JPS6352879A - Tissue plasminogen activator precursor - Google Patents

Abstract

PURPOSE:To produce plasminogen activator precursor (pro-t-PA) capable of developing more effective thromboplastinogen action than melanoma t-PA, etc., by cultivating a cultivated cell of human womb muscular tissue, etc., in a serum-free medium containing various protein decomposition products. CONSTITUTION:Mormal muscular tissue is separated from womb obtained in extraction of womb is subjected to primary culture and further subculture is repeated to give estabilished cell KW strain of human womb muscular normall cell. The strain is cultivated in a serum-free medium containing various kinds of protein decomposition products (e.g. lactoalbumin hydrolyzate, proteose peptone, etc.) preferably by high-density culture method using microbeads and Pro-tPA is obtained from the culture solution by purification method of combination of various affinity chromatography and gel filtration method. The precursor is activated by bringing it into contact with plasmin Sepharose and has high affinity for fibrin and lysine.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a Mi-woven plasminogen actichator precursor (hereinafter abbreviated as pro-t-PA).

[Conventional technology]

Plasminogen activators (hereinafter abbreviated as P and A) are widely distributed in animal tissues and body fluids, and have the effect of activating blood plasminogen into plasmin. This plasmin breaks down fibrin, which is the main component of blood clots, so PA is medicinally
It has been researched and developed as a thrombolytic enzyme.

As a PA, urokinase (hereinafter abbreviated as UK) purified from human urine and human kidney cell culture fluid is well known and has already been used clinically. However, when administered in large doses, tJK degrades not only fibrin but also fibrinogen, a precursor of fibrin, which can lead to serious side effects such as disrupting the balance of the coagulation system and resulting in bleeding tendencies. It has been pointed out. On the other hand, UK
(hereinafter abbreviated as pro-UK) and tissue PA (hereinafter abbreviated as t-PA) that is produced by vascular endothelial cells and is immunologically completely different from UK. As the fibrinolytic activity is expressed only on the solid phase of fibrin, the enzyme is actively researched and developed as an enzyme with better plug-dissolving properties without any of the side effects seen in the UK. is in progress.

[Problem that the invention seeks to solve]

Although the isolation and purification of pro-t-PA and the identification of its properties have not been previously reported, the present inventor has conducted extensive research on pro-t-PA, which is a precursor of t-PA. As a result, we succeeded in isolating and purifying a novel pro-t-PA from the culture solution of a cultured cell line such as human uterine muscle tissue, leading to the completion of the present invention.

The t-PA according to the present invention is obtained in the form of a precursor, and by limited decomposition treatment with plasmin or the like, the precursor with a main chain structure is converted into a double-stranded structure, and its activity is expressed and enhanced. be. That is, the present invention relates to the following (1) to QQ produced in the culture medium of cultured cell lines derived from human normal tissue.
Concerning pro-t-PA with characteristics of I.

fllSDS-polyacrylamide gel electrophoresis showed a single protein band, with a molecular weight of approximately 70.00
0±2,000. Furthermore, according to the gel filtration method, the molecular weight is approximately 67,000±5,000.

(2) The presence of a reducing agent does not change the apparent molecular weight.

(3) Through plasmin treatment, etc., the molecular weight is reduced to approximately 3.
2,000 and about 38,000 subunits are S-S
In addition to converting into a double-stranded structure bound by a bond,
It is a precursor of plasminogen activator whose enzyme activity is expressed and enhanced.

(4) It has immunological properties that do not react with anti-urokinase antibodies but react with anti-tissue plasminogen activator antibodies.

(5) Decompose synthetic substrate S-2288 more specifically than synthetic substrate S-2444.

(6) It has an affinity for fibrin.

(7) Stronger affinity for lysine than tissue plasminogen activator.

(8) It has an isoelectric point in the range of 5 to 8.

(9) Optimum pt+ is in the range of 9 to 10.

Stable for 10 hours or more at 0.045°C.

(Preparation of production mother) As a means for supplying pro-t-PA according to the present invention, human uterine muscle cells are used, for example.

For example, human uterine muscle cells are produced by separating normal muscle tissue from the uterus obtained during hysterectomy and performing primary culture, and as they are repeatedly subcultured, they become proliferative and reach a limit. Since a cultured cell line that is capable of dividing and that produces a high amount of pro-t-PA is generated, this cell line is selected and utilized. As such an established cell line, the established cell line KW (hereinafter also referred to as KW line) derived from normal human uterine muscle cells is suitable.

The KW strain was obtained by the present inventor and is publicly known. Its acquisition method, cytological properties, etc. are described in "Cell Culture Research", Volume 5, No. 1, Japan & U Textile Culture Society, No. 59.
The disclosure is in the collection of conference lecture abstracts (1986), page 197, and a patent application filed in 1986-29 related to the invention by the present inventors.
No. 2481 also discloses the same, and the strain can be obtained repeatedly and reproducibly by the method disclosed in the publication. In addition, when filing Japanese Patent Application No. 60-292481, an application was made to deposit the strain in the Institute of Microbial Technology, Agency of Industrial Science and Technology, but the applicant was notified that the deposit was refused.

For example, the method for acquiring KW shares is as follows.

<KW strain acquisition experiment> The normal stroma was removed from a surgically removed human uterus, washed with Eagle's minimum essential medium (hereinafter abbreviated as MEM), cut into 1-2 mm pieces with scissors, washed with MEM, and centrifuged. (800-1.OOOrpm, 5 minutes) and collected the U-weave.

About 5 times the weight of enzyme ti, (collagenase 2
00 units/ll11. ) Lipsin 0.05-0.1
%), stir at 37℃, and add 2% while watching the digestion.
After digestion for ~4 hours, undigested tissue was removed with a mouthwash, and the filtrate was centrifuged to collect cells.

After washing the cells several times with MEM, they were implanted into a φ601 plastic dish, and the cells were washed with MEM containing 10% tallow serum.
was added and cultured at 37°C in 5% CO2-95% air. After the cells have grown sufficiently, they are treated with the enzyme solution mentioned above, and the cells are peeled off from the culture medium and collected.
011 plastic jar, added MEMloIIll containing 109< fetal bovine serum, and incubated at 37°C for 5 minutes.
Cultured in %COz 95% air. This subculture was repeated several dozen times, and cells with high proliferative ability were collected.

Normal human cells usually have a finite number of divisions of up to 50 ± 10 divisions, but the sorted cells show good proliferative ability even after dividing more than 500 times, making them a natural product that can be expected to be subcultured endlessly. It turned out to be a transformation method.

It was also confirmed that the culture supernatant of these cells secreted t-PA, which is immunochemically different from UK.

In addition, this KW strain uses MEM containing 10% dimethyl sulfoxide and 10% tallow serum as a protective agent,
It can be frozen by a slow freezing method in which the temperature is lowered by 1 to 1.5° C. per minute, and stored in liquid nitrogen for a long period of time.

The KW strain of the present invention thus obtained exhibits the following cytological properties.

(Cytological properties of KW strain) Substance producing ability It produces t-PA, which is immunochemically different from UK, and pro-t-PA of the present invention, which is a precursor of 8-PA.

Receptors Uterine muscle cells have characteristic estrogen receptors, the number of which varies depending on the cell cycle.

Cell proliferation phase: approx. 2.7X105/cell steady! 11! : approx.], 0X104/cell subculture
Can be subcultured without limit.

Culture time: 1.4-1.6 days. (In MEM containing 10% fetal bovine serum) (Culture conditions for producing pro-t-PA) The medium includes, for example, Eagle MEM, Hanks 19
9. Add 2m of glutamine to the basal medium of Dulbecco, Ham, etc.
M and live fetal blood +'n (manufactured by G I B CO) 5
-10% is used for cell growth. pr
A serum-free medium is used during o-t-PA production. Preferably, a serum-free medium to which various protein decomposition products, such as lactalbumin lysate, proteose beptone, meat extract, and various amino acids, are added is used.

For culture, culture flasks and Petri diffusers (all manufactured by CORN ING) are usually used for plate culture, but plastic or glass ones are also suitable.

Evening') Altray (1,200ad, N U N C
can be cultured in large quantities using Furthermore, high-density culture using microbeads (Cytodex-1, manufactured by Pharmacia) is also possible, and is suitable for mass production of pro-t-PA. Both are preferably cultured at 37°C under 5% CO2 partial pressure.

(Recovery of pro-t-PA) Recovery of pro-t-PA from the culture medium was performed using Ryken (D,
C, Rijken) and Colen (D, Co11en)
) et al. [Journal of Biological Chemistry]
al Chemistry) Sword 6, 7035-704
1, (1981)) with some modifications. Namely, zinc chelate sepharose, concanavalin A
・Various affinity chromatography and gel filtration (e.g., Sepharose, Lysine Sepharose, etc.)
Sephacryl S-200) is used in appropriate combination. This entire purification step was carried out in the presence of protease inhibitors, detergents, and pro-
PA is a 5DS-polyacrylamide gel electrophoresis method [Nature, 227.680-68
5 (1970)) to show a single protein band.

(Activity measurement method) pro-t-PA is activated by, for example, a method in which it is brought into contact with plasmin/sepharose, and the activity value is determined by the fibrin plate method [Acta haematologica 0 japonica (A
cta Haematologica Japonic
a), Dish, 298-305 (1976)'),
and methods using synthetic substrates [Journal of Clinical Laboratory Investigation (Jo
Urnal of CIinical Laborato
ry Investigation) 162+ 3
68-374 (1982)). The t-PA international unit was used as the unit of activity value.

(Characteristics of pro-t-PA) The pro-tPA obtained as described above has the characteristics described below.

fllsDs-polyacrylamide gel electrophoresis (Nach + - (Nature), dish, 680-685 (
(1970)) showed a single protein band, and the molecular weight determined therefrom was approximately 70,000±2,000. In addition, Sephadex G 150 (Pbar
The molecular weight determined by the gel filtration method using Macia Inc. is approximately 67,000±5,000.

(2) When reduction treatment was performed at 100° C. for 5 minutes in the presence of 5% SDS and 2.5% 2-mercaptoethanol, the apparent molecular weight did not change.

(3) Due to plasmin treatment, etc., the molecular weight is about 32,
4.000 and about 38,000 subunits are converted into a Miki chain structure in which they are linked by S-8 bonds. It is a precursor of plasminogen actichetae, whose enzyme activity is expressed and enhanced. The confirmation was performed as follows. That is, CNB ractivation Sepharose (Pharmac
A plasmin-Sepharose column was prepared by campling human plasminogen with IA (Company) and then contacting it with UK. pro-t-PA was added to this column at 37°C.
The activated enzyme was activated by contacting the enzyme with the filtrate, and the active enzyme of pro-t-PA was obtained in the flow-through liquid. The activity of pro-t-PA before and after activation was measured using synthetic substrates S-2444 and S-2288.

(4) It has immunological properties that do not react with anti-UK antibodies but react with anti-malignant t-PA antibodies.

- That is, UK and melanoma cells (Bo
p
React with rot-PA, enzymography [Analytical Biochemistry (Analyti
cal Biochemistry), 122, 16
4-172 (19B2)), the above antigenicity was revealed.

(5) Decompose synthetic substrate S-2288 more specifically than synthetic substrate S-2444.

That is, synthetic substrates S-2444 and S-2288 were each 0
Reactions were carried out using a constant amount of pro-L-PA in the concentration range of ~5mM, and the results were compared with Lineweber and Paak (
Substrate specificity and reaction rate constant were determined by Lineweaver-Burk plot.

(6) It has an affinity for fibrin.

That is, after CNBr-activated Sepharose was used to camp human fibrinogen, it was brought into contact with thrombin, and the fibrin-Sepharose column was prepared once. pro-t-PA is 0.15M NaCl! Contains 0.1 M Tris-HCl buffer (pH.

7.5), and the activity of the pass-through fraction and the fraction eluted with 0.2 M arginine and 2 M KSCN was measured, and the affinity for fibrin was determined by adsorption/non-adsorption on the fibrin-Sepharose column. I looked into gender.

(7) Stronger affinity for lysine than tissue t-PA. That is, lysine-5PW [5PW is a support manufactured by Toyo Soda Kogyo Co., Ltd. (Journal of Chromatography)
pt+y)2 511. (1986) ) was used to elute lysine using a linear concentration gradient of 0 to 0.3M using arginine, and the strength of lysine affinity was determined from the arginine concentration required for elution. .

(8) It has an isoelectric point in the range of 5 to 8. The isoelectric point is mo
Chromatofocusing was performed using a no-P column (Pharmacia), and fast protein
Analyzed by liquid chromatography.

(9) It has an optimum pH in the range of 9 to 10. Optimal p
+i was measured as follows. Adjust the pif of the reaction system of synthetic substrate S-2288 using the buffer solution of each po, add a certain amount of this enzyme, let it react, and then adjust the pif of the reaction solution.
By adjusting if to 7.0 and measuring enzyme activity,
The optimum pH was determined.

In phosphoric acid solution overnight (pH 9,0), at 4℃~
Thermal stability was investigated in a temperature range of 90°C. As a result pr.

-t-PA is stable at 45°C for more than 10 hours.

[Action/Effect]

As mentioned above, this enzyme is pro-t-PA, which has a -terminal molecular structure, and the peptide bond at a specific site is cleaved by plasmin treatment etc., converting it into a double-stranded molecular structure, and the activity is expressed. , is estimated to be enhanced. pro
-t-PA is pr.

Unlike -1JK, both the pro and active forms have high affinity for fibrin. In addition, t-PA purified from the culture medium of human melanoma cells (Bowes), (hereinafter referred to as melanoma t-PA), has an affinity for lysine.
-Abbreviated as PA), pro-
The fibrin affinity of t-PA is considered to be stronger than that of melanoma L-PA.

In melanoma t-PA, this phenomenon of expression and enhancement of activity due to plasmin treatment is hardly observed, and pr
o-t-PA is a new PA.

Analysis of enzyme reaction rate constants using synthetic substrate S-2444 and synthetic substrate S-2288 was compared with that of melanoma t-PA, and the results are shown in Table 1. pro-t-p after activation, 6.
The reaction rate constant for pro-t-P is similar to that for melanoma t-P, but when actually administered into the blood, pro-t-P
In the case of AO type, the active site is masked, so there is no deactivation by various PA vein bitters present in the blood during the process until it reaches the solid phase called thrombus (fibrin) and exhibits activity. It is believed that more effective thrombolytic action can be achieved.

As mentioned above, this enzyme is pro-t-PA and has the potential to exhibit a more stable and specific thrombolytic effect than the conventional active type of melanoma t-PA. It is highly anticipated as a new Mi-woven PA.

〔Example〕

Example 1 A serum-free culture soil solution of cultured cell line KW strain derived from human uterine muscle Mi tissue was collected, and aprotinin was added to a final concentration of 10/-
ml, added Tween 80 to a final concentration of 0.01%,
The starting material (approximately 50 international units/m ) was taken as the starting material.

The starting material was adjusted to pif 8.0 and zinc chelate
After contacting with Sepharose, 1-I NaC14
0.1 M Tris-HCl buffer (pif 5.5
) The adsorbed pro-t-PA was eluted. pro
The elution fractions containing -L-PA were collected, adjusted to pit 7.5, and adsorbed on concanavalin A Sepharose, followed by LM NaC1-containing 0.0. pr with 1M Tris-HCl buffer (pif 7.5).

-t-PA was eluted. The eluate was purified by ultrafiltration.
This was gel-filtered using Sephacryl S-200.

The pro-L-PA fractionated by gel II filtration was brought into contact with lysine Sepharose, and the adsorbed pro-L-PA
-t-PA with IM Na containing 0.3M arginine
0.1 M Tris-HCl buffer containing C1 (pif 7
．． 5′). Is the eluate desalted? ;
We were able to obtain a highly purified enzyme with a low specific activity (1,365,000 international units/mg) with a recovery rate of over 40%. Molecular weight approximately 70.00 by gel electrophoresis
A single protein band was shown at 0±2,000.

Experimental Example 1 Structural Change in Pro-t-PA Due to Plasmin Treatment Pro-t-PA was passed through plasmin Sepharose, and samples before and after passing were subjected to molecular weight analysis by 5DS-polyacrylamide gel electrophoresis. Pro-t-PA before plasmin sepharose treatment showed a single protein band under both non-reducing and reducing conditions, and the molecular weight was 70,000. The sample after plasmin treatment showed a single protein band with a molecular weight of 70,000 under non-reducing conditions, but under reducing conditions (e.g. 2-
(reacted with mercaptoethanol), the subunits with molecular weights of about 32,000 and about 38,000 are S-S
The structure was converted into a double-stranded structure in which the two strands were bonded together. Therefore, pro-tPA undergoes limited proteolysis by plasmin, and one peptide bond is cleaved, and each S
It is thought that the structure is converted into a double-stranded structure bound by a -8 bond.

Experimental Example 2 The conversion of pro-t-PA into active t-PA by plasmin treatment was demonstrated as an enhancement of the degree of decomposition of the synthetic substrate S-2444 or S-2288.

Pro-t-PA or melanoma t-PA as a control was activated with plasmin sepharose, and sample 50P1 before and after activation was mixed with various concentrations (
0-5mM) S-2444 or S-2288 solution 50μ, and 50mM) Lis-HCl buffer (pH 8,
4) (contains 0.1M NaCl) 100. J in a 96-well plate. Due to the action of enzymes, S-
The pNA released from 2444 or S-2288 was
The absorbance was measured at 0.05 ns. The results were analyzed by Lineweaver and Berk.
Analysis was performed by plotting to determine substrate specificity and reaction rate constant, and the results are shown in Table 1.

(See margins below) As a result, the enzymatic action of melanoma t-PA was hardly changed before and after plasmin treatment. Therefore, Km and Kcat values were also almost the same before and after plasmin treatment. On the other hand, pr.

The enzyme action of -t-PA was significantly enhanced before and after plasmin treatment. That is, when S-2444 is used as a substrate,
The cat value of pro-tPA is 0.0.
63(S-1) to 3.70(S-1) after plasmin treatment
1), it was enhanced by about 6 times (Table 1), and S-2288
When using the substrate as a substrate, 1.45 (S-
1) to 5.10 (S-1) after plasmin treatment, an increase of about 4 times. As described above, since the activity of pro-t-PA is markedly enhanced by plasmin treatment, it is concluded that pro-t-PA is an enzyme precursor. In addition, S-2 of pro-t-PA
From the difference in Km value with respect to 444 or S-2288,
pro-t-PA and its active enzyme are also S-2288
has a strong affinity for

Experimental Example 3 The conversion of pro-t-PA into activated PA by plasmin was proven by the incorporation of tritium-labeled diisopropylfluorophosphate (hereinafter abbreviated as [3■ (]DFP). Because it is specifically incorporated into the active site of serine enzymes,
Uptake occurs when the pro form is converted to the active form.

For pro-t-PA before and after plasmin treatment, (3
H) After adding DFP and allowing sufficient reaction, unreacted (3H
) DFP was removed by desalting, and the enzyme was subjected to 5DS-polyacrylamide gel electrophoresis. This gel was sliced in the direction of electrophoresis, and the radioactivity contained in each slice was measured using a scintillation counter to determine the molecular weight position at which uptake occurred. The results are shown in Figure 1.

As shown in FIG. 1, pro-t-PA has a molecular weight of about 70.
000, and after activation, enhancement of DFP uptake was observed only at the same molecular weight position (3) 1), which clearly indicates that pro-t-PA itself was activated and an active site appeared. It is supported. As is clear from Figure 1, the molecular weight is approximately 70,000 even before activation (3H).
DFP uptake was observed, which suggests that the final purified pro-t-PA fraction was mixed with partially activated t-PA.

Experimental Example 4 Affinity for lysine The affinity for lysine was investigated by high performance liquid chromatography using lysine-5PW. First, after applying the sample to lysine-5PW, elution with arginine was performed linearly from 0 to 0.3M. ; The arginine concentration required for elution was determined using a gradient gradient. When pro-t-PA was applied, it was eluted with 0.28 M arginine, and there was only one peak. pro-t-PA
When applied to lysine-5PW after treatment with plasmin, it was eluted as two peaks, with respective arginine concentrations of 0.23 M and 0.08 M. When melanoma t-PA was applied, an elution pattern with two peaks was obtained, and the respective arginine concentrations were 0.2M and 0.13M.

Thus, pro-t-PA has a stronger affinity for lysine than melanoma t-PA, and therefore pro-t-PA is considered to have a stronger affinity for fibrin than melanoma t-PA.

[Brief explanation of drawings]

Figure 1 shows the uptake of 3HIDFP according to the position of the molecular weight of this enzyme before and after plasmin treatment. Shows: Patent Jaw Man Osamu Matsuo

Claims (1)

[Scope of Claims] A tissue plasminogen activator precursor having the following characteristics, which is produced in the culture medium of a cultured cell line derived from human normal tissue. (1) SDS-polyacrylamide gel electrophoresis showed a single protein band with a molecular weight of approximately 70,000.
±2,000. Further, the molecular weight determined by gel filtration method is approximately 67,000±5,000. (2) The presence of a reducing agent does not change the apparent molecular weight. (3) Due to plasmin treatment, the molecular weight is about 32,
It is a precursor of plasminogen activator, which converts into a double-stranded structure in which approximately 38,000 and 38,000 subunits are linked by SS bonds, and the enzyme activity is expressed and enhanced. (4) It has immunological properties that do not react with anti-urokinase antibodies but react with anti-tissue plasminogen activator antibodies. (5) Decompose synthetic substrate S-2288 more specifically than synthetic substrate S-2444. (6) It has an affinity for fibrin. (7) Stronger affinity for lysine than tissue plasminogen activator. (8) It has an isoelectric point in the range of 5 to 8. (9) It has an optimum pH in the range of 9 to 10. (10) Stable at 45°C for 10 hours or more.