JP5373346B2 - Novel transporter molecule of sphingosine 1-phosphate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new transporter molecule which migrates S1P (sphingosine-1-phosphate) from cell inside to cell outside, to provide a cell which can express the S1P transporter molecule, and to provide a method for screening the antagonist and/or agonist of the S1P transporter. <P>SOLUTION: Vertebrate Spin2 acts as a new transporter molecule of S1P. The method for screening the antagonist and/or agonist of the S1P transporter comprises interacting a test substance with the transporter molecule to detect a substance reinforcing or reducing the functions of the transporter. By the screening method, an agent for improving diseases caused by the function potentiation or attenuation of S1P or diseases potentiating or attenuating the functions of S1P as symptoms can be screened. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a novel transporter molecule that moves sphingosine 1-phosphate from the inside of the cell to the outside of the cell. Specifically, it relates to a novel transporter molecule of sphingosine 1-phosphate consisting of vertebrate Spin2. The present invention also relates to a cell capable of expressing the sphingosine 1-phosphate transporter molecule, and further relates to a method for screening an antagonist and / or agonist of the sphingosine 1-phosphate transporter.

  Sphingosine 1-phosphate (hereinafter referred to as “S1P”) is produced from cell membrane sphingomyelin, and the hydroxyl group at the 1-position of sphingosine acts phosphorylated by sphingokinase and acts outside the cell. Ceramide and sphingosine cause apoptosis, whereas S1P is said to inhibit apoptosis and act on cell proliferation. Thus, since sphingosine and S1P have completely different physiological activities, it is considered that the balance of the amounts in vivo is important for maintaining the balance of in vivo functions.

S1P interacts with S1P receptor 1 (S1P ₁ ) -5 (S1P ₅ ), which is a G protein-coupled receptor present in the cell membrane, and acts on vascular endothelial cells, vascular smooth muscle cells, etc., and vascular endothelial cells Regulates cell proliferation, smooth muscle contraction, and cell migration. In particular, the function of S1P during thrombus formation has attracted attention, and a new function as an immunomodulator has been clarified (Non-patent Document 1). The physiological significance of S1P and its receptor in the vascular system and immune system is being elucidated. Since S1P is present in a high concentration in the blood, the importance of S1P in the vascular system is predicted. In addition, the importance of S1P and its receptor in the immune system is being clarified by a new immunosuppressant (FTY720, Novartis Pharma Co., Ltd.) under clinical evaluation. FTY720, rather than acting directly in vivo, FTY720-P becomes phosphorylated, is said to act as agonists of SlP ₂ other than the receptor. Thus, the action of S1P and its receptor is being clarified.

  Although the role as a receptor agonist is becoming clear, there are many unclear parts about the intracellular action of S1P. It is considered that a part of S1P produced in the cell is released and acts on the receptor. On the other hand, intracellular S1P actions may include cell proliferation, apoptosis inhibition, calcium mobilization, etc., but these actions are similar to actions mediated by the S1P receptor, and the involvement of the signal transduction pathway is unknown. There are many points.

It is not clear by what mechanism the S1P generated in the cell is transported outside the cell (Non-patent Document 1). ABCA1 and ABCC1 belonging to extracellular ABC transporters have been reported to have S1P transporter activity (Non-patent Documents 2 and 3), but whether or not they function as S1P transporters due to differences in assay methods. It is not yet fully understood. Thus, there are many unclear points about S1P and its transporters.
Nat. Rev. Mol. Cell Biol., 9, 139-150 (2008) Proc. Natl. Acad. Sci. USA, 103, 16394-16399 (2006) Prostaglandins Other Lipid Mediat., 84, 154-162 (2007)

  An object of the present invention is to provide a novel transporter molecule that moves S1P from the inside of a cell to the outside of the cell. Another object of the present invention is to provide a cell capable of expressing the S1P transporter molecule, and further to provide a screening method for an S1P transporter antagonist and / or agonist.

  The inventors of the present invention have made extensive studies to solve the above-mentioned problems, and as a result of identifying the responsible gene and analyzing the function by analyzing the mutant of zebrafish, the vertebrate Spin2 can function as a transporter of S1P. The present invention has been completed.

That is, this invention consists of the following.
1. The S1P transporter molecule, which is a polypeptide consisting of any amino acid sequence selected from the following 1) to 4):
1) the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;
2) Arginine located at position 199 of the amino acid sequence shown in SEQ ID NO: 1 is substituted, deleted, added or introduced in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. An amino acid sequence comprising at least and having a transport function of S1P;
3) A partial amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, comprising at least arginine located at position 199 of the amino acid sequence shown in SEQ ID NO: 1, and having a transport function of S1P Amino acid sequence;
4) Among the amino acid sequences shown in 3) above, 1 to 5 amino acids are substituted, deleted, added or introduced, and at least arginine located at position 199 of the amino acid sequence shown in SEQ ID NO: 1 Contains amino acid sequence.
2. A cell for expressing an S1P transporter molecule, comprising cDNA encoding the S1P transporter molecule according to item 1.
3. The S1P transporter molecule-expressing cell according to item 2, further comprising cDNA encoding sphingosine kinase.
4). A method for screening an S1P transporter antagonist and / or agonist, wherein a test substance and the S1P transporter molecule described in the preceding item 1 are allowed to interact to detect a substance capable of enhancing or reducing the function of the transporter.
5. 5. The method according to item 4 above, wherein the method of causing the test substance to interact with the S1P transporter molecule according to item 1 above comprises contacting the sample with the cell for expressing the S1P transporter molecule according to item 2 or 3 above. Screening method.
6). A method for screening an antagonist and / or agonist of an S1P transporter comprising the following steps:
1) a step of expressing an S1P transporter molecule and a sphingosine kinase in a cell for expressing the S1P transporter molecule according to item 3;
2) The labeled sphingosine is added to the cell culture medium of 1) above and cultured, the labeled sphingosine is introduced into the cell, and the sphingosine kinase expressed in the cell of 1) is reacted to produce labeled S1P. Process;
3) Further, a step of adding a test substance to the cell culture medium of 2) above, culturing, and allowing the test substance to interact with an S1P transporter molecule expressed in the cell;
4) A step of measuring the amount of S1P inside and outside the cell after culturing for a certain time.
7). 7. The screening method according to any one of items 4 to 6, wherein the S1P transporter antagonist or agonist is an ameliorating agent for immune diseases or vascular diseases.
8). 7. The screening method according to any one of 4 to 6 above, wherein the S1P transporter antagonist or agonist is an ameliorating agent for a disease caused by S1P hyperactivity or attenuation, or a disease in which S1P function is enhanced or diminished as a symptom. .
9. A reagent for screening an S1P transporter antagonist and / or agonist, comprising the S1P transporter molecule according to item 1 or the cell for expressing the S1P transporter molecule according to item 2 or 3.

  By analyzing the interaction between the S1P transporter molecule of the present invention and S1P, it is possible to elucidate the function of S1P in more detail and to contribute to the elucidation of diseases associated with S1P function enhancement and attenuation. It is. Furthermore, by the screening method of the present invention, it is possible to develop an agent for improving a disease caused by S1P function enhancement or attenuation, or a disease in which S1P function is enhanced or attenuated as a symptom.

  S1P is produced from cell membrane sphingomyelin as described in the background section above, and the hydroxyl group at the 1-position of sphingosine is phosphorylated by sphingokinase and acts extracellularly. The transporter molecule of S1P in the present invention refers to a molecule having a function of transporting S1P generated in the cell from the inside of the cell to the outside of the cell in the process of the sphingolipid and the cell information transmission mechanism.

  The transporter molecule of S1P specified in the present invention is a vertebrate-derived Spin2, or a portion thereof, or a partial vertebrate-derived Spin2 amino acid substituted, deleted, added or introduced, and comprising an S1P trans Any device having a port function may be used. Specific examples of vertebrate-derived Spin2 include human Spin2 represented by SEQ ID NO: 1 in the sequence listing and zebrafish Spin2 represented by SEQ ID NO: 2. The amino acid sequences described in SEQ ID NOs: 1 and 2 are registered in DDJB Accession Nos. AB441165 and AB441164, respectively.

The S1P transporter molecule specified in the present invention is specifically composed of a polypeptide consisting of any amino acid sequence selected from the following 1) to 4).
1) the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;
2) Arginine located at position 199 of the amino acid sequence shown in SEQ ID NO: 1 is substituted, deleted, added or introduced in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. An amino acid sequence comprising at least and having a transport function of S1P;
3) A partial amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, comprising at least arginine located at position 199 of the amino acid sequence shown in SEQ ID NO: 1 and having an S1P transport function Sequence;
4) Among the amino acid sequences shown in 3) above, 1 to 5 amino acids are substituted, deleted, added or introduced, and at least arginine located at position 199 of the amino acid sequence shown in SEQ ID NO: 1 Contains amino acid sequence.

Another transporter molecule of S1P specified in the present invention may be composed of a polypeptide consisting of any amino acid sequence selected from the following 5) to 8).
5) the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;
6) Arginine located at position 153 of the amino acid sequence shown in SEQ ID NO: 2 in which 1 to 5 amino acids are substituted, deleted, added or introduced in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing An amino acid sequence comprising at least and having a transport function of S1P;
7) A partial amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, comprising at least arginine located at position 153 of the amino acid sequence shown in SEQ ID NO: 2, and having a transport function of S1P Amino acid sequence;
8) Among the amino acid sequences shown in the above 7), 1-5 amino acids are substituted, deleted, added or introduced, and at least arginine located at position 153 of the amino acid sequence shown in SEQ ID NO: 2 Contains amino acid sequence.

S1P and its receptor 1 (S1P ₁ ) -5 (S1P ₅ ) interact to act on the vascular system and / or the immune system. When S1P function is enhanced or attenuated, the balance of S1P function in vivo is lost, leading to disease. It is considered that the S1P transporter molecule of the present invention affects S1P function enhancement or attenuation. Therefore, the S1P transporter antagonist or agonist of the present invention controls S1P dysfunction, that is, S1P hyperactivity or attenuation, thereby causing a disease caused by S1P hyperfunction or attenuation, or as a result of the disease. It is conceivable to improve diseases that increase or decrease function.

  Examples of diseases caused by S1P hyperactivity or attenuation, or diseases that result from S1P hyperfunction or attenuation as a result of the disease include immune diseases and vascular diseases.

  In addition to so-called immune diseases, immune diseases include all diseases in which immune functions act. Specific examples include inflammatory diseases, autoimmune diseases and cancer-related diseases. More specifically, rheumatoid arthritis, psoriasis, lung inflammation, nephrotic syndrome, Crohn's disease, colitis, chronic diarrhea, renal fibrosis, pulmonary fibrosis, liver fibrosis, chronic bronchial asthma, adult respiratory distress syndrome (ARDS) , Chronic obstructive pulmonary disease (COPD), interstitial pneumonia, idiopathic interstitial pneumonia, hypersensitivity pneumonitis, pleurisy and solid tumor, solid tumor metastasis, angiofibroma, myeloma, multiple myeloma, Kaposi's sarcoma Etc. Among the above-mentioned solid tumors are, for example, breast cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, liver cancer, ovarian cancer, follicular cell carcinoma, cervical cancer, endometrial cancer, prostate cancer, kidney cancer, skin Examples include cancer, osteosarcoma, pancreatic cancer, urinary tract cancer, thyroid cancer, and brain tumor. In addition, as an immune disease, for example, rejection associated with various organ transplants such as heart transplantation, kidney transplantation, skin transplantation, liver transplantation, and bone marrow transplantation can be included.

  Vascular diseases include arteriosclerosis, obstructive thromboangiitis, Buerger's disease, peripheral arterial disease of diabetic neuropathy, sepsis, vasculitis, cerebral infarction, myocardial infarction, varicose vein, dissecting aortic aneurysm, angina, DIC, congestive heart failure, multiple organ failure, and other diseases accompanied by abnormal angiogenesis (eg restenosis, diabetic retinopathy, angiogenic glaucoma, posterior lens fiber hyperplasia, atherosclerosis) Capillary proliferation of plaques, thyroid hyperplasia, pulmonary inflammation, nephrotic syndrome) and the like can also be included in vascular diseases.

The invention also extends to cells capable of expressing the S1P transporter molecule of the invention. The cell contains a cDNA encoding the S1P transporter molecule. Specifically, constructing a cell capable of expressing an S1P transporter molecule by constructing a plasmid having the cDNA downstream of the promoter of the expression vector, preparing an expression vector, and introducing it into a host cell. Can do. Here, the expression vector is not particularly limited as long as it is a vector capable of expressing the S1P transporter molecule. Specific examples include a vector containing a CMV promoter and the like, and for example, commercially available pcDNA3.1 (Invitrogen) can be used. For confirmation of S1P transporter molecule expression, an expression vector that expresses a fusion protein with a reporter protein may be used.
A method for introducing a gene is not particularly limited, and a method known per se or a method developed in the future can be applied. For example, a gene introduction reagent (Lipofectamine ^™ -200 reagent, Invitogen) can be used.
The host cell is not particularly limited, and for example, bacteria, yeast, animal cells, plant cells and the like can be used. When cells are used to screen for S1P transporter agonists and / or antagonists described later, the cells can be made to transport S1P extracellularly by expressing the S1P transporter of the present invention. It is necessary to use animal cells, and more specifically, CHO (Chinese Hamster Ovary) cells and the like can be used.

  The cells capable of expressing the S1P transporter molecule of the present invention can be used for screening for S1P transporter agonists and / or antagonists described below. In order to be used for the screening method for agonists and / or antagonists of the S1P transporter, cells that can confirm the function of the S1P transporter are preferable. The function of the S1P transporter can be confirmed, for example, by measuring the amount of S1P inside and outside the cell. For example, when S1P is not observed outside the cell, the function of the S1P transporter is considered to be low, and when more S1P is found outside the cell than inside the cell, the S1P transporter is considered to be functioning. .

  Therefore, in the present invention, the cell capable of expressing the S1P transporter molecule may further contain cDNA encoding sphingosine kinase. Each expression vector may be any vector as long as each protein can be expressed separately, and may be a different vector, or may include an expression cassette capable of expressing each in one vector. For the sequence of cDNA encoding sphingosine kinase, the sequence disclosed in Accession No. NP — 079643 can be referred to.

  The present invention also extends to methods for screening for S1P transporter antagonists and / or agonists. As a screening method, a test substance which is a candidate substance interacts with the transporter molecule of the present invention, and a substance capable of enhancing or reducing the transport function is detected.

The screening method of the present invention can be performed, for example, by the following steps 1) to 4).
1) a step of expressing an S1P transporter molecule and sphingosine kinase in a cell for expressing the S1P transporter molecule;
2) A step of adding Sphingosine to the cell culture medium of 1) above, culturing it, introducing the sphingosine into the cell, and reacting the sphingosine kinase expressed in the cell of 1) above to generate S1P;
3) Further, a step of adding a test substance to the cell culture medium of 2) above, culturing, and allowing the test substance to interact with an S1P transporter molecule expressed in the cell;
4) A step of measuring the amount of S1P inside and outside the cell after culturing for a certain time.

More specifically, the screening method of the present invention can be performed by the following steps 1) to 5).
1) A step of constructing cells expressing sphingosine kinase and the S1P transporter molecule of the present invention.
2) A step of adding sphingosine to the constructed cell culture medium and culturing, and introducing sphingosine into the cell. The introduced sphingosine becomes S1P by intracellular sphingosine kinase. Here, the sphingosine to be introduced may be labeled and labeled for later detection.
3) A step of detecting S1P inside and outside the cell by allowing the above-mentioned cells to interact with the test substance, culturing for a certain period of time, separating the cells and the culture solution by centrifugation or the like. The detection method is not particularly limited as long as S1P can be detected. For example, it can be detected by chromatography, mass spectrometry, or a combination thereof, or the label can be detected if it is a labeled S1P produced from labeled sphingosine.
4) A step of comparing S1P inside and outside the cells when the test substance and cells interact with each other with the amount of S1P inside and outside the cells when the test substance and cells do not interact as a control.
5) When extracellular S1P is increased when the test substance interacts with cells relative to the amount of control extracellular S1P, the test substance is an S1P transporter agonist and the extracellular S1P is decreased And determining the test substance as an S1P transporter antagonist.

In the above screening method, a cell that expresses sphingosine kinase and the S1P transporter molecule of the present invention may be constructed in advance. In addition, a pre-labeled sphingosine may be used as the label-labeled sphingosine. A method for labeling sphingosine is not particularly limited, and a labeling method known per se can be applied. Considering ease of determination, it can be labeled with a radioactive substance or a fluorescent substance, for example, with ³ H.

More specifically, S1P transporter agonists and / or antagonists can be screened by the following method.
1) A plasmid having a cDNA encoding sphingosine kinase is constructed downstream of the promoter of the expression vector, an expression vector is prepared and introduced into CHO (Chinese Hamster Ovary) cells, and CHO cells expressing sphingosine kinase 1 (CHO −) SphK) is constructed. Furthermore, an expression vector that expresses the S1P transporter molecule of the present invention is prepared and introduced into CHO-SphK. For confirmation of S1P transporter molecule expression, an expression vector that expresses a fusion protein with a reporter protein may be used. For gene introduction, for example, a gene introduction reagent (Lipofectamine ^™ -200 reagent, Invitogen) can be used.
2) After culturing CHO-SphK for about 6 to 16 hours, the culture solution is exchanged and labeled sphingosine is added. Lipid-soluble sphingosine is phosphorylated by mouse sphingosine kinase 1 expressed in cells and converted to labeled S1P.
3) A test substance was added to the culture solution, and after about 30 minutes at 37 ° C. in a CO ₂ incubator, the medium (supernatant: S) and the cells (precipitation: P) were obtained for CHO-SphK into which each gene was introduced. The extracted labeled S1P is developed by thin layer chromatography and detected using a bioimaging analyzer.
4) Using the amount of S1P inside and outside the cell when the test substance and cells do not interact as a control, compare the S1P inside and outside the cell when the test substance and cells interact.
5) When extracellular S1P is increased when the test substance interacts with cells relative to the amount of control extracellular S1P, the test substance is an S1P transporter agonist and the extracellular S1P is decreased In addition, the test substance is determined as an S1P transporter antagonist.

  The S1P transporter agonist or antagonist screened by the screening method of the present invention can be an active ingredient of an immunity disease or vascular disease ameliorating agent, or a disease caused by S1P hyperfunction or attenuation, or as a symptom of S1P It can be used as an active ingredient of a disease ameliorating agent whose function is enhanced or attenuated.

  The improving agent comprising an S1P transporter agonist or antagonist obtained by the screening method of the present invention as an active ingredient is a pharmaceutically acceptable carrier, diluent, excipient, It can be formulated and manufactured with an agent and the like. As the auxiliaries for the preparation, those well known to those skilled in the art can be used. The improving agent of the present invention can be expected to achieve a desired therapeutic effect by setting a preferable administration route such as intravaginal administration or oral administration.

  The present invention also extends to reagents used for the above screening. Here, as a reagent used for screening, in addition to a reagent containing an S1P transporter molecule, a cell capable of expressing the S1P transporter molecule can be used as the reagent. Furthermore, another screening reagent of the present invention includes a screening reagent containing sphingosine, and a screening kit containing a cell capable of expressing the S1P transporter molecule and sphingosine can also be provided. Here, when detecting the label S1P, the label-labeled sphingosine can be used as a reagent.

  Examples of the present invention will be described below in more detail, but the present invention is not limited to these, and various applications are possible without departing from the technical idea of the present invention. It is.

Morphological features Sebura fish according (Example 1) Spin2, were compared morphological mutant of wild-type zebrafish, SlP ₂ deficient zebrafish and mutant Spin2 held zebrafish from each embryo. A mutant zebrafish carrying the mutant Spin2 was prepared by administration of a chemical mutagen (ENU, N-ethyl N-nitrosourea) according to the method described in Development, 123, 1-36 (1996). When the second generation (F2 fish) became an adult fish, pair mating was carried out, and mutants showing abnormalities in heart formation were screened to obtain mutants having an allele of ko157, that is, Spin2 mutant embryos. SlP ₂ functional inhibition embryos, antisense morpholinos for SlP _2; were produced by administration of (S1P ₂ MO 15ng).

As a result, the wild-type zebrafish embryos while the one heart, the SlP ₂ function blocking embryos and Spin2 mutant embryos, two hearts were observed (Figure 1). Therefore, the Spin2 mutant embryos, S1P function similarly to S1P ₂ functional inhibition embryos is not sufficient, as a result, was considered as an admission morphologically abnormal. Confirmation of the developmental process of the heart is obtained for each embryo from a transgenic line having a reporter gene in which monomeric red fluorescent protein (mRFP) is connected to the promoter region of the heart-specific gene cmlc2 (cardiac myosin light chain 2). was embryo administered antisense morpholinos for Spin2 and SlP _2, hearts were observed visualized in red.

  As a result of analyzing the cDNA of the mutant Spin2 in the Spin2 mutant embryo using the ABI Genetic Analyzer 3130, the 153rd arginine in the amino acid sequence (SEQ ID NO: 2) constituting the zebrafish Spin2 (zSpin2) was mutated to serine. It was confirmed. The 153rd amino acid of sebrafish Spin2 corresponds to the 199th amino acid (arginine) of human Spin2 (SEQ ID NO: 1) (see FIG. 2).

(Example 2) Confirmation of the function as an S1P transporter by Spin2 According to the following steps, each gene of zebrafish wild-type Spin2, zebrafish mutant Spin2, human wild-type Spin1, human wild-type Spin2, and human mutant Spin2 The amount of S1P secreted when introduced was measured.

A plasmid having a gene encoding mouse sphingosine kinase 1 downstream of the CMV promoter of an expression vector (pcDNA3) having a neomycin (G418) resistance gene was prepared, and a gene introduction reagent (Lipofectamine ^™ -2000 reagent, Invitogen) was used. The cells were introduced into CHO (Chinese Hamster Ovary) cells. Among cells exhibiting resistance to G418, cells having high S1P synthesis activity were selected and used as CHO-SphK in the experiment.

CHO-SphK includes zebrafish wild-type Spin2 (zSpins2), zebrafish mutant Spin2 (zSpins2-R153S), human wild-type Spin1 (hSpin1), human wild-type Spin2 (hSpin2), human mutant Spin2 (hSpin2-R199S) Each gene was introduced.
A mammalian cell expression vector expressing each of the above spin proteins and green fluorescent protein (EGFP) as a fusion protein was prepared and introduced into CHO-SphK using a gene transfer reagent (Lipofectamine ^™ -2000 reagent, Invitogen). Spin-related genes were not included, and CHO-SphK into which only a gene expressing EGFP was introduced was used as a control.

After culturing CHO-SphK into which each of the genes was introduced for 6 to 16 hours, the culture solution was exchanged and [ ³ H] sphingosine was added. Fat-soluble [ ³ H] sphingosine is phosphorylated by mouse sphingosine kinase 1 expressed in cells and converted to [ ³ H] S1P.

After 30 minutes at 37 ° C. in a CO ₂ incubator, the cells into which each gene was introduced were separated into a medium (supernatant: S) and cells (precipitate: P) by centrifugation, and extracted into each fraction. [ ³ H] S1P was developed by thin layer chromatography and then detected using a bioimaging analyzer (FLA-3000 Bioimaging Analyzer (Fuji Film)) (FIGS. 3 and 4).

  As a result, in the control cells, the amount of S1P extracted outside the cell was very small, and it was considered that there was almost no substance having a function of transporting S1P from the inside of the cell to the outside of the CHO cell. In the system into which zSpins2 or hSpin2 was introduced, S1P was observed outside the cell, but in the system into which zSpins2-R153S, hSpin1 or hSpin2-R199S was introduced, only S1P comparable to the control was observed outside the cell. As a result, when a zebrafish wild-type Spin2 gene or human wild-type Spin2 gene was introduced into CHO cells, a large amount of extracellular S1P was observed, and it was confirmed that wild-type Spin2 has an S1P transport function. It was done.

  As described in detail above, by analyzing the interaction between the transporter molecule of the present invention and S1P, it is possible to elucidate the function of S1P in more detail, and also to elucidate diseases associated with S1P enhancement and attenuation. It seems to contribute. Furthermore, by the screening method of the present invention, it is possible to develop an ameliorating agent for a disease caused by S1P hyperactivity or attenuation, or a disease that causes S1P hyperfunction or attenuation as a symptom.

It is a figure which shows the morphological effect which gives Spin2 to zebrafish. In zebrafish or Spin2 mutant embryos have a zebrafish mutant Spin2, it showed two heart like the SlP ₂ deficient zebrafish. S1P ₂ MO (15 ng) or spin ₂ MO (10 ng) was administered to zebrafish embryos that can monitor the developmental process of the heart by the expression of monomeric red fluorescent protein. Example 1 It is a figure which shows the mutation site | part of a zebrafish mutant type Spin2 protein. Spin2 was mapped to the vicinity of the z9419 and z63525 marker genes present on chromosome 5 (LG5). TM indicates a transmembrane domain. The 153rd arginine (R) of Spin2 was mutated to serine (S). Example 1 The ^[3 H] SlP inside and outside the cells in various Spin transfected cells (CHO-SphK), is a diagram showing the result of detection using the Bio-imaging Analyzer. P represents a cell fraction, and S represents a culture solution. Sph represents sphingosine, and Cer represents ceramide. Ceramide is a precursor of sphingosine. (Example 2) It is a figure which shows the amount of extracellular S1P secretion in the cell (CHO-SphK) which introduce | transduced various Spin genes. (Example 2)

Claims (9)

Sphingosine 1-phosphate transporter molecule, which is a polypeptide consisting of any amino acid sequence selected from the following 1) to 2):
1) the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;
2) Arginine located at position 199 of the amino acid sequence shown in SEQ ID NO: 1 is substituted, deleted, added or introduced in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. An amino acid sequence comprising at least a sphingosine 1-phosphate transport function. A cell for expressing a sphingosine 1-phosphate transporter molecule, comprising cDNA encoding the sphingosine 1-phosphate transporter molecule according to claim 1. Furthermore, the cell for a sphingosine 1-phosphate transporter molecule expression of Claim 2 containing cDNA which codes a sphingosine kinase. An antagonist of a sphingosine 1-phosphate transporter that detects a substance that interacts with the test substance and the sphingosine 1-phosphate transporter molecule according to claim 1 to enhance or reduce the function of the transporter; A method for screening for agonists. The method for causing the test substance to interact with the sphingosine 1-phosphate transporter molecule according to claim 1 comprises the test substance and the sphingosine 1-phosphate transporter molecule expression cell according to claim 2 or 3; The screening method of Claim 4 by making it contact. A method for screening an antagonist and / or agonist of sphingosine 1-phosphate transporter, comprising the following steps:
1) a step of expressing a sphingosine 1-phosphate transporter molecule and a sphingosine kinase in a cell for expressing the sphingosine 1-phosphate transporter molecule according to claim 3;
2) The labeled sphingosine is added to the cell culture medium of 1) above and cultured, the labeled sphingosine is introduced into the cells, and the sphingosine kinase expressed in the cells of 1) is reacted to give labeled sphingosine 1-phosphorus Generating an acid;
3) Further, a step of adding a test substance to the cell culture medium of 2) above, culturing, and allowing the test substance to interact with a sphingosine 1-phosphate transporter molecule expressed in the cell;
4) A step of measuring the amount of sphingosine 1-phosphate inside and outside the cell after culturing for a certain time. The screening method according to any one of claims 4 to 6, wherein the sphingosine 1-phosphate transporter antagonist or agonist is an ameliorating agent for immune diseases or vascular diseases. The sphingosine 1-phosphate transporter antagonist or agonist is an ameliorating agent for diseases caused by sphingosine 1-phosphate function enhancement or attenuation, or diseases in which sphingosine 1-phosphate function is enhanced or attenuated as a symptom. Item 7. The screening method according to any one of Items 4 to 6. An antagonist of sphingosine 1-phosphate transporter comprising the sphingosine 1-phosphate transporter molecule according to claim 1 or the cell for expressing the sphingosine 1-phosphate transporter molecule according to claim 2 or 3, and / or Agonist screening reagents.