JPH104959A - Alpha1 - 6 fucosyltransferase derived from human - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new fucosyltransferase useful for the saccharide chain engineering such as modification or synthesis, etc., of a saccharide chain and/or diagnosis of diseases such as cancer by purifying a human cell culture solution having activities of an α 1 → 6 fucosyltransferase. SOLUTION: This a 1-6 fucosyltransferase derived from a human is obtained by concentrating or desalting a human cell culture solution as a crude enzymic solution having activities of the a 1→6 fucosyltransferase such as a serumfree culture solution of a human gastric cancer cell and then purifying the resultant product by an ion exchange chromatography, etc., and has the following physicochemical properties: (1) transferring fucose from guanosine diphosphate-fucose to N-acetylglucosamine bonded to Asn in the basic part of an asparagine type saccharide chain through an a 1 → 6 bond, (2) optimal pH about 7.5, (3) about 30-37 deg.C optimal temperature and (4) about 60000 molecular weight [measured by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)].

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to human-derived α1 → 6
Regarding fucosyltransferase, in particular, N-linked to Asn in the base of asparagine-type sugar chain (Asn-type sugar chain)
-Α1 → 6 to acetylglucosamine (GlcNAc)
Guanosine diphosphate (GDP)
The present invention relates to a novel human-derived α1 → 6 fucosyltransferase which is an enzyme that transfers fucose from fucose and is useful for sugar chain engineering such as sugar chain modification and synthesis and / or diagnosis of diseases such as cancer.

[0002]

2. Description of the Related Art In recent years, interest in the structure and function of sugar chains in complex carbohydrates such as glycoproteins and glycolipids derived from higher organisms has been increasing, and research has been actively conducted. Sugar chains are formed by the action of sugar hydrolases and glycosyltransferases, of which the glycosyltransferases are the major contributors.
Glycosyltransferases are enzymes that use sugar nucleotides as sugar donors to transfer sugars to sugar chains serving as acceptors and extend sugar chains.
Its specificity for the receptor sugar chain structure is rigorous, and usually one glycosidic bond is formed by one corresponding transferase. Therefore, glycosyltransferases are used for structural studies of the sugar chain portion of glycoconjugates, convenient synthesis of specific sugar chain structures, and modification of natural sugar chain structures. Further, it is expected to be used for modifying the properties of glycoconjugates or cells by artificial modification of sugar chains. For these reasons, development of various glycosyltransferases whose substrate characteristics are clear is desired.

[0003] α1 → 6 fucosyltransferase is
It is an enzyme that exists in the Golgi apparatus of organelles and is an important enzyme that is considered to be one of the enzymes that control the processing of asparagine-linked sugar chains. The action of the enzyme on an asparagine-linked sugar chain is thought to be useful for elucidation of its control mechanism, control of sugar chain structure formation, and the like.

It is also known that α1 → 6 fucosyltransferase activity is increased in some diseases such as liver cancer and cystic fibrosis, and the ratio of the enzyme reaction product is increased. Immediate development is desired.

[0005] α1 → 6 fucosyltransferase is
Although the activity was detected in cultured cells of various animals in the body fluids or organs of various animals, purified enzyme preparations include α1 → 6 fucosyltransferase purified from pig brain in 1995. It has already been announced at the 68th Annual Meeting of the Biochemical Society of Japan. However, human cystic fibrosis cell debris is still not available from humans [Journal
Journal of Biological Chemistry
266), 21572-21577
(1991)] is known, but this enzyme is obtained as a membrane-bound enzyme, and since bovine serum is required for cell culture, it is difficult to purify the enzyme. Because of the enormous cost of culturing such cells, it is practically difficult to stably supply the enzyme preparation.

[0006]

An object of the present invention is to provide a human α1 → 6 fucosyltransferase that can be stably supplied as a reagent for sugar chain structure analysis, sugar chain engineering or a diagnostic agent.

[0007]

Means for Solving the Problems The present inventors have conducted various studies in order to achieve the above object, and as a result, purified a protein having α1 → 6 fucosyltransferase activity from a human cell culture, and obtained an enzymatic property. Elucidated, and reached the present invention.

That is, the present invention is a human α1 → 6 fucosyltransferase having the following physicochemical properties. (1) Action: (GlcNAcβ1-2Manα1-6)
(GlcNAcβ1 {2Manα1-3) Manβ1}
4GlcNAcβ1-4GlucNAc-R (where R
Represents a peptide chain) as a receptor, and transfers fucose from guanosine diphosphate-fucose to a hydroxyl group at position 6 of GluNAc closest to the peptide chain of the receptor,
(GlcNAcβ1-2Manα1-6) (GlcNAc
cβ1─2Manα1-3) Manβ1─4GlcNA
Generate cβ1-4 (Fucα1-6) GlucNAc-R. (2) Optimum pH: about 7.5 (3) pH stability: pH 4.0 after treatment at 4 ° C. for 5 hours.
It is stable in the range of up to 10.0. (4) Optimum temperature: about 30 to 37 ° C. (5) Inhibition or activation: The activity does not require a divalent metal for its expression, and its activity is not inhibited in the presence of 5 mM EDTA. (6) Molecular weight: about 60,000 (SDS-polyacrylamide gel electrophoresis)

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION As a starting material for purifying the enzyme of the present invention, any human cell culture having α1 → 6 fucosyltransferase activity may be used. α1 →
Specific examples of cells having 6-fucosyltransferase activity include, for example, human pancreatic cancer cells, human gastric cancer cells, human myeloma cells, and the like. The α1 → 6 fucosyltransferase of the present invention is present in the cell membrane as a membrane-bound enzyme, but is released into the culture solution as a soluble enzyme by being cleaved by a protease at a site that does not affect the enzyme activity. The culture solution can be used as a crude enzyme solution without complicated operations such as cell disruption and enzyme solubilization. Further, by using cells capable of serum-free culture, a crude enzyme solution of high purity can be obtained at low cost. After concentrating and desalting the culture solution, a purified enzyme sample having no contaminating transferase and glycosidase activities can be obtained by ion exchange chromatography, affinity chromatography, or the like.

In the present invention, for example, human gastric cancer cells MKN4
The serum-free culture solution of No. 5 was concentrated by filtration with an ultrafiltration membrane, and 5 mM 2
-Mercaptoethanol and 0.1% CHAPS [3
-((3-cholamidopropyl) dimethylammonio)-
1-propanesulfonate] and a buffer solution exchange with a Tris-HCl buffer solution (pH 7.5) to obtain a crude enzyme solution.

Further, this enzyme solution is used in Q-Sepharose,
GDP-hexanolamine sepharose, (GlcN
Acβ1-2Manα1-6) (GlcNAcβ1-2
Manα1-3) Manβ1-4GlucNAcβ1-
The fucosyltransferase of the present invention can be purified by subjecting it to column chromatography such as 4GlucNAc-asparagine sepharose and collecting the active fraction.

The enzymatic properties of the α1 → 6 fucosyltransferase of the present invention are as follows. (1) Action: (GlcNAcβ1-2Manα1-6)
(GlcNAcβ1 {2Manα1-3) Manβ1}
4GlcNAcβ1-4GlucNAc-R (where R
Represents a peptide chain) as a receptor, and transfers fucose from guanosine diphosphate-fucose to a hydroxyl group at position 6 of GluNAc closest to the peptide chain of the receptor,
(GlcNAcβ1-2Manα1-6) (GlcNAc
cβ1─2Manα1-3) Manβ1─4GlcNA
Generate cβ1-4 (Fucα1-6) GlucNAc-R.

(2) Measurement of Enzyme Activity The α1 → 6 fucosyltransferase activity of the present invention was measured as follows. That is, asparagine at the sugar chain terminal is converted to 4- (2-pyridylamino) butylamine [PABA: -NH ₂ (CH ₂ ) ₄ -NH-pyrid.
Ine], a compound represented by the following formula 1 was used as a substrate for measuring the enzyme activity. By using the substrate, the product of the enzyme reaction transferred at the α1 → 6 bond of fucose can be subjected to fluorescence detection by high performance liquid chromatography.

[0014]

Embedded image

A specific measuring method will be described below. 250 containing 62.5 μM of the acceptor fluorescently labeled substrate represented by the above formula 1 and 625 μM of the donor substrate (GDP-fucose)
mM female (MES) buffer, pH 7.0, 40 μl,
An enzyme solution (10 μl) was added and mixed, and reacted at 37 ° C. for 1 hour. After stopping the reaction by boiling for 5 minutes, the reaction solution is subjected to high performance liquid chromatography, and the peak of the product is quantified with a fluorescence detector. One unit of the enzyme amount is 1 pmole of GlcNAcβ1-2 Ma per minute under these conditions.
nα1-6 (GlcNAcβ1-2Manα1-3) M
anβ1 → 4GlcNAcβ1-4 (Fucα1-6)
GlcNAc-R [R is _{Asn-NH- (CH 2) 4} -
PA and PA mean a 2-pyridylamino group. ].

(3) Optimum pH As shown by the curve in FIG. 1, the enzyme of the present invention has a high activity at a pH of about 7.0 to 7.5. In FIG. 1, pH 4.5 to 7.5 was measured using a 500 mM female (MES) buffer (black circles), and pH 7.0 to 9.0 was measured using a 100 mM Tris-HCl buffer (white circles).

(4) pH Stability As shown in FIG. 2, the pH stability of the enzyme of the present invention is about 4 to 10, especially between 5 and 9. The buffer used for the measurement was 50 m at pH 3.5 to 5.5.
M acetate buffer (closed triangle), pH 5.5-7.5 at 50
mM female (MES) buffer (filled circle) at pH 7.5
9.0: 50 mM Tris-HCl buffer (open circles), pH
For 9.0 to 11.5, a sodium bicarbonate buffer (open triangle) was used. The residual activity was measured after treating the enzyme of the present invention in each buffer at 4 ° C. for 5 hours at each pH. FIG. 1 shows the α-value obtained by the present invention.
FIG. 2 is a graph showing the relationship between the pH (horizontal axis) and the relative activity (%, vertical axis) of 1,6-fucosyltransferase.
It is a graph which shows activity of (horizontal axis) and residual activity (%, vertical axis).

(5) Optimal temperature The optimal temperature of the enzyme of the present invention is, as shown in FIG.
Around, and can be used in the range of 20 to 40 ° C. The frozen product is stable at -20 ° C for at least several months.

(6) Requirement of divalent metal ion Many glycosyltransferases require a divalent metal such as magnesium or manganese for their activity. The enzyme of the present invention is used in the absence of a divalent metal or with a chelating agent. It shows sufficient activity in the presence of certain EDTA and does not show divalent metal ion requirement.

(7) Molecular weight The purified sample of the enzyme of the present invention shows a single band at a molecular weight of about 60,000 in SDS-polyacrylamide gel electrophoresis.

(8) Morphology The enzyme of the present invention originally exists in the cell membrane as a membrane-bound enzyme, but porcine α1 → 6 has been reported so far.
Unlike fucosyltransferase and α1 → 6 fucosyltransferase of human cystic fibrosis cells,
Since it is released into the culture solution by being cleaved at a site where the enzyme activity is not affected by proteolytic enzymes in the cultured cells, it exists as a soluble enzyme that is easy to handle.
In view of the above properties, the α1 → 6 fucosyltransferase of the present invention has an optimum pH, metal requirement, and molecular weight.
Conventional α1 → 6 fucosyltransferase derived from human cystic fibrosis cells (optimum pH 6.5, molecular weight 34,000
0 and 39,000).

Using the α1 → 6 fucosyltransferase of the present invention, the following matters can be elucidated. (1) Fucose can be newly introduced into an asparagine-linked sugar chain to artificially modify the sugar chain structure. By doing so, it is possible to elucidate the mechanism of controlling the processing of sugar chains of cell devices and glycoconjugates, and to elucidate the role of sugar chains. (2) Various diseases can be diagnosed by measuring the enzyme activity of the present invention. (3) Various diseases can be diagnosed by using an antibody induced by the enzyme of the present invention.

[0023]

Next, the present invention will be described specifically with reference to examples. Example 1 (1) Preparation of Crude Enzyme Solution from Serum-Free Culture Solution of Human Gastric Cancer Cell MKN45 Human RPMI 1640 medium containing sodium selenite and kanamycin: Ham's
The cells were cultured in a serum-free medium of F-12 medium = 1: 1 at 37 ° C. and a carbon dioxide concentration of 5%. 100 liters of the resulting serum-free culture was concentrated to 2 liters by ultrafiltration, and 5 mM 2-mercaptoethanol and 0.1 liters were added.
% CHAPS [3-((3-cholamidopropyl) dimethylammonio) -1-propanesulfonate] containing Tris-HCl buffer, pH 7.5, buffer exchange,
A crude enzyme solution was used. Further, this crude enzyme solution was used for Q-sepharose, GDP hexanolamine sepharose, (Gl
cNAcβ1-2Manα1-6) (GlcNAcβ1
-2Manα1-3) Manβ1-4GlcNAcβ1
The resultant was subjected to column chromatography such as -4GlcNAc-asparagine sepharose, and the active fractions were collected to purify the fucosyltransferase of the present invention.

(2) Preparation of enzyme The crude enzyme extract obtained in the above (1) was used for the following purification. 5 mM 2-mercaptoethanol and 0.1%
It was applied to a column of Q-Sepharose equilibrated with a Tris-HCl buffer containing CHAPS, pH 7.5. After washing the column with 5 volumes of the same buffer, 0.1 M
The active fraction eluted with the same buffer containing NaCl was collected. The active fraction was concentrated with an ultrafiltration membrane, and buffer exchange was performed with a Tris-HCl buffer containing 5 mM 2-mercaptoethanol and 0.7% CHAPS, pH 7.5,
The sample was applied to a column of GDP hexanolamine sepharose equilibrated with the same buffer. Elution is from 0 to 0.5M N
Performed by a linear concentration gradient of aCl. 0.15-0.3
The active fraction of M was collected, concentrated with an ultrafiltration membrane, desalted, and then treated with 5 mM 2-mercaptoethanol and 0.7 mM.
Equilibrated with Tris-HCl buffer containing 7.5% CHAPS, pH 7.5 (GlcNAcβ1-2Manα1-6)
(GlcNAcβ1-2Manα1-3) Manβ1-
The sample was applied to a column of 4GlcNAcβ1-4GlcNAc-asparagine sepharose. Elution was performed with a linear gradient of NaCl from 0 to 0.5M. 0.2-0.
The 5M active fraction was collected, concentrated by an ultrafiltration membrane, and desalted to obtain α1 → 6 fucosyltransferase. The obtained α1 → 6 fucosyltransferase fraction showed a single band at a molecular weight of 60,000 by SDS polyacrylamide gel electrophoresis,
There was no activity of other transferases and glycosidases, and the purified enzyme preparation was sufficiently usable as a sugar chain research reagent.

FIG. 1 shows the results obtained by determining the optimum pH of the enzyme of the present invention by changing the pH of the buffer solution. The enzyme has a pH
High activity was shown around 7.0 to 7.5. In the figure, black circles indicate the case where a female buffer was used, and white circles indicate the case where a Tris-HCl buffer was used. The pH stability of the enzyme of the present invention was similarly examined. FIG. 2 shows the residual activity after treating the enzyme in each buffer at the respective pH at 4 ° C. for 5 hours. The enzyme is relatively stable at pH 4 to 10, particularly at pH 5 to 9. Showed better stability between In the figure, the closed triangles indicate the case where the acetate buffer, the closed circles the case where the female buffer, the open circles where the Tris-HCl buffer, and the open triangle the case where the sodium bicarbonate buffer were used. The optimum temperature of the enzyme of the present invention was found around 37 ° C. as shown in FIG. Further, it was considered that sufficient action was maintained in the range of 20 to 40 ° C. The frozen product stably retained its activity at -20 ° C for at least several months. Further, the enzyme of the present invention exhibited a sufficient activity even in the absence of divalent metal ions. Furthermore, since sufficient activity was exhibited even in the presence of 5 mM EDTA as a chelating agent, it was concluded that the requirement for divalent metal ions was not exhibited.

[0026]

EFFECT OF THE INVENTION The human α1 → 6 fucosyltransferase of the present invention differs from known human α1 → 6 fucosyltransferases in various points in physicochemical properties and under optimal reaction conditions closer to physiological conditions. Show action. Therefore, the present invention can provide a novel α1 → 6 fucosyltransferase useful for sugar chain engineering such as sugar chain modification or synthesis and / or diagnosis of diseases such as cancer.

[Brief description of the drawings]

FIG. 1 shows the optimum pH of α1 → 6 fucosyltransferase of the present invention.

FIG. 2 shows the pH stability of α1 → 6 fucosyltransferase of the present invention.

FIG. 3 shows the optimum temperature of α1 → 6 fucosyltransferase of the present invention.

Claims (3)

[Claims] 1. A human-derived α1 having the following physicochemical properties:
→ 6 Fucosyltransferase. (1) Action: (GlcNAcβ1-2Manα1-6)
(GlcNAcβ1 {2Manα1-3) Manβ1}
4GlcNAcβ1-4GlucNAc-R (where R
Represents a peptide chain) as a receptor, and transfers fucose from guanosine diphosphate-fucose to a hydroxyl group at position 6 of GluNAc closest to the peptide chain of the receptor,
(GlcNAcβ1-2Manα1-6) (GlcNAc
cβ1─2Manα1-3) Manβ1─4GlcNA
Generate cβ1-4 (Fucα1-6) GlucNAc-R. (2) Optimum pH: about 7.5 (3) pH stability: pH 4.0 after treatment at 4 ° C. for 5 hours.
It is stable in the range of up to 10.0. (4) Optimum temperature: about 30 to 37 ° C. (5) Inhibition or activation: The activity does not require a divalent metal for its expression, and its activity is not inhibited in the presence of 5 mM EDTA. (6) Molecular weight: about 60,000 (SDS-polyacrylamide gel electrophoresis) 2. The method according to claim 1, which is purified from a human cell culture solution.
The human-derived α1 → 6 fucosyltransferase according to the above. 3. The human α1 → 6 fucosyltransferase according to claim 2, wherein the human cell culture is a serum-free culture of human gastric cancer cells.