showing the effects of the hemolytic proteins POH-A and POH-B of the present invention on human erythrocytes. Activity curve A (○): POH-A was allowed to act on erythrocytes alone. Activity curve B (●): POH-B was allowed to act on erythrocytes alone. Activity curve C (△): A certain amount of POH-B (2830 ng / ml) and various concentrations of POH-A were allowed to act simultaneously on erythrocytes. Activity curve D (▲): A fixed amount of POH-A (72.8 ng / ml) and various concentrations of POH-B were allowed to act simultaneously on erythrocytes. Activity measurements were performed in a second system (see Experimental Methods). FIG. 3 shows the protein POH-A of the present invention (295 ng / ml) and POH-A.
B is a curve showing the inhibitory effect of erythrocyte ghost on the hemolytic activity when ghosts of B (181 ng / ml) and various concentrations act on erythrocytes. I went in. FIG. 4 is a graph showing the sensitivity of red blood cells to hemolysis. A: A fixed amount of POH-B (2.00 μg / ml) and various concentrations of POH-
And A simultaneously acted on red blood cells. B: fixed amount of POH-A (23.4 μg / ml) and various concentrations of POH-A
B and E simultaneously acted on red blood cells. The red blood cells used here are sheep red blood cells (○), human red blood cells (●), rabbit red blood cells (△), dog red blood cells (▲), and horse red blood cells (□). Activity measurements were performed in a second system (see Experimental Methods). FIG. 5 is a graph showing the change over time of hemolysis. Curve A (○): POH-A (1.37 μg / ml) and POH-B (1.51
(μg / ml) was added to the mixture to start the reaction. Curve B (△): POH-A (1.37 μg / ml) was allowed to act on erythrocytes, and then POH-B (1.51 μg / ml) was added to start the reaction. Curve C (□): POH-B (1.51 μg / ml) was allowed to act on erythrocytes, and then POH-A (1.37 μg / ml) was added to start the reaction. Activity measurements were performed in the first system (see Experimental Methods). FIG. 6 is a graph showing a quantitative relationship between POH-A and POH-B with respect to hemolysis. Curve A (○): POH-A 0.547 μg = 3.04 × 10 ^-11 moles
And POH-B 0.511 μg = 1.22 × 10 ⁻¹¹ moles were allowed to act on erythrocytes (molar ratio, 2.5: 1.0). Curve B (●): POH-A 1.37 μg = 7.61 × 10 ^-11 moles
0.511 μg of POH-B = 1.22 × 10 ⁻¹¹ moles was allowed to act on erythrocytes (molar ratio, 6.25: 1.0). Curve C (△): POH-A 0.547 μg = 3.04 × 10 ⁻¹¹ moles
And 1.28 μg of POH-B = 3.04 × 10 ⁻¹¹ moles were allowed to act on erythrocytes (molar ratio, 2.5: 2.5). Curve D (▲): POH-A 1.37 μg = 7.61 × 10 ^-11 moles
1.28 μg of POH-B = 3.04 × 10 ⁻¹¹ moles was allowed to act on erythrocytes (molar ratio, 6.25: 2.5). The molecular weights of POH-A and POH-B are 18 kDa and 42 kDa, respectively. FIG. 7 is a graph showing the effect of the hemolysin of the present invention on sphingomyelin-liposomes. ：: POH-A was allowed to act alone on the liposome. ●: POH-B was allowed to act alone on the liposome. Δ: fixed amount of POH-B (5.67 μg / ml) and various concentrations of POH
-A acted simultaneously. ▲: fixed amount of POH-A (2.18 μg / ml) and various concentrations of POH
-B acted simultaneously. Liposomes are sphingomyelin and cholesterol at a molar ratio of 1: 1. CF: carboxyfluorescein Figure 8 shows POH-A and POH- required for 50% CF release.
6 is a graph showing the correlation between the amounts of proteins in B. The liposomes used here were sphingomyelin from egg yolk (（), sphingomyelin from bovine spinal cord (●), cardiolipin from bovine heart muscle (心 筋), phosphatidylinositol from soybean (□), and phosphatidylglycerol from yolk ( 1) and phosphatidylserine (▲) derived from egg yolk. Each liposome contains cholesterol in a molar ratio of 1: 1. The broken lines (...) indicate POH-A and POH-B required for hemolysis.
Of the protein.

Claims (2)

(57) [Claims] 1. A hemolytic protein derived from the fruit body of Pleurotus ostreatus having a molecular weight of 18 kDa and an isoelectric point of 6.3 and binding to a sphingomyelin-containing biological membrane. 2. It comprises the following proteins (a) and (b):
A mixture in which both act synergistically to exhibit hemolytic activity. (A) a hemolytic protein derived from the fruit body of Pleurotus ostreatus having a molecular weight of 18 kDa and an isoelectric point of 6.3 and binding to a sphingomyelin-containing biological membrane; Proteins from fruiting bodies