JPWO2003037930A1 - Novel human N-methyl-D-aspartate (NMDA) glutamate receptor polypeptide and gene encoding the same - Google Patents

Abstract

To find a novel substance relating to human NMDA-type glutamate receptor subunit 3A (NR3A). A novel cDNA derived from human brain was isolated and the entire nucleotide sequence was analyzed. Then, when this base sequence was translated into an amino acid sequence and subjected to homology search, it was confirmed that it had 93% homology with rat NR3A, and it was found that this was human NR3A. Furthermore, in the brain of schizophrenic patients, it was confirmed that the gene encoding NR3A was significantly increased, and it was found that NR3A is a substance involved in the pathology of schizophrenia, thereby completing the present invention. It came.

Description

（実施例４）（脳組織からのｔｏｔａｌ ＲＮＡの抽出）
精神分裂病患者および非精神分裂病患者（コントロール）の死後脳からｔｏｔａｌ ＲＮＡを抽出した。すなわち、−８０℃に保存してあるヒト前頭皮質サンプルをドライアイス上で小断片（約４０ｍｇ）に切断し、１．５ｍｌの遠心チューブに移した。Ｔｏｔａｌ ＲＮＡの抽出はＡｂｓｏｌｕｔｅｌｙ ＲＮＡ ＲＴ−ＰＣＲ Ｍｉｎｉｐｒｅｐ Ｋｉｔ（Ｓｔｒａｔａｇｅｎｅ社）を用いた。抽出したｔｏｔａｌ ＲＮＡの一部を取り、ＯＤ２６０の値から濃度を求めた。また２５０ｎｇのｔｏｔａｌ ＲＮＡを０．１ｍｇ／ｍｌのエチジウムブロミドを含む１％アガロースゲルにて電気泳動した。泳動後のゲルをＵＶ照射し、２８ＳリボソームＲＮＡ、１８ＳリボソームＲＮＡのバンドを確認した。
（実施例５）（逆転写酵素反応）
約０．５μｇのｔｏｔａｌ ＲＮＡを鋳型とし、ＳｕｐｅｒＳｃｒｉｐｔ^ＴＭ Ｆｉｒｓｔ−Ｓｔｒａｎｄ Ｓｙｎｔｈｅｓｉｓ Ｓｙｓｔｅｍ ｆｏｒ ＲＴ−ＰＣＲ（Ｇｉｂｃｏ ＢＲＬ社）を用い、ｃＤＮＡを合成した。また検量線を作成するための標準サンプルの鋳型としてはＨｕｍａｎ Ｂｒａｉｎ Ｔｏｔａｌ ＲＮＡ（Ｃｌｏｎｔｅｃｈ社）を用いた。
（実施例６）（リアルタイムＰＣＲ）
１／１００量のｃＤＮＡを鋳型として、ＮＲ３Ａの増幅にはＳＹＢＲ Ｇｒｅｅｎ ＰＣＲ Ｍａｓｔｅｒ Ｍｉｘ（ＰＥ Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社）、内部標準としてのβ−ａｃｔｉｎの増幅にはＴａｑＭａｎ β−ａｃｔｉｎ Ｃｏｎｔｒｏｌ Ｒｅａｇｅｎｔｓ（ＰＥ Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社）を用いた。ＰＣＲの条件は以下の通りである。

５０℃で２分間、さらに９５℃で１０分間保温した後、９５℃で１５秒間、６０℃で１分間のサイクルを４０回繰り返した。ＰＣＲ装置はＡＢＩ ＰＲＩＳＭ ７７００ Ｓｅｑｕｅｎｃｅ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ（ＰＥ Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社）を用いた。
（実施例７）（ＮＲ３Ａ遺伝子の測定）
系列希釈した標準サンプルの値を基に検量線を作成し、この検量線から各サンプルのＮＲ３Ａおよびβ−ａｃｔｉｎ遺伝子の初期量を算出した。さらに初期のｔｏｔａｌ ＲＮＡ量を補正するためにＮＲ３Ａ量をβ−ａｃｔｉｎ量で割った。最終的にコントロール群の値を１とした時の精神分裂病群の値を算出した。
その結果、図１に示すように、精神分裂病患者の群では、コントロール群に比し、脳内ＮＲ３Ａ遺伝子が有意（ｐ＜０．０５）に増加していた。従って、ＮＲ３Ａは精神分裂病の病態に関連する遺伝子であることが明らかとなった。
産業上の利用可能性
以上説明したように、本発明は精神分裂病の病態に関与する新規なヒトＮＲ３Ａタンパク質およびその遺伝子を提供するものであり、この特性を利用した新規医薬組成物、診断手段の提供はＮＲ３Ａが関与する疾患、特に精神分裂病の臨床、医用領域において有用である。
【配列表】

【図面の簡単な説明】
第１図は、リアルタイムＰＣＲによりコントロール（ｎ＝１０）と精神分裂病患者（ｎ＝９）の脳内ＮＲ３Ａ遺伝子の発現を定量した結果（平均±ＳＥ）を示す図である。＊ｐ＜０．０５（ｔ検定）Technical field
The present invention relates to a human NMDA type glutamate receptor subunit 3A polypeptide or protein, and a polynucleotide encoding the polypeptide or protein. More specifically, a peptide having all or part of the amino acid sequence of the polypeptide or protein, a polynucleotide encoding the peptide or polypeptide, a recombinant vector containing the polynucleotide, and a recombinant vector transformed with the recombinant vector Transformant, peptide or polypeptide production method using the transformant, antibody against the peptide or polypeptide, compound screening method using the same, the screened compound, the polypeptide or the polynucleotide The present invention relates to an activity-inhibiting compound or activity-stimulating compound that acts on a drug, a pharmaceutical composition related thereto, and a disease diagnosis method related thereto.
Background art
N-methyl-D-aspartate (NMDA) glutamate receptors are thought to be involved in various physiological functions such as developmental brain plasticity, memory / learning, neurotransmission in the sensory system, respiration and blood pressure control. (Collingridge GL and Bliss TVP (1987) Trends Neurosci., 10, 288-293).
The NMDA type glutamate receptor is a kind of glutamate receptor, and three subunits (NR1, NR2, NR3A) have been reported in rats and mice. The NR1 subunit and the NR2 subunit are tetramer to pentamer to form a ligand-gated ion channel. Usually Mg in the synaptic cleft ²⁺ If the membrane potential is in a depolarized state due to frequent excitation, etc., Mg ²⁺ The blockage is released and activated. Ion channel is Na ⁺ And Ca ²⁺ It is permeable and contains Ca in the postsynaptic cell. ²⁺ A physiological role is to increase the concentration. In vivo ligands are glutamate and aspartate, but are strongly activated by NMDA.
When Xenopus oocytes are coexpressed with rat NR1 and NR2 subunits, ion channels are formed and current responses are recorded. In addition, when the rat NR1 subunit, NR2 subunit and NR3A subunit were co-expressed, the amount of current decreased. Thus, NR3A is considered an inhibitory subunit (Das S. et al. (1998) Nature, 393, 377-381).
In mice lacking NR1 only in hippocampal CA1, long-term potentiation (LTP), spatial memory, and temporal memory were both impaired (Shimizu E. (2000) Science 290, 1170-4). In mice overexpressing NR2D, LTP and long-term suppression (LTD) decreased (Okabe S. et al. (1998) J. Neurosci., 18, 4177-88). On the other hand, in the NR2B overexpressing mouse, LTD did not change, but LTP increased 5-fold and learning ability increased (Tang YP et al. (1999) Nature, 401, 63-9). Mice deficient in NR3A had increased sensitivity to NMDA and increased spinous dendrites (Das S. et al. (1998) Nature, 393, 377-381). Therefore, it is considered that the NMDA glutamate receptor is necessary for synapse formation, synaptic plasticity, and memory formation.
NMDA glutamate receptors and schizophrenia
Phencyclidine (PCP), a dissociative anesthetic, has been distributed secretly since 1970 under the popular name of angel dust, and has become a typical abused drug along with cocaine in the United States. Symptoms similar to schizophrenia are manifested by continuous use of PCP, but this causes not only so-called positive symptoms such as hallucinations and delusions, but also negative symptoms such as emotional dullness and poor communication, attracting interest as a model of schizophrenia. . However, it has been found that this PCP acts as an antagonist of the NMDA type glutamate receptor, and has attracted attention as indicating a connection between the glutamate receptor and schizophrenia (Wrobleski JT and Danysz W. (1989) Annu). Rev. Pharmacol. Toxicol., 29, 441-474). In addition, there are reports that antagonists of NMDA glutamate receptors have neurotoxicity to the zonal gyrus of rats and the like, and that toxicity is antagonized by antipsychotic drugs (Fix AS et al. (1993). ) Exp. Neurol., 123, 204-215).
On the other hand, mutant mice with NR1 expression reduced to about 5% of normal were prepared, and their behaviors were observed. Schizophrenia-like behaviors such as translocation movement, increased stereotypical behavior, and avoidance of social and sexual behavior. The behavioral pattern was shown. Furthermore, these symptoms were improved by the antipsychotic drugs haloperidol and clozapine, and clozapine was more effective (Mohn AR et al. (1999) Cell, 98, 427-36).
NR3A subunit and schizophrenia
Analysis of schizophrenia symptoms caused by PCP abuse and analysis of animals administered NMDA glutamate receptor antagonists, and generation of animals with altered expression of NMDA glutamate receptor subunits, dysfunction of NMDA glutamate receptors It may be closely related. The NR3A subunit binds to the NR1 / NR2 heteropolymer and inhibits the function as a channel.
However, the NR3A subunit has not yet been found in humans, and its relevance to schizophrenia has not been demonstrated.
(Problems to be solved by the invention)
One of the problems to be solved by the present invention is to find a novel substance relating to human NMDA type glutamate receptor subunit 3A (NR3A) as described above. Another object of the present invention is to clarify the physiological role and function of human NR3A in vivo, to diagnose a disease involving human NR3A, a useful therapeutic agent / therapeutic method, a therapeutic drug screening method, etc. Is to provide.
(Means for solving the problem)
As a result of intensive studies, the present inventors have succeeded in isolating a novel cDNA derived from human brain (management code: PF00154) and analyzing the entire nucleotide sequence. When this base sequence was translated into an amino acid sequence and a homology search was performed, it was confirmed that it had 93% homology with rat NR3A and found to be human NR3A. Furthermore, the present inventors significantly increased the gene encoding NR3A in the brain of schizophrenic patients by comparing the expression level of the NR3A gene in postmortem brains of schizophrenic patients and non-schizophrenic patients. As a result, it was found that NR3A is a substance involved in the pathology of schizophrenia, and the present invention was completed.
Disclosure of the invention
That is, the present invention
1. A polypeptide related to a human N-methyl-D-aspartate (NMDA) -type glutamate receptor selected from the following group or a protein having the polypeptide;
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing,
(2) A polypeptide containing the polypeptide of (1) above,
(3) A polypeptide having at least about 50% amino acid sequence homology with the polypeptide of (1),
and
(4) A polypeptide having a mutation such as deletion, substitution, addition or insertion of one to several amino acids in the amino acid sequence of the above (1) to (3).
2. A partial peptide having a part of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing and having at least about 8 consecutive amino acid sequences.
3. A polynucleotide encoding the polypeptide according to item 1 or a complementary strand thereof.
4). A polynucleotide comprising the sequence of 602 to 3949 in the coding region of the base sequence set forth in SEQ ID NO: 2 in the sequence listing or a complementary strand thereof.
5. 5. A polynucleotide that hybridizes with the polynucleotide according to item 3 or 4 or a complementary strand thereof under stringent conditions.
6). A polynucleotide encoding the peptide according to item 2 or a complementary strand thereof.
7. A polynucleotide comprising the sequence of 602 to 3949 in the coding region of the base sequence set forth in SEQ ID NO: 2 in the sequence listing or a polynucleotide comprising at least about 15 consecutive base sequences of its complementary base sequence.
8). 8. A polynucleotide that hybridizes with the polynucleotide according to item 6 or 7 or a complementary strand thereof under stringent conditions.
9. 9. A recombinant vector comprising any one of the polynucleotides according to 3 to 8 above or a complementary strand thereof.
10. A transformant transformed with the recombinant vector according to item 9 above.
11. 11. A method for producing the polypeptide according to item 1 or 2, or a protein or peptide having the polypeptide, comprising culturing the transformant according to item 10.
12 3. An antibody that immunologically recognizes the polypeptide according to item 1 or 2 or a protein or peptide having the polypeptide.
13. 13. The antibody according to item 12 above, which recognizes at least an NMDA type glutamate receptor subunit.
14 6. A compound having the action of interacting with the polypeptide of the preceding item 1 or a protein having the polypeptide to inhibit or promote its activity, and / or interacting with any one of the polynucleotides of the preceding item 3 to 5. A method for screening a compound having an action of inhibiting or promoting the expression thereof, comprising the polypeptide according to item 1 or 2, the protein or peptide having the polypeptide, or any one of items 3 to 8 above 14. A screening method for an antipsychotic drug, comprising using at least one of a polynucleotide, the vector according to 9 above, the transformant according to 10 above, and the antibody according to 12 or 13 above.
15. 6. A compound that interacts with the polypeptide according to item 1 or a protein having the polypeptide to inhibit or activate the activity, or interacts with any one of the polynucleotides according to items 3 to 5 to express the compound. A method for screening a compound that inhibits or promotes the interaction of a compound by contacting the polypeptide or protein with the compound to be screened under conditions that allow the interaction between the compound and the polypeptide or protein. Action is assessed (such interaction is associated with a second component that can provide a detectable signal in response to the interaction of the polypeptide or protein with the compound), and then the compound and polypeptide or protein The presence or absence or change of signals caused by the interaction of By detecting, compound interacts with a polypeptide or protein, the screening method of antipsychotics include determining whether to activate or inhibit the activity.
16. 16. A compound that is screened by the method for screening an antipsychotic drug according to item 14 or 15, which interacts with the polypeptide according to item 1 or a protein having the polypeptide to inhibit or promote its activity, or Its salt.
17. 16. A compound or a salt thereof, which is screened by the method according to item 14 or 15, and which inhibits or promotes expression by interacting with any one of the polynucleotides according to items 3 to 5.
18. The polypeptide according to item 1 or 2, or the protein or peptide having the polypeptide, the polynucleotide according to any one of items 3 to 8, the vector according to item 9, the transformant according to item 10 above, 18. A pharmaceutical composition comprising an antipsychotic agent comprising at least one of the antibody according to 12 or 13, or the compound according to 16 or 17 above.
19. A method for diagnosing a disease (schizophrenia) associated with the expression or activity of the polypeptide or the protein having the polypeptide according to item 1, comprising: (a) a nucleic acid encoding the polypeptide or protein; and (B) A method comprising analyzing the polypeptide or protein in a sample as a marker.
20. 14. The polypeptide according to item 1 or 2, or a protein or peptide having the polypeptide, any one polynucleotide according to item 3 to 8, or the antibody according to item 12 or 13, Diagnosis of schizophrenia for use in a method comprising analyzing (a) a nucleic acid encoding the polypeptide or protein, and / or (b) analyzing the polypeptide or protein in a sample as a marker Reagent kit.
It is about.
BEST MODE FOR CARRYING OUT THE INVENTION
(New human NR3A protein)
The novel human NR3A protein provided in the present invention is related to the N-methyl-D-aspartate (NMDA) type glutamate receptor, and is cloned from mRNA in human brain cells. The cDNA was obtained as a substance having a sequence. The amino acid sequence encoded by the cDNA clone is shown in SEQ ID NO: 1 in the sequence listing. The novel NR3A cDNA of the present invention was highly expressed in the brain, followed by high expression in the spinal cord, ovary, liver, heart, testis, kidney and spleen. In the brain, expression was highest in the amygdala, followed by expression in the subthalamic nucleus, hippocampus, thalamus, substantia nigra, and the like. In particular, high expression of the gene encoding the protein of the present invention was confirmed in the brain of schizophrenic patients.
The novel NR3A protein according to the present invention consists of 1115 amino acid residues. The gene has an open reading frame of 3348 base pairs, and 93% homology with rat N-methyl-D-aspartate receptor homolog NMDAR-L (EMBL protein accession number: T31068) over the entire deduced amino acid sequence. There is. In addition, when the transmembrane domain was predicted by the membrane protein prediction program, the five positions from the eighth to the 29th, the 674th to the 695th, the 712th to the 734th, the 744th to the 766th, and the 929th to the 951th A transmembrane domain is expected.
(Protein, polypeptide or peptide)
The novel human NR3A protein according to the present invention is a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing. The novel human NR3A protein may be a protein derived from any tissue present in human cells, or may be a synthetic protein.
As an amino acid sequence substantially identical to the amino acid sequence described in SEQ ID NO: 1 in the sequence listing, at least the N-methyl-D-aspartate (NMDA) type glutamate receptor is maintained, and in Examples 3 and 7, It is not particularly limited as long as it retains the observed expression characteristics. For example, it is not less than about 50%, preferably not less than about 70%, more preferably not less than about 80%, more preferably not more than the amino acid sequence set forth in SEQ ID NO: 1 Include amino acid sequences having homology of about 90% or more, most preferably about 95% or more.
Moreover, as a protein containing substantially the same amino acid sequence as SEQ ID NO: 1 in the sequence listing, for example, it contains an amino acid sequence substantially identical to the amino acid sequence described in SEQ ID NO: 1 in the sequence listing, Examples thereof include proteins having functional properties substantially the same as those of proteins containing amino acid sequences substantially the same as the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. Substantially homogeneous activity can include maintaining an association with at least N-methyl-D-aspartate (NMDA) glutamate receptors.
Techniques for determining amino acid sequence homology are known per se. For example, a method for directly determining an amino acid sequence, a method for determining a base sequence of cDNA, and estimating an amino acid sequence encoded by this can be used.
A protein comprising a novel NR3A polypeptide according to the present invention may be in the form of a “mature” protein, or may be part of a larger protein such as a fusion protein. It may contain secretory or leader sequences, pro sequences, sequences that facilitate purification, such as multiple histidine residues, or additional sequences for stability during recombinant production.
The polypeptide or peptide according to the present invention includes a polypeptide or peptide having a partial sequence of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, and these are used as, for example, reagents, standard substances, and immunogens. it can. The minimum unit consists of an amino acid sequence composed of 8 or more amino acids, preferably 10 or more amino acids, more preferably 12 or more, and even more preferably 15 or more consecutive amino acids, preferably immunological Polypeptides or peptides that can be identified are subject of the present invention. These peptides may be used alone or as a carrier (eg, keyhole limpato hemocyanin or ovalbumin) as an antigen for preparing an antibody specific to a reagent or standard substance, or a novel human NR3A protein according to the present invention as described later. ) And other types of proteins or substances bound thereto are also included in the scope of the present invention. A partial sequence may be “free standing” or contained within a larger polypeptide, in which the partial sequence may form part or a region. Good. Most preferably, it is contained in a larger polypeptide as a single continuous region.
Furthermore, based on the polypeptide or peptide thus identified, by using as an index the relationship with N-methyl-D-aspartate (NMDA) type glutamate receptors, one or more, for example 1 to 100, Preferably, a poly consisting of an amino acid sequence having a mutation such as deletion, substitution, addition or insertion of 1 to 30 amino acids, more preferably 1 to 20, even more preferably 1 to 10 and particularly preferably 1 to several amino acids. A peptide or peptide is also provided. Deletion, substitution, addition or insertion means are known per se, for example, site-directed mutagenesis, gene homologous recombination, primer extension or polymerase chain amplification (PCR), alone or in combination, for example, , Molecular Cloning; A Laboratory Manual, 2nd edition, edited by Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; [Lab Manual Genetic Engineering], edited by Masami Muramatsu, Maruzen Stock Company, 1988; [PCR technology, principles and applications of DNA amplification], Ehrlich, HE. Can be carried out in accordance with the methods described in the book of Hen, Stockton Press, 1989, etc., or by modifying those methods. For example, Ulmer's technology (Science (1983) 219: 666) is used. be able to. For example, a series of residues including the N-terminal is deleted, a series of residues including the C-terminal is deleted, or two series of residues including one N-terminal and the other including the C-terminal are deleted. The truncated polypeptide having the amino acid sequence of the novel human NR3A protein is included.
Also, partial sequences characterized by structural or functional attributes such as alpha helix and alpha helix forming region, beta sheet and beta sheet forming region, turn and turn forming region, coil and coil forming region, hydrophilic region, hydrophobic A partial sequence including a region, an alpha amphipathic region, a beta amphipathic region, a variable region, a surface forming region, a substrate binding region, five transmembrane domains, and a highly antigenic indicator region is also preferable. Biologically active regions are also preferred, such as subsequences with similar or improved activity, or reduced undesirable activity. Also included are subsequences that are antigenic or immunogenic in animals, particularly humans.
The amino acid sequences of the proteins, polypeptides and partial sequence peptides thereof according to the present invention include those altered by conservative amino acid substitution. Such typical substitutions are between Ala, Val, Leu and Ile; between Ser and Thr; between Asp and Glu; between Asn and Gln; and between the basic residues Lys and Arg; or the aromatic residues Phe, Trp and This is between Tyr. Particularly preferred are those in which several, 5 to 10, 1 to 5, or 1 to 2 amino acids are substituted, deleted or added in any combination.
From the viewpoint of not changing the basic properties (physical properties, activity, immunological activity, etc.) of the protein in the introduction of mutations as described above, for example, homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobicity) Mutual substitution between amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) is readily envisaged.
The novel human NR3A protein according to the present invention means any peptide containing two or more amino acids linked to each other by peptide bonds or modified peptide bonds. In the present specification, a short chain, also referred to as a peptide, oligopeptide or oligomer, may be referred to as a polypeptide, and a long chain may be referred to as a protein.
The novel human NR3A protein according to the present invention can also contain amino acids different from those encoded by 20 genes. “Proteins” include those modified by natural processing, such as processing and other post-translational modifications, but are also modified by chemical modification techniques well known to those skilled in the art. Such modifications are well described in basic references and more detailed papers and numerous research literatures, which are well known to those skilled in the art.
Modifications can be made at any site of the polypeptide such as the peptide backbone, amino acid side chains and N-terminal or C-terminal side. The same type of modification may be present in the same or varying degrees at several sites in the polypeptide. A protein may also contain many types of modifications. The protein may be branched as a result of ubiquitination or may be cyclic with or without branching. Cyclic, branched and branched and cyclic proteins may be produced by post-translation natural processing or may be produced by synthetic methods.
Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol covalent bond , Cross-linking, cyclization, disulfide bond formation, demethylation, cross-linking covalent bond formation, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation Addition of amino acids to transfer RNA-mediated proteins such as myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid binding, sulfation, and selenoylation, arginylation, and Ubiquitation etc. A.
For example, Proteins-Structure and Molecular Properties, 2nd edition, T.A. E. Creighton, W.C. H. Freeman and Company, New York, 1993, and Post-translational Covalent Modification of Proteins, B.C. C. Edited by Johnson, Academic Press, New York, 1983. , Posttranslational Protein Modifications: Perspective and Prospects, pages 1-12; Seifter et al. , Meth. Enzymol. (1990) 182: 626-646 and Rattan et al. , Protein Synthesis: Post-translational Modifications and Aging, Ann. N. Y. Acad. Sci. (1992) 663: 48-62.
Furthermore, in order to facilitate the detection or purification of the polypeptide or the like according to the present invention, or to add another function, another protein such as alkaline phosphatase, β-galactosidase, N-terminal side or C-terminal side, It is easy for those skilled in the art to add an immunoglobulin Fc fragment such as IgG or a peptide such as FLAG-tag directly or indirectly through a linker peptide using a genetic engineering technique, etc. Polypeptides to which substances are bound are also included in the scope of the present invention. Naturally, homologous gene products of animal species other than humans are also included in the scope of the present invention.
(Polynucleotide)
In one embodiment, the polynucleotide according to the present invention and its complementary strand are a polynucleotide encoding the polypeptide according to the present invention, such as a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing, and the polynucleotide. Means the complementary strand to. These provide, for example, gene information useful for the production of the above-described novel human NR3A protein, or can be used as reagents / standards for nucleic acids. A preferable polynucleotide base sequence is described in the sequence of 602 to 3949 of the coding region in SEQ ID NO: 2 in the sequence listing.
In another aspect, the present invention relates to a polynucleotide encoding a polypeptide comprising the amino acid sequence of the polypeptide or peptide according to the present invention, for example, the amino acid sequence of SEQ ID NO: 1 in the sequence listing, preferably the base of SEQ ID NO: 2 in the sequence listing Provided is a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising the sequence of 602 to 3949 in the coding region or a corresponding region of its complementary strand. Hybridization conditions can be in accordance with, for example, Molecular Cloning; A Laboratory Manual, 2nd edition, edited by Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, and the like. Stringent hybridization conditions are generally known, and examples include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate, pH 7.6, 5 ×. Washing in a solution containing Denhart's solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon / sperm DNA overnight at 42 ° C. and then at about 65 ° C. in 0.1 × SSC. Also good.
These polynucleotides are not necessarily complementary so long as they hybridize to the polynucleotide of interest, particularly the polynucleotide consisting of the sequence of 602 to 3949 in the coding region of the nucleotide sequence of SEQ ID NO: 2 in the sequence listing or its complementary strand. It doesn't have to be an array. For example, at least about 40%, for example, about 70% or more, preferably about 80% or more in homology to the sequence of 602 to 3949 in the coding region or its complementary sequence in the base sequence of SEQ ID NO: 2 in the sequence listing More preferably, it is about 90% or more, more preferably about 95% or more. The polynucleotide according to the present invention is a polynucleotide or oligonucleotide consisting of 10 or more, preferably 15 or more, more preferably 20 or more consecutive nucleotides corresponding to a designated nucleotide sequence region, and these. Of complementary strands. Here, the nucleotide sequence of a polynucleotide encoding the novel human NR3A protein according to the present invention or a polypeptide having the same activity as this is determined by, for example, confirming the expressed protein using a known protein expression system. The screening can be carried out by selecting the physiological activity, particularly maintaining the association with the NMDA glutamate receptor as an index. In the case of using a cell-free protein expression system, for example, a ribosome technology derived from wheat germ, rabbit reticulocyte or the like can be used (Nature (1957) 179: 160-161).
The polynucleotide according to the present invention is a probe or primer for detecting a nucleic acid encoding the novel human NR3A protein according to the present invention, for example, its gene, or mRNA, in the production of the polypeptide according to the present invention ( primer) or as an antisense oligonucleotide or the like for regulating gene expression. In that sense, the polynucleotides and oligonucleotides according to the present invention include not only translated regions but also those corresponding to untranslated regions. For example, in order to specifically inhibit the expression of the novel NR3A-containing protein according to the present invention by antisense, it is assumed that the base sequence of a region unique to the NR3A protein is used. On the other hand, it is considered possible to simultaneously suppress the expression of the protein containing the NR3A protein by using a conserved sequence.
The polynucleotide according to the present invention may generally be unmodified RNA or DNA, or modified RNA or DNA. “RNA or DNA” encompasses DNA or RNA having modified bases, as well as DNA or RNA having a modified backbone, for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications have been made to DNA and RNA, typically chemically, enzymatically or metabolically modified forms of polynucleotides found in nature, and DNA and RNA chemistry characteristic of viruses and cells. Including typical forms. “RNA or DNA” also encompasses short polynucleotides often referred to as “oligonucleotides”.
The polynucleotide according to the present invention may change the amino acid sequence encoded by the target RNA or DNA, and substitution, addition, deletion, fusion and truncation of amino acids in the polypeptide encoded by the target sequence However, the present invention also includes these. These changes can change the amino acid sequence by one or more substitutions, additions, deletions in any combination, and these changes can be performed by mutation techniques, direct synthesis, and those skilled in the art. It can be produced by other known recombinant techniques.
The identity of the base sequence of the polynucleotide according to the present invention and the amino acid sequence of the polypeptide is a measure of the identity of the RNA, DNA or protein according to the present invention. In general, the sequences that match the maximum are aligned. Identity itself has an art-recognized meaning and can be calculated using published methods.
See, for example, Computational Molecular Biology, Lesk, A .; M.M. Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D .; W. Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A .; M.M. And Griffin, H .; G. Hen, Human Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heijne, G. Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M .; And Devereux, J. et al. Hen, M Stockton Press, New York, 1994, can be used. There are many ways to measure the identity of two sequences, but the term “identity” is well known to those skilled in the art (Sequence Analysis in Molecular Biology, von Heijne, G., Academic Press, 1987; Sequence; Analysis Primer, Gribskov, M. and Devereux, J., M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math. (1988) 48: 1073).
A commonly used method for measuring identity or similarity between sequences is the method of Carillo, H .; and Lipman, D.M. , SIAM J. et al. Applied Math. (1998) 48: 1073, etc., but is not limited thereto. Methods for determining identity and similarity have been assembled into publicly available computer programs. Preferred computer program methods for measuring identity and similarity between two sequences include the GCG program package (Devereux, J., et al., Nucleic Acids Research (1984) 12 (1): 387), BLASTP, BLASTN , And FASTA (Atschul, SF et al., J. Molec. Biol. (1990) 215: 403), etc., but are not limited thereto.
(Transformant)
The present invention can be applied to a cell-free protein using a gene recombination technique using a known host such as Escherichia coli, yeast, Bacillus subtilis, insect cells, animal cells, plant cells, etc., or using RNA derived from the DNA according to the present invention. Peptides and polypeptides comprising the novel human NR3A protein and its derivatives according to the present invention can be provided by synthetic methods.
For transformation, means known per se are applied. For example, transformation of a host is performed using a plasmid, chromosome, virus or the like as a replicon. A more preferable system is an integration method into a chromosome in consideration of gene stability, but it is simply an autonomous replication system using an extranuclear gene. The vector is selected according to the type of the selected host, and comprises a gene sequence intended for expression and a gene sequence carrying information on replication and control. Combinations are sorted by prokaryotic cells, eukaryotic cells, non-promoters such as promoters, ribosome binding sites, terminators, signal sequences, enhancers, marker sequences, transcribed sequences, untranslated sequences, splicing and polyadenylation signals, sequences that stabilize mRNA. Coding 5 'and 3' sequences and the like can be used in combination by a method known per se. The marker sequence is described in a hexahistidine peptide (Gentz et al., Proc. Natl. Acad. Sci., USA (1989) 86: 821-824) as provided in a pQE vector (manufactured by QIAGEN). Or HA tag (Wilson et al., Cell (1984) 37: 767).
Introduction of DNA into host cells is described, for example, by Davis et al., Basic Methods in Molecular Biology, 1986; Molecular Cloning; A Laboratory Manual, 2nd edition, edited by Sambrook et al., Cold Spring Harbor Laboratory Press. Can be performed by methods described in many standard laboratory manuals such as Spring Harbor, New York, 1989, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid mediated Transfection, electroporation, transduction, scrape loading ), There is a ballistic introduction (ballistic introduction) and infection.
Representative of suitable hosts are bacterial cells such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis. Cells; fungal cells such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK 293 and Bowes melanoma cells; and plant cells.
Vectors include chromosomal, episomal and viral vectors such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements such as baculoviruses, papovaviruses such as SV40, vaccinia virus, Examples include vectors derived from viruses such as adenovirus, fowlpox virus, pseudorabies virus and retrovirus, and vectors combining them, such as vectors derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
Expression system constructs may contain regulatory regions that control and cause expression. Usually, in order to retain, extend or express DNA in the host, it is necessary to select any system or vector suitable for expressing the protein. These can insert an appropriate DNA sequence into an expression system by well-known techniques. For example, Molecular Cloning; A Laboratory Manual, 2nd edition, edited by Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring. • Harbor, New York, 1989. Alternatively, the translated protein can be incorporated into a protein that expresses an appropriate secretion signal for secretion into the endoplasmic reticulum lumen, periplasmic space, or extracellular environment. These signals may be intrinsic to the protein or may be heterogeneous signals.
The transformant is cultivated by selecting the optimum conditions for the culture conditions of each host known per se. The culture may be performed using the novel human NR3A protein according to the present invention that is expressed and produced and a specific sequence of a peptide or polypeptide comprising the derivative thereof as a marker, but using the amount of transformant in the medium as an index. You may perform by subculture or batch culture.
When the target protein, polypeptide or peptide is secreted into the culture medium of the transformant, it can be obtained by recovering the medium and purifying it from the medium. Furthermore, when they are produced in the cells of the transformant, the target protein, polypeptide or peptide can be obtained by first lysing the cells and then recovering the protein. In addition, when a target protein, polypeptide or peptide is expressed for a screening assay, it is generally preferable to produce the protein on the cell surface. In this case, the cells may be collected prior to use in a screening assay.
(Purification and recovery of novel NR3A domain-containing protein and its derivatives)
Peptides and polypeptides comprising the novel human NR3A protein and its derivatives according to the present invention include human and other mammalian cells, tissues, cultured cells or culture media thereof (cell cultures), or the above transformations It can be obtained from body cell cultures by well-known purification methods. The methods include, for example, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. . These methods may be appropriately combined. Preferably, a method is used in which an antibody against the amino acid sequence is prepared on the basis of information on the amino acid sequence and specifically adsorbed and recovered by a polyclonal antibody or a monoclonal antibody. Most preferably, high performance liquid chromatography is used for purification. Conveniently, affinity chromatography using heparin can be used. Furthermore, in the purification process, purification can be confirmed using as an index the maintenance of the association with the NMDA type glutamate receptor. If the protein is denatured during isolation and / or purification, well-known techniques for protein regeneration can be used to re-activate the active conformation.
(antibody)
The antibody is prepared by selecting a peptide or polypeptide comprising the novel human NR3A protein and its derivative according to the present invention, or a peptide containing an antigenic determinant thereof, and using them as an antigen. The antigen may be the NR3A protein or a fragment thereof, and is composed of at least 8, preferably at least 10, more preferably at least 12, even more preferably 15 or more amino acids. In order to produce an antibody specific for the NR3A protein, it is preferable to use a region consisting of a sequence unique to the novel human NR3A protein. This amino acid sequence does not necessarily have to be homologous to SEQ ID NO: 1 in the sequence listing, and an externally exposed site on the three-dimensional structure of the protein is preferable, and the exposed site is a discontinuous site on the primary sequence of amino acids. As long as it is a continuous amino acid sequence for the exposed site. The antibody is not particularly limited as long as it immunologically binds or recognizes the peptide or polypeptide comprising the NR3A protein and its derivatives. The presence or absence of this binding or recognition is determined by a known antigen-antibody binding reaction. “Immunospecific” means that the affinity for the NR3A protein is substantially greater than its affinity for other related proteins of the prior art.
Antibody production involves the humoral response to animals by linking a novel human NR3A protein according to the present invention, a peptide or polypeptide comprising the protein, alone or to a carrier in the presence or absence of an adjuvant. And / or by inducing immunity such as a cellular response. The carrier is not particularly limited as long as it does not adversely affect the host itself, and examples thereof include cellulose, polymerized amino acid, albumin (including those derived from plasma or produced by gene recombination techniques) and the like. As the animal to be immunized, a mouse, rat, rabbit, goat, horse or the like is preferably used. Polyclonal antibodies are obtained by a method for recovering antibodies from sera known per se. A preferred means is immunoaffinity chromatography.
For the production of monoclonal antibodies, antibody-producing cells (for example, derived from spleen or lymph nodes) are collected from animals that have been subjected to the above-mentioned immunization means, and are known per se (for example, myeloma strains such as the P3X63Ag8 strain). It is carried out by introducing the transformation means. For example, a hybridoma prepared from the antibody-producing cell and the permanently proliferating cell is cloned, a hybridoma producing an antibody that specifically recognizes the novel human NR3A protein according to the present invention is selected, and the hybridoma The antibody is recovered from the culture medium. Illustrative examples are the hybridoma method (Kohler, G. and Milstein, C., Nature (1975) 256: 495-497); the trioma method (Kozbor et al., Immunology Today (1983) 4:72); and the EBV-hybridoma There are various techniques as described in the method (Cole et al., Monoclonal Antibodies and Cancer Therapies, Alan R Liss, Inc., (1985): 77-96).
Techniques described for the production of single chain antibodies (US Pat. No. 4,946,778) can be applied to produce single chain antibodies against the proteins of the invention. Alternatively, the humanized antibody may be expressed by a method of inducing an immunological reaction in a mammal using a transgenic mouse or other organisms including other mammals.
The above antibody can be used for isolation or identification of a clone expressing the novel human NR3A protein of the present invention, or for purification of the protein by affinity chromatography.
The above antibodies may also be used to treat schizophrenia related diseases, among others.
(Screened and screened compounds)
Peptides or polypeptides comprising the novel human NR3A protein and its derivatives thus prepared, polynucleotides encoding them and their complementary strands, cells transformed based on information on their amino acid sequences and base sequences, or these A protein synthesis system using the above and an antibody that immunologically recognizes a peptide or polypeptide consisting of the NR3A protein and its derivatives can be used alone or in combination of a plurality of means to produce a peptide consisting of the NR3A protein and its derivatives and Provided is an effective means for screening for an activity inhibitor or activity activator for a polypeptide. For example, selection of antagonists by drug design based on the three-dimensional structure of a peptide or polypeptide, selection of expression regulators at the gene level using a protein synthesis system, selection of antibody recognition substances using antibodies, etc. are known per se. It can be implemented using a pharmaceutical screening system. The human NR3A protein of the present invention strongly suggests a relationship with schizophrenia diseases. Therefore, it is useful to use the novel human NR3A protein according to the present invention for screening to find a compound having an action of inhibiting or promoting its activity.
Specifically, by using the peptide or polypeptide comprising the novel human NR3A protein and its derivative according to the present invention, the interaction between the compound to be screened and these peptides or polypeptides is enabled. Introducing a system that uses a signal and / or marker (second component) that can be screened to detect the presence or absence of this interaction and then the presence or absence of this signal and / or marker or By detecting the change (including quantitative change and / or qualitative change), it is possible to screen for compounds that activate or inhibit the activity of peptides and polypeptides comprising the NR3A protein and its derivatives. For example, the binding of a small molecule substrate and a ligand in a compound to be screened may be evaluated using the NR3A protein. These substrates and ligands may be natural substrates and ligands, or may mimic structures or functions (see Coligan et al., Current Protocols in Immunology (1991) 1 (2): Chapter 5). ).
Furthermore, using the novel human NR3A protein cDNA, protein and antibody against the protein according to the present invention, a screening assay was conducted to examine the effect of the added compound on the mRNA and protein production of the NR3A protein in cells. Also good. For example, an enzyme-linked immunosorbent assay (ELISA) for measuring secretion or cell binding levels of the NR3A-containing protein using monoclonal and polyclonal antibodies by common methods known in the art. And can be used to find factors that can inhibit or promote production of the NR3A protein from appropriately treated cells or tissues. General methods for conducting screening assays are well known in the art.
Examples of compounds to be screened include cells, products prepared from cell-free, chemical libraries, natural product mixtures, and the like.
The thus screened compounds can be used as candidate compounds for activity inhibitors, activity antagonists, and activity activators for peptides and polypeptides comprising the novel human NR3A protein and its derivatives according to the present invention. . It can also be used as a candidate compound for expression inhibitors, expression antagonists, and expression activators for the NR3A protein and its derivatives at the gene level. They can be expected to prevent or treat various symptoms derived from NR3A protein. Candidate compounds selected by the above screening method can be prepared as a pharmaceutical composition by further selecting considering the balance between biological usefulness and toxicity.
(Pharmaceutical composition)
A peptide or polypeptide comprising the novel human NR3A protein and its derivatives provided in the present invention, a polynucleotide encoding them and their complementary strands, a vector comprising these base sequences, the NR3A protein and its derivatives An antibody that immunologically recognizes a peptide or polypeptide, and a compound selected by the above screening method can be used as a pharmaceutical means such as a therapeutic drug utilizing functions such as activity inhibition, antagonism, and activation of the NR3A-containing protein. It is. In formulating, a formulation method corresponding to each known peptide or polypeptide, protein, polynucleotide, antibody or the like may be introduced.
For example, the present invention provides a therapeutic, mental, and therapeutic method by inoculating a mammal with the novel human NR3A protein or fragment thereof sufficient to produce antibodies and / or T cells to induce an immunological response in the mammal. It can also be used to protect the animal from diseases associated with schizophrenia.
The present invention further comprises delivering a nucleic acid vector for in vivo expression of the novel human NR3A protein according to the present invention to generate antibodies that induce such immunological response and protect the animal from disease. It can also be used.
Furthermore, the present invention can also be used in immunological vaccine formulations (compositions) that, when introduced into a mammalian host, induce a mammalian immunological response to the novel human NR3A protein. The composition comprises the novel human NR3A protein or a gene encoding the protein. The vaccine formulation may further comprise a suitable carrier. Since the NR3A protein may be degraded in the stomach, it is preferably administered parenterally. Examples of parenteral administration include administration by injection into, for example, subcutaneous intramuscular, intravenous, intradermal, intraperitoneal and the like. Formulations suitable for parenteral administration are aqueous or non-aqueous sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents and solutes that render the formulation isotonic with the blood of the recipient; and suspensions Or an aqueous or non-aqueous sterile suspension which may contain a thickening agent. Formulations may be provided in single or multiple doses in containers, for example, in sealed ampoules and vials, or frozen requiring only the addition of a sterile liquid carrier just prior to use It may be stored as a dry state. The vaccine formulation may also include an adjuvant system to enhance the immunogenicity of the formulation, such as oil-in-water systems and other systems known in the art. The dose depends on the activity of the individual vaccine and can be readily determined by routine experimentation.
The present invention is expected to be very useful in elucidating biological functions related to schizophrenia, elucidating, preventing, treating and diagnosing diseases related to schizophrenia.
When the novel human NR3A protein according to the present invention and its activity are excessive, an effective amount of the inhibitor compound is administered to the subject together with a pharmaceutically acceptable carrier to inhibit the activity of the NR3A protein, thereby Abnormal symptoms can also be improved.
Furthermore, expression of the gene encoding the endogenous NR3A protein may be inhibited using an expression blocking method. Intracellularly generated or separately administered antisense sequences are used in such known methods (eg, Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988), Coccor, J. Neurochem (1991) 56: 560). Alternatively, oligonucleotides that form a triple helix with the gene may be used (eg, Lee et al., Nucleic Acids Res (1979) 6: 3073; Cooney et al., Science (1988) 241: 456; Dervan et al., Science (1991) 251: 1360). These oligonucleotides can be administered per se or the relevant oligomers can be expressed in vivo.
For treatment of abnormal symptoms related to the novel expression of human NR3A protein and its activity according to the present invention, as one method, a therapeutically effective amount of activating the NR3A protein itself or a gene encoding this protein is used. Examples include a method characterized in that a compound (accelerator) is administered together with a pharmaceutically acceptable carrier, thereby improving abnormal symptoms. Alternatively, gene therapy may be used to validate the intracellular production of the NR3A protein by cells in the subject. For example, as described above, the polynucleotide according to the present invention may be processed and expressed in a replication-defective retrovirus vector. The retroviral expression construct is then isolated and introduced into packaging cells transduced with a retroviral plasmid vector containing RNA encoding a novel human NR3A protein according to the present invention, this time the packaging cells Infectious virus particles containing the target gene may be generated. These producer cells may be administered to a subject for processing of the cells in vivo and expression of the protein in vivo. For a review of gene therapy, see Human Molecular Genetics, T .; Strachan and A.M. P. See Read, BIOS Scientific Publishers Ltd (1986), Chapter 20, Gene Therapeutic and other Molecular Genetic-based Therapeutic Approaches (and references cited therein).
The formulation and dosage form of the novel human NR3A protein according to the present invention and a peptide or polypeptide or compound comprising the derivative thereof are preferably formulated in combination with an appropriate pharmaceutical carrier. Such formulations comprise a therapeutically effective amount of the protein, compound or antibody, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and mixtures thereof. The formulation should be appropriate for the route of administration and will be familiar to those skilled in the art. The present invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the above-described components of the present invention.
Peptides or polypeptides, compounds and antibodies comprising the protein according to the present invention and its derivatives may be used alone or together with other compounds required for treatment. The preferred form of systemic administration of the pharmaceutical composition is injection, especially intravenous injection. Other injection routes such as subcutaneous, intramuscular or intraperitoneal can also be used. Another means for systemic administration is transmucosal or transdermal administration using penetrants such as bile salts or fusidic acids or other surfactants. Furthermore, oral administration is possible if enteric or capsule formulations are well formulated. Administration of these proteins, compounds or antibodies may be local or in the form of a salve, pasta, gel or the like.
The required dosage range depends on the peptide, compound or antibody as the active ingredient, the route of administration, the nature of the formulation, the nature of the subject's symptoms, and the judgment of the attending physician. However, suitable doses are in the range of 0.1-100 μg / kg of subject body weight. However, the required doses are even wider when considering the different effectiveness of different proteins, compounds and antibodies, different routes of administration. For example, oral administration requires a higher dose than intravenous injection. These dosage changes can be made using general routine experimentation for optimization well known in the art.
In gene therapy, proteins used for treatment can also be produced in a subject. For example, cells derived from a subject can be processed ex vivo using, for example, a retroviral plasmid vector with DNA or RNA encoding the protein, and then the cells can be introduced into the subject. .
(Diagnostics and diagnostic methods)
In addition, the peptide or polypeptide comprising the novel human NR3A protein and its derivative according to the present invention, the polynucleotide encoding it and its complementary strand, and the peptide or polypeptide comprising the NR3A protein and its derivative are immunologically isolated. Can be used alone as a diagnostic marker, reagent or the like. Also provided are reagent kits comprising one or more containers filled with one or more of these. In the formulation, formulation means corresponding to each known peptide or polypeptide, protein, polynucleotide, antibody or the like may be introduced.
As a diagnostic method, for example, using the interaction / reactivity with a nucleic acid encoding a peptide or polypeptide comprising the novel human NR3A protein and its derivative according to the present invention, the abundance of the corresponding nucleic acid, And / or the biodistribution in the individual and / or the presence in the sample derived from the individual and / or the abundance in the sample derived from the individual known per se for the peptide or polypeptide comprising the protein and its derivatives Measured by the method, and related to the expression or activity of the peptide or polypeptide comprising the NR3A protein and its derivatives according to the present invention by the presence or absence and / or change (increase or decrease) of the presence The presence or absence and / or change (exacerbation or reduction) of the disease can be determined. Specifically, the NR3A protein and / or nucleic acid is examined as a marker. As the measurement method, a known antigen-antibody reaction system, enzyme reaction system, PCR reaction system, or the like may be used.
Nucleic acids for diagnosis may be obtained from cells from an individual, such as blood, urine, saliva, tissue biopsy or autopsy material. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification methods prior to analysis. RNA or cDNA may be used as well. In comparison with the normal genotype, deletions and insertions can be detected by a change in size of the amplified product. Point mutations can be identified by hybridizing amplified DNA to labeled NR3A protein-encoding DNA. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences can also be detected by detecting changes in electrophoretic mobility of DNA fragments in gels with or without denaturing substances, or by direct DNA sequencing (see, eg, Meyers et al. , Science (1985) 230: 1242). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S1 protection or chemical cleavage methods (eg, Cotton et al., Proc. Natl. Acad. Sci. , USA (1985) 85: 4397-4401). In addition, an array of oligonucleotide probes containing a DNA encoding the NR3A protein or a fragment thereof can be constructed to effectively screen for genetic variation, for example. Array methods are well known and have a wide range of applications and can be used to investigate various problems in molecular genetics, including gene expression, genetic linkage, and genetic variability (eg, M. Chee et al., Science (1996) 274: 610).
In addition, a method for diagnosing a disease associated with schizophrenia is provided by detecting mutation, decrease, or increase in the DNA encoding the NR3A protein by the method described herein.
Further, a method for diagnosing a disease associated with schizophrenia is provided by a method characterized by examining the NR3A protein abnormally increased or decreased in a sample derived from a subject or the mRNA level of the NR3A protein.
The increase or decrease in the expression of DNA encoding the novel human NR3A protein according to the present invention can be determined by any method known in the art as a polynucleotide quantification method, such as amplification, PCR, RTPCR, RNase protection, Northern Blotting and other hybridization methods can be used to measure at the RNA level. Also, assay methods that can be used to determine the NR3A protein level in a sample are well known to those of skill in the art. Such assay methods include radioimmunoassays, competitive binding assays, Western blot analysis and ELISA assays.
The identification (chromosome assay) of the chromosome of the polynucleotide according to the present invention is also valuable. The polynucleotide sequence can target and hybridize to a specific location on an individual human chromosome. The mapping of important sequences to chromosomes according to the present invention is an important first step in associating those sequences with gene-related diseases. Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data includes, for example, V.I. McKusick, Mendrian Inheritance in Man (available online from Johns Hopkins University Welch Medical Library). Then, by analyzing the linkage (co-inheritance of physically adjacent genes), the relationship between the gene mapped to the same chromosomal region and the disease is identified. Differences in the cDNA or genomic sequence between affected and unaffected individuals can also be examined. If a mutation is observed in some or all of the affected individuals but not in normal individuals, the mutation may be responsible for the disease.
(Example)
EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to the following Example.
(Example 1)
(Construction of human brain-derived cDNA library and gene sorting)
Cloning of human brain hippocampal cDNA
The human brain hippocampus-derived cDNA library was constructed according to the method of Ohara et al. (Ohara O. et al. (1997) DNA Res. 4, 53-59). That is, an oligonucleotide having a Not I site (SEQ ID NO: 3: GACTAGTTCTAGATCGCGAGCGGCCCCC (T) ₁₅ ) (Invitrogen) was used as a primer, and human brain hippocampal mRNA (Clontech) was used as a template to synthesize a double-stranded cDNA with Superscript II reverse transcriptase kit (Invitrogen). An adapter having a Sal I site (Invitrogen) was ligated with cDNA and then digested with Not I. A DNA fragment of 3 kb or more was purified by 1% concentration low melting point agarose electrophoresis. PBluescript II SK treated with Sal I-Not I restriction enzyme from the purified cDNA fragment ₊ Ligation with a plasmid (Stratagene) was performed. A recombinant plasmid was introduced into E. coli and ElectroMaxDH10B (Invitrogen) by electroporation. About 6,000 recombinants were selected from the cDNA library thus constructed, and the DNA sequences at both ends of these clones were determined. Among these, the 5 ′ terminal sequence (about 500 bp) of a clone having a novel sequence at both ends was analyzed by a program called GeneMark (Borodovsky M. et al. (1995) Nucleic Acids Res., 23, 3554-3562). . About 160 independent clones with predicted protein-coding regions were selected. Furthermore, using these clones as templates, proteins encoded on cDNA were produced using an in vitro transcription / translation system (Promega), and the molecular weight was measured by SDS-denaturing gel electrophoresis. Full-length sequence analysis was performed on about 60 clones capable of producing a protein having a molecular weight of 50 kDa or more. A DNA sequencer, ABI377 (PE Applied Biosystems) was used for sequencing. Most of the sequences were determined by shotgun clones using the dye terminator method. For some base sequences, oligonucleotides were synthesized based on the determined base sequences and determined by the primer walking method.
(Example 2) (Method and result of homology search)
For the amino acid sequence deduced from the entire base sequence obtained, homology search between proteins is performed on the FASTA program (Pearson WR (1990) Methods Enzymol., 183, against a known sequence database (nr database, NCBI)). 63-98). As a result, plasmid PF00154 contained 7770 base pairs of cDNA and had a 3348 base pair open reading frame encoding a novel protein consisting of 1115 amino acid residues. There was 93% homology with rat N-methyl-D-aspartate receptor homolog NMDAR-L (EMBL protein accession number: T31068) over the entire deduced amino acid sequence. Furthermore, as a result of performing a domain search on the Pfam database Release 6.6 (Bateman A. et al. (2000) Nucleic Acids Res., 28, 263-26) using the predicted amino acid sequence, 674th to 952 There was a sequence similar to the Ligand-gated ion channel domain at the amino acid residue. In addition, when the transmembrane domain was predicted by a membrane protein prediction program, SOSUI (Hirokawa T. et al. (1998) Bioinformatics, 14, 378-379), the 8th to 29th, 674th to 695th, Transmembrane domains were predicted at five positions from 712 to 734, 744 to 766, and 929 to 951.
(Example 3) (Expression analysis in human tissue by RT-PCR ELISA method)
Expression of the gene for PF00154 in human tissues and brain sites is performed using the RT-PCR method (DNA Research 4, pp. 141-150 (1997)), and the PCR method is used to quantify the PCR product (DNA Research 5, pp. 277-286 (1998)) was applied. Specifically, it is as follows.
13 types of human tissue-derived mRNA (heart, brain, lung, liver, skeletal muscle, kidney, pancreas, spleen, testis, ovary, spinal cord, fetal liver and fetal brain) purchased from Clontech and 8 parts of brain Single-stranded cDNA was synthesized using reverse transcriptase Superscript II (Invitrogen) and random primer (Takara Shuzo) using the mRNA of amygdala, corpus callosum, cerebellum, caudate nucleus, hippocampus, substantia nigra, hypothalamus and thalamus as a template. did. Next, using these cDNAs as templates, PF00154 gene-specific primers (SEQ ID NO: 5′-CAGTGCTGAAGATTATTGGAG-3 ′ and SEQ ID NO: 5′-CACAGTGAGAAGTTGCAGTC-3 ′) and digoxigenin (DIG) -labeled dUTP Direct labeling of RT-PCR products was performed. At this time, in order to control the subsequent gene expression quantification, similar PCR was carried out using PF00154 plasmid DNA serially diluted 10-fold from 0.05 fg to 500 fg as a template to prepare a DIG-labeled control PCR product. . On the other hand, using the same primer set and biotin-labeled dUTP, a large amount of biotin-labeled PCR product was prepared using PF00154 plasmid DNA as a template. Next, hybridization was performed using a DIG-labeled PCR product and an excess biotin-labeled PCR product for quantification of expression, and then a DIG / biotin-hetero PCR product was captured on a streptavidin plate (Roche). The excess was washed away.
The supplemented DIG-labeled PCR product is reacted with a peroxidase (POD) -labeled anti-DIG antibody (Roche), and 2,2′-azino-bis (3-ethylbenzothizoline-6-sulfonic acid) (Roche) is used as a substrate. The color was developed and quantified using a microtiter plate reader (Melcular Devices). Finally, using the standard line created based on the control PCR value with clear template DNA concentration, the numerical value obtained in each tissue, etc. is converted, and the RNA expression level of the gene is determined as a specific amount derived from the tissue. It was expressed as the relative amount of cDNA among the cDNAs reversely transcribed from RNA.
The results are shown in the following table.
Tissue relative expression level

(Example 4) (Extraction of total RNA from brain tissue)
Total RNA was extracted from postmortem brains of schizophrenic patients and non-schizophrenic patients (control). That is, a human frontal cortex sample stored at −80 ° C. was cut into small fragments (about 40 mg) on dry ice and transferred to a 1.5 ml centrifuge tube. The extraction of total RNA was performed using Absolutely RNA RT-PCR Miniprep Kit (Stratagene). A part of the extracted total RNA was taken and the concentration was determined from the value of OD260. Further, 250 ng of total RNA was electrophoresed on a 1% agarose gel containing 0.1 mg / ml ethidium bromide. The gel after electrophoresis was irradiated with UV, and bands of 28S ribosomal RNA and 18S ribosomal RNA were confirmed.
(Example 5) (Reverse transcriptase reaction)
About 0.5 μg of total RNA as a template, SuperScript ^TM CDNA was synthesized using First-Strand Synthesis System for RT-PCR (Gibco BRL). In addition, Human Brain Total RNA (Clontech) was used as a template for a standard sample for preparing a calibration curve.
(Example 6) (Real-time PCR)
SYBR Green PCR Master Mix (PE Applied Biosystems) for amplification of NR3A using 1/100 amount of cDNA as a template, and TaqMan β-actin Control Reagents (PE Applied Biosystems) for amplification of β-actin as an internal standard. Was used. The conditions for PCR are as follows.

After keeping at 50 ° C. for 2 minutes and further at 95 ° C. for 10 minutes, a cycle of 95 ° C. for 15 seconds and 60 ° C. for 1 minute was repeated 40 times. As the PCR apparatus, ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems) was used.
(Example 7) (Measurement of NR3A gene)
A calibration curve was prepared based on the values of serially diluted standard samples, and the initial amounts of NR3A and β-actin gene of each sample were calculated from the calibration curve. Further, in order to correct the initial total RNA amount, the NR3A amount was divided by the β-actin amount. Finally, the value of the schizophrenia group was calculated when the value of the control group was 1.
As a result, as shown in FIG. 1, the NR3A gene in the brain was significantly increased (p <0.05) in the schizophrenic patient group as compared to the control group. Therefore, it was revealed that NR3A is a gene related to the pathology of schizophrenia.
Industrial applicability
As described above, the present invention provides a novel human NR3A protein and its gene involved in the pathogenesis of schizophrenia, and the provision of a novel pharmaceutical composition and diagnostic means utilizing this characteristic involves NR3A. It is useful in the clinical and medical fields of diseases that cause schizophrenia.
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a graph showing the results (mean ± SE) of quantifying the expression of NR3A gene in the brain of control (n = 10) and schizophrenic patients (n = 9) by real-time PCR. * P <0.05 (t test)

Claims (20)

A polypeptide related to an N-methyl-D-aspartate (NMDA) glutamate receptor selected from the group below or a protein containing the polypeptide;
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing,
(2) A polypeptide containing the polypeptide of (1) above,
(3) A polypeptide having at least about 50% amino acid sequence homology with the polypeptide of (1),
And (4) a polypeptide having a mutation such as deletion, substitution, addition or insertion of one to several amino acids in the amino acid sequence of (1) to (3). A partial peptide having a part of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, wherein the peptide has at least about 8 consecutive amino acid sequences. A polynucleotide encoding the polypeptide according to claim 1 or a complementary strand thereof. A polynucleotide comprising the sequence of 602 to 3949 in the coding region of the base sequence set forth in SEQ ID NO: 2 in the sequence listing or a complementary strand thereof. A polynucleotide that hybridizes with the polynucleotide according to claim 3 or 4 or a complementary strand thereof under stringent conditions. A polynucleotide encoding the peptide according to claim 2 or a complementary strand thereof. A polynucleotide comprising the sequence of 602 to 3949 in the coding region of the base sequence set forth in SEQ ID NO: 2 in the sequence listing or a polynucleotide comprising at least about 15 consecutive base sequences of its complementary base sequence. A polynucleotide that hybridizes with the polynucleotide according to claim 6 or 7 or a complementary strand thereof under stringent conditions. A recombinant vector comprising the polynucleotide according to any one of claims 3 to 8 or a complementary strand thereof. A transformant transformed with the recombinant vector according to claim 9. A method for producing the polypeptide according to claim 1 or 2, or a protein or peptide having the polypeptide, comprising culturing the transformant according to claim 10. An antibody that immunologically recognizes the polypeptide according to claim 1 or 2 or a protein or peptide having the polypeptide. The antibody according to claim 12, which recognizes at least an NMDA type glutamate receptor subunit. A compound having an action of inhibiting or promoting the activity by interacting with the polypeptide of claim 1 or a protein having the polypeptide, and / or the claims of claims 3 to 5. A method for screening a compound having an action of interacting with any one of the polynucleotides and inhibiting or promoting the expression thereof, comprising the polypeptide according to claim 1 or 2, or the polypeptide according to claim 1. A protein or peptide, a polynucleotide according to any one of claims 3 to 8, a vector according to claim 9, a transformant according to claim 10, and a claim An antipsychotic drug characterized by using at least any one of the antibodies described in the above paragraphs 12 or 13 Cleaning method. 6. A compound that interacts with the polypeptide according to claim 1 or a protein having the polypeptide to inhibit or activate the activity, or any one of claims 3 to 5. A method of screening for a compound that interacts with one polynucleotide to inhibit or promote its expression and screens with the polypeptide or protein under conditions that allow the interaction between the compound and the polypeptide or protein Contacting the compound to be evaluated and evaluating the interaction of the compound (such interaction is associated with a second component that may provide a detectable signal in response to the interaction of the polypeptide or protein with the compound) Then the presence of a signal resulting from the interaction of the compound with the polypeptide or protein. Or by detecting the absence or change, compound interacts with a polypeptide or protein, the screening method of antipsychotics include determining whether to activate or inhibit the activity. A compound to be screened by the screening method for an antipsychotic drug according to claim 14 or 15, which interacts with the polypeptide according to claim 1 or a protein having the polypeptide. Or a salt or a salt thereof that inhibits or promotes the activity. A compound screened by the method according to claim 14 or 15, wherein the compound interacts with any one of the polynucleotides according to claims 3 to 5 to express its expression. Inhibiting or promoting compounds or salts thereof. The polypeptide according to claim 1 or 2, or a protein or peptide having the polypeptide, any one polynucleotide according to claims 3 to 8, and claim 9 The vector according to claim 10, the transformant according to claim 10, the antibody according to claim 12 or 13, or the compound according to claim 16 or 17. A pharmaceutical composition comprising an antipsychotic drug, comprising at least one of them. A method for diagnosing a disease (schizophrenia) related to the expression or activity of the polypeptide according to claim 1 or a protein having the polypeptide, comprising: (a) encoding the polypeptide or protein And / or (b) analyzing the polypeptide or protein in the sample as a marker. The polypeptide according to claim 1 or 2, or a protein or peptide having the polypeptide, any one polynucleotide according to claims 3 to 8, or claim 14. (a) a nucleic acid encoding the polypeptide or protein, and / or (b) the polypeptide in a sample, comprising at least one of the antibodies of paragraphs 12 or 13 Alternatively, a reagent kit for diagnosing schizophrenia for use in a method comprising analyzing a protein as a marker.

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