JP2003210185A - Exocrine gland tight junction-constituting protein jeap family - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new exocrine gland TJ (tight junction)-constituting protein. <P>SOLUTION: A mouse cDNA library from gene fragments encoding proteins localizing at cell - cell junctions is screened by a technique visualizing localization of a protein to identify a junction-enriched and -associated protein (JEAP). GenBank homology search was performed based on the sequence. Based on the obtained sequence, a mouse cDNA library is screened to identify JEAP-2. By using prepared antibodies against these proteins, it is revealed that these proteins express specifically at tight junctions, in particular, tight junctions in exocrine glands. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel constituent protein of a tight junction (hereinafter referred to as TJ) of an exocrine gland and a method of using the same.

[0002]

2. Description of the Related Art In multicellular animals, information on adhesion with adjacent cells is deeply related to the regulation and maintenance of life phenomena such as cell proliferation, differentiation, inflammation and cancer metastasis. Intercellular adhesion molecules that are involved in adhesion often assemble at the cell surface to create specially differentiated membrane regions for adhesion. In particular, in epithelial cells, it is known that intercellular adhesion molecules such as cadherin are strongly bound to the cytoskeleton through their cytoplasmic domain.

Such a membrane region is called an intercellular adhesion device and is mainly classified into the following four types. That is,
Gap junction (GJ), adhesive bonding (adher
ensjunction (AJ), desmosome and tight junction (TJ).

TJ is one of the intercellular adhesion devices in the epithelial or endothelial cell layer. It acts as a physical barrier (barrier function) that prevents solutes and water from freely passing through the extracellular space and constitutes a continuous peripheral seal surrounding the cells. Further, TJ is considered to play a role of forming and maintaining cell polarity as a boundary between the apical plasma membrane region and the basolateral plasma membrane region (barrier function).

TJ is a membrane protein molecule such as claudin, occludin, JAM and ZO-1, -2,
-3, cingulin, 7H6, symplekin
kin), Rab3B, Sec6 / Sec8 homolog, ASIP / PAR-3, PAR-6,
It is composed of proteins directly under the cell membrane such as MAGI-1. Claudin and occludin form the skeleton of the TJ strand and are involved in the barrier function of TJ.

[0006] JAM is involved in cell-cell adhesion and adhesion formation of endothelial cells and epithelial cells, as well as chemokine-induced infiltration of monocytes through intercellular spaces of endothelial cells. ZO-1,
-2 and -3 are scaffold proteins containing the PDZ region, which directly bind to TJ
Binds to claudin and occludin on the cytoplasmic surface of. ZO-1, -2 and -3 bind to F-actin,
It is believed to control TJ function by linking the TJ strand to the actin cytoskeleton. Some PDZ domain-containing proteins localized in other TJs are thought to function as markers for transporting cytoskeletal proteins and proteins involved in signal transduction to TJ strands. The F-actin non-binding scaffold protein MAGI-1 / 2/3 is T
It localizes to J and interacts with signaling molecules such as the tumor suppressor gene product PTEN and the Rap small G protein GDP / GTP exchange protein. ASIP / PAR-3 and PAR-6 are cell polarity-related molecules containing a PDZ region, and interact with atypical PKC. ASIP / PAR-3 also interacts with JAM. T
PDZ among the proteins localized under the cell membrane of J-strand
For region-less proteins, Rab3B, a Sec6 / Sec8 homolog, is involved in vesicle transport. In addition, chingulin, 7H6 antigen, and symplekin are known to be localized in TJ, but their functions have not been clarified. Tingling interacts with ZO-1, -2, -3, occludin, AF-6, JAM.

[0007] In order to more clearly understand the molecular system of TJ, identification and provision of a new constituent protein of TJ are desired.

On the other hand, as a gene whose function is unknown, mouse cD
NA clone (GenBank accession number BAB30287)
Is reported by RIKEN (see Non-Patent Document 1).

[0009]

[Non-Patent Document 1] GenBank, accession number BAB30287

[0010]

It is an object of the present invention to provide a new constituent protein of exocrine gland TJ.

[0011]

[Means for Solving the Problems] In order to clarify the molecular mechanism of cell-cell adhesion in detail, the present inventors have conducted a cloning method using localization of a fluorescent labeled protein as an index.
We attempted to identify a novel protein localized at the cell-cell adhesion site.

First, a cDNA fragment derived from mouse vascular endothelial cell line MS-1 cells was expressed as a fusion protein with a GFP protein, the localization of the fluorescent labeled protein in the cell was observed, and it was localized at the intercellular adhesion site. The cDNA fragment encoding the existing gene product was isolated. A full-length cDNA clone was then isolated from the MS-1 cell cDNA library.

Further, as a result of homology search from GenBank / EMBL / DDBJ, one kind of cDNA clone of unknown function was found, and using this sequence, full-length cDNA was extracted from mouse MS-1 cell cDNA library. Isolated. Structural analysis of these two interrelated cDNAs was performed. Encodes these proteins
The cDNA was introduced into MDCK cells (Madin-Darby canine kidney cells), MDCK cells stably expressing the protein were used, and the intracellular distribution of these proteins was subjected to immunostaining with a specific antibody,
When it was examined by microscopic observation, it was confirmed that it existed specifically in TJ, and they were named JEAP and JEAP-2, respectively.

As a result of examining the tissue distribution of JEAP by immunohistochemical staining, JEAP was found to be found in the pancreas, submandibular gland, lacrimal gland, parotid gland,
Expression was observed in the exocrine glands such as the sublingual gland, but not in the brain, heart, liver, kidney, spleen, gallbladder, and small intestine. In the exocrine gland, JEAP was expressed at the terminal part of the serous gland, and showed a stained image similar to that of ZO-1.
As a result of immunoelectron microscopic observation, in the lacrimal gland, JEAP was actually present in TJ, but not in AJ or desmosome. JEAP-2 was also expressed in exocrine glands such as the pancreas, submandibular gland, and lacrimal gland, but not in the small intestine.

From the above results, it was found that JEAP and JEAP-2 form a gene family constituting TJ of the exocrine gland, and the present invention has been completed.

That is, the present invention relates to the following: 1. The following protein (a) or (b) (excluding the protein having the amino acid sequence shown in SEQ ID NO: 2): (A) A protein having the amino acid sequence shown in SEQ ID NO: 4. (B) A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 and localized at a tight junction.

DNA encoding the protein described in 2.1
(However, the DNA having the nucleotide sequence shown in SEQ ID NO: 1 is excluded).

3. 2 having the base sequence shown in SEQ ID NO: 3
DNA described in.

4. DNA of the following (a) or (b) (excluding the DNA having the nucleotide sequence shown in SEQ ID NO: 1). (A) A DNA having the base sequence shown in SEQ ID NO: 3. (B) a DNA that hybridizes with a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions and encodes a protein localized at a tight junction.

5. DNA of the following (a) or (b) (excluding the DNA having the nucleotide sequence shown in SEQ ID NO: 1). (A) A DNA having the base sequence shown in SEQ ID NO: 3. (B) A DNA encoding a protein having a nucleotide sequence having a homology of 90% or more with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and localized at a tight junction.

A protein encoded by the DNA according to any one of 6.4 to 5.

A recombinant vector containing the DNA according to any one of 7.2 to 5.

A transformant obtained by transforming a host with the DNA according to any one of 8.2 to 5.

Transformant described in 9.8 or SEQ ID NO: 1
Culturing a transformant obtained by transforming a host with a DNA having the nucleotide sequence shown in, and collecting the protein localized in the tight junction expressed by the transformant from the culture, A method for producing a localized protein.

An antibody which reacts with the protein described in 10.1.

A method for measuring the protein according to 1, characterized in that the antibody according to 11.10 is used.

12. A DNA or RNA encoding the protein according to 1, which comprises using an oligonucleotide having at least 15 consecutive base sequences of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 as a primer or a probe. How to measure.

13. (1) a step of mixing the test substance with the protein described in 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2, or a partial peptide thereof, and
(2) the step of measuring the amount of the test substance bound or not bound to the protein or partial peptide, and reacting with the protein according to 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2. A method for screening a substance having:

14. (1) A step of culturing cells expressing the protein described in 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2 in the presence of a test substance, and (2)
The protein according to 1, comprising the protein described in 1 expressed in cells or a protein having the amino acid sequence shown in SEQ ID NO: 2 or the step of measuring the mRNA encoding the protein in the cell. Alternatively, a method of screening a substance that affects the expression of a protein having the amino acid sequence shown in SEQ ID NO: 2.

15. (1) a step of identifying a promoter region that controls the expression of the protein described in 1 or a protein having the amino acid sequence shown in SEQ ID NO: 2, and (2) a step of measuring the effect of a test substance on the promoter activity,
A method for screening a substance which affects the expression of the protein according to 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2, which comprises:

16. (1) A step of culturing cells expressing the protein described in 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2 in the presence of a test substance, and (2)
Measuring the distribution of the protein described in 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2.
A method for screening a substance that affects the distribution of the protein described in 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2.

[0032]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
For convenience of explanation, the protein revealed by the present inventors to be localized in tight junction is
The protein having the amino acid sequence shown in SEQ ID NO: 2 is referred to as the “protein of the present invention”.

Among the proteins of the present invention, the protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 is a protein identified as a protein localized in tight junctions, as described in Examples below. . It is predicted that there will be variants with the same function in the protein, and
By appropriately modifying the amino acid sequence of the protein, a mutant having the same function can be obtained. Therefore,
A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 and localized at a tight junction is also included in the protein of the present invention. .

The modification of the amino acid sequence of the protein can be carried out by modifying the base sequence of the DNA encoding the protein by well-known means such as site-directed mutagenesis and expressing the modified DNA of the base sequence. it can. Localization at a tight junction can be easily confirmed by those skilled in the art by a fluorescent antibody method, immunoelectron microscopy, or the like.

The protein of the present invention may be made into a fusion protein by fusing it with another protein such as glutathione transferase (GST) or His tag.

The DNA of the present invention is a DNA encoding the protein of the present invention. Examples of the DNA of the present invention include DNAs having the nucleotide sequences shown in SEQ ID NO: 1 or SEQ ID NO: 3. This DNA is the DNA whose nucleotide sequence was determined in the examples described below. Genes that encode the same product but have different base sequences or genes that encode mutants that have the same function are predicted to exist, and by modifying the base sequence, the same product or the same function can be obtained. It is possible to obtain a gene encoding a mutant having Therefore,
The DNA of the present invention also includes a DNA having a base sequence similar to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and encoding a protein localized at a tight junction. As a DNA having a similar nucleotide sequence, SEQ ID NO: 1
Alternatively, a DNA that hybridizes with a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 3 under stringent conditions, or a base having a homology of 90% or more with the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. An example is a DNA having a sequence.

Here, the stringent conditions are, for example, hybridization at 65 ° C. 4 × SSC, and then washing at 65 ° C. for 1 hour in 0.1 × SSC. Alternatively, stringent conditions are 42% in 50% formamide.
℃ 4xSSC. Also, in PerfectHyb ^™ (TOYOBO) solution
Hybridization at 65 ℃ for 2.5 hours, then 1). 2xSS
C, 0.05% SDS solution: 25 ℃ for 5 minutes, 2). 2xSSC, 0.05% SDS solution: 25 ℃ for 15 minutes, 3). 0.1xSSC, 0.1% SDS solution: 50 ℃ for 20 minutes.

The homology is the homology calculated by the ClustalW method.

The fact that the DNA encodes a protein localized at a tight junction means that the DNA is expressed in an appropriate cell capable of forming a tight junction, and that the expressed protein is present at the tight junction, as described above. It can be easily confirmed by confirming with a fluorescent antibody method or immunoelectron microscopy.

The DNA of the present invention can be used for gene analysis of JEAP family proteins and gene expression analysis by using a part of the DNA as a primer or a probe. The term "part" means that the oligonucleotide used as a primer or probe contains at least 15 consecutive corresponding base sequences based on the DNA sequence of the present invention, preferably at least 20 base sequences, and It preferably means the corresponding polynucleotide consisting of those containing at least about 20 to 30 base sequences. In addition, as the probe, higher molecular
DNA can also be used.

The DNA of the present invention can be obtained by a conventional method based on the revealed nucleotide sequence. For example, it may be synthesized by a chemical synthesis method, or by using an appropriately set primer, an RT-PCR method from mRNA prepared from cells or tissues expressing the protein localized at the tight junction of the present invention. May be obtained by

Gene manipulation methods are described in the manuscript (Maniatis et al., Mo.
lecular Cloing: A Laboratory Manual, Cold Spring Ha
rbor Laboratory, 1989).

The vector of the present invention is a recombinant vector containing the DNA of the present invention. The vector of the present invention can be obtained by inserting the DNA of the present invention into a vector such as an expression vector.

The transformant of the present invention is a transformant obtained by transforming a host with the DNA of the present invention, and expresses the protein of the present invention. The host is not particularly limited and includes animal cells, bacterial cells, yeast cells, insect cells and the like. Transformation may be performed by a conventional method. For example,
Introducing into the host the vector of the present invention that expresses the DNA of the present invention in the host.

The production method of the present invention is a method for producing a protein localized in a tight junction of the present invention, which comprises culturing the transformant of the present invention, and expressing the protein localized in a tight junction expressed by the transformant. From the culture.

Cultivation may be carried out under the condition that the transformant expresses the protein of the present invention, and the protein of the present invention can be collected from the culture by various chromatographic and electrophoretic methods commonly used for protein purification. It may be carried out by appropriately combining methods such as electrophoresis and gel filtration. The protein of the present invention is added to GST or His.
When expressed as a fusion protein with a tag, it can be purified using a glutathione sepharose column or a nickel sepharose column, respectively.

All or part of the protein of the present invention can be used as an epitope in the preparation of antibodies, and can also be used in research and diagnostic reagents using the antibodies. Epitope means an antigenic determinant of a polypeptide, generally composed of at least 6 amino acids and
It is known that a polypeptide composed of individual amino acids binds to an antibody (Published Patent Publication No. 60-500684). Based on the amino acid sequence of the present invention, the antigenic peptide of the present protein comprises at least 6 continuous amino acids, preferably at least 8 continuous amino acids, and more preferably at least about 15 continuous amino acids. By amino acid, and more preferably, is meant a polypeptide consisting of at least about 20 consecutive amino acids.

The antibody of the present invention can be obtained from an animal immunized with the protein of the present invention obtained by the production method of the present invention or an antigenic peptide thereof. In the case of a polyclonal antibody, the antibody-producing cells obtained from the sera of the above-mentioned immunized animal are fused with the myeloma cells, and in the case of a monoclonal antibody, the antibody-producing cells obtained from the spleen or lymph node of the above-mentioned immunized animal are fused with myeloma cells, and show a strong specificity for the protein of the present invention It is prepared by selecting a hybridoma that produces an antibody.

As the immunogen, the protein of the present invention obtained by the production method of the present invention can be used. Alternatively, the immunogen is SEQ ID NO: 2 or SEQ ID NO: 4.
It may be a fragment or peptide having a partial structure appropriately selected from the amino acid sequences shown in. Various condensing agents can be used to prepare the complex of the antigen and the carrier protein, and glutaraldehyde, carbodiimide, maleimide active ester and the like can be used. The carrier protein may be a commonly used carrier protein such as bovine serum albumin, thyroglobulin, hemocyanin, etc., and a method of coupling at a ratio of 1 to 5 times is usually used.

Animals to be immunized include mice, rats,
Rabbits, guinea pigs, hamsters and the like can be mentioned, and the inoculation method includes subcutaneous, intramuscular or intraperitoneal administration. Upon administration, it may be mixed with complete Freund's adjuvant or incomplete Freund's adjuvant,
Administration is usually once every 2 to 5 weeks. In the case of a monoclonal antibody, antibody-producing cells obtained from the spleen or lymph node of an immunized animal are fused with myeloma cells and isolated as a hybridoma. As the myeloma cells, those derived from mice, rats, humans, etc. are used,
It is preferably derived from the same species as the antibody-producing cells, but it may be possible between different species.

The operation of cell fusion is known, for example, the method of Kohler and Milstein (Nature, 256, 495, 197).
It can be carried out according to 5). Examples of fusion promoters include polyethylene glycol and Sendai virus,
Usually, polyethylene glycol (average molecular weight 1000-4000) with a concentration of about 20-50% is used at 20-40 ° C, preferably 30
The cell fusion can be carried out by reacting at a temperature of ~ 37 ° C with a ratio of the number of antibody-producing cells to the number of myeloma cells of usually about 1: 1 to 10: 1 for about 1 to 10 minutes.

Various immunochemical methods can be used for screening antibody-producing hybridomas. For example, EL using a microplate coated with the protein of the present invention
ISA (Enzyme-linked immunosorbent assay) method, using a microplate coated with anti-immunoglobulin antibody
Examples include EIA (Enzyme immunoassay) method, immunoblotting method using a nitrocellulose transfer membrane after electrophoresis of a sample containing the protein of the present invention.

From such wells, cloning is further performed by, for example, the limiting dilution method to obtain clones. Selection and breeding of hybridomas are usually performed in an animal cell culture medium (eg, RPMI1640) containing 10 to 20% fetal bovine serum with the addition of HAT (hypoxanthine, aminopterin, thymidine). The clones thus obtained were transplanted into the peritoneal cavity of SCID mice pre-administered pristane,
Ascites fluid containing a high concentration of monoclonal antibody can be collected after a day and used as a raw material for antibody purification. Alternatively, the clone can be cultured and the culture can be used as a raw material for antibody purification. The monoclonal antibody may be collected by a known method for immunoglobulin purification, for example,
It can be easily achieved by means such as ammonium sulfate fractionation method, PEG fractionation method, ethanol fractionation method, use of anion exchanger, and affinity chromatography.

The protein of the present invention can be measured by using the antibody of the present invention. The measurement may be performed according to a usual measuring method using an antibody, for example, qualitative determination of the protein of the present invention in a biological sample by an immunological method using the antibody of the present invention (preferably a monoclonal antibody),
Quantitation can be done. As an immunological method, a biological sample is appropriately treated as necessary, for example, separation of cells,
Known methods such as an immunohistological staining method, an enzyme immunoassay method, an agglutination method, a competition method, and a sandwich method can be applied to the sample subjected to the extraction operation. The immunohistological staining method can be performed by, for example, a direct method using a labeled antibody, an indirect method using a labeled antibody against the antibody, or the like.
Fluorescent substances, radioactive substances, enzymes, metals,
Any known labeling substance such as a dye can be used.

As the antibody of the present invention, Fc ′ or Fab ′ or Fab fraction from which the Fc region has been removed, or a polymer thereof may be used. Further, it may be a chimeric antibody or a humanized antibody thereof.

Screening for substances (such as drugs) such as compounds that influence the expression of the protein of the present invention is carried out by culturing cells expressing the protein of the present invention in the presence of a test substance, and then culturing the cells. It can be carried out by measuring the protein of the present invention expressed in, or mRNA encoding the protein.

Cell lines expressing the protein of the present invention are:
It can be selected by Northern blotting, RT-PCR, etc.
Alternatively, the antibody obtained by the method described above may be used for selection by a fluorescent antibody method, an enzyme antibody method, or the like.

The selected cells are cultured in the presence of a test substance (for example, the test substance is added to the cells and cultured), and mRNA is expressed.
The expression level is quantified by Northern blotting, slot blot hybridization, RT-PCR, or the like, or the protein expression level is quantified by the fluorescent antibody method, enzyme antibody method, or the like to measure the effect of the test drug on the expression of the protein of the present invention. For example, it can be measured according to the methods described in Examples 3 and 6.

Further, in order to more easily screen a large amount of substances, a promoter region which controls the expression of the protein of the present invention may be identified and the influence of the test substance on the promoter activity may be measured.

Identification of the promoter region and measurement of the influence on the promoter activity can be carried out, for example, as follows. From the human DNA library, a clone that hybridizes with the cDNA 5'region of the protein of the present invention is selected and inserted into an appropriate promoter screening system to select a clone having a promoter activity. In some cases, a region essential for promoter activity may be narrowed down here.

A DNA having a promoter region of the protein of the present invention selected here is inserted upstream of a DNA encoding an enzyme whose activity can be easily measured, for example, luciferase / alkaline phosphatase, to construct a reporter gene. After introducing this reporter gene into cells that can be cultured together with an appropriate resistance gene such as Neo ^r and hyg ^r , for example, HeLa cells, etc., the activity of the promoter that expresses the protein of the present invention is selected by a drug corresponding to the resistance gene Establish a cell line that can measure The activity of the enzyme introduced by acting a drug on this cell line is measured, and the drug that affects the expression of the protein of the present invention is screened.

As another screening system, it can be used to select a substance that affects the intracellular localization of the protein of the present invention. This screening can be carried out by culturing cells expressing the protein of the present invention in the presence of a test substance and measuring the distribution of the protein of the present invention in the cells.

As an example, the cell line expressing the protein of the present invention or the transformant of the present invention is stained with the antibody obtained by the above-mentioned method by a fluorescent antibody method, an enzyme antibody method or the like. The effect of the test drug on the intracellular localization of the protein of the present invention can be measured by observing under a microscope. For example, it can be carried out according to the methods described in Examples 3 and 6.

Further, as another screening system, it can be used for selection of a substance that binds to the protein of the present invention (that is, a substance having reactivity with the protein of the present invention). This screening can be carried out by mixing the protein of the present invention or its partial peptide with a test substance, and measuring the amount of the test substance bound or not bound to the protein or partial peptide.

In this system, a substance reactive with the protein of the present invention is considered to bind to a part of the protein of the present invention, so a partial peptide of the protein of the present invention can be used. Therefore, the partial peptide only needs to be a portion sufficient for binding to the partial peptide to be equivalent to binding to the protein of the present invention.

The protein of the present invention may be in the state of being expressed on the cell membrane of the transformant of the present invention. Therefore, mixing the protein of the present invention with the test substance also includes mixing the transformant of the present invention or its cell membrane with the test substance.

The amount of the test substance bound or not bound to the protein of the present invention or its partial peptide is measured by reacting the protein of the present invention with the test substance under appropriate conditions. The presence or absence of can be measured. The measuring method can be measured, for example, by appropriately using a labeling substance.

Since a substance having reactivity has a possibility of affecting the function of the protein of the present invention, and as shown in Examples, the exocrine gland specificity is observed in the distribution of the protein of the present invention, Any substance having specificity for the binding ability to the protein of the present invention may exhibit organ-specific action.

Although the JEAP family of which the structure of the present invention has been analyzed is of mouse origin, the JEAP family gene analysis method and screening method of the present invention are of human origin.
The use of families is also within the scope of the invention.

[0070]

The present invention will be specifically described with reference to the following examples, but the present invention is not limited thereto. The antibodies and cells used in the examples were as follows. The rat anti-mouse JEAP monoclonal antibody immunizes a rat with a GST-JEAP fusion protein containing the amino acid sequence 808-882 of JEAP (amino acid number in SEQ ID NO: 2), and the rat anti-mouse JEAP-2 monoclonal antibody is JEAP-2. Rats were immunized with the GST-JEAP-2 fusion protein containing the amino acid sequence 726-772 (amino acid number in SEQ ID NO: 4) of SEQ ID NO. Rabbit anti-ZO-1 polyclonal antibody,
Rabbit anti-claudin polyclonal antibody, mouse anti-Z
O-1 monoclonal antibody and mouse anti-occludin monoclonal antibody were purchased from Zymed. Mouse anti-E-cadherin monoclonal antibody was purchased from Takara Shuzo.

MDCK cells (Madin-Darby canine kidney cells) are 10%
The cells were grown in DMEM culture medium containing fetal bovine serum. Mouse vascular endothelial cell line MS-1 was purchased from ATCC and used in DMEM / 5% FC
Cultured in S.

[0072]

Example 1 Identification of cDNA Encoding Protein Localized at Intercellular Adhesion Site To clarify the molecular mechanism of intercellular adhesion in detail, Kitamura et al. (Misawa, K. et al., Proc. Natl. Acad. Sci. USA
97, 3062-3066, 2000) to identify a novel cDNA encoding a protein localized at the cell-cell adhesion site by applying the cloning method using localization of fluorescently labeled protein as an index (Fig. 1). Tried.

First, a preliminary experiment was carried out in which various recombinant cell lines were infected with an expression recombinant retrovirus expressing a fusion protein of VE-cadherin localized in cell-cell adhesion and a fluorescent protein EGFP. Observed, however, no cell line showing a clear fluorescent signal with the naked eye was present as far as examined. Therefore, we tried to make MDCK cells with high cell length and clear cell-cell adhesion site infectable with ecotropic retrovirus, and to use them in experiments. MDCK cells capable of infecting ecotropic retrovirus (MDCK /
EcoVR) was obtained as follows. The ecotropic virus receptor (EcoVR) cDNA was inserted into pCAGGS-puro and the lipofectamine reagent (Life Techn
(introduced by ologies) was used to introduce into MDCK cells. 24 hours later,
Culture was started in the presence of 5 μg / ml puromycin, and resistant clones were selected. Isolate each clone, pMXII-EG
Infectious clones were obtained by infecting FP expressing recombinant retrovirus. EcoVR cDNA and pMX expression vector were kindly provided by Dr. Toshio Kitamura. Infection of MDCK / EcoVR cells with a recombinant retrovirus expressing VE-cadherin-EGFP showed a bright fluorescent signal, making this cell line suitable for visual screening of proteins localized at cell-cell adhesion sites. I understood that.

The cDNA-GFP fusion library was prepared from the mouse endothelial cell line MS-1 based on the method of Kitamura et al. The result was an average length of 1,500 with 3 x 10 ⁵ independent clones.
A bp library was obtained. The expression library was transferred to the 293 / EBNA-1 cells (Invitrogen) together with the viral packaging vector pCL-Eco (Imgenex) into the TransIT LT1 (M
irus) was used to prepare a recombinant retrovirus. MDCK / EcoVR cells were infected with various concentrations of diluted retrovirus to obtain individually infected cells. As a result of FACS, the frequency of EGFP-positive cells after infection was about 4%. After culturing, EGFP-positive cells were separated with a cell sorter, and cultivated so that each well of the 96-well plate had 50 cells.
When the cells became confluent, 10 plates were screened by a fluorescence microscope, and 6 wells containing cells in which EGFP fluorescence was observed specifically at the intercellular adhesion site were selected. These cells were re-plated in a 10-cm dish to obtain a single clone. Each clone has 24
Culture in a well plate and PCR from genomic DNA
The cDNA fragment integrated into the chromosome was recovered. as a result,
From the cDNA fragments encoding proteins showing a specific staining pattern at the intercellular adhesion site, we obtained cDNA fragments encoding proteins not known to be localized at the intercellular adhesion site.

[0075]

[Example 2] Cloning of full-length cDNA encoding mouse JEAP and JEAP-2 Using the cDNA fragment obtained in Example 1 as a probe, mouse
CDNA was cloned from a cDNA library derived from MS-1 cells. For cloning of cDNA, total RNA of mouse MS-1 cells was extracted using Trizol (Life Technologies), and poly A (+) RNA was prepared using oligo dT beads (Miltenyi). . The cDNA library is a superscript based on this poly A (+) RNA.
(Superscript) II cDNA Synthesis Kit (Life Technologies
(Manufactured by K.K.) and pMXII vector, and prepared for cDNA screening. The nucleotide sequence is based on the Dye Terminater Cycle Sequence Kit.
t) (manufactured by Applied Biosystems). Details of cloning are shown in (1) below. The obtained cDN
The protein encoded by A was named JEAP.

A human cDNA clone KIAA09 was obtained by homology search using the amino acid sequence deduced from the JEAP cDNA.
89 were identified as homologous over the entire length. This clone has been reported by Kazusa DNA Research Laboratories as a gene of unknown function. Furthermore, by homology search using the amino acid sequence deduced from KIAA0989 cDNA, mouse EST clone (BF536192) and mouse cDNA clone (AF17
5967, AF175968) were identified as partially homologous. Using these nucleotide sequences, mouse MS-1 cell cDNA
Another cDNA was isolated from NA. Single-stranded cDNA is Superscript II reverse transcriptase (Life Technologies) based on poly A (+) RNA of MS-1 cells.
And prepared for PCR. The base sequence was determined using a dye terminator cycle sequence kit (manufactured by Applied Biosystems). Details of cloning are shown in (2) below. The protein encoded by the obtained cDNA was named JEAP-2.

(1) Determination of JEAP cDNA Using the cDNA fragment obtained in Example 1 as a template, a DIG-labeled probe was prepared by PCR and used for screening a pMXII MS-1 cell cDNA library. As a result, a plurality of positive clones were obtained and the nucleotide sequences of clones # 2 and # 5 were determined. The nucleotide sequence of the portion containing all ORFs was determined and the structure was analyzed.
Encodes a 882 amino acid polypeptide with d
It contained the ORF (SEQ ID NO: 1). JEAP contained a polyglutamic acid repeat sequence in the N-terminal region, a coiled-coil region in the intermediate region, and a sequence containing a consensus motif for binding to the PDZ region at the C-terminus (Fig. 2). .

Mouse cDNA clone identical to JEAP (BAB302
87) has been reported by RIKEN as a gene whose function is unknown.

(2) Determination of cDNA of JEAP-2 As a result of structural correlation search by Genbank / EMBL / DDBJ database, human cDNA clone KIAA0989 was identified as having homology with JEAP over the entire length. Furthermore, a homology search using an amino acid sequence deduced from the KIAA0989 cDNA identified that the mouse EST clone (BF536192) and the mouse cDNA clones (AF175967, AF175968) have partial homology. Therefore, using the primers of the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 designed based on these mouse sequences, cDNA containing all ORFs from MS-1 cell cDNA
Was amplified by the PCR method. pSPORT1 vector (Life tec
sub-cloning using hnology).
It was named SPORT-JEAP-2. As a result of nucleotide sequence analysis, JEAP-2
CDNA contained an ORF encoding a 772 amino acid polypeptide having a predicted molecular weight of 85.3 kd (SEQ ID NO: 3). The JEAP-2 polypeptides showed JEAP sequence similarity (42% identity at the amino acid sequence level). Furthermore, as a result of the structural analysis, it was speculated that JEAP-2 contains a sequence containing a consensus motif for binding to the coiled coil region in the intermediate region and the PDZ region at the C-terminal, as in JEAP.

[0080]

Example 3 Preparation of Monoclonal Antibody Against Mouse JEAP and JEAP-2 Preparation of a monoclonal antibody against mouse JEAP was carried out as follows. First, the clone # obtained in Example 2
The region encoding the amino acid sequence 808-882 of JEAP (amino acid number in SEQ ID NO: 2) was amplified by PCR using 2 as a template and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8 as a primer. The recombinant protein was expressed by introducing it into a fusion protein expression vector (manufactured by Amersham) and an MBP fusion protein expression vector (manufactured by NEB). The GST-JEAP fusion protein was mixed with an adjuvant to immunize WKY rats. Lymphocytes were separated from the immunized rat, P3 myeloma cells and lymphocytes were mixed at a ratio of 1: 5, and cell fusion was performed using a PEG1,500 solution (Boehringer) to prepare hybridomas. The prepared hybridoma was cultured in a 96-well plate for one week, and the positive well was identified by ELISA using a culture supernatant in which an MBP-JEAP fusion protein-immobilized ELISA plate was used. The hybridoma contained in the positive well was cloned to finally obtain a rat anti-mouse JEAP monoclonal antibody.

The rat anti-mouse JEAP-2 monoclonal antibody was prepared in the same manner as the JEAP monoclonal antibody.
By using pSPORT-JEAP-2 obtained in Example 2 as a template and PCR with the oligonucleotides having the nucleotide sequences of SEQ ID NO: 6 and SEQ ID NO: 9 as primers, the amino acid sequence 726-772 of JEAP-2 (SEQ ID NO: 4 GST fusion protein expression vector
(Manufactured by Amersham) and MBP fusion protein expression vector (manufactured by NEB) to prepare a recombinant protein. GST
-JEAP-2 fusion protein was mixed with an adjuvant to immunize WKY rats. Lymphocytes were separated from the immunized rat, P3 myeloma cells and lymphocytes were mixed at a ratio of 1: 5, and cell fusion was performed using a PEG1,500 solution (Boehringer) to prepare hybridomas. The prepared hybridoma was cultured in a 96-well plate for 1 week, and the positive well was identified by ELISA using the ELISA plate in which the culture supernatant was immobilized with MBP-JEAP-2 fusion protein. The hybridoma contained in the positive well was cloned to finally obtain a rat anti-mouse JEAP-2 monoclonal antibody.

[0082]

[Example 4] Expression and intracellular distribution of mouse JEAP and JEAP-2 Using the monoclonal antibody prepared in Example 3,
It was confirmed that the isolated cDNA clone contained all ORFs of JEAP and JEAP-2.

First, the JEAP expression vector (pMXII JEAP IRES
SEQ ID NO: 8 and SEQ ID NO: 1 to generate EGFP)
The entire ORF of JEAP was amplified by PCR using the oligonucleotide having the nucleotide sequence described in 0 as a primer. S of this PCR product
The alI-NotI fragment was introduced into the SalI-NotI site of pMXII IRES EGFP to construct pMXII JEAP IRES EGFP. JEAP-2 expression vector pMXII JEAP-2 IRES EGFP is from pSPORT1-JEAP-2
The SalI-NotI fragment containing the entire ORF of JEAP-2 was excised and pMXII
It was constructed by introducing it into the SalI-NotI site of IRES EGFP.

The expression vector was 293 / EBNA-1 cells (Invitro
gen) was transfected with TransIT LT1 (Mirus). SDS polyacrylamide electrophoresis is Lae
Perform one-dimensional SDS-PAGE according to the method of mmli (1970). For immunoblotting, transfer from gel to nitrocellulose membrane, and then react with antibody, and bound antibody is peroxidase-labeled secondary antibody (Amersham). , ECL (Amersham) was detected as an enzyme substrate.

In the 293 / EBNA-1 cells introduced with the JEAP expression vector, a 105 kDa band was detected by Western blotting using an anti-JEAP monoclonal antibody. Parent's 293
No band was observed in / EBNA-1 cells. The apparent molecular weight of JEAP expressed was consistent with that of JEAP expressed endogenously in MS-1 cells, and thus the isolated cD
It has been clarified that NA includes all JEAP ORFs.
Similarly, in the 293 / EBNA-1 cells into which the JEAP-2 expression vector was introduced, a 105 kDa band was detected by Western blotting using an anti-JEAP-2 monoclonal antibody. Parent's 293
In / EBNA-1 cells, a thin band that was probably derived from endogenous JEAP-2 was observed. The apparent molecular weight of JEAP-2 expressed is JEAP-2 expressed endogenously in 293 / EBNA-1 cells.
Which is identical to that of
It was revealed to include all JEAP-2 ORFs.

Next, in order to examine the expression and distribution of mouse JEAP and JEAP-2, JEAP and JEAP-2 were expressed in MDCK / EcoVR cells and cell staining was performed using a monoclonal antibody.

The expression vector was 293 / EBNA-1 cells together with the virus packaging vector pCL-Eco (Imgenex).
(Invitrogen) was transfected with TransIT LT1 (manufactured by Mirus) to prepare a recombinant retrovirus. Infect MDCK / EcoVR cells with the recombinant virus and
MDCK / EcoVR cells stably expressing AP and JEAP-2 were obtained. Cells expressing JEAP are either GFP fluorescent or anti-JEA.
It confirmed by the fluorescence microscope using P antibody. Cells expressing JEAP-2 were also confirmed.

Next, the intracellular distribution of these proteins was determined by
Confocal microscopy was performed comparing with that of ZO-1 and E-cadherin. As shown in Figure 3, introduced JEAP and JE
Both AP-2 were accurately juxtaposed with ZO-1 at TJ. From the cross-sectional image created by computer, it was revealed that JEAP and JEAP-2 were dense and juxtaposed with ZO-1 at the apical portion of the gene-transferred MDCK cell membrane, but E-cadherin was localized further below. Seemed to be present, indicating that it did not overlap with the distribution of JEAP or JEAP-2.
These findings indicated that both JEAP and JEAP-2 were enriched in TJ.

[0089]

Example 5 Expression of Mouse JEAP and JEAP-2 in In Vivo Tissues Next, the inventors of the present invention examined JEAP and JEAP in various tissues.
The expression of -2 was examined.

First, the tissue distribution of JEAP was examined by detecting the expression of JEAP in various mouse tissues (liver, brain, lung, kidney, spleen, testis, ovary, heart) by Western blot. JEAP was not detected in the tissues. Therefore, the tissue distribution of JEAP was examined by immunohistological staining. Expression of JEAP was observed in exocrine glands such as pancreas, submandibular gland, lacrimal gland (Fig. 4), parotid gland, and sublingual gland, but it was expressed in brain, heart, liver, kidney, spleen, gallbladder, and small intestine. Was not recognized. In the exocrine gland, JEAP
Was found at the terminal part of the serous glands. At the terminal end, JEAP showed a stained image similar to ZO-1. As a result of immunoelectron microscopy in the lacrimal gland, JEAP was actually present in TJ, but not in AJ or desmosome (DS).
(Figure 4).

Next, the tissue distribution of JEAP-2 was examined by immunohistological staining. For JEAP-2, the pancreas, submandibular gland,
Expression was observed in exocrine glands such as the lacrimal gland (Fig. 5), but not in the small intestine.

These findings indicate that JEAP and JEAP-2 have tissue specificity and are localized in large amounts especially in TJ of exocrine glands.

[0093]

Example 6 Transition Kinetics to Intercellular Adhesion Sites Similar to Other TJ Components of JEAP The behavior of JEAP and other components of AJ and TJ during disruption and remodeling of intercellular adhesion was observed. An experiment was performed using MDCK / EcoVR cells stably expressing JEAP prepared in Example 4. It is known that when MDCK cells are cultured for 2 hours in the presence of 2 μM Ca ²⁺ , AJ and TJ are destroyed and the components of AJ and TJ disappear from the vicinity of the cell membrane except ZO-1. There is. Furthermore, from this state, in the presence of 2 mM Ca ²⁺ , 2
AJ and TJ are reconstructed by culturing for a long time, and all AJ and TJ are reconstructed.
It is known that the components of TJ are localized again at the intercellular adhesion site.

In the case of culturing in a normal medium, JEAP is localized at the intercellular adhesion site together with ZO-1, as in Example 4. When cultured for 2 hours in the presence of 2 μM Ca ^2+, the fluorescence indicating the localization of JEAP disappears, but the fluorescence indicating the localization of ZO-1 decreases in intensity, but a part To remain. From this state, when the cells were cultured in the presence of 2 mM Ca ²⁺ for 2 hours, JEAP was accumulated again at the intercellular adhesion site together with ZO-1. Also, after culturing in the presence of 2 μM Ca ²⁺ for 2 hours, 100 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) was added and further culturing for 1 hour, similar to TJ. It is known that the structure is reconstructed, but the AJ is not. Previously TJ
The constituents claudin, occludin, JAM, nectin, ZO-1, and afadin accumulate at structural sites similar to TJ in TPA-induced cell-cell adhesion, but are components of AJ. E-cadherin, α-catenin (ca
tenin) and β-catenin have been shown not to accumulate. JEAP, along with ZO-1, accumulated at TJ-like structural sites of TPA-induced cell-cell adhesion. Similarly, JEAP
Both claudin-1 and occludin accumulated in a structural site similar to TJ in TPA-induced cell-cell adhesion.

These findings indicated that JEAP was incorporated into TJ, along with other TJ components.

[0096]

INDUSTRIAL APPLICABILITY The present invention provides a novel constituent protein of exocrine gland TJ and a screening method for useful substances using this protein.

[0097]

[Sequence list] <110> Eisai Co., Ltd. <120> Exocrine gland tight junction protein JEAP family <130> P-B0275 <150> JP 2001-352241 <151> 2001-11-16 <160> 10 <170> PatentIn Ver. 2.1 <210> 1 <211> 2649 <212> DNA <213> Mus musculus <220> <221> CDS <222> (1) .. (2649) <400> 1 atg aga ggt tct gag gat gta gcc tct gga aga gtg tta cag cgg ctg 48 Met Arg Gly Ser Glu Asp Val Ala Ser Gly Arg Val Leu Gln Arg Leu   1 5 10 15 atc cag gaa caa ctg cgc tat ggc acc cca act gag aac atg aac ctg 96 Ile Gln Glu Gln Leu Arg Tyr Gly Thr Pro Thr Glu Asn Met Asn Leu              20 25 30 ctg gcc att cag cac cag gcc aca ggg agt gca ggg cca gcc cac gcc 144 Leu Ala Ile Gln His Gln Ala Thr Gly Ser Ala Gly Pro Ala His Ala          35 40 45 acc acc aac ttt tct tcc acg gaa acc ctc act caa gaa gat cca caa 192 Thr Thr Asn Phe Ser Ser Thr Glu Thr Leu Thr Gln Glu Asp Pro Gln      50 55 60 atg gtc tat cag tcg gcc cgc caa gaa ccg cag ggt caa gag cat cag 240 Met Val Tyr Gln Ser Ala Arg Gln Glu Pro Gln Gly Gln Glu His Gln  65 70 75 80 gga gac aat acg gtg atg gag aag cag gtc cgg tcc aca cag cct cag 288 Gly Asp Asn Thr Val Met Glu Lys Gln Val Arg Ser Thr Gln Pro Gln                  85 90 95 cag aac aac gag gag ctc ccc acg tat gag gaa gcc aag gcc cag tcc 336 Gln Asn Asn Glu Glu Leu Pro Thr Tyr Glu Glu Ala Lys Ala Gln Ser             100 105 110 cag ttc ttc agg gga cag cag cag cag cag caa cag cag cag cag caa 384 Gln Phe Phe Arg Gly Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln         115 120 125 cag cag cag caa cag cag cag gga cag ggg ccc ctt agc cac act tat 432 Gln Gln Gln Gln Gln Gln Gln Gly Gln Gly Pro Leu Ser His Thr Tyr     130 135 140 tac atg gcc gga ggt acc agt cag aag tcc cgc act gag ggg agg ccc 480 Tyr Met Ala Gly Gly Thr Ser Gln Lys Ser Arg Thr Glu Gly Arg Pro 145 150 155 160 aca gtg aac cgg gcc aac agt gga cag gcg cat aag gat gag gcc ctg 528 Thr Val Asn Arg Ala Asn Ser Gly Gln Ala His Lys Asp Glu Ala Leu                 165 170 175 aaa gaa cta aag cag ggc cat gtc cgg tcc ctc agt gag agg atc atg 576 Lys Glu Leu Lys Gln Gly His Val Arg Ser Leu Ser Glu Arg Ile Met             180 185 190 cag ctg tca ctg gag agg aat ggg gct aag caa cac ctc ccc agc tct 624 Gln Leu Ser Leu Glu Arg Asn Gly Ala Lys Gln His Leu Pro Ser Ser         195 200 205 gga aat gga aag agc ttc aaa gct gga gga gag cca tcc cca gct cag 672 Gly Asn Gly Lys Ser Phe Lys Ala Gly Gly Glu Pro Ser Pro Ala Gln     210 215 220 cct gtc tgt aaa gca ctg gac cct cgt ggc cct cca cct gaa tac ccc 720 Pro Val Cys Lys Ala Leu Asp Pro Arg Gly Pro Pro Pro Glu Tyr Pro 225 230 235 240 ttc aag acc aag cca atg aaa tcc cca gtc agc aag aac caa gat cac 768 Phe Lys Thr Lys Pro Met Lys Ser Pro Val Ser Lys Asn Gln Asp His                 245 250 255 ggt ctt tac tac aat gac cag cac cct ggg gta ctc cat gag atg gtc 816 Gly Leu Tyr Tyr Asn Asp Gln His Pro Gly Val Leu His Glu Met Val             260 265 270 aaa cct tac cca gca cct cag cct gcg aga aca gaa gtg gcc gtc ctg 864 Lys Pro Tyr Pro Ala Pro Gln Pro Ala Arg Thr Glu Val Ala Val Leu         275 280 285 agg tat cag cca ccc ccg gag tat ggc gtc acc agc cgg ccc tgc cag 912 Arg Tyr Gln Pro Pro Pro Glu Tyr Gly Val Thr Ser Arg Pro Cys Gln     290 295 300 ctg cct ttt cca tct acg gtg cag cag cat agc ccc atg tcc tct cag 960 Leu Pro Phe Pro Ser Thr Val Gln Gln His Ser Pro Met Ser Ser Gln 305 310 315 320 acc tcc tcc atc ggt ggt act ctg cac tcc gtc tcc ctg cct ctt cca 1008 Thr Ser Ser Ile Gly Gly Thr Leu His Ser Val Ser Leu Pro Leu Pro                 325 330 335 ctt ccc ata agc ctg gcg gct tca cag ccc cta cca gcc tcc ccc aac 1056 Leu Pro Ile Ser Leu Ala Ala Ser Gln Pro Leu Pro Ala Ser Pro Asn             340 345 350 cag cag ctt gga ccg gat gcc ttt gcg att gtg gag cga gcc cag caa 1104 Gln Gln Leu Gly Pro Asp Ala Phe Ala Ile Val Glu Arg Ala Gln Gln         355 360 365 atg gta gag atc ctg aca gag gag aac cgt gtg ctt cac cag gag ctt 1152 Met Val Glu Ile Leu Thr Glu Glu Asn Arg Val Leu His Gln Glu Leu     370 375 380 cag ggc tgc tat gac aac gct gac aag ctc cac aag ttt gaa aaa gag 1200 Gln Gly Cys Tyr Asp Asn Ala Asp Lys Leu His Lys Phe Glu Lys Glu 385 390 395 400 ctg cag agt att tcg gag gcc tac gag agc ctg gtc aag tcc acc acc 1248 Leu Gln Ser Ile Ser Glu Ala Tyr Glu Ser Leu Val Lys Ser Thr Thr                 405 410 415 aag cgt gag tct ctg gac aag gca atg aga acc aag ctc gaa ggc gag 1296 Lys Arg Glu Ser Leu Asp Lys Ala Met Arg Thr Lys Leu Glu Gly Glu             420 425 430 ata agg aga ctt cat gac ttc aac aga gat ctc cga gat cga ctg gag 1344 Ile Arg Arg Leu His Asp Phe Asn Arg Asp Leu Arg Asp Arg Leu Glu         435 440 445 aca gcc aac agg cag ctg tcc agc agg gaa tac gat ggg cat gaa gac 1392 Thr Ala Asn Arg Gln Leu Ser Ser Arg Glu Tyr Asp Gly His Glu Asp     450 455 460 aaa gct gca gag agc cat tac gtg tcc cag aac aaa gaa ttc ttg aag 1440 Lys Ala Ala Glu Ser His Tyr Val Ser Gln Asn Lys Glu Phe Leu Lys 465 470 475 480 gaa aag gaa aag ttg gaa atg gag ttg gca gca gtg cgt acg gca agt 1488 Glu Lys Glu Lys Leu Glu Met Glu Leu Ala Ala Val Arg Thr Ala Ser                 485 490 495 gag gac cat cgg agg cac atc gag atc ctg gac cag gct ttg agc aat 1536 Glu Asp His Arg Arg His Ile Glu Ile Leu Asp Gln Ala Leu Ser Asn             500 505 510 gcg cag gct aga gtg atc aaa ctg gag gaa gag tta cga gag aag caa 1584 Ala Gln Ala Arg Val Ile Lys Leu Glu Glu Glu Leu Arg Glu Lys Gln         515 520 525 gcc tat gtg gaa aag gtg gag aag ctg cag cag gcc ctg acc cag ctg 1632 Ala Tyr Val Glu Lys Val Glu Lys Leu Gln Gln Ala Leu Thr Gln Leu     530 535 540 cag tca gcg tgc gag aag cga ggg cag atg gaa cgc agg ttg cgg acc 1680 Gln Ser Ala Cys Glu Lys Arg Gly Gln Met Glu Arg Arg Leu Arg Thr 545 550 555 560 tgg ctg gag agg gag cta gac gct ctg agg aca cag cag aaa cat ggc 1728 Trp Leu Glu Arg Glu Leu Asp Ala Leu Arg Thr Gln Gln Lys His Gly                 565 570 575 aca ggc cct cca gtc agt ctc cca gaa tgt aat gct cct gcc ctc atg 1776 Thr Gly Pro Pro Val Ser Leu Pro Glu Cys Asn Ala Pro Ala Leu Met             580 585 590 gag ctg gtg agg gag aag gag gag cgg atc ctc gcc ttg gag gcc gac 1824 Glu Leu Val Arg Glu Lys Glu Glu Arg Ile Leu Ala Leu Glu Ala Asp         595 600 605 atg acc aag tgg gag cag aag tac ctg gaa gag agc acc atc cgg cac 1872 Met Thr Lys Trp Glu Gln Lys Tyr Leu Glu Glu Ser Thr Ile Arg His     610 615 620 ttt gcc atg agc gca gct gcc gct gcc aca gcc gag agg gac acc acc 1920 Phe Ala Met Ser Ala Ala Ala Ala Ala Thr Ala Glu Arg Asp Thr Thr 625 630 635 640 atc agc aac cac tcg agg aat ggc agc tat ggg gag agc tcc ctg gag 1968 Ile Ser Asn His Ser Arg Asn Gly Ser Tyr Gly Glu Ser Ser Leu Glu                 645 650 655 gcc cac atc tgg cca gag gaa gaa gaa gtg gtg cag gcc aac agg agg 2016 Ala His Ile Trp Pro Glu Glu Glu Glu Val Val Gln Ala Asn Arg Arg             660 665 670 tgt cag gac atg gag tac act att aaa aac ctc cat gcc aaa atc ata 2064 Cys Gln Asp Met Glu Tyr Thr Ile Lys Asn Leu His Ala Lys Ile Ile         675 680 685 gag aag gat gcc atg ata aag gtc ctg cag cag cga tcc cgt aag gat 2112 Glu Lys Asp Ala Met Ile Lys Val Leu Gln Gln Arg Ser Arg Lys Asp     690 695 700 gct ggg aag acg gac tct gcc agc ctg agg cct gcc cgc tct gtc cca 2160 Ala Gly Lys Thr Asp Ser Ala Ser Leu Arg Pro Ala Arg Ser Val Pro 705 710 715 720 tcc atc gct gcg gcc act ggg aca cat tct cgc cag act tct ctt acc 2208 Ser Ile Ala Ala Ala Thr Gly Thr His Ser Arg Gln Thr Ser Leu Thr                 725 730 735 agc agc cag ctg aca gaa gag aaa aag gaa gag aag acg acc tgg aaa 2256 Ser Ser Gln Leu Thr Glu Glu Lys Lys Glu Glu Lys Thr Thr Trp Lys             740 745 750 ggg agt ata gga ttc ctg ctg gga aag gaa cac cag gga cag gca tct 2304 Gly Ser Ile Gly Phe Leu Leu Gly Lys Glu His Gln Gly Gln Ala Ser         755 760 765 gcc cct ctg ctg ccg acc aca cct gcc tct gca ttg tcc ctt ccg gcc 2352 Ala Pro Leu Leu Pro Thr Thr Pro Ala Ser Ala Leu Ser Leu Pro Ala     770 775 780 tct acc aca tcg gcc agc agc acc cac gcc aag acg ggc agc aag gac 2400 Ser Thr Thr Ser Ala Ser Ser Thr His Ala Lys Thr Gly Ser Lys Asp 785 790 795 800 agc agc aca cag acc gac aag agc acg gag ctc ttc tgg ccc agc atg 2448 Ser Ser Thr Gln Thr Asp Lys Ser Thr Glu Leu Phe Trp Pro Ser Met                 805 810 815 gct tcc ctc ccc agc cgt ggc agg ctg agc aca gcc cct tcc aac agc 2496 Ala Ser Leu Pro Ser Arg Gly Arg Leu Ser Thr Ala Pro Ser Asn Ser             820 825 830 ccc atc cta aag cat cca gct gcc aaa gga gcc gtg gag aag cag gag 2544 Pro Ile Leu Lys His Pro Ala Ala Lys Gly Ala Val Glu Lys Gln Glu         835 840 845 aac tct cct ggt cat ggg aag gca tcg gag cac aga ggc cga gtc agc 2592 Asn Ser Pro Gly His Gly Lys Ala Ser Glu His Arg Gly Arg Val Ser     850 855 860 aac ttg ctg cac aag cct gag ttc cca gat ggc gag atg atg gaa gtg 2640 Asn Leu Leu His Lys Pro Glu Phe Pro Asp Gly Glu Met Met Glu Val 865 870 875 880 ctc atc tag 2649 Leu Ile <210> 2 <211> 882 <212> PRT <213> Mus musculus <400> 2 Met Arg Gly Ser Glu Asp Val Ala Ser Gly Arg Val Leu Gln Arg Leu   1 5 10 15 Ile Gln Glu Gln Leu Arg Tyr Gly Thr Pro Thr Glu Asn Met Asn Leu              20 25 30 Leu Ala Ile Gln His Gln Ala Thr Gly Ser Ala Gly Pro Ala His Ala          35 40 45 Thr Thr Asn Phe Ser Ser Thr Glu Thr Leu Thr Gln Glu Asp Pro Gln      50 55 60 Met Val Tyr Gln Ser Ala Arg Gln Glu Pro Gln Gly Gln Glu His Gln  65 70 75 80 Gly Asp Asn Thr Val Met Glu Lys Gln Val Arg Ser Thr Gln Pro Gln                  85 90 95 Gln Asn Asn Glu Glu Leu Pro Thr Tyr Glu Glu Ala Lys Ala Gln Ser             100 105 110 Gln Phe Phe Arg Gly Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln         115 120 125 Gln Gln Gln Gln Gln Gln Gln Gly Gln Gly Pro Leu Ser His Thr Tyr     130 135 140 Tyr Met Ala Gly Gly Thr Ser Gln Lys Ser Arg Thr Glu Gly Arg Pro 145 150 155 160 Thr Val Asn Arg Ala Asn Ser Gly Gln Ala His Lys Asp Glu Ala Leu                 165 170 175 Lys Glu Leu Lys Gln Gly His Val Arg Ser Leu Ser Glu Arg Ile Met             180 185 190 Gln Leu Ser Leu Glu Arg Asn Gly Ala Lys Gln His Leu Pro Ser Ser         195 200 205 Gly Asn Gly Lys Ser Phe Lys Ala Gly Gly Glu Pro Ser Pro Ala Gln     210 215 220 Pro Val Cys Lys Ala Leu Asp Pro Arg Gly Pro Pro Pro Glu Tyr Pro 225 230 235 240 Phe Lys Thr Lys Pro Met Lys Ser Pro Val Ser Lys Asn Gln Asp His                 245 250 255 Gly Leu Tyr Tyr Asn Asp Gln His Pro Gly Val Leu His Glu Met Val             260 265 270 Lys Pro Tyr Pro Ala Pro Gln Pro Ala Arg Thr Glu Val Ala Val Leu         275 280 285 Arg Tyr Gln Pro Pro Pro Glu Tyr Gly Val Thr Ser Arg Pro Cys Gln     290 295 300 Leu Pro Phe Pro Ser Thr Val Gln Gln His Ser Pro Met Ser Ser Gln 305 310 315 320 Thr Ser Ser Ile Gly Gly Thr Leu His Ser Val Ser Leu Pro Leu Pro                 325 330 335 Leu Pro Ile Ser Leu Ala Ala Ser Gln Pro Leu Pro Ala Ser Pro Asn             340 345 350 Gln Gln Leu Gly Pro Asp Ala Phe Ala Ile Val Glu Arg Ala Gln Gln         355 360 365 Met Val Glu Ile Leu Thr Glu Glu Asn Arg Val Leu His Gln Glu Leu     370 375 380 Gln Gly Cys Tyr Asp Asn Ala Asp Lys Leu His Lys Phe Glu Lys Glu 385 390 395 400 Leu Gln Ser Ile Ser Glu Ala Tyr Glu Ser Leu Val Lys Ser Thr Thr                 405 410 415 Lys Arg Glu Ser Leu Asp Lys Ala Met Arg Thr Lys Leu Glu Gly Glu             420 425 430 Ile Arg Arg Leu His Asp Phe Asn Arg Asp Leu Arg Asp Arg Leu Glu         435 440 445 Thr Ala Asn Arg Gln Leu Ser Ser Arg Glu Tyr Asp Gly His Glu Asp     450 455 460 Lys Ala Ala Glu Ser His Tyr Val Ser Gln Asn Lys Glu Phe Leu Lys 465 470 475 480 Glu Lys Glu Lys Leu Glu Met Glu Leu Ala Ala Val Arg Thr Ala Ser                 485 490 495 Glu Asp His Arg Arg His Ile Glu Ile Leu Asp Gln Ala Leu Ser Asn             500 505 510 Ala Gln Ala Arg Val Ile Lys Leu Glu Glu Glu Leu Arg Glu Lys Gln         515 520 525 Ala Tyr Val Glu Lys Val Glu Lys Leu Gln Gln Ala Leu Thr Gln Leu     530 535 540 Gln Ser Ala Cys Glu Lys Arg Gly Gln Met Glu Arg Arg Leu Arg Thr 545 550 555 560 Trp Leu Glu Arg Glu Leu Asp Ala Leu Arg Thr Gln Gln Lys His Gly                 565 570 575 Thr Gly Pro Pro Val Ser Leu Pro Glu Cys Asn Ala Pro Ala Leu Met             580 585 590 Glu Leu Val Arg Glu Lys Glu Glu Arg Ile Leu Ala Leu Glu Ala Asp         595 600 605 Met Thr Lys Trp Glu Gln Lys Tyr Leu Glu Glu Ser Thr Ile Arg His     610 615 620 Phe Ala Met Ser Ala Ala Ala Ala Ala Thr Ala Glu Arg Asp Thr Thr 625 630 635 640 Ile Ser Asn His Ser Arg Asn Gly Ser Tyr Gly Glu Ser Ser Leu Glu                 645 650 655 Ala His Ile Trp Pro Glu Glu Glu Glu Val Val Gln Ala Asn Arg Arg             660 665 670 Cys Gln Asp Met Glu Tyr Thr Ile Lys Asn Leu His Ala Lys Ile Ile         675 680 685 Glu Lys Asp Ala Met Ile Lys Val Leu Gln Gln Arg Ser Arg Lys Asp     690 695 700 Ala Gly Lys Thr Asp Ser Ala Ser Leu Arg Pro Ala Arg Ser Val Pro 705 710 715 720 Ser Ile Ala Ala Ala Thr Gly Thr His Ser Arg Gln Thr Ser Leu Thr                 725 730 735 Ser Ser Gln Leu Thr Glu Glu Lys Lys Glu Glu Lys Thr Thr Trp Lys             740 745 750 Gly Ser Ile Gly Phe Leu Leu Gly Lys Glu His Gln Gly Gln Ala Ser         755 760 765 Ala Pro Leu Leu Pro Thr Thr Pro Ala Ser Ala Leu Ser Leu Pro Ala     770 775 780 Ser Thr Thr Ser Ala Ser Ser Thr His Ala Lys Thr Gly Ser Lys Asp 785 790 795 800 Ser Ser Thr Gln Thr Asp Lys Ser Thr Glu Leu Phe Trp Pro Ser Met                 805 810 815 Ala Ser Leu Pro Ser Arg Gly Arg Leu Ser Thr Ala Pro Ser Asn Ser             820 825 830 Pro Ile Leu Lys His Pro Ala Ala Lys Gly Ala Val Glu Lys Gln Glu         835 840 845 Asn Ser Pro Gly His Gly Lys Ala Ser Glu His Arg Gly Arg Val Ser     850 855 860 Asn Leu Leu His Lys Pro Glu Phe Pro Asp Gly Glu Met Met Glu Val 865 870 875 880 Leu Ile <210> 3 <211> 2319 <212> DNA <213> Mus musculus <220> <221> CDS <222> (1) .. (2319) <400> 3 atg agg aca ctg gaa gac tcc tca ggg aca gtc ctg cac cgt ctc atc 48 Met Arg Thr Leu Glu Asp Ser Ser Gly Thr Val Leu His Arg Leu Ile   1 5 10 15 caa gag cag ctg cgc tac ggc aac ctg aca gag act cgc act ctg ctg 96 Gln Glu Gln Leu Arg Tyr Gly Asn Leu Thr Glu Thr Arg Thr Leu Leu              20 25 30 gct atc cag cag cag gcc ctg cgg ggt ggg gct gga gct ggg ggc acg 144 Ala Ile Gln Gln Gln Ala Leu Arg Gly Gly Ala Gly Ala Gly Gly Thr          35 40 45 ggg agc ccc cag gct tcc ttg gag atc gga gca ccc gag gac agt cag 192 Gly Ser Pro Gln Ala Ser Leu Glu Ile Gly Ala Pro Glu Asp Ser Gln      50 55 60 gtg ctg cag caa gcc acc agg cag gag ccc cag ggc cag gag cat cag 240 Val Leu Gln Gln Ala Thr Arg Gln Glu Pro Gln Gly Gln Glu His Gln  65 70 75 80 ggt gga gag acc cac ctg gca gag aac agg ctg tac cgg ctg tgc cca 288 Gly Gly Glu Thr His Leu Ala Glu Asn Arg Leu Tyr Arg Leu Cys Pro                  85 90 95 cag ccc agc aaa gga gaa gag ttg ccc acc tat gag gag gcc aaa gcc 336 Gln Pro Ser Lys Gly Glu Glu Leu Pro Thr Tyr Glu Glu Ala Lys Ala             100 105 110 cat tcg cag tac tac gca gcg cag cag gca ggg tcc cgg ccg cat gtt 384 His Ser Gln Tyr Tyr Ala Ala Gln Gln Ala Gly Ser Arg Pro His Val         115 120 125 ggg gac cgg gat cct aga gga ggg gtg tcc gga ggc ggc cgg cga cag 432 Gly Asp Arg Asp Pro Arg Gly Gly Val Ser Gly Gly Gly Arg Arg Gln     130 135 140 gat gaa gct ctt cga gag ctg agg cat ggc cat gtg cgc tcc ttg agt 480 Asp Glu Ala Leu Arg Glu Leu Arg His Gly His Val Arg Ser Leu Ser 145 150 155 160 gaa cgg ctt ctg caa ctg tcc ctg gaa aga aac ggt gct cgg gtc ccc 528 Glu Arg Leu Leu Gln Leu Ser Leu Glu Arg Asn Gly Ala Arg Val Pro                 165 170 175 agc cac atg agc tct tcc cac agc ttc cct cag ctg gcc cgc agc cag 576 Ser His Met Ser Ser Ser His Ser Phe Pro Gln Leu Ala Arg Ser Gln             180 185 190 cag ggc ccc caa ccc cga ggg ccc cca gct gag ggc cca gag ccc cgc 624 Gln Gly Pro Gln Pro Arg Gly Pro Pro Ala Glu Gly Pro Glu Pro Arg         195 200 205 ggg cca cca cct cag tac cca cac gct gta atg gct cag gag act gcg 672 Gly Pro Pro Pro Gln Tyr Pro His Ala Val Met Ala Gln Glu Thr Ala     210 215 220 gct gtc act gac cca aga tac cga ccc cga agc agc cca cac ttc cag 720 Ala Val Thr Asp Pro Arg Tyr Arg Pro Arg Ser Ser Pro His Phe Gln 225 230 235 240 cat gcc gaa gtc agg atc ctg cag gcc cag gta cca ccg gtg ttc ctc 768 His Ala Glu Val Arg Ile Leu Gln Ala Gln Val Pro Pro Val Phe Leu                 245 250 255 cag cag cag cag tac cag tac ctg cca cag ccc cag gag cac tct cca 816 Gln Gln Gln Gln Tyr Gln Tyr Leu Pro Gln Pro Gln Glu His Ser Pro             260 265 270 ccc ctc cac ccg gca gct ctg ggc cat gga ccc cca agc tcc ttt ggt 864 Pro Leu His Pro Ala Ala Leu Gly His Gly Pro Pro Ser Ser Phe Gly         275 280 285 cca cct gca gtg gag gga cca ccc agt gcc cag gcc acc ttg ggc agt 912 Pro Pro Ala Val Glu Gly Pro Pro Ser Ala Gln Ala Thr Leu Gly Ser     290 295 300 gcc cac ctg gcc cag atg gag act gta ctg agg gag aat gcc agg ctg 960 Ala His Leu Ala Gln Met Glu Thr Val Leu Arg Glu Asn Ala Arg Leu 305 310 315 320 cag agg gac aat gag cga ttg cag aga gag ctg gag agc act tca gag 1008 Gln Arg Asp Asn Glu Arg Leu Gln Arg Glu Leu Glu Ser Thr Ser Glu                 325 330 335 aag gct ggc cgc ata gaa aag ctg gaa aat gaa atc cag cgg ctc tct 1056 Lys Ala Gly Arg Ile Glu Lys Leu Glu Asn Glu Ile Gln Arg Leu Ser             340 345 350 gag gcc cac gag agc ctg atg agg acc tct tcc aag cgt gag gcc ctg 1104 Glu Ala His Glu Ser Leu Met Arg Thr Ser Ser Lys Arg Glu Ala Leu         355 360 365 gag aag acc atg agg aac aag atg gac ggt gag atg aga cgg ttg cag 1152 Glu Lys Thr Met Arg Asn Lys Met Asp Gly Glu Met Arg Arg Leu Gln     370 375 380 gac ttc aac cga gac ctt aga gag aga ttg gaa tcg gca aac cgc cac 1200 Asp Phe Asn Arg Asp Leu Arg Glu Arg Leu Glu Ser Ala Asn Arg His 385 390 395 400 ctg gca agc aag acc cag gaa gcc cag gcg ggc agt cag gac atg gtg 1248 Leu Ala Ser Lys Thr Gln Glu Ala Gln Ala Gly Ser Gln Asp Met Val                 405 410 415 gcg aaa ctg ctt gcc cag agc tat gag cag caa cag gaa cag gag aag 1296 Ala Lys Leu Leu Ala Gln Ser Tyr Glu Gln Gln Gln Glu Gln Glu Lys             420 425 430 ctg gag cgg gag atg gca ctg ctg cgt ggt gcc atc gag gac cag cgg 1344 Leu Glu Arg Glu Met Ala Leu Leu Arg Gly Ala Ile Glu Asp Gln Arg         435 440 445 cga cat gct gaa ctg ctg gag cag gct ctg ggc aat gca caa agc cgt 1392 Arg His Ala Glu Leu Leu Glu Gln Ala Leu Gly Asn Ala Gln Ser Arg     450 455 460 gcc gcc cgg gct gaa gag gag cta cgc aaa aag cag gcc tat gtg gag 1440 Ala Ala Arg Ala Glu Glu Glu Leu Arg Lys Lys Gln Ala Tyr Val Glu 465 470 475 480 aag gtg gag cgg ctg cag cag gca ctg ggg cag ttg cag gct gcc tgt 1488 Lys Val Glu Arg Leu Gln Gln Ala Leu Gly Gln Leu Gln Ala Ala Cys                 485 490 495 gaa aag cga gag cag ttg gag ctg cgt ctg cgc acg cgc ctg gag cag 1536 Glu Lys Arg Glu Gln Leu Glu Leu Arg Leu Arg Thr Arg Leu Glu Gln             500 505 510 gaa ctc aaa gcc ttg cgt gca cag cag agg cag aca ggc acc ctc gca 1584 Glu Leu Lys Ala Leu Arg Ala Gln Gln Arg Gln Thr Gly Thr Leu Ala         515 520 525 ggt ggt ggc ggc agc cat ggt ggg tcc gcc gag ctc agt gcc ctg cgg 1632 Gly Gly Gly Gly Ser His Gly Gly Ser Ala Glu Leu Ser Ala Leu Arg     530 535 540 ctg tct gaa cag ctg cgg gag aag gag gaa cag atc ctg gct cta gag 1680 Leu Ser Glu Gln Leu Arg Glu Lys Glu Glu Gln Ile Leu Ala Leu Glu 545 550 555 560 gcg gac atg acc aag tgg gaa cag aag tat ttg gaa gaa cgg gct atg 1728 Ala Asp Met Thr Lys Trp Glu Gln Lys Tyr Leu Glu Glu Arg Ala Met                 565 570 575 agg cag ttc gcc atg gac gcg gct gcc act gcg gct gcc cag cgc gat 1776 Arg Gln Phe Ala Met Asp Ala Ala Ala Thr Ala Ala Ala Gln Arg Asp             580 585 590 acc act ctc atc cgg cac tcc ccc cag ccc tcg ccc agc agc agt ttc 1824 Thr Thr Leu Ile Arg His Ser Pro Gln Pro Ser Pro Ser Ser Ser Phe         595 600 605 aac gag ggc ctg ctg cca ggc aac cac agg cac cag gag atg gag agc 1872 Asn Glu Gly Leu Leu Pro Gly Asn His Arg His Gln Glu Met Glu Ser     610 615 620 aga ttg aag gtg ctc cat gct cag atc cta gag aag gat gcg gtg atc 1920 Arg Leu Lys Val Leu His Ala Gln Ile Leu Glu Lys Asp Ala Val Ile 625 630 635 640 aag gtc ctt cag cag cgc tcc agg aaa gac cct ggc aag gcc acc cag 1968 Lys Val Leu Gln Gln Arg Ser Arg Lys Asp Pro Gly Lys Ala Thr Gln                 645 650 655 ggc acc cta cgg cct gcc aag tcg gtg ccg tcc atc ttc gcg gct gca 2016 Gly Thr Leu Arg Pro Ala Lys Ser Val Pro Ser Ile Phe Ala Ala Ala             660 665 670 gtg gga act cag ggc tgg caa ggg ctc gta tcc agt gag cgg caa act 2064 Val Gly Thr Gln Gly Trp Gln Gly Leu Val Ser Ser Glu Arg Gln Thr         675 680 685 gat gca cgg cca gcg gga gac cgg gtc cca gca gag gag cct ccg gcc 2112 Asp Ala Arg Pro Ala Gly Asp Arg Val Pro Ala Glu Glu Pro Pro Ala     690 695 700 aca gct cct ctc cct gcc cac acc aaa cat ggc agc aga gac ggg agc 2160 Thr Ala Pro Leu Pro Ala His Thr Lys His Gly Ser Arg Asp Gly Ser 705 710 715 720 acc cag acg gat ggc cct gca gac aac acc tct gcc tgc ttg gcc tca 2208 Thr Gln Thr Asp Gly Pro Ala Asp Asn Thr Ser Ala Cys Leu Ala Ser                 725 730 735 gaa ccc gat ggc ctc ctg ggg tgc aac agt agc cag agg aca ccc tct 2256 Glu Pro Asp Gly Leu Leu Gly Cys Asn Ser Ser Gln Arg Thr Pro Ser             740 745 750 ctg gac tct ata gct gca acc aga gtc cag gat ctg tca gac atg gta 2304 Leu Asp Ser Ile Ala Ala Thr Arg Val Gln Asp Leu Ser Asp Met Val         755 760 765 gaa ata ctg atc tga 2319 Glu Ile Leu Ile     770 <210> 4 <211> 772 <212> PRT <213> Mus musculus <400> 4 Met Arg Thr Leu Glu Asp Ser Ser Gly Thr Val Leu His Arg Leu Ile   1 5 10 15 Gln Glu Gln Leu Arg Tyr Gly Asn Leu Thr Glu Thr Arg Thr Leu Leu              20 25 30 Ala Ile Gln Gln Gln Ala Leu Arg Gly Gly Ala Gly Ala Gly Gly Thr          35 40 45 Gly Ser Pro Gln Ala Ser Leu Glu Ile Gly Ala Pro Glu Asp Ser Gln      50 55 60 Val Leu Gln Gln Ala Thr Arg Gln Glu Pro Gln Gly Gln Glu His Gln  65 70 75 80 Gly Gly Glu Thr His Leu Ala Glu Asn Arg Leu Tyr Arg Leu Cys Pro                  85 90 95 Gln Pro Ser Lys Gly Glu Glu Leu Pro Thr Tyr Glu Glu Ala Lys Ala             100 105 110 His Ser Gln Tyr Tyr Ala Ala Gln Gln Ala Gly Ser Arg Pro His Val         115 120 125 Gly Asp Arg Asp Pro Arg Gly Gly Val Ser Gly Gly Gly Arg Arg Gln     130 135 140 Asp Glu Ala Leu Arg Glu Leu Arg His Gly His Val Arg Ser Leu Ser 145 150 155 160 Glu Arg Leu Leu Gln Leu Ser Leu Glu Arg Asn Gly Ala Arg Val Pro                 165 170 175 Ser His Met Ser Ser Ser His Ser Phe Pro Gln Leu Ala Arg Ser Gln             180 185 190 Gln Gly Pro Gln Pro Arg Gly Pro Pro Ala Glu Gly Pro Glu Pro Arg         195 200 205 Gly Pro Pro Pro Gln Tyr Pro His Ala Val Met Ala Gln Glu Thr Ala     210 215 220 Ala Val Thr Asp Pro Arg Tyr Arg Pro Arg Ser Ser Pro His Phe Gln 225 230 235 240 His Ala Glu Val Arg Ile Leu Gln Ala Gln Val Pro Pro Val Phe Leu                 245 250 255 Gln Gln Gln Gln Tyr Gln Tyr Leu Pro Gln Pro Gln Glu His Ser Pro             260 265 270 Pro Leu His Pro Ala Ala Leu Gly His Gly Pro Pro Ser Ser Phe Gly         275 280 285 Pro Pro Ala Val Glu Gly Pro Pro Ser Ala Gln Ala Thr Leu Gly Ser     290 295 300 Ala His Leu Ala Gln Met Glu Thr Val Leu Arg Glu Asn Ala Arg Leu 305 310 315 320 Gln Arg Asp Asn Glu Arg Leu Gln Arg Glu Leu Glu Ser Thr Ser Glu                 325 330 335 Lys Ala Gly Arg Ile Glu Lys Leu Glu Asn Glu Ile Gln Arg Leu Ser             340 345 350 Glu Ala His Glu Ser Leu Met Arg Thr Ser Ser Lys Arg Glu Ala Leu         355 360 365 Glu Lys Thr Met Arg Asn Lys Met Asp Gly Glu Met Arg Arg Leu Gln     370 375 380 Asp Phe Asn Arg Asp Leu Arg Glu Arg Leu Glu Ser Ala Asn Arg His 385 390 395 400 Leu Ala Ser Lys Thr Gln Glu Ala Gln Ala Gly Ser Gln Asp Met Val                 405 410 415 Ala Lys Leu Leu Ala Gln Ser Tyr Glu Gln Gln Gln Glu Gln Glu Lys             420 425 430 Leu Glu Arg Glu Met Ala Leu Leu Arg Gly Ala Ile Glu Asp Gln Arg         435 440 445 Arg His Ala Glu Leu Leu Glu Gln Ala Leu Gly Asn Ala Gln Ser Arg     450 455 460 Ala Ala Arg Ala Glu Glu Glu Leu Arg Lys Lys Gln Ala Tyr Val Glu 465 470 475 480 Lys Val Glu Arg Leu Gln Gln Ala Leu Gly Gln Leu Gln Ala Ala Cys                 485 490 495 Glu Lys Arg Glu Gln Leu Glu Leu Arg Leu Arg Thr Arg Leu Glu Gln             500 505 510 Glu Leu Lys Ala Leu Arg Ala Gln Gln Arg Gln Thr Gly Thr Leu Ala         515 520 525 Gly Gly Gly Gly Ser His Gly Gly Ser Ala Glu Leu Ser Ala Leu Arg     530 535 540 Leu Ser Glu Gln Leu Arg Glu Lys Glu Glu Gln Ile Leu Ala Leu Glu 545 550 555 560 Ala Asp Met Thr Lys Trp Glu Gln Lys Tyr Leu Glu Glu Arg Ala Met                 565 570 575 Arg Gln Phe Ala Met Asp Ala Ala Ala Thr Ala Ala Ala Gln Arg Asp             580 585 590 Thr Thr Leu Ile Arg His Ser Pro Gln Pro Ser Pro Ser Ser Ser Phe         595 600 605 Asn Glu Gly Leu Leu Pro Gly Asn His Arg His Gln Glu Met Glu Ser     610 615 620 Arg Leu Lys Val Leu His Ala Gln Ile Leu Glu Lys Asp Ala Val Ile 625 630 635 640 Lys Val Leu Gln Gln Arg Ser Arg Lys Asp Pro Gly Lys Ala Thr Gln                 645 650 655 Gly Thr Leu Arg Pro Ala Lys Ser Val Pro Ser Ile Phe Ala Ala Ala             660 665 670 Val Gly Thr Gln Gly Trp Gln Gly Leu Val Ser Ser Glu Arg Gln Thr         675 680 685 Asp Ala Arg Pro Ala Gly Asp Arg Val Pro Ala Glu Glu Pro Pro Ala     690 695 700 Thr Ala Pro Leu Pro Ala His Thr Lys His Gly Ser Arg Asp Gly Ser 705 710 715 720 Thr Gln Thr Asp Gly Pro Ala Asp Asn Thr Ser Ala Cys Leu Ala Ser                 725 730 735 Glu Pro Asp Gly Leu Leu Gly Cys Asn Ser Ser Gln Arg Thr Pro Ser             740 745 750 Leu Asp Ser Ile Ala Ala Thr Arg Val Gln Asp Leu Ser Asp Met Val         755 760 765 Glu Ile Leu Ile     770 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cgcgtcgaca tgaggacact ggaagactcc tc 32 <210> 6 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gcggcggccg ctcagatcag tatttctacc atgtc 35 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cgcgtcgaca gcacggagct cttctggccc agc 33 <210> 8 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gcggcggccg cctagatgag cacttccatc atctcgcc 38 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cgcgtcgaca tgaggacact ggaagactcc tc 32 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 cgcgtcgaca tgagaggttc tgaggatgta gcc 33

[Brief description of drawings]

FIG. 1 is an outline of cDNA cloning of a protein localized at an intercellular adhesion site.

FIG. 2 is a schematic diagram showing the structural characteristics of JEAP and JEAP-2. Polyglutamate repeats are shown in grey, coiled coil regions are shown in white, and regions similar to the protein consensus motifs that bind to PDZ regions are shown in black.

FIG. 3 is a diagram (micrograph) of an MDCK cell stained image showing the distribution of JEAP, JEAP-2, ZO-1, and E-cadherin.

FIG. 4 is a diagram of a tissue-stained image showing the distribution of JEAP and ZO-1 (micrograph) and a diagram of an immunoelectron microscopic image showing the distribution of JEAP in the cell gap of the exocrine gland (micrograph).

FIG. 5 is a diagram (micrograph) of a tissue-stained image showing the distribution of JEAP-2 and ZO-1.

FIG. 6 is a diagram (micrograph) of an MDCK cell stained image showing the distribution of JEAP and ZO-1.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/21 C12P 21/02 C 4H045 5/10 C12Q 1/68 A C12P 21/02 G01N 33/15 Z C12Q 1/68 33/50 Z G01N 33/15 33/53 D 33/50 M 33/53 33/566 C12N 15/00 ZNAA 33/566 5/00 A (72) Inventor Yuichi Ono Ibaraki City, Osaka Prefecture Hozumi 1-2-30 Noble Yamaoka No. 403 (72) Inventor Koji Morimoto 40-7 Ikedakitamachi, Neyagawa City, Osaka Prefecture (72) Inventor Katsuichi Takeuchi 33-17 Nishiishijoishiimachi, Shimogyo-ku, Kyoto Prefecture Kyoto Prefecture 33 202 (72) Inventor Yoko Inoue 109 Koji Heights Omiya 303, 109 Takatsuji Omiyacho, Shimogyo-ku, Kyoto Prefecture Kyoto (72) Inventor Toshio Imai 688 Daikokucho, Kamigyo-ku, Kyoto Prefecture Kyoto Prefecture Yoshimi Takai Kobe City, Hyogo Prefecture Ward Gakuen Higashimachi 2-5-73 F Term (reference) 2G045 AA34 AA35 BB20 CB01 DA13 DA36 FB02 FB03 FB12 GC15 4B024 AA01 AA11 BA80 CA01 DA02 GA11 HA12 HA20 4B063 QA18 QQ42 QQ79 QQ96 QR08 QR32 QR42 QR19 QR0 QR24 QR55 QR34 QR25 QR34 QR01 QR25 QR34 QR25 QR34 QR25 QR34 QR25 QR34 DA01 DA13 4B065 AA91Y AB01 AC14 BA02 CA24 CA25 CA44 CA60 4H045 AA10 AA11 AA20 BA10 CA40 DA50 EA20 EA50 FA74

Claims (16)

[Claims] 1. A protein of the following (a) or (b) (excluding the protein having the amino acid sequence shown in SEQ ID NO: 2): (A) A protein having the amino acid sequence shown in SEQ ID NO: 4. (B) A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 and localized at a tight junction. 2. A DNA encoding the protein according to claim 1.
(However, the DNA having the nucleotide sequence shown in SEQ ID NO: 1 is excluded). 3. The DNA according to claim 2, which has the base sequence shown in SEQ ID NO: 3. 4. A DNA of (a) or (b) below (excluding the DNA having the nucleotide sequence shown in SEQ ID NO: 1). (A) A DNA having the base sequence shown in SEQ ID NO: 3. (B) a DNA that hybridizes with a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions and encodes a protein localized at a tight junction. 5. The following DNA (a) or (b) (excluding the DNA having the nucleotide sequence shown in SEQ ID NO: 1): (A) A DNA having the base sequence shown in SEQ ID NO: 3. (B) A DNA having a nucleotide sequence having a homology of 90% or more with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and encoding a protein localized at a tight junction. 6. The DNA according to any one of claims 4 to 5.
The protein encoded by. 7. The DNA according to any one of claims 2 to 5.
A recombinant vector containing. 8. The DNA according to any one of claims 2 to 5.
A transformant obtained by transforming a host with. 9. A transformant according to claim 8 or a transformant obtained by transforming a host with the DNA having the nucleotide sequence shown in SEQ ID NO: 1 is cultivated, and a tight junction expressed by the transformant is cultured. A method for producing a protein localized at a tight junction, comprising collecting the localized protein from a culture. 10. An antibody which reacts with the protein according to claim 1. 11. The method for measuring the protein according to claim 1, which comprises using the antibody according to claim 10. 12. The protein according to claim 1, wherein an oligonucleotide having at least 15 consecutive base sequences of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 is used as a primer or a probe. A method for measuring DNA or RNA that encodes. 13. A method of mixing (1) the protein according to claim 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2 or a partial peptide thereof with a test substance, and (2) the protein or 3. A substance having reactivity with the protein according to claim 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2, which comprises the step of measuring the amount of a test substance bound or not bound to the partial peptide. To screen. 14. (1) A step of culturing cells expressing the protein according to claim 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2 in the presence of a test substance, and
(2) The method according to claim 1, which is expressed in cells or a protein having the amino acid sequence shown in SEQ ID NO: 2, or the step of measuring the mRNA encoding the protein in the cells. A method for screening a substance that affects the expression of the protein according to claim 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2. 15. (1) A step of identifying a promoter region which controls the expression of the protein according to claim 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2, and (2) the test substance has a promoter activity. A method of screening a substance that affects the expression of the protein according to claim 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2, which comprises the step of measuring the effect. 16. (1) A step of culturing cells expressing the protein according to claim 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2 in the presence of a test substance, and
(2) measuring the distribution of the protein of claim 1 or the protein having the amino acid sequence of SEQ ID NO: 2 in cells, the amino acid of claim 1 or the amino acid of SEQ ID NO: 2 A method for screening a substance that affects the distribution of a protein having a sequence.