JP2001178467A - New phosphodiesterase and gene encoding the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new phosphodiesterase (PDE) and a gene encoding PDE which are useful for studying the complicated mechanism of the intracellular signal transduction and can be target molecules against a medicine for a new disease. SOLUTION: Phosphodiesterase 11 (PDE11), more in detail a phosphodiesterase selected from (A) a protein having an amino acid sequence shown by sequence 2, 4 or 6 in the specification and (B) a protein which has an amino acid sequence modified by deleting, substituting or adding an amino acid or more from, in or into the amino acid sequence shown by sequence 2, 4 or 6 and has an activity of hydrolyzing a cyclic nucleotide and a gene encoding PDE11 are provided. Methods for characterizing, identifying, and selecting an inhibitor against phosphodiesterase are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel phosphodiesterase and its gene.

[0002]

BACKGROUND ART Cyclic nucleotides such as cAMP and cGMP are involved in the regulation of many functions in vivo as second messengers of intracellular signal transduction (Kukove).
tz et al., Naunyn Schmiedeberg's Arch. Pharmacol., 31.
0, 129-138, 1979; Schram et al., Science, 225.
Vol. 1350-1356, 1984; Ignarro et al., Annu. Rev. Ph.
armacol. Toxicol., 25, pp. 171-191, 1985; Ma
rtin et al., J. Pharmacol. Exp., 237, 539-547, 1
986).

[0003] The concentration of intracellular cAMP and cGMP, which fluctuates in response to extracellular signals, is regulated by the balance between adenyl cyclase and guanyl cyclase, which are involved in their synthesis, and phosphodiesterase (PDE), which is involved in cyclic nucleotide degradation. ing.

To date, many phosphodiesterases that degrade cyclic nucleotides have been found in mammalian tissues, and are classified into multiple types based on amino acid sequence homology, biochemical properties, and characterization by inhibitors. (Beavo, Physiol. Rev., 75, 725-748, 199
5 years).

[0005] For example, PDE1 is a Ca ²⁺ / calmodulin-dependent PDE, and hydrolyzes both cAMP and cGMP. PDE2 is activated by cGMP and c
Hydrolyzes both AMP and cGMP. PDEs classified as PDE3 are inhibited by cGMP. PDE4
Uses cAMP specifically as a substrate, and rolipram (Ro
lipram) sensitive. PDE5 specifically uses cGMP as a substrate. PDE6 is a photoreceptor cGMP
-PDE. PDE7 uses cAMP specifically as a substrate and is insensitive to rolipram.

[0006] More recently, the existence of three new types of PDEs has been reported. One is called PDE8, cA
MP is specifically used as a substrate and another is called PDE9, and cGMP is specifically used as a substrate (Soderling et al., Pro.
c. Natl. Acad. Sci. USA, Vol. 95, pp. 8991-8996, 1998
Year; Fisher et al., Biochem. Biophys. Res. Commun., 246.
Vol. 570-577, 1998; Soderling et al., J. Biol. Che.
m., 273: 15553-15558, 1998; Fisher et al.
J. Biol. Chem., 273, 15559-15564, 1998; H.
ayashi et al., Biochem. Biophys. Res. Commun., Volume 250, Vol.
751-756, 1998). These two PDEs are IBMX
It has been reported that it is insensitive to (3-isobutyl-1-methylxanthine). Still another is P
It is called DE10, and uses both cAMP and cGMP as substrates, but has been reported to have a stronger affinity for cAMP (Fujishige et al., J. Biol. Che.
m., 274, 18438-18445, 1999; Kotera et al., B.
iochem.Biophys.Res.Commun., Vol.261, No.551-557
P. 1999).

[0007] PDE is also an important target molecule in drug development research, and inhibitor research is being vigorously pursued. Some known drugs have been found to have PDE inhibitory effects, and it has been found that specific PDE inhibitors can be useful therapeutic agents.

For example, the inotropic agent Milrinone (Milrin)
one) and Zaprinast are PDE3 and P respectively
DE5 inhibitors (Harrison et al., Mol. Pharmacol.,
29, 506-514, 1986; Gillespie et al., Mol. Pha.
rmacol., 36, 773-781, 1989). Rolipram, an antidepressant, is a PDE4 inhibitor (Schn
eider et al., Eur. J. Pharmacol., Vol. 127, pp. 105-115,
1986). PDE4 inhibitors have also been developed as anti-inflammatory agents or asthma therapeutics. Also, selective inhibitors of PDE5 have been developed as therapeutics for erectile dysfunction.

[0009] In addition, IBMX is known as a non-selective inhibitor acting on many types of PDEs. Vinpocetin is a PDE1 inhibitor, EHNA [erythro-9- (2-hydroxy-3-nonyl) adenine]
Is a PDE2 inhibitor; Dipyridamole is a PDE5 and PDE6 inhibitor; SCH51866 is a PD
E1 and PDE5 inhibitors and E4021 are known as PDE5 inhibitors.

In order to develop an excellent drug having a high therapeutic effect and few side effects, it is desired to select an inhibitor which is highly selective for a certain type of PDE to be targeted.

Furthermore, finding a new type of PDE, which is a different molecular species from the conventional one, is useful for studying the complex mechanism of intracellular signal transduction and also as a target molecule for a new therapeutic agent. It was also desired from the possibility.

[0012]

An object of the present invention is to provide a novel type of phosphodiesterase [type 11 phosphodiesterase (PDE11)] and its gene. Another object of the present invention is to provide a new method for characterizing, identifying and selecting a phosphodiesterase inhibitor. The purpose other than the above is clear from the following description.

[0013]

Means for Solving the Problems The present inventors have isolated full-length cDNAs encoding a new type of phosphodiesterase (also referred to as PDE11 or PDE11A) which is a different molecular species from humans and rats. In addition, human phosphodiesterase (PDE11 or PD
Also referred to as E11A. ) By genetic engineering technology
It was successfully expressed and isolated in S cells. Further, the present invention was characterized as an enzyme, and the present invention was completed.

That is, the present invention relates to type 11 phosphodiesterase (PDE11) and its gene. More specifically, it is a phosphodiesterase selected from the following (A) and (B), and a gene or nucleic acid encoding the phosphodiesterase.

(A) a protein having the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, and (B) a protein having the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 A protein having an amino acid sequence in which a plurality of amino acids have been deleted, substituted or added, and having an activity of hydrolyzing cyclic nucleotides.

The gene or nucleic acid encoding the phosphodiesterase of the present invention includes a gene or nucleic acid selected from the following (a) and (b):

(A) a gene or nucleic acid comprising a DNA having the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, and (b) a gene or nucleic acid represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: A gene or nucleic acid comprising a DNA which hybridizes with a DNA having a base sequence under stringent conditions, and which encodes a protein having an activity of hydrolyzing cyclic nucleotides.

Furthermore, the present invention is a recombinant vector and a host cell containing these genes or nucleic acids. Furthermore, it is a method of using these to characterize, identify and select phosphodiesterase inhibitors.

SEQ ID NO: 1 in the sequence listing is a human homolog of the novel PDE gene isolated by the inventors (human PD).
E11 gene. Specifically, it is also referred to as a human PDE11A gene. ) Represents the nucleotide sequence of the cDNA including the full length of the translation region, and SEQ ID NO: 2 is a novel PDE (human PDE11; specifically, human PDE11A1) encoded by the full length cDNA.
Also called. ) Represents the amino acid sequence.

[0020] SEQ ID NO: 3 in the Sequence Listing also shows the cDNA of the novel PDE gene, isolated from the human, which contains the entire translation region of the human homologue of the PDE gene (human PDE11 gene, specifically also referred to as human PDE11A gene). SEQ ID NO: 4 represents a novel PDE (human PDE11, specifically human PDE11) encoded by the full-length cDNA.
Also referred to as A2. ) Represents the amino acid sequence.

SEQ ID NOs: 1 and 3 are cDNAs derived from two types of splice variants of the human PDE11 gene.
This is the NA base sequence, and SEQ ID NOs: 2 and 4 are the amino acid sequences of PDE proteins derived from each variant.

SEQ ID NO: 5 in the sequence listing below shows the nucleotide sequence of the cDNA containing the entire translation region of the rat-derived homolog of the novel PDE gene isolated by the inventors (rat PDE11 gene, more specifically also referred to as rat PDE11A gene). SEQ ID NO: 6 represents a novel PDE encoded by the full-length cDNA (rat PDE11;
Also referred to as E11A. ) Represents the amino acid sequence.

The nucleotide sequences shown in SEQ ID Nos. 1, 3 and 5 and the amino acid sequences shown in SEQ ID Nos. 2, 4 and 6 are known DNA databases (GenB
and EST (Genbank / EMBL IDN) as a result of performing a homology search on the protein databases (NBRF and SWISS-PROT) and protein databases (NBRF and SWISS-PROT).
With the exception of o: AI025081), none of those considered to be derived from the same molecular species were found.

The human PDE1 shown in SEQ ID NO: 4
As a result of comparing the respective amino acid sequences of rat PDE11 and rat PDE11 shown in SEQ ID NO: 6, as high as about 93% homology was recognized.

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION The protein of the present invention includes those having the amino acid sequence shown in SEQ ID NO: 2, 4 or 6. In addition, those having an amino acid sequence represented by SEQ ID NO: 2, 4 or 6 in which one or more amino acids have been deleted, substituted or added.

The amino acid may be deleted, substituted or added in such a manner that the activity of hydrolyzing the cyclic nucleotide is not lost, and it is usually 1 to about 420, preferably 1 to about 3
10, more preferably 1 to about 165, even more preferably 1 to about 80, even more preferably 1 to about 40.

The region responsible for the activity of hydrolyzing cyclic nucleotides in PDE11 (PDE11A), ie, the catalytic region of PDE11 (PDE11A), is, for example, the 640th to 881st amino acid sequence in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing below. No. 390 in the amino acid sequence shown in SEQ ID NO: 4
And a region corresponding to the 631st amino acid residue.

In order not to lose the activity of hydrolyzing the cyclic nucleotide of PDE11 (PDE11A), it is necessary to use PD
It is desirable that the amino acid sequence is more highly conserved in the region responsible for the activity of E11 (PDE11A), that is, in the catalytic region of PDE11 (PDE11A) than in other regions.

Deletion, substitution or addition of amino acids in the catalytic region of PDE11 (PDE11A)
About 20, preferably 1 to about 10, more preferably 1
~ About 5. The catalytic region of such a protein differs from the catalytic region present in the amino acid sequence shown in SEQ ID NO: 2 or 4 usually by about 90% or more, preferably by about 95% or more.
More preferably, it has about 97% or more amino acid level homology.

On the other hand, amino acid deletion, substitution or addition in the non-catalytic region of PDE11 (PDE11A)
Usually 1 to about 400, preferably 1 to about 300, more preferably 1 to about 160, and still more preferably 1 to about 8
0, and even more preferably 1 to about 40.

Such proteins include, in addition to mutant proteins found in nature, artificially modified mutant proteins,
It includes proteins and the like derived from heterologous organisms.

Examples of the gene or nucleic acid of the present invention include those containing DNA having the base sequence shown in SEQ ID NO: 1, 3 or 5. Further, there may be mentioned those containing DNAs that can hybridize under stringent conditions with DNAs having the base sequence shown in SEQ ID NO: 1, 3 or 5. Such hybridizable DNA may be any that encodes a protein having an activity of hydrolyzing cyclic nucleotides. Such a DNA is usually about 70% or more, preferably about 80% or more, more preferably about 90%,
It has the above homology. Such genes or nucleic acids include mutant genes found in nature, artificially modified mutant genes, homologous genes derived from heterologous organisms, and the like.

In the present invention, the hybridization under stringent conditions is carried out under a normal stringent condition in a hybridization solution having a salt concentration of 6 × SSC or equivalent thereof at a temperature of 50 to 70 ° C. Hybridization for about 16 hours,
Preliminary washing may be performed as necessary with a solution of × SSC or a salt concentration equivalent thereto, followed by washing in a solution of 1 × SSC or a salt concentration equivalent thereto.
Further, under conditions having higher stringency (high stringency conditions), the washing can be performed in the above-mentioned manner in a solution having a salt concentration of 0.1 × SSC or an equivalent salt concentration.

The gene or nucleic acid of the present invention can be isolated and obtained by performing screening using mammalian tissues or cells as a gene source. As mammals, dogs, cows,
Non-human animals such as horses, goats, sheep, monkeys, pigs, rabbits, rats and mice, as well as humans.

The gene or nucleic acid of the present invention can be obtained by utilizing the sequence information disclosed in the present specification (SEQ ID NO: 1, 3 or 5 in the Sequence Listing described later). For example, primers and probes are designed based on the disclosed base sequence information, and PCR (polymerase chain reactant) using them is designed.
ion), colony hybridization, and plaque hybridization.
You can select and obtain from A library.

For example, cDNA is synthesized from mRNA prepared from mammalian cells or tissues, and using this as a template,
A cDNA fragment is obtained by the PCR method. Obtained cDNA
Was used as a probe, and cD was detected by colony hybridization or plaque hybridization.
Screen the NA library for full-length cDNA
Can be obtained. In addition, genomic genes can be isolated by screening a genomic DNA library. Further, by screening a DNA library of another mammal, a homologous gene derived from a heterologous organism can be isolated. Such mammals include, for example, dogs, cows, horses, goats, sheep,
Examples include non-human animals such as monkeys, pigs, rabbits and rats, as well as humans.

CDNA library and genomic D
DNA libraries such as NA libraries are, for example,
"Molecular Cloning" (Sambrook,
J., Fritsch, EF and Maniatis, T., Cold Spring
Harbor Laboratory Press, published in 1989). Alternatively, if there is a commercially available library, this may be used.

By determining the nucleotide sequence of the obtained cDNA, the translation region encoding the protein of the gene product can be determined, and the amino acid sequence of this protein can be obtained.

The PDE of the present invention can be produced by overexpression by a conventional gene recombination technique. It can also be expressed and produced in the form of a fusion protein with another protein or peptide.

For example, an expression vector is constructed by inserting a DNA encoding PDE into a vector in a form ligated downstream of an appropriate promoter. Next, the obtained expression vector is introduced into a host cell.

The expression system (host-vector system) includes, for example, bacterial, yeast, insect cell and mammalian cell expression systems. Of these, insect cells (Spodoptera frugiperd) are needed to obtain proteins with well-preserved functions.
a SF9, SF21, etc.) and mammalian cells (monkey COS-7)
Cells, Chinese hamster CHO cells, human HeL
a) is preferably used as a host.

As a vector, a retrovirus vector, a papilloma virus vector, a vaccinia virus vector, an SV40 vector and the like can be used for a mammalian cell system, and a baculovirus vector and the like can be used for an insect cell system.

As a promoter, an SV40 promoter, an LTR promoter, an elongation 1α promoter and the like can be used in a mammalian cell system, and a polyhedrin promoter and the like can be used in an insect cell system.

As the DNA encoding PDE, a cDNA corresponding to an mRNA existing in nature (for example, a cDNA having the base sequence shown in SEQ ID NO: 1, 3 or 5)
Can be used, but is not limited thereto. A DNA corresponding to the amino acid sequence of the target protein can be designed and used. In this case, codons encoding one amino acid are each known in 1 to 6 types, and the codon to be used may be selected arbitrarily. For example, in consideration of the frequency of codon usage of the host used for expression, the expression efficiency can be increased. Tall arrays can be designed. D with designed base sequence
NA can be obtained by chemical synthesis of DNA, fragmentation and binding of the cDNA, partial modification of the nucleotide sequence, and the like. Partial alteration or mutation of an artificial base sequence is performed using a site-specific mutagene (primer) consisting of a synthetic oligonucleotide encoding a desired modification.
sis) (Proceedings of National Academy of Science
s, Vol. 81, pp. 5662-5666, 1984).

The PDE of the present invention can be prepared from a culture of cells into which an expression vector has been introduced by a known purification method (salting out with inorganic salts, fractional precipitation with an organic solvent, ion exchange resin column chromatography, affinity column chromatography). , Gel filtration, etc.) can be separated and purified.

A nucleic acid (oligonucleotide or polynucleotide) that hybridizes with the gene or nucleic acid of the present invention under stringent conditions can be used as a probe for detecting the gene of the present invention. In addition, in order to modulate gene expression, it can be used as, for example, an antisense oligonucleotide, a ribozyme, or a decoy. As such a nucleic acid, for example, a nucleotide having a continuous partial sequence of 14 or more bases or a complementary sequence thereof in the base sequence represented by SEQ ID NO: 1, 3, or 5 can be used, for example.

An antibody recognizing the PDE of the present invention is obtained using the PDE of the present invention or a protein or peptide having immunological equivalence to the PDE (a synthetic peptide having a protein fragment or a partial sequence) as an antigen. be able to. Having immunological equivalence means, for example, that P
Means cross-reactive with antibodies to DE.

The polyclonal antibody can be produced by a usual method of inoculating a host animal (for example, rat or rabbit) with an antigen and collecting an immune serum. Monoclonal antibodies can be produced by conventional techniques such as the hybridoma method. In addition, a humanized monoclonal antibody or the like can be prepared by modifying the gene of the monoclonal antibody.

Using the antibody obtained above, the expression of the PDE of the present invention in cells or tissues can be detected by a conventional immunochemical method (enzyme immunoassay or the like). Alternatively, the PDE of the present invention can be purified by affinity chromatography using an antibody.

The PDE of the present invention comprises a cyclic nucleotide (c
Having the activity of hydrolyzing AMP or cGMP can be determined by a generally known method for measuring PDE activity (Th
ompson et al., Adv. Cyclic Nucleotide Res., Volume 10, 69
-92, 1979; Yanaka et al., Eur. J. Biochem., 255.
Vol., Pp. 391-399, 1998).

As substrates for the enzymatic reaction, cAMP and c
Cyclic nucleotides such as GMP and derivatives thereof can be used. The PDE of the present invention comprises cAMP and cGM.
Each of P is used as a substrate to hydrolyze them.

The PDEs of the present invention can be used for characterizing, identifying or selecting phosphodiesterase inhibitors.

For example, an enzyme reaction is carried out in a system containing the PDE of the present invention, an enzyme substrate and a test substance (preferably a low-molecular compound or the like), and inhibition of the test substance on enzyme activity (activity of hydrolyzing cyclic nucleotides). Test the effect.

Alternatively, a binding reaction is performed in a system containing the PDE of the present invention and a test substance (preferably a low-molecular compound or the like), and it is determined whether the test substance has a binding ability to the PDE of the present invention. . A test substance (ligand) having binding ability is likely to be an inhibitor.

Further, the test substance (preferably a low molecular weight compound or the like) is examined for its inhibitory effect (or binding ability) on PDE of the present invention, and is compared with the inhibitory action (or binding ability) on other types of PDE. The selectivity of the inhibitory action (or binding ability) can be determined. Thereby, an inhibitor having a relatively high effect on a specific type of PDE (selective inhibitor) can be selected. In addition, inhibitors can be identified and characterized.

Hereinafter, the present invention will be described in more detail by way of examples, but these examples do not limit the present invention.

In the following Examples, unless otherwise specified, each operation is referred to as “molecular cloning (Molecu
lar Cloning) "(Sambrook, J., Fritsch, EF and Ma
niatis, T., Cold Spring Harbor Laboratory Press
Published in 1989), or when a commercially available reagent or kit is used, it was used according to the instructions of a commercially available product.

[0058]

EXAMPLES Example 1 Isolation of cDNA for New Human PDE (PDE11) (I) (1) By comparing the amino acid sequences of the catalytic regions of various known PDE molecules, a portion having high conservation was selected and selected. Based on the nucleotide sequence encoding, sense primers and antisense primers for PCR were designed.
As a sense primer, an oligonucleotide having the sequence shown in SEQ ID NO: 7 in the Sequence Listing was designed, and as an antisense primer, an oligonucleotide having the sequence shown in SEQ ID NO: 8 was designed.

RT- using these PCR primers
PCR (reverse transcript-polymerase chain reac
) was performed to isolate a cDNA fragment from human testis-derived mRNA.

That is, human testis-derived mRNA (Clonte
ch), RNA PCR kit (PEBiosystems GeneAm
Reverse transcription was performed using pRNA PCR Core kit) and random primers (hexamers) to obtain cDNA. Using the obtained cDNA as a template, SEQ.
PCR was performed using the oligonucleotides having the nucleotide sequences shown in FIGS. 8 and 9 as sense primers and antisense primers, respectively. One cycle of the PCR reaction was performed at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 3 seconds.
A total of 30 cycles were performed under the condition of 0 seconds.

The obtained PCR product is used as a vector plasmid.
It was ligated to pGEM-T Easy (Promega) and its nucleotide sequence was determined. The nucleotide sequence is determined using an automatic DNA sequencer (PE B
Using the dideoxy method (BigDyeterminator cycle sequenc manufactured by PE Biosystems) using ABI PRISM 310 manufactured by iosystems
ing reaction kit).
same). The nucleotide sequence of the cDNA fragment thus obtained is
It was a novel nucleotide sequence that had not been reported before, and had a high homology with a part of the cDNA encoding PDE5.

(2) From the cDNA fragment obtained in the above (1), RACE (rapid amplification)
The full-length cDNA was obtained by the method of cation of cDNA ends).

First, based on the nucleotide sequence information of the cDNA fragment obtained in the above (1), an oligonucleotide having the sequence shown in SEQ ID NO: 9 in the Sequence Listing is designed as an antisense primer. A cDNA fragment was prepared from human testis-derived mRNA (Clontech) using a kit for RACE (5'-RACE) for 5 'extension (5'-Full RACE Core Set, manufactured by Takara Shuzo).

Further, LA PCR (long and accurate PCR) (using a Takara Shuzo LA PCR Kit) was performed using the prepared cDNA fragment as a template. As the PCR primers, two types of oligonucleotides having the sequences shown in SEQ ID NOS: 10 and 11 in the sequence listing below were used for the first amplification, and two types of oligonucleotides having the sequences shown in SEQ ID NOS: 12 and 13 were used in the second amplification. Oligonucleotides were used as sense and antisense primers, respectively. These four types of PCR primers were all designed based on the nucleotide sequence information of the cDNA fragment obtained in the above (1).

The nucleotide sequence of the obtained PCR product was determined. As a result, it was revealed that a part of the missing 5′-side region was repaired by the 5′-RACE, and that the base sequence of the repaired 5′-side region was found.

Next, a further 5′-RACE was performed using human prostate-derived cDNA as a template (Marathon-R manufactured by Clontech).
Using eady cDNA (human prostate), the 5 'region was completely repaired. As a primer, a first primer (AP1 primer) corresponding to a linker portion in the first amplification and a primer having the sequence shown in SEQ ID NO: 14 in the sequence listing described later, and a second primer corresponding to the linker portion in the second amplification (AP2 primer) and a primer having the sequence shown in SEQ ID NO: 15 were used as a sense primer and an antisense primer, respectively. The two kinds of antisense primers were designed based on the base sequence information of the 5'-side region repaired by 5'-RACE.

Next, RACE (3′-
RACE) Kit (Clontech SMART RACE cDNA Amp)
lification kit) and mRNA derived from human thyroid (Clontech
3′-RACE was performed using the method described above, and the 3′-side region was completely repaired. As a PCR primer in 3′-RACE, a primer having a sequence shown in SEQ ID NO: 16 in the sequence listing and a first primer (UPM primer) corresponding to a linker portion in the first amplification, and a SEQ ID NO: A primer having the sequence shown in No. 17 and a second primer (NUP primer) corresponding to the linker portion were used as a sense primer and an antisense primer, respectively. The two types of sense primers were designed based on the nucleotide sequence information of the cDNA fragment obtained in the above (1).

In each of the above RACE, PC
The reaction of R was performed at 94 ° C. for 1 minute, followed by 5 cycles at 94 ° C. for 30 seconds and 72 ° C. for 3 minutes, 94 ° C. for 30 seconds,
5 cycles at 0 ° C. for 30 seconds and 72 ° C. for 3 minutes, 9
25 at 4 ° C. for 30 seconds, 68 ° C. for 30 seconds and 72 ° C. for 3 minutes
Cycled.

(3) After analyzing the nucleotide sequence of the full-length cDNA thus obtained, an open reading frame was identified by comparison with the amino acid sequence of a known PDE.

Further, PCR primers surrounding the open reading frame were designed based on the nucleotide sequence information, and RT-PCR using these primers and human prostate-derived mRNA was performed as follows.

That is, human prostate-derived mRNA (Clon
tech), RNA PCR kit (PE Biosystems Gen
Reverse transcription was performed using eAmp RNA PCR Core kit) and random primers (hexamers) to obtain cDNA. PCR was performed using the obtained cDNA as a template. P
As the CR primer, oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 18 and 19 in the Sequence Listing described later were used as sense primers and antisense primers, respectively. One cycle of the PCR reaction was performed at 94 ° C.3
The test was performed under the conditions of 0 second, 55 ° C. for 30 seconds, and 72 ° C. for 5 minutes.

The base sequence of the cDNA fragment (about 3 kb) was determined for a plurality of clones obtained by PCR, and the comparison was performed to eliminate errors due to PCR.
The nucleotide sequence of the full-length cDNA was determined.

(4) The full-length cDNA (447) thus obtained
6 bp) was considered to be the full-length cDNA of the gene for a novel human PDE (referred to as human PDE11 or human PDE11A). The nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing below, and the protein encoded thereby, ie, human PDE11 (specifically, human PDE11A1)
Is shown in SEQ ID NO: 2. Human PDE11 deduced from amino acid sequence (934 amino acid residues)
The molecular weight of (human PDE11A1) was about 105 kDa.

Further, when the homology of the amino acid sequence with the known PDE was examined, the catalytic region of the obtained human PDE11 (human PDE11A1) was found to correspond to the 640th to 881st amino acid residues based on the sequence similarity. Region, the literature (McAllister-Lucus et al., J. Biol. Chem., 268).
22863-22873, 1993), the region having a sequence highly homologous to the cGMP binding region (hereinafter referred to as the cGMP binding region) is 195-4.
It was presumed to be a region corresponding to the 03rd and 379-591th amino acid residues.

The amino acid sequence of human PDE11 (human PDE11A1) was replaced with various known cGMP-binding human PDEs.
In the catalyst region, PDE2A and 42
%, PDE5A and 51%, PDE6A and 44%, PDE
It showed a homology of 44% with 6B and 43% with PDE10A. Also, in the two cGMP binding regions, PD
E2A, PDE5A, PDE6A, PDE6B and PD
E10A showed a homology of 19 to 47% each.

Example 2 New human PDE (PDE11)
(1) A cDNA fragment was obtained in the same manner as in Example 1 (1).

(2) In order to obtain a full-length cDNA from the cDNA fragment obtained in the above (1), R
ACE was performed.

First, in the same manner as in Example 1, a part of the 5′-side region was restored.

Next, further 5′-RACE was performed using human testis-derived cDNA as a template (Marathon-R manufactured by Clontech).
eady cDNA (human testis)). As a primer, a first primer (AP1 primer) corresponding to the linker portion in the first amplification and a primer having the sequence shown in SEQ ID NO: 15 in the sequence listing described later, and a second primer corresponding to the linker portion in the second amplification (AP2 primer) and a primer having the sequence shown in SEQ ID NO: 20 were used as a sense primer and an antisense primer, respectively. The two types of antisense primers were designed based on the base sequence information of the 5'-side region repaired as described above.

Further, the 5′-RA
CE was performed to completely repair the 5 'region. As a primer, an AP1 primer and a primer having a sequence shown in SEQ ID NO: 21 in the Sequence Listing described later were used for the first amplification, and an AP2 primer and a SEQ ID
Primers having the sequence shown in No. 2 were used as sense primers and antisense primers, respectively.
In addition, the two types of antisense primers correspond to the 5′-
It is designed based on the base sequence information of the 5 'side region repaired by RACE.

Next, the 3′-R
ACE was performed to completely repair the 3 'region.

(3) After analyzing the nucleotide sequence of the full-length cDNA thus obtained, an open reading frame was identified by comparing and examining the amino acid sequence of a known PDE.

Further, PCR primers surrounding the open reading frame were designed based on the nucleotide sequence information, and RT-PCR using these primers and human testis-derived mRNA was performed as follows.

That is, human testis-derived mRNA (Clonte
ch), RNA PCR kit (PEBiosystems GeneAm
Reverse transcription was performed using pRNA PCR Core kit) and random primers (hexamers) to obtain cDNA. PCR was performed using the obtained cDNA as a template. Examples of the PCR primers include SEQ ID NOS: 23 and 19 in the sequence listing below.
Were used as sense primers and antisense primers, respectively. One cycle of the PCR reaction is 95 ° C. for 30 seconds,
The test was performed at 55 ° C. for 30 seconds and at 72 ° C. for 3 minutes.

The nucleotide sequence of the cDNA fragment (about 2.4 kb) was determined for a plurality of clones obtained by PCR, and by comparing each, errors due to PCR were eliminated to determine the nucleotide sequence of the full-length cDNA.

(4) The full-length cDNA (350
7 bp) was considered to be the full-length cDNA of the gene for human PDE11 (human PDE11A). The nucleotide sequence is shown in SEQ ID NO: 3 in the sequence listing below, and the protein encoded thereby, ie, human PDE11 (specifically, human PDE11)
11A2. ) Is shown in SEQ ID NO: 4. The molecular weight of human PDE11 (human PDE11A2) deduced from the amino acid sequence (684 amino acid residues) was about 78 kDa.

The human PDE11 obtained in this example (human PD
The cDNA of E11A2) was obtained from human P obtained in Example 1.
It was presumed to correspond to a splicing variant of the cDNA of DE11 (human PDE11A1). Two Ps thought to be derived from two splicing variants
DE, ie, human PDE11A1 and human PDE11A
2 is the N-terminal side of the amino acid sequence (first to
The amino acid residue at position 304, the amino acid residues at positions 1 to 54 of SEQ ID NO: 4) and their corresponding cDNA sequences are different.

When the homology of the amino acid sequence with the known PDE was examined, the similarity of the sequence to human PD
The catalytic region of E11A2 is a region corresponding to the 390th to 631st amino acid residues, and is a c-region of human PDE11A2.
The GMP binding region was estimated to be a region corresponding to the 129th to 341st amino acid residues. Human PDE11A
The catalytic region of No. 2 has the same amino acid sequence as the catalytic region of human PDE11A1. In addition, the amino acid sequence of the cGMP-binding region of human PDE11A2 matches that of the two cGMP-binding regions present in human PDE11A1 that are present downstream (3 ′ side).

Example 3 COS of human PDE11A1
Expression and Purification in Cells (1) Construction of PDE11A1 Expression Vector Plasmid For human PDE11A1 obtained in Example 1 above,
The cDNA fragment amplified in Example 1 (4) was ligated to a vector plasmid pGEM-T Easy (Promega) to obtain a plasmid pGEM-PDE11PF. This pGEM-P
PCR was performed using DE11PF as a template and oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 24 and 25 in the sequence listing below as sense primers and antisense primers, respectively. These PCR primers were designed based on the nucleotide sequences at positions 319 to 684 of SEQ ID NO: 1 (that is, the nucleotide sequences corresponding to the amino acid residues at positions 1 to 122 of SEQ ID NO: 2). is there.

The cDNA fragment amplified by PCR is
ligated to pGEM-T Easy (Promega)
EM-PDE11PBM was obtained and its base sequence was confirmed.

Furthermore, cDNA obtained by treating pGEM-PDE11PF with restriction enzymes KpnI and SalI.
The fragment and the cDNA fragment obtained by treating pGEM-PDE11PBM with the restriction enzymes BamHI and KpnI were used to convert the expression vector pcDNA4 / HisMaxC (Invitroge
n: hereinafter referred to as pHis. BamHI-Xh)
The vector was inserted into the oI site to obtain a vector plasmid for expression of PDE11A1 pHis-PDE11P.

(2) Transfection into COS cells COS-7 cells (ATCC CRL1651) were prepared from 10% fetal bovine serum, 100 units / ml penicillin and 100 μM /
Dulbecco's modified Eagle's medium supplemented with streptomycin (Life Techn)
subcultures at 37 ° C. and 5% carbon dioxide.

[0093] The pHis-PDE was added to COS-7 cells.
11P (or the vector pHis as a control) was transfected. Transfection is performed using a polycationic liposome reagent (LipofectAMINE PLUS: Life Tec
hnologies).

(3) Purification of Recombinant Human PDE11A1 Twenty-four hours after transfection, the cells were washed with ice-cold phosphate buffer, and then ice-cold homogenization buffer (40 mM Tris-HCl, pH 7.5). , 15m
M benzamidine, 5 μg / ml pepstatin A, 5 μ
(g / ml leupeptin).
The obtained homogenate was centrifuged (100,000 g, 60 minutes), and the supernatant was collected.

The supernatant obtained above was applied to a nickel-nitrilotriacetate resin (manufactured by Qiagen) equilibrated with a buffer, and incubated at 4 ° C. for 4 hours. After packing the resin in a column (0.8 × 5 cm), the resin in the column was washed with a washing buffer (40 mM Tris-HCl, pH 7.0).
5, 15 mM benzamidine, 200 mM sodium chloride, 5 mM imidazole, 5 μg / ml pepstatin A, 5 μg / ml leupeptin), and further, elution buffer (40 mM Tris-HCl, pH 7.5,
15 mM benzamidine, 200 mM sodium chloride,
The protein was eluted with 200 mM imidazole, 5 μg / ml pepstatin A, 5 μg / ml leupeptin).

[0096] The obtained protein was cAMP and c
When the hydrolysis activity (PDE activity) using GMP as a substrate was measured, it was found to have an activity of hydrolyzing both cAMP and cGMP.

The PDE activity was measured by the radiolabeled nucleic acid method. That is, 1 μM unlabeled cAM
P (or cGMP) and 22nM ^[3 H] -cAMP
(Or [ ³ H] -cGMP) (Amersham Pharmacia Biot
ech) (50 mM Tris
-HCl, pH 8.0, 5 mM magnesium chloride, 4
mM 2-mercaptoethanol, 0.33 mg / ml
Bovine serum albumin (Sigma)]
The reaction was started by adding 〜１０10 μl of enzyme solution. 37
After the reaction was carried out by keeping the temperature at 30 ° C. for 30 minutes, the reaction was stopped by boiling for 2 minutes, and further 1 mg / ml of snake venom (Crotal
us atrox snake venom) and incubated at 37 ° C for 30 minutes. Then, 500 μl of methanol was added, and the reaction solution was subjected to a Dowex column (1 × 8-400).
After treatment with, a liquid scintillation cocktail was added to each eluate, and radioactivity was measured.

Example 4 COS of human PDE11A2
Expression and Purification in Cells (1) Construction of PDE11A2 Expression Vector Plasmid For human PDE11A2 obtained in Example 2 above,
The cDNA fragment amplified in Example 2 (4) was used for pGEM-T Easy
(Promega) and plasmid pGEM-PDE
11TF was obtained. Using this pGEM-PDE11TF as a template, PCR was performed using oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 26 and 27 in the Sequence Listing below as sense primers and antisense primers, respectively. In addition, these PCR primers were designed based on the 100th to 417th nucleotide sequences of SEQ ID NO: 3 (the nucleotide sequences corresponding to the 1st to 106th amino acid residues of SEQ ID NO: 4).

The cDNA fragment amplified by PCR was
ligated to pGEM-T Easy (Promega)
EM-PDE11TBM was obtained and its nucleotide sequence was confirmed.

Further, pGEM-PDE11TF was converted to restriction enzymes SacI and EcoRV, and EcoRV and SaR.
cDNA fragment obtained by treatment with II and pGE
M-PDE11TBM was replaced with restriction enzymes BamHI and Sac
The cDNA fragment obtained by the treatment with I was inserted into the BamHI-XhoI site of the expression vector pHis,
Vector plasmid pHis-P for PDE11A2 expression
DE11T was obtained.

(2) Transfection into COS cells COS-subcultured under the same conditions as in Example 3 (2) above
Seven cells were transfected with the pHis-PDE11T (or the vector pHis as a control) in the same manner as in Example 3 (2) above.

(3) Purification of Recombinant Human PDE11A2 In the same manner as in Example 3 (3), recombinant human PDE1A2
1A2 was purified. When the PDE activity was measured in the same manner as in Example 3 (3), cAMP was determined.
And cGMP were found to have an activity of hydrolyzing both.

Example 5 Analysis of Enzymatic Properties of Human PDE11 Using the purified recombinant human PDE11A1 obtained in Example 3 above and the purified recombinant human PDE11A2 obtained in Example 4 above, the following procedures were performed. The various enzymatic properties of human PDE11 were analyzed.

(1) Kinetic Analysis of Enzyme Reaction An enzyme reaction was carried out using various substrate (cAMP or cGMP) concentrations, and PDE activity (initial rate of the reaction) was measured. The enzyme reaction and PDE activity measurement were performed in the same manner as in Example 3 (3). However, the concentration of unlabeled cAMP (or cGMP) in the reaction solution was 0.1 to 10 μM.

The results (Lineweaver-Burk plot) are shown in FIG. 1 for human PDE11A1 and human PDE11A.
2 is shown in FIG.

From the results of the analysis, it was found that human PDE11A1 c
The Km value using AMP as a substrate is 2.96 ± 0.457.
μM and Vmax was 267 ± 47.9 pmol / min / μg. On the other hand, cGMP of human PDE11A1
The value of Km is 1.43 ± 0.109 μM, and Vmax is 121 ± 8.08 pmol / min / μg.
Met.

The Km value of cAMP of human PDE11A2 as a substrate is 2.99 ± 0.488 μM, and Vmax is 9.63 ± 1.88 pmol / min / μg.
Met. On the other hand, the Km value of human PDE11A2 using cGMP as a substrate is 1.47 ± 0.115 μM,
Vmax was 4.02 ± 0.214 pmol / min / μg.

[0108] From these results, human PDE11 is c
It was found to have an activity of hydrolyzing both AMP and cGMP, but to have a stronger affinity for cGMP.

(2) cGMP for cAMP degradation activity
Of cAMP on cGMP-degrading activity and cGMP at various concentrations in the reaction mixture
The effect of MP degradation activity was examined. The effect of cGMP degrading activity when various concentrations of cAMP were added to the reaction solution was also examined.

The enzymatic reaction and PDE activity measurement were performed in the same manner as in Example 3 (3). However, in an experiment for examining cAMP degradation activity, unlabeled cAMP was used in the reaction solution.
Was added at 3.5 μM, and cGMP (unlabeled) was added at 0.
01 to 100 μM was added (or not added). on the other hand,
In experiments for examining cGMP-degrading activity, unlabeled cG
MP was added at 1.3 μM, and cAMP (unlabeled) was further added (or not added) at 0.01 to 100 μM.

Regarding the cAMP degrading activity, the activity without cGMP was taken as 100% and the relative value (%) of the activity
Was calculated. As for cGMP degradation activity, cA
The relative value (%) of the activity was calculated with the activity when MP was not added as 100%. These results are shown in FIG. FIG.
As is apparent from the above, in human PDE11, cG
A decrease in cAMP degradation activity was observed depending on the concentration of added MP, while cGMP was decreased depending on the concentration of added cAMP.
Degradation activity was reduced.

(3) Inhibition of activity by various known PDE inhibitors Various known PDE inhibitors against cAMP and cGMP hydrolysis activity (PDE activity) of human PDE11 [IBM
X, Vinpocetine, EHNA, Milrinone, Rolipram, Zaprinast
(Zaprinast), Dipyridamole, SCH
51866 and E4021] were examined as follows.

An enzymatic reaction was carried out using cAMP or cGMP as a substrate, and various known PDE inhibitors were added to the reaction solution, and the hydrolysis activity was measured. The enzymatic reaction and PDE activity measurement were performed in the same manner as in Example 3 (3). However,
In the reaction solution, the concentration when adding unlabeled cAMP is 3.
5 μM, the concentration when unlabeled cGMP was added was 1.3 μM, and various PDE inhibitors were added to the reaction solution at 0 to 1 μM.
00 μM was added.

Various PDEs for the activity of human PDE11
The results obtained by expressing the inhibitory action of the inhibitor by IC50 were determined using human PD.
Table 1 shows E11A1 and Table 2 shows human PDE11A2.

[0115]

[Table 1]

[0116]

[Table 2]

Example 6 PDEs in Various Human Tissues
Expression of 11 The presence or absence of PDE11 gene expression in various human tissues was examined as follows.

Various human tissue-derived mRNAs (Human Mult
Dot blot analysis was performed using an iple Tissue Expression Array (manufactured by Clontech). As a probe, a DNA fragment encoding a part of human PDE11A obtained in Examples 1 and 2 above (the nucleotide sequence of the fragment is SEQ ID NO: 1)
Nos. 1237-1 of the human PDE11A1 cDNA shown in
SEQ ID NO: 3 corresponds to the nucleotide sequence at position 801
Nos. 268-83 of the human PDE11A2 cDNA shown in
This corresponds to the second base sequence. ) Was used after being labeled with ³² P. In addition, the amount of mRNA is the cDNA of human ubiquitin.
And the major histocompatibility complex class Ic as a probe, and the amount adjusted so that the signal for them was constant was used.

Hybridization was performed under the following conditions. That is, the nylon membrane, ³²
Hybridization solution containing a probe labeled with P (50% formamide, 4 × SSC, 0.5% SD
S, 5x Denhardt's solution, 100 μg / ml salmon sperm DNA) at 42 ° C. for 20 hours for hybridization. Then, the cleaning solution A (0.5 × SS
C, 0.1% SDS) for 3 minutes at room temperature, and further, washing solution B (0.2 × SSC, 0.1% SDS)
Was performed twice at 60 ° C. for 30 minutes. Thereafter, autoradiography was performed at −80 ° C. for 3 days.

As a result of dot blot, as shown in FIG. 4, strong expression of PDE11 mRNA was detected in the prostate and testis. Also, weak expression was observed in the salivary gland, pituitary gland, thyroid and liver (the order of expression intensity was as described).

[0121] Northern blot analysis was performed on mRNA derived from human testis and human prostate using the same probe as described above under the same conditions.

As a result of Northern blotting, a band of about 3 kb was detected in the testis, and bands of about 2 kb, about 6 kb and about 10 kb were observed in the prostate.

Example 7 New rat PDE (PDE1
1) Isolation of cDNA (1) Human PDE11A obtained in Examples 1 and 2 above
PCR primers were designed on the basis of the cDNA base sequence information, and RT-PCR using the primers was performed to isolate a PDE11A cDNA fragment from rat testis-derived mRNA.

That is, mRNA from rat testis (manufactured by Wako Pure Chemical Industries), RNA PCR kit (Gen
Reverse transcription was performed using eAmp RNA PCR Core kit) and random primers (hexamers) to obtain cDNA. PCR was performed using the obtained cDNA as a template. P
As the CR primer, oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 28 and 29 in the Sequence Listing described below were used as sense primers and antisense primers, respectively. One cycle of the PCR reaction was 95 ° C 3
A total of 30 cycles were performed under the conditions of 0 second, 54 ° C. for 30 seconds, and 72 ° C. for 2 minutes.

Further, the nucleotide sequence of the cDNA fragment amplified by PCR was determined.

(2) In order to obtain a full-length cDNA from the cDNA fragment obtained in the above (1), rat testis-derived mRN was used.
Using A (manufactured by Wako Pure Chemical Industries), 5 ′
-RACE and 3'-RACE were performed. RACE kits (Clontech SMART RACE cDNA Amplif
ication kit).

First, 5′-RACE was performed to repair a part of the 5′-side region. As a primer, a first primer (UPM primer) corresponding to a linker portion in the first amplification and a primer having the sequence shown in SEQ ID NO: 30 in the sequence listing described later, and a second primer corresponding to the linker portion in the second amplification (NUP primer) and a primer having the sequence shown in SEQ ID NO: 31 were used as a sense primer and an antisense primer, respectively. The two types of antisense primers were designed based on the nucleotide sequence information of the cDNA obtained in the above (1).

Further, 5′-RACE was performed to completely restore the 5 ′ side region. As a primer, a UPM primer and an oligonucleotide having the sequence shown in SEQ ID NO: 32 of the Sequence Listing described later in the first amplification, and a NUP primer and an oligonucleotide having the sequence shown in SEQ ID NO: 33 in the second amplification, Used as sense and antisense primers. In addition, 2
The kinds of antisense primers are designed based on the base sequence information of the 5'-side region repaired as described above.

Next, 3′-RACE was performed to completely restore the 3 ′ side region. As a primer, an oligonucleotide having the sequence shown in SEQ ID NO: 34 of the sequence listing below and an UPM primer in the first amplification, and an oligonucleotide and a NUP primer having the sequence shown in SEQ ID NO: 35 in the second amplification, respectively Used as sense and antisense primers. The two types of sense primers are the cDNs obtained in the above (1).
It is designed based on the nucleotide sequence information of fragment A.

(3) After analyzing the nucleotide sequence of the full-length cDNA thus obtained, an open reading frame was identified by comparison with the amino acid sequence of a known PDE.

Further, PCR primers surrounding the open reading frame were designed based on the nucleotide sequence information, and these PCR primers and rat testis-derived mR
RT-PCR using NA was performed as follows.

Specifically, rat testis-derived mRNA (manufactured by Wako Pure Chemical Industries, Ltd.), RNA PCR kit (Genome manufactured by PE Biosystems)
Reverse transcription reaction was performed using eAmp RNA PCR Core kit) and poly dT tailed primer to obtain cDNA. PCR was performed using the obtained cDNA as a template. As PCR primers, oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 36 and 37 in the Sequence Listing described later were used as sense primers and antisense primers, respectively. One cycle of the PCR reaction was performed at 95 ° C. for 30 seconds, 5
30 seconds at 7 ° C. and 3 minutes at 72 ° C. for a total of 30
Cycled.

The nucleotide sequence of the cDNA fragment (about 2.3 kb) was determined for a plurality of clones obtained by PCR, and by comparing each, errors due to PCR were eliminated to determine the nucleotide sequence of the full-length cDNA.

(4) The cDNA (3492b
p) is the full-length cDNA of the gene of the novel rat PDE (referred to as rat PDE11 or rat PDE11A)
Was thought to be. The nucleotide sequence is shown in SEQ ID NO: 5 in the Sequence Listing, and the protein encoded thereby, ie, the amino acid sequence of rat PDE11 (rat PDE11A) is shown in SEQ ID NO: 6. Rat PDE11 (rat PDE) deduced from the amino acid sequence (685 amino acid residues)
The molecular weight of 11A) was about 78 kDa.

Rat PDE11 (rat PDE11A)
The amino acid sequence of human PDE11 obtained in Example 2
When compared with the amino acid sequence of A2, homology as high as about 93% was observed.

[0136]

The novel PDE of the present invention and its gene are
It is useful for studying multiple mechanisms of intracellular signal transduction. In addition, it can be a target molecule of a therapeutic drug for a new disease.

Further, the novel PDE of the present invention and a method for characterizing, identifying and selecting an inhibitor utilizing the gene thereof are described in the following.
It is useful for developing highly selective inhibitors and excellent drugs with high therapeutic effects and few side effects.

"Sequence List Free Text" SEQ ID NOs: 7 to 3
7 free text <223> Artificially synthesized primer sequence (Artifici
ally Synthesized Primer Sequence)

[0139]

[Sequence List] SEQUENCE LISTING <110> TANABE SEIYAKU CO., LTD. <120> Novel Phosphodiesterase and DNA Thereof <130> A00-4685 <160> 37 <170> PatentIn Ver. 2.0 <210> 1 <211> 4476 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (319) .. (3120) <400> 1 gcagcggcgg cagccagaac aggagcagcg atagctcggg tttccggaac aggagccggg 60 gcagcggcgg cagctcagtg ctgggcacct gtgcggagca ggagtagcag gaccacgggg 120 tggggtcggc gccagccact ctgagccaga gaaggaaggg gcatctccca gattccactg 180 ctgggaataa tctccagggg aggtggcgct gaactgggaa tactggtggg ggtgaacatg 240 tgcaggaaca gctagaggcc tcggggcagg aaaacatttg gttcacgtgt aaacaggcaa 300 ggaaagctgt ctgggacc atg gca gcc tcc cgc ctg gac ttt ggg gag gtg 351 Met Ala Ala Ser Arg Leu Asp Phe Gly Glu Val 1 5 10 gaa act ttc ctg gac agg cac cca gag ttg ttt gaa gat tac ttg atg 399 Glu Thr Phe Leu Asp Arg His Pro Glu Leu Phe Glu Asp Tyr Leu M 15 20 25 cgg aag ggg aag cag gag atg gtt gaa aag tgg ctg cag agg cac agt 447 Arg Lys Gly Lys Gln Glu Met Val Glu Lys Trp Leu Gln Arg His Ser 30 35 40 cag ggt cag ggg gct tta ggt cca agg ccc tct ttg gct ggt acc agc 495 Gln Gly Gln Gly Ala Leu Gly Pro Arg Pro Ser Leu Ala Gly Thr Ser 45 50 55 agc ttg gct cac agc acc tgc aga ggt ggc agc agc gtt ggt ggt ggc 543 Ser Leu Ala His Ser Thr Cys Arg G ly Gly Ser Ser Val Gly Gly Gly 60 65 70 75 act gga cca aat ggc tct gcc cac agc cag ccc ctt ccc ggt ggc ggg 591 Thr Gly Pro Asn Gly Ser Ala His Ser Gln Pro Leu Pro Gly Gly Gly 80 85 90 gac tgt ggt ggg gtt ccc ttg agt ccc agc tgg gcc ggt ggc agc agg 639 Asp Cys Gly Gly Val Pro Leu Ser Pro Ser Trp Ala Gly Gly Ser Arg 95 100 105 ggc gat ggg aac ctg cag cgg aga gct tct cag aaa gag cta agg aag 687 Gly Asp Gly Asn Leu Gln Arg Arg Ala Ser Gln Lys Glu Leu Arg Lys 110 115 120 agt ttt gcc cgc tcc aag gcc atc cac gtg aac agg acc tac gat gaa 735 Ser Phe Ala Arg Ser Lys Ala Ile His Val Asn Arg Thr Tyr Asp Glu 125 130 135 cag gtg acc tcc cgg gct cag gaa ccc ctg agt agt gta cga cgg agg 783 Gln Val Thr Ser Arg Ala Gln Glu Pro Leu Ser Ser Val Arg Arg Arg 140 145 150 155 gca ctt ctc cgg aag gca agc tcc ctg ccc ccc acc aca gcc cat att 831 Ala Leu Leu Arg Lys Ala Ser Ser Leu Pro Pro Thr Thr Ala His Ile 160 165 170 ctc agt gcg ctg ctg gaa tcg aga gtg aat ctg cct cag tat ccc cct 879 Leu Ser Alau Leu Glu S er Arg Val Asn Leu Pro Gln Tyr Pro Pro 175 180 185 aca gcc atc gac tac aag tgc cat ctg aaa aag cat aat gag cgt cag 927 Thr Ala Ile Asp Tyr Lys Cys His Leu Lys Lys His Asn Glu Arg Gln 190 195 200 ttc ttt ctg gaa ttg gtc aaa gat atc tcc aat gac ctt gac ctc acc 975 Phe Phe Leu Glu Leu Val Lys Asp Ile Ser Asn Asp Leu Asp Leu Thr 205 210 215 agc ctg agc tac aag att ctc atc ttt gtc gc gct 1023 Ser Leu Ser Tyr Lys Ile Leu Ile Phe Val Cys Leu Met Val Asp Ala 220 225 230 235 gac cgc tgc tct ctt ttc ctg gtg gaa ggg gca gct gct ggc aag aag 1071 Asp Arg Cys Ser Leu Phe Leu Val Glu Gly Ala Ala Gly Lys Lys 240 245 250 acc ttg gtc tcc aaa ttc ttt gat gtg cat gca gga acc cct ctg ctg 1119 Thr Leu Val Ser Lys Phe Phe Asp Val His Ala Gly Thr Pro Leu Leu 255 260 265 cct tgc agc agc aca gag aac tca aat gag gtg cag gtc ccc tgg ggc 1167 Pro Cys Ser Ser Thr Glu Asn Ser Asn Glu Val Gln Val Pro Trp Gly 270 275 280 aaa ggt atc att ggc tat gtc ggg gag cat gga gaa acg gtc aac att 1215 Lys Gly Ile Ile Gly Tyr Val Gly Glu His Gly Glu Thr Val Asn Ile 285 290 295 cct gat gcc tac cag gat cga cga ttc aat gat gaa atc gac aag cta 1263 Pro Asp Ala Tyr Gln Asp Arg Arg Phe Asn Asp Glu Ile Asp Lysu 300 305 310 315 act gga tac aag aca aaa tca tta ttg tgc atg cct atc cga agc agt 1311 Thr Gly Tyr Lys Thr Lys Ser Leu Leu Cys Met Pro Ile Arg Ser Ser 320 325 330 gat ggt gag att att ggt gtg gcc caa gcg ata aat aag att cct gaa 1359 Asp Gly Glu Ile Ile Gly Val Ala Gln Ala Ile Asn Lys Ile Pro Glu 335 340 345 gga gct cca ttt act gaa gat gat gaa aaa gtt atg cag atg tat ctt 1407 Gly Ala Pro Phe Thr Glu Asp Glu Lys Val Met Gln Met Tyr Leu 350 355 360 cca ttt tgt gga atc gcc ata tct aac gct cag ctc ttt gct gcc tca 1455 Pro Phe Cys Gly Ile Ala Ile Ser Asn Ala Gln Leu Phe Ala Ala Ser 365 370 375 agg aaa gaa tat gaa aga agc aga gct ttg cta gag gtg gtt aat gac 1503 Arg Lys Glu Tyr Glu Arg Ser Arg Ala Leu Leu Glu Val Val Asn Asp 380 385 390 395 395 ctc ttt gaa gaa cag act gac ctg gag ag aaatt a ata atg 1551 Leu Phe Glu Glu Gln Thr Asp Leu Glu Lys Ile Val Lys Lys Ile Met 400 405 410 cat cgg gcc caa act ctg ctg aaa tgt gaa cgc tgt tct gtt tta ctc 1599 His Arg Ala Gln Thr Leu Leu Lys Cys Glu Arg Cys Ser Val Leu Leu 415 420 425 cta gag gac atc gaa tca cca gtg gtg aaa ttt acc aaa tcc ttt gaa 1647 Leu Glu Asp Ile Glu Ser Pro Val Val Lys Phe Thr Lys Ser Phe Glu 430 435 440 440 ttg atg tcc cca aag tgc agt gct gat gct gag aac agt ttc aaa gaa 1695 Leu Met Ser Pro Lys Cys Ser Ala Asp Ala Glu Asn Ser Phe Lys Glu 445 450 455 agc atg gag aaa tca tca tac tcc gac tgg cta ata aat aac agc att 1743 Ser Glu Lys Ser Ser Tyr Ser Asp Trp Leu Ile Asn Asn Ser Ile 460 465 470 475 gct gag ctg gtt gct tca aca ggc ctt cca gtg aac atc agt gat gcc 1791 Ala Glu Leu Val Ala Ser Thr Gly Leu Pro Val Asn Ile Ser Asp Ala 480 485 490 tac cag gat ccg cgc ttt gat gca gag gca gac cag ata tct ggt ttt 1839 Tyr Gln Asp Pro Arg Phe Asp Ala Glu Ala Asp Gln Ile Ser Gly Phe 495 500 505 cac ata aga tct gtt ctt tgt gtc ctc att tgg aat agc aac cac caa 1887 His Ile Arg Ser Val Leu Cys Val Pro Ile Trp Asn Ser Asn His Gln 510 515 520 ata att gga gtg gct caa gtg tta aac aga ctt gat ggg aaa cct ttt 1935 Ile Ile Gly Val Ala Gln Val Leu Asn Arg Leu Asp Gly Lys Pro Phe 525 530 535 gat gat gca gat caa cga ctt ttt gag gct ttt gtc atc ttt tgt gga 1983 Asp Asp Ala Asp Gln Arg Leu Phe Glu Ala Phe Val Ile Phe Cys Gly 540 550 550 545 ctt ggc atc aac aac aca att atg tat gat caa gtg aag aag tcc tgg 2031 Leu Gly Ile Asn Asn Thr Ile Met Tyr Asp Gln Val Lys Lys Ser Trp 560 565 570 570 gcc aag cag tct gtg gct ctt gat gtg cat gca tca tac aca tgt 2079 Ala Lys Gln Ser Val Ala Leu Asp Val Leu Ser Tyr His Ala Thr Cys 575 580 585 tca aaa gct gaa gtt gac aag ttt aag gca gcc aac atc cct ctg gtg 2127 Ser Lys Ala Glu Val Asp Lys Phe Lys Ala Ala Asn Ile Pro Leu Val 590 595 600 tca gaa ctt gcc atc gat gac att cat ttt gat gac ttt tct ctc gac 2175 Ser Glu Leu Ala Ile Asp Asp Ile His Phe Asp Asp Phe Ser Leu Asp 605 610 615 gtt gat gcc atg atc aca gct gct ctc cgg atg ttc atg gag ctg ggg 2223 Val Asp Ala Met Ile Thr Ala Ala Leu Arg Met Phe Met Glu Leu Gly 620 625 630 635 atg gta cag aaa ttt aaa att gac tat gag aca ctg tgt agg tgg Met Val Gln Lys Phe Lys Ile Asp Tyr Glu Thr Leu Cys Arg Trp Leu 640 645 650 ttg aca gtg agg aaa aac tat cgg atg gtt cta tac cac aac tgg aga 2319 Leu Thr Val Arg Lys Asn Tyr Arg Met Val Leu Tyr His Asn Trp Arg 655 660 665 cat gcc ttc aac gtg tgt cag ctg atg ttc gcg atg tta acc act gct 2367 His Ala Phe Asn Val Cys Gln Leu Met Phe Ala Met Leu Thr Thr Ala 670 675 680 ggg ttt caa gac att ctg acc gag gaa att tta gcg gtg att gtg 2415 Gly Phe Gln Asp Ile Leu Thr Glu Val Glu Ile Leu Ala Val Ile Val 685 690 695 gga tgc ctg tgt cat gac ctc gac cac agg gga acc aac aat gcc ttc 2463 Gly Cys Leu Cys Leu Asp His Arg Gly Thr Asn Asn Ala Phe 700 705 710 715 caa gct aag agt ggc tct gcc ctg gcc caa ctc tat gga acc tct gct 2511 Gln Ala Lys Ser Gly Ser Ala Leu Ala Gln Leu Tyr Gly Thr Ser Ala 720 7 25 730 acc ttg gag cat cac cat ttc aac cac gcc gtg atg atc ctt caa agt 2559 Thr Leu Glu His His His Phe Asn His Ala Val Met Ile Leu Gln Ser 735 740 745 gag ggt cac aat atc ttt gct aac ctg tcc tcc aag gaa tat agt gac 2607 Glu Gly His Asn Ile Phe Ala Asn Leu Ser Ser Lys Glu Tyr Ser Asp 750 755 760 ctt atg cag ctt ttg aag cag tca ata ttg gca aca gac ctc acg ctg 2655 Leu Met Gln Leu Leu Lys Gln Leu Ala Thr Asp Leu Thr Leu 765 770 775 tac ttt gag agg aga act gaa ttc ttt gaa ctt gtc agt aaa gga gaa 2703 Tyr Phe Glu Arg Arg Thr Glu Phe Phe Glu Leu Val Ser Lys Gly Glu 780 785 790 790 tgg gat aac atc aaa aac cat cgt gat ata ttt cga tca atg tta 2751 Tyr Asp Trp Asn Ile Lys Asn His Arg Asp Ile Phe Arg Ser Met Leu 800 805 810 atg aca gcc tgt gac ctt gga gcc gtg acc aaa ccg tgg gag atc tcc 2799 Met Thr Ala Cys Asp Leu Gly Ala Val Thr Lys Pro Trp Glu Ile Ser 815 820 825 aga cag gtg gca gaa ctt gta acc agt gag ttc ttc gaa caa gga gat 2847 Arg Gln Val Ala Glu Leu Val Thr Ser Glu Phe Phe Gl u Gln Gly Asp 830 835 840 cgg gag aga tta gag ctc aaa ctc act cct tca gca att ttt gat cgg 2895 Arg Glu Arg Leu Glu Leu Lys Leu Thr Pro Ser Ala Ile Phe Asp Arg 845 850 855 aac cgg aag gat gaactg cgg ttg caa ctg gag tgg att gat agc 2943 Asn Arg Lys Asp Glu Leu Pro Arg Leu Gln Leu Glu Trp Ile Asp Ser 860 865 870 875 atc tgc atg cct ttg tat cag gca ctg gtg aag gtc Aac gtg aaa gtg aaa Pro Leu Tyr Gln Ala Leu Val Lys Val Asn Val Lys Leu 880 885 890 aag ccg atg cta gat tca gta gct aca aac aga agt aag tgg gaa gag 3039 Lys Pro Met Leu Asp Ser Val Ala Thr Asn Arg Ser Lys Trp Glu Glu 895 900 905 cta cac caa aaa cga ctg ctg gcc tca act gcc tca tcc tcc tcc cct 3087 Leu His Gln Lys Arg Leu Leu Ala Ser Thr Ala Ser Ser Ser Ser Pro 910 915 920 gcc agt gtt atg gta gcc aag gaa gac agg aac taaacctcc ggtcagctgc 3140 Ala Ser Val Met Val Ala Lys Glu Asp Arg Asn 925 930 agctgcaaaa tgactacagc ctgaagggcc attttcagtc cagcaatgtc atccttttgt 3200 tcttttagct cagaaagacc taacatctca aggatgcact gggaacc atg cctgggcttt 3260 caccttgaag catggtcagc agcagagaga gcaacgggaa ggacaaagaa agaggtgggg 3320 cagggagcac accccaggac cctcactttt ccctaatgaa cacgcatggg ctgaaatgaa 3380 ggctctgggt aggggactgt tttggatcca aggacctgtg gacagtcggc ctacttactc 3440 tgagctgagg gaacactgaa cagtaaaagc gtcattagcg ctgcttcatt ttgtataggg 3500 cttttctgtt tgttacaagc caaacattgc ctgtctttgc ttcccgtccc tgaatgcctt 3560 tttgtgccag actgtcccaa gaatcctaat ttgtattcca tagaggtatt ttatttttaa 3620 tcctagagct tcttattgat ggatccttta gaattgccta cctaaaaggt aaactatact 3680 atccttataa atactgatca atcccagttc tccccctaaa aatgaataca tagtaggact 3740 atagcaaatg tgtttgatgg gtaattctag actgggacta tggtaccctt ttccagagtt 3800 ttaaaattca accttcatta cagacaaagt tttctcccag aaggaatgga ttgatagatt 3860 ttgattaaag taagggtgga aggaaatctg tagctggatt taccacaagt gacatctaga 3920 aactatagtt cacaggacag agcagagcca tggagaataa gcattgacta ccttgagttc 3980 tcctagtgag gagttctggt ataaaattta agattactac cagtaaccaa cttaaagcaa 4040 actatagggg tccctaattt tggatttttc cttaagtgta agaaacaatg ct tcaaatgt 4100 taagaaataa cagtctgggc aaagaacgca tattctatag gaagccaggt ttacaatagg 4160 taagaataaa ctgtattaag tagatgtaat gactagaaag ctgctttgct ccctatattg 4220 agaaattgtg gacatggtat gtgttatcca aagaacattg ggctagaaga tagatttcta 4280 tccttagctt tggcattatt gactggattg acttgaacaa gtcgcttaac ttctacaagc 4340 ttgtttcctt atttgtcaaa ttagattaca ctaggaaacg attctcgaac atgttttaac 4400 cttacaactc tttgttcaaa taaatctttc aatgaatccc caacataaaa aaaaaaaaaa 4460 aaaaaaaaaa aaaaaa 4476 <210> 2 <211> 934 <212> PRT <213> Homo sapiens <400> 2 Met Ala Ala Ser Arg Leu Asp Phe Gly Glu Val Glu Thr Phe Leu Asp 1 5 10 15 Arg His Pro Glu Leu Phe Glu Asp Tyr Leu Met Arg Lys Gly Lys Gln 20 25 30 Glu Met Val Glu Lys Trp Leu Gln Arg His Ser Gln Gly Gln Gly Ala 35 40 45 Leu Gly Pro Arg Pro Ser Leu Ala Gly Thr Ser Ser Leu Ala His Ser 50 55 60 Thr Cys Arg Gly Gly Ser Ser Val Gly Gly Gly Thr Gly Pro Asn Gly 65 70 75 80 Ser Ala His Ser Gln Pro Leu Pro Gly Gly Gly Asp Cys Gly Gly Val 85 90 95 Pro Leu Ser Pro Ser Trp Ala Gly Gly Ser Arg Gly Asp Gly Asn Leu 100 105 110 Gln Arg Arg Ala Ser Gln Lys Glu Leu Arg Lys Ser Phe Ala Arg Ser 115 120 125 Lys Ala Ile His Val Asn Arg Thr Tyr Asp Glu Gln Val Thr Ser Arg 130 135 140 Ala Gln Glu Pro Leu Ser Ser Val Arg Arg Arg Ala Leu Leu Arg Lys 145 150 155 160 Ala Ser Ser Leu Pro Pro Thr Thr Ala His Ile Leu Ser Ala Leu Leu 165 170 175 Glu Ser Arg Val Asn Leu Pro Gln Tyr Pro Pro Thr Ala Ile Asp Tyr 180 185 190 Lys Cys His Leu Lys Lys His Asn Glu Arg Gln Phe Phe Leu Glu Leu 195 200 205 Val Lys Asp I le Ser Asn Asp Leu Asp Leu Thr Ser Leu Ser Tyr Lys 210 215 220 Ile Leu Ile Phe Val Cys Leu Met Val Asp Ala Asp Arg Cys Ser Leu 225 230 235 240 Phe Leu Val Glu Gly Ala Ala Ala Gly Lys Lys Thr Leu Val Ser Lys 245 250 255 Phe Phe Asp Val His Ala Gly Thr Pro Leu Leu Pro Cys Ser Ser Thr 260 265 270 270 Glu Asn Ser Asn Glu Val Gln Val Pro Trp Gly Lys Gly Ile Ile Gly 275 280 285 Tyr Val Gly Glu His Gly Glu Thr Val Asn Ile Pro Asp Ala Tyr Gln 290 295 300 Asp Arg Arg Phe Asn Asp Glu Ile Asp Lys Leu Thr Gly Tyr Lys Thr 305 310 315 320 Lys Ser Leu Leu Leu Cys Met Pro Ile Arg Ser Ser Asp Gly Glu Ile Ile 325 330 335 Gly Val Ala Gln Ala Ile Asn Lys Ile Pro Glu Gly Ala Pro Phe Thr 340 345 350 Glu Asp Asp Glu Lys Val Met Gln Met Tyr Leu Pro Phe Cys Gly Ile 355 360 365 Ala Ile Ser Asn Ala Gln Leu Phe Ala Ala Ser Arg Lys Glu Tyr Glu 370 375 380 Arg Ser Arg Ala Leu Leu Glu Val Val Asn Asp Leu Phe Glu Glu Gln 385 390 395 400 Thr Asp Leu Glu Lys Ile Val Lys Lys Ile Met His Arg Ala Gln Thr 405 410 415 Leu Leu Lys C ys Glu Arg Cys Ser Val Leu Leu Leu Glu Asp Ile Glu 420 425 430 Ser Pro Val Val Lys Phe Thr Lys Ser Phe Glu Leu Met Ser Pro Lys 435 440 445 Cys Ser Ala Asp Ala Glu Asn Ser Phe Lys Glu Ser Met Glu Lys Ser 450 455 460 Ser Tyr Ser Asp Trp Leu Ile Asn Asn Ser Ile Ala Glu Leu Val Ala 465 470 475 480 Ser Thr Gly Leu Pro Val Asn Ile Ser Asp Ala Tyr Gln Asp Pro Arg 485 490 495 Phe Asp Ala Glu Ala Asp Gln Ile Ser Gly Phe His Ile Arg Ser Val 500 505 510 510 Leu Cys Val Pro Ile Trp Asn Ser Asn His Gln Ile Ile Gly Val Ala 515 520 525 Gln Val Leu Asn Arg Leu Asp Gly Lys Pro Phe Asp Asp Ala Asp Gln 530 535 540 Arg Leu Phe Glu Ala Phe Val Ile Phe Cys Gly Leu Gly Ile Asn Asn 545 550 555 560 Thr Ile Met Tyr Asp Gln Val Lys Lys Ser Trp Ala Lys Gln Ser Val 565 570 575 Ala Leu Asp Val Leu Ser Tyr His Ala Thr Cys Ser Lys Ala Glu Val 580 585 590 Asp Lys Phe Lys Ala Ala Asn Ile Pro Leu Val Ser Glu Leu Ala Ile 595 600 605 Asp Asp Ile His Phe Asp Asp Phe Ser Leu Asp Val Asp Ala Met Ile 610 615 620 620 Thr Ala Ala Leu A rg Met Phe Met Glu Leu Gly Met Val Gln Lys Phe 625 630 635 640 Lys Ile Asp Tyr Glu Thr Leu Cys Arg Trp Leu Leu Thr Val Arg Lys 645 650 655 Asn Tyr Arg Met Val Leu Tyr His Asn Trp Arg His Ala Phe Asn Val 660 665 670 Cys Gln Leu Met Phe Ala Met Leu Thr Thr Ala Gly Phe Gln Asp Ile 675 680 685 Leu Thr Glu Val Glu Ile Leu Ala Val Ile Val Gly Cys Leu Cys His 690 695 700 Asp Leu Asp His Arg Gly Thr Asn Asn Ala Phe Gln Ala Lys Ser Gly 705 710 710 715 720 Ser Ala Leu Ala Gln Leu Tyr Gly Thr Ser Ala Thr Leu Glu His His 725 730 735 His Phe Asn His Ala Val Met Ile Leu Gln Ser Glu Gly His Asn Ile 740 745 750 Phe Ala Asn Leu Ser Ser Lys Glu Tyr Ser Asp Leu Met Gln Leu Leu 755 760 765 Lys Gln Ser Ile Leu Ala Thr Asp Leu Thr Leu Tyr Phe Glu Arg Arg 770 775 780 Thr Glu Phe Phe Glu Leu Val Ser Lys Gly Glu Tyr Asp Trp Asn Ile 785 790 795 800 Lys Asn His Arg Asp Ile Phe Arg Ser Met Leu Met Thr Ala Cys Asp 805 810 815 Leu Gly Ala Val Thr Lys Pro Trp Glu Ile Ser Arg Gln Val Ala Glu 820 825 830 Leu Val Thr Ser G lu Phe Phe Glu Gln Gly Asp Arg Glu Arg Leu Glu 835 840 845 Leu Lys Leu Thr Pro Ser Ala Ile Phe Asp Arg Asn Arg Lys Asp Glu 850 855 860 Leu Pro Arg Leu Gln Leu Glu Trp Ile Asp Ser Ile Cys Met Pro Leu 865 870 875 880 Tyr Gln Ala Leu Val Lys Val Asn Val Lys Leu Lys Pro Met Leu Asp 885 890 895 Ser Val Ala Thr Asn Arg Ser Lys Trp Glu Glu Leu His Gln Lys Arg 900 905 910 Leu Leu Ala Ser Thr Ala Ser Ser Ser Ser Pro Ala Ser Val Met Val 915 920 925 Ala Lys Glu Asp Arg Asn 930 <210> 3 <211> 3507 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (100) .. (2151) <400> 3 cgcttgcagc ccagggcgtg aggtgctctt tctggatcgc cagcctcgaa gttgaggcgt 60 ggactctggc caggtggctg ttccagagca acatgggag atg ctg aag cag gca 114 Met Leu Lys Gln Ag a c ag ag ag ag c ag ag ag Arg Asn Val Leu Ser Ala Thr Gln Trp Lys Lys 10 15 20 gtg aaa atc aca aga ctg gtc caa atc tct ggg gcc tct ttg gct gaa 210 Val Lys Ile Thr Arg Leu Val Gln Ile Ser Gly Ala Ser Leu Ala Glu 25 30 35 aaa cag gaa aag cac cag gat ttt ctt ata cag agg caa aca aaa aca 258 Lys Gln Glu Lys His Gln Asp Phe Leu Ile Gln Arg Gln Thr Lys Thr 40 45 50 aag gat cga cga ttc aat gat gaa atc gac aag cta act gga tac aag 306 Lys Asp Arg Arg Phe Asn Asp Glu Ile Asp Lys Leu Thr Gly Tyr Lys 55 60 65 aca aaa tca tta ttg tgc atg cct atc cga agc agt gat ggt gag att 354 Thr Lys Ser Leu Leu Cys Met Pro Ile Arg Ser Ser Asp Gly Glu Ile 70 75 80 85 att ggt gtg gcc caa gcg ata aat aag att cct gaa gga gct cca ttt 402 Ile Gly Val Ala Gln Ala Ile Asn Lys Ile Pro Glu Gly Ala Pro Phe 90 95 100 act gaa gat gat gaa aaa gtt atg cag atg tat ctt cca ttt tgt gga 450 Thr Glu Asp Asp Glu Lys Val Met Gln Met Tyr Leu Pro Phe Cys Gly 105 110 115 atc gcc ata tct aac gct cag ctc ttt gct gcc tca agg aaa gaa tat 498 Ile Ala Ile Ser Asn Ala Gln Leu Phe Ala Ala Ser Arg Lys Glu Tyr 120 125 130 gaa aga agc aga gct ttg cta gag gtg gtt aat gac ctc ttt gaa gaa 546 Glu Arg Ser Ar Leu Glu Val Val Asn Asp Leu Phe Glu Glu 135 140 145 cag act gac ctg gag aaa att gtc aag aaa ata atg cat cgg gcc caa 594 Gln Thr Asp Leu Glu Lys Ile Val Lys Lys Ile Met His Arg Ala Gln 150 155 160 165 act ctg ctg aaa tgt gag cgc tgt tct gtt tta ctc cta gag gac atc 642 Thr Leu Leu Lys Cys Glu Arg Cys Ser Val Leu Leu Leu Glu Asp Ile 170 175 180 gaa tca cca gtg gtg aaa ttt acc aaa tcc tttgaa tcc cca 690 Glu Ser Pro Val Val Lys Phe Thr Lys Ser Phe Glu Leu Met Ser Pro 185 190 195 aag tgc agt gct gat gct gag aac agt ttc aaa gaa agc atg gag aaa 738 Lys Cys Ser Ala Asp Ala Glu Asn Ser Phe Lys Gl u Ser Met Glu Lys 200 205 210 tca tca tac tcc gac tgg cta ata aat aac agc att gct gag ctg gtt 786 Ser Ser Tyr Ser Asp Trp Leu Ile Asn Asn Ser Ile Ala Glu Leu Val 215 220 225 gct tca aca ggc ctt cca gtg aac atc agt gat gcc tac cag gat ccg 834 Ala Ser Thr Gly Leu Pro Val Asn Ile Ser Asp Ala Tyr Gln Asp Pro 230 235 240 245 cgc ttt gat gca gag gca gac cag ata tct ggt ttt cac ata aga tct 882 Arg Phe Asp Ala Glu Ala Asp Gln Ile Ser Gly Phe His Ile Arg Ser 250 255 260 gtt ctt tgt gtc cct att tgg aat agc aac cac caa ata att gga gtg 930 Val Leu Cys Val Pro Ile Trp Asn Ser Asn His Gln Ile Ile Gly Val 265 270 275 gct caa gtg tta aac aga ctt gat ggg aaa cct ttt gat gat gca gat 978 Ala Gln Val Leu Asn Arg Leu Asp Gly Lys Pro Phe Asp Asp Ala Asp 280 285 290 290 caa cga ctt ttt gag gct ttt gtc atc ttt gga ctt ggc atc aac 1026 Gln Arg Leu Phe Glu Ala Phe Val Ile Phe Cys Gly Leu Gly Ile Asn 295 300 305 aac aca att atg tat gat caa gtg aag aag tcc tgg gcc aag cag tct 1074 Asn Thr Ile Met Tyr Asp GlnLys Lys Ser Trp Ala Lys Gln Ser 310 315 320 325 gtg gct ctt gat gtg cta tca tac cat gca aca tgt tca aaa gct gaa 1122 Val Ala Leu Asp Val Leu Ser Tyr His Ala Thr Cys Ser Lys Ala Glu 330 335 340 340 gtt gac aag ttt aag gca gcc aac atc cct ctg gtg tca gaa ctt gcc 1170 Val Asp Lys Phe Lys Ala Ala Asn Ile Pro Leu Val Ser Glu Leu Ala 345 350 355 atc gat gac att cat ttt gat gac ttt tct ctc gac gtt gat gcc att 1218 Ile Asp Asp Ile His Phe Asp Asp Phe Ser Leu Asp Val Asp Ala Met 360 365 370 atc aca gct gct ctc cgg atg ttc atg gag ctg ggg atg gta cag aaa 1266 Ile Thr Ala Ala Leu Arg Met Phe Met Glu Leu Gly Met Val Gln Lys 375 380 385 ttt aaa att gac tat gag aca ctg tgt agg tgg ctt ttg aca gtg agg 1314 Phe Lys Ile Asp Tyr Glu Thr Leu Cys Arg Trp Leu Leu Thr Val Arg 390 395 400 405 aaa aac tat cgg atg gtt tac cac aac tgg aga cat gcc ttc aac 1362 Lys Asn Tyr Arg Met Val Leu Tyr His Asn Trp Arg His Ala Phe Asn 410 415 420 gtg tgt cag ctg atg ttc gcg atg tta acc act gct ggg ttt caa gac 1410 Val Cys G ln Leu Met Phe Ala Met Leu Thr Thr Ala Gly Phe Gln Asp 425 430 435 att ctg acc gag gtg gaa att tta gcg gtg att gtg gga tgc ctg tgt 1458 Ile Leu Thr Glu Val Glu Ile Leu Ala Val Ile Val Gly Cys 440 445 450 cat gac ctc gac cac agg gga acc aac aat gcc ttc caa gct aag agt 1506 His Asp Leu Asp His Arg Gly Thr Asn Asn Ala Phe Gln Ala Lys Ser 455 460 465 ggc tct gcc ctg gcc caa ctc tat gga acct gct acc ttg gag cat 1554 Gly Ser Ala Leu Ala Gln Leu Tyr Gly Thr Ser Ala Thr Leu Glu His 470 475 480 485 cac cat ttc aac cac gcc gtg atg atc ctt caa agt gag ggt cac aat 1602 His His Phe Asn His Ala Val Met Ile Leu Gln Ser Glu Gly His Asn 490 495 500 atc ttt gct aac ctg tcc tcc aag gaa tat agt gac ctt atg cag ctt 1650 Ile Phe Ala Asn Leu Ser Ser Lys Glu Tyr Ser Asp Leu Met Gln Leu 505 510 515 ttg aag cag tca ata ttg gca aca gac ctc acg ctg tac ttt gag agg 1698 Leu Lys Gln Ser Ile Leu Ala Thr Asp Leu Thr Leu Tyr Phe Glu Arg 520 525 530 aga act gaa ttc ttt gaa ctt gtc agt aaa gga gaa tac aac 1746 Arg Thr Glu Phe Phe Glu Leu Val Ser Lys Gly Glu Tyr Asp Trp Asn 535 540 545 atc aaa aac cat cgt gat ata ttt cga tca atg tta atg aca gcc tgt 1794 Ile Lys Asn His Arg Asp Ile Phe Arg Ser Met Leu Met Thr Ala Cys 550 555 560 565 gac ctt gga gcc gtg acc aaa ccg tgg gag atc tcc aga cag gtg gca 1842 Asp Leu Gly Ala Val Thr Lys Pro Trp Glu Ile Ser Arg Gln Val Ala 570 575 580 580 gaa ctt gta acc agt gag ttc ttc gaa caa gga gat cgg gag aga tta 1890 Glu Leu Val Thr Ser Glu Phe Phe Glu Gln Gly Asp Arg Glu Arg Leu 585 590 595 gag ctc aaa ctc act cct tca gca att ttt gat cgg aac cgg aag gat 1938 Glu Leu Thr Pro Ser Ala Ile Phe Asp Arg Asn Arg Lys Asp 600 605 610 gaa ctg cct cgg ttg caa ctg gag tgg att gat agc atc tgc atg cct 1986 Glu Leu Pro Arg Leu Gln Leu Glu Trp Ile Asp Ser Ile Cys Met Pro 620 625 ttg tat cag gca ctg gtg aag gtc aac gtg aaa ctg aag ccg atg cta 2034 Leu Tyr Gln Ala Leu Val Lys Val Asn Val Lys Leu Lys Pro Met Leu 630 635 640 645 gat tca gta gct aca aac aga agt tgg gaa gag cta cac caa aaa 2082 Asp Ser Val Ala Thr Asn Arg Ser Lys Trp Glu Glu Leu His Gln Lys 650 655 660 cga ctg ctg gcc tca act gcc tca tcc tcc tcc cct gcc agt gtt atg 2130 Arg Leu Leu Ala Ser Thr Ala Ser Ser Ser Ser Pro Ala Ser Val Met 665 670 675 gta gcc aag gaa gac agg aac taaacctcca ggtcagctgc agctgcaaaa 2181 Val Ala Lys Glu Asp Arg Asn 680 tgactacagc ctgaagggcc attttcagtc cagcaatgtc atccttttgt tcttttagct 2241 cagaaagacc taacatctca aggatgcact gggaaccatg cctgggcttt caccttgaag 2301 catggtcagc agcagagaga gcaacgggaa ggacaaagaa agaggtgggg cagggagcac 2361 accccaggac cctcactttt ccctaatgaa cacgcatggg ctgaaatgaa ggctctgggt 2421 aggggactgt tttggatcca aggacctgtg gacagtcggc ctacttactc tgagctgagg 2481 gaacactgaa cagtaaaagc gtcattagcg ctgcttcatt ttgtataggg cttttctgtt 2541 tgttacaagc caaacattgc ctgtctttgc ttcccgtccc tgaatgcctt tttgtgccag 2601 actgtcccaa gaatcctaat ttgtattcca tagaggtatt ttatttttaa tcctagagct 2661 tcttattgat ggatccttta gaattgccta cctaaaaggt aaactatact atccttataa 2721 atactgatc a atcccagttc tccccctaaa aatgaataca tagtaggact atagcaaatg 2781 tgtttgatgg gtaattctag actgggacta tggtaccctt ttccagagtt ttaaaattca 2841 accttcatta cagacaaagt tttctcccag aaggaatgga ttgatagatt ttgattaaag 2901 taagggtgga aggaaatctg tagctggatt taccacaagt gacatctaga aactatagtt 2961 cacaggacag agcagagcca tggagaataa gcattgacta ccttgagttc tcctagtgag 3021 gagttctggt ataaaattta agattactac cagtaaccaa cttaaagcaa actatagggg 3081 tccctaattt tggatttttc cttaagtgta agaaacaatg cttcaaatgt taagaaataa 3141 cagtctgggc aaagaacgca tattctatag gaagccaggt ttacaatagg taagaataaa 3201 ctgtattaag tagatgtaat gactagaaag ctgctttgct ccctatattg agaaattgtg 3261 gacatggtat gtgttatcca aagaacattg ggctagaaga tagatttcta tccttagctt 3321 tggcattatt gactggattg acttgaacaa gtcgcttaac ttctacaagc ttgtttcctt 3381 atttgtcaaa ttagattaca ctaggaaacg attctcgaac atgttttaac cttacaactc 3441 tttgttcaaa taaatctttc aatgaatccc caacataaaa aaaaaaaaaa aaaaaaaaaa 3501 aaaaaa 3507 <210> 4 <211> 684 <212> PRT <213> Homo sapiens <400> 4 Met Leu Lys Gln Ala Arg Arg Pro Leu Phe Arg Asn Val Leu Ser Ala 1 5 10 15 Thr Gln Trp Lys Lys Val Lys Ile Thr Arg Leu Val Gln Ile Ser Gly 20 25 30 Ala Ser Leu Ala Glu Lys Gln Glu Lys His Gln Asp Phe Leu Ile Gln 35 40 45 Arg Gln Thr Lys Thr Lys Asp Arg Arg Phe Asn Asp Glu Ile Asp Lys 50 55 60 Leu Thr Gly Tyr Lys Thr Lys Ser Leu Leu Cys Met Pro Ile Arg Ser 65 70 75 80 Ser Asp Gly Glu Ile Ile Gly Val Ala Gln Ala Ile Asn Lys Ile Pro 85 90 95 Glu Gly Ala Pro Phe Thr Glu Asp Asp Glu Lys Val Met Gln Met Tyr 100 105 110 Leu Pro Phe Cys Gly Ile Ala Ile Ser Asn Ala Gln Leu Phe Ala Ala 115 120 125 Ser Arg Lys Glu Tyr Glu Arg Ser Arg Ala Leu Leu Glu Val Val Asn 130 135 140 Asp Leu Phe Glu Glu Gln Thr Asp Leu Glu Lys Ile Val Lys Lys Ile 145 150 155 160 Met His Arg Ala Gln Thr Leu Leu Lys Cys Glu Arg Cys Ser Val Leu 165 170 175 Leu Leu Glu Asp Ile Glu Ser Pro Val Val Lys Phe Thr Lys Ser Phe 180 185 190 Glu Leu Met Ser Pro Lys Cys Ser Ala Asp Ala Glu Asn Ser Phe Lys 195 200 205 Glu Ser Met G lu Lys Ser Ser Tyr Ser Asp Trp Leu Ile Asn Asn Ser 210 215 220 Ile Ala Glu Leu Val Ala Ser Thr Gly Leu Pro Val Asn Ile Ser Asp 225 230 235 240 Ala Tyr Gln Asp Pro Arg Phe Asp Ala Glu Ala Asp Gln Ile Ser Gly 245 250 255 Phe His Ile Arg Ser Val Leu Cys Val Pro Ile Trp Asn Ser Asn His 260 265 270 Gln Ile Ile Gly Val Ala Gln Val Leu Asn Arg Leu Asp Gly Lys Pro 275 280 285 Phe Asp Asp Ala Asp Gln Arg Leu Phe Glu Ala Phe Val Ile Phe Cys 290 295 300 Gly Leu Gly Ile Asn Asn Thr Ile Met Tyr Asp Gln Val Lys Lys Ser 305 310 315 320 Trp Ala Lys Gln Ser Val Ala Leu Asp Val Leu Ser Tyr His Ala Thr 325 330 335 Cys Ser Lys Ala Glu Val Asp Lys Phe Lys Ala Ala Asn Ile Pro Leu 340 345 350 Val Ser Glu Leu Ala Ile Asp Asp Ile His Phe Asp Asp Phe Ser Leu 355 360 365 Asp Val Asp Ala Met Ile Thr Ala Ala Leu Arg Met Phe Met Glu Leu 370 375 380 Gly Met Val Gln Lys Phe Lys Ile Asp Tyr Glu Thr Leu Cys Arg Trp 385 390 395 400 Leu Leu Thr Val Arg Lys Asn Tyr Arg Met Val Leu Tyr His Asn Trp 405 410 415 Arg His Ala P he Asn Val Cys Gln Leu Met Phe Ala Met Leu Thr Thr 420 425 430 Ala Gly Phe Gln Asp Ile Leu Thr Glu Val Glu Ile Leu Ala Val Ile 435 440 445 Val Gly Cys Leu Cys His Asp Leu Asp His Arg Gly Thr Asn Asn Ala 450 455 460 Phe Gln Ala Lys Ser Gly Ser Ala Leu Ala Gln Leu Tyr Gly Thr Ser 465 470 475 480 Ala Thr Leu Glu His His His Phe Asn His Ala Val Met Ile Leu Gln 485 490 495 Ser Glu Gly His Asn Ile Phe Ala Asn Leu Ser Ser Lys Glu Tyr Ser 500 505 510 510 Asp Leu Met Gln Leu Leu Lys Gln Ser Ile Leu Ala Thr Asp Leu Thr 515 520 525 Leu Tyr Phe Glu Arg Arg Thr Glu Phe Phe Glu Leu Val Ser Lys Gly 530 535 540 Glu Tyr Asp Trp Asn Ile Lys Asn His Arg Asp Ile Phe Arg Ser Met 545 550 555 560 Leu Met Thr Ala Cys Asp Leu Gly Ala Val Thr Lys Pro Trp Glu Ile 565 570 575 Ser Arg Gln Val Ala Glu Leu Val Thr Ser Glu Phe Phe Glu Gln Gly 580 585 590 Asp Arg Glu Arg Leu Glu Leu Lys Leu Thr Pro Ser Ala Ile Phe Asp 595 600 605 Arg Asn Arg Lys Asp Glu Leu Pro Arg Leu Gln Leu Glu Trp Ile Asp 610 615 620 Ser Ile Cys Met P ro Leu Tyr Gln Ala Leu Val Lys Val Asn Val Lys 625 630 635 640 Leu Lys Pro Met Leu Asp Ser Val Ala Thr Asn Arg Ser Lys Trp Glu 645 650 655 Glu Leu His Gln Lys Arg Leu Leu Ala Ser Thr Ala Ser Ser Ser Ser 660 665 670 Pro Ala Ser Val Met Val Ala Lys Glu Asp Arg Asn 675 680 <210> 5 <211> 3492 <212> DNA <213> Rat <220> <221> CDS <222> (241) .. (2295) <400> 5 gcggggagcg aggatggagc ccgccatcag ctggtgcatg atccggcctg taagcggagt 60 cctcgggcac agcagtcgcg ccttaaatct tggcctcact gcatcagtgg accaggggtg 120 gctcagggca gcgctgctgt gatcgtgaaa acaaagaata gggaaaccaa gtccaggttt 180 gcaaatctga gacttttttt ggttggtgat ttaaggtttt attgccgagc aaaatgggag 240 atg ctg aag cag gca aga aga ttt tca ttc aga aat gtg cgc agt gcc 288 Met Leu Lys Gln Ala Arg Arg Phe Ser Phe Arg Asn Val Arg Ser Ala 1 5 10 15 aca cag tgg aga aag gtg gga agc aca aga cag ggc caa atc tct ggg 336 Thr Gln Trp Arg Lys Val Gly Ser Thrr Arg Gln Gly Gln Ile Ser Gly 20 25 30 gcc ttt ttg gcc gaa aga ctg gac aag cac cag gat ttt ctt aca cgg 384 Ala Phe Leu Ala Glu Arg Leu Asp Lys His Gln Asp Phe Leu Thr Arg 35 40 45 atg caa aca aga aca aag gac cga aga ttc aat gat gaa att gac aag 432 Met Gln Thr Arg Thr Lys Asp Arg Arg Phe Asn Asp Glu Ile Asp Lys 50 55 60 ctg act gga tac aag aca aaa tca cta ttg tgc atg cct atc cgg aac 480 Leu Thr Gly Tyr Lys Thr Lys Ser Leu Leu Cys Met Pro Ile Arg Asn 65 70 75 80 agt gac ggt gag att atc ggt gtg gcc cag gcg ata aat aag gtt cct 528 Ser Asp Gly Glu Ile Ile Gly Val Ala Gln Ala Ile Asn Lys Val Pro 85 90 95 gag ggt gct cca ttt aca gaa gac gac gaa aaa gtt atg cag atg tat 576 Glu Gly Ala Pro Phe Thr Glu Asp Asp Glu Lys Val Met Gln Met Tyr 100 105 110 ctt cca ttc tgt ggc atc gcc ata tct aat gct cag ctc ttc gcc gcc 624 Leu Pro Phe Cys Gly Ile Ala Ile Ser Asn Ala Gln Leu Phe Ala Ala 115 120 125 tcg agg aaa gaa tat gaa aga agt agg gcc ttg ctg gag gtg gtc aat 672 Ser Arg Lys Glu Tyr Glu Arg Ser Arg Ala Leu Leu Glu Val Val Asn 130 135 140 gac ctc ttt gaa gag gac ctg gaa aag att gtc aag aaa ata 720 Asp Leu Phe Glu Glu Gln Thr Asp Leu Glu Lys Ile Val Lys Lys Ile 145 150 155 160 atg cat cgg gcc caa act ctg ttg aaa tgt gaa cgc tgt tcc gtt tta Met His 768 Ala Gln Thr Leu Leu Lys Cys Glu Arg Cys Ser Val Leu 165 170 175 ctt cta gaa gac att gaa tca cca gtg gtg aag ttt acc aaa tcc ttt 816 Leu Leu Glu Asp Ile Glu Ser Pro Val Val Lys Phe Thr Lys Ser Phe 180 185 190 gaa ctg atg tcc cca aag tgc agt gcg gac gcg gag aac agt ttc aaa 864 Glu Leu Met Ser Pro Lys Cys Ser Ala Asp Ala Glu Asn Ser Phe Lys 195 200 205 gaa agt gtg gag aag tca tct tac tcc gac tgg aat aac agt 912 Glu Ser Val Glu Lys Ser Ser Tyr Ser Asp Trp Leu Ile Asn Asn Ser 210 215 220 atc gct gag ctg gtt gct tcg aca ggc ctt cct gtg aat gtc agc gat 960 Ile Ala Glu Leu Val Ala Ser Thr Gly Leu Pro Val Asn Val Ser Asp 225 230 235 240 gcc tac cag gac cca cgc ttt gac gct gag gct gac cag ata tct ggc 1008 Ala Tyr Gln Asp Pro Arg Phe Asp Ala Glu Ala Asp Gln Ile Ser Gly 245 250 255 ttt cat ata aga tct gtt ctc tgt gtc cct att tgg aac agc aac cac 1056 Phe His Ile Arg Ser Val Leu Cys Val Pro Ile Trp Asn Ser Asn His 260 265 270 caa ata ata ggg gtc gct caa gtg ctg aac aga ctc gat ggg aaa cct 1104 Ile Ile Gly Val Ala Gln Val Leu Asn Arg Leu Asp Gly Lys Pro 275 280 285 ttt gat gat gct gac caa agg ctt ttt gag gcc ttt gtc atc ttt tgt 1152 Phe Asp Asp Ala Asp Gln Arg Leu Phe Glu Ala Phe Val Ile Phe Cys 290 295 300 ggc ctt ggt att aac aac acg att atg tat gac caa gtg aag aag tcc 1200 Gly Leu Gly Ile Asn Asn Thr Ile Met Tyr Asp Gln Val Lys Lys Ser 305 310 315 320 tgg gcc aag cag tcc gtg gct ctt gat gtg ctg tcc tac cac gcc acg 1248 Trp Ala Lys Gln Ser Val Ala Leu Asp Val Leu Ser Tyr His Ala Thr 325 330 335 tgt tcc aag gct gaa gtt gac aag ttt aag gca gcc aac atc ccc ctg 1296 Cys Ser Lys Ala Glu Val Asp Lys Phe Lys Ala Ala Asn Ile Pro Leu 340 345 350 gtg tcg gaa ctg gcc atc gat gac atc cat ttt gat gac ttt tcc ctt 1344 Val Ser Glu Leu Ala Ile Asp Asp Ile His Phe Asp Asp Phe Ser Leu 355 360 365 gat gtt gat gcc atg atc aca gcc gct cta cgg atg ttc atg gag ctg 1392 Asp Val Asp Ala Met Ile Thr Ala Ala Leu Arg Met Phe Met Glu Leu 370 375 380 ggg atg gta cag aaa ttt aaa atc gac tat gg acc ct g agg tgg 1440 Gly Met Val Gln Lys Phe Lys Ile Asp Tyr Glu Thr Leu Cys Arg Trp 385 390 395 400 ctt ctg aca gta agg aaa aac tat cgg atg gtt ctc tac cac aac tgg 1488 Leu Leu Thr Val Arg Lys Asn Tyr Arg Met Val Leu Tyr His Asn Trp 405 410 415 aga cat gcc ttc aac gtg tgc cag ctg atg ttt gcc atg cta act act 1536 Arg His Ala Phe Asn Val Cys Gln Leu Met Phe Ala Met Leu Thr Thr 420 425 430 430 gct ggg ttt caa gag att ctg acc gag gtg gaa att tta gcg gtg att 1584 Ala Gly Phe Gln Glu Ile Leu Thr Glu Val Glu Ile Leu Ala Val Ile 435 440 445 gtg gga tgc ctg tgt cat gac ctc gac cac agg gga acc32 Val Gly Cys Leu Cys His Asp Leu Asp His Arg Gly Thr Asn Asn Ala 450 455 460 ttc caa gct aag agt gac tct gca ctg gcc cag ctc tat ggg acc tca 1680 Phe Gln Ala Lys Ser Asp Ser Ala Leu Ala Gln Leu Tyr Gly Thr Ser 465 470 475 480 gcg acc tta gag cat cac cac ttt aac cac gcc gtg atg atc ctt cag 1728 Ala Thr Leu Glu His His His Phe Asn His Ala Val Met Ile Leu Gln 485 490 495 agt gag ggt cac aac atc ttt gct aat ttg tcc tcc aag gaa tac agc 1776 Ser Glu Gly His Asn Ile Phe Ala Asn Leu Ser Ser Lys Glu Tyr Ser 500 505 510 gac ctc atg cag ctc ctg aag cag tcg ata cta gcc act gac ctc acg 1824 Asp Leu Met ln Leu Leu Lys Gln Ser Ile Leu Ala Thr Asp Leu Thr 515 520 525 ctg tac ttc gag aga aga act gag ttc ttc gag ctt gtc agt aaa gga 1872 Leu Tyr Phe Glu Arg Arg Thr Glu Phe Phe Glu Leu Val Ser Lys Gly 535 540 gcc tat gat tgg agc atc aca agt cac cgc gat gtg ttt cga tca atg 1920 Ala Tyr Asp Trp Ser Ile Thr Ser His Arg Asp Val Phe Arg Ser Met 545 550 555 560 tta tta atg aca gct tgt gac ctt gga gcc gtg acc aaa ccg tgg gag atc 1968 Leu Met Thr Ala Cys Asp Leu Gly Ala Val Thr Lys Pro Trp Glu Ile 565 570 575 tcc aga cag gtg gct gaa ctt gtc acc agc gag ttc ttc gaa caa gga 2016 Ser Arg Gln Val Ala Glu Le Thr Ser Glu Phe Phe Glu Gln Gly 580 585 590 gat cgg gag agg tcg gaa ctc aag ctc acc ccc tct gct att ttt gac 2064 Asp Arg Glu Arg Ser Glu Leu Lys Leu Thr Pro Ser Ala Ile Phe Asp 595 600 605 cgg aac cgg aaa gat gag ctg cct cgg ctg caa ctg gag tgg att gac 2112 Arg Asn Arg Lys Asp Glu Leu Pro Arg Leu Gln Leu Glu Trp Ile Asp 610 615 620 agc atc tgc atg cct ttg tat cag gcc ttg gtg aaa 2160 Ser Ile Cys Met Pro Leu Tyr Gln Ala Leu Val Lys Val Asn Ala Lys 625 630 635 640 ctg aag ccg atg ctg gac tca gtg gcc gcc aac cgc agg aag tgg gaa 2208 Leu Lys Pro Met Leu Asp Ser Val Ala Ala Asn Arg Lys Trp Glu 645 650 655 gag ttg cac caa aaa aga cta cag gtc tct gct gcc tcc cca gtc cct 2256 Glu Leu His Gln Lys Arg Leu Gln Val Ser Ala Ala Ser Pro Val Pro 660 665 670 tcc agt ccc agc cca gg gc gcc gga gag gac aga ctg taaaccaccc 2305 Ser Ser Pro Ser Pro Ala Val Ala Gly Glu Asp Arg Leu 675 680 685 agagctgctg cgccaccctg tggcctggag gaccctctgc atcctgaact atctgctttg 2365 gttgaatagc atcaccctct tcgttcagct cggacagtcc taaagctttg actggatcag 2425 gaagcacaca aggaagtgtg cttggcggca gagaaacgga aggatgaaga gaatacgacc 2485 ctcaactttg tcatgaaccc atgctgctgg atttggatcc gtggacctga aggccatcac 2545 cccactcgtg ctgagttgag aggacaccgt tgtaaaagtg tcactatggc tgcttcctgt 2605 atggcacttt tctgttatga gcatttcctt tctgtgcagc ctctccggag caggctccag 2665 ccggtcccaa gaatgcgat gtatcgtatc gtatgtgtt tt 2725 gtaatgcacc agacccttcg tctcctttgc tagtggataa gcctgctccc tttattagag 2785 ccaggtaacc ccaatcttgc ttgccacagt agtaatatag tgtgtttgtc taattggtgt 2845 tacacaggag ctgtaactct gcagtgagat agatcatggt tcagcttttt aattcagcct 2905 ttgttgcaaa caaagccttt ttctaggggg aaacagatta ataattttat taaagcaaag 2965 gtgaaaagga atctaaggcc acatgtacaa gtgacatgta tgaacaccag aaatgcttcc 3025 aacaaagaaa tatttcacag gtgcagagga accctgtgac cagtcgtgga atagtttgag 3085 gtgaagggga tgaaggtttg gggataaaat ttaaggaacc ctgattttac atttttcctt 3145 acatgtaaaa aagaatgttc tagaaaggcc aggaaataac acctgcacaa cctatatcca 3205 gtaagaagtc agatctacag cacgtgaaaa cagttttaaa gaggtatgct cactagaaag 3265 ctgcctgaga cactaaggac aggatctgtc ttacccaaag aacattgggc ttcctgtccc 3325 tcactttgca ttactagttg agtgagtttg gacctagatt tctctgtgtc aagcgagatt 3385 acactaggaa tttatttgca gtaatcttag cattaaaatc ttttgtccaa ataaacttcc 3445 aaggattccc cagggcacaa ggaaaaaaaa aaaaaaaaaa aaaaaaa 3492 <210> 6 <211> 685 <212> PRT <213> Rat <400> 6 Met Leu Lys Gln Ala Arg Arg Phe Ser Phe Arg Asn Val Arg Ser Ala 1 5 10 15 Thr Gln Trp Arg Lys Val Gly Ser Thr Arg Gln Gly Gln Ile Ser Gly 20 25 30 Ala Phe Leu Ala Glu Arg Leu Asp Lys His Gln Asp Phe Leu Thr Arg 35 40 45 Met Gln Thr Arg Thr Lys Asp Arg Arg Phe Asn Asp Glu Ile Asp Lys 50 55 60 Leu Thr Gly Tyr Lys Thr Lys Ser Leu Leu Cys Met Pro Ile Arg Asn 65 70 75 80 Ser Asp Gly Glu Ile Ile Gly Val Ala Gln Ala Ile Asn Lys Val Pro 85 90 95 Glu Gly Ala Pro Phe Thr Glu Asp Asp Glu Lys Val Met Gln Met Tyr 100 105 110 Leu Pro Phe Cys Gly Ile Ala Ile Ser Asn Ala Gln Leu Phe Ala Ala 115 120 125 Ser Arg Lys Glu Tyr Glu Arg Ser Arg Ala Leu Leu Glu Val Val Asn 130 135 140 Asp Leu Phe Glu Glu Gln Thr Asp Leu Glu Lys Ile Val Lys Lys Ile 145 150 155 160 Met His Arg Ala Gln Thr Leu Leu Lys Cys Glu Arg Cys Ser Val Leu 165 170 175 Leu Leu Glu Asp Ile Glu Ser Pro Val Val Lys Phe Thr Lys Ser Phe 180 185 190 Glu Leu Met Ser Pro Lys Cys Ser Ala Asp Ala Glu Asn Ser Phe Lys 195 200 205 Glu Ser Val Gl u Lys Ser Ser Tyr Ser Asp Trp Leu Ile Asn Asn Ser 210 215 220 Ile Ala Glu Leu Val Ala Ser Thr Gly Leu Pro Val Asn Val Ser Asp 225 230 235 240 Ala Tyr Gln Asp Pro Arg Phe Asp Ala Glu Ala Asp Gln Ile Ser Gly 245 250 255 Phe His Ile Arg Ser Val Leu Cys Val Pro Ile Trp Asn Ser Asn His 260 265 270 Gln Ile Ile Gly Val Ala Gln Val Leu Asn Arg Leu Asp Gly Lys Pro 275 280 285 Phe Asp Asp Ala Asp Gln Arg Leu Phe Glu Ala Phe Val Ile Phe Cys 290 295 300 Gly Leu Gly Ile Asn Asn Thr Ile Met Tyr Asp Gln Val Lys Lys Ser 305 310 315 320 Trp Ala Lys Gln Ser Val Ala Leu Asp Val Leu Ser Tyr His Ala Thr 325 330 335 Cys Ser Lys Ala Glu Val Asp Lys Phe Lys Ala Ala Asn Ile Pro Leu 340 345 350 Val Ser Glu Leu Ala Ile Asp Asp Ile His Phe Asp Asp Phe Ser Leu 355 360 365 Asp Val Asp Ala Met Ile Thr Ala Ala Leu Arg Met Phe Met Glu Leu 370 375 380 Gly Met Val Gln Lys Phe Lys Ile Asp Tyr Glu Thr Leu Cys Arg Trp 385 390 395 400 Leu Leu Thr Val Arg Lys Asn Tyr Arg Met Val Leu Tyr His Asn Trp 405 410 415 Arg His Ala Ph e Asn Val Cys Gln Leu Met Phe Ala Met Leu Thr Thr 420 425 430 Ala Gly Phe Gln Glu Ile Leu Thr Glu Val Glu Ile Leu Ala Val Ile 435 440 445 Val Gly Cys Leu Cys His Asp Leu Asp His Arg Gly Thr Asn Asn Ala 450 455 460 Phe Gln Ala Lys Ser Asp Ser Ala Leu Ala Gln Leu Tyr Gly Thr Ser 465 470 475 480 Ala Thr Leu Glu His His His Phe Asn His Ala Val Met Ile Leu Gln 485 490 495 Ser Glu Gly His Asn Ile Phe Ala Asn Leu Ser Ser Lys Glu Tyr Ser 500 505 510 510 Asp Leu Met Gln Leu Leu Lys Gln Ser Ile Leu Ala Thr Asp Leu Thr 515 520 525 Leu Tyr Phe Glu Arg Arg Thr Glu Phe Phe Glu Leu Val Ser Lys Gly 530 535 540 Ala Tyr Asp Trp Ser Ile Thr Ser His Arg Asp Val Phe Arg Ser Met 545 550 555 560 Leu Met Thr Ala Cys Asp Leu Gly Ala Val Thr Lys Pro Trp Glu Ile 565 570 575 Ser Arg Gln Val Ala Glu Leu Val Thr Ser Glu Phe Phe Glu Gln Gly 580 585 590 Asp Arg Glu Arg Ser Glu Leu Lys Leu Thr Pro Ser Ala Ile Phe Asp 595 600 605 Arg Asn Arg Lys Asp Glu Leu Pro Arg Leu Gln Leu Glu Trp Ile Asp 610 615 620 620 Ser Ile Cys Met P ro Leu Tyr Gln Ala Leu Val Lys Val Asn Ala Lys 625 630 635 640 Leu Lys Pro Met Leu Asp Ser Val Ala Ala Asn Arg Arg Lys Trp Glu 645 650 655 Glu Leu His Gln Lys Arg Leu Gln Val Ser Ala Ala Ser Pro Val Pro 660 665 670 Ser Ser Pro Ser Pro Ala Val Ala Gly Glu Asp Arg Leu 675 680 685 <210> 7 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 7 caygaystgg aycayag 17 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 8 cakgtcwgtk gcyaaka 17 <210> 9 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 9 ctgcttcaaa agctg 15 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 10 atgatccttc aaagtgaggg tcac 24 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 11 ggtagcagag gttccataga gttg 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 12 ggtcacaata tctttgctaa cctg 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 13 cttagcttgg aaggcattgt tggt 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 14 tctgacacca gagggatgtt ggct 24 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 15 gtcagtctgt tcttcaaaga ggtc 24 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 16 accaacaatg ccttccaagc taag 24 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 17 ttccaagcta agagtggctc tgcc 24 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 18 tggcgctgaa ctgggaatac tggtg 25 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 19 tcaggctgta gtcattttgc agc 23 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 20 tgaggcagca aagagctgag cgtt 24 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 21 agcgttagat atggcgattc caca 24 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 22 tgtcttgtat ccagttagct tgtc 24 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 23 gcgcttgcag cccagggc 18 <210> 24 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 24 ggatccatgg cagcctcc 18 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 25 ccttagctct ttctgagaag ctc 23 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 26 ggatccatgc tgaagcaggc aag 23 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 27 ttcatcatct tcagtaaatg g 21 <210> 28 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 28 ataatgcatc gggcccaaac tctg 24 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 29 ctgtctggag atctcccacg g 21 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 30 gtcgacttca gccttggaac acgt 24 <210> 31 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 31 atcgctgaca ttcacaggaa ggcc 24 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 32 attgaccacc tccagcaagg c 21 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 33 agcattagat atggcgatgc c 21 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 34 tcgatcaatg ttaatgacag c 21 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 35 tgtgaccttg gagccgtgac c 21 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 36 atcttggcct cactgcatca gtgg 24 <210> 37 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Artificially Synthesized Primer Sequence <400> 37 agcagatagt tcaggatgca gagg 24.

[Brief description of the drawings]

FIG. 1. cAMP or cG by human PDE11A1
Kinetic analysis results of MP hydrolysis reaction (Lineweaver-Bur
k plot).

FIG. 2. cAMP or cG by human PDE11A2
Kinetic analysis results of MP hydrolysis reaction (Lineweaver-Bur
k plot).

FIG. 3. Human PDE11A1 and human PDE11A2
FIG. 3 is a graph showing the results of examining the effect of cGMP on cAMP degradation activity and the effect of cAMP on cGMP degradation activity. The cAMP degradation activity when no cGMP was added or the cGMP degradation activity when no cAMP was added was 10
The relative value (%) of the activity was shown as 0%.

FIG. 4 shows the expression of PDE11 gene in human tissues (the results of dot blot).

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C12N 5/10 C12Q 1/44 4H045 9/16 1/68 A C12Q 1/44 G01N 33/15 Z 1/68 33 / 50Z G01N 33/15 33/573 A 33/50 C12N 15/00 ZNAA 33/573 5/00 A (72) Inventor Kotomi Fujishige 1-41-8 Akabane, Kita-ku, Tokyo Maison de Clarte 403 (72) Invention Person Hideo Michibata 2-3-8 Kawagishi, Toda City, Saitama Prefecture Tanabe Seiyaku Toda Dormitory F-term (reference) 2G045 AA34 AA35 CB01 DA12 DA13 DA20 FB01 FB02 FB08 4B024 AA01 AA11 BA11 BA43 BA44 DA02 HA15 4B050 CC03 DD11 FF11Q06 QQ08 QQ33 QR42 QX07 4B065 AA90X AA91Y AA93Y AB01 BA02 CA25 CA31 CA44 CA46 4H045 AA10 AA11 CA40 DA75 DA89 EA26 FA74

Claims (22)

[Claims] 1. A type 11 phosphodiesterase. 2. The phosphodiesterase according to claim 1, which is a protein selected from the following (A) and (B): (A) a protein having an amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; (B) a protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, and A protein having hydrolytic activity. 3. The phosphodiesterase according to claim 1, which is a protein selected from the following (A) and (B): (A) a protein having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; (B) a protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, and an activity of hydrolyzing cyclic nucleotides A protein having 4. The phosphodiesterase according to claim 1, which is derived from human. 5. The phosphodiesterase according to claim 1, which is derived from a rat. 6. A gene or nucleic acid encoding the phosphodiesterase according to claim 1, 2 or 3. 7. A gene or nucleic acid selected from the following (a) and (b): (A) A gene or nucleic acid consisting of a DNA having the base sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. (B) a gene or nucleic acid consisting of a DNA that hybridizes under stringent conditions with a DNA having the base sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, and hydrolyzes cyclic nucleotides A gene or nucleic acid encoding a protein having activity. 8. A gene or nucleic acid selected from the following (a) and (b): (A) A gene or nucleic acid consisting of a DNA having the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. (B) a gene or nucleic acid consisting of a DNA that hybridizes under stringent conditions with a DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and a protein having an activity of hydrolyzing cyclic nucleotides A gene or nucleic acid encoding 9. The gene or nucleic acid according to claim 7, which is derived from human. 10. The gene or nucleic acid according to claim 7, which is derived from a rat. 11. A recombinant vector containing the gene or nucleic acid according to claim 6, 7, or 8. 12. The recombinant vector according to claim 11, which is an expression vector. A host cell into which the recombinant vector according to claim 12 has been introduced. 14. A step of carrying out an enzymatic reaction in a system containing the phosphodiesterase according to claim 1, an enzyme substrate and a test substance, and a step of assaying an inhibitory effect of the test substance on the enzyme activity of the phosphodiesterase according to claim 1. A method for characterizing, identifying or selecting a phosphodiesterase inhibitor, including: 15. The enzyme substrate is cAMP and cGMP.
The method according to claim 14, which is a cyclic nucleotide selected from the group consisting of: 16. The enzyme activity of a phosphodiesterase,
15. The method of claim 14, which is a cyclic nucleotide hydrolytic activity. 17. A step of performing a binding reaction in a system containing the phosphodiesterase according to claim 1 and a test substance, and a step of determining whether the test substance has a binding ability to the phosphodiesterase according to claim 1. For characterizing, identifying or selecting phosphodiesterase inhibitors. 18. The method according to claim 14 or 17, which is used to characterize, identify or select by the selectivity of an inhibitory effect on a plurality of types of phosphodiesterases. 19. A nucleic acid having a nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 which hybridizes under stringent conditions with a nucleic acid as a probe or primer. A method for detecting the described gene or nucleic acid. 20. A DNA having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, which is used for suppressing the expression of the gene according to claim 6, 7 or 8 in a cell. Nucleic acid that hybridizes under stringent conditions. 21. An antibody that recognizes the phosphodiesterase according to claim 1. 22. A method for detecting the expression of the phosphodiesterase of claim 1 in a cell or tissue using an antibody that recognizes the phosphodiesterase of claim 1.