JPH10234378A - Protein analogous to sub-unit bound to myosin light chain and its gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject protein MLP which is the homologue of a myosin-bound subunit(MBS) and wherein a base sequence partial fragment encoding the protein is useful as a diagnostic medicine for familial hypertrophic cardiomyopathy (FHCM) or familial dilated cardiomyopathy (FDCM). SOLUTION: This human MLP is strongly expressed in brains and hearts, (1) has an active Rho protein-binding ability, (2) stimulates the phosphatase activity of a myosin light chain phosphatase catalyst subunit, (3) has its gene at the 1q32.1 of human chromosome and (4) has a mol.wt. of about 125kDa measured by SDA-PAGE. The protein is obtained by expressing a cDNA sequence of the formula in a host. The cDNA sequence of the formula is obtained e.g. by a method comprising screening a DNA library originated from a human brain by a method conventional in the genetic engineering field.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a protein having the ability to bind to active Rho protein and enhancing the phosphatase activity of the catalytic subunit of myosin light chain phosphatase. Regarding the cDNA sequence.

[0002] Type 1 protein phosphatase (PP1) is one of those that are the most well-studied among phosphatase. PP1 exists as a complex of a catalytic subunit (PP1c) and a regulatory subunit, and recent studies have revealed that its function and activity are controlled by various regulatory subunits.

[0003] PP1 exists in cells in the form of a holoenzyme in which a regulatory subunit is bound to PP1c. PP1
It is thought that the substrate specificity and activity regulation mechanism characteristic of various holoenzymes are expressed by the difference in the regulatory subunit that binds to c.

As holoenzymes, PP1S, PP1G, P
P1E, PP1M, and PP1N are known, which are present in the cytosolic soluble fraction, glycogen granules, sarcoplasmic reticulum membrane, myofibrils, and nucleus, respectively (Masaaki Ito, Kazuto Ichikawa, Takeshi Nakano, Cell Engineering, 16,187-193 (1997)).

Among them, PP1M is a PP1 holoenzyme that catalyzes a dephosphorylation reaction of myosin light chain and the like, and PP1cδ
And two regulatory subunits. The electrophoretic molecular weights of the two regulatory subunits are:
It is about 130 kDa (M130) and 20 kDa (M20) (Masaaki Ito, Takeshi Nakano, Biochemistry, 68,147-151 (199)
6)).

[0006] Myosin light chain phosphatase is known to be a type of type 1 protein phosphatase (PP1). Myosin light chain phosphatase is composed of a myosin binding subunit and a catalytic subunit, and it is believed that the myosin binding subunit directly binds to phosphorylated myosin, thereby enhancing the phosphatase activity of the phosphatase catalytic subunit for myosin ( Ichikawa, K. et al., Bioc
hemistry, 35, 6313-6320 (1996)).

This myosin-binding subunit is composed of the M
At 130, the catalytic subunits are thought to correspond to PP1cδ above (Shimizu, H. et al.,
J. Biol. Chem. 269: 30407-30411 (1994); Chen, YH
et al., FEBS Lett. 356: 51-55 (1994)).

The amino acid sequence of this myosin binding subunit has been deduced from chicken and rat-derived cDNAs (Shimizu, H. et al., J. Biol. Chem. 269, 30407-3).
0411 (1994), Chen, YH et al., FEBS Lett. 356, 51-55 (1
994)).

However, no homologue of the myosin binding subunit has been reported to the best of the present inventors.

[0010]

SUMMARY OF THE INVENTION The present inventors have now determined the cDNA sequence and amino acid sequence of the human homolog of the myosin binding subunit (hereinafter sometimes referred to as "MBS"), and
A recombinant fusion protein with T was made. The protein having this amino acid sequence was named "MLP".

[0011] The present inventors have also found that, as a result of Western blotting with an anti-MLP antibody and Northern blotting with a cDNA fragment, human MLP is strongly expressed in the brain and heart, and human MLP is myosin light chain phosphatase. , The human MLP binds to the active Rho protein, and the human MLP gene is located at q32.1 on human chromosome 1.

Accordingly, the present invention provides a protein having an amino acid sequence of an MLP protein, a base sequence encoding the same, a vector containing the base sequence, a host transformed with the vector, and an MLP comprising culturing the host. A method for producing a protein, a method for screening a substance that inhibits the binding between an MLP protein and an activated Rho protein, and a method for screening a substance that inhibits the enhancement of phosphatase activity of the catalytic subunit of myosin light chain phosphatase. Aim.

Another object of the present invention is to provide a probe containing the partial fragment of the nucleotide sequence, and a diagnostic agent containing the partial fragment of the nucleotide sequence or the probe.

The protein according to the present invention has the following properties: (1) It has an activity of binding to an active Rho protein; (2)
Enhances the phosphatase activity of the catalytic subunit of myosin light chain phosphatase, (3) its gene is located on human chromosome 1q32.1, (4) its molecular weight is SD
It is about 125 kDa as measured by S-PAGE.

Relationship between MLP and MBS The present inventors confirmed that human MLP is a homolog of rat and chicken MBS and not a counterpart, as described in (1) to (3) below. I have. (1) The present inventors used rat MBS cDNA as a probe to derive MLP c from human brain cDNA library.
The DNA was cloned and the amino acid sequence encoded by the human MLP cDNA was compared to the rat and chicken M
The identity with the amino acid sequence of BS is 54% and 50%, respectively, not very high (Example 1). (2) Human MLP is strongly expressed in brain and heart,
Little expression was observed in lung, aorta, vascular smooth muscle (chicken gizzard), liver and kidney. On the other hand, MBS
Has been reported to be widely expressed in organs where MLP expression was not observed (Okubo, S. et al., Bi
ochem. Biophys. Res. Commun., 200, 429-434 (1994)). (3) Important biochemical functions of MBS (enhancing action of phosphatase activity and active RhoA binding activity) (Ichi
kawa K. et al. (1996) Biochemistory, 35: 6313-6320
And Kimura, K. et al. (1996) .Science 273: 245-2.
48) was also found in human MLP (Examples 6 and 7).

[0016]

DETAILED DESCRIPTION OF THE INVENTION Definitions In the present invention, the term "amino acid" is used to include both optical isomers, that is, both L-form and D-form. Therefore, in the present invention, the term “peptide” means not only a peptide composed of only L-form amino acids but also a peptide containing some or all of D-form amino acids.

In the present invention, the term "amino acid" means not only the 20 kinds of α-amino acids constituting natural proteins, but also other α-amino acids, β-,
γ- and δ-amino acids, unnatural amino acids and the like are used. Thus, amino acids that are substituted in a peptide or inserted into a peptide as described below include the 20
It is not limited only to species α-amino acids, but may be other α-amino acids and β-, γ-, δ-amino acids and unnatural amino acids. Such β
-, Γ- or δ-amino acids, β-alanine,
γ-aminobutyric acid or ornithine; and amino acids other than those constituting natural proteins or unnatural amino acids include 3,4-dihydroxyphenylalanine, phenylglycine, cyclohexylglycine, 1,2,3,4-tetraethyl Hydroisoquinoline-3
-Carboxylic acid or nipecotic acid.

In the present specification, the term "protein according to the present invention" is used to include its derivatives.

Furthermore, in the present specification, "base sequence"
Means both the DNA sequence and the RNA sequence.

Protein In the present invention, the "catalytic subunit of myosin light chain phosphatase" can be PP1cδ (see Example 6).

In the present invention, the term “protein having the ability to bind to active Rho protein” refers to a protein which is evaluated by those skilled in the art to have binding to active Rho protein. It means a protein that is evaluated to have been found to bind to the active Rho protein when tested under the same conditions as in the hybrid system.

Here, Rh protein includes Rh protein.
oA, RhoB, RhoC, or RhoG proteins.

[0023] In the present specification, the Rho protein also includes a Rho protein modified so that the binding between the Rho protein and the protein according to the present invention is not substantially impaired. Such a modified Rho protein includes a RhoA mutant in which the 14th amino acid is substituted with valine (RhoA ^Val14 ).

The protein according to the present invention comprises, in addition to the Rho protein binding region, an ankyrin repeat structure (amino acid sequence at positions 57 to 313 of SEQ ID NO: 2) and a leucine zipper structure (amino acid sequence at positions 885 to 982 of SEQ ID NO: 2) (Example 1). Further, the protein according to the present invention is characterized in that it is strongly expressed in human brain and heart but not in lung or aorta (Example 4).

In the present invention, "a protein that enhances the phosphatase activity of the catalytic subunit of myosin light chain phosphatase" is evaluated by those skilled in the art as having been found to enhance the phosphatase activity of the catalytic subunit of myosin light chain phosphatase. For example, a protein evaluated to have been found to enhance the phosphatase activity of the catalytic subunit of myosin light chain phosphatase when tested under the same conditions as in Example 6.

According to the present invention, it is also provided that (a) SEQ ID NO: 2
Or (b) the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been added and / or inserted and / or one or more amino acids have been substituted and / or deleted, wherein the active Rho protein Provided is a protein or a derivative thereof having an amino acid sequence that has binding ability and enhances the phosphatase activity of the catalytic subunit of myosin light chain phosphatase. The addition, insertion, substitution and deletion referred to herein substantially impair the Rho protein binding ability of the protein consisting of the amino acid sequence of SEQ ID NO: 2 and the ability of the catalytic subunit of myosin light chain phosphatase to enhance the phosphatase activity. Something that doesn't exist.

The origin of the protein according to the present invention is not particularly limited, and it may be derived from mammals including humans, or may be derived from other sources.

The molecular weight of the protein consisting of the amino acid sequence of SEQ ID NO: 2 was about 12 as measured by SDS-PAGE.
5 kDa.

As used herein, the term “protein derivative” refers to a part or all of the amino group at the amino terminus (N-terminus) of the protein or the amino group on the side chain of each amino acid, and / or the carboxyl terminus of the peptide ( A part or all of the carboxyl group at the C-terminus or the side chain of each amino acid and / or a functional group other than the amino group and the carboxyl group of the side chain of each amino acid of the peptide (for example, hydrogen group, thiol group) , An amide group, etc.) are partially or entirely modified with other appropriate substituents. Modification with an appropriate other substituent is performed, for example, for the purpose of protecting a functional group present in the peptide, improving safety and tissue transferability, or enhancing activity.

Specific examples of the protein derivative include (1) the amino group at the amino terminus (N-terminus) of the protein or part or all of the hydrogen atoms in the amino group in the side chain of each amino acid are substituted or non-substituted; Substituted alkyl groups (which may be linear, branched or cyclic) (e.g., methyl, ethyl, propyl, isopropyl, isobutyl, butyl, t-butyl, cyclopropyl,
Cyclohexyl group, benzyl group), substituted or unsubstituted acyl group (for example, formyl group, acetyl group, caproyl group, cyclohexylcarbonyl group, benzoyl group, phthaloyl group, tosyl group, nicotinoyl group, piperidinecarbonyl group), urethane-type protection Group (for example, p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, p-biphenylisopropyloxycarbonyl group, t-butoxycarbonyl group) or a urea-type substituent (for example, methylaminocarbonyl group, phenylcarbonyl group) , Cyclohexylaminocarbonyl group), and (2) a carboxyl group at the carboxyl terminal (C-terminal) of the protein or part or all of the carboxyl group at the side chain of each amino acid is an ester type Decorated (e.g., those whose hydrogen atoms have been replaced by methyl, ethyl, isopropyl, cyclohexyl, phenyl, benzyl, t-butyl, 4-picolyl), those which have been modified by amide type (e.g., , Unsubstituted amides, those forming C1-C6 alkylamides (eg, methylamide, ethylamide, isopropylamide), and (3) functional groups other than the amino group and carboxyl group of the side chain of each amino acid of the protein (eg, , A hydrogen group, a thiol group, an amino group, etc.) in which a part or the whole is modified with the same substituent as the above-mentioned amino group or a trityl group.

The protein according to the present invention can be obtained, for example, by expressing the cDNA sequence of SEQ ID NO: 1 in a host (Example 2). Acquisition of the sequence of SEQ ID NO: 1 and expression in the host will be described later.

According to the present invention, there is provided the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids are added and / or inserted, and / or one or more amino acids are substituted and / or deleted, wherein the active form is Rho. A protein comprising an amino acid sequence having protein binding ability or a derivative thereof is provided.

That is, the addition, insertion, substitution,
The term "deletion" and "deletion" refer to those which do not substantially impair the ability of a protein consisting of the amino acid sequence of SEQ ID NO: 2 to bind to the active Rho protein.

Examples of the amino acid sequences deleted include SEQ ID NOs: 613 to 982, 613 to 913, 71
Nos. 3 to 982, 813 to 982, and 913 to 98
No. 2 amino acid sequence.

According to the present invention, there is also provided SEQ ID NO: 2 wherein one or more amino acids have been added and / or inserted and / or one or more amino acids have been substituted and / or deleted.
Or a derivative thereof comprising an amino acid sequence that enhances the phosphatase activity of the catalytic subunit of myosin light chain phosphatase. The addition, insertion, substitution, and deletion referred to herein are
A protein consisting of the amino acid sequence of SEQ ID NO: 2 that does not substantially impair the ability of the catalytic subunit of myosin light chain phosphatase to enhance phosphatase activity.

An example of the deleted amino acid sequence is the amino acid sequence of positions 1 to 648 of SEQ ID NO: 2.

As described below, the protein according to the present invention has been shown to be involved in familial hypertrophic cardiomyopathy (FHCM) and familial dilated cardiomyopathy (FDCM). Therefore, the protein according to the present invention is useful for elucidating the mechanism of these diseases.

The protein according to the present invention is an active form of Rho
It has both or either protein binding ability and ability to enhance phosphatase activity of the catalytic subunit of myosin light chain phosphatase. In addition, Rho protein, including hypertrophy of cardiomyocytes, smooth muscle contraction, tumor formation,
It is closely related to the expression of cell functions such as metastasis, cell morphology, cell motility, cell adhesion, and cytokinesis (Sar, VP et
al., J. Biol. Chem., 271, 31185-31190 (1996), Noda, M. et.
al., FEBS Lett., 367, 246-250 (1995), Symons, M., Curr.
ent Opinion in Biotechnology, 6,668-674 (1995), Naru
Miya, S., J. Biochem. 129, 215-228 (1996)). Therefore, the protein according to the present invention is also useful for elucidating the mechanism of myocardial hypertrophy, contraction, expansion and the like.

Nucleotide Sequence According to the present invention, a nucleotide sequence encoding the protein of the present invention is provided. A typical sequence of this nucleotide sequence is
It has part or all of the DNA sequence of SEQ ID NO: 1.

The DNA sequence of SEQ ID NO: 1 was obtained from a cDNA library derived from human brain. The sequence at positions 164 to 3109 in SEQ ID NO: 1 corresponds to the MLP open reading frame.

Given the amino acid sequence of the protein according to the present invention, the nucleotide sequence encoding it is easily determined, and various nucleotide sequences encoding the amino acid sequence of SEQ ID NO: 2 can be selected. Accordingly, the nucleotide sequence encoding the protein according to the present invention is defined as DN of SEQ ID NO: 1.
A DNA sequence encoding the same amino acid in addition to part or all of the A sequence
A sequence having the A sequence is also meant, and an RNA sequence corresponding thereto is also included.

The base sequence according to the present invention may be of natural origin or totally synthesized. In addition, a product synthesized using a part of a product derived from a natural product may be used.

The nucleotide sequence is, for example, a commercially available c
It can be obtained by amplifying a specific region using a base sequence of a DNA library as a template and appropriate primers sandwiching the open reading frame. Also, a method commonly used in the field of genetic engineering from a chromosome library or a cDNA library, for example, an appropriate D prepared based on partial amino acid sequence information.
It can also be obtained by a screening method using an NA probe, and the like.

When the nucleotide sequence is of natural origin, its origin is not particularly limited, and it may be derived from mammals including humans, or may be derived from other sources.

Examples of the nucleotide sequence encoding the protein according to the present invention include the nucleotide sequence of SEQ ID NO: 1 and a part of the DNA sequence of SEQ ID NO: 1 (for example, 164 to 31 of SEQ ID NO: 1).
09th, 2000-3109, 2000-2902
Nos. 2300-3109, 2600-3109 and 2900-3109).

Vector and Host Cell According to the present invention, there is provided a vector comprising the above-described nucleotide sequence of the present invention in a state capable of replicating in a host cell and expressing a protein encoded by the nucleotide sequence. You. Further, according to the present invention, there is provided a host cell transformed with the vector. The host-vector system is not particularly limited, and a fusion protein expression system with another protein can be used. Examples of fusion protein expression systems include MBP (maltose binding protein), GST (glutathione S transferase), HA (hemagglutinin), polyhistidine, my
c, Fas and the like.

Examples of the vector include a plasmid vector (for example, an expression vector in prokaryotic cells, yeast, insect cells, animal cells, etc.), a viral vector (for example, a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, Sendai virus vector, HIV vector, baculovirus vector), liposome vector (eg, cationic liposome vector) and the like.

In order to express a desired protein by actually introducing the vector into a host cell, the vector according to the present invention may contain, in addition to the base sequence according to the present invention, a sequence controlling its expression (for example, a promoter). Sequences, terminator sequences, enhancer sequences) and genetic markers for selecting host cells (eg, neomycin resistance gene,
(A kanamycin resistance gene) and the like. In addition, this vector may contain the nucleotide sequence according to the present invention in a repeated form (for example, in tandem). These may be made to exist in a vector according to a conventional method, and a method of transforming a host cell with this vector may be one commonly used in this field.

The procedure and method for constructing a vector according to the present invention may be those commonly used in the field of genetic engineering.

Examples of host cells include Escherichia coli, yeast, insect cells, and animal cells (eg, COS cells,
Lymphocytes, fibroblasts, CHO cells, blood cells, tumor cells, etc.).

The transformed host cells are cultured in a suitable medium, and the above-mentioned protein according to the present invention can be obtained from the culture. Thus, according to another aspect of the present invention, there is provided a method for producing a protein according to the present invention. The culture of the transformed host cell and its conditions are as follows:
It may be essentially the same as that for the cells used. The recovery and purification of the protein according to the present invention from the culture solution can also be performed according to a conventional method.

[0052] According to the screening method the present invention, (1) screening of the subject, is present in the screening system comprising an active Rho protein, a protein according to the present invention having the activated Rho protein binding activity, and (2) A substance that inhibits the binding between the active Rho protein and the protein according to the present invention, which has the ability to bind to the active Rho protein, including measuring the degree of inhibition of the binding between the active Rho protein and the protein according to the present invention. Is provided.

Here, the “degree of inhibition of binding” is defined as
Hybrid system (two hybrid system) (M.Ka
wabata Experimental Medicine 13,2111-2120 (1995), ABVojetk et
al. Cell 74, 205-214 (1993)), a method using immunoblotting with an antibody against the protein according to the present invention, and a method using an overlay assay (Ishizaki, T. et al., EMBO J. , 15, 1885-1
893 (1996)), a method of measuring using a protein translated in vitro (Shibata, H. et al., FEBS Lett. 38)
5, 221-224 (1996)), Amano, M., et al., Science 27.
1, 648-650 (1996).

In the present invention, "substance inhibiting binding"
The term “substance” refers to a substance evaluated to be inhibited by a person skilled in the art. For example, when an experiment is performed under the same conditions as in the yeast two-hybrid system of Example 7, no binding to the active Rho protein is observed. It means the case where is evaluated.

In the present specification, “determining the degree of inhibition of binding” is used in a sense including the measurement of the presence or absence of binding. The term “screening” is used to include an assay.

The screening system may be either a cell system or a cell-free system. Examples of the cell system include yeast cells, COS cells, Escherichia coli, insect cells, nematode cells, lymph cells, fibroblasts, and CHO cells. Cells, blood cells, and tumor cells.

The target of the screening is not particularly limited, but examples include peptides, peptide analogs, microorganism cultures, and organic compounds.

According to the present invention, (1) the objects of screening include the catalytic subunit of myosin light chain phosphatase and the protein of the present invention which enhances the phosphatase activity of the catalytic subunit of myosin light chain phosphatase. Inhibits the enhancement of the phosphatase activity of the catalytic subunit of myosin light chain phosphatase, including presence in a screening system and (2) measuring the degree of inhibition of the enhancement of the phosphatase activity of the catalytic subunit of myosin light chain phosphatase. Methods for screening substances are provided.

Here, “the degree of inhibition of the enhanced activity”
Examples include a method for measuring the degree of dephosphorylation of the phosphorylated myosin light chain. For example, the degree of inhibition of phosphatase activity can be measured according to the method described in Example 6.

In the present invention, the term “substance that inhibits the enhancement of activity” refers to a substance which is evaluated by those skilled in the art as inhibiting the enhancement of activity. For example, an experiment was conducted under the same conditions as in Example 6. In this case, it means that it is evaluated that inhibition of the enhancement of phosphatase activity is recognized.

The screening system can include a phosphatase substrate. Such substrates include those containing orthophosphate, polyphosphoric acid, etc., and in particular, phosphorylation (monophosphorylated or diphosylated).
phorylated) -myosin light chain and myosin. Phosphorylated myosin light chain and myosin are commercially available, Shimizu, H. et al., J. Biol. Chem., 26
Those prepared according to the description of 9,30407-30411 (1994) may be used. For phosphorylation, a method commonly used by those skilled in the art can be used.

The phosphatase substrate has its phosphate ester bond hydrolyzed by the action of phosphatase. By using a radioisotope such as ³² P for phosphoric acid, the degree of dephosphorylation can be measured.

In the above screening system, compounds having a phosphate group such as ATP and ATPγS can be present.

The myosin light chain phosphatase may be of natural origin extracted from a living body (eg, human, chicken, rat, bovine) or commercially available, but is not limited thereto. . For example, Shimiz
u, H. et al., J. Biol. Chem., 269, 30407-30411 (1994)
And PP1cδ prepared according to the method described in Chen, YH et al., FEBS Lett. 356, 51-55 (1994).

The screening system and the object of the screening include those similar to the above-mentioned screening method.

As described above, it has been confirmed that the active Rho protein is closely involved in myocardial hypertrophy, contraction, dilation, and the like.

Therefore, the above-mentioned screening method can also be used as a method for screening a substance for preventing or treating cardiomyopathy and the like.

Human MLP Locus Mutations in the human MLP gene can cause certain heart diseases, such as familial hypertrophic cardiomyopathy.
iomyopathy (FHCM)) or familial dilated cardiomyopathy (family
lial dilated cardiomyopathy (FDCM)). The grounds that human MLPs are mutated in FHCM and FDCM patients and do not function properly are described below.

First, the present inventors have found that the position of the gene of human MLP coincides with the position of the gene mutation responsible for FHCM and FDCM. As shown in Example 8, the present inventors have found by FISH analysis that the human MLP gene is present at the long arm of chromosome 1 at address 32.1 (1q32.1). FHCM and FDCM are mapped to this 1q32. That is, Watkins, H. et al.
To 1q32 (Watkins, H. et al. (1993). Nature Genet.
3: 333-337), Durand, J.-B. et al.
32 (Durand, J.-B. et al. (1995).
Circulation. 92: 3387-3389). Therefore, FHCM and F
In order to examine the possibility that the human MLP gene is mutated in DCM patients, the present inventors examined the chromosomal location of the human MLP gene in more detail.

The present inventors, as shown in Example 9,
Using radiation hybrid method, human MLP gene was
Was mapped to the 4.29 cR telomere side from the chromosome marker WI-9272. WI-9272 is compatible with markers D1S477 and D1S504
(875-883 cR from the end of the short arm of chromosome 1). Recently, the gene for troponin T2 (heart-type isoform of troponin T; TNNT2) has been mapped to WI-9272 at 1q32 (http://www3.ncbi.nlm.nih.gov/htbin-pos).
t / Omim / disamim? 191045; Townsend, PJ etal. (199
4) .Genomics 21: 311-316; Mesnard, L. et al. (199
5). Circ. Res. 76: 687-692). In addition, Theirfelder,
L. et al. Reported that TNNT2 was mutated in some patients in families with FHCM (Thierfelder, L. et al. (199).
4). Cell77: 701-712). However, subsequent studies have revealed that not all FHCM patients have TNNT2 mutations, but only up to 15% of those mutations (Watkins, H . et
al. (1995) .New England J. Med. 332, 1058-1064.
). From these facts, (1) About 85% of FHCM
Mutations in other genes, not T2 mutations, are responsible for the disease. (2) Some such genes are present in 1q32, near the location of the TNNT2 gene (WI-9272), it is conceivable that. Furthermore, the human MLP gene is TNNT2
Mapped to the same chromosomal marker as TNNT
2 In at least some of the FHCM patients without mutations,
It is considered that the human MLP gene is mutated.

On the other hand, the FDCM shows that the markers D1S456 and D1S of 1q32
It was reported to be linked to seven markers during S490 (Durand, J.-B. et al. (1995). Circulation. 92: 33).
87-3389). This region is from the end of the short arm of chromosome 1.
This corresponds to the position of 888-902cR. As mentioned above, human ML
The P gene is 3.98 to WI-9272 between markers D1S477 and D1S504 (875-883 cR from the end of the short arm of chromosome 1).
4.29cR Telomere side (approximately 87 from the end of the short arm of chromosome 1)
9cR). The human MLP gene is located very close to the above D1S456, and is located only 5 cR away from the centromere. 5cR distance is approx.
It is thought to correspond to a distance of 3 cM, ie about 3 Mb (3000 kb). Since the mapping of the FDCM to this region was performed using linkage analysis, there was some error (±
M). Thus, the above results do not rule out, but rather support, the possibility that mutations in the human MLP gene are responsible for FDCM.
That is, it is considered that the mutation of the MLP gene causes not only FDCM but also FHCM.

Second, both FHCM and FDCM are diseases specific to the heart (myocardium). FHCM is characterized by reduced left ventricular diastolic compliance associated with abnormal left ventricular myocardial hypertrophy. FDCM is also characterized by ventricular insufficiency and ventricular dilation. In any case, since the heart (heart muscle) is specifically damaged, it is considered that in these diseases, genes of important proteins specifically expressed in the heart are mutated.

On the other hand, the present inventors have shown that human MLP is specifically and strongly expressed in the heart by Western blotting analysis (Example 4) and Northern blotting analysis (Example 5). From this,
Human MLPs have shown an important role, particularly in the heart. From these facts, it is considered that mutation of the human MLP gene results in impairment of the function of human MLP in the heart.

Third, the present inventors biochemically analyzed the function of human MLP and found that it could play an important role in the contractile mechanism of the heart. We believe that human ML
P was found to function as a regulatory subunit of myosin light chain phosphatase (Example 6). Myosin light chain phosphatase induces depolymerization of actin and myosin through dephosphorylation of the myosin light chain. It is known that the polymerization and depolymerization of actin and myosin play important roles in remodeling the skeleton of cells and contracting and relaxing cells. Thus, the finding that human MLP functions as a subunit of myosin light chain phosphatase,
It has been shown that human MLP plays an important role in cardiomyocyte contraction and relaxation. From this, when a mutation occurs in the human MLP gene, it is expected that the contraction and expansion of the heart (myocardium) will be impaired. Therefore, the symptoms of FDCM (ventricular insufficiency and ventricular dilation) are thought to be due to mutations in the human MLP gene.

Fourth, the present inventors have found that human MLP binds to an active RhoA protein using a two-hybrid system (Example 7). Activated RhoA protein is a type of low molecular weight GTPase, but is known to be involved in cytoskeleton remodeling and cell contraction (Narumiya, S., J. Biochem. 129, 215-228).
(1996)). Interestingly, the RhoA protein regulates cytoskeletal remodeling and cell contraction by regulating the phosphorylation level of myosin light chain via myosin binding protein (MBS) (Kimura, K. et a
l., Science 273, 245-248 (1996)). On the other hand, the present inventors have found that human MLP is a homolog of MBS that is specifically expressed in the heart (Examples 1, 4 and 5), and that human MLP binds to the active RhoA protein ( Example 7). From these facts, it is considered that the role of MBS in other organs (cytoskeleton remodeling and cell contraction / relaxation) is played by human MLP in the heart. From this, it is expected that when a mutation occurs in the human MLP gene, the contraction and expansion of the heart (myocardium) are impaired. Therefore, the symptoms of FDCM (ventricular insufficiency and ventricular dilation) are thought to be due to mutations in the human MLP gene.

Recently, it has been revealed that the RhoA protein regulates cardiomyocyte proliferation and gene expression (Sar, VP et al., J. Biol. Chem. 271, 31185).
-31190 (1996)). In cardiomyocytes, phenylephrine (P
E) α1-adrenergic receptor stimulation or GTPase-deficie
atrial natriuretic facto induced by ntGαq
The RhoA protein positively regulates the expression of the r (ANF) receptor gene and the myosin light chain 2 gene. This indicates that the RhoA protein promotes cardiomyocyte hypertrophy. As described above, human MLPs are activated R
By binding to the hoA protein, human MLPs are thought to be involved in downstream signaling. Therefore, it is thought that human MLP is involved in cardiomyocyte hypertrophy downstream of the activated Rho protein. Therefore, the symptoms characteristic of FHCM (abnormal hypertrophy of the left ventricular myocardium) are considered to be caused by mutation of the human MLP gene.

As described above, in FHCM and FDCM, human ML
It is considered to be a genetic disease in which the P gene is mutated. FH
Mutations in the human MLP gene in CM and FDCM include full-length or partial deletion, amplification, translocation and the like of the gene, as well as point mutation. Such mutations cause human MLPs to malfunction in the heart and FHCM
Or it is thought to cause FDCM.

Therefore, the nucleotide sequences and probes according to the present invention described below are useful for diagnosis of the above-mentioned diseases.

Probe According to the present invention, (a) a nucleotide sequence containing a DNA sequence of at least 20 consecutive nucleotides in the sequence of SEQ ID NO: 1 and a sequence complementary thereto, and (b) a nucleotide sequence of (a) And a base sequence selected from base sequences that hybridize under stringent conditions with the present invention.

As the above sequence, for example, SEQ ID NO: 1 from No. 1 to 1135 (M1 partial cDNA), 575 to 287
No. 9 (M2 partial cDNA), No. 692-3763 (ML
P-8 partial cDNA), base sequences 3423 to 3442 (SEQ ID NO: 4), and base sequences 3513 to 3532 and their complementary sequences.

The nucleotide sequence of SEQ ID NO: 3 is a complementary strand of the nucleotide sequence of SEQ ID NO: 1. Therefore, the complementary strands of the above-mentioned base sequences are 2629 to 3763 of SEQ ID NO: 3, respectively.
No., 835-3189, 1-3072, 322-3
No. 41, and the nucleotide sequences of Nos. 232 to 251 (SEQ ID NO: 5).

In the present invention, “stringent conditions” in the hybridization refers to the Southern hybridization method, the PCR method (Examples 1 and 2) in which these sequences are used as probes and primers.
And 9), and hybridization conditions of the FISH method (Example 8).

According to the present invention, there is provided a probe comprising the above base sequence and a detectable label. The detectable label can be selected from those known to those skilled in the art, for example, interacting substances (eg, avidin and biotin), enzymes (eg, peroxidase, alkaline phosphatase), radioisotopes (eg, ³² P and ^35). S
Etc.), fluorescence (eg, FITC, europium), antigen (eg, digoxigenin) and the like.

The method for labeling the nucleotide sequence may be determined depending on the labeling molecule, and can be performed by, for example, nick translation, chemical (or photochemical) crosslinking, oligonucleotide chemical synthesis, chelation, or the like.

The method of detecting the label may be determined depending on the label molecule, for example, fluorescence, enzyme or ferritin-
The label can be detected using a labeled antibody, avidin-FITC, β-galactosidase, colloidal gold, or the like.

[0086] As described above, the MLP gene is
And FDCM.
Therefore, the nucleotide sequence and the probe according to the present invention (hereinafter, referred to as
These diseases can be diagnosed (including a presymptomatic diagnosis, such as a prenatal diagnosis) using a “probe”.

According to the present invention, there is also provided a diagnostic agent comprising the above-mentioned probe.

The diagnosis can be made by removing a gene sample (chromosome or genomic DNA, etc.) from the patient and measuring the degree of hybridization between the probe and the gene sample. Methods for measuring the degree of such hybridization include the FISH method,
Examples include the Southern hybridization method and the PCR method.

(1) FISH Method When the FISH method is used, the probe is hybridized with a sample in which chromosomes of cells (for example, lymphocytes) isolated from humans are fixed and the MLP gene of the chromosomes is mapped. In normal human cells, the MLP gene is mapped only to the q32.1 site (1q32.1) on the long arm of a pair of chromosomes 1, as described in the Examples below.

On the other hand, among the FHCM and FDCM patients, those in which no signal of the MLP gene is detected (ML
P gene is deleted), a patient whose MLP gene signal is strongly detected (MLP gene is amplified) or a patient whose MLP gene signal is detected at a site other than 1q32.1. Translocation) is found. Such a mutation (deletion, amplification or translocation) of the MLP gene is a characteristic change in any of the above diseases. Therefore, the disease can be diagnosed by measuring the degree of hybridization by the FISH method or detecting the position of the MLP gene on the chromosome.

The FISH method can be performed, for example, under the conditions described in Example 8. Actually, hybridization is carried out under the optimum conditions within these conditions, depending on the purpose of the experiment, the type of the probe and the DNA to be hybridized with the probe, and the like. The conditions of the FISH method are described in Heng HHQ et al., Proc. Natl. Acad. Sci. USA, 89,
9509-9513 (1992); Heng HHQ & Tsui LC, In situ
hybridization protocols: Methods in Molecular Biol
ogy. Choo KHA (ed) .Humana Press: New Jersey, pp.10
9-122 (1994) Choo, KHA, ed., Methods in Molecu
lar Biology: Insitu hybridization protocoles. (Cho
o, KHA, ed.). pp35-49 (1994) Humana Press, Cl
ifton, NJ., USA and Gerhard, DSet al., Proc. Nat
l. Acad. Sci. USA 78, 3755-3759 (1981).

(2) Southern hybridization method Southern hybridization (J. Sambrook et. Al.,
Molecular Cloning 2nd ed.Ch. 9 (1989) Cold Spring
When using Harbor Laboratory Press, New York, genomic DNA is isolated from cells (eg, lymphocytes) isolated from humans, digested with appropriate restriction enzymes, and then subjected to gel electrophoresis for digestion. Separated DNA fragments. Thereafter, the separated DNA fragment is transferred to a filter and hybridized with the probe on the filter. Among the above patients, hybridization is not observed as compared to healthy individuals, or even if it is observed, the amount of hybridization is small (part or all of the MLP gene is deleted). Patients with a large amount of hybridization (where the MLP gene is amplified) or patients whose DNA fragments to be hybridized with the probe differ in size and number from those of healthy individuals are found. In addition, by combining Southern hybridization with a human / rodent somatic cell hybrid panel, it is possible to detect the chromosomal location of the patient's MLP gene (Macera, MJet a).
l., Genomics 13,829-831 (1992)). Thereby, the presence or absence of MLP gene deletion or translocation in the patient can be detected.

Such mutation (deletion, amplification, translocation, etc.) of the MLP gene is a change characteristic of any of the above-mentioned diseases. Therefore, the disease can be diagnosed by measuring the pattern of the band that hybridizes with the probe by Southern hybridization.

When a DNA fragment is used as a probe, 2-6 × SSC (0.15 M sodium chloride solution,
0.015M sodium citrate solution), 65 ° C-70
Southern hybridization can be performed under the condition of ° C.

In practice, hybridization is carried out under optimum conditions according to the purpose of the experiment, the type of the probe and the DNA to be hybridized with the probe, and the like. The conditions for hybridization can be referred to “Gene manipulation manual” edited by Yasutaka Takagi, Kodansha, Tokyo, Japan (1982).

(3) PCR method A gene sample (chromosome or genomic DNA, etc.) is taken out from a patient, and P is used instead of Southern hybridization.
CR method (Saiki, RK et. Al., Science 239 (1988) 48
7-491), diagnosis can be performed by measuring the degree of amplification of a gene fragment. In the PCR method, a pair of primers (primer pair) is hybridized to DNA or RNA serving as a template, and a DNA fragment is amplified using a polymerase. The above probes can be paired and used as primers for PCR. By using such a primer pair and performing PCR using the extracted patient gene sample as a template, all or part of the MLP gene is amplified. By analyzing the amplified DNA by electrophoresis or nucleotide sequence analysis, mutation (deletion, amplification, recombination, translocation, base substitution, etc.) of the MLP gene can be detected.

For example, when a gene sample of a patient in which the MLP gene is deleted is applied to the PCR method, the specific region is not amplified. Therefore, deletion of the MLP gene can be detected by examining the presence or absence of the amplified fragment.

When a gene sample of a patient whose MLP gene is amplified is applied to the PCR method, the specific region is amplified more than in a healthy person. Therefore, amplification of the MLP gene can be detected by checking the amount of the amplified fragment.

The PCR method is described, for example, in Takara LA taq kit
Using ver.2, the reaction can be performed 30 times at 95 ° C for 15 seconds, 55 ° C for 30 seconds, and 72 ° C for 2 minutes. In practice, optimal conditions are selected according to the purpose of the experiment, the type of gene sample used as a probe or its template, etc.
I do. PCR conditions are described in Saiki, RK et. Al., Scie
nce, 239, 487-491 (1988).

Mutations in the MLP gene are characteristic of familial hypertrophic cardiomyopathy and familial dilated cardiomyopathy as described above. Therefore, the above diagnostic agents can be used as diagnostic agents for these diseases.

According to the present invention, there is provided a method for detecting a mutation in the MLP gene, which comprises hybridizing the probe with a mammalian gene sample and measuring the degree of hybridization. The above detection method can be carried out in the same manner as in the use mode of the above diagnostic agent.

[0102]

The present invention will be described in detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Example 1 Cloning of Human MLP cDNA
Myosin binding subunit (MBS) of Ning rat myosin phosphatase (sometimes called the regulatory subunit of smooth muscle protein phosphatase 1M or M130) (Chen, YH et al., FEBS Lett., 356:
51-55, (1994) cDNA fragment encoding the N-terminal region (1-698 amino acids) of GenBank ac.
en, YH et al. (1994).
No.4) as a probe to screen a human brain λgt10cDNA library (CLONTECH, Inc.) 1.0 × 10 ⁵ plaques.

The screening was performed according to J. Sambrook et. Al., M.
olecular Cloning: A Laboratory Manual: Cold Spring
Performed according to Habor Laboratory, Cold Spring Harbor, NY (1989). Specifically, it is as described below. 6 x SSC, 10 x Denhardt'ssolution, 1% SDS, s
Prehybridization was performed in sDNA at 200 μg / ml at 65 ° C. for 3 hours. DNA fragment is STRATAGENE Primi
³² P using e-it II Random PrimerLabeling Kit
Labeled. Using this as a probe, 6 × SSC,
10 × Denhardt's solution, 1% SDS, ssDNA 200 μg /
Hybridization was performed overnight at 65 ° C. in ml. The plate was washed four times at room temperature using 2 × SSC and 0.1% SDS. The analysis was performed using Fuji Bio-image Analyzer. The inserted DNA fragment was cut out from the positive clone, and the nucleotide sequence was determined. The nucleotide sequence is determined by Pharmaci
A double-stranded nested deletion kit was used to create an alignment deletion, and the deletion was performed using an ABI 377 DNA sequencer. The composition of the solution used above was 1 ×
SSC solution (0.15M NaCl, 15mM sodium citrate (pH7.
0)) and 1 × Denhardt's solution (0.02% ficoll, 0.02% polyvinylpyrrolidone 0.02% bovine serum albumin (BSA)).

As a result, positive clones M1 (the nucleotide sequence of nucleotides 1 to 1135 of SEQ ID NO: 1) and M2 (the nucleotide sequence of nucleotides 575 to 2879 of SEQ ID NO: 1) were obtained. Further, 1.0 × 10 ⁶ human brain λgt10 cDNA library (manufactured by CLONTECH) was screened using M2 as a probe.
Screening and nucleotide sequence were performed as described above. As a result, MLP-8 (692-376 of SEQ ID NO: 1)
No. 3 base sequence) was obtained.

By combining the above three clones, the nucleotide sequences of Nos. 1 to 3763 of SEQ ID NO: 1 were obtained. This nucleotide sequence encoded a protein consisting of 982 amino acids (amino acid sequence of Nos. 1 to 982 of SEQ ID NO: 2; hereinafter referred to as human MLP). The positions of the sequences are as shown in FIG.

As a result of a database search, the amino acid sequence of human MLP was a novel amino acid sequence which had not been reported before. The estimated molecular weight of MLP was 110.4 kDa.
In the amino acid sequence of MLP, an ankyrin repeat (AR) structure (amino acid sequence from No. 220 to No. 476 of SEQ ID NO: 1) is arranged on the N-terminal side, and a leucine zipper (L
Z) The structure (amino acid sequence at positions 885 to 982 of SEQ ID NO: 2) was present.

The deduced amino acid sequence of human MLP (amino acid sequence Nos. 1 to 982 of SEQ ID NO: 2) is the deduced amino acid sequence of rat MBS (Chen, YH et al., FEBS Lett.,
No. 356, 51-55 (1994), GenBank ac. No .: S74907) and deduced amino acid sequence of chicken MBS (Shimizu, H. et al., J. Biol. Chem. 26)
9, 30407-30411 (1994), GenBank ac.no .: D37985-D379
Amino acid sequence 1 to 963) described in 87, and 5
It showed 4% and 50% identity. Because of the low amino acid sequence identity, the human MLPs identified by the present inventors are similar proteins (homologs or isoforms) to human MBS corresponding to rat and chicken MBS, but do not match. Obviously (ie not the counterpart).

Example 2 Expression of recombinant MLP in E. coli
And preparation of recombinant protein N-terminal region of human MLP (MLP-N-terminal: 1 to 1 of SEQ ID NO: 2)
648 amino acid sequence) recombinant protein (rG-hML
P [1-648]) is a GST (glutathione S-transferase) fusion protein, and is used for E. coli BL21 (DE3) p
It was expressed in Lys-S and BL21 (DE3). In particular,
It is as follows.

First, an rG-hMLP [1-648] expression vector (pG
EX-hMLP [1-648]) (Fig. 2)
The target site of the cDNA fragment of MLP cloned in tII was amplified by PCR, purified from gel,
The DNA was reconstituted into one piece of DNA by the PCR method. This D
A restriction enzyme recognition site was inserted at the end of NA at the time of primer design, and ligation was performed with an expression vector (pGEX4T-1) using this restriction enzyme recognition site.

Specifically, a sense primer (5'-GAG) was prepared using M1 (base sequence from No. 1 to 1135 of SEQ ID NO: 1) cloned in pBluescript II as a template.
GCCGGAGCAATGGGGAGATG-3 ')
And antisense primer (5'-GGCAGCAG
[M1] PCR product was obtained by amplification by PCR method using CCACATGAAG-3 ′).

The PCR conditions used were as follows. Perkin Elmer Cetus as Thermal cycler
PJ2000-Model480 was used. TaKa as Taq polymerase
Ra LA PCR kit (LA Taq) was used. The total volume of the PCR reaction solution was 50 μl, and its composition was 1 μl of the template (about 10 μl).
0ng), sense primer 1μl (final concentration 0.5μl)
M), antisense primer 1 μl (final concentration
0.5μM), 10 × LAPCRBuffer 5μl, dNTP mixture
4 μl (final concentration 0.2 mM), LA Taq 0.5 μl (2.5
U), 27.5 μl of distilled water. The PCR cycle is
Pre-PCR was performed at 94 ° C. for 1 minute. The PCR was performed for 30 cycles of 94 ° C. for 30 seconds, 52 ° C. for 30 seconds, and 72 ° C. for 1 minute, and then a final extension was performed at 72 ° C. for 10 minutes.
Soaking was performed at 4 ° C.

The above PCR product was electrophoresed on a 0.8% agarose gel (using FMC Bioproduct, SeaKem GTG), the target band was cut out, and the QIAGEN QIAEX was used.
DNA was extracted with a II gel extraction kit and confirmed by agarose electrophoresis, and then used as [M1] PCR product.

Next, a sense primer (5'-C) was prepared using MLP-8 (base sequence Nos. 692 to 3763 of SEQ ID NO: 1) cloned in pBluescript II as a template.
TTCATGTGGCTGCTGCC-3 ') and antisense primer (5'-AGTAGACCTTC
[8A] PCR product was obtained by amplification by PCR using GGTTCTGCCGAGC-3 ′).

Next, the above-mentioned M1 and 8A were reconstituted into one DNA by applying the PCR method. The aforementioned sense primer (5'-GAGGCCGGAGCAATGGCGGA
ACTG-3 ') and antisense primer (5'
-AGTAGACCTTCGTGTCTGCCGGAGC
-3 ') was used. [M1] PCR as a template
Both the product and the [8A] PCR product were used. The composition of the reaction solution (49.5 μl total) was as follows:
1 μl of template [M1] (about 100 ng), 1 μl of template [8A]
l (about 100 ng), sense primer 1 μl (final concentration 0.5 μM), antisense primer 1 μl (final concentration 0.5 μM), 10 × LAPCR Buffer 5 μl, dNTP mix
ture 4μl (final concentration 0.2mM), distilled water 26.5μl
.

The above reaction solution was first incubated at 95 ° C. for 10 minutes, and cooled on ice immediately after completion. After cooling for 5 minutes, 0.5 μl of LA Taq was added, the mixture was stirred, and mineral oil was overlaid to perform a PCR cycle. PCR conditions were as described below. Pre-PCR was performed at 94 ° C. for 1 minute, and PCR was performed 30 cycles of 94 ° C. for 1 minute, 52 ° C. for 1.5 minutes, and 72 ° C. for 2 minutes.
After that, final extension was performed at 72 ° C. for 10 minutes. Soaking was performed at 4 ° C.

The above PCR product was subjected to electrophoresis on a 1% agarose gel (using FMC Bioproduct, SeaKem GTG), the target band was cut out, and QIAEXII g
The DNA was extracted with an el extraction kit and confirmed by agarose gel electrophoresis, and then the [MLP-N] PCR product (in this state, BamHI at the N-terminus of this DNA and Eco
RI restriction site is included). The obtained [MLP-N] was treated with restriction enzymes using BamH1 and EcoRI, and the resulting fragment was treated with BamH1 and EcoRI to obtain pGEX 4T-1 (Pharmacia Bi).
otech Cat. 27-4580-01)
A vector for expression of -hMLP [1-648] (pGEX-hMLP [1-648]) (FIG. 2) was constructed.

Escherichia coli was transformed with the above pGEX-hMLP [1-648], cultured in LB containing 50 mg / ml ABPC at 37 ° C. until OD600 = 0.8 to 1.0, and 0.1 mM IPTG was added. Incubated for 4 hours. Then, centrifuge at 6500 rpm × 10 minutes to precipitate the precipitate in PBS.
(About 30 ml). The precipitate was washed with a homogenizing buffer [30 mM Tris / HCl (pH 7.5), 50 mM EDTA, 1 mM EGT
A, 10% sucrose, 10 mg / ml leupeptin, 10 mM
(p-Amidinophenyl) methanesulfonyl fluoride
(aPMSF), 1 mM DTT] After dissolving in 20 ml, add tritonX-100
00 ml was added and crushed with a sonicator. Ultracentrifuge at 50,000 rpm for 30 minutes, and wash the supernatant with 30 mM Tris / HCl (pH 7.5), 30 mM KC
l, dialyzed against 0.2 mM DTT. Next, it was applied to a SP Sepharose column (size 1.6 × 11 cm) and extracted with a 0 to 0.5 M NaCl concentration gradient. Collect fractions containing protein of interest
After dialysis against 30 mM Tris / HCl (pH 7.5), 30 mM KCl, and 0.2 mM DTT, purification was performed using a Mono Q HR5 / 5 column. rG-hMLP [1-
648] was further purified by gel filtration using Superdex 200 HR 10/30. The purified recombinant protein is 30 mM Tris / H
After dialysis against Cl (pH 7.5), 30 mM KCl, 0.2 mM DTT, -8
Stored at 0 ° C.

To obtain a recombinant protein (rG-hMLP [652-863]) of the C-terminal region of human MLP (MLP-C-terminal: amino acid sequence from 652 to 863 of SEQ ID NO: 2) PCR was carried out according to the procedure to amplify the target cDNA.
After restriction enzyme treatment with EcoRI (TaKaRa), it was incorporated into pGEX4T-1 and transformed into BL21 (DE3). Then, rG-hMLP [1-
648], expressed, purified and stored.

Example 3 Preparation of Anti-Human MLP Antibody C-terminal region of human MLP (MLP-C terminal: 65 of SEQ ID NO: 2)
Rabbits were immunized using a recombinant fusion protein (rG-hMLP [652-863]) of GST with the amino acid sequence of amino acids 2 to 863) as an antigen to prepare anti-human MLP antiserum. Specifically, it was carried out according to the method described below.

For the first immunization, 50 mg of the above antigen and 1 ml of complete adjuvant (FCA) [manufactured by DIFCO] were mixed and stirred until an emulsion was obtained.
Each ml was injected subcutaneously. The second immunization was injected 3 weeks after the first immunization in the same manner as the first immunization. For the third and subsequent immunizations, 50 mg of antigen and incomplete adjuvant (FIA) [DIFCO
1 ml), stirred until an emulsion was obtained, and injected about 0.1 ml subcutaneously into the back of a New Zealand white female. Thereafter, the same injection was repeated twice every two weeks.

One week after the last injection,
Collect 50 ml of blood and store at 4 ° C for 24 hours.
The serum was collected by centrifugation for 1 minute and sterilized by filtration through a 0.45 μm filter. For antibody affinity purification, 1 mg of antigen (rG-hMLP [652-863]) was added to CNBr-activated Sepharose.
This was performed using a column coupled to 0.3 g of 4B (Pharmacia Biotech) (coupling ratio: 45%). Similarly, an affinity column of GST (1 mg) was prepared (in this case, the coupling ratio was 100%). First, 10 ml of the antiserum was reacted with a GST affinity column, and the serum from which the anti-GST antibody was removed was reacted with the affinity column to which rG-hMLP [652-863] was coupled (24 hours at 4 ° C.).
Tumble and mix). Then 10 ml TBS, then 30 ml wash buffer (20 mM Tris / HCl pH7.5, 1 M NaCl, 1% trit
on X-100) and another 30 ml of Tris-buffered saline (TBS
) And wash with elution buffer (0.1M glycine / HCl pH
2.5) The antibody was eluted with 1.5 ml. This elution was repeated four times. The eluted antibody was neutralized with 2M Tris / HCl pH 8.0.

Example 4 Western blotting method
Examination of the tissue distribution of MLP expression by using Western blotting using the anti-human MLP antibody prepared in Example 3 was performed to examine in which tissues the human MLP was expressed. Specifically, it was carried out according to the method described below.

3-4 g of each of human brain, lung, heart, aorta (each autopsy specimen) and chicken gizzard were mixed with a homogenizing buffer [500 mM NaCl, 20 mM Tris / HCl (pH 7.
5), 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine, 1
0 mg / ml leupeptin, 10 mM aPMSF, 0.1 mM diisopropylfluorophosphate], 3 ml, homogenized, ultracentrifuged at 20,000 xg for 30 minutes, and the supernatant was subjected to BCA method (PIER
(Manufactured by CE)). This was converted to SDS and applied to a 7.5% acrylamide gel at 100 μg each. After electrophoresis, the cells were transferred to a nitrocellulose membrane, and reacted with an anti-human MLP antibody as a primary antibody. After reaction with the secondary antibody, detection was performed by the ECL method (manufactured by PIERCE).

The results were as shown in FIG. A 125 kDa band cross-reactive with anti-MLP antibody was detected in human brain and human heart. This 125kDa
Band is considered to correspond to human MLP. on the other hand,
A 135 kDa band was found in human brain, lung, heart and aorta, and a 130 kDa band was found in human lung and aorta.
Cross-reactive with MLP antibody. These bands also show cross-reactivity with the anti-myosin binding subunit (MBS) antibody (data not shown), and are presumed to correspond to human MBS. From the above, it was revealed that human MLP is specifically expressed at least in the brain and heart, but not in the lung and aorta. Thus, human M
The expression pattern of LP is ubiquitous (ubiquitou) of MBS.
s) Expression was distinctly different.

The anti-human MLP antibody also crossed the 130 kDa band (corresponding to chicken MBS) in chicken gizzard, which is rich in vascular smooth muscle.

Example 5 By Northern blotting
Examination of tissue distribution of MLP expression by using a DNA fragment corresponding to the nucleotide sequence of Nos. 2033 to 3087 of SEQ ID NO: 1 as a probe, Human Mult of CLONTECH
Northern blotting analysis was performed using iple Tissue Northern Blot. DNA fragment was purchased from STRATAGENE
Use the mie-it II RandomPrimer Labeling Kit to label the probe with ³² P, and use this probe with 50% formamide, 5 × SSC, 10 × Denhardt's solution, 1% SD
S, 200 μg / ml of ssDNA, 50 mM phosphate buffer, at 42 ° C.,
Hybridization was performed overnight. 2 x SSC, 0.1
Wash once at room temperature with 2% SDS, 0.1% SD
2 times at 42 ° C using S, using 0.2 × SSC, 0.1% SDS
Washed twice at ° C. Analysis was performed using the Fuji Bio-image Analyzer
This was performed using

The results were as shown in FIG. About 12kb
Transcript (human MLP mRNA) is expressed in the heart,
Very strong in skeletal muscle, very weak in brain, placenta and pancreas, but not in lung, liver and kidney. The results of Northern blotting and Western blotting (Example 4) revealed that human MLP is expressed not only as mRNA but also as a protein at least in the heart and brain. From the results of Northern blotting, expression of human MLP mRNA was observed in skeletal muscle, but further investigation is required to determine whether MLP is expressed as a protein in skeletal muscle.

Meanwhile, it has already been reported that myosin-binding protein (MBS), a homolog of human MLP, is widely expressed not only in smooth muscle but also in organs such as myocardium, spleen, brain, lung and kidney ( Miyadera, K. et
al., Cancer Res., 55, 1687-1690 (1995)). From this, it is clear that the human MLP identified by the present inventors is not a human counterpart of MBS but a homolog or an isoform.

Example 6 PP1 catalytic subunit (PP1c
δ) and phosphatase activity in human MLP reconstruction
The holoenzyme for detection myosin light chain phosphatase consists of three subunits: a catalytic subunit (PP1cδ), a 130 kDa regulatory subunit (M130), and a 20 kDa regulatory subunit (M20). Among these, the 130 kDa regulatory subunit (M130) is synonymous with the myosin binding subunit (MBS) or the smooth muscle protein phosphatase 1M regulatory subunit (Shimizu, H. et al., J. Biol. Chem. 269). , 30407-304
11 (1994) and Chen, YH et al., FEBS Lett., 356, 5
1-55 (1994).).

The present inventors investigated whether MLP increases myosin light chain phosphatase activity when mixed with PP1cδ. The phosphatase assay is described in Ichikawa K. et al., Biochemistry, 35, 6313-632.
0 (1996) and performed as described below. A total of 40 μl of the reaction buffer (37.5 mM Tris / HCl (pH 7.
5) In 125 mM KCl, 0.25 mg / ml BSA), PP1cδ (1.38
ng) and rG-hMLP [1-648] (up to 5 times the molar ratio with respect to PP1cδ), and pre-incubated at 30 ° C. for 10 minutes. 5 μl of a mixed solution of 10 mM ATP and 10 mM MgCl ₂
(Final concentration 1 mM ATP, 1 mM MgCl ₂ ) and add another ³² PL
C20 (5 mM) was added to make a total of 50 μl. Reaction solution
After incubation at 0 ° C for 5 minutes, the phosphatase activity was measured (Ichikawa K. et al., Biochemistor
y, 35, 6313-6320 (1996)). Reaction was performed with 50% 25% TCA.
The reaction was stopped by adding μl. The mixture was centrifuged at 5000 rpm for 5 minutes, and the supernatant was measured by the Cherenkov method. PP1cδ, and
³² P-LC20 was purified from chicken gizzard smooth muscle (Sh
imizu, H. et al., J. Biol. Chem., 269, 30407-30411
(1994)).

As a result, a fusion protein of the N-terminus of human MLP and GST (rG-hMLP [1-648]) (Example 2)
Increased myosin light chain phosphatase activity of 1cδ.
The degree of activation was up to about 4-fold (FIG. 5).

Example 7 [0133] Using a two-hybrid method,
Detection of binding between activated Mho and Rho protein Two hybrid system using yeast (Vojtek,
AB et. Al., Cell, 74, 205-214 (1993)), co-expressed human MLP and active Rho protein in yeast, and found that human MLP binds to active Rho protein. The active Rho protein binding region in the MLP was determined. Specifically, it was carried out according to the method described below.

Various lengths of partial cDNA fragments encoding the C-terminal region of the human MLP protein were amplified by PCR using MLP-8 (FIG. 1) as a template and the following as primers. The prepared MLP partial cDNA fragment was amplified using the primers described below. Partial cDN encoding amino acids 613 to 982
The A fragment is a primer 5 'TTG CGG CCG CTA AGG GAG AAG A
GG AGG TCC TAT C and 5'TAG CGG CCG CAA CTT GGA CAG TT
Using T GCT GAT GAC, a partial cDNA fragment encoding amino acids 613 to 713 was synthesized using primer 5 ′ TTG CGG C
CG CTA AGG GAGAAG AGG AGG TCC TAT C and 5'ATC TAG CGG
CCG CAA ATC CAG ACT CCT GCC CCA GGG CTG CTG GC
Is used to encode amino acids 613-813
NA fragment is primer 5 'TTG CGG CCG CTA AGG GAG AAG
AGG AGG TCC TAT C and 5 'ATC TAGCGG CCG CAA CCA GAA
Using ATT GAT GCC TGT GCC TCT TCG TC, a partial cDNA fragment encoding amino acids 613 to 913 was prepared using primers 5 ′ TTG CGG CCG CTAAGG GAG AAG AGG AGG TCC TA
TC and 5 'ATC TAG CGG CCG CAA CTT GGA CTT TAT ATC TG
Using C TAG CTC TA 3 ', amino acids 713-982
The partial cDNA fragment encoding No. 5 is primer 5 'CAA T
TG CGG CCG CTA GAT GAA GAG CCT ATC TGT CAT CGC and TA
Using G CGG CCG CAA CTT GGA CAG TTT GCT GAT GAC, partial cDN encoding amino acids 813 to 982
A fragment is primer 5 'CAA TTG CGG CCGCTA TGG ACA AA
G GAT GAG GAT GAA ACT and 5 'TAG CGG CCG CAA CTT GGA
Using CAG TTTGCT GAT GAC, amino acids 913-982
The partial cDNA fragment encoding No. 5 is primer 5 'CAA T
TG CGG CCG CTA AAG CTT GAG AAG GTG GCC CAG CAG and 5 '
Each was amplified using TAG CGG CCG CAA CTT GGA CAG TTT GCT GAT GAC. Each of the obtained cDNA fragments was converted to plasmid pVP16 (Vojtek, AB et. Al. (1993) supra).
Was inserted into the NotI site located upstream of the VP16 transcription activation region sequence to prepare a vector for expressing the MLP-VP16 fusion protein.

Wild type RhoA protein or activated R
A vector for expressing a fusion protein of hoA protein (Rho ^Val14 ) and LexA is RhoA or Rh
o A construct constructed by inserting the ^Val14 cDNA in-frame upstream of the LexA DNA binding region sequence of the plasmid pBTM116 (Vojtek, AB et. al. (1993), supra) (Shibata, H. et al., FEBS Lett., 385, 22
1-224 (1996)). Wild-type H-Ras protein or active H-Ras protein (H-Ras
^A vector for expressing a fusion protein of ^Val12 ) and LexA is a plasmid pBTM116 (Vojtek, AB, supra).
et. al. (1993)), which was constructed by inserting H-Ras or H-Ras ^Val12 in-frame upstream of the LexA DNA binding region sequence.

In order to co-express the above VP16 fusion protein expression vector and LexA fusion protein expression vector in yeast, they were expressed in budding yeast (S. cerev
isiae) L40 strain (Mat a trp1 leu2 his3 ade2 LYS
2: :( LexAop) 4-HIS3 URA3: :( LexAop) 8-LacZ), LiCL
(Ito, H. et. Al. (1983). J. Bacteriol., 153: 163
-168). The transfected yeast strain was cultured on a selective medium (Leu-, Trp-, His-) and the histidine requirement was examined.

The results were as shown in FIG. Human M
Amino acids 613 to 982, 713 to 982 of LP,
Nos. 813 to 982 or 913 to 982 and RhoA
It was found that the yeast strain co-expressed with ^Val14 grows on the selective medium. In comparison, amino acids 613-913, 613-813 and 6 of human MLP
In the 13th to 713th, the degree of growth was weak. In addition, yeast strains in which wild-type H-Ras or wild-type Rho coexisted with any partial fragment of human MLP did not grow on the selective medium.

From the above results, it was found that human MLP was activated Rh
It was found to bind to oA, and that its active RhoA binding region exists within amino acids 613-913. Furthermore, in the partial fragment of human MLP lacking amino acid 813 and subsequent amino acids, the active RhoA binding activity is significantly attenuated.
Portions 13 to 982 were shown to play an important role in binding.

By the way, within this region of human MLP,
Leucine zipper (LZ) structure (885 to SEQ ID NO: 2)
982) (Example 1). It has also been reported that the LZ structure plays an important role in binding to active RhoA in other active Rho-binding proteins (protein kinase N) (Shibat
a, H. et al., FEBS Lett., 385, 221-224 (1996)). These facts suggest that the LZ structure plays an important role in binding to activated RhoA also in human MLP.

Example 8 Fluorescence in situ hybridization
Of the human MLP locus by FISH analysis (FISH)
Analysis was performed. Lymphocytes isolated from human blood can be obtained by adding 10% fetal bovine serum and phytohemagglutinin (PHA) to α-
The cells were cultured at 37 ° C. in a minimum essential medium (MEM) for 68 to 72 hours. The cultured lymphocytes were treated with BrdU (0.18 mg / ml Sigma) and synchronized. Synchronized lymphocytes were washed three times in serum-free medium and thymidine (2.5 mg / ml Sigma) containing α-M
The cells were cultured in EM at 37 ° C for 6 hours. The cells were collected to prepare a slide. CDNA clone M containing C-terminal sequence of human MLP
LP-8 (base sequence Nos. 692 to 3763 of SEQ ID NO: 1)
Was biotinylated using a BRL BioNick labeling kit (Heng, HHQ, Pro. Nati. Acad. Sci. USA, 89, 9509-9).
513 (1992)). The method of detecting FISH is described in Heng et al., (19)
92) and Heng, HHQ & Tuji, L.-C., Chromosoma, 102, 32
5-332 (1993). Slides were heat treated at 55 ° C for 1 hour. After RNase treatment, slides were denatured with 2XSSC containing 70% formamide for 2 minutes and dehydrated with ethanol at 70 ° C. Probes were denatured in hybridization solution (50% formamide, 10% dextran sulfate) at 75 ° C. for 5 minutes. The probe was placed on a denatured chromosome slide and placed at 37 ° C. overnight. The slides were washed three times with 50% formamide and 2 × SSC for 3 minutes, and then washed with 2 × SSC
And washed three times at 43 ° C. for 3 minutes, and a signal was detected. The FISH signal and DAPI band pattern were separately photographed and superimposed to determine their chromosomal location. As a result of FISH analysis, signals were detected on a set of chromosomes in 93 chromosomes out of 100 dividing chromosomes. Based on the DAPI band, the signal was found to be on the long arm of chromosome 1. Detailed chromosomal locations were determined by combining the results of the ten photographs (FIG. 7). The human MLP gene is q32.1 on chromosome 1.
Was found to exist.

Example 9 Radiation of human MLP gene
Determination of chromosomal location using Panel To determine the chromosomal location of the human MLP gene,
Research Genetics GeneBridge 4 Radiation Hybri
d Analysis was performed using Panel. Primers specific to human MLP, CAG CTA CTT, using DNA 25ng as template
TGT CCA CAT TG (base sequence from 3423 to 3442 of SEQ ID NO: 1; similar to SEQ ID NO: 4) and AAG ACT CAC CTA TAT
PCR was performed using CAG AG (the nucleotide sequence of 232 to 251 in SEQ ID NO: 3; the same as in SEQ ID NO: 5) and determined. PCR
Was prepared according to the method described in Examples 1 and 2 by Takara LA
Using PCR kit Ver.2, 98 ℃ 15 seconds, 55 ℃ 30 seconds, 72 ℃ 2
The minute was repeated 30 times. The reaction solution was subjected to 8% polyacrylamide gel electrophoresis. As a result, a human MLP-specific
The DNA fragment had been amplified. WI / MIT Radiati
on Hybrid Mapper (http: // www- genome.wi.mit.ed
u / cgi-bin / contig / rhmapper.pl # instructions).

As a result, the MLP was 3.98 to WI-9272.
4.29cR was found to be on the telomere side.

[0143]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 3763 Sequence type: nucleic acid Number of strands: single strand Topology: linear Antisense: NO Sequence type: cDNA to mRNA Sequence characteristics Characteristic symbol: CDS Location: 164 . . 3112 SEQ GAGGATCCGG GTACCATGGG GCGGGAGGAG TAAAGATGGC GGCGCGAGGG TCTCCGCCCT 60 CTGCTCCGGG CTGAAGCGCT CTGAGAGAGG CGGCAGCGGC AACTCGAGCC CCAACAGTAA 120 TTTAGTGTTG GTAGTTTTGG CAGCAGCTGC CGAGGCCGGA GCA ATG GCG GAA CTG 175 Met Ala Glu Leu 1 GAG CAC CTA GGA GGG AAG CGG GCA GAG TCG GCG CGA ATG CGG CGG GCA 223 Glu His Leu Gly Gly Lys Arg Ala Glu Ser Ala Arg Met Arg Arg Ala 5 10 15 20 GAG CAG CTT CGG CGC TGG CGG GGC TCG CTG ACA GAG CAG GAG CCT GCG 271 Glu Gln Leu Arg Arg Trp Arg Gly Ser Leu Thr Glu Gln Glu Pro Ala 25 30 35 GAG CGA CGA GGC GCG GGG CGG CAG CCG CTG ACC AGG CGC GGG AGC CCC 319 Glu Arg Arg Gly Ala Gly Arg Gln Pro Leu Thr Arg Arg Gly Ser Pro 40 45 50 AGG GTC CGC TTC GAG GAC GGT GCT GTC TTT CTG GCC GCC TGC TCT AGC 367 Arg Val Arg Phe Glu Asp Gly Ala Val Phe Leu Ala Ala Cys Ser Ser 55 60 65 GGG GAC ACC GAC GAG GTG AGA AAG CTT CTG GCA AGA GGT GCT GAT ATC 415 Gly Asp Thr Asp Glu Val Arg Lys Leu Leu Ala Arg Gly Ala Asp Ile 70 75 80 AAC ACG GTC AAC GTG GAC GGC TTG ACA GCC CTG CAC CAG GCA TGT ATT 463 Asn Thr Val Asn Val Asp Gly Leu Thr Ala Leu His Gln Ala Cys Ile 85 90 95 100 GAT GAA AAT TTG GAC ATG GTG AAG TTT CTG GTG GAG AAC AGA GCC AAT 511 Asp Glu Asn Leu Asp Met Val Lys Phe Leu Val Glu Asn Arg Ala Asn 105 110 115 GTA AAC CAG CAA GAC AAC GAG GGC TGG ACA CCC CTT CAT GCA GCA GCT 559 Val Asn Gln Gln Asp Asn Glu Gly Trp Thr Pro Leu His Ala Ala Ala 120 125 130 TCC TGT GGC TAT CTC AAC ATA GCA GAG TAT TTC ATT AAT CAC GGA GCC 607 Ser Cys Gly Tyr Leu Asn Ile Ala Glu Tyr Phe Ile Asn His Gly Ala 135 140 145 AGT GTA GGT ATT GTC AAT AGT GAA GGT GAA GTT CCC TCT GAC CTT GCA 655 Ser Val Gly Ile Val Asn Ser Glu Gly Glu Val Pro Ser Asp Leu Ala 150 155 160 GAA GAG CCA GCC ATG AAG GAT CTT CTT CTG GAG CAA GTA AAG AAG CAA 703 Glu Glu Pro Ala Met Lys Asp Leu Leu Leu Glu Gln Val Lys Lys Gln 165 170 175 180 GGA ATT GAT CTA GAG CAG TCA AGA AAA GAA GAA GAG CAG CAG ATG TTG 751 Gly Ile Asp Leu Glu Gln Ser Arg Lys Glu Glu Glu Gln Gln Met Leu 185 190 195 CAG GAT GCC CGC CAG TGG CTC AAC AG T GGG AAA ATA GAG GAT GTG AGG 799 Gln Asp Ala Arg Gln Trp Leu Asn Ser Gly Lys Ile Glu Asp Val Arg 200 205 210 CAG GCT CGC TCA GGG GCT ACA GCC CTT CAT GTG GCT GCT GCC AAG GGC 847 Gln Ala Arg Ser Gly Ala Thr Ala Leu His Val Ala Ala Ala Lys Gly 215 220 225 TAC TCT GAA GTC CTC AGA CTT TTA ATT CAG GCT GGC TAT GAA CTC AAT 895 Tyr Ser Glu Val Leu Arg Leu Leu Ile Gln Ala Gly Tyr Glu Leu Asn 230 235 240 GTT CAG GAT TAT GAT GGC TGG ACT CCC CTC CAT GCT GCT GCA CAC TGG 943 Val Gln Asp Tyr Asp Gly Trp Thr Pro Leu His Ala Ala Ala His Trp 245 250 255 260 GGA GTG AAG GAG GCT TGC TCC ATC CTG GCA GAA GCA CTT TGT GAC ATG 991 Gly Val Lys Glu Ala Cys Ser Ile Leu Ala Glu Ala Leu Cys Asp Met 265 270 275 GAT ATT CGA AAT AAA CTG GGC CAG ACA CCA TTT GAT GTG GCT GAT GAG 1039 Asp Ile Arg Asn Lys Leu Gly Gln Thr Pro Phe Asp Val Ala Asp Glu 280 285 290 GGT CTC GTG GAG CAT TTG GAG TTG CTC CAG AAG AAG CAG AAT GTG CTT 1087 Gly Leu Val Glu His Leu Glu Leu Leu Gln Lys Lys Gln Asn Val Leu 295 300 305 CGA AGT GAA AAG GAG ACA CGG AAT AAA CTC ATT GAG TCA GAT CTG AAC 1135 Arg Ser Glu Lys Glu Thr Arg Asn Lys Leu Ile Glu Ser Asp Leu Asn 310 315 320 AGC AAG ATT CAG AGT GGG TTC TTT AAG AAC AAA GAG AAG ATG CTC TAT 1183 Ser Lys Ile Gln Ser Gly Phe Phe Lys Asn Lys Glu Lys Met Leu Tyr 325 330 335 340 GAG GAG GAG ACA CCT AAG TCC CAA GAA ATG GAG GAA GAA AAT AAA GAA 1231 Glu Glu Glu Thr Pro Lys Ser Gln Glu Met Glu Glu Glu Asn Lys Glu 345 350 355 TCT AGT AGC TCC AGC TCA GAG GAG GAG GAA GGT GAA GAT GAA GCT TCT 1279 Ser Ser Ser Ser Ser Ser Glu Glu Glu Glu Gly Glu Asp Glu Ala Ser 360 365 370 GAG TCA GAA ACT GAG AAG GAG GCA GAT AAA AAG CCA GAA GCC TTT GTC 1327 Glu Ser Glu Thr Glu Lys Glu Ala Asp Lys Lys Pro Glu Ala Phe Val 375 380 385 AAT CAT TCC AAC TCT GAA AGC AAG AGT AGT ATC ACA GAG CAG ATA CCA 1375 Asn His Ser Asn Ser Glu Ser Lys Ser Ser Ile Thr Glu Gln Ile Pro 390 395 400 GCA CCA GCT CAA AAT ACC TTC TCT GCC TCT TCT GCT AGG AGG TTC TCT 1423 Ala Pro Ala Gln Asn Thr Phe Ser Ala Ser Ser Ala Arg Arg Phe Ser 405 410 415 420 TCT GGC CTT TTT AAC AAG CCA GAA GAG CCC AAA GAT GAA TCT CCT TCT 1471 Ser Gly Leu Phe Asn Lys Pro Glu Glu Pro Lys Asp Glu Ser Pro Ser 425 430 435 435 TCA TGG AGA TTG GGA CTG AGA AAA ACT GGC AGC CAC AAC ATG CTG AGT 1519 Ser Trp Arg Leu Gly Leu Arg Lys Thr Gly Ser His Asn Met Leu Ser 440 445 450 GAG GTG GCC AAT TCC AGG GAA CCT ATA AGG GAC CGA GGC TCT TCC ATC 1567 Glu Val Ala Asn Ser Arg Glu Pro Ile Arg Asp Arg Gly Ser Ser Ile 455 460 465 TAT CGC TCC TCT TCA AGC CCT CGA ATT TCT GCT CTA CTG GAC AAC AAA 1615 Tyr Arg Ser Ser Ser Ser Pro Arg Ile Ser Ala Leu Leu Asp Asn Lys 470 475 480 GAT AAG GAG AGA GAA AAC AAA AGC TAT ATT AGT TCA CTA GCA CCC CGG 1663 Asp Lys Glu Arg Glu Asn Lys Ser Tyr Ile Ser Ser Leu Ala Pro Arg 485 490 495 500 AAG CTC AAC AGC ACA AGT GAT ATT GAA GAA AAG GAG AAC AGA GAA TCA 1711 Lys Leu Asn Ser Thr Ser Asp Ile Glu Glu Lys Glu Asn Arg Glu Ser 505 510 515 GCT GTT AAT CTA GTG AGG AGT GGC TCC TAT ACC CGG CAG CTA TGG AGG 1759 Ala Val Asn Leu Val Arg Ser Gly Ser Tyr Thr Arg Gln Leu Trp Arg 520 525 530 GAT GAA GCA AAA GGA AAT GAA ATC CCA CAG ACA ATT GCT CCC TCC ACC 1807 Asp Glu Ala Lys Gly Asn Glu Ile Pro Gln Thr Ile Ala Pro Ser Thr 535 540 545 545 TAT GTA TCA ACT TAC TTG AAA AGG ACT CCT CAC AAA TCC CAG GCC GAC 1855 Tyr Val Ser Thr Tyr Leu Lys Arg Thr Pro His Lys Ser Gln Ala Asp 550 555 560 ACA ACA GCA GAG AAA ACA GCA GAC AAT GTC TCT TCT AGC ACC CCG CTC 1903 Thr Thr Ala Glu Lys Thr Ala Asp Asn Val Ser Ser Ser Thr Pro Leu 565 570 570 575 580 TGT GTG ATC ACC AAT CGC CCT CTT CCT AGC ACT GCC AAT GGG GTT ACA 1951 Cys Val Ile Thr Asn Arg Pro Leu Pro Ser Thr Ala Asn Gly Val Thr 585 590 595 GCT ACT CCT GTG CTC TCC ATT ACT GGA ACA GAT TCC TCT GTG GAA GCC 1999 Ala Thr Pro Val Leu Ser Ile Thr Gly Thr Asp Ser Ser Val Glu Ala 600 605 610 AGG GAG AAG AGG AGG TCC TAT CTG ACT CCT GTA CGG GAT GAG GAA GCA 2047 Arg Glu Lys Arg Arg Ser Tyr Leu Thr Pro Val Arg Asp Glu Glu Ala 615 620 625 GAG TCT TTA CGG AAA GCA CGC TCC AGA CAA GCT CGG CAG ACA CGA AGG 2095 Glu Ser Leu Arg Lys Ala Arg Ser A rg Gln Ala Arg Gln Thr Arg Arg 630 635 640 TCT ACT CAA GGT GTC ACC CTA ACA GAC CTT CAA GAA GCA GAA AGG ACA 2143 Ser Thr Gln Gly Val Thr Leu Thr Asp Leu Gln Glu Ala Glu Arg Thr 645 650 655 655 660 TTC AGC CGG TCG AGG GCA GAG AGG CAA GCT CAG GAG CAG CCT CGT GAG 2191 Phe Ser Arg Ser Arg Ala Glu Arg Gln Ala Gln Glu Gln Pro Arg Glu 665 670 675 AAG CCC ACA GAC ACT GAA GGG CTT GAG GGG AGC CCT GAG AAG CAT GAG 2239 Lys Pro Thr Asp Thr Glu Gly Leu Glu Gly Ser Pro Glu Lys His Glu 680 685 690 CCC TCA GCA GTT CCA GCA ACA GAA GCT GGG GAG GGC CAG CAG CCC TGG 2287 Pro Ser Ala Val Pro Ala Thr Glu Ala Gly Glu Gly Gln Gln Pro Trp 695 700 705 GGC AGG AGT CTG GAT GAA GAG CCT ATC TGT CAT CGC CTG AGG TGC CCA 2335 Gly Arg Ser Leu Asp Glu Glu Glu Pro Ile Cys His Arg Leu Arg Cys Pro 710 715 720 GCT CAG CCA GAC AAA CCC ACC ACG CCA GCA TCT CCT TCT ACG TCA AGA 2383 Ala Gln Pro Asp Lys Pro Thr Thr Pro Ala Ser Pro Ser Thr Ser Arg 725 730 735 740 740 CCC TCA CTC TAC ACC AGT TCC CAC CTG CTA TGG ACA AAT AGA TTT TCA 2431 Pro Ser Le u Tyr Thr Ser Ser His Leu Leu Trp Thr Asn Arg Phe Ser 745 750 755 GTC CCT GAT TCT GAG AGT TCA GAG ACT ACC ACA AAC ACT ACA ACT GCA 2479 Val Pro Asp Ser Glu Ser Ser Glu Thr Thr Thr Asn Thr Thr Thr Ala 760 765 770 AAG GAA ATG GAC AAA AAT GAG AAT GAA GAA GCA GAT TTG GAT GAG CAG 2527 Lys Glu Met Asp Lys Asn Glu Asn Glu Glu Ala Asp Leu Asp Glu Gln 775 780 780 785 TCC TCT AAG AGG CTG TCC ATC CGA GAG AGG CGG CCC AAG GAA CGA 2575 Ser Ser Lys Arg Leu Ser Ile Arg Glu Arg Arg Arg Pro Lys Glu Arg 790 795 800 CGA AGA GGC ACA GGC ATC AAT TTC TGG ACA AAG GAT GAG GAT GAA ACT 2623 Arg Arg Gly Thr Gly Ile Asn Phe Trp Thr Lys Asp Glu Asp Glu Thr 805 810 815 820 820 GAT GGC TCT GAA GAG GTC AAA GAA ACG TGG CAT GAA AGA CTT TCT AGG 2671 Asp Gly Ser Glu Glu Val Glu Val Lys Glu Thr Trp His Glu Arg Leu Ser Arg 825 830 835 TTG GAA TCG GGA GGT AGT AAT CCT ACA ACC AGT GAT TCT TAC GGT GAC 2719 Leu Glu Ser Gly Gly Ser Asn Pro Thr Thr Ser Asp Ser Tyr Gly Asp 840 845 850 CGG GCT TCA GCA AGA GCC CGT CGG GAG GCC CGG GAG GCC CGC CTA GCC 2767 Arg Ala Ser Ala Arg Ala Arg Arg Glu Ala Arg Glu Ala Arg Leu Ala 855 860 865 ACC CTG ACC AGC CGT GTA GAA GAA GAC AGC AAC AGA GAT TAT AAA AAA 2815 Thr Leu Thr Ser Arg Val Glu Glu Asp Ser Asn Arg Asp Tyr Lys Lys 870 875 880 CTC TAT GAG AGT GCT CTG ACT GAA AAC CAA AAA CTG AAA ACA AAA CTT 2863 Leu Tyr Glu Ser Ala Leu Thr Glu Asn Gln Lys Leu Lys Thr Lys Leu 885 890 895 900 CAG GAA GCC CAG CTA GAG CTA GCA GAT ATA AAG TCC AAG CTT GAG AAG 2911 Gln Glu Ala Gln Leu Glu Leu Ala Asp Ile Lys Ser Lys Leu Glu Lys 905 910 915 GTG GCC CAG CAG AAA CAA GAA AAG ACC TCT GAC CGA TCA TCA GTG CTG 2959 Val Ala Gln Gln Lys Gln Glu Lys Thr Ser Asp Arg Ser Ser Val Leu 920 925 930 GAG ATG GAG AAA CGG GAG AGG CGA GCC TTG GAG CGC AAA ATG TCA GAA 3007 Glu Met Glu Lys Arg Glu Arg Arg Ala Leu Glu Arg Lys Met Ser Glu 935 940 945 ATG GAG GAA GAA ATG AAG GTG TTA ACA GAA CTG AAA TCC GAC AAC CAG 3055 Met Glu Glu Glu Met Lys Val Leu Thr Glu Leu Lys Ser Asp Asn Gln 950 955 960 AGG CTG AAA GAT GAA AAT GGT GCC CTC ATC AGA GTC ATC AGC AAA CTG 3103 Arg Leu Lys Asp Glu Asn Gly Ala Leu Ile Arg Val Ile Ser Lys Leu 965 970 975 980 TCC AAG TAG GCTAGGCTCC AGATTTATGA GGAAAGAAAG GGACAGCATT 3152 Ser Lys * TGCTGCCCCC ACCCCTCTTT TCCAGTCCTT GCCTTCCAAC CAAAAGAAAT GGATGTTTTG 3212 GTGGAAGGAC ACTTCTTTCT ATCACCCTCT TTAGTCACCT CTATACACTC TACATTTTCT 3272 CTGCACTTTC AATGCCCTGT TCTTCCAAAC CCCTATCCCA AGTTTTATGA CAGTTTTAAT 3332 TGAAGCATGA TTGTGGTAAT TCGAGCCATC TGGAGAATGC TCTGGGGAGT ACACCAGGCT 3392 CAGCTGTGGA CCCCTCAACT TCCTGCTGCT CAGCTACTTT GTCCACATTG GATTTGGTCC 3452 AAACATGTAA GACTTCTACC CTAATCAGTA TCCTTCAGCT TTTTACATTA ACCCAGTGTC 3512 CTCTGATATA GGTGAGTCTT GTGGTAGCCA CTCCAGGATC CTGATTGGGG TGCCAAGAGA 3572 AACAGCAGGA TGTTGAATTG ATCATCAGAT GCCCTCTGGA ATGGTTAGCA TCCAAGGTGA 3632 CAGTGACTGC ATTGAGGCGC TCTATTCTTC TTCACCTCTC AGGAACTGAC TTTTATTTTT 3692 TCTATCAACA CCCAGTAATC TCCCTAACTA GTTTTAACCC TTATTCCTCC CTCATACCTA 3752 GCCATTTCCC G 3763

SEQ ID NO: 2 Sequence length: 982 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: protein sequence Met Ala Glu Leu Glu His Leu Gly Gly Lys Arg Ala Glu Ser Ala Arg 1 5 10 15 Met Arg Arg Ala Glu Gln Leu Arg Arg Trp Arg Gly Ser Leu Thr Glu 20 25 30 Gln Glu Pro Ala Glu Arg Arg Gly Ala Gly Arg Gln Pro Leu Thr Arg 35 40 45 Arg Gly Ser Pro Arg Val Arg Phe Glu Asp Gly Ala Val Phe Leu Ala 50 55 60 Ala Cys Ser Ser Gly Asp Thr Asp Glu Val Arg Lys Leu Leu Ala Arg 65 70 75 80 Gly Ala Asp Ile Asn Thr Val Asn Val Asp Gly Leu Thr Ala Leu His 85 90 95 Gln Ala Cys Ile Asp Glu Asn Leu Asp Met Val Lys Phe Leu Val Glu 100 105 110 Asn Arg Ala Asn Val Asn Gln Gln Asp Asn Glu Gly Trp Thr Pro Leu 115 120 125 His Ala Ala Ala Ser Cys Gly Tyr Leu Asn Ile Ala Glu Tyr Phe Ile 130 135 140 Asn His Gly Ala Ser Val Gly Ile Val Asn Ser Glu Gly Glu Val Pro 145 150 155 160 Ser Asp Leu Ala Glu Glu Pro Ala Met Lys Asp Leu Leu Leu Glu Gln 165 1 70 175 Val Lys Lys Gln Gly Ile Asp Leu Glu Gln Ser Arg Lys Glu Glu Glu 180 185 190 Gln Gln Met Leu Gln Asp Ala Arg Gln Trp Leu Asn Ser Gly Lys Ile 195 200 205 Glu Asp Val Arg Gln Ala Arg Ser Gly Ala Thr Ala Leu His Val Ala 210 215 220 Ala Ala Lys Gly Tyr Ser Glu Val Leu Arg Leu Leu Ile Gln Ala Gly 225 230 235 240 Tyr Glu Leu Asn Val Gln Asp Tyr Asp Gly Trp Thr Pro Leu His Ala 245 250 255 Ala Ala His Trp Gly Val Lys Glu Ala Cys Ser Ile Leu Ala Glu Ala 260 265 270 Leu Cys Asp Met Asp Ile Arg Asn Lys Leu Gly Gln Thr Pro Phe Asp 275 280 285 Val Ala Asp Glu Gly Leu Val Glu His Leu Glu Leu Leu Gln Lys Lys 290 295 300 Gln Asn Val Leu Arg Ser Glu Lys Glu Thr Arg Asn Lys Leu Ile Glu 305 310 315 320 Ser Asp Leu Asn Ser Lys Ile Gln Ser Gly Phe Phe Lys Asn Lys Glu 325 330 335 Lys Met Leu Tyr Glu Glu Glu Thr Pro Lys Ser Gln Glu Met Glu Glu 340 345 350 Glu Asn Lys Glu Ser Ser Ser Ser Ser Ser Glu Glu Glu Glu Gly Glu 355 360 365 Asp Glu Ala Ser Glu Ser Glu Thr Glu Lys Glu Ala Asp Lys Lys Pro 370 375380 Glu Ala Phe Val Asn His Ser Asn Ser Glu Ser Lys Ser Ser Ile Thr 385 390 395 400 Glu Gln Ile Pro Ala Pro Ala Gln Asn Thr Phe Ser Ala Ser Ser Ala 405 410 415 Arg Arg Phe Ser Ser Gly Leu Phe Asn Lys Pro Glu Glu Pro Lys Asp 420 425 430 Glu Ser Pro Ser Ser Trp Arg Leu Gly Leu Arg Lys Thr Gly Ser His 435 440 445 Asn Met Leu Ser Glu Val Ala Asn Ser Arg Glu Pro Ile Arg Asp Arg 450 455 460 Gly Ser Ser Ile Tyr Arg Ser Ser Ser Ser Pro Arg Ile Ser Ala Leu 465 470 475 480 Leu Asp Asn Lys Asp Lys Glu Arg Glu Asn Lys Ser Tyr Ile Ser Ser 485 490 495 Leu Ala Pro Arg Lys Leu Asn Ser Thr Ser Asp Ile Glu Glu Lys Glu 500 505 510 Asn Arg Glu Ser Ala Val Asn Leu Val Arg Ser Gly Ser Tyr Thr Arg 515 520 525 Gln Leu Trp Arg Asp Glu Ala Lys Gly Asn Glu Ile Pro Gln Thr Ile 530 535 540 Ala Pro Ser Thr Tyr Val Ser Thr Tyr Leu Lys Arg Thr Pro His Lys 545 550 555 560 Ser Gln Ala Asp Thr Thr Ala Glu Lys Thr Ala Asp Asn Val Ser Ser 565 570 575 Ser Thr Pro Leu Cys Val Ile Thr Asn Arg Pro Leu Pro Ser Thr Ala 580 585590 Asn Gly Val Thr Ala Thr Pro Val Leu Ser Ile Thr Gly Thr Asp Ser 595 600 605 Ser Val Glu Ala Arg Glu Lys Arg Arg Ser Tyr Leu Thr Pro Val Arg 610 615 620 Asp Glu Glu Ala Glu Ser Leu Arg Lys Ala Arg Ser Arg Gln Ala Arg 625 630 635 640 Gln Thr Arg Arg Ser Thr Gln Gly Val Thr Leu Thr Asp Leu Gln Glu 645 650 655 Ala Glu Arg Thr Phe Ser Arg Ser Arg Ala Glu Arg Gln Ala Gln Glu 660 665 670 Gln Pro Arg Glu Lys Pro Thr Asp Thr Glu Gly Leu Glu Gly Ser Pro 675 680 685 Glu Lys His Glu Pro Ser Ala Val Pro Ala Thr Glu Ala Gly Glu Gly 690 695 700 Gln Gln Pro Trp Gly Arg Ser Leu Asp Glu Glu Pro Ile Cys His Arg 705 710 715 720 Leu Arg Cys Pro Ala Gln Pro Asp Lys Pro Thr Thr Pro Ala Ser Pro 725 730 735 Ser Thr Ser Arg Pro Ser Leu Tyr Thr Ser Ser His Leu Leu Trp Thr 740 745 750 Asn Arg Phe Ser Val Pro Asp Ser Glu Ser Ser Glu Thr Thr Thr Asn 755 760 765 Thr Thr Thr Ala Lys Glu Met Asp Lys Asn Glu Asn Glu Glu Ala Asp 770 775 780 Leu Asp Glu Gln Ser Ser Lys Arg Leu Ser Ile Arg Glu Arg Arg Arg 785 790 795 800 Pro Lys Glu Arg Arg Arg Gly Thr Gly Ile Asn Phe Trp Thr Lys Asp 805 810 815 Glu Asp Glu Thr Asp Gly Ser Glu Glu Val Lys Glu Thr Trp His Glu 820 825 830 Arg Leu Ser Arg Leu Glu Ser Gly Gly Ser Asn Pro Thr Thr Ser Asp 835 840 845 Ser Tyr Gly Asp Arg Ala Ser Ala Arg Ala Arg Arg Glu Ala Arg Glu 850 855 860 Ala Arg Leu Ala Thr Leu Thr Ser Arg Val Glu Glu Asp Ser Asn Arg 865 870 875 880 Asp Tyr Lys Lys Leu Tyr Glu Ser Ala Leu Thr Glu Asn Gln Lys Leu 885 890 895 Lys Thr Lys Leu Gln Glu Ala Gln Leu Glu Leu Ala Asp Ile Lys Ser 900 905 910 Lys Leu Glu Lys Val Ala Gln Gln Lys Gln Glu Lys Thr Ser Asp Arg 915 920 925 Ser Ser Val Leu Glu Met Glu Lys Arg Glu Arg Arg Ala Leu Glu Arg 930 935 940 Lys Met Ser Glu Met Glu Glu Glu Met Lys Val Leu Thr Glu Leu Lys 945 950 955 960 Ser Asp Asn Gln Arg Leu Lys Asp Glu Asn Gly Ala Leu Ile Arg Val 965 970 975 Ile Ser Lys Leu Ser Lys * 980

SEQ ID NO: 3 Sequence length: 3763 Sequence type: nucleic acid Number of strands: single strand Topology: linear Antisense: YES Sequence type: cDNA to mRNA Sequence characteristics Other information: SEQ ID NO: 1 minus strand sequence CGGGAAATGG CTAGGTATGA GGGAGGAATA AGGGTTAAAA CTAGTTAGGG AGATTACTGG 60 of SEQ GTGTTGATAG AAAAAATAAA AGTCAGTTCC TGAGAGGTGA AGAAGAATAG AGCGCCTCAA 120 TGCAGTCACT GTCACCTTGG ATGCTAACCA TTCCAGAGGG CATCTGATGA TCAATTCAAC 180 ATCCTGCTGT TTCTCTTGGC ACCCCAATCA GGATCCTGGA GTGGCTACCA CAAGACTCAC 240 CTATATCAGA GGACACTGGG TTAATGTAAA AAGCTGAAGG ATACTGATTA GGGTAGAAGT 300 CTTACATGTT TGGACCAAAT CCAATGTGGA CAAAGTAGCT GAGCAGCAGG AAGTTGAGGG 360 GTCCACAGCT GAGCCTGGTG TACTCCCCAG AGCATTCTCC AGATGGCTCG AATTACCACA 420 ATCATGCTTC AATTAAAACT GTCATAAAAC TTGGGATAGG GGTTTGGAAG AACAGGGCAT 480 TGAAAGTGCA GAGAAAATGT AGAGTGTATA GAGGTGACTA AAGAGGGTGA TAGAAAGAAG 540 TGTCCTTCGTCAGGAACATG GGGGGCAGC AAATGCTGTC CCTTTCTTTC CTCATAAATC TGGAGCCTAG CCTACTTGGA 660 CAGTTTGCTG ATGACTCTGA TGAGGGCACC ATTTTCATCT TTCAGCCTCT GGTTGTCGGA 720 TTTCAGTTCT GTTAACACCT TCATTTCTTC CTCCATTTCT GACATTTTGC GCTCCAAGGC 780 TCGCCTCTCC CGTTTCTCCA TCTCCAGCAC TGATGATCGG TCAGAGGTCT TTTCTTGTTT 840 CTGCTGGGCC ACCTTCTCAA GCTTGGACTT TATATCTGCT AGCTCTAGCT GGGCTTCCTG 900 AAGTTTTGTT TTCAGTTTTT GGTTTTCAGT CAGAGCACTC TCATAGAGTT TTTTATAATC 960 TCTGTTGCTG TCTTCTTCTA CACGGCTGGT CAGGGTGGCT AGGCGGGCCT CCCGGGCCTC 1020 CCGACGGGCT CTTGCTGAAG CCCGGTCACC GTAAGAATCA CTGGTTGTAG GATTACTACC 1080 TCCCGATTCC AACCTAGAAA GTCTTTCATG CCACGTTTCT TTGACCTCTT CAGAGCCATC 1140 AGTTTCATCC TCATCCTTTG TCCAGAAATT GATGCCTGTG CCTCTTCGTC GTTCCTTGGG 1200 CCGCCTCCTC TCTCGGATGG ACAGCCTCTT AGAGGACTGC TCATCCAAAT CTGCTTCTTC 1260 ATTCTCATTT TTGTCCATTT CCTTTGCAGT TGTAGTGTTT GTGGTAGTCT CTGAACTCTC 1320 AGAATCAGGG ACTGAAAATC TATTTGTCCA TAGCAGGTGG GAACTGGTGT AGAGTGAGGG 1380 TCTTGACGTA GAAGGAGATG CTGGCGTGGT GGGTTTGTCT GGCTGAGCTG GGCACCTCAG 1440 GCGATGACAG A TAGGCTCTT CATCCAGACT CCTGCCCCAG GGCTGCTGGC CCTCCCCAGC 1500 TTCTGTTGCT GGAACTGCTG AGGGCTCATG CTTCTCAGGG CTCCCCTCAA GCCCTTCAGT 1560 GTCTGTGGGC TTCTCACGAG GCTGCTCCTG AGCTTGCCTC TCTGCCCTCG ACCGGCTGAA 1620 TGTCCTTTCT GCTTCTTGAA GGTCTGTTAG GGTGACACCT TGAGTAGACC TTCGTGTCTG 1680 CCGAGCTTGT CTGGAGCGTG CTTTCCGTAA AGACTCTGCT TCCTCATCCC GTACAGGAGT 1740 CAGATAGGAC CTCCTCTTCT CCCTGGCTTC CACAGAGGAA TCTGTTCCAG TAATGGAGAG 1800 CACAGGAGTA GCTGTAACCC CATTGGCAGT GCTAGGAAGA GGGCGATTGG TGATCACACA 1860 GAGCGGGGTG CTAGAAGAGA CATTGTCTGC TGTTTTCTCT GCTGTTGTGT CGGCCTGGGA 1920 TTTGTGAGGA GTCCTTTTCA AGTAAGTTGA TACATAGGTG GAGGGAGCAA TTGTCTGTGG 1980 GATTTCATTT CCTTTTGCTT CATCCCTCCA TAGCTGCCGG GTATAGGAGC CACTCCTCAC 2040 TAGATTAACA GCTGATTCTC TGTTCTCCTT TTCTTCAATA TCACTTGTGC TGTTGAGCTT 2100 CCGGGGTGCT AGTGAACTAA TATAGCTTTT GTTTTCTCTC TCCTTATCTT TGTTGTCCAG 2160 TAGAGCAGAA ATTCGAGGGC TTGAAGAGGA GCGATAGATG GAAGAGCCTC GGTCCCTTAT 2220 AGGTTCCCTG GAATTGGCCA CCTCACTCAG CATGTTGTGG CTGCCAGTTT TTCTCAGTCC 2280 CAATCTCCAT GAAGAAG GAG ATTCATCTTT GGGCTCTTCT GGCTTGTTAA AAAGGCCAGA 2340 AGAGAACCTC CTAGCAGAAG AGGCAGAGAA GGTATTTTGA GCTGGTGCTG GTATCTGCTC 2400 TGTGATACTA CTCTTGCTTT CAGAGTTGGA ATGATTGACA AAGGCTTCTG GCTTTTTATC 2460 TGCCTCCTTC TCAGTTTCTG ACTCAGAAGC TTCATCTTCA CCTTCCTCCT CCTCTGAGCT 2520 GGAGCTACTA GATTCTTTAT TTTCTTCCTC CATTTCTTGG GACTTAGGTG TCTCCTCCTC 2580 ATAGAGCATC TTCTCTTTGT TCTTAAAGAA CCCACTCTGA ATCTTGCTGT TCAGATCTGA 2640 CTCAATGAGT TTATTCCGTG TCTCCTTTTC ACTTCGAAGC ACATTCTGCT TCTTCTGGAG 2700 CAACTCCAAA TGCTCCACGA GACCCTCATC AGCCACATCA AATGGTGTCT GGCCCAGTTT 2760 ATTTCGAATA TCCATGTCAC AAAGTGCTTC TGCCAGGATG GAGCAAGCCT CCTTCACTCC 2820 CCAGTGTGCA GCAGCATGGA GGGGAGTCCA GCCATCATAA TCCTGAACAT TGAGTTCATA 2880 GCCAGCCTGA ATTAAAAGTC TGAGGACTTC AGAGTAGCCC TTGGCAGCAG CCACATGAAG 2940 GGCTGTAGCC CCTGAGCGAG CCTGCCTCAC ATCCTCTATT TTCCCACTGT TGAGCCACTG 3000 GCGGGCATCC TGCAACATCT GCTGCTCTTC TTCTTTTCTT GACTGCTCTA GATCAATTCC 3060 TTGCTTCTTT ACTTGCTCCA GAAGAAGATC CTTCATGGCT GGCTCTTCTG CAAGGTCAGA 3120 GGGAACTTCA CCTTCACTAT TG ACAATACC TACACTGGCT CCGTGATTAA TGAAATACTC 3180 TGCTATGTTG AGATAGCCAC AGGAAGCTGC TGCATGAAGG GGTGTCCAGC CCTCGTTGTC 3240 TTGCTGGTTT ACATTGGCTC TGTTCTCCAC CAGAAACTTC ACCATGTCCA AATTTTCATC 3300 AATACATGCC TGGTGCAGGG CTGTCAAGCC GTCCACGTTG ACCGTGTTGA TATCAGCACC 3360 TCTTGCCAGA AGCTTTCTCA CCTCGTCGGT GTCCCCGCTA GAGCAGGCGG CCAGAAAGAC 3420 AGCACCGTCC TCGAAGCGGA CCCTGGGGCT CCCGCGCCTG GTCAGCGGCT GCCGCCCCGC 3480 GCCTCGTCGC TCCGCAGGCT CCTGCTCTGT CAGCGAGCCC CGCCAGCGCC GAAGCTGCTC 3540 TGCCCGCCGC ATTCGCGCCG ACTCTGCCCG CTTCCCTCCT AGGTGCTCCA GTTCCGCCAT 3600 TGCTCCGGCC TCGGCAGCTG CTGCCAAAAC TACCAACACT AAATTACTGT TGGGGCTCGA 3660 GTTGCCGCTG CCGCCTCTCT CAGAGCGCTT CAGCCCGGAG CAGAGGGCGG AGACCCTCGC 3720 GCCGCCATCT TTACTCCCCCGCCCTCTCTCCCCCGCCCTCTCCCCCGCCCTC

SEQ ID NO: 4 Sequence length: 20 Sequence type: nucleic acid Number of strands: single strand Topology: linear Antisense: NO Sequence type: cDNA to mRNA sequence CAGCTACTTT GTCCACATTG 20

SEQ ID NO: 5 Sequence length: 20 Sequence type: nucleic acid Number of strands: single strand Topology: linear Antisense: YES Sequence type: cDNA to mRNA Sequence characteristics Other information: SEQ ID NO: 1 AAGACTCACC TATATCAGAG 20

[Brief description of the drawings]

FIG. 1 is a view showing the position of a cDNA fragment of Example 1.

FIG. 2 shows a vector (pGEX-hMLP [1-64] for expressing and producing a fusion protein of human MLP (amino acid sequence of positions 1 to 648 of SEQ ID NO: 2) and GST in Escherichia coli.
8]) is a diagram showing the structure of FIG. Plasmid pGEX4T
BamH1 site downstream of the GST coding region of E-1 and E
Utilizing the coRI site, human MLP (SEQ ID NO: 1
ア ミ ノ 酸 648 amino acid sequence) coding region is inserted in frame.

FIG. 3 is an electrophoretic photograph showing the result of Western blotting analysis using an anti-human MLP antibody. Bands corresponding to human MLP (arrow) and human MBS (horizontal line) are shown on the right of the photograph. Brain: Human brain, Lun
g: human lung, Heart: human heart, Aorta: human artery, M
BP: chicken gizzard. A molecular weight marker is shown on the left of the photograph.

FIG. 4. Northern and human MLP cDNA fragments
It is an electrophoresis photograph which showed the result of blotting analysis. heart: heart, brain: brain, placenta: placenta, live
r: liver, skeletal muscle: skeletal muscle, kidney: kidney, p
ancreas: Pancreas. A molecular weight marker is shown on the left of the photograph.

FIG. 5 is a diagram showing enhancement of phosphatase activity of PP1cδ by human MLP. ●: rG-hMLP [1-648].

FIG. 6 is a photograph showing co-expression of various human MLP partial fragments and active RhoA protein in yeast using a two-hybrid system, and showing the binding between the two.

FIG. 7 is a photograph showing the position (1q32.1) of the human MLP gene on the chromosome by the FISH method.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/53 G01N 33/53 D 33/566 33/566 33/573 33/573 A // (C12N 1/21 C12R 1: 19) (C12P 21/02 C12R 1:19)

Claims (25)

[Claims] 1. A protein or a derivative thereof having the following properties: (1) having an active Rho protein binding ability;
Enhances the phosphatase activity of the catalytic subunit of myosin light chain phosphatase, (3) its gene is located on human chromosome 1q32.1, (4) its molecular weight is SD
It is about 125 kDa as measured by S-PAGE. 2. A protein comprising the following amino acid sequence or a derivative thereof: (a) the amino acid sequence of SEQ ID NO: 2, or (b) one or more amino acids are added and / or inserted, and / or one or more amino acids are added. A substituted and / or deleted amino acid sequence of SEQ ID NO: 2, which has an activity of binding to an active Rho protein and enhances the phosphatase activity of a catalytic subunit of myosin light chain phosphatase. 3. A protein comprising the amino acid sequence of SEQ ID NO: 2 wherein one or more amino acids have been added and / or inserted and / or one or more amino acids have been substituted and / or deleted, or a derivative thereof, A protein whose amino acid sequence has an active Rho protein binding ability or a derivative thereof. (4) the amino acid sequence of 613 to 9 of SEQ ID NO: 2;
No. 82, 613-913, 713-982, 813
The protein of claim 3, wherein the protein is selected from the amino acid sequence of positions # 982 and 913 to 982. 5. A protein comprising the amino acid sequence of SEQ ID NO: 2 wherein one or more amino acids have been added and / or inserted, and / or one or more amino acids have been substituted and / or deleted, or a derivative thereof, A protein whose amino acid sequence enhances the phosphatase activity of the catalytic subunit of myosin light chain phosphatase or a derivative thereof. (6) the amino acid sequence of SEQ ID NO: 2
The protein according to claim 5, which is the amino acid sequence of No. 7. A nucleotide sequence encoding the protein according to any one of claims 1 to 6. 8. A nucleic acid comprising the nucleotide sequence according to claim 7,
vector. 9. A plasmid vector, a virus vector,
And a liposome vector.
A vector according to claim 8. 10. A host cell transformed with the vector according to claim 8 or 9 (however, in the case of a human cell, it is limited to a cell isolated from a human). 11. Escherichia coli, yeast, insect cells, COS cells,
The host cell according to claim 10, which is selected from the group consisting of lymph cells, fibroblasts, CHO cells, blood cells, and tumor cells. 12. A method comprising culturing the host cell according to claim 10 or 11, and isolating the protein according to any one of claims 1 to 6 or a derivative thereof from the culture. A method for producing the protein according to any one of claims 1 to 6 or a derivative thereof. 13. A screening target comprising: (1) a screening target containing an active Rho protein and the protein or a derivative thereof according to any one of claims 1 to 4; An active Rho protein, comprising measuring the degree of inhibition of binding between the type Rho protein and the protein or a derivative thereof according to any one of claims 1 to 4, and any one of claims 1 to 4. A method for screening a substance that inhibits binding to the protein or a derivative thereof according to claim 1. 14. The screening method according to claim 13, wherein the screening system is a yeast two-hybrid system. 15. The method according to claim 1, wherein (1) the subject of the screening is a catalytic subunit of myosin light chain phosphatase.
2, 5, and 6, the protein or derivative thereof according to any one of the above,
And (2) a method for screening a substance that inhibits the enhancement of the phosphatase activity of the catalytic subunit of myosin light chain phosphatase, which comprises measuring the degree of enhancement of the phosphatase activity of the catalytic subunit of myosin light chain phosphatase. 16. The screening method according to claim 15, wherein the screening system comprises a phosphatase substrate. 17. The screening method according to claim 16, wherein the phosphatase substrate is a phosphorylated-myosin light chain. 18. An antibody against the amino acid sequence of positions 652 to 863 of SEQ ID NO: 2. 19. The method according to claim 18, which is a polyclonal antibody.
The antibody according to 1. 20. A base sequence selected from the following: (a) a base sequence containing a continuous DNA sequence of at least 20 bases in the sequence of SEQ ID NO: 1, a sequence complementary thereto, and (b) (a) A) a base sequence which hybridizes with the base sequence under stringent conditions. 21. Sequence Nos. 1 to 1135, 575
2879, 692-3763 and 3513-35
The nucleotide sequence of No. 32, and 2629-37 of SEQ ID NO: 3
No. 63, No. 835-3189, No. 1-3072, 322
The nucleotide sequence according to claim 20, which is selected from the group consisting of the nucleotide sequences of Nos. 341 to 341 and 232 to 251. 22. A probe comprising the base sequence according to claim 20 and a label. 23. The probe according to claim 22, wherein the label is selected from the group consisting of an enzyme, a radioisotope, fluorescence, and an antigen. (24) A method comprising the nucleotide sequence according to (20) or (21) or the probe according to (22) or (23).
Diagnostics. 25. The diagnostic agent according to claim 24, which is used for diagnosis of familial hypertrophic cardiomyopathy or familial dilated cardiomyopathy.