WO1999031237A1

WO1999031237A1 - Apoptosis inhibitory factor

Info

Publication number: WO1999031237A1
Application number: PCT/JP1998/005609
Authority: WO
Inventors: Yasuo Uchiyama; Yoshiyuki Ohsawa
Original assignee: Taisho Pharmaceutical Co., Ltd.
Priority date: 1997-12-12
Filing date: 1998-12-11
Publication date: 1999-06-24
Also published as: AU1506399A; JPH11225771A

Abstract

A protein obtained from a culture medium of a pheochromocytoma transformed by a bcl2 gene as a protooncogene and having the activity of inhibiting apoptosis; and a method for cloning a gene encoding the protein. This protein can contribute to the development of remedies for diseases caused by apoptosis.

Description

Description Cell death inhibitor Technical field

The present invention relates to a factor having an activity of suppressing cell death (cell death suppressing factor, hereinafter referred to as PCTF35), a gene encoding the same, and a pharmaceutical use thereof.

Background art

Many neurodegenerative diseases are said to cause neurons to die and develop due to abnormalities in nerve cells or signal transmission between nerve cells.

Representative examples of such neurodegenerative diseases include Parkinson's disease and Alzheimer's disease. Parkinsonism is said to be caused by the degeneration of the substantia nigra neurons and the cessation of production of dopamine, one of the neurotransmitters, and the death of dopaminergic neurons. On the other hand, Alzheimer's disease is said to cause dementia mainly due to death of neurons in the cerebral cortex and hippocampus.

For the diseases related to nerve cell death described above, it is assumed that substances that suppress the nerve cell death, which is the cause of the disease, or promote the growth or growth of nerve cells, etc., can be effective therapeutic agents. Research is being actively conducted.

Recently, the proto-oncogene bc2 gene (Tsujimoto et al., Science, Vol. 228, pl440-1443, 1985) has been reported to suppress cell death (Vaux D. et al., Nature, Vol. 335, p440-442, 1988). Various mechanisms have been proposed for its function to suppress cell death, but they have not been fully proved.

Identification of a protein that suppresses nerve cell death, which is thought to be the cause of neurological disease, or that promotes nerve cell growth, and the gene that encodes it, will provide an effective therapeutic agent for diseases caused by nerve cell death. It can be provided, and is extremely important in searching for compounds that mimic it. That is, a protein having an activity of suppressing the death of nerve cells can itself be an effective medicine, and at the same time, has a substance having a function similar to that of the protein, and has an action of inhibiting or promoting the function. It is also very useful when developing substances as drugs. Disclosure of the invention

The present inventors have focused on the b _C 2 gene suppresses cell death, assuming inducible expression of proteins involved in curbing cell death, Toko filtrate was examined for the presence of such protein, By introducing the b2 gene, it was found that a factor (PCT F35) having the activity of suppressing cell death was secreted out of the transformed cells. Then, the PCTF 35 was purified and isolated, and further, based on the purified partial amino acid sequence of PCTF 35, the gene encoding the same (hereinafter referred to as pctf 35) was cloned, and the present invention was carried out. completed.

That is, the present invention relates to a protein comprising the amino acid sequence of SEQ ID NO: 1 or a sequence comprising the amino acid sequence of SEQ ID NO: 1 in which one or several amino acids have been deleted, replaced or added. It is a protein that has the activity of inhibiting cell death.

Further, the present invention relates to a gene encoding a protein comprising the amino acid sequence of SEQ ID NO: 1.

Furthermore, the present invention relates to a DNA comprising the nucleotide sequence of SEQ ID NO: 2, or a protein that hybridizes with the DNA of SEQ ID NO: 2 under stringent conditions and has an activity of suppressing cell death. The DNA to be loaded.

Further, the present invention relates to a cell death inhibitory factor characterized by being a proteinaceous substance having the following physicochemical properties and physiological activities (1) to (4).

(1) It can be obtained from a culture of a pheochromocytoma transformed with the proto-oncogene bc £ 2 gene;

(2) Estimated molecular weight by reduced SDS-PAGE is about 35,000;

③ has the activity of suppressing cell death;

アミノ酸 The amino acid sequence from the 1st to 22nd amino acid on the N-terminal side is the following amino acid sequence; Va1etG1ySerG1yAspSperVa1

ProG1yG1yVa1CysTrpLeuGin

G in G 1 y L y s G 1 u A 1 a T h r

⑤ The amino acid sequence at the N-terminal 1 38 to 14 1 is the following amino acid sequence Ty r A rg G ly A rg

Further, the present invention is a pharmaceutical composition for suppressing cell death, comprising the protein or the cell death inhibitor as an active ingredient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a photograph showing the morphology of an organism, showing wild type PC12 cells in a growing state (the cell density is 2500 cells).

Figure 2 is a photograph showing the form of the biologically, wild-type PC 1 2 cells (cell density 2 500. ₁ 11 2) after 24 hours was shifted to serum-free medium, killing conditions 1 shows wild-type PC12 cells.

Figure 3 is a photograph showing the morphology of the organism. Wild-type PC12 cells (cell density of 2500 cells _< 1112) were transferred to a serum-free medium containing the culture supernatant of PC12bc2 cells. The figure shows wild-type PC12 cells 24 hours after the transfer.

FIG. 4 shows a reversed-phase chromatograph of a culture supernatant of PC12 / bc £ 2 cells using 60% acetonitrile containing 0.05% of choline as a gradient solvent. Ordinate left and bar one graph the cell death suppressing activity (% NGF) the activity of each fraction, the longitudinal axis right and line shows the solvent gradient (Asetonitoriru concentration. / _0).

FIG. 5 is a photograph showing the results of electrophoresis, and shows a gel stained with silver after fractionation 11 obtained by reverse phase chromatography was subjected to SDS-PAGE.

Figure 6 is a photograph showing the results of electrophoresis. The fraction 11 obtained by reversed-phase chromatography was subjected to Natent-PAGE, followed by CBB-stained gel and the position of the confirmed protein. Or — as shown in 4.

FIG. 7 shows the cell death inhibitory activities of the proteins corresponding to -1 to -4 in FIG. 6 (the vertical axis represents% activity).

FIG. 8 is a photograph showing the result of electrophoresis, and is a diagram of a gel of a protein corresponding to -1 in FIG. 6 in which cell death inhibitory activity was confirmed, which was subjected to silver staining after SDS-PAGE. FIG. 9 shows the N-terminal amino acid sequence of PCTF35 determined by the amino acid sequencer. In the figure? Means an unidentified residue.

FIG. 10 shows the amino acid sequence at the N-terminus of a 28 kd protein, which is a limited digest of PCTF 35, determined by the amino acid sequencer. FIG. 11 is a photograph showing the results of electrophoresis and shows the results of exposure of Northern hybridization to mRNA extracted from a transformant containing the gene pctf35. Lanes 1 to 3 show each clone of PCI 2Zp CTF cells, lane 4 shows PC12 / pc DNA3 cells, and lane 5 shows wild-type PC12 cells.

FIG. 12 shows the survival rate of PC 12 / p CTF cells in serum-free medium.

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors transduced the b2 gene into rat PC12 cells, which are pheochromocytomas, and analyzed the transformed cells (hereinafter referred to as PC12Zbc2 cells) for functional analysis of the b2 gene. The behavior was examined in detail.

Wild-type PC12 cells will die if serum is removed from a suitable growth medium. However, PC12 / bc2 cells transformed with the b2 gene continued to grow without cell death even in a serum-free medium, and exhibited the behavior of extending projections.

The present inventors have paid attention to the behavior of the PC12 / b2 cells and assumed that a factor having an activity of suppressing cell death may be secreted out of the cells. Therefore, a culture supernatant was prepared after culturing PC 12Zb c £ 2 cells, and this was added to a serum-free medium, that is, wild-type PC 12 placed in an environment where the cells died. It was confirmed that the wild-type PC12 continued to grow similarly to the PC12 / b2 cells, and extended the protrusion. This is, PC 12Zb _C f 2 cells, showing that the releasing agent having activity of inhibiting the death of the other PC I 2 cells culture ground.

The present inventors investigated the factors that suppress cell death, which are thought to be present in this medium, by using reversed-phase high-performance liquid chromatography (RP-HPLC) using 60% acetonitrile as a developing solvent, and polyacrylamide electrophoresis. (PAGE) was used for purification. As a result, a protein having a molecular weight of 35 kDa in SDS-PAGE could be recovered. By confirming that the recovered protein had an activity to suppress cell death, it was determined that the protein was the target cell death inhibitory factor, that is, PCTF35.

In addition, the present inventors have studied the N-terminal 21 residues of PCTF35 purified by the above method. , And the N-terminal amino acid sequence of the limited digest obtained by cutting the peptide chain of PCTF 35 with Bromcian were analyzed (FIGS. 9 and 10). Oligonucleotides consisting of the deduced nucleotide sequence were synthesized based on these amino acid sequences, and messenger RNA (mRNA) prepared from PC12 cells by the polymerase chain reaction (PCR) method using this as a probe. From the cDNA library prepared from the above, the gene pctf35 encoding PCTF35 could be cloned.

From the amino acid sequence of PCTF35 determined from the cloned gene, the N-terminal amino acid sequences shown in FIGS. V a 1 Met G ly SerG 1 yAspSerVa1ProGlyGlyGlyVa1CysTrpLeuGinGinGlyLysG1uA laThr) 138-141 amino acid sequence at the N-terminal side (TyrArgGly

A r g).

The gene pctf35 corresponds to a region encoding a PCTF35 consisting of a total of 256 amino acid residues including a signal peptide in the 1645 base pair (bp) base sequence shown in SEQ ID NO: 3. Is a gene that Further, based on the analysis results of the nucleotide sequence and the N-terminal sequence of PCTF35, PCTF35 of the present invention is a secreted protein secreted extracellularly by a sidanal peptide consisting of 23 amino acid residues. This secreted PCTF 35 is a protein with an estimated molecular weight of 24.9 kd consisting of 233 amino acid residues.According to the measurement of the molecular weight by SDS_PAGE, the molecular weight of secreted PCTF 35 is 35 kd. Therefore, it is considered that PCTF35 has undergone some modification such as a sugar chain.

The activity of PCTF 35 of the present invention to suppress cell death means that when PCTF 35 is added to cells, the cells continue to grow without being killed even in the absence of NGF and serum. Means that. When acted on PC12 cells, PCTF35 of the present invention exhibits an activity of suppressing cell death and an activity of promoting extension of projections. In other words, wild-type PC12 cells were prepared from serum or NGF (Nurve

Growth Factor: Nerve cell growth factor, Greene L A., J. Cel 1. Biol., Vol. 78, Continue growth in a suitable medium containing p747 (1978)) (Fig. 1). Transfer cells in this state to a medium without serum and NGF at a low density of 2500 cells / c. Eventually, the PC12 cells shrink and 90 to 100% of the whole die (Fig. 2). When the culture supernatant of PC12 / bc12 cells in serum-free medium was added to the medium from which serum and NGF had been removed, PC12 cells survived in the same manner as when NGF was added (Fig. 3)

Such activity of PCTF 35 to suppress cell death can be confirmed by observing cell survival. In addition, to measure more easily, a simple method for measuring the activity of NGF, which exhibits cell death-inhibiting activity similar to PCTF35 (Sasaki et al., Abstracts of the 4th Annual Meeting of the Japan Society for Alternatives to Animal Experiments, 1990, According to p. 85), it is also possible to confirm the change in lactate dehydrogenase (LDH) activity in the cells after adding PCTF 35 to the cells.

Thus, it is thought that PCTF 35, which is a protein that suppresses cell death, can be an effective therapeutic agent for diseases caused by cell death. In addition, PCTF 35 and the gene encoding it, Pctf 35, are extremely useful in developing a substance having a function similar to that of the protein, a substance having an action of inhibiting or promoting the function, or the like as a medicine. In particular, the gene pctf35 is indispensable for mass-producing PCTF35 using genetic recombination technology and the like.

In the present invention, any gene that encodes a protein having the amino acid sequence shown in SEQ ID NO: 1 is within the scope of the present invention. In the present invention, in addition to the DNA sequence shown in SEQ ID NO: 2, a DNA that hybridizes with the DNA and encodes a bioactive protein having an activity of suppressing cell death is also included in the scope of the present invention.內.

That is, the full-length sequence of the gene pctf35 is partially modified by various artificial treatments, for example, site-directed mutagenesis, random mutation by treatment with a mutagen, and mutation / deletion / ligation of DNA fragments by restriction enzyme cleavage. Even if the DNA sequence is changed, it is a DNA that hybridizes with the gene pctf35 under stringent conditions and encodes a bioactive protein having cell death inhibitory activity, even if the DNA sequence is changed. Irrespective of the difference from the DNA sequence shown in SEQ ID NO: 2 Are within the range.

The degree of the above-mentioned DNA mutation is 90 ° / 90 ° with the DNA sequence of the gene pctf35. A substance having the above homology is acceptable. The degree of hybridization with the gene P ctf 35 can be determined under normal conditions (for example, when the probe is labeled with DIG DNA Labeling kit (Cat No. 1175033 manufactured by Boehringer Mannheim) at 32 ° C). In DIGE asy Hy b solution (Berlinger's Mannheim Co., Cat No. 1 603558) in 0.5 x SSC solution (0.1% [w // v] SDS at 50 ° C) Hybridization to the gene pctf35 with Southern hybridization under washing conditions (1 XS 3 is 0.15 M NaCl, 0.015 M sodium citrate) in Further, even a protein having a sequence different from the amino acid sequence shown in SEQ ID NO: 1 is a protein encoded by a mutant gene having high homology to gene pctf35 as described above. Physiology with cell death inhibitory activity Active proteins are within the scope of the present invention.

That is, even if a mutant in which one or more amino acids of the amino acid sequence of PCTF 35 of the present invention is deleted, substituted or added, the mutant is a protein having cell death inhibitory activity. Variants are within the scope of the present invention. The side chains of the amino acids that are the constituents of proteins differ in hydrophobicity, charge, size, etc., but do not substantially affect the three-dimensional structure (also called three-dimensional structure) of the entire protein In this sense, some highly conservative relationships are known empirically and by physicochemical measurements. For example, for substitution of amino acid residues, glycine (G1y) and proline (Pro), G1y and alanine (A1a) or valine (Va1), leucine (Leu) and isoloicin (I 1 _e ), glutamic acid (G1u) and glutamine (Gin), aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), Thr and serine (S _e r) or a 1 a, lysine (L ys) and arginine (a rg), and the like.

Accordingly, substitutions, insertions, and substitutions in the amino acid sequence of PCTF 35 shown in SEQ ID NO: 1 Even if it is a mutant protein due to deletion or the like, the mutation is a highly conserved mutation in the three-dimensional structure of PCTF 35, and the mutant protein has a cell death inhibitory activity like PCTF 35 If so, they can be said to be within the scope of the present invention. The degree of mutation is within a range in which homology with the amino acid sequence shown in SEQ ID NO: 1 is 90% or more.

Examples of such a mutant protein include a protein consisting of an amino acid sequence obtained by removing the amino acid sequence corresponding to the fern peptide from the amino acid sequence shown in SEQ ID NO: 4. The amino acid sequence shown in SEQ ID NO: 4 was obtained as follows. That is, as a result of searching a human fetal brain cDNA library using a part of the gene shown in SEQ ID NO: 3 as a probe, a sequence having high homology to the gene shown in SEQ ID NO: 2 was obtained. A gene which is considered to encode a human-derived cell death inhibitory factor shown in No. 5 was obtained. The amino acid sequence shown in SEQ ID NO: 4 was obtained from the nucleotide sequence of this gene, and a protein having the amino acid sequence of this amino acid sequence excluding the amino acid sequence corresponding to the signal peptide was obtained. Is a mutant protein of the protein consisting of the amino acid sequence shown in Fig. 1 and is considered to be a human-derived cell death inhibitor.

As described above, PCTF 35 of the present invention can be produced in a medium by culturing rat PC12 cells transformed with the b2 gene in an appropriate medium. The bc2 gene is recombined into a shuttle vector that can be replicated and maintained in both PC12 cells and Escherichia coli or Bacillus subtilis, such as pcDNA3, pAGE123, etc. It can be introduced into PC12 cells by a general gene transfer method such as the Yong method. In addition, the transformed PC12 / bc £ 2 cells may be cultured in an appropriate medium such as RPMI1640 medium or HDMEM medium, and require no special culture method or means. do not do.

The PCTF 35 of the present invention is prepared by linking the gene pctf 35 of the present invention to a vector for recombination by a general gene recombination technique to prepare a recombinant gene pctf 35, It can also be produced by expression in a single host vector system. A suitable vector is a plasmid derived from E. coli (eg, pBR32, pUCll8, etc., Bacillus subtilis-derived plasmids (eg, pUB110, pC194, etc.), yeast-derived plasmids (eg, pSH19, etc.), and more Animal viruses such as bacteriophage II retrovirus and vaccinia virus can be used. Upon recombination, it is also possible to add a translation start codon and a translation stop codon using an appropriate synthetic DNA adapter. Furthermore, in order to express the gene, an appropriate expression promoter can be connected upstream of the gene. The promoter to be used may be appropriately selected depending on the host. For example, when the host is Escherichia coli, a T7 promoter, a lac promoter, a trp promoter, a p-promoter, etc., and when the host is a Bacillus genus, an SPO-based promoter, etc. When the host is a yeast, a PHO5 promoter, a GAP promoter, an ADH promoter, or the like can be used. When the host is an animal cell, an SV40-derived promoter, a retrovirus promoter, or the like can be used.

The PCTF35 of the present invention can also be expressed as a so-called fusion protein by linking the gene pctf35 to a gene encoding another protein (eg, glutathione S transferase, protein A, etc.). It is possible. The fused PCTF35 expressed in this manner can be excised using an appropriate protease (eg, thrombin or the like).

Examples of the host that can be used for expression of the PCTF 35 of the present invention include various strains of Escherichiaco 1 i that is a genus Escherichia, various strains of Bacillus aci 1 1 uss υ bti 1 is, and yeasts. Various strains of S accharo my cescerevisiae and animal cells include COS-7 cells, CHO cells, PC12 cells and the like.

As a method for transforming the above host cells using the recombinant gene pctf35, a transformation method generally used in accordance with the host cells to be used, such as a calcium chloride method or an electroporation method, can be applied. it can.

The protein of the present invention, for example, a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or a mutant protein thereof, for example, the amino acid sequence shown in SEQ ID NO: 4 excluding the amino acid sequence corresponding to the signal peptide, Proteins consisting of amino acids It is useful as a drug that suppresses For example, it can be specifically applied to the treatment of diseases related to nerve cell death, such as Parkinson's disease and Alzheimer's disease.

When applied to these diseases, the protein of the present invention can be prepared as a lyophilized product by a conventional method and applied to humans, for example, as an injection. The dose may vary depending on the subject to be applied, the administration route and the like, but is usually from lg to l001718 _/ body weight 1 ^ g · day.

Hereinafter, the present invention will be described more specifically with reference to Examples and Experimental Examples.

Various operations in the examples described below are performed according to the instructions attached to the kit when a commercially available experimental kit is used, and various operations known to those skilled in the art unless otherwise specified. Operation method (Mo lecular

C loning, 2nd. Ed., Old Spring Harb. Lab. Press, 1998, and other methods described in technical manuals introducing standard methods for those skilled in the art. ).

Example 1

Preparation of PCTF 35

1) Introducing the bc12 gene into PC12 cells

Using wild type PC12 (Mah SP et al., J. Neurochem., Vol. 63, p. 1183-1186, 1993) cells provided by RIKEN, the method of Sato et al. (J. Neurobio., Vol. 25 (10), ρ1227-1234), the bcf2 gene was introduced by the following procedure.

PC12 cells were cultured in a 6-well plate containing HDMEM medium for 24 hours, and then 1.5 μg of the expression vector pAGE123 obtained by recombining the human b2 gene was added, followed by the calcium phosphate method. Marker substance G4 18 (Wako Pure Chemical Industries) 4 PC12 cells transformed with pAGE123 containing gene bc (2 by culturing for 14 days in HDMEM medium containing 50 _g mL ( PCl SZb cf Z cells) were prepared.

2) Preparation of culture supernatant of transformed cells

1) Transfer the PC 12 bci-2 cells obtained in 1) to a Petri dish with a diameter of 100 mm. / ₀ horse serum, 5% fetal bovine serum in HDMEM medium (Gibco) And pre-cultured to 80-90% confluence.

Inoculate bc £ 2 // ρ C cells equivalent to three plates of the above Petri dish into 50 mL of the same culture as the preculture prepared in a 245 X 245 X 25 mm culture tray (Sumitomo Beichik). Then, the cells were cultured again until they became 90% confluent. The cells after the culture were washed three times in a tray using a serum-free HDMEM medium, and 5 OmL of the same serum-free medium was added, followed by culturing for 2 days. The serum-free medium after this culture was collected, and the medium after 2 additional days of addition of 5 OmL of the same serum-free medium was also collected.

The above operation was repeated to prepare a total of 100 OmL as the culture supernatant.

3) Activity measurement of PCTF 35

The cell death inhibitory activity of PCTF35 of the present invention was measured according to the method of Sasaki et al. (Described above), and the lactate deoxygenase in wild-type PC12 cells when PCTF35 was allowed to act on wild-type PC12 cells. The method for measuring the activity of the drogenase (LDH) was specifically measured by the following procedure.

A sample containing PCTF 35 was lyophilized and then dissolved in HDMEM medium to obtain a test sample. To each well of a 24 well plate, prepared HDMEM media 200 mu L containing about 5000 wild-type PC 1 2 cells, there was added a test sample 200 mu 1, C0 ₂ cell culture vessel For 24 hours. After the culture, each sample was collected in an Etbendorf tube, and wild-type PC12 cells were spun down and collected at 3000 X g for 5 minutes. After washing the collected PC12 cells with physiological saline (PBS) adjusted to pH 6.8 with phosphate buffer, the PC12 cells were solubilized with PBS containing 0.2% Tween. The eluted LDH activity was measured using MTX "LDH" (manufactured by Kyokuto Pharmaceutical). When PCTF 35 was changed to NGFOOng Zml and the same operation was performed, the LDH activity in PC12 cells was also measured and set as a standard value, and the ratio (% NGF) to the standard value was calculated. The cell death inhibitory activity of PCTF 35 contained in the test sample was determined.

4) Purification of PCTF 35

a) High-performance liquid chromatograph

Using a stirring cell (manufactured by Amicon), add 10 ° OmL of the culture supernatant obtained in 2) 10 kc as protein molecular weight! The fraction corresponding to 5050 kd was purified and concentrated 50-fold (2 OmL). The concentrated fraction was subjected to reversed-phase high-performance liquid chromatography (RP-HPLC) under the following conditions.

Equipment: High-speed liquid mouth chromatography manufactured by Hitachi, Ltd. (L6009, L6299, L4000, D2,500)

Detection; UV (220 nm)

Column: Tosoichi Phenyl-5 PWRP (7.5 cm long), room temperature

Developing solution: A buffer solution 0.05% trifluoroacetic acid

B buffer solution 0.05% trifluoroacetic acid / 60% acetonitrile Development condition; development speed l mLZ min

0 to 50 minutes B buffer 0 to 20% linear gradient

50-56 minutes B buffer 20-35% linear gradient

56-68 minutes B buffer 35-47% linear gradient

68-73 minutes B buffer 47-52. /. Linear gradient of

73 minutes to 78 minutes B buffer 100%

Recovery; fractionated and collected in lmL

When the activity of PCTF 35 was measured for each collected fraction, fraction 11 eluted from the column at around 25% of acetonitrile showed the highest activity. Figure 4 shows the results of this chromatography.

b) SDS-polyacrylamide electrophoresis (SDS-PAGE)

The fraction 11 obtained in a) was freeze-dried and then dissolved in 100 L of 20 mM Tris-HCl buffer (pH 8.0). Add 4 μL of 5X sampling buffer to 16 μL of this dissolved sample, and heat for 5 minutes in boiling water to denature the sample. Perform SDS-PAGE under the following conditions, and fractionate The molecular weight of the protein contained in 11 was measured.

Concentrated gel: 0.45mL 30% T-2.7% C acrylamide solution

0.75mL 0.4% SDS-containing 0.5M Tris-HCl buffer

(p H6.8)

1.8 mL purified water 1 8 ML 1 0% ammonium persulfate solution

6.0 μL TEMED

Separation gel: Mini slab gel (1 mm thick)

3 mL 30% T-2.7% C acrylamide solution 0.75 mL 0.4. /. 1.5M Tris-HCl buffer containing 303

(p H 8.8)

3.7 5 mL purified water

70 μL 10. /. Ammonium persulfate

10 μL TEMED

Running buffer: 25 mM Tris, 0.192 M glycine, 0.1% SDS Electrophoresis conditions; Concentrated gel 50 V, Separation gel 150 V

The gel after electrophoresis was stained with Coomassie Blue (CBB) and the presence of protein was confirmed by the blue band. The protein with the highest content in the recovered fraction was confirmed to have a molecular weight of 35 kd. Was done. The gel after this CBB staining is shown in FIG.

c) Polyacrylamide electrophoresis (Na t i v e— PAGE)

For the fraction 11 of a), Nativ e-PAGE was performed under the following conditions. Concentrated gel; l mL 12.5% T—2% C acrylamide solution

0.5 mL 0.5 M Tris-HCl buffer (pH 6.8)

2 mL purified water

0.5 mL 4% riboflavin solution

3 μL TEMED

Separation gel; Mini slab gel (1 mm thick)

3 mL 30% T-2.7% C acrylamide solution

3 mL 1.5 M Tris-HCl buffer (pH 8.8) 6 mL Purified water

53.3 μL 20% ammonium persulfate

6.7 μL TEMED

Running buffer: 25 mM Tris, 0.192 M glycine

Running conditions: Concentrated gel 50 V, Separation gel 1 50 V Fraction 11 was lyophilized and then dissolved in 16 / iL of 20 mM Tris-HCl buffer (pH 8.0). To 32 μl of the dissolved sample, 8 / iL of 5X sampling buffer was added to prepare a sample for electrophoresis. Two of the above gels were prepared, and electrophoresis was performed by placing 16 of the electrophoresis data on each gel. One of the gels after the electrophoresis was stained with CBB to confirm the protein position (Fig. 6, -1 to -4). Portions corresponding to positions 1 to 4 confirmed by CBB staining were cut out from another gel. The excised gel is immersed in 200 // L 20 mM Tris buffer solution, pH 8.0 for 24 hours at 4 ° C to extract the protein in the gel, and the gel is centrifuged to recover the supernatant. did. Each of the collected supernatants was dialyzed against HDMEM medium for 12 hours at 4 ° C., and the cell death inhibitory activity of each sample was confirmed. As a result, this activity was observed in the protein recovered from the gel corresponding to -1. Figure showing the cell death inhibitory activity of each recovered protein

See Figure 7.

When the recovered protein was subjected to SDS-PAGE again according to the method of b), a protein of 35 kd was confirmed (FIG. 8). From the above, it was determined that this protein was PCTF35.

5) Determination of N-terminal amino acid sequence of PCTF 35

After performing SDS-PAGE electrophoresis in 3) c), PC TF 35 located at 35 kd was transferred from immobilized gel to Immobilon P SQ membrane (Millipore) with 10 mM methanol containing 10% methanol. Using PS (pH 11) buffer, transcription was performed at 18 OmA for 90 minutes. The membrane after the transfer was immobilized by immersing it in a 50% methanol Z 10% acetic acid solution for 5 minutes, and stained with a Rapid Stain CBB Kit (Nakarai) for 20 minutes. Thereafter, the part where PCTF 35 is located is cut out by decolorization and drying, and the amino acid sequencer (HPG 1005A) manufactured by Hulett Packard Co., Ltd. is used to remove the PCTF 35 on the PSQ membrane according to the operation manual of the equipment. The N-terminal amino acid sequence was analyzed. The N-terminal amino acid sequence is shown in FIG.

6) Determination of partial amino acid sequence of PCTF 35

After performing SDS-PAGE electrophoresis in c) of 3), the electrophoresis gel where PCTF 35 was located was cut out and the gel was homogenized-containing 20 / ig bromocyan 70. / ₀ formate 100 mu L was added and reacted at room temperature for 3 hours, The gel homogenized at 15000 X g was recovered. Using the recovered gel as a sample, SDS-PAGE was performed according to the method of 3) b). When the gel after the electrophoresis was stained with CBB, a band of a new protein, that is, a limited degradation product of PCTF 35 by Bromcyan was confirmed at a position corresponding to about 28 kd. The same operation as in 5) was performed on this limited digest, and the N-terminal amino acid sequence was analyzed. As a result, the sequence shown in FIG. 10 was confirmed.

Example 2

Cloning of gene pctf35

1) RT—PCR amplification of gene

The oligo DNA (sequence 11 below) corresponding to the sequence QQGKE AT, which is a part of the N-terminal 21 residues of PCTF 35 determined in Example 1 (FIG. 9), was designated as SENSE PRIMER. The oligo DNA (the following sequence-2) corresponding to the amino acid sequence YRGRSVP (Fig. 10) at the N-terminus of the limited degradation product of PCTF 35 by bromocyan obtained in 6) was used as an ANTISENSE PRIMER, each of which was manufactured by PE Applied Biosystems. It was synthesized using a DNA synthesizer (AB I 380 B).

Sequence — 1; 5 '— CA (A / G) C A (A / G) GG (A / C / G / T) AA

(A / G) GA (A / G) GC (A / G / C / T) AC— 3 'sequence— 2; 5'—GG (A / G / C / T) AC (A / C / G / T) (C / G)

(A / T) (A / G / C / T) C (G / T) (A / G / C / T) CC (A / G / C / T) C (G / T) (A / G) All RNAs to be TA-3 ′ type II were extracted from PC cells using an ISOGEN kit (manufactured by Futatsu Gene) according to the instructions of the kit. A reverse transcription reaction and PCR (RT-PCR) were carried out using TAKARA RNA PCR kit (AMV) (manufactured by Takara Shuzo Co., Ltd.) under the following conditions using all the RNAs as type II.

Type 全 total RNA 5 μ1 (1 μg)

10 X PCR buffer 5 μ

2.5 mM d NT P 1 μ

10 μM oligonucleotide (sequence-1) 2 μ

10 μ μ Oligonucleotide (sequence-1 2) 2 μ Water 29 μ 1 Reverse transcriptase 0.5 μ 1

T a q Polymerase 0.5 1

2 5 mM magnesium chloride 5 μ 1

Total volume 50 μ 1

After holding the above reaction solution at 94 for 30 seconds, cool at a rate of 12.5 ° CZ for 2 seconds to 55 ° C, and cool to 55 ° C. After heating for 30 seconds at +2.5 ° C / 2 seconds, the process of reacting at 72 ° C for 90 seconds is repeated 30 times to amplify the target sequence. Was.

After completion of the reaction, acrylamide gel electrophoresis (gel concentration: 10%) was performed according to a conventional method, and the gel was stained with ethidium / methylene chloride, and then irradiated with ultraviolet light, and the amplified DNA was located at a position corresponding to about 390 bp. Was confirmed, and a gel containing the band of the amplified DNA was cut out. After homogenizing this gel, add 350 μL of Maxam Gilbert buffer (0.5 mM ammonium sulfate, 0.1% SDS, 1 mM EDTA, 1 OmM magnesium acetate) and leave at room temperature for 24 hours. After extracting the DNA fragment from, the supernatant was collected by centrifugation at 15000 X g for 10 minutes. The DNA fragment was purified from the recovered supernatant by ethanol precipitation.

2) Determination of nucleotide sequence of amplified DNA

The amplified DNA purified in 1) was transferred to pBluescriptll (manufactured by Stratagene), a vector sequencing primer, using a Ligation Pack manufactured by Futatsu Gene Co., Ltd. at 16 ° C under the following conditions. The reaction was carried out for 18 hours and ligated to prepare a recombinant vector. Purified amplified DNA 4 μ1 (200 ng)

pBluescriptll 1 μ 1 (50 ng)

B AS 1.2 5 1

Hexamine cobalt chloride 0.5 μL

Spermidine 0.5 μL

0.75 μL water

D N ALigase 5 μ 1

Using 5 μL of the above reaction solution containing the recombinant vector, heat shock E. coli DH5 was transformed. Transformants were treated with ampicillin (Amp) 50 μg / mI 5—Bromo—4—C hioro—3—indo 1 y1—j3—D—galactoside (X—gal) 40 μg / m1 I sopropyl-] 3-DT hio -G a1 actopyranoside (IPTG) was plated on an LB agar medium containing 100 µΜ, and cultured at 37 ° C at-晚 ° C. After inoculating the white colonies on the plate into 10 ml of LB liquid medium containing 50 μg / ml of Amp and culturing overnight at 37 ° C, collect the cells by centrifugation. The recombinant DNA was purified using FlexiPrep (Pharmacia).

1 μg of the purified recombinant DNA was denatured according to a conventional method, and the base sequence of the amplified DNA was determined by the dideoxy method according to the description of the ThermoSequenase Pre-mix cycle sequencing Kit (manufactured by Manjam). The total length of the amplified DNA was 3466 bp.

3) Acquisition of gene pctf35

Based on the nucleotide sequence of 346 bp determined in 2), the entire target gene was obtained by the following RACE (Rapid Amplification of cDNA Ends) method (Frohman. A., et.al., Proc. Natl. Acad. Sci. USA, Vol. 85, p8998 (1988)).

As reaction primers, sequence 13 was synthesized for 3 ′ RACE, and sequence 14 was synthesized for 5 ′ RACE.

GGTDAGC- 3 '

Sequence—4; 5'-GACTCTAGAGCTCGAGGACG CGC

AGGTCTGGGTGTCC- 3 '

1 μg of the total RNA obtained in 1) was subjected to PCR under the following conditions according to the instruction of Marathon cDNA amplification kit (manufactured by Clonetech) using type 铸. Type 全 total RNA 5 μ (1 μ g)

1 0 X PCR buffer 5 μ

2.5 m d NT P 1 μ

10 μΜ oligonucleotide (sequence-1 3) 2 μ

\ 0 μΜ oligonucleotide (sequence 1-4) 2 μ Water 34.5 μ 1

Reverse transcriptase

T a q Polymerase 0.5 μ 1

Total volume 50 μ 1

After holding the above reaction mixture at 94 ° C for 30 seconds, cool at -2.5 ° C for 2 seconds to 72 ° C, hold for 4 minutes, and +2.5 ° C for 2 seconds. Return to 94 ° C five times, then 94. After holding for 30 seconds, -2.5. Cool down to 70 ° C at a speed of 2 seconds, hold for 4 minutes, return to +94 ° C at a speed of +2.5 ° C / 2 seconds. After holding for 0 seconds, one-2.5. The process of cooling to 68 ° C at a rate of C / 2 seconds, holding for 4 minutes, and returning to 94 ° C at a rate of + 2.5 ° C / 2 seconds was repeated 30 times.

After completion of the reaction, agarose gel electrophoresis (gel concentration: 1%) was performed according to a conventional method. The gel was stained with ethidium gel, and then irradiated with ultraviolet light. It was confirmed. Purification of amplified DNA from the gel containing the amplified DNA, integration into a vector, and sequencing were performed in the same manner as in 2) to determine the entire nucleotide sequence of the amplified DNA.

As a result, the amplified DNA was a DNA consisting of a total of 1,645 bp, and contained a 7 lbp ORF (Open Reading Flame) containing a termination codon in the amplified DNA. The amino acid sequence encoded by this ORF contains the N-terminal amino acid sequence of PCTF 35 and a sequence fragment which partially matches the amino acid sequence at the N-terminus of 23 kd of its limited digest. Therefore, it was determined that the target gene Pctf35 was cloned. SEQ ID NO: 3 shows the entire nucleotide sequence of 1645 bp, which is amplified DNA containing the gene pct35.

The amino acid sequence determined from the cloned gene is shown in SEQ ID NO: 3. From this amino acid sequence, the N-terminal amino acid sequence of PCTF35 shown in FIG. 9 is the amino acid sequence of the 1st to 22nd amino acids on the N-terminal side of PCTF35: Va1MetGlySerG1y Asp SerVa1ProGlyG1yVa1CysTrpLeuGinGinGlyLysGluAlaThr. It was also found that the N-terminal amino acid sequence shown in FIG. 10 corresponds to the N-terminal 138 to 141st amino acid sequence of PCTF35: TyrArgG1yArg.

Example 3

Preparation of PCTF 35 by recombinant gene technique

1) Construction of recombinant expression vector pCTF35 and transformation of PC12 cells

1 μg of a recombination vector pcDNA3 (Invitrogen) opened with the restriction enzymes EcoRI and XhoI was prepared, and the recombinant vector pcDNA35 containing the gene pctf35 obtained in Example 2-3) was added. 1 μg of 645 bp amplified DNA was added, and ligation, transformation and preparation of recombinant DNA were performed in the same manner as in 2) of Example 2 to obtain recombinant expression vector pCTF35.

After culturing wild-type PC12 cells provided by RIKEN in a 6-well plate containing HDM EM medium for 24 hours, add 1.5 μg of recombinant expression vector pCTF35, and use the calcium phosphate method. By culturing the marker vector substance G418 (Dainippon Pharmaceutical Co., Ltd.) in HDMEM medium containing 400 μg ZmL for 14 days, PC 12 cells (PC I 2 / p CTF cells) transformed with the recombinant vector 1 were isolated. Three clones were prepared. At the same time, PC12 cells (mock • PC12 cells) transformed with pcDNA3 were also prepared.

2) Northern hybridization

HDMEM containing 3 clones of PC12 / p CTF cells, mock ^ PC12 cells, and wild-type PCI2 cells in a Petri dish 10 mm in diameter and 10% horse serum and 5% fetal bovine serum, respectively. Medium (Gibco) Add 1 OmL, culture to 80-90% confluence, and use ISOGEN kit (Futtsubon Gene) and oligotex dT30 (Nippon Synthetic Rubber). mRNA was extracted. 2 / g mRNA was fractionated by agarose gel electrophoresis according to a conventional method, and then transferred to a membrane (NEN Gene Screen Plus) using 10 XSSC buffer at room temperature for 18 hours, followed by Northern hybridization. Was done. As a probe, a fragment of 1462 bp from the 5 'end of DNA shown in SEQ ID NO: 3 was used as type I and Ramdom Primer DNA Labeling Kit (manufactured by Takara Shuzo) using [α-32P] d C A TP-labeled DNA fragment was prepared. Hybridization was carried out at 65 ° C for 16 hours in a solution having the following composition (all concentrations were final concentrations).

10% dextran sulfate

1% S DS

1 M N a C 1

2.4 x 105 cpm / mL labeled probe

After the hybridization, the membrane was washed twice with 2 XSSC buffer at room temperature for 5 minutes, then with 2 XSSC buffer containing 1% SDS at 60 ° C, twice for 30 minutes, and then with 0.1 XSSC buffer. Using 0.1% SDS at room temperature for 5 minutes once and washing at 51 ° C. Detection of signal after membrane washing is -80. For 24 hours using Hyperfi1m ^^-ECL (Amersham) film.

As a result, significant expression was confirmed in three PC12Zp CTF cells as compared with the moCK PC12 cells and the wild-type PC12 cells. The exposure results after this hybridization are shown in FIG.

3) Confirmation of viability of recombinant PC 12p CTF cells

Wild type PC12 cells were placed in a 100 mm diameter Petri dish, and 5,000 cells / cm ³ , 10,000 cells cm ³ , 10 mL of serum-free HDMEM medium (Gibco) containing 250 μg / mL of NGF The cells were added at a cell density of 25,000 cells / cm ³ and cultured for 24 and 48 hours, and the number of viable cells was counted by the trepan pull-one dye exclusion test (Gibco). %. The PC12 / p CTF cells and wild-type PCI2 cells obtained in 1) were 5,000 cells per 10 mL of serum-free HDMEM medium (Gibco) prepared in a Petri dish with a diameter of 10 mm. / cm ³ , 10000 cells / cm ³ , and 25,000 cells cm ³ , and the number of viable cells after culturing for 24 hours and 48 hours was counted, and PC12 / pCTF 35 Was confirmed to have a cell death inhibitory effect. As a result, PC 12Zp CTF cells had more viable cells than wild type PC 12 cells (Fig. 12) ₌

Also, a concentrated solution of the supernatant obtained by culturing PC 12Zp CTF cells in the same manner as in Example 1. In addition, a cell death inhibitory effect was observed, and when this was added to wild-type PC12 cells in a serum-free medium, it was confirmed that cell death was suppressed.

Example 4

Cloning of the human pctf35 gene and its amino acid sequencing.

To isolate the human homologue of pctf35 (hpctf35) obtained in Example 1, a part of the nucleotide sequence shown in SEQ ID NO: 3 (the 7th to 777th nucleotide sequence) was pro Brain5'-STRETCH PLUS cDNA Library (manufactured by CLONTECH) was screened. Four hundred positive clones were obtained with a screen jungle of about 500,000 clones. Three of them were found to be genomic fragments, but one was a cDNA fragment containing a poly (A) tail, which partially had high homology with the pctf35 gene. 5'-RACE was performed based on the above sequence. In addition, PCR was performed using the ORF region sequence expected to be contained in the previously obtained genomic fragment, and the structure of the hpctf35 gene ORF was determined using the same library and cDNA derived from human placenta. Were determined. The nucleotide sequence of the hpctf35 gene ORF encoding hpctf35 comprising a total of 263 amino acid residues including the signal peptide is shown in SEQ ID NO: 5. The amino acid sequence of hpct35 including the signal peptide deduced therefrom is shown in SEQ ID NO: 4.

Industrial applicability

According to the present invention, a protein that suppresses cell death was obtained. Further, according to the present invention, the gene encoding the gene was successfully cloned. The protein for suppressing cell death according to the present invention is useful for treating diseases associated with nerve cell death, and has a function similar to that of the protein, and has an effect of inhibiting or promoting the function of the protein. It is also very useful when developing substances as drugs.

Claims

The scope of the claims

1. The following protein (a) or (b):

(a) a protein consisting of the amino acid sequence of SEQ ID NO: 1;

(b) a protein consisting of the amino acid sequence of SEQ ID NO: 1 with one or several amino acids deleted, substituted or added, and having an activity of suppressing cell death;

2. A gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1.

3. A gene consisting of the following (a) or (b):

(a) DNA consisting of the nucleotide sequence of SEQ ID NO: 2:

(b) a DNA that hybridizes with the DNA of SEQ ID NO: 2 under stringent conditions and encodes a protein having an activity of suppressing cell death.

4. A cell death inhibitory factor characterized in that it is a proteinaceous substance having the following physicochemical properties and physiological activities of ① to ：:

(1) It can be obtained from a culture of a pheochromocytoma transformed with the proto-oncogene b2 gene;

(2) The estimated molecular weight by reduced SDS-PAGE is about 35,000;

③ has the activity of suppressing cell death;

アミノ酸 The 1st to 22nd amino acid sequence on the N-terminal side is the following amino acid sequence; Va1MetG1ySerG1yAspSeRVa1

P r o G l y G 1 y V a 1 Cys T r p L e u G In

G i n G l y L y s G 1 u A l a Th r

⑤ The amino acid sequence at positions 138 to 14 1 on the N-terminal side is the following amino acid sequence.

Ty r A r g G l y A r g

5. A pharmaceutical composition for suppressing cell death, comprising the protein of claim 1 or the cell death inhibitor of claim 4 as an active ingredient.

6. The pharmaceutical composition according to claim 5, wherein the protein is the protein of (b) according to claim 1.

7. The pharmaceutical composition according to claim 6, wherein the protein comprises an amino acid sequence obtained by removing the amino acid sequence corresponding to the signal peptide from the amino acid sequence of SEQ ID NO: 4.

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