JP2721236B2 - Lentin from Oyster mushroom and its collection method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] (Field of Industrial Application) The present invention relates to oyster mushroom-derived lectin and a method for collecting the same, and more specifically, a oyster mushroom-derived lectin having Ga ²⁺ requirement and a method for collecting the same. About.

(Problems to be solved by the prior art and the invention) Lectin is a non-immune sugar-binding protein having at least two sugar-binding sites and agglutinating animal or plant cells or precipitating polysaccharides and complex carbohydrates. And its specificity is determined by the structure of the monosaccharide or oligosaccharide.

Extensive searches performed on fruiting body extracts have shown that
It has been revealed that there are many basidiomycetes having lectins, and lectins have been isolated from fruiting bodies of various basidiomycetes (for example, J. Biol. Chem., 242 , 120 (1967)).

The presence of lectin was unknown because the fruit body of Oyster mushroom contains hemolysin, but Kogure (Vox Sang., 2
9 , 221 (1975)) inactivates hemolysin by heat treatment, obtains a crude preparation of lectin, and conducts agglutination reaction of human erythrocytes and agglutination reaction inhibition test with monosaccharide or oligosaccharide. L-fucose has no inhibitory activity, but 2'-fucosyllactose inhibits the agglutination reaction, suggesting that it is a Cytisus-type agglutinin.

The present inventors have conducted intensive studies on the means for isolating and purifying oyster mushroom-derived lectins, and have found that affinity chromatography using a compound having a D-galactose residue or an N-acyl-D-galactosamine residue as a ligand is carried out. The lectin was successfully isolated by using it, and this lectin is a glycoprotein, consisting of subunits linked by disulfide bonds,
^{In view} of the need for ²⁺ , the present inventors have found that this is a new type of lectin that has not been found in fungal lectins and completed the present invention.

[Constitution of the Invention] (Means and Actions for Solving the Problems) The lectin derived from Oyster mushroom of the present invention has the following physicochemical properties: molecular weight of subunit: 45,000, 47,000 and 49,000 (SDS polyacrylamide gel electrophoresis), etc. Electric point: 10.3 (isoelectric focusing method) Sugar specificity: D-galactose residue or N-acetyl-D-
It binds to a compound having a galactosamine residue. Stable pH range: 3 to 10 (Stable pH range even when left at 4 ° C. for 24 hours) Metal ion requirement: Ca ²⁺ required It is.

The oyster mushroom-derived lectin of the present invention (hereinafter referred to as “POA”) can be collected as follows.

That is, after crushing the fruit body of Oyster mushroom in a saline solution,
Ammonium sulfate was added to the supernatant obtained by centrifugation, and the resulting precipitate was washed with D-galactose residues or N-acyl-D.
-Can be collected by purification by affinity chromatography using a compound having a galactosamine residue as a ligand.

In the present invention, oyster mushrooms used as a raw material include, for example, oyster mushrooms: Pleurotus ostreatus, stalk mushrooms: Pleurotus curnucopiae, kantake mushrooms: Pleurotus spodo
leucus, oyster mushroom: Pleurotus salmoneo-stram
ineus and Pleurotus mushroom: Pleurotus dryinus.

The crushing treatment can be performed by using a brander or the like according to a conventional method. Examples of the saline solution include Tris-HCl buffer, phosphate buffered saline, borate buffer, and Good's buffer.
Is preferably 6 to 8. In the aqueous salt solution, a metal chelating agent such as ethylenediaminetetraacetic acid (hereinafter referred to as “EDTA”) and its sodium salt and potassium salt; β-mercaptoethanol, glutathione, dithiothreitol, dithioerythritol, cysteine, thioglycolic acid, etc. May be added as appropriate.

In the above-mentioned affinity chromatography,
Examples of the compound having a D-galactose residue used as a ligand include lactose, melibiose, raffinose, phenyl β-D-galactoside, thiodigalactoside, asialofetuin, and the like, and also N-acyl-D-galactosamine residue. As the compound having a group,
N-acetyl-D-galactosamine, N-succinyl-
D-galactosamine, D-galactose, lactose,
p-Aminophenyl α (β) -N-acetyl-D-galactosaminide, p-aminophenyl α (β) -D-galactoside, melibiose, raffinose, asialofetuin, asia roushi submandibular gland mucin and the like.

In the collection method of the present invention, prior to the affinity chromatography, the precipitate is washed with Tris-HCl buffer, Good (Good) buffer, borate buffer, cacodylate buffer, Tris-maleate buffer and the like. After dissolving in an appropriate buffer (preferably pH 6 to 8), dialyzing the buffer, subjecting the buffer to DEAE-cellulose column chromatography, and subjecting the obtained non-adsorbed fraction to the affinity chromatography. Is preferred.

The POA of the present invention is expected to be used as a test reagent, an immunomodulator, and a specific adsorbent for separation and analysis of carbohydrates, as well as the known lectins PHA, concanavalin A, PWM, and the like. The application of anticancer drugs to missile therapy is also expected.

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, which do not limit the scope of the present invention.

Example 1 All purification operations described below were performed at 4 ° C.

(1) Extract Oyster mushroom : The fruiting body of Pleurotus ostreatus is purchased directly from a farmer in Ogo Town, Gunma Prefecture, and is at -80 ° C until immediately before use.
And stored frozen.

214 g (fresh weight) of the fruiting body was crushed with a blender together with 20 mM Tris-HCl buffer, pH 8.0 (buffer 1) containing 3 times the amount of 2 mM EDTA to extract POA. The homogenate was centrifuged at 13,000 × g for 30 minutes and the supernatant was collected. This supernatant was used as an extract.

(2) Ammonium sulfate fractionation Powdered solid ammonium sulfate (ammonium sulfate) in the extract
Was gradually added to 60% saturation, and the mixture was allowed to stand for 5 hours. The resulting precipitate was collected by centrifugation at 13,000 × g for 30 minutes, dissolved in 10 mM Tris-HCl buffer, pH 8.0 (buffer 2), and dialyzed against buffer 2 overnight. The dialyzed solution was centrifuged at 28,000 × g for 20 minutes, and the supernatant was used as an ammonium sulfate precipitation fraction.

(3) DEAE-cellulose column chromatography When the ammonium sulfate precipitated fraction was passed through a DEAE-cellulose column (3.6 × 20.7 cm) which had been equilibrated with buffer 2, POA was recovered as a non-adsorbed fraction. This non-adsorbed fraction is collected and DEAE
-The cellulose fraction.

(4) in affinity chromatography DEAE- cellulose fraction, NaCl solid and CaCl ₂ · 2
H ₂ O was added to a final concentration of 130 mM and 2 mM, respectively.
Stir gently for hours. This sample was previously prepared with 130 mM NaCl and
It was adsorbed on N-succinyl-D-galactosamine sepharose (2.3 × 6.5 cm) equilibrated with 10 mM Tris-HCl buffer containing ₂ mM CaCl ₂ , pH 8.0 (buffer 3). Sufficient Buffer 3, then 10 mM with 500 mM NaCl and ₂ mM CaCl ₂
Wash the resin with Tris-HCl buffer, pH 8.0 (buffer 4) to remove non-adsorbed proteins. After thoroughly washing the resin with buffer 3, POA was eluted with 10 mM Tris-HCl buffer, pH 8.0 (buffer 5) containing 130 mM NaCl and 2 mM EDTA. Flow velocity is 6
Performed at 0 ml / h and collected 3 mM fractions. FIG. 1 shows the elution pattern of POA. In FIG. 1, −- ○ indicates hemagglutination activity, Represents absorption at 280 nm. Collect the active fraction and add 2 mM Ca
20 mM Tris-HCl buffer containing Cl ₂ , pH 7.4 (buffer 6)
Dialyzed against. Diaflow Membrane for POA in dialysate
It was concentrated by ultrafiltration using YM-5 to obtain a final purified sample.

Table 1 summarizes the purification process. Extract, ammonium sulfate fractionation and DE
The measurement of hemagglutinating activity in the AE-cellulose fraction was performed after treating the sample at 56 ° C. for 30 minutes to inactivate hemolysin. From the three-step purification, POA was purified 341 times with a yield of 74%.

Test Example 1 (1) By polyacrylamide gel electrophoresis (PAGE)
Analysis of POA The analysis of the protein by PAGE was performed using a 7.5% gel of pH 4.3 under the condition of a constant voltage of 300V. Methyl green was used as a front marker. The protein band in the gel was 0.25% (W / V) Coomassie Brilliant Blue R-250.
7.5% (V / V) acetic acid in a 50% (V / V) methanol mixture at 60 ° C for 1 hour.
The test was performed using a solution containing 5% (V / V) methanol.

A purified sample of POA (14 μg) was run on a pH 4.3, 7.5% gel and stained with protein. As a result, a single protein band was observed.

In addition, a purified sample of POA incubated at 37 ° C for 24 hours in the presence of 0.1% NaN ₃ was subjected to protein staining and PAS
Upon staining, two bands were detected in addition to the main band, all of which were positive for PAS staining, indicating that they were glycoproteins.

(2) Analysis of POA by SDS-PAGE SDS-PAGE was performed according to the method of Laemmli (Nature, 227 , 680 (197
6)). As the separation gel, a uniform gel having a concentration of 12.5% and a gel having a concentration gradient of 4 to 20% were used. In the measurement of molecular weight, the following molecular weight markers were used.

Ferritin (Mr = 220,000), phosphorylase b (Mr
= 92,500), bovine serum albumin (Mr = 66,200), catalase (Mr = 60,000), ovalbumin (Mr = 45,000)
0), lactate dehydrogenase (Mr = 36,000), carbonic acid
Anhydrase (Mr = 31,000), soybean trypsin inhibitor (Mr = 21,500), lysozyme (Mr = 14,400) Protein staining and decolorization were performed under the same conditions as PAGE.

When the purified POA was analyzed by SDS-PAGE using a 12.5% gel, two major protein bands and one weak protein band were detected. All three protein bands were positive for PAS staining, indicating that all of them contained sugar. When the apparent molecular weight was measured by SDS-PAGE using a 12.5% gel, it was found that the molecular weights of the main protein components were 45,000 and 47,000, and the molecular weight of one trace protein component was 49,000. When the ratios of the protein components of 45,000, 47,000 and 49,000 were determined using a densitometer, they were 40%, 53% and 7%, respectively. When SDS-PAGE was performed on POA under non-reducing conditions, it migrated as a single protein band. Sephadex G-10
POA from the molecular weight measurement by gel permeation method using 0 column
Has an apparent molecular weight of 90,000. These experimental results indicate that POA is a dimer in which identical subunits differing in sugar content are linked by disulfide bonds.

After treating the purified POA in the presence of 0.1% NaN ₃ at 37 ° C. for 24 hours, SDS-PAG using a concentration gradient gel (4-20%) was used.
E was performed in the absence of 2-mercaptoethanol. One broad protein band (molecular weight, ~ 90,000), plus two weak but high molecular weights (180,000 and 220,000)
Bands were detected. This high molecular weight protein indicates that the subunits are tetramers or pentamers linked by disulfide bonds.

(3) Measurement of isoelectric point The isoelectric point was determined by the method of Vesterberg (Methods Enzymol., 22 ,
389 (1971)) using an LKB8101 isoelectric focusing apparatus. The electrophoretic carrier ampholine is
Using LKB3.0-10.5, the test was performed at 4 ° C. and a constant voltage of 300 V for 2 days. After completion of the electrophoresis, a 1 ml sample was collected, and the pH, absorption at 280 nm, and hemagglutinating activity of each fraction were measured.

 The isoelectric point (pI) of POA was measured at pI 10.3.

(4) POA assay using immunological technique (a) Preparation of antibody A physiological saline containing 2 mg of purified hemagglutinin (POA),
Emulsions were made by mixing equal amounts of Freund's complete adjuvant. This emulsion was subcutaneously injected into two rabbits in equal amounts. Injections were performed three times every two weeks, with the fourth injection injecting the antigen directly into the vein. One week later, whole blood was collected from the carotid artery. The titer of the antibody at that time was 128. The antigen-antibody reaction of the serum before immunization and at the time of test blood collection was examined by the overlay method.

(B) Immunological method For detection of antigen by the in-gel diffusion method, Ouchterlony's immunodouble diffusion method (Ouchterlony, O., and Nilson, LA (19)
78) Handbook of Experimental Immunology, 3rd Ed., We
ir, DM (Ed), Vol.1, Chapter 19, ppl-44, Blackwell S
cientific Pub. Co., Oxford & Edinburgh).
First glass plate 0.1% (W / V) 0.2 % containing NaN ₃ (W
/ V) Dipped in agarose GP-36 solution, left on a horizontal plate at room temperature, dried, and pre-coated. After the gel has dried, 0.1 M lactose, 1% (W / V) PBS solution of agarose GP-36 containing 0.01% (W / V) NaN 3, so that the gel surfaces precoated plates is horizontal And left at room temperature to solidify the gel. A hole was formed at an appropriate position in this gel, and an antiserum was placed in the center and a sample was placed in the periphery. The sedimentation reaction in the gel was carried out in a container keeping moisture sufficiently.
C., 6-18 hours, and 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 proteins in the gel, and then washed in distilled water at 4 ° C. for 6 hours. After washing, it was immersed in ethanol at 4 ° C. overnight to dehydrate. Thereafter, the plate was dried and stored.

Immunoelectrophoresis (Biochim. Biophys. Acta, 10 , 193 (195
3)) heated 0.1 M lactose, 0.1% (W / V) Na
1.2 including N ₃ , barbital buffer, pH 8.6 (I = 0.10)
% (W / V) agarose GP-36 solution was poured onto the precoated glass precoat and allowed to solidify at room temperature. Make a hole and groove in this gel, put the sample in the hole, 10 ℃,
Electrophoresis was performed at 10 V / cm for 1 hour. After the electrophoresis was completed, antiserum was put in the groove, left in a wet box at 4 ° C. for 6 to 18 hours, and a sedimentation line was detected. The gel plate was stored according to the method described above.

In the immunodiffusion method, a single sedimentation line was formed, and the antiserum reacted with 0.588 μg or more of POA. In immunoelectrophoresis,
POA formed a single sedimentation line upon reaction with the antiserum, confirming that POA was highly pure.

(5) Analysis of amino acid composition The sample dried under reduced pressure was added with 1 ml of 4N methanesulfonic acid, sealed under reduced pressure, and hydrolyzed at 105 ° C. for 24, 48 and 72 hours. After the hydrolysis was completed, 3.5N sodium hydroxide was added to each sample to adjust the pH to 4. In the analysis of cysteine,
After cysteine in the protein was formic acid oxidized to cysteic acid, 1 ml of 6N hydrochloric acid was added, and the tube was sealed under reduced pressure.
For 24 hours.

Amino acids in each sample were analyzed by Hitachi High Performance Liquid Chromatography.
655 type orthophthalaldehyde and amino acid analysis system.
Analysis was performed using citrate buffer.

 Table 2 shows the amino acid composition of POA.

From Table 2, POA has a high content of aspartic acid or asparagine, threonine, alanine, and valine,
It is clear that the content is low in the contents of the basic amino acids lysine, histidine, and arginine, and that they lack methionine.

(6) Analysis of sugar composition Dissolve 5 mg of purified POA in 0.4 ml of distilled water, and add 0.4 ml of 4N
Trifluoroacetic acid (TFA) was added, sealed, and hydrolyzed at 121 ° C. for 1 hour. Remove the TFA by vacuum drying to
Add 2 ml of distilled water, add a drop of 2N ammonia water,
5 mg of NaBH ₄ was added and left at room temperature for more than 2 hours. NaBH ₄
After decomposition and removal, 0.3 ml of acetic anhydride was added,
After heating for a period of time and drying under reduced pressure, the sugar was converted to an alditol acetate derivative. For analysis of sugar, Shimadzu gas chromatograph
Performed using GC-6A. Neutral sugar analysis is performed by Gas-chrom Q
Performed at 190 ° C. using a 3% ECNSS-M column using
In addition, for neutral sugar and amino sugar analysis, a 0V-17 column using Gas-chrom Q as a carrier was used.
The temperature was raised under the following conditions.

The content of neutral sugar was determined as follows. First, the purified POA was dialyzed against a 500 mM aqueous NaCl solution containing 0.01% (W / V) NaN ₃ for 2 days. Then, it was dialyzed against distilled water for 3 days. At this time, the distilled water is frequently exchanged and the stirring is performed. Thereafter, the samples were lyophilized. After lyophilization, 5.0 mg was weighed and dissolved in 1.0 ml of distilled water. As a result of analyzing the sugar content of this sample by the phenol sulfate method, the neutral sugar content of POA was 9.0% when glucose was used as a standard substance.
Met.

As a result of analyzing the sugar composition by gas chromatography, mannose, glucose, and glucosamine were detected, and the molar ratio was mannose: glucose: glucosamine = 10: 1: 2.

(7) Stability of POA After treating POA for 10 minutes at various temperatures, the remaining hemagglutinating activity was measured. POA is stable up to 70 ° C, but 8
It was rapidly deactivated at 0 ° C. and completely deactivated at 90 ° C.

The pH stability was determined by allowing samples to stand in various buffers ranging from pH 3.0 to 10.0 at 4 ° C. for 24 hours, adding 0.5 M Tris-HCl buffer, pH 7.4, and measuring hemagglutination activity. . The buffers used here are as follows.

pH3.0-6.5: citrate buffer, pH3.5-6.0: acetate buffer, pH7.0-9.0: Tris-HCl buffer, pH8.5-10.0: borate buffer In the pH range of 10.0, POA was extremely stable to pH changes and retained its activity without deactivation.

(8) Requirement of metal cation POA (titer = 64) sample (25 µl) (20 mM containing 2 mM Ca ²⁺ )
mM Tris-HCl buffer, pH 7.4) was mixed with a colleague's 20 mM Tris-HCl buffer, pH 7.4, containing 4 mM ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (hereinafter referred to as “EGTA”). When left at 4 ° C. for 60 minutes, POA was completely inactivated. 25 μl of a 10 mM divalent metal solution was added to the inactivated POA, the mixture was allowed to stand at 4 ° C. for 60 minutes, and the hemagglutination activity was measured. Table 3 summarizes the effects on the activity of various divalent metal ions, with Ca ²⁺ being the most effective,
Activity is completely restored. Also, 50,25% of the activity was detected in the presence of Ca ²⁺ , Cd ²⁺ and Ni ²⁺ , but the recovery of activity was
^At less than 50% of ²⁺ , POA appeared to be a lectin that required Ca ²⁺ for its hemagglutinating activity. As a control, 20 mM Tris-HCl buffer, pH 7.4 containing 2 mM ^{Ca2 +} instead of EGTA and metal ions was used.

Furthermore, after inactivation with EGTA, 20 mM containing 130 mM NaCl
To a Tris-HCl buffer, pH 7.4, dialyzed against POA solution (25 μl, titer = 32) from which EGTA was removed, various concentrations (0-1000 μM) of Ca ²⁺ solutions (25 μl) were added. And the activity was measured. POA inactivated by EGTA treatment was 31.3 μM C
The activity was restored in the presence of a ²⁺ . Ca ²⁺ below this concentration
Did not give a clear hemagglutination reaction.

(9) Aggregation activity of POA on various erythrocytes Table 4 shows the results of comparison of agglutination activity on erythrocytes of humans, rabbits, dogs, sheep and horses. POA agglutinates human blood cells irrespective of ABO type, but O-type blood cells (titer = 512: number of individuals = 6, titer = 256: number of individuals = 4) have A-type blood cells (titer = 256: individual). Equation 4, titer = 128: number of individuals = 6) and B
Type blood cells (titer = 256: number of individuals = 5, titer = 128: number of individuals =
There were blood cells that were agglutinated at twice the titer of 5), and the tendency of POA to agglutinate human blood cells was that type O blood cells> type A blood cells = type B blood cells. In trypsin-sensitized blood cells, O
The titers increased by a factor of 16 for type blood cells and by a factor of 8 for type A and type B blood cells, indicating a fundamental difference in the aggregation of these blood cells.

Significant differences were observed in the erythrocytes of the four mammals,
Rabbit blood cells have twice the agglutination property of human O-type blood cells, but only agglutination is observed in dog and sheep blood cells, and horse blood cells were not aggregated at all by POA.

(10) Sugar-binding specificity The sugar-binding specificity of POA was examined by using various monosaccharides, oligosaccharides, and glycosides and measuring the inhibitory activity of the O-type hemagglutination reaction. Table 5 shows the concentrations (mM) required for various sugars to inhibit the agglutination reaction by 50%. Among various sugars, N
-Acetyl-D-galactosamine has the most cooperative inhibitory activity, and its glycosides, D-galactose and its glycosides, and oligosaccharides having D-galactosyl residues have inhibitory activities. Thus, this lectin belongs to the D-galactose, N-acetyl-D-galactosamine type in basic sugar binding specificity. D-Fucose (6
-Deoxy-D-galactose) has inhibitory activity,
L-arabinopyranose did not inhibit the agglutination reaction.
This fact suggests that the sugar linkage of POA contains two of D-galactose.
Position (C ₂ ), position ₃ (C ₃ ), and position ₄ (C ₄ ) are required, and position ₆ (C ₆ ) is reduced by reduction of the hydroxymethyl group. It shows that methyl groups have no effect, but pentoses cannot bind. OH group of the C ₂ rather affinity becomes the acetamido group is increased.

Further, comparison of the inhibitory activities of the glycoside and the oligosaccharide having an α- or β-D-galactosyl residue revealed that the specificity of the POA for the anomer was not very strict.
In glycosides, aglycones affect POA sugar linkages,
The effect of inhibition decreased in the order of methyl group> phenyl group> p-nitrophenyl group.

(11) Binding of FITC-lectin to glycolipid (a) Preparation of human erythrocyte membrane Human erythrocyte membrane was prepared by Marchesi, Palade, and Dodg
e et al. (J. Cell Biol., 35 , 385 (1967); Arch. Bioche.
m. Biophys., 100 , 131 (1963)). Human type A blood was centrifuged at 1,200 × g for 15 minutes to remove the supernatant. The precipitated erythrocytes were suspended in physiological saline, centrifuged at 1,200 × g for 15 minutes to remove the supernatant, and this operation was repeated. The resulting precipitate was added with 10 volumes of 5 mM phosphate buffer, pH 7.2, stirred for 30 minutes, and centrifuged at 25,000 × g for 30 minutes. This operation was repeated to wash the erythrocyte membrane. The membrane is then
Suspended in 50 mM phosphate buffer containing Cl, pH 7.2, 25,000x
g, and centrifuged for 30 minutes. After repeating this operation, the precipitate was dialyzed against distilled water, freeze-dried, and stored at 4 ° C. in a desiccator.

(B) Extraction of glycolipids from human erythrocyte membrane Extraction of glycolipids from human erythrocyte membrane was performed by Svennerholm and F.
Redman's method (Biochim. Biophys. Acta, 617 , 97 (198
0)). 5 ml of distilled water was added to 250 mg of human erythrocyte membrane and crushed at 4 ° C. for 1 minute. 13 to this homogenate
To the mixture, 6.5 ml of chloroform was added dropwise while stirring, and stirring was further continued for 30 minutes to extract glycolipids. The extract was centrifuged at 2,000 × g for 20 minutes to collect the supernatant. The precipitate was extracted by the above operation. The mixed extract was dried under reduced pressure, and a crude glycolipid was suspended in chloroform: methanol = 2: 1 (hereinafter referred to as “CM”) and dried completely over P ₂ H ₅ . Next, 1 ml of methanol containing 0.1N NaOH was added, and the mixture was allowed to stand at 37 ° C. for 1 hour, and then neutralized with acetic acid. This was dialyzed against distilled water at 4 ° C. overnight, dried under reduced pressure, and dissolved in chloroform.

(C) Separation of glycolipid Acid glycolipid and neutral glycolipid were separated using DEAE-cellulose (DE-52). After immersing DE-52 in 1.0 M acetic acid for 30 minutes, it was washed with distilled water until neutral. The acetic acid type DE-52 was suspended in methanol, decanted several times, and then suspended in CM kneading. The resin was packed in a column and equilibrated with chloroform. The mixture of glycolipids was allowed to flow down the column, washed sufficiently with chloroform, and then CM was allowed to flow down. The fraction not adsorbed to the resin was collected as the neutral glycolipid fraction. The column was eluted with CM containing 0.01 M ammonium acetate, and fractions positive for the phenol sulfate reaction were collected to obtain acidic glycolipid fractions.

(D) Labeling of POA with FITC Labeling of lectin with FITC was performed according to the method of Marshall et al.
oc.Soc.Exper.Biol. & Med. (NY), 98 , 898 (195
8)). A 500 mM galactose solution was added to the solution containing 7 mg of the purified POA to adjust the final concentration to 300 mM. After leaving this solution at 4 ° C. for 24 hours, 1/10 volume of 0.5 M carbonate buffer,
pH 9.5 was added. On the other hand, the same amount of 0.02% (W / V) including FITC
A 50 mM carbonate buffer, pH 9.5 was prepared, mixed with the above POA solution, and gently stirred at 8 ° C. for 6 hours while protecting from light. This solution was dialyzed against 20 mM Tris-HCl buffer containing ₂ mM CaCl ₂ and pH 7.4, and then adsorbed on N-succinylgalactosamine Sepharose equilibrated with the same buffer.After washing sufficiently, 2 mM EDTA was added. 20 mM Tris-HCl buffer, pH 7.4
Was used to elute POTC labeled with FITC (hereinafter referred to as "FITC-POA"), stored at -20 ° C, protected from light.

(E) Binding of FITC-POA to sugar substance Neutral glycolipids were developed on a thin plate using chloroform: methanol: water = 60: 35: 8 (V / V / V) as a solvent, Lipid is chloroform: methanol: 0.02% CaC
Using l ₂ = 60: 35: 8 (V / V / V) as a solvent, the mixture was developed twice and separated.

Binding of glycolipids to FITC-POA was performed by the method of Magnani et al.
s Enzymol., 83 , 235 (1982)). Expand the glycolipid and dry the thin layer plate with 0.15M NaCl, 1%
Polyvinylpyrrolidone -40, and 10mM containing 0.1% NaN ₃
It was immersed in a phosphate buffer, pH 7.2, at 4 ° C. for 10 minutes. Place the wet plate horizontally at 4 ° C under humid conditions,
TC-POA was allowed to act directly. After standing for 16 hours in this state, the plate was washed with 10 mM phosphate buffer containing 0.15 M NaCl, pH
After washing with 7.2 times and drying, fluorescence was detected using a UV lamp.

As a result, FITC-POA is of human origin globoside, Forssman antigen, and be firmly bonded such as asialo · G _M1 shown. On the other hand, binding to human-derived glucosylceramide, lactosylceramide, and pig-derived globoside was weak. No binding to acidic glycolipid was detected.

[Effects of the Invention] According to the present invention, a new type of lectin not found in conventional fungal lectins can be provided.

[Brief description of the drawings]

 FIG. 1 is a diagram showing an elution pattern of POA.

Claims (2)

(57) [Claims] 1. The following physicochemical properties: Molecular weight of subunit: 45,000, 47,000 and 49,000 (SDS polyacrylamide gel electrophoresis) Isoelectric point: 10.3 (Isoelectric point electrophoresis) Sugar specificity: D-galactose Residue or N-acetyl-D-
Binds to a compound having a galactosamine residue. Stable pH range: 3 to 10 (Stable pH range even when left at 4 ° C. for 24 hours) Metallic ion requirement: Ca ²⁺ requirement Lentin derived from Pleurotus ostreatus. 2. An oyster mushroom fruit body is crushed in an aqueous salt solution, and then ammonium sulfate is added to a supernatant obtained by centrifugation.
2. The method for collecting oyster mushroom-derived lectin according to claim 1, wherein the resulting precipitate is purified by affinity chromatography using a compound having a D-galactose residue or an N-acyl-D-galactosamine residue as a ligand. .