JPH0867697A - Bone resorption inhibiting albuminous substance - Google Patents

Abstract

PURPOSE: To obtain a new bone resorption inhibiting protein substance useful as a therapeutic agent for bone diseases such as osteoporosis, fracture of bone and hypercalcemia, derived from bone cells of chicken calvaria, having a specific N-end amino acid sequence and inhibitory action on bone resorption. CONSTITUTION: This new bone resorption inhibiting protein substance is derived from bone cells of chicken calvaria, has about 18,500 estimated molecular weight by nonreduced SDS-PAG and reduced SDS-PAGE, shows physiological activity with bone resorption inhibiting action, loses the activity by trypsin treatment, is adsorbed on an ion exchange resin and Q sepharose at >=pH7.5, is not adsorbed on heparin in the presence of 0.15M NaCl, has an amino acid sequence of the formula at the N-end in the molecule and is useful as a therapeutic agent for bone diseases such as osteoporosis, fracture of bone and hypercalcemia. The protein substance is obtained by homogenizing chicken calvaria in a buffer solution for cell separation containing collagenase and purifying the centrifuged supernatant liquid by a heparin-cellulofine column and a gel filtration column.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel proteinaceous substance derived from chicken bone cells which has a bone resorption inhibitory action.
More specifically, the present invention is effective for treating bone diseases in which bone metabolism function is improved by suppressing the bone resorption activity of osteoclasts in many bone diseases such as osteoporosis, bone fracture and hypercalcemia. Regarding proteinaceous substances. In particular, by suppressing the leakage of bone calcium from the bone, it can be an effective means for preventing the progression of osteoporosis and further for treating osteoporosis. Further, it can be considered to be used as an osteoclast culture test, a bone function test in clinical / basic research, and a clinical diagnostic reagent / research reagent using this substance and an antibody / receptor / receptor antibody of this substance.

[0002]

2. Description of the Related Art Bone causes osteoporosis due to a decrease in bone mass per unit volume with age. Bone functions to efficiently turn over the cycle of remodeling (bone remodeling), that is, the cycle in which old bone is absorbed and new bone is formed, supplying calcium into the blood and maintaining homeostasis of the body ( Has a coupling phenomenon). When this balance is lost and bone resorption exceeds bone formation, bone mass decreases and osteoporosis occurs (Mebio 7 (9) 22-27 (199).
0). From such a turnover mechanism, osteoporosis can be treated or prevented by activating bone formation or suppressing bone resorption. Calcitonin preparations are currently used as agents that suppress bone resorption. However,
It is known that this drug has an escape phenomenon (drug resistance) caused by continuous injection, and that many side effects such as stomach discomfort, fever, and local pain are frequently observed (Clin.
ical Calcium 2 716-7 21 (1992)).

[0003]

It is desired to develop a drug which can avoid such an escape phenomenon and has few side effects. The present inventors have searched for substances that suppress bone resorption from cells and organs of many animals, and succeeded in extracting a novel bone resorption inhibitor from bone cells of chicken calvaria. Therefore, an object of the present invention is to provide a novel physiologically active substance having a bone resorption inhibiting effect.

[0004]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors decided to isolate and purify a proteinaceous factor having a bone resorption inhibitory action which is produced in a very small amount in bone cells of chicken calvaria. Successfully, the N-terminal amino acid sequence of this substance was clarified and the present invention was completed.

That is, the present invention is a proteinaceous substance derived from chicken bone cells and having the following physicochemical properties and physiological activities. Non-reducing SDS-PAGE and reducing SDS-PAGE
The estimated molecular weight by E is about 18,500; it exhibits a biological activity having an inhibitory effect on bone resorption; loses the above biological activity by trypsin treatment; adsorbs it on an ion exchange resin, Q sepharose at pH 7.5 or higher; It does not adsorb to heparin in the presence of 15 M NaCl; and has the following amino acid sequence at the N-terminal in the molecule.

Embedded image (In the formula, A is alanine, D is aspartic acid, G is glycine, K is lysine, L is leucine, and N is
Represents asparagine, P represents proline, T represents threonine, and V represents valine. The alphabets shown in the two columns indicate the amino acids in the upper column or the amino acids in the lower column, but which cannot be specified. )

The process of isolating and purifying the proteinaceous substance of the present invention will be described in detail below. (1) Extraction of this substance from chicken calvaria 100 ml of αMEM solution per 10 g of calvaria / 10m
Wash with MHEPES buffer. After washing, remove the supernatant,
Buffer for cell separation (0.1% collagenase solution) 100
Add ml and incubate at 37 ° C for 30 minutes. 15 ml after thoroughly washing with 30 ml of PBS (phosphate buffer / saline)
PBS (phosphate buffer solution / physiological saline) was added and the sample was cooled in ice while using a homogenizer at 15 krpm for 1 hour.
Stir for minutes. This stirring operation is performed 5 times while cooling the sample. Next, this solution is centrifuged, and the supernatant is dialyzed against 10 mM Tris-HCl buffer (pH 7.0), and the internal solution is used as the extraction substance.

(2) Purification of this substance The above extracted substance was preliminarily treated with 10 mM Tris-HCl buffer (pH 7.
0) equilibrated with Q-Sepharose column. The column is washed with 10 mM Tris-HCl buffer (pH 7.0) and eluted with 0-1.0 M NaCl / 10 mM Tris-HCl buffer (pH 7.0). 0.15-0.40MN
Bone resorption inhibiting activity is observed in the aCl-eluted fraction. The bone resorption inhibitory activity is measured by the method of Test Examples described below. Concentrate by freeze-drying, Sephadex G-
At 50, gel filtration operation is performed. Molecular weight 10,000-2
Bone resorption inhibitory activity is present in the 10,000 fraction. This fraction is lyophilized, dissolved in a small amount of water, and dialyzed against 10 mM Tris-HCl buffer (pH 7.0). (3) Electrophoresis of this substance and transfer to PVDF membrane This substance obtained by dialysis is subjected to electrophoresis, that is, SDS.
-Separate by PAGE (8% polyacrylamide). Transfer to a PVDF membrane (Millipore) and stain with Coomassie Brilliant Blue (CBB). (4) Peptide sequence of this substance The band confirmed to match the bone resorption inhibitory activity in advance is cut out, and the N-terminal amino acid sequence of this substance can be determined by a protein sequencer.

From the steps shown above and the examples described below, it is understood that the proteinaceous substance of the present invention has the following physicochemical properties and physiological activities. Non-reducing SDS-PAGE and reducing SDS-PAGE
The estimated molecular weight by E is about 18,500; it exhibits a biological activity having an inhibitory effect on bone resorption; loses the above biological activity by trypsin treatment; adsorbs it on an ion exchange resin, Q sepharose at pH 7.5 or higher; It does not adsorb to heparin in the presence of 15 M NaCl; and has the following amino acid sequence at the N-terminal in the molecule.

[Chemical 3] (In the formula, A is alanine, D is aspartic acid, G is glycine, K is lysine, L is leucine, and N is
Represents asparagine, P represents proline, T represents threonine, and V represents valine. The alphabets shown in the two columns indicate the amino acids in the upper column or the amino acids in the lower column, but which cannot be specified. )

[0009]

According to the present invention, a novel proteinaceous substance that suppresses bone resorption is provided. This substance can be an extremely useful means as a therapeutic or prophylactic drug for diseases caused by abnormal bone metabolism such as osteoporosis and hypercalcemia. In addition, basic research such as osteoclast culture and measuring the in vivo concentration of this substance can be useful as a clinical diagnostic reagent for osteoporosis functional tests and the like.

[0010]

EXAMPLES The present invention will be described in more detail with reference to Examples and Test Examples. Example 1 Hereinafter, an example of a bone resorption inhibitor derived from chicken will be described. (1) Extraction of this substance In order to clarify the structure of this substance and to know its physicochemical and biological properties, a chicken (16-day-old fertilized) calvaria was used as a material. The protein was extracted from this by the following procedure. 100 ml per 1 g of chicken calvaria
10 mM HEPES buffer / αMEM medium (containing 5 mg / ml of kanamycin) (manufactured by Irvine).
The supernatant is removed, and a cell separation buffer (25 mM HEPE
S, 10 mM NaHCO ₃ , 100 mM NaCl, 3 m
MK ₂ HPO ₄ , 1 mM CaCl ₂ , 30 mM KCl,
1 mg / ml BSA, 5 mg / ml glose, 5μ
After washing with Mα-TLCK (protease inhibitor), 1m
The above-mentioned cell separation buffer containing g / ml collagenase was added, and the mixture was gently stirred at 37 ° C for 30 minutes. After washing 10 times with 30 ml of PBS (Dulbecco's phosphate buffer), 1
5 mM EDTA, 2MN containing 5 mM 1 ml PMSF
An aCl solution (pH 7.0) was added and the sample was stirred in ice while using a homogenizer for 1 minute. This stirring operation was repeated three times while cooling the sample. Next, this solution was centrifuged at 10,000 × g, the supernatant was dialyzed against 20 mM Tris-HCl buffer (pH 8.0), and filtered through a 0.22 μm pore size filter. This solution was used as the extraction substance. The extract thus obtained was dissolved in 20 mM phosphate buffer (pH 7.0) /0.15 M NaCl and added to a heparin-cellulofine column equilibrated with the same solution in advance. Wash with the same solution, 20 mM phosphate buffer (pH
It was eluted with 7.0) /0.2 M NaCl, but this substance was present only in the non-adsorbed fraction and not in the eluted fraction.
The bone resorption suppressing activity was recovered in 95% of the non-adsorbed fraction and not in the eluted fraction.

(2) Purification of this substance The above-mentioned extracted substance was previously prepared in 10 mM Tris-HCl buffer (pH 8.
0) equilibrated with Q-Sepharose Fast Flow
Add to a column (Pharmacia). The unadsorbed substance was washed with 10 mM Tris buffer and eluted with a linear concentration gradient of 10 mM Tris-HCl buffer (pH 8.0) and 0 M to 1.0 M NaCl. 0.15M to 0.40M Na
Bone resorption inhibiting activity was observed in the Cl-eluted fraction, which was designated as the Q-sepharose-eluted fraction. The bone resorption inhibiting activity was measured according to the method of Test Example 1 shown below. This Q-Sepharose eluate fraction was concentrated by freeze-drying, and the concentration was adjusted to 0.
20 mM phosphate buffer containing 15 M sodium chloride (pH
Sephadex G-50 that had been equilibrated in 7.0)
Gel filtration was performed using a column (Pharmacia).
Bone resorption inhibiting activity was observed in the fractions having a molecular weight of 10,000 to 20,000. 20% of this G-50 gel filtration fraction
M diluted to 4 times volume with mM phosphate buffer (pH 7.0) and equilibrated with 20 mM phosphate buffer (pH 7.0) in advance
It was added to the onoQ column (Pharmacia). Two
It was washed with 0 mM phosphate buffer (pH 7.0) to obtain an unadsorbed substance. Bone resorption inhibitory activity was observed in this unadsorbed fraction.
The MonoQ-unadsorbed fraction was concentrated by freeze-drying,
Superose 12 pre-equilibrated with 20 mM phosphate buffer (pH 7.0) containing 0.15 M sodium chloride
-A gel filtration operation was performed using a column (Pharmacia). As a result of measurement based on the method described in Test Example 1 at a molecular weight of around 18,500, bone resorption inhibitory activity was observed. The bone resorption inhibiting substance obtained on the Superose 12 column was used at a concentration of 1 μg / ml, 10 μg / ml of tribucin (manufactured by Sigma) and 5 mM phosphate buffer (pH 7.
When the reaction was carried out in 0) at 37 ° C. for 60 minutes, the bone resorption inhibiting activity possessed before the reaction disappeared.

(3) Electrophoresis of this substance and transfer to PVDF membrane Superose 12 gel filtration fraction was lyophilized, dissolved in a small amount of water, and dialyzed against 5 mM phosphate buffer (pH 7.0). The dialyzed substance was reduced with 2-mercaptoethanol and subjected to SDS-polyacrylamide electrophoresis. After electrophoresis, 0.8mA on PVDF membrane (Bio-Rad)
/ Cm ² for 2 hours and stained with Coomassie Brilliant Blue. (Protein structure analysis for gene cloning, p41-62, Hirano Hisa, 1993 Tokyo Kagaku Dojinsha). The obtained results are shown in FIG. (4) Peptide sequence of this substance A band which was previously confirmed to match the bone resorption inhibitory activity was cut out from the PVDF membrane, and the protein sequencer (Applied Biosystems: 473
The N-terminal amino acid sequence of this substance was determined by A type).

Test Example 1 Measurement of Bone Resorption Inhibitory Action A femur and a tibia are excised from a 5-day-old rabbit and cut into small pieces with medium using scissors. The mixture is stirred for 30 seconds in α-MEM (minimum essential medium) containing 5% fetal bovine serum, and the supernatant after standing for 3 minutes is used as a cell suspension. 4 × of this cell suspension was put into a 96-well plate containing ivory pieces sliced into a diameter of 6 mm and a thickness of 150 μm.
Seeded so that the 10 ⁵ / 250μl / well.
After culturing for 2 hours, the supernatant was removed and the test substance was added.
Α-MEM containing% FBS (fetal bovine serum) was added to 100
Add μl / well. After culturing for 24 hours, the supernatant and cells on the ivory pieces are removed, and the resorption pits formed on the dent pieces are stained with acid hematoxylin solution for 5 minutes. The number of stained resorption pits is measured under a microscope, and the presence or absence of the bone resorption inhibitory effect is determined by comparison with the number of resorption pits formed in the control group without addition of the test substance.

Example 2 The biological activity of the substance obtained in Example 1 was examined below. Examination of the bone resorption inhibitory effect of this substance This substance was measured by the method described in Test Example 1 and measured at 1 ng / ml.
The above concentrations showed a strong inhibitory effect on the formation of absorption cavities.

[Brief description of drawings]

FIG. 1 is a photograph showing the results of subjecting each fraction of Superose 12 column chromatography to SDS-polyacrylamide gel electrophoresis.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Mari Aoki 3-24-1 Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd. (72) Toshio Miyoshi 3-24-1 Takada, Toshima-ku, Tokyo Taisho Pharmaceuticals Co., Ltd. (72) Inventor Hiroshi Hara 3-24-1 Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd.

Claims (1)

[Claims] 1. A proteinaceous substance derived from chicken bone cells and having the following physicochemical properties and physiological activities: non-reducing SDS-PAGE and reducing SDS-PAG.
The estimated molecular weight by E is about 18,500; it exhibits a biological activity having an inhibitory effect on bone resorption; loses the above biological activity by trypsin treatment; adsorbs it on an ion exchange resin, Q sepharose at pH 7.5 or higher; It does not adsorb to heparin in the presence of 15 M NaCl; and has the following amino acid sequence at the N-terminal in the molecule. Embedded image (In the formula, A is alanine, D is aspartic acid, G is glycine, K is lysine, L is leucine, and N is
Represents asparagine, P represents proline, T represents threonine, and V represents valine. The alphabets shown in the two columns indicate amino acids that cannot be specified as either the upper amino acid or the lower amino acid. )