JP3368909B2 - Novel cystatin polypeptide, method for producing the same, and enzyme inhibitor containing the polypeptide as an active ingredient - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel cystatin, a process for producing the same, and use thereof. Cystatin is a polypeptide that inhibits enzymes such as papain and cathepsin, which have cysteine as an active center, which is present in the living body, and thus is expected to have wide applications in the fields of food and pharmaceuticals.

[0002]

2. Description of the Related Art Cystatin contains cysteine as an active center.
Enzymes, such as plant-derived papain, in animal cells
It is an enzyme inhibitor that specifically inhibits cathepsin, etc.
To date, present in human, bovine, rat, and chicken tissues
It has been reported to do. For example, Takio, K. et al.
Biochem. Biophys. Res. Commun.,115, 902 (198
3), Biochem. Biophys. Res. Comm by Takio, K. et al.
un.,121, 149 (1984), Bioche by Ritonja, A. et al.
m. Biophys. Res. Commun.,131, 1187 (1985), Isemu
ra, S. et al., J. Biochem.,96, 489, (1984), Ab
e, K. et al., J. Biol. Chem.,262, 16793 (1987), O
Biochemistry, by hkubo, I. et al.twenty three, 5691 (1984),
Lee, C.-C. et al. Proc. Natl. Acad. Sci. USA.8
Four: 4403 (1987) and so on. According to these reports,
Tatin differs in structure and inhibitory activity from its origin
There is. However, an example of extracting and purifying cystatin from fish
There is no.

[0003]

[Problems to be Solved by the Invention] Fresh foods, especially fishes, are subject to self-digestion due to enzymes in the body and are easily deteriorated, and maintaining their freshness is a major problem. Therefore, preservatives and the like suppress autolysis. However, various synthetic preservatives used as preservatives have been pointed out to have various problems in terms of long-term safety. For this purpose, the use of an enzyme inhibitor that suppresses autolysis as a preservative has been investigated.

On the other hand, in recent years, viruses relating to diseases caused by human viral infections have synthesized preproproteins in their own protein synthesis in human cells and obtained mature proteins by processing enzymes such as cysteine as an active center. I understand. From this, an inhibitor of an enzyme having cysteine as an active center is expected as a drug that suppresses virus growth.

For these purposes, application of cystatin is considered. On the other hand, when comparing the inhibitory activities of enzymes having a cysteine as the active center of known cystatin polypeptides whose amino acid sequences have been determined, there are differences in the titers.
Therefore, the development of higher titer cystatin polypeptides is desired.

[0006]

The novel polypeptide of the present invention is shown in SEQ ID NO: 1. The polypeptide of the present invention can be produced by extracting and purifying from the pituitary gland of salmon, for example, chum salmon. In this production method, ethanol or the like is used as the extraction solvent. As the purification method, various chromatographies are used, in which case the cystatin activity serves as an index.

Further, the present invention resides in an enzyme inhibitor containing the polypeptide represented by SEQ ID NO: 1 as an active ingredient. The cystatin polypeptide of the present invention was obtained for the first time from fish. Moreover, it is the first time that cystatin polypeptide found in mammals and the like is produced from the pituitary gland from the brain, skin epidermis, blood, and cerebrospinal fluid. Therefore, the titers for many types of cysteine enzymes are
Differences between the cystatin polypeptides of the invention and known cystatin polypeptides are possible.

[0008]

EXAMPLES The present invention will be described in more detail with reference to Examples and Reference Examples, but the technical scope of the present invention is not limited thereto.

[0009]

[Reference Example] Activity measurement method Cystatin activity was measured by the method of Barrett et al.
Enzymology, Vol 80, pp 771-778) was used. That is, papain was used as the enzyme and Bz-DL-Arg-p-nitroanilide was used as the substrate. Substrate solution 1 ml (43.5 mg of Bz-DL-Arg-p-nitroanilide was dissolved in 1 ml of DMSO and 2 mM ethylenediaminotetraacetic acid (EDTA) and 5 mM cysteine were added to 50 m.
M Tris-HCl buffer, pH 7.5 was made up to 100 ml. ) To papain solution 0.1 ml (papain concentration: papain 0.5 mg / ml
Water) and cystatin solution 0.1 ml (cystatin concentration: cystatin 0.05-0.1 mg / ml water) are added, reacted at 37 ° C for 25 minutes, and stopped by adding 0.2 ml of 30% acetic acid aqueous solution.
The absorbance was measured at 410 nm.

[0010]

Example 1 (1) Extraction / Purification Method Based on the reference example, cystatin was purified using the activity of cystatin as an index. Frozen in liquid nitrogen, -80
30 g of female salmon pituitary gland frozen at ℃
ml ethanol extract (35% ethanol, 10% ammonium acetate, 1.5 mM EDTA, 1.5 mM PMSF (phenylmethylsulfonyl fluoride), pH 6.1) Extraction was performed at 4 ° C for 14 hours,
The supernatant was added to 300 ml of cold ethanol, and the mixture was stirred and allowed to stand at 4 ° C. for 24 hours, and the precipitate was freeze-dried to obtain an ethanol extract.

The ethanol extract was subjected to DE-52 anion exchange chromatography (column size: 1 × 30 cm). Elution was performed with 0.05 M ammonium bicarbonate, pH 9.0, the flow rate was 60 ml per hour, the absorbance was measured at 280 nm, and each fraction was freeze-dried (Fig. 1). The cystatin activity was confirmed specifically in 170 mg of DE-52 flow-through fraction, fraction 1 (see Fig. 1). This fraction was used as CM-Sephadex C-50 (column size: 1 x 30 cm) cation. Subjected to exchange chromatography. Elution was carried out by the stepwise method of 0.05 M ammonium acetate (pH 4.6), 0.1 M (pH 4.6) and 0.2 M (pH 9.0). Flow rate is 60 ml per hour, absorbance is 28
Each fraction was freeze-dried, measured at 0 nm (Figure 2).

Since cystatin activity was specifically confirmed in 51.7 mg of CM-Sephadex C-50 column fraction, fraction 5 (see FIG. 2), this fraction was equilibrated with 0.05 M ammonium bicarbonate, pH 9.0. Gel filtration was carried out using the separated Sephadex G-75SF (column size: 2.2 × 90 cm). The elution was 0.05 M ammonium bicarbonate, pH 9.0, the flow rate was 20 ml per hour, and 120 ml of the front flow was collected. Each fraction was freeze-dried (Fig. 3).

Cystatin activity was specifically confirmed in 1 mg of Sephadex G-75SF column fraction, fraction 3 (see FIG. 3), and this fraction was dissolved in 0.1% trifluoroacetic acid (TFA) to prepare TSK. Gel ODS-120T (column size: 0.46 × 25 cm) was subjected to reverse phase high performance liquid chromatography. Column temperature was 40 ℃, elution was with 0.1% TFA
A linear gradient of 20% to 50% acetonitrile solution containing The flow rate was 60 ml per hour, the absorbance was measured at 220 nm, and each fraction was freeze-dried (Fig. 4). Cystatin activity was confirmed specifically in fraction 15. Fraction 15
(See Figure 4) inhibited the enzymatic activity of papain by 50%.
(Figure 5). (2) Structural determination method-1 Pyridylethylation of the cystatin polypeptide isolated according to the above (1) was performed to obtain 100 μg of the cystatin polypeptide.
1.5M Tris-HCl buffer containing EDTA adjusted to a concentration of 0.2% and guanidine hydrochloride adjusted to a concentration of 6M (pH 8.6)
Add 30 μl to dissolve and DTT (dithiothreitol) 10 μl
After adding 1 l and reducing for 4 hours, 1 μl of 4-vinylpyridine was added. Desalting was carried out by adding TSKgel ODS-120T (0.45 × 25 cm) reverse phase high performance liquid chromatography after adding 4 μl of formic acid to the reaction solution. Elution contains 0.1% TFA 10
It was carried out by a linear gradient method of acetonitrile solution of 80% to 80%. (3) Structure determination method-2 The cystatin polypeptide reduced-pyridylethylated according to the above (2) was added with 0.1M ammonium bicarbonate (pH8.0) 200
It was dissolved in μl, lysyl endopeptidase (Wako Pure Chemical Industries Lot NO. CTP9276) was added so that the substrate / enzyme ratio was 1/60, and the mixture was digested at 37 ° C. for 18 hours. After digestion, the digest was subjected to TSK gel ODS-120T (0.46 x 25 cm) reverse phase high performance liquid chromatography. Elution of each fragment peptide was 0.1
A linear gradient of 90 minutes of a 5 to 50% isopropanol solution containing TFA was used. The column temperature was 40 ° C., the flow rate was 30 ml per hour, the absorbance was measured at 210 nm, and each fraction was freeze-dried (FIG. 6). (4) Structure determination method-3 The cystatin polypeptide was dissolved in 200 μl of 70% formic acid aqueous solution, 100 μg of cyanogen bromide was added, and the mixture was incubated at room temperature for 24 hours. Degradation product is TSK gel ODS-120T (0.46 × 25c
m) Subjected to reverse phase high performance liquid chromatography. Elution of each fragment peptide is 5-50% with 0.1% TFA
A 90 minute linear gradient of isopropanol solution was used.
Column temperature is 40 ℃, flow rate is 30ml per hour, absorbance is 210n
Measured in m, each fraction was lyophilized. (5) Structure determination method-4 Obtained by cystatin reduced with pyridylethylated according to the above (2), each fragment obtained by digestion with lysyl endopeptidase according to the above (3) and cyanogen bromide decomposition according to the above (4). The entire structure of the amino acid sequence of each fragment was determined by a gas phase automatic amino acid sequence analyzer (Shimadzu PSQ-1) (Fig. 7).

[0014]

INDUSTRIAL APPLICABILITY According to the present invention, a novel cystatin polypeptide useful as an enzyme inhibitor can be provided.

[0015]

[Sequence list]

SEQ ID NO: 1 Array length: 110 Sequence type: Amino acid Topology: linear Sequence Type: Protein Array: Gly Leu Ile Gly Gly Pro Met Asp Ala Asn Met Asn Asp Gln Gly 15 Thr Arg Gln Ala Leu Gln Phe Ala Val Val Glu His Asn Lys Lys 30 Thr Asn Asp Met Phe Val Arg Gln Val Ala Lys Val Val Asn Ala 45 Gln Lys Gln Val Val Ser Gly Met Lys Tyr Ile Phe Thr Val Gln 60 Met Gly Arg Thr Pro Cys Arg Lys Gly Gly Asn Glu Lys Ile Cys 75 Ser Val His Lys Asp Pro Gln Met Ala Val Pro Tyr Lys Cys Thr 90 Phe Glu Val Trp Ser Arg Pro Trp Met Ser Gly Ile Lys Met Val 105 Lys Asn Gln Cys Glu 110

[Brief description of drawings]

FIG. 1 shows the results of DE-52 anion exchange chromatography.

FIG. 2 shows the results of CM-Sephadex C-50 cation exchange chromatography.

FIG. 3 shows the results of gel filtration using Sephadex G-75SF.

FIG. 4 shows the results of TSK gel ODS-120T reverse phase high performance liquid chromatography.

FIG. 5 shows the inhibitory effect of the cystatin polypeptide of the present invention on papain.

FIG. 6 shows the results of TSK gel ODS-120T reverse phase high performance liquid chromatography.

FIG. 7 is a view showing a result of structure determination by a gas phase automatic amino acid sequence analyzer.

Continuation of the front page (58) Fields investigated (Int.Cl. ⁷ , DB name) C07K 14/00-14/825 C12N 15/00-15/90 BIOSIS / WPI (DIALOG) SwissProt / PIR / GeneS eq

Claims (4)

(57) [Claims] 1. A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 and inhibiting the enzymatic activity of papain. 2. The method for producing the polypeptide according to claim 1, which comprises extracting and purifying from the salmon pituitary gland. 3. The method according to claim 2, wherein the salmon is chum salmon. 4. An enzyme inhibitor comprising the polypeptide according to claim 1 as an active ingredient.