JPH07215890A - New polypeptide, its production, dna encoding the same, vector, host cell, antibody against the same, pharmaceutical preparation containing the polypeptide or the antibody, prevention and treatment of diabetes, preventing and treating agent and screening method of the agent - Google Patents

Abstract

PURPOSE:To obtain a protein p140 polypeptide useful for a prevention and treatment of an insulin independent diabetes, having a specific amino acid sequence as a key protein in an insulin signal transmitting system, and capable of reducing a blood sugar level. CONSTITUTION:This protein p140 polypeptide, its homologue, its fragment or a homologue of the fragment is composed of a specific amino acid sequence as a substantially pure form expressed by the formula. Further, the polypeptide is obtained by culturing a host cell transformed by a replication or an expression vector consisting of a DNA encoding this polypeptide, under a condition for expressing the polypeptide, and by isolating the polypeptide.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a p140 polypeptide which is a key protein in the signal transduction system of insulin,
A method for producing the same, a DNA encoding the polypeptide,
Vector comprising the DNA, host cell transformed with the vector, antibody of the polypeptide, pharmaceutical composition containing the peptide or antibody, protein p140
Phosphorylation of tyrosine (hereinafter, may be simply referred to as phosphorylation in the present specification), a preventive and / or therapeutic method for diabetes, and diabetes containing a substance that phosphorylates protein p140 as tyrosine as an active ingredient. The present invention relates to a prophylactic and / or therapeutic agent and a method for screening the prophylactic and / or therapeutic agent.

[0002]

BACKGROUND OF THE INVENTION Diabetes mellitus is a metabolic disorder in which glucose metabolism is impaired and blood glucose levels rise abnormally. Glucose metabolism in the normal state is performed by the following mechanism. That is, the carbohydrate ingested as food is digested to glucose in the small intestine and taken into the blood. The β cells of the pancreas sense the rise in blood sugar and secrete insulin. Insulin acts on target peripheral tissues (muscle and liver) along the bloodstream, promotes absorption of glucose in blood and accumulates as glycogen, thereby lowering blood glucose level.

Diabetes is classified into two types depending on the cause of the disorder. One is a type in which insulin is not secreted or the amount of secretion is very small even if the blood sugar level rises due to food intake, and it is called insulin-dependent diabetes mellitus (IDDM or type I diabetes). Insulin-dependent diabetes has been found to be caused by the destruction of pancreatic beta cells. As a therapeutic method, insulin is supplemented from outside the body. The other is that although insulin is secreted normally,
It is a type in which the response mechanism of peripheral tissues does not operate normally and the blood glucose level does not decrease, and non-insulin-dependent diabetes mellitus (N
It is called IDDM or type II diabetes). In the United States, 5% of the population over the age of 40 is so common that it is said to have non-insulin dependent diabetes,
The cause has not yet been clarified.

[0004]

BACKGROUND OF THE INVENTION The cause of non-insulin-dependent diabetes mellitus is to elucidate the mechanism of glucose uptake by insulin into peripheral tissue cells. However, the insulin signaling mechanism remains largely unknown at present. Secreted insulin binds to the insulin receptor present on the cell membrane of peripheral tissue cells. Information transmission after that is still under study based on the phosphate cascade theory and the second messenger theory. Next, two theories will be briefly explained.

Phosphate Cascade Theory: When insulin binds to the α-subunit of the insulin receptor, the β-subunit present inside the cell membrane is phosphorylated and at the same time the tyrosine kinase site internal to the receptor is activated.
The substrate phosphorylated by this kinase is 3
A variety of proteins have been identified.

[0006] One is insulin receptor substrate-1 (IRS-) consisting of 1235 amino acids and having a molecular weight of 185 kD.
1). When IRS-1 is phosphorylated on tyrosine,
It is known that PI3-kinase, which is an enzyme that phosphorylates phosphatidylinositol, binds to and is activated. However, how information is transduced after that, localization of glucose transporter to the membrane and It is unclear whether ruffling of the membrane occurs. IRS
In addition to -1, two substrate proteins (Shc and PTP-1
The existence of C) has been confirmed, but the mechanism after that has not been clarified at all.

Second messenger theory: When insulin binds to the insulin receptor, a specific phospholipase C is activated, and phosphatidylinositol glycan (PIG), which is a cell membrane phospholipid, is hydrolyzed to cause inositol glycan (IG) and diacyl glycerol (IG). DA
G) is generated. IG has been reported to exhibit various insulin actions, but no report has been made regarding a typical action of glucose uptake. On the other hand, DAG is known to activate C kinase and promote localization of C kinase to the cell membrane. It is also suggested that DAG may phosphorylate intracellular proteins one after another via C kinase to eventually cause sugar uptake, but the details thereof are not known at present.

As described above, two theories have been advocated, but each of them is only partially clarified, particularly the early part of information transmission. In 1988, Cooper (GJS) and others discovered a hormone secreted by pancreatic β-cells like insulin in association with hyperglycemia, and named it amylin. They pointed out that amylin is an inducer of insulin resistance because it inhibits insulin action as a physiological action of amylin. 1991,
There has been a definitive report that overexpression of amylin in transgenic mice causes non-insulin dependent diabetes. However, it is currently unknown how amylin is involved in the insulin signal transduction system.

[0009]

[Means for Solving the Problems] The present inventors have focused their attention on the insulin resistance of amylin and have conducted intensive studies on the effect of amylin on the insulin signal transduction system. We identified the inhibitory site for the first time and discovered the key proteins involved in it (pp140 and pp70). The present invention has been completed by clarifying the structures (DNA base sequence and amino acid sequence) and functions of these proteins and clarifying the whole insulin signal transduction system based on these findings.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides a protein p140 consisting of the amino acid sequence represented by SEQ ID NO: 1 in a substantially pure form.
It relates to polypeptides, homologues thereof, fragments of the sequences and homologues thereof. The invention further relates to DNA encoding those polypeptides. More specifically, it relates to a DNA having the base sequence represented by SEQ ID NO: 2 or 3, and a DNA having a fragment selectively hybridizing to the base sequence represented by SEQ ID NO: 2 or 3. Further, the present invention provides a method for preventing and / or treating diabetes, which comprises phosphorylating p140 on tyrosine, and a prophylactic and / or therapeutic agent for diabetes containing a substance that phosphorylates protein p140 on tyrosine as an active ingredient, and The present invention relates to a method for screening prophylactic and / or therapeutic agents.

In particular, the present invention relates to (1) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, (2) a DNA encoding the polypeptide described in (1) above,
(3) DNA having the base sequence shown in SEQ ID NO: 2,
(4) DNA having the nucleotide sequence represented by SEQ ID NO: 3,
(5) A method for preventing and / or treating diabetes which is characterized by phosphorylating protein p140 with tyrosine, (6) An agent for preventing and / or treating diabetes which comprises as an active ingredient a substance which phosphorylates protein p140 with tyrosine. ) A method for screening a preventive and / or therapeutic agent for diabetes, which comprises using the protein p140.

Administration of amylin to healthy rats (0.1 mg / kg,
ip, tid 7 days), the expression level of insulin receptor and the amount of secreted insulin are significantly decreased, and the expression level of glucose transporter 4 (hereinafter abbreviated as Glut4) and the synthesis amount of glycogen are controlled. Less than half of the blood glucose level
It was observed to increase 1.7 times. Furthermore, L6 which is a rat muscle cell (rat skeletal muscle myoblasts)
In an experiment using cells (ATCC deposit number, CRL1458), amylin administration was also found to reduce the glucose uptake amount of cells.

Next, in order to confirm what kind of changes occur in the tyrosine phosphate cascade system by insulin in muscle cells by amylin treatment,
It was examined by Western blotting using an anti-phosphotyrosine antibody. That is, in the experiment using L6 cells, it was confirmed that tyrosine phosphorylation was promoted when incubated with insulin, but there were two types of proteins whose phosphorylation was suppressed when incubated with insulin after pre-addition of amylin. . . Hereinafter, these proteins will be referred to as pp140 and pp70, respectively, based on their molecular weights, and the precursor proteins before phosphorylation will be referred to as p140 and p70, respectively.

We have found that pp140 and pp70.
Was purified and the partial amino acid sequence was determined. When the sequence was examined for similarity to the amino acid sequence of a known polypeptide registered in Swiss Prot Release 2.0, pp70 was found to be a known glucose-regulated protein (Gluc).
ose-regulated protein 70), but pp1
It was estimated that 40 is a completely unknown novel protein.
Therefore, the present inventors have examined p140 from rat myoblasts and the like.
Isolate protein mRNA and use this as starting material
NA was obtained, its entire base sequence was determined, and its entire amino acid sequence was determined. As a result, they succeeded in finding a completely novel polypeptide and full-length DNA encoding it,
The present invention has been completed.

From the above experimental results, amylin was p140
And p70 are phosphorylated to inhibit pp140 and pp70, respectively. Conversely, considering that amylin acts suppressively on the process from insulin receptor binding to glucose uptake, p140 and p70 are pp14 and pp14, respectively.
It is suggested that phosphorylation to 0 and pp70 may play an important role in the mechanism of cellular glucose uptake. The present inventors then p1
It was decided to elucidate the action mechanism of 40 and p70.

When myoblasts (rat L6 cells) are cultured in an insulin-containing medium, a band of pp140 appears from the 3rd day of culture, and the production of pp140 continues until the 9th day. At the same time, it was found that the expression of Glut4 appeared around the 3rd day, and the expression level gradually increased with the differentiation of myoblasts. In the same system, multinucleation of myoblasts was observed from the 7th day, showing that myoblasts differentiate into muscle cells. It was found that pp70 appeared from the 7th day and continued to be produced until the 14th day.

On the other hand, localization of pp140 in cells was examined. When insulin was added to serum-free L6 cells, pp140 appeared in the cytoplasmic microsomal membrane (MM) within 10 minutes after culturing, but then disappeared, and in the cell membrane (PM), 1 to pp140 after culturing. It was found that pp140 appeared only after 2 hours. This means that pp140 produced in the cytoplasm immediately after the addition of insulin.
Is supposed to migrate to the cell membrane after 1-2 hours. Furthermore, localization of pp70 was also examined in a system using L6 cells. In MM, pp70 appeared immediately after culturing, the phosphorylation amount reached a peak after 10 minutes, and then gradually disappeared, and in the nucleus, pp7 immediately after culturing.
0 showed expression, but then gradually increased and reached a peak 3 hours later. From this, pp7
0 is present in MM in the absence of insulin,
It was found that the addition of insulin transferred to the nuclear fraction within 3 hours.

From the results of the above experiments, the information transmission mechanism of pp140 is assumed as follows. That is, when insulin binds to the receptor, the receptor is autophosphorylated and activated. The information is that after several steps of activation of protein kinase by phosphorylation, p140 is phosphorylated to pp140, and the activated pp140 localizes to the cell membrane surface and at the same time And then phosphorylates p70. Phosphorylated pp70 is activated and translocates into the nucleus, activating the Glut4 expression gene in the nucleus. It is considered that Glut4 synthesized in the cytoplasm based on this information is localized on the cell membrane surface and glucose is taken up. The correctness of the above information transmission mechanism will be experimentally proven in the future. In any case, it is concluded that activation of p140 is an essential step for glucose uptake and thus for lowering blood glucose levels.

Therefore, the present invention relates to a method for preventing and / or treating diabetes, especially non-insulin-dependent diabetes, which is characterized by phosphorylating protein p140 with tyrosine. Here, the method for preventing and / or treating diabetes which is characterized by phosphorylating protein p140 with tyrosine includes all methods for preventing and / or treating diabetes whose main mechanism of action is to phosphorylate protein p140 with tyrosine. It is a waste. Furthermore, the present invention relates to a preventive and / or therapeutic agent for diabetes, especially non-insulin-dependent diabetes mellitus, which comprises as an active ingredient a substance that phosphorylates protein p140 on tyrosine phosphorylation. Here, the substance that phosphorylates protein p140 on tyrosine is not only a substance that is currently confirmed to have that activity, but also any substance that will be newly confirmed to have that activity in the future. Is included.

The cells that tyrosine phosphorylate the protein p140 of the present invention include not only myoblasts (rat L6 cells) but all cells that are confirmed to have the above-mentioned action. For example, it was confirmed that rat FaO hepatocytes, human A673 myocytes and human HepG2 hepatocytes have this effect. Furthermore, since expression of the p140 mRNA of the present invention has been confirmed in the heart, brain, spleen, lung, kidney, testis, placenta and pancreas other than muscle and liver, the action of phosphorylating p140 on tyrosine is
It is expected to spread widely not only to muscle and liver. Therefore, the action of the present invention includes not only muscle and liver cells but also heart, brain, spleen, lung, kidney, testis, placenta and pancreatic cells.

Swiss Plot (Swiss Prot Release 2.
The amino acid sequence of the known polypeptide registered in 0) was examined, but none of them had the same sequence as that of the full-length polypeptide of the present invention. Furthermore, the nucleotide sequence registered in GeneBank (GenBank Release 70) was also examined, but none of them had the same sequence as the cDNA encoding the full-length polypeptide of the present invention. Therefore, it was confirmed that the polypeptide of the present invention is completely novel. In addition, as a result of investigating the amino acid sequences of known polypeptides registered in Swiss Prot Release 2.0, about 40% homology with epithelial cell kines (hereinafter abbreviated as Eck) was confirmed. It was From this, it was inferred that the novel protein of the present invention belongs to the Eck family.

In the present invention, the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 in substantially pure form generally means 90% or more of the polypeptide at the time of production, for example, 95, 98 or 99. % Means a polypeptide having the amino acid sequence shown by SEQ ID NO: 1.

The homologue of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 generally means at least 2
0, preferably at least 30, for example 40, 60
Or a homology of at least 70%, preferably at least 80 or 90%, more preferably 95% or more in a region of 100 contiguous amino acids, and such homologues are hereinafter described as the polypeptide of the present invention. To be done. Furthermore, a fragment of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, or a homologue thereof, means at least 10 amino acids, preferably at least 15 amino acids, for example 20,
Means 25, 30, 40, 50 or 60 amino acid moieties.

The DNA which selectively hybridizes to the DNA having the base sequence shown in SEQ ID NO: 2 or 3 generally means at least 20, preferably at least 30, for example 40, 60 or 100 consecutive bases. At least 70%, preferably at least 80 or 90%, and more preferably 95% or more homology in the sequence region, and such DNA is hereinafter described as the DNA of the present invention.

The DNA fragment having the base sequence shown in SEQ ID NO: 2 or 3 means at least 10 bases, preferably at least 15 bases, for example 20, 2 bases.
It refers to a 5, 30 or 40 base portion and such fragments are also included in the DNA of the present invention. Further, the present invention includes a replication or expression vector comprising the DNA of the present invention. The vector includes, for example, a plasmid comprising an ori region and, if necessary, a promoter for expressing the above DNA, a promoter regulatory factor, and the like,
Examples include virus or phage vectors. The vector may include one or more selectable marker genes, such as the ampicillin resistance gene. Vectors are in vitro, eg D
It can be used for production of RNA corresponding to NA and transformation of host cells.

Further, in the present invention, SEQ ID NO: 2 or 3
Also included is a host cell transformed with a vector for replicating or expressing the DNA of the present invention containing the DNA having the nucleotide sequence represented by or the open reading frame thereof. Examples of cells include bacteria, yeast,
Insect cells or mammalian cells are mentioned. Furthermore, the present invention also includes a method for producing the polypeptide of the present invention, which comprises culturing the host cell of the present invention under conditions for expressing the polypeptide of the present invention. Culturing is preferably carried out under conditions in which the polypeptide of the present invention is expressed and produced by a host cell.

The DNA of the present invention is inserted into the antisense region of the vector as described above to obtain an antisense RN.
A can also be produced. Such antisense R
NA can also be used to control the levels of the polypeptides of the invention in cells. The present invention also includes monoclonal or polyclonal antibodies of the polypeptides of the present invention. Furthermore, it also includes a method for producing a monoclonal or polyclonal antibody of the polypeptide of the present invention. The monoclonal antibody is the peptide of the present invention or
Using the fragment as an antigen, it can be produced by a usual hybridoma technique. The polyclonal antibody can be produced by a usual method in which a host animal (for example, rat or rabbit) is inoculated with the polypeptide of the present invention and immune serum is collected.

The present invention also includes a pharmaceutical composition containing the polypeptide of the present invention, its antibody and a pharmaceutically acceptable excipient and / or carrier. As the polypeptide of the present invention, in addition to the polypeptide having the amino acid sequence shown by SEQ ID NO: 1, a part thereof is deleted (for example, a polypeptide consisting of only a part essential for the expression of biological activity in SEQ ID NO: 1). Peptide), a part of which is replaced with another amino acid (for example, a part of which is substituted with an amino acid having similar physical properties),
And those in which other amino acids are added or inserted in part thereof. As is well known, 1 to 6 types of codons encoding one amino acid (for example, Me
1 is known as t and 6 types as Leu). Therefore,
The base sequence of DNA can be changed without changing the amino acid sequence of the polypeptide.

The DNA of the present invention specified in (2) includes
It includes all the base sequence groups encoding the polypeptide represented by SEQ ID NO: 1 in (1). The productivity of the polypeptide may be improved by changing the base sequence. The DNA specified in (3) is the D shown in (2).
It is one aspect of NA and represents the native sequence. The DNA shown in (4) shows the sequence obtained by adding the natural untranslated portion to the DNA specified in (3). The DNA having the nucleotide sequence represented by SEQ ID NO: 3 is prepared according to the following method.

That is, (i) cells that produce the polypeptide of the present invention, such as rat myoblast L6 cells, are expressed as mR
NA was separated, and (ii) the first strand (single-stranded DNA) and then the second strand (2) were separated from the mRNA.
Double-stranded DNA) (synthesis of cDNA), and (iii) the c
Incorporating the DNA into an appropriate plasmid vector, (iv)
A host cell is transformed with the obtained recombinant vector (c
(Preparation of DNA library), (v) A large amount of cloning and sequencing of an average of 300 bases from the 5 ′ side were performed from the obtained cDNA library, (vi)
A novel clone having a base sequence can be prepared by sequencing the entire length.

More specifically, in the step (i), rat myoblast L6 cells are brought to a logarithmic growth phase and then subjected to a method such as Okayama (H.) [Methods in Enzymolog].
y, 154 , 3 (1987)]. Cells that produce this polypeptide include rat and human muscle, liver, heart, brain, spleen, lung, kidney, testis, placenta and pancreatic cells, preferably rat L6 myoblasts (ATCC deposit no. CRL-1458), rat F
aO hepatocytes, human A673 myocytes and human HepG2
Examples include hepatocytes. The steps (ii), (iii) and (iv) are steps for preparing a cDNA library, and the modified Gub
It is performed according to the Ler & Hoffman method [described in Gene, 25 , 263 (1983)].

The plasmid vector used in the step (iii) is one that functions in E. coli (for example, pBR).
322) or those that function in Bacillus subtilis (eg, pUB1
Although 10) is known in large numbers, pGEM-3Zf (+) [3199 bp, sold by Promega] which functions in E. coli is preferably used. Many host cells are already known in the step (iv), and any of them may be used, but preferably DH5 competent cells [Gen
e, 96 , 23 (1990). ]. The cloning in step (v) is performed by a known method. Sequencing is performed by the Maxam-Gilbert method or dideoxy terminator method. Step (vi) is based on Molecular Cloning
[Sambrook, J., Fritsch, EF and Maniatis, T.
Published by Cold Spring Harbor Laboratory Press in 1989].

The DNA thus obtained is (I)
It is necessary to convert the DNA sequence into an amino acid sequence in a possible frame and (II) confirm that the DNA is full length or almost full length. These confirmations may be performed after the step (vi), but it is more efficient to perform them between the step (v) and the step (vi). Considering the above,
Confirmation of (II) is performed by Northern analysis.

Once the base sequences shown in SEQ ID NOS: 2 and 3 have been determined, thereafter, by chemical synthesis or by PCR (Polymerase Chain Reaction) method.
Alternatively, the DNA of the present invention can be obtained by hybridizing with a fragment of the base sequence as a probe. Furthermore, the desired amount of the desired DNA of the present invention can be obtained by introducing the vector DNA containing the present DNA into a suitable host and allowing it to grow.

As the method for obtaining the polypeptide of the present invention, (1) a method of purifying and isolating from a living body or a cultured cell, (2) a method of synthesizing a peptide, or (3) a method of producing using a gene recombination technique , And the like, but the method described in (3) is industrially preferable. Examples of the expression system (host-vector system) for producing a polypeptide using a gene recombination technique include bacteria,
Expression systems for yeast, insect cells and mammalian cells are included.

For example, in the case of expression in Escherichia coli, 5'of DNA encoding the nucleotide sequence shown in SEQ ID NO: 3.
DNA obtained by adding a start codon (ATG) to the end
Is connected to the downstream of an appropriate promoter (eg, trp promoter, lac promoter, λpL promoter, T7 promoter, etc.), and a vector (eg, pBR322, pUC18, pUC1) that functions in E. coli is obtained.
9) to prepare an expression vector. Next, E. coli transformed with this expression vector (for example, E. coli
DH5, E. Coli JM109, E. Coli HB101 strain, etc.) can be cultured in an appropriate medium to obtain the desired polypeptide from the cells. In addition, by using a bacterial signal peptide (for example, pelB signal peptide), the target polypeptide can be secreted into the periplasm. In addition, fusion proteins with other polypeptides
Can also be produced.

When expressed in mammalian cells, for example, a DNA encoding the nucleotide sequence shown in Sequence Listing 3 is used as a suitable vector (eg, retrovirus vector, papillomavirus vector, vaccinia virus vector, SV40 system). An expression vector is prepared by inserting it into the downstream of a suitable promoter (eg, SV40 promoter, LTR promoter, metallothionein promoter, etc.) in the vector).

Next, an appropriate mammalian cell (eg, monkey COS-7 cell, Chinese hamster CHO cell, mouse L cell) is transformed with the obtained expression vector, and the transformant is cultured in an appropriate medium. By,
The desired polypeptide is produced in the cell. The polypeptide obtained as described above can be isolated and purified by a general biochemical method. The protein of the present invention includes the polypeptide obtained by the present invention to which a sugar chain and a phosphate are bound. That is, in the present invention, a p140 protein in which a sugar chain is bound to a p140 polypeptide and a tyrosine phosphorylated p140 protein (pp
140) are included.

[0039]

The protein p140 of the present invention is presumed to have the above-described mechanism of action. Therefore,
By tyrosine phosphorylating the polypeptide of the protein p140 of the present invention, it is possible to improve the state of hyperglycemia and prevent and / or treat diabetes, especially non-insulin-dependent diabetes. In addition, using a polyclonal antibody or a monoclonal antibody of p140 polypeptide,
Quantification of the polypeptide in the living body can be performed. As a result, it can be used for research on the relationship between the polypeptide and a disease or diagnosis of a disease. Polyclonal antibodies and monoclonal antibodies can be prepared by a conventional method using the pp140 polypeptide or a fragment thereof as an antigen.

The DNA of the present invention not only serves as an important and essential template for producing the polypeptide of the present invention, which is expected to have great utility, but also can be used for diagnosis and treatment of genetic diseases (treatment of gene deficiency). Alternatively, it can be used for the treatment by stopping the expression of the polypeptide by antisense DNA (RNA). Further, genomic DNA can be separated using the DNA of the present invention as a probe. Similarly, it is also possible to isolate a gene of a human related polypeptide having high homology with the DNA of the present invention or a gene of a polypeptide highly homologous to the polypeptide of the present invention in an organism other than human.

The present invention also relates to a preventive and / or therapeutic agent for diabetes which contains, as an active ingredient, a substance that phosphorylates protein p140 on tyrosine phosphorylation. Where protein p1
The substance that phosphorylates 40 on tyrosine includes not only substances that are currently confirmed to have the activity but also all substances that will be newly confirmed to have the activity in the future. Examples of substances that are currently confirmed to have tyrosine phosphorylation activity include, for example, (1) general formula (I):

[0042]

[Chemical 1]

(In the formula, each of n R ^{1 s} is independently a hydrogen atom, a C1-4 alkyl group, a hydroxy group, an amino group, or a formula COOR ² (wherein R ² is a hydrogen atom or C1-4). 4 alkyl group), and n represents an integer of 1 to 3), a benzene or naphthalene derivative represented by the formula (4), and a non-toxic salt thereof.
And their non-toxic acid addition salts, (2) the general formula (I
I):

[0044]

[Chemical 2]

(In the formula, each of m R ^3's is independently a hydrogen atom, a C1-12 alkyl group, a C1-4 alkoxy group, a C1-4 alkylthio group, a hydroxy group, a halogen, a phenyl group, or a halogen. Represents a phenyl group substituted with, and m represents an integer of 1 to 4), a benzoquinone or naphthoquinone derivative represented by the formula (3):

[0046]

[Chemical 3]

(In the formula, X represents an oxygen atom or a sulfur atom, and R ⁴ and R ⁵ are each independently a hydrogen atom, a phenyl group, or a C1-4 alkyl group, C1-8.
Represents a phenyl group substituted with an alkoxy group, a halogen atom or a nitro group, or R ⁴ and R ⁵ together form a benzylidene group, or a C1-4 alkyl group, a C1-8 alkoxy group, a halogen atom or a nitro group. Represents a benzylidene group substituted with a group or a β-methylcinnamylidene group, and R ⁶ represents a hydrogen atom or a C1-4 alkyl group. And a non-toxic salt thereof and a non-toxic acid addition salt thereof.

More specifically, examples of the compound represented by the general formula (I) include 4-amino-2-hydroxybenzoic acid, 4-amino-1-naphthol, 4-amino-2-naphthol and 1-amino. Naphthalene, 1,4-dihydroxynaphthalene, 4-amino-2-methyl-1-naphthol (hereinafter referred to as vitamin K ₅ ), 1,4-dihydroxy-2-naphthoic acid and the like,

As the compound represented by the general formula (II),
2-methyl-1,4-benzoquinone, 2,6-di-tert
-Butyl-1,4-benzoquinone, 2,6-dibromo-
1,4-benzoquinone, 2,3,5,6-tetrafluoro-1,4-benzoquinone, 1,4-naphthoquinone, 2
- methyl-1,4-naphthoquinone (hereinafter, vitamin K ₃
, 2-hydroxy-3-methyl-1,4-naphthoquinone, 2- (3,7-dimethyloctyl) -3-hydroxy-1,4-naphthoquinone, 2-methoxy-3-.
Methyl-1,4-naphthoquinone, 2-hydroxy-1,
4-naphthoquinone, 3- (4-chlorophenyl) -2-
Hydroxy-1,4-naphthoquinone, 2-propylthio-1,4-naphthoquinone and the like,

Examples of the compound represented by the general formula (III) include 5-phenylrhodanine, 5-phenyl-1,3-thiazolidine-2,4-dione, 5-benzylidene-rodanine and 5-benzylidene-1,3. -Thiazolidine-2,4
-Dione, 5,5-diphenylrhodanine, 5,5-diphenyl-1,3-thiazolidine-2,4-dione, 5-
(4-Isoamyloxybenzylidene) Rhodanine, 5-
Compounds such as (4-isoamyloxybenzylidene) -1,3-thiazolidine-2,4-dione and 5- (β-methylcinnamylidene) rhodanin-3-acetic acid, and their non-toxic salts, and their non-toxic salts Examples include toxic acid addition salts.

Here, suitable non-toxic salts include salts of alkali metals (potassium, sodium, etc.), salts of alkaline earth metals (calcium, magnesium, etc.), ammonium salts, pharmaceutically acceptable organic amines ( Tetramethylammonium, triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine,
Examples thereof include salts of diethanolamine, tris (hydroxymethyl) aminomethane, lysine, arginine, N-methyl-D-glucamine and the like.

Suitable non-toxic acid addition salts include
Inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, or acetate, lactate, tartrate, oxalate, fumarate, maleate, citrate, benzoate, Organic acid salts such as methane sulfonate, ethane sulfonate, benzene sulfonate, toluene sulfonate, isethionate, glucuronate and gluconate are mentioned. The compounds represented by the general formulas (I), (II) and (III) are known per se, or can be easily produced from known starting materials by a known production method.

[0053]

[Application to Pharmaceuticals] Since the substance used in the present invention phosphorylates protein p140 by tyrosine, it is useful for improving hyperglycemia and preventing and / or treating diabetes, especially non-insulin-dependent diabetes. It has been confirmed that the toxicity of each active ingredient and non-toxic salts thereof used in the present invention is extremely low. Therefore, it can be judged that all of them are sufficiently safe and suitable for use as a medicine. In order to use the respective active ingredients used in the present invention and their non-toxic acid addition salts for the above purpose, they are usually administered systemically or locally, orally or parenterally.

The dose depends on age, body weight, symptoms, therapeutic effect,
Although it varies depending on the administration method, treatment time, etc., it is usually within the range of 10 μg to 1000 mg per adult per dose.
Orally administered once to several times a day, or in the range of 10 μg to 100 mg per adult,
It is administered once to several times a day by parenteral administration (preferably intravenous administration), or is continuously administered intravenously in the range of 1 hour to 24 hours a day. Of course, as described above, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient in some cases, or a dose exceeding the range may be necessary in some cases. When administering the compound of the present invention,
It is used as a solid composition for oral administration, a liquid composition and other compositions, an injection for parenteral administration, an external preparation, a suppository and the like. Solid compositions for oral administration include
It includes tablets, pills, capsules, powders, granules and the like.
Capsules include hard capsules and soft capsules.

In such solid compositions, the one or more active substances comprise at least one inert diluent such as lactose, mannitol, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, It is mixed with polyvinylpyrrolidone and magnesium aluminometasilicate. According to a conventional method, the composition contains an additive other than an inert diluent, for example, a lubricant such as magnesium stearate, a disintegrating agent such as calcium fibrin glycolate, and a solubilizing agent such as glutamic acid or aspartic acid. It may be contained. If necessary, tablets or pills should be sucrose, gelatin,
It may be coated with a film of a gastric or enteric substance such as hydroxypropylcellulose or hydroxypropylmethylcellulose phthalate, or may be coated with two or more layers. Also included are capsules of absorbable material such as gelatin.

Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, syrups, elixirs and the like. In such a liquid composition, one or more active substances are contained in a generally used inert diluent (eg, purified water, ethanol).
The composition may contain, in addition to an inert diluent, auxiliary agents such as a wetting agent and a suspending agent, a sweetening agent, a flavoring agent, an aromatic agent, and a preservative.

Other compositions for oral administration include spray formulations which contain one or more active substances and are formulated in a manner known per se. This composition contains, in addition to an inert diluent, a stabilizer such as sodium bisulfite and a stabilizer that imparts isotonicity, an isotonic agent such as sodium chloride, sodium citrate or citric acid. May be. The manufacturing method of the spray agent is, for example, U.S. Patent Nos. 2,868,691 and 3,095,3.
No. 55 is described in detail.

The injections for parenteral administration according to the present invention include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of the aqueous solution and suspension include distilled water for injection and physiological saline. Examples of the water-insoluble solution and suspension include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, Polysorbate 80 (registered trademark) and the like. Such a composition may further contain adjuvants such as preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizing agents (eg glutamic acid, aspartic acid).

These are sterilized by filtration through a bacteria-retaining filter, blending of a bactericide or irradiation.
They can also be used by preparing a sterile solid composition and dissolving it in sterilized or sterile distilled water for injection or other solvent before use. Other compositions for parenteral administration include external solutions, ointments, coatings, suppositories for rectal administration and vaginal administration which contain one or more active substances and are prescribed by a conventional method. Includes a pessary, etc.

[0060]

The present invention will be described in detail below with reference to examples and experimental examples, but these do not limit the present invention.

Example 1: Purification method of pp140 Rat L6 cells were used in a cell tray (225 cm ² ).
The cells were cultured at 37 ° C for 7 to 10 days in a 5% CO ₂ incubator.
Dulbecco's modified Eagle medium (containing 10% fetal calf serum) was used as the medium, and the medium was changed every 3 days. The cells differentiated from myoblasts to myocytes were treated with serum-free Dulbecco's modified Eagle medium for 2 hours, and then 500 μM vanadic acid was added and reacted for 10 minutes. The cells were suspended in 25 mM Tris buffer (400 μM vanadic acid, containing a protease inhibitor), solubilized, and the supernatant was collected by centrifugation.

A final concentration of 0.1% octa (ethylene glycol) ether (C ₁₂ E ₈ ) was added, and the mixture was subjected to Millipore membrane filtration. Protein G bound with anti-phosphotyrosine antibody
A sepharose gel was prepared and filled with the sample solution treated as above. Tyrosine phosphorylated protein (pp140) was adsorbed on the gel. After washing the column with 25 mM Tris buffer,
Pp140 was eluted with a 10 mM phenyl phosphate solution. The eluate was concentrated with Centricon 30, and pp140 was precipitated by the acetone precipitation method.

Example 2: Phosphorylation of p140 tyrosine in each tissue Each cell (1 × 10 ⁵ cells / dish) was treated with Dulbecco's modified Eagle medium (containing 10% fetal bovine serum) at 5%.
Incubated in a CO ₂ incubator at 37 ° C for 5-8 days. The cells differentiated from myoblasts to myocytes. The differentiated cells were treated with serum-free Dulbecco's modified Eagle medium for 4 hours, and then cultured for 1 day with or without addition of amylin (100 pM). Further, insulin (100 nM) was added, and the cells were cultured for a certain period of time (10 minutes or 60 minutes). Then, the cells were washed with an ice-cooled phosphate buffer solution, and then the cells were solubilized with a phosphate buffer solution containing 0.5% octa (ethylene glycol) ether (C ₁₂ E ₈ ). pp140 was recovered by Sepharose beads to which a phosphotyrosine antibody (manufactured by Transformation) was bound, eluted with phenyl phosphate, and detected by Western blotting. With purified pp140 as the standard, p
The amount of p140 was measured with a densitometer. The results are shown in Table 1.

[0064]

[Table 1]

In Table 1, the amount of insulin added is 100 n.
The amount of M and amylin added was 100 pM, and the addition of amylin was performed 24 hours before the addition of insulin.

[Discussion] In rat L6 cells, insulin causes pp140 to appear within 10 minutes, and amylin treatment antagonizes this. This phenomenon was also confirmed in FaO cells, which are rat liver cells. Furthermore, this phenomenon is not limited to the rat but to human muscle cells A
It was also confirmed in 673 cells and HepG2 cells which are liver cells, and it was predicted that amylin suppresses the phosphorylation signal of insulin before the step of phosphorylating p140.

Experimental Example 3: p140 Tyrosine Phosphorylation of Each Test Drug Rat L6 cells (1 × 10 ⁵ cells / dish) were cultured in Dulbecco's modified Eagle medium (containing 10% fetal calf serum) at 5% CO _2. The cells were cultured at 37 ° C for 8 days in an incubator. The cells differentiated from myoblasts to myocytes. Differentiated cells
After treatment with serum-free Dulbecco's modified Eagle medium for 4 hours, various test drugs (10 mM, only insulin 1 mM) shown in Table 1 were added, and the cells were further cultured for a certain period of time. Then, after washing with ice-cooled phosphate buffer, 0.5% octa (ethylene glycol) ether (C ₁₂
The cells were solubilized with a phosphate buffer containing E ₈ ). pp
140 was recovered by Sepharose beads to which a phosphotyrosine antibody (manufactured by Transformation) was bound, and then eluted with phenyl phosphate and detected by Western blotting. Using purified pp140 as a standard
The amount of pp140 of the band was measured with a densitometer.
The results are shown in Table 2.

[0068]

[Table 2]

Experimental Example 4 Glucose Uptake Promoting Activity Rat L6 cells (1 × 10 ⁵ cells / dish) were cultured in a 5% CO ₂ incubator using Dulbecco's modified Eagle medium (containing 10% fetal calf serum). Culture was performed at 8 ° C for 8 days. The cells differentiated from myoblasts to myocytes. Differentiated cells
After treatment with serum-free Dulbecco's modified Eagle medium for 2 hours, various test drugs (10 mM, only insulin 1 mM) shown in Table 2 were added, and the cells were further cultured for 2 hours. Then Krebs Ringer phosphate buffer (pH
After culturing at 7.4) for 20 minutes, ³ H-2-deoxyglucose (0.05 mCi / ml, 5 mM) was added. After addition
The amount of label incorporated into the cells in the initial 3 minutes was measured with a liquid scintillation counter. The results are shown in Table 3.

[0070]

[Table 3]

[Discussion] From Tables 2 and 3, it was confirmed that all the compounds that promote tyrosine phosphorylation of p140 have glucose uptake activity.

Experimental Example 5 Effect of Vitamin K ₅ on Diabetes Male Wistar rats were treated with streptozotocin (STZ) to prepare diabetic models (STZ rats). ST
Various concentrations (30 mg / Kg and 3 mg / K /
Vitamin K _{5 (} Kg) was intraperitoneally administered 3 times a day for 3 days, and then blood glucose, neutral lipid and cholesterol concentrations were measured. In addition, physiological saline was administered to STZ rats and normal rats 3 times a day for 3 days, and various blood parameters were measured as in the drug administration group. As a positive control, insulin was 8 U / Kg1
What was subcutaneously administered once a day for 3 days was used. The results are shown in FIGS. 1 to 3.

[Discussion] In any of glucose, neutral lipid and cholesterol, vitamin K
_{In case of 5} , ₅ returned to the normal state on the 3rd day after administration.

Example 6 Partial Amino Acid Sequence Analysis of pp140 The pp140 purified in Example 1 was separated by electrophoresis, transferred to a PVDF membrane, treated with trypsin, and separated by liquid chromatography. The pp140 fragment thus obtained was used as an ABI model 470A Automated Gas Phase Proteen Sequencer / 120A PTH analyzer (ABI model 470A au from ABI).
tomated gas-phase protein sequencer / 120A PTH ana
lyzer) and the partial amino acid sequence was determined by sequencing by a known method (Edman degradation method). The sequences are shown in Sequence Listing 5 to Sequence Listing 7.

Example 7: PCR Method Using Partial Amino Acid Sequence of pp140 Various primers were prepared from the obtained partial amino acid fragments, each was combined, and PCR was carried out by a known method.
Was carried out. As a result, the combination of the sense primer of Sequence Listing 5 and the antisense primer of Sequence Listing 6 was about 4
A specifically amplified fragment with a length of 00 bp was observed.

Example 8: Separation and Purification of mRNA 3 × 10 ⁷ rat myoblast L6 cells (ATCC deposit number, CRL-1458) in the logarithmic growth phase were treated with Okayama (O
kayama, H.) et al. [Methods in Enzymology, 154 ,
3 (1987)]. That is, after solubilizing cells with a 5.5 M GTC solution (5.5 M guanidine thiocyanate, 25 mM sodium citrate, 0.5% sodium lauryl sarcosine), cell lysate (cell lysate) was mixed with cesium trifluoroacetate (density 1.51). CsTF
A) Place on a cushion of solution and perform ultracentrifugation (120,000 ×
g, 20 hours) to recover the total RNA in the precipitate. This is passed through an oligo dT cellulose column twice and 106
μg of poly (A) ⁺ RNA was purified and collected.

Example 9: Distribution of each tissue of p140 mRNA From each tissue, a poly was obtained by the same method as in Example 8.
(A) ⁺ RNA was purified. Poly derived from each tissue
2 μg of (A) ⁺ RNA was electrophoresed on an agarose gel and transferred to a filter. The open reading frame of p140 was labeled with an isotope (32P) by the random primer labeling method, and the β-actin open reading frame of 2 kb was also labeled as an internal control. Hybridization was carried out by a conventional method, a specific binding labeled probe was taken on an autoradiogram, and densitometric analysis was performed using an imaging analyzer. Β-actin m of each tissue
Table 4 shows the p140 mRNA expression level when the RNA expression level was 100.

[0078]

[Table 4]

[Discussion] The expression of mRNA is observed in the tissues examined, and it is considered that it may act not only in liver and muscle tissues but also in a wide range of tissues. In particular, it is highly expressed in the human pancreas.

Example 10: Preparation of cDNA library The cDNA library was prepared by the Gubler & Hoffman method [Gene,
25 , 263 (1983)]. Example 2
The first strand was synthesized from the poly (A) ⁺ RNA (5 μg) prepared in step 1 above with a reverse transcriptase using a random hexanucleotide primer. Subsequently, second strands were synthesized, ligated with EcoRI adapter, and then gel filtration column chromatography [Sephacryl S-500HR (sold by Pharmacia) column was used. ], 1620 ng of cDNA fraction was recovered by removing excess adapter and primer.

The above-mentioned cDNA synthesis step is carried out according to λgt1.
0 cloning system (sold by Amersham) was used. Then, λgt10phage (Amers
ligated to EcoRI-treated arm of λZAPII phage (sold by Stra Tagene), average length 1.8 kb, number of independent clones 3
A x10 ⁵ phage cDNA library was obtained.

Example 11: Cloning and Sequencing From the phage cDNA library prepared in Example 10, 15 cm × 15 cm plates were seeded with clones at a density of about 100,000 plaques / plate and obtained in Example 7 above. Screening was performed using the thus obtained about 400 bp fragment as a probe. Among the positive clones, the one with long insert was used as a plasmid vector pGEM-3Zf.
(+) Subcloning into the EcoRI site of [3199 bp, sold by Promega Corp.], T7
Alternatively, SP6 was used as a primer for sequencing.

Sequencing of DNA was carried out by the cycle sequencing method using a fluorescent dye-terminator of ABI (Applied Biosystems Inc.) based on the dideoxy terminator method of Sanger (Sanger, F.) and the like. Also, to read the sequence,
A DNA sequencer (Model 373A) manufactured by ABI was used. Thus, a nucleotide sequence with an average length of 300 bases was obtained from the 5'side or 3'side of each clone.

Example 12: Analysis of partial sequence data The nucleotide sequence obtained in Example 11 was Lippman (DJ) and Pearson,
The FASTA program of (WR) was used to perform a homology search against all nucleotide sequences contained in the known databases (GenBank and EMBL). Thereby, the sequenced clone was identified as a clone having a new sequence. The nucleotide sequence of the identified clone was converted into an amino acid sequence in three possible frames. It was also found that the amino acid sequence was a novel amino acid sequence.

However, the cloned cDNA clone does not always cover the entire length of mRNA. If it is not full-length, it is unlikely to include the N-terminal amino acid sequence portion. Therefore, Northern analysis was performed to determine whether or not the obtained clone was full length. That is, poly (A) ⁺ RNA obtained by the operation of Example 8 → Example 9 was electrophoresed and then blotted on a nylon membrane. When hybridized with the subcoulomb cDNA insert as a probe, one band was observed at a position of about 4400 bp. Since the insert size of the clone was about 2200 bp, 5'-side and 3'-side PCR was performed to obtain the full-length cDNA. This PCR is 3'-RACE (BR
L company) and 5'-RACE (CLONTECH) system kits.

Example 13: Determination of full-length sequence of cDNA and determination of open reading frame The full-length sequence of cDNA was prepared by Molecular Cloning.
[Sambrook, J., Fritsch, EF and Maniatis, T.
Published by Cold Spring Harbor Laboratory Press in 1989] and determined by random sequencing.

That is, the plasmid was recovered from the clone, and the cDNA insert was separated and purified. This was ligated and fragmented, the ends of the DNA fragment were blunted with T4 polymerase, and a DNA fragment having a length of about 400 bp was recovered by agarose electrophoresis. The obtained DNA fragment is a plasmid vector,
pGEM-3Zf (+) [3199bp, Promega (Prom
ega Corp.)] and then transformed into E. Coli. Eighty colonies were picked up randomly and plasmid D
After preparing NA, these 80 plasmids (all of which have cDNA fragment as insert)
DNA sequencing was performed. DNA sequencing and sequence reading were performed by the method described in Example 11. The sequence data of the cDNA fragment was edited into a continuous sequence by using the DNA sequence ligation program of DNASIS to obtain the nucleotide sequence shown in SEQ ID NO: 3. From this cDNA full-length sequence data, open reading frame (O
RF) was determined and further translated into an amino acid sequence to obtain the sequence shown in SEQ ID NO: 1. Since one frame has an open reading frame of 2982 bp and the ORF of the Eck family has a total length of about 3000 bp, it is presumed that the polypeptide of the present invention is also 2982 bp in full length.

Further, the protein p140 was found to be a typical type I type membrane protein from the hydrophobicity data (see FIG. 4. In the figure, the + side is a region with strong hydrophobicity, and the − side is Indicates that the region is weak hydrophobicity). That is, the p140 polypeptide is a typical type I membrane protein having an open reading frame of 2982 bp and 993 amino acids, and has an estimated molecular weight of 109,860D.
Although it is a, it was expected to show 140 kD due to sugar chain binding.

Example 14: Construction of a plasmid vector for producing an expression vector As a vector for expression, a derivative of pUC-SRαML-1 (disclosed in JP-A-6-56895 together with its production method) was used. This derivative was constructed using the two insert fragments shown below.

[0090]

[Chemical 4]

[0091]

[Chemical 5]

The pUCSRαML1 vector was digested with PstI and SacI, and the resulting product was subjected to agarose gel electrophoresis to recover a fragment of about 4.1 kpb, and the phosphate group at the 5'end was removed by treatment with BAP (bacterial alkaline phosphatase). It was The phosphorylated DNA fragment T7 was ligated with the approximately 4.1 kbp fragment obtained from pUCSRαML1 and circularized.

The obtained vector further contains SpeI and K
Digested with pnI and subjected to agarose gel electrophoresis, about 4.
A 1 kbp fragment was recovered, and the phosphate group at the 5'end was BAP
(Bacterial alkaline phosphatase) treatment removed. Phosphorylated DNA fragment SP6 was added to pUCSR
It was ligated with the approximately 4.1 kbp fragment obtained from αML1 and circularized. The vector constructed in this way was transformed into pUC
It was named SRαML2 (see FIG. 5).

Example 15: Construction of expression vector The following primers X, Y and YH (SEQ ID NOs: 8, 9 and 10) which anneal to rat p140 cDNA were synthesized.

Primer X: 5'-A ATA TAG TCG ACC ACC ATG GAG AAC CCC TAC GT
T GGG CGA GCG A -3 '

Primer Y: 5'-CGG CGG ACT AGT TCA GAC CTG CAC GGG CAG TGT
CTG G -3 '

Primer YH: 5'-GCC GCC ACT AGT TCA GTG GTG GTG GTG GTG GTG
GAC CTG CAC GGG CAG TGT CTG G -3 ′

P using the above synthetic oligonucleotides X and Y with a plasmid containing p140 cDNA as a template
I went to CR. The resulting PCR fragment contains the sequence flanking the 5'end of the start codon known to those skilled in the art as the Kozak sequence and the cDNA encoding the protein molecule that constitutes the p140 protein. PCR fragment is S
The sal of pUCSRαML2 prepared in Example 14 prepared by digesting with alI-SpeI, separating and purifying the resulting product.
The expression vector p inserted into the I-SpeI site
UC-SRαML2-p140-A was obtained.

Further, using the plasmid containing p140 cDNA as a template, the above-synthesized oligonucleotides X and YH
Was subjected to PCR. The resulting PCR fragment is present at the sequence known to those skilled in the art as the Kozak sequence adjacent to the 5'end of the start codon, the cDNA encoding the protein molecule constituting the p140 protein, and the C-terminus.
Contains histidine residues. PCR fragment is Sal
SalI- of pUCSRαML2 prepared in Example 14 by digesting with I-SpeI, separating and purifying the obtained product.
An expression vector pUC inserted into the SpeI site
-SRαML2-p140-B was obtained.

Further, the following primers Z and ZH
(SEQ ID NOS: 11 and 12) were synthesized. These are c
Adjacent to the amino terminus of the transmembrane region in DNA.

Primer Z: 5'-CGG CGG ACT AGT TCA TGA GCC TCT TTC ACT CGT
GGT CTC AAA CT-3 ′

Primer ZH: 5'-GCC GCC ACT AGT TCA GTG GTG GTG GTG GTG GTG
TGA GCC TCT TTC ACT CGT GGT CTC AAA CT-3 ′

PCR was carried out using the above-synthesized oligonucleotides X and Z using a plasmid containing p140 cDNA as a template. The resulting PCR fragment contains a sequence flanking the 5'end of the start codon known to those skilled in the art as a Kozak sequence and a cDNA encoding a polypeptide that constitutes the extracellular portion of the p140 protein.
The PCR fragment was digested with SalI-SpeI, and the resulting product was separated and purified to prepare pUCSRα prepared in Example 14.
It was inserted into the SalI-SpeI site of ML2 to obtain an expression vector pUC-SRαML2-p140-C.

Further, using the plasmid containing p140 cDNA as a template, the above-synthesized oligonucleotides X and ZH
Was subjected to PCR. The resulting PCR fragment was present at the sequence known as a Kozak sequence by those skilled in the art adjacent to the 5'end of the start codon, the cDNA encoding the polypeptide constituting the extracellular portion of the p140 protein, and the C-terminus. It contains 6 histidine residues that PC
The R fragment was digested with SalI-SpeI, and the obtained product was separated and purified to prepare pUCSRαML prepared in Example 14.
2 was inserted into the SalI-SpeI site to obtain an expression vector pUC-SRαML2-p140-D.

PUC-SRαML2-p140-A, p
UC-SRαML2-p140-B and pUC-SR
αML2-p140-C, pUC-SRαML2-p1
40-D was transformed into E. coli DH5, and the plasmid was separated from the culture broth (100 ml) of the transformed bacterium.
The CsCl density gradient method was repeated twice for purification.

Example 16: Expression in COS cells Plasmid DNA, pUC-SRαML2 and pUC-SR were added to COS-7 cells [described in Cell, 23 , 175 (1981)].
αML2-p140-A, pUC-SRαML2-p1
40-B, pUC-SRαML2-p140-C and pUC-SRαML2-p140-D were respectively subjected to the diethylaminoethyl (DEAE) dextran method [J. Immun.
ology, 136 , 4291 (1986)].

That is, about 1.8 × 10 ⁶ COS-7 cells were mixed with 50 ml of a growth medium (Dulbecco's modified MEM medium containing 10% inactivated fetal bovine serum) for 225c.
It was inoculated into a m ² flask (manufactured by Corning) and cultured overnight in a carbon dioxide gas incubator (37 ° C., 5% CO ₂ ). The next day, after removing the culture solution, 12m per flask
l DNA cocktail (each plasmid DNA15
μg, 50 mM Tris-HCl buffer (pH 7.4), 400
Dulbecco's modified MEM culture medium containing μg / ml DEAE-dextran) was added, and the mixture was reacted at 37 ° C. in 5% CO ₂ for 3 hours. Then, the DNA cocktail was removed, 15 ml of chloroquine solution (Dulbecco's modified MEM culture medium containing 150 μM chloroquine, 7% inactivated fetal bovine serum) was added, and the mixture was further reacted for 3 hours.

After removing the chloroquine solution, a growth medium (50 ml) was added, and the cells were cultured for 72 hours in an incubator at 37 ° C. and 5% CO ₂ to grow the cells to a nearly monolayer. Next, after removing the culture solution and washing once with a serum-free culture solution (trade name: SFM-101, manufactured by Nissui Pharmaceutical Co., Ltd.), a new serum-free culture solution (75 ml) was added and the culture was continued for 72 hours. did. The culture broth was collected, and the cells were solubilized with the cell lysate solubilized according to Example 1 (sterilization filter: trade name: STERIVEX-GS,
It was filtered through Millipore. These culture medium cell lysates were stored at 4 ° C. and used for the subsequent experiments. Of the obtained samples, c of p140-A and p140-B
C transduced with a plasmid containing a DNA insert
The mature protein portion of the polypeptide corresponding to p140 should be expressed in the cell lysate of OS cells. In addition, cDNA of p140-C and p140-D
The culture medium of COS cells transduced with the plasmid containing the insert should secrete the polypeptide corresponding to the extracellular portion of p140.

Example 17: Confirmation of Expression 2 ml of each transduced COS cell culture supernatant (obtained in Example 16) was taken, and this was concentrated by a centrifugal concentration filter (trade name: Centricon-10, Millipore). (Manufactured by the company). 1 μl each
An equal volume of loading buffer for SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) [0.125M Tris-HCl buffer (pH 6.
8) 4% sodium dodecyl sulfate, 30% glycerol] and treated at 90 ° C. for 3 minutes, then S
It was subjected to DS-PAGE.

Furthermore, not only for COS cell culture supernatant and cell lysate but also for the p140-B and p140-D proteins having His hexamer introduced at the C-terminal,
The purified product was also analyzed by SDS-PAGE. Purification is based on the fact that His forms complexes with various transition metal ions, and the method of metal chelate affinity chromatography [Biotechnology, 9 , 273 (1
991)]]. That is, to a culture supernatant (350 ml) obtained from COS cells or a cell lysate (100 ml), an aqueous sodium chloride solution was added so that the final concentration was 1 M, and a zinc-bound chelating sepharose column [trade name: chelating・ Chelating Sepharose Fa
st-Flow), manufactured by Pharmacia, 4 ml]. 50 containing 1M sodium chloride aqueous solution (40 ml)
After washing the column with mM phosphate buffer (pH 7.0), 1M
Elution was performed with a 50 mM phosphate buffer (pH 7.0) containing an aqueous sodium chloride solution and 0.4 M imidazole.
The eluate was concentrated to 100 μl, 1 μl of which was SDS
-Analyzed by PAGE.

SDS-PAGE is SDS10 / 20 gra
pUC-SRαML2-p14 using dient
For 0-A and p140-B introduced cell samples, p
The product corresponding to a molecular weight of 140 is pUC-SRαML
It was detected in the purified cell lysate of the 2-p140-B-introduced cell sample. On the other hand, pUC-SRαML2-p
For 140-C and p140-D-introduced cell samples, the expected molecular weight of the polypeptide corresponding to the extracellular portion of p140 was detected in the culture supernatant and its purified product.

Formulation Example 1 The following components were admixed in a conventional method and punched out to give 100 tablets each containing 5 mg of the active ingredient. Vitamin K ₅ …………………………………………………………
…………… 500mg carboxymethylcellulose calcium ………………
…………… 200mg Magnesium stearate ……………………………………
…………… 100mg Microcrystalline cellulose ………………………………………………
……………… 9.2g

[0113]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 993 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide Origin organism name: rat Strain name: rat skeletal muscle myoblasts Cell line: L6 Sequence: Met Glu Asn Pro Tyr Val Gly Arg Ala Arg Ala Ala Ala Glu Arg Ala 1 5 10 15 Ala Ala Glu Ala Thr Asn Ser Leu Ser Ile Leu Val Arg Pro Thr Ser 20 25 30 Glu Gly Ser Arg Ile Asp Ser Glu Phe Val Glu Leu Ala Trp Thr Ser 35 40 45 His Pro Glu Ser Gly Trp Glu Glu Val Ser Ala Tyr Asp Glu Ala Met 50 55 60 Asn Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Arg Glu Ser Ser Gln 65 70 75 80 Asn Asn Trp Leu Arg Thr Gly Phe Ile Trp Arg Arg Glu Val Gln Arg 85 90 95 Val Tyr Val Glu Leu Lys Phe Thr Val Valg Asp Cys Asn Ser Ile Pro 100 105 110 Asn Ile Pro Gly Ser Cys Lys Glu Thr Phe Asn Leu Phe Tyr Tyr Glu 115 120 125 Ala Asp Ser Asp Val Ala Ser Ala Ser Ser Pro Phe Trp Met Glu Asn 130 135 140 Pro Tyr Val Lys Val Asp Thr Ile Ala Pro Asp Glu Ser Phe Ser Arg 145 150 155 160 Leu Asp Ala Gly Arg Val Asn Thr Lys Val Arg Ser Phe Gly Pro Leu 165 170 175 Ser Lys Ala Gly Phe Tyr Leu Ala Phe Gln Asp Gln Gly Ala Cys Met 180 185 190 Ser Leu Ile Ser Val Arg Ala Phe Tyr Lys Lys Cys Ala Ser Thr Thr 195 200 205 Ala Gly Phe Ala Leu Phe Pro Glu Thr Leu Thr Gly Ala Glu Pro Thr 210 215 220 Ser Leu Val Ile Ala Pro Gly Thr Cys Ile Ala Asn Ala Val Glu Val 225 230 235 240 Ser Val Pro Leu Lys Leu Tyr Cys Asn Gly Asp Gly Glu Trp Met Val 245 250 255 Pro Val Gly Ala Cys Thr Cys Ala Thr Gly His Glu Pro Ala Ala Lys 260 265 270 Glu Thr Gln Cys Arg Ala Cys Pro Pro Gly Ser Tyr Lys Ala Lys Gln 275 280 285 Gly Glu Gly Pro Cys Leu Pro Cys Pro Pro Asn Ser Arg Thr Thr Ser 290 295 300 Pro Ala Ala Ser Ile Cys Thr Cys His Asn Asn Phe Tyr Arg Ala Asp 305 310 315 320 Ser Asp Thr Ala Asp Ser Ala Cys Thr Thr Val Pro Ser Pro Pro Arg 325 330 335 Gly Val Ile Ser Asn Val Asn Glu Thr Ser Leu Ile Leu Glu Trp Ser 340 345 350 Glu Pro Arg Asp Leu Gly Gly Arg Asp Asp Leu Leu Tyr Asn Val Ile 355 360 365 Cys Lys Lys CysArg Gly Ser Ser Gly Ala Gly Gly Pro Ala Thr Cys 370 375 380 Ser Arg Cys Asp Asp Asn Val Glu Phe Glu Pro Arg Gln Leu Gly Leu 385 390 395 400 Thr Glu Arg Arg Val His Ile Ser His Leu Leu Ala His Thr Arg Tyr 405 410 415 Thr Phe Glu Val Gln Ala Val Asn Gly Val Ser Gly Lys Ser Pro Leu 420 425 430 Pro Pro Arg Tyr Ala Ala Val Asn Ile Thr Thr Asn Gln Ala Ala Pro 435 440 445 Ser Glu Val Pro Thr Leu His Leu His Ser Ser Ser Gly Ser Ser Leu 450 455 460 Thr Leu Ser Trp Ala Pro Pro Glu Arg Pro Asn Gly Val Ile Leu Asp 465 470 475 480 Tyr Glu Met Lys Tyr Phe Glu Lys Ser Lys Gly Ile Ala Ser Thr Val 485 490 495 Thr Ser Gln Lys Asn Ser Val Gln Leu Asp Gly Leu Gln Pro Asp Ala 500 505 510 Arg Tyr Val Val Gln Val Arg Ala Arg Thr Val Ala Gly Tyr Gly Gln 515 520 525 Tyr Ser Arg Pro Ala Glu Phe Glu Thr Thr Ser Glu Arg Gly Ser Gly 530 535 540 Ala Gln Gln Leu Gln Glu Gln Leu Pro Leu Ile Val Gly Ser Thr Val 545 550 555 560 Ala Gly Phe Val Phe Met Val Val Val Val Ile Ala Leu Val Cys 565 570 575 Leu Arg Lys Gln Arg Gln Gly Pro Asp Ala Glu Tyr Thr Glu Lys Leu 580 585 590 Gln Gln Tyr Val Ala Pro Arg Met Lys Val Tyr Ile Asp Pro Phe Thr 595 600 605 Tyr Glu Asp Pro Asn Glu Ala Val Arg Glu Phe Ala Lys Glu Ile Asp 610 615 620 Val Ser Cys Val Lys Ile Glu Glu Val Ile Gly Ala Gly Glu Phe Gly 625 630 635 640 Glu Val Cys Arg Gly Arg Leu Lys Leu Pro Gly Arg Arg Glu Val Phe 645 655 655 Val Ala Ile Lys Thr Leu Lys Val Gly Tyr Thr Glu Arg Gln Arg Arg 660 665 670 Asp Phe Leu Ser Glu Ala Ser Ile Met Gly Gln Phe Asp His Pro Asn 675 680 685 Ile Ile Arg Leu Glu Gly Val Val Thr Lys Ser Arg Pro Val Met Ile 690 695 700 Leu Thr Glu Phe Met Glu Asn Cys Ala Leu Asp Ser Phe Leu Arg Leu 705 710 715 720 Asn Asp Gly Gln Phe Thr Val Ile Gln Leu Val Gly Met Leu Arg Gly 725 730 735 Ile Ala Ala Gly Met Lys Tyr Leu Ser Glu Met Asn Tyr Val His Arg 740 745 750 Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys 755 760 765 Val Ser Asp Phe Gly Leu Ser Arg Phe Leu Glu Asp Asp Pro Ser Asp 770 775 780 Pro Thr Tyr Thr Ser Ser Leu Gly Gly Lys Ile Pro Ile Arg Trp Thr 785 790 795 800 Ala Pro Glu Ala Ile Asp Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val 805 810 815 Trp Ser Tyr Gly Ile Val Met Trp Glu Val Met Ser Tyr Gly Glu Arg 820 825 830 Pro Tyr Trp Asp Met Ser Asn Gln Asp Val Ile Asn Ala Val Glu Gln 835 840 845 Asp Tyr Arg Leu Pro Pro Pro Met Asp Cys Pro Ala Ala Leu His Gln 850 855 860 Leu Met Leu Asp Cys Trp Val Arg Asp Arg Asn Leu Arg Pro Lys Phe 865 870 875 880 Ser Gln Ile Val Asn Thr Leu Asp Lys Leu Ile Arg Asn Ala Ala Ser 885 890 895 Leu Lys Val Ile Ala Ser Ala Pro Ser Gly Met Ser Gln Pro Leu Leu 900 905 910 Asp Arg Thr Val Pro Asp Tyr Thr Thr Phe Thr Thr Val Gly Asp Trp 915 920 925 Leu Asp Ala Ile Lys Met Gly Arg Tyr Lys Glu Ser Phe Val Gly Ala 930 935 940 Gly Phe Ala Ser Phe Asp Leu Val Ala Gln Met Thr Ala Glu Asp Leu 945 950 955 960 Leu Arg Ile Gly Val Thr Leu Ala Gly His Gln Lys Lys Ile Leu Ser 965 970 975 Ser Ile Gln Asp Met Arg Leu Gln Met Asn Gln Thr Leu Pro Val Gln 980 985 990 Val

SEQ ID NO: 2 Sequence length: 2982 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: rat Strain name: rat skeletal muscle myoblasts cell line: L6 sequence: ATGGAGAACC CCTACGTTGG GCGAGCGAGA GCAGCAGCGG AGCGAGCAGC GGCAGAAGCC 60 ACGAATTCAC TATCGATCCT GGTTCGGCCC ACCTCTGAAG GTTCCAGAAT CGATAGTGAA 120 TTCGTGGAGC TGGCATGGAC ATCTCATCCA GAGAGTGGGT GGGAAGAAGT GAGCGCCTAC 180 GATGAAGCCA TGAATCCTAT CCGCACGTAT CAGGTGTGTA ACGTGCGCGA GTCCAGCCAG 240 AACAACTGGC TGCGGACCGG TTTCATCTGG CGGCGGGAAG TCCAGCGCGT CTACGTGGAG 300 CTGAAGTTTA CCGTGAGAGA TTGCAACAGC ATCCCCAACA TCCCTGGCTC CTGCAAGGAA 360 ACCTTCAACC TTTTTTACTA CGAGGCTGAT AGCGATGTGG CGTCAGCCTC CTCTCCCTTC 420 TGGATGGAGA ACCCCTACGT GAAAGTGGAC ACCATTGCGC CAGATGAGAG CTTCTCGCGG 480 CTAGACGCTG GGCGCGTTAA CACCAAAGTG CGCAGCTTCGGGGCGCTTTC CAAAGCCGGC 540 TTCTACTTGGCCTGTCCTCCACTCTCCAATCGCAATC TTCGCACTCT TCCCCGAGAC CCTCACGGGG 660 GCTGAGCCCA CTTCGCTGGT CATTGCCCCT GGCACCTGCA TCGCTAACGC TGTGGAGGTG 720 TCTGTACCGC TCAAGCTCTA CTGCAATGGC GACGGGGAGT GGATGGTGCC CGTTGGTGCC 780 TGCACCTGCG CTACTGGCCA TGAGCCAGCC GCCAAGGAGA CCCAGTGCCG CGCCTGTCCC 840 CCTGGGAGCT ACAAGGCAAA GCAAGGAGAG GGGCCCTGCC TCCCCTGTCC CCCCAATAGC 900 CGCACCACCT CGCCGGCTGC CAGCATCTGC ACCTGTCACA ATAATTTCTA CCGCGCAGAC 960 TCAGACACAG CGGACAGCGC CTGCACCACG GTGCCGTCTC CCCCCCGGGG TGTGATCTCC 1020 AATGTGAATG AGACCTCGCT GATCCTCGAG TGGAGTGAGC CCCGGGACCT TGGCGGACGA 1080 GATGACCTCC TTTATAATGT TATCTGTAAG AAGTGCCGTG GCAGCTCTGG GGCTGGAGGT 1140 CCGGCGACCT GTTCACGCTG TGATGACAAC GTGGAGTTCG AGCCCCGACA GCTGGGCCTG 1200 ACCGAGCGCC GGGTCCACAT CAGCCACCTG TTGGCCCACA CCCGCTACAC CTTTGAGGTG 1260 CAGGCTGTCA ACGGCGTCTC TGGCAAAAGC CCTTTGCCGC CCCGCTATGC AGCTGTGAAT 1320 ATCACCACCA ACCAGGCCGC CCCATCAGAA GTGCCTACGC TCCACTTGCA CAGCAGTTCA 1380 GGGAGCAGCC TGACCCTGTC CTGGGCACCC CCGGAGCGGC CTAACGGAGT CATCTTGGAC 1440 TATGAGATGA AGTACTTTGA GAAGAGTAAA GGCATCGCCT C CACTGTCAC CAGCCAGAAG 1500 AACTCTGTAC AACTGGACGG ACTGCAGCCC GACGCCCGCT ATGTAGTTCA GGTCCGGGCT 1560 CGCACAGTAG CAGGTTACGG ACAGTATAGC CGCCCAGCTG AGTTTGAGAC CACGAGTGAA 1620 AGAGGCTCAG GGGCCCAGCA GCTTCAAGAG CAGCTTCCCC TAATTGTGGG ATCCACCGTA 1680 GCTGGCTTTG TCTTCATGGT GGTCGTCGTG GTCATTGCTC TTGTCTGCCT CAGGAAGCAG 1740 CGCCAGGGCC CTGATGCAGA ATACACGGAG AAGTTGCAGC AATACGTTGC CCCCAGGATG 1800 AAAGTTTACA TTGACCCCTT TACCTACGAG GATCCCAATG AGGCCGTCCG AGAGTTCGCC 1860 AAGGAGATCG ATGTGTCCTG CGTCAAGATC GAGGAGGTGA TTGGAGCTGG GGAGTTTGGG 1920 GAAGTGTGCC GGGGTCGGCT GAAACTGCCC GGCCGCCGGG AGGTGTTCGT GGCCATCAAG 1980 ACACTGAAGG TGGGATACAC GGAGAGGCAG CGGCGGGACT TCCTGAGTGA GGCTTCCATC 2040 ATGGGTCAAT TTGACCATCC AAATATAATC CGTCTAGAGG GCGTGGTCAC CAAAAGTCGT 2100 CCAGTCATGA TCCTCACTGA GTTCATGGAG AACTGTGCCC TGGACTCCTT CCTACGGCTC 2160 AATGACGGGC AGTTCACAGT CATCCAGCTT GTGGGCATGT TGCGTGGCAT TGCTGCCGGC 2220 ATGAAGTACT TGTCTGAGAT GAACTACGTG CACCGTGACC TCGCTGCCCG CAACATCCTT 2280 GTCAACAGTA ACTTGGTCTG CAAAGTATCT GACTTTGGGC TCTCCCG CTT CCTGGAGGAC 2340 GACCCCTCAG ACCCCACCTA CACCAGCTCC CTGGGTGGGA AGATCCCTAT CCGTTGGACC 2400 GCCCCAGAGG CCATAGACTA TCGGAAGTTC ACGTCTGCCA GCGATGTCTG GAGCTACGGG 2460 ATCGTCATGT GGGAGGTCAT GAGCTACGGA GAGCGACCAT ACTGGGACAT GAGCAACCAG 2520 GATGTCATCA ATGCCGTAGA GCAAGACTAT CGGTTACCAC CCCCCATGGA CTGCCCAGCG 2580 GCGCTGCACC AGCTCATGCT GGACTGTTGG GTGCGGGACC GGAACCTCAG GCCCAAGTTC 2640 TCCCAAATCG TCAACACGCT AGACAAGCTT ATCCGCAATG CTGCCAGCCT CAAGGTCATC 2700 GCCAGTGCCC CATCTGGCAT GTCCCAGCCC CTCCTAGACC GCACGGTCCC AGATTATACG 2760 ACCTTCACGA CGGTGGGCGA CTGGCTAGAT GCCATCAAGA TGGGGAGGTA TAAAGAGAGC 2820 TTCGTCGGTG CGGGTTTTGC CTCCTTTGAC CTGGTGGCCC AGATGACTGC AGAAGATCTGGAGCGCCACAGCAGCTATCAGCAGCACGATCACT CAGCAGAG ATGACT

SEQ ID NO: 3 Sequence length: 4027 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: rat Strain name: rat skeletal muscle myoblasts cell line: L6 sequence: GAAAAATGAA GATCTATACC GACAGCAGAT CAGTGGCTGC CTGGGGCAAA GTTGGAGGGA 60 CATGTTATTT TGATTGTGAT GACATAATAC ATGCAAACAC GGCTAATCCT CTCAAAGCAT 120 ACACTTATAC ATGTGCAGCT TGGTATACAT AAATTATCCA TTACAAAACT ATGAGAAAGC 180 TATCACCACT ATGAAGCACC ACTCACAGTA TGTGAATCTC CACCCCCCTT CCACTGCTGA 240 GACACAGAAA TCCTAGACTG GATGGAGAAC CCCTACGTTG GGCGAGCGAG AGCAGCAGCG 300 GAGCGAGCAG CGGCAGAAGC CACGAATTCA CTATCGATCC TGGTTCGGCC CACCTCTGAA 360 GGTTCCAGAA TCGATAGTGA ATTCGTGGAG CTGGCATGGA CATCTCATCC AGAGAGTGGG 420 TGGGAAGAAG TGAGCGCCTA CGATGAAGCC ATGAATCCTA TCCGCACGTA TCAGGTGTGT 480 AACGTGCGCGCGCGAGTCCAGCCA GAACAACTGG CTGCGGACCGAGTCATCCATCCATCACTCTCGTACAGACCATCAGTCTAC ATCGACTGCTAG CTTTTTTACT ACGAGGCTGA TAGCGATGTG 660 GCGTCAGCCT CCTCTCCCTT CTGGATGGAG AACCCCTACG TGAAAGTGGA CACCATTGCG 720 CCAGATGAGA GCTTCTCGCG GCTAGACGCT GGGCGCGTTA ACACCAAAGT GCGCAGCTTC 780 GGGCCGCTTT CCAAAGCCGG CTTCTACTTG GCCTTCCAGG ACCAGGGTGC CTGCATGTCA 840 CTCATCTCTG TGCGCGCCTT CTACAAGAAG TGTGCATCCA CCACTGCAGG CTTCGCACTC 900 TTCCCCGAGA CCCTCACGGG GGCTGAGCCC ACTTCGCTGG TCATTGCCCC TGGCACCTGC 960 ATCGCTAACG CTGTGGAGGT GTCTGTACCG CTCAAGCTCT ACTGCAATGG CGACGGGGAG 1020 TGGATGGTGC CCGTTGGTGC CTGCACCTGC GCTACTGGCC ATGAGCCAGC CGCCAAGGAG 1080 ACCCAGTGCC GCGCCTGTCC CCCTGGGAGC TACAAGGCAA AGCAAGGAGA GGGGCCCTGC 1140 CTCCCCTGTC CCCCCAATAG CCGCACCACC TCGCCGGCTG CCAGCATCTG CACCTGTCAC 1200 AATAATTTCT ACCGCGCAGA CTCAGACACA GCGGACAGCG CCTGCACCAC GGTGCCGTCT 1260 CCCCCCCGGG GTGTGATCTC CAATGTGAAT GAGACCTCGC TGATCCTCGA GTGGAGTGAG 1320 CCCCGGGACC TTGGCGGACG AGATGACCTC CTTTATAATG TTATCTGTAA GAAGTGCCGT 1380 GGCAGCTCTG GGGCTGGAGG TCCGGCGACC TGTTCACGCT GTGATGACAA CGTGGAGTTC 1440 GAGCCCCGAC AGCTGGGCCT GACCGAGCGC CGGGTCCACA T CAGCCACCT GTTGGCCCAC 1500 ACCCGCTACA CCTTTGAGGT GCAGGCTGTC AACGGCGTCT CTGGCAAAAG CCCTTTGCCG 1560 CCCCGCTATG CAGCTGTGAA TATCACCACC AACCAGGCCG CCCCATCAGA AGTGCCTACG 1620 CTCCACTTGC ACAGCAGTTC AGGGAGCAGC CTGACCCTGT CCTGGGCACC CCCGGAGCGG 1680 CCTAACGGAG TCATCTTGGA CTATGAGATG AAGTACTTTG AGAAGAGTAA AGGCATCGCC 1740 TCCACTGTCA CCAGCCAGAA GAACTCTGTA CAACTGGACG GACTGCAGCC CGACGCCCGC 1800 TATGTAGTTC AGGTCCGGGC TCGCACAGTA GCAGGTTACG GACAGTATAG CCGCCCAGCT 1860 GAGTTTGAGA CCACGAGTGA AAGAGGCTCA GGGGCCCAGC AGCTTCAAGA GCAGCTTCCC 1920 CTAATTGTGG GATCCACCGT AGCTGGCTTT GTCTTCATGG TGGTCGTCGT GGTCATTGCT 1980 CTTGTCTGCC TCAGGAAGCA GCGCCAGGGC CCTGATGCAG AATACACGGA GAAGTTGCAG 2040 CAATACGTTG CCCCCAGGAT GAAAGTTTAC ATTGACCCCT TTACCTACGA GGATCCCAAT 2100 GAGGCCGTCC GAGAGTTCGC CAAGGAGATC GATGTGTCCT GCGTCAAGAT CGAGGAGGTG 2160 ATTGGAGCTG GGGAGTTTGG GGAAGTGTGC CGGGGTCGGC TGAAACTGCC CGGCCGCCGG 2220 GAGGTGTTCG TGGCCATCAA GACACTGAAG GTGGGATACA CGGAGAGGCA GCGGCGGGAC 2280 TTCCTGAGTG AGGCTTCCAT CATGGGTCAA TTTGACCATC CAAATAT AAT CCGTCTAGAG 2340 GGCGTGGTCA CCAAAAGTCG TCCAGTCATG ATCCTCACTG AGTTCATGGA GAACTGTGCC 2400 CTGGACTCCT TCCTACGGCT CAATGACGGG CAGTTCACAG TCATCCAGCT TGTGGGCATG 2460 TTGCGTGGCA TTGCTGCCGG CATGAAGTAC TTGTCTGAGA TGAACTACGT GCACCGTGAC 2520 CTCGCTGCCC GCAACATCCT TGTCAACAGT AACTTGGTCT GCAAAGTATC TGACTTTGGG 2580 CTCTCCCGCT TCCTGGAGGA CGACCCCTCA GACCCCACCT ACACCAGCTC CCTGGGTGGG 2640 AAGATCCCTA TCCGTTGGAC CGCCCCAGAG GCCATAGACT ATCGGAAGTT CACGTCTGCC 2700 AGCGATGTCT GGAGCTACGG GATCGTCATG TGGGAGGTCA TGAGCTACGG AGAGCGACCA 2760 TACTGGGACA TGAGCAACCA GGATGTCATC AATGCCGTAG AGCAAGACTA TCGGTTACCA 2820 CCCCCCATGG ACTGCCCAGC GGCGCTGCAC CAGCTCATGC TGGACTGTTG GGTGCGGGAC 2880 CGGAACCTCA GGCCCAAGTT CTCCCAAATC GTCAACACGC TAGACAAGCT TATCCGCAAT 2940 GCTGCCAGCC TCAAGGTCAT CGCCAGTGCC CCATCTGGCA TGTCCCAGCC CCTCCTAGAC 3000 CGCACGGTCC CAGATTATAC GACCTTCACG ACGGTGGGCG ACTGGCTAGA TGCCATCAAG 3060 ATGGGGAGGT ATAAAGAGAG CTTCGTCGGT GCGGGTTTTG CCTCCTTTGA CCTGGTGGCC 3120 CAGATGACTG CAGAAGATCT GCTAAGGATC GGGGTCACTT TGGCCGGCCA CC AGAAGAAG 3180 ATCCTCAGCA GTATCCAGGA CATGCGGCTG CAGATGAACC AGACACTGCC CGTGCAGGTC 3240 TGACGCTCAG CTCCAGCGAG GGGCGTGGCC CCCCGGGACT GCACAAGGAT TCTGACCAGC 3300 CAGCTGGACT TTTGGATACC TGGCCTTTGG CTGTGGCCCA GAAGACAGAA GTTCGGGGGA 3360 GAACCCTAGC TGTGACTTCT CCAAGCCTGT GCTCCCTCCC AGGAAGTGTG CCCCAAACCT 3420 CTTCATATTG AAGATGGATT AGAAGAGGGG GTGATATCCC CTCCCCAGAT GCCTCAGGGC 3480 CCAGGCCTGC CTGCTCTCCA GTCGGGGATC TTCACAACTC AGATTTGGTT GTGCTTCAGT 3540 AGTGGAGGTC CTGGTAGGGT CGGGTGGGGA TAAGCCTGGG TTCTTCAGGC CCCAGCCCTG 3600 GCAGGGGTCT GACCCCAGCA GGTAAGCAGA GAGTACTCCC TCCCCAGGAA GTGGAGGAGG 3660 GGACTCTGGG AATGGGGAAA TATGGTGCCC CATCCTGAAG CCAGCTGGTA CCTCCAGTTT 3720 GCACAGGGAC TTGTTGGGGG CTGAGGGCCC TGCCTACCCT TGGTGCTGTC ATAAAAGGGC 3780 AGGCGGGAGC GGGCTGAGAA ACAGCCTGTG CCTCCCAGAG ACTGACTCAG AGAGCCAGAG 3840 ACGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGAAA GACGGGGGTG 3900 GGGTATGTAT GCGTGTGTTG TGCACATGCT TGCCTGCACA GAGAGCATGA GTGTGTACAA 3960 GCTTAGCCCT GTGCCCTGTA GTGGGGCCAG CTGGGCAGAC AGCGAAATAA AAGGCAAT AA 4020 GTTGAAA 4027

SEQ ID NO: 4 Sequence length: 4027 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: rat Strain name: rat skeletal muscle myoblasts cell Line: L6 Sequence feature Symbol representing the feature: CDS Location: 262..3243 Method of determining the feature: P sequence: GAAAAATGAA GATCTATACC GACAGCAGAT CAGTGGCTGC CTGGGGCAAA GTTGGAGGGA 60 CATGTTATTGA TGATTGTGAT GACATAACATAT ACATACTACT ACTACTACATCT ACTACTCTTACACTTACACTCTTACACTTACACTCTCAA ATGAAGCACC ACTCACAGTA TGTGAATCTC CACCCCCCTT CCACTGCTGA 240 GACACAGAAA TCCTAGACTG G ATG GAG AAC CCC TAC GTT GGG CGA GCG AGA 291 Met Glu Asn Pro Tyr Val Gly Arg Ala Arg 1 5 10 GCA GCA GCG GAG CGA GCA GCC ACC GCA GCA GCC ACA Ala Ala Ala Glu Arg Ala Ala Ala Glu Ala Thr Asn Ser Leu Ser Ile 15 20 25 CTG GTT CGG CCC ACC TCT GAA GGT TCC AGA ATC GAT AGT GAA TTC GTG 387 Leu Val Arg Pro Thr Ser Glu Gly Ser Arg Ile Asp Ser Glu Phe Val 30 35 40 GAG CTG GCA TGG ACA TCT CAT CCA GAG AGT GGG TGG GAA GAA GTG AGC 435 Glu Leu Ala Trp Thr Ser His Pro Glu Ser Gly Trp Glu Glu Val Ser 45 50 55 GCC TAC GAT GAA GCC ATG AAT CCT ATC CGC ACG TAT CAG GTG TGT AAC 483 Ala Tyr Asp Glu Ala Met Asn Pro Ile Arg Thr Tyr Gln Val Cys Asn 60 65 70 GTG CGC GAG TCC AGC CAG AAC AAC TGG CTG CGG ACC GGT TTC ATC TGG 531 Val Arg Glu Ser Ser Gln Asn Asn Trp Leu Arg Thr Gly Phe Ile Trp 75 80 85 90 CGG CGG GAA GTC CAG CGC GTC TAC GTG GAG CTG AAG TTT ACC GTG AGA 579 Arg Arg Glu Val Gln Arg Val Tyr Val Glu Leu Lys Phe Thr Val Arg 95 100 105 GAT TGC AAC AGC ATC CCC AAC ATC CCT GGC TCC TGC AAG GAA ACC TTC 627 Asp Cys Asn Ser Ile Pro Asn Ile Pro Gly Ser Cys Lys Glu Thr Phe 110 115 120 AAC CTT TTT TAC TAC GAG GCT GAT AGC GAT GTG GCG TCA GCC TCC TCT 675 Asn Leu Phe Tyr Tyr Glu Ala Asp Ser Asp Val Ala Ser Ala Ser Ser 125 130 135 CCC TTC TGG ATG GAG AAC CCC TAC GTG AAA GTG GAC ACC ATT GCG CCA 723 Pro Phe Trp Met Glu Asn P ro Tyr Val Lys Val Asp Thr Ile Ala Pro 140 145 150 GAT GAG AGC TTC TCG CGG CTA GAC GCT GGG CGC GTT AAC ACC AAA GTG 771 Asp Glu Ser Phe Ser Arg Leu Asp Ala Gly Arg Val Asn Thr Lys Val 155 160 165 170 CGC AGC TTC GGG CCG CTT TCC AAA GCC GGC TTC TAC TTG GCC TTC CAG 819 Arg Ser Phe Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu Ala Phe Gln 175 180 185 GAC CAG GGT GCC TGC ATG TCA CTC ATC TCT GTG CGC GCC TTC TAC AAG 867 Asp Gln Gly Ala Cys Met Ser Leu Ile Ser Val Arg Ala Phe Tyr Lys 190 195 200 AAG TGT GCA TCC ACC ACT GCA GGC TTC GCA CTC TTC CCC GAG ACC CTC 915 Lys Cys Ala Ser Thr Thr Ala Gly Phe Ala Leu Phe Pro Glu Thr Leu 205 210 215 ACG GGG GCT GAG CCC ACT TCG CTG GTC ATT GCC CCT GGC ACC TGC ATC 963 Thr Gly Ala Glu Pro Thr Ser Leu Val Ile Ala Pro Gly Thr Cys Ile 220 225 230 GCT AAC GCT GTG GAG GTG TCT GTA CCG CTC AAG CTC TAC TGC AAT GGC 1011 Ala Asn Ala Val Glu Val Ser Val Pro Leu Lys Leu Tyr Cys Asn Gly 235 240 245 250 GAC GGG GAG TGG ATG GTG CCC GTT GGT GCC TGC ACC TGC GCT ACT GGC 1059 Asp Gly Glu Trp Met Val Pro Val Gly Ala Cys Thr Cys Ala Thr Gly 255 260 265 CAT GAG CCA GCC GCC AAG GAG ACC CAG TGC CGC GCC TGT CCC CCT GGG 1107 His Glu Pro Ala Ala Lys Glu Thr Gln Cys Arg Ala Cys Pro Pro Gly 270 275 280 AGC TAC AAG GCA AAG CAA GGA GAG GGG CCC TGC CTC CCC TGT CCC CCC 1155 Ser Tyr Lys Ala Lys Gln Gly Glu Gly Pro Cys Leu Pro Cys Pro Pro 285 290 295 AAT AGC CGC ACC ACC TCG CCG GCT GCC AGC ATC TGC ACC TGT CAC AAT 1203 Asn Ser Arg Thr Thr Ser Pro Ala Ala Ser Ile Cys Thr Cys His Asn 300 305 310 AAT TTC TAC CGC GCA GAC TCA GAC ACA GCG GAC AGC GCC TGC ACC ACG 1251 Asn Phe Tyr Arg Ala Asp Ser Asp Thr Ala Asp Ser Ala Cys Thr Thr 315 320 325 330 GTG CCG TCT CCC CCC CGG GGT GTG ATC TCC AAT GTG AAT GAG ACC TCG 1299 Val Pro Ser Pro Pro Arg Gly Val Ile Ser Asn Val Asn Glu Thr Ser 335 340 345 CTG ATC CTC GAG TGG AGT GAG CCC CGG GAC CTT GGC GGA CGA GAT GAC 1347 Leu Ile Leu Glu Trp Ser Glu Pro Arg Asp Leu Gly Gly Arg Asp Asp 350 355 360 CTC CTT TAT AAT GTT ATC TGT AAG AAG TGC CGT GGC AGC TCT G GG GCT 1395 Leu Leu Tyr Asn Val Ile Cys Lys Lys Cys Arg Gly Ser Ser Gly Ala 365 370 375 GGA GGT CCG GCG ACC TGT TCA CGC TGT GAT GAC AAC GTG GAG TTC GAG 1443 Gly Gly Pro Ala Thr Cys Ser Arg Cys Asp Asp Asp Asn Val Glu Phe Glu 380 385 390 CCC CGA CAG CTG GGC CTG ACC GAG CGC CGG GTC CAC ATC AGC CAC CTG 1491 Pro Arg Gln Leu Gly Leu Thr Glu Arg Arg Val His Ile Ser His Leu 395 400 405 410 TTG GCC CAC ACC CGC TAC ACC TTT GAG GTG CAG GCT GTC AAC GGC GTC 1539 Leu Ala His Thr Arg Tyr Thr Phe Glu Val Gln Ala Val Asn Gly Val 415 420 425 TCT GGC AAA AGC CCT TTG CCG CCC CGC TAT GCA GCT GTG AAT ATC ACC 1587 Ser Gly Lys Ser Pro Leu Pro Pro Arg Tyr Ala Ala Val Asn Ile Thr 430 435 440 ACC AAC CAG GCC GCC CCA TCA GAA GTG CCT ACG CTC CAC TTG CAC AGC 1635 Thr Asn Gln Ala Ala Pro Ser Glu Val Pro Thr Leu His Leu His Ser 445 450 455 AGT TCA GGG AGC AGC CTG ACC CTG TCC TGG GCA CCC CCG GAG CGG CCT 1683 Ser Ser Gly Ser Ser Leu Thr Leu Ser Trp Ala Pro Pro Glu Arg Pro 460 465 470 AAC GGA GTC ATC TTG GAC TAT GAG ATG AA G TAC TTT GAG AAG AGT AAA 1731 Asn Gly Val Ile Leu Asp Tyr Glu Met Lys Tyr Phe Glu Lys Ser Lys 475 480 485 490 GGC ATC GCC TCC ACT GTC ACC AGC CAG AAG AAC TCT GTA CAA CTG GAC 1779 Gly Ile Ala Ser Thr Thr Val Thr Ser Gln Lys Asn Ser Val Gln Leu Asp 495 500 505 GGA CTG CAG CCC GAC GCC CGC TAT GTA GTT CAG GTC CGG GCT CGC ACA 1827 Gly Leu Gln Pro Asp Ala Arg Tyr Val Val Gln Val Arg Ala Arg Thr 510 515 520 GTA GCA GGT TAC GGA CAG TAT AGC CGC CCA GCT GAG TTT GAG ACC ACG 1875 Val Ala Gly Tyr Gly Gln Tyr Ser Arg Pro Ala Glu Phe Glu Thr Thr 525 530 535 AGT GAA AGA GGC TCA GGG GCC CAG CAG CTT CAA GAG CAG CTT CCC CTA 1923 Ser Glu Arg Gly Ser Gly Ala Gln Gln Leu Gln Glu Gln Leu Pro Leu 540 545 550 ATT GTG GGA TCC ACC GTA GCT GGC TTT GTC TTC ATG GTG GTC GTC GTG 1971 Ile Val Gly Ser Thr Val Ala Gly Phe Val Phe Met Val Val Val Val 555 560 565 570 GTC ATT GCT CTT GTC TGC CTC AGG AAG CAG CGC CAG GGC CCT GAT GCA 2019 Val Ile Ala Leu Val Cys Leu Arg Lys Gln Arg Gln Gly Pro Asp Ala 575 580 585 GAA TAC ACG GA G AAG TTG CAG CAA TAC GTT GCC CCC AGG ATG AAA GTT 2067 Glu Tyr Thr Glu Lys Leu Gln Gln Tyr Val Ala Pro Arg Met Lys Val 590 595 600 TAC ATT GAC CCC TTT ACC TAC GAG GAT CCC AAT GAG GCC GTC CGA GAG 2115 Tyr Ile Asp Pro Phe Thr Tyr Glu Asp Pro Asn Glu Ala Val Arg Glu 605 610 615 TTC GCC AAG GAG ATC GAT GTG TCC TGC GTC AAG ATC GAG GAG GTG ATT 2163 Phe Ala Lys Glu Ile Asp Val Ser Cys Val Lys Ile Glu Glu Val Ile 620 625 630 GGA GCT GGG GAG TTT GGG GAA GTG TGC CGG GGT CGG CTG AAA CTG CCC 2211 Gly Ala Gly Glu Phe Gly Glu Val Cys Arg Gly Arg Leu Lys Leu Pro 635 640 645 650 GGC CGC CGG GAG GTG TTC GTG GCC ATC AAG ACA CTG AAG GTG GGA TAC 2259 Gly Arg Arg Glu Val Phe Val Ala Ile Lys Thr Leu Lys Val Gly Tyr 655 660 665 ACG GAG AGG CAG CGG CGG GAC TTC CTG AGT GAG GCT TCC ATC ATG GGT 2307 Thr Glu Arg Gln Arg Arg Asp Phe Leu Ser Glu Ala Ser Ile Met Gly 670 675 680 CAA TTT GAC CAT CCA AAT ATA ATC CGT CTA GAG GGC GTG GTC ACC AAA 2355 Gln Phe Asp His Pro Asn Ile Ile Arg Leu Glu Gly Val Val Thr Lys 685 690 695 AGT CGT CCA GTC ATG ATC CTC ACT GAG TTC ATG GAG AAC TGT GCC CTG 2403 Ser Arg Pro Val Met Ile Leu Thr Glu Phe Met Glu Asn Cys Ala Leu 700 705 710 GAC TCC TTC CTA CGG CTC AAT GAC GGG CAG TTC ACA GTC ATC CAG CTT 2451 Asp Ser Phe Leu Arg Leu Asn Asp Gly Gln Phe Thr Val Ile Gln Leu 715 720 725 730 GTG GGC ATG TTG CGT GGC ATT GCT GCC GGC ATG AAG TAC TTG TCT GAG 2499 Val Gly Met Leu Arg Gly Ile Ala Ala Gly Met Lys Tyr Leu Ser Glu 735 740 745 ATG AAC TAC GTG CAC CGT GAC CTC GCT GCC CGC AAC ATC CTT GTC AAC 2547 Met Asn Tyr Val His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Asn 750 755 760 AGT AAC TTG GTC TGC AAA GTA TCT GAC TTT GGG CTC TCC CGC TTC CTG 2595 Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Phe Leu 765 770 775 GAG GAC GAC CCC TCA GAC CCC ACC TAC ACC ACC AGC TCC CTG GGT GGG AAG 2643 Glu Asp Asp Pro Ser Asp Pro Thr Tyr Thr Ser Ser Leu Gly Gly Lys 780 785 790 ATC CCT ATC CGT TGG ACC GCC CCA GAG GCC ATA GAC TAT CGG AAG TTC 2691 Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Asp Tyr Arg Lys Phe 795 800 805 810 ACG TCT GCC AGC GAT GTC TGG AGC TAC GGG ATC GTC ATG TGG GAG GTC 2739 Thr Ser Ala Ser Asp Val Trp Ser Tyr Gly Ile Val Met Trp Glu Val 815 820 825 ATG AGC TAC GGA GAG CGA CCA TAC TGG GAC ATG AGC AAC CAG GAT GTC 2787 Met Ser Tyr Gly Glu Arg Pro Tyr Trp Asp Met Ser Asn Gln Asp Val 830 835 840 ATC AAT GCC GTA GAG CAA GAC TAT CGG TTA CCA CCC CCC ATG GAC TGC 2835 Ile Asn Ala Val Glu Gln Asp Tyr Arg Leu Pro Pro Pro Met Asp Cys 845 850 855 CCA GCG GCG CTG CAC CAG CTC ATG CTG GAC TGT TGG GTG CGG GAC CGG 2883 Pro Ala Ala Leu His Gln Leu Met Leu Asp Cys Trp Val Arg Asp Arg 860 865 870 AAC CTC AGG CCC AAG TTC TCC CAA ATC GTC AAC ACG CTA GAC AAG CTT 2931 Asn Leu Arg Pro Lys Phe Ser Gln Ile Val Asn Thr Leu Asp Lys Leu 875 880 885 890 ATC CGC AAT GCT GCC AGC CTC AAG GTC ATC GCC AGT GCC CCA TCT GGC 2979 Ile Arg Asn Ala Ala Ser Leu Lys Val Ile Ala Ser Ala Pro Ser Gly 895 900 905 ATG TCC CAG CCC CTC CTA GAC CGC ACG GTC CCA GAT TAT ACG ACC TTC 3027 Met Ser Gln Pro Leu Leu Asp Arg Thr Val Pro Asp Tyr Thr Thr Phe 910 915 920 ACG ACG GTG GGC GAC TGG CTA GAT GCC ATC AAG ATG GGG AGG TAT AAA 3075 Thr Thr Val Gly Asp Trp Leu Asp Ala Ile Lys Met Gly Arg Tyr Lys 925 930 935 GAG AGC TTC GTC GGT GCG GGT TTT GCC TCC TTT GAC CTG GTG GCC CAG 3123 Glu Ser Phe Val Gly Ala Gly Phe Ala Ser Phe Asp Leu Val Ala Gln 940 945 950 ATG ACT GCA GAA GAT CTG CTA AGG ATC GGG GTC ACT TTG GCC GGC CAC 3171 Met Thr Ala Glu Asp Leu Leu Arg Ile Gly Val Thr Leu Ala Gly His 955 960 965 970 CAG AAG AAG ATC CTC AGC AGT ATC CAG GAC ATG CGG CTG CAG ATG AAC 3219 Gln Lys Lys Ile Leu Ser Ser Ile Gln Asp Met Arg Leu Gln Met Asn 975 980 985 CAG ACA CTG CCC GTG CAG GTC TGA CGCTCAG CTCCAGCGAG GGGCGTGGCC 3270 Gln Thr Leu Pro Val Gln Val 990 CCCCGGGACT GCACAAGGAT TCTGACCAGC CAGCTGGACT TTTGGATACC TGGCCTTTGG 3330 CTGTGGCCCA GAAGACAGAA GTTCGGGGGA GAACCCTAGC TGTGACTTCT CCAAGCCTGT 3390 GCTCCCTCCC AGGAAGTGTG CCCCAAACCT CTTCATATTG AAGATGGATT AGAAGAGGGG 3450 GTGATATCCC CTCCCCAGAT GCCTCAGGGC CCAGGCCTGC CTGCTCTCCA G TCGGGGATC 3510 TTCACAACTC AGATTTGGTT GTGCTTCAGT AGTGGAGGTC CTGGTAGGGT CGGGTGGGGA 3570 TAAGCCTGGG TTCTTCAGGC CCCAGCCCTG GCAGGGGTCT GACCCCAGCA GGTAAGCAGA 3630 GAGTACTCCC TCCCCAGGAA GTGGAGGAGG GGACTCTGGG AATGGGGAAA TATGGTGCCC 3690 CATCCTGAAG CCAGCTGGTA CCTCCAGTTT GCACAGGGAC TTGTTGGGGG CTGAGGGCCC 3750 TGCCTACCCT TGGTGCTGTC ATAAAAGGGC AGGCGGGAGC GGGCTGAGAA ACAGCCTGTG 3810 CCTCCCAGAG ACTGACTCAG AGAGCCAGAG ACGTGTGTGT GTGTGTGTGT GTGTGTGTGT 3870 GTGTGTGTGT GTGTGTGAAA GACGGGGGTG GGGTATGTAT GCGTGTGTTG TGCACATGCT 3930 TGCCTGCACA GAGAGCATGA GTGTGTACAA GCTTAGCCCT GTGCCCTGTA GTGGGGCCAG 3990 CTGGGCAGAC AGCGAAATAA AAGGCAATAA GTTGAAA 4027

SEQ ID NO: 5 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide Fragment type: Intermediate fragment Origin Biological name: rat Strain name: rat skeletal muscle myoblasts Cell line: L6

SEQ ID NO: 6 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide Fragment type: Intermediate fragment Origin Biological name: rat Strain name: rat skeletal muscle myoblasts Cell line: L6

SEQ ID NO: 7 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Polypeptide Fragment type: Intermediate fragment Origin Biological name: rat Strain name: rat skeletal muscle myoblasts Cell line: L6

SEQ ID NO: 8 Sequence length: 44 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: AATATAGTCG ACCACCATGG AGAACCCCTA CGTTGGGCGA GCGA

SEQ ID NO: 9 Sequence Length: 37 Sequence Type: Nucleic Acid Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: CGGCGGACTA GTTCAGACCT GCACGGGCAG TGTCTGG

SEQ ID NO: 10 Sequence length: 55 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: GCCGCCACTA GTTCAGTGGT GGTGGTGGTG GTGGACCTGC ACGGGC
AGTG TCTGG

SEQ ID NO: 11 Sequence length: 44 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: CGGCGGACTA GTTCATGAGC CTCTTTCACT CGTGGTCTCA AACT

SEQ ID NO: 12 Sequence length: 62 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: GCCGCCACTA GTTCAGTGGT GGTGGTGGTG GTGTGAGCCT CTTTCA
CTCG TGGTCTCAAA CT

[Brief description of drawings]

FIG. 1 Vitamin K ₅ in STZ diabetic rats
₃ is a graph showing the effect of (VK ₅ ) on blood glucose level.

FIG. 2 Vitamin K ₅ in STZ diabetic rats
₃ is a graph showing the effect of (VK ₅ ) on the amount of neutral lipids in blood.

FIG. 3 Vitamin K ₅ in STZ diabetic rats
₃ is a graph showing the effect of (VK ₅ ) on blood cholesterol level.

FIG. 4 is a hydrophobicity profile of a polypeptide of the present invention.

FIG. 5: Recombinant D with p140 cDNA inserted
It is a construction drawing of NA.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07K 14/575 8318-4H C12N 15/09 ZNA C12P 21/02 C 9282-4B 21/08 9161- 4B G01N 33/577 B // A61K 31/12 AED 9454-4C 31/425 (C12P 21/02 C12R 1:91) (54) [Title of Invention] Novel polypeptide, its production method and its polypeptide Encoding DNA, vector, host cell, antibody of the polypeptide thereof, pharmaceutical composition containing the polypeptide or antibody, preventive / therapeutic method and preventive / therapeutic agent for diabetes, and screening method of the preventive / therapeutic agent

Claims (14)

[Claims] 1. A protein p140 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, in a substantially pure form, a homologue thereof, a fragment thereof or a homologue of the fragment. 2. The polypeptide according to claim 1, which consists of the amino acid sequence represented by SEQ ID NO: 1. 3. A DNA encoding the polypeptide according to claim 1. 4. The DNA according to claim 3, which has the base sequence represented by SEQ ID NO: 2, or a fragment which selectively hybridizes to the sequence. 5. The DNA according to claim 3, which has the nucleotide sequence represented by SEQ ID NO: 3, or a fragment which selectively hybridizes to the sequence. 6. A replication or expression vector comprising the DNA according to any one of claims 3 to 5. 7. A host cell transformed with the replication or expression vector of claim 6. 8. A polypeptide comprising culturing the host cell according to claim 7 under conditions for expressing the polypeptide according to claim 1 or 2 and isolating the polypeptide. Production method. 9. A monoclonal or polyclonal antibody of the polypeptide according to claim 1 or 2. 10. A pharmaceutical composition comprising the polypeptide according to claim 1 or 2 or the antibody according to claim 9 and a pharmaceutically acceptable excipient and / or carrier. 11. A preventive and / or therapeutic agent for diabetes containing the polypeptide according to claim 1 or 2. 12. A method for preventing and / or treating diabetes, which comprises phosphorylating protein p140 with tyrosine. 13. A preventive and / or therapeutic agent for diabetes, which comprises, as an active ingredient, a substance that phosphorylates protein p140 on tyrosine. 14. A method for screening a preventive and / or therapeutic agent for diabetes, which comprises using the protein p140.