JP2003033190A - Calcium-independent new phospholipase a2, gene encoding the same and promoter for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a calcium-independent new phospholipase A2, to provide a gene encoding the enzyme, and to provide an antibody to the enzyme. SOLUTION: This calcium-independent new phospholipase A2 is, more specifically, the calcium-independent phospholipase A2 having specific three kinds of amino acid sequences, or those wherein at least one amino acid is deleted, substituted by another amino acid, or added. The 2nd gene encodes the enzyme and has a base sequence present in the intron and enabling the transcription initiation of an RNA by external stimulation such as kainic acid stimulation or electric stimulation. The 3rd method for regulating the expression using the gene, and the other organisms each transferred with the gene, are also provided, respectively.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel phospholipase A2, which is calcium-independent, more specifically, calcium-independent phospholipase A2, which can be induced by external stimulation such as kainic acid or electrical stimulation. Phospholipase A2 that is specifically expressed, wherein the amino acid sequence shown in SEQ ID NO: 1, 5 or 8 of the sequence listing, or one or more amino acids in those amino acid sequences is replaced with another amino acid, and is deleted. The present invention relates to a novel phospholipase A2 having an amino acid sequence that is lost and has one or more amino acids added. The present invention also relates to a gene having a nucleotide sequence existing in an intron, which can enable RNA transcription initiation by external stimuli such as kainic acid stimulation or electrical stimulation, and regulates expression using the gene. And a living organism into which it is introduced. Furthermore, the present invention relates to a pharmaceutical composition containing the phospholipase A2 of the present invention.

[0002]

2. Description of the Related Art In eukaryotic genes, genetic information defining the amino acid sequences of proteins is often written. The portion of the protein that has the genetic information of the amino acid sequence is called an exon, and the portion that does not have the genetic information of the amino acid sequence is called an intron. Gene DN
After A is transcribed to form an mRNA precursor, it is spliced and the intron portion is excised, resulting in a mature mRNA. The reason why such an intron part exists in eukaryotes has not been clarified yet. However, in most cases, one exon is encoded as a specific domain (functional region) of a protein, and when a new protein with a similar function is required in the evolution process, different exons are It is believed that the combination has made it possible to produce the required protein.

It is known that splicing of an mRNA precursor before being subjected to splicing not only cuts out an intron but also cuts out an exon to obtain mRNAs encoding different proteins having the same function. For example, calcitonin is inherited as A, B, C,
D, calcitonin CCP, CGRP (Calcitonin gene
It has 6 exons of related peptide). Exon A and exon B are untranslated regions, and the translated region is the remaining four exons. When transcribed in the nucleus of a cell, it contains all exons, but the process of splicing varies from organ to organ. For example, in thyroid C cells, the 6th CGRP exon is also spliced, and as a result, the protein of the translation product becomes a peptide consisting of CD-calcitonin CCP, which mainly acts to lower serum Ca. Further, in the cells of the hypothalamus, the exon of the fifth calcitonin CCP is also spliced, and as a result, the protein of the translation product becomes a peptide consisting of CD-CGRP, which mainly serves to control pain and autonomic nervous activity. .

As described above, by dividing the exon portion into several parts, it becomes possible to generate different proteins having several exons linked thereto, if necessary. Although the division of exons for this purpose has been described, little is known about the need for introns other than the creation of exon moieties. Most introns have 5'-GT and AG-3 'sequences at their ends and are known to have a pyrimidine-rich region in the middle. Splicing is performed by recognizing the sequences at both ends. It is believed that.

Phospholipase A2 is widely distributed in mammals and microorganisms, most of which are membrane-bound enzymes and are involved in the metabolism of membrane phospholipids. The 85-kDa cytoplasmic phospholipase A2 (cPLA2α) is one kind of phospholipase A2, which mainly excises arachidonic acid from membrane phospholipids and induces arachidonic acid-derived prostaglandins, thromboxane, leukotrienes, and other physiological activities by an arachidonic acid cascade. Produces a substance. In addition, it is known that arachidonic acid released in the brain is involved in various nerve functions, and thus far, the present inventors have used the Northern plot method and the in situ hybridization method to determine that cPLA2α It has been shown to be abundantly expressed in brain neurons.

On the other hand, it is a kind of kainic acid amino acid, which has been isolated as an anthelmintic component in linseed. Since kainic acid has a chemical structure similar to glutamate, it is known as a substance that binds to glutamate receptors in animal brains and nerve cells and causes neuron excitatory action.

The present inventors applied kainic acid stimulation or electrical stimulation to investigate the function of phospholipase A2 in the brain, and as a result, a novel phospholipase A2 (transiently expressed in the dentate gyrus of the hippocampus). Amino acid 455, molecular weight about 50K) was discovered. This enzyme is a partial protein starting from the 308th methionine of cytosolic phospholipase A2α (85K), but since it also appears in genetically deficient mice of this enzyme, it has a unique promoter that can be expressed site-specifically in response to stimulation. It was included. This enzyme does not exist in the unstimulated state, but it is expressed by electrical stimulation or kainic acid stimulation, is calcium-independent unlike conventional phospholipase A2, produces eicosanoids, regulates brain function, and nerve cells. Since it is involved in the degeneration, apoptosis, and regeneration of, it is thought to be a molecule that holds the key to these brain functions.

[0008] The novel phospholipase A2 (amino acid 455, molecular weight about 50K) is a partial protein translated from the 308th methionine of the known cytoplasmic phospholipase A2α (85K). There is a unique promoter region for expressing a protein, and the present inventors have found that there is an intron having a function of enabling RNA transcription initiation in the intron. In a normal state, this intron does not have the function of initiating RNA transcription, but by setting certain conditions, the R from the part of the base sequence of this intron is not from the original transcription position.
It has a function of initiating the transcription of NA.

[0009]

DISCLOSURE OF THE INVENTION The present invention provides a novel calcium-independent phospholipase A2 (amino acid 45).
5, a molecular weight of about 50K), a gene encoding the same, and an antibody thereto. The present invention also provides a unique promoter or regulatory gene consisting of a nucleotide sequence present in an intron, which is site-specifically expressed in response to an external stimulus. Furthermore, the present invention provides a method for expressing a protein of interest in response to an external stimulus, and an organism into which the protein has been introduced. Furthermore, the present invention provides a method for specifically searching neural stem cells, since KIDS cPLA2 of the present invention is specifically expressed in neural stem cells.

[0010]

The present invention provides a novel calcium-independent phospholipase A2, more specifically a calcium-independent, hippocampal-specific phospholipase A2, which is represented by SEQ ID NO: 1 in the sequence listing, 5 or 8, the amino acid sequence described, or one or more amino acids in those amino acid sequences are replaced with other amino acids, deleted,
Phospholipase A2 having an amino acid sequence in which one or more amino acids are added, genes encoding them,
And an antibody having the full length or a fragment thereof as an antigen. The present invention also relates to a gene having a base sequence existing in an intron, which can enable RNA transcription initiation by external stimulation such as kainic acid stimulation or electrical stimulation, more specifically, a site-specific gene. In particular, it relates to a gene capable of initiating RNA transcription. Examples of preferred genes of the present invention include SEQ ID NO: 12, 13 or 14 in the sequence listing.
Examples of the gene include a base sequence described in 1) or a base sequence consisting of a partial sequence obtained by deleting, adding, or substituting a part thereof.

The present invention also relates to a promoter, which is a nucleotide sequence existing in an intron, and is capable of initiating RNA transcription by an external stimulus such as kainic acid stimulation or electrical stimulation, more specifically, RNA transcription. It relates to said promoters whose start is site-specific and to regulatory genes having regulatory elements upstream of said promoters. Furthermore, the present invention, by introducing any of the above-mentioned gene, the above-mentioned promoter, or the above-mentioned regulatory gene upstream of the gene encoding the protein, by external stimulation such as kainic acid stimulation or electrical stimulation, A method for initiating RNA transcription preferably in a site-specific manner to express the protein in response to an external stimulus, and the above gene, the above promoter, or the above regulation upstream of the gene encoding the protein It relates to an organism into which any of the genes has been introduced. The present invention also relates to the KIDS cP of the present invention.
The present invention relates to a method for specifically searching neural stem cells by the expression of LA2. That is, the present invention detects neural stem cells, which comprises stimulating neural cells by external stimulation to detect or identify the expression of mRNA encoding phospholipase A2 according to any one of claims 1 to 3. Alternatively, it relates to a method for identifying. Furthermore, the present invention relates to the KIDS cP of the present invention.
It relates to a pharmaceutical composition comprising LA2 and a pharmaceutically acceptable carrier. More particularly, the invention relates to the KIDS of the invention.
It relates to a phospholipid level regulator such as lysophosphatidylcholine (LPC) containing cPLA2.

The inventors of the present invention, as part of a study for investigating the function of phospholipase A2 in the brain, prepared a brain section of a rat into which kainic acid was intraperitoneally injected, and histochemically analyzed mRNA using cPLA2 as a probe (searcher). Has been investigated. Usually cPLA2 for probe selection
It was confirmed by Northern blotting using different sites of cPLA when specific site (5 'end) was used.
It was found that mRNA having a length shorter than 2 (about 1.8 kilobase pairs) was induced.

The result of the Northern blot is shown in FIG. 1 by a photograph as a drawing. The upper row of FIG. 1 shows the base sequence of cPLA2. The left end is the translation initiation codon (ATG), and the portions used as probes are indicated by A, B, C and D. That is, the probe A is BamHI to Bal.
In part I, probe B is RsaI to RsaIBal
In part I, probe C is from RsaI to BalI, and probe D is from RsaI to the stop codon (TG
Up to A). The middle part of FIG. 1 shows the case where kainic acid treatment is not carried out (KA (−)). The lower part of FIG. 1 shows the case of 3 hours after the treatment with kainic acid (KA (+), 3 h). When kainic acid treatment is not performed (KA (-)) (middle row of Fig. 1),
Plots can be seen only at the position of cPLA2α, but when the kainic acid treatment was performed 3 hours after the treatment (KA
In (+), it was possible to observe not only the position of cPLA2α for the probes B, C and D on the 5′-terminal side but also a plot of shorter chain lengths below it.

Next, the northern blot of the hippocampus and cerebellum was performed over time. The result is shown in FIG. 2 by a photograph instead of a drawing. The hippocampus is on the left side of FIG. 2, and the cerebellum is on the right side.
After each kainic acid treatment (0 hours), 0.5 hours later,
Blots after 3 hours, 8 hours, 14 hours, and 18 hours are shown. CPLA2 anywhere in Figure 2
A plot can be seen at the position of α, but the hippocampus (left side of Fig. 2)
CPLA only after 3 hours of kainic acid treatment
It was possible to observe a plot of shorter chain length not only at the position of 2α but also below it.

Furthermore, when in situ hybridization was performed, specific expression was recognized in the dentate gyrus of the hippocampus. The result is shown in FIG. 3 by a photograph as a drawing. The left side of FIG. 3 is a cross section of the brain, and the right side of FIG. 3 is from a vertical cross section of the brain. Figure 3
The part that looks black in is the part that is colored. The colored area is the dentate gyrus of the hippocampus. An enlarged view of this result is a photograph replacing the drawing of FIG. The left side of FIG. 4 is not treated with kainic acid, and the right side is after 3 hours of kainic acid treatment. Color development can be observed along the dentate gyrus of the hippocampus. This color is strongly developed outside the dentate gyrus of the hippocampus, and since there are many neural stem cells in the dentate gyrus of the hippocampus, this is attributed to neural stem cells present in the dentate gyrus of the hippocampus. Presumed.

Then, the cDNA of interest was obtained from the hippocampal dentate gyrus library using the full-length cPLA2 as a probe. When this cDNA was translated into a protein and the enzyme activity was examined, phospholipase A2 activity was recognized. From this structural analysis, cytosolic phospholipase A2 (cytosolic phos
It was found to be a truncated phospholipase A2 molecule (abbreviated as pholipaseA2, cPLA2). The rat cDNA has 1,842 base pairs and the translation region has 1,335 base pairs.
It was a protein consisting of 45 amino acids and having a molecular weight of 50810.6. Since this truncated phospholipase A2 is specifically expressed in the hippocampal dentate gyrus of the brain after stimulation with kainic acid, it is kainate-inducible dentate gyrus specific.
It was named cPLA2 (KIDS cPLA2).

The amino acid sequence of the obtained KIDS cPLA2 is shown below in the one-letter code of amino acids. Human KI
DS cPLA2 is

[0018]         MNTTLSSLEKVNTAQCPLP 20         LFTCLHVKPDVSELMFADWV 40         EFSPYEIGMAKYGTFMAPDL 60         FGSKFFMGTVVKKYEENPLH 80         FLMGVWGSASFSILFNRVLGV 100         SGSQSRGSTMEEELENITK 120         HIVSNDSSDSDDESHEPKGT 140         ENEDAGSDYQSDNQASWIHR 160         MIMALVSDSALFNTREGRAG 180         KVHNFMLGLNLNTSYPLSPL 200         SDFATQDSFDDDDELDAAVAD 220         PDEFERIYEPLDVKSKKIHV 240         VDSGLTFNLPYPLILRPQRG 260         VDLIISFDFSARPSDSSPPF 280         KELLLAEKWAKMNKLPFPKI 300         DPYVFDREGLKECYVFKPKN 320         PDMEKDCPTIIHFVLANINF 340         RKYKAPG VPREEEEEKEIAD 360         FDIFDDPESPFSTFNFQYPN 380         QAFKRLHDLMHFNTNLNNIDV 400         IKEAMVESIEYRRQNPSRCS 420         VSLSNVEARRFFNKEFLSP 440         KA 442

[0019]   KIDS cPLA2 from rat         MSTTLSSLEKVSAARCPLP 20         LFTCLHVKPDVSELMFADWV 40         EFSPYEIGMAKYGTFMTPDL 60         FGSKFFMGTVVKKYEENPLH 80         FLMGVWGSASFSILFNRVLGV 100         SGSQNKGSTMEEELENITAK 120         HIVSNDSSDSDDEAQGPKGT 140         ENEDAEREYQNDNQASWVHR 160         MLMALVSDSALFNTREGRAG 180         KEHNFMLGLNLNTSYPLSPL 200         RDFSPQDSFDDDDELDAAVAD 220         PDEFERIYEPLDVKSKKIHV 240         VDSGLTFNLPYPLILRPQRG 260         VDLIISFDFSARPSDTSPPF 280         KELLLAEKWAKMNKLPFPKI 300         DPYVFDREGLKECYVFKPKN 320         PDVEKDCPTIIHFVLANINF 340         RKYKAPGVLRETKEEKEIAD 360         FDIFDDPESPFSTFNFQYPN 380         QAFKRLHDLMYFNTLNNIDV 400         IKDAIVESIEYRRQNPSRCS 420         VSLSNVEARKFFNKEFLSP 440         TAESI 445

[0020]   Mouse KIDS cPLA2         MSMTLSSSLEKVNAARCPLP 20         LFTCLHVKPDVSELM FADW 40         VEFSPYEIGMAKYGTFMAPD 60         LFGSKFFMGTVVKKYEENPL 80         HFLMGVWGSASFSILFNRVLG 100         VSGSQNKGSTMEEELENITA 120         KHIVSNDSSDSDDEAQGPKG 140         TENEEAEKEYQSDNQASWVH 160         RMLMALVSDSALFNTREGRA 180         GKVHNFMLGLNLNTSYPLSP 200         LRDFSSQDSFDDELDAAVAD 220         PDEFERIYEPLDVKSKKIHV 240         VDSGLTFNLPYPLILRPQRG 260         VDLIISFDFSARPSDTSPPF 280         KELLLAEKWAKMNKLPFPKI 300         DPYVFDREGLKECYVFKPKN 320         PDVEKDCPTIIHFVLANINF 340         RKYKAPGVLRETKEEKEIAD 360         FDIFDDPESPFSTFNFQYPN 380         QAFKRLHDLMYFNTLNNIDV 400         IKDAIVESIEYRRQNPSRCS 420         VSLSNVEARKFFNKEFLSP 440         TV 442

The amino acid sequence of human KIDS cPLA2 is shown in SEQ ID NO: 1 in the sequence listing. Human KIDS cP
The nucleotide sequences of the translation region of the LA2 cDNA are shown in SEQ ID NOs: 2, 3 and 4 in the sequence listing. SEQ ID NO: 2 is a 5'UTR sequence of type I, and SEQ ID NO: 3 is 5'UT
The R sequence is type II, and SEQ ID NO: 4 is the case where the 5'UTR sequence is not divided into type I and type II. The amino acid sequence of rat KIDS cPLA2 is shown in SEQ ID NO: 5 in the sequence listing. KIDS of rat
The nucleotide sequences of the translation region of the cPLA2 cDNA are shown in SEQ ID NOs: 6 and 7 in the sequence listing. SEQ ID NO: 6 is of type I and SEQ ID NO: 7 is of type II. The amino acid sequence of mouse KIDS cPLA2 is shown in SEQ ID NO: 8 in the sequence listing. Mouse KIDS cPLA2 cDNA
The nucleotide sequences of the translation region of A are shown in SEQ ID NOs: 9, 10 and 11 of the sequence listing. SEQ ID NO: 9 is a 5'UTR sequence of type I, SEQ ID NO: 10 is a 5'UTR sequence of type II, and SEQ ID NO: 11 is 5'UT.
This is the case where the R sequence is not divided into type I and type II.

A polyclonal antibody (a stump antibody) that specifically recognizes KIDS cPLA2 of the present invention was prepared. The expression of the target protein was confirmed by immunohistochemical analysis using this antibody. The results are shown in FIG. 5 by a photograph instead of a drawing. The left side of the upper part of FIG. 5 is the case where kainic acid is not treated,
The right side of the upper row is for 3 hours after the kainic acid treatment. Color development by the antibody can be confirmed. The left side of the lower part of FIG. 5 is a control in the case of no treatment with anti-KIDS cPLA2 antibody (IgG). The right side of the lower part of FIG. 5 shows the presence of anti-KIDS cPLA2 antibody (IgG) in the absence ((-) on the left side of the lower part of FIG. 5) and the presence ((+) on the right side thereof) 3 hours after the kainic acid treatment. Is the result of chromatography.

Next, cPLA2 and KIDS of the present invention
cDNA encoding cPLA2 is expressed in expression vector pTr
It was incorporated into acerEF and its expression was examined. The result is shown in FIG. 6 by a photograph instead of a drawing. Lane 1 in FIG. 6 is for the control vector, lane 2 is cPLA2.
In case of α / pTracerEF, lane 3 is KI
This is the case of DS cPLA2 / pTracerEF.
The left side of FIG. 6 shows the case where anti-V5 epitope IgG was used, the middle part shows the replication using anti-cPLA2αIgY, and the right side shows the case where anti-KIDS cPLA2 IgG was used. Each spot with anti-V5 epitope IgG and KIDS cP with anti-KIDS cPLA2 IgG
The LA2 spot was confirmed, and the expression of KIDS cPLA2 was confirmed.

Next, cPLA2α and KIDS of the present invention
The enzymatic activity of cPLA2 was examined. As a substrate, 1-
Pam-2- [ ^14C ] arachidonoyl-PC (black circles (●) in FIG. 7), 1-Pam-2- [ ^14C ].
Linole oil-PC (black triangle mark (▲) in FIG. 7), 1-Pam-2- [ ¹⁴ C] oleoyl-P
C (black square mark (■) in FIG. 7) and 1-Pam
Each enzyme activity was tested using -2- [ ¹⁴ C] palmitoyl-PC (asterisk mark (*) in FIG. 7). The results are shown in Fig. 7. The left side of FIG. 7 is that of KIDS cPLA2 of the present invention, and the right side is cPLA.
2α. Both enzymes have extremely high enzyme activity for arachidonic acid phospholipids, and phospholipase A2
As a result, it can be seen that they have almost the same activity.
The values of these enzyme activities (pmol / min) are shown in Table 1 below.

Table 1 Enzyme activity of KIDS cPLA2 and cPLA2α Phospholipase A2 activity (pmol / min) substrate KIDS cPLA2 cPLA2α 1-Pam-2- [ ¹⁴ C] arachidonoyl-PC 35.6 ± 3.8 24.4 ± 1.4 1-Pam-2- [ ¹⁴ C] Linole oil-PC 20.1 ± 1.7 11.9 ± 1.8 1-Pam-2- [ ¹⁴ C] oleoyl-PC 14.3 ± 1.5 9.1 ± 1.1 1-Pam-2- [ ¹⁴ C] palmitoyl-PC 9.4 ± 1.0 9.8 ± 1.7 The phospholipase A2 activity is due to the difference from the control.

Next, as a substrate, 1-Pam-2- [ ¹⁴
[C] Arachidonoyl-PC was used to examine the calcium dependence on the enzymatic activity of cPLA2α and KIDS cPLA2 of the present invention. The results are shown in FIGS. 8 and 9.
The solid line in FIG. 8 shows the case of KIDS cPLA2 of the present invention, and the broken line shows the case of cPLA2α. The black circles (●) are in the absence of EDTA and the white circles (∘) are in the presence of EDTA, respectively. In the case of cPLA2α, a sharp decrease in activity is observed due to the absence of calcium by EDTA.
It can be seen that in the case of cPLA2, the activity is not significantly reduced. FIG. 9 shows the above results in a relative ratio. In the case of KIDS cPLA2 of the present invention, an activity of about 40% is maintained even in the absence of calcium, but in the case of cPLA2α, the activity is 10% in the absence of calcium. It can be seen that it has dropped to about 15%. Thus, the KI of the present invention
DS cPLA2 is characterized by being calcium-independent as compared to conventional cPLA2α.

Next, the expression of KIDS cPLA2 of the present invention in the cPLA-deficient mouse (Uozumi, N. et al. Nature 390, 618-622, 1997) prepared by Shimizu et al. Was examined. The results are shown in the photograph in place of the drawing in FIG. The upper row of FIG. 10 is for a knockout mouse (+ / +), and the lower row is for a knockout mouse (-/-). The left side of FIG. 10 shows kainic acid untreated (KA (−)), and the right side shows kainic acid treated 3 hours (KA (+)). In all knockout mice, the expression of this enzyme could be confirmed by kainic acid treatment. This means that the KIDS cPLA2 of the present invention has a total length of cPL
It is shown that expression is performed using a promoter different from A2.

FIG. 11 shows cPLA2 and KIDS cPL.
It is the figure which showed the expression condition of A2. The upper part of FIG. 11 schematically shows the exons and introns of cPLA2 in the genomic gene. Full-length cPLA2 was generated from all exons, and a regulatory gene containing a promoter region is present upstream of the first exon. In contrast, the KIDS cPLA of the present invention
No. 2 is a protein starting from the 308th methionine described as “M308” in FIG. 11, and this protein has cPLA-deficient mouse, that is, the function of a regulatory gene containing a promoter region upstream of the first exon is destroyed. Since the expression was confirmed in mice, it was found that KIDS cPLA2 of the present invention has a regulatory gene region including a promoter region upstream of “M-308”. However, this regulatory gene does not function under normal conditions, and it can be seen that it functions only in response to stimulation such as kainic acid stimulation.

Therefore, the nucleotide sequence of the intron upstream of "M-308" was analyzed in rat, mouse and human. The results are shown side by side in FIG. The human base sequence of this intron is shown in SEQ ID NO: 7 in the sequence listing. The base sequence of rat is shown in SEQ ID NO: 8 in the sequence listing. Furthermore, the nucleotide sequence of mouse is shown in SEQ ID NO: 9 in the sequence listing. FIG. 12 shows rat (upper), mouse (middle), and human (lower) "M-".
The base sequence from the first base of the intron immediately before the exon containing "308" is numbered with the base starting from the exon region of the full-length cPLA2 as number 1.
ATG from the 92nd (human) with this number is KIDS
It is the translation initiation codon of cPLA2.

Next, the expression of KIDS cPLA2 of the present invention was examined using neurons in the dentate gyrus of the hippocampus of the brain. The result is shown in FIG. 13 by a photograph instead of a drawing. The upper row of FIG. 13 shows Nestin as a control, the middle row shows the case of using neural stem cells, and the lower row shows the case of using mature nerve cells. A on the left shows the position of each cell,
B in the center is color development indicating the expression of KIDS cPLA2 of the present invention, and on the right side, A on the left side and B in the center are overlapped to confirm the positions of both. As a result, it can be seen that KIDS cPLA2 of the present invention was not clearly expressed in neural mature cells, but could be clearly observed in neural stem cells. This is the KIDS of the present invention
This suggests that cPLA2 is a substance specifically expressed in neural stem cells, and that introns in mature cells specifically play a role of promoter in neural stem cells.

Next, neural stem cells were used to stimulate kainic acid (10 μM), and stimulated with kainic acid and CNQX (KA).
10 μM + CNQX 20 μM), and glutamic acid (5
Stimulation with 0 μM) and its expression were examined. The result is shown in FIG.
Shown in the picture instead of the drawing. The upper part of FIG. 14 is a P90-P27 252 bp probe (this sequence is a part of the sequence common to the full-length cPLA2). The second line from the top is a P19-P27 290 bp probe (this sequence is The present invention contains a sequence specific to KIDS cPLA2 of the invention.), The lower two rows are G for control.
3PDH and Nestin. FIG. 14 shows the transcription initiation position on the 5 ′ side of KIDS cPLA2 and the sequence position of the probe used in the upper two rows of FIG. 14 below it. Each lane in FIG. 14 shows control, kainic acid stimulation (KA (10 μM)), kainic acid and CNQX from the left.
Stimulation (KA (10 μM) + CNQX (20 μ
M)) and glutamic acid (Glu (50 μM)). As a result, the expression of KIDS cPLA2 of the present invention was specifically confirmed when stimulated with kainic acid (10 μM). Therefore, the present invention relates to the KIDS of the present invention.
A method for specifically searching neural stem cells by the expression of cPLA2 is provided. That is, according to this method of the present invention, the candidate cells are stimulated with kainic acid, and the K of the present invention is stimulated.
By observing the expression in IDS cPLA2, neural stem cells can be specifically and easily searched for.

Furthermore, the present inventors have found that the KIDS of the present invention.
It was found that cPLA2 has phospholipase A1 activity. Phospholipase A1 is an enzyme that specifically decomposes the acyl group by the saturated fatty acid at the 1-position of glycerophospholipid. Therefore, the present inventors measured the phospholipase A1 activity of KIDS cPLA2 of the present invention using lysophosphatidylcholine (lysoPC) labeled with the ^14C palmitoyl group of glycerophospholipid as a substrate. The results are shown in Fig. 15. The vertical axis of FIG. 15 is dm
phospholipase A1 activity represented by p (mean ±
Standard deviation, n = 3), and the horizontal axis is time (minutes). From the left, the activity values at each time are cPLA2α (black coating), KIDScPLA2 of the present invention (light black), and lacZ.
(Gray). The right end (white) at the time of 40 minutes on the horizontal axis indicates the buffer. The value of p in FIG. 15 shows that there is a significant difference between KIDS cPLA2 (light black) of the present invention and lacZ by t-test.
As a result, it was found that KIDS cPLA2 of the present invention has an excellent phospholipase A1 activity.

Next, the phospholipase A1 activity of KIDS cPLA2 of the present invention was analyzed dynamically. The results are shown in Fig. 16. The vertical axis of FIG. 16 represents the phospholipase A1 activity represented by dmp (mean value ± standard deviation, n =
3), the horizontal axis is the substrate lysophosphatidylcholine (lys)
oPC) concentration (μM) is shown. The black circle in Figure 16 is cP
LA2α, a thin black circle indicates KIDS cP of the present invention
LA2, dark gray circles indicate lacZ, light gray circles indicate buffer. The small graph on the upper side of FIG. 16 shows the results of kinetic analysis.
The upper side y = 85.784x + 0.378 is the KIDS cPLA2 of the present invention, and the lower side y =
The formula of 59.441x + 0.898 is cPL
A2α.

Further, the influence of calcium ion and AEBSF (4- (2-aminoethyl) -benzenethronylfluoride: serine protease inhibitor) on the phospholipase A1 activity of KIDS cPLA2 of the present invention was examined. The results of the test for the effect of calcium ions are shown in FIG. The ordinate of FIG. 17 represents the phospholipase A1 activity represented by dmp (mean ± standard deviation, n
= 3), the horizontal axis shows the case of absence (-) and the presence (+) of 5 mM EDTA. The activity values in each case are from the left, cPLA2α (black coating), KIDS c of the present invention.
PLA2 (light black), lacZ (grey), and buffer (white). The values of p in FIG. 17 are the values of KIDS cPLA2 (light black) and lacZ of the present invention determined by t-test.
It shows that there is a significant difference between. As a result, cP
The activity of LA2α (black coating) decreases when calcium ions are absent due to the presence (+) of EDTA, whereas KIDS cPLA2 (light black) of the present invention has calcium ions present (absence of EDTA (absent (−)). ))
It can be seen that the same level of activity is maintained in both cases (absence of EDTA (+)).
Next, FIG. 18 shows the result of the influence of the presence of AEBSF. The vertical axis of FIG. 18 represents phospholipase A represented by dmp.
1 activity (mean ± standard deviation, n = 3), horizontal axis is A
The EBSF concentration (mM) is shown. The black circle in Figure 18 is cP
LA2α, a thin black circle indicates KIDS cP of the present invention
LA2, dark gray circles indicate lacZ, light gray circles indicate buffer. As a result, it was found that both enzymes lost their activity due to the presence of AEBSF.

Recently, lysophosphatidylcholine (hereinafter LPC) was found to be a ligand for G2A, an immunomodulatory receptor (Janusz H., et al., Sci.
ence, 293, 702-705 (2001)). In addition, studies in mice lacking the G2A immunoregulatory receptor have revealed that G2A plays an important role in controlling homeostasis of peripheral lymphocytes (Lu Q. Le, et al., Im.
munity, 14, 561-571 (2001)). Therefore, LPC is considered to be a regulatory factor for immunity, cell proliferation, etc. in vivo, especially in the peripheral system, and when LPC becomes excessive, cell death occurs, and arteriosclerosis, deterioration of immunocompetence, etc. occur. The KIDS cPLA2 of the present invention has phospholipase A2 activity and phospholipase A1 activity.
It has an effect of appropriately controlling the level of PC. The KIDS cPLA2 of the present invention can remove excess LPC, and can be used for suppression of apoptosis, improvement of immune function, prevention and treatment of arteriosclerosis and the like. Accordingly, the present invention provides a lysophosphatidylcholine (LPC) modulator by KIDS cPLA2 of the present invention.

[0036]

BEST MODE FOR CARRYING OUT THE INVENTION KIDS cPLA2 of the present invention
Is a partial length of full-length cPLA2, which is characterized by retaining phospholipase A2 activity and being calcium-independent, and is not necessarily SEQ ID NO: 1, 3 or It is not limited to those having the amino acid sequence described in 5. If the phospholipase A2 retains the activity and is calcium-independent, 1 to 200, preferably 1 of the amino acid sequences shown in SEQ ID NO: 1, 3 or 5 of the sequence listing is preferable.
About 100, 1 to 50, or 1 to 20 amino acids may be substituted with other amino acids, may be deleted, or may be added. Alternatively, these substitutions, deletions, and additions may be performed in combination at the same time.

The KIDS cPLA2 of the present invention can be expressed and produced according to the method disclosed in the present specification, but the cD of the KIDS cPLA2 of the present invention can be produced.
NA can be used to produce by a conventional gene recombination technique.

The KIDS cPLA2 of the present invention can be used to produce an antibody against the full length or a part of the peptide, preferably a peptide having a partial length of 5 amino acids or more, or 10 amino acids or more as an antigen. The antibody of the present invention can be produced by a conventional method, and can be a polyclonal antibody or a monoclonal antibody, if necessary. It is also known that kainic acid stimulation and epileptic seizures cause cell death specific to the dentate gyrus of the hippocampus.
The present invention has found that KIDS cPLA2 is expressed in the hippocampal dentate gyrus due to kainic acid stimulation, epileptic seizures, and the like. Therefore, it is possible to prevent cell death in the hippocampal dentate gyrus by preparing an inhibitor of the present enzyme, and the present enzyme is also useful for the development of the inhibitor.

Further, the present invention was for the first time found that some of the introns have a function as a regulatory gene that is activated in response to an external stimulus, and the intron has an external sequence in the base sequence. It was clarified that at least a nucleotide sequence that also functions as a promoter in response to stimulation exists. Therefore, the present invention
The present invention provides a gene having a nucleotide sequence existing in an intron, which nucleotide sequence can enable RNA transcription initiation by an external stimulus. The gene of the present invention is present in an intron consisting of at least 6 bases, preferably consisting of at least 4 bases or more, preferably 6 bases or more in the base sequence shown in SEQ ID NO: 7, 8 or 9. It is an oligonucleotide having a base sequence that allows the initiation of RNA transcription by an external stimulus.

The gene of the present invention has at least R
Since the present invention has a function as a promoter involved in NA transcription initiation, the present invention provides a promoter capable of activating RNA transcription by an external stimulus comprising the gene or a partial length thereof. The promoter of the present invention is an RNA under normal conditions of mature cells.
It is characterized in that it does not cause initiation of transcription of RNA, but can initiate initiation of RNA transcription only by a specific external stimulus. Furthermore, the promoter of the present invention is characterized in that its base sequence is a base sequence existing in an intron. More preferably, the site that allows the initiation of RNA transcription is specific. The promoter of the present invention preferably has a length of 4 to 20 bases or more, preferably 6 to 20 bases or more, but is not limited thereto.

The promoter of the present invention may be used alone, but is preferably used in combination with a regulatory element such as an enhancer. The regulatory elements are preferably located in cis, but may be trans. The present invention also provides a regulatory gene in which the promoter and regulatory elements of the present invention are combined. Such a regulatory gene may be single-stranded or double-stranded when the regulatory element is a cis element. When the regulatory element is a trans element, it is used as a double strand. When it is found that an intron has a nucleotide sequence capable of initiating transcription of RNA by external stimulus, which nucleotide sequence in the intron plays a role such as a promoter? When it is not clear enough to say, the entire length of the nucleotide sequence of the intron can be used as the regulatory gene of the present invention.

The "external stimulus" in the present invention is a stimulus that does not occur under normal growth conditions of mature cells,
The stimulus is preferably such that cell death is induced by these stimuli. For example, stimulation by chemical substances such as kainic acid, physical stimulation such as electric shock or temperature change,
Examples include stimulation caused by abnormalities in other organs such as epileptic seizures. Further, the term "site-specific" in the present invention means
It is specific to a part of a living body that can be distinguished from others according to the type, state, or degree of growth of tissues, organs, or cells. The promoter or gene capable of initiating RNA transcription by the external stimulus of the present invention is not necessarily site-specific, but may be site-specific. The nucleotide sequences of the introns shown in SEQ ID NOS: 7, 8 and 9 in the sequence listing of the present specification are considered to be specific to the dentate gyrus of the hippocampus, but the promoter and gene of the present invention are not limited thereto. Not a thing.

The present invention reveals that there is a nucleotide sequence capable of initiating transcription of RNA by an external stimulus in the intron of an organism, and the range of utilization of such a gene of the present invention is Extremely wide. The first feature is that it exists as an intron, so that the introduction of this gene normally functions only as an intron and does not affect the normal growth of the organism. The second feature is that the regulatory gene of the present invention is inactive under normal conditions for RNA transcription and does not express the protein encoded downstream thereof. The third feature is that it can be expressed site-specifically. Since the promoter and the regulatory gene of the present invention have such characteristics, they can be applied according to their purpose. For example, in the case of expressing a partial length of a certain protein by a specific external stimulus and observing its physiological activity in vivo, the gene of the present invention is added immediately before the exon containing methionine as a start codon. By introducing the protein, it is possible to promote the expression of a protein having a target partial length by giving a specific external stimulus to the living body. In addition, when there is no suitable methionine, it is possible to introduce a nucleotide sequence encoding methionine into the intron region.

According to the second feature, by introducing a specific external stimulus into the living body by introducing the gene having the promoter and the regulatory gene of the present invention bound to the upstream of the target protein, the target introduced protein can be obtained. Can be expressed only when external stimuli are given. For example, it is also possible to attach a physiologically active protein to the end of the promoter of the present invention, express the physiologically active protein only when a specific external stimulus is given, and temporarily give the physiological activity to cells. If a toxin such as diphtheria toxin is used as the physiologically active protein, it is possible to transiently kill cells. Alternatively, a gene linked to the CRE gene is introduced downstream of the promoter of the present invention, and a specific gene such as glutamate receptor is lo
Create transgenic mice surrounding the x-P sequence. By doing this, when a specific external stimulus is given,
Since the CRE gene is expressed and a specific gene such as the glutamate receptor surrounded by the lox-P sequence is removed by homologous recombination, the glutamate receptor or the like can be removed from a specific external stimulus. Mice deficient in a particular gene can be created. With such a transgenic mouse, it becomes possible to analyze in detail a pathological condition in which a specific gene such as glutamate receptor is deleted in a mature organism.

Further, according to the above-mentioned third characteristic, it becomes possible to cause the above-mentioned characteristic in a site-specific manner in the living body. For example, it is possible to specifically destroy a specific gene in the dentate gyrus of the hippocampus.

Therefore, the present invention provides a method for controlling the expression of a gene encoding a protein introduced into a living body using the promoter and the regulatory gene of the present invention by a specific external stimulus. As described above, according to this method of the present invention, it becomes possible to control the length of expression of the introduced protein, the timing of expression, and the site of expression. The protein introduced in this method of the present invention is not particularly limited as long as it is a protein having some physiological activity, and it can be introduced in the state of genome or the state of cDNA. The physiologically active protein may be, for example, a so-called physiologically active protein such as hormone or cytokine, a toxin such as diphtheria toxin, or a homologous recombination such as CRE gene. May be induced.

The present invention also provides an organism in which the promoter and regulatory gene of the present invention are introduced upstream of a gene encoding a protein. The organism of the present invention is useful as a test animal, and can be applied to, for example, mice, rats, rabbits, monkeys and the like. It can also be applied to plants. Conventionally, transgenic mice and knockout mice have been developed as such test animals. Knockout mice are required to develop conditional targeting methods, and tissue-specific and time-specific promoters are being developed, such as tissue-specific expression promoters and tetracycline-sensitive promoters. It has been demanded. Since the promoter and regulatory gene of the present invention satisfy such requirements and also have a function as an intron, the promoter and regulatory gene of the present invention can be widely applied to test animals.

When the KIDS cPLA2 of the present invention is used as a level-adjusting agent for phospholipids such as lysophosphatidylcholine (LPC), it can be administered by methods such as intravenous administration, intramuscular administration and mucosal administration. The dose depends on the patient's condition and disease symptoms, but it is 10 nM-1.
00 mM is administered in several divided doses. The pharmaceutical composition of the present invention can be formulated into an intravenous administration preparation or an intramuscular administration preparation by using a commonly used pharmaceutical carrier. It can also be a freeze-dried preparation. The pharmaceutical composition of the present invention for a phospholipid level regulator can be used for suppressing apoptosis, improving immune function, and preventing or treating arteriosclerosis.

[0049]

EXAMPLES The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (Northern Blot Using Various Probes of cPLA2) Rat cPLA2a cDNA was cloned from the 5'end to 4 clones.
It was divided into two areas A, B, C and D. The length of each region is about 300-500 bp, and these cD
After incorporating the NA fragment into the riboprobe synthesis vector, in vitro transcription (in vitro
A radiolabeled riboprobe was synthesized by the transcription method. A hybridization reaction was carried out using a membrane in which poly (A) ⁺ RNA of hippocampus and hippocampus dentate gyrus of which rat was stimulated with kainic acid was blotted, and riboprobes of A, B, C, and D respectively. It was confirmed whether the probe could detect KIDS cPLA2. As a result, KIDScPLA2 mRNA excludes A,
It was found to be detected by B, C, and D riboprobes. The results are shown in FIGS. 1 and 2.

Example 2 (In Situ Hybridization) The brains of 3-week-old Wistar rats stimulated with kainic acid were fixed with 4% paraformaldehyde, and frozen sections were prepared. The radiolabeled riboprobes B, C, and D were separately subjected to a hybridization reaction with a frozen section, and it was confirmed that the labeled images of the sections by the respective probes were the same. Next, using the riboprobe C, a brain frozen section was again prepared with or without kainic acid stimulation, and KIDScPLA2 mR was prepared.
The expression pattern of NA was examined. As a result, KIDScP
LA2 mRNA was found to be dramatically induced in the hippocampal dentate gyrus. When observing the strong magnified image with a microscope, KID
It was found that ScPLA2 mRNA was abundantly expressed in the innermost layer of the dentate gyrus. The results are shown in FIG. 3 and FIG.
Shown in.

Example 3 (Immunohistochemical staining) 3 week-old Wistar rats stimulated with kainic acid, and 6-10 week old C57 / Black6J mice, cPLA2.
After fixing the brain of a knockout mouse with 4% paraformaldehyde, frozen sections were prepared. Anti-KIDS cPL
The immune reaction between the A2-specific stump antibody and the brain slice was carried out overnight at 4 ° C., and then with the secondary antibody labeled with gold colloid, KIDScPLA.
The expression of 2 proteins was confirmed. As a result, KIDScPLA
2 was found to be dramatically induced in the hippocampal dentate gyrus like mRNA, and its expression was also found in the hippocampal dentate gyrus of cPLA2a knockout mice. From this,
It was suggested that the KIDS cPLA2 promoter is present downstream of the eighth exon of cPLA2a disrupted in the cPLA2a knockout mouse, and it was found that acute neurostimulation induces the cPLA2a isoform. Results are shown in FIG.

Example 4 (KIDS cPLA of rat
Cloning of 2 cDNA) The presence was confirmed by two methods, and a clone was isolated. (1) Po purified from rat hippocampus after kainic acid stimulation
cPLA2a capable of detecting KIDS cPLA2 by preparing a cDNA library using ly (A) ⁺ RNA
From the translation start point of 1,365 (Rsa I) -1,92
A positive clone was selected using the cDNA sequence of 5 (Bal I) as a probe. Twelve positive clones were selected from 4 million clones. Two of them
The ones are full-length phospholipase A2, and the remaining 10
6 and 4 of them are of different types, type the former
II, and the latter was type I.

(2) Rat KIDS cPLA2 cDN
In order to confirm the 5 ′ end of A, 5 ′ was obtained using poly (A) ⁺ RNA purified from rat hippocampus after kainic acid stimulation.
RACE method (5'-rapid amplification of cDNA ends)
I went. The above, 1,365 (Rsa I) -1,92
The sequence amplified 5'upstream from the primer present within the sequence of 5 (Bal I) matched the sequence of the clone selected from the cDNA library. From the above, it was found that KIDScPLA2 is a novel gene induced in the hippocampus after kainic acid stimulation. The amino acid sequence of the obtained rat KIDS cPLA2 is shown in SEQ ID NO: 5 in the sequence listing. C of rat KIDS cPLA2
The nucleotide sequences of the translated region of DNA are shown in SEQ ID NO: 6 (type I) and SEQ ID NO: 7 (type II) in the sequence listing.

The amino acid sequence of mouse KIDS cPLA2 is shown in SEQ ID NO: 8 in the sequence listing. KI of mouse
The nucleotide sequence of the translation region of the DS cPLA2 cDNA is shown in SEQ ID NO: 9 (when 5 ′ UTR is type I), SEQ ID NO: 10 (when 5 ′ UTR is type II) and SEQ ID NO: 11 (5 ′). UTR type I, type
If not divided into II). Human KIDS cPLA
The amino acid sequence of 2 is shown in SEQ ID NO: 1 in the sequence listing. The nucleotide sequence of the translation region of human KIDS cPLA2 cDNA is SEQ ID NO: 2 (when 5 ′ UTR is type I) and SEQ ID NO: 3 (when 5 ′ UTR is type II) in the sequence listing.
And SEQ ID NO: 4 (when 5'UTR is not divided into type I and type II). These sequences were obtained by subjecting human, mouse, and rat cDNA sequences to an alignment program at the same time, and one of the nucleotides presumed to be the transcription start positions of the transcription start position, type I and type II, the type I sequence and the type II sequence A junction array that connects
It has been selected from the most appropriate sequences by applying conditions such as comprehensive sequence homology.

Example 5 (Production of KIDS cPLA2 Antibody) In order to specifically detect KIDScPLA2 having a sequence identical to the sequence of rat cPLA2a Met-308, 7 amino acid sequences starting from this Met-308 were detected. Rabbits were immunized with a synthetic peptide having (MSTTLSS), and the serum fraction thereof was adjusted. Further, immunoglobulin (IgG) was purified from this fraction and used as the final preparation. It has been confirmed that this stump antibody specifically recognizes not only rat KIDScPLA2 but also mouse KIDScPLA2.

Example 6 (Analysis of intron base sequence) The intron sequence of rat and mouse KIDScPLA2 (probable promoter region) was analyzed by the following method. Rat and mouse KIDScPLA
A region (cP containing about 5 kb upstream including the 5'UTR of 2
Regarding the region up to the exon that is destroyed in the LAα knockout mouse), it was examined whether there is a basic transcriptional activity. First, a sequence of about 9 kb in this region and a sequence containing about 1,000 bp upstream of 5'UTR, about 500 bp with high homology between human, rat and mouse.
Was inserted into the upstream region of the luciferase gene to construct a reporter vector. After introducing these reporter vectors into a cultured cell line, the cell supernatant was prepared and its luciferase activity was measured as an indicator of basal transcription activity. As a result, it was found that the approximately 700 bp sequence containing the 5'UTR had a particularly high transcription activity. Results are shown in FIG. In addition, the nucleotide sequence of each animal is shown in the sequence listing as SEQ ID NO: 12 (human), 13 (rat) and 14 (mouse)
Shown in.

[0058] Example 7 (Measurement of lysophospholipase A1 activity) ^1-(1-14 C-palmitoyl) - lysophosphatidylcholine, standard buffer (50 mM Tris Cl,
(PH 8.0), 2 mM EDTA (pH 8.0), 10
mM CaCl ₂ , 30% glycerin, 1 mM Triton X-100) adjusted to 10 μM, 61,400 dp
Rat recombinant KID at m / 0.5 nmol / 50 μL
Each enzymatic reaction of ScPLA2, cPLA2α, and lacZ was performed. The reaction time was 4 minutes at 0 minutes, 10 minutes, 20 minutes, and 40 minutes, and the reaction was stopped at each time to perform the assay. To stop the reaction, stop solution (isopropanol / n-heptane / 1 for ice-cooled reaction system of 50 mL) was used.
NH ₂ SO ₄ : 78/20/2) 250 mL,
I went vortexing. Then, 150 mL of n-heptane and 100 mL of H ₂ O were enthusiastically vortexed for 5 minutes. This solution at 15,000 rpm for 5 minutes,
After centrifugation, the upper organic layer is activated silica gel (20
mg) to the tube. After adding 150 mL of n-heptane and performing vigorous PORTEX again for 5 minutes, centrifugation was performed at 15,000 rpm for 5 minutes, and an equal amount of organic layer was taken from each reaction tube and subjected to cauting. The results are shown in Fig. 15.

[0059] Example 8 (a lysophospholipase A1 activity kinetic measurements) standard buffer the substrate using ^1-(1-14 C-palmitoyl) - the concentration of the lysophosphatidylcholine, 0,7Myu
M was changed to 10 to 100 μM, and each enzyme was reacted in the same manner as in Example 7. The results are shown in Fig. 16.

Example 9 (Measurement of Effect of Ca Ion on Lysophospholipase A1 Activity) A buffer containing 5 mM CaCl ₂ was used, and no EDTA was added and 5 mM EDTA was added. Each enzymatic reaction was carried out in the same manner as in Example 7. Results are shown in FIG.

Example 10 (Measurement of Effect of AEBSF on Lysophospholipase A1 Activity) A standard buffer solution was used as a buffer solution, without addition of AEBSF, and at 1 mM, 2 mM, 20 mM, and 55 mM.
Each enzyme reaction was carried out in the same manner as in Example 7 except that AEBSF was added. The results are shown in Fig. 18.

EFFECTS OF THE INVENTION The present invention provides a novel enzyme that is considered to be the cause of cell death specific to the hippocampal dentate gyrus due to kainic acid stimulation, epileptic seizures and the like. By making an inhibitor of this enzyme, it becomes possible to prevent cell death. The present invention also provides a novel enzyme that has phospholipase A2 activity and is calcium-independent. Furthermore, the present invention makes it clear for the first time that the intron has a function that allows transcription initiation of RNA by external stimulation. While elucidating the new function of introns in the genome, it also provides a new type of gene that functions as a promoter or regulatory gene by external stimuli. The novel type of gene that functions as a promoter or a regulatory gene of the present invention not only has a function as an intron, but also enables the expression of a target gene of a specific time in response to an external stimulus, It enables the site-specific expression of a target gene. Therefore, the promoter or regulatory gene of the present invention can be used for the regulation of gene expression, and can be applied to transgenic animals, knockout animals and the like. Further, KIDScPLA2 of the present invention has a lysophospholipase A1 activity in a calcium-independent manner, is useful as a phospholipid level regulator, and is useful for the prevention / treatment of immune diseases and arteriosclerosis.

[Sequence list]                             SEQUENCE LISTING <110> Japan Science and Technology Corporation <120> <130> PA905783 <160> 12 <210> 1 <211> 442 <212> PRT <213> Human <400> 1 Met Asn Thr Thr Leu Ser Ser Leu Lys Glu Lys Val Asn Thr Ala Gln   1 5 10 15 Cys Pro Leu Pro Leu Phe Thr Cys Leu His Val Lys Pro Asp Val Ser              20 25 30 Glu Leu Met Phe Ala Asp Trp Val Glu Phe Ser Pro Tyr Glu Ile Gly          35 40 45 Met Ala Lys Tyr Gly Thr Phe Met Ala Pro Asp Leu Phe Gly Ser Lys      50 55 60 Phe Phe Met Gly Thr Val Val Lys Lys Tyr Glu Glu Asn Pro Leu His  65 70 75 80 Phe Leu Met Gly Val Trp Gly Ser Ala Phe Ser Ile Leu Phe Asn Arg                  85 90 95 Val Leu Gly Val Ser Gly Ser Gln Ser Arg Gly Ser Thr Met Glu Glu              100 105 110 Glu Leu Glu Asn Ile Thr Thr Lys His Ile Val Ser Asn Asp Ser Ser          115 120 125 Asp Ser Asp Asp Glu Ser His Glu Pro Lys Gly Thr Glu Asn Glu Asp      130 135 140 Ala Gly Ser Asp Tyr Gln Ser Asp Asn Gln Ala Ser Trp Ile His Arg 145 150 155 160 Met Ile Met Ala Leu Val Ser Asp Ser Ala Leu Phe Asn Thr Arg Glu                  165 170 175 Gly Arg Ala Gly Lys Val His Asn Phe Met Leu Gly Leu Asn Leu Asn              180 185 190 Thr Ser Tyr Pro Leu Ser Pro Leu Ser Asp Phe Ala Thr Gln Asp Ser          195 200 205 Phe Asp Asp Asp Glu Leu Asp Ala Ala Val Ala Asp Pro Asp Glu Phe      210 215 220 Glu Arg Ile Tyr Glu Pro Leu Asp Val Lys Ser Lys Lys Ile His Val 225 230 235 240 Val Asp Ser Gly Leu Thr Phe Asn Leu Pro Tyr Pro Leu Ile Leu Arg                  245 250 255 Pro Gln Arg Gly Val Asp Leu Ile Ile Ser Phe Asp Phe Ser Ala Arg              260 265 270 Pro Ser Asp Ser Ser Pro Pro Phe Lys Glu Leu Leu Leu Ala Glu Lys          275 280 285 Trp Ala Lys Met Asn Lys Leu Pro Phe Pro Lys Ile Asp Pro Tyr Val      290 295 300 Phe Asp Arg Glu Gly Leu Lys Glu Cys Tyr Val Phe Lys Pro Lys Asn 305 310 315 320 Pro Asp Met Glu Lys Asp Cys Pro Thr Ile Ile His Phe Val Leu Ala                  325 330 335 Asn Ile Asn Phe Arg Lys Tyr Lys Ala Pro Gly Val Pro Arg Glu Thr              340 345 350 Glu Glu Glu Lys Glu Ile Ala Asp Phe Asp Ile Phe Asp Asp Pro Glu          355 360 365 Ser Pro Phe Ser Thr Phe Asn Phe Gln Tyr Pro Asn Gln Ala Phe Lys      370 375 380 Arg Leu His Asp Leu Met His Phe Asn Thr Leu Asn Asn Ile Asp Val 385 390 395 400 Ile Lys Glu Ala Met Val Glu Ser Ile Glu Tyr Arg Arg Gln Asn Pro                  405 410 415 Ser Arg Cys Ser Val Ser Leu Ser Asn Val Glu Ala Arg Arg Phe Phe              420 425 430 Asn Lys Glu Phe Leu Ser Lys Pro Lys Ala          435 440 <210> 2 <211> 1918 <212> DNA <213> Human <400> 2 gattttgatt ggaagtacta ttttgaatag cattctttct gtgtctgttt ataaatttaa 60 agtcatcttt ttctttcttc tgtggacaga gaatgaatac tactctgagc agtttgaagg 120 aaaaagttaa tactgcacaa tgccctttac ctcttttcac ctgtcttcat gtcaaacctg 180 acgtttcaga gctgatgttt gcagattggg ttgaatttag tccatacgaa attggcatgg 240 ctaaatatgg tacttttatg gctcccgact tatttggaag caaatttttt atgggaacag 300 tcgttaagaa gtatgaagaa aaccccttgc atttcttaat gggtgtctgg ggcagtgcct 360 tttccatatt gttcaacaga gttttgggcg tttctggttc acaaagcaga ggctccacaa 420 tggaggaaga attagaaaat attaccacaa agcatattgt gagtaatgat agctcggaca 480 gtgatgatga atcacacgaa cccaaaggca ctgaaaatga agatgctgga agtgactatc 540 aaagtgataa tcaagcaagt tggattcatc gtatgataat ggccttggtg agtgattcag 600 ctttattcaa taccagagaa ggacgtgctg ggaaggtaca caacttcatg ctgggcttga 660 atctcaatac atcttatcca ctgtctcctt tgagtgactt tgccacacag gactcctttg 720 atgatgatga actggatgca gctgtagcag atcctgatga atttgagcga atatatgagc 780 ctctggatgt caaaagtaaa aagattcatg tagtggacag tgggctcaca tttaacctgc 840 cgtatccctt gatactgaga cctcagagag gggttgatct cataatctcc tttgactttt 900 ctgcaaggcc aagtgactct agtcctccgt tcaaggaact tctacttgca gaaaagtggg 960 ctaaaatgaa caagctcccc tttccaaaga ttgatcctta tgtgtttgat cgggaagggc 1020 tgaaggagtg ctatgtcttt aaacccaaga atcctgatat ggagaaagat tgcccaacca 1080 tcatccactt tgttctggcc aacatcaact tcagaaagta caaggctcca ggtgttccaa 1140 gggaaactga ggaagagaaa gaaatcgctg actttgatat ttttgatgac ccagaatcac 1200 cattttcaac cttcaatttt caatatccaa atcaagcatt caaaagacta catgatctta 1260 tgcacttcaa tactctgaac aacattgatg tgataaaaga agccatggtt gaaagcattg 1320 aatatagaag acagaatcca tctcgttgct ctgtttccct tagtaatgtt gaggcaagaa 1380 gatttttcaa caaggagttt ctaagtaaac ccaaagcata gttcatgtac tggaaatggc 1440 agcagtttct gatgctgagg cagtttgcaa tcccatgaca actggattta aaagtacagt 1500 acagatagtc gtactgatca tgagagactg gctgatactc aaagttgcag ttacttagct 1560 gcatgagaat aatactatta taagttaggt gacaaatgat gttgattatg taaggatata 1620 cttagctaca ttttcagtca gtatgaactt cctgatacaa atgtagggat atatactgta 1680 tttttaaaca tttctcacca actttcttat gtgtgttctt tttaaaaatt ttttttcttt 1740 taaaatattt aacagttcaa tctcaataag acctcgcatt atgtatgaat gttattcact 1800 gactagattt attcatacca tgagacaaca ctatttttat ttatatatgc atatatatac 1860 atacatgaaa taaatacatc aatataaaaa taaaaaaaaa cggaattc 1918 <210> 3 <211> 1925 <212> DNA <213> Human <400> 3 gattattttt taaatgaaga tagttacttc catagagctt attttttgtt gttcattcag 60 gacctagtaa tttctagaag taataagact tatttttatt ataaagagaa tgaatactac 120 tctgagcagt ttgaaggaaa aagttaatac tgcacaatgc cctttacctc ttttcacctg 180 tcttcatgtc aaacctgacg tttcagagct gatgtttgca gattgggttg aatttagtcc 240 atacgaaatt ggcatggcta aatatggtac ttttatggct cccgacttat ttggaagcaa 300 attttttatg ggaacagtcg ttaagaagta tgaagaaaac cccttgcatt tcttaatggg 360 tgtctggggc agtgcctttt ccatattgtt caacagagtt ttgggcgttt ctggttcaca 420 aagcagaggc tccacaatgg aggaagaatt agaaaatatt accacaaagc atattgtgag 480 taatgatagc tcggacagtg atgatgaatc acacgaaccc aaaggcactg aaaatgaaga 540 tgctggaagt gactatcaaa gtgataatca agcaagttgg attcatcgta tgataatggc 600 cttggtgagt gattcagctt tattcaatac cagagaagga cgtgctggga aggtacacaa 660 cttcatgctg ggcttgaatc tcaatacatc ttatccactg tctcctttga gtgactttgc 720 cacacaggac tcctttgatg atgatgaact ggatgcagct gtagcagatc ctgatgaatt 780 tgagcgaata tatgagcctc tggatgtcaa aagtaaaaag attcatgtag tggacagtgg 840 gctcacattt aacctgccgt atcccttgat actgagacct cagagagggg ttgatctcat 900 aatctccttt gacttttctg caaggccaag tgactctagt cctccgttca aggaacttct 960 acttgcagaa aagtgggcta aaatgaacaa gctccccttt ccaaagattg atccttatgt 1020 gtttgatcgg gaagggctga aggagtgcta tgtctttaaa cccaagaatc ctgatatgga 1080 gaaagattgc ccaaccatca tccactttgt tctggccaac atcaacttca gaaagtacaa 1140 ggctccaggt gttccaaggg aaactgagga agagaaagaa atcgctgact ttgatatttt 1200 tgatgaccca gaatcaccat tttcaacctt caattttcaa tatccaaatc aagcattcaa 1260 aagactacat gatcttatgc acttcaatac tctgaacaac attgatgtga taaaagaagc 1320 catggttgaa agcattgaat atagaagaca gaatccatct cgttgctctg tttcccttag 1380 taatgttgag gcaagaagat ttttcaacaa ggagtttcta agtaaaccca aagcatagtt 1440 catgtactgg aaatggcagc agtttctgat gctgaggcag tttgcaatcc catgacaact 1500 ggatttaaaa gtacagtaca gatagtcgta ctgatcatga gagactggct gatactcaaa 1560 gttgcagtta cttagctgca tgagaataat actattataa gttaggtgac aaatgatgtt 1620 gattatgtaa ggatatactt agctacattt tcagtcagta tgaacttcct gatacaaatg 1680 tagggatata tactgtattt ttaaacattt ctcaccaact ttcttatgtg tgttcttttt 1740 aaaaattttt tttcttttaa aatatttaac agttcaatct caataagacc tcgcattatg 1800 tatgaatgtt attcactgac tagatttatt cataccatga gacaacacta tttttattta 1860 tatatgcata tatatacata catgaaataa atacatcaat ataaaaataa aaaaaaacgg 1920 aattc 1925 <210> 4 <211> 2020 <212> DNA <213> Human <400> 4 gattattttt taaatgaaga tagttacttc catagagctt attttttgtt gttcattcag 60 gacctagtaa tttctagaag taataagact tatttttatt ataaagttat aagattttga 120 ttggaagtac tattttgaat agcattcttt ctgtgtctgt ttataaattt aaagtcatct 180 ttttctttct tctgtggaca gagaatgaat actactctga gcagtttgaa ggaaaaagtt 240 aatactgcac aatgcccttt acctcttttc acctgtcttc atgtcaaacc tgacgtttca 300 gagctgatgt ttgcagattg ggttgaattt agtccatacg aaattggcat ggctaaatat 360 ggtactttta tggctcccga cttatttgga agcaaatttt ttatgggaac agtcgttaag 420 aagtatgaag aaaacccctt gcatttctta atgggtgtct ggggcagtgc cttttccata 480 ttgttcaaca gagttttggg cgtttctggt tcacaaagca gaggctccac aatggaggaa 540 gaattagaaa atattaccac aaagcatatt gtgagtaatg atagctcgga cagtgatgat 600 gaatcacacg aacccaaagg cactgaaaat gaagatgctg gaagtgacta tcaaagtgat 660 aatcaagcaa gttggattca tcgtatgata atggccttgg tgagtgattc agctttattc 720 aataccagag aaggacgtgc tgggaaggta cacaacttca tgctgggctt gaatctcaat 780 acatcttatc cactgtctcc tttgagtgac tttgccacac aggactcctt tgatgatgat 840 gaactggatg cagctgtagc agatcctgat gaatttgagc gaatatatga gcctctggat 900 gtcaaaagta aaaagattca tgtagtggac agtgggctca catttaacct gccgtatccc 960 ttgatactga gacctcagag aggggttgat ctcataatct cctttgactt ttctgcaagg 1020 ccaagtgact ctagtcctcc gttcaaggaa cttctacttg cagaaaagtg ggctaaaatg 1080 aacaagctcc cctttccaaa gattgatcct tatgtgtttg atcgggaagg gctgaaggag 1140 tgctatgtct ttaaacccaa gaatcctgat atggagaaag attgcccaac catcatccac 1200 tttgttctgg ccaacatcaa cttcagaaag tacaaggctc caggtgttcc aagggaaact 1260 gaggaagaga aagaaatcgc tgactttgat atttttgatg acccagaatc accattttca 1320 accttcaatt ttcaatatcc aaatcaagca ttcaaaagac tacatgatct tatgcacttc 1380 aatactctga acaacattga tgtgataaaa gaagccatgg ttgaaagcat tgaatataga 1440 agacagaatc catctcgttg ctctgtttcc cttagtaatg ttgaggcaag aagatttttc 1500 aacaaggagt ttctaagtaa acccaaagca tagttcatgt actggaaatg gcagcagttt 1560 ctgatgctga ggcagtttgc aatcccatga caactggatt taaaagtaca gtacagatag 1620 tcgtactgat catgagagac tggctgatac tcaaagttgc agttacttag ctgcatgaga 1680 ataatactat tataagttag gtgacaaatg atgttgatta tgtaaggata tacttagcta 1740 cattttcagt cagtatgaac ttcctgatac aaatgtaggg atatatactg tatttttaaa 1800 catttctcac caactttctt atgtgtgtgttc tttttaaaaa ttttttttct tttaaaatat 1860 ttaacagttc aatctcaata agacctcgca ttatgtatga atgttattca ctgactagat 1920 ttattcatac catgagacaa cactattttt atttatatat gcatatatat acatacatga 1980 aataaataca tcaatataaa aataaaaaaa aacggaattc 2020 <210> 5 <211> 445 <212> PRT <213> rat <400> 5 Met Ser Thr Thr Leu Ser Ser Leu Lys Glu Lys Val Ser Ala Ala Arg   1 5 10 15 Cys Pro Leu Pro Leu Phe Thr Cys Leu His Val Lys Pro Asp Val Ser              20 25 30 Glu Leu Met Phe Ala Asp Trp Val Glu Phe Ser Pro Tyr Glu Ile Gly          35 40 45 Met Ala Lys Tyr Gly Thr Phe Met Thr Pro Asp Leu Phe Gly Ser Lys      50 55 60 Phe Phe Met Gly Thr Val Val Lys Lys Tyr Glu Glu Asn Pro Leu His  65 70 75 80 Phe Leu Met Gly Val Trp Gly Ser Ala Phe Ser Ile Leu Phe Asn Arg                  85 90 95 Val Leu Gly Val Ser Gly Ser Gln Asn Lys Gly Ser Thr Met Glu Glu              100 105 110 Glu Leu Glu Asn Ile Thr Ala Lys His Ile Val Ser Asn Asp Ser Ser          115 120 125 Asp Ser Asp Asp Glu Ala Gln Gly Pro Lys Gly Thr Glu Asn Glu Asp      130 135 140 Ala Glu Arg Glu Tyr Gln Asn Asp Asn Gln Ala Ser Trp Val His Arg 145 150 155 160 Met Leu Met Ala Leu Val Ser Asp Ser Ala Leu Phe Asn Thr Arg Glu                  165 170 175 Gly Arg Ala Gly Lys Glu His Asn Phe Met Leu Gly Leu Asn Leu Asn              180 185 190 Thr Ser Tyr Pro Leu Ser Pro Leu Arg Asp Phe Ser Pro Gln Asp Ser          195 200 205 Phe Asp Asp Asp Glu Leu Asp Ala Ala Val Ala Asp Pro Asp Glu Phe      210 215 220 Glu Arg Ile Tyr Glu Pro Leu Asp Val Lys Ser Lys Lys Ile His Val 225 230 235 240 Val Asp Ser Gly Leu Thr Phe Asn Leu Pro Tyr Pro Leu Ile Leu Arg                  245 250 255 Pro Gln Arg Gly Val Asp Leu Ile Ile Ser Phe Asp Phe Ser Ala Arg              260 265 270 Pro Ser Asp Thr Ser Pro Pro Phe Lys Glu Leu Leu Leu Ala Glu Lys          275 280 285 Trp Ala Lys Met Asn Lys Leu Pro Phe Pro Lys Ile Asp Pro Tyr Val      290 295 300 Phe Asp Arg Glu Gly Leu Lys Glu Cys Tyr Val Phe Lys Pro Lys Asn 305 310 315 320 Pro Asp Val Glu Lys Asp Cys Pro Thr Ile Ile His Phe Val Leu Ala                  325 330 335 Asn Ile Asn Phe Arg Lys Tyr Lys Ala Pro Gly Val Leu Arg Glu Thr              340 345 350 Lys Glu Glu Lys Glu Ile Ala Asp Phe Asp Ile Phe Asp Asp Pro Glu          355 360 365 Ser Pro Phe Ser Thr Phe Asn Phe Gln Tyr Pro Asn Gln Ala Phe Lys      370 375 380 Arg Leu His Asp Leu Met Tyr Phe Asn Thr Leu Asn Asn Ile Asp Val 385 390 395 400 Ile Lys Asp Ala Ile Val Glu Ser Ile Glu Tyr Arg Arg Gln Asn Pro                  405 410 415 Ser Arg Cys Ser Val Ser Leu Ser Asn Val Glu Ala Arg Lys Phe Phe              420 425 430 Asn Lys Glu Phe Leu Ser Lys Pro Thr Ala Glu Ser Ile          435 440 445 <210> 6 <211> 1782 <212> DNA <213> rat <400> 6 cgatttggtt agacatatta tttcaaatag cttttatctg tgtccatgtc tatgtattta 60 aagccacctt attctttttg tgtgtgtgtgtg tgaaaagaga atgagtacga ccttgagtag 120 cttgaaggaa aaggtcagcg ccgcccggtg tcctctgcct ctcttcacct gtctccatgt 180 caaaccggac gtgtcagagc tgatgtttgc cgattgggta gaatttagtc catacgaaat 240 tggcatggca aaatatggta cctttatgac tcctgacttg tttggaagca aattttttat 300 gggaacagtt gtaaaaaaat atgaagaaaa ccccttgcat ttcttaatgg gtgtctgggg 360 cagtgccttt tctatactgt tcaacagagt tttgggagtt tctggctcac agaataaagg 420 ttctacaatg gaggaggaat tagaaaatat tacagcaaag cacattgtga gtaacgacag 480 ctctgacagc gatgacgagg cccaaggacc caaaggcacc gagaatgaag atgcggaaag 540 agagtaccaa aatgacaacc aagcaagttg ggtccatcgg atgctaatgg ccttggtgag 600 tgactcagct ttattcaata cccgagaagg acgtgctggg aaggagcata acttcatgtt 660 gggcttgaat ctcaacacat cgtatccact gtctcccctg agagacttca gcccccaaga 720 ttccttcgat gatgatgaac tcgacgcagc ggtagcagat ccagatgaat ttgaacgaat 780 atatgaacca ctggatgtca aaagtaaaaa gattcatgtt gtagacagtg ggctcacgtt 840 taacctgccg tatcccttga ttctgcgacc tcagagaggt gtggatctca tcatttcctt 900 tgacttttct gcaaggccaa gtgacaccag ccctccattc aaggaacttc tgcttgcaga 960 gaagtgggct aaaatgaaca agctcccttt tccaaagatt gatccttacg tgtttgatcg 1020 ggaaggattg aaggaatgct atgtgtttatta acctaagaat cctgatgtgg aaaaggattg 1080 cccaaccatt atccactttg ttctggccaa catcaacttc agaaagtaca aggccccagg 1140 tgttctgagg gaaaccaaag aagagaaaga aatagctgac tttgacattt tcgatgaccc 1200 cgaatcgcca ttttcaacct tcaacttcca gtatccaaat caagcattca aaaggctaca 1260 tgatctgatg tacttcaaca cactgaacaa cattgatgtg ataaaggatg ccattgttga 1320 gagcattgaa tacagaagac agaacccatc tcgttgctct gtttccctca gtaatgttga 1380 ggcaagaaaa ttcttcaaca aggagttcct aagtaaaccc acagcggagt ccatttgaat 1440 tccatgacta ctggagttca gagccacatg agagactcat cttactatgc acaagagact 1500 gactgctact cagagttgct ggggacggag gcgtgtgtta ggtgaaaatg gtgttgatta 1560 tgcaatactt ggcaacagtt tctgacagta tgaatttttt gtacataagc atagggctat 1620 atactgtatt ttaaacattc ctcacatttt tacctgagca tttttatata tataaaaata 1680 tcctttcctt ttataaatat ttaatagtta actcagtaaa aaaaagcttc ccattgtgtg 1740 tgaatgttat tctgaactag atttgttcat gccatgttac aa 1782 <210> 7 <211> 1792 <212> DNA <213> rat <400> 7 caattgtttt agaatacaga catctatttc cagggagctt tctttctgtt gtctaatcga 60 gaccacagat tgccagaaat aataggactt cgtttcatta taaaaagaga atgagtacga 120 ccttgagtag cttgaaggaa aaggtcagcg ccgcccggtg tcctctgcct ctcttcacct 180 gtctccatgt caaaccggac gtgtcagagc tgatgtttgc cgattgggta gaatttagtc 240 catacgaaat tggcatggca aaatatggta cctttatgac tcctgacttg tttggaagca 300 aattttttat gggaacagtt gtaaaaaaat atgaagaaaa ccccttgcat ttcttaatgg 360 gtgtctgggg cagtgccttt tctatactgt tcaacagagt tttgggagtt tctggctcac 420 agaataaagg ttctacaatg gaggaggaat tagaaaatat tacagcaaag cacattgtga 480 gtaacgacag ctctgacagc gatgacgagg cccaaggacc caaaggcacc gagaatgaag 540 atgcggaaag agagtaccaa aatgacaacc aagcaagttg ggtccatcgg atgctaatgg 600 ccttggtgag tgactcagct ttattcaata cccgagaagg acgtgctggg aaggagcata 660 acttcatgtt gggcttgaat ctcaacacat cgtatccact gtctcccctg agagacttca 720 gcccccaaga ttccttcgat gatgatgaac tcgacgcagc ggtagcagat ccagatgaat 780 ttgaacgaat atatgaacca ctggatgtca aaagtaaaaa gattcatgtt gtagacagtg 840 ggctcacgtt taacctgccg tatcccttga ttctgcgacc tcagagaggt gtggatctca 900 tcatttcctt tgacttttct gcaaggccaa gtgacaccag ccctccattc aaggaacttc 960 tgcttgcaga gaagtgggct aaaatgaaca agctcccttt tccaaagatt gatccttacg 1020 tgtttgatcg ggaaggattg aaggaatgct atgtgtttaa acctaagaat cctgatgtgg 1080 aaaaggattg cccaaccatt atccactttg ttctggccaa catcaacttc agaaagtaca 1140 aggccccagg tgttctgagg gaaaccaaag aagagaaaga aatagctgac tttgacattt 1200 tcgatgaccc cgaatcgcca ttttcaacct tcaacttcca gtatccaaat caagcattca 1260 aaaggctaca tgatctgatg tacttcaaca cactgaacaa cattgatgtg ataaaggatg 1320 ccattgttga gagcattgaa tacagaagac agaacccatc tcgttgctct gtttccctca 1380 gtaatgttga ggcaagaaaa ttcttcaaca aggagttcct aagtaaaccc acagcggagt 1440 ccatttgaat tccatgacta ctggagttca gagccacatg agagactcat cttactatgc 1500 acaagagact gactgctact cagagttgct ggggacggag gcgtgtgtta ggtgaaaatg 1560 gtgttgatta tgcaatactt ggcaacagtt tctgacagta tgaatttttt gtacataagc 1620 atagggctat atactgtatt ttaaacattc ctcacatttt tacctgagca tttttatata 1680 tataaaaata tcctttcctt ttataaatat ttaatagtta actcagtaaa aaaaagcttc 1740 ccattgtgtg tgaatgttat tctgaactag atttgttcat gccatgttac aa 1792 <210> 8 <211> 441 <212> PRT <213> mouse <400> 8 Met Ser Met Thr Leu Ser Ser Leu Lys Glu Lys Val Asn Ala Ala Arg   1 5 10 15 Cys Pro Leu Pro Leu Phe Thr Cys Leu His Val Lys Pro Asp Val Ser              20 25 30 Glu Leu Met Phe Ala Asp Trp Val Glu Phe Ser Pro Tyr Glu Ile Gly          35 40 45 Met Ala Lys Tyr Gly Thr Phe Met Ala Pro Asp Leu Phe Gly Ser Lys      50 55 60 Phe Phe Met Gly Thr Val Val Lys Lys Tyr Glu Glu Asn Pro Leu His  65 70 75 80 Phe Leu Met Gly Val Trp Gly Ser Ala Phe Ser Ile Leu Phe Asn Arg                  85 90 95 Val Leu Gly Val Ser Gly Ser Gln Asn Lys Gly Ser Thr Met Glu Glu              100 105 110 Glu Leu Glu Asn Ile Thr Ala Lys His Ile Val Ser Asn Asp Ser Ser          115 120 125 Asp Ser Asp Asp Glu Ala Gln Gly Pro Lys Gly Thr Glu Asn Glu Glu      130 135 140 Ala Glu Lys Glu Tyr Gln Ser Asp Asn Gln Ala Ser Trp Val His Arg 145 150 155 160 Met Leu Met Ala Leu Val Ser Asp Ser Ala Leu Phe Asn Thr Arg Glu                  165 170 175 Gly Arg Ala Gly Lys Val His Asn Phe Met Leu Gly Leu Asn Leu Asn              180 185 190 Thr Ser Tyr Pro Leu Ser Pro Leu Arg Asp Phe Ser Ser Gln Asp Ser          195 200 205 Phe Asp Asp Glu Leu Asp Ala Ala Val Ala Asp Pro Asp Glu Phe Glu      210 215 220 Arg Ile Tyr Glu Pro Leu Asp Val Lys Ser Lys Lys Ile His Val Val 225 230 235 240 Asp Ser Gly Leu Thr Phe Asn Leu Pro Tyr Pro Leu Ile Leu Arg Pro                  245 250 255 Gln Arg Gly Val Asp Leu Ile Ile Ser Phe Asp Phe Ser Ala Arg Pro              260 265 270 Ser Asp Thr Ser Pro Pro Phe Lys Glu Leu Leu Leu Ala Glu Lys Trp          275 280 285 Ala Lys Met Asn Lys Leu Pro Phe Pro Lys Ile Asp Pro Tyr Val Phe      290 295 300 Asp Arg Glu Gly Leu Lys Glu Cys Tyr Val Phe Lys Pro Lys Asn Pro 305 310 315 320 Asp Val Glu Lys Asp Cys Pro Thr Ile Ile His Phe Val Leu Ala Asn                  325 330 335 Ile Asn Phe Arg Lys Tyr Lys Ala Pro Gly Val Leu Arg Glu Thr Lys              340 345 350 Glu Glu Lys Glu Ile Ala Asp Phe Asp Ile Phe Asp Asp Pro Glu Ser          355 360 365 Pro Phe Ser Thr Phe Asn Phe Gln Tyr Pro Asn Gln Ala Phe Lys Arg      370 375 380 Leu His Asp Leu Met Tyr Phe Asn Thr Leu Asn Asn Ile Asp Val Ile 385 390 395 400 Lys Asp Ala Ile Val Glu Ser Ile Glu Tyr Arg Arg Gln Asn Pro Ser                  405 410 415 Arg Cys Ser Val Ser Leu Ser Asn Val Glu Ala Arg Lys Phe Phe Asn              420 425 430 Lys Glu Phe Leu Ser Lys Pro Thr Val          435 440 <210> 9 <211> 1861 <212> DNA <213> mouse <400> 9 gaaaatgatt tgcttagata tgttattttg aataactttt atctgtgccc catgcctatg 60 tatttaaagc catctcttct tttcttatgt ttgtggacag aggatgagca tgaccctgag 120 tagtttgaag gaaaaggtca atgccgcccg gtgtcctttg cctctcttca cgtgtctcca 180 cgtcaaacct gatgtgtcag agctgatgtt tgccgattgg gtggaattta gtccatatga 240 gattggcatg gcaaaatatg gtacctttat ggctcctgac ctatttggaa gcaagttttt 300 tatgggaaca gttgtaaaaa aatatgaaga aaaccccttg catttcttga tgggtgtctg 360 gggcagtgcc ttttctatac tgttcaacag agttttggga gtttctggct cacagaataa 420 aggctctaca atggaagagg aattagaaaa tattacagca aagcacatcg tgagtaatga 480 cagctccgac agtgatgatg aggctcaagg acccaaaggc accgagaatg aagaagctga 540 aaaagagtac caaagcgaca accaagcaag ttgggtccat cggatgctaa tggccttggt 600 gagcgactcg gctttattca atacccgaga aggacgtgcc ggaaaggtgc ataacttcat 660 gctgggcttg aatctcaaca catcatatcc actgtctccc ctgagagact tcagctctca 720 ggattccttc gatgacgagc tcgacgcagc ggtagcagat ccagatgaat ttgaacgaat 780 atatgaacca ctggatgtca aaagtaagaa gattcatgtg gtagatagtg ggctcacatt 840 taacctgcca tatcccttga ttcttcgacc tcagagaggt gtggatctta tcatctcctt 900 tgacttttct gcaaggccga gtgacaccag tccccctttc aaggaacttc tgcttgcaga 960 gaagtgggcg aaaatgaaca agcttccctt tccaaagatc gatccttatg tgtttgatcg 1020 ggaaggatta aaggaatgct atgtttttaa acctaagaat cctgatgtgg agaaggattg 1080 cccaaccatt atccactttg ttctggccaa catcaacttc agaaagtaca aggccccagg 1140 tgttctaagg gaaaccaaag aagagaaaga aattgctgac tttgacattt ttgatgaccc 1200 cgaatcgcca ttttcaacct tcaactttca gtatcccaat caagcattca aaaggcttca 1260 cgatttgatg tacttcaaca cactgaacaa cattgatgtg ataaaggatg ccattgttga 1320 gagcattgaa tacagaagac agaacccatc tcgttgctct gtttccctca gtaatgttga 1380 agcaagaaaa ttcttcaata aggagtttct aagtaaaccc actgtgtaat ttctgtgctg 1440 ggatgatcaa gccatttgaa ttccatgaca atttgagttc agaagacatt agaggtcatc 1500 ttactatgca gaagagactg gctgctactc aaagttgtgg agatttagcc atgtgttagg 1560 tgaaaatgat gttgattatg taatacttag caacagtttc tgacagtatg aattttttga 1620 cattagcata gagctatata ctgtatttta aacattcctc acatttttta cctgtacttt 1680 ttatataaat atgacatgtc ttttcttttg aaaatattta atagtttaac tcagtaaagg 1740 agacttccca ttgtgtgtga atgttattct gaactagatt tgttcatgcc atgttacaac 1800 actattttta tttaaatgtt tatatttaca catacgaaat aaatactttg ctgtacaaat 1860 t 1861 <210> 10 <211> 1860 <212> DNA <213> mouse <400> 10 agttgtttta aaatacacac atctttttcc ctggaacttt atttctgttg tctacttgag 60 accacagatt tccaggaata ataggacttc atttcattaa ggatgagcat gaccctgagt 120 agtttgaagg aaaaggtcaa tgccgcccgg tgtcctttgc ctctcttcac gtgtctccac 180 gtcaaacctg atgtgtcaga gctgatgttt gccgattggg tggaatttag tccatatgag 240 attggcatgg caaaatatgg tacctttatg gctcctgacc tatttggaag caagtttttt 300 atgggaacag ttgtaaaaaa atatgaagaa aaccccttgc atttcttgat gggtgtctgg 360 ggcagtgcct tttctatact gttcaacaga gttttgggag tttctggctc acagaataaa 420 ggctctacaa tggaagagga attagaaaat attacagcaa agcacatcgt gagtaatgac 480 agctccgaca gtgatgatga ggctcaagga cccaaaggca ccgagaatga agaagctgaa 540 aaagagtacc aaagcgacaa ccaagcaagt tgggtccatc ggatgctaat ggccttggtg 600 agcgactcgg ctttattcaa tacccgagaa ggacgtgccg gaaaggtgca taacttcatg 660 ctgggcttga atctcaacac atcatatcca ctgtctcccc tgagagactt cagctctcag 720 gattccttcg atgacgagct cgacgcagcg gtagcagatc cagatgaatt tgaacgaata 780 tatgaaccac tggatgtcaa aagtaagaag attcatgtgg tagatagtgg gctcacattt 840 aacctgccat atcccttgat tcttcgacct cagagaggtg tggatcttat catctccttt 900 gacttttctg caaggccgag tgacaccagt ccccctttca aggaacttct gcttgcagag 960 aagtgggcga aaatgaacaa gcttcccttt ccaaagatcg atccttatgt gtttgatcgg 1020 gaaggattaa aggaatgcta tgtttttaaa cctaagaatc ctgatgtgga gaaggattgc 1080 ccaaccatta tccactttgt tctggccaac atcaacttca gaaagtacaa ggccccaggt 1140 gttctaaggg aaaccaaaga agagaaagaa attgctgact ttgacatttt tgatgacccc 1200 gaatcgccat tttcaacctt caactttcag tatcccaatc aagcattcaa aaggcttcac 1260 gatttgatgt acttcaacac actgaacaac attgatgtga taaaggatgc cattgttgag 1320 agcattgaat acagaagaca gaacccatct cgttgctctg tttccctcag taatgttgaa 1380 gcaagaaaat tcttcaataa ggagtttcta agtaaaccca ctgtgtaatt tctgtgctgg 1440 gatgatcaag ccatttgaat tccatgacaa tttgagttca gaagacatta gaggtcatct 1500 tactatgcag aagagactgg ctgctactca aagttgtgga gatttagcca tgtgttaggt 1560 gaaaatgatg ttgattatgt aatacttagc aacagtttct gacagtatga attttttgac 1620 attagcatag agctatatac tgtattttaa acattcctca cattttttac ctgtactttt 1680 tatataaata tgacatgtct tttcttttga aaatatttaa tagtttaact cagtaaagga 1740 gacttcccat tgtgtgtgaa tgttattctg aactagattt gttcatgcca tgttacaaca 1800 ctatttttat ttaaatgttt atatttacac atacgaaata aatactttgc tgtacaaatt 1860 <210> 11 <211> 1966 <212> DNA <213> mouse <400> 11 agttgtttta aaatacacac atctttttcc ctggaacttt atttctgttg tctacttgag 60 accacagatt tccaggaata ataggacttc atttcattat aaaatgaaaa tgatttgctt 120 agatatgtta ttttgaataa cttttatctg tgccccatgc ctatgtattt aaagccatct 180 cttcttttct tatgtttgtg gacagaggat gagcatgacc ctgagtagtt tgaaggaaaa 240 ggtcaatgcc gcccggtgtc ctttgcctct cttcacgtgt ctccacgtca aacctgatgt 300 gtcagagctg atgtttgccg attgggtgga atttagtcca tatgagattg gcatggcaaa 360 atatggtacc tttatggctc ctgacctatt tggaagcaag ttttttatgg gaacagttgt 420 aaaaaaatat gaagaaaacc ccttgcattt cttgatgggt gtctggggca gtgccttttc 480 tatactgttc aacagagttt tgggagtttc tggctcacag aataaaggct ctacaatgga 540 agaggaatta gaaaatatta cagcaaagca catcgtgagt aatgacagct ccgacagtga 600 tgatgaggct caaggaccca aaggcaccga gaatgaagaa gctgaaaaag agtaccaaag 660 cgacaaccaa gcaagttggg tccatcggat gctaatggcc ttggtgagcg actcggcttt 720 attcaatacc cgagaaggac gtgccggaaa ggtgcataac ttcatgctgg gcttgaatct 780 caacacatca tatccactgt ctcccctgag agacttcagc tctcaggatt ccttcgatga 840 cgagctcgac gcagcggtag cagatccaga tgaatttgaa cgaatatatg aaccactgga 900 tgtcaaaagt aagaagattc atgtggtaga tagtgggctc acatttaacc tgccatatcc 960 cttgattctt cgacctcaga gaggtgtgga tcttatcatc tcctttgact tttctgcaag 1020 gccgagtgac accagtcccc ctttcaagga acttctgctt gcagagaagt gggcgaaaat 1080 gaacaagctt ccctttccaa agatcgatcc ttatgtgttt gatcgggaag gattaaagga 1140 atgctatgtt tttaaaccta agaatcctga tgtggagaag gattgcccaa ccattatcca 1200 ctttgttctg gccaacatca acttcagaaa gtacaaggcc ccaggtgttc taagggaaac 1260 caaagaagag aaagaaattg ctgactttga catttttgat gaccccgaat cgccattttc 1320 aaccttcaac tttcagtatc ccaatcaagc attcaaaagg cttcacgatt tgatgtactt 1380 caacacactg aacaacattg atgtgataaa ggatgccatt gttgagagca ttgaatacag 1440 aagacagaac ccatctcgtt gctctgtttc cctcagtaat gttgaagcaa gaaaattctt 1500 caataaggag tttctaagta aacccactgt gtaatttctg tgctgggatg atcaagccat 1560 ttgaattcca tgacaatttg agttcagaag acattagagg tcatcttact atgcagaaga 1620 gactggctgc tactcaaagt tgtggagatt tagccatgtg ttaggtgaaa atgatgttga 1680 ttatgtaata cttagcaaca gtttctgaca gtatgaattt tttgacatta gcatagagct 1740 atatactgta ttttaaacat tcctcacatt ttttacctgt actttttata taaatatgac 1800 atgtcttttc ttttgaaaat atttaatagt ttaactcagt aaaggagact tcccattgtg 1860 tgtgaatgtt attctgaact agatttgttc atgccatgtt acaacactat ttttatttaa 1920 atgtttatat ttacacatac gaaataaata ctttgctgta caaatt 1966 <210> 12 <211> 560 <212> DNA <213> Human <400> 12 taattcattt caatgatgta aagattttga atgtgtgagg aagtgctttt gtattccttt 60 tctctggaaa aaaaaaaaaa aaaaaaaatt cacattttaa cccttaactg cccattccct 120 ccaagaatgg taacattttt agatgaggaa gaatgaagtt tgcctgaata gagtcaagaa 180 aggaagggga tcgcatagaa cagactcgct tgatgcatga ttgcattgat gtttcgttga 240 agataaagca gaggagcgcc tgtgacaggg agtccagggg ctaagtttct tccaggctcc 300 acagttgcta attcattctc cagttcagat gtagacatat aatctagagt tatgattatt 360 ttttaaatga agatagttac ttccatagag cttatttttt gttgttcatt caggacctag 420 taatttctag aagtaataag acttattttt attataaagt tataagattt tgattggaag 480 tactattttg aatagcattc tttctgtgtc tgtttataaa tttaaagtca tctttttctt 540 tcttctgtgg acagagaatg 560 <210> 13 <211> 667 <212> DNA <213> rat <400> 13 taaaaaaagt aatatttagg tgagatggta agactatgca ttgcttttga gggggatgtg 60 agttcagtag tcagcaccta catctggcag cgcacaactg cctgtaactc cagctttagg 120 aggagccgat acctctggcc tttatgaaca cctacactca catgcatata tccacacaca 180 gataatatac atatgcatat tttacttttt atattcatat tttaaaataa tagaagtggt 240 agaaaaaata ctctttgcct agtaagagtc aaataaggaa atggatcata ggaaacaaat 300 gtacttgatg tgtcaccaga gggtgacatt tcatctgaag ataaagcagg agagacggga 360 caacctgtgc cagggacacc agctgagaga attagttccc agaactatag ctgccaaatc 420 ttcccccact tcaaatgttg acacagtccc cagagattca attgttttag aatacagaca 480 tctatttcca gggagctttc tttctgttgt ctaatcgaga ccacagattg ccagaaataa 540 taggacttcg tttcattata aaaaggcaag cgatttggtt agacatatta tttcaaatag 600 cttttatctg tgtccatgtc tatgtattta aagccacctt attctttttg tgtgtgtgtg 660 tgaaaag 667 <210> 14 <211> 739 <212> DNA <213> mouse <400> 14 tggtaagatg gtgaatagaa gtgctattta ggtgagatcg tgaatacaag tgatattaaa 60 agcaggaagg aggaggtttt cgctcctcag cagataagcc catgcactgc tcttgtgggg 120 acatgagttc aacaaccagc acctatgtct gacagctcat aactacctgt aattccagat 180 tcaggaggtg ccaatacctc tggcctctgt gaatatctgc actcacaatc atatatccac 240 acacagatac atattcatat acatatttta ttttttatat tcacatttta aaataataaa 300 ttgaaatggt agaagaacac tctttgcctc ataagagaca aataaggaaa tggaccatgg 360 gaaacaaatg gacttgatgt gtcaccagag ggtgacattt catctgaaga taaagcaggg 420 gagaaaggac agctgtgcca gggaacgcca gctgagagaa ctagttctca acactctagt 480 tgccaaatct tcctcnnctt caaatgttga cacagtcttc agagattcag ttgttttaaa 540 atacacacat ctttttccct ggaactttat ttctgttgtc tacttgagac cacagatttc 600 caggaataat aggacttcat ttcattataa aatgaaaatg atttgcttag atatgttatt 660 ttgaataact tttatctgtg ccccatgcct atgtatttaa agccatctct tcttttctta 720 tgtttgtgga cagaggatg 739

[Brief description of drawings]

FIG. 1 is a photograph as a substitute for a drawing, which shows the result of Northern blotting when various sites of cPro Leu Ala 2 were used as probes. The top row of Figure 1 shows cPro Le
The base sequence of uAla 2 is shown. Ala, B, Cys, and Asp represent probes. The middle part of FIG. 1 is the case without kainic acid treatment (Lys Ala (−)),
The lower part of FIG. 1 shows the case of 3 hours after the kainic acid treatment (Lys Ala (+), 3 h).

FIG. 2 is a photograph instead of a drawing, which shows the results of Northern blotting of the hippocampus and cerebellum over time. The hippocampus is on the left side of FIG. 2, and the cerebellum is on the right side. Blots after kainic acid treatment (0 hour), 0.5 hour, 3 hours, 8 hours, 14 hours and 18 hours are shown.

FIG. 3 is a photograph instead of a drawing, which shows the result of in situ hybridization in rat brain. The left side of FIG. 3 is a cross section of the brain, and the right side of FIG. 3 is from a vertical cross section of the brain. In FIG. 3, the part that appears black is the part that is colored.

FIG. 4 is a photograph, instead of a drawing, showing an enlarged part of the dentate gyrus of the hippocampus in the result of FIG. 3. The left side of FIG. 4 is not treated with kainic acid, and the right side is after 3 hours of kainic acid treatment.

FIG. 5 shows the Lys Ile Asp Ser cPro Le of the present invention.
The results of confirming the expression of the target protein by immunohistochemical analysis using an antibody that specifically recognizes u Ala 2 are shown.
It is a photograph replacing a drawing. The left side of the upper part of FIG. 5 is the case without kainic acid treatment, and the right side of the upper part is after kainic acid treatment.
It's time. The left side of the lower part of Fig. 5 is anti-Lys Ile Asp
SercPro Leu Ala 2 antibody (IlegGly) untreated control. The right side of the lower part of Fig. 5 shows the absence of the anti-Lys Ile Asp Ser cPro Leu Ala 2 antibody (IlegGly) 3 hours after the kainic acid treatment ((-) on the left side of the lower part of the Fig. 5) and the presence (their). It is the result of chromatography at (+) on the right side.

FIG. 6 shows the Lys Ile Asp Ser cPro Le of the present invention.
cAsp As which encodes u Ala 2 and cPro Leu Ala 2α
It is a photograph instead of a drawing, which shows the result of examining the expression by incorporating n Ala into an expression vector pThr racer Glu Phe. Lane 1 in FIG. 6 is for the control vector, lane 2 is cPro Leu Ala 2α / pThrrac.
For erGlu Phe, lane 3 is Lys Ile Asp Se
This is the case of r cPro Leu Ala 2 / pThr racerGlu Phe. The left side of FIG. 6 shows the anti-Val 5 epitope IlegGl.
When using y, the middle is anti-cPro Leu Ala 2α
The replication using Ile gTyr, the right side is anti-Lys Ile Asp S
This is the case where er cPro Leu Ala 2Ile gGly was used.

FIG. 7: Lys Ile Asp Ser cPro Le of the present invention
The result of the enzyme activity of uAla2 and cPro LeuAla2 (alpha) is shown. The left side of FIG. 7 shows the Lys Ile Asp Ser cProL of the present invention.
eu Ala 2 and on the right is that of cPro Leu Ala 2α. As a substrate, 1-Proam-2- [ ¹⁴ Cy
s] Arachidonoyl-Pro Cys (black circle (●)), 1-
Pro am-2- [ ¹⁴ Cys] Linoleoyl-Pro Cys
(Black triangle (▲)), 1-Pro am-2- [ ¹⁴ Cys]
Oleoyl-Pro Cys (black square (■)), and 1-Pr
o am-2- [ ¹⁴ Cys] palmitoyl-Pro Cys (asterisk (*)) was used.

FIG. 8 shows 1-Pro am-2- [ ¹⁴ as a substrate.
Cys] arachidonoyl-Pro Cys was used to
s Ile Asp Ser cPro Leu Ala 2 and cPro Leu Ala
The result of the test of calcium dependence on the enzyme activity of 2α is shown. The solid line in FIG. 8 is the Lys Ile Asp Ser cPr of the present invention.
o In case of Leu Ala 2, the broken line is cPro Leu Ala 2α
Is the case. Each black circle (●) is Glu Asp Thr
In the absence of Ala-Cys a, the white circles (○)
Glu Asp Thr Ala-Cys a.

FIG. 9 shows the results shown in FIG. 8 above in a relative ratio.

FIG. 10 is a photograph replacing a drawing, which shows the result of examining the expression of Lys Ile Asp Ser cProLeu Ala 2 of the present invention in cPro Leu Ala deficient mice. The upper row of FIG. 10 is for a knockout mouse (+ / +), and the lower row is for a knockout mouse (-/-). Figure 1
The left side of 0 shows kainic acid untreated (Lys Ala (−)), and the right side shows 3 hours after kainic acid treatment (Lys Ala (+)).

FIG. 11 shows cPro Leu Ala 2 and Lys Ile Asp.
It is the figure which showed the expression situation of Ser cPro Leu Ala 2. The upper part of FIG. 11 shows cPro Leu in the genomic gene.
The exons and introns of Ala 2 are schematically shown.

FIG. 12 shows the nucleotide sequence from the first base of the intron immediately before the exon containing “Met-308” in rat (upper row), mouse (middle row), and human (lower row), with full-length cPro Leu It is numbered with the base starting from the exon region of Ala 2 as number 1.

FIG. 13 is a photograph instead of a drawing, which shows the results of examining the expression of KIDS cPLA2 of the present invention using neural stem cells and mature neurons. The upper row of FIG. 13 shows Nestin as a control, the middle row shows the case of using neural stem cells, and the lower row shows the case of using mature nerve cells. A on the left side shows the position of each cell, B at the center is the color development indicating the expression of KIDS cPLA2 of the present invention, and on the right side, the positions of both A and the center B are confirmed by overlapping. .

FIG. 14 shows KIDS cPLA2 of the present invention in kainic acid stimulation, kainic acid and CNQX stimulation, and glutamate stimulation using neural stem cells.
Is a photograph as a substitute for a drawing, which shows the result of examining the expression of E. coli. The upper part of FIG. 14 shows 2 of P90-P27 as a probe.
52 bp, the second row from the top is 29 of P19-P27.
0 bp, lower two rows are G3P for control
DH and Nestin. The bottom of FIG. 14 is K
The transcription start position on the 5'side of IDS cPLA2 and the sequence position of the probe used in the upper two rows of FIG. 14 are shown. Figure 1
Each lane of 4 shows control, kainic acid stimulation (KA (10 μM)), stimulation with kainic acid and CNQX (KA (10 μM) + CNQX (20 μM)), and glutamic acid (Glu (50 μM)) from the left.

FIG. 15 shows the results of measuring the lysophospholipase A1 activity of KIDS cPLA2 of the present invention. The vertical axis in FIG. 15 represents the lysophospholipase A1 activity represented by dmp (mean ± standard deviation, n = 3),
The horizontal axis is time (minutes). From the left, the activity values at each time are cPLA2α (black coating), KIDScPL of the present invention.
A2 (light black) and lacZ (grey). The right end (white) at the time of 40 minutes on the horizontal axis indicates the buffer. The value of p in FIG. 15 shows that there is a significant difference between KIDS cPLA2 (light black) of the present invention and lacZ by t-test.

FIG. 16 shows the results of kinetic analysis of the lysophospholipase A1 activity of KIDS cPLA2 of the present invention. The vertical axis of FIG. 16 represents the lysophospholipase A1 activity represented by dmp (mean ± standard deviation, n = 3), and the horizontal axis represents the concentration (μM) of the substrate lysophosphatidylcholine (lysoPC). The black circles in FIG. 16 indicate cPLA2α, and the thin black circles indicate the KID of the present invention.
ScPLA2, dark gray circles indicate lacZ, light gray circles indicate buffer. FIG.
The small graph on the upper side of FIG. 1 shows the results of kinetic analysis, and the upper side of the equation, KIDS cPLA2 of the present invention, is represented by the formula of y = 85.784x + 0.378, and the lower side y = 59.441x + 0. The expression of .898 is cPLA2α.

FIG. 17 shows the results of a test for examining the effect of calcium ions on the lysophospholipase A1 activity of KIDS cPLA2 of the present invention.
The vertical axis of FIG. 17 indicates the lysophospholipase A indicated by dmp.
1 activity (mean ± standard deviation, n = 3), horizontal axis is 5
The cases of absence (-) and presence (+) of mM EDTA are shown. The activity value in each case is from the left, cPLA
2α (black coating), KIDS cPLA2 of the present invention (light black), lacZ (grey), and buffer (white). The value of p in FIG. 17 is the KI of the present invention by the t-test.
It shows that there is a significant difference between DS cPLA2 (light black) and lacZ.

FIG. 18 shows AEBSF (4-) in lysophospholipase A1 activity of KIDS cPLA2 of the present invention.
Fig. 2 shows the results of a test for examining the influence of (2-aminoethyl) -benzenethronylfluoride: an inhibitor of serine protease). The vertical axis of FIG. 18 is dm
lysophospholipase A1 activity represented by p (mean ± standard deviation, n = 3), the horizontal axis represents the concentration of AEBSF (m
M) is shown. The black circles in FIG. 18 represent cPLA2α, the light black circles represent the KIDS cPLA2 of the present invention, the dark gray circles represent lacZ, and the light gray circles represent buffers.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] April 9, 2002 (2002.4.9)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0032

[Correction method] Change

[Correction content]

Furthermore, the present inventors have found that the KIDS of the present invention.
It was found that cPLA2 has lysophospholipase A1 activity. Lysophospholipase A1 is an enzyme that specifically decomposes the acyl group by the saturated fatty acid at the 1-position of glycerophospholipid. Accordingly, the present inventors have lysophosphatidylcholine, labeled at the 1-position of the palmitoyl group of glycerophospholipids in ¹⁴ C a (lysoPC) as a substrate to measure the lyso phospholipase <br/> peptidase A1 activity of KIDS cPLA2 of the present invention. The results are shown in Fig. 15. The vertical axis of FIG. 15 represents lysophospholipase A1 activity represented by dmp (mean value ± standard deviation, n = 3), and the horizontal axis represents time (minutes). From the left, the activity values at each time are cPLA2α
(Black coating), KIDS cPLA2 (light black) of the present invention,
And lacZ (gray). The right end (white) at the time of 40 minutes on the horizontal axis indicates the buffer. Figure 15
The value of p is KIDS cP of the present invention by t-test.
It shows that there is a significant difference between LA2 (light black) and lacZ. As a result, KIDS cPLA2 of the present invention
Was found to have excellent lysophospholipase A1 activity.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0033

[Correction method] Change

[Correction content]

Next, a re- installation of the KIDS cPLA2 of the present invention.
The zone phospholipase A1 activity was performed kinetic analysis. The results are shown in Fig. 16. The vertical axis in FIG. 16 represents the lysophospholipase A1 activity represented by dmp (mean ± standard deviation, n =).
3), the horizontal axis is the substrate lysophosphatidylcholine (lys)
oPC) concentration (μM) is shown. The black circle in Figure 16 is cP
LA2α, a thin black circle indicates KIDS cP of the present invention
LA2, dark gray circles indicate lacZ, light gray circles indicate buffer. The small graph on the upper side of FIG. 16 shows the results of kinetic analysis.
The upper side y = 85.784x + 0.378 is the KIDS cPLA2 of the present invention, and the lower side y =
The formula of 59.441x + 0.898 is cPL
A2α.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0034

[Correction method] Change

[Correction content]

Furthermore, the KIDS cPLA2 of the present invention
The effect of calcium ions and AEBSF (4- (2-aminoethyl) -benzenethronylfluoride: an inhibitor of serine protease) on lysophospholipase A1 activity was examined. The results of the test for the effect of calcium ions are shown in FIG. The ordinate of FIG. 17 is the lysophospholipase A1 activity represented by dmp (mean ± standard deviation, n = 3), and the abscissa is the absence of 5 mM EDTA (−).
And the case of existence (+) are shown. The activity values in each case are from the left, cPLA2α (black coating), KID of the present invention.
ScPLA2 (light black), lacZ (grey), and buffer (white). The value of p in FIG. 17 is the same as that of KIDS cPLA2 (light black) of the present invention by t-test.
It shows that there is a significant difference between acZ. As a result, the activity of cPLA2α (black coating) decreases when calcium ions are absent due to the presence (+) of EDTA, whereas KIDS cPLA2 (light black) of the present invention.
It is understood that the same level of activity is maintained in the presence or absence of calcium ions (absence of EDTA (−)) and the absence thereof (presence of EDTA (+)). Next, FIG. 18 shows the result of the influence of the presence of AEBSF. The vertical axis is the lysophospholipase <br/> Horipaze A1 activity represented by dmp (mean ± standard deviation of FIG. 18, n =
3), the horizontal axis represents the AEBSF concentration (mM). FIG.
Black circles indicate cPLA2α, light black circles indicate KIDS cPLA2 of the present invention, and dark gray circles indicate lac.
Z, light gray circles indicate buffer.
As a result, it was found that both enzymes lost their activity due to the presence of AEBSF.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0035

[Correction method] Change

[Correction content]

Recently, lysophosphatidylcholine (hereinafter LPC) was found to be a ligand for G2A, an immunomodulatory receptor (Janusz H., et al., Sci.
ence, 293, 702-705 (2001)). In addition, studies in mice lacking the G2A immunoregulatory receptor have revealed that G2A plays an important role in controlling homeostasis of peripheral lymphocytes (Lu Q. Le, et al., Im.
munity, 14, 561-571 (2001)). Therefore, LPC is considered to be a regulatory factor for immunity, cell proliferation, etc. in vivo, especially in the peripheral system, and when LPC becomes excessive, cell death occurs, and arteriosclerosis, deterioration of immunocompetence, etc. occur. The KIDS cPLA2 of the present invention has a phospholipase A2 activity and a lysophospholipase A1 activity, and has an action of appropriately controlling the LPC level. The KIDS cPLA2 of the present invention can remove excess LPC, and can be used for suppression of apoptosis, improvement of immune function, prevention and treatment of arteriosclerosis and the like. Accordingly, the present invention provides a lysophosphatidylcholine (LPC) modulator by KIDS cPLA2 of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 43/00 105 C07K 16/40 4H045 111 C12N 9/16 D C07K 16/40 C12Q 1/02 C12N 9 / 16 1/68 A C12Q 1/02 C12P 21/08 1/68 C12N 15/00 ZNAA // C12P 21/08 A61K 37/54 F term (reference) 4B024 AA01 BA11 CA04 CA12 DA02 EA04 FA02 FA20 GA11 HA01 4B050 CC03 CC04 DD11 LL01 4B063 QA01 QQ08 QQ53 QR41 4B064 AG27 CC24 DA13 4C084 AA02 AA07 BA01 BA08 BA22 BA23 CA28 DC22 NA14 ZA452 ZB092 ZB212 ZC022 4H045 AA10 AA11 BA10 CA45 DA76 DA89 EA20

Claims (26)

[Claims] 1. A phospholipase A2 that can be expressed in the hippocampus by external stimulation, is calcium-independent, and has a shorter amino acid sequence than a normal phospholipase A2. 2. The phospholipase A2 according to claim 1, wherein the external stimulation is kainic acid stimulation or electrical stimulation. 3. Calcium-independent phospholipase A
2, wherein one or more amino acids in the amino acid sequences set forth in SEQ ID NOS: 1, 5 or 8 in the sequence listing, or one or more amino acids in those amino acid sequences are substituted with another amino acid and deleted, The phospholipase A2 according to claim 1 or 2, which has an amino acid sequence obtained by adding the amino acid of. 4. A gene having a nucleotide sequence encoding the phospholipase A2 according to claim 1. 5. The gene according to claim 4, wherein the gene is DNA. 6. An antibody using the full-length or fragment of phospholipase A2 according to any one of claims 1 to 3 as an antigen. 7. The antibody according to claim 6, wherein the antibody is a monoclonal antibody. 8. A gene having a base sequence existing in an intron, which has a base sequence capable of initiating RNA transcription by an external stimulus. 9. The gene according to claim 8, wherein the transcription initiation of RNA is site-specific. 10. The gene according to claim 8, wherein the external stimulation is kainic acid stimulation or electrical stimulation. 11. The gene has SEQ ID Nos. 12 and 1 in the sequence listing.
The gene according to any one of claims 8 to 10, which has a base sequence described in 3 or 14, or a base sequence consisting of a partial sequence in which a part thereof is deleted, added, or substituted. 12. A promoter which is a nucleotide sequence present in an intron and which can initiate RNA transcription by an external stimulus. 13. The promoter according to claim 12, wherein the transcription initiation of RNA is site-specific. 14. The promoter according to claim 12, wherein the external stimulation is kainic acid stimulation or electrical stimulation. 15. A regulatory gene having a regulatory element upstream of the promoter according to claims 12-14. 16. A gene encoding any one of claims 8 to 15 is introduced upstream of a gene encoding the protein to initiate RNA transcription by an external stimulus to externally stimulate the protein. The method of expressing according to. 17. The method according to claim 16, wherein the gene encoding the protein is CRE. 18. The gene encoding a protein is a gene encoding a toxic protein.
The method according to 6. 19. The method of claim 18, wherein the toxic protein is diphtheria venom. 20. An organism obtained by introducing the gene according to any one of claims 8 to 15 upstream of a gene encoding a protein. 21. The organism according to claim 20, wherein the organism is a test animal. 22. The organism according to claim 20 or 21, wherein the gene encoding the protein is CRE. 23. Stimulating a nerve cell with an external stimulus,
A method for detecting or identifying neural stem cells, which comprises detecting or identifying the expression of mRNA encoding phospholipase A2 according to claim 1. 24. The method according to claim 23, wherein the external stimulation is kainic acid stimulation or electrical stimulation. 25. A pharmaceutical composition comprising the phospholipase A2 according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier. 26. The pharmaceutical composition according to claim 25, which is a phospholipid level regulator.