JPH05170800A - Lectin a obtained from tachypleus tridentatus or limuluspolyphemus and method for separating and purifying the same - Google Patents

Abstract

PURPOSE:To obtain a new lectin A useful as a reagent for detecting complex carbohydrate containing a liberation type N-acetylamino sugar, a sialic acid such as N-acetylneuraminic acid and N-glycolylneuraminic acid and a bond type sialic acid in a test specimen of organism. CONSTITUTION:A lectin A obtained from blood lymph of Tachypleus tridentatus or Limuluspolyphemus, having affinity for N-acetylamino sugar, sialic acid, its complex carbohydrate and human and animal erythrocyte, showing the following properties. Emagglutination: showing agglutination reaction in the presence and absence of Ca<2+>. Human erythrocyte +++. Equine erythrocyte ++. Chicken erythrocyte +. The lectin A is produced from Tachypleus tridentatus or Limuluspolyphemus by using affinity chromatography.

Description

Detailed Description of the Invention

[0001]

The present invention relates to Japanese horseshoe crab (Tach
ypleus tridentatus) or American horseshoe crab (Li
The present invention relates to a new lectin A obtained from hemolymph fluid of (Mulus polyphemus) and a method for producing the same.

[0002]

2. Description of the Related Art Horseshoe crab is currently known as four genera of three genera. Among them, the horseshoe crab (Limulus poly
phemus), Black-headed crab (Carcinoscorpius rotund)
icauda), Japanese horseshoe crab (Tachypleus tridentatu)
Hemagglutinin (lectin) has been isolated from the three species (s).

[0003]

An object of the present invention is to provide novel and useful lectins having properties different from those of the conventional lectins. The present inventors have found that lectin A, which is clearly different in property from conventional lectins, is present in the hemolymph of Japanese and American horseshoe crabs, and succeeded in separating and further partially purifying it. The invention has been completed.

[0004]

The present invention is obtained from the hemolymph of Japanese horseshoe crab (Tachypleus tridentatus) and has an affinity for N-acetylamino sugar, sialic acid and its complex carbohydrate, and human and animal red blood cells. Which is a lectin A having the following properties and shows an agglutination reaction in the presence and absence of hemagglutinating Ca ²⁺ .

Human red blood cells +++ Horse red blood cells ++ Chicken red blood cells + and American horseshoe crab (Limulus polyphemus)
Lectin A, which is obtained from the hemolymph fluid of Escherichia coli and has an affinity for N-acetylamino sugar, sialic acid and its complex carbohydrate, and human and animal red blood cells, and has the following properties: Lectin A hemagglutinating Ca ² Aggregation reaction is shown both in the presence and absence of ⁺ .

[0006] Human red blood cells +++ Horse red blood cells ++ It is aimed to provide chicken red blood cells +. Furthermore, the present invention uses the affinity chromatography using a sialoglycoprotein as a fixed ligand and a gel filtration method to extract the lectin of the present invention from the hemolymph of Japanese horseshoe crab (Tachypleus tridententatus) or American horseshoe crab (Limulus polyphemus). It is an object of the present invention to provide a method for producing lectin A characterized in that A is obtained.

The present invention will be described in detail below. The lectin A of the present invention is, for example, separated from the hemolymph of Japanese or American horseshoe crab by affinity chromatography using a human erythrocyte membrane sialoglycoprotein as a fixed ligand, and further combined with gel filtration using sephacryl as a support. Partially purified by. As the eluate for gel filtration, a tris-hydrochloric acid buffer containing potassium thiocyanate is preferably used.

When human red blood cell membrane sialoglycoprotein is used as a ligand, a lectin having the following properties is isolated. N-acetylamino sugar + N-acetylneuraminic acid ++ N-glycolylneuraminic acid + In addition, the lectin A of the present invention causes Ca ^{2+ in} the binding reaction with the sugar chain and the agglutination reaction of human and animal red blood cells. The known lectins obtained from the same species and other species of horseshoe crab, which do not require ions, clearly differ in that they require Ca ²⁺ ions.

[0009]

EXAMPLES Next, the present invention will be described with reference to examples. [Example 1] (1) Lis-phenol extraction method (VT Marc
50 mg of sialoglycoprotein (glycophorin HA) obtained from human erythrocyte membrane by hesi and EP Andrews, 174 , 1247 (1971) was bound to 25 ml of Affigel 10 (Bio-Rad Laboratories) to obtain a support for affinity chromatography. It was made. The column using this support was washed with 50 mM Tris-HCl buffer (hereinafter referred to as “Tris-HCl”) (pH 7.2) containing 1.0 M KSCN and 0.3 M NaCl, and 1
50 mM Tris-HCl (pH 7.2) containing 0 mM CaCl ₂ and 0.3 M NaCl
After sufficiently equilibrating with, hemolymph fluid (10 to 50 ml) of Japanese or American horseshoe crab was added to the column. After the non-adsorbed substance was made to flow out from the column using the same buffer solution, the adsorbed substance was extracted from the column with 2 to 10 mM EDTA, 0.3 M NaCl,
It was eluted with 50 mM Tris-HCl (pH 7.2) to obtain a Ca ion-dependent adsorption fraction. Next, 0.5M potassium thiocyanate (KSC
The target adsorbed substance was eluted with 0.3 M NaCl, 50 mM Tris-HCl (pH 7.2) containing N) (hereinafter referred to as “KSCN fraction” (see FIG. 1)).

The KSCN fraction was concentrated using a molecular sieve membrane (PM-30, molecular weight cut off: 30,000), and the concentrated solution was preliminarily diluted with 0.5 M KSCN in 0.3 M NaCl and 50 mM Tris-HCl (pH).
7.2) It was added to a Sephacryl S-300 (Pharmacia) column (1.0 × 70 cm) equilibrated with a buffer solution. Elution was performed using the same buffer solution, and the fractions showing agglutinating activity on human erythrocytes were collected to obtain lectin A of the present invention (see FIG. 2).

(2) The lectin A of the present invention has a molecular weight of 0.5.
Estimated by gel filtration method using Sephacryl S-300 equilibrated with 0.3 M NaCl, 50 mM Tris-HCl (pH7.2) buffer containing M KSCN, the total was about 1.7 million. The molecular weights of horseshoe crab lectin B produced in Southeast Asia and known lectin are clearly different (Fig. 3).

(3) Both lectins A of the present invention are neutral sugars such as glucose, mannose, galactose and fucose,
It did not react with amino sugars such as glucosamine and galactosamine, but N-acetylglucosamine, N-acetylamino sugars such as N-acetylgalactosamine, N-acetylneuraminic acid, N-glycolylneuraminic acid, and bovine jaw It reacted with mucins of the lower gland, fetuin, etc. (Table 1).
Table 1 shows the results when using lectin A having an agglutinating activity of 2 ^{3 on} human erythrocytes.

(4) Both lectins A of the present invention remarkably agglutinate human erythrocytes more strongly than equine erythrocytes and do not require Ca ⁺³ ion for agglutination reaction. There is a clear difference in that the horseshoe crab lectin B produced in Southeast Asia and the known lectin require Ca ²⁺ ions for the agglutination reaction (Table 2).
In Table 2, the aggregation activity was measured under the following conditions. In the absence of Ca ²⁺ : 0.3M KCl, 50mM Tris-HCl (pH7.2) In the presence of Ca ²⁺ : 0.3M KCl, 50mM Tris-HCl (pH7.2) 10mM CaCl ₂ or more, both of the present invention Lectin A is a commercially available lectin or a known lectin obtained from Japanese horseshoe crab (JP-A-53-928).
No. 00), and further American horseshoe crab lectin B
(Japanese Patent Application No.3-110631) and Japanese horseshoe crab lectin B
(Japanese Patent Application No. 3-246463) and the horseshoe crab lectin B from Southeast Asia (Japanese Patent Application No. 3-280813) are also clearly different.
It is a novel lectin having the property of not requiring Ca ²⁺ ions for the reaction with sugar chains and hemagglutination reaction and having the binding properties to free and bound sialic acid and N-acetylamino sugar.

[0014]

[Table 1]

[0015]

[Table 2]

[0016]

INDUSTRIAL APPLICABILITY The lectin A of the present invention is novel, and is free of N-acetylamino sugars in biological samples and sialic acids such as N-acetylneuraminic acid and N-glycolylneuraminic acid. It is also useful as a reagent for detecting glycoconjugates having bound sialic acid. Also,
A large number of physiologically active substances are known for glycoconjugates having bound sialic acid, and the lectin A of the present invention is also effective as an adsorption carrier for their separation and purification and affinity chromatography.

In general, animal-derived lectins require metal ions such as Ca ²⁺ ions for their action expression, but the fact that the lectin A of the present invention does not require Ca ²⁺ ions means that animal lectin molecules It has a very important property in elucidating the mechanism.

[Brief description of drawings]

FIG. 1 shows the agglutination activity of an affinity chromatography elution fraction using human erythrocyte membrane sialoglycoprotein as a fixed ligand [agglutination activity in human erythrocytes (in the absence of Ca ²⁺ )]. A: 0.5M KSCN, 0.3M NaCl, 50mM Tris-HCl (pH7.2)

FIG. 2 shows the agglutination activity of gel filtration elution fractions using Sephacryl S-300 as a support [human erythrocytes (in the absence of Ca ²⁺ ).
Aggregating activity in]. V. : Void volume

FIG. 3 shows the estimated molecular weight from the gel filtration method.

Claims (4)

[Claims] 1. A Japanese horseshoe crab (Tachypleus trident)
Lactin A having affinities for N-acetylamino sugar, sialic acid and its complex carbohydrate, and human and animal erythrocytes, which are obtained from the hemolymph of Atus). In the presence of hemagglutinating Ca ²⁺ ,
Aggregation reaction is shown even in the absence. Human red blood cells +++ Horse red blood cells ++ Chicken red blood cells + 2. American horseshoe crab (Limulus polyph)
Lectin A having affinity for N-acetylamino sugar, sialic acid and its complex carbohydrate, and human and animal erythrocytes, which is obtained from the hemolymph fluid of E. In the presence of hemagglutinating Ca ²⁺ ,
Aggregation reaction is shown even in the absence. Human red blood cells +++ Horse red blood cells ++ Chicken red blood cells + 3. Japanese horseshoe crab (Tachypleus tridentatu) using affinity chromatography using sialoglycoprotein as a fixed ligand and gel filtration.
A method for producing lectin A, wherein the lectin A according to claim 1 is obtained from the hemolymph fluid of s). 4. American horseshoe crab (Limulus polyphemus) using affinity chromatography using sialoglycoprotein as a fixed ligand and gel filtration method.
A method for producing lectin A, comprising obtaining the lectin A according to claim 2 from the hemolymph fluid of.