JPH08313531A - Seaweed lectin and its manufacture - Google Patents

Abstract

PURPOSE: To obtain a novel seaweed lectin having characteristic agglutination activity and a specific sugar binding specificity by extracting the lectin from algae of Geldium amansii genus. CONSTITUTION: A seaweed lectin is extracted from algae of Geldium amansii genus by dipping the algae in a water-based medium prepared by adding a phosphate buffer to a physiological saline for about 12-24 hours at a temperature of 4-25 deg.C. The extracted solution is subjected to affinity chromatography using a chitin column and the adsorption fraction is eluted with 50-100mM NH4 OH, separated, and refined. The obtained lectin shows strong agglutination activity against the Group O human, rabbit, and rat red blood cells, but the agglutination disappears or decreases when the red blood cells are treated with pronase. In addition, the lectin does not show any agglutination activity against Group A and B human and sheep red blood cells. Moreover, the lectin strongly labels N-acetylchitotetraose, fetuin, asialofetuin, etc., and has a specific sugar binding property.

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 the red algae Genus Astragalus and Genus Astragalus 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]

BACKGROUND OF THE INVENTION Lectins are glycoproteins found in animals and plants or bacteria, which bind to sugars or sugar chains having a specific structure and aggregate or precipitate animal or plant cells or complex sugars, or glycoproteins other than immunological products. It is a protein.

With the recent 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, carcinogenesis, etc.
The biological property of recognizing each unique sugar structure of lectins has been noticed and widely studied.

Regarding lectins of land plants, the presence of legumes is recognized mainly in legumes, and various lectins are commercially available, and they are extremely important substances in the medical field and biochemical field. On the other hand, lectins derived from marine organisms have few studies compared to those derived from terrestrial organisms,
It is widely distributed in seaweed, and 205 species have been revealed from 326 species of seaweed. However, most of the seaweed lectins are not blocked by monosaccharides and oligosaccharides, so that it is difficult to purify them by affinity chromatography using an inhibitory monosaccharide as a ligand or eluent like land lectins. Lectins have been isolated from 10 species of green algae and 13 species of red algae, and partially purified from 5 species of red algae, and these are all subjected to the usual protein purification method [Chemistry and Biology, Vol. 35. , No. 9, p. 586 (1994)]. However, such a purification method is not suitable for mass production because it has a long process and requires a long time. This is the reason why few were isolated and purified while recognizing the presence of many seaweed lectins.

Extraction from seaweed has been carried out by stirring with dry powder or homogenate at 4 ° C. for 24 to 48 hours. However, when seaweed is pulverized or homogenized and extracted, a large amount of contaminant proteins such as pigment proteins are simultaneously extracted, which makes lectin isolation and purification difficult. For extraction at 4 ° C,
Since the lectin is a glycoprotein or a protein, it is a means for extracting under safer temperature conditions. However, such extraction conditions must be said to be a disadvantageous method in terms of equipment when considering actual production, and short-time extraction at room temperature using untreated raw algae that does not powderize or homogenize seaweed is desirable. .

As the lectin of seaweed, several ones obtained from the genus Ogonori or Ogonori species have been reported. They are listed below.

[0007] A lectin having a molecular weight of 52,000, which has a strong activity against rabbit erythrocytes from the genus Gracilaria sp., But has virtually no activity against erythrocytes of other animals.
(JP-A-3-279396)

A lectin that shows strong activity against horse erythrocytes from Gracilaria verrucosa. This lectin is stable below 40 ° C, but deactivates above 60 ° C.
[Proceedings of the 46th Annual Meeting of the Food Sanitation Society of Japan, page 2 (1983)]

Three lectins from Gracilaria verrucosa having activity against equine erythrocytes having molecular weights of 300,000 to 400,000, 100,000 and 45,000. [Nippon Sui
san Gakkaishi. 53, 2133 (1987)]

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

A lectin with a molecular weight of 26,000 derived from Gracilaria verrucosa. (Proceedings of Spring Meeting of the Fisheries Society of Japan, 1992, p. 314)

A lectin which is stable up to 40 ° C. against heat from Gramolia bursa-pastoris and is stable at a pH of 8, but its activity is reduced by acidity and alkalinity. [Bulletin of the Japanese Society of Scient
ific Fisheries. 52, p. 323, (1986)]

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

A lectin with a molecular weight of 150,000 from O. chinensis (Gracilaria tikvahiae). [Carbohydrate Research.
207, 319 (1990)]

As described above, terrestrial organism lectins are used in the fields of medicine and biochemistry, and it is desired to search for and develop lectins having a novel type of sugar-binding specificity that has never existed before. Many of the land plant lectins are
Its activity is blocked by simple monosaccharides and oligosaccharides, but there are few seaweed lectins whose activity is blocked by monosaccharides and oligosaccharides, and it is known that the two clearly have different sugar-binding specificities. .

[0016]

DISCLOSURE OF THE INVENTION The present invention is directed to resource-rich agar genus (Gelidium sp.) And genus Gracilaria (Gracilaria sp.) Among seaweeds, and has a sugar-binding specificity different from known seaweed lectins. The purpose of the present invention is to provide a novel lectin having properties and a method for producing the same.

[0017]

[Means for Solving the Problems] As a result of intensive studies to develop a novel lectin derived from seaweed, the present inventors have found that
We have found a novel lectin that is clearly different from the previously reported ones. Furthermore, the inventors have invented an efficient extraction method, isolation method, and purification method assuming this lectin for industrial production, and completed the present invention.

That is, the seaweed lectin according to claim 1 of the present invention is a substance extracted from the algae of the red alga Gelidium sp. And exhibits strong agglutinating activity against human O-type, rabbit and rat erythrocytes. , Human A-type, B-type and sheep erythrocytes, showing no agglutinating activity, N-acetylchitotetraose, fetuin, asialofetuin, BSM
(Bovine submandibular gland mucin), has a sugar-binding specificity that strongly recognizes thyroglobulin and lactoferrin, and has a molecular weight of 26,000.
It is characterized in that it is a monomer of.

The seaweed lectin according to claim 2 of the present invention is a substance extracted from the red alga Gracilaria sp. Algae and shows a strong agglutinating activity on rabbit erythrocytes, and human A-type and B-type. , O-type, horse, sheep and rat erythrocytes show virtually no agglutination activity, and fetuin,
Asialofetuin, BSM (bovine submandibular gland mucin), asialo BSM, thyroglobulin and lactoferrin have strong sugar-binding specificity and molecular weights of 29,000 and 16,0.
It is characterized by being a dimer having a molecular weight of 45,000 and consisting of two subunits having no disulfide bond of 00.

Further, the method for producing a seaweed lectin according to claim 3 of the present invention is the method for producing a seaweed lectin according to claim 1 or 2, wherein an aqueous medium is used from raw algae which is not subjected to pulverization or homogenization treatment. It is characterized in that it is extracted at room temperature for 12 to 24 hours, and then isolated and purified from the extract by affinity chromatography using a chitin column and gel filtration.

[0021]

The present invention will be described in detail below. The lectin of the present invention is obtained from the algae of the genus Astragali (Gelidium sp.) And the genus Gracilaria sp.

The lectin obtained from the alga of the genus Amanita is
It has the following sugar-binding specificity. (1) It shows strong agglutination activity against human O-type, rabbit and rat erythrocytes, but the pronase treatment of erythrocytes eliminates the agglutination of human O-type erythrocytes and reduces the agglutination of rabbit and rat erythrocytes. Furthermore, no agglutination activity was observed with human A type, B type and sheep erythrocytes without treatment or with pronase treated erythrocytes. (2) Strongly recognize N-acetylchitotetraose, fetuin, asialofetuin, BSM (bovine submaxillary mucin), thyroglobulin and lactoferrin from hemagglutination inhibition test using various monosaccharides, oligosaccharides and glycoproteins. . In addition, the lectin obtained from algae of the genus Amanita has the following physicochemical properties. (1) Glycoprotein containing 67.6% of neutral sugar having a molecular weight of about 26,000. (2) The stability of the solution is as follows:
By holding for 3 hours between H4.0 and 10.5, the hemagglutination activity was stable in all cases. The lectin derived from the algae of the genus Amaranthus is a novel lectin that is different from the known seaweed lectin in view of the sugar-binding specificity, the molecular weight, and the stability as described above.

Lectins obtained from algae of the genus Ogonori are
It has the following sugar-binding specificity. (1) Human A showing strong agglutination activity against rabbit erythrocytes
It is virtually inactive against type B, type B, type O, horse, sheep and rat erythrocytes. However, any erythrocyte aggregates by the pronase treatment. (2) From hemagglutination inhibition test using various monosaccharides, oligosaccharides and glycoproteins, fetuin, asialofetuin, B
Strongly recognizes SM (bovine submandibular mucin), asialo BSM, thyroglobulin and lactoferrin. In addition, the lectin obtained from the alga of the genus Agonori 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 as follows:
By holding for 3 hours between H4.0 and 10.5, the hemagglutination activity was stable in all cases. The lectin derived from the alga of the genus Ogonoli is a novel lectin that is different from the known seaweed lectin in view of the sugar bond specificity, the molecular weight and the stability.

These lectins can be obtained as follows. Traditionally, lectin extraction from seaweed
It is carried out with dry powder or homogenate by stirring extraction at 4 ° C. for 24-28 hours. In addition, for the isolation and purification methods, the usual protein purification method is used because affinity chromatography is difficult, and the purification process is long and requires a long time, which is a disadvantage in terms of equipment when considering actual production. is there.

The lectin extraction, isolation and purification methods of the present invention described below were developed by the present inventors to solve this problem and are epoch-making as a method for producing a lectin from seaweed. It is a thing.

In the extraction, by using untreated raw algae, a large amount of chromoprotein which is extracted at the same time when powdered or homogenized is not extracted, and the isolation and purification by chromatography in the next step is very easy. I decided. Further, the purified lectin thus obtained does not change the hemagglutination activity even at 70 ° C. by heating for 30 minutes and is very stable, so that it can be extracted, isolated and purified at room temperature as described below. It has become possible.

First, as the seaweed, untreated raw algae is used and extracted with an aqueous medium. The aqueous medium is preferably physiological saline to which a buffer solution such as a phosphate buffer solution or a Tris-hydrochloric acid buffer solution is added. The extraction is preferably at room temperature between 4 and 25 ° C.
It is carried out by immersion 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, Marinkaito: manufactured by Fuji Spinning Co., Ltd.) can be used. The chitin column was prepared by packing chitin into a suitable chromatography column and using NH ₄ O at a concentration of 100 mM.
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 at a concentration of 50 mM to 100 mM. It can be isolated and purified by purification by gel filtration chromatography.

[0030]

The lectin derived from the seaweed of the present invention is a novel lectin, and the lectin of the genus Amanita (Gelidium sp.) Is
It shows strong agglutination activity against human O-type, rabbit and rat erythrocytes, and no activity against human A-type, B-type and sheep erythrocytes. In addition, human O type,
Rabbit and rat erythrocytes have sugar specificity that their agglutinating activity disappears or diminishes.
cilaria sp.) lectin has a strong agglutinating activity on rabbit erythrocytes, and has substantially no agglutinating activity on human A, B, O, horse, sheep and rat erythrocytes. And both lectins have heat and broad p
It is stable in the H range. Therefore, it has a wide range of applications in the medical field and biochemical field by utilizing this characteristic. Further, the manufacturing method of the present invention has a significantly shortened process as compared with the previously reported method, and since it can be manufactured at room temperature, it brings great advantages in terms of equipment.

[0031]

[Examples] The following is the case of Gelidium, which belongs to the genus Astragalus of the red algae.
amansii) and the red algae genus O.
The present invention will be described in more detail with reference to Examples and Test Examples carried out in the same manner with the use of verrucosa), but these do not limit the scope of the present invention in any way.

Example (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 was 20 to 25 ° C. It was extracted with stirring for 24 hours. Then, the algal cells were removed and the extract was filtered.

(2) affinity chromatography,
A chitin column was prepared by equilibrating chitin with PBS, packing it in a column for chromatography, eluting and washing with 100 mM NH ₄ OH, and then eluting and washing with PBS.
The above extract was passed through this chitin column, and washed with PBS until elution of the protein component was no longer observed. The adsorbed fraction on the chitin column was eluted with 100 mM NH ₄ OH, dialyzed against pure water, and lyophilized.

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

Test Example: Molecular weight measurement, SDS-polyacrylamide gel electrophoresis (PAGE) under reducing and non-reducing conditions was performed by Laemmli.
Method. The molecular weight was measured by gel filtration using Toyopearl HW55F. Lectins derived from scorpion are SDS-P under reducing and non-reducing conditions.
A single band having a molecular weight of 26,000 was detected by AGE, indicating that the monomer was a monomer having a molecular weight of 26,000. The lectins derived from Gogonori have molecular weights of 29,000 and 16,000 by SDS-PAGE under reducing and non-reducing conditions.
2 bands were detected and the molecular weight was 45,0 by gel filtration method.
Since it was calculated to be 00, the lectin derived from Ogonori is
It is shown to be a dimer having a molecular weight of 45,000, which is composed of two subunits having a molecular weight of 29,000 and 16,000 having no disulfide bond.

Measurement of Neutral Sugar Content Neutral sugar content is measured by
The orcinol-sulfuric acid method was used. The neutral sugar contents of the lectins derived from the mulberry and hornworm were 67.6% and 4 respectively.
It was 3.8%.

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

Table 1 shows the blood specificity of the lectins derived from Astragalus serrata and A. chinensis.

[Table 1]

The lectin derived from Maxa showed strong agglutinating activity only on O-type erythrocytes in human erythrocytes, and this activity was abolished by pronase treatment of erythrocytes. It showed strong agglutination activity against rabbit erythrocytes and rat erythrocytes against animal erythrocytes, and weak agglutination activity against horse erythrocytes. This activity was attenuated in rabbit and rat erythrocytes and enhanced in horse erythrocytes by pronase treatment of erythrocytes. . It also showed no agglutination activity on sheep red blood cells.

The lectin derived from Ogonori showed a strong agglutinating activity only on rabbit erythrocytes, and substantially no agglutinating activity on human A-type, B-type, O-type, horse, sheep and rat erythrocytes. However, this lectin showed agglutinating activity on erythrocytes treated with pronase.

Table 2 shows the results of the hemagglutination inhibition test of lectins derived from Scutellaria and S.

[Table 2]

The hemagglutination activity of the lectins derived from Maxa was found to be neutral sugars such as D-glucose, D-galactose, D-mannose and D-fucose, N-acetyl-D-glucosamine and N among monosaccharides and oligosaccharides. -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, It was inhibited by amino sugars such as D-glucosamine, D-galactosamine and D-mannosamine, and N-acetylchitotetraose. Glycoprotein was inhibited by fetuin, asialofetuin, BSM (bovine submandibular mucin), thyroglobulin and lactoferrin. From these results, the lectin derived from Maxa of the present invention does not bind to D-fucose, but binds to N-acetylchitotetraose, and as described above, it specifically agglutinates human O-type red blood cells. , And that it is a cytisus type anti-H lectin.

The hemagglutination activity of the lectins derived from Porphyra angustifolia was not inhibited by all monosaccharides and oligosaccharides tested. Among glycoproteins, fetuin, asialo fetuin, BSM (bovine submandibular mucin), asialo BSM,
It was strongly inhibited by thyroglobulin and lactoferrin, and weakly inhibited by α ₁ -acid glycoprotein, glycophorin and asialoglycophorin.

The stability of the solution, and the stability of the lectin of the present invention, which is derived from maksa and hornwort, in the solution, were carried out by heating from 30 ° C. to 90 ° C. and maintaining the pH at 4.0 to 10.5.

The temperature stability of the lectins derived from Maxa and Ogonori was dissolved in 100 mM phosphate buffered saline, and they were dissolved 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 the temperature stability test of lectins derived from Astragalus serrata and A. chinensis.

Up to 90 ° C. of the lectin from Maxa and up to 70 ° C., the lectin from Scutellariae was stable with no change in the agglutinating activity of erythrocytes.

As the pH stabilizer, the lectins derived from Astragalus serrata and A. chinensis were dissolved in 0.15 M NaCl to adjust the pH from 4.0 to 10.5.
Up to 7 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-hydrochloric acid buffer, pH 9.0 and 10.5; glycine-NaOH buffer) were mixed and allowed to stand for 3 hours, after which hemagglutination activity was measured.

PH of lectins derived from Astragalus serrata
The stability test results are shown in FIG.

Both lectins derived from Astragalus serrata and P. chinensis
The hemagglutination activity was stable and stable in a wide range from H4.0 to 10.5.

The lectins of the present invention, which were derived from the cornweed and the corn borer, were very stable with no change in their hemagglutination activity under heat and a wide pH range.

[Brief description of drawings]

FIG. 1 is a graph showing the results of a temperature stability test of lectins derived from scorpion grass and hornworm.

FIG. 2 is a graph showing the results of a pH stability test of lectins derived from scorpion grass and hornworm.

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[Procedure amendment]

[Submission date] June 23, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction content]

In the extraction, by using untreated raw algae, a large amount of chromoprotein which is extracted at the same time when powdered or homogenized is not extracted, and the isolation and purification by chromatography in the next step is very easy. I decided. In addition, the purified lectin thus obtained was stored for 30 minutes.
The hemagglutination activity did not change even when heated to 70 ° C, and it was extremely stable, which enabled extraction, isolation and purification at room temperature as described below.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0041

[Correction method] Change

[Correction content]

Table 2 shows the results of the hemagglutination inhibition test of lectins derived from Scutellaria and S.

[Table 2]

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Claims (3)

[Claims] 1. A substance extracted from the red alga Gelidium sp. Algae, which shows a strong agglutinating activity on human O-type, rabbit and rat erythrocytes, and on human A-type, B-type and sheep erythrocytes. In contrast, it does not show agglutinating activity, and N-acetylchitotetraose, fetuin, asialofetuin, B
It has a sugar-binding specificity that strongly recognizes SM (bovine submandibular mucin), thyroglobulin and lactoferrin, and has a molecular weight of 2
A seaweed lectin that is a monomer of 6,000. 2. A substance extracted from the red alga Gracilaria sp. Algae, which shows strong agglutinating activity against rabbit erythrocytes and exhibits human A, B, O, horse, sheep and rat erythrocytes. It does not show agglutination activity, and 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 with a molecular weight of 45,000 consisting of two subunits without disulfide bonds of 16,000. 3. The method for producing a seaweed lectin according to claim 1 or 2, wherein the raw alga that has not been powdered or homogenized is extracted with an aqueous medium at room temperature for 12 to 24 hours, and the extract is extracted. A method for producing a seaweed lectin, which comprises isolating and purifying by affinity chromatography using a chitin column and gel filtration.