JPH0759561A - Fucosyltransferase - Google Patents

Abstract

PURPOSE:To obtain an alpha-1,3-fucosyltransferase useful as a reagent for the saccharide chain engineering. CONSTITUTION:This alpha-1,3-fucosyltransferase has the following physico-chemical properties: action on production of Galbeta1-4 (Fucalpha1-3)GlcNAcbeta1-R (R denotes a saccharide residue) from GDP fucose using Galbeta1-4GlcNAcbeta1-R as a receptor; substrate specificity in which Galbeta1-3GlcNAcbeta1-R and NeuAcalpha2-3Gabeta1-4 GlcNAcbeta1-R do not become receptors without tranferring fucose to hydroxyl group at the 2-position of any Gal in the Galbeta1-4GlcNAcbeta1-R or Galbeta1-3 GlcNAcbeta1-R; optimum pH: pH 7.0; pH stability: stable at pH 6.0-11.0 by treatment at 37 deg.C for 5hr and temperature stability: stable at about 45 deg.C by treatment for 20min. The alpha-1,3-fucosyltransferase is isolated from an extracted solution from a calf liver.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an α-1,3-fucosyltransferase useful for analysis of sugar chain structure and function, sugar chain structure modification, sugar chain synthesis, and other sugar chain engineering.

[0002]

2. Description of the Related Art In recent years, the structure and function of the sugar chain portion in glycoconjugates such as glycoproteins and glycolipids derived from higher animals have been studied. And glycosyltransferases play an important role. Glycosyltransferases require a disaccharide unit as an acceptor and not only have specificity for their structure strictly, but usually one glycosidic bond is formed by one corresponding transferase. It is used for structural studies of chain parts. Furthermore, glycosyltransferase can modify a natural sugar chain structure or can easily synthesize a specific sugar chain structure. For example, Galβ1-4GlcNAc is often attached to the non-reducing end of a sugar chain.
A β-structure is found. Conventionally, analysis methods such as methylation analysis and nuclear magnetic resonance spectroscopy have been used for such sugar chain structure analysis, but they have the drawback of requiring a large amount of sample. However, using α-1,3-fucosyltransferase that specifically transfers fucose to the 3-position of GlcNAc of this structure, the enzyme is reacted with GDP-fucose and a sugar chain of unknown structure to form a sugar chain. If fucose is transferred, it can be easily determined that the Galβ1-4GlcNAcβ-structure is present at the non-reducing end of this sugar chain. Also, in the so-called sugar chain remodeling technology, which artificially modifies the sugar chain structure in glycoproteins and glycolipids, if fucose is added to a specific binding position by using the enzyme as a method of adding fucose to sugar chains. It becomes possible to introduce. Thus, in the analysis of sugar chain structure, modification of sugar chain structure, and synthesis of sugar chain, development of a glycosyltransferase having a clear substrate specificity is desired. α-1,
Although 3-fucosyltransferase has been detected in body fluids and organs of various animals, human serum [Journal of Biological Chemistry] was used as a purified enzyme preparation.
emistry), vol. 267, pp. 2745-2754 (19
92)] and cells derived from human neuroblastoma [Journal
Little is known other than the enzyme derived from The Biological Chemistry, Volume 266, Pages 3526 to 3531 (1991)], and it is practically difficult to stably supply this enzyme preparation.

[0003]

Therefore, an object of the present invention is to provide α-1, which has a clear substrate specificity and can be stably supplied as a sugar chain engineering reagent for performing sugar chain structure analysis and sugar chain remodeling. Providing 3-fucosyltransferase.

[0004]

The present invention will be described in brief. The present invention is characterized by having the following physicochemical properties.
-1,3-fucosyltransferase. 1) Action Using Galβ1-4GlcNAcβ1-R (R represents a sugar residue, and the sugar residue is hereinafter referred to as R) as an acceptor, fucose is transferred from GDP fucose to the hydroxyl group at the 3-position of GlcNAc to form Galβ1-4 (Fucα1). -3) GlcNAcβ
Generate 1-R. 2) Substrate specificity As Galβ1-4GlcNAcβ1-R as a receptor,
Transfer of fucose from GDP fucose to 3-hydroxyl group of GlcNAc to form Galβ1-4 (Fucα1-3) Glc
Produces NAcβ1-R, but Galβ1-3GlcN
Acβ1-R does not become a receptor. Galβ1-4Gl
cNAcβ1-R or Galβ1-3GlcNAc
It does not transfer fucose to the hydroxyl group at the 2-position of Gal of β1-R. NeuAcα2-3Galβ1-4GlcNAc
β1-R does not become a receptor. 3) Optimum pH: pH 7.0 4) pH stability: pH 6.0-37 after 5 hours of treatment.
Stable in the range of 11.0 5) Temperature stability: Stable up to around 45 ° C after 20 minutes of treatment

In view of the above situation, the present inventors have been pursuing fucosyltransferase, and have purified the present enzyme from a calf liver extract to elucidate its enzymatic properties and have arrived at the present invention.

The present invention will be described in detail below. The enzyme source used in the present invention may be any animal organ having fucosyltransferase activity. It may also be collected from animal body fluids. Specific examples of organs having fucosyltransferase activity include bovine liver, pig liver, horse liver, sheep liver, rabbit liver and the like. Alternatively, the enzyme may be collected from these animal body fluids such as serum. The fucosyltransferase of the present invention can be obtained, for example, by preparing a crude extract from bovine liver, such as calf liver, and separating the enzyme from the extract.

Since fucosyltransferase in calf liver is a membrane-bound enzyme, a crude enzyme extract can be obtained from a crushed liver using a suitable surfactant. Then, a purified enzyme preparation can be obtained from this extract by a commonly used purification means. For example, purification is carried out by salting out, organic solvent precipitation, ion exchange column chromatography, hydrophobic binding column chromatography, affinity column chromatography, gel filtration, lyophilization, etc., and other contaminating transferase and glycosidase activity-free purified enzyme samples are purified. You can get the goods.

For example, calf liver was crushed in a phosphate buffer using a Waring blender, the enzyme was extracted from the crushed product with the same buffer containing Triton CF-54, and the supernatant was obtained by centrifugation. Can be collected to obtain a crude enzyme extract. Furthermore, this crude enzyme extract was added to DEAE-
Cellulose, S-Sepharose, Biogel (Bioge
l) It can be subjected to column chromatography using P-6DG, GDP-agarose, etc., and the active fractions can be collected to purify the fucosyltransferase of the present invention.

The enzymatic and physicochemical properties of α-1,3-fucosyltransferase obtained by the present invention are as follows. 1) Action As Galβ1-4GlcNAcβ1-R as a receptor,
Transfer of fucose from GDP fucose to 3-hydroxyl group of GlcNAc to form Galβ1-4 (Fucα1-3) Glc
An enzyme that produces NAcβ1-R.

2) Measurement of enzyme activity The fucosyltransferase activity was measured as follows. The following formula (Formula 1) as a substrate:

[0011]

Embedded image Galβ1-4GlcNAcβ1-3Glcβ
1-4Glc-PA

A pyridyl aminated sugar chain having a structure represented by [wherein PA means a 2-pyridylamino group] was used. Since the substrate and the enzyme reaction product with fucose transferred to it are labeled at the reducing end with a pyridylamino group, they can be directly analyzed by high-performance liquid chromatography using a reversed-phase or normal-phase column equipped with a fluorescence detector. Qualitative quantitative analysis is possible. 300 pmol of the above substrate and 500 pmol of GDP fucose, 5 mM MnCl ₂ , 100 m
4 μl of the enzyme solution was added to 6 μl of 50 mM MOPS buffer (MOPS) buffer containing M NaCl and 1 mg / ml BSA, pH 7.0, and mixed, and reacted at 37 ° C. for 1 hour. After 10 μl of acetonitrile was added to stop the reaction, the reaction solution was subjected to high performance liquid chromatography. Under these conditions, 1 μmol of pyridyl aminated lacto-per minute
The amount of the enzyme that produces N-fucopentaose III is 1 unit.

3) Substrate specificity The α-1,3-fucosyltransferase obtained according to the present invention is used as a receptor substrate in the method of 2), [1] human milk-derived lacto-N-neotetraose (Ga).
1β1-4GlcNAcβ1-3Glcβ1-4Gl
When c) is used, fucose is transferred from GDP fucose to the hydroxyl group at the 3-position of GlcNAc to give Lewis X pentasaccharide (lacto-N-fucopentaose III, Gal.
β1-4 (Fucα1-3) GlcNAcβ1-3Ga
lβ1-4Glc). [2] Lacto-N-tetraose (Galβ1-3Gl
When cNAcβ1-3Glcβ1-4Glc) is used, fucose is not transferred at all. [3] When lacto-N-neotetraose is used, G
The fucose is not transferred from the DP fucose to the 2-position hydroxyl group of the non-reducing terminal Gal. [4] α-2,3-sialylacto-N-neotetraose (NeuAcα2-3Galβ1-4GlcNAc
β1-3Gal β1-4Glc) is used, GDP
It does not transfer fucose from the fucose to the hydroxyl group at the 3-position of GlcNAc.

4) Optimum pH and pH stability The optimum pH of this enzyme is pH as shown by the curve in FIG.
It has a high activity around 6.0 to 7.5. In the figure, pH 4 to 5.5 are 100 mM acetate buffer (black squares), pH 6 is 100 mM female (MES) buffer (white triangles), pH 7 is 100 mM mops buffer (white circles), and pH 7 to 8 are 100 mM Hepes (HEPE
S) buffer solution (white square), pH 9-12 is 100 mM
The measurement was performed using a sodium hydrogen carbonate buffer solution (black circle).

The pH stability of the present enzyme is pH 5.5 to 11.5 as shown in FIG. 2, and is particularly stable between pH 6 and 11. The buffer used for the measurement is the same as that shown in FIG. 1, and the enzyme was added to each buffer at each pH.
The residual activity was measured after treatment at 37 ° C. for 5 hours.
In addition, FIG. 1 shows pH (horizontal axis) and relative activity (%, of α-1,3-fucosyltransferase obtained by the present invention).
2 is a graph showing the relationship between pH (horizontal axis) and residual activity (%, vertical axis).

5) Optimum temperature and thermostability The optimum temperature of action of this enzyme is around 37 ° C.
It is applicable in the range of ° C. The thermostability of this enzyme is 37 ℃
Stable for at least overnight at 45 ° C for at least 20
Stable for minutes. Frozen products are stable at -20 ° C for at least several months.

The following items can be elucidated using the fucosyltransferase of the present invention. (1) It is possible to artificially modify the sugar chain structure by newly introducing fucose into the sugar chain of the glycoconjugate. This makes it possible to elucidate the role of sugar chains in cell devices and glycoconjugates, particularly fucose-containing sugar chains such as Lewis X and sialyl Lewis X. (2) By using this enzyme, the presence or absence of the N-acetyllactosamine structure in the complex carbohydrate can be directly estimated, and the type 1 sugar chain and the type 2 sugar chain can be directly distinguished. (3) The reducing end of the sugar chain is preliminarily subjected to a reductive pyridyl amination method [Journal of Biochemistry (Journal
of Biochemistry), 95, 197-203 (1
984)] using a fluorescently labeled sugar chain, and the two-dimensional sugar chain mapping method [Analytical Biochemistry (Analytical Biochemistry), Volume 171, above].
73-90 (1988)], the entire sugar chain structure including N-acetyllactosamine can be estimated with a sensitivity several hundred times that of the conventional method. (4) Using the sugar chain whose reducing end is labeled with [ ³ H] or the unlabeled sugar chain, the enzymatic digestion of (2) is performed, and the enzymatic digest is analyzed by gel filtration chromatography or ion exchange chromatography. By doing so, the sugar chain structure can be estimated.

[0018]

EXAMPLES Next, the present invention will be described with reference to examples.
The present invention is not limited to the scope of the following examples.

Example 1 (1) Crush of calf liver and preparation of crude extract Calf liver, 125 g, 250 mM sucrose, 25 m
10 mM potassium phosphate buffer containing M potassium chloride, 5 mM EDTA, and a protease inhibitor, pH
After crushing with a Waring blender in 7.5,
The enzyme was extracted with the same buffer containing Triton CF-54 at a final concentration of 1.4%. After the extraction operation, the supernatant was collected by centrifugation to obtain a crude enzyme extract.

(2) Preparation of Enzyme 12 ml of 400 ml of the crude enzyme extract obtained above was used for the following purification. 0.05% Triton CF-5
It was applied to a column of DEAE-cellulose equilibrated with 10 mM potassium phosphate buffer, pH 7.5, containing 4,5 mM EDTA and protease inhibitors. The column was washed with 3 times its volume of the same buffer, and the eluted non-adsorbed fraction was collected. Add 0.1% Triton C to the active fraction.
After diluting with 10 mM MOPS buffer containing F-54 and protease inhibitor, pH 7.0, it was applied to an S-Sepharose column equilibrated with the same buffer. Elution is 0 to 1M
By a linear gradient of NaCl up to. 0.3 ~
After collecting 0.8 M active fractions and concentrating with an ultrafiltration membrane,
10 mM with 01% Triton CF-54, 2 mM 2-mercaptoethanol and 10% glycerol
It was desalted by applying it to Biogel P-6DG equilibrated with Mops buffer, pH 7.0. The eluate was applied to a GDP-agarose column equilibrated with the same buffer. 0.2 m
By eluting with the same buffer containing M GDP and 0.4 M sodium chloride and collecting the active fractions, the α of the present invention was collected.
It is possible to obtain -1,3-fucosyltransferase. The fucosyltransferase fraction thus obtained had no activity of other transferases and glycosidases, and thus the purified sample was sufficiently usable as a reagent for sugar chain research.

[0021]

INDUSTRIAL APPLICABILITY The present invention provides a novel fucosyltransferase useful for sugar chain engineering such as elucidation of sugar chain structure and function.

[Brief description of drawings]

FIG. 1 is a graph showing the optimum pH of α-1,3-fucosyltransferase obtained by the present invention.

FIG. 2 is a diagram showing pH stability of α-1,3-fucosyltransferase obtained by the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ikunoshin Kato 3-4-1, Seta, Otsu City, Shiga Prefecture

Claims (1)

[Claims] 1. An α-1,3-fucosyltransferase having the following physicochemical properties. 1) Action Using Galβ1-4GlcNAcβ1-R (R represents a sugar residue, and the sugar residue is hereinafter referred to as R) as an acceptor, fucose is transferred from GDP fucose to the hydroxyl group at the 3-position of GlcNAc to form Galβ1-4 (Fucα1). -3) GlcNAcβ
Generate 1-R. 2) Substrate specificity As Galβ1-4GlcNAcβ1-R as a receptor,
Transfer of fucose from GDP fucose to 3-hydroxyl group of GlcNAc to form Galβ1-4 (Fucα1-3) Glc
Produces NAcβ1-R, but Galβ1-3GlcN
Acβ1-R does not become a receptor. Galβ1-4Gl
cNAcβ1-R or Galβ1-3GlcNAc
It does not transfer fucose to the hydroxyl group at the 2-position of Gal of β1-R. NeuAcα2-3Galβ1-4GlcNAc
β1-R does not become a receptor. 3) Optimum pH: pH 7.0 4) pH stability: pH 6.0-37 after 5 hours of treatment.
Stable in the range of 11.0 5) Temperature stability: Stable up to around 45 ° C after 20 minutes of treatment