JP3711356B2 - Kurokawa-derived lectin and its isolation and purification method - Google Patents

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel black lectin protein and a method for separating and purifying the same.
[0002]
[Prior art]
A lectin is a protein that recognizes a sugar chain, and its production has high industrial significance and there are many patents. The following are some examples.
The lectin active protein derived from JP 09-059298 Aloe leaf bark relates to allelectin.
The lectin derived from Japanese Patent Application Laid-Open No. 08-1119994 and its purification method relate to the purification of mannose / glucose affinity lectin and the separation and purification method by affinity chromatography using maltose as a ligand.
JP 2000-505643 A compound having a lectin-like property and biological application thereof are disclosed in nucleotide sequences capable of decoding a polypeptide having a lectin-like property, a similar sarcolectin-type polypeptide, and treatment thereof. Is about the use of.
The method of isolating isolectin from mistletoe 10-504287 relates to a method of isolating MLI-type isolectin by performing chromatography on lactosyl sepharose from mistletoe.
Table 95/018149 The novel lectin and the method for producing the same relate to the separation and purification of a novel lectin from a seaweed belonging to the genus Kirinzai and obtaining it in large quantities.
As described above, there are various materials and methods, and it is considered that there are still undeveloped useful lectins, and the development of these lectins, particularly food-derived lectins and appropriate production methods remain undeveloped. It is left.
[0003]
[Problems to be solved by the invention]
There are many types of lectins, and the development of cell separation reagents, clinical test reagents, clinical therapeutics, etc. using sugar chain binding specificity, blood group specificity, anticancer action, etc. is underway. Furthermore, the development of lectins with new characteristics is required to meet various purposes. In particular, food-derived lectins are expected to have advantageous applications because of their high safety.
[0004]
[Means for Solving the Problems]
The purpose of this study is to conduct research on lectins from edible mushrooms, Kurokawa by buffer extraction, and to provide a novel lectin production method that has not been known so far.
The present inventors have conducted intensive research to solve the above-mentioned object, and found that lectins can be efficiently separated and purified by affinity chromatography using N, N′-dichitobiose as a ligand. That is, Kurokawa was extracted with a buffer solution, and the extract was adsorbed, separated and fractionated by affinity chromatography using ammonium sulfate precipitation and N, N′-diacetylchitobiose as a ligand. A lectin derived from Kurokawa is obtained.
[0005]
The resulting Kurokawa-derived lectin has a molecular weight of about 15,000 as determined by SDS-polyacrylamide electrophoresis, the amino group of the N-terminal amino acid is blocked, and the amino acid sequence from the N-terminal is degraded by cyanogen bromide decomposition. A peptide which is Gly-Gly-Ser-Gly-Thr-Ser-Gly-Thr-Ile-Arg- is obtained and has the activity of aggregating rabbit erythrocytes at a concentration of about 50 ng / ml, and N, N′-diacetyl Has binding activity to chitobiose carrier. Kurokawa lectin is a novel lectin that has not been described in the literature, and its sugar chain binding specificity is not yet clear. However, general monosaccharides and disaccharides do not inhibit their hemagglutination activity, so there is a characteristic sugar chain binding specificity. It is considered a thing.
[0006]
Furthermore, the present inventors have found that kurokawa lectin suppresses the growth of cultured human cancer cells, induces apoptosis and kills it. That is, the obtained Kurokawa-derived lectin is a lectin that suppresses the growth of human monocytic leukemia U937 cells and mouse lung cancer LL2 cells and exhibits apoptosis-inducing activity.
In animal experiments, it has also been found to have a life-prolonging effect on cancer-bearing animals. Although it is not certain at present, anti-cancer effects are expected by food, and Kurokawa may be positioned as a functional food. is there.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Kurokawa (Boletopsis leucomelas), an edible mushroom used in this study, can be collected from the forest in Yamanashi Prefecture.
As a preferred method for separating and purifying Kurokawa lectin, Kurokawa is ground in a Tris buffer (pH 7.4) using a homogenizer to obtain an extract. A 70% precipitate was separated by an ammonium sulfate fractionation method, the dissolved fraction was applied to an affinity column using N, N′-diacetylchitobiose-sepharose, washed thoroughly with Tris buffer, and then an N-acetylglucosamine-containing solvent. To obtain kurokawa lectin.
The kurokawa lectin thus obtained has an activity of agglutinating rabbit erythrocytes.
[0008]
The black lectin produced according to the present invention suppresses the growth of human monocytic leukemia U937 cells and has U937 cell apoptosis-inducing activity, and thus is considered to have an anticancer effect. In addition, it suppresses the growth of mouse lung cancer cells, and in animal experiments using mice, brings about a life-prolonging effect by intraperitoneal administration to cancer-bearing animals transplanted with cancer cells.
[0009]
【Example】
The following examples are not meant to be limiting and are merely illustrative.
Add 100 ml of Tris-HCl buffer (pH 7.5) to 100 g of Kurokawa fruit body collected from the mountain forest in Yamanashi Prefecture, homogenize, centrifuge at 13,000 xg for 10 minutes, and then 70% saturation in the supernatant After adding ammonium sulfate, the mixture was allowed to stand for 1 hour and centrifuged to collect the precipitate. The precipitate was dissolved by adding 50 ml of Tris-HCl buffer, and after dialysis, the centrifuged supernatant was applied to a gel bed 2 ml N, N′-diacetylchitobiose-immobilized Sepharose 4B column, thoroughly washed with Tris-HCl buffer, Elution with 250 mM N-acetylglucosamine gave 7 mg of bound protein.
This protein migrates to the position of molecular weight 15,000 by SDS-polyacrylamide gel electrophoresis as shown in FIG. 1 and shows the same molecular weight as shown in FIG. 2 regardless of the presence or absence of mercaptoethanol. This revealed that the subunit was not a protein bound by an SS bond.
[0010]
Since this protein was considered to be one of the lectins that recognize sugar chains from the separation method, as a result of examining the hemagglutination reaction, as shown in FIG. 3, the protein was agglutinated at a very low concentration of 50 ng / ml. Showed activity. Therefore, it was confirmed that this protein is a kind of lectin. This hemagglutination reaction was not inhibited by 250 mM N-acetylglucosamine, methyl α-mannoside, glucose, mannose, lactose, or sucrose.
[0011]
Although an attempt was made to determine the amino acid sequence of the present kurokawa lectin, no results relating to the amino acid sequence could be obtained as it was, so it was considered that the N-terminal amino acid residue was blocked. Therefore, cyanogen bromide treatment that specifically cleaves at the carboxyl side of the methionine residue was performed, and from the analysis of the peptide obtained by this method, the present kurokawa lectin has Gly-Gly-Ser-Gly-Thr-Ser-Gly. It was revealed that the sequence of -Thr-Ile-Arg- exists. As a result of computer search of the amino acid sequence database, no protein having the same sequence has been reported, and this kurokawa lectin was determined to be a novel protein.
[0012]
Next, in order to examine the effect on cancer cells, the present kurokawa lectin was added to 2 × 10 ⁴ human monocytic leukemia U937 cell culture solution, cultured for 24 hours, and then added with alamar blue to increase cell proliferation. When compared with a control to which no lectin was added, as shown in FIG. 4, kurokawa lectin inhibited the growth of U937 cells in a concentration-dependent manner.
[0013]
When cells treated with kurokawa lectin were stained with Hoechst 33342, chromatin condensation characteristic of apoptosis was observed as shown in FIG. Furthermore, DNA was extracted from cells treated with black lectin, separated by agarose gel electrophoresis, and then stained with cyber green. As a result, as shown in FIG. 6, kurokawa lectin formed a DNA ladder characteristic of apoptosis in a concentration-dependent manner. Induced. Therefore, it was revealed that the present black lectin has apoptosis-inducing activity against U937 cells.
[0014]
In order to examine the effect on mouse cancer cells, this kurokawa lectin was added to 2 × 10 ⁴ mouse lung cancer LL2 cell cultures, cultured for 24 hours, added with trypan blue to count the number of viable cells, and When compared with the control without addition of kurokawa lectin, as shown in FIG. 7, kurokawa lectin inhibited the growth of LL2 cells in a concentration-dependent manner.
[0015]
In animal experiments using mice, intraperitoneal administration of this kurokawa lectin showed a life-prolonging effect in mice transplanted intraperitoneally with mouse Lewis lung cancer LL2 cells. That is, 1 × 10 ⁶ LL2 cells were transplanted into the abdominal cavity of syngeneic mouse C57BL / 6 mice, physiological saline was administered to the control group, and kurokawa lectin was administered to the experimental group every two days, and the mice died. The number of days until it was measured. As a result, as shown in FIG. 8, it was recognized that there was a life-prolonging effect because 2 animals were alive after 7 control groups died. Thus, it was confirmed in animal experiments that the present lectin induced apoptosis of cultured cells and suppressed proliferation.
[0016]
【The invention's effect】
As described above, Kurokawa is extracted with a buffer solution, and the extract is adsorbed, separated and fractionated using ammonium sulfate precipitation and affinity chromatography with N, N′-diacetylchitobiose as a ligand. A lectin derived from Kurokawa could be produced by the characteristic separation and purification method. The lectin derived from Kurokawa produced according to the present invention has (a) a molecular weight of about 15,000 by SDS-polyacrylamide electrophoresis, (b) the amino group of the N-terminal amino acid is blocked, (c) Peptide whose amino acid sequence from the N-terminus is Gly-Gly-Ser-Gly-Thr-Ser-Gly-Thr-Ile-Arg- was obtained by cyanogen bromide decomposition, (d) at a concentration of about 50 ng / ml It has an activity of agglutinating rabbit erythrocytes, and (e) has a characteristic of binding to an N, N′-diacetylchitobiose carrier.
[0017]
The lectin derived from Kurokawa of the present invention suppresses the growth of human monocytic leukemia U937 cells and mouse lung cancer LL2 cells and exhibits apoptosis-inducing activity. Conventionally, wheat germ lectin is known as a lectin isolated from food that induces apoptosis in cancer cells and has a cancer suppressive effect. However, hemagglutination by wheat germ lectin is inhibited by N-acetylglucosamine. In contrast, kurokawa lectin is not inhibited, so there is a difference in sugar chain binding specificity between the two. Therefore, among food-derived lectins, kurokawa lectin is considered to be a novel one having cancer cell apoptosis-inducing activity and novel sugar chain binding specificity. From the above, contributions to the industry can be expected, such as development as a new anticancer agent based on anticancer activity and development of a test drug utilizing sugar chain binding specificity.
[0018]
[Brief description of the drawings]
FIG. 1 SDS-polyacrylamide gel electrophoresis of kurokawa lectin having a molecular weight of about 15,000 (15 kDa) separated and purified by affinity chromatography using N, N′-diacetylchitobiose-immobilized sepharose of Kurokawa extract. The electrophoresis pattern (3 in the figure) is shown together with the marker protein (1 in the figure) and the Kurokawa extract before separation (2 in the figure).
FIG. 2 shows that kurokawa lectin migrates to the same molecular weight of about 15,000 (15 kDa) by SDS-polyacrylamide gel electrophoresis regardless of the presence or absence of mercaptoethanol, which is a reducing agent. It is a figure which shows 6) with marker protein (4 in a figure).
FIG. 3 is a view showing that kurokawa lectin separated and purified by affinity chromatography using N, N′-diacetylchitobiose-immobilized sepharose aggregates rabbit erythrocytes (7 and 8 in the figure).
FIG. 4 shows that kurokawa lectin suppresses cell proliferation when cultured in addition to the culture solution of human monocytic leukemia U937 cells.
FIG. 5 shows a control when the kawakawa lectin is not present when the kawakawa lectin is added to the culture medium of human monocytic leukemia U937 cells (right diagram in the figure). It is a figure which shows that the chromatin condensation characteristic of aportis is not observed.
[Fig. 6] When kurokawa lectin is added to the culture medium of human monocytic leukemia U937 cells and cultured, it is not seen in the control in the absence of kurokawa lectin (11 in the figure). It is a figure which shows that fragmentation of DNA (DNA ladder) has occurred in a concentration-dependent manner (12, 13, 14 in the figure).
FIG. 7 shows that kurokawa lectin suppresses cell proliferation when cultured in addition to a culture solution of mouse lung cancer LL2 cells.
FIG. 8 is a graph showing that when clokawalectin is administered intraperitoneally to mice transplanted intraperitoneally with mouse lung cancer LL2 cells, the number of days of death is increased compared to the group administered with physiological saline. .
[Explanation of symbols]
1 Marker protein mixture 2 Kurokawa crude extract 3 Isolated Kurokawa lectin 4 Marker protein mixture 5 Kurokawa lectin 6 under non-reducing conditions Kurokawa lectin 7 under reducing conditions Erythrocyte aggregation with 100 ng / ml Kurokawa lectin 8 Kurokawa lectin 50 ng / ml Hemagglutination by 9 erythrocyte aggregation by 25 ng / ml kurokawa lectin 10 control erythrocyte 11 DNA from cells not treated with kurokawa lectin
12 DNA from cells treated with 20 μg / ml kurokawa lectin
13 DNA from cells treated with 10 μg / ml kurokawa lectin
14 DNA from cells treated with 5 μg / ml kurokawa lectin

Claims (3)

A lectin from Kurokawa (Boletopsis leucomelas) having the following characteristics: (a) a molecular weight of about 15,000 by SDS-polyacrylamide electrophoresis, and (b) an amino group of an N-terminal amino acid (C) Cyanogen bromide decomposition gives a peptide whose amino acid sequence from the N-terminus is Gly-Gly-Ser-Gly-Thr-Ser-Gly-Thr-Ile-Arg- d) has an activity of aggregating rabbit erythrocytes at a concentration of about 50 ng / ml, and (e) has an activity of binding to an N, N′-diacetylchitobiose carrier. The lectin according to claim 1, wherein the obtained lectin suppresses proliferation of human monocytic leukemia U937 cells and mouse lung cancer LL2 cells and exhibits apoptosis-inducing activity. Extracting Kurokawa with a buffer solution, and adsorbing, separating, and fractionating the lectin of claim 1 using affinity chromatography using ammonium sulfate precipitation and N, N'-diacetylchitobiose as a ligand. The method for separating and purifying Kurokawa-derived lectin according to any one of claims 1 and 2.

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