JPH0692995A - Human masking protein and polymer unit - Google Patents

Abstract

PURPOSE:To obtain a human masking protein for masking transforming growth factor beta (TGF-beta) and deactivating TGF-beta, a polypeptide to become a polymer subunit and a precursor of the human masking protein and a nucleotide encoding the human masking protein, etc. CONSTITUTION:A polypeptide is recorded in a sequence number 1 or 4, hMPU and hMP comprise the polypeptide and a polynucleotide is recorded in a sequence number 5 or 8. The polypeptide, MPU and hMPU bonded to a low- molecular-weight subunit are bonded to TGF-beta by themselves or optionally through linkage to a saccharide chain to show various kinds of physiological activities such as multiplication inhibition of tumor cell. Polypeptide of the sequence number 1 and the polynucleotide of this invention can produce the polypeptide of this invention.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to transforming growth factor beta.
hFactor-β, hereinafter referred to as “TGF-β”) and inactivates the human masking protein and its polymeric subunit, a precursor polypeptide, and nucleotides encoding them. .

[0002]

Transforming growth factor beta T
GF-β is a factor that promotes the growth of NRK cells derived from normal rat kidney, which originally does not grow in soft agar, in the soft agar, and is 1978 from the culture supernatant of cells transformed with MuSV (mouse RNA sarcoma virus). A protein discovered in 1980, named transforming growth factor in the sense that it is related to transformation, and was considered to be a substance that specifically exists in tumor cells. As a result of the subsequent research, it was clarified that TGF-β is abundant in normal tissues, especially in platelets, and it was revealed that it is involved in various physiological phenomena in vivo.

TGF-first purified from human platelets
β is currently named TGF-β1, and five types of β1 to β5 have been discovered so far from human, rat, pig, chicken and the like as TGF-β analogs. Hereinafter, in the present invention, these TGF-β analogs are referred to as “TGF-β”.
Collectively. TGF-β shows some action on almost all cells of mammals, and is expected to be therapeutically applied to various diseases. The main actions of TGF-β are to promote proliferation of mesenchymal cells such as normal cultured fibroblasts, suppress proliferation of epithelial cells such as hepatocytes, control cell differentiation, suppress proliferation of immune system cells such as T cells, and hematopoiesis. Inhibition of proliferation of system cells
It has been confirmed by tro, and its activity in the living body has been found to be involved in promotion of wound healing, bone metabolism, stop of liver regeneration and the like.

It has been elucidated so far that TGF-β1 is produced as a precursor (latent type) consisting of 390 amino acids, and is cleaved into 112 amino acids on the C-terminal side to become a dimer and becomes the active form. . TGF-β or its partial peptides can be produced by purification from animal cells or expression in genetically modified cells, and clinical application development thereof, for example, burns utilizing the growth promoting activity of epithelial cells, after surgery It has been attempted to be used as a wound healing agent, an eczema therapeutic agent, an osteoporosis therapeutic agent due to its involvement in bone formation, an immunosuppressive agent for organ transplantation and immune diseases due to suppression of immune system cell proliferation.

It is known that cancer cells produce large amounts of TGF-β by themselves and repeat vigorous growth by promoting the growth of cancer cells themselves and suppressing the growth of immune system cells such as killer T cells and helper T cells. .

On the other hand, TGF-β produced by normal cells is
As described below, it was clarified that all were inactive forms that formed a complex with the masking protein. On the other hand, it is known that many cancer cells secrete TGF-β in an active form.

Therefore, a factor that inactivates TGF-β is very promising as an anticancer agent. This factor is the only substance that the inventor discovered in rat platelets and named TGF-β masking protein, specifically reacts with and inactivates TGF-β that has been discovered so far. Was (Biochem BiophysRe
S Commun, 141 , 176-184, 198.
6). Further, in order to analyze various activities of TGF-β, it is important to elucidate the mechanism that regulates TGF-β in vivo, and for that purpose, it is necessary to analyze the detailed structure of human masking protein and It has been desired to obtain it as a purified protein.

The present inventors have found that TGF-
High molecular weight subunit MP of a protein that specifically binds to β
U, its precursor MPU-P, its prepro structure M
PU-PP, the gene MPU-N encoding them and the polynucleotide MPU-PN, MPU-PPN were purified, isolated and specified, and these have been filed as Japanese Patent Application No. 2-173679.

[0009]

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention
Since GF-β masking protein specifically reacts with TGF-β which promotes the growth of cancer cells and inactivates it, it is obtained from human tissues as a purified protein, and its detailed structure is obtained. It has been an issue to analyze and contribute to the production of a substance that suppresses the growth of cancer cells.

[0010]

The present invention provides a transforming growth factor beta (TransformingG).
human masking protein h that specifically binds to row Factor-β) (“TGF-β”)
MP, its polymeric subunit hMPU, its precursor hMPU-P, and the gene h encoding them
It is the invention of MPU-N and the polynucleotide hMPU-PN.

The present inventor has elucidated the existence of a protein which inactivates TGF-β as follows. That is, TG
The molecular weight of F in non-reducing SDS-PAGE is 26 kD, whereas that of latent TGF-β is 400 to 500 kD.
Based on its high molecular weight, the existence of a high molecular weight protein that inactivates TGF-β was predicted and repeated researches revealed that a protein that specifically binds to TGF-β was partially purified from rat platelets. It was successful, and this was confirmed to be TGF-β masking protein (TGF-β maskingpr
(Otein) (Biochem Bioph)
ys Res Commun, 141 176-18
4, 1986). TGF-β masking protein (hereinafter referred to as “MP”) is an atypical trimer consisting of two 39 kD subunits and one 110 KD subunit, of which the amino acid sequence of the N-terminal 15 bases of the 39 kD subunit is When determined, it was in agreement with the amino acid sequence of the N-terminal region following the signal sequence of the TGF-β precursor (Proc Natl Acad Sci, 83 , 64).
89-6493, 1986. FEBS Lett, 24
2 , 240-244, 1989). This makes MP 3
It has been elucidated that the 9 kD subunit is only the precursor protein part of TGF-β.

On the other hand, the present inventor conducted research on a 110 kD subunit (hereinafter referred to as "polymer subunit"), and clarified that this plays an important role in inactivating TGF-β, and rat platelets. By further analyzing the amino acid sequence of the protein isolated and purified and the nucleotide sequence of the gene encoding the protein, it was revealed that the protein was a completely novel protein, and this novel polymer subunit was named MPU. . Thereby, the present inventor
Transforming growth factor beta (Transfo
rming Growth Factor-β), a high molecular weight subunit MPU of a protein that specifically binds to rat platelets was isolated, purified, and identified from the above-mentioned Japanese Patent Application No. 2-17.
Completed No. 3679.

[0013] Further, as a result of continuing diligent research, the present inventor completed the present invention by isolating and purifying TGF-β human masking protein from human platelets and identifying it. That is, the present invention is
Transforming growth factor beta (Transfo
The invention is the invention of a masking protein hMP that specifically binds to an illuminating Growth Factor-β), a mature high molecular weight subunit hMPU of a protein, and a gene that produces this. We succeeded in isolating and purifying hMPU-1, 2, 3 and its precursor hMPU-P. Furthermore, the present inventors have found that human placenta-derived cD
By analyzing the NA library, mature hMP
The nucleotide sequences of genes encoding U-1, 2, 3 and precursor hMPU-P have been elucidated to complete the present invention.

That is, the present invention is an invention of polypeptide hMPU-1 having the amino acid sequence of SEQ ID NO: 1.

The present invention is an invention of polypeptide hMPU-2 having the amino acid sequence of SEQ ID NO: 2.

The present invention is an invention of a polypeptide hMPU-3 having the amino acid sequence of SEQ ID NO: 3.

The present invention provides polypeptide hMPU-1, hMP having the amino acid sequences of SEQ ID NOs: 1 to 3.
It is an invention of a mixed polypeptide hMPU of U-2 and hMPU-3.

The present invention is an invention of a human masking protein hMP obtained by S—S bond of the polypeptide having the amino acid sequence of SEQ ID NO: 9 and the polypeptide hMPU of the preceding paragraph.

The present invention is an invention of a polypeptide hMPU-P having the amino acid sequence of SEQ ID NO: 4.

The present invention is an invention of the polynucleotide hMPU-1N having the nucleotide sequence of SEQ ID NO: 5.

The present invention is an invention of the polynucleotide hMPU-2N having the nucleotide sequence of SEQ ID NO: 6.

The present invention is an invention of the polynucleotide hMPU-3N having the nucleotide sequence of SEQ ID NO: 7.

The present invention is an invention of the polynucleotide hMPU-PN having the nucleotide sequence of SEQ ID NO: 8.

Polypeptides and polynucleotides of the present invention include those having the above-mentioned amino acid sequences and base sequences, but naturally the present invention is not limited to those having the same sequences, but partially Substances having substantially the same sequence even though having different differences are included in the scope of the present invention.

Since the present invention is an invention of useful polypeptides and polynucleotides per se, they are included in the scope of the present invention regardless of their manufacturing methods and uses.

The polypeptide and polynucleotide of the present invention can be extracted and purified from human tissues such as stomach, lung, kidney, thymus, serum, blood serum, etc., for example, according to the procedure shown in Example 1. .

For example, after crushing human platelets by ultrasonic waves, a protease inhibitor is added and the mixture is centrifuged,
The supernatant is purified by various gel columns according to the method for purifying the latent TGF-β complex, separated by SDS-PAGE, and then transferred to a membrane to obtain hMP of the present invention.
U-1, 2, 3, P can be isolated.

As another method for producing the polypeptide of the present invention, the polynucleotide having the nucleotide sequence of the present invention is extracted from human tissue, purified, or synthesized, and then incorporated into an appropriate expression vector, It can be obtained by introducing the gene into a cultured cell such as Escherichia coli and then from the culture supernatant.

More specifically, it can be produced according to a commonly used genetic engineering technique, for example, the following procedure. That is, cDNA is synthesized using the PCR (polymerase chain reaction) method using mRNA extracted from human tissue cells, serum or blood scaffold as a template, and this cDNA is used as a set of E. coli-derived plasmid pBR322 or bacteriophage λgt11. E. coli Escherichia coli NM51
No. 4 or the like, which is incorporated into a host cell to be expressed and expressed by a colony hybridization method or a plaque hybridization method using a partial oligonucleotide (radiolabeled) probe synthesized based on the nucleotide sequence revealed by the present invention. Select cDNA. In addition, Maxam and Gilbert's chemical method (Proc
Natl Acad Sci, 74 , 560, 197.
7) etc. to determine the nucleotide sequence of the cDNA, and the cDNA is excised from the vector containing the cDNA encoding the entire amino acid sequence of the human MP macromolecular subunit with a restriction enzyme, and an appropriate expression vector, for example, Escherichia coli-derived plasmid pBR322. Alternatively, it can be obtained by incorporating it into tumor virus SV40 and the like using a restriction enzyme and DNA ligase and introducing it into mouse C127 cells or monkey COS cells to express it.

[0030]

The polymer subunit hMPU-1 of the present invention,
HMPU consisting of 2, 3 and these is a small molecule subunit obtained by purification or expression separately from this (an example of its amino acid sequence is shown in SEQ ID NO: 9, but it has a partial difference from this). Have substantially the same sequence,
HMPU of the present invention can be obtained by chemically or enzymatically coupling with those having the same activity). hMP itself has an activity of specifically binding to TGF-β by binding to a sugar chain as necessary.

According to the study of the present inventor, the DNA synthesis inhibitory activity of mature rat primary culture hepatocytes and NRK49F
From the results of the cell colony forming activity bioassay system, hMPU in which the hMPU consisting of the polymer subunits hMPU-1, 2, and 3 of the present invention is bound to a low molecular subunit is shown.
Is not only human-derived TGF-β, but also rat TGF-
It was confirmed that β activity was also completely suppressed. Thus, the hMP of the present invention and the polymer subunits hMPU, hMPU-1, 2, and 3 thereof can suppress TGF-β activity in various animals including humans.

Further, the polymer subunit hMP of the present invention
UP is a precursor of hMPU-1,2,3, and from this, hMPU-1,2,3 can be produced.

Further, hMPU-1N, 2N, 3 of the present invention
For N and PN, hMPU-1, 2, 3, and P of the present invention can be produced by using a known genetic engineering technique.

[0034]

EXAMPLES Examples will be described below to explain the present invention in detail, but the present invention is not limited to these examples.

Example 1 Determination of partial amino acid sequence The latent TGF-β complex was purified from human platelets according to the following method. Human platelets were treated with 50 mM Tris-H
The cells were suspended in Cl buffer, sonicated and then added with a protease inhibitor such as antipain and bestatin and centrifuged. Mix the supernatant with S Sepharose gel,
It was centrifuged. Use the supernatant as starting material,
DEAE-Toyopearl 650s, Phenyl Sepharose CL-4B, Chelating Sepharose,
Purification was performed using each gel column of Cu chelating Sepharose and Sepharose 6 (J Biochem, 1
06 , 304, -310, 1989).

The latent TGF-β complex fraction obtained by separating the masking protein and the free mature high molecular weight subunit hMPU was subjected to reverse phase liquid chromatography with a concentration gradient in the elution solvent. It showed a pattern and was separated into 3 peaks. Separate each peak and SD under non-reducing and reducing
The results of S-PAGE analysis are shown in FIG. 2 (a: under non-reduction, b: under reduction). Rat-derived latent TGF-β complex (including rat MP) used as a control (lane 1)
From the comparison with, the peak 1 is free mature polymer subunit hMPU (lane 2), and the peak 2 is free TGF-β.
(Lane 3), peak 3 was estimated to be hMP (lane 4). The peak 1 fraction was reduced, subjected to SDS-PAGE, and then transferred to a polyvinylidene-difluoride membrane to obtain hMPU on the membrane.

Regarding the hMPU on the membrane obtained by the determination of the partial amino acid sequence, the N-terminal amino acid sequence was determined by a conventional method.
IHLHPQFPVVVE, HPQFPVVVEKK,
It was confirmed that there are three kinds of polypeptide having PQFPVVVEK, and hMPU-1, 2, 3 respectively.
I named it. On the other hand, hMPU on the membrane was subjected to cyanogen bromide decomposition, followed by lysine endopeptidase treatment, and the amino acid sequences of the three peptides obtained after the enzyme treatment were determined (single-letteramine).
o-acid code); peptide 1, QTEGX
ERTXTXG; Peptide 2, GFVPAGESSY
E: Peptide 3, EGTYYDPV. These amino acid sequences have homology with rat platelet-derived MP polymer subunit disclosed in Japanese Patent Application No. 2-173679,
It was suggested that hMPU is a protein similar to rat MP high molecular subunit. Also, at SDS-PAG
A band of about 120 kDa corresponding to hMPU was excised from the human MP reduced product obtained from E (lane 4 in FIG. 2 (b)), and the peptide obtained by transferring to a membrane was analyzed. As a result, it was confirmed that this peptide was the same substance as hMPU obtained in.

CDNA screening A cDNA library was constructed from megakaryocyte cells purified from rat bone marrow, and the obtained 4054 bp cDNA fragment of the rat MP polymer subunit was used as a probe for screening the MP polymer subunit cDNA clone. did. Use a polynucleotide probe with T4 polynucleotide kinase (Takara Biochemicals) [γ
^The 5'end was phosphorylated with ³² P] ATP (Amersham) and radioactively labeled. The cDNA was synthesized from human placenta using oligo (dT) as a primer. c
The construction of the DNA library was performed using the λZAP11 vector (Stratagene). The recombinant phage was plated on E. coli and transferred to a nitrocellulose filter (Hybond N, Amersham). These replicas were inspected with an inosine-containing radiolabel probe to obtain 0.2 × SSC (0.9M NaCl, 90 mM
A hybridization reaction was carried out with trisodium citrate), 0.05% sodium phosphate, 100 μg / ml tRNA, and 6 × Denhardt's solution at 65 ° C. for 15 hours. After the reaction, use a nitrocellulose filter with 0.1% S
Washing with 0.2 × SSC buffer containing DS at 65 ° C. for 20 minutes was repeated 3 times.

Determination of cDNA Nucleotide Sequence A plurality of the obtained clones were cut out with a restriction enzyme, and the nucleotide sequence of hMPUcDA was determined by the dideoxy method using Sequenase (United State Biochemicals). As a result, the obtained cDNA is rat M
It covers 2,535 nucleotides including the coding region corresponding to the mature polymer subunit in P, and the coding region of free mature hMPU-1 is a polypeptide starting from the 268th base in this example. It was confirmed.

Example 2. Example of Obtaining hMPU-1N, 2N, 3N Among the clones obtained in Example 1, hMP3-1 having a 3.1 kb insert was used to prepare hMPU-P.
A plasmid containing N, 1N, 2N and 3N was prepared.

Plasmid hMP3-1 is a restriction enzyme Sty
Digested with I and SphI and 104 bp to 2264 bp 2.
After a 1 kb fragment, DN was added to the 5'end and 3'end.
The following DNA fragment prepared by the A synthesizer was used as a linker and ligated with T4 DNA ligase. The 5'-terminal linker has 104 bp of the constituent nucleotides of hMPU-P, a start codon ATG, and a SalI digestion site. The 3'-terminal side linker is 78 bp of the constituent nucleotides of hMPU-P and stop codons TGA and HindI
II Has a digestion site. Expression vector pKK223-3
Was digested with restriction enzymes SalI and HindIII, and then a DNA fragment to which a linker was bound was inserted by T4 DNA ligase to obtain a plasmid pKK223-3 [hMPU-P] having hMPU-P as an insert.

A plasmid having hMPU-1N as an insert was obtained as follows. Plasmid hMP3
-1 was digested with the restriction enzymes HindIII and SphI.
5'after the formation of a 1.9 kb fragment of 5 bp to 2264 bp
77 bp of the constituent nucleotides of hMPU-1 and a start codon ATG, and a linker having a SalI digestion site on the terminal side, and 78 bp of the constituent nucleotides of hMPU-1 and the stop codons TGA and HindII on the 3'terminal side.
The linker with the I digestion site was ligated with T4 DNA ligase. The expression vector pKK223-3 was digested with restriction enzymes SalI and HindIII, and the linker-ligated DNA fragment was inserted with T4 DNA ligase to obtain pKK223-3 [hMPU-1].

Similarly, for the plasmid having hMPU-2N, 3N as an insert, the plasmid hMPU3-1 was used.
Was digested with HindIII and SalI, and then prepared using a linker containing 65 bp and 62 bp hMPU-2 and 3 constituent nucleotides at the 5'end, and pKK22 and pKK22, respectively.
3-3 [hMPU-2], pKK223-3 [hMPU
-3].

Example 3. Example of Obtaining hMPU-P, 1, 2, 3 E. E. coli (DH5-α strain) was cultured in L-Broth medium, and the cells were collected and suspended in 100 mM CaCl ₂ .
Calcium-activated E. coli was transformed into pKK223-
The cells were transformed with 3 [hMPU-P] and cultured in L-Broth medium. The culture solution was purified, subjected to SDS-PAGE, transferred to a polyvinylidene-difluoride membrane, and hMP
I got U. Similarly, pKK223-3 [hMPU-
1], pKK223-3 [hMPU-2], pKK22
E. coli using 3-3 [hMPU-3]. coli (DH5
-Α strain) and hMPU-
1,2,3 were obtained by purification.

Example 4. Inhibitory effect of human MP on human TGF-β activity Human MP having hMPU of the present invention as a constituent protein is human T
The inhibition of GF-β activity was confirmed as follows. In Example 1-hM was obtained from human platelets and hM was obtained from the reduced product.
Human MP, which was confirmed to contain PU-1, 2, and 3 as constituent components, was used in the following examples. TG of human MP
The F-β inhibitory effect was measured using rat primary culture hepatocytes. Liver parenchymal cells were separated and purified from Wistar rats by the collagen perfusion method. 5 hepatocytes obtained
The suspension was suspended in Williams E medium (Flow Laboratories, Inc.) containing 0.5% bovine calf serum at a concentration of 2.5 × 10 ⁵ cells / ml, and seeded at 0.5 ml / well on a 24-well multiplate. After culturing at 37 ° C. for 20 hours under the conditions of 5% CO ₂ , 30% O ₂ and 65% N ₂ , 1 × 10 ⁻⁷ M insulin (Sigma), 10 ng / ml EGF (recombinant human E
(GF, Earth Chemical Co., Ltd.) and serum-free Williams E medium was exchanged, and 45 μl of a solution prepared by adding a predetermined amount of MP to a phosphate buffer containing 2.5 mg / ml bovine serum albumin was added to the same buffer. 1. Each of TGF-β1 and β2.
5 μl of the solution containing 8 ng was mixed, and the solution incubated at room temperature for 1 hour was added. 1.2 after 12 hours of culture
10 μl of 5 μCi / ml [ ³ H] deoxythymidine
/ Well, and to the control group, 5 μg / ml of aphidicolin was added 15 minutes before the addition of [ ³ H] deoxythymidine. After further culturing for 24 hours and labeling with tritium, the cells were washed twice with a phosphate buffer solution of pH 7.4 and fixed with a cold 10% trichloroacetic acid aqueous solution. The cells were solubilized with 0.5 ml of 1N aqueous sodium hydroxide solution per well, and the radioactivity thereof was measured by a gamma counter. The amount of tritium incorporated into the liver parenchymal cells when the test sample was added was determined as a control count ratio, and defined as the DNA synthesis activity (%). As a result, as shown in FIG. 3 (◯: TGF-β1, ●: TGF-β2), human MP containing hMPU of the present invention is human TGF-β.
Dose-dependently suppress the hepatocyte proliferation inhibitory activity of
It was revealed that 40 to 100 ng / ml (20 to 55 parts by weight relative to 1 part by weight of TGF-β) almost completely suppressed the activity of TGF-β.

[0046]

The polypeptide hMPU-1 of the present invention,
HMPU having a few components can be bound to a low molecular weight subunit to obtain the hMP of the present invention. h
MP binds to TGF-β derived from humans and various animals by binding to the sugar chain itself or, if necessary, to sugar chains, and exerts various physiological activities such as inhibition of tumor cell growth. The polypeptide hMPU-P of the present invention is
PU-1, 2, 3 can be manufactured. The polynucleotide hMPU-1N, 2N, 3N, PN of the present invention comprises
The polypeptide of the present invention can be produced by subjecting it to a genetic engineering technique.

As described above, the polypeptide and polynucleotide of the present invention are indispensable and important substances for producing a masking protein that binds to TGF-β.

[0048]

[Brief description of drawings]

FIG. 1: Reversed phase liquid chromatography fractionation of latent TGF-β complex fractions. The solid line is the absorbance at 280 nm,
The broken line shows the acetonyl concentration.

FIG. 2 SDS-PA under non-reducing and reducing of each peak
GE analysis.

FIG. 3: Suppression of TGF-β1, β2 activity of hMPU. ◯ indicates TGF-β1 and ● indicates TGF-β2.

[Sequence list]

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] August 26, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Detailed explanation of the invention

[Correction method] Change

[Correction content]

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to transforming growth factor beta.
hFactor-β, hereinafter referred to as “TGF-β”) and inactivates the human masking protein and its polymeric subunit, a precursor polypeptide, and nucleotides encoding them. .

[0002]

Transforming growth factor beta T
GF-β is a factor that promotes the growth of NRK cells derived from normal rat kidney, which originally does not grow in soft agar, in the soft agar, and is 1978 from the culture supernatant of cells transformed with MuSV (mouse RNA sarcoma virus). A protein discovered in 1980, named transforming growth factor in the sense that it is related to transformation, and was considered to be a substance that specifically exists in tumor cells. As a result of the subsequent research, it was clarified that TGF-β is abundant in normal tissues, especially in platelets, and it was revealed that it is involved in various physiological phenomena in vivo.

TGF-first purified from human platelets
β is currently named TGF-β1, and five types of β1 to β5 have been discovered so far from human, rat, pig, chicken and the like as TGF-β analogs. Hereinafter, in the present invention, these TGF-β analogs are referred to as “TGF-β”.
Collectively. TGF-β shows some action on almost all cells of mammals, and is expected to be therapeutically applied to various diseases. The main actions of TGF-β are to promote proliferation of mesenchymal cells such as normal cultured fibroblasts, suppress proliferation of epithelial cells such as hepatocytes, control cell differentiation, suppress proliferation of immune system cells such as T cells, and hematopoiesis. Inhibition of proliferation of system cells
It has been confirmed by tro, and its activity in the living body has been found to be involved in promotion of wound healing, bone metabolism, stop of liver regeneration and the like.

It has been elucidated so far that TGF-β1 is produced as a precursor (latent type) consisting of 390 amino acids, and is cleaved into 112 amino acids on the C-terminal side to become a dimer and becomes the active form. . TGF-β or its partial peptides can be produced by purification from animal cells or expression in genetically modified cells, and clinical application development thereof, for example, burns utilizing the growth promoting activity of epithelial cells, after surgery It has been attempted to be used as a wound healing agent, an eczema therapeutic agent, an osteoporosis therapeutic agent due to its involvement in bone formation, an immunosuppressive agent for organ transplantation and immune diseases due to suppression of immune system cell proliferation.

It is known that cancer cells produce large amounts of TGF-β by themselves and repeat vigorous growth by promoting the growth of cancer cells themselves and suppressing the growth of immune system cells such as killer T cells and helper T cells. .

On the other hand, TGF-β produced by normal cells is
As described below, it was clarified that all were inactive forms that formed a complex with the masking protein. On the other hand, it is known that many cancer cells secrete TGF-β in an active form.

Therefore, a factor that inactivates TGF-β is very promising as an anticancer agent. This factor is the only substance that the inventor discovered in rat platelets and named TGF-β masking protein, specifically reacts with and inactivates TGF-β that has been discovered so far. Was (Biochem BiophysRe
S Commun, 141 , 176-184, 198.
6). Further, in order to analyze various activities of TGF-β, it is important to elucidate the mechanism that regulates TGF-β in vivo, and for that purpose, it is necessary to analyze the detailed structure of human masking protein and It has been desired to obtain it as a purified protein.

The present inventors have found that TGF-
High molecular weight subunit MP of a protein that specifically binds to β
U, its precursor MPU-P, its prepro structure M
PU-PP, the gene MPU-N encoding them and the polynucleotide MPU-PN, MPU-PPN were purified, isolated and specified, and these have been filed as Japanese Patent Application No. 2-173679.

[0009]

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention
Since GF-β masking protein specifically reacts with TGF-β which promotes the growth of cancer cells and inactivates it, it is obtained from human tissues as a purified protein, and its detailed structure is obtained. It has been an issue to analyze and contribute to the production of a substance that suppresses the growth of cancer cells.

[0010]

The present invention provides a transforming growth factor beta (TransformingG).
human masking protein h that specifically binds to row Factor-β) (“TGF-β”)
MP, its polymeric subunit hMPU, its precursor hMPU-P, and the gene h encoding them
It is the invention of MPU-N and the polynucleotide hMPU-PN.

The present inventor has elucidated the existence of a protein which inactivates TGF-β as follows. That is, TG
The molecular weight of F in non-reducing SDS-PAGE is 26 kD, whereas that of latent TGF-β is 400 to 500 kD.
Based on its high molecular weight, the existence of a high molecular weight protein that inactivates TGF-β was predicted and repeated researches revealed that a protein that specifically binds to TGF-β was partially purified from rat platelets. It was successful, and this was confirmed to be TGF-β masking protein (TGF-β maskingpr
(Otein) (Biochem Bioph)
ys Res Commun, 141 176-18
4, 1986). TGF-β masking protein (hereinafter referred to as “MP”) is an atypical trimer consisting of two 39 kD subunits and one 110 KD subunit, of which the amino acid sequence of the N-terminal 15 bases of the 39 kD subunit is When determined, it was in agreement with the amino acid sequence of the N-terminal region following the signal sequence of the TGF-β precursor (Proc Natl Acad Sci, 83 , 64).
89-6493, 1986. FEBS Lett, 24
2 , 240-244, 1989). This makes MP 3
It has been elucidated that the 9 kD subunit is only the precursor protein part of TGF-β.

On the other hand, the present inventor conducted research on a 110 kD subunit (hereinafter referred to as "polymer subunit"), and clarified that this plays an important role in inactivating TGF-β, and rat platelets. By further analyzing the amino acid sequence of the protein isolated and purified and the nucleotide sequence of the gene encoding the protein, it can be revealed that the protein is a completely novel protein. This novel polymer subunit is named MPU. did. Thereby, the present inventor
Transforming growth factor beta (Transfo
rming Growth Factor-β), a high molecular weight subunit MPU of a protein that specifically binds to rat platelets was isolated, purified, and identified from the above-mentioned Japanese Patent Application No. 2-17.
Completed No. 3679.

[0013] Further, as a result of continuing diligent research, the present inventor completed the present invention by isolating and purifying TGF-β human masking protein from human platelets and identifying it. That is, the present invention is
Transforming growth factor beta (Transfo
The invention is the invention of a masking protein hMP that specifically binds to an illuminating Growth Factor-β), a mature high molecular weight subunit hMPU of a protein, and a gene that produces this. We succeeded in isolating and purifying hMPU-1, 2, 3 and its precursor hMPU-P. Furthermore, the present inventors have found that human placenta-derived cD
By analyzing the NA library, mature hMP
The nucleotide sequences of genes encoding U-1, 2, 3 and precursor hMPU-P have been elucidated to complete the present invention.

That is, the present invention is an invention of polypeptide hMPU-1 having the amino acid sequence of SEQ ID NO: 1.

The present invention is an invention of polypeptide hMPU-2 having the amino acid sequence of SEQ ID NO: 2.

The present invention is an invention of a polypeptide hMPU-3 having the amino acid sequence of SEQ ID NO: 3.

The present invention provides polypeptide hMPU-1, hMP having the amino acid sequences of SEQ ID NOs: 1 to 3.
It is an invention of a mixed polypeptide hMPU of U-2 and hMPU-3.

The present invention is an invention of a human masking protein hMP obtained by S—S bond of the polypeptide having the amino acid sequence of SEQ ID NO: 9 and the polypeptide hMPU of the preceding paragraph.

The present invention is an invention of a polypeptide hMPU-P having the amino acid sequence of SEQ ID NO: 4.

The present invention is an invention of the polynucleotide hMPU-1N having the nucleotide sequence of SEQ ID NO: 5.

The present invention is an invention of the polynucleotide hMPU-2N having the nucleotide sequence of SEQ ID NO: 6.

The present invention is an invention of the polynucleotide hMPU-3N having the nucleotide sequence of SEQ ID NO: 7.

The present invention is an invention of the polynucleotide hMPU-PN having the nucleotide sequence of SEQ ID NO: 8.

Polypeptides and polynucleotides of the present invention include those having the above-mentioned amino acid sequences and base sequences, but naturally the present invention is not limited to those having the same sequences, but partially Substances having substantially the same sequence even though having different differences are included in the scope of the present invention.

Since the present invention is an invention of useful polypeptides and polynucleotides per se, they are included in the scope of the present invention regardless of their manufacturing methods and uses.

The polypeptide and polynucleotide of the present invention can be extracted and purified from human tissues such as stomach, lung, kidney and thymus, or serum and plasma, for example, according to the procedure shown in Example 1.

For example, after crushing human platelets by ultrasonic waves, a protease inhibitor is added and the mixture is centrifuged,
The supernatant is purified by various gel columns according to the method for purifying latent TGF-β complex, separated by SDS-PAGE, and then transferred to a membrane to obtain hMP of the present invention.
U-1, 2, 3, P can be isolated.

As another method for producing the polypeptide of the present invention, the polynucleotide having the nucleotide sequence of the present invention is extracted from human tissue, purified, or synthesized, and then incorporated into an appropriate expression vector to prepare E. coli. It can be obtained by introducing the gene into cultured cells such as the above, and then from the culture supernatant.

More specifically, it can be produced according to a commonly used genetic engineering technique, for example, the following procedure. That is, cDNA is synthesized using the PCR (polymerase chain reaction) method using mRNA extracted from human tissue cells, serum, or plasma as a template, and this cDNA is recombined with Escherichia coli-derived plasmid pBR322 or bacteriophage λgt11. E. coli Escherichia coli NM51
No. 4 or the like, which is incorporated into a host cell to be expressed and expressed by a colony hybridization method or a plaque hybridization method using a partial oligonucleotide (radiolabeled) probe synthesized based on the nucleotide sequence revealed by the present invention. Select cDNA. In addition, Maxam and Gilbert's chemical method (Proc
Natl Acad Sci, 74 , 560, 197.
7) etc. to determine the nucleotide sequence of the cDNA, and the cDNA is excised from the vector containing the cDNA encoding the entire amino acid sequence of the human MP macromolecular subunit with a restriction enzyme, and an appropriate expression vector, for example, Escherichia coli-derived plasmid pBR322. Alternatively, it can be obtained by incorporating it into tumor virus SV40 and the like using a restriction enzyme and DNA ligase and introducing it into mouse C127 cells or monkey COS cells to express it.

[0030]

The polymer subunit hMPU-1 of the present invention,
HMPU consisting of 2, 3 and these is a small molecule subunit obtained by purification or expression separately from this (an example of its amino acid sequence is shown in SEQ ID NO: 9, but it has a partial difference from this). Have substantially the same sequence,
HMPU of the present invention can be obtained by chemically or enzymatically coupling with those having the same activity). hMP itself has an activity of specifically binding to TGF-β by binding to a sugar chain as necessary.

According to the study of the present inventor, the DNA synthesis supine inhibitory activity and NRK49F of adult rat primary culture hepatocytes were studied.
From the results of the cell colony forming activity bioassay system, hMPU in which the hMPU consisting of the polymer subunits hMPU-1, 2, and 3 of the present invention is bound to a low molecular subunit is shown.
Is not only human-derived TGF-β, but also rat TGF-
It was confirmed that β activity was also completely suppressed. Thus, the hMP of the present invention and the polymer subunits hMPU, hMPU-1, 2, and 3 thereof can suppress TGF-β activity in various animals including humans.

Further, the polymer subunit hMP of the present invention
UP is a precursor of hMPU-1,2,3, and from this, hMPU-1,2,3 can be produced.

Further, hMPU-1N, 2N, 3 of the present invention
For N and PN, hMPU-1, 2, 3, and P of the present invention can be produced by using a known genetic engineering technique.

[0034]

EXAMPLES Examples will be described below to explain the present invention in detail, but the present invention is not limited to these examples.

Example 1 Determination of partial amino acid sequence The latent TGF-β complex was purified from human platelets according to the following method. Human platelets were treated with 50 mM Tris-H
The cells were suspended in Cl buffer, sonicated and then added with a protease inhibitor such as antipain and bestatin and centrifuged. Mix the supernatant with S Sepharose gel,
It was centrifuged. Use the supernatant as starting material,
DEAE-Toyopearl 650s, Phenyl Sepharose CL-4B, Chelating Sepharose,
Purification was performed using each gel column of Cu chelating Sepharose and Sepharose 6 (J Biochem, 1
06 , 304-310, 1989).

The latent TGF-β complex fraction obtained by separating the masking protein and the free mature high molecular weight subunit hMPU was subjected to reverse phase liquid chromatography with a concentration gradient in the elution solvent. It showed a pattern and was separated into 3 peaks. Separate each peak and SD under non-reducing and reducing
The results of S-PAGE analysis are shown in FIG. 2 (a: under non-reduction, b: under reduction). Rat-derived latent TGF-β complex (including rat MP) used as a control (lane 1)
From the comparison with, the peak 1 is free mature polymer subunit hMPU (lane 2), and the peak 2 is free TGF-β.
(Lane 3), peak 3 was estimated to be hMP (lane 4). The peak 1 fraction was reduced, subjected to SDS-PAGE, and then transferred to a polyvinylidene-difluoride membrane to obtain hMPU on the membrane.

Regarding the hMPU on the membrane obtained by the determination of the partial amino acid sequence, the N-terminal amino acid sequence was determined by a conventional method.
IHLHPQFPVVVE, HPQFPVVVEKK,
It was confirmed that there are three kinds of polypeptide having PQFPVVVEK, and hMPU-1, 2, 3 respectively.
I named it. On the other hand, hMPU on the membrane was subjected to cyanogen bromide decomposition, followed by lysine endopeptidase treatment, and the amino acid sequences of the three peptides obtained after the enzyme treatment were determined (single-letteramine).
o-acid code); peptide 1, QTEGX
ERTXTXG; Peptide 2, GFVPAGESSY
E: Peptide 3, EGTYYDPV. These amino acid sequences have homology with rat platelet-derived MP polymer subunit disclosed in Japanese Patent Application No. 2-173679,
It was suggested that hMPU is a protein similar to rat MP high molecular subunit. Also, at SDS-PAG
A band of about 120 kDa corresponding to hMPU was excised from the human MP reduced product obtained from E (lane 4 in FIG. 2 (b)), and the peptide obtained by transferring to a membrane was analyzed. As a result, it was confirmed that this peptide was the same substance as hMPU obtained in.

CDNA screening A cDNA library was constructed from megakaryocyte cells purified from rat bone marrow, and the obtained 4054 bp cDNA fragment of the rat MP polymer subunit was used as a probe for screening the MP polymer subunit cDNA clone. did. Use a polynucleotide probe with T4 polynucleotide kinase (Takara Biochemicals) [γ
^The 5'end was phosphorylated with ³² P] ATP (Amersham) and radioactively labeled. The cDNA was synthesized from human placenta using oligo (dT) as a primer. c
The construction of the DNA library was performed using the λZAP11 vector (Stratagene). The recombinant phage was plated on E. coli and transferred to a nitrocellulose filter (Hybond N, Amersham). These replicas were inspected with an inosine-containing radiolabel probe to obtain 0.2 × SSC (0.9M NaCl, 90 mM).
A hybridization reaction was carried out with trisodium citrate), 0.05% sodium phosphate, 100 μg / ml tRNA, and 6 × Denhardt's solution at 65 ° C. for 15 hours. After the reaction, use a nitrocellulose filter with 0.1% S
Washing with 0.2 × SSC buffer containing DS at 65 ° C. for 20 minutes was repeated 3 times.

Determination of cDNA Nucleotide Sequence The obtained plural clones were cut out with a restriction enzyme and the nucleotide sequence of hMPUcDNA was determined by the dideoxy method using Sequenase (United State Biochemicals). As a result, the obtained cDNA covers 2,535 nucleotides including the coding region corresponding to the mature polymer subunit in rat MP, and the coding region of free mature hMPU-1 is the same as that of the present example. In this case, it was confirmed to be a polypeptide starting from the 268th base.

Example 2. Example of Obtaining hMPU-1N, 2N, 3N Among the clones obtained in Example 1, hMP3-1 having a 3.1 kb insert was used to prepare hMPU-P.
A plasmid containing N, 1N, 2N and 3N was prepared.

Plasmid hMP3-1 is a restriction enzyme Sty
Digested with I and SphI and 104 bp to 2264 bp 2.
After a 1 kb fragment, DN was added to the 5'end and 3'end.
The following DNA fragment prepared by the A synthesizer was used as a linker and ligated with T4 DNA ligase. The 5'-terminal linker has 104 bp of the constituent nucleotides of hMPU-P, a start codon ATG, and a SalI digestion site. The 3'-terminal side linker is 78 bp of the constituent nucleotides of hMPU-P and stop codons TGA and HindI.
II Has a digestion site. Expression vector PKK223-3
Was digested with restriction enzymes SalI and HindIII, and then a DNA fragment to which a linker was bound was inserted by T4 DNA ligase to obtain a plasmid pKK223-3 [hMPU-P] having hMPU-P as an insert.

A plasmid having hMPU-1N as an insert was obtained as follows. Plasmid hMP3
-1 was digested with the restriction enzymes HindIII and SphI.
5'after the formation of a 1.9 kb fragment of 5 bp to 2264 bp
77 bp of the constituent nucleotides of hMPU-1 and a start codon ATG, and a linker having a SalI digestion site on the terminal side, and 78 bp of the constituent nucleotides of hMPU-1 and the stop codons TGA and HindII on the 3'terminal side.
The linker with the I digestion site was ligated with T4 DNA ligase. The expression vector pKK223-3 was digested with restriction enzymes SalI and HindIII, and the linker-ligated DNA fragment was inserted with T4 DNA ligase to obtain pKK223-3 [hMPU-1].

Similarly, for the plasmid having hMPU-2N, 3N as an insert, the plasmid hMPU3-1 was used.
Was digested with HindIII and SalI, and then prepared using a linker containing 65 bp and 62 bp hMPU-2 and 3 constituent nucleotides at the 5'end, and pKK22 and pKK22, respectively.
3-3 [hMPU-2], pKK223-3 [hMPU
-3].

Example 3. Example of Obtaining hMPU-P, 1, 2, 3 E. E. coli (DH5-α strain) was cultured in L-Broth medium, and the cells were collected and suspended in 100 mM CaCl ₂ .
Calcium-activated E. coli was transformed into pKK223-
The cells were transformed with 3 [hMPU-P] and cultured in L-Broth medium. The culture solution was purified, subjected to SDS-PAGE, transferred to a polyvinylidene-difluoride membrane, and hMP
I got U. Similarly, pKK223-3 [hMPU-
1], pKK223-3 [hMPU-2], pKK22
E. coli using 3-3 [hMPU-3]. coli (DH5
-Α strain) and hMPU-
1,2,3 were obtained by purification.

Example 4. Inhibitory effect of human MP on human TGF-β activity Human MP having hMPU of the present invention as a constituent protein is human T
It was confirmed that GF-β activity was suppressed as follows. In Example 1-hM was obtained from human platelets and hM was obtained from the reduced product.
Human MP, which was confirmed to contain PU-1, 2, and 3 as constituent components, was used in the following examples. TG of human MP
For F-β elevation production, rat primary culture liver parenchymal cells were used for measurement. Liver parenchymal cells were separated and purified from Wistar rats by the collagen perfusion method. 5 hepatocytes obtained
The suspension was suspended in Williams E medium (Flow Laboratories) containing 0.5% fetal bovine serum at a concentration of 2.5 × 10 ⁵ cells / ml, and seeded in 24-well multiplates at 0.5 ml / well. After culturing at 37 ° C. for 20 hours under the conditions of 5% CO ₂ , 30% O ₂ and 65% N ₂ , 1 × 10 ⁻⁷ M insulin (Sigma), 10 ng / ml EGF (recombinant human E
(GF, Earth Chemical Co., Ltd.) and serum-free Williams E medium was exchanged, and 45 μl of a solution prepared by adding a predetermined amount of MP to a phosphate buffer containing 2.5 mg / ml bovine serum albumin was added to the same buffer. 1. Each of TGF-β1 and β2.
5 μl of the solution containing 8 ng was mixed, and the solution incubated at room temperature for 1 hour was added. 1.2 after 12 hours of culture
10 μl of 5 μCi / ml [ ³ H] deoxythymidine
/ Well, and to the control group, 5 μg / ml of aphidicolin was added 15 minutes before the addition of [ ³ H] deoxythymidine. After further culturing for 24 hours and labeling with tritium, the cells were washed twice with a phosphate buffer solution of pH 7.4 and fixed with a cold 10% trichloroacetic acid aqueous solution. The cells were solubilized with 0.5 ml of 1N aqueous sodium hydroxide solution per well, and the radioactivity thereof was measured by a gamma counter. Counting the amount of tritium incorporated in hepatocytes when a test sample was added, as a control count The hepatocyte proliferation inhibitory activity of TGF-β was suppressed in a dose-dependent manner, and 40 to 100 ng / ml (20 to 55 parts by weight relative to 1 part by weight of TGF-β) almost completely enhanced the activity of TGF-β. It became clear to control.

[0046]

The polypeptide hMPU-1 of the present invention,
HMPU having a few components can be bound to a low molecular weight subunit to obtain the hMP of the present invention. h
MP binds to TGF-β derived from humans and various animals by binding to the sugar chain itself or, if necessary, to sugar chains, and exerts various physiological activities such as inhibition of tumor cell growth. The polypeptide hMPU-P of the present invention is
PU-1, 2, 3 can be manufactured. The polynucleotide hMPU-1N, 2N, 3N, PN of the present invention comprises
The polypeptide of the present invention can be produced by subjecting it to a genetic engineering technique.

As described above, the polypeptide and polynucleotide of the present invention are indispensable and important substances for producing a masking protein that binds to TGF-β.

[0048]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 756 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence

SEQ ID NO: 2 Sequence length: 752 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence

SEQ ID NO: 3 Sequence length: 751 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence

SEQ ID NO: 4 Sequence length: 845 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence

SEQ ID NO: 5 Sequence Length: 2268 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: cDNA to genomic DNA Characterization Method: E Sequence

SEQ ID NO: 6 Sequence Length: 2256 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: cDNA to genomic DNA Characterization Method: E Sequence

SEQ ID NO: 7 Sequence Length: 2253 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: cDNA to genomic DNA Characterization Method: E Sequence

SEQ ID NO: 8 Sequence Length: 2535 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: cDNA to genomic DNA Characterization Method: E Sequence

SEQ ID NO: 9 Sequence length: 278 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1: Reversed phase liquid chromatography fractionation of latent TGF-β complex fractions. The solid line is the absorbance at 280 nm,
The broken line shows the acetonyl concentration.

FIG. 2 SDS-PA under non-reducing and reducing of each peak
GE analysis.

FIG. 3: Suppression of TGF-β1, β2 activity of hMPU.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1:19)

Claims (10)

[Claims] 1. A polypeptide hMPU-1 having the amino acid sequence of SEQ ID NO: 1. 2. A polypeptide hMPU-2 having the amino acid sequence set forth in SEQ ID NO: 2. 3. A polypeptide hMPU-3 having the amino acid sequence of SEQ ID NO: 3. 4. Polypeptides hMPU-1, hMPU-2, h having the amino acid sequences of SEQ ID NOs: 1 to 3.
Mixed polypeptide hMPU of MPU-3. 5. A human masking protein hMP, which is an S—S bond of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 9 and the polypeptide set forth in claims 1 to 4. 6. A polypeptide hMPU-P having the amino acid sequence of SEQ ID NO: 4. 7. A polynucleotide hMPU-1N having the nucleotide sequence of SEQ ID NO: 5. 8. A polynucleotide hMPU-2N having the nucleotide sequence of SEQ ID NO: 6. 9. A polynucleotide hMPU-3N having the nucleotide sequence of SEQ ID NO: 7. 10. A polynucleotide hMPU-PN having the nucleotide sequence of SEQ ID NO: 8.