JPS5945679B2 - Captoganillectin and its isolation and purification method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to Japanese horseshoe crab (Tachypleust).
ridentatus), and specifically, using affinity chromatography using Japanese horseshoe crab hemolymph as a starting material and N-acetylamino sugar and/or N-acetylamino sugar-containing substance as a ligand. The present invention relates to a method for separating and purifying horseshoe crab lectin, which exhibits a wide affinity for N-acetylamino sugars.

Lectin is a protein derived from animals, plants, and microorganisms, and is a general term for substances that bind specifically to red blood cells, and hemagglutinin, which is derived from plants, has been known for a long time.

This hemagglutination reaction is said to occur when lectins specifically bind to sugar chains present on blood cells.
Furthermore, it has recently been known that certain lectins specifically aggregate not only red blood cells, but also white blood cells, cancer cells, and the like.

Recently, lectins have been discovered not only in plants but also in animals, and their physiological significance and activity have been attracting attention. The present inventors have discovered that the Japanese horseshoe crab (Tachyple-Ustridentatus)
), we discovered the existence of a lectin with sugar-binding specificity not found in conventional lectins, and arrived at the present invention.

Horseshoe crabs are said to be living fossils, and are valuable creatures that have existed since ancient times. Their habitat is limited to the east coast of North America and the eastern coast of Asia, and there are currently three genera and four species known.

Japanese horseshoe crab JapaneseHOrseshOe
Crab (scientific name: Tachypleustridsnta)
tus) only inhabits the Seto Inland Sea and Kitakyushu, and is similar to the North American horseshoe crab AmericaHOrseshO.
eCrab (scientific name: Limulus POlypheOSu)
Although the blood components and properties of s) have been studied, almost no physiological or chemical studies have been conducted.

The hemolymph of horseshoe crabs from North America contains N-acetylneuraminic acid (to NAN) and N-glycolylneuraminic acid (to NAN).
The existence of a lectin that has an affinity for NGNA) is known, and its chemical properties have also been reported {ROche,
A. C. , B.B.A. 37l242 (1974)}.

The present inventors discovered the presence of a new lectin in the hemolymph of a Japanese horseshoe crab, which has properties not found in the lectin derived from the North American horseshoe crab, and fractionated and purified this lectin. Namely, the Japanese horseshoe crab (Tachypleus
An agarose-based carrier for affinity chromatography, which uses the blood fluid of a cow, horse, sheep, etc., as a ligand, and N-acetylamino sugar-containing substances such as chitin. One fraction of NANAsN-acetylglucosamine (GlcNAc), N-acetylgalactosamine (Gl'NAc), and N-acetylmannosamine (M
The lectin is purified using an affinity chromatography carrier having N-acetylamino sugars as a binding ligand, such as anNAc), to obtain a lectin that shows affinity for these N-acetylamino sugars. The lectin of the present invention thus obtained is L
Electrofocus ink using KB Ampholine acrylamide gel sheet ◆Koyoru's isoelectric point is 5.8
It showed a single peak, and in 7.5% gel disk electrophoresis, it was so high that it did not enter the gel, and Sepha
Gel filtration using rOse4B8 showed a molecular weight of about 500,000. Furthermore, SDS electrophoresis showed a single band with a molecular weight of 22,000, and it is therefore considered that this substance exists as an aggregate of subunits with a molecular weight of 22,000 under normal conditions. Furthermore, as is clear from the examples described below, the results of amino acid analysis of the lectin of the present invention show that Pr
O, Met, and 1/2Cys were not found, and the presence of Tyr was very small, indicating that this lectin was different from the conventionally known lectin derived from the American horseshoe crab. Furthermore, from a comparison of its behavior with horse red blood cells, the lectin of the present invention is a novel lectin that is clearly different from the lectin derived from American horseshoe crabs. The Japanese horseshoe crab lectin of the present invention contains glucose (
GIc), galactose (Gal), mannose (Ma
n), neutral sugars such as fucose (Fuc), sugar acids such as glucuronic acid, and sugar amines such as glucosamine (GlcNH) and galactosamine (GalNH2), but N-acetylneu Laminic acid (NAN)
A), N-acetylglucosamine (GlcNAc), N
-Acetylgalactosamine (Ga-1NAc) and N-
It shows a strong affinity for N-acetylamino sugars such as acetylmanonesamine (ManNAc).

Utilizing this specific affinity for N-acetylaminosaccharides, the lectin of the present invention can be applied as a means to clarify the distribution and role of N-acetylaminosaccharides in vivo. Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Bovine submandibular gland mucin (Sigma reagent) 500719 was added to Br
A column for Affinity chromatography is prepared by binding to CN-activated Sepharose 4B (manufactured by Pharmacia Fine Chemicals) 4007711 and using NANA-containing bovine submandibular gland mucin as a ligand.

This column was 0.15M Nacl, 20mMcac12.
0.05M Tris-HCl buffer (pH 7.
After thorough washing with O), Japanese horseshoe crab hemolymph fluid 11 is poured into this column to adsorb bovine submandibular gland mucin-binding substances. This column was 40,15M NaCl
Contains 0.1M borate buffer (PH8.5), 650mM
N-acetylglucosamine, 0.15M NaCl, 20
0.05M Tris buffer containing mMCaC12 (PH7.
O), 0.05M Tris buffer containing 61M NaCl (P
H7. Elution was performed sequentially using each of the solvents listed above. The chromatogram at this time is shown in FIG. 1, and the analytical values of the obtained fractions are shown in Table 1. The agglutination activity was measured by diluting the indicated animal blood cells twice using a microtiter according to the method described in Immunology Experimental Procedures edited by the Japanese Society of Immunology (authored by Yoshitami Kimura, 1971), page 67.

The agglutination titer was expressed as the reciprocal of the maximum dilution value indicating agglutination activity.
Example 21. Affinity chromatography using N-acetylgalactosamine as a ligand was carried out by binding N-acetylgalactosamine of 39 to commercially available epoxy activated Sepharose 6B (manufactured by Pharmacia Fine Chemicals) 25Tn1 according to a known method. Prepare a single carrier and use it as a column.

An amount equivalent to 9.2% of the M3 fraction obtained in Example 1 was treated with this affinity chromatography column to bind the lectin of interest.

After washing with water, elution was performed using a linear concentration gradient method using N-acetylmannosamine O to 0.5M to obtain the chromatogram shown in FIG. By N-acetylmanonesamine elution, 80% of the agglutination activity against human type O red blood cells was recovered. This active fraction was desalted by gel filtration and then lyophilized.
Japanese horseshoe crab lectin 7.8W9 was obtained. Amino acid analysis of this lectin (compared with the amino acid composition of lectin derived from American horseshoe crabs) is shown in Table 2.

The results of amino acid analysis of the Japanese horseshoe crab lectin of the present invention show that PrO. Met. l/2Cys was not found T
There were very few ry. Table 3 shows the inhibition of various monosaccharides on the human O-type hemagglutination activity of the lectin of the present invention.

Table 4 shows the inhibition of various sialic acid-containing glycopeptides on the human O-type hemagglutination activity of the lectin of the present invention.

Concentrations of glycopeptides are expressed in terms of sialic acid. Table 5 shows the agglutination titers of the lectin of the present invention against human type O red blood cells and horse red blood cells, and the corresponding agglutination titers after treating each red blood cell with neuraminidase.

As is clear from Table 5, the agglutination titer of human type O red blood cells is drastically reduced by neuraminidase treatment.

However, no decrease in titer was observed in horse red blood cells. This suggests that sialic acid plays a major role in the agglutination of human type O red blood cells with the lectin of the present invention.

[Brief explanation of the drawing]

FIG. 1 shows an affinity chromatogram of the hemolymph of a Japanese horseshoe crab. A: Elution start point with 0.1M borate buffer containing 0.15M NaCl, B: 0.0 containing 50mM N-acetylglucosamine, 0.15M NaCl, 20mMCaC12
Elution start point with 5M Tris buffer, C: 1M NaCl
Elution start point with 0.05M Tris buffer containing. FIG. 2 shows the affinity chromatogram of the M3 fraction obtained in Example 1 (N-acetylmannosamine O~0.
By 5M linear concentration gradient method.

Claims (1)

[Claims] 1. The following physicochemical properties: Isoelectric point: 5.8 (electrofocus ink using an acrylamide gel sheet) Molecular weight of subunit: 22,000 Amino acid composition (mol%): Lys7.6 His4 .5 Arg1.9 Asp(n)19.1 Thr6.7 Ser7.5 Glu(n)8.0 Pro0.0 Gly13.5 Ala4.9 1/2Cys0.0 Val5.9 Met0.0 Ile7.2 Leu8. Japanese horseshoe crab Ta-ch characterized by having 2 Tyr0.4 Phe4.5
N- obtained from ypleus tridentatus
Acetylaminosugar affinity lectin. 2 Japanese horseshoe crab (Tachypleustrid)
N-
1. A method for separating and purifying a lectin with affinity for N-acetylamino sugars, which comprises using affinity chromatography using acetylamino sugars and/or N-acetylamino sugar-containing substances as ligands.