JPH0421700A - Novel matrix protein - Google Patents

Abstract

NEW MATERIAL:The matrix protein separated from tooth and bone and having the following physical and chemical properties and a partial peptide sequence expressed by formulas I to V. Molecular weight, 23-25K (SDS-PAGE process); solubility, soluble in 4M guanidine hydrochloride, and insoluble in 6M urea/0.01M sodium acetate buffer solution (pH4.8) and 0.5M guanidine hydrochloride and water. USE:Diagnostic for osteoporosis and assistant for tissue culture. PREPARATION:The objective matrix peptide having partial peptide sequence of formula I to V can be produced e.g. by collecting cutting tooth from freshly slaughtered cattle beef, mechanically separating the soft tissue and dental pulp, defatting, drying, freezing and crushing the material, deashing with 0.6N hydrochloric acid, washing and drying the deashed product, extracting with 4M guanidine hydrochloride, freeze-drying the extract after dialysis, treating with 6M urea/0.01M sodium acetate buffer solution to separate insoluble fraction and purifying the product by gel-filtration chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel matrix protein isolated from teeth and bones, which is expected to be useful as a diagnostic agent or a tissue culture aid.

[Conventional technology]

In diagnosing osteoporosis, the amount of bone mineral in bones such as vertebrae, bones, arms, etc. was measured by X-ray transparency and gamma-ray absorption. Additionally, various types of collagen have been used as tissue culture aids.

[Problems to be solved by the present invention]

Diagnosing osteoporosis requires expensive equipment and has problems with the accuracy of measuring small changes. Also,
There are hopes for the development of a method to speed up cell culture in osteoblast cell culture.

[Failure to solve the problem]

The present invention provides novel matrix proteins isolated from teeth and bones having the following physicochemical properties and main partial peptide sequences, extracted from bovine demineralized tooth matrix and bone matrix.
It was successfully purified.

(1) Molecular weight: 23-25K (SDS-PAGE
(2) Solubility.

4M guanidine hydrochloride soluble 6M urea 10.
01M sodium acetate buffer (pH 4,8)
Insoluble 0.5M guanidine hydrochloride
Insoluble water Insoluble (3)
Main partial peptide sequence ■ Val-Lys-Pro-Gly-Asn-Tyr
-11e-Leu-Lys-ValSer-Val-A
sn-Pro-3er-Tyr-Leu-Val-Pr
o-GluSer-Asp-Tyr-3er-Asn-
Asn-Val-Val-Arg-CysGlu-11
e-Arg-Tyr-Thr-Gly-His-His
-Ala-TyrAla-3er-Gly-Cys-T
hr-I 1e-3er-Pro-Tyr■ Tyr-
Glu-Arg-Pro-Arg-Pro-Gly-3
er-Arg-TyrArg-Pro-Gly-Tyr
-Gly-Thr-Gly-Tyr-Phe-GlnT
yr-Gly-Leu-Pro-Asp-Leu-Va
l-Pro-Asp-Pr.

Tyr ■ His-8er-Met-Asp-Glu-Phe
-3er-His-Tyr-AspLeu-Leu-A
sp-Ala-3er-Thr-Gln-Arg-Ar
g-Va 1Ala-Glu ■ Asn-Gin-Gly-Thr-3er-Asp
-Phe-Leu-Pro-3erArg-Pro-A
rg-Tyr OThr-Arg-Gly-Asp-Val-Arg-
Asp-Tyr-Asp A homology search for the main partial peptide sequence of the matrix protein of the present invention revealed that it was completely novel. Furthermore, as a result of various studies on the uses of the novel matrix protein (hereinafter referred to as MP) of the present invention, it was discovered that it has potential as a diagnostic agent and an adjuvant for tissue culture.

The possibility of this as a diagnostic agent was investigated using an antibody prepared by immunizing rabbits with MP. In other words, an enzyme immunoassay method was developed to measure the amount of MP antigen in human blood using an anti-MP antibody, and when osteoporosis patients were examined, it was found that simply measuring the amount of MP antigen in the blood was enough to detect bone loss. It was discovered that there was a possibility of diagnosing inflammatory disease.

Furthermore, its potential as a tissue culture adjuvant was demonstrated by culturing osteoblasts on MP-coated tissue culture plates. That is, osteoblast MC3T3-
Cell adhesion of El cells was improved depending on the coated MP concentration, and cell proliferation was promoted.

The novel tooth and bone matrix protein (MP) of the present invention can be obtained as follows.

Fresh bovine teeth ↓ Grind to 1+r+m3 using a horn mill ↓ Chloroform Degrease with methanol (1:1) (12 hours) Step 1 Step 2 Deash with 0.6N hydrochloric acid (4°C 196 hours) ↓ Chloroform:methanol (1:1) ) (12 hours) (DDM) ↓ Extraction with 4M guanidine hydrochloride containing a neutral protease inhibitor ↓ ↓ Dissolved substances Insoluble substances (]-2) ↓ Dialysis against ion-exchanged water (4°C) ↓ Centrifugation Separation - Supernatant liquid ↓ Sediment Precipitate ↓ Freeze drying (1-1) ↓ 6M urea 10.01M sodium acetate Step 3 Dissolve in (CH3COONa-3820) buffer ↓ Centrifugation → Supernatant liquid (2-1) ↓ Precipitation Sludge (2-2) ↓ Re-extraction with 4M guanidine hydrochloride (4°C, 24 hours) ↓ Centrifugation → Precipitation ↓ Supernatant liquid ↓ Dialysis against 7 times deionized water (4°C) ↓ Centrifugation → Supernatant liquid (3-1) ↓ Precipitate (3-2) ↓ Freeze-drying step 4 Gel filtration with Sephacryl S-200 (4-1~
4-4) ↓ Step 5 Chromatographic purification with HA-Ultrogel (5-1
~5-2) Materials and Method 1: Preparation of Bovine DDM A large amount of incisors were collected from beef cattle immediately after slaughter at a slaughterhouse.

Immediately after mechanically removing the soft tissue and pulp, the specimen was degreased at room temperature and thoroughly dried.

In this experiment, 5 kg of dried tooth was used. After freezing with liquid nitrogen, use a Wiley-type crusher (Yoshida Seisakusho) to crush approximately 1 m.
It was crushed into m square pieces. After re-defatting at room temperature, it was further dried thoroughly. Deashing was done at 0.6N according to the method of “Llrist”.
The test was carried out using hydrochloric acid for 4 days. In addition, the demineralization solution is 10% of the sample.
The amount (weight) was doubled and the exchange was performed 5 times. After deashing, the sample was thoroughly washed with deionized water and freeze-dried. The following extraction operation was performed using the obtained sample as DDM.

Reference 1) 5ciense 150:893-899
1965°2, extraction of crude MP, extraction from purification steps 1 to 5, and purification in a cold room (4°C)
It was carried out according to the method of Mizutani et al.

Step 1: An extraction operation was performed using 4M guanidine hydrochloride (GuHCl) (manufactured by Sigma) in an amount 7 times the amount (weight) of DDM. In addition, as a protease inhibitor, 2mMN
-x ethylmaleimide
(NEM) (manufactured by Sigma), 1mM iodoacetic acid (IAA) (Si
manufactured by GMA), 1mM sodium azide (sodiu
m azide) (NaN3) (manufactured by Sig+r+a)
and 1mM phenyl-methyl-sulfonyl-fluoride (phenyl methyl 5ulfon
yl fluoride) (PMSF) (manufactured by Sigma) was dissolved. Extraction was repeated a total of three times using the same DDM. The soluble fraction was dialyzed against deionized water, and the precipitate was freeze-dried (1-1 fraction). Further, the extracted DDM was washed with water and then freeze-dried (1-2 fractions).

Step 2 The 1-1 fraction was added to 100 times the amount (weight) of 6M urea (
Schwarz/Mann)/0,01M sodium acetate buffer (manufactured by Katayama Chemical Co., Ltd.) (pH 4,8). The soluble fraction was dialyzed against deionized water and the precipitate was lyophilized (fraction 2-1). The insoluble fraction was washed with deionized water and lyophilized (2-2 fractions).

Step 3: The 2-2 fraction was redissolved in 4M GuHCI and dialyzed against 7 volumes of deionized water. The precipitate (0.5M GuHC1 insoluble fraction) was washed with deionized water and lyophilized (3-2 fraction). In addition, the 0.5M GuHC1 soluble fraction in the dialysis tube was also dialyzed against deionized water, and the precipitate was freeze-dried (3-1 fraction).
.

Reference 2) Cl1n 0rthop 171:213
-223.19823, Cephacryl (5ephacr
yl ■) 1200 i: Yoru Gelquamadography (
Step 4) 50 mg of the 3-2 fraction was dissolved in 4 M GuHCl 5- and sephacryl (5ephac) equilibrated with the same solution.
ry l■) S-200 (manufactured by Pharmacia)
Gel chromatography was performed on a column (25 mm x 850 mm). The flow rate is 1.68 m 11 minutes (PUMP
P-5oo + GRADIENT PROGRAMM
ERGP-250: Pharmacia), 280n
Absorbance measurement at m (SINGLE PATHMONIT
ORUV-]: Phar-macia),
Fraction collector (FAC-300: Phar
(Manufactured by Macia) 1.68 mj/tube was collected. The elution curve was divided into 7 fractions, and each fraction was dialyzed against deionized water. The precipitate was collected by centrifugation and freeze-dried (4
-1 fraction to 4-4 fraction).

4. Chromatography using HA-Ultrogel (Step 5) 100+ng of the 4-5 fraction was dissolved in 6 ml of 6M urea 15mM phosphate buffer (manufactured by Wako Pure Chemical Industries, Ltd.) (pH 6,8), and HA equilibrated with the same solution. -Ultrogel (HA-
ULTROGEU■ (manufactured by I B'F) column (20 m
m x 250 mm), and chromatography was performed. The flow rate was 97 nl/min, the absorbance was measured at 2800 m, and the fraction collector was 3.0 mt.
'/tube was collected. The non-adsorbed fraction that was adsorbed on the column and passed through the soot was designated as the 5-1 fraction, and after the non-adsorbed fraction was exhausted, the adsorbed fraction eluted with 6M urea and 10.2M phosphate buffer (pH 6, 8) was designated as 5-1 fraction. -2 fractions. These fractions were concentrated using an ultrafiltration membrane YM-10 manufactured by Amicon, and then each fraction was dialyzed against pure water to precipitate. After centrifugation, the precipitate is
Lyophilized.

5. Osteogenic activity assay (BMP activity assay) 1-1 to 3-
For the two fractions, 5 mg each, 4-1 to 4-4 and 5-1 to 5
-2 fractions: 1 mg and 5 mg each, N
o. After encapsulating in a 5-seratin capsule and sterilizing it with ethylene oxide gas, it was implanted under the left femoral abdominal fascia of an AKR mouse. In addition, bovine plasma albumin (Sig) was used as a control.
5 mg manufactured by MA was transplanted to the gold side and the right side. After 3 weeks, the layer was removed and Softex was taken. Madoxylin-eosin (H-
E) Observed by staining.

6.3DS-polyacrylamide gel electrophoresis (SD
S-PAGE method) 1-2 fractions were removed (both fractions were examined for molecular weight by SDS-PAGE).

A 12.5% gel was prepared according to the method of Laemml et al. 3, and electrophoresis was performed at a constant current of 35 mA (POWER
5UPPLY type-207 engineering equipment) (SO
P 1500: Toyo). Molecular weight marker (Phosph
orylase: 92.5 to 1 Bovine
serumalbumin: 66.2K, O
Valbumin: 45K, carboni
canhydrase: 31K, 5oybean
trypsin 1nhibitor21.5K
, Lysozyme: 14.3K) (BIO-
RAD) was also run at the same time. Staining was performed using 0.044% Coomassie brilliant blue according to the method of 5cott et al.
) was used.

Reference 3) Nature 227: 680-68
5.1970゜4) Anal, Biochem,
70:251-257. 1976.7, Osteoblast differentiation promotion activity assay (ALP activity assay) 1000 mouse parietal bone-derived osteoblasts MC3T3-El cells were added to a 96-well Mic D plate at 1000 cells/100 μl/well, and 5% fetal bovine serum was added. α-MEM (medium) containing (Fe2) was added and cultured for 2 days to grow to subconfluence, followed by washing with α-MBM. Each fraction contains 6M urea 15[1
0.1% after dissolving in 1M phosphate buffer (p) (6,8)
A solution diluted with α-MEM containing FC3 was added to the cells and incubated for an additional 2 days.

After removing the medium from the plate, it was frozen and stored until measurement of alkaline phosphatase activity (ALP activity).

ALP activity was measured by adding 0.7M 2-amino-2-methylpropatur buffer pH 10.3 (AM
After adding 50 μl of AMP buffer, 100 μl of a substrate solution containing 20 mM p-trophenyl phosphate (Wako Pure Chemical Industries) dissolved in AMP buffer was added and reacted at 37° C. for 1 hour. After stopping the reaction by adding 50 μl of IM NaOH, the absorbance at 405 nm was measured using a microplate reader.

8. Measurement of partial peptide sequence 5-] The N-terminus of both components was thought to be blocked, and the amino acid sequence could not be determined directly. Therefore, prior to sequence analysis, it was hydrolyzed into peptide fragments by various enzyme treatments.

That is, 0. OIM Dissolved in HCI and digested with pepsin, 2%
It was dissolved in formic acid and digested with trypsin, v8 protease, and chymotrypsin. The obtained peptide fragment was loaded onto a Hydac C4 (300A) column (4,6x
250 mm) by reverse phase chromatography. The mobile phase contains 0.05% TFA to 0.025% TFA.
Elution was performed using a gradient to % TFA/acetonitrile.

The amino acid sequence of each peptide fragment was analyzed by the Edman degradation method using a gas phase sequencer (manufactured by Applied Biosystems).

9. Amino acid analysis Prior to amino acid analysis, 1 mg of the 5-1 fraction was treated with 6NHC1.
After hydrolysis at 1 mI at 110° C. for 122 hours and drying, amino acid composition analysis was performed using an amino acid analyzer (JLC-300 JEOL Ltd.).

Result 1: By extraction of crude MP and demineralization with purified 0.6N hydrochloric acid, the dry weight of bovine teeth of 5 kg was reduced to about 1/8 (620 g). Subsequent extraction and purification steps were performed using 4 M GuHC1 soluble fraction (1-1 fraction).
BMP activity and ALP activity were observed in 4 M Gu
It was not observed in the HC1-insoluble fraction (1-2 fractions). In step 2.3, BMP activity and ALP activity are each 2.
-2 fractions and 3-2 fractions, i.e. 4 M GuHC
] Soluble, 6M urea, 0.01M sodium acetate buffer (pH 4,8) insoluble, 0.5M GuHC1 insoluble and water insoluble.

2. Sephacryl (5ephacry 1■) S-20
0 gel chromatography 3-2 fractions of Sephacryl S
The elution curve in -200 gel chromatography showed many peaks, suggesting that it was still in a crude state and that further purification was required.

The eluates were divided into four fractions and assayed for BMP activity and ALP activity. As a result, strong activity was observed in the 4-2 fraction and weak activity was observed in the 4-3 fraction.

3.8A-Utrogel (HA-ULTROGEL■
) Chromatography Sephacryl S-200 gel chromatography The 4-2 fraction was separated into a non-adsorbed 5-1 fraction (MP) and an adsorbed 5-2 fraction by HA-bovine trogel chromatography, Both fractions are BMP
It no longer showed any activity. On the other hand, ALP activity was strongly accumulated only in the 5-2 fraction, and could be completely separated using HA-bovine trogel.

When both fractions were analyzed by 5DS-PAGE method, 5
One portion (MP) had been purified to a single/<< molecular weight of 23-25. On the other hand, in the 5-2 fraction, a large number of bands were observed in the range of several thousand to 1,000 to 1,000, which suggests that it is still in its primitive state or contains factors that increase ALP activity.

4. Bone formation activity assay (BMP activity assay) As mentioned above,
Also, as shown in Table 1, BMP activity is 1-1.2-2.3-2
．． It was observed in both cases of 4-2 and 4-3. However, 1-
One fraction was 315.2-2 fraction was 215 and was inactive. On the other hand, the 3-2 fraction and 4-3 fraction are 5mg, 4-
In the second fraction, active bone formation on the gold side was observed after transplantation of only 1 mg. Similar to the control, no bone formation was observed in the 5-15-1 (~5-2 fraction).

Soft X-ray photographs showed that ossicles with trabecular structures were formed 3 weeks after implantation in all active fractions.
An opaque image as shown in e) was confirmed.

In the H-E stained histological findings, in the specimen in which the activity of the 1-1.2-2 fraction was observed, delayed bone formation with partial cartilage tissue and osteoid tissue was observed. In the BMP active fractions after the 3-2 fraction, formation of mature lamellar bone with bone marrow inside was observed.

1 - 1 6595 +
+2-2 288
5 + +3-2
1150 +
+4 - 2 110
← ←4 - 3
55 +
+5-1 (MP) 335-2
57-
←5.5DS-polyacrylamide gel electrophoresis method (SDS-PAGE method) When the fractions of each purification step were analyzed by SDS-PAGE method, except for the 5-1 fraction, a significantly large number of hunts was observed in the others. It was done. Only fraction 5-1 was purified to a single band with a molecular weight of 23 to 25.

6. Osteoblast differentiation promoting activity assay (ALP activity assay) As shown in Table 1, the ALP activity of each two-fold diluted fraction was purified at each refinement step, and the 5-2 fraction had the highest specific activity (per protein). (ALP activity) increased. 5-1 fraction (
No activity was found in MP).

7. Measurement of partial peptide sequences The sequences of the main fragments of the peptides hydrolyzed by pepsin, trypsin, ■8 protease, and chymotrypsin in the 5-1 fraction were determined, and the sequences were extended by overlapping the common sequences, and the results are shown in Table 2. I got it.

Table 2.5-Partial peptide sequence of fraction 1: ■ Va
l-Lys-Pro-Gly-Asn-Tyr-1]e
-Leu-Lys-ValSer-Val-Asn-P
ro-3er-Tyr-Leu-Val-Pro-Gl
uSer-Asp-Tyr-3er-Asn-Asn-
Val-Val-Arg-Cys-Glu-11e-A
rg-Tyr-Thr-Gly-His-His-Al
a-TyrAla-3er-Gly-Cys-Thr-
11e-3er-Pro-Tyr■ Tyr-Glu-
Arg-Pro-Arg-Pro-Gly-3er-A
rg-Tyr-Arg-Pro-Gly-Tyr-Gl
y-Thr-Gly-Tyr-Phe-Gln-Tyr
-Gly-Leu-Pro-Asp-Leu-Val-
Pro-Asp-Pro-Tyr ■ His-Set-Met-Asp-Glu-Phe
-3er-His-Tyr-Asp-Leu-Leu-
Asp-Ala-3er-Thr-Gln-Arg-A
rg-ValAla-Glu ■ Asn-Gln-Gly-Thr-3er-Asp
-Phe-Leu-Pro-3erArg-Pro-A
rg-Tyr @ Thr-Arg-Gly-Asp-Val-Ar
g-Asp-Tyr-Asp8, Amino Acid Analysis The results of the amino acid analysis of the 5-1 fraction are shown in Table 3. As
The contents of p, Leu, cly, and Arg were high.

Table 3 Amino acid composition Amino acid mole% Aspartic acid (Asp) 14.3 Threonine (Thr) 5.9 Serine
(Ser) 7.8 Glutamic acid
(Glu) 14.8 Proline
(Pro) 6.2 Glycine
(Gly) 9.7 Alanine
(Ala) 8.1 Valine (V
al) 6.8 Cysteine (Cys
Cys) Methionine (Met)
1.6 isoleucine ([1e)
3.80 Leu
10.2 Tyrosine (Tyr)
7.1 Phenylalanine (Phe) 7
．． 6 Histidine (His) 3.
9 Lysine (Lys) 6.1
Arginine (Arg) 8.69
．． Preparation of antibody against 5-1 fraction (MP) and analysis of pre-extracted proteins 1 mg of 5-1 fraction (MP) was emulsified with Freund's complete adjuvant (Wako Pure Chemical Industries, Ltd.), and rabbits were immunized three times with anti-5-1 fraction (MP). One fraction of antibody was created. In addition, a pre-demineralized extract was prepared by extracting bovine bone with 4M guanidine hydrochloride in the same manner as in the case of bovine teeth. Bovine tooth 5-1 fraction (MP
) was analyzed using the 5O5-PAGE method, transferred to a nitrocellulose membrane, and immunostained with the prepared anti-5-1 fraction antibody.
It was found that a corresponding amount of protein exists.

〔result〕

Novel tooth and bone matrix protein of the present invention (5-
Fraction 1) alone does not show either osteogenic activity or osteoblast differentiation promoting activity, or the osteoblast differentiation promoting active component (5-2
fraction) provides a site for bone formation and exhibits a remarkable osteogenic effect.

Patent applicant: Nippon Kayaku Co., Ltd.

Claims (1)

[Claims] 1. A novel matrix protein isolated from teeth and bones having the following physicochemical properties and partial peptide sequence (1) Molecular weight: 23-25K (by SDS-PAGE method) (2) Solubility : 4M guanidine hydrochloride soluble 6M urea/0.01M sodium acetate buffer (pH 4.
8) Insoluble 0.5M guanidine hydrochloride Insoluble Water insoluble (3) Main partial peptide sequence: [1] [Gene sequence available] [2] [Gene sequence available] [3] [Gene sequence available] [4] [ There is a gene sequence] [5] [There is a gene sequence]