# Patent

## Title
Human apoptosis-associated genes and human apoptosis-associated proteins produced thereby

## Abstract

As novel human apoptosis-related protein inducing apoptosis, a protein comprising the amino acid sequence of SEQ ID No. 2, 4 or 6 is provided. Moreover, a polynucleotide encoding the protein, an antibody against the protein, etc. are provided. These protein, polynucleotide and antibody are useful in, for example, diagnosis and treatment of cancers, autoimmune diseases, etc.

## Background
This application is a U.S. national stage of PCT/JP02/00413 filed Jan. 22, 2002.
TECHNICAL FIELD
The inventions of the present application relate to novel human apoptosis-related proteins that induce apoptosis, polynucleotides encoding the proteins, and antibodies against the proteins. The proteins and the antibodies of the inventions are useful in diagnosis and treatment of various diseases, and the polynucleotides are useful as probes for gene diagnosis and gene sources for gene therapy. Further, the polynucleotides can be used as gene sources for mass production of the proteins of the invention.
BACKGROUND ART
Apoptosis is known to have an important role in morphogenesis, homeostasis, immune protection, etc. of individuals [Bosman F T, Visser B C, van Oeveren J. (1996) Pathol. Res. Pract. 192(7): 676-83]. Further, human TGF-β is known to have various functions related with regulation of development, differentiation, growth and oncogenic transformation of cells [Massague, J. (1990). Annu. Rev. Cell Biol. 6:597-641] and to show a useful role in morphogenesis [Akhurst R J, FitzPatrick D R, Gatherer D. Lehnert S A, Millan S A, Millan F A. Prog. Growth Factor Res. (1990) 2(3):153-68]. Accordingly, proteins induced by human TGF-β and having apoptotic activity are considered to participate in morphogenesis, as well as onset and progression of cancers, immunological diseases, etc.
Proteins inducing apoptosis have a potential as target proteins for developing low-molecular pharmaceuticals intended for apoptosis, and it is required to obtain as many apoptosis-related proteins as possible.
The object of the invention is to provide novel apoptosis-related proteins inducing apoptosis of cells and genes encoding the apoptosis-related proteins, which genes are useful for development of pharmaceuticals, etc. Other object of the invention is to provide genetic manipulation materials such as polynucleotides encoding the proteins, antibodies against the proteins, etc.
DISCLOSURE OF INVENTION
The invention of the application provides a human apoptosis-related gene that encodes a human apoptosis-related protein having the amino acid sequence of SEQ ID No. 2, 4 or 6. From mRNA transcribed from this gene, cDNA having the base sequence of SEQ ID No. 1, 3 or 5 is synthesized.
The invention provides a polynucleotide purified from genomic DNA, mRNA or cDNA of the human apoptosis-related gene or a complementary sequence thereof.
The invention provides a probe hybridizing with the human apoptosis-related gene or the purified polynucleotide under stringent conditions, and a primer set for PCR amplification of the human apoptosis-related gene of claim 1 or the purified polynucleotide.
The invention provides a recombinant vector having the polynucleotide, and a transformant with the recombinant vector.
The invention provides a purified human apoptosis-related protein, which is an expression product of the human apoptosis-related gene, and has the amino acid sequence of SEQ ID No. 2, 4 or 6. As one embodiment, the protein is produced from the transformant of claim 7.
The invention provides a purified or synthesized peptide, which is a part of the human apoptosis-related protein. A preferred embodiment is a peptide consisting of at least five continuous amino acid residues of the 1st-328th amino acid sequence in SEQ ID No. 2, a peptide consisting of at least five continuous amino acid residues of the 1st-326th amino acid sequence in SEQ ID No. 4, a peptide comprising at least the 385th-389th amino acid sequence in SEQ ID No. 4, and a peptide consisting of at least five continuous amino acid residues of the 1st-301st amino acid sequence in SEQ ID No. 6.
The invention provides an antibody against the purified human apoptosis-related protein.
The invention provides a method for screening a molecule that controls an apoptotic activity of the purified human apoptosis-related protein, which comprises identifying the target molecule by using, as an index, a cleavage activity to the peptide comprising at least the 385th-389th amino acid sequence in SEQ ID No. 4.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is phase-contrast and fluorescent micrographs of HEK293, a human embryo kidney cell line, to which the polynucleotides encoding SEQ ID No. 2, 4 and 6 in vector (TAIP-3, TAIP-2, TAIP-12 respectively) or empty vector (vector) were transfected, and Annexin V-FITC was treated. The left panels are the phase-contrast and the right panels are fluorescent micrograph.
FIG. 2 is phase-contrast and fluorescent micrographs as same as FIG. 1 except that NTERA-2, a human pluripotent embryonal carcinoma cell line was used.
FIG. 3, the upper panels are phase-contrast and fluorescent micrograph of HEK293 cells, which were transfected with a plasmid encoding green fluorescent protein (GFP). The lower panels are light and fluorescent micrograph of HEK293 cells, which was transfected with plasmids encoding GFP fusion proteins with a full-length (Full), a N-terminal half (N-hall), and a C-terminal half (C-half) of SEQ ID No. 4 amino acid sequences. The left panels are the phase-contrast and the right panels are fluorescent micrographs.
FIG. 4 is phase-contrast and fluorescent micrographs of HEK293, which were transfected with plasmids encoding GFP fusion proteins with a full-length (Full), a N-terminal half (N-half), and a C-terminal half (C-half) of SEQ ID No. 2 amino acid sequences. The left panels are the phase-contrast and the right panels are fluorescent micrographs.
FIG. 5 is phase-contrast and fluorescent micrographs of HEK293, which were transfected with plasmids encoding GFP fusion proteins with a full-length (Full), a N-terminal half (N-half), and a C-terminal half (C-half) of SEQ ID No. 6 amino acid sequences. The left panels are the phase-contrast and the right panels are fluorescent micrographs.
FIG. 6 is the results of northern blotting of poly (A)+RNA from Human tissues and human cancer cell lines, which were blotted on nylon membrane (Clontech) and hybridized with radiolabeled polynucleotide encoding C-terminus of TAIP-3 or -12 as probe. The transcripts of TAIP-3 and -12 (about 3.3 kb and 4.3 kb, respectively) were shown.
FIG. 7 is the result of FACS analysis for DNA contents of GFP expressing HEK239 cells, to which the plasmids encoding GFP fusion protein with various length of TAIP protein were transfected before 24 hours of the analysis.
FIG. 8 is the result of western blotting for detecting active form of caspase-3, in which the plasmids encoding GFP fusion protein with TAIPs were transfected into HEK293 cells and cell lysates were prepared 24 h later. Each sample was electrophoresed on a 20% polyacrylamide gel, blotted onto a nitrocellulose filter and detected with an anti-caspase-3 polyclonal antibody.
FIG. 9, the upper panel shows TAIP3 protein expression in A549, a human lung adenocarcinoma cell line, which were treated with TGF-β for 0, 3, 6, 12 or 24 hrs, lysed and analyzed with western blotting with anti-TAIP3 antibody. The lower panel showed TAIP3 protein expression in normal human hepatocytes, which were treated with TGF-β for 0, 3, 6, 12, 36 or 60 hrs, lysed and analyzed with western blotting with anti-TAIP3 antibody.
FIG. 10, the upper panel shows a confocal micrographs of A549 cells, which were cultured with TGF-β for 12 hr, fixed, permealized with Triton-X100 and treated with anti-TAIP3 antibody followed by Cy3-conjugated secondary antibody. The lower panel shows a confocal micrographs of a human hepatoma cell line, which was cultured with TGF-β for 48 or 60 hr, fixed, permealized with Triton-X100 and treated with anti-TAIP3 antibody followed by Cy3-conjugated secondary antibody.
FIG. 11 is the result of FACS analysis for caspase-3 activity, in which normal human hepatocytes were treated with TGF-β for 24 h and were further treated with PhiPhiLuxTMG1D2kit. Blue line shows caspase-3 activity before treatment of TGF-β and red line shows that of after treatment of TGF-β.
FIG. 12 shows TAIP-3 expression by western blotting. HLF cells were cultured with TGF-β for 24 h. Untreated cells (N) and TGF-β-treated total cell (T), flat cell (F) and round cell (R) populations were analyzed with anti-TAIP-3 antibody.
FIG. 13 is photographs of a brain section, which was prepared from the brain with focal ischemic damage, fixed onto a slideglass, treated with preimmuno serum (left) or anti-TAIP-2 polyclonal antibody (right) and then treated with LSAB kit (DAKO) for detecting the antibody. The portions recognized with the antibody were stained as reddish-brown.



BEST MODE FOR CARRYING OUT THE INVENTION
The human apoptosis-related genes (hereinafter sometimes referred to as TAIP genes) of the invention are genomic genes identified from human Chromosome 3, Chromosome 2 and Chromosome 12, and encoding three types of proteins having amino acid sequences of SEQ ID Nos. 2, 4 and 6 (hereinafter sometimes referred to as TAIP proteins or TAIP-3, TAIP-2 and TAIP-12 respectively). And cDNA of TAIP-3 gene has a base sequence of SEQ ID No. 1, cDNA of TAIP-2 gene has a base sequence of SEQ ID No. 3, and cDNA of TAIP-12 has a base sequence of SEQ ID No. 5.
The TAIP genes contain expression-regulating regions (promoter/enhancer and suppressor sequences, etc.) to protein encoding regions. These expression-regulating sequences are useful as, for example, materials for screening substances that regulate the in vivo expression of TAIP proteins.
The TAIP genes may be isolated by screening a human genomic DNA library using probes provided respectively by the invention. As the probes, for example, partial sequences (15 bp or more) or their complementary sequences of the purified polynucleotides (for example, cDNAs) provided by the invention can be used. The screening with the probes can be conducted under “stringent conditions” enabling specific hybridization of genomic DNAs and the probes. The stringent conditions can be defined by a salt concentration, a concentration of an organic solvent (formaldehyde, etc.), temperature conditions, etc. in hybridization and washing steps. For example, the conditions disclosed in U.S. Pat. No. 6,100,037, etc. can be employed.
The TAIP genes can also be amplified by the PCR (Polymerase Chain Reaction) method using the primer set provided by the invention with genomic DNAs as templates. The primer set can be formed by a combination of at least two partial sequences (15 bp or more) selected from the purified polynucleotides (for example, cDNAs) provided by the invention. Further, the upstream of the genes can also be PCR-amplified by the 5′ RACE method using 1 primer on the 5′ side of cDNAs and the downstream region of the genes by the 3′RACE method using 1 primer on the 3′ side of cDNAs. For example, the following can be indicated as points to be considered in primer designing. The size (number of bases) of the primer is from 15 to 40 bases, preferably from 15 to 30 bases in consideration of satisfying the specific annealing with template DNAs, provided at least 30 bases are effective in conducting LA (long accurate) PCR. A complementary sequence between both primers is avoided lest a set or a pair (two) of primers comprising a sense chain (on the 5′ end) and an antisense chain (on the 3′ end) are mutually annealed, and a self complementary sequence is also avoided for inhibiting formation of a hairpin structure in the primers. Further, the GC content is set at approximately 50% for securing stable binding with template DNAs lest the GC-rich or the AT-rich be present in the primers. Since the annealing temperature depends on Tm (melting temperature), primers approximate to each other with a Tm value of from 55 to 65° C. are selected to obtain PCR products having a high specificity. Moreover, it has to be considered that the final concentration in using the primers in PCR is adjusted to from approximately 0.1 to approximately 1 μm. In addition, commercial software for primer designing, for example, OligoTM [National Bioscience Inc. (U.S.A.)] or GENETYX [Software Kaihatsu K.K. (Japan)] can also be used.
The thus-obtained full-length genomic genes can be amplified by ordinary gene amplification methods such as a PCR method, an NASBN (Nucleic acid sequence based amplification) method, a TMA (Transcription-mediated amplification) method and an SDA (Strand Displacement Amplification) method.
The purified polynucleotides (DNA fragments or RNA fragments) can be prepared from TAIP genomic genes, mRNAs transcribed from these genes and cDNAs synthesized from the mRNAs. For example, cDNAs can be synthesized using Poly(A)+RNA extracted from human cells as a template. The human cells may be cells extracted from the human body by, for example, surgical operation or culture cells. Alternatively, desired cDNAs can also be synthesized by the RT-PCR method with mRNAs isolated from human cells as templates using the primer set provided by the invention. The thus-prepared cDNAs have specifically base sequences of SEQ ID Nos. 1, 3 and 5 respectively. These polynucleotides can be used in the genetic engineering production of TAIP proteins.
Polymorphism due to individual differences is generally often observed in human genes. Accordingly, polynucleotides with addition and deletion of one or more nucleotides and/or replacement with other nucleotides in SEQ ID Nos. 1, 3 and 5 are also included in the scope of the invention. Likewise, proteins with addition and deletion of one or more amino acids and/or replacement with other amino acids, occurring according to these changes in polynucleotides, are also included in the scope of the proteins of the invention so long as they possess an activity of proteins having amino acid sequences represented by SEQ ID Nos. 2, 4 and 6.
The recombinant vector of the invention is a cloning vector or an expression vector, and appropriate vector is used according to the type of the polynucleotide as an insert, the use purpose thereof and the like. For example, when TAIP protein is produced using cDNA or ORF region thereof as an insert, an expression vector for in vitro transcription or an expression vector suited for prokaryotic cells such as Escherichia coli and Bacillus subtilis or eukaryotic cells such as yeasts, insect cells and mammal cells can be used. Further, when genomic DNA of the TAIP gene is used as an insert, BAC (Bacterial Artificial Chromosome) vectors, cosmid vectors or the like can also be used.
As the transformant of the invention, prokaryotic cells such as Escherichia coli and Bacillus subtilis, eukaryotic cells such as yeasts, insect cells, mammal cells and the like can be used. This transformant can be prepared by introducing the recombinant vector into cells by a known method such as an electroporation method, a calcium phosphate method, a ribosome method and a DEAE dextran method.
The TAIP protein of the invention can be obtained by isolating from human organs, cell lines, etc., by chemical synthesis to prepare a peptide based on the amino acid sequence of SEQ ID No. 2, 4 or 6, and by a recombinant DNA technology using the purified polynucleotides (cDNAs or translational regions thereof) provided by the invention. The recombinant DNA technology is preferably used to obtain the protein. For example, preparing RNA from the vector having the polynucleotide through in vitro transcription and conducting in vitro translation using it as a template can express the protein in vitro. Further, when the polynucleotide is recombined into an appropriate expression vector by a known method, the protein can be mass-expressed in prokaryotic cells such as Escherichia coli and Bacillus subtilis or eukaryotic cells such as yeasts, insect cells and mammal cells.
In case of producing the TAIP protein by expressing DNA through in vitro translation, the polynucleotide is inserted into a vector having a promoter for an RNA polymerase to form a recombinant vector, and this vector is added to an in vitro translation system containing the RNA polymerase corresponding to the promoter, such as rabbit reticulocyte lysate, wheat germ extract or the like to produce the TAIP protein in vitro. Examples of the RNA polymerase promoter include T7, T3 and SP6. Examples of the vectors containing these RNA polymerase promoters can include pKA1, pCDM8, pT3/T7 18, pT7/3 19 and pBluescript II.
In case of producing the TAIP protein by expressing DNA in microorganisms such as Escherichia coli, an expression vector having an origin replicable in the microorganism, promoter, ribosome binding site, DNA cloning sites, terminator or the like is recombined with the foregoing polynucleotide to form a recombinant expression vector. A host cell is transformed with this expression vector, and the obtained transformant is then cultured, whereby the protein can be mass-produced in microorganisms. At this time, when the expression is conducted by adding an initiation codon and a termination codon before and after an optional translational region, a protein fragment containing the optional region can be obtained. Alternatively, it can also be expressed as a fusion protein with another protein. Only the protein portion that the polynucleotide encodes can also be obtained by cleaving this fusion protein with an appropriate protease. Examples of the expression vector for Escherichia coli can include pUC series, pBluescript II, pET expression system and pGEX expression system.
In case of producing the TAIP protein by expressing DNA in eukaryotic cells, the polynucleotide is inserted into an expression vector for eukaryotic cells having a promoter, a splicing region, a Poly(A) addition site, etc. to form a recombinant vector, and this recombinant vector is transfected into eukaryotic cells. Then, the TAIP protein can be produced in eukaryotic cells. Examples of the expression vector can include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS and pYES2. Further, when pIND/V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1 and the like are used as an expression vector, the TAIP protein can also be expressed as fusion proteins with various tags such as His tag, FLAG tag and GFP added. As eukaryotic cells, mammal culture cells such as monkey renal cell line COS7 and Chinese hamster ovary cell line CHO, budding yeast, fission yeast, Bombyx mori cells, Xenopus egg cells and the like are generally used. However, any eukaryotic cells are available so long as they can express the TAIP protein. For the expression vector to be transfected into eukaryotic cells, a known method such as an electroporation method, a calcium phosphate method, a ribosome method or a DEAE dextran method can be used.
After the expression of the TAIP protein in prokaryotic cells or eukaryotic cells, isolation and purification of the desired protein from the culture can be conducted by a combination of known separation procedures. Examples thereof include treatment with a denaturant such as urea or a surfactant, ultrasonication, enzymatic digestion, salting, solvent precipitation, dialysis, centrifugal separation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, affinity chromatography and reverse phase chromatography.
Incidentally, the recombinant TAIP protein obtained by the foregoing method also includes fusion proteins with any other proteins. Examples thereof can include fusion proteins with glutathine-S-transferase (GST) and green fluorescent protein (GFP). Further, the proteins of the invention sometimes undergo various modifications in cells after translation. Accordingly, the modified proteins are also included in the range of the proteins of the invention. Examples of the modifications after translation can include elimination of N-terminal methionine, N-terminal acetylation, sugar chain addition, limited degradation with an intracellular protease, myristoylation, isoprenylation and phosphorylation.
The peptide of the invention is peptide fragment comprising a part of TAIP protein. This peptide can be prepared by cleaving the TAIP protein with any restriction enzyme. Further, it can also be formed by a known peptide synthesis method based on the amino acid sequences of SEQ ID No. 2, 4 or 6. Such peptide can be used as, for example, antigen for producing the antibody of the invention.
Further, as preferable embodiments, the peptide of the invention include is a peptide consisting of at least five continuous amino acid residues of the 1st-328th amino acid sequence in SEQ ID No. 2, a peptide consisting of at least five continuous amino acid residues of the 1st-326th amino acid sequence in SEQ ID No. 4, and a peptide consisting of at least five continuous amino acid residues of the 1st-301st amino acid sequence in SEQ ID No. 6. That is, as shown in Example 6 to be described later, each of the TAIP proteins has an apoptosis induction activity on the N-terminal. Specifically, TAIP-3 has an apoptosis activity in a peptide of 1st-328th amino acid residues in SEQ ID No. 2, TAIP-2 in 1st-326th amino acid residues in SEQ ID No. 4 and TAIP-12 in a peptide of 1st-301st amino acid residues in SEQ ID No. 6 (FIGS. 3, 4 and 5). Accordingly, the peptide of at least five continuous amino acid residues in these regions can be used as a drug component of an apoptosis inducing agent or the like by combination with cell membrane penetration peptides such as HIV-TAT.
The other embodiment of the peptides of the invention is a peptide comprising at least 385th-389th amino acid sequence in SEQ ID No. 4. That is, the amino acid sequence (Asp-Asp-Asp-Asp-Lys) is known to be a recognition sequence of a bovine enterokinase (Biochemistry 16(15): 3354-3360, 1977). A molecule controlling an activity of the human apoptosis-related protein TAIP can be specified by screening a human enterokinase family with the peptide containing this sequence as a reporter.
The antibody of the invention is polyclonal antibody or monoclonal antibody which recognizes the purified human TAIP protein. Specifically, it is the antibody prepared using the purified TAIP protein or partial peptide thereof as antigen. The antibody of the invention includes the whole molecules Fab, F(ab′)2 and Fv fragments and the like that all can bind to epitopes of the TAIP protein. Such antibody can be obtained from a serum after animal immunized with the TAIP protein or the peptide as antigens. Alternatively, introducing the expression vector for eukaryotic cells into the muscle or the skin of animal through injection or a gene gun and then collecting the serum can produce the antibody. As animals, mice, rats, rabbits, goats, chickens and the like can be used. When B cells collected from the spleen of immunized animal are fused with myelomas to form hybridomas, monoclonal antibody can be produced. This antibody can be used, for example, to detect the expression of the TAIP gene.
EXAMPLES
The present inventions are described in more detail and more specifically with reference to Examples below, but are not to be constructed to be limited thereto. Basic manipulations and enzymatic reactions for DNA recombination are performed according to the reference [“Molecular Cloning, Laboratory Manual” Cold Spring Harbor Laboratory, 1989]. Restriction enzymes and various modification enzymes produced by Lifetech Oriental Inc. were used unless otherwise specified. Composition of buffer and conditions employed in each enzymatic reaction followed the attached instructions.
Example 1
Cloning of TAIP-3
According to the method described in the reference [Aidyama N. et al. Mol. Cell. Biology p.3266-3273, (2000)], human lung adenocarcinoma cell line (A549) were subjected to the gene trap method to allow to select cell clones in which expression is enhanced by TGF-β. From one of these cell clones, the gene sequences of 5′-untranslated region and 3′-untranslated region of cDNA clone (TAIP-3) were obtained as a trapped gene.
On the basis of the information, using 27mer sense primer (oligonucleotide of SEQ ID NO 7) and 30mer antisense primer (oligonucleotide of SEQ ID NO 8), the regions were amplified by PCR using cDNAs prepared from mRNAs extracted from human fibroblast MRC-5 cells as templates. After blunting the PCR product with T4 polymerase, the resultant was subject to 5′phosphorylation by T4 polynucleotide kinase and then inserted into pBluescriptII SK- EcoRV site to make transformation to host Escherichia coli DH-5 α FT (Gibco BRL). The nucleotide sequence of the obtained clone was determined and the clone caused no amplification error during the reverse transcription reaction and PCR reaction was selected. The obtained clone TAIP-3 consisted of a total length of 2593 bp having a structure comprising 106 bp of 5′-untranslated region, 1767 bp of open reading frame (ORF) and 720 bp of 3′-untranslated region (SEQ ID NO. 1). The ORF encoded a protein comprising 589 amino acid residues (SEQ ID NO. 2).
Example 2
Cloning of TAIP-2
In the case of making a search of human genomic database using cDNA of TAIP-3 determined in Example 1 (SEQ ID NO. 1), genomic DNA clone of Chromosome 2 (GenBank: AC023867) showed homology. Using 28mer sense primer (oligonucleotide of SEQ ID NO. 9) and 30mer antisense primer (oligonucleotide of SEQ ID NO. 10), the homologous cDNA fragment was amplified by PCR using plasmid DNA extracted from commercial library prepared from human fetal brain (ProQuest Two-Hybrid Human Fetal Brain cDNA Library: Life Technologies Inc., catalogue number: 11386-018) as a template. After blunting the PCR product with T4 polymerase, the resultant was subject to 5′phosphorylation by T4 polynucleotide kinase and then inserted into pBluescriptII SK-EcoRV site to make transformation to host Escherichia coli DH-5 α FT (Gibco BRL). The nucleotide sequence of the obtained clone was determined and the clone caused no amplification error during the reverse transcription reaction and PCR reaction was selected. The obtained clone TAIP-2-part consisted of a total length of 301 bp and corresponded with the partial sequence of the database clone (GenBank: AC023867). The obtained insert was used as a probe, commercial library prepared from human fetal brain (ProQuest Two-Hybrid Human Fetal Brain cDNA Library: Life Technologies Inc., catalogue number: 11386-018) was subject to screening by hybridization using a radioisotope to obtain a clone (TAIP-2). The obtained clone consisted of a total length of 2975 bp having a structure comprising 263 bp of 5′-untranslated region, 1755 bp of ORF, and 957 bp of 3′-untranslated region (SEQ ID NO .3). The ORF encoded a protein comprising 585 amino acid residues (SEQ ID NO. 4).
Example 3
Cloning of TAIP-12
In the case of making a search of human genomic database using cDNA of TAIP-3 determined in Example 1 (SEQ ID NO. 1), genomic DNA clone of Chromosome 12 (GenBank: AC023648) showed homology. In the case of making a search again of human EST database using information from 5′-end sequence of the genomic clone, the clone (AA305194) was thought to include 5′-untranslated region of a cDNA homologous to TAIP-3.
In addition, two gene-specific 30mer oligonucleotide (SEQ ID NO. 11 and 12) were prepared for sense primer toward downstream of ORF from the sequences in the region of the genomic DNA clone of Chromosome 12 (GenBank AC023648) having homology with TAIP-3. Using these primers, 3′-RACE was performed by the method described in the reference [Akiyama N., et al Mol. Cell. Biology p.3266-3273, (2000)] using cDNA prepared from mRNA extracted from human lung adenocarcinoma cell line A549 as templates. To amplify all regions including ORF from these obtained sequences, using 30mer sense primer (oligonucleotide of SEQ ID NO. 13) and 29mer antisense primer (oligonucleotide of SEQ ID NO. 14) synthesized the regions were amplified by PCR using cDNA prepared from mRNA extracted from human fibroblast MRC-5 cells as a template. After blunting the PCR product with T4 polymerase, the resultant was subject to 5′phosphorylation by T4 polynucleotide kinase and then inserted into pBluescriptII SK-EcoRV site to make transformation to host Escherichia coli DH-5 α FT (Gibco BRL). The nucleotide sequence of the obtained clone was determined and the clone caused no amplification error during the reverse transcription reaction and PCR reaction was selected. The obtained clone TAIP-12 consisted of a total length of 1682 bp having a structure comprising 27 bp of 5′-untranslated region, 1629 bp of ORF and 26 bp of 3′-untranslated region (SEQ ID NO. 5). The ORF encoded a protein comprising 543 amino acid residues (SEQ ID NO. 6).
Example 4
Confirmation of Apoptosis by Expression of Tag Fusion Proteins on Human Cells
Using cDNAs of TAIP-3, TAIP-2, and TAIP-12 specified in Example 1, Example 2, and Example 3 respectively as templates, the translational region was amplified by the PCR method. The PCR primers used were as follows: a 36mer sense primer (oligonucleotide of SEQ ID NO. 15) and a 33mer antisense primer which was added XhoI site to immediately after the termination codon (oligonucleotide of SEQ ID NO. 16) in the case of TAIP-3, a 36mer sense primer to which added BamHI site (oligonucleotide of SEQ ID NO. 17) and a 33mer antisense primer, which was added XhoI site to immediately after the termination codon (oligonucleotide of SEQ ID NO. 18) in the case of TAIP-2, and a 36mer sense primer to which is added BamHI site (oligonucleotide of SEQ ID NO. 19) and a 33mer antisense primer added SalI site immediately after the termination codon (oligonucleotide of SEQ ID NO. 20) in the case of TAIP-12.
The PCR product was purified and cleaved with a restriction enzyme in which a recognition site is added to each primer. After the obtained insert was inserted into an unique site from BamHI site to XhoI site of a modified vector (pFMH-3.1), in which tags and multicloning sites from NheI site to AflII site of a Tag fusion protein expression vector (pcDNA3.1/myc-His(−)B: Invitrogen) are substituted for cDNA of SEQ ID NO. 21, the resultant plasmid was transformed to host Escherichia coli DH-5αFT (GibcoBRL). The nucleotide sequence was confirmed and a clone expressing Myc tag and His tag as a fusion protein on the C-terminal of ORF in each cDNA was selected. The clone was cultured in LB culture medium at 37° C. for 16 hours to purify an expression plasmid DNA.
The plasmids were introduced into human lung adenocarcinoma cell line A549 cells, human embryo kidney cell line HEK293, and human teratocarcinoma cell line NTERA-2 according to attached instructions of lipofectamin2000 (GibcoBRL), 24 hours thereafter the cells were treated with an reagent detecting apoptotic cells, fluorescent-labeled Annexin V (Medical and Biological Laboratory) and a marker pigment for dead cells, PI (propidium iodide), and 15 minutes thereafter observations under a fluorescence microscope revealed the cells cause apoptosis (FIGS. 1 and 2).
Example 5
Confirmation of Apoptosis by Expression of Fusion Protein Comprising Green Fluorescent Protein (GFP) and Full Length Protein
Using cDNAs of TAIP-3, TAIP-2, and TAIP-12 specified in Example 1, Example 2, and Example 3 respectively as templates, the tanslational region was amplified by the PCR method. The PCR primers used were as follows: a 46mer sense primer to which added Kozak initiation sequence and BamHI site (oligonucleotide of SEQ ID NO. 22) and a 33mer antisense primer which was added XhoI site to immediately after the termination codon (oligonucleotide of SEQ ID NO. 23) in the case of TAIP-3, a 43mer sense primer to which added Kozak initiation sequence and BamHI site (oligonucleotide of SEQ ID NO. 24) and a 33mer antisense primer which was added XhoI site to immediately after the termination codon (oligonucleotide of SEQ ID NO.25) in the case of TAIP-2, and a 43mer sense primer to which is added Kozak initiation sequence and BamHI site (oligonucleotide of SEQ ID NO. 26) and a 34mer antisense primer added SalI site immediately after the termination codon (oligonucleotide of SEQ ID NO. 27) in the case of TAIP-12. The obtained PCR product was purified and cleaved with a restriction enzyme in which a recognition site was added to each primer. After the obtained insert was inserted into from BamHI site to XhoI site of a GFP expression vector (pEGFP-N3: Clontech), the resultant plasmid was transformed to host Escherichia coli DH-5αFT (GibcoBRL). The nucleotide sequence was confirmed and a clone expressing a protein allowing GFP to fuse to the C-terminal of ORF in each cDNA was selected. The clone was cultured in LB culture medium at 37° C. for 16 hours to purify an expression plasmid DNA.
The plasmids were introduced into human fetal hepatocyte line HEK293 and human teratocarcinoma cell line NTERA-2 according to attached instructions of lipofectamin2000 (GibcoBRL) and then 24 hours thereafter observations under a fluorescence microscope revealed the cells in which the fusion protein expressed showed typical image for apoptosis (FIGS. 3, 4, and 5).
Example 6
Confirmation of Apoptosis by Expression of Fusion Protein Comprising Green Fluorescent Protein (GFP) and N-Terminal or C-Terminal Region of Each Protein
Using cDNAs of TAIP-3, TAIP-2, and TAIP-12 specified in Example 1, Example 2, and Example 3, respectively, as templates, the N-terminal and C-terminal regions, respectively, were amplified by PCR method. In the case of N-terminal region of TAIP-3 (amino acid position 1-328 of SEQ ID NO. 2), a 46mer sense primer to which added Kozak initiation sequence and BamHI site (oligonucleotide of SEQ ID NO. 22) and a 34mer antisense primer which was added XhoI site (oligonucleotide of SEQ ID NO. 28) were used and in the case of C-terminal region of TAIP-3 (position 328-589 of SEQ ID NO. 2) a 46mer sense primer to which added Kozak initiation sequence and BamHI site (oligonucleotide of SEQ ID NO. 29) and a 33mer primer which was added XhoI site to immediately after the terminatin codon (oligonucleotide of SEQ ID NO. 23) were used. In the case of N-terminal region of TAIP-2 (amino acid position 1-326 of SEQ ID NO. 4) a 43mer sense primer to which is added Kozak initiation sequence and BamHI site (oligonucleotide of SEQ ID NO. 24) and a 34mer antisense primer added XhoI site (oligonucleotide of SEQ ID NO. 30) were used and in the case of C-terminal region of TAIP-2 (position 327-585 of SEQ ID NO. 4) a 46mer sense primer to which added Kozak initiation sequence and BamHI site (oligonucleotide of SEQ ID NO. 31) and a 34mer antisense primer which was added XhoI site to immediately after the termination codon (SEQ ID NO. 25) were used. Further, in the case of N-terminal region of TAIP-12 (position 1-301 of SEQ ID NO. 6) a 43mer sense primer to which is added Kozak initiation sequence and BamHI site (oligonucleotide of SEQ ID NO. 26) and a 34mer antisense primer added SalI site (oligonucleotide of SEQ ID NO. 32) were used and in the case of C-terminal region (position 302-543 of SEQ ID NO. 6) a 46mer sense primer to which added Kozak initiation sequence and BamHI site (oligonucleotide of SEQ ID NO. 33) and a 34mer antisense primer which was added SalI site to immediately after the termination codon (oligonucleotide of SEQ ID NO. 27) were used. The obtained PCR product was purified and cleaved with a restriction enzyme in which a recognition site was added to each primer. After the obtained insert was inserted into from BgIII site to SalI site of a GFP expression vector (pEGFP-N3: Clontech), the resultant plasmid was transformed to host Escherichia coli DH-5αFT (GibcoBRL). The nucleotide sequence was confirmed and a clone expressing a protein allowing GFP to fuse to the C-terminal region or C-terminus of N-terminal region of each protein was selected. The clone was cultured in LB culture medium at 37° C. for 16 hours to purify an expression plasmid DNA.
The each plasmid was transfected to human embryo kidney cells HEK293 and human teratocarcinoma cell line NTERA-2 according to attached instructions of lipofectamin2000 (GibcoBRL) and then 20 hours thereafter observations under a fluorescence microscope confirmed the cells in which the fusion protein of N-terminal region and GFP expressed showed typical image for apoptosis. Moreover, it was confirmed that no typical image for apoptosis was observed in the cells in which the fusion protein of C-terminal region and GFP were expressed. Therefore, it was turned out that even partial polypeptide of N-terminal region in each protein had a function inducing apoptosis. (FIGS. 3, 4, and 5)
Example 7
Induction of Expression by TGF-β
RNA was extracted from A549 cells (1×108 cells), which were untreated or treated with TGF-β for 48 hours and analyzed by the Northern blot analysis. Using a 30mer sense primer initiating from the initiation codon (oligonucleotide of SEQ ID NO. 34) and a 30mer antisense primer up to immediately before the termination codon (oligonucleotide of SEQ ID NO. 35) of TAIP-3 cDNA (SEQ ID NO. 1), the translational region was amplified by PCR, the obtained product was used as a probe to allow hybridization, and it was confirmed that approximately 3 kb of mRNA was expressed in A549 cells and that expression of TAIP-3 was enhanced by TGF-β treatment.
Example 8
Tissue Distribution of TAIP
Multiple Tissue Northern blots (Clontech), which are blots of mRNA extracted from human tissues and human cancer cell lines on nylon membranes was hybridized with [32P]dCTP-labeled probe prepared with Taip-2, 3 and 12 polynucleotides encoding C-terminal region of their products.
According to the results, Taip-3 is expressed high in lung, peripheral blood leukocytes, adrenal gland, mammary gland, and Taip-12 is expressed all the tissues examined and relatively high expression was observed in brain, heart, skeletal muscle, kidney, placenta and adrenal gland. Expression of Taip-2 was not detected in any tissue examined (FIG. 6 and data not shown).
Example 9
DNA Degradation Induced by TAIP
The plasmids encoding GFP fusion protein with full-length, N- or C-halves of Taip genes products or GFP only (control) were transfected to human embryo kidney cell line HEK293 with lipofectamin2000 (Gibco BRL) according to attached instructions. Twenty-four hours later, DNA contents of GFP expressing cells were analyzed with FACSCalibur (BD Bioscience).
According to the results, in the cells expressing fill-length or N-terminal region of TAIP proteins, DNA degradation were confirmed (FIG. 7).
Example 10
Caspase-3 Activation in TAIP Expressing Cells
The plasmids encoding GFP fusion protein with N-halves of Taip-2, 3, 12 gene products or GFP only (control) were transfected into human embryo kidney cell line HEK293 with lipofectamin2000 (Gibco BRL) according to attached instructions and cell lysates were prepared 24 h later. Each sample (30 μg) was electrophoresed on a 20% polyacrylamide gel, blotted onto a nitrocellulose filter and detected with an anti-caspase-3 polyclonal antibody (BD Bioscience). Active form of caspase-3 was detected as bands near 17 kDa in Taip gene transfectants. The results indicated that caspase-3 was activated in the transfectants of these plasmids (FIG. 8).
Example 11
Preparation of Anti-TAIP-3 Antibody and Endogenous TAIP-3 Protein Induction by TGF-β
Rabbit polyclonal antibody against TAIP-3 was prepared by using SEQ ID No. 36 as antigen according to a standard protocol. A549, a human lung adenocarcinoma cell line were treated with TGF-β for 0, 3, 6, 12 or 24 hrs and normal human hepatocytes were treated with TGF-β for 0, 3, 6, 12, 36 or 60 hrs. The cells treated with TGF-□ for indicated time were lysed and analyzed with western blotting with anti-TAIP3 antibody.
The results indicated that TAIP-3 was not detected in human lung adenocarcinoma cell line A549 before TGF-β treatment, and detected 3 to 5 hrs after TGF-β treatment (FIG. 9). In A549 cells, which do not undergo apoptosis by TGF-β, TAIP-3 expression decreased at 24 hr after TGF-β addition (FIG. 9, Upper panel) while in normal human hepatocytes, which undergo apoptosis by TGF-β, TAIP3 expression can be detected even 60 hrs after TGF-β addition (FIG. 9, Lower panel).
Example 12
Subcellular Localization of TAIP-3 in TGF-β Treated Cells
A549 cells, normal human hepatocytes and HLF a non-differentiated hepatoma cell line (obtained from Health Science Research Resources Bank) were treated with TGF-β, fixed and permealized with Triton-X100. The subcellular localization of TAIP-3 in these cells was observed by conforcal microscope after treatment with anti-TAIP3 and Cy3-conjugated secondary antibodies (Jackson ImmunoResearch).
In A549 cells, TAIP3 was observed around the nucleus after 12 hr TGF-β treatment (FIG. 10, Upper panel) but gradually became undetectable thereafter. On the other hand, in normal human hepatocytes and HLF cells, TAIP3 was detected as small lumps around the nucleus and then the cells with crumpled nuclei were observed as small aggregates (FIG. 10, Lower panel), resulted in apoptosis.
Example 13
Caspase-3 Activation in TAIP-3 Expressing Cells
Normal human hepatocytes and HLF human hepatoma cell line were treated with PhiPhiLuxTMG1D2kit (OncoImmunin) after culturing in the presence or absence of TGF-β for 24 hr and caspase-3 activation in the cells treated with TGF-β were detected with flow cytometry (FIG. 11). In these cells with activated caspase-3, higher level of TAIP-3 expression was detected by western blot analysis with anti-TAIP-3 antibody (FIG. 12, Upper panel). The lower panel shows proteins on the immunoblot filter stained with amid black.
From the results shown in examples 11 to 13 revealed the strong correlation between TAIP-3 expression and TGF-β-induced apoptosis.
Example 14
Anti-TAIP Antibody and Detection of TAIP-2 in a Brain Disorder
Rabbit polyclonal antibody against TAIP-2 was prepared by using SEQ ID No. 37 according to a standard protocol. The section prepared from the brain with focal ischemic damage was Immunohistochemiclly analyzed with anti-TAIP2 antibody by using LSAB kit (DAKO). Although no significant staining was observed with preimmuno serum, significant spotted staining was observed with anti-TAIP-2 antibody (FIG. 13). These results indicate that TAIP2 can be expressed in the brain with focal ischemic damage and suggested that anti-TAIP-2 antibody may be useful for diagnosis of the brain disorders induced by apoptosis.
INDUSTIAL APPLICABILITY
As mentioned above in detail, the present invention provides novel human proteins relating to apoptosis and the antibodies useful for the diagnosis, therapy, and etc. of apoptosis-related diseases such as cancer and autoimmune disease. Also, the utilization of the cDNA of the invention enables the mass production of the above proteins. And a screening of low molecular compounds bound to the proteins enables the search for pharmaceuticals such as a new type of antitumor drug.

## Claims

1. A purified human apoptosis-related protein comprising the amino acid sequence of SEQ ID NO: 2.

2. The purified human apoptosis-related protein of claim 1, which is an expression product of a human apoptosis-related gene that encodes a human apoptosis-related protein, and said protein is produced by a transformant, which has been transformed with a recombinant vector comprising a polynucleotide encoding the human anoptosis-related protein having the amino acid sequence of SEQ ID NO: 2.

3. A purified or synthesized peptide, 1st328th amino acids of SEQ ID NO:2.