JPH10279597A - Seaweed lectin and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a seaweed lectin from the genus Gracilaria sp. which is rich as a resource in seaweeds. SOLUTION: This seaweed lectin is an extract from a seaweed belonging to the genus Gracilaria sp. of red alga and manifests strong agglutination activities to rabbit erythrocyte, substantially does not manifest the agglutination activities to human blood types of A, B and O, and equine, ovine and rat erythrocytes. The seaweed lectin has a saccharide combining specificity strongly recognizing fetuin, asialofetuin, BSM(bovine submandibular gland mucin), asialo BSM, thyroglobulin and lactoferrin, and is a dimer having 45,000 molecular weight comprising two subunits of 29,000 and 16,000 molecular weights having no disulfide bond.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a lectin extracted from a red algae, Ogonori, and a method for producing the same. The lectin according to the present invention is used as a clinical diagnostic reagent, a biochemical reagent and the like.

[0002]

2. Description of the Related Art Lectins are found in animals and plants or bacteria and bind to sugars or sugar chains having a specific structure to aggregate or precipitate animal and plant cells or complex carbohydrates. It is a protein.

In recent years, with the development of biochemistry and cell biology, it has become clear that the structure of sugar chains plays an important role in various cell-cell interactions, differentiation, canceration, etc.
The biological properties of lectins that recognize their unique sugar structures have attracted attention and have been widely studied.

[0004] Lectins of land plants are recognized mainly in leguminous plants, and various kinds of lectins are commercially available, which are extremely important substances in the fields of medicine and biochemistry. On the other hand, lectins derived from marine organisms have few research examples compared to those derived from terrestrial organisms,
It is also widely distributed in seaweed, and 205 species have been identified from 326 species of seaweed. However, since most of the seaweed lectins are monosaccharides and oligosaccharides and their activity is not blocked, it is difficult to purify by affinity chromatography using inhibitory monosaccharides as ligands or eluents like land plant lectins. Lectins have been isolated from 10 species of green algae and 13 species of red algae, and those of 5 species of red algae have been partially purified, and all of them have been subjected to conventional protein purification methods [Chemistry and Biology Vol. 35 9, No. 586 (1994)]. However, such a purification method is not suitable for mass production because the steps are long and require a long time. This is the reason that few of them have been isolated and purified while recognizing the existence of many seaweed lectins.

[0005] The extraction from seaweed is carried out by stirring at 4 ° C for 24 to 48 hours using a dry powder or a homogenate. However, when seaweed is powdered or homogenized and extracted, a large amount of contaminating proteins such as chromoproteins are simultaneously extracted, which makes isolation and purification of lectin difficult. For extraction at 4 ° C,
Since lectin is a glycoprotein or a protein, it is a means for extracting under safer temperature conditions. However, such extraction conditions have to be said to be a disadvantageous technique in terms of equipment in view of actual production, and it is desirable to use seaweed untreated original algae that does not powder or homogenize, and to extract at room temperature for a short time. .

Some lectins of seaweed have been reported from the genus Ogonori or Ogonori species. These are listed below.

A lectin having a molecular weight of 52,000, which has a strong activity on rabbit erythrocytes from the genus Gracilaria sp. But has substantially no activity on other animal erythrocytes.
(JP-A-3-279396)

[0008] A lectin showing strong activity on horse erythrocytes from Ogonori (Gracilaria verrucosa). This lectin is stable below 40 ° C but deactivated above 60 ° C.
[Proceedings of the 46th Annual Meeting of the Japan Society for Food Hygiene, 2 pages (1983)]

Three lectins from Gracilaria verrucosa with activity on equine erythrocytes of molecular weight 300,000-400,000, 100,000 and 45,000. [Nippon Sui
san Gakkaishi. 53, 2133 (1987)]

A lectin having a molecular weight of 41,000, which has the strongest activity on horse erythrocytes from Ogonori (Gracilaria verrucosa), but also has a considerably strong activity on rabbit, guinea pig and sheep erythrocytes. [Bulletin of the Japane
se Society of Scientific Fisheries. 47, 1079 (1
981)]

A lectin having a molecular weight of 26,000 from Gracilaria verrucosa. (Abstracts of the 1992 Spring Meeting of the Japanese Society of Fisheries Science, 314 pages)

A lectin which is stable up to 40.degree. C. against heating from squid (Gracilaria bursa-pastoris) and has a pH of 8, but is more acidic and alkaline and has lower activity. [Bulletin of the Japanese Society of Scient
ific Fisheries. 52, 323, (1986)]

A lectin having a molecular weight of 15,500 and a neutral sugar content of 20% from shiramo (Gracilaria bursa-pastoris). [Experi
entia. 46, 975 (1990)]

A lectin having a molecular weight of 150,000 from a species of Ogonori (Gracilaria tikvahiae). [Carbohydrate Research.
207, 319 (1990)]

As described above, terrestrial biological lectins are used in the fields of medicine and biochemistry, but it is desired to search for and develop lectins having a new type of sugar-binding specificity. Many land plant lectins are
The activity is blocked by simple monosaccharides and oligosaccharides, but there are few seaweed lectins whose activity is blocked by monosaccharides and oligosaccharides, and both are known to have distinctly different sugar-binding specificities. .

[0016]

DISCLOSURE OF THE INVENTION The present invention relates to a novel lectin having a different sugar-binding specificity from known seaweed lectins, targeting Gracilaria sp. The purpose of the present invention is to provide a manufacturing method thereof.

[0017]

Means for Solving the Problems The present inventors have made intensive studies to develop a novel lectin derived from seaweed.
A novel lectin has been found that is distinctly different from the previously reported ones. Furthermore, the present inventors have invented an efficient extraction method and an isolation and purification method assuming the case of industrial production of this lectin, and have completed the present invention.

That is, the seaweed lectin according to claim 1 of the present invention is a substance extracted from the red alga Gracilaria sp. Algae, which shows strong agglutinating activity on rabbit erythrocytes, Type, type O, horse, sheep, and rat erythrocytes have substantially no agglutinating activity and strongly recognize fetuin, asialofetuin, BSM (bovine submandibular gland mucin), asialo BSM, thyroglobulin, and lactoferrin. Binding specificity, molecular weight 29,000
And a dimer having a molecular weight of 45,000 comprising two subunits having no disulfide bond of 16,000.

Further, the method for producing a seaweed lectin according to claim 2 of the present invention is the method for producing a seaweed lectin according to claim 1, wherein the raw algae which is not powdered or homogenized is treated with an aqueous medium at room temperature. And extract for 12-24 hours,
It is characterized in that it is isolated and purified from the extract by affinity chromatography using a chitin column and gel filtration.

[0020]

Hereinafter, the present invention will be described in detail. The lectin of the present invention is obtained from the red alga Gracilaria sp.

Lectins obtained from Ogonori alga are:
It has the following sugar-binding specificity. (1) It shows strong agglutinating activity on rabbit erythrocytes and human A
It shows virtually no activity on type B, type B, type O, horse, sheep and rat erythrocytes. However, all erythrocytes are aggregated by pronase treatment. (2) From hemagglutination inhibition tests using various monosaccharides, oligosaccharides and glycoproteins, fetuin, asialofetuin, B
Strongly recognizes SM (bovine submandibular gland mucin), asialo BSM, thyroglobulin and lactoferrin. The lectin obtained from the genus Algae has the following physicochemical properties. (1) A glycoprotein containing 43.8% of neutral sugars having a molecular weight of about 45,000 and consisting of two subunits having a molecular weight of 29,000 and 16,000 and having no disulfide bond. (2) The stability of the solution is up to 70 ° C by heating for 30 minutes,
After holding for 3 hours between H4.0 and 10.5, the hemagglutination activity is stable without any change. In view of the above-mentioned sugar binding specificity, molecular weight and stability, lectins derived from Alga spp. Are novel lectins different from known seaweed lectins.

The lectin of the present invention can be obtained as follows. Conventionally, extraction of lectin from seaweed
It has been carried out by stirring with dry powder or homogenate at 4 ° C. for 24 to 28 hours. In addition, isolation and purification methods are usually protein purification methods because affinity chromatography is difficult, and the purification process is long and requires a long time. is there.

The lectin extraction, isolation and purification methods of the present invention described below have been developed by the present inventor to solve this problem, and are an epoch-making method for producing lectins from seaweed. Things.

In the extraction, since untreated original algae is used, a large amount of chromoprotein which is simultaneously extracted when powdered or homogenized is not extracted, and isolation and purification by chromatography in the next step are very easy. It was taken. In addition, the purified lectin thus obtained does not change its hemagglutination activity even at 70 ° C. by heating for 30 minutes, and is extremely stable, so that the following extraction, isolation and purification at room temperature can be performed. It has become possible.

First, untreated raw algae are extracted with an aqueous medium. As the aqueous medium, a medium obtained by adding a buffer such as a phosphate buffer or a Tris-HCl buffer to physiological saline is preferable. The extraction is preferably performed at room temperature between 4 and 25 ° C.
It is performed by soaking or stirring for 12 to 24 hours.

On the other hand, a column filled with chitin (chitin column) is prepared. As chitin, commercially available powdered chitin or porous granular chitin (for example, marine kite: manufactured by Fuji Boseki) can be used. The chitin column is prepared by packing chitin into a suitable chromatography column and adding 100 mM NH4O
After washing with H 2, it is prepared by washing and equilibrating with the solution used for the above extraction.

The lectin of the present invention is isolated by subjecting the above extract to affinity chromatography using a chitin column and eluting the adsorbed fraction with, for example, NH 4 OH having a concentration of 50 mM to 100 mM, followed by gel filtration. Isolation and purification can be achieved by purification by chromatography.

[0028]

The lectin derived from seaweeds of the present invention is a novel lectin, which exhibits a strong agglutinating activity on rabbit erythrocytes and has an effect on human A, B, O, horse, sheep and rat erythrocytes. And has a sugar-binding specificity of exhibiting substantially no aggregating activity, and is stable at heat and in a wide pH range. Therefore, there is a use which can be widely used in the medical field and the biochemical field by utilizing this property. In addition, the production method of the present invention has significantly reduced steps as compared with the previously reported method,
And since it can be manufactured at room temperature, there is a great advantage in terms of equipment.

[0029]

EXAMPLES The red alga Ogonori (Gracila) is described below.
The present invention will be described in more detail with reference to Examples and Test Examples conducted using ria verrucosa), but these do not limit the scope of the present invention in any way. Also described below are the red alga Tengusa spp.
The test examples using the above-mentioned mamansii) are also described.

Examples (1) To 100 g of extracted and sun-dried original algae, 2 L of 100 mM phosphate buffered saline, pH 7.4 (hereinafter referred to as PBS) was added, and room temperature at 20 to 25 ° C. For 24 hours. Then, the algal cells were removed, and the extract was filtered.

(2) affinity chromatography,
The chitin column was prepared by equilibrating chitin with PBS, filling the column for chromatography, eluting and washing with 100 mM NH4OH, and then eluting and washing with PBS.
The extract was passed through this chitin column, and eluted and washed with PBS until no elution of protein components was observed. The fraction adsorbed on the chitin column was eluted with 100 mM NH4OH, dialyzed against pure water, and lyophilized.

(3) Gel filtration chromatography, the lyophilized product was dissolved in PBS, and purified by Toyopearl HW65F which had been equilibrated with PBS in advance. The active fraction was dialyzed against pure water and lyophilized to obtain a lectin.

Test Example: Measurement of molecular weight, SDS-polyacrylamide gel electrophoresis (PAGE) under reducing and non-reducing conditions was performed by the method of Laemmli. The measurement of the molecular weight by the gel filtration method was performed using Toyopearl HW55F. The lectin derived from maxa had a molecular weight of 26,000 by SDS-PAGE under reducing and non-reducing conditions.
Was detected, indicating that the monomer had a molecular weight of 26,000. As for the lectin derived from Ogonori, two bands having a molecular weight of 29,000 and 16,000 were detected by SDS-PAGE under reducing and non-reducing conditions.
The molecular weight was calculated to be 45,000 by the gel filtration method, indicating that the lectin derived from Ogonori is a dimer having a molecular weight of 45,000 and two subunits having a molecular weight of 29,000 and 16,000 and having no disulfide bond.

Measurement of Neutral Sugar Content
Performed by the orcinol-sulfuric acid method. The neutral sugar content of lectins from Maxa and Ogonori was 67.6% and 4%, respectively.
It was 3.8%.

Sugar binding specificity The sugar binding specificity was examined by blood specificity and hemagglutination inhibition test using various monosaccharides, oligosaccharides and glycoproteins.

Table 1 shows the blood specificity of lectins derived from Maxa and Ogonori.

[Table 1]

The lectin derived from maxa showed strong agglutinating activity only on type O erythrocytes in human erythrocytes, and this activity was lost by pronase treatment of erythrocytes. It shows strong agglutination activity against rabbit and rat erythrocytes and weak agglutination against horse erythrocytes on animal erythrocytes, and this activity was attenuated by pronase treatment of erythrocytes in rabbit and rat erythrocytes, and horse erythrocytes were enhanced. . It did not show any agglutinating activity on sheep erythrocytes.

The lectin derived from Ogonori showed strong agglutinating activity only on rabbit erythrocytes and substantially no agglutinating activity on human A, B, O, horse, sheep and rat erythrocytes. However, all of the lectins showed agglutinating activity on erythrocytes treated with pronase.

Table 2 shows the results of the hemagglutination inhibition test of lectins derived from Maxa and Ogonori.

[Table 2]

The hemagglutinating activity of lectins derived from maxa is determined from the monosaccharides and oligosaccharides such as neutral sugars such as D-glucose, D-galactose, D-mannose and D-fucose, N-acetyl-D-glucosamine, -Acetyl-
Not inhibited by N-acetylamino sugars such as D-galactosamine and N-acetyl-D-mannosamine, N-acetylneuraminic acid, N-acetyllactosamine, N-acetylchitobiose and N-acetylchitotriose; Inhibited by amino sugars such as D-glucosamine, D-galactosamine and D-mannosamine and N-acetylchitotetraose. The glycoprotein was inhibited by fetuin, asialofetuin, BSM (bovine submandibular gland mucin), thyroglobulin, and lactoferrin. From these results, the lectin derived from maxa is D
-Does not bind to fucose, but binds to N-acetylchitotetraose, and specifically agglutinates human O-type erythrocytes as described above, indicating that it is a Sitisus-type anti-H lectin.

The hemagglutinating activity of the lectin from Ogonori was not inhibited by any of the monosaccharides and oligosaccharides tested. Glycoproteins include fetuin, asialofetuin, BSM (bovine submandibular gland mucin), asialo BSM,
It was strongly inhibited by thyroglobulin and lactoferrin, and weakly inhibited by α1-acid glycoprotein, glycophorin and asialoglycophorin.

The stability of the lectin derived from Maxa and Ogonori in solution was determined by heating the solution to 30 ° C. to 90 ° C. and maintaining the pH at 4.0 to 10.5.

The temperature stability was determined by dissolving both lectins derived from Maxa and Ogonori in 100 mM phosphate-buffered saline at 30 ° C., 40 ° C., 50 ° C., 60 ° C., 70 ° C., 80 ° C. and 90 ° C., respectively.
After heating for 30 minutes and cooling, the hemagglutination activity was measured.

FIG. 1 shows the results of a temperature stability test of lectins derived from Maxa and Ogonori.

The lectin derived from maxa was stable up to 90 ° C, and the lectin derived from Ogonori was stable up to 70 ° C, with no change in the agglutinating activity of erythrocytes.

The pH stabilizer was dissolved in 0.15 M NaCl together with the lectins derived from Maxa and Ogonori, and had a pH of 4.0 to 10.5.
Up to seven buffers (pH 4.0 and 5.0; 20 mM acetate buffer, pH 6.0 and 7.0; 20 mM phosphate buffer, pH 8.
0; Tris-HCl buffer, pH 9.0 and 10.5; glycine-NaOH buffer), and allowed to stand for 3 hours, after which hemagglutination activity was measured.

PH of Lectins from Maxa and Ogonori
The results of the stability test are shown in FIG.

Both lectins derived from maxa and Ogonori have p
There was no change in the hemagglutinating activity over a wide range from H4.0 to 10.5, and it was stable.

The lectin derived from Ogonori of the present invention was very stable together with the lectin derived from Maxa without change in the hemagglutinating activity in heat and in a wide pH range.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of a temperature stability test of lectins derived from maxa and Ogonori.

FIG. 2 is a graph showing the pH stability test results of lectins derived from maxa and Ogonori.

Claims (2)

[Claims] 1. A substance extracted from the red alga Gracilaria sp. Algae, which has strong agglutinating activity on rabbit erythrocytes and has human A, B, O, horse, sheep and rat erythrocytes. Has a sugar-binding specificity that strongly recognizes fetuin, asialofetuin, BSM (bovine submandibular gland mucin), asialo BSM, thyroglobulin, and lactoferrin, and has a molecular weight of 29,000.
And a seaweed lectin which is a dimer having a molecular weight of 45,000 and consisting of two subunits having no disulfide bond of 16,000. 2. The method for producing a seaweed lectin according to claim 1, wherein the raw algae that has not been powdered or homogenized is extracted with an aqueous medium at room temperature for 12 to 24 hours.
A method for producing a seaweed lectin, comprising isolating and purifying an extract from the extract by affinity chromatography using a chitin column and gel filtration.