WO2003031627A1

WO2003031627A1 - Platelet-origin polypeptides having sphingosine kinase activity and sphingosine kinase genes encoding the same

Info

Publication number: WO2003031627A1
Application number: PCT/JP2001/008537
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

Abstract

Platelet-origin polypeptides having a sphingosine kinase activity. In particular, human platelet-origin sphingosine kinase 4 (SPHK4) and a sphingosine kinase gene encoding the same.

Description

Satsuki Itoda »Platelet-derived polypeptide with sphingosine kinase activity and sphingosine kinase gene encoding it

The present invention relates to a platelet-derived polypeptide having sphingosine kinase activity and a sphingosine kinase gene encoding the same, and in particular, to a human platelet-derived sphingosine kinase 4 (SPHK4) and encoding the same. It relates to the sphingosine kinase gene. Dagger

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

The sphingosine 1-phosphoric acid produced by phosphorylation of sphingosine is accumulated in platelets in the blood and released with the activation of platelets. This sphingosine 1-phosphate is known to have various physiological and pathological roles such as promotion of platelet aggregation, wound healing of blood vessel walls, metastasis of pile cancer, 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 sphingosine 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 50 times lower than that of SPHK1. Increases and inhibitions of activity were observed depending on the concentration of surfactant, NaCl, and KC1, and in this respect, the properties were opposite to those of SPHK1. Dehydrosfingosine is a better substrate than sphingosine. In addition, mRNA expression is strongly expressed in kidney, liver and brain.

SPHK1 and SPHK2 are both enzymes that catalyze the phosphorylation of sphingosine, but both are specifically expressed in the aforementioned organs and are suitable for drug development targeting SPHK in blood. It was not what it was.

Therefore, an object of the present invention is to provide a novel sphingosine kinase, which is a main enzyme responsible for sphingosine-1-phosphate synthesis in platelets different from SPHK1 and SPHK2. Day S ^

Means for Solving the Problems The present inventors conducted intensive research to solve the above problems, and found a novel sphingosine kinase 4 (SPHK4 or P-SPHK) different from SPHK1 and SPHK2 from human platelets. The invention has been completed.

SPHK4 is a protein that is specifically expressed in platelets, and is the main synthase of sphingosine-1-phosphate in platelets. Therefore, SPHK4 is a novel sphingosine kinase derived from human platelets that enables drug development targeting SPHK in blood.

That is, the present invention relates to a polypeptide having a platelet-derived sphingosine kinase activity.

Further, the present invention provides a polypeptide of the following (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;

About.

Further, the present invention relates to a sphingosine kinase gene encoding a polypeptide having platelet-derived sphingosine kinase activity. The present invention also provides a nucleic acid molecule of the following (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 base sequence having a codon sequence different from that of SEQ ID NO: 2 due to degeneracy of the genetic code but having the same amino acid sequence as (a),

About.

Further, the present invention relates to a sphingosine kinase gene encoding the above-mentioned polypeptide.

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

Furthermore, the present invention relates to an expression vector comprising the above gene operably linked to a promoter.

The present invention also relates to a sphingosine kinase-expressing host cell transformed or transfected with the above 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 polypeptide having sphingosine kinase activity as described above. The present invention relates to a medicament for treating a sphingosine-related disease, comprising a tide 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 nucleic acid molecule as a medicament for treating a sphingosine-related disease.

Further, the present invention relates to the 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 drug for treating a sphingosine kinase-related disease, which comprises labeling sphingosine and a drug candidate compound in a medium of a host cell expressing sphingosine kinase 4. And quantifying the labeled sphingosine 1-phosphate, and evaluating the effect of the compound on sphingosine kinase activity.

The present invention further provides a medicament for treating a sphingosine-related disease obtained by the above-mentioned screening method. ] ¾ί な ^ ^

FIG. 1 is a graph showing the elution pattern of Hi-trap Q anion exchange chromatography.

FIG. 2 is a graph showing an elution pattern of Heparin-Sepharose column chromatography.

FIG. 3 is a graph showing one elution pattern of hide mouth xyapatite column chromatography. FIG. 4 shows the amino acid sequence of human SPHK4.

FIG. 5 is a photograph of electrophoresis showing expression of SPHK4 mRNA in platelets.

FIG. 6 is a photograph showing the sphingosine kinase activity of SPHK4 expressed by the E. coli expression system.昍》 Shape for skewering

In the present invention, sphingosine kinase activity refers to an enzyme activity that phosphorylates sphingosine and catalyzes the production of sphingosine 1-phosphate. As a polypeptide having a sphingosine kinase activity, sphingosine kinase 1 type (SPHK1) and sphingosine kinase type 2 (SPHK2) are known, and the sphingosine kinase activity of the present invention is known. The polypeptide having is typically sphingosine kinase 4 (SPHK4; SEQ ID NO: 1).

Amino acid deletion refers to partial deletion of one or more amino acids from any 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 sphingosine kinase activity of the present invention includes sphingosine as a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 in which amino acid is deleted, substituted or added. It is included as long as it has kinase activity.

The sphingosine kinase gene of the present invention is a gene encoding a polypeptide having sphingosine kinase activity, and is typically a gene encoding sphingosine kinase 4 consisting of the amino acid sequence of SEQ ID NO: 1. is there. The gene encoding sphingosine kinase 4 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. Genes consisting of the same nucleic acid molecules are also included. Here, the genetic code is a base sequence having information for defining the amino acid sequence of the polypeptide. The nucleotide 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, each of these six codons encoding a leucine residue For the purpose of 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 SPHK4 gene, and the expression and transcription of a gene of the coding sequence are regulated by the regulatory sequence. 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 results in transcription of the coding sequence. And the ligation between the two DNA sequences can enhance the ability of the promoter region to (1) not introduce a frameshift mutation and (2) direct transcription of the coding sequence. Two DNA sequences are operably linked if they do not, or (3) do not interfere with the ability of the corresponding RNA transcript to be translated into the protein.

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

The vector should contain one or more unique sequences suitable for use in identifying whether a cell has been transformed or transfected in the cell. Can be. For example, genes encoding proteins that increase or decrease resistance or sensitivity to an antibody or other compound (eg, a kanamycin resistance gene, an ampicillin resistance gene, etc.), may have a standard activity. Genes encoding enzymes that can be detected by conventional methods (eg, galactosidase, alkaline phosphatase, luciferase, etc.) and transformed or transfected cells, hosts, colonies or Genes that can visually detect the black phenotype (eg, green fluorescent protein (GFP)).

Examples of such vectors include pBluescript II SK (+), pBlueScript II SK (-), pcDNA3-FLAG1, pcDM3-HAl, pEGFP-N pEGFP-C pcDNA, etc. . Examples of expression vectors include pGEX-2T and pMAL-C2. Of these, from the viewpoints of expression efficiency, recovery rate after expression, detection efficiency, and solubilization of expressed protein, pcDNA3-FLAG pMA or C2 is preferred.

As the marker, an anti-FLAG M2 antibody, an anti-FLAG M5 antibody, GFP and the like are preferable.

The “stringent conditions” refer to conditions under which nucleic acid hybridization known in the art can be performed. For example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., Eds. Second Eaiti on, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Ne York, 1989 or Current Protocols in Molecular Biology, FM Ausubel, et al., Eds., John Wiley & Sons, Inc., New York, etc. I have.

In particular the scan Application Benefits Nji down preparative conditions herein, for example, Haibu Ridizeshiyon solution (5 XSSC, 1 O x Denhardt (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 hybridis can also be used in a method for diagnosing sphingosine-related diseases.

The host cell used in the present invention may be any of prokaryotic cells and eukaryotic cells as long as sphingosine kinase 4 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, C0S7, HEK293, He, CH0, H.end FB, Jurkat MEG-O CMK and the like can be mentioned. A particularly preferred cell is C0S7 from the viewpoint of high expression level when transfected. Culture of the host cell may be performed by a generally known method, and is not particularly limited. If C0S7 or HeLa is used as the host cell, use Dulbecco's Modified Eagle Medium (DMEM) (composition: 2 mM glumin, 100 zg / ml streptomycin, 100 U / ml penicillin, 10% fetal serum). , 37 ° C, 5% C 0 cultured in ₂ conditions.

In the present specification, a sphingosine-related disease refers to a disease or disorder caused by excessive, insufficient or defective phosphorylation of sphingosine. It also includes diseases and disorders in which phosphorylation of sphingosine is caused by causes other than excess, deficiency or deficiency, and which can be cured by regulating phosphorylation of sphingosine.

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 the active ingredient of the medicament of the present invention, a polypeptide having sphingosine kinase activity derived from platelets such as SPHK4, a nucleic acid molecule encoding the same, or a host cell expressing secreted sphingosine kinase can be used.

As used herein, a pharmaceutically acceptable carrier is 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 resins, 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., but usually the upper limit is 10 mg to 20 mg and the lower limit is 1 mg to 2 mg per day for adults. Yes, the preferred dosage is 5 mg. The method for screening a medicament for treating a sphingosine kinase-related disease of the present invention includes, for example, preparing a specific antibody against SPHK from the amino acid sequence of the polypeptide shown in SEQ ID NO: 1, It is possible to perform Western blotting, immunoprecipitation and the like using the antibody.

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.

In screening Sufi coral thin kinase activity inhibitor, media play me ³ H of SP HK4 expressing cells - to quantify the sphingosine 1-phosphate by TLC or HPLC - was added Sufi Ngoshin, 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 excessive sphingosine 1-phosphate in the blood, such as platelet transfusion toxicity inhibitor, platelet stabilizer And so on.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. (Example 1) Fractionation of cytosol and membrane and preparation of 1M NaCl extract from blood platelets

A platelet concentrated platelet solution obtained from the Hokkaido Red Cross Center (Hokkaido, Japan) was centrifuged at 2000 xg for 15 minutes. Perez DOO shaped platelet with Hepes / Tyrode buffer one (129 m NaCl, 8.9 mM NaHC0 3, 0.8 mM KH 2 P0 4, 0.8mM MgCl 2, lOm Hepes, pH 7.4, 2 mM EGTA) and washed 3 times with Was. The washed platelets were resuspended in buffer A (20 mM Tris-HCK pH 7.5, 0.25 mM EDTA, 1 mM dithiothreitol), sonicated, and centrifuged at 4 ° C, 100,000 xg for 30 minutes.

The membrane fraction was resuspended in buffer A containing 1 M NaCl, stirred on ice for 1 hour, and centrifuged at 100,000 xg for 30 minutes. The obtained supernatant was dialyzed, concentrated with a Centriprep-10 Consentrator, diluted with a buffer A containing 10% sucrose, and re-concentrated until the final salt concentration became 100 mM or less. The cytosol fraction and the 1M NaCl-extracted fraction were stored at -80 ° C until use.

(Example 2) Immunoprecipitation and SPHK analysis

The platelet fraction was dissolved in lysis buffer containing 1 mM phenylmethyl fluoride and 1 mM EDTA (20 mM Tris-HCK pH 7.5, 1% (w / v) Triton X-100 (sig ma), l¾ (w / v) Suspended in Tween-40, 0.85% NaCl). The lysate was sonicated and centrifuged at 100,000 xg for 30 minutes, and the resulting supernatant (300 zg protein) and MBP-SPHK1 (control protein) were ligated with lg anti-SPHKla antibody and 501 Incubation with Protein A Sepharose CL-4B 1: 1 suspension (Amersham Pharmacia Biotech) at 4 ° C. for 12 hours. Anti-SPHKla antibody was provided by Dr. Sakano (Gifu University, Gifu, Japan). The immunoconjugate beads were recovered by centrifugation at 3,000 xg for 5 minutes and washed three times with lysis buffer. The supernatant and pellet were assayed for SPHK activity.

(Example 3) Atsushi activity of sphingosine kinase activity Sample and 10 1 of 1 mM sphingosine (dissolved in 5% Triton X-100) in Buffer 1 B (20 mM Tris-HCK pH 7.5, 10% glycerol, 1 mM dithiothreitol, 0.25 mM EDTA, 1 mM orthovanadium NaOH, 20 mM? -Glycephate phosphate, 5 mM NaF, 1 mM phenylmethyl fluoride and 1 mM 4-deoxypyridoxine) and mix to a total volume of 1901, containing MgCl ₂ (200 mM) The reaction was started by adding [ ³² P] ATP (10 Ci, 20 mM) to IOJLI and incubated at 37 ° C for 15 minutes. After that, the reaction was stopped by adding 200 μl of 1 M HC1, and then 0.8 ml of chloroform / methanol / HCl (100: 200: 1, v / v) was added. After vigorous stirring, black form 240/1 and 1 M KC1 240-1 were added and the suspension was separated by centrifugation at 2,000 × g for 1 minute. Separate and concentrate the lipids in the organic layer, redissolve in 1-butanol / ethanol / acetic acid / water (80: 20: 10: 20, v / v) on a silica gel 60HP TLC plate, and perform radiography. Visualized by one. Radioactive spots corresponding to sphingosine 1-phosphate were quantified by Phospho Imager BAS-2000 (Fuji Film, Japan). Sphingosine kinase-specific activity is expressed as pmol sphingosine 1-phosphate formed per minute per mg protein.

(Example 4) Partial purification of SPHK activity capable of extracting NaCl from blood platelets Hitrap-Q anion Partial roasting by thumb chromatography

The above 1M NaCl extract was connected to FPLC equipment (Amersham Pharmacia Biotech), and applied to Hitrap-Q sepharose Fast Frow column Amersham Pharmacia Biotech previously equilibrated with buffer B at a flow rate of 0.5 ml / min. did. The eluate was washed with buffer B until the absorbance at 280 nm was less than 0.05. Bound protein was eluted with a 10 ml linear gradient of 0-0.5 M NaCl at a flow rate of 0.5 ml / min. One ml fractions were collected and each fraction was analyzed for SPHK activity.

The results are shown in Figure 1. SPHK activity eluted around 0.3 M NaCl. Heparin-Sepharose column chromatography

Active fractions. Dissolve and concentrate using Centriprep-10 Consentrator (Amicon). 0.3 ml / flow rate into a Hitrap-Heparin sepharose Fast Frow column (Amersham Pharmacia Biotech) pre-equilibrated with sodium, 20 mM? -Glycemic phosphate, 1 mM phenylmethyl fluoride, 1 mM 4-deoxypyridoxine). Loaded with min. The eluate was washed with buffer C until the absorbance at 280 nm was less than 0.05. Bound proteins were eluted with a 10 ml linear gradient of 0-1 M NaCl at a flow rate of 0.3 ml / min. 0.5 ml fractions were collected and the SPHK activity of each fraction was determined.

The result is shown in figure 2. SPHK4 has a strong affinity for heparin, and strong SPHK activity is concentrated and eluted around 0.5 to 0.6 M NaCl. These results indicate that this protein may interact with heparin, and the presence of a heparin-binding motif is expected. Manufactured by Hydroxyapatite column chromatography

The active fractions are pooled and buffer D (potassium phosphate, pH 6.8, 5¾ glycerol, 5 mM NaFs 1 m dithiothreitol, 1 mM sodium orthononadimate, 20 mM? -Glycerol phosphate, 1 mM phenol The mixture was injected at a flow rate of 0.5 ml / min into a hydroxyapatite column (Bio-Rad) pre-equilibrated with dimethyl fluoride and 1 mM 4-deoxypyridoxine. 0.5 ml fractions were collected and each fraction was analyzed for SPHK activity. The results are shown in Figure 3. The activity of this enzyme is very sharply separated by the hydroxyapatite column. Elution was performed by a concentration gradient method using potassium phosphate. The active fraction eluted at this stage was used for amino acid sequence analysis. (Example 5) Determination of amino acid sequence of SPHK4

Active fractions were pooled and loaded at a flow rate of 0.5 ml / min onto a Resource-Q Column (Amersham Pharmacia Biotech) pre-equilibrated with buffer B. 0.25 ml fractions were collected and each fraction was analyzed for SPHK activity.

The fraction containing the peak of SPHK activity was subjected to SDS-containing polyacrylamide gel electrophoresis (SDS-PEGE). The protein separated by SDS-PAGE was transferred to a polyvinylidene difluoride membrane (I-band obilon P, Millipore AB) and stained with Coomassie Blue. The band was cut out, immersed in a medium, and subjected to amino acid sequence analysis using a Procise 492 protein sequencing system (PE Applied Biosystems). The amino acid sequence was determined according to the method of the manufacturer.

The results are shown in FIG. The shaded portion in the figure indicates the portion where the N-terminal amino acid sequence matches with the known sphingosine kinase.

SPHK4 (SEQ ID NO: 1) is a basic protein composed of 416 amino acids and having an isoelectric point of 8.6 (theoretical value). In addition, it has a PKC phosphorylation site, a casein kinase site, a cAMP and a cGMP protein kinase site as phosphorylation sites. It also has a calmodulin binding domain, a heparin binding motif, and an ATP binding domain.

In particular, the heparin-binding motif does not have a known SPHK and is a major feature of SPHK4. The heparin-binding property of SPHK4 was inferred from the fact that a heparin affinity column could be used in the partial purification of SPHK4 described above. The presence of the motif was identified.

When SPHK is released from platelets, it has been suggested that extracellular matrix, heparin and heparan sulfate on the cell surface reflect this heparin-binding property, and act as reservoirs. This is an important property when considering various drugs derived from platelets. (Example 6) Cloning of human SPHK4 cDNA

The N-terminal sequence of SPHK4 corresponds to the deduced amino acid sequence of human K IAA clone646 (clone: BA B33316) in GenBank. This clone has been deposited by the Kazusa DNA Research Institute (Chiba, Japan). Dr. Nagase distributed pBluescript I I SK +, which contains a 4171 bp fragment inserted into the Sal I-Not I site. 5, the forward primer (5, p-SPHK primer: 5, -GAATCCATGCTGGAGAAGCTG-3 ′; SEQ ID NO: 3) is based on the sequence of the N-terminal amino acid sequence peptide (MLEKL; SEQ ID NO: 5), Reverse primer — (3, p-SPHK primer 1: 5, -GAATCCTCAGCTGTGTGAGTC-3 ′; SEQ ID NO: 4) is based on the sequence of the stop codon of the KIAA 1646 clone. CDNA was amplified using Taq DNA polymerase, 5, p-SPHK primer and 3, P-SPHK primer. The denaturation at 94 ° C for 45 seconds, annealing at 60 ° C for 1 minute, and extension at 72 ° C for 45 seconds were used as one cycle, and 30 cycles of PCR reaction were performed. As a result, a PCR product of 1251 base pairs was obtained. The amplified PCR product was purified from agarose gel using Mag Extractor (Toyobo), ligated into pGEM-T easy vector (Promega), and used to transform E. coli XLl-Blue strain. . Positive clones were treated with 50 g / ml ampicillin, 40 g / ml X-gal (5-bromo-4 -chloro-3 -indoleyl? -D-galactpranoside), 100 M isopropyl-/?-D-thiogalactoside (these Drugs were selected on LB agar (Difco) containing Sigma). Plasmid DNA was purified from positive clones and digested with EcoRI (Sigma) to confirm the presence of the cloned PCR product. The cloned DNA was sequenced using the ABI 377 DNA sequencer.

The result is shown in SEQ ID NO: 2.

(Example 7) Expression of human SPHK4 cDNA

The expression vector pMAL-c2 (New England Biolabs) was used, which expresses a protein containing an MBP (maltose binding protein) tag at the N-terminus. It is designed to be. This expression vector was digested with the restriction enzyme BamHI at 37 ° C. for 1 hour, linearized, and treated with alkaline phosphatase. Thereafter, it was purified from agarose gel. Following ligation of the prepared SPHK4 cDNA to pMA or c2, the vector was used for transformation of the E. coli XL1-Blue line. When the positive clone was confirmed using the ABI 377 DNA sequencer, it was confirmed that the clone had the normal frame and coordination of SPHK4 cDNA.

Positive clones were inoculated into LB broth medium containing 50 µg / ml ampicillin. Incubate the culture at 37 ° C until the absorbance A6 _{() Q} reaches 0.5-0.7, then add isopropyl-D-thiogalactoside (Sigma) to a final concentration of 0.5 mM. In addition, incubation was continued for 3 hours under the same conditions. The E. coli precipitate was collected, disrupted by sonication, and cell debris was removed by centrifugation. The supernatant was loaded on amylose resin (New England Biolabs) to capture the MBP fusion protein. After washing with buffer B until absorbance A _{28 fl} became 0.005, recombinant SPHK4 was eluted using equilibrated 10 mM maltose.

The expression was induced by IPTG according to a conventional method (37 ° C, 4 hours, final concentration 0.5 mM), and the SPHK activity of the induced SPHK4 was assayed in the same manner as in Example 3. Figure 6 shows the results. The sphingosine-111-phosphoric acid spot (indicated by the arrow in the figure), which is hardly seen in those without IPTG-induced SPHK4 gene expression, clearly appears in the IPTG-induced one. It was confirmed that the polypeptide encoding the inserted SPHK4 gene had SPHK activity.

(Example 7) RT-PCR analysis

For the isolation of mRM, platelets were prepared using Sepacel PLX-5A-ILS (Asahi Medical Co., Ltd.) in the same manner as described above except that leukocytes were not mixed. The flow-through sample was centrifuged at 2,000 X g for 10 minutes at 22 ° C, and the supernatant was carefully removed from the platelet pellet. QuickPrep Micro mRNA It was isolated from human platelets using an mRNA purification kit (Amersham Pharmacia Biotech). The mMA preparation was aliquoted until use and frozen at -80 ° C.

De - human _SPHK family of evening in pace - on the basis of the nucleotide sequence of (SPHK1 and SPHK2) and platelet factor 4 (PF-4), it was prepared Origonukureo Chidopuraima scratch. Π-PCR was performed using a Super Script One-Step RT-PCR system (Life Tecnnologies, In) on template mRNA (50 ng) on a thermal cycler (GeneAmp PCR system 9700, PE Applied Biosystems). Performed under conditions.

Reaction conditions: (1) 50 ° C, 30 minutes, (2) 94 ° C, 2 minutes, (3) 94 ° C, 15 seconds, (4) 58 ° C, 30 seconds, (5) 72 ° C (6) (3) through (5) for 40 cycles, and (7) 72 ° C for 1 minute.

The RT-PCR product was visualized on 1% agarose gel electrophoresis and its nucleotide sequence was confirmed on an ABI 377 DNA sequencer.

To clarify whether the SPHK4 mA was expressed in platelets, total RNA from platelets was extracted and RT-PCR was performed using the primers (SEQ ID NOS: 3 and 4). Fig. 5 shows the results. As a control, a PCR reaction was similarly performed for PF-4, and a sample without the addition of reverse transcriptase (represented as RT (-) in the figure) was also examined. As shown in FIG. 5, it was confirmed that the platelet expresses SPHK4 mMA (the position of the arrow in the figure). Invitation Hell II T calf

According to the present invention, a novel sphingosine kinase 4 (SPHK4) different from SPHK1 and SPHK2 was obtained from human platelets. This SPHK4 has sphingosine kinase activity and can be used as a medicament for treating sphingosine-related diseases.

In particular, it is superior to the known SPHK1 and SPHK2 in that it is specifically expressed on platelets, and is useful for direct drug action in blood. Specifically Is suitable for use as a platelet transfusion toxicity inhibitor, a platelet stabilizing agent, and the like. It can also be applied to the diagnosis of sphingosine-related diseases and the like using the SPHK4 and SPHK4 genes of the present invention.

Claims

Scope of word blue

I. Polypeptides with platelet-derived sphingosine kinase activity

2. 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 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 and having sphingosine kinase activity.

3. A sphingosine kinase gene encoding a polypeptide having platelet-derived sphingosine kinase activity.

4. The nucleic acid molecule of (a) or (b) below:

(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).

5. A sphingosine kinase gene encoding the polypeptide of claim 2.

6. A sphingosine kinase gene comprising the nucleic acid molecule according to claim 4.

7. An expression vector comprising the gene according to claim 3 operably linked to a promoter.

8. A sphingosine kinase-expressing host cell transformed or transfected with the expression vector of claim 7.

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 4 and / or a nucleic acid molecule that hybridizes with the nucleic acid molecule under stringent conditions.

II. Use of the nucleic acid molecule according to claim 4 and Z or a nucleic acid molecule that hybridizes with the nucleic acid molecule under stringent conditions. A diagnostic method for a sphingosine-related disease.

12. A medicament for treating a sphingosine-related disease, comprising the polypeptide having sphingosine kinase activity according to claim 1 or 2, and a pharmaceutically acceptable carrier.

13. A medicament for treating sphingosine-related diseases, comprising the nucleic acid molecule according to claim 3 or 4 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 claim 1 or 2 as a medicament for treating a sphingosine-related disease.

16. Use of the nucleic acid molecule according to claim 3 or 4 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 4 and a compound as a drug candidate to a medium of a host cell that expresses sphingosine kinase 4, and labeling the compound. The method according to claim 1, further comprising quantifying sphingosine 1-phosphate 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.