JP2001161379A - Cyclic nucleotide phosphodiesterase gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new cyclic nucleotide phosphodiesterase, a gene encoding the enzyme, a method for producing these, and uses of these. SOLUTION: A full-length cDNA encoding a new cyclic nucleotide phosphodiesterase (PDE10A) was successfully isolated from a human cDNA library. Since PDE10A has structure different from known PDE, PDE10A was assumed to belong to a new PDE family. PDE10A has an ability of hydrolyzing both cAMP and cGMP, a Km value lower than known PDE families, and sensitivity properties to various PDE inhibitors different from known PDE families. Expression of PDE10A was high especially in the putamen and caudate nucleus. PDE10A is useful as a target for developing a medicine having a new effect.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel protein belonging to the cyclic nucleotide phosphodiesterase (PDE) family. The protein of the present invention
For example, it can be used as a target molecule for drug development.

[0002]

2. Description of the Related Art Cyclic nucleotide phosphodiesterase (PDE) is an enzyme that hydrolyzes cAMP or cGMP to convert it to the corresponding nucleoside 5'-monophosphate. cAMP and cGMP are intracellular second messengers and are involved in the regulation of signal transduction (MD Houslay
and G. Milligan (1997) Trends Biochem. Sci. 22: 2
17-224). So far known cyclic nucleotide phosphodiesterases have similarity in amino acid sequence,
It is classified into seven PDE families based on substrate specificity, substrate affinity, sensitivity to cofactors and sensitivity to inhibitors (JA Beavo (1995) Physiol.
Rev. 75: 725-748). PDE1 is a Ca ²⁺ / calmodulin-dependent PDE, and PDE2 is a cGMP-stimulated PDE (cGMP-stimulated PD
E), PDE3 is cGMP-inhibited PDE, PDE4
Is a cAMP-specific PDE, PDE5 is a cGMP-specific PDE, PDE6 is a photoreceptor PDE, and PDE7 is a high-affinity cAMP-specific, rolipram-insensitive PDE. In addition, two families, PDE8
And PDE9 were recently reported. PDE8 is a high-affinity cAMP-specific, IBMX-insensitive PDE (Fisher, DA et al., 1998, Bi
ochem. Biophys. Res. Commun. 246: 570-577; Hayashi
et al., 1998, Biochem. Biophys. Res. Commun. 250:
751-756), and PDE9 is a high-affinity cGMP-specific PDE (Fis
her, DA et al., 1998, J. Biol. Chem. 273: 15559-
15564; Soderling, SH et al., 1998, J. Biol. Che
m. 273: 15553-15558). All of these PDE families conserve approximately 270 amino acids at the carboxyl terminus and constitute the catalytic domain. On the other hand, the amino terminus of the catalytic domain is rich in diversity and is expected to be responsible for regulation specific to individual families (Charbonnea
u, H., 1990, in Cyclic Nucleotide Phosphodiesteras
es: Structure, Regulation AndDrug Avtion. vol.2, p
p. 267-296, John Wiley & Sons, Ltd., Chichester; M
anganiello, VC et al., 1995, Arch. Biochem. Biop
hys. 322: 1-13). The region outside the catalytic domain is diverse in all families, but PDE2, PDE5, and PD
The three families of E6 have conserved segments towards the N-terminus. This is said to constitute an allosteric cGMP binding domain separate from the cyclic nucleotide hydrolysis site. The cyclic nucleotide phosphodiesterase family has been attracting attention as a target molecule for drug discovery. Have been. Therefore, the isolation of new molecules belonging to the cyclic nucleotide phosphodiesterase family is considered to be an important step for the development of new drugs.

[0003]

An object of the present invention is to provide a novel cyclic nucleotide phosphodiesterase and a gene thereof, and their production and use.

[0004]

Means for Solving the Problems In order to isolate a novel gene belonging to the cyclic nucleotide phosphodiesterase family, the present inventors first used a human nucleotide sequence as a probe by using the nucleotide sequence of the catalytic region of cyclic nucleotide phosphodiesterase as a probe. We conducted a homology search of the EST database and found a new EST whose primary structure is different from that of the conventional PDE family proteins. Next, using primers prepared based on the sequence information of this EST, using a human brain cDNA library as a template, 5′-RAC
E and 3'-RACE were performed, and the full-length cDNA corresponding to this EST was successfully isolated. The present inventor called this "PDE10A"
I named it. The protein encoded by the isolated cDNA has a cGMP binding domain and catalytic region that are highly similar to other PDE family proteins, and
Since it had two cGMP binding motifs characteristic of the family protein, it is considered to be a novel protein belonging to the PDE family.

[0005] Due to the homology of the amino acid sequences of the catalytic domains, PDE10A is a cGMP-linked PDE, ie, PDE2, PDE5,
And PDE6 (42-47% amino acid identity) and the lowest with PDE8 (23% amino acid identity). Like other cGMP-binding PDEs, PDE10A
Has a non-catalytic cGMP binding domain at the N-terminus outside the catalytic domain. By Northern blot analysis, the mRNA encoding PDE10A was approximately 9.5 kb in size and was expressed in many different tissues, including brain, heart, placenta, and kidney. Furthermore, quantitative analysis of PDE10A mRNA in 50 different human tissues revealed that the most highly expressed tissues were putamen and caudate nucleus. The baculovirus system was used to express human PDE10A with a histidine tag at the N-terminus. In a kinetic analysis of the enzyme expressed in the baculovirus system, this enzyme was identified as cA
Both MP and cGMP were able to hydrolyze, with Km values of 0.033 ± 0.004 and 2.7 ± 0.3 μM, respectively. cAM
The hydrolytic capacity of P was not affected by 1 μM cGMP, but the hydrolytic capacity of cGMP was inhibited by 1 μM cAMP. Vmax of cAMP and cGMP was 57.1 ± 5.7, respectively.
And 833 ± 67 pmol / min / mg protein. PDE10A
Has the ability to hydrolyze both cAMP and cGMP, vinpocetine, EHNA (erythro-9- (2-hydroxy-3-
nonyl) -adenine), milrinone, rolipram, and IBMX (3-isobutyl-1-methylx)
anthine) (up to 100 μM) but not dipyridamole (dipyri
damole) (IC ₅₀ = 7.1 ± 0.6 μM). These findings suggest that PDE10A is structurally different and has different kinetics from any known PDE family.

Various useful drugs have been developed for the PDE family proteins by targeting them, and these proteins are also useful as targets for the development of drugs having new effects.

[0007] The present invention relates to a novel cyclic nucleotide phosphodiesterase (PDE) and its gene, and their production and use, and more specifically, (1)
A protein comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in the protein in which one or more amino acids are substituted, deleted, inserted, and / or added; A protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 2, and (2) a protein encoded by a DNA hybridizing with the DNA consisting of the nucleotide sequence of SEQ ID NO: 1, A protein functionally equivalent to a protein consisting of an amino acid sequence, (3) (1)
Or a DNA encoding the protein of (2),
(4) a vector into which the DNA of (3) has been inserted,
(5) a host cell carrying the vector according to (4),
(6) The method for producing a protein according to (1) or (2), comprising culturing the host cell according to (5) and recovering the protein expressed from the host cell or a culture supernatant thereof. (7) an antibody that binds to the protein according to (1) or (2); (8) a partial peptide of the protein according to (1) or (2); A polynucleotide comprising at least 15 nucleotides complementary to the DNA or its complementary strand, (1
0) A method for screening a compound that binds to the protein according to (1) or (2), wherein (a) a step of bringing a test sample into contact with the protein or a partial peptide thereof, (b) the protein or a portion thereof A method comprising: detecting a binding activity between a peptide and a test sample; (c) selecting a compound having an activity of binding to the protein or a partial peptide thereof; (11) a method according to (10). A method for screening a compound that can be isolated, which binds to the protein according to (1) or (2), or (12) a compound that promotes or inhibits the activity of the protein according to (1) or (2), (A) contacting the protein with 3 ′, 5′-cyclic nucleotide monophosphate in the presence of a test sample; (b) 3 ′, 5′-servo of the protein Detecting the hydrolytic activity against click nucleotide monophosphate, (c)
Selecting a compound that increases or decreases the hydrolysis activity, as compared with the case where the compound is detected in the absence of the test sample,
And (13) a compound that promotes or inhibits the activity of the protein according to (1) or (2), which can be isolated by the method according to (12).

[0008]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel cyclic nucleotide phosphodiesterase protein.
The nucleotide sequence of the cDNA of the human cyclic nucleotide phosphodiesterase “PDE10A” isolated by the present inventors is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO: 2. The primary structure of the PDE10A protein has high homology to known cyclic nucleotide phosphodiesterase proteins, especially in its cGMP binding and catalytic regions. Also, PDE10A protein has two cGMPs characteristic of cyclic nucleotide phosphodiesterase protein.
It retains the binding motif. Therefore, the PDE10A protein discovered by the present inventors is considered to be a novel protein belonging to the cyclic nucleotide phosphodiesterase (PDE) family. As a result of RT-PCR and Northern analysis, the gene encoding the protein is:
Expression is observed in tissues including the brain, heart, placenta, and kidney, especially in the putamen and caudate nucleus (caudate n) of the brain.
ucleus). In these tissues, the PDE10A protein hydrolyzes the intracellular second messengers cAMP and cGMP to form the corresponding nucleoside.
By converting to 5'-monophosphate, it is thought to be involved in the control of intracellular signal transduction. It is also conceivable that it may have a new biological activity through binding to a selective target molecule. Human PDEA10A of the present invention
Is supposed to be involved in human development-related diseases, neurological diseases, cardiopulmonary diseases, and kidney diseases, and is useful for drug development for prevention and treatment of these diseases.

[0009] The present invention also includes proteins that are functionally equivalent to the human PDE10A protein. Such proteins include, for example, homologous proteins of other organisms corresponding to the human PDE10A protein and mutants of the human PDE10A protein. "Functionally equivalent" in the present invention
Means that the protein of interest has cyclic nucleotide phosphodiesterase activity. What is cyclic nucleotide phosphodiesterase activity?
Cyclic nucleotides (eg, cAMP or cGMP)
Refers to the activity of hydrolyzing the phosphodiester bond of Such activity can be determined, for example, by a two-step assay (Mukai et a
l., Br. J. Pharmacol. (1994) 111, 389-390).

[0010] As a method well known to those skilled in the art for preparing a protein functionally equivalent to a certain protein, a method for introducing a mutation into a protein is known.
For example, those skilled in the art will recognize site-directed mutagenesis (Hash
imoto-Gotoh, T. et al. (1995) Gene 152, 271-275; Z
oller, MJ and Smith, M. (1983) Methods Enzymol.
100, 468-500, Kramer, W. et al. (1984) Nucleic Ac
ids Res. 12, 9441-9456; Kramer, W. and Fritz, H.
J. (1987) Methods Enzymol. 154, 350-367; Kunkel,
TA (1985) Proc. Natl. Acad. Sci. US A. 82, 488
-492; Kunkel TA (1988) Methods Enzymol. 85, 2763
Using -2766) and the like, mutations can be appropriately introduced into amino acids of human PDE10A protein to prepare variants functionally equivalent to human PDE10A protein.
Amino acid mutations in proteins can also occur in nature. Thus, a protein having an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence of human PDE10A protein (SEQ ID NO: 2) and functionally equivalent to human PDE10A protein is also included in the protein of the present invention. . In such a mutant, the number of amino acids to be mutated is usually within 100 amino acids, preferably within 50 amino acids, more preferably within 20 amino acids, further preferably within 10 amino acids, and still more preferably It is considered to be within 5 amino acids.

The amino acid residue to be mutated is preferably mutated to another amino acid in which the properties of the amino acid side chain are conserved. For example, the properties of amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),
Hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S,
T), amino acids having aliphatic side chains (G, A, V, L, I,
P), an amino acid having a hydroxyl group-containing side chain (S, T, Y), an amino acid having a sulfur atom-containing side chain (C, M), an amino acid having a carboxylic acid and amide-containing side chain (D, N, E,
Q), an amino group having a base-containing side chain (R, K, H) and an amino acid having an aromatic-containing side chain (H, F, Y, W). Represents the title).

It is already known that a protein having an amino acid sequence modified by deletion, addition and / or substitution of one or more amino acid residues relative to an amino acid sequence maintains its biological activity. (Mark, DF etal. (1984) Proc. Natl. A
cad.Sci. USA 81, 5662-5666; Zoller, MJ and Smit
h, M. (1982) Nucleic Acids Res. 10, 6487-6500; Wan
g, A. et al. (1984) Science 224, 1431-1433; Dalbad
ie-McFarland, G. et al. (1982) Proc. Natl. Acad. S
ci. USA 79, 6409-6413).

[0013] Examples of the protein in which one or more amino acid residues are added to the amino acid sequence of human PDE10A protein (SEQ ID NO: 2) include a fusion protein containing human PDE10A protein. The fusion protein is a fusion of the human PDE10A protein and another peptide or protein, and such a fusion protein is also included in the present invention. The method for producing the fusion protein is based on the DN encoding the human PDE10A protein of the present invention.
A and DNA encoding another peptide or protein may be ligated in such a manner that their frames coincide with each other, introduced into an expression vector, and expressed in a host, and a method known to those skilled in the art can be used. Other peptides or proteins to be fused with the human PDE10A protein of the present invention are not particularly limited.

Other peptides to be fused with the protein of the present invention include, for example, FLAG (Hopp, TP et a
l. (1988) BioTechnology 6, 1204-1210), 6 Hiss
6 × His, 10 × His composed of (histidine) residues, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag , SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protei
Known peptides such as fragments of n C can be used. Other proteins to be fused with the protein of the present invention include, for example, GST (glutathione S-transferase), HA (influenza agglutinin), immunoglobulin constant region, β-galactosidase, MBP
(Maltose binding protein) and the like. Encode these commercially available peptides or proteins
A fusion protein can be prepared by fusing DNA with DNA encoding the protein of the present invention and expressing the prepared fusion DNA.

Other methods well known to those skilled in the art for preparing proteins functionally equivalent to a protein include hybridization techniques (Sambrook,
J. et al. (1989) Molecular Cloning 2nd ed., 9.47-
9.58, Cold Spring Harbor Lab. Press). That is, those skilled in the art can isolate a DNA highly homologous to the human PDE10A protein based on the DNA sequence encoding the human PDE10A protein (SEQ ID NO: 1) or a part thereof, and perform functional and functional It is also usually possible to prepare a protein that is equivalent to the target.
Thus, a protein that is encoded by a DNA that hybridizes to a DNA sequence encoding the human PDE10A protein (SEQ ID NO: 1) or a DNA consisting of a part thereof and that is functionally equivalent to the human PDE10A protein is also used. Included in the protein of the present invention. Such proteins include, for example, homologs or orthologs of non-human eukaryotes (eg, monkeys, rats, mice,
Proteins encoded by guinea pigs, nematodes, yeast, flies, etc.).

Hybridization conditions for isolating a DNA encoding a protein functionally equivalent to the human PDE10A protein can be appropriately selected by those skilled in the art. For example, if the stringency is 10
% Formamide, 5x SSPE, 1x Denhardt's solution, 1x salmon sperm DNA. More preferable conditions (more stringent conditions) include 25% formamide, 5xSSPE, 1x Denhardt's solution, and 1x salmon sperm DNA, and more preferable conditions (more stringent conditions) include 50% formamide and 5xSSPE. , 1x
Denhardt's solution, 1x salmon sperm DNA conditions. Also,
Preferred temperature conditions are 45 ° C., more preferably 65 ° C.
° C. However, a plurality of factors that influence the stringency of hybridization can be considered in addition to the above-mentioned formamide concentration and temperature, and those skilled in the art can realize the same stringency by appropriately selecting these factors. .

Further, instead of the above-mentioned method using the hybridization technique, a gene amplification method using a primer synthesized based on the sequence information of DNA (SEQ ID NO: 1) encoding human PDE10A protein, for example, polymerase chain reaction It is also possible to isolate using (PCR) method.

The human P encoded by the DNA isolated by these hybridization techniques or gene amplification techniques
Proteins functionally equivalent to the DE10A protein usually have high homology in amino acid sequence to human PDE10A protein. The protein of the present invention includes human PDE10A
Also included are proteins that are functionally equivalent to the protein and have high homology to the amino acid sequence shown in SEQ ID NO: 2. High homology usually refers to an identity of 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more. These proteins have a cGMP binding region and a catalytic region characteristic of a cyclic nucleotide phosphodiesterase protein, and in particular, are considered to have high homology to human PDE10A protein in these regions. To determine protein homology, refer to the literature (Wilbu
r, WJ and Lipman, DJ Proc. Natl. Acad. Sci.
USA (1983) 80, 726-730).

The protein of the present invention may differ in amino acid sequence, molecular weight, isoelectric point, presence / absence and form of sugar chains, etc., depending on the cell, host, or purification method for producing the protein as described below. However, the obtained protein is included in the present invention as long as it has a function equivalent to that of the human PDE10A protein of the present invention (SEQ ID NO: 2). For example, when the protein of the present invention is expressed in a prokaryotic cell, for example, Escherichia coli, a methionine residue can be added to the N-terminal of the amino acid sequence of the original protein. The proteins of the present invention also include such proteins.

The protein of the present invention can be prepared as a recombinant protein or a natural protein by methods known to those skilled in the art. DNA that encodes the protein of the present invention if it is a recombinant protein
(E.g., a DNA having the nucleotide sequence of SEQ ID NO: 1) is inserted into an appropriate expression vector, and introduced into an appropriate host cell to express the protein of the present invention in the cell. When the cells are collected, the extract is obtained, and then subjected to chromatography such as ion exchange, reverse phase, gel filtration, or affinity chromatography in which an antibody against the protein of the present invention is immobilized on a column. Alternatively, purification can be performed by further combining a plurality of these columns. In addition, the protein of the present invention is expressed by glutathione S
-Expression in host cells (eg, animal cells and E. coli) as a fusion protein with a transferase protein or as a recombinant protein with multiple histidines, and purify the expressed recombinant protein using a glutathione column or a nickel column be able to. After purification of the fusion protein, if necessary, a region other than the target protein in the fusion protein is cut with thrombin or Factor Xa, etc.
It is also possible to remove it. If it is a natural protein, a method known to those skilled in the art, for example, for an extract of a tissue or a cell expressing the protein of the present invention, an affinity column to which an antibody that binds to the protein of the present invention described below is bound. It can be isolated by acting and purifying.

The present invention also includes a partial peptide of the protein of the present invention. The partial peptide having an amino acid sequence specific to the protein of the present invention has at least 7 amino acids, preferably at least 8 amino acids, and more preferably at least 9 amino acids.
It consists of an amino acid sequence of amino acids or more. The partial peptide is, for example, preparation of an antibody against the protein of the present invention,
It can be used for screening for a compound that binds to the protein of the present invention, and for screening for promoters and inhibitors of the protein of the present invention. Further, a partial peptide which has a binding ability to 3 ′, 5′-cyclic nucleotide monophosphate but does not have its hydrolysis activity may be used as a competitive inhibitor of the protein of the present invention. Also,
Regulatory peptides within PDE10A may be used as activators or inhibitors of PDE10A activity by other mechanisms. The partial peptide of the present invention can be produced by genetic engineering techniques, known peptide synthesis methods, or by cleaving the protein of the present invention with an appropriate peptidase. The peptide may be synthesized by, for example, either a solid phase synthesis method or a liquid phase synthesis method.

[0022] The present invention also relates to a DNA encoding the protein of the present invention. The DNA of the present invention can be used for producing the protein of the present invention as a recombinant protein. For example, it is used for in vivo or in vitro production of the protein of the present invention as described above. When the DNA encoding the protein of the present invention is defective, application to antisense function inhibition, gene therapy to replace a normal gene, and the like can be considered. The DNA of the present invention may be in any form as long as it can encode the protein of the present invention. That is, mRNA
It does not matter whether it is cDNA synthesized from DNA, genomic DNA, or chemically synthesized DNA. In addition, DNAs having any base sequence based on the degeneracy of the genetic code are included as long as they can encode the protein of the present invention.

The DNA of the present invention can be prepared by a method known to those skilled in the art. For example, it can be prepared by preparing a cDNA library from cells expressing the protein of the present invention and performing hybridization using a part of the sequence of the DNA of the present invention (for example, SEQ ID NO: 1) as a probe. cDNA libraries are described, for example, in Sambrook, J .;
et al., Molecular Cloning, Cold Spring Harbor Labo
ratory Press (1989), or a commercially available library may be used. Also, preparing RNA from cells expressing the protein of the present invention,
After being converted into cDNA by reverse transcriptase, an oligo DNA is synthesized based on the sequence of the DNA of the present invention (for example, SEQ ID NO: 1), and PCR is performed using the oligo DNA as a primer to obtain a cDNA encoding the protein of the present invention. Can also be prepared by amplifying.

Further, by determining the nucleotide sequence of the obtained cDNA, the translation region encoded thereby can be determined, and the amino acid sequence of the protein of the present invention can be obtained.
Genomic DNA can be isolated by screening a genomic DNA library using the obtained cDNA as a probe.

Specifically, the following may be performed. First, mRNA is isolated from cells, tissues, and organs that express the protein of the present invention (eg, tissues such as brain, heart, lung, kidney, skeletal muscle, thymus, or placenta). mRNA can be isolated by a known method, for example, guanidine ultracentrifugation (Chirgwin, J.
M. et al. (1979) Biochemistry 18, 5294-5299), AG
PC method (Chomczynski, P. and Sacchi, N. (1987) Anal.
Biochem. 162, 156-159) to prepare total RNA
Purification can be performed by purifying mRNA from total RNA using Purification Kit (Pharmacia) or the like. Also,
MRNA can also be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia).

CDNA is obtained from the obtained mRNA using reverse transcriptase.
Are synthesized. For cDNA synthesis, AMV Reverse Transcriptas
e It can also be performed using the First-strand cDNA Synthesis Kit (Seikagaku Corporation) or the like. In addition, using the primers and the like described in the present specification, 5'-Ampli FINDER RACE Kit (Cl
ontech) and polymerase chain reaction (polymerasech)
ain reaction; PCR) using the 5'-RACE method (Frohman, M .;
A. et al. (1988) Proc. Natl. Acad. Sci. USA 85,
8998-9002; Belyavsky, A. et al. (1989) Nucleic Ac
ids Res. 17, 2919-2932), cDNA can be synthesized and amplified.

A target DNA fragment is prepared from the obtained PCR product and ligated to a vector DNA. Further, a recombinant vector is prepared therefrom, and introduced into Escherichia coli or the like, and colonies are selected to prepare a desired recombinant vector. Aim
The base sequence of DNA can be confirmed by a known method, for example, a dideoxynucleotide chain termination method.

In the DNA of the present invention, a nucleotide sequence having a higher expression efficiency can be designed in consideration of the codon usage of the host used for expression (Grantham, R. et al., J. Am.
etal. (1981) Nucleic Acids Res. 9, r43-74). The DNA of the present invention can be modified by a commercially available kit or a known method. Modifications include, for example, digestion with restriction enzymes, synthetic oligonucleotides and appropriate DNA
Insert fragment, add linker, start codon (AT
G) and / or stop codon (TAA, TGA, or TAG)
And the like.

[0029] Specifically, the DNA of the present invention comprises SEQ ID NO:
And a DNA consisting of base A at position 516 to base T at position 2933 in the base sequence of No. 1. The DNA of the present invention is a DNA that hybridizes with a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1, and includes a DNA that encodes a protein functionally equivalent to the above-described protein of the present invention. Hybridization conditions can be appropriately selected, and for example, the conditions described above can be used. The DNA to be hybridized is preferably a naturally occurring DN.
A, for example, cDNA or chromosomal DNA.

The present invention also relates to a vector into which the DNA of the present invention has been inserted. The vector of the present invention is not particularly limited as long as it can introduce the DNA of the present invention into a host cell. The vector of the present invention is useful for retaining or amplifying the DNA of the present invention in a host cell or expressing the protein of the present invention. As a vector, for example, when E. coli is used as a host, the vector may be E. coli (eg, JM109, DH5α, HB101, XL1-Blue).
In order to amplify and prepare a large amount in E. coli, etc., it has an "ori" to be amplified in Escherichia coli and further has a selected gene for transformed Escherichia coli (for example, any drug (ampicillin, tetracycline, kanamycin, chloramphenic acid). Drug resistance gene that can be identified by
There is no particular limitation as long as it has. Examples of vectors include, for example, M13-based vectors, pUC-based vectors, pBR322, pBlues
cript, pCR-Script and the like. In addition, in the case of subcloning and excision of cDNA, for example, pGEM-T, pDIRECT, pT7 and the like can be mentioned in addition to the above vectors. When a vector is used for the purpose of producing the protein of the present invention, an expression vector is particularly useful. As an expression vector, for example, when the purpose is expression in Escherichia coli, in addition to having the above-described characteristics such that the vector is amplified in Escherichia coli, the host may be JM109, DH5α, HB10
1.When E. coli such as XL1-Blue is used, a promoter that can be efficiently expressed in E. coli, such as la
cZ promoter (Ward, ES et al. (1989) Nature 3
41, 544-546; Ward, ES (1992) FASEB J. 6, 2422-2
427), araB promoter (Better, M. et al. (1988)
Science 240, 1041-1043), or have a T7 promoter. Such vectors include pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP,
Or pET (in this case, BL21 expressing T7 RNA polymerase is preferred as the host) and the like.

Further, the vector may include a signal sequence for polypeptide secretion. As a signal sequence for protein secretion, a pelB signal sequence (Lei, SP
et al (1987) J. Bacteriol. 169, 4379). Introduction of a vector into a host cell can be performed using, for example, a calcium chloride method or an electroporation method. Other than Escherichia coli, for example, as a vector for producing the protein of the present invention, a mammalian-derived expression vector (for example, pcDNA3 (manufactured by Invitrogen)) or pEF-BOS (Mizushima, S. and Nagata, S. 199
0) Nucleic Acids Res. 18, 5322), pEF, pCDM8), insect cell-derived expression vectors (eg, “BAC-TO-BAC Bacul
ovirus Expression Systems "(GIBCO BRL), pBac
PAK8), plant-derived expression vectors (eg, pMH1, pMH
2), animal virus-derived expression vectors (eg, pHS
V, pMV, pAdexLcw), an expression vector derived from a retrovirus (eg, pZIPneo), an expression vector derived from a yeast (eg, “Pichia Expression Kit” (manufactured by Invitrogen), pNV11, SP-Q01), expression from Bacillus subtilis Vectors (eg, pPL608, pKTH50).

When the expression is intended for expression in animal cells such as CHO cells, COS cells, and NIH3T3 cells, a promoter necessary for expression in cells, for example, SV40 promoter (Mulligan, RC et al. 1979) Nature 277, 10
8-114), MMLV-LTR promoter, EF1α promoter (Mizushima, S. and Nagata, S. (1990) Nucleic Acid
s Res. 18, 5322), it is essential to have a CMV promoter, etc., and a gene for selecting transformation into cells (for example, a drug resistance gene which can be identified by a drug (neomycin, G418, etc.)) ) Is more preferable. Examples of vectors having such properties include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13
And the like.

Furthermore, in a host vector system for the purpose of amplifying the copy number in a cell, when obtaining a stable production cell line, a vector having a DHFR gene complementary to a CHO cell lacking a nucleic acid synthesis pathway is used. (Eg, pCHOI) and a method of amplifying with methotrexate (MTX). For the purpose of transient expression of a gene, a method is used in which COS cells having a gene expressing the SV40 T antigen on the chromosome are transformed with a vector having an SV40 origin of replication (such as pcD). Is mentioned. As the replication origin, those derived from polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can also be used. Furthermore, in order to amplify the gene copy number in the host cell system, the expression vector is selected as an aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine guanine phosphoribosyl transferase (Ecogpt) as a selection marker.
Gene, dihydrofolate reductase (dhfr) gene and the like.

On the other hand, as a method for expressing the DNA of the present invention in an animal body, the DNA of the present invention is incorporated into an appropriate vector, for example, a retrovirus method, a liposome method, or the like.
Examples thereof include a method of introducing into a living body by a cationic liposome method, an adenovirus method, and the like. Thereby, diseases caused by mutation of the PDE10A gene of the present invention, and PDE1
Gene therapy for diseases and the like that can be treated by the expression of 0A can also be performed. As a vector to be used,
Examples include, but are not limited to, adenovirus vectors (eg, pAdexlcw) and retrovirus vectors (eg, pZIPneo). Vector of the present invention
General genetic manipulations such as DNA insertion can be performed according to conventional methods (Sambrook, J. et al. (1989) M
olecular Cloning 2nd ed., 5.61-5.63, Cold Spring H
arbor Lab. press). Administration into a living body may be an ex vivo method or an in vivo method.

[0035] The present invention also relates to a host cell into which the vector of the present invention has been introduced. The host cell into which the vector of the present invention is introduced is not particularly limited, and for example, Escherichia coli and various animal cells can be used. Escherichia coli includes, for example, JM109, DH5α, HB101, and the like, and animal cells include, for example, CHO cells, COS cells, 3T3 cells, and HeLa cells. The host cell of the present invention
For example, it can be used as a production system for producing or expressing the protein of the present invention. Production systems for protein production include in vitro and in vivo production systems. i
Examples of the in vitro production system include a production system using eukaryotic cells and a production system using prokaryotic cells. CHO cells are particularly preferred for the purpose of large-scale expression in animal cells. Introduction of a vector into a host cell can be performed by, for example, a calcium phosphate method, a DEAE dextran method, electroporation, lipofection, or the like.

When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as hosts.
As animal cells, mammalian cells such as CHO, COS, 3T
3, myeloma, BHK (baby hamster kidney), HeLa, V
ero, amphibian cells such as Xenopus oocytes (Valle, et al. (1981) Nature 291, 358-340), or insect cells such as Sf9, Sf21 and Tn5 are known. In particular, CHO cells lacking the DHFR gene
Dhfr-CHO (Urlaub, G. and Chasin, LA (1
980) Proc. Natl. Acad. Sci. USA 77, 4216-4220) and C
HO K-1 (Kao, FT and Puck, TT (1968) Proc. Na
Acad. Sci. USA 60, 1275-1281) can be suitably used. CHO cells are particularly preferred for the purpose of large-scale expression in animal cells. The introduction of the vector into the host cell can be performed by, for example, a calcium phosphate method, a DEAE dextran method, a method using cationic liposome DOTAP (manufactured by Boehringer Mannheim), an electroporation method, a lipofection method, or the like.

As plant cells, for example, Nicotiana
Cells derived from tabacum (Nicotiana tabacum) are known as a protein production system, which may be callus cultured. As fungal cells, yeasts, for example, genus Saccharomyces, for example, Saccharomyces cerevisiae, and filamentous fungi, for example, genus Aspergillus, for example, Aspergillus niger are known. .

When using prokaryotic cells, there is a production system using bacterial cells. As bacterial cells, E. coli (E. col)
i), for example, JM109, DH5α, HB101 and the like, and Bacillus subtilis are known.

[0039] These cells are transformed with the desired DNA, and the transformed cells are cultured in vitro to obtain the protein. Culture can be performed according to a known method. As a culture solution of animal cells, for example, DMEM, MEM, RPMI1640, IMDM can be used. At that time, a serum replacement solution such as fetal calf serum (FCS) may be used in combination, or serum-free culture may be performed. PH during culture
Is preferably about 6-8. Culture is usually about 30-
Perform at 40 ° C for about 15 to 200 hours, replace the medium if necessary,
Add aeration and agitation.

On the other hand, examples of a system for producing a protein in vivo include a production system using animals and a production system using plants. The target DNA is introduced into these animals or plants, and proteins are produced and recovered in the animals or plants. "Host" in the present invention
The term includes these animals and plants.

When animals are used, there are production systems using mammals or insects. Goats, pigs, sheep, mice, and cows can be used as mammals (Vick
i Glaser, SPECTRUM Biotechnology Applications, 199
3). When a mammal is used, a transgenic animal can be used. For example, the target DNA
Is prepared as a fusion gene with a gene encoding a protein specifically produced in milk, such as goat β casein. Next, the DNA fragment containing the fusion gene is injected into a goat embryo, and the embryo is transplanted into a female goat. A target protein can be obtained from milk produced by a transgenic goat born from a goat that has received an embryo or a progeny thereof. Hormones may optionally be used in transgenic goats to increase the amount of milk containing proteins produced by the transgenic goats (Ebert,
KM et al. (1994) Bio / Technology 12, 699-70
2).

As an insect, for example, a silkworm can be used. When a silkworm is used, the target protein can be obtained from the body fluid of the silkworm by infecting the silkworm with a baculovirus into which DNA encoding the protein of interest has been inserted (Susumu, M. et al.
(1985) Nature 315, 592-594).

When using plants, for example, tobacco can be used. When tobacco is used, DNA encoding the protein of interest is inserted into a plant expression vector, for example, pMON530, and this vector is inserted into Agrobacterium tumefaciens.
ciens). This bacterium is transferred to tobacco, for example, Nicotian Tabacum.
a tabacum), and the desired polypeptide can be obtained from the leaves of this tobacco (Ma, JK et al. (1994)
Eur. J. Immunol. 24, 131-138).

The thus-obtained protein of the present invention can be isolated from the inside or outside of a host cell (such as a medium) and purified as a substantially pure and homogeneous protein. The separation and purification of the protein may be performed by the same separation and purification methods used in ordinary protein purification, and is not limited at all. For example, chromatography columns, filters, ultrafiltration, salting-out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. are appropriately selected and combined. Then, the protein can be separated and purified. Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography and the like (Strategies for Protein Purification and Chlorination).
aracterization: A Laboratory Course Manual. Ed Dan
iel R. Marshak et al., Cold Spring Harbor Laborato
ry Press, 1996). These chromatographys can be performed using liquid-phase chromatography, for example, liquid-phase chromatography such as HPLC and FPLC. The present invention
Highly purified proteins using these purification methods are also included.

The protein can be arbitrarily modified or partially removed by applying an appropriate protein modifying enzyme before or after the protein is purified. As the protein modifying enzyme, for example, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, glucosidase and the like are used.

The present invention also relates to an antibody that binds to the protein of the present invention. The form of the antibody of the present invention is not particularly limited, and includes a monoclonal antibody in addition to a polyclonal antibody. Also included are antisera obtained by immunizing an immunized animal such as a rabbit with the protein of the present invention, polyclonal antibodies and monoclonal antibodies of all classes, as well as human antibodies and humanized antibodies obtained by genetic recombination.

The protein of the present invention used as a sensitizing antigen for obtaining an antibody is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammal, for example, a human, a mouse or a rat, and particularly preferably a protein derived from a human. Is preferred. Human-derived proteins can be obtained using the gene sequences or amino acid sequences disclosed herein. In the present invention, the protein used as the sensitizing antigen may be a complete protein or a partial peptide of the protein. Examples of the partial peptide of the protein include an amino (N) terminal fragment and a carboxy (C) terminal fragment of the protein. As used herein, “antibody” refers to an antibody that reacts with the full length or fragment of a protein.

A gene encoding the protein of the present invention or a fragment thereof is inserted into a known expression vector system, and the vector is used to transform a host cell described herein.
A target protein or a fragment thereof may be obtained from inside or outside the host cell by a known method, and these may be used as a sensitizing antigen. Alternatively, a cell expressing the protein, a lysate thereof, or a chemically synthesized protein of the present invention may be used as the sensitizing antigen. The short peptide can be used as an antigen by appropriately binding to a carrier protein such as keyhole limpet hemocyanin, bovine serum albumin, and ovalbumin.

As a mammal immunized with a sensitizing antigen,
Although not particularly limited, it is preferable to select in consideration of compatibility with the parent cell used for cell fusion. Generally, rodents, lagomorphs, and primates are used. . As rodent animals, for example, mice, rats, hamsters and the like are used. As an animal of the order Lagomorpha, for example, a rabbit is used. As a primate animal, for example, a monkey is used. As the monkeys, monkeys with lower nose (old world monkeys), for example, cynomolgus monkeys, rhesus monkeys, baboons, chimpanzees, and the like are used.

Immunization of an animal with a sensitizing antigen is performed according to a known method. As a general method, the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. In particular,
After sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, a normal adjuvant, such as Freund's complete adjuvant, is mixed if necessary, if desired, and emulsified. To be administered. Further, thereafter, it is preferable that the sensitizing antigen mixed with an appropriate amount of Freund's incomplete adjuvant be administered several times every 4 to 21 days. In addition, a suitable carrier can be used at the time of immunization with the sensitizing antigen. Immunization is performed in this manner, and it is confirmed by a conventional method that the level of a desired antibody in serum is increased.

Here, in order to obtain a polyclonal antibody against the protein of the present invention, for example, a small animal such as a rabbit or other mammals is immunized with the protein of the present invention and the desired antibody level in the serum is increased. After confirming the above, the blood of the mammal sensitized with the antigen is taken out. The serum is separated from the blood by a known method. As the polyclonal antibody, serum containing the polyclonal antibody may be used, or if necessary, a fraction containing the polyclonal antibody may be further isolated from the serum and used. For example, using an affinity column to which the protein of the present invention is coupled, a fraction that recognizes only the protein of the present invention is obtained, and this fraction is further purified by using a protein A or protein G column. And immunoglobulin G or M can be prepared.

In order to obtain a monoclonal antibody, after confirming that the level of the desired antibody is increased in the serum of the mammal sensitized with the above antigen, the immune cells may be removed from the mammal and subjected to cell fusion. . At this time, spleen cells are particularly preferable as the immune cells used for cell fusion. As the other parent cell fused with the immune cell,
Preferably, a mammalian myeloma cell, more preferably, a myeloma cell that has acquired characteristics for selecting fused cells by a drug is used. The cell fusion of the immune cells and myeloma cells is basically performed by a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, C. (198
1) It can be carried out according to Methods Enzymol. 73, 3-46).

More specifically, for example, a small animal such as a mouse is immunized with the protein of the present invention, and a spleen is excised from the mouse, crushed into cells, and fused with mouse myeloma cells using a reagent such as polyethylene glycol. To obtain a fused cell (hybridoma).

Hybridomas obtained by cell fusion can be used in a conventional selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine).
Selected by culturing. Culturing in the HAT culture solution is performed for a period of time sufficient to kill cells (non-fused cells) other than the target hybridoma, usually for several days to several weeks. Next, a conventional limiting dilution method is performed to screen and clone a hybridoma producing the desired antibody.

Further, in addition to obtaining the above-mentioned hybridoma by immunizing a non-human animal with an antigen, human lymphocytes such as EB
Human lymphocytes infected with the virus are sensitized in vitro with a protein, a protein-expressing cell or a lysate thereof, and the sensitized lymphocytes are fused with a human-derived, permanently dividing myeloma cell, such as U266, to bind to the protein. A hybridoma producing a desired human antibody having activity can also be obtained (Japanese Patent Application Laid-Open No. Sho 63-17688).

Next, the obtained hybridoma was transplanted into a mouse intraperitoneal cavity, ascites was recovered from the mouse, and the obtained monoclonal antibody was subjected to, for example, ammonium sulfate precipitation, protein
A, protein G column, DEAE ion exchange chromatography, protein of the present invention can be prepared by purification using an affinity column coupled thereto. The antibody of the present invention is used for purification and detection of the protein of the present invention, and is also a candidate for an agonist or antagonist of the protein of the present invention. It is also conceivable to apply this antibody to antibody therapy for diseases involving the protein of the present invention. When the obtained antibody is used for the purpose of administration to the human body (antibody therapy), a human antibody or a humanized antibody is preferable in order to reduce immunogenicity.

For example, a transgenic animal having a human antibody gene repertoire is immunized with a protein serving as an antigen, a protein-expressing cell or a lysate thereof to obtain an antibody-producing cell, and a hybridoma obtained by fusing the antibody-producing cell with a myeloma cell is used. To obtain a human antibody against the target protein (see International Patent Application Publication No.
WO92-03918, WO93-2227, WO94-02602, WO94-25585, WO9
6-33735 and WO96-34096).

In addition to producing antibodies using hybridomas, cells in which immune cells such as sensitized lymphocytes producing antibodies are immortalized by oncogenes may be used. The monoclonal antibody thus obtained can also be obtained as a recombinant antibody produced using a gene recombination technique (for example, Borrebaeck, CAKand).
Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBODIES,
Published in the United Kingdom by MACMILLAN PUBLI
SHERS LTD, 1990). Recombinant antibodies are produced by cloning DNA encoding the same from immune cells such as hybridomas or sensitized lymphocytes that produce the antibody, incorporating the DNA into an appropriate vector, and introducing it into a host. The present invention includes this recombinant antibody.

Furthermore, the antibody of the present invention may be an antibody fragment or a modified antibody thereof as long as it binds to the protein of the present invention. For example, as antibody fragments, Fab, F (ab ') 2,
Fv or single chain Fv (scFv) in which Hv and Lv Fv are linked by an appropriate linker (Huston, JS et al. (198
8) Proc. Natl. Acad. Sci. USA 85, 5879-5883). Specifically, an antibody is treated with an enzyme, for example, papain, pepsin or the like, to generate an antibody fragment, or a gene encoding these antibody fragment is constructed, and after introducing this into an expression vector, an appropriate host Expressed in cells (eg, Co, MS et al. (1994) J. Immunol.
152, 2968-2976; Better, M. and Horwitz, AH (198
9) Methods Enzymol. 178, 476-496; Pluckthun, A. an
d Skerra, A. (1989) Methods Enzymol. 178, 497-515;
Lamoyi, E. (1986) Methods Enzymol. 121, 652-663;
Rousseaux, J. et al. (1986) Methods Enzymol. 121,
663-669; Bird, RE and Walker, BW (1991) Tren
ds Biotechnol. 9, 132-137). As the modified antibody, an antibody bound to various molecules such as polyethylene glycol (PEG) can be used. "Antibody" of the present invention
Also includes these modified antibodies. Such a modified antibody can be obtained by subjecting the obtained antibody to chemical modification. These methods are already established in this field.

Furthermore, the antibody of the present invention can be prepared by using a chimeric antibody composed of a variable region derived from a non-human antibody and a constant region derived from a human antibody or a CDR (complementarity determining region) derived from a non-human antibody using known techniques. It can be obtained as a humanized antibody consisting of a human antibody-derived FR (framework region) and a constant region.

The antibody obtained as described above can be purified to homogeneity. The separation and purification of the antibody used in the present invention may be performed by the separation and purification methods used for ordinary proteins. For example, if appropriate selection and combination of chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc., antibodies can be separated and purified. Can be (Antibodi
es: A Laboratory Manual. Ed Harlow and David Lan
e, Cold Spring Harbor Laboratory, 1988). The concentration of the antibody obtained above can be measured by measuring absorbance or enzyme-linked immunosorbent assay (En
zyme-linked immunosorbent assay (ELISA) or the like.

Columns used for affinity chromatography include a protein A column and a protein G column. For example, as a column using a protein A column, Hyper D, POROS, Sepharose FF (P
harmacia) and the like. Examples of chromatography other than affinity chromatography include, for example,
Examples include ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein).
Purification and Characterization: A Laboratory
Course Manual. Ed Daniel R. Marshak et al., Cold S
pring Harbor Laboratory Press, 1996). These chromatographys can be performed using liquid phase chromatography such as HPLC and FPLC.

As a method for measuring the antigen-binding activity of the antibody of the present invention, for example, measurement of absorbance, Enzyme-linked immunosorbent assay (ELI)
SA), EIA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay)
Alternatively, a fluorescent antibody method can be used. When using ELISA, the protein of the present invention is added to a plate on which the antibody of the present invention is immobilized, and then a sample containing the target antibody,
For example, a culture supernatant of antibody-producing cells or a purified antibody is added.
An enzyme, for example, a secondary antibody recognizing an antibody labeled with alkaline phosphatase or the like is added, the plate is incubated, and then washed. Binding activity can be evaluated. As the protein, a protein fragment, for example, a C-terminal fragment or an N-terminal fragment thereof may be used. BIAcore (Pharmacia) can be used to evaluate the activity of the antibody of the present invention. By using these techniques, the antibody of the present invention is brought into contact with a sample expected to contain the protein of the present invention, and the method comprises detecting or measuring an immune complex of the antibody and the protein. The method for detecting or measuring the protein of the present invention can be carried out. Since the protein detection or measurement method of the present invention can specifically detect or measure a protein, it is useful for various experiments and the like using proteins.

The present invention also provides a polynucleotide comprising at least 15 nucleotides complementary to DNA encoding human PDE10A protein (eg, SEQ ID NO: 1) or its complementary strand. Here, “complementary strand” means G: C, A: T
(Or U) refers to one strand of a double-stranded polynucleotide consisting of base pairs of the other strand. Further, `` complementary '' is not limited to a completely complementary sequence in at least 15 consecutive nucleotide regions, at least 70%, preferably at least 80%, more preferably 90% or more, more preferably 95% or more It suffices that the homologous sequence has a homology of not less than%. The algorithm described in this specification may be used as an algorithm for determining homology.

Such polynucleotides include probes and primers used for detection and amplification of the DNA encoding the protein of the present invention, probes and primers used for detecting the expression of the DNA, and expression of the protein of the present invention. A nucleotide or a nucleotide derivative (for example, an antisense oligonucleotide or a ribozyme, or a DNA encoding the same) for suppression is included. Such a polynucleotide can also be used for producing a DNA chip. The polynucleotide of the present invention includes DNA and RNA. It also includes sense nucleotides and antisense nucleotides. When used as a primer, the 3 '
A restriction enzyme recognition sequence, a tag or the like can be added to the side.

The antisense oligonucleotide includes, for example, an antisense oligonucleotide that hybridizes at any position in the nucleotide sequence of SEQ ID NO: 1. This antisense oligonucleotide is preferably an antisense oligonucleotide for at least 15 consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 1. More preferably, it is an antisense oligonucleotide in which at least 15 consecutive nucleotides contain a translation initiation codon.

As the antisense oligonucleotide, derivatives and modifications thereof can be used.
Examples of the modified form include a modified lower alkyl phosphonate such as a methylphosphonate type or an ethylphosphonate type, a phosphorothioate modified form, a phosphoroamidate modified form, and the like.

Antisense oligonucleotides are DNA
Alternatively, not only those whose nucleotides corresponding to the nucleotides constituting the predetermined region of the mRNA are all complementary sequences, but also those which can specifically hybridize the DNA or mRNA and the oligonucleotide to the base sequence shown in SEQ ID NO: 1 , One or more nucleotide mismatches.

The antisense oligonucleotide derivative of the present invention acts on a cell producing the protein of the present invention,
By binding to DNA or mRNA encoding the protein, the transcription or translation thereof is inhibited, or the degradation of the mRNA is promoted, and the expression of the protein of the present invention is suppressed, resulting in the present invention Has the effect of suppressing the action of the protein.

The antisense oligonucleotide derivative of the present invention can be mixed with a suitable base material which is inactive against the derivative to prepare an external preparation such as a coating agent or a poultice. If necessary, excipients, isotonic agents, solubilizing agents, stabilizing agents, preservatives, soothing agents, etc. are added to tablets, splinters, granules, capsules, liposome capsules, injections. , A liquid preparation, a nasal drop and the like, and a freeze-dried preparation.
These can be prepared according to a conventional method. The antisense oligonucleotide derivative of the present invention is applied directly to the affected area of the patient, or is applied to the patient so as to be able to reach the affected area as a result of intravenous administration or the like. Further, an antisense encapsulating material that enhances durability and membrane permeability can be used. For example, liposomes, poly-L-lysine, lipids, cholesterol, lipofectin or derivatives thereof can be mentioned. The dose of the antisense oligonucleotide derivative of the present invention can be appropriately adjusted according to the patient's condition, and a preferred amount can be used. For example, it can be administered in the range of 0.1 to 100 mg / kg, preferably 0.1 to 50 mg / kg.

The antisense oligonucleotides of the invention inhibit the expression of the protein of the invention and are therefore useful in inhibiting the biological activity of the protein of the invention. Moreover, an expression inhibitor containing the antisense oligonucleotide of the present invention is useful in that it can suppress the biological activity of the protein of the present invention.

The present invention also relates to a method for screening for a compound that binds to the protein of the present invention, using the protein of the present invention. That is, the protein of the present invention is useful for screening for a compound that binds to the protein. This screening method comprises: (a) a step of bringing a test sample into contact with the protein or its partial peptide of the present invention; (b) a step of detecting the binding activity between the protein or its partial peptide of the present invention and the test sample; c) selecting a compound that binds to the protein of the present invention or a partial peptide thereof.

The protein of the present invention used for screening may be a recombinant protein or a protein of natural origin. Further, it may be a partial peptide. The test sample is not particularly limited and includes, for example, a cell extract, a cell culture supernatant, a fermentation microorganism product, a marine organism extract, a plant extract, a purified or crude protein, a peptide, a non-peptide compound, Synthetic low molecular weight compounds and natural compounds are exemplified. The protein of the present invention to be brought into contact with a test sample may be, for example, a purified protein, a soluble protein, a form bound to a carrier, or a fusion protein with another protein, on a cell surface or a cell membrane. As the expressed form,
Alternatively, it can be brought into contact with a test sample as a membrane fraction.

As a method for screening a protein binding to the protein using the protein of the present invention, for example, many methods known to those skilled in the art can be used. Such screening can be performed, for example, by immunoprecipitation. Specifically, it can be performed as follows. The gene encoding the protein of the present invention is inserted into a vector for exogenous gene expression such as pSV2neo, pcDNA I, pCD8 or the like, whereby the gene is expressed in animal cells or the like. SV40 early promoter (Rigby In Williamson (ed.),
Genetic Engineering, Vol.3. Academic Press, Londo
n, p.83-141 (1982)), EF-1α promoter (Kim, DM
et al. (1990) Gene 91, 217-223), CAG promoter (Ni
wa, H. et al. (1991) Gene 108, 193-200), RSV LTR p
romoter (Cullen, BR (1987) Methods in Enzymology
152, 684-704), SRα promoter (Takebe, Y. et al.
(1988) Mol. Cell. Biol. 8, 466-472), CMV immediat
e early promoter (Seed, B. and Aruffo, A. (1987) Pr
Natl. Acad. Sci. USA 84, 3365-3369), SV40 lat.
e promoter (Gheysen, D. and Fiers, W. (1982) J. Mo
l. Appl. Genet. 1, 385-394), Adenovirus late promo
ter (Kaufman, RJ et al. (1989) Mol. Cell. Biol.
9, 946-958), and any commonly used promoter such as the HSV TK promoter.

In order to express a foreign gene by introducing the gene into animal cells, electroporation (Chu, G. et al. (1987) Nucleic Acid Res. 15, 1311)
-1326), calcium phosphate method (Chen, C and Okayama,
H. (1987) Mol. Cell. Biol. 7, 2745-2752), DEAE dextran method (Lopata, MA et al. (1984) NucleicAci)
ds Res. 12, 5707-5717; Sussman, DJ and Milman,
G. (1985) Mol. Cell. Biol. 4, 1642-1643), lipofectin method (Derijard, B. (1994) Cell 7, 1025-1037;
Lamb, BT et al. (1993) Nature Genetics 5, 22-3
0; Rabindran, SK et al. (1993) Science 259, 230-
234) etc., but any method may be used.

By introducing a recognition site (epitope) of the monoclonal antibody of which specificity is known to the N-terminus or C-terminus of the protein of the present invention, the protein of the present invention can be used as a fusion protein having the recognition site of the monoclonal antibody. Can be expressed. A commercially available epitope-antibody system can be used (Experimental Medicine 13, 85-90 (1995)). Vectors capable of expressing a fusion protein with β-galactosidase, maltose binding protein, glutathione S-transferase, green fluorescent protein (GFP), and the like via a multicloning site are commercially available.

A method for preparing a fusion protein by introducing only a small epitope portion consisting of several to several tens of amino acids in order to minimize the properties of the protein of the present invention by changing it into a fusion protein Have also been reported. For example, polyhistidine (His-
tag), influenza agglutinin HA, human c-myc, FLAG, V
esicular stomatitis virus glycoprotein (VSV-G
P), T7 gene10 protein (T7-tag), human herpes simplex virus glycoprotein (HSV-tag), E-tag (epitope on monoclonal phage) and other monoclonal antibodies that recognize it, Can be used as an epitope-antibody system for screening proteins that bind to (Experimental Medicine 13, 85-90
(1995)).

In immunoprecipitation, an immunocomplex is formed by adding these antibodies to a cell lysate prepared using a suitable surfactant. This immune complex comprises the protein of the present invention, a protein capable of binding thereto, and an antibody. In addition to using antibodies against the above epitopes, immunoprecipitation can be performed using antibodies against the protein of the present invention. Antibodies against the protein of the present invention include, for example, a gene encoding the protein of the present invention introduced into an appropriate Escherichia coli expression vector, expressed in Escherichia coli, and the expressed protein is purified. It can be prepared by immunizing goats, chickens and the like. Alternatively, it can be prepared by immunizing the above animal with the synthesized partial peptide of the protein of the present invention.

[0079] The immunocomplex may be, for example, a mouse IgG
For antibodies, use Protein A Sepharose or Protein G Sepha
It can be settled using rose. When the protein of the present invention is prepared, for example, as a fusion protein with an epitope such as GST, glutathione-Sepha
Using a substance that specifically binds to these epitopes such as rose 4B, an immune complex can be formed in the same manner as in the case of using the antibody of the protein of the present invention. For general methods of immunoprecipitation, see, for example, the literature (Harlo).
w, E. and Lane, D .: Antibodies, pp.511-552, Cold Sp
ring Harbor Laboratory publications, New York (198
8)) The method may be performed according to or according to the method described.

For analysis of the immunoprecipitated protein, SDS-
PAGE is common, and the bound protein can be analyzed based on the molecular weight of the protein by using an appropriate concentration gel. In addition, at this time, it is generally difficult to detect the protein bound to the protein of the present invention by a normal staining method of the protein such as Coomassie staining or silver staining, so that ³⁵ S-
By culturing cells in a culture solution containing methionine or ³⁵ S-cysteine, labeling proteins in the cells, and detecting them, detection sensitivity can be improved. Once the molecular weight of the protein is known, the target protein can be directly purified from the SDS-polyacrylamide gel and its sequence can be determined.

As a method for isolating a protein binding to the protein using the protein of the present invention, for example, a West Western blotting method (Sk
olnik, EY et al. (1991) Cell 65, 83-90). That is, a phage vector (λgt11, ZAP) is prepared from cells, tissues, and organs that are expected to express a binding protein that binds to the protein of the present invention.
A cDNA library using LB-
The protein expressed on agarose and expressed on a filter are immobilized, and the protein of the present invention, which is purified and labeled, is reacted with the filter, and the plaque expressing the protein bound to the protein of the present invention is detected by the label. I just need. Methods for labeling the protein of the present invention include biotin labeling and other proteins (for example, GST
And a method utilizing the binding property of biotin and avidin, and an antibody that specifically binds to the protein of the present invention or a peptide or polypeptide (eg, GST or the like) fused to the protein of the present invention. It can be detected by the method used. Labeling may be performed by a method using a radioisotope, a method using fluorescence, or the like.

As another embodiment of the screening method of the present invention, a two-hybrid system using cells (Fields, S., and Sternglanz, R. (1994) Trends Gen.
et. 10, 286-292; Dalton S, and Treisman R (1992) Ch
aracterization of SAP-1, a protein recruited by ser
um response factor to the c-fos serum responseelem
ent, Cell 68, 597-612, `` MATCHMAKER Two-Hybrid Sys
tem '', `` Mammalian MATCHMAKER Two-Hybrid Assay Ki
t '', `` MATCHMAKER One-Hybrid System '' (both Clon
tech); `` HybriZAP Two-Hybrid Vector System '' (St
ratagene))). In the two-hybrid system, the protein of the present invention or a partial peptide thereof is ligated to the SRF DNA binding region or GAL4 DN.
Fused with A-binding region and expressed in yeast cells, and expressed in a form fused with VP16 or GAL4 transcription activation region from cells expected to express a protein that binds to the protein of the present invention Such a cDNA library is prepared, introduced into the above yeast cell, and the cDNA derived from the library is isolated from the detected positive clone (when the protein binding to the protein of the present invention is expressed in the yeast cell, Binds to activate the reporter gene, and a positive clone can be confirmed). The protein encoded by the cDNA can be obtained by introducing the isolated cDNA into E. coli and expressing it. As a result, it is possible to prepare a protein that binds to the protein of the present invention or a gene thereof. As a reporter gene used in the two-hybrid system, for example,
In addition to HIS3 gene, Ade2 gene, LacZ gene, CAT gene, luciferase gene, PAI-1 (Plasminogen activ
ator inhibitor type1) gene and the like, but are not limited thereto. Screening by the two-hybrid method can be performed using mammalian cells and the like in addition to yeast.

Further, the screening for a compound that binds to the protein of the present invention can be carried out by using affinity chromatography. For example, the protein of the present invention is immobilized on a carrier of an affinity column, and a test sample which is expected to express a protein that binds to the protein of the present invention is applied thereto. The test sample in this case includes, for example, a cell extract, a cell lysate, and the like. After applying the test sample, the column is washed, and the protein that specifically binds to the column is purified, whereby the protein that binds to the protein of the present invention can be prepared.

The obtained protein is analyzed for its amino acid sequence, oligo DNA is synthesized based on the amino acid sequence, and a cDNA library is screened using the DNA as a probe to obtain a DNA encoding the protein. it can.

In the present invention, a biosensor utilizing the surface plasmon resonance phenomenon can be used as a means for detecting or measuring a bound compound. A biosensor utilizing the surface plasmon resonance phenomenon enables real-time observation of the interaction between the protein of the present invention and a test compound as a surface plasmon resonance signal using a trace amount of protein and without labeling. (Eg, BIAcore, Pharmacia). Therefore, BIAcor
By using a biosensor such as e, it is possible to evaluate the binding between the protein of the present invention and the test compound.

The method for isolating not only proteins but also compounds (including agonists and antagonists) that bind to the protein of the present invention includes, for example, immobilizing the compound of the present invention, A method for screening a molecule that binds to the protein of the present invention by acting on a product bank or a random phage peptide display library or the like, or a screening method using high throughput by combinatorial chemistry technology (Wrighton, NC et al. (1996) S
mall peptides as potent mimetics of the protein ho
rmone erythropoietin, Science 273, 458-64; Verdin
e, GL (1996) The combinatorial chemistry of nat
ure, Nature 384, 11-13; Hogan, JC Jr. (1996) Di
rected combinatorial chemistry, Nature 384, 17-1
9) is known to those skilled in the art. The compound isolated by the screening of the present invention is a candidate for a compound that promotes or inhibits the activity of the protein of the present invention. Such compounds can be applied to the treatment of diseases caused by abnormal expression or dysfunction of the protein of the present invention, and diseases that can be treated by controlling the activity of the protein of the present invention.
A substance obtained by using the screening method of the present invention and having a part of the structure of a compound having an activity of binding to the protein of the present invention, which is converted by addition, deletion and / or substitution also binds to the protein of the present invention. Included in the compound.

The present invention also relates to a method for screening a compound that promotes or inhibits the activity of the protein of the present invention. Since the protein of the present invention has an activity as a cyclic nucleotide phosphodiesterase, it is possible to screen for a compound that promotes or inhibits the activity of the protein of the present invention using this activity as an index.

This screening method comprises the steps of: (a) contacting the protein of the present invention with 3 ′, 5′-cyclic nucleotide monophosphate in the presence of a test sample;
(B) a step of detecting the hydrolysis activity of the protein on 3 ', 5'-cyclic nucleotide monophosphate, (c) an increase in the hydrolysis activity as compared to the case where the protein is detected in the absence of a test sample Or selecting a compound to be reduced.

The protein of the present invention used for screening may be a recombinant protein or a protein of natural origin. Further, it may be a partial peptide. The test sample is not particularly limited and includes, for example, a cell extract, a cell culture supernatant, a protein, a peptide, a synthetic low-molecular compound, and a natural compound. Further, a compound obtained by screening for a compound that binds to the protein of the present invention can be used as a test compound. As the 3 ′, 5′-cyclic nucleotide monophosphate serving as a substrate of the protein of the present invention, for example, cAMP, cGMP or cCMP can be used.

The detection of the hydrolysis activity of 3 ′, 5′-cyclic nucleotide monophosphate by the protein of the present invention was carried out by a two-step assay (Mukai et al., Br. J. Pharmaco.
l. (1994) 111, 389-390). Compounds that promote or inhibit the activity of the protein of the present invention isolated by this screening can be used for the treatment of diseases associated with the protein of the present invention (eg, diseases involving development and differentiation, neurological diseases, cardiopulmonary diseases, kidney diseases). It can be applied to medicines for prevention.

When the compound isolated by the screening method of the present invention, or the protein or its partial peptide of the present invention is used as a drug, the compound may be used in combination with other solutes or solvents as appropriate in addition to using the isolated compound itself. To form a composition. For example, a compound isolated by the screening method of the present invention, or a protein or a partial peptide thereof of the present invention can be used for humans or animals, such as mice, rats, guinea pigs, rabbits, chickens, cats, dogs, sheep, pigs, cows, monkeys, When used as a medicine for baboons and chimpanzees,
In addition to directly administering a protein or an isolated compound itself to a patient, it is possible to formulate and administer it by a known pharmaceutical method. For example, tablets, capsules, elixirs, microcapsules, or a sterile solution with water or other pharmaceutically acceptable liquid or suspension, if desired, may be sugar coated. It can be used parenterally in the form of injections. Also, for example, a pharmacologically acceptable carrier or medium, specifically, sterilized water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavor, excipient, vehicle, It is conceivable to formulate a formulation by appropriately combining it with a preservative, a binder and the like and mixing it in a unit dose form required for generally accepted pharmaceutical practice. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.

Examples of additives that can be mixed with tablets and capsules include binders such as gelatin, corn starch, tragacanth gum, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin,
Swelling agents such as alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry are used. When the unit dosage form is a capsule, the above-mentioned materials may further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.

Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other adjuvants, for example,
Sorbitol, D-mannose, D-mannitol, sodium chloride, and a suitable solubilizing agent, for example, alcohol, specifically ethanol, polyalcohol, for example, propylene glycol, polyethylene glycol, a nonionic surfactant, for example, polysorbate 80 (TM), HC
May be used with O-50.

The oily liquid includes sesame oil and soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Also, buffers, such as phosphate buffers, sodium acetate buffers, soothing agents, such as
Procaine hydrochloride, stabilizers such as benzyl alcohol,
You may mix with phenol and an antioxidant. The prepared injection is usually filled into a suitable ampoule.

The administration to a patient can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., or intranasally, transbronchially, intramuscularly, transdermally, or orally by a person skilled in the art. It can be done by a method. The dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose. In addition, the compound
As long as it can be encoded by DNA, the DNA may be incorporated into a vector for gene therapy to perform gene therapy. The dose and administration method vary depending on the patient's weight, age, symptoms, and the like, but can be appropriately selected by those skilled in the art.

For example, the dose of a compound that binds to the protein of the present invention or a compound that promotes or inhibits the activity of the protein of the present invention varies depending on the condition.
In the case of oral administration, it is generally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day for an adult (with a body weight of 60 kg). In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms and administration method.
About 0.01 to 100 mg per day, preferably about 0.1 to 20 mg,
More preferably, about 0.1 to 10 mg is administered by intravenous injection. For other animals, weight 60 kg
It can be administered in an amount converted per unit or an amount converted per body surface area.

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

[0098]

[Example 1] Cloning of human PDE10A cDNA
G cyclic nucleotide phosphodiesterase family
First, to isolate a new molecule belonging to
EST database using amino acid sequence of catalytic domain of E4
(dbEST) homology search (BLAST search; Altschul, S.F. et
 al., 1990, J. Mol. Biol. 215: 403-410) [World Wide
 Web (http://www.blast.genome.ad.jp/)]
Was. As a result, a cDNA with EST number W04835
Primary structure differs from any of the DE family isozymes
And PDE motif (HDX _TwoHX_FourN)
And found. So, first, the I.M.A.G.E.consortia
Human cDNA clone 298975 containing W04835
It was purchased and its full-length nucleotide sequence was determined (base 6 in FIG. 2).
34-2320). From sequence analysis, this clone was identified as P
CGMP binding PDE, not only DE motif sequence, ie PDE
2, including the cGMP binding motif found in PDE5 and PDE6
It turned out to be. But this clone has 5 '
And did not appear to contain both the 3 'end. cDNA
Specific for internal sequence within clone 298975 to obtain full length
Sense and antisense oligonucleotide primers
5'- and 3'-RACE were performed using immers (Frohma
n, M.A., 1993, Methods Enzymol. 218: 340-356).

A human brain cDNA library for RACE-PCR (Clontech) was used as a template cDNA. Gene-specific primers prepared based on the base sequence of EST (5 ′ RACE primer, 5′-GGCAG CATGG GTGGA GTTGT GGTAG G-3 ′ / SEQ ID NO: 3; 3 ′ RACE primer, 5′-TTGGA AAGGA AAGA
G TAAAG GGAAG C-3 '/ SEQ ID NO: 4)
Using an AP1 primer (manufactured by Clontech) complementary to the adapter sequence added to both ends of the cDNA fragment of the above library, 0.2 mM dNTP, 10 pmol each primer, 2.5 units TAKARA LA Taq (Takara) PCR) was performed in a 50 μl reaction solution containing 1 × buffer attached to the enzyme. PCR should be performed at 94 ° C for 30 seconds and at 72 ° C for 4 minutes.
The cycle was performed, followed by 5 cycles of 94 ° C. for 30 seconds and 70 ° C. for 4 minutes, followed by 25 cycles of 94 ° C. for 30 seconds and 68 ° C. for 4 minutes. The amplified fragment was ligated into pGE by TA ligation.
It was cloned into an MT vector (Promega) and both strands were sequenced.

As a result of ligation of the obtained cDNA with the original EST clone, PDE10A having a total length of 3631 bases was obtained (FIG. 1).
The isolated PDE10A had an open reading frame encoding 806 amino acids (FIG. 2). PDE10A
Is shown in SEQ ID NO: 1, and the predicted amino acid sequence is shown in SEQ ID NO: 2. On the C-terminal side of PDE10A, a known P
A catalytic region conserved in DE was identified. When a database search was performed for the determined nucleotide sequence, an EST (EST number AA973152) corresponding to the 3 ′ portion of PDE10A was found in addition to W04835 described above. That is, it was found that W04835 and AA973152 were derived from the same gene.

The nucleotide sequence around the first methionine codon is a consensus sequence of the translation initiation site of eukaryotes (Kozak, M.,
1986, Cell 44: 283-292).
There are three stop codons upstream of TG. In the 3 'non-coding region, 15 nucleotides upstream of the poly A tail,
A typical AATAAA polyadenylation signal was included.

[Example 2] Human PDE10A and known human cGMP
Comparison with bound PDEs Other known PDs of the predicted amino acid sequence of human PDE10A
A homology search for the catalytic domain of E revealed that this sequence showed three cyclic nucleotide phosphodiesterase families that hydrolyze cGMP, namely PDE2, a cGMP stimulated PDE.
(43% identity; Rosman, GJ et al., 1997, Gene 191:
89-95), PDE5, a cGMP-specific PDE (47% identity; St
acey, P. et al., 1998, Biochem. Biophys. Res. Comm
un. 247: 249-254) and the photoreceptor PDE PDE6 (4
0% identity; Pittler, SJ et al., 1990, Genomics
6: 272-283; Collins, C., 1992, Genomics 13: 698-70
4; Viczian, AS, 1995, Gene 166: 205-211).

Human PDE10A and cG reported to date
The amino acid sequence of the MP-binding PDE was compared and examined using the Clustal W program (FIG. 3). Gen of PDE used for comparison
The registration numbers at Bank are 2A: U67733 and 5A: AJ, respectively.
004865, 6A: M26061, 6B: S41458, 6C: U31973.
The cGMP binding and catalytic domains showed relatively high homology to known PDEs. Two cGMP binding motifs were also conserved. Human PDE10A has three cGMP-bound PDs
Like E, it contains a non-catalytic cGMP binding domain spanning about 380 amino acid residues, and the entire domain contains PDE2, PDE
5, and 31%, 27%, and 22%, respectively, with PDE6. Among known PDE family members, the homology within the catalytic domain is 80-95% and is not limited to this region, but spans almost the entire coding region. Thus, the sequence homology indicated that the isolated cDNA was the first isozyme of the novel PDE family, which was named PDE10A.

As a result of searching the GenBank ^™ database of PDE10A, it was found that C. elegans showed the highest homology with PDE10A.
Amino acid sequence of cosmid clone CELC32E12. The identity and homology of the catalytic domain amino acids were 51% and 75%, respectively. Furthermore, the identity and homology outside the catalytic domain is high,
They were 61% and 74%, respectively. Drosophili (Drosophi) is considered a homolog of mammalian PDE4
dnc, a learning and memory gene product of la melanogaster)
Shows about 60% identity with the human PDE4 family. Human
The highly conserved amino acid sequence between PDE10A and CELC32E12 suggests that CELC32E12 encodes a homolog of human PDE10A.

All cGMP-bound PDEs (ie, PDE2, PDE
5, and PDE6), it has been proposed that two internal homologous repeats are conserved within the cGMP binding region and that they form two different cGMP binding sites (McAllister-Lucas, LM, 1993). , J. Biol. Chem. 26
8: 22863-22873). Tandem repeat pattern is cAMP
It is also found in the sequences of the protein-dependent and cGMP-dependent protein kinases, which represent two different cyclic nucleotide binding sites in both kinases (Shabb, JB & Corbin, JD, 1992, J. Bio.
l. Chem. 267: 5723-5726). cGMP-specific PDE (PDE5)
Studies of the binding and dissociation patterns of cGMP suggest that there is more than one type of cGMP binding site (Th
omas, MKet al., 1990, J. Biol. Chem. 265: 14964-
14970). Furthermore, from Scatchard analysis of cGMP binding, cGS
-PDE (Stroop, SD et al., 1991, J. Biol. Chem. 26
6: 23802-23809), cGB-PDE (McAllister-Lucas LM, 1
995, J. Biol. Chem. 270: 30671-30679), and ROS-P
DE (Gillespie, PG & Beavo, JA, 1989, Proc. Nat
l. Acad. Sci. USA 86: 4311-4315) has shown that two classes of non-catalytic sites exist for cGMP binding. The two different cGMP binding sites share a common sequence in the amino acid sequence of all published mammalian cGMP binding PDEs, LX ₂ P (V / I) X _5-7 (V / I / L) (V / I / L) (G / A) (V / I / L)
X ₄ N (K / R) X _{5-10 with} FX ₃ DE. Site-directed mutagenesis of bovine lung cGB-PDE has shown that the conserved Asp residues at all cGMP-binding PDE sites are important for interaction with cGMP (McAllister-Lucas LM). , 1995,
J. Biol. Chem. 270: 30671-30679). PDE10A also has two internal homologous repeat sequences, but all cGMP-binding PDEs
Are not conserved in the first repeat of PDE10A. Amino acids involved in cGMP binding, such as Arg-235 and His-236, differ from PDE10A
Has a different specificity from other cGMP-binding PDEs.

PDE10A shares some features with PDE2, both contain a cGMP binding sequence at the amino terminus, and contain cAMP and cGMP.
It has hydrolytic resolution for both MPs (described below). In the PDE2 family, cAMP hydrolytic activity is reduced at concentrations from 0.5 μM.
Stimulated by cGMP, the Km of cAMP and cGMP is 40
μM and 25 μM (Pyne, NJ et al., 1986, Bio
chem. J. 234: 325-334). On the other hand, PDE10A does not stimulate its cAMP hydrolysis activity even with cGMP at a concentration of up to 100 μM (described later). Furthermore, compared to PDE2, PDE10A
The Km of the hydrolysis activity of cAMP is 100 times lower than that of cGMP (described later). In addition, EHNA, a PDE2-specific inhibitor, does not inhibit PDE10A in inhibitor assays (see below). These findings clearly indicate that PDE10A is not a member of the PDE2 family, but the first isozyme of the new family.

[Example 3] Tissue distribution of human PDE10A mRNA <RT-PCR> The tissue distribution of human PDE10A mRNA was examined by RT-PCR. As templates, cDNAs of various tissues of Clontech with quantitative correction were used. Primer is 5'-ATGATGGACA
GAGACAAGAA-3 ′ (SEQ ID NO: 5) and 5′-AATGTTGCTTTG
Using ATGTTGTTGG-3 '(SEQ ID NO: 6), after 95 ° C for 10 minutes, 34 cycles of “94 ° C for 20 seconds, 50 ° C for 10 seconds, and 72 ° C for 1 minute” were performed, followed by 5 minutes at 72 ° C and 4 ° C. The reaction was stopped. As a result, RT-PCR products were detected in tissues such as brain, heart, lung, kidney, skeletal muscle, thymus, and placenta (FIG. 4). In particular, strong expression was observed in the fetal brain, heart, kidney, and adult placenta.

<Northern blot analysis> To further examine the tissue distribution of PDE10A mRNA, Northern blot analysis was performed. Multiple human tissue Northern blots (Clon
tech) and human RNA Master Blot ^TM of poly
Using (A) + (Clontech), ³² P-labeled PDE10
ExpressHyb hybridization sol using AcDNA as a probe
hybridization (Clontech) at 68 ° C. for 1 hour. 0.2 × SSC / 0.1% SDS After washing at 52 ° C for 30 minutes, the filter is applied to X-ray film,
Exposure was performed at ℃. As a result, as shown in FIG.
The 10A mRNA was found to be expressed as a 9.5 kb transcript. Its expression level is highest in fetal brain and kidney,
Expression was also observed in adult brain, kidney, and placenta. PDE1
To analyze the tissue specificity of 0A mRNA in more detail, quantitative analysis was performed using human RNA Master Blot ^™ (Clontech), which standardized and blotted polyA RNA from 50 different human tissues. (FIG. 5B). PDE10A mRNA was ubiquitously expressed at low levels in all tissues examined. However, transcript expression levels were very high in the extrapyramidal tract putamen and caudate nuclei.

Example 4 Expression of Human PDE10A in Insect Cells 711 amino acids at the carboxyl terminal side of PDE10A (bases 801-29)
93; amino acids 96-806) as primers
5'- GGATCC TACG AATATGCAGG GAGTTGTAT-3 '(base 801-822
(SEQ ID NO: 7) and 5'- AAGCTT GTCA AAGAAG
It amplified by PCR using CAAG ATGAGGAT-3 '(corresponding to base 2972-2993) (SEQ ID NO: 8). The underlined sequences are restriction enzyme linkers for BamHI and HindIII, respectively. The fragment was subcloned into a T vector (manufactured by Pramega Biotech), and after confirming the sequence, a 2.2 kb BamHI-DNA containing a coding region of 711 amino acids at the carboxyl terminal of PDE10A.
A baculovirus transfer vector pBlue modified from a HindIII fragment to include a tag for six histidine residues and an enterokinase cleavage site (Xpress ^™ tag)
It was cloned into BAC-His2B (Invitrogen). Recombinant virus stock was prepared according to the attached instructions. Sf9 cells were cultured at 27 ° C. in Grace's insect medium (Gibco BRL) containing 10% fetal calf serum. For expression, 1 × 10 ⁸ Sf9 cells were infected with the virus and the multiplicity of infection (moi)
At 5, infected in a final volume of 50 ml. Two days after infection, the transfected Sf9 cells were collected by centrifugation, and homogenized buffer (20 mM Tris-HCl, 1 mM EDTA, 20 mM sucr).
ose, 100μM phenylmethylsulfonyl fluoride, pH7.5)
Was resuspended to 1 × 10 ⁷ cells / ml, and the cells were disrupted by sonication. Remove cell debris by centrifugation at 14,000 xg for 10 minutes, and remove supernatant (cytoplasmic fraction) and precipitate (granule (pa
rticulate) fraction until dispensing and use for the next analysis-
Stored at 70 ° C.

<Western Analysis> Lysates (corresponding to 11 × 10 ⁴ cells) of cells infected with PDE10A and mock-infected cells were separated by denaturing PAGE, the gel was stained with Coomassie dye, and the band of the expressed protein was separated. It was confirmed. Alternatively, a PVDF membrane (Millipore
And Western analysis was performed. Western analysis was performed using the enhanced chemiluminescence method (Amersham) according to the instructions. Anti-primary antibody
Xpress ^™ antibody (manufactured by Invitrogen), horseradish peroxidase-conjugated anti-mouse IgG (Life scie
nce) were used at a dilution of 1: 1000 and 1: 500, respectively. As a result of Western analysis, a clear band having a molecular weight of 84.4 kDa was detected by SDS-polyacrylamide gel electrophoresis in the cytoplasmic fraction and the particulate fraction (insoluble fraction) prepared from infected Sf9 cells, and a histidine tag was detected. Was consistent with the expected molecular weight of the PDE10A fusion protein with the addition of (FIG. 6A). In addition, this band
Anti-Xpress histidine-tagged Western blot positive, showing the same mobility as the only major protein
(FIG. 6B).

Example 5 Measurement of PDE Activity of Human PDE10A <Kinetics of PDE10A> To examine the properties of human PDE10A as an enzyme, a cytoplasmic extract was prepared from transfected Sf9 cells, and 1 μM cGMP or 1 μM was prepared. Assay of cyclic nucleotide hydrolysis activity was performed using cAMP. PDE activity was measured by the method described previously (M
ukai, J. et al., 1994, Br. J. Pharmacol. 111: 389-
390) with some modifications. The sample was 50 mM Tris
(pH 8.0), 2 mM EGTA, 5 mM MgCl ₂ , 0.1 mg / ml bovine serum albumin, 0.064-0.1 μM [ ³ H] cGMP or [ ³ H] cAMP,
Various concentrations of unlabeled cyclic nucleotides [final concentrations of cGMP (0.064-2 μM) or cAMP (3-30
0 μM) in a 0.5 ml reaction solution,
Incubated at 30 ° C. for 10 minutes. Boil for 5 minutes to stop the reaction, add 50 μl of 2.5 mg / ml snake venom,
Incubation was further performed at 30 ° C. for 10 minutes. [ ³ H] cAMP or [ ³ H] cGMP was applied to a BioRad AG50W-X4 resin column (Bio-Rad
Company, Mississauger, ON, Canada) and Beckman
Using LS 5000TA, were measured levels of ³ H in ACS scintillation solution of 10 ml. Blanks were measured with buffer only. Inhibition experiments used dimethyl sulfoxide (DM
Various inhibitors were dissolved in SO) 10 ^{- 7} ~10 ^-4 were measured in addition to the sample so that the M. In the PDE assay, the solvent concentration of the added inhibitor did not exceed 2% (v / v). All assays were performed in triplicate.

Sf9 cells expressing PDE10A had 50-fold higher cGMP and cAMP hydrolysis activity than mock-transfected cells (FIG. 7). CG of recombinant PDE10A
To determine whether the hydrolysis activity of MP and cAMP could be regulated by the other cyclic nucleotide, as seen in the PDE2 and PDE3 families, experiments were performed in which cGMP or cAMP was added. 1 μM cAMP with recombinant PDE10A
Was not affected by the addition of 1 μM cGMP. However, the hydrolysis of 1 μM cGMP by PDE10A was significantly inhibited by the addition of 1 μM cAMP. PDE1
The level of PDE activity in OA infected cells is sufficiently high, and the amount of cyclic nucleotide hydrolysis in basal insect cells is considered negligible.

To determine the Km and Vmax of this enzyme,
CGMP and cA at various substrate concentrations using dilute enzymes
The hydrolysis rate of MP was measured. As shown in FIG. 8A, P
CGMP hydrolysis activity of DE10A is standard Michaelis-Menten
Kinetic (γ ² = 0.99), Km is 2.7 ± 0.
At 3 μM, Vmax was 833 ± 67 pmol / min / mg protein.
Furthermore, the Km and Vmax of human PDE10A for cAMP are 0.033 ± 0.004 μM and 57.1 ± 5.7 pmol / min / mg pr, respectively.
otein (FIG. 8B). This is the lowest Km value for cAMP observed to date with phosphodiesterases, 2-4 times lower than that of PDE8,
100 to 200 times lower than that of PDE4.

<Inhibitor Properties of PDE10A> The activity of PDE10A
50 times higher than the intrinsic activity of Sf9 PDE. So, inhibition
The background of endogenous PDE activity
The effect of the inhibitor carrier was also subtracted as well.
Table 1 shows the results. I c₅₀ Value (concentration at 50% inhibition)
Is 1 μM unlabeled cGMP and 22 nM [^ThreeH] Using cGMP,
Calculated by linear regression in the presence of various inhibitors. Day
Data represent mean ± S.D. Of triplicate assays. PDE10A is cAMP
Shows both PDE and cGMP PDE activity, but non-specific inhibition
IBMX (IC ₅₀ Was greater than 100 μM)
Is not sufficiently inhibited by most PDE inhibitors
(Table 1). PDE2-specific inhibitor EHNA (erythro-
9- (2-hydroxy-3-nonyl) -adenine)
Do not inhibit this PDE in the range of
It is noteworthy that it was caught. PDE5 and PDE6 inhibitors
One dipyridamole is relatively dense
Inhibited PDE10A in degrees.

[0115]

[Table 1]

[0116]

According to the present invention, a novel protein belonging to the cyclic nucleotide phosphodiesterase (PDE) family and its gene have been provided. The protein of the present invention plays an important role in the control of intracellular signal transduction by hydrolyzing cAMP and cGMP, which are intracellular second messengers, in the living body and converting them into the corresponding nucleoside 5'-monophosphate. It is considered to be playing. The structural and pharmacological features of the PDE10A of the present invention suggest that it is the first member of a new PDE family. The homology between the predicted amino acid sequence of human PDE10A and the catalytic domains of the other nine PDE families does not exceed 50%, supporting this new PDE family. Regulation of phosphorylation of specific substrate proteins by cAMP-dependent protein kinases appears to be a general mechanism for neurotransmitters to produce physiological effects in specific target nerves.
Localization of PDE10A in the striatum, putamen and caudate, and PDE
Because 10A has high affinity for cAMP, PDE
10A may play an important role in these regions controlling the extrapyramidal system.
Therefore, the protein or gene of the present invention can be used as a useful tool for purifying and cloning new factors involved in biological functions including the function of the central nervous system, and for developing pharmaceuticals.

[0117]

[Sequence List] SEQUENCE LISTING <110> TANAKA, TOSHIO <120> Cyclic nucleotide phosphodiesterase gene <130> C1-103DP1 <140> <141> <150> JP 1999-276957 <151> 1999-09-29 <160> 8 <170> PatentIn Ver. 2.0 <210> 1 <211> 3631 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (516) .. (2933) <400> 1 agcacatgca cccccaaatg tgaagtgcac tcggacacac acacacacac gcacgcacac 60 acaagcactc atgttctaaa gagagaggtc tgagtccgaa gttgctgctg ctgggagctt 120 gagctgagat ggactggtct tcatgggcgc ccaaggcgct gggtgcagct ttccccgaga 180 cccccagatg gaaaggaggg aaggaggaac cccacacact cgccttttgc gagaagatcg 240 gcgcgcaccc cagagtgccc caagcctttg gaatctgcct gctgagcgga gcgcgcgagc 300 gtggtggaca ggtcccgaac ttggccagcg ggctttcttg gcaacttgct ttgcgcagtt 360 ctccatggaa ccctggaccc actgtgctcc cggcgccttg cctttttttt tctttttctt 420 tctctcactg tctcttttta aatttatgaa ctcgaaatga agcggaaagc agatatgcgc 480 gtcagcatac tttggcggcc agccgcctcc ccttc atg ctt aaa ggt ggg agc 533 Met Leu Lys Gly Gly Ser 15 aaa gga ccc gct ttg ttg gct ttg agg agc ggg aca gac ccc cgg gcg 581 Lys Gly Pro Ala Leu Leu Ala Leu Arg Ser Gly Thr Asp Pro Arg Ala 10 15 20 cag atg agc gat gac cca gca gaa ggc acc ctg gac ccc tgc agt gcg 629 Gln Met Ser Asp Asp Pro Ala Glu Gly Thr Leu Asp Pro Cys Ser Ala 25 30 35 cca ggt ttg aca gat gaa aaa gtg aag gca tat ctt tct ctt cac ccc 677 Pro Gly Leu Thr Asp Glu Lys Val Lys Ala Tyr Leu Ser Leu His Pro 40 45 50 cag gta tta gat gaa ttt gta tct gaa agt gtt agt gcg gag aca gta 725 Gln Val Leu Asp Glu Phe Val Ser Glu Ser Val Ser Ala Glu Thr Val 55 60 65 70 gag aaa tgg ctg aag agg aag aac cac aaa tca gaa gat gaa tca gct 773 Glu Lys Trp Leu Lys Arg Lys Asn His Lys Ser Glu Asp Glu Ser Ala 75 80 85 cct aag gaa gtc agc agg tac caa gat acg aat atg cag gga gtt gta 821 Pro Lys Glu Val Ser Arg Tyr Gln Asp Thr Asn Met Gln Gly Val Val 90 95 100 tat gaa cta aac agc tat ata gaa caa cgg ttg gac aca gga gga gac 869 Tyr Glu Leu Asn Ser Tyr Ile Glu Gln Arg Leu Asp Thr Gly Gly Asp 105 110 115 aac cag cta ctc ctc tat gaa ctg ag atc att aaa ata gc c aca 917 Asn Gln Leu Leu Leu Tyr Glu Leu Ser Ser Ile Ile Lys Ile Ala Thr 120 125 130 aaa gcc gat gga ttt gca ctg tat ttc ctt gga gag tgc aat aat agc 965 Lys Ala Asp Gly Phe Ala Leu Tyr Phe Leu Gly Glu Cys Asn Asn Ser 135 140 145 150 ctg tgt ata ttc acg cca cct ggg ata aag gaa gga aaa ccc cgc ctc 1013 Leu Cys Ile Phe Thr Pro Pro Gly Ile Lys Glu Gly Lys Pro Arg Leu 155 160 165 atc cct gct ggg ccc atc act cag ggc acc acc gtc tct gct tat gtg 1061 Ile Pro Ala Gly Pro Ile Thr Gln Gly Thr Thr Val Ser Ala Tyr Val 170 175 180 gcc aag tcc agg aaa aca ctg cta gta gaa gac atc ctt gga gat gaa 1109 Ala Lys Ser Arg Lys Thr Leu Leu Val Glu Asp Ile Leu Gly Asp Glu 185 190 195 cga ttt cca aga ggt act gga ctg gaa tca ggg act cgt atc cag tct 1157 Arg Phe Pro Arg Gly Thr Gly Leu Glu Ser Gly Thr Arg Ile Gln Ser 200 205 210 gtt ctt tgc tta cca att gtc act gca att ggt gac ttg att ggt att 1205 Val Leu Cys Leu Pro Ile Val Thr Ala Ile Gly Asp Leu Ile Gly Ile 215 220 225 230 ctc gag ctg tat cgg cac tgg ggc aaa gaa gcc ttc tgt ctt agt cac 1253 Leu Glu Leu Tyr Arg His Trp Gly Lys Glu Ala Phe Cys Leu Ser His 235 240 245 cag gag gtt gca aca gca aat ctt gcc tgg gct tca gta gca ata cat 1301 Gln Glu Val Ala Thr Ala Asn Leu Ala Trp Ala Ser Val Ala Ile His 250 255 260 cag gtg cag gta tgc aga ggc ctt gcc aaa cag aca gaa ttg aat gac 1349 Gln Val Gln Val Cys Arg Gly Leu Ala Lys Gln Thr Glu Leu Asn Asp 265 270 275 ttc cta ctc gac gta tca aaa aca tat ttt gat aac ata gtt gca ata 1397 Phe Leu Leu Asp Val Ser Lys Thr Tyr Phe Asp Asn Ile Val Ala Ile 280 285 290 gat tct cta ctt gaa cac ata atg ata tat gca aaa aac ctg aat 1445 Asp Ser Leu Leu Glu His Ile Met Ile Tyr Ala Lys Asn Leu Val Asn 295 300 305 310 gcc gat cgt tgt gcg ctt ttc cag gtg gac cat aag aac aag gag tta 1493 Ala Asp Arg Cys Ala Leu Phe Gln Val Asp His Lys Asn Lys Glu Leu 315 320 325 tat tca gac ctt ttt gat att gga gag gaa aag gaa gga aaa cct gtc 1541 Tyr Ser Asp Leu Phe Asp Ile Gly Glu Glu Lys Glu Gly Lys Pro Val 330 335 340 ttc aag aag acc a aa gag ata aga ttt tca att gag aaa gga att gct 1589 Phe Lys Lys Thr Lys Glu Ile Arg Phe Ser Ile Glu Lys Gly Ile Ala 345 350 355 ggc caa gta gca aga aca ggg gaa gtc ctg aac att cca gat gcctat 16 Gln Val Ala Arg Thr Gly Glu Val Leu Asn Ile Pro Asp Ala Tyr 360 365 370 gca gac cca cgc ttt aac aga gaa gta gac ttg tac aca ggc tac acc 1685 Ala Asp Pro Arg Phe Asn Arg Glu Val Asp Leu Tyr Thr Gly Tyr Thr 375 380 385 390 acg cgg aac atc ctg tgc atg ccc atc gtc agc cga ggc agc gtg ata 1733 Thr Arg Asn Ile Leu Cys Met Pro Ile Val Ser Arg Gly Ser Val Ile 395 400 405 ggt gtg gtg cag atg ttcac agt ggc agt gcc ttc tct aaa 1781 Gly Val Val Gln Met Phe Asn Lys Ile Ser Gly Ser Ala Phe Ser Lys 410 415 420 aca gat gaa aac aac ttc aaa atg ttt gcc gtc ttt tgt gct tta gcc 1829 Thr Asp Glu Asn Asn Phe Lys Met Phe Ala Val Phe Cys Ala Leu Ala 425 430 435 tta cac tgt gct aat atg tat cat aga att cgc cac tca gag tgc att 1877 Leu His Cys Ala Asn Met Tyr His Arg Ile Arg His Ser Glu Cys Ile 440 445 45 0 tac cgg gta acg atg gaa aag ctg tcc tac cat agc att tgt act tca 1925 Tyr Arg Val Thr Met Glu Lys Leu Ser Tyr His Ser Ile Cys Thr Ser 455 460 465 470 470 gaa gag tgg caa ggt ctc atg caa ttc acc ctt ccc gtg cgt ctc tgc 1973 Glu Glu Trp Gln Gly Leu Met Gln Phe Thr Leu Pro Val Arg Leu Cys 475 480 485 aaa gaa att gaa tta ttc cac ttt gac att ggt cct ttt gaa aac atg 2021 Lys Glu Ile Glu Leu Phe Ile Gly Pro Phe Glu Asn Met 490 495 500 tgg cct gga att ttt gtc tac atg gtt cat cgg tcc tgt ggg aca tcc 2069 Trp Pro Gly Ile Phe Val Tyr Met Val His Arg Ser Cys Gly Thr Ser 505 510 515 tgc ttt gag ctt gaa aag ttg tgt cgt ttt att atg tct gtg aag aag 2117 Cys Phe Glu Leu Glu Lys Leu Cys Arg Phe Ile Met Ser Val Lys Lys 520 525 530 530 aac tat cgg cgg gtt cct tat cac aac tgg aag cat gcg gtc act gta Tyr Arg Arg Val Pro Tyr His Asn Trp Lys His Ala Val Thr Val 535 540 545 550 gca cac tgc atg tat gcc ata ctt cag aac aat cac acg ctt ttc aca 2213 Ala His Cys Met Tyr Ala Ile Leu Gln Asn Asn His T hr Leu Phe Thr 555 560 565 gac ctt gag cgc aaa gga ctg ctg att gcg tgt ctg tgt cat gac ctg 2261 Asp Leu Glu Arg Lys Gly Leu Leu Ile Ala Cys Leu Cys His Asp Leu 570 575 580 gac cac agg ggc agc tac ctg cag aag ttc gac cac cct 2309 Asp His Arg Gly Phe Ser Asn Ser Tyr Leu Gln Lys Phe Asp His Pro 585 590 595 ctg gcc gct ctc tac tcc act tcc acc atg gag cag cac cac ttc tcc 2357 Leu Ala Ala Tyr Ser Thr Ser Thr Met Glu Gln His His Phe Ser 600 605 610 cag act gtg tcc atc ctc cag ttg gaa ggg cac aat atc ttc tcc act 2405 Gln Thr Val Ser Ser Ile Leu Gln Leu Glu Gly His Asn Ile Phe Ser Thr 615 620 625 630 ctg agc tcc agt gaa tat gag cag gtg ctt gag atc atc cgc aaa gcc 2453 Leu Ser Ser Ser Glu Tyr Glu Gln Val Leu Glu Ile Ile Arg Lys Ala 635 640 645 atc att gcc aca gac ctt gct tta tac tg agg aag cag ttg 2501 Ile Ile Ala Thr Asp Leu Ala Leu Tyr Phe Gly Asn Arg Lys Gln Leu 650 655 660 gaa gag atg tac cag acc gga tca cta aac ctt aat aat caa tca cat 2549 Glu Glu Met Tyr Gln Thr Gly Se r Leu Asn Leu Asn Asn Gln Ser His 665 670 675 aga gac cgt gta att ggt ttg atg atg act gcc tgt gac ctt tgt tct 2597 Arg Asp Arg Val Ile Gly Leu Met Met Thr Ala Cys Asp Leu Cys Ser 680 685 690 gtg aca aaa ctg tgg ccc gtt aca aaa ttg acg gca aat gat ata tat 2645 Val Thr Lys Leu Trp Pro Val Thr Lys Leu Thr Ala Asn Asp Ile Tyr 695 700 705 710 gca gaa ttc tgg gct gag ggt gat gaa atg aag aaa ttg gat cag 2693 Ala Glu Phe Trp Ala Glu Gly Asp Glu Met Lys Lys Leu Gly Ile Gln 715 720 725 cct att cct atg atg gac aga gac aag aag gat gaa gtc ccc caa ggc 2741 Pro Ile Pro Met Met Asp Arg Asp Lylu Lys As Val Pro Gln Gly 730 735 740 cag ctt ggg ttc tac aat gcc gtg gcc att ccc tgc tat aca acc ctt 2789 Gln Leu Gly Phe Tyr Asn Ala Val Ala Ile Pro Cys Tyr Thr Thr Leu 745 750 755 acc cag atc ctc cct ccc acg gag cct ctt ctg aaa gca tgc agg gat 2837 Thr Gln Ile Leu Pro Pro Thr Glu Pro Leu Leu Lys Ala Cys Arg Asp 760 765 770 aat ctc agt cag tgg gag aag gtg att cga ggg gag gag act gca acc 2885 Asn Leu S er Gln Trp Glu Lys Val Ile Arg Gly Glu Glu Thr Ala Thr 775 780 785 790 tgg att tca tcc cca tcc gtg gct cag aag gca gct gca tct gaa gat 2933 Trp Ile Ser Ser Pro Ser Val Ala Gln Lys Ala Ala Ala Ser Glu Asp 795 800 805 tgagcactgg tcaccctgac acgctgtccc acctacagat cctcatcttg cttctttgac 2993 attcttttcc tttttttggg gggggtgggg agaacctgca cctggtaact ggggtgcaaa 3053 cctcttcaag aaggtaacat caaataaata agtcaagcag aggacttcct gccaatctct 3113 tctgtgaggc atcatagaca ctgagcaacc aggaccatcc ccacgttcag aaatcagctg 3173 gccaagtgac tccatttgac ttgcaaacca gccttttcta ataggctaat attgctgagg 3233 ccctaaagga aatggacaaa aattatccag aaggggtact tttccattgt atctttctaa 3293 taagggttta aaatggtact attatggtat tgtacttggg ctttaacatc aatgttgctt 3353 tgatgttgtt ggatataaat aggaattttt acacattact attgtgaatg gtgaatgttc 3413 atgtatgacc tacttgtaat taacttgagt tgtagtccac agcctcagga caaatgtcgt 3473 tgaggttaca gagtaagaaa tgatggcaaa acgtcaaact cttatttcag agcttcatga 3533 atttagttag actaaacata attctttaag ttcaacctaa agggctgaga tcaataaatt 3593 taa cactaga cggaaaaaaa aaaaaaaaaa aaaaaaaa 3631 <210> 2 <211> 806 <212> PRT <213> Homo sapiens <400> 2 Met Leu Lys Gly Gly Ser Lys Gly Pro Ala Leu Leu Ala Leu Arg Ser 1 5 10 15 Gly Thr Asp Pro Arg Ala Gln Met Ser Asp Asp Pro Ala Glu Gly Thr 20 25 30 Leu Asp Pro Cys Ser Ala Pro Gly Leu Thr Asp Glu Lys Val Lys Ala 35 40 45 Tyr Leu Ser Leu His Pro Gln Val Leu Asp Glu Phe Val Ser Glu Ser 50 55 60 Val Ser Ala Glu Thr Val Glu Lys Trp Leu Lys Arg Lys Asn His Lys 65 70 75 80 Ser Glu Asp Glu Ser Ala Pro Lys Glu Val Ser Arg Tyr Gln Asp Thr 85 90 95 Asn Met Gln Gly Val Val Tyr Glu Leu Asn Ser Tyr Ile Glu Gln Arg 100 105 110 Leu Asp Thr Gly Gly Asp Asn Gln Leu Leu Leu Tyr Glu Leu Ser Ser 115 120 125 Ile Ile Lys Ile Ala Thr Lys Ala Asp Gly Phe Ala Leu Tyr Phe Leu 130 135 140 Gly Glu Cys Asn Asn Ser Leu Cys Ile Phe Thr Pro Pro Gly Ile Lys 145 150 155 160 Glu Gly Lys Pro Arg Leu Ile Pro Ala Gly Pro Ile Thr Gln Gly Thr 165 170 175 Thr Val Ser Ala Tyr Val Ala Lys Ser Arg Lys Thr Leu Leu Val Glu 180 185 19 0 Asp Ile Leu Gly Asp Glu Arg Phe Pro Arg Gly Thr Gly Leu Glu Ser 195 200 205 Gly Thr Arg Ile Gln Ser Val Leu Cys Leu Pro Ile Val Thr Ala Ile 210 215 220 Gly Asp Leu Ile Gly Ile Leu Glu Leu Tyr Arg His Trp Gly Lys Glu 225 230 235 240 Ala Phe Cys Leu Ser His Gln Glu Val Ala Thr Ala Asn Leu Ala Trp 245 250 255 Ala Ser Val Ala Ile His Gln Val Gln Val Cys Arg Gly Leu Ala Lys 260 265 270 Gln Thr Glu Leu Asn Asp Phe Leu Leu Asp Val Ser Lys Thr Tyr Phe 275 280 285 Asp Asn Ile Val Ala Ile Asp Ser Leu Leu Glu His Ile Met Ile Tyr 290 295 300 Ala Lys Asn Leu Val Asn Ala Asp Arg Cys Ala Leu Phe Gln Val Asp 305 310 315 320 His Lys Asn Lys Glu Leu Tyr Ser Asp Leu Phe Asp Ile Gly Glu Glu 325 330 335 Lys Glu Gly Lys Pro Val Phe Lys Lys Thr Lys Glu Ile Arg Phe Ser 340 345 350 Ile Glu Lys Gly Ile Ala Gly Gln Val Ala Arg Thr Gly Glu Val Leu 355 360 365 Asn Ile Pro Asp Ala Tyr Ala Asp Pro Arg Phe Asn Arg Glu Val Asp 370 375 380 Leu Tyr Thr Gly Tyr Thr Arg Asn Ile Leu Cys Met Pro Ile Val 385 390 395 40 0 Ser Arg Gly Ser Val Ile Gly Val Val Gln Met Phe Asn Lys Ile Ser 405 410 415 Gly Ser Ala Phe Ser Lys Thr Asp Glu Asn Asn Phe Lys Met Phe Ala 420 425 430 Val Phe Cys Ala Leu Ala Leu His Cys Ala Asn Met Tyr His Arg Ile 435 440 445 Arg His Ser Glu Cys Ile Tyr Arg Val Thr Met Glu Lys Leu Ser Tyr 450 455 460 His Ser Ile Cys Thr Ser Glu Glu Trp Gln Gly Leu Met Gln Phe Thr 465 470 475 480 Leu Pro Val Arg Leu Cys Lys Glu Ile Glu Leu Phe His Phe Asp Ile 485 490 495 Gly Pro Phe Glu Asn Met Trp Pro Gly Ile Phe Val Tyr Met Val His 500 505 510 Arg Ser Cys Gly Thr Ser Cys Phe Glu Leu Glu Lys Leu Cys Arg Phe 515 520 525 Ile Met Ser Val Lys Lys Asn Tyr Arg Arg Val Pro Tyr His Asn Trp 530 535 540 Lys His Ala Val Thr Val Ala His Cys Met Tyr Ala Ile Leu Gln Asn 545 550 555 560 Asn His Thr Leu Phe Thr Asp Leu Glu Arg Lys Gly Leu Leu Ile Ala 565 570 575 Cys Leu Cys His Asp Leu Asp His Arg Gly Phe Ser Asn Ser Tyr Leu 580 585 590 Gln Lys Phe Asp His Pro Leu Ala Ala Leu Tyr Ser Thr Ser Thr Met 595 600 605 G lu Gln His His Phe Ser Gln Thr Val Ser Ile Leu Gln Leu Glu Gly 610 615 620 His Asn Ile Phe Ser Thr Leu Ser Ser Ser Glu Tyr Glu Gln Val Leu 625 630 635 640 Glu Ile Ile Arg Lys Ala Ile Ile Ala Thr Asp Leu Ala Leu Tyr Phe 645 650 655 Gly Asn Arg Lys Gln Leu Glu Glu Met Tyr Gln Thr Gly Ser Leu Asn 660 665 670 Leu Asn Asn Gln Ser His Arg Asp Arg Val Ile Gly Leu Met Met Thr 675 680 685 Ala Cys Asp Leu Cys Ser Val Thr Lys Leu Trp Pro Val Thr Lys Leu 690 695 700 Thr Ala Asn Asp Ile Tyr Ala Glu Phe Trp Ala Glu Gly Asp Glu Met 705 710 715 720 Lys Lys Leu Gly Ile Gln Pro Ile Pro Met Met Asp Arg Asp Lys Lys 725 730 735 Asp Glu Val Pro Gln Gly Gln Leu Gly Phe Tyr Asn Ala Val Ala Ile 740 745 750 Pro Cys Tyr Thr Thr Leu Thr Gln Ile Leu Pro Pro Thr Glu Pro Leu 755 760 765 Leu Lys Ala Cys Arg Asp Asn Leu Ser Gln Trp Glu Lys Val Ile Arg 770 775 780 Gly Glu Glu Thr Ala Thr Trp Ile Ser Ser Pro Ser Val Ala Gln Lys 785 790 795 800 Ala Ala Ala Ser Glu Asp 805 <210> 3 <211> 26 <212> DNA <213> Artificial Seque nce <220> <223> Description of Artificial Sequence: Artificially synthesized oligonucleotide sequence <400> 3 ggcagcatgg gtggagttgt ggtagg 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially synthesized oligonucleotide sequence <400> 4 ttggaaagga aagagtaaag ggaagc 26 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially synthesized oligonucleotide sequence <400> 5 atgatggaca gagacaagaa 20 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially synthesized oligonucleotide sequence <400> 6 aatgttgctt tgatgttgtt gg 22 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially synthesized oligonucleotide sequence <400> 7 ggatcctacg aatatgcagg gagttgtat 29 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: A rtificially synthesized oligonucleotide sequence <400> 8 aagcttgtca aagaagcaag atgaggat 28

[Brief description of the drawings]

FIG. 1 is a schematic diagram of an overlapping cDNA clone of human PDE10A cDNA. The alignment of the fragments isolated by cDNA cloning and RACE is shown. The box at the top of the figure indicates the translation region, and the hatched box therein indicates the catalytic region. PDE cDNA, 5 'and 3' RACE cDNA fragments cloned under PDE mRNA are shown. Gene-specific primers are shown as black squares. Arrows indicate position, length, and direction of EST and RACE products. The GenBank accession number is shown above the arrow. The scale in the figure represents the number of bases. No association between the EST clone (IMAGE: 1564242) and IMAGE: 298975 was identified in the initial homology search.

FIG. 2 shows the nucleotide sequence and predicted amino acid sequence of human PDE 10A. Human PDE10A has a total length of 3631 bases,
Encodes 806 amino acids. The predicted amino acid sequence is shown below the base sequence. The stop codon is indicated by an asterisk, the three stop codons present in frame in the 5 'noncoding region are indicated by boxes, and the expected polyadenylation signal (AATAAA) is indicated by underlining.

FIG. 3 shows a comparison of amino acid sequences between PDE10A and other human cGMP-binding PDEs. PDE10A and other human cGMP binding PD
The amino acid sequence of E was aligned with the Clustal W program. The registration numbers of the PDEs used in the comparison in GenBank are 2A: U67733, 5A: AJ004865, and 6A: M2, respectively.
6061, 6B: S41458, 6C: U31973. An asterisk indicates that all amino acid sequences are identical, and a dot indicates that the property is conserved. Gaps are represented by lines. The upper and lower boxes represent the cGMP binding domain and the catalytic domain, respectively. In addition, two tandem cGMP binding motifs are shown in bold.

FIG. 4 is a diagram showing tissue distribution of human PDE10A mRNA. R
The tissue distribution of human PDE10A mRNA was examined by T-PCR. Templates are Clontech quantitatively corrected cDNAs from various tissues
It was used. Lanes 1-8 contain fetal tissue, lanes 9-16
Indicates an adult tissue. In addition, lane 1: brain, lane 2: heart, lane 3: lung, lane 4: liver, lane 5:
Kidney, lane 6: skeletal muscle, lane 7: spleen, lane 8: thymus, lane 9: brain, lane 10: heart, lane 11: lung,
Lane 12: Liver, Lane 13: Kidney, Lane 14: Skeletal muscle,
Lane 15: pancreas, lane 16: placenta.

FIG. 5 is a diagram showing tissue distribution of human PDE10A mRNA.
A shows three human mRNAs containing poly (A) ⁺ RNA (about 2 μg / lane)
The results of Northern analysis of ultiple tissue Northern blots are shown. The organization is illustrated. RNA size markers from 9.5 kb to 1.35 kb are shown on the left of the figure. B shows the results of Northern analysis of a human RNA master blot in which each dot was standardized and blotted with poly (A) ⁺ RNA derived from 50 different human tissues. The type and position of poly (A) ⁺ RNA and the position of the control are shown on the left.

FIG. 6 shows the expression of human PDE10A in insect cells. A shows Coomassie brilliant blue (CBB) staining of recombinant PDE10A. Mock-infected Sf9 cells and Sf9 cells infected with baculovirus carrying PDE10A were prepared. Lanes 1 and 3 are cytoplasmic fractions, lanes 2 and 4
Represents a granule fraction (insoluble fraction). The arrows in lanes 3 and 4 indicate PDE10A which is not observed in control Sf9 cells, lanes 1 and 2, but is observed only in Sf9 cells infected with baculovirus incorporating PDE10A. Molecular weight markers (MW) are shown on the right of the figure. B shows Western blot analysis of recombinant PDE10A. Lanes 3 and 4 show Western blots of duplicate gels shown in A with anti-Xpress tag antibody.

FIG. 7 shows the PDE activity of the cytoplasmic extract of SDE cells infected with PDE10A baculovirus. A shows the results of assaying the cAMP hydrolysis activity of the extract of infected cells in the presence (black bar) or absence (white bar) of 1 μM cGMP using 1 μM [ ³ H] cAMP as a substrate. . B shows the cGMP hydrolysis activity of the extract of infected cells as a substrate at 1 μM [ ³ H].
The results of assays using cGMP in the presence (black bars) or absence (white bars) of 1 μM cAMP are shown. All assays were performed in triplicate.

FIG. 8 shows the kinetics of PDE10A. cAMP
Concentration 0.022-10.0 μM (Panel A) or cGMP concentration 0.
Shown are Lineweaver-Burk plots at 1-10.0 μM (Panel B). Human PDE10A protein was prepared from the cytoplasmic fraction of infected Sf9 cells and used. The enzyme concentration and incubation time were optimized so that the conversion of cyclic nucleotides did not exceed 15%.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 45/00 A61P 9/00 48/00 11/00 A61P 9/00 13/12 11/00 25/00 13/12 43/00 111 25/00 C07K 16/40 43/00 111 C12N 1/15 C07K 16/40 1/19 C12N 1/15 1/21 1/19 9/16 C 1/21 C12Q 1/44 5/10 G01N 33/15 Z 9/16 33/50 Z C12Q 1/44 33/53 D G01N 33/15 33/573 A 33/50 C12P 21/08 33/53 C12N 15/00 ZNAA 33/573 A61K 37/02 // C12P 21/08 C12N 5/00 A

Claims (13)

[Claims] Claims 1. A protein comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in the protein, wherein one or more amino acids are substituted, deleted, inserted, and / or added, A protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 2. 2. It consists of the nucleotide sequence of SEQ ID NO: 1.
A protein encoded by a DNA hybridizing with the DNA, the protein being functionally equivalent to the protein having the amino acid sequence of SEQ ID NO: 2. 3. A DNA encoding the protein according to claim 1 or 2. A vector into which the DNA according to claim 3 has been inserted. 5. A host cell carrying the vector according to claim 4. 6. The method for producing a protein according to claim 1, comprising a step of culturing the host cell according to claim 5, and recovering the expressed protein from the host cell or a culture supernatant thereof. 7. An antibody that binds to the protein according to claim 1 or 2. 8. A partial peptide of the protein according to claim 1 or 2. 9. It consists of the nucleotide sequence of SEQ ID NO: 1.
A polynucleotide comprising at least 15 nucleotides complementary to DNA or its complementary strand. 10. A method for screening a compound that binds to the protein according to claim 1 or 2, wherein (a) a step of bringing a test sample into contact with the protein or a partial peptide thereof, (b) the protein or a protein thereof. A method comprising: detecting a binding activity between a partial peptide and a test sample; and (c) selecting a compound having an activity of binding to the protein or the partial peptide thereof. 11. A compound that binds to the protein according to claim 1 or 2, which can be isolated by the method according to claim 10. 12. A method for screening a compound that promotes or inhibits the activity of a protein according to claim 1 or 2, wherein (a) the protein and 3 ′, 5′-cysteine in the presence of a test sample. Contacting with a click nucleotide monophosphate; (b) detecting the hydrolysis activity of the protein on 3 ′, 5′-cyclic nucleotide monophosphate; (c) detecting the protein in the absence of a test sample; Selecting a compound that increases or decreases the hydrolysis activity in comparison. 13. A compound that promotes or inhibits the activity of the protein according to claim 1 or 2, which can be isolated by the method according to claim 12.