Patent Grant
7824676
The present application shares at least one common inventor with, and hereby claims priority to U.S. patent application Ser. No. 10/298,417, filed Nov. 15, 2002, which issued on Oct. 10, 2006 as U.S. Pat. No. 7,129,063; and which was co-pending with, shared at least one common inventor with, and claimed priority to Japanese Patent Application Serial Number JP2001-352241, filed Nov. 16, 2001. Each of these applications is incorporated herein by reference in its entirety.
The present invention relates to a novel component protein of tight junctions (hereinafter referred to as TJs) of exocrine glands and uses of the protein.
In multicellular animals, information on junction of adjacent cells is deeply related to control and maintenance of life phenomena such as proliferation and differentiation of cells, inflammation, and metastasis of cancers. In many cases, intercellular adhesion molecules that participate in adhesion gather on the surface of cells to form a membrane region uniquely differentiated for adhesion. In particular, in epithelial cells, it has been known that intercellular adhesion molecules such as cadherin are strongly bound to cytoskeleton in the cytoplasm domain.
Such a membrane region, which is called an intercellular adhesion mechanism, is generally classified into four types: gap junction (GJ), adherens junction (AJ), desmosome and tight junction (TJ).
TJ is one of intercellular adhesion mechanisms in the epithelial or endothelial cell layer. It plays a role of a physical barrier for preventing free passage of solutes and water through extracellular space (barrier function), and constitutes a continuous peripheral seal surrounding the cells. Also, it is considered that TJ plays a role of a boundary between apical and basolateral cytomembrane regions for forming and maintaining cell polarity (fence function).
TJs comprise transmembrane protein molecules such as claudin, occludin and JAM and peripheral membrane proteins, such as ZO-1, -2, and -3, cingulin, 7H6, symplekin, Rab3B, Sec6/Sec8 homolog, ASIP/PAR-3, PAR-6, and MAGI-1. Claudin and occludin constitute the backbone of TJ strands and are involved in the barrier function of TJs.
JAM is involved in cell-cell adhesion and/or junctional assembly of endothelial and epithelial cells, as well as infiltration of monocytes through interstices between endothelial cells induced by chemokines. ZO-1, -2 and -3 are scaffold proteins containing PDZ domains and directly bind to claudin and occludin at the cytoplasmic surface of TJ strands. ZO-1, -2 and -3 also bind to F-actin and might regulate TJ functions via cross-linking 1.1 strands and the actin cytoskeleton. Several other PDZ domains—containing proteins localized at TJs might also serve as landmarks to recruit cytoskeletal and signaling proteins to TJ strands. As a non-F-actin binding scaffold protein, MAGI-1/2/3 localizes at TJs and interacts with signaling molecules such as a tumor suppressor gene product, PTEN, and a GDP/GTP exchange protein for Rap small G protein. ASIP/PAR-3 and PAR-6 are cell polarity-related molecules containing PDZ domains and interact with a typical protein kinase C (PKC). ASIP/PAR-3 interacts with JAM. Among peripheral membrane proteins devoid of PDZ domains at TJ strands, Rab3B and Sec6/Sec8 homologs are involved in vesicular transport. Furthermore, cingulin, 7H6 antigen, and symplekin have been known to localize at TJs. However, their functions have not been elucidated yet. Cingulin interacts with ZO-1, -2 and -3, occludin, AF-6, and JAM.
To further characterize the molecular organization of TJ, identification and provision of novel TJ-constituting proteins has been desired.
Meanwhile, a mouse cDNA clone (GenBank accession No. BAB30287) has been reported by RIKEN as a gene whose function is unknown.
An object of the present invention is to provide a novel component protein of TJs in exocrine glands.
To clarify the details of molecular organization of intercellular adhesion, the inventors of the present invention have attempted to screen for novel proteins localized at cell-cell junctions by a fluorescence-labeled protein localization-based expression cloning method.
First, several cDNA fragments derived from mouse vascular endothelial cell line MS-1 cells were expressed as GFP fusion proteins, and a cDNA fragment that encodes a gene product localized at the cell-cell junctions was isolated based on observation of subcellular distribution of the fluorescence-labeled proteins observed in exocrine glands such as pancreas, submandibular gland and lacrimal gland but not in small intestine.
From the above results, it has been found that JEAP and JEAP-2 form a gene family that constitutes TJs of exocrine glands, thereby achieving the present invention.
Therefore, the present invention relates to the following items.
1. A protein of the following (a) or (b):
2. A DNA encoding the protein as defined in the item 1.
3. A DNA according to the item 2, having a nucleotide sequence shown in or SEQ ID NO: 3.
4. A DNA of the following (a) or (b):
5. A DNA of the following (a) or (b):
6. A protein encoded by the DNA as defined in the item 4 or 5.
7. A recombinant vector comprising the DNA as defined in any one of the items 2 to 5.
8. A transformant obtained by transforming a host with the DNA as defined in any one of the items 2 to 5.
9. A method for producing a protein localizing at tight junctions, comprising:
10. An antibody reacting with the protein as defined in the item 1.
11. A method for measuring the protein as defined in the item 1, comprising using the antibody as defined in the item 10 to measure the protein.
12. A method for measuring a DNA or RNA encoding the protein as defined in the item 1, comprising using an oligonucleotide having a nucleotide sequence composed of at least 15 consecutive nucleotides in a nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 as a primer or probe to measure the DNA or RNA.
13. A method for screening for a substance having a reactivity with the protein as defined in the item 1 or a protein having an amino acid sequence shown in SEQ ID NO: 2, comprising the steps of:
14. A method for screening for a substance affecting expression of the protein as defined in the item 1 or a protein having an amino acid sequence shown in SEQ ID NO: 2, comprising the steps of:
15. A method for screening a substance affecting expression of the protein as defined in the item 1 or a protein having an amino acid sequence shown in SEQ ID NO: 2, comprising the steps of:
(1) identifying a promoter region controlling expression of the protein as defined in item 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2; and
(2) measuring an influence of a test substance on a promoter activity of the promoter region, thereby screening for the substance.
16. A method for screening for a substance affecting distribution of the protein as defined in the item 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2, comprising the steps of:
(1) culturing a cell expressing the protein as defined in the item 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 distribution of the protein as defined in claim 1 or the protein having the amino acid sequence shown in SEQ ID NO: 2 in the cell, thereby screening for the substance.
Hereinafter, the present invention will be described in detail. For convenience's sake, the protein which is revealed by the present inventors that they localize at tight junctions, including the protein having the amino acid sequence shown in SEQ ID NO: 2, are referred to as “the protein of the present invention”.
Of the proteins of the present invention, those proteins having the amino acid sequence shown in SEQ ID NO: 2 or 4 are proteins identified as ones localized at tight junctions, as will be described in Examples below. Generally, proteins are expected to have mutants having the identical function and appropriate modification of the amino acid sequence of a protein can give rise to a mutant having the identical function. Therefore, those proteins that have an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 and that localize at tight junctions are also encompassed by the proteins of the present invention.
The modification of the amino acid sequence of a protein can be performed by modifying the nucleotide sequence of DNA encoding the protein by a well-known means such as site-specific mutation inducing method and expressing the DNA having the modified nucleotide sequence. One skilled in the art can readily confirm localization of the protein at tight junctions by means of, for example, a fluorescent antibody technique or immuno-electron microscopy.
The protein of the present invention may be converted into fused proteins by fusing it with other proteins such as glutathione transferase (GST) or His tag.
The DNA of the present invention is a DNA that encodes the protein of the present invention. The DNA of the present invention includes DNA having a nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. The DNA was one whose nucleotide sequence was determined in Examples described below. Generally, for a gene, there are expected to exist genes that encode the same product but have different nucleotide sequences or genes that encode mutants having the same function as that of the gene concerned. Also, modification of the nucleotide sequence of the gene can give rise to a gene that encodes a mutant producing the same product or exhibiting the same function as that of the gene concerned. Therefore, the DNA of the present invention encompasses DNAs having nucleotide sequences similar to that of SEQ ID NO: 1 or SEQ ID NO: 3 and encoding proteins localized at tight junctions. DNAs having similar nucleotide sequences include DNAs that hybridize with a DNA having a nucleotide sequence complementary to that of SEQ ID NO: 1 or SEQ ID NO: 3 under a stringent condition and DNAs having nucleotide sequences with 90% or more homologies to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.
Here, the term “stringent condition” comprises, for example, hybridization at 65° C. in 4×SSC followed by washing at 65° C. for 1 hour in 0.1×SSC. As an alternative method, the stringent condition comprises 42° C. in 4×SSC in 5000 formamide. Also, conditions of performing hybridization at 65° C. for 2.5 hours in PERFECTHYB™ (TOYOBO), followed by washings 1) 2×SSC, 0.05% SDS solution: 25° C., 5 minutes, 2) 2×SSC, 0.05% SDS solution: 25° C., 15 minutes, and 3) 0.1×SSC, 0.1 SDS solution: 50° C., 20 minutes may be used.
The homology as used herein is the homology calculated by the Clustal W method.
Whether a DNA encodes protein localizing at tight junctions may be easily confirmed by expressing the DNA in a suitable cell which can form tight junctions, and determining the presence of the expressed protein in tight junctions by a fluorescent antibody technique, immuno-electron microscopy and the like as described above.
The DNA of the present invention can be used in the analysis of the gene of JEAP family protein and in the analysis of the expression of genes, by using a part of it as a primer or a probe. By the term “a part” is meant that the oligonucleotide used as a primer or a probe comprises a polynucleotide having a nucleotide sequence composed of at least consecutive 15 bases, preferably at least 20 bases, and more preferably at least about 20 to 30 bases corresponding to the DNA sequence of the present invention. As a probe, polynucleotides having larger molecular weights and whole DNA may 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 can be synthesized by a chemical synthesis method, or by means of mRNA prepared from cells or tissues expressing the protein localized at the tight junction of the present invention or by an RT-PCR method.
The gene manipulation can be performed by the method described in the literature (Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, (1989).
The vector of the present invention is a recombinant vector comprising the DNA of the present invention. The vector of the present invention may 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 that can be 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, bacteria cells, yeast cells, insect cells, and the like. The transformation may be performed by a conventional method. For example, it may be performed by introduction into a host, of the vector of the present invention which expresses the protein of the present invention in the host.
The production method of the present invention is a method for producing the protein localized at the tight junction of the present invention. The method includes culturing the transformant of the present invention and collecting the protein expressed by the transformant and localized at the tight junction from the culture.
The culture may be performed under the condition where the transformant expresses the protein of the present invention. The collection of the protein of the present invention from the culture may be performed by appropriate combinations of various chromatographies, electrophoreses, gel filtrations and the like that have been usually used for the purification of proteins. In the case where the protein of the present invention is to be expressed as a fused protein with a tag such as GST or His tag, the protein can be purified by using a glutathione Sepharose column or a nickel Sepharose column, respectively.
The whole or a part of the JEAP protein of the present invention can be used as an epitope for preparing antibodies, which in turn can be used as reagents for investigation and diagnosis. By the term “epitope” is meant an antigenic determinant of a polypeptide, which generally comprises at least six amino acids. It is known that polypeptides composed of six amino acids bind with antibodies (JP-60-500684 A). The antigenic peptide of the protein of the present invention means a polypeptide composed of at least six consecutive amino acids, preferably at least eight consecutive amino acids, more preferably at least about 15 consecutive amino acids and still more preferably at least about 20 consecutive amino acids based on the amino acid sequence of the present invention.
The antibody of the present invention can be obtained from animals immunized with the protein of the present invention obtained by the production method of the present invention or an antigenic peptide thereof. Polyclonal antibodies can be prepared from sera of the immunized animals while monoclonal antibodies can be prepared by fusing antibody-producing cells obtained from the spleen or lymph nodes of the immunized animals with myeloma cells and screening hybridomas producing antibodies showing strong specificity to the protein of the present invention.
As the immunogen, the protein of the present invention obtained by the production method of the present invention can be used. As an alternative method, the immunogen may be a fragment or peptide having a partial structure appropriately selected from amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 4. A complex between an antigen and a carrier protein can be prepared by using various coupling agents. For example, glutaraldehyde, carbodiimide, maleimide active ester, and the like can be used. The carrier protein may be those conventionally used such as bovine serum albumin, thyroglobulin, and hemocyanin. Usually, a method is used in which the carrier protein is coupled with the antigen in a proportion of 1- to 5-fold amount.
The animals to be immunized include mice, rats, rabbits, guinea pigs, hamsters and the like. The inoculation method includes subcutaneous, intramuscular and intraperitoneal administrations. Upon the administration, the complex may be mixed with Freund's complete adjuvant or Freund's incomplete adjuvant before it can be administered. The administration is performed usually once in 2 to 5 weeks. The antibody-producing cells obtained from the spleen or lymph node of the immunized animal are subjected to cell fusion with myeloma cells and isolated as hybridomas. As the myeloma cells, those derived from mice, rats, humans and the like are used. They are preferably derived from the same species as the antibody-producing cells but in some cases the immunization is possible between different species.
The procedures of cell fusion can be practiced by a known method, for example, a method of Kohler, G. and Milstein. C., Nature 256, 495, 1975. The fusion accelerator includes polyethylene glycol, Sendai virus and the like. The cell fusion can be usually practiced by reacting the cells at 20 to 40° C., preferably at 30 to 37° C. for about 1 to 10 minutes in a ratio of the number of antibody-producing cells to that of the myeloma cells of usually about 1:1 to about 10:1 by using polyethylene glycol (average molecular weight: 1,000 to 4,000) in a concentration of usually about 20 to 50%.
Various immunochemical methods can be used for the screening of antibody-producing hybridomas. The immunochemical methods include, for example, an ELISA (enzyme-linked immunosorbent assay) method using a microplate having coated thereon the protein of the present invention, an EIA (enzyme immunoassay) method using a microplate having coated thereon an anti-immunoglobulin antibody, an immunoblot method using a nitrocellulose transfer membrane after electrophoresing a sample containing the protein of the present invention, and the like.
Further cloning from such wells may be performed, for example, by a limiting dilution method to obtain a clone. Screening and breeding of hybridomas are performed in a medium for animal cells (for example, RPMI1640), usually containing HAT (hypoxanthine, aminopterin, thymidine) through addition and 10 to 20% fetal calf serum. The clone thus obtained can be transplanted into abdominal cavity of an SCID mouse preliminarily administered with pristane and the ascites containing a monoclonal antibody in a high concentration is collected therefrom after 10 to 14 days to obtain a raw material for purifying antibody. Also, the clone can be cultured and the culture may be used as a raw material for purifying antibody. The recovery of monoclonal antibody may be achieved without difficulty by using a known method as a purification method for immunoglobulins, for example, an ammonium sulfate fractionation method, a PEG fractionation method, an ethanol fractionation method, use of an anion exchange member, and further affinity chromatography and other means.
By using the antibody of the present invention, the protein of the present invention can be measured. The measurement can be performed by a conventional method using an antibody. For example, by the immunological method using the antibody (preferably monoclonal antibody) of the present invention, qualitative and quantitative determination of the protein of the present invention in biological samples can be performed. As the immunological method, a known method such as an immune tissue stain method, an enzyme immunoassay, an agglutination method, a competitive method, or a sandwich method may be applied to samples obtained by subjecting biological samples to appropriate treatment, for example, separation of cells, extraction operation or the like, as necessary. The immune tissue stain method can be performed, for example, by a direct method using a labeled antibody, and an indirect method using a labeled antibody against the antibody. As the labeling agent, any known labeling substances, such as fluorescent substances, radioactive substances, enzymes, metals, and dyestuffs can be used.
The antibody of the present invention may be used in the form of Fab′ or Fab fraction free of Fc′ or Fc region or polymers thereof. Alternatively, the antibody of the invention may be a chimera antibody or a humanized antibody thereof.
The screening for a substance such as a compound (such as drug) that affects the expression of the protein of the present invention can be practiced by culturing a cell expressing the protein of the present invention in the presence of a test substance, and then measuring the protein of the present invention expressed in the cell or mRNA encoding the protein in the cell.
The cell that expresses the protein of the present invention may be selected by Northern blotting, RT-PCR or the like. Also, the cell line may be selected by a fluorescent antibody technique, an enzyme antibody method or the like using the antibody obtained by the method described above.
The selected cell is cultured in the presence of a test substance (for example, the cell is cultured by adding a test substance to the cell) and then the mRNA expression amount is determined by Northern blotting, slot blot hybridization, RT-PCR, and the like, or the protein expression amount is determined by fluorescent antibody technique, an enzyme antibody method, and the like, thereby measuring the influence of the test substance on the expression of the protein of the present invention. For example, this can be done in accordance with the methods described in Example 3 and Example 6.
Furthermore, in order to enable screening of a large number of kinds of test substances more readily, a promoter region controlling expression of the protein of the invention may be identified and then an influence of a test substance on a promoter activity may be measured.
The identification of the promoter region and the measurement of the influence of the test substance on the promoter activity may be performed as follows. A clone that hybridizes with the 5′-region of cDNA encoding the protein of the present invention is selected out of a human DNA library and is inserted into an appropriate promoter screening system to select a clone having a promoter activity. In some cases, the region indispensable for the promoter activity may be narrowed.
The DNA selected here that has a promoter region for the protein of the present invention is inserted on the upstream side of the DNA encoding an enzyme whose activity can be readily measured, such as luciferase or alkaline phosphatase, to construct a reporter gene. The reporter gene is introduced into a cell, for example, HeLa cell, that can be cultured together with a suitable resistant gene, such as Neor or hygr and the cells are selected with a substance corresponding to the resistant gene to establish a cell line enabling measurement of the activity of the promoter that expresses the protein of the present invention. By causing the substance to act on the cell line, the activity of the introduced enzyme is measured, thereby practicing screening for the substance that affects the expression of the protein of the present invention.
The protein of the present invention can be used as another screening system for selecting a substance that affects the intracellular localization or distribution of the protein of the present invention. The screening may be performed by culturing a cell expressing the protein of the present invention in the presence of a test substance and measuring distribution of the protein of the present invention in the cell.
An example of the screening method includes staining the cell line that expresses the protein of the present invention or transformant of the present invention by a fluorescent antibody technique, an enzyme antibody method or the like by using the antibody obtained by the above-mentioned method and observing the stained cells under a microscope to measure the influence of the test substance on the intracellular localization of the protein of the present invention. For example, this can be performed in accordance with the methods described in Example 3 and Example 6.
Furthermore, the protein of the present invention can be used as still another screening system for selecting a substance that binds to the protein of the present invention (that is, a substance having a reactivity with the protein of the present invention). The screening may be performed by mixing the protein of the present invention or a partial peptide thereof with a test substance, and measuring an amount of the test substance bound or not bound to the protein or the partial peptide thereof.
In this system, because the substance having reactivity with the protein of the present invention is considered to bind to a part of the protein of the present invention, a partial peptide of the protein of the present invention may be used. Therefore, the partial peptide is sufficient to have such a part that binding to the partial peptide becomes equivalent to binding to the protein of the present invention.
Also, the protein of the present invention may be in a form in which the protein is expressed in cell membrane of the transformant of the present invention. Therefore, mixing the protein of the present invention with the test substance includes mixing the transformant of the present invention or cell membrane thereof with the test substance.
The measurement of the test substance bound or not bound to the protein of the present invention or the partial peptide thereof may be performed by reacting the protein of the present invention and a test substance under appropriate conditions and determine presence or absence of the binding between the two. The determination can be performed by using, for example, a labeling substance as necessary.
Since the substance having reactivity with the protein of the present invention have a possibility that they affects the function of the protein of the present invention and there is observed exocrine specificity in distribution of the protein of the present invention as described in Examples, it is expected that substances having specificity to binding ability to the protein of the present invention have a possibility that they exhibit an organ-specific action.
Note that although the JEAP family whose structure has been actually analyzed by the present invention is derived from a mouse, the method of analyzing JEAP family gene and method of screening it of the invention are also encompassed by the scope of the present invention when human-derived JEAP family is used.
The present invention will be described in more detail by the following examples. However, the present invention should not be considered as being limited thereto. The antibodies and cells used in Examples were as follows. Rat anti-mouse JEAP monoclonal antibody and rat anti-mouse JEAP-2 monoclonal antibody were prepared by a conventional method by immunizing a rat with GST-JEAP fused protein containing amino acid sequence 808-882 (amino acid numbers in SEQ ID NO: 2) of JEAP and GST-JEAP-2 fused protein containing amino acid sequence 726-772 (amino acid numbers in SEQ ID NO: 4) of JEAP-2, respectively. Rabbit anti-ZO-1 polyclonal antibody, rabbit anti-claudin polyclonal-antibody, mouse anti-ZO-1 monoclonal antibody, and mouse anti-occludin monoclonal antibody were purchased from Zymed, Inc. Mouse anti-E-cadherin monoclonal antibody was purchased from Takara Shuzo Co., Ltd.
MDCK cells (Madin-Darby canine kidney cells) were cultured in a DMEM medium with 10% fetal calf serum (FCS). Mouse vascular endothelial cell line MS-1 was purchased from American Type Culture Collection (ATCC) and cultured in DMEM/5% FCS.
To further characterize the molecular organization of intercellular adhesion, identification of novel cDNAs encoding proteins localized at cell-cell junctions was attempted by the localization-based expression cloning method using a fluorescence-labeled protein (
First, a pilot experiment was carried out in which an expression recombinant retrovirus expressing a fused protein between VF-cadherin localized at cell-cell junctions and a fluorescence protein EGFP was infected to various cell lines and fluorescent stained images were observed under a fluorescence microscope. As a result, no cell line that showed a clear fluorescent signal as viewed with naked eye existed so far as was examined. Accordingly, it was attempted to make MDCK cells having high cell height and showing clear cell-cell junctions competent for ecotropic retrovirus and use the competent cells in experiments. The ecotropic retrovirus competent MDCK cell (MDCK/EcoVR) was obtained as follows. The ecotropic virus receptor (EcoVR) cDNA was inserted into pCAGGS-puro and it was transfected to the MDCK cells by using LipofectAMINE reagent (Life Technologies. Inc.). After 24 hours, culture of the cells in the presence of 5 μg/ml of puromycin was started and resistant clones were selected. Each clone was isolated and infected with pMXII-EGFPN expression recombinant retrovirus and a high competent clone was obtained. EcoVR cDNA and pMX expression vector were kindly provided by Dr. T. Kitamura (Tokyo University, Tokyo, Japan). Transfection of VE-cadherin-EGFP expression recombinant retrovirus to MDCK/ErcoVR cells gave rise to bright fluorescent signals, which indicated that the cell line was suitable for visual screening of proteins localized at cell-cell junctions.
A cDNA-GFP fusion library was created from a mouse endothelial cell line MS-1 based on a method of Kitamura et al. The resulting library contained 3×105 independent clones and showed an average insert size of cDNA with 1,500 bp. The expression library was then co-transfected into 293/EBNA-1 cells (Invitrogen Corporation) with a virus packaging vector, pCL-Eco (Imgenex Corporation), by using TransiT LT1 (Mirus Corporation) to prepare a recombinant retrovirus. MDCK/EcoVR cells were infected with variously diluted retrovirus supernatants to obtain singly infected cells. The initial frequency of the EGFP-positive cells was about 4% as determined by fluorescence-activated cell sorting (FACS) analysis. After culture, EGFP-positive cells were sorted by a cell sorter and cultured at 50 cells per well in 96 well plates. When the cells became confluent, screening of 10 plates was performed under a fluorescent microscope and there were selected 6 wells containing cells with junction-specific fluorescence of EGFP. These cells were replated into 10-cm dishes and a single clone was obtained. Each clone was expanded in 24-well plates, and cDNA integrated into the chromosome was recovered by PCR from the genomic DNA. Among the cDNAs encoding proteins showing the junction-specific staining pattern, a novel cDNA encoding protein unknown to be localized at cell-cell junctions was obtained.
Using the cDNA fragment obtained in Example 1 as a probe, cDNA was cloned from a mouse MS-1 cell-derived cDNA library. The cloning of cDNA was performed as follows. The total RNA of mouse MS-1 cell was extracted with Trizol (Life Technologies, Inc.) and poly A(+) RNA was prepared with oligo dT beads (Miltenyi Biotec GmbH). The cDNA library was prepared based on this poly A (+)RNA with a Superscript II cDNA synthesis kit (Life Technologies, Inc.) and a PMXII vector and used for screening cDNAs. The nucleotide sequences were determined by use of Dye Terminator Cycle Sequence Kit (Applied Biosystems, Inc.). The detail of the cloning is described in (1) below. A protein encoded by the obtained cDNA was named JEAP.
Homology search using an amino acid sequence deduced from JEAP cDNA revealed that human cDNA clone KIAA0989 has homology thereto over the full length thereof. This clone has been reported by Kazusa DNA Research Institute as a gene whose function is unknown. Further, as a result of homology search using an amino acid sequence deduced from KIAA0989 cDNA, mouse EST clone (BF536192) and mouse cDNA clones (AF175967, AF175968) were identified to be partially homologous to each other. Using these nucleotide sequences, another cDNA was separated from mouse MS-1 cell cDNA. Single strand cDNA was prepared with Superscript 11 reverse transcriptase (Life Technologies, Inc.) based on the poly A (+)RNA of MS-1 cell and used for PCR. The nucleotide sequence of it was determined by use of Dye Terminator Cycle Sequence Kit (Applied Biosystems, Inc.). The detail of the cloning is described in (2) below. A protein encoded by the obtained cDNA was named JEAP-2.
(1) Determination of cDNA of JEAP
A digoxigenin (DIG)-labeled probe was prepared by PCR by using the cDNA fragment obtained in Example 1 as a template and used for screening a pMXII MS-1 cell cDNA library. As a result, a plurality of positive clones were obtained, from which the nucleotide sequences of clones #2 and #5 were determined. As a result of structural analysis by determining the nucleotide sequence of the part including the entire open reading frame (ORF), JEAP cDNA contained ORF encoding a polypeptide with 882 amino acids with a calculated molecular weight of 98.4 kD (SEQ ID NO: 1). JEAP contained a polyglutamic acid repeat at the N-terminal region, a coiled-coil domain at the middle region and a consensus motif for binding to PDZ domains at the C-terminal region (
RIKEN (the Institute of Physical and Chemical Research) reported the same mouse cDNA clone (BAB30287) as JEAP to be a gene with unknown functions.
(2) Determination of cDNA of JEAP-2
A structural correlation search on Genbank/EMBL/DDBJ database resulted in identification of homology between human cDNA clone KIAA0989 and JEAP over the full length thereof. Furthermore, homology search using the amino acid sequence deduced from KIAA0989 cDNA identified mouse EST clone (BF536192) and mouse cDNA clones (AF175967, AF175968) to be partially homologous to each other. Then, a cDNA containing the entire ORF was amplified from MS-1 cell cDNA by a PCR method by use of two kinds of primers of nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 designed based on these mouse sequences. The cDNA was subcloned to pSPORT1 vector (Life Technology, Inc.) and it was named PSPORT-JEAP-2. Nucleotide sequence analysis indicated that cDNA of JEAP-2 contained ORF encoding a polypeptide with 772 amino acids having a calculated molecular weight of 85.3 kD (SEQ ID NO: 3). The JEAP-2 polypeptide showed a high similarity to the sequence of JEAP (42% identity on the level of amino acid sequence). Structural analysis suggested that JEAP-2, like JEAP, contains a sequence containing a coiled-coil domain at the middle region, a consensus motif for binding to PDZ domains at the C-terminal region.
A monoclonal antibody to mouse JEAP was prepared as follows. First, a region encoding the amino acid sequence 808-882 (amino acid number in SEQ ID NO: 2) of JEAP was amplified by PCR using the clone #2 obtained in Example 2 as a template and oligonucleotides of nucleotide sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 as primers and introduced into GST fusion protein expressing vector (Amersham) and MBP fusion protein expressing vector (NEB) to express recombinant proteins. The GST-JEAP fusion protein was mixed with an adjuvant and a WKY rat was immunized therewith. Lymphocytes were isolated from the immunized rat and mixed with P3 myeloma cells in a mixing ratio of 1:5 (myeloma/lymphocyte), followed by cell fusion using PEG 1,500 solution (Boehringer) to prepare hybridomas. The prepared hybridomas were cultured in a 96-well plate for 1 week and the supernatant was assayed by ELISA with an ELISA plate having immobilized thereon MBP-JEAP fusion protein to identify positive wells. The hybridomas contained in the positive wells were cloned and finally a rat anti-mouse JEAP monoclonal antibody was obtained.
The rat anti-mouse JEAP-2 monoclonal antibody was prepared in the same manner as the JEAP monoclonal antibody. That is, a region encoding the amino acid sequence 726-772 (amino acid number in SEQ ID NO: 4) of JEAP-2 was amplified by PCR using the pSPORT-JEAP-2 obtained in Example 2 as a template and oligonucleotides of nucleotide sequences shown in SEQ ID NO: 6 and SEQ ID NO: 9 as primers and introduced into GST fusion protein expressing vector (Amersham) and MBP fusion protein expressing vector (NEB) to prepare recombinant proteins. The GST-JEAP-2 fusion protein was mixed with an adjuvant and a WKY rat was immunized therewith. Lymphocytes were isolated from the immunized rat and mixed with P3 myeloma cells in a mixing ratio of 1:5 (myeloma/lymphocyte), followed by cell fusion using PEG 1,500 solution (Boehringer) to prepare hybridomas. The prepared hybridomas were cultured in a 96-well plate for 1 week and the supernatant was assayed by ELISA with an ELISA plate having immobilized thereon MBP-JEAP-2 fusion protein to identify positive wells. The hybridomas contained in the positive wells were cloned and finally a rat anti-mouse JEAP-2 monoclonal antibody was obtained.
Whether the isolated cDNA clones contain the entire ORFs of JEAP and JEAP-2, respectively, was confirmed as follows by using the monoclonal antibody prepared in Example 3.
First, to prepare a JEAP expressing vector (pMXII JEAP IRES EGFP), the entire ORF of JEAP was amplified by PCR using oligonucleotides of nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 10 as primers. SalI-NotI fragment of the PCR product was introduced into the SalI-NotI site of PMXII IRES EGFP to construct pMXII JEAP IRES EGFP. A JEAP-2 expressing vector, pMXII JEAP-2 IRES EGFP, was constructed by excising a SalI-NotI fragment containing the entire ORF of JEAP-2 from pSPORT1-JEAP-2 and introducing it into the SalI-NotI site of PMXII IRES EGFP.
The expression vector was transfected to 293/EBNA-1 cells (Invitrogen Corporation) by use of TransIT LT1 (Mirus Corporation). As SDS polyacrylamide electrophoresis, one-dimensional SDS-PAGE was performed in accordance with the method of Laemmli (1970), followed by immunoblotting, which was performed as follows. After transfer from the gel to a nitrocellulose membrane, the expressed protein was reacted with an antibody and the bound antibody was detected with peroxidase-labeled second antibody (Amersham) and ECL (Amersham) as a substrate for the enzyme.
A single band of 105-kDa was detected by Western blotting with an anti-JEAP monoclonal antibody in the extract of 293/EBNA-1 cells transfected with JEAP expressing vector, but not in that from nontransfected 293/EBNA-1 cells. The apparent molecular weight of the expressed JEAP was identical with that of JEAP endogenously expressed in MS-1 cells. Therefore, it revealed that the isolated cDNA contain the full-length of JEAP. Similarly, a single band of 105 kDa was detected by Western blotting with an anti-JEAP-2 monoclonal antibody in the extracts of 293/EBNA-1 cells transfected with JEAP-2 expressing vector. In the extracts of nontransfected 293/EBNA-1 cells, a thin band that is thought to be derived from endogenous JEAP-2 was contain. The apparent molecular weight of the expressed JEAP-2 was identical with that of JEAP-2 endogenously expressed in 293/EBNA-1 cells. Therefore, it revealed that the isolated cDNA contained the entire ORF of JEAP-2.
Next, to examine expression and distribution of mouse JEAP and JEAP-2, JEAP and JEAP-2 were each expressed in MDCK/EcoVR cells and the cells were stained with a monoclonal antibody.
The expressing vector was cotransfected together with virus packaging vector pCL-Eco (Imgenex Corporation) to 293/EBNA-1 cells (Invitrogen Corporation) by use of TransIT LT1 (Mirus Corporation) to prepare a recombinant retrovirus. The recombinant virus was transfected to MDCK/EcoVR cells to obtain MDCK/EcoVR cells stably expressing JEAP or JEAP-2. The JEAP-expressing cells were confirmed by fluorescence of GFP or with an anti-JEAP antibody under a fluorescence microscope. JEAP-2-expressing cells were also confirmed similarity.
Then, intracellular distribution of these proteins was examined by confocal microscopy comparing with those of ZO-1 and E-cadherin. As shown in
Next, expression of JEAP and JEAP-2 in various tissues was examined.
First, tissue distribution of JEAP was examined by detecting expression of JEAP in various mouse tissues (liver, brain, lung, kidney, spleen, testis, ovary, and heart) by Western blotting, and no JEAP was detected in any of these tissues. Then, the tissue distribution of JEAP was examined by immunohistochemical staining. JEAP was detected specifically in exocrine glands including pancreas, submandibular gland, lacrymal gland (
Then, the tissue distribution of JEAP-2 was examined by immunohistochemical staining. For JEAP-2 too, expression in exocrine glands such as pancreas, submandibular gland, and lacrymal gland (
These findings indicate that JEAP and JEAP-2 have tissue specificity and localize particularly at TJs of exocrine glands in large amounts.
Finally, the behaviors of JEAP and other AJs and TJs components during the disruption and reformation of cell-cell junctions were monitored. For this purpose, MDCK/EcoVR cells stably expressing JEAP as prepared in Example 4 were used to perform the experiment. It has been known that when MDCK cells are cultured in the presence of 2 μM Ca2+ for 2 hours, AJs and TJs are disrupted and the staining of the AJ and TJ components except ZO-1 disappear from the periphery of the plasma membrane. Further, it has also been known that when the cells in this state are recultured in the presence of 2 mM Ca for 2 hours, AJs and TJs relocalize where all the AJ and TJ components reconcentrate at the cell-cell junctions.
In culturing in an ordinary medium, JEAP co-localized at the cell-cell junctions together with ZO-1 in the same manner as shown in Example 4. When the cells were cultured in the presence of 2 μM Ca2+ for 2 hours, the immunofluorescence signal for JEAP disappeared, whereas the fluorescence signal for ZO-1 partially remained near the plasma membrane though the intensity decreased. When the cells in this state were cultured in the presence of 2 mM Ca2+ for 2 hours, JEAP reconcentrate at the cell-cell junctions together with ZO-1. Furthermore, it has been known that culturing at 2 μM Ca2+ for 2 hours followed by further culturing for 1 hour with addition of 100 nM 12-O-tetradecanolylphorbol 13-acetate (TPA) results in reformation of a structure similar to that of TJs but does not result in reformation of AJs. It has been shown that TJ components, i.e., claudin, occludin, JAM, nectin, ZO-1 and afadin concentrate at the structural site similar to TJs of cell-cell junctions induced with TPA but AJ components, i.e., E-cadherin, α-catenin and β-catenin, do not concentrate there. Here, JEAP along with ZO-1 concentrated at the structural site similar to TJs of cell-cell junctions induced with TPA. In the same manner, JEAP concentrated at the structural site similar to TJs of cell-cell junction induced with TPA along with claudin-1 and accludin.
These findings indicate that JEAP is incorporated into TJs along with other components of TJs.
The foregoing description is to be understood as being representative only and is not intended to be limiting. Alternative methods and materials for implementing the invention and also additional applications will be apparent to one of ordinary skill in the art, and are intended to be included within the scope of the accompanying claims.
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