WO1996003497A1

WO1996003497A1 - Novel hyaluronidase

Info

Publication number: WO1996003497A1
Application number: PCT/JP1995/001452
Authority: WO
Inventors: Tatsuya Yamagata; Ryu Miura; Sadako Yamagata
Original assignee: Seikagaku Corporation
Priority date: 1994-07-22
Filing date: 1995-07-21
Publication date: 1996-02-08

Abstract

A novel hyaluronidase (HAase) isolated from a supernatant of a human uterine body cancer tissue culture by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis using hyaluronic acid, modified chondroitin sulfate or the like as the substrate. The obtained HAase hydrolyzes hyaluronic acid at a pH of 3.5 to 7.5, but not chondroitin sulfate, and is useful for, for example, searching remedies for cerebral edema, myocardial infarct and the like, drug additives, and hyaluronidase inhibitors.

Description

New Hear-Lonidase Technology Field

The present invention relates to a novel hyaluronidase, and in particular, to a novel hyaluronidase useful for pharmaceuticals, quasi-drugs, or screening thereof. Background technology

Hyaluronidase (hereinafter abbreviated as “HA ase”) is a generic name given to enzymes that degrade hyaluronic acid into molecules. (1) Hyaluronic acid / chondroitin '/ 9-N-acetyl-D of chondroitin sulfate Endohexosaminidase-type enzyme (EC 3.2.1.35), which hydrolyzes hexosaminide bond, (2) Hydrolyzes; 3-—D-glucuronide bond of hyaluronic acid Endoglucidin enzyme (EC 3.2.1.36), (3) End dalcosamine cleavage that degrades the / S—N-acetyl-D-glucosaminide bond of hyaluronic acid -Type enzymes (EC 4.2.2.1) and (4) endosomes which degrades the N-acetyl-D-hexosaminide bond of hyaluronic acid.chondroitin / chondroitin sulfate. Different types such as cleavage type enzymes are known. There. These differ in the end products of the reactions, but all have been widely used as mucopolysaccharide structural studies and as reagents for identification and quantification.

On the other hand, HAase is used as a drug in the treatment of acute phase such as cerebral edema and myocardial infarction, and is also used as an additive for subcutaneous administration or as a drug for local treatment of pleural effusion. Have been.

Furthermore, HAase is also useful in searching for a substance having an action of inhibiting the activity of HAase. Hyaluronic acid retains moisture in the intercellular space of the human body, forms a matrix in the tissue to retain cells, keeps the skin lubricious and flexible, and prevents mechanical damage. It works to prevent external force and bacterial infection. However, HAase has the activity of decomposing such hyaluronic acid. So hyal Substances that suppress the activity of HA ase, which degrades lonic acid, can be used as agents and cosmetics that keep the skin moist, prevent skin roughness, small blemishes, and dryness. Thus, various uses of HAase have been proposed, but not all of its functions have been elucidated, and the emergence of a new HAase has been awaited. Disclosure of the invention

The present inventors have conducted a search to find a new HAase, and as a result, for the first time, have found that a novel HAase is produced in the culture supernatant of human endometrial cancer tissue, and have completed the present invention. Reached.

That is, the gist of the present invention resides in a hyalinidase derived from human endometrial cancer tissue having the following physicochemical properties.

Substrate specificity:

Degrades hyaluronic acid in the pH range of 3.5 to 7.5 and does not degrade chondroitin sulfate in the pH range of 3.5 to 7.5

Optimal pH:

around pH 5.0

8.8

The hyaluronidase of the present invention was confirmed to have a molecule S of about 94 kilodalton as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using hyaluronic acid as a substrate. Was done.

Hereinafter, the present invention will be described in detail.

The novel HAase of the present invention can be isolated and purified from human endometrial cancer tissue according to various treatment procedures utilizing the physical and chemical properties of the target HAase. That is, treatment with a normal protein precipitant, salting, ultrafiltration, molecular sieve chromatography (gel filtration), centrifugation, electrophoresis, ion exchange chromatography, affinity chromatography, reverse Processing operations such as phase chromatography, adsorptive pore chromatography, dialysis, and a combination thereof are mentioned. Concrete Specifically, from the culture of human endometrial cancer tissue, Markusw GuntenhOner, et al., Et al. According to the method of [Matrix Vol. 12.38 88-39 (1992)], Vol. 12, No. 3, pages 388-396 (Trix) Can be separated.

For the synthesis of a carrier for gel electrophoresis, a conventional method for preparing a polyacrylamide gel {Laemmli, UK, UK, Nayiya, Vol. 227, pp. 68-68 (1970) [Nature, 227, 680-680 (1970)]]}. That is, hyaluronic acid or modified monochondroitin sulfate is added to an aqueous solution containing acrylamide and N, N'-methylenebisacrylamide, and the mixture is uniformly mixed, for example, by placing it in a cool and dark place for 24 hours. Polymerization is carried out using a polymerization initiator such as a peroxide such as persulfate or benzoyl peroxide, an azo compound such as azobisisobutyronitrile, or a redox initiator composed of an oxidizing agent and a reducing agent. Further, N, N, N ′, N′-tetramethylethylenediamine may be added as a polymerization accelerator. The amount of N, N'-methylenebisacrylamide used for acrylamide is the same as that used for general polyacrylamide gels, and the amount of hyaluronic acid or modified monochondroitin sulfate used is usually 1-2. It is selected from the range of 0 ig / m1. Further, the polymerization reaction is performed in a cell suitable for a carrier having a desired size.

Using the thus prepared hyaluronic acid mixed or modified-chondroitin sulfate-binding polyacrylamide gel as a carrier, the culture supernatant of human endometrial cancer tissue is degraded by the enzyme contained in the culture supernatant. Perform electrophoresis under the conditions not used. Next, the carrier after swimming is washed with a buffer solution or the like as necessary, and then placed under ordinary oxygen reaction conditions, and the carrier after the enzyme reaction is colored with a coloring reagent such as Alshan blue or Toluidine blue. . When the enzyme contained in the culture medium decomposes the group K immobilized on the polyacrylamide gel, only the decomposed part is detected as white without coloration, and the activity and molecular weight of the enzyme must be identified. Can be. The enzyme thus obtained is the HAase of the present invention. In addition, the optimal pH of the enzyme of the present invention can be confirmed by measuring the HAase activity while varying the pH during the enzyme reaction. You.

Regarding the above method and the nature of the HAase of the present invention, the inventor's own paper published on March 1, 1995, the date of priority of the present application, Analytical Biochemistry, No. 25 Vol., Pp. 333-340 (Analytical Biochemistry, 222, 333-340). BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a drawing showing a synthetic pathway of modified monochondroitin sulfate.

FIG. 2 is a drawing showing the results of (a) H A ase of the present invention detected by SDS-P AGE using hyaluronic acid as a substrate. In the figure, lanes 1, 2, 3, and 4 represent the electrophoresis patterns at pH 3.5, 5.0, 6.0, and 7.5, respectively. (b) A drawing showing the results of detection of HA a se of the present invention by SDS-PAGE using modified-chondroitin sulfate as a substrate. In the figure, lanes 1, 2, 3, and 4 represent the electrophoresis patterns at pH 3.5, 5.0, 6.0, and 7.5, respectively. <Best mode for carrying out the invention>

Hereinafter, the present invention will be described with reference to specific examples, but the present invention is not limited to the following without departing from the gist thereof.

Reference example

Chondroitin sulfate (I) derived from shark cartilage (molecular weight 40,000-48,000)

0; 4 monosulfuric acid: 6-sulfuric acid = 1: 5) The reducing end of R. J. Rajya et al. (RH Rajaeta 1.) Anal. Biochem. 77 page (1994) [Anal. Biochem., 13 9,

1 6 8 - 1 7 7 (1 9 8 4)] is reduced with N a beta Eta ₄ according to the method of, and partial oxidation with N a I 0 _4, to obtain a reaction was § Mi Roh of using ethylenediamine Was. The reactants are

Purified by dialysis of 4. 100 mg of the purified reduced terminal aminated chondroitin sulfate (IV) was dissolved in 2 ml of distilled water, and 1 ml of ethanol was added. The mixture was incubated with 1 ml of aryl glycidyl ether at 40 ° C for 2 hours, dialyzed against distilled water at 4 ° C, and lyophilized. Thus modified one chondroitin Sulfuric acid (V) was obtained. Figure 1 shows the above synthetic route.

Example 1

A 1 mm thick 8% sodium dodecyl sulfate (SDS) -polyacrylamide gel was prepared. At the time of preparation of this gel, lmggZml of hyaluronic acid (derived from cockscomb, molecule S824,000) stored at 4 ° C overnight was added to a final concentration of 17gZm1.

On the other hand, culture supernatant of human endometrial cancer tissue (available from Dr. Koji Tamakoshi of Nagoya University) was added to SDS and Laemmli buffer [Nachiya, Vol. 227, Vol. -685 (1970) [Nature, 227, 680-680 (1970)] was added and the gel was used.

Electrophoresis was performed at room temperature. After electrophoresis, the gel was washed with 2.5% Triton X—100 for 1 hour at room temperature. Gel fresh buffer (5 0 mM click E down monobasic N a ₂ HP 0 ₄ and 0. 1 5 MN a C l ( p H 5. 0, 6. 0. 7. 5), or 0. 1 MHCO OH--HCO ON a and 0.15 MN a C 1 (pH 3.5))), incubate at 37 ° C for 16 hours, and then incubate at 37 ° C for 2 hours. 1 mg / m 1 pronase solution [20 mM Tris—HCl (pH 8.0): Pronase is derived from Streptomyces griseus, and is derived from Calnochem Coop. or). The incubated gel was washed with 20% ethanol and 10% acetic acid for 20 minutes, and then washed with 20% ethanol and 10% acetic acid containing 0.5% Alcian blue (A1cianblue) for 1 hour. After staining, the cells were washed with 20% ethanol—10% acetic acid.

The results are shown in Fig. 2 (a). A clear band was observed around 94 kilodaltons in the pH range of 3.5 to 7.5. It was also confirmed that the band had high hyaluronic acid-degrading activity, especially at around pH 5.0.

Example 2

In the same manner as in Example 1, except that the modified-chondroitin sulfate prepared in Reference Example was used instead of hyaluronic acid and added so that the final concentration in the gel was 8.5 ug / m1, The electrophoresis of the culture supernatant of human endometrial cancer tissue was performed. The results are shown in Fig. 2 (b). Within the range of pH 3.5 to 7.5, no particularly characteristic band was observed.

From the above results, it was found that the 94-kilodalton protein has a hyaluronidase activity that acts on hyaluronic acid and has no effect on chondroitin sulfate.

Example 3

The isoelectric point of the 94 kilodalton protein was determined by two-dimensional electrophoresis under the following conditions.

First dimension:

Upper gel (1% thick 3% acrylic gel)

30% Acrylamide 0.8% Bisacrylamide 0.2 m

40% glycerin 0.25 m distilled water 1.5 3 m ammonium peroxodisulfate (ammonium persulfate)

(100 mg / ml) 20

N, N.N ', N' Tetramethylethylenediamine

Isoelectric focusing gel (1% thick 6% acrylamide gel)

30% acrylamide 0.8% bisacrylamide 2.0 m

40% glycerin 2.0 m distilled water 5.5 m ampholine (LKB), pH 3.5 to 100 0.5 m ammonium peroxodisulfate (ammonium persulfate)

(1 0 0 mg / m 1) 4 0

N, N. N ', N' Tetramethylethylene diamine 7.0 running buffer

Cathode solution: 20 mM Na 0 H

Anode solution: 10 mM H 3 P 04

Sample buffer

Isoelectric focusing gel solution: 30% Acrylamide 0.8% Bisacrylamide 2.0m1

40% glycerin 2.0m1 distilled water 5.5m1 ampholine (L KB) .pH 3.5 to 1000.5m1 and an equal amount of 40% glycerin Thing

sample

Endometrial tissue culture supernatant 1 0 1

Marker (BI0—RAD) 5 1

Running conditions

400 V, 2 hours 30 minutes, 0 ° C

Dimension:

Separation gel (0.17 mg gZm 1 Hyaluronic acid-containing, 1 mm thick 3% acrylamide gel)

30% Acrylamide 0.8% Bisacrylamide 2.2 4 ml 1.5 M Tris-H C 1 (pH 8.3)-0.4% S DS

2.10 ml

1 mg gZm 1 Hyaluronic acid aqueous solution 1.44 ml ml distilled water 2.59 ml ammonia peroxodisulfate (ammonium persulfate)

(100 mg / m1) 8 4 1

N, N, N ', N' Tetramethylethylenediamine 2.101 Running buffer

0.02 5 M T r i s

0.192 M glycine (pH 8.3)-0.1% S DS

Sample buffer

Laemli (Laemm1i) buffer solution

sample

Endometrial cancer tissue culture supernatant on isoelectric focusing

Running conditions 20 mA, 1 hour 40 minutes

Washing

2.5% Triton (Triton) X—100 for 1 hour

Incubation

16 hours at 37 ° C (pH 5.0)

Protease treatment

0.1 mg gZm 1 pronase (Calbiochem Corp. (Calb

1 o c he m C o r p) company, 37, 2 hours

Fixed

20% ethanol—20% acetic acid for 20 minutes

Dyeing

1 hour in 0.5% Alcian blue (Alcianblue) dissolved in 20% ethanol / 10% acetic acid

Bleaching

Twice for one hour with 20% ethanol and 10% acetic acid

As a result, it was confirmed that the isoelectric point of the HA A se of the present invention was 8.8, and industrial applicability.

The HAase of the present invention is a novel enzyme and, like the conventional HAase, is useful as a drug for cerebral edema, myocardial infarction, etc., as a drug additive, and in searching for a substance that inhibits the activity of HAase. .

Claims

The scope of the claims

1. Hyaluronidase derived from human endometrial cancer tissue having the following physicochemical properties. Substrate specificity:

Optimal PH:

around pH 5.0

-·

8.8

2. The hyaluronidase according to claim 1, wherein the molecular weight measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis using hyaluronic acid as a substrate is about 94 kDa.