WO2003072779A1 - Method of using pituitary-specific genes - Google Patents

Abstract

Genes expressed specifically in the pituitary and amino acids. As the results of studies on genes actively expressed in human pituitary, genes highly expressed in human pituitary tissues are specified. Then it is found out that genes of SEQ ID NOS:1 to 4 are expressed specifically in human pituitary. Based on these findings, methods of effectively using these genes and proteins are established. Namely, utilization of a base sequence represented by any of SEQ ID NOS:1 to 4 or a fragment thereof in examinations relating to human pituitary or treatments of human pituitary-associated diseases.

Description

 Description Use of pituitary-specific genes

 The present invention relates to genes and amino acids specifically expressed in the pituitary. Conventional technology

 The pituitary gland consists mainly of endocrine cells that make pituitary hormones. These hormones are stored in secretory granules and released by regulated exocytosis (extracellular release). These secretory granules also contain processing enzymes that melt into cell membranes and release hormones and other components (Arvan P & Castle D Biochemical Journal 332 593-610 (1998))

This characteristic function of the pituitary gland is maintained by proteins such as the pituitary hormone ゃ transcription factor, which is unique to the pituitary gland. Abnormalities in these genes result in both a deficiency of isolated anterior pituitary hormone and a deficiency of bound pituitary hormone (.Phillips III JA et al. Proceeding of the National Academy of Sciences of the United States of America 78 6372-6375 (1981); Tatsumi K et al. Nature Genetics 1 56-58 (1992); Cushman LJ & Camper, Mammalian Genome 12 485—494 (2001)). Tannoヽ⁰ click modifying enzymes (this ί or GalNAc-4-pituitary as sulfotransferase specifically intended to express (Okuda T et al. Journal of Biological Chemistry 51 40605- 40613 (2000)), prohormone convertase synthetase (PC) 1/3, PC 2, carboxypeptidase E (CPE) and neuroendocrine protein 7B2 (Iguchi H et al. Neuroendocrinology 39 453-458 (1984)) some force ^s are also expressed in且織(Gorr SU et al. Molecular and Cellular Endocrinology 1721-6 (2001)). a CPE of and 7 B 2 abnormality, secretion of ACTH (畐lj renal cortical stimulating hormone) is controlled (Gorr SU et al. Molecular and Cellular Endocrinoloay 172 1-6 (2001)). Abnormalities have already been proven to be useful markers of autoimmune diseases. Pituitary-specific antibodies to lymphocytic pituititis have been reported, but the corresponding ίΐ ^ ίΐ: ¾ て ヽ ヽ (Crock PA, Metabolism 83 609-618 (1998); Nishiki M challenges et al. Clinical Endocrinology (Oxford) 54 327- 333 (2001)) o the invention is to you it'll solve

It is important to isolate pituitary-specific transcripts in order to elucidate the major molecules of pituitary-specific tissues and elucidate the mechanism of pituitary disease. In an attempt to identify molecules that were specifically expressed in the human pituitary gland, the inventors used the gene expression profiling method and the gene expression profiling method (Okubo K et al. Nature Genetics 2 173-179). (1992)) ₀ This method constructs a 3′-directional cDNA library that faithfully represents the original composition of the mRNA species, randomly arranges the cDNA clones, and uses GATC (MboI recognition sequence). A 3 ′ EST consisting of a short nucleotide sequence located just upstream of poly (A) adjacent to) is obtained. Since these 3 'ESTs are unique to individual genes, they are called gene signatures (GSs) (okubo K et al. Nature Genetics 2 173-179 (1992)). The expressed genes are identified by their GS species, and the relative abundance of each transcript can be estimated by the frequency of the corresponding GS species in the library. The resulting gene expression profile represents the amount of gene transcription in the human pituitary.

A profile of similar gene expression in 64 human tissues constitutes a molecular anatomical database called the Body Map database (Japanese Clinical Journal Vol. 52 No. 4 (4, 1994); Hishiki T et al. Nucleic Acids Research 28 136-138 (2000); Kawamoto S et al. Genome Research 10 1817-1827 (2000)). By comparing gene expression profiles obtained from various tissues in the body map database, genes specific to many tissues and diseases caused by abnormalities of the genes have been identified (okubo K et al. Nature Genetics). 2173-179 (1992); Kita H et al. DNA Research 31-7 (1996); Nishida K et al. Investigative Ophthalmology & Visual Science 37 1800-1809 (1996); Yokoyama M et al. DNA Research 3 311-320 (1996); Maeda K et al. Gene 190 227-235 (1997); Nishida K et al. American Journal of Human Genetics 61 1268-1275 (1997); Shimizu-Matsumoto A et al. Blood 92 1432-1441 (1998)). Thus, this method is an effective alternative to other methods, such as mechanical, homologous, and positional closing, for identifying novel genes. Means for solving the problem

 The present inventors have identified genes that are abundantly expressed in human pituitary tissues by examining genes that are actively expressed in human pituitary. By comparing the expression profile of this gene in the human pituitary gland with a body map database, we found genes that are specifically expressed in the human pituitary gland and found that they can be used for the detection and treatment of diseases in the human pituitary gland .

 In the present invention, it has been found that the following four genes are specifically expressed in human pituitary.

 1. PGSF 1a (splice variant of GS 9544): SEQ ID NO: 1

 2. PGSF lb (splice variant of GS 9544): SEQ ID NO: 2

 3. PGSF2 (same as GS 9589): SEQ ID NO: 3

 4. Pi—a (GS 9573, same as KI AA 0512): SEQ ID NO: 4 Therefore, the present invention provides that the amino acid sequences (SEQ ID NOs: 5 to 8) encoded by these are proteins derived from the pituitary gland. Reveals.

 The nucleotide sequence and amino acid sequence of pi-a have already been registered (AX127740, AX036667, AK026832.AB037745), PGSFla (AB058892), PGSFlb (AB058893) and PGSF2 (AB058894). The inventors registered S14—APR—2001. Further, the present invention is any one of the nucleotide sequences of SEQ ID NOS: 1 to 3, and it can be said that the present invention is any one of the amino acid sequences of SEQ ID NOS: 5 to 7.

In the present invention, based on such a finding, a method for effectively utilizing these genes and proteins has been found. That is, the present invention relates to the use of any one of SEQ ID NOs: 1 to 4 or a portion thereof for testing for human pituitary gland or human joint or treating human pituitary gland-related disease or human joint-related disease.

 That is, the present invention includes the following inventions.

 1) A kit for diagnosing a genetic abnormality in a human pituitary-related disease or a human joint-related disease, comprising a primer for PCR amplifying any one of the protein coding regions of SEQ ID NOs: 1 to 4. Furthermore, a method for diagnosing a genetic abnormality in a human pituitary-related disease or a human joint-related disease using a primer for PCR amplifying any one of the protein coding regions of SEQ ID NOs: 1 to 4.

 2) A diagnostic agent for gene expression of a human pituitary-related disease or a human joint-related disease, using a partial sequence of any one of SEQ ID NOs: 1 to 4 as a probe. Furthermore, SEQ ID NO: 1

A method for diagnosing gene expression of a human pituitary-related disease or a human joint-related disease, using a partial sequence of any one of the base sequences of any one of (1) to (4) as a probe.

 3) SEQ ID NO :! A therapeutic agent for a human pituitary gland-related disease or a human joint-related disease in which low expression of a protein having any of SEQ ID NOS: 5 to 8 is an exacerbation factor, comprising an expression vector containing any of the base sequences of SEQ ID NOS: to 4. Furthermore, a human pituitary-related disease in which low expression of the protein of any of SEQ ID NOs: 5 to 8 is an exacerbation factor using an expression vector containing any of the nucleotide sequences of SEQ ID NOs:! Or a method for treating a human joint-related disease.

 4) SEQ ID NOs: 1-4, consisting of an antisense nucleic acid to any of the base sequences, SEQ ID NOs: 5-8, a human pituitary-related disease or human whose overexpression of any of the proteins is an exacerbating factor Treatment for joint-related diseases. Further, a human pituitary-related disease or human disease in which overexpression of any of the proteins of SEQ ID NOS: 5 to 8 is a negative factor using an antisense nucleic acid against any of the base sequences of SEQ ID NOs: 1 to 4 Treatment method for joint-related diseases.

 In addition, the present invention relates to a test for a human pituitary gland or a human joint or a treatment for a human pituitary gland-related disease or a human joint-related disease, using a protein having any of SEQ ID NOS: 5 to 8 or an antibody against the protein. Is for use.

That is, the present invention includes the following inventions. 1) A diagnostic agent for a human pituitary-associated autoimmune disease comprising a protein having any one of SEQ ID NOs: 5 to 8 as an antigen. Further, a method for diagnosing a human pituitary gland or a human joint-related autoimmune disease, using the protein of any one of SEQ ID NOs: 5 to 8 as an antigen. The antigen may be labeled, and the autoimmune disease may be autoimmune pituititis or rheumatoid arthritis.

 2) A diagnostic agent for human pituitary function, comprising an antibody against a protein having any one of SEQ ID NOs: 5 to 8. Furthermore, a method for diagnosing human pituitary or human joint function using an antibody against a protein having any one of SEQ ID NOs: 5 to 8.

 3) A therapeutic agent for a human pituitary-related disease or a human joint-related disease, which comprises a protein having any of the sequences of SEQ ID NOS: 5 to 8, wherein low expression of the protein of any of the sequences of SEQ ID NOs: 5 to 8 is an exacerbating factor. Furthermore, treatment of a human pituitary-related disease or a human joint-related disease in which low expression of any of the sequence proteins of SEQ ID NOS: 5 to 8 becomes a worsening factor, using a protein of any of the sequences of SEQ ID NOs: 5 to 8. Method.

 4) An agent for treating a human pituitary-related disease, which comprises an antibody against a protein having any of the sequences of SEQ ID NOS: 5 to 8, and whose overexpression of a protein having any of the sequences of SEQ ID NOs: 5 to 8 becomes a worsening factor. Further, a method for treating a human pituitary gland-related disease in which overexpression of a protein having any of the sequences of SEQ ID NOS: 5 to 8 is an exacerbating factor, using an antibody against a protein having any of the sequences of SEQ ID NOs: 5 to 8. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows the structure of PGSF1. The upper represents the PGSF1 genome, and the lower represents the cDNA structure of PGSF1. The tall and short boxes in the cDNA structure represent the protein coding and noncoding regions, respectively. Closed squares indicate regions conserved in the soybean nodulin family (see Figure 4). AW583046 differs in splice at Exon 2 and has a 53 bp insert that results in a frameshift in the pituitary ORF.

 FIG. 2 shows an amino acid sequence alignment of PGSF1.

FIG. 3 shows multi-tissue Northern hybridization of PGSF1 and actin. FIG. 4 shows an amino acid sequence alignment of PGSF1 and soybean nodulin family. Amino acids corresponding to PGSF1 are shown in black. N〇2B—S0YBN represents soybean nodulin 26 B, 023 S0YBN represents soybean nodulin 23, and N04 SOYBN represents soybean nodulin 44.

 FIG. 5 shows the structures of PGSF2 and IGDC1. The tall squares and the short squares represent the protein coding and non-coding regions, respectively. Shaded cells indicate different nucleotide sequences. The signal peptide, the Ig-like region, the transmembrane region (TMs), and the probe used in Northern hybridization are shown.

 FIG. 6 shows the amino acid sequence alignment of PGSF2I GDC1 and InhBP-s. Matching amino acids are shown in black.

 FIG. 7 shows multi-tissue northern hybridization using 3 and 5′-probes of PGSF2.

 FIG. 8 shows multi-tissue northern hybridization of KIAA0512. FIG. 9 shows the inhibitory effect of recombinant human PGSFla on positive samples. FIG. 10 shows the inhibitory effect of recombinant human PGSFla on positive and negative samples. Embodiment of the Invention

 The gene found to be specifically expressed in human pituitary gland found in the present invention can be used as follows.

 1. A method for diagnosing a genetic abnormality in congenital pituitary hormone deficiency.

By comparing the cDNA sequence (SEQ ID NO: 14) encoding the protein of the present invention (SEQ ID NO: 58) with a known human genomic sequence, the exon structure of each gene can be determined. Based on this, a primer is prepared to amplify the protein coding region by PCR, and DNA extracted from patient blood is amplified by PCR, the base sequence is determined, and the difference from the normal sequence is examined. That is, primers for PCR-amplifying any of the protein coding regions of SEQ ID NO: 14 are useful for such diagnosis. For example, in PGSFla and PGSFlb, the nucleotide sequence can be determined by PCR amplification using the following three sets of primers. exl-U 5'-TGGGCCACAACCTATGAGGTAATC-3 '(Rooster column number 9) humPGSFlb.ex2 + L 5'-TCAGACCACCCTCTCTCGTCC-3' Ex4 + L 5 '-CGTCAGAGACATGGGATTTGGAGT-3' (SEQ ID NO 1 2) humPGSFlb .ex5-U 5 '-CCAATTCTCTGCAACACCCTAATC-3' (Distribution number 13) humPGSFlb .ex5 + L 5'-CTTTGATGCCTTTATTGATTCAACAC-3 '(SEQ ID NO: 14)

 2. Diagnosis of gene expression in the pituitary gland using the partial sequence of cDNA.

 Tissues such as pituitary tumors are determined by examining gene expression using the quantitative and qualitative methods such as Northern method, PCR method, DNA tip, etc. using the partial sequence of the cDNA of SEQ ID NOs: 1-4. Diagnosis of gene expression. That is, the partial sequence of the cDNA of SEQ ID NOs: 1 to 4 can be used as a diagnostic probe for such diagnosis.

 3. Diagnosis of autoimmune diseases such as autoimmune pituititis and rheumatoid arthritis.

 Using an antigen consisting of the protein having the sequence of SEQ ID NOs: 5 to 8, the autoantibodies against it are measured to determine the autoimmune diseases such as autoimmune pituititis, rheumatoid arthritis, and tissue destruction such as the pituitary gland. Used for diagnosis. As an example, in Example 2, a method of reacting a patient's serum with a labeled antigen (each of the novel proteins) to form a labeled antigen / autoantibody complex and then detecting this complex (radioligand assay) is described. Shown.

 4. Make antibodies and measure protein in the serum and tissues of patients to diagnose pituitary, endocrine and exocrine functions.

 For the diagnosis of pituitary, endocrine and exocrine diseases, an antibody against the protein having the sequence of SEQ ID NOS: 5 to 8 is prepared, and the protein content of each new protein in serum and tissue is measured by the immunoassay.

5. SEQ ID Nos. 5 to 8! By administering proteins of any sequence or DNAs encoding these proteins (SEQ ID NOS: 1-4), low expression of these proteins can be exacerbating factors, including pituitary diseases. To treat diseases with abnormal endocrine secretions and rheumatoid arthritis. Example 1) Intra-articular injection of PGSFla protein to replace and treat rheumatoid arthritis patients.

 Example 2) In a disease in which low expression of a novel protein (pi-a, PGSFla, PGSFlb, PGSF2) is an exacerbating factor, replacement or treatment is performed by intravenous or oral administration of the novel protein.

 Example 3) New tanno II. Low pituitary expression of proteins (pi-a, PGSFla, PGSFlb, PGSF2) is a negative factor, and expression vectors containing DNAs encoding these proteins (SEQ ID NOS: 1 to 4) Replacement and treatment by administration to the pituitary gland. 6. Overexpression of a protein of any one of SEQ ID NOs: 5 to 8 is a disease of exacerbation factor. Antibodies against these proteins or antisense nucleic acids are administered to neutralize the effect and pituitary gland. Treat diseases such as diseases with endocrine and exocrine abnormalities and chronic rheumatoid arthritis.

 Example 1) Anti-PGSFla antibody is injected intra-articularly into rheumatoid arthritis patients to suppress the amount of protein and treat it.

 Example 2) In a disease in which overexpression of a novel protein (pi-a, PGSFla, PGSFlb, PGSF2) is an exacerbating factor, the amount of the protein is suppressed and treated by intravenous administration of an antibody against the novel protein.

 Example 3) A disease in which the overexpression of novel proteins (pi-a, PGSFla, PGSFlb, PGSF2) in the pituitary gland is an exacerbation factor. Antisense nucleic acid is administered to the pituitary gland to suppress the expression and treat it.

 7. Produce a protein of any one of SEQ ID NOs: 5 to 8 and use it as a material for functional and structural analysis studies. Produce antibodies against new proteins and analyze their functions and structures.

Example 1) Novel protein (pi-a, PGSFla, PGSFlb, PGSF2) is synthesized in vitro or chemically in various cells such as animal cells, insect cells, yeast, and Escherichia coli, and administered to cultured cells or animals. It is used for research on functional analysis by overexpression experiments. Example 2) An antibody against a novel protein is prepared to obtain an antibody that recognizes the primary structure and tertiary structure of the protein and a neutralizing antibody that neutralizes the activity. Recognition of primary structure and three-dimensional structure Antibodies that we know are useful for studying the structure of protein structures. Neutralizing antibodies suppress protein activity when administered to cultured cells or animals, and are useful for functional analysis studies.

 Example 3) A protein expressed in the living body or in an expression system where the activity is obtained is purified using the antibody as the indicator of activity and activity, and the sugar chain structure is analyzed using the purified protein, or the solid is analyzed by crystal analysis. Examine the structure. Hereinafter, the present invention will be illustrated by way of examples, but these are not intended to limit the present invention.

Example

 Hereinafter, the present invention will be illustrated by examples, but is not intended to limit the present invention.

 The test method used in this example is described below.

Construction and sequence analysis of 3'-directional cDNA library

 16 Human pituitary poly (A) RNA samples from 21 persons aged 70 years were purchased from CLONTEC (Palo Alto, CA, USA). From these, a three-way cDNA library was constructed, and Escherichia coli was transformed according to the report (Okubo K et al. Nature Genetics 2 173-179 (1992)). Briefly, the procedure was as follows: cDNA was synthesized from the above sample using a pUC19-based vector primer digested with MboI, and it was cyclized and introduced into E. coli. This was spread on a plate, and the cDNA insert of a randomly selected transformant was amplified with PCR to produce sequence type II. Using a fluorescence cycle sequencing kit (Perkin-Elmer, Norwalk, Conn., USA) and an ABI PRISM373 gene analyzer (Perkin-Elmer), the 3 ′ terminal nucleotide sequence of this amplified product (cDNA) was determined.

The resulting 3 'terminal base sequence (hereinafter referred to as "GS s".) Compared to each other Rere by BLAST method to cluster I spoon (Altschul SF et al. Nature Genetics 6 119-129 (1994)) 0 One representative GS from each cluster was selected and compared to a representative sequence from a previously generated cluster. One for each independent cluster GS number. The representative sequence of this group of GS clusters (GS species) was searched for in the DDBJ / GenBank / EMBL database using the regenerated BLAST method (Altschul SF et al. Nature Genetics 6 119-129 (1994)). Comparison of RNA expression in a computer (in silico)

 The frequency of each GS was compared with that of the body map database. The specific expression of each GS species in the human pituitary is represented by the “pituitary TS ratio” in the following formula.

 Frequency of GS species in pituitary gland

 Total number of GS in the pituitary

 Pituitary TS ratio =

 All Pod One Map Database

 Of GS species in Japan

 All body one map database

 Of GS cDNA screen in DNA

Using the Uni-ZAP XR vector, the human pituitary cDNA library constructed using the Zap-cDNA synthesis kit (STRATAGENE, La Jolla, CA, USA) was screened by a standard method. The probe, labeled with alkaline phosphate for the GS sequence, was synthesized by Alphos Graphicsystems (Amersham Pharmacia, Arlington Height, IL, USA). After the phage plaque was transferred to a nylon membrane (Hybond N +, Amersham Pharmacia) by the usual alkaline transfer method, the membrane was heated at 80 ° C for 2 hours. Next, the membrane was hybridized at 55 ° C. with each probe. After hybridization, wash the membrane twice at 55 ° C for 10 minutes with a primary washing solution (2M urea, 0.1% SDS, 50mM sodium phosphate buffer pH 7.0, 150mM NaCl, lmM MgCl _2> 0.2 blocker) Subsequently, it was washed twice with a secondary washing solution (50 mM Tris base, 100 mM NaCl, 2 mM MgCl ₂ ) at room temperature for 5 minutes. Positive sample was illuminated with CDP-Star detection drug (Amersham Pharmacia), and 日 1 day at room temperature using 增 sensitive membrane # Diagnostic finolem (RX-ϋ, Fuji Film, Tokyo, Japan) did. The double-stranded plasmid DNA of this positive clone was recovered, and the DNA sequence was determined using an ABI PRISM373 gene analyzer (Perkin-Elmer). Rapid amplification of cDNA ends (5 and RACE)

Using the Marathon cDNA amplification kit (CLONTECH), the 5 'end arrangement of GS9544, GS9589 and GS9573 was determined. Using ReverTraAce (TOYOBO, Osaka, Japan), 1 μg of human pituitary poly (A) RNA was reverse-transcribed by a random hexamer. It was synthesized double stranded DN A, the adapter (5'-CTAATACGAC TCACTATAGG GCTCGAGCGG CCGCCCGGGC AGGT -3 '( Hai歹lj No. 1 5), 3' - attached to _{H 2 N-CCCGTCCA-P0 4} -5. Adapter primer 1 and primer for GS9544 (5'-CTCAGACCAC CCCTCCTCCC ACGCA-3 '(SEQ ID NO: 16)), primer for GS9589 (5 for TGTCTCTTC CAGTAGCAGC ACCGGTAAA-3' (Tokimi IJ PCR was performed using primers (5'-AGACAGACCC TCCAAAGATT CA-3 '(J No. 18)) for GS9573. Primers specific to AP1 or genes for this PCR product The sequence was determined using DNA.

Poly (A) RNA from human pituitary and liver was fractionated on formaldehyde-agagarose gel and transferred to Hybond N + membrane by the usual alkaline transfer method. This membrane or the human heart, moon, liver, pancreas, skeletal muscle and lung mRNA containing Northern LIGHT Human Multiple mRNA Blot I (Life Technologies, Gaithersburg, MD, USA) was screened using a cDNA library. The protein coding region of the cDNA of the obtained positive clone was hybridized with a cDNA probe labeled with an ECL direct system (Amersham Pharmacia) and washed. This film was exposed to the RX-U diagnostic film at room temperature for 1 hour or more using a sensitizing film. These cDNA probes were obtained by PCR using the primers shown in Table 1. GS sense primer antisense primer

GS9544 (PGSF1) GACAGGACGGCGAAGTTTGA TCCTTATTGCCTCACCATTTCC

 (SEQ ID NO: 19) (SEQ ID NO: 20)

GS9589 (PGSF2) 3 'AGGACTTATATGCCAGQCAC TGTTATTTTTATGTGTGGAA

 (SEQ ID NO: 21) (SEQ ID NO: 22)

GS9589 (PGSF2) 5 'CGCTCTAGAACTAGTQGATC TT GTQTCTTATCCTTCAGCAGCAG

 (SEQ ID NO: 23) (SEQ ID NO: 24)

GS9573 (KIAA05I2) TGCTGAGTTCAGGTTAGAGQCC GTTACTGCTTTTCCACTTGCTC

 (SEQ ID NO: 25) (SEQ ID NO: 26)

Hereinafter, the test results of this example are described. Human pituitary gene expression profile file

 In order to understand the synthesis and secretion of pituitary hormone from a molecular aspect, the inventors constructed a human pituitary gene expression profile as described above. This profile shows the relative activity of the gene in retaining the specificity of the tissue. A GS of 1015 was obtained from the human pituitary 3'-directional cDNA library (Table 2).

Table 2

Position GS species GSs Frequency

1, Known genes 263 658 64.8 i.Extracellular proteins 16 23S 23.4

Secretory proteins 12 234 23.1

Pituitary hortyiofics 5 219 21,6

Other secretory proteins 7 14 ΙΑ

Matrix proteins 4 5 0,5 i \ _t Plasma membrane proteins 12 20 2,0 in.Intracellular proteins 172 301

Secretorv granule oteitis f, 2 "5-3 j4t1) ^a (2 * 5-3 * 4) ^a

E --Golct nroteins 18 21 1

Nuclear nt tetns 32 49 4 Q

Fifteen

 en

 Ribosomal proteins 50 100 9.9

Signal transducers (J5-16) ^a (17-20) ^a (1.7-2.6) ³

Cytoskeletal proteins 8 17 1.7

Others 6 IS 1.5 iv.Uncharactedzed 63 98 9.7

II.Novel genes 264 357 35.2

Total 527 1015 100.0 In the table, a is a variant of the GNAS1 gene (see GS1007 in Table 3).

 The sequences were compared and 527 independent GS species were selected. From this, the GS GS of 527, and the genetic abilities registered in the DDBJ / GenBank / EMBL database, and the like, 263 GS species representing 658 GS were derived. These 263 GS species represent 357 GS derived from new genes.

In Tables 3 and 4 (continuation of Table 3), the frequency of genes frequently occurring in the human pituitary gland was compared to that of the other 63 tissues in the body map database. Their specificity to the pituitary is indicated by the “pituitary TS ratio”. To characterize the genes active in the human pituitary gland, these GS species were localized to their cells, as shown in Table 2. Classification was based on gender and frequency in the pituitary gland.

 Table 3 Location / Definition All in Pituitary Blood Epithelium

 #ACC Occurrence TS ratio Nerve Connective tissue Complex

Extracellular proteins

 Secretory proteins

 Pituitary hormones

 PRL 181 85.53 185 4 0 0 0 0 NM 000948

GH 20 87.42 20 0 0 0 0 0 NM—000515

LH beta 7 87.45 7 0 0 0 0 0 N „000737 glycoprotein alpha 6 B7.42 6 0 0 0 0 0 NM— 000735

FSH beta 5 87,42 5 0 0 0 0 0 NM—000510

Other secretory proteins

 B2M 4 0.74 471 20 151 33 218 45 NM— 004048

SEPPl 4 6.60 53 6 0 7 29 7 NM— 005410

Plasma membrane proteins

 HLA-C 4 2.59 135 7 41 12 55 16 NM_002117

TNFRSF12 3 23.84 11 3 2 0 2 1 NM— 003790

IntracBllular proteins

 Secretoiy granule proteins

 CHGB 12 61.71 17 4 0 1 0 0 NM— 001819

(NESP55) (9) (6.15) (32) (8) (36) (29) (H) (N „01 592)

CPE 3 5.96 44 30 0 8 0 3 NH_001873

7B2 3 26.23 10 7 0 0 0 0 NM— 003020

Nuclear proteins

 RBt 5 33.62 13 1 3 3 \ 0 NM— 000321

CDC2L1 3 0.66 399 s 27 70 223 6S NM— 001787

TB4X 3 0.81 322 72 106 40 64 37 NM_021109

DOC-1 3 4.86 54 21 3 4 13 10 NM— 00464 itochoaanal proteins

 ATP5 3 2.62 100 19 11 39 20 8 NM_001685

Cytoplasmic proteins

 Ribosomal proteins

 Component proteins

RPS8 6 1 0 477 60 85 78 181 67 N _0010I2 Table 4

RPS24 5 2.01 218 45 36 39 73 20 NM „0OIO26

RPS20 5 2.99 146 17 32 27 49 16 NM-001023

RPS27A 4 2.13】 64 16 43 21 56 24 already 002954

RPL34 3 33 197 39 22 39 73 21 NM_000995

Proteins related to peptide synthesis

 TPT1 6 0.91 577 72 79 113 232 75 NM— 003295

NACA 6 2.19 239 58 44 43 63 25 NM.005594

EEF1A1 5 0.67 652 151 145 76 208 67: N — 001402

MAT2A 4 4.32 81 20 16 12 18 11 M-0059n

EIF4G2 3 3.80 69 3 22 14 27 3 NM_001418

Signal transducers

 (Qs-alpha) (9) (6,15) (128) (32) (8) (36) (29) (14) (N _000516)

(XL-alpha-s) (9) (6.15) (128) (32) (8) (36) (29) (14) (AJ251790)

RALBP1 4 26.90 13 0 2 4 3 0 NMJ30678S

PBP 3 3.32 79 41 3 8 17 7 NH.002567

ANXA6 3 3.50 75 37 4 24 2 5 NM— 001155

PRKACA 3 87.42 3 0 0 0 0 0 NM_002730

C toskeletal proteins

 6.81 77 29 4 8 29 1 NM— 003746

ACTGI 3 2.02 130 32 12 30 30 23 NM— 0016M

Others

 ubtquitin C 4 1.75 200 23 37 49 58 24 26 SS0

PSMB4 3 5.96 44 10 5 9 13 4 NM.002796

PSMB5 3 y. / 1 * 7 c ri _UU y /

Uncharacterized

 PUM1 3 9.37 28 3 1 5 10 6 NM— 014676

MLN51 3 12.49 21 4 4 8 0 2 NM 007359

BM-002 3 14.57 18 5 2 4 2 2 NM_016617 galanin-related peptidi e 3 23.84 11 0 1 0 7 0 NMJ15973 1AA0343 3 29.14 9 6 0 0 0 0 AB002341 IAA0512 3 65.57 4 1 0 0 0 0 NM— 014782 Poddy Map database

 Each profile in

 1015 88732 22537 7112 19113 28917 10299

 In the table, the protein and number in parentheses indicate variants of the GNAS1 gene. The GNAS1 gene has three promoters, one of which is a secretory granule protein (NESP55),

'Your (Gs-alpha ヽ XL-alpha-s). The PRL gene was most actively expressed, followed by the GH gene. All of the pituitary hormone genes listed in the table were specifically expressed in the pituitary and most frequently in the human pituitary (23.4 Table 2).

 The third most common gene was chromogranin B (CHGB). CHGB is a secretory granule protein involved in the regulated secretory pathway. The other two secretory granule protein genes (CPE and 7B2) were mainly expressed in the pituitary and neural tissues. These secretory granule protein genes belong to the fourth most highly expressed group (2.3-3.4, Table 2).

 All of the genes already known for genes specific to both the pituitary and nervous tissues are either pituitary hormones or secretory granule proteins, with the exception of the cAMP-dependent catalytic alpha subunit (PRKACA). Is. Thus, other less well-known genes that are predominantly expressed in the pituitary and nervous tissues, such as KI 0343 and KI 0512, may be involved in regulated secretory pathways.

The fourth most common gene is the GNAS 1 gene. This gene produces three proteins, Gs—hi, extra-large G protein (XL-α-s) and NESP 55, transcribed by three different promoters (Kozasa T et al. Proceeding of the National Academy of Sciences of the United States of America 85 2081-2085 (1988); Kehlenbach RH et al. Nature 372 804-809 (1994); Ischia R et al. Journal of Biological Chemistry 27211657-11662 (1997).ぴ NE SP55 expression is restricted to neuroendocrine tissue, but activity on G s-α is detected in various tissues, and conversion between its activity and inactivity is due to abnormal endocrine disorders, such as Albright's genetic bone malformation. (Albright hereditary osteodis trophy) (Patten JL et al. Ew England Journal of Medicine 322 1412-1419 (1990); Weinstein LS, et al. Genomics 131319-1321 (1992)) and McCune-Albright syndrome (Schwindinger WF et al. Proceeding of the National Academy of Sciences of the United States of America 89 5152-5156 (199 2)), consistent with the activity of G s—o; and their expression as a whole in the bodymap database. Were not restricted to the pituitary and nervous tissue (Tables 3 and 4).

 Most of the other frequently occurring genes do not express specifically in the pituitary or nervous tissue and are therefore considered housekeeping genes. Ribosomal protein genes (9.9%) and nucleoprotein genes (4.8%) are frequently expressed. Since the two commonly used standards, i3-actin and glyceraldehyde-3-phosphate dehydrogenase, are not expressed uniformly or frequently in tissues in the Body Map Database, some of these genes are expressed in tissues. It will be a good internal standard when comparing levels.

Hu et al. Published the expression profile of the bacterial pituitary gland (Hu RM et al. Proceeding of the National Academy of Sciences of the United States of America 97 9543-9548 (2000)). It is based on 7,000 5'-EST from this protein cDNA, one third of which contains the first ATG. They compared it to the hypothalamic and adrenal expression profiles. Consistent with our observations, GH (growth hormone) and PRL (prolactin) were expressed in more than 1% of all ESTs analyzed. Other frequently occurring known Tables 3 and 4 of the genes force ^s observed in their Purofuainore, SEPP1, NACA, and EIF4G2, TNFRSF12, PBP, ANXA6, PRKACA gene, Pumilio homolog 1, MLN51, KIAA0343 , Some uncharacterized genes, such as KIAA0512, were not observed. Among the pituitary hormones, TSHiS and P0MC did not appear in our profile, PRL was 10 times more in our profile, and FSH did not appear in their profile. To assess the quality of these profiles, a comparison was made of well-characterized pituitary homons.

The TSH (thyroid stimulating hormone) i3 cDNA lacks the MboI recognition sequence (Tatsumi K et al. Gene 73 489-497 (1988)) and does not appear as GS. The very high expression of PRL in our profile is not due to specific endocrine conditions. The reason is as follows. Since the human pituitary poly (A) RNA used here is derived from 21 humans, tissues from one or two individuals may contain PRL as a result of either prolactinoma or endocrine conditions. Even with high expression, this large amount of PRL Because of the dilution of more than two-fold, the exceptional individuals expressing very high levels of PRL used in our profile express PRL in more than 100% of the total mRNA, a virtually impossible situation. Become. The absence of POMC reflects low levels of ACTH (adrenocorticotropic hormone) in the anterior pituitary gland (Matsuyama H et al. Endocrinology 88 692-695 (1971); Moldow R & Yalow RS Proceeding of the National Academy of Sciences of the United States of America 75 994-998 (1978); shi shikawa J et al. Endocrinologia japonica 34 755-767 (1987)). Previously measured expression levels of FSH j8 and LHi3 showed similar results to our profile (Dalkin AC et al. Endocrinology 125 917-924 (1989)).

 Our method has two advantages over other gene expression profiling methods. First, compared to gene expression profiling methods from single or multiple tissues, the body map database of 63 different human tissues is currently available, and the method used for the body map database Using the same method and comparing the constructed pituitary expression profiles, tissue-specific transcript candidates can be immediately identified. Second, most GSs are less than 1 Kb in length and more than 30 bp, making it easier to prepare probes for further analysis. In fact, this method was effective in isolating tissue-specific genes in various other tissues (Okubo K et al. Genomics 30 178-186 (1995); Kita H et al. DNA Research 31). -7 (1996); Nishida K et al. Investigative Ophthalmology & Visual Science 37 1800-1809 (1996)); Yokoyama M et al. DNA Research 3 311-320 (1996); Shimizu-Matsumoto A et al. Investigative Ophthalmology & Visual Science 38 25 f 6-2585 (199 f)); Itoh K et al. Blood 92 1432—1441 (1998)). We concluded that our method was reliable, although further analysis of other methods and other gene expression profiles obtained in other organisms was needed. Human pituitary-specific gene expression profile

To identify genes that may play important roles in the pituitary, we GS species that are frequently and specifically expressed in the pituitary gland were selected (Table 5). Of the 11 GS species, 8 were known genes. Five of them (PRL, GH, LHJ3, glycoprotein alpha, FSHJ3) are anterior pituitary hormones, two (CHGB, PRKACA) are specific to endocrine tissues, and one (ΚΙΑΑΟδΙ) ^{In 2} ), the characteristics were not determined.

 Table 5 Pituitary

 GS type definition Expression analysis

 Number of occurrences

9703 PRL 181

 9496 Gil 20

 7687 CHGB 12

 9512 glycoprotein alpha 7

 9535 LHbeta 6

 9544 PGSFla * 6 PS

 PGSFltf ps

9504 FSHbeta

 9651 4

9589 PGSF2 ¹ 3 ps 9606 PRKACA 3

 9573 KJAA0512 PPd In the table, s-pamashita® represents pituitary-specific, ppd represents pituitary-specific pituitary-predominant.

By screening the human pituitary cDNA library with the GS sequence, cDNA clones of GS9544, GS9589 and GS9651 were obtained. Although GS9651 was a unique sequence in the human genome draft sequence (International Human Genome Sequencing Consortium 2001, Venter et al 2001, Nature 409 860-921 (2001)), screening of cDNA clones was unsuccessful. According to Northern hybridization, GS9544 and GS9589 were pituitary-specific, and GS9573 (KIAA0512) was the most prominent of the pituitary. Characterization of a novel pituitary-specific transcript

 For GS9544, two separate variants of 0.7 kb and 0.9 kb were isolated from a human pituitary cDNA library. They were predicted to encode two proteins, a pituitary-specific gene 1a consisting of 128 amino acids (PGSFla) and a pituitary-specific gene 1b consisting of 91 amino acids (PGSFlb) (No. 1 and 2). A BLAST search of the human genome draft sequence revealed that these PGSF1 genes were present on 9 chromosomes (ACC # NT-0111255).

(International Human Genome Sequencing Consortium 2001, Nature 409 860-921 (2001)). In the EST database, a single partial human cDNA from the Langerhans Island DNA library represented another splice variant (ACC # AW583046) (FIG. 1). The cDNA probes for the two PGSF1s (PGSFla and PGSFlb) strongly hybridize with about 1 kb of pituitary (Pituitary) transcripts in multi-tissue northern hybridizations, resulting in a size of about 1.4 kb. It weakly hybridized to the pancreas transcript (Fig. 3). These are probably endocrine-specific transcripts that are specifically expressed in the pituitary gland. These may be proteins related to secretory granules, one of the most abundant in the gene expression profile of the human pituitary gland. These PGSF1s have 33% homology within 51 amino acids with the nodulin 26B-carboxy terminal domain of soybean (Jacobs FA et al. Ucleic Acids Research 15 1271-1280 (1987)) (FIG. 4). nodulin is a protein associated with the bacterial membrane of the soybean root nodule, whose carboxy-terminal domain is located in the cytoplasmic portion of the nodule. This domain's machine! Identified and rare Reka S (Panter S et al. Molecular Plant-Microbe Interactions 13 325-333 (2000)), the fact that this domain force is retained between S and D Indicates that it has a new function.

The full length cDNA of GS9589 is 2 kb long and is predicted to encode a PGSF2 protein of 242 amino acids. By BLAST search, this cDNA was identical to immunoglobulin (Ig) -like domain exons 1-5 containing 0.8 kb force 1 (IGDC1) in 5 (Frattini A et al. Gene 214 1- 6 (1998)) and found to be a human homologous protein to the recently identified short splice product of rat inhibin-binding protein, InhBP-S (Bernard DJ & Woodruff K, Molecular Endocrinoloay 15 654). -667 (2001)) (Figs. 5 and 6). This IGGD1 protein consists of an N-terminal single peptide, 12 Ig-like domains and a C-terminal transmembrane domain. PGSF2 is a splice variant of IGDC1 and is probably secreted. The probe for PGSF2 hybridizes with a transcript unique to the pituitary (Pituitary) of about 2 kb by multi-tissue Northern hybridization (Fig. 7). The 3'-probe hybridizes with pituitary-specific transcripts, while the 5'-probe hybridizes with the 2 kb band encoding PGS F2 and IGDC1, respectively, and longer bands (primary). 7). InhBP-L, a long splice product of InhBP, was identified as a homologous protein of IGDC1 and a unique receptor for Inhibin A (Bernard DJ & Woodruff TK, Molecular Endocrinology 15 654-667 (2001)). InhBP-S and InhBP-: Ln mRNA are specifically detected in rat pituitary gland and testis (Bernard DJ & Woodruff TK, Molecular Endocrinology 15 654-667 (2001)), and IGDC1 mRNA is human testis and prostate. PGSF2 / InhBP-S and IGDCl / InhBP-L are both specifically detected in human and rat tissues with secretory granules (Frattini A et al. Gene 214 1-6 (1998)). Is expressed. A BLAST search on the draft human genome sequence revealed that PGSF2 was located in the q25-26.2 region of chromosome X. The gene for recessive pituitary dysfunction dwarfism linked to X is present in this region (Lagerstrom-Fermer M et al. American Journal of Human Genetics 60 910-916 (1997)). If PGSF2 is associated with this disease, it may be related to pituitary development and function.

The full-length GS9573 cDNA is identical to the cDNA encoding KIAA0512, except that it has a 44 bp insert in the 5'-noncoding region. KIAA0512 is predicted to encode a protein consisting of 632 amino acids, has an armadillo] 3-force tenin-like repeat motif, and has 61% homology with pituitary protein (ACC # AF211175) within 307 amino acids. Multi-organized Northern Hanoko " According to Zession, KIAA0512 was expressed as 3.2 kb and 4.5 kb transcripts in all tissues tested. Although its expression level is different, it was most strongly expressed in pituitary (Pituitary) and heart (Heart) (Fig. 8). This pituitary protein (ACC # AF211175) binds in vivo to human pectin, which is expressed outside of erythrocytes. Since the sctulin family is a structural protein that exists in association with the membrane or Golgi apparatus, the KIAA0512 protein interacts with sctulin or other spectrin-binding proteins (eg, ankyrin, actin or band 4.1). It may form a complex and may be involved in the transport of parcels.

 As a result, a gene specifically expressed in human pituitary gland was able to be isolated from the expression profile of human pituitary gland and the expression profile of other tissues. Increasing the number of GSs can also identify small amounts of pituitary-specific genes. In this experiment, three specific transcripts were obtained from two GSs specifically expressed in the human pituitary. A recently developed oligonucleotide-based PCR-based multiple tissue quantitative expression analysis method (Heid CA et al. Genome Research 6 986-994 (1996); Kato K, Nucleic Acids Research 25 4694-4696 (1997); Hall; By using LL et al. Biotechniques 24 652-658 (1998); Kawamoto S et al. Genome Research 9 1305-1312 (1999)), it will be easier to select pituitary-specific genes in the future. I can do it. These genes will provide effective treatments for pituitary diseases such as congenital pituitary anomalies and self-antigens in lymphocytic pituititis. Example 2

In this example, the radioligand assay (RLA) was used to examine the autoantibody titer to the pituitary-specific protein PGSFla present in the serum of patients with rheumatoid arthritis. To identify autoantibodies in patients with rheumatoid arthritis, rheumatoid factor, which detects antibodies to denatured IgG, has been mainly used, but rheumatoid factor has a low positive rate in the early stages of rheumatoid arthritis. Recently, an RLA was developed that can be used as an antigen while maintaining its three-dimensional structure if cDNA is present, and that it can also detect three-dimensional structure-recognizing antibodies. Was done. Table 6 shows the patient samples used in this example.

 Table 6

Disease Name Number of Cases Male / Female Age (Average Sat SD) Rheumatoid Arthritis 41

 Rheumatoid factor (RF) exposure 37 1/36 53.9 Sat 13.4

RF negative 4 0/4 48.8 Sat 2.5 Osteoarthritis 10 1/9 69.0 Sat 13.7 Autoimmune pituitary and related diseases 34 14/18 50.5 Sat 16.7

SLE 14 2/12 37.8 Sat 12.6 PSS 6 0/6 55.2 Sat 15.0

MCTD 80/8 45.4 Sat 10.1 Healthy person 36 18/18 49.9 Sat 13.8 In this example, the following method was used.

Radio ligand assay

1. Car · Banba! ^ Sam (NT coupled in vitro transcription / translation system, Promega) ί This PGSFla expression plasmid was reacted with ³⁵ S-methionine for 30 minutes at 30 ° C.

2. After the reaction, the ³⁵ S-GH fraction was separated using a column.

3.Reaction buffer (NaCl 150 band ol / L, Tris 50 band ol / L, H 7.4, Tween-20 lml / L, bovine serum albumin 4g / L, and NaNg lg / L)

³⁵ S—PGSFla was diluted to 20,000 cpm / 20 μl. '

4. Serum 4 [mu] 1 was diluted with Reaction buffer (as ^{above) 26 ^ 1 35 S - 4} ° C De晚reaction mixed with GH 20μ1. 96-well filter plate (Millipore) with Blocking buffer (aCl 150 marl ol / L, Tris 50 marl ol / L, pH 7.4, Tween-20 lml / L, bovine serum albumin 30 g / L, and Na 3 lg / L) Blocking was performed at room temperature for 3 半 hours and at 4 ° C.

5. Protein G Sepharose (Amersham) 4. Then, the cells were blocked with a blocking buffer (same as above) for 1 hour. 6.4 The “Serum I ³⁵ S-PGSFla” mixture was transferred to a blocked 96-well filter plate, and 10 μl of Protein G Sepharose (5) was mixed and reacted at room temperature for 45 minutes.

 7.React 6 reactions 10 times with Wash buffer (NaCl 150 ol / L, Tris 50 marl ol / L, pH 7.4, Tween-20 10 ml / L) for 96-well filtration system (Millipore). Washed.

8. The liquid scintillator was measured for calo and β counter (Micro Beta, Amersham) 35 _S- PGSFla I antibody I Protein G complex.

 9. Based on the obtained counts, the accuracy was examined using the following indices.

 anti-PGSFla Ab index (unit)

 = ,, the count of unknown sample (cpm) / the count of NPS

(cpm)

 Recombinant PGSFla was prepared as follows. That is, PGSFla with His tag was expressed in a recombinant protein poor in Escherichia coli (pET expression system, Novaaen) and purified with an affinity column of ALON Metal Affinity Resins (CLONTECH).

 The suppression test was performed as follows. At the time of dilution of the positive sample, the recombinant PGSFla or ovalbumin (negative control) was reacted with calo;

 The results are shown below. The intra-assay variation error was 3.1 (n = 6), and the inter-assay variation error was 3.3% (n = 4).

 Table 7 shows the frequency of anti-PGSFla antibody positive in serum.

 Table 7

Disease name Number of cases m k (yeast) “'Rheumatoid arthritis 41

 RF positive 37 31 (83.8%)

RF negative 4 3 (75.0%) Osteoarthritis 10 2 (20.0%) Autoimmune pituitary and related diseases 34 3 (8.8%) SLE 14 1 (7.1%)

PSS 6 0

MCTD 8 1 (12.5%) Healthy 36 0

 To examine the specificity of these positive samples, competition tests were performed with recombinant human PGSFla expressed in E. coli. Positive samples were suppressed by recombinant human PGSFla (Fig. 9), but not by ovalbumin (Fig. 10), and were PGSFla-specific.

 In this study, anti-PGSFla antibodies were detected in the serum of patients with rheumatoid arthritis at a frequency of about 80%. Not found in 36 healthy subjects, 20.0% for related osteoarthritis, 0 to 12.5% for autoimmune disease, significantly lower in positivity, especially high in RF-negative patients with rheumatoid arthritis Therefore, it is considered to be effective for supplementary diagnosis of patients with rheumatoid arthritis, which is equal to or better than the measurement of RF.

Claims

The scope of the claims I. Use of any one of SEQ ID NOs: 1 to 4 or a portion thereof for testing a human pituitary or human joint or treating a human pituitary-related disease or a human joint-related disease.  2. A diagnostic kit for a genetic abnormality of a human pituitary-related disease or a human joint-related disease, comprising a primer for PCR amplifying any one of the nucleotide sequences of SEQ ID NOs: 1 to 4 or a portion thereof.  3. A diagnostic agent for gene expression of a human pituitary-related disease or a human joint-related disease, using a partial sequence of any one of SEQ ID NOs: 1 to 4 as a probe.  4. A human pituitary gland-related disease or an expression vector comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 4, wherein the low expression of the protein of any of SEQ ID NOs: 5 to 8 is a worsening factor or A therapeutic agent for human joint-related diseases.  5. A human pituitary-related disease or a human joint-related disease comprising an antisense nucleic acid corresponding to any of the nucleotide sequences of SEQ ID NOS: 1 to 4 and overexpression of any of the proteins of SEQ ID NOs: 5 to 8 being a worsening factor Therapeutic drugs.  6. Use of a protein of any one of SEQ ID NOs: 5 to 8 or an antibody against the protein for testing for human pituitary gland or human joint or treating human pituitary gland-related disease or human joint-related disease. .  7. A diagnostic agent for a human pituitary or human joint-related autoimmune disease comprising a protein having any one of SEQ ID NOs: 5 to 8 as an antigen.  8. The diagnostic agent according to claim 7, wherein the antigen is labeled.  9. The diagnostic agent according to claim 7 or 8, wherein the autoimmune disease is autoimmune pituititis or rheumatoid arthritis.  10. A diagnostic agent for human pituitary or human joint function, comprising an antibody against a protein having any of SEQ ID NOs: 5 to 8. II. A therapeutic agent for a human pituitary-related disease or a human joint-related disease, which comprises a protein having any of the sequences of SEQ ID NOS: 5 to 8, and in which low expression of the protein of any of the sequences of SEQ ID NOs: 5 to 8 is a worsening factor. 1 2. A human pituitary-related disease or human joint in which an overexpression of a protein of any of SEQ ID NOs: 5 to 8 is a worsening factor, comprising an antibody against a protein of any of SEQ ID NOs: 5 to 8 Drugs for related diseases.