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WO2003031627A1 - Polypeptides a origine plaquettaire a activite sphingosine kinase et genes de sphingosine kinase codant pour ces polypeptides - Google Patents

Polypeptides a origine plaquettaire a activite sphingosine kinase et genes de sphingosine kinase codant pour ces polypeptides Download PDF

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Publication number
WO2003031627A1
WO2003031627A1 PCT/JP2001/008537 JP0108537W WO03031627A1 WO 2003031627 A1 WO2003031627 A1 WO 2003031627A1 JP 0108537 W JP0108537 W JP 0108537W WO 03031627 A1 WO03031627 A1 WO 03031627A1
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sphingosine
sphingosine kinase
nucleic acid
acid molecule
medicament
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PCT/JP2001/008537
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English (en)
Japanese (ja)
Inventor
Yasuyuki Igarashi
Akio Kihara
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Hokkaido Technology Licensing Office Co., Ltd.
Chemical Biology Institute
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Priority to PCT/JP2001/008537 priority Critical patent/WO2003031627A1/fr
Publication of WO2003031627A1 publication Critical patent/WO2003031627A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • 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 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.
  • SPHK1 sphingosine kinase 1
  • 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
  • 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.
  • 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.
  • SPHK4 or P-SPHK novel sphingosine kinase 4
  • 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.
  • the present invention relates to a polypeptide having a platelet-derived sphingosine kinase activity.
  • 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):
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. ] 3 ⁇ 4 ⁇ ⁇ ⁇ ⁇
  • 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. ⁇ Shape for skewering
  • sphingosine kinase activity refers to an enzyme activity that phosphorylates sphingosine and catalyzes the production of sphingosine 1-phosphate.
  • 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.
  • amino acid substitution means that one or more amino acids are replaced with another amino acid in an arbitrary amino acid sequence.
  • 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.
  • 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.
  • a leucine residue can be encoded by codons CTA, CTC, CTG, CTT, TTA and TTG, each of these six codons encoding a leucine residue
  • 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.
  • 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.
  • 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.
  • the coding sequence be translated into a polypeptide
  • induction of a promoter results in transcription of the coding sequence.
  • 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.
  • 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.
  • genes encoding proteins that increase or decrease resistance or sensitivity to an antibody or other compound may have a standard activity.
  • Genes encoding enzymes that can be detected by conventional methods eg, galactosidase, alkaline phosphatase, luciferase, etc.
  • transformed or transfected cells hosts, colonies or Genes that can visually detect the black phenotype (eg, green fluorescent protein (GFP)).
  • GFP green fluorescent protein
  • 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.
  • an anti-FLAG M2 antibody, an anti-FLAG M5 antibody, GFP and the like are preferable.
  • stringent conditions refer to conditions under which nucleic acid hybridization known in the art can be performed.
  • 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.
  • 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.
  • 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.
  • DMEM Dulbecco's Modified Eagle Medium
  • 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.
  • sphingosine-related diseases include arteriosclerosis, cancer, allergy, and myocardial infarction.
  • 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.
  • angiogenesis promoter 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.
  • 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.
  • 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.
  • the adjuvant examples 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.
  • 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.
  • the following method is exemplified.
  • 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 .
  • 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.
  • Example 1 Fractionation of cytosol and membrane and preparation of 1M NaCl extract from blood platelets
  • 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
  • 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.
  • 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), l3 ⁇ 4 (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.
  • 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?
  • 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 active fractions are pooled and buffer D (potassium phosphate, pH 6.8, 53 ⁇ 4 glycerol, 5 mM NaFs 1 m dithiothreitol, 1 mM sodium orthononadimate, 20 mM? -Glycerol phosphate, 1 mM phenol
  • buffer D potassium phosphate
  • 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).
  • SDS-PEGE polyacrylamide gel electrophoresis
  • 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.
  • 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.
  • 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.
  • 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?
  • 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 ⁇ g / 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.
  • 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.
  • 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.
  • RNA from platelets was extracted and RT-PCR was performed using the primers (SEQ ID NOS: 3 and 4).
  • Fig. 5 shows the results.
  • 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.
  • RT (-) the reverse transcriptase
  • SPHK4 novel sphingosine kinase 4
  • SPHK4 has sphingosine kinase activity and can be used as a medicament for treating sphingosine-related diseases.
  • SPHK1 and SPHK2 are superior to the known SPHK1 and SPHK2 in that it is specifically expressed on platelets, and is useful for direct drug action in blood.
  • SPHK1 and SPHK2 are 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.

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Abstract

L'invention concerne des polypeptides d'origine plaquettaire possédant une activité sphingosine kinase. Elle concerne, en particulier une sphingosine kinase 4 d'origine plaquettaire et un gène de sphingosine kinase la codant.
PCT/JP2001/008537 2001-09-28 2001-09-28 Polypeptides a origine plaquettaire a activite sphingosine kinase et genes de sphingosine kinase codant pour ces polypeptides WO2003031627A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000052173A2 (fr) * 1999-03-02 2000-09-08 Nps Allelix Corp. Homologues de sphingosine kinase humaine clones
WO2000058473A2 (fr) * 1999-03-31 2000-10-05 Curagen Corporation Acides nucleiques comprenant des phases de lecture ouverte codant des polypeptides; «orfx»
WO2001060990A2 (fr) * 2000-02-14 2001-08-23 Curagen Corporation Nouvelles kinases de sphingosine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000052173A2 (fr) * 1999-03-02 2000-09-08 Nps Allelix Corp. Homologues de sphingosine kinase humaine clones
WO2000058473A2 (fr) * 1999-03-31 2000-10-05 Curagen Corporation Acides nucleiques comprenant des phases de lecture ouverte codant des polypeptides; «orfx»
WO2001060990A2 (fr) * 2000-02-14 2001-08-23 Curagen Corporation Nouvelles kinases de sphingosine

Non-Patent Citations (3)

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Title
HIROSAWA M. ET AL.: "Identification of novel transcribed sequences on human chromosome 22 by expressed sequence tag mapping", DNA RES., vol. 8, no. 1, February 2001 (2001-02-01), pages 1 - 9, XP002906347 *
HONG LIU ET AL.: "Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 275, no. 26, June 2000 (2000-06-01), pages 19513 - 19520, XP002945744 *
TAKAFUMI KOHAMA ET AL.: "Molecular cloning and functional characterization of murine sphingosine kinase", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 37, September 1998 (1998-09-01), pages 23722 - 23728, XP002150781 *

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