WO2003031628A1

WO2003031628A1 - Secretory polypeptides having sphingosine kinase activity and sphingosine kinase genes encoding the same

Info

Publication number: WO2003031628A1
Application number: PCT/JP2001/008538
Authority: WO
Inventors: Yasuyuki Igarashi; Akio Kihara
Original assignee: Hokkaido Technology Licensing Office Co., Ltd.; Chemical Biology Institute
Priority date: 2001-09-28
Filing date: 2001-09-28
Publication date: 2003-04-17
Also published as: JPWO2003031628A1

Abstract

Secretory polypeptides having a sphingosine kinase activity. In particular, human-origin sphingosine kinase 3 (SPHK3) and a sphingosine kinase gene encoding the same.

Description

Satsuki Itoda 分泌 Secreted polypeptide with sphingosine kinase activity and sphingosine kinase gene encoding it

The present invention relates to a secreted polypeptide having sphingosine kinase activity (SPHK activity) and a sphingosine kinase gene encoding the same, and particularly to human-derived secreted sphingosine kinase 3 (SPHK3) and It relates to the sphingosine kinase gene encoding. Background art

Sphingosine kinase (SPHK) is an enzyme that phosphorylates sphingosine and catalyzes the production of sphingosine 1-phosphate, which is mainly present in platelets.

Sphingosine 1-phosphate, generated by phosphorylation of sphingosine, is accumulated in platelets in the blood and released with the activation of platelets. This sphingosine: I-phosphate is known to have various physiological and pathological roles such as promotion of platelet aggregation, wound healing of blood vessel walls, anticancer metastasis, and prevention of arteriosclerosis.

So far, sphingosine kinase has been known as sphingosine kinase 1 (SPHK1) (Olivera, A., et al., J. Biol. Chem. 273, 12576-125).

83. (1998), Kohama, T., et al., J. Biol. Chem. 273, 23722-23728.

(1998)) and sufingosine kinase 2 (SPHK2) (Hong L., et al.,

J. Biol. Chem. 275, 19513-19520. (2000)).

SPHK1 is a protein with a molecular weight of 43 kDa, and shows the strongest SPHK activity among conventionally known SPHKs. It is abundant in the cytoplasm and has high affinity for calmodulin. As a splicing variant, SPHK la

(Molecular weight: 42.3 kDa) and SPHK lb (molecular weight: 43.3 kDa) However, they do not have signal peptides and transmembrane regions. In addition, mRNA expression is strongly expressed in spleen and lung.

SPHK2 is a protein with a molecular weight of 65 kDa, and its SPHK activity is about 1/50 that of SPM1. Increases and inhibitions of activity were observed depending on the concentration of detergent, NaCl, and KC1. Dehydrosfingosine is a better substrate than sphingosine. In addition, mRNA expression is strongly expressed in kidney, liver and brain.

Both SPHK1 and SPHK2 are enzymes that catalyze the phosphorylation of sphingosine, but these have been shown to have intracellular effects, but have been found to produce extracellular sphingosine-1-phosphate. Not been. In other words, these enzymes were not enzymes responsible for the production of sphingosine-1-phosphate in blood, and were not suitable for drug development targeting SPHK in blood.

Therefore, an object of the present invention is to provide a novel sphingosine kinase that controls the production of sphingosine-1-phosphate present in blood different from SPHK1 and SPHK2. Day S

The present inventors conducted intensive research to solve the above-mentioned problems, and found a novel secretory sphingosine kinase 3 (SPHK3) different from SPHK1 and SPHK2 by homology analysis of SPHK1 based on EST data. The present invention has been completed.

That is, the present invention relates to a secretory polypeptide having sphingosine kinase activity.

The present invention also relates to the secretory polypeptide, which is derived from human.

Further, the present invention relates to the following polypeptide (a) or (b).

(a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1

(b) one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 A polypeptide having an amino acid sequence in which amino acids have been deleted, substituted or added, and having sphingosine kinase activity.

The present invention also relates to a sphingosine kinase gene encoding the above-mentioned polypeptide.

Further, the present invention relates to the following nucleic acid molecule (a) or (b).

(a) a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 2

(b) A nucleic acid molecule consisting of a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO: 2 due to the degeneracy of the genetic code, but encodes the same amino acid sequence as in (a).

The present invention also relates to a sphingosine kinase gene containing the above nucleic acid molecule.

Further, the present invention relates to an expression vector comprising the sphingosine kinase gene operably linked to a promoter.

The present invention also relates to a sphingosine kinase-expressing host cell transformed or transfected using the above-described expression vector.

Furthermore, the present invention relates to a method for producing sphingosine kinase, which comprises culturing the host cell.

The present invention also relates to a method for detecting a sphingosine kinase gene, which comprises using the nucleic acid molecule and / or a nucleic acid molecule that hybridizes with the nucleic acid molecule under stringent conditions.

Furthermore, the present invention relates to a method for diagnosing a sphingosine-related disease, which comprises using the nucleic acid molecule and / or a nucleic acid molecule that hybridizes with the nucleic acid molecule under stringent conditions.

The present invention also relates to a medicament for treating a sphingosine-related disease, comprising the polypeptide having sphingosine kinase activity and a pharmaceutically acceptable carrier.

Furthermore, the present invention relates to a medicament for treating a sphingosine-related disease, comprising the nucleic acid molecule and a pharmaceutically acceptable carrier. The present invention also relates to a medicament for treating a sphingosine-related disease, comprising the host cell and a pharmaceutically acceptable carrier.

Furthermore, the present invention relates to the use of the polypeptide having sphingosine kinase activity as a medicament for treating a sphingosine-related disease.

The present invention also relates to the use of the above-described nucleic acid molecule in a medicament for treating a sphingosine-related disease.

Further, the present invention relates to use of the above-mentioned host cell in a medicament for treating a sphingosine-related disease.

The present invention also relates to a method for screening a medicament for treating a sphingosine kinase-related disease, which is a method for labeling sphingosine and a drug in a medium of a host cell that expresses sphingosine kinase 3. The present invention relates to the above method, comprising adding a compound, quantifying labeled sphingosine 1-phosphate, and evaluating the effect of the compound on sphingosine kinase activity.

Further, the present invention relates to a medicament for treating a sphingosine-related disease obtained by the above-mentioned screening method.

The secretory polypeptide having sphingosine kinase activity of the present invention is typically a polypeptide represented by the amino acid sequence shown in FIG. It is secreted extracellularly.

A brief description of the screen

FIG. 1 shows the amino acid sequence of human-derived SPHK3 (hSPHK3) of the present invention.

FIG. 2 is a diagram showing an outline of pcDNA3-Flagl and its restriction enzyme map. FIG. 3 is a diagram showing the results of Western blotting of SPHK3 transiently expressed in C0S7 cells.

FIG. 4 is a diagram showing spots of TLC showing SPHK activity of SPHK3 transiently expressed in C0S7 cells. FIG. 5 is a graph showing the SPHK activity of SPHK3 transiently expressed in C0S7 cells.

FIG. 6 is a graph showing the pH dependence of SPHK activity of SPHK3.

FIG. 7 is a graph showing the substrate specificity of SPHK3. Day fl ^ ..

In the present invention, sphingosine kinase activity (SPHK activity) refers to an enzyme activity that phosphorylates sphingosine and catalyzes the production of sphingosine 1-phosphate. Sphingosine kinase 1 (SPHK1), sphingosine kinase 2 (SPHK2), and the like are known as polypeptides having SPHK activity, but polypeptides having SPHK activity of the present invention are typically Is sphingosine kinase 3 (SPHK3; SEQ ID NO: 1). Amino acid deletion refers to partial deletion of one or more amino acids from an arbitrary amino acid sequence. In addition, amino acid substitution means that one or more amino acids are replaced with another amino acid in an arbitrary amino acid sequence. Further, addition of an amino acid means that one or more amino acids are added to an arbitrary amino acid sequence.

The polypeptide having the SPHK activity of the present invention includes a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 in which amino acids are deleted, substituted or added, as long as the polypeptide has the SPHK activity. It is.

The sphingosine kinase gene of the present invention is a gene that encodes a polypeptide having SPHK activity, and is typically a gene that encodes sphingosine kinase 3 consisting of the amino acid sequence of SEQ ID NO: 1. . The gene encoding sphingosine kinase 3 is typically a gene consisting of a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 2.

The sphingosine kinase gene of the present invention has a codon sequence different from the nucleotide sequence of SEQ ID NO: 2 due to the degeneracy of the genetic code. Nucleic acids that are the same Genes consisting of molecules are also included. Here, the genetic code is a base sequence having information for defining the amino acid sequence of the polypeptide. The base sequence of the present invention includes DNA, RNA and cDNA sequences. Here, the degeneracy of the genetic code will be described. For example, a leucine residue can be encoded by codons CTA, CTC, CTG, CTT, TTA and TTG, and each of these six codons encodes a leucine residue. They are equivalent in purpose. Such a relationship between amino acid residues and nucleotide sequences is well known to those skilled in the art. In a situation in which a plurality of codons exist for such an amino acid, a gene encoding one amino acid sequence can be composed of a plurality of base sequences. Includes nucleic acid molecules consisting of sequences.

The promoter that can be used in the present invention is not particularly limited as long as it can transcribe an operably linked nucleic acid molecule. Specific promotions include CMV, SV40, PGK, EF321, MC1, TK, Plac, Pt ac, etc. In particular, Ptac and Plac are preferred for expression in bacteria such as E. coli. From the viewpoint of the expression level, CMV and EF321 are preferred.

As used herein, "operably linked" means that a regulatory sequence such as a promoter is directly or indirectly linked to a coding sequence such as a SPHK3 gene, and expression / transcription of a gene of the coding sequence is regulated by the Functionally linked so as to be under influence or control.

Where it is desired that the coding sequence be translated into a polypeptide, for example, induction of a promoter will result in transcription of the coding sequence. And the linkage between the two DNA sequences does not interfere with (1) the introduction of frameshift mutations, (2) the ability of the promoter region to direct the transcription of the coding sequence, or (3) Two DNA sequences are operably linked if they do not interfere with the ability of the corresponding RNA transcript to be translated. As used herein, the term "vector" refers to a vector into which a desired sequence can be inserted by restriction enzyme treatment, ligation, or the like, for transfer between different gene environments or for expression in a host cell. is there. Vector

— Typically consists of DNA, but RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids, and viral genomes. Cloning vectors are capable of replication in a host cell, either autonomously or after integration into the genome of the host cell. An expression vector is one in which a coding sequence is operably linked to a regulatory sequence so that the gene for the code sequence can be expressed as an RNA transcript.

A vector can include one or more marker sequences suitable for use in identifying whether a cell has been transformed or transfected with the vector. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibodies or other compounds (eg, kanamycin resistance gene, ampicillin resistance gene, etc.), and activity detected by standard methods Genes encoding possible enzymes (eg, monogalactosidase, alkaline phosphatase, luciferase, etc.) and phenotypes of transformed or transfected cells, hosts, colonies or plaques (eg, green fluorescent protein) (GFP), etc.).

Examples of such vectors include pBlueScriptl l SK (+), pB lueScript l I SK (-), pcDNA3-FLAGl, pcDNA3-HAl, pEGFP-N pEGFP-C pcDNA, etc. Can be Examples of expression vectors include pGEX-2T and pMA and C2. Among them, pcDNA3-FLAGl and pMAL-C2 are preferred from the viewpoints of expression efficiency, recovery rate after expression, detection efficiency, solubilization of the expressed protein, and the like.

In addition, preferred examples of the anti-FLAG M2 antibody, anti-FLAG M5 antibody, and GFP are preferred. "Stringent conditions" refers to nucleic acid hybridases that are commonly known.

— Refers to conditions under which chilling can take place, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., Eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 1989, or Current Protocols in Molecular Biology, FM Ausubel, et al., Eds., John Wiley & Sons, Inc., New York, and the like.

In particular the scan Application Benefits Njiwen preparative conditions herein, for example, Haibu Ridize one Chillon solution (5 XSSC, 1 O x Den'no, belt (Denhart's) _{solution, 1 OmM N a H 2 P} 0 4 (pH 6.5) , 0.5% SDS, 50% formamide) at 68 ° C.

In the method for detecting a sphingosine kinase gene of the present invention, for example, a probe labeled with an appropriate marker is prepared from the nucleotide sequence represented by SEQ ID NO: 2, and the probe is hybridized with a nucleic acid obtained from a sample. This can be done. Such hybridization can also be used in a method for diagnosing a sphingosine-related disease.

The host cell used in the present invention may be any of prokaryotic cells and eukaryotic cells as long as sphingosine kinase 3 can be expressed, and is not particularly limited. In addition, any cells such as animal cells, plant cells, filamentous fungi, and bacteria may be used. More specifically, COS 7, HEK293, HeLa, CH0, H.end FB, Jurkat, EG-OK CMK and the like can be mentioned. Particularly preferred cells are C0S7 and HeLa, from the viewpoint of high expression level when transfused and production of endogenous SPHK3.

Culture of the host cell may be performed by a generally known method, and is not particularly limited. When C0S7 or HeLa is used as the host cell, Dulbecco's modified Eagle's medium (DMEM) (2 mM glutamine,

〃G / ml scan streptomycin, added 100 U / ml penicillin and 10% © shea calf serum) is used to culture at 37 ° C, 5% C0 ₂ conditions.

In the present specification, a sphingosine-related disease is defined as Diseases / disorders caused by excessive, insufficient or defective phosphorylation. Also included are diseases caused by causes other than excess, deficiency or deficiency of sphingosine phosphorylation, which can be cured by regulating sphingosine phosphorylation.

Specific examples of such sphingosine-related diseases include arteriosclerosis, cancer, allergy, and myocardial infarction.

Therefore, the medicament of the present invention can be used as a therapeutic agent for sphingosine-related diseases. Further, it can be used as an angiogenesis promoter, an anti-cancer metastasis agent, an arteriosclerosis inhibitor and the like. As an active ingredient of the medicament of the present invention,

A secretory sphingosine kinase such as SPHK3, a nucleic acid molecule encoding the same, or a host cell expressing the secretory sphingosine kinase can be used.

As used herein, a pharmaceutically acceptable carrier refers to a carrier in which the polypeptides, nucleic acid molecules and host cells of the present invention are stably maintained, enable pharmaceutically effective administration, and are substantially non-toxic. Means Specific carriers include water, serine, mineral oil, vegetable oils, animal oils, organic or inorganic oils, xanthan, gelatin, natural polymers such as cellulose or gum arabic, synthetic polymers, alcohols and the like.

The medicament of the present invention can be administered in a dosage form such as oral administration, intravenous administration, subcutaneous administration, intramuscular administration, and the like. The dosage form can be appropriately selected according to the dosage form. For example, dosage forms suitable for oral administration such as powders, granules, pellets or tablets, coatings, capsules, solutions, syrups, etc., or injections, drops, suppositories, eye drops, nose drops, sprays It can be made into a dosage form suitable for parenteral administration. These preparations can be prepared using known adjuvants usually used in the technical field of pharmaceutical preparations. Examples of the adjuvant include an excipient, a binder, a disintegrant, a lubricant, a flavoring agent, a solubilizing agent, a suspending agent, a coating agent and the like.

The dosage of these pharmaceutical preparations varies depending on symptoms, age, body weight, administration method, dosage form, etc. 0 mg, with a lower limit of lmg to 2 mg, with a preferred dosage of 5 mg. The method for screening a medicament for treating a sphingosine kinase-related disease of the present invention includes, for example, preparing a SPHK-specific antibody from the amino acid sequence of the polypeptide shown in SEQ ID NO: 1, This can be done by Western blotting, immunoprecipitation, etc. using antibodies.

Alternatively, a specific labeled probe for SPHK can be prepared from the nucleotide sequence shown in SEQ ID NO: 2, and the sample can be subjected to Northern blot or the like.

The drugs obtained by these methods are further selected in an in vitro system based on desired properties (for example, properties inhibiting SPHK activity) as an index, and then examined for various diseases through animal experiments. Drugs having desired properties can be screened.

As the screening method, the following method is exemplified.

Sufi In coral screening thin kinase activity inhibitor, media play me ³ H of SP HK3 expressing cells - to quantify the sphingosine 1-phosphate by TLC or HPLC - was added sphingosine, ³ H to be generated in the medium. In this system, a compound that is a candidate for an inhibitor can be added to the medium, and a compound having an inhibitory effect can be screened based on the level of the activity. Labeling of Sufui Ngoshin is not limited to by ³ H, if also the become labeled, can be selected as appropriate, in the case of using the antibodies, it is possible to quantify the activity by using Imunoatsusi. Inhibitors obtained by such screening can control the production of excess sphingosine triphosphate in the blood, such as platelet transfusion toxicity inhibitor, platelet stabilizer, etc. Can be used.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

(Example 1) Cloning of SPHK3

Human derived EST database (http: 〃www.ncbi.nlm.nih.gov / dbES) From the T / (provided by the National Center for Biotechnology Information) (NCBI)), the base sequence of the known mSPHKl gene (mouse sphingosine kinase 1) (GenBank accession number AF068748, J. Biol. Chem., Vol. 273, Issue 37, 23722-23728, September 11, 1998) using a BLAST search (http: www. Ncbi.nlm.nih.gov/BLAST/ (provided by NCBI)). I did it. As a result, it was found that a clone homologous to the mSPHKl gene (GenBank accession number AA075971, IMAGE cloneD 531456) was present.

The nucleotide sequence of the ₀ E ST clone purchased from ATCC (American Type Culture Collection (http://www.atcc.org) for the clone 531456 homologous to this mSPHKl gene is only partially disclosed. Therefore, to determine the open reading frame (0RF) of the homologous clone, the entire nucleotide sequence of the EST clone was determined. From the estimated molecular weight of the polypeptide, we found 0RF, which is estimated to encode the target polypeptide.

FIG. 1 shows the amino acid sequence of the polypeptide encoded by the 0RF. This amino acid sequence is obtained from the signal sequence and its sequence by the prediction program of http: 〃www.cbs.dtu.dk / servers / SignalP-2.0 / (provided by Center for Biological Sequence Analysis, BioCentrum-DTU, Technica 1 University of Denmark). A cleavage site was expected.

In order to clone the 0RF from the entire nucleotide sequence of the determined EST clone, the following primers including BamHI site and EcoRI site were designed.

Primer 1 (SEQ ID NO: 3):

5,-CGC GGAT C CATGACGGTGT T C T T TAAAA C G-3 '

Primer 2 (SEQ ID NO: 4):

5,-CC GGAAT TCTCTTCTGCTGCT CA CT GGG T-3,

The PCR reaction was performed using KOD dash (Toyobo) according to the manufacturer's protocol, using the cDNA prepared from clone AA075971 as a template, and using primers 1 and 2. The reaction mixture was 2.5 U / / 1 K0D dash (1〃1), 2 mM dNTP mix (5〃1), 25 mM magnesium chloride (2〃1), and 10X reaction buffer (5〃1). 〃1.

The PCR reaction was performed 25 cycles, each cycle consisting of 98 ° C for 15 seconds, 65 ° C for 2 seconds and 74 ° C for 30 seconds. When the obtained PCR product was confirmed by agarose gel electrophoresis, it was a fragment of about 1300 bp. The nucleotide sequence was determined by a conventional method (SEQ ID NO: 2).

The resulting PCR product was digested with BamHI and EcoRI, and the transcript was digested with BamHI and EcoRI so that the transcript had a FLAG HA tag at the N-terminus.pcDNA3-FLAG1 (purchased pcDNA3 from Invitrogen, It was prepared by inserting the FLAG expression sequence at the MAP position, and was incorporated into Figure 2).

(Example 2) Expression of human sphingosine kinase 3 (hSPHK3)

Seed 2 x 10 ⁵ C0S7 cells (purchased from RIKEN Life Science Tsukuba Research Center, Cell Development Bank (RCB)) into a 35 mm dish, 24 hours after seeding, pcDNA3-Flagl alone and hSPHK3 gene (Example Lipofect AMINE plus (Lifetime PCR product)

Technologies) Transient transfection using reagents. Cultivation of C0S7 cells was performed using Dulbecco's modified

Glue medium (DMEM (Dulbecco, modified Eagle's mudium, purchased from SIGMA) with 10% fetal serum, lOU / ml penicillin, 100 μg / ml streptomycin) at 37 ° C, 5% It was carried out in the C0 ₂ conditions.

24 hours after seeding 100〃 1 IX sample buffer (0.0125M Tris-HC

0.4% SDS, 2% glycerol, 0.0002% BPB (Promoenol Blue),

C0S7 cells were collected using 1¾-mercaptoethanol). Collect The protein was separated by SDS-PAGE using a 1/10 amount of the cultured cells as a sample by a conventional method. The separated protein is applied to a polyvinylidene difluoride membrane (I

P, Millipore AB), and bands were confirmed by Western blotting using an anti-FLAG polyclonal antibody (SIGM A) (Fig. 3). The color was developed using an ECL western blotting detection reagent (Amersharm Pharmacia Biotech).

As shown in Fig. 3, two bands indicating SPHK3 were detected from the cells transfected with pcDNA3-Flagl into which the hSPHK3 gene was inserted, but these bands were immature (Fig. PSPHK3), mature type

(Referred to as mSPHK3 in the figure). SPHK3 has a signal sequence (underlined in Fig. 1) at the N-terminus, which is cleaved at the cleavage site shown in Fig. 1 in the endoplasmic reticulum (ER) to convert from immature to mature .

(Example 3) Measurement of sphingosine kinase activity

Inoculate 2 x 10 ⁶ C0S7 cells in a 100 mm dish, and 24 hours after seeding, pass _pcDNA3-FLAG1 alone and pcDNA3-Flagl into which the hSPHK3 gene was inserted using the Lipofect AMINE plus (Life Technologies) reagent. I did a transfection.

After 24 hours, collect the cells, disrupt the cells by sonication, add Triton X-100 to a final concentration of 1%, and solubilize by rotating at 4 ° C for 1 hour. did. After centrifugation at 100,000 xg for 60 minutes, anti-FLAG polyclonal antibody (SIGMA) and protein A sepharose (Pharmacia Biotech) were added to the obtained supernatant, followed by antibody precipitation reaction. The precipitate is collected, and an Assay buffer containing 0.01% TritonX-100, 50 M sphingosine, ImM [r-32P] ATP (composition: 15 mM MOPS, ImM phenylmethylsulfonyl fluoride, 50 mM NaCl, 5 mM mM M sodium orthovanadate, 2 mM MgCl ₂ , ImM 4-deoxypyridoxin, ImM EDTA, 0.5% aprotinin, pH 7.5) were added and incubated at 37 ° C. for 30 minutes. Then, the method of Olivera et al. (Olivera, A., Kohama, T., Tu, Ζ., Milstien, S., and Spiegel, S. (1998) J Biol Chem 273 (20), 12576-8 3.) According to the method, sphingosine 1-phosphoric acid extracted and extracted with lipids by ³² P was developed by TLC using a developing solvent of toluene / acetic acid / water (3: 1: 1), separated and analyzed by an image analyzer. (Imaging Analyzer BAS 2000, Fuji Film) Figures 4 and 5 show the analysis results.

Figure 4 shows spots of TLC, but sphingosine 1-1, which is hardly found in the control (pcDNA3-FLAG1 alone) from cells transfected with pcDNA3-Flagl into which the hSPHK3 gene has been inserted. Phosphoric acid spots (positions indicated by arrows in the figure) clearly appeared, confirming that the inserted polypeptide encoded by the hSPHK3 gene had SPHK activity.

Fig. 5 is a graph of the spots in Fig. 4 quantified.It can be seen that the cells transfected with PCDNA3-Flagl into which the hSPHK3 gene was inserted have 63 times higher SPHK activity than the control. Was.

(Example 4) pH dependence of hSPHK3

To see the effect of pH on SPHK activity, SPHK activity at each pH was measured. The measurement was carried out in the same manner as in Example 3, and 50 mM triethylamine was used for pH 3 to 6 and 50 mM MOPS was used for pH 5.5 to 8.5 as the assay buffer at each pH. Figure 6 shows the measurement results.

As shown in FIG. 6, SPHK3 has an optimum pH around neutrality, especially around pH 6.5-8.

(Example 5) Substrate specificity of hSPHK3

To examine the substrate specificity of hSPHK3, kinase activity was measured using a lipid other than sphingosine as a substrate. The measuring method is the same as in Example 3. The lipids used (10 μg each) were D-erythro- sphingosine (SPH), D-erythro-dihydrosphingosine (DHS), N, N_ Dimethyl sphingosine (N, N-dimethyl l-sphingosine (DMS)), ceramide (ceramide (CER)), C2-ceramide (C2-Ceramide (C2-CER)) (Matreya), diacilglycerol (diacylgly cerol (DG)) (Avanti) and phosphatidylinositol (PI) (SIGMA). Fig. 7 shows the results.

As shown in FIG. 7, hSPH has the strongest activity on sphingosine, but also has activity on DHS, DMS, DG and PI.卜禾 ilffl calf

According to the present invention, a novel sphingosine kinase 3 (SPHK3) different from SPHK1 and SPHK2 was obtained from the EST database. This SPHK 3 has SPHK activity and can be used as a medicament for treating sphingosine-related diseases.

In particular, it is superior in terms of extracellular secretion compared to the known SPHK1 and SPHK2, and is useful for direct drug action in the blood. Specifically, it is suitable for use as a platelet transfusion toxicity inhibitor, platelet stabilizer, and the like.

Further, the SPHK3 of the present invention and the SPHK3 gene can be applied to diagnosis of a sphingosine-related disease and the like.

Claims

Scope of Word

1. A secreted polypeptide having sphingosine kinase activity.

2. The secretory polypeptide according to claim 1, which is derived from human.

3. The following (a) or (b) polypeptides:

(a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1

(b) A polypeptide having sphingosine kinase activity, comprising 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: 1.

4. A sphingosine kinase gene encoding the polypeptide according to any one of claims 1 to 3.

5. The following nucleic acid molecules of (a) or (b):

6. A sphingosine kinase gene comprising the nucleic acid molecule according to claim 5.

7. An expression vector comprising the sphingosine kinase gene according to claim 4 or 6 operably linked to a promoter.

8. A sphingosine kinase-expressing host cell transformed or transfected with the expression vector according to claim Ί.

9. A method for producing sphingosine kinase, comprising culturing the host cell according to claim 8.

10. A method for detecting a sphingosine kinase gene, comprising using the nucleic acid molecule according to claim 5 and / or a nucleic acid molecule that hybridizes with the nucleic acid molecule under stringent conditions.

11. Use of the nucleic acid molecule according to claim 5 and / or a nucleic acid molecule that hybridizes with the nucleic acid molecule under stringent conditions. A method for diagnosing a sphingosine-related disease.

12. A medicament for treating a sphingosine-related disease, comprising the polypeptide having sphingosine kinase activity according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

13. A medicament for treating a sphingosine-related disease, comprising the nucleic acid molecule according to claim 5 and a pharmaceutically acceptable carrier.

14. A medicament for treating a sphingosine-related disease, comprising the host cell according to claim 8 and a pharmaceutically acceptable carrier.

15. Use of the polypeptide having sphingosine kinase activity according to any one of claims 1 to 3 as a medicament for treating a sphingosine-related disease.

16. Use of the nucleic acid molecule according to claim 5 for a medicament for treating a sphingosine-related disease.

17. Use of the host cell according to claim 8 for a medicament for treating a sphingosine-related disease.

18. A method for screening a medicament for treating a sphingosine kinase-related disease, comprising adding sphingosine kinase 3 and a drug candidate compound to a medium of a host cell expressing sphingosine kinase 3. The method, wherein the method comprises quantifying labeled sphingosine monophosphate and evaluating the effect of the compound on sphingosine kinase activity.

19. A medicament for treating a sphingosine-related disease obtained by the screening method according to claim 18.