JPH06116299A - Gene-recombinant tcf exhibiting high bioactivity owing to specified sugar chain structure - Google Patents

Abstract

PURPOSE:To provide a gene-recombinant TCF exhibiting a growth promoting action on the hepatocyte and useful for treating hepatic diseases. CONSTITUTION:A TCF having a sugar chain structure and specified by the following properties; (1) Molecular weight (SDS electrolysis method) 78000+ or -2000 dalton in nonreduction state, 52000+ or -2000 dalton (alpha chain), 30000+ or -2000 dalton (beta chain) and 26000+ or -2000 dalton (beta' chain) in reduction state. (2) Neutral saccharides, aminosaccharides and acid saccharides are contained and most of the acid saccharides are N-acetylneuraminic acid. (3) To most of the N- bonded saccharides, one or more cyanuric acids are bonded. (4) Most of asialo N-bonded sugar chains are double stranded composite N-bonded saccharides to which a fucose is bonded.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a recombinant glycoprotein TCF (r) having high biological activity due to a specific sugar chain structure.
TCF). The rTCF having a sugar chain structure obtained by the present invention has proliferative activity on liver parenchymal cells and is useful as a therapeutic agent for liver diseases.

[0002]

BACKGROUND ART β-interferon is widely known as a tumor cytotoxic factor produced by human-derived fibroblasts. The substances produced by fibroblasts are disclosed in JP-A-58-146293 and JP-A-61-33120.
No. 6/1872, 62-103021
And Japanese Patent Laid-Open No. 64-10998. In the process of studying human fibroblast-derived antitumor protein, the present inventors discovered a novel antitumor substance that is completely different from these previously reported proteins, and further investigated the cDNA encoding this protein. The cloning was successful, the entire amino acid sequence was confirmed, and the usefulness was confirmed. This novel antitumor protein and its gene are disclosed as WO90 / 10651. This novel antitumor protein is named TCF-II. In the present invention, the glycoprotein having the amino acid sequence disclosed in WO90 / 10651 is referred to as TCF. This TCF has a strong antitumor activity and a growth activity of normal cells, and further, H which is a growth factor of liver parenchymal cells.
It was confirmed to be a member of a diverse family of GFs. TCF is 780 by molecular weight measurement by SDS electrophoresis.
Shows a molecular weight of 00 ± 2000 daltons, when reduced 52000
The α-chain, which is the band of ± 2000 daltons, the β-chain, which is the band of 30000 ± 2000 daltons, and the β′-chain, which is the band of 26000 ± 2000 daltons, are shown. Two N-linked sugar chains are bound to the β chain and one N-linked sugar chain is bound to the β ′ chain.

Since TCF is a growth factor for liver parenchymal cells, its use for the purpose of liver regeneration after hepatectomy has been studied. However, the relationship between its activity and the sugar chain structure of glycoprotein has not been known. Although sugar chains may bind in the production of recombinant proteins using eukaryotic cells, the structure of this sugar chain is not determined by the cell alone, but is known to differ depending on the type of gene. It is also known that in some glycoproteins such as human erythropoietin, there are subtle differences in the sugar chain structure of the obtained glycoprotein even in the gene encoding the same protein or in the producing cell, and the biological activity is different. (Goto M. et al., Bio / TECHNOLOG
Y, Vol. 6, 67-71, 1988). At present, little is known about the relationship between sugar chain structure and physiological activity. Nakamura et al. Have published the sugar chain structure of HGF obtained by expressing a gene encoding HGF, which is one of the liver growth factors, in CHO cells derived from hamsters (Nakamura et al., Journal of the Japan Society for Agricultural Chemistry, Vol. 66 3 No., p. 302, 1992). However, the relationship between the sugar chain structure and the proliferative activity of hepatocytes has not been clarified. The present invention is, for the first time, to clarify the relationship between the liver parenchymal cell proliferation effect of rTCF and the sugar chain structure.

[0004]

The present inventors have paid attention to the usefulness of TCF, and have investigated its use as an antitumor agent and its use as a marker for diagnosis of diseases. However, TCF
The relationship between the sugar chain structure and the activity has not been confirmed. From the process of studying the action of rTCF on hepatocytes, the present inventors have for the first time found that the sugar chain structure plays an important role when rTCF expresses its activity. INDUSTRIAL APPLICABILITY The present invention has a sugar chain structure different from that of the hepatocyte growth factor that has been reported so far and is useful as a hepatocyte growth factor.
The challenge is to provide F.

[0005]

The rTCF having a sugar chain structure according to the present invention has the following physicochemical properties. The molecular weight measured by electrophoresis showed a molecular weight of 78000 ± 2000 daltons, and when reduced, the α chain, which is the band of 52000 ± 2000 daltons, the β chain, which is the band of 30000 ± 2000 daltons, and the β'band of 26000 ± 2000 daltons. Indicates a chain. It contains N-acetylgalactosamine, N-acetylglucosamine, mannose, fucose and galactose as neutral sugars and amino sugars. The acid sugar contains about 3.5 to 6.5 mol of sialic acid per mol of rTCF, most of which is N-acetylneuraminic acid. About 50% or more of N-linked sugar chains have 1 sialic acid, 20% or more have 2 sialic acids, and 5% or more have 3 sialic acids. About 40% or more of the N-linked sugar chain is a double-chain complex sugar chain in which fucose is bound, and about 5% is a double-chain complex sugar chain in which fucose having a bisect glucosamine structure is bound. It is a chain.

[0006] TCF, rTCF, HGF or rHGF having such a sugar chain structure has not been known so far. In particular, the rTCF having a sugar chain structure of the present invention is characterized by higher biological activity as compared with asialo-type rTCF. The in vitro biological activity of recombinant erythropoietin is higher than that of untreated erythropoietin in the asialo form (Tsuda E. et al., Eu.
r, J. Biochem., Vol. 188, 405-411, 1990), an inverse correlation was observed with TCF. Such a correlation between sugar chain structure and biological activity is a new finding that has not been known so far.

The method for obtaining rTCF having a sugar chain structure according to the present invention is as follows. TCF is the WO shown above
90/10651 discloses its entire amino acid sequence and the coding DNA sequence. Furthermore, based on the gene sequence described in the publication, r having the sugar chain structure of the present invention
Similarly, in order to produce TCF, WO92 / 010
It is produced by the method published as No. 53. WO92
The production of Namalwa cells described in No. 01053 can produce the biologically active rTCF having the sugar chain structure of the present invention.

TCF can be further concentrated and purified by a conventional isolation and purification method. For example, salting out, gel filtration chromatography, affinity chromatography using a monoclonal antibody, electrophoresis method and the like can be mentioned. Regarding the affinity chromatography using a monoclonal antibody among these purification methods, the present inventor proposes Japanese Patent Application No. 3-17723.
It can be purified using the monoclonal antibody filed as No. 6. The purified TCF obtained can be freeze-dried or stored frozen.

The present invention will be described in more detail with reference to the following examples.

Example 1 Production of TCF having a sugar chain structure contributing to expression of biological activity
It was carried out according to the method disclosed in the production WO92 / 01053. TCF mass expression plasmid pCDTCFdh (see above WO9
10 μg (disclosed in 2/01053) and pMCIneo (Funakoshi) in 10 μl of TE buffer (10 mM Tris, 1 mM E
DNA was dissolved in DTA, pH 7.5) and 1.5 ml of OPTI-MEM (manufactured by Gibco) was added to prepare a DNA. This was introduced into human Namalwa cells (ATCC CRL 1432) by the lipofectin method (Focus, 11 , (2), 37 (1989)), and transformants were selected by a conventional method to obtain rTCF-producing cell lines. The cells into which the TCF gene had been incorporated were cultured to obtain purified rTCF. The transformed Namalwa cells were cultured to obtain 20 liters of the culture solution. This culture solution was added to CM-Sephadex C-50 chromatograph, Mo
HPLC with a noS column and HPLC with a heparin 5Pw column were carried out in this order to obtain about 11 mg of rTCF. The cell line used for this production has been deposited as Microbial Research Institute No. 3480 (FERM BP-3480).

[0010]

Example 2 Analysis Example of Sugar Chain Structure of rTCF In this Example, an analysis example of sugar chain structure of rTCF having the sugar chain structure obtained in Example 1 will be shown. [Method] 1. Monosaccharide composition analysis 1-1. Quantification of amino sugar and neutral sugar Recombinant TCF expressed and purified in Namalwa cells
(Hereinafter, rTCF) 1 nmole was dissolved in 100 μl of 2M hydrochloric acid containing 2M trifluoroacetic acid, and the solution was mixed at 100 ° C. under reduced pressure for 6
Heated for hours. After cooling to room temperature, ribose (20 nmole) was added as an internal standard and the mixture was concentrated to dryness. 55 μl of a pyridine / methanol / water (30:15:10) solution and 2 μl of acetic anhydride were added to dissolve the sample, which was allowed to stand at room temperature for 30 minutes and then concentrated to dryness. For this sample, the sugar PA analyzer PALSTATION Mode
The monosaccharide was labeled with 2-aminopyridine using l4000 (Takara Shuzo Co., Ltd.) and a reagent kit for monosaccharide analysis (Takara Shuzo Co., Ltd.). That is, the coupling reagent is added to the analysis sample.
After adding 10 μl and reacting at 90 ° C for 10 minutes, in a nitrogen stream
It was concentrated to dryness at 60 ° C. 10 μl of a reducing reagent was added to the residue, and the mixture was reacted at 90 ° C. for 35 minutes. Methanol in this reaction mixture
After adding 20 μl and stirring well, 40 μl of toluene was added, and after stirring, the mixture was concentrated to dryness for 10 minutes at 50 ° C. under a nitrogen stream.
After repeating this concentration to dryness operation, 50 μl of toluene was added, well stirred, and concentrated to dryness for 10 minutes at 50 ° C. under a nitrogen stream. N-acetylglucosamine, N-acetylgalactosamine, mannose, galactose, fucose 20 nmol each
The above operation was performed on the sample containing e as well, and the sample was labeled with 2-aminopyridine. 200 of these analytical samples
Dissolve in 5 μl of water and add 5 μl of the sample to TSKgel Sugar AXI
It was analyzed with a column (0.46 x 15 cm; manufactured by Tosoh Corporation).
The analysis conditions by column chromatography are described below. Mobile phase; 0.7 M potassium borate buffer (pH 9.0): Acetonitrile = 9: 1 Flow rate; 0.3 ml / min Column temperature; 65 ° C Detector; Fluorometer Ex. 310nm, Em. 380nm

1-2. Determination of sialic acid 25 μg of rTCF was dissolved in 20 μl of 60 mM sodium acetate buffer (pH 6.5) containing 4 mM calcium chloride, and 2 U / ml of sialidase (from Streptococcus sp .; Seikagaku Corporation) 5 μl) was added and reacted at 37 ° C. for 3 hours. Free sialic acid in this reaction solution was quantified by the periodic acid-thiobarbituric acid method. That is, 25 μl of 0.1 M sodium periodate solution containing 9 M phosphoric acid was added, and the mixture was allowed to stand at room temperature for 20 minutes. 125 μl of O.77M sodium arsenite solution containing 0.5M sodium sulfate and O.O5M sulfuric acid was added, and the mixture was stirred well.
Thiobarbituric acid solution with 5M sodium sulfate 375
μl was added, well stirred, and heated at 100 ° C. for 10 minutes. After the reaction solution was cooled to room temperature, 625 μl of cyclohexanone was added and stirred well to extract a red reaction product. The absorption at 549 nm of the cyclohexanone layer was measured. 0-2 μg of N-acetylneuraminic acid (manufactured by Seikagaku Corporation) was treated by the periodate-thiobarbituric acid method in the same manner as the test sample to prepare a calibration curve.

1-3. Determination of sialic acid molecular species 250 μg of rTCF was dissolved in 100 μl of 2M acetic acid solution,
Sialic acid was released by heating at 80 ° C for 3 hours. The solution was cooled to room temperature and dissolved in 1.4M acetic acid, 0.75M 2-mercaptoethanol, 18mM sodium hydrosulfite solution 7mM DMB (1,2-diamino-4,5-methylene-dioxybe).
nzene) solution (100 μl) and heated at 50 ℃ for 2 hours and 30 minutes,
Free sialic acid was DMB labeled. The sample was cooled in ice water to stop the reaction. N-acetylneuraminic acid
(Seikagaku Corporation) and N-glycolylneuraminic acid (Sigma) (100 μg) were similarly labeled with DMB. These DMB labeled samples were analyzed by high performance liquid chromatography using an ODS-80 ™ column (0.46 x 25 cm; manufactured by Tosoh Corporation). The analysis conditions are described below. Mobile phase; acetonitrile: methanol: water = 9: 7:
84 (v / v) flow rate; 1 ml / min column temperature; room temperature detector; fluorometer Ex. 373nm, Em. 448nm

2. Structure analysis of N-linked sugar chain 2-1. Preparation of 2-aminopyridine-labeled sialo N-linked sugar chain 5 mg of rTCF was used as a glycoprotein sugar chain preparation system, Hydraclub S-204 (manufactured by Honen Corporation). And hydrazine decomposition reagent
(Manufactured by Hornen Co., Ltd.) was used to decompose hydrazine to release the sialo N-linked sugar chain. That is, freeze-dried r
5 mg of TCF were further dried at 50 ° C. under vacuum pump suction. After reducing the pressure with a vacuum pump, a hydrazine reagent was injected and the reaction was carried out at 110 ° C. for 1 hour. After completion of the reaction, suction was performed with a vacuum pump to remove hydrazine. The hydrazine-decomposed sample was subjected to N-acetylation using an acetylation reagent (made by Hornen Co., Ltd.). That is, after adding 2.5 ml of ammonium acetate reagent (Hornen Co., Ltd.) to the hydrazine-decomposed sample and stirring well, add 250 μl of acetic anhydride reagent (Hornen Co., Ltd.) and stir well at room temperature.
Let stand for 30 minutes. After repeating this operation, the sample was freeze-dried. The hydrazine-decomposed and N-acetylated sample was fluorescently labeled with 2-aminopyridine. That is, the freeze-dried sample was transferred to a tube with a sealed lid, and 2-aminopyridine reagent (2-aminopyridine 1 g was dissolved in concentrated hydrochloric acid 0.65 ml).
After adding 0.4 ml and stirring well, it heated at 90 degreeC for 10 minutes.
After cooling the sample to room temperature, sodium cyanoborohydride solution (80 mg of sodium cyanoborohydride
(dissolved in μl of water) 40 μl was added and heated at 90 ° C. for 1 hour.
This sample was subjected to gel filtration chromatography using a Toyopearl HW-40F (Tosoh Corporation) column (1.6x40 cm) as a carrier and a 10 mM ammonium bicarbonate solution as a mobile phase to perform 2-aminopyridine-labeled sialo N-bonding. The sugar chain was purified.

2-2. Analysis of the number of sialic acid bonds in the sialo N-linked sugar chain The purified 2-aminopyridine labeled sialo N-linked sugar chain was analyzed.
Analysis was performed using a TSKgel DEAE-5PW column (0.75 x 7.5 cm; manufactured by Tosoh Corporation). The analysis conditions are described below. Mobile phase; Mobile phase A (water), Mobile phase B (0.1M sodium chloride solution) gradient; Linear gradient flow rate from 100% mobile phase A to 100% mobile phase B over 60 minutes; 0.6 ml / min Column temperature; Room temperature detector; Fluorometer Ex. 320 nm, Em. 400 nm A 2-aminopyridine-labeled sialo N-linked sugar chain was prepared from human α ₁ -acid glycoprotein according to the method described in Section 2-1. Using this sample as a standard, rTCF sialo N-
The number of sialic acid bonds in the sugar chain was determined.

2-3. Structural analysis of asialo N-linked sugar chain A 60 mM sodium acetate buffer solution prepared by adding 2-aminopyridine-labeled sialo N-linked sugar chain preparation to 4 mM calcium chloride.
(pH 6.5) Dissolve in 20 μl and add 2 U / ml sialidase (Str
eptococcus sp. origin; Seikagaku Corporation) 5 μl was added and reacted at 37 ° C. for 3 hours to prepare an asialo N-linked sugar chain preparation. This sample was applied to a Shimpack CLC-ODS column (0.
The product was subjected to reverse phase high performance liquid chromatography using 46 × 15 cm (manufactured by Shimadzu Corp.) to purify and separate 10 main types of asialo N-linked sugar chains. The chromatographic conditions are described below. Mobile phase A; 10 mM sodium phosphate buffer (pH 3.8) Mobile phase B; 10 mM sodium phosphate buffer (pH 3.8), 0.5
% n-Butanol Mobile phase A, B concentration gradient time (min) B (%) 0 18 20 18 40 28 140 58 Flow rate; 1 ml / min Column temperature; 55 ° C Detector; Fluorometer Ex. 320nm, Em .400 nm

The purified and fractionated asialo N-linked sugar chain preparation was subjected to β-galactosidase digestion or α-fucosidase digestion under the conditions described below. β-Galactosidase digestion conditions Test sample was diluted with 0.1M citrate-sodium phosphate buffer (pH
It was dissolved in 4.1), β-galactosidase (derived from jack bean; manufactured by Seikagaku Corporation) was added to a concentration of 2 U / ml, and the mixture was reacted at 37 ° C overnight. After heating at 100 ° C for 10 minutes, centrifugation was performed at 12,000 rpm for 10 minutes, and the supernatant was used as a test sample for analysis.

Α-Fucosidase digestion conditions The test sample was treated with 0.1 M citrate-sodium phosphate buffer (pH
6.0) and α-fucosidase (from bovine kidney;
Boehringer Mannheim Yamanouchi Co., Ltd.) was added to a concentration of 5 U / ml, and the mixture was reacted at 37 ° C. for 2 hours. After heating at 100 ° C for 10 minutes, centrifugation was performed at 12,000 rpm for 10 minutes, and the supernatant was used as a test sample for analysis.

Purified and fractionated asialo N-linked sugar chains and exoglycosidase-digested asialo N-linked sugar chain samples were prepared for Shimpack CLC-ODS column and Amido-80 column (0.46 × 25 cm; manufactured by Tosoh Corporation). Was performed. The analytical conditions using the Shimpack CLC-ODS column are the same as those already described. The analytical conditions using the Amido-80 column are described below. Mobile phase A; 3% triethylamine acetate buffer (pH 7.3): acetonitrile = 35:65 Mobile phase B;
3% triethylamine acetate buffer (pH 7.3): acetonitrile = 50:50 gradient; mobile phase A 100% to mobile phase
B Linear gradient flow rate up to 100% for 50 minutes; 1 ml / min Column temperature; 45 ° C Detector; Fluorometer Ex. 320nm, Em. 400nm Elution time of each sugar chain is 2-aminopyridine labeled glucose oligomer standard The elution time of the product (manufactured by Hornen Co., Ltd.) was compared, and the elution time was converted into glucose oligomer units. The elution position of each sugar chain in terms of glucose oligomer unit was compared with the elution position of the asialo N-linked sugar chain standard to determine the sugar chain structure.

[Results] 1. Monosaccharide composition analysis 1-1. Quantification of amino sugars and neutral sugars rTCF by hydrolysis with trifluoroacetic acid and hydrochloric acid
The monosaccharides were released from and the released monosaccharides were fluorescently labeled with 2-aminopyridine. Use this analytical sample for TSKgel Sugar
Amino and neutral sugars were quantified by high performance liquid chromatography using AXI column. The analysis results are shown in Table 1.

From this analysis result, it was suggested that the sugar chain of rTCF is mainly a complex N-linked sugar chain. Further, it was speculated that the complex type N-linked sugar chain is a sugar chain of about double chain type. Since a small amount of N-acetylgalactosamine was detected, the possibility that O-linked sugar chains were present was also shown.

[0021]

[Table 1]

1-2. Determination of sialic acid Sialic acid was released from rTCF by sialidase digestion,
Free sialic acid was quantified by the periodate-thiobarbituric acid method. The sialic acid content of rTCF was 5.2 mole / mole.

1-3. Determination of sialic acid molecular species Sialic acid is released from rTCF by an acid hydrolysis method, and free sialic acid is converted to DMB (1,2-diamino-4,5-methylene-dioxyb).
enzene) and fluorescently labeled. DMB-labeled sialic acid was analyzed by high performance liquid chromatography using an ODS-80 ™ column. The chromatogram is shown in FIG. rTCF
Was confirmed to be N-acetylneuraminic acid. Also, trace amounts of N-glycolylneuraminic acid were detected.

2. Structural analysis of N-linked sugar chain 2-1. Analysis of sialic acid bond number of sialo N-linked sugar chain Sialo N-linked sugar of rTCF released by hydrazine decomposition method and fluorescently labeled with 2-aminopyridine Chain the TSKgel D
It was analyzed by high performance liquid chromatography using an EAE-5PW column. The chromatogram is shown in FIG. The number of sialic acid bonds at each peak of the sialo-glycan was determined by using a sialo-N-linked sugar chain prepared from human α ₁ -acid glycoprotein as a standard. Table 2 shows the number of sialic acid bonds in the N-linked sugar chain of rTCF analyzed by high performance liquid chromatography using a TSKgel DEAE-5PW column.

[0025]

[Table 2]

2-2. Structural analysis of asialo N-linked sugar chain A 2-aminopyridine-labeled sialo N-linked sugar chain preparation was digested with sialidase to prepare an asialo N-linked sugar chain preparation. This sample was subjected to high performance liquid chromatography using a Shimpack CLC-ODS column to purify and separate asialo N-linked sugar chains. The chromatogram is shown in FIG. The sugar chains with symbols a to j in the chromatogram were collected and their structures were analyzed by the method described in Section 2-3. The structure determined as the content of asialo N-linked sugar chains a to j is shown in FIG. Sugar chains occupying about 20% of N-linked sugar chains of rTCF
For af, we have not made a complete structural determination.

The asialo N-linked sugar chain of rTCF is characterized in that a double-chain type complex N-linked sugar chain to which fucose is bonded occupies about 50% of all sugar chains. Further, it is also a characteristic that about 5% of fucose-bonded double-chain complex N-linked sugar chains having a so-called bisected glucosamine structure shown in j are present. TCF and HGF having such a sugar chain structure are considered to be completely novel substances. In particular, it has been reported that a sugar chain having a structure of j exists in human IgG. Namalwa cells used for the production of rTCF are cell lines derived from Burkitt's lymphoma patients, and cells in which normal B cell surface antigens are mostly expressed. Therefore, Na
It is considered that rTCF expressed in malwa cells also expresses sugar chains similar to IgG produced in B cell-derived plasma cells.

[0028]

Example 3 Examination of in vitro biological activity of asialo rTCF In this example, it was confirmed that the biological activity of TCF having the novel sugar chain structure obtained in Example 1 is affected by the change of sugar chain structure. Here is an example. [Method] Examination of in vitro biological activity of asialo rTCF 1. Method for preparing asialo rTCF Asialo rTCF was prepared by treating rTCF with sialidase. That is, 0.5 mg of rTCF was dissolved in 400 μl of 0.1 M sodium acetate buffer containing 10 mM calcium chloride,
100 μl of 2 U / ml sialidase (from Streptococcus sp .; manufactured by Seikagaku Corporation) was added, and the mixture was reacted at 37 ° C. for 3 hours. Add 2% bovine serum albumin and 0.01% Twe to this sample.
After adding 500 μl of phosphate buffered saline (pH 7.2) to which en 20 was added, the solution was sterilized by filtration with a sterilizing filter (Millex GV; manufactured by Nippon Millipore Limited). To 700 μl of the filtered sample was added 2.8 ml of phosphate buffered saline (pH 7.2) containing 1% bovine serum albumin and 0.01% Tween 20, and the mixture was mixed well and frozen in a sterilized tube at -80 ° C. saved. RTC treated in the same manner without the addition of sialidase
F was prepared as a control sample.

2. Confirmation of removal of sialic acid by sialidase treatment Removal of sialic acid from rTCF used in the test was carried out by comparing the amount of sialic acid released by the sialidase treatment with the amount of sialic acid released by the acid hydrolysis method. It was That is, 50 μg of rTCF plus 10 mM calcium chloride was added to 0.1
It was dissolved in 40 μl of M sodium acetate buffer, 10 μl of 2 U / ml sialidase (from Streptococcus sp .; manufactured by Seikagaku Corporation) was added, and the mixture was reacted at 37 ° C. for 3 hours. Again 50
μg of rTCF was placed in a tube with a sealed lid, dissolved in 50 μl of 0.1N sulfuric acid solution, and hydrolyzed at 80 ° C. for 1 hour. These samples were quantified by the periodic acid-thiobarbituric acid method described in Section 1-2. Periodic acid-
The amount of each reagent used in the thiobarbituric acid method was twice the amount described in Section 1-2.

3. In vitro biological activity measurement method Mature rat hepatocytes were prepared according to a conventional method (Gchda
E. et al., Exp. CellRes., Vol. 166, 139-150, 198
6). That is, liver Wistar parenchymal cells were obtained by perfusing Wistar adult rat liver weighing about 200 g with collagenase (manufactured by Wako Pure Chemical Industries, Ltd.) and centrifuging cells at 50 xg for 1 minute 5 times under cooling at 5 ° C. Was prepared. The obtained cells were treated with Williams E supplemented with 10 nM dexamethasone and 10% fetal bovine serum.
The cells were suspended in a medium (manufactured by GIBCO) (hereinafter, activity measurement medium) and seeded in a 96-well multiwell (10 ⁴ cells / 0.1 ml / well).
). In a CO ₂ incubator adjusted to 5% CO ₂ concentration
After culturing for 20 hours, 0 to 80 ng / ml of rTCF sample was added, and the cells were further cultured for 24 hours. The uptake of deoxybromouridine was measured by partially modifying the protocol of the cell proliferation assay kit (manufactured by Amersham Japan KK). That is, 20 μl of the labeling reagent diluted 10-fold with the activity measuring medium was added to each well, and the cells were further cultured in a CO ₂ incubator for 24 hours. After the culturing, each well was washed with phosphate buffered saline (pH 7.2), 200 μl of a fixative (acetic acid: ethanol: water = 5: 90: 5) was added, and the cells were fixed for 30 minutes. Each well was washed three times with phosphate buffered saline (pH 7.2) containing 0.1% Tween 20, and then phosphate buffered saline containing 3% bovine serum albumin and 0.1% Tween 20 (pH 7. 2) 100 μl was added and left for 15 minutes. After removing this solution, 50 μl of nuclease / anti-5-bromo-2′-deoxyuridine antibody solution was added to each well and left for 1 hour. Each well was washed 3 times with phosphate buffered saline (pH 7.2) containing 0.1% Tween 20, then 50 μl of peroxidase-labeled anti-mouse IgG antibody solution was added, and the mixture was left for 30 minutes. Each well was washed three times with phosphate buffered saline (pH 7.2) containing 0.1% Tween 20, and 100 μl of peroxidase substrate solution was added and reacted for 30 minutes. 0.01
50 μl of 2.1% citric acid solution with% sodium azide
Was added to stop the reaction, and then the absorbance at 405 nm was measured with an ELISA plate reader.

[Results] 1. Confirmation of removal of sialic acid by treatment with sialidase The free sialic acid from rTCF treated with sialidase under the conditions used for the study of in vitro biological activity and free sialic acid released from sialic acid by the acid hydrolysis method was used. It was measured. The results are shown in Table 3. As shown in Table 3, the analysis values of sialic acid by both analysis methods showed almost the same numerical values, indicating that sialic acid was completely removed from rTCF by the sialidase treatment. The reason why the quantified value of sialic acid released by sulfuric acid hydrolysis is rather low is considered to be that some sialic acid is decomposed by the hydrolysis operation.

[0032]

[Table 3]

2. Examination of in vitro biological activity of asialo rTCF Using adult rat liver parenchymal cells as target cells, the in vitro biological activity of asialo rTCF and untreated rTCF was compared using the DNA uptake into the cells as an index. . The dose-dependent curve is shown in FIG. Asialo rTCF in vi
The tro biological activity was reduced to about 50% compared to untreated rTCF. From these results, it was shown that the addition of a sugar chain having an appropriate structure is important for the in vitro biological activity expression of rTCF.

[0034]

INDUSTRIAL APPLICABILITY The present invention provides rTCF having high biological activity due to a specific sugar chain structure. The rTCF having a sugar chain structure according to the present invention has a higher proliferative effect on liver parenchymal cells than that of asialo form.

[Brief description of drawings]

FIG. 1 shows an HPLC pattern of a substance obtained by fluorescently labeling sialic acid released from rTCF.

FIG. 2 HP of 2-aminopyridine-labeled sialo N-linked sugar chain released from rTCF by hydrazine decomposition method.
LC pattern is shown.

FIG. 3 shows an HPLC pattern of asialo N-linked sugar chains obtained by digesting 2-aminopyridine-labeled sialo N-linked sugar chains with sialidase.

FIG. 4 shows the structure of asialo N-linked sugar chains separated by HPLC and the composition ratio in rTCF.

FIG. 5 shows DN of rTCF having an asialo-type sugar chain and rTCF having the sugar chain structure of the present invention to mature rat hepatocytes.
The capacity dependence curve which made the uptake of A an index is shown.

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Claims (2)

[Claims] 1. A non-reducing 78000 in a molecular weight measurement by recombinant TCF (rTCF) SDS electrophoresis having a sugar chain structure specified by the following characteristics.
Shows a molecular weight of ± 2000 daltons, when reduced 52000 ±
The α chain, which is the band of 2000 daltons, the β chain, which is the band of 30000 ± 2000 daltons, and the β ′ chain, which is the band of 26000 ± 2000 daltons, are shown. As neutral sugars and amino sugars, N-acetylgalactosamine, N-acetylglucosamine, mannose, fucose, and galactose are contained. The acidic sugar contains about 3.5 to 6.5 mol of sialic acid per mol of TCF, most of which is N-acetylneuraminic acid. About 50% or more of N-linked sugar chains have 1 sialic acid, 20% or more have 2 sialic acids, and 5% or more have 3 sialic acids. About 40% or more of the N-linked sugar chain is a double-chain complex sugar chain in which fucose is bound, and about 5% is a double-chain complex sugar chain in which fucose having a bisect glucosamine structure is bound. It is a chain. 2. The rTCF having a sugar chain structure according to claim 1, which is obtained by culturing Namalwa cells transformed with a vector incorporating the human TCF gene.