JP2009034096A - Transporter selectively transporting sulfate conjugate, and gene of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a liver specific organic anion transporter transporting a sulfate conjugate selectively, and a gene of the same. <P>SOLUTION: This method for modifying pharmacodynamics is provided by using an organic anion transporter protein having a capacity of selectively transporting the sulfate conjugate. The gene encoding the same, a protein associated with transportation of the liver sulfate conjugate, a specific antibody against the same and a function promotor or a function inhibitor for modifying the capacity of selectively transporting the sulfate conjugate of the protein are also provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a protein involved in the transport of a liver sulfate conjugate, and a gene encoding the protein. The present invention also uses a protein involved in the transport of a liver sulfate conjugate, a specific antibody thereof, a function-promoting substance, or a function-inhibiting substance to modulate the ability of the protein to selectively transport the sulfate conjugate. To a method for modifying pharmacokinetics.

Conventional technology

  The liver plays an important role in the metabolism and elimination of xenobiotics and drugs. Hepatocytes are polar epithelial cells that are in contact with blood through the basolateral membrane (similar side membrane) and exchange various substances. Drugs in portal vein blood are taken into liver cells via the basolateral membrane via a transport carrier (transporter) or by passive diffusion via a cell membrane lipid bilayer, and undergo a conjugation reaction by metabolism within the cell. Receive and become a conjugate.

The conjugation reaction includes glutathione conjugation, glucuronidation conjugation, sulfate conjugation, etc., and the resulting conjugate is in the form of an anion (anion) and is recognized by the organic anion transporter present in the liver cell membrane, Discharged. The glutathione conjugate and the glucuronic acid conjugate are mainly discharged from the luminal side (biliary duct side) membrane of hepatocytes into the bile duct. The sulfate conjugate is excreted into the blood mainly from the basolateral membrane of hepatocytes.
Exogenous foreign substances other than drugs and steroid hormones are metabolized in hepatocytes by the same mechanism as drugs and are excreted as conjugates.

  Sulfate conjugates such as drugs, foreign substances, and steroid hormones excreted into the blood are taken up by the organic anion transporter present in the basolateral membrane of the proximal tubular epithelial cells of the kidney, and the tubes of the tubular epithelial cells It is excreted in the urine via the organic anion transporter in the cavity side membrane.

  As an organic anion transporter on the luminal side of hepatocytes, a multi-bile duct side multi-selective organic anion transporter (cMOAT) has already been cloned.

Uptake of organic anions through the basolateral membrane of hepatocytes has been studied by an experimental system using an isolated organ perfusion method or an isolated cell membrane vesicle system.
However, with conventional methods, it is difficult to analyze in detail the transport mechanism responsible for transport of organic anions through the cell membrane, especially the discharge of organic anions produced by metabolism in the cell. It has been desired to analyze in detail.

Based on these backgrounds, the molecular entity of the organic anion transporter in the basolateral membrane of the liver was actively searched in the 1990s. As a result, the organic anion transporter oatp of the basolateral membrane of the liver was isolated. (Hagenbuch, B. et al. Proc Natl Acad Sci USA 88, 10629-33, 1991, Jacquemin, E. et al., Proc Natl Acad Sci USA, 91, 133-7, 1994). However, this is responsible for the transport of bile acids and the like, and was different from that for transporting drug conjugates.
Furthermore, in 1997, an organic anion transporter OAT1 responsible for organic anion transport in the kidney was isolated (Sekine, T. et al., J Biol Chem 272, 18526-9, 1997). OAT1 transports some conjugate drugs but does not show expression in the liver.
Following OAT1, an organic anion transporter OAT2 having a structure similar to OAT1 and expressed in the liver was isolated (Sekine, T. et al., Biochem. Biophys. Res. Commun. 251: 586-591, 1998). However, OAT2 had low transport activity for the conjugate drug.

Furthermore, an organic anion transporter OAT3 having a structure similar to that of OAT1 and expressed in the kidney, liver and brain has been postulated (Kusuhara, H. et al., J. Biol. Chem., 274: 13675-13680, 1999). . OAT3 is a multi-selective transporter that exhibits extremely wide substrate selectivity using a wide variety of organic anions as substrates, including drug conjugates, and excretion of drug conjugates efficiently through the basolateral membrane of hepatocytes. In order to avoid competition with other organic anions, it is desirable to have an organic anion transporter dedicated to the drug conjugate in addition to OAT3.
Following OAT3, an organic anion transporter OAT4 having a structure similar to OAT1 and expressed in the kidney and placenta was isolated (Cha, SH et al., J. Biol. Chem. 275: 4507-4512 (2000). Shows a relatively sulfate-selective substrate selectivity, including some organic anions centered on sulfate conjugates, but is not expressed in the liver.

  As already mentioned, the transport of organic anions in the basolateral membrane of the liver is complicated, and the outflow route into the blood of conjugates (mostly organic anions) that are produced in large amounts in hepatocytes is still unknown. is there. The above-mentioned organic anion transporter expressed in the liver cannot be explained alone, and the presence of further unknown transporters is expected.

  An object of the present invention is to identify and provide a novel organic anion transporter gene that selectively transports a sulfate conjugate of hepatocyte basolateral membrane and an organic anion transporter that is a polypeptide encoded by the gene. It is in. Other purposes will be clear from the following description.

The present inventors used EST (expressed) using the base sequence of the translation region of cDNA of OAT1.
sequence tag) A database was searched and a base sequence similar to OAT1 was identified. A corresponding probe was prepared, a cDNA library was screened, and a gene encoding a novel protein was cloned. Furthermore, the gene product was expressed in Xenopus oocytes, revealing that the gene product is a novel organic anion transporter that selectively transports sulfate conjugates. Furthermore, a specific antibody against the product of this gene was prepared, it was clarified that the product of this gene was present in the hepatocyte basolateral membrane, and the present invention was completed.

That is, the present invention is a protein selected from the following (A) and (B), which has the ability to selectively transport sulfate conjugates.
(A) Protein comprising the amino acid sequence represented by SEQ ID NO: 1 (B) Protein comprising the amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1

In addition, the present invention is a gene comprising a DNA selected from the following (a) and (b), which encodes a protein having an ability to selectively transport a sulfate conjugate.
(A) DNA consisting of the base sequence represented by SEQ ID NO: 2.
(B) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence represented by SEQ ID NO: 2.

The novel protein of the present invention having the ability to selectively transport sulfate conjugates [hereinafter referred to as organic anion transporter 7]. ] Has the ability to selectively transport (incorporate) sulfate conjugates.
The liver-specific organic anion transporter OAT7 that selectively transports sulfate conjugates is expressed only in the liver in the living body and is present in the basolateral membrane of hepatocytes. OAT7 is considered to be a transporter that excretes sulfate conjugates such as drugs, foreign substances, and steroid hormones generated by intracellular metabolism in hepatocytes into the blood.

  The liver-specific organic anion transporter that selectively transports a sulfate conjugate of the present invention and its gene are used for the transport of a drug, a foreign substance, a steroid hormone, and a similar compound thereof in the liver of the transporter. It enables in vitro studies and in vitro prediction of the pharmacokinetics of drugs and foreign substances, steroid hormones and similar compounds based on them. Furthermore, it is considered useful for the development of a drug that efficiently permeates the transporter. In addition, it enables in vitro prediction of drug-drug interactions involving the transporter. Furthermore, by modulating the ability of the transporter to selectively transport sulfate conjugates, methods for modifying the pharmacokinetics of drugs, toxic substances or foreign substances, steroid hormones, etc., side effects of drugs and exogenous It can be used to develop a method for reducing the toxic effects of foreign substances and a method for protecting hepatocytes from drugs and foreign substances.

BEST MODE FOR CARRYING OUT THE INVENTION

  Sequence number 2 of a postscript sequence table is the full length cDNA base sequence (about 2.3 kbp) of the gene of the liver specific organic anion transporter (human OAT7) which selectively conveys the sulfate conjugate derived from human liver, and its translation region Represents the amino acid sequence of the protein encoded by (533 amino acids).

  As the protein of the present invention, in addition to the protein having the amino acid sequence represented by SEQ ID NO: 1, for example, an amino acid sequence obtained by deleting, substituting or adding one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 The protein which has. Deletion, substitution, or addition of amino acids may be performed so long as the organic anion transport activity is not lost, and is usually 1 to about 110, preferably 1 to about 55. Such a protein usually has 1-80%, preferably 1-90% amino acid sequence homology with the amino acid sequence shown in SEQ ID NO: 1.

The gene of the present invention includes a gene having a base sequence represented by SEQ ID NO: 2 and a DNA capable of hybridizing under stringent conditions with a DNA comprising the base sequence represented by SEQ ID NO: 2. Is mentioned. The DNA that can be hybridized in this way is not limited as long as the protein encoded by the DNA has the ability to transport organic anions. Such DNA has a homology of 70% or more, preferably 80% or more of the base sequence shown by SEQ ID NO: 2 in general. Such DNA includes mutant genes found in nature, artificially modified mutant genes, homologous genes derived from different organisms, and the like.
In the present invention, hybridization under stringent conditions is usually performed in a hybridization solution of 5 × SSC or an equivalent salt concentration at a temperature of 37-42 ° C. for about 12 hours. Preliminary washing can be performed with 5 × SSC or a solution having a salt concentration equivalent thereto, if necessary, followed by washing in a solution having a salt concentration equivalent to 1 × SSC or the like.

The liver-specific organic anion transporter gene that selectively transports the sulfate conjugate of the present invention can be isolated and obtained by screening using an appropriate mammalian organ, tissue or cultured cell as a gene source. Mammals include non-human animals such as dogs, cows, horses, goats, sheep, monkeys, pigs, rabbits, rats and mice, as well as humans.
Screening and isolation of the gene can be suitably performed by a homology cloning method or the like.

For example, using mouse or human liver as a gene source, mRNA (poly (A) + RNA) is prepared therefrom. A cDNA library is constructed from this, and EST (expressed
sequence tag) To obtain a clone containing cDNA of OAT7 gene by screening a cDNA library using a probe corresponding to an OAT7-like sequence obtained by searching a database (for example, GenBankTM / EBI / DDBJ accession No. AA705512) Can do.
Regarding the obtained cDNA, the base sequence can be determined by a conventional method, the translation region can be analyzed, and the amino acid sequence of the protein encoded thereby, that is, OAT7 can be determined.

  The obtained cDNA is a cDNA of an organic anion transporter gene that selectively transports sulfate conjugates, that is, the gene product encoded by the cDNA is a transporter that selectively transports sulfate conjugates. Can be verified, for example, as follows. That is, RNA (cRNA) (capacitated) prepared from the obtained cDNA of OAT7 gene, which is complementary to the cDNA, is introduced into the oocyte and expressed, and the sulfate conjugate is transported into the cell. The ability of (incorporation) to measure the uptake of a substrate into a cell by a normal uptake test using an appropriate sulfate conjugate as a substrate (Kanai and Hediger, Nature, Vol. 360, paragraphs 467-471, 1992) Can be confirmed.

  By using in vitro translation method [Hediger et al., Biochim. Biophys. Acta, 1064, 360, 1991] using a complementary RNA (cRNA) prepared from the obtained cDNA of OAT7 gene, Proteins can be synthesized and the size of the protein, presence or absence of sugar addition, etc. can be examined by electrophoresis.

  In addition, for the expressed cells, the same uptake experiment can be applied to examine the characteristics of OAT7, such as OAT7 substrate selectivity and pH dependency.

Isolation of homologous genes and chromosomal genes from different tissues and different organisms by screening appropriate cDNA libraries or genomic DNA libraries prepared with different gene sources using the cDNA of the obtained OAT7 gene can do.
In addition, by searching various gene protein databases using the disclosed nucleotide sequence of the gene of the present invention or the amino acid sequence of its gene product, homologous genes and chromosomal genes derived from different tissues and organisms are identified. Can do.
In addition, a general PCR (Polymerase Chain Reaction) method using a synthetic primer designed based on the information of the disclosed base sequence of the gene of the present invention (base sequence shown in SEQ ID NO: 2 or a part thereof) A gene can be isolated from a cDNA library or a genomic DNA library.
A DNA library such as a cDNA library or a genomic DNA library is, for example, a method described in “Molecular cloning” [Sambrook, J., Fritsh, EF and Manitis, T., published by Cold Spring Harbor Press, 1989]. Can be prepared. Alternatively, if there is a commercially available library, it may be used.

  The liver-specific organic anion transporter (OAT7) that selectively transports a sulfate conjugate of the present invention can be produced by a gene recombination technique using, for example, cDNA encoding it. For example, DNA encoding OAT7 (such as cDNA) can be incorporated into an appropriate expression vector, and the resulting recombinant DNA can be introduced into an appropriate host cell. Examples of the expression system (host-vector system) for producing a polypeptide include bacterial, yeast, insect cell and mammalian cell expression systems. Of these, insect cells and mammalian cells are preferably used to obtain functional proteins.

  For example, when a polypeptide is expressed in a mammalian cell, a DNA encoding the liver-specific organic anion transporter OAT7 that selectively transports a sulfate conjugate is converted into an appropriate expression vector (for example, an adenovirus vector, retrovirus). An expression vector inserted downstream of an appropriate promoter (eg, cytomegalovirus promoter, SV40 promoter, LTR promoter, elongation 1a promoter, etc.) in a vector vector, papilloma virus vector, vaccinia virus vector, SV40 vector, etc. To construct. Next, an appropriate animal cell is transformed with the obtained expression vector, and the transformant is cultured in an appropriate medium to produce the desired polypeptide. Examples of mammalian cells used as a host include cell lines such as mouse S2 cells, monkey COS-7 cells, Chinese hamster CHO cells, or human HeLa cells.

  As a DNA encoding the liver-specific organic anion transporter OAT7 that selectively transports a sulfate conjugate, for example, a cDNA having the base sequence represented by SEQ ID NO: 2 can be used, and the DNA sequence is limited to the above cDNA sequence. Alternatively, DNA corresponding to the amino acid sequence can be designed and used as DNA encoding the polypeptide. In this case, 1 to 6 types of codons encoding one amino acid are known, and the selection of codons to be used may be arbitrary. For example, considering the frequency of codon usage of the host used for expression, more efficient expression can be achieved. High arrays can be designed. DNA having the designed base sequence can be obtained by chemical synthesis of DNA, fragmentation and binding of the cDNA, partial modification of the base sequence, and the like. Artificial modification of the base sequence and mutagenesis can be achieved by using site-specific mutagenesis [Mark, DF et al., Proceedings] using primers consisting of synthetic oligonucleotides that encode the desired modifications. of National Academy of Sciences, Vol. 81, Paragraph 5562 (1984)].

The present invention also provides a nucleotide comprising a partial sequence of 14 bases or more, preferably 20 bases or more, or a complementary sequence thereof in the base sequence represented by SEQ ID NO: 2.
The nucleotide of the present invention can be used as a probe for detecting a gene encoding a protein having an ability to selectively transport a sulfate conjugate, and also has a high homology with the gene encoding the protein. Can be used as a primer for obtaining a protein-encoding gene and the like, and to modulate the expression of a gene encoding a protein having the ability to selectively transport a sulfate conjugate by its antisense strand, etc. Can be used for

  The antibody can be obtained by using the liver-specific organic anion transporter that selectively transports the sulfate conjugate of the present invention or a polypeptide having immunological equivalent thereto. The antibody can be used for detection or purification of a liver-specific organic anion transporter that selectively transports a sulfate conjugate. The antibody can be produced using the liver-specific organic anion transporter that selectively transports a sulfate conjugate of the present invention, a fragment thereof, or a synthetic peptide having a partial sequence thereof as an antigen. A polyclonal antibody can be produced by a conventional method in which a host animal (eg, rat or rabbit) is inoculated with an antigen and immune serum is collected, and a monoclonal antibody is produced by a technique such as a usual hybridoma method. it can.

The liver-specific organic anion transporter OAT7 that selectively transports a sulfate conjugate of the present invention, its gene, and its expression cell are related to the permeation efficiency at the cell membrane where OAT7 is present or at the site where OAT7 is expected to be present. Can be used for in vitro testing. That is, using the protein of the present invention, the action of the test substance as a substrate on the ability to selectively transport the sulfate conjugate of the protein can be detected.
In addition, liver-specific organic anion transporter OAT7 that selectively transports sulfate conjugates, its gene, and its expression cells are developed as a compound that efficiently permeates the cell membrane where OAT7 is present and the site where OAT7 is expected to be present. Can be used for

The liver-specific organic anion transporter OAT7 that selectively transports a sulfate conjugate of the present invention, its gene, and its expression cell are the sites where a compound transported by OAT7 is expected to pass through the cell membrane or exist. It can be used to develop inhibitors that limit permeation (such as specific inhibitors of OAT7).
For example, using an oocyte into which human OAT7 gene cRNA has been injected, an uptake experiment of a substance transported by OAT7 (eg, sulfate conjugate of steroid hormone) is performed. This experiment is performed in the presence of various drugs and the absorption rate of the substance transported by OAT7 is measured. A drug that reduces the absorption rate of a substance transported by OAT7 is obtained as an inhibitor of the ability to selectively transport a sulfate conjugate possessed by OAT7.
In addition, liver-specific organic anion transporter OAT7 that selectively transports sulfate conjugates, its gene, and its expression cells are expressed in vitro in cell membranes where OAT7 is present and drug interactions at sites where OAT7 is expected to exist. Can be used for testing at

The ability of the present invention to selectively transport the sulfate conjugate of the protein using the liver-specific organic anion transporter that selectively transports the sulfate conjugate, its specific antibody, its function promoter or function inhibitor It is possible to modulate the pharmacokinetics of drugs, toxicants, foreign substances or steroid hormones transported by the transporter of the present invention.
The transporter of the present invention can be produced by the gene recombination technique described above, and can be used as a transconjugate active agent for sulfate conjugates. In addition, by using the organic anion transporter of the present invention and a test using cells expressing the same, it is possible to develop a substance that promotes or suppresses the function of a liver-specific organic anion transporter that selectively transports a sulfate conjugate. By using a drug, it is possible to modify the pharmacokinetics of a drug, poison, foreign substance or steroid hormone transported by the transporter of the present invention.
Furthermore, by modifying the pharmacokinetics of the drug transported by the transporter of the present invention, it is possible to reduce the side effects of the drug and the toxic effects of foreign substances. In addition, it is possible to protect hepatocytes from drugs and foreign substances by modifying the pharmacokinetics of the compound transported by the transporter of the present invention.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, these Examples do not restrict | limit this invention.
In the following examples, unless otherwise specified, each operation is performed according to the method described in “Molecular cloning” [Sambrook, J., Fritsh, EF and Manitis, T., published by Cold Spring Harbor Press, 1989]. Or when using commercially available reagents and kits, they were used according to the instructions for commercial products.

Example 1 Cloning of human cDNA of liver-specific organic anion transporter gene that selectively transports sulfate conjugate (1) Isolation of human cDNA of liver-specific organic anion transporter OAT7 that selectively transports sulfate conjugate Preparation of cRNA An EST (expressed sequence tag) using the base sequence of the translation region of OAT1 A sequence corresponding to an OAT1-like sequence (GenBankTM / EBI / DDBJ accession No. AA705512) obtained by searching a database is derived from a human liver-derived poly ( A) Amplification was performed by reverse transcription-PCR using + RNA as a template, and the amplified product was labeled with ³² P-dCTP and a human liver cDNA library was screened as a probe.
A cDNA library was prepared from poly (A) + RNA derived from human liver using a cDNA synthesis kit (trade name: Superscript Choice System, manufactured by Gibco), and digested with the restriction enzyme EcoRI of the phage vector lZipLox (produced by Gibco). Incorporated into the site. Hybridization with the probe labeled with ³² P-dCTP was performed overnight in a hybridization solution at 37 ° C., and the filter membrane was washed with 0.1 × SSC / 0.1% SDS at 37 ° C. As a solution for hybridization, 5 × SSC, 3 × Denhard's solution 0.2% SDS, 10% dextran sulfate, 50% formamide, 0.01% Abformform B (trade name, Sigma) (antifoam) Agent), 0.2 mg / ml salmon sperm denatured DNA, 2.5 mM sodium pyrophosphate, pH 6.5 buffer containing 25 mM MES was used. The cDNA portion of lZipLox phage into which cDNA was incorporated was incorporated into plasmid pZL1.

For the obtained clone, ie, a clone containing human OAT7 cDNA, the nucleotide sequence of the cDNA was determined by the dye terminator cycle sequencing method (Applied Biosystems) using synthetic primers for nucleotide sequencing.
Thereby, the base sequence of the human OAT7 gene was obtained. Further, the cDNA base sequence was analyzed by a conventional method to determine the cDNA translation region and the amino acid sequence of human OAT7 encoded therein.
These sequences are shown in SEQ ID NO: 2 in the Sequence Listing described later.
OAT7 has 42% amino acid sequence homology with human organic anion transporter OAT1 and rat organic anion transporter OAT2 with 35%, human organic anion transporter OAT3 with 41%, and human organic anion transporter OAT4 with 46%. Was.
A comparison of the amino acid sequences of OAT7 and human OAT1, rat OAT2, human OAT3 and human OAT4 is shown in FIG. In FIG. 1, the expected transmembrane site is indicated by an attached line. The predicted glycosylation site is indicated by *, and the expected C-kinase-dependent phosphorylation site is indicated by #. In addition, the molecular evolution of human OAT7, human OAT1, rat OAT2, human OAT2, human OAT3, human OAT4 and similar organic cation transporters human OCT1, human OCT2, and human OCT3. The phylogenetic tree is shown in FIG.
TopPred2 algorithm [von Heijne, G. et al., J. Mol. Biol., 225, 487 (1992); Cserzo, M. et al., Protein Eng, 10, 673 ( 1997)], as a result of analyzing the amino acid sequence of OAT7, 12 membrane-spanning domains were predicted as shown in FIG. In addition, there were sugar chain addition sites in the first hydrophilic loop, and sites considered as protein kinase C-dependent phosphorylation sites in the second, fourth, and sixth hydrophilic loops.

(2) Identification of human OAT7 chromosomal gene and determination of exon-intron construction As a result of searching the NCBI human genome sequence database using the nucleotide sequence of human OAT7 cDNA, Contic RP11-151E18 mapped on chromosome 11 (Accession No. AP002367) revealed that it contains the same sequence as the base sequence of human OAT7. The exon-intron construction of human OAT7 was determined by comparing the base sequence of this contic with the base sequence of human OAT7 cDNA. The results are shown in FIG.

(3) Expression of OAT7 gene in various human tissues (analysis by Northern blotting)
The full length of the OAT7 cDNA was excised with EcoRI, labeled with ³² P-dCTP, and used as a probe with Human Blot (Clontech), followed by hybridization according to the attached protocol.
As a result of Northern blotting (FIG. 4A), bands were detected in the vicinity of 2.4, 3.0, and 4.4 kb only in the liver. Further, in Northern blotting of fetal tissue, fetal liver, fetal kidney, fetal lung, and fetal brain were compared, and expression was detected only in fetal liver (FIG. 4B). The band size was the same as that of adult liver.

(4) Expression of OAT7 protein in human liver A specific antibody against a synthetic oligopeptide [CKQEDPRVEVTQ] corresponding to human OAT7 542-552 was prepared according to the method of Altman et al. [Altman et al., Proc. Natl. Acad. Sci. USA 81, 2176-2180, 1984]. An N-terminal cysteine residue was introduced for KLH conjugation.
Human liver total protein and human liver protein membrane fraction (both purchased from BIOCHAIN) were electrophoresed on SDS-polyacrylamide gel, blotted onto Hybond-P PVDV transfer membrane, and peptide affinity purified anti-OAT7 antibody (1: 100) ).
As a result, as shown in FIG. 5A, a band was detected in the vicinity of 50 kDa with anti-OAT7 antibody in both human liver total protein and human liver protein membrane fraction.

(5) Immunohistochemical analysis of OAT7 protein in human liver According to a conventional method, human liver paraffin sections (purchased from BIOCHAIN) were treated with peptide affinity purified anti-OAT7 antibody (1: 100) and developed with diaminobenzidine. . For the purpose of examining the specificity of staining, an experiment was also conducted in which a peptide affinity-purified anti-OAT7 antibody (1: 100) was treated in the presence of 200 mg / ml antigenic peptide.
As a result, as shown in FIG. 5C, staining was observed in hepatocytes. This staining was not detected when anti-OAT7 antiserum was allowed to act in the presence of the antigenic peptide, indicating the specificity of the staining (FIG. 5E). Further observation with strong magnification revealed that OAT7 protein was present in the basolateral membrane of hepatocytes (FIG. 5D).

Example 2 Characterization of liver-specific organic anion transporter OAT7 that selectively transports sulfate conjugates (1) Transport activity of OAT7 Human OAT7 gene cRNA was expressed in oocytes and [ ³ H] estrone-sulfate conjugates ([ ³ H] estrone sulfate) and [ ³ H] dehydroepiandrosterone-sulfate conjugate ([ ³ H] dehydroepiandrosterone sulfate) were measured.
It was expressed by injecting 20 ng of human OAT7 gene cRNA into oocytes and cultured for 3 days.
For an oocyte injected with human OAT7 gene cRNA and an oocyte injected with water as a control, [ ³ H] estrone-sulfate conjugate and [ ³ H] dehydroepiandrosterone-sulfate conjugate were used as substrates. In accordance with the method of Kanai et al. (Kanai and Hediger, Nature, Vol. 360, 467-471, 1992). ND96 solution [96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM containing [ ³ H] estrone-sulfate conjugate (50 nM) and [ ³ H] dehydroepiandrosterone-sulfate conjugate (100 nM) as substrates The oocytes were left in magnesium chloride, 5 mM HEPES, pH 7.4] for 60 minutes, and the substrate uptake rate was measured by counting the radioactivity incorporated into the cells.
As a result (FIG. 6A), [ ³ H] estrone-sulfate conjugate uptake and [ ³ H] dehydroepiandrosterone-sulfate conjugate uptake were injected with water as a control in the oocytes expressing OAT7. Compared with the oocyte, it showed a significantly large value.

Using sulfuric acid conjugate (50nM), - (2) for OAT7 the transport activity of time-dependent human OAT7 oocytes gene cRNA was injected and oocytes water was injected as a control, as a substrate ^[3 H] estrone According to the method of Example 2 (1), the time course of substrate uptake was observed.
As a result (FIG. 6B), it was revealed that the uptake of [ ³ H] estrone-sulfate conjugate via OAT7 increases almost linearly in a time-dependent manner up to 120 minutes.

(3) Sodium ion dependence of transport activity of OAT7 In the [ ³ H] estrone-sulfate conjugate (50 nM) uptake experiment, oocytes injected with human OAT7 gene cRNA and oocytes injected with water as a control The effect of the salt added to was investigated.
The [ ³ H] estrone-sulfate conjugate uptake experiment was performed using oocytes injected with human OAT7 gene cRNA according to the method described in Example 2 (1). However, for the purpose of examining the influence of sodium ions, a sodium ion-free absorption solution (96 mM sodium chloride was changed to 96 mM choline chloride) was used instead of the standard absorption solution.
As a result (FIG. 6C), even by changing the sodium extracellular choline, [3 ^H] estrone - did not have any effect on the sulfate conjugate (50 nM) uptake. This indicates that OAT7 is a transporter that works independently of sodium ions.

(4) Michaelis-Menten kinetic test of OAT7 Michaelis-Menten kinetic test of liver-specific organic anion transporter OAT7 that selectively transports sulfate conjugates was performed. Substrate ^{[3 H]} estrone - sulfate conjugate and ^{[3 H]} dehydroepiandrosterone - the substrate due to the difference in the concentration of sulfate conjugate ^{[3 H]} estrone - sulfate conjugate and ^{[3 H]} dehydroepiandrosterone - sulfate Idaku The Michaelis-Menten kinetics test for OAT7 was performed by examining the change in incorporation rate.
The uptake experiment of [ ³ H] estrone-sulfate conjugate and [ ³ H] dehydroepiandrosterone-sulfate conjugate was carried out using the oocyte injected with human OAT7 gene cRNA in the method described in Example 2 (1). It carried out according to. As a result (FIGS. 7A and 7B), the Km values were 8.7 ± 1.1 mM and 2.2 ± 0.3 mM (mean ± standard error), respectively.

(5) Substrate selectivity of OAT7 (inhibition experiment by adding various organic anions and organic cations)
In the uptake experiment of [ ³ H] estrone-sulfate conjugate by oocytes injected with human OAT7 gene cRNA, the influence of various organic anions and organic cations added to the system was examined.
The [ ³ H] estrone-sulfate conjugate uptake experiment was performed using oocytes injected with human OAT7 gene cRNA according to the method described in Example 2 (1). However, the uptake of [ ³ H] estrone-sulfate conjugate (1 mM) was measured in the presence and absence of 100 mM of various compounds (unlabeled).
As a result, among the examined organic anions and organic cations shown in FIG. 8, estrone sulfate, estrone sulfate, dehydroepiandrosterone sulfate, and β-estradiol-sulfate conjugates ( A significant cis-inhibitory effect was observed with β-estradiol sulfate). However, no significant cis-inhibitory effect was observed for the other compounds shown in FIG.
Since OAT7 is an organic anion transporter existing in the liver, the effect of various organic anions transported in hepatocytes on the uptake of [ ³ H] estrone-sulfate conjugates via OAT7 was examined. did.
As a result, among the examined organic anions shown in FIG. 9, a significant cis-inhibiting effect was observed with sulfobromophthalein (BSP) and indocyanin green (ICG). However, no significant cis-inhibiting effect was observed for the other compounds shown in FIG.
OAT7 incorporates [ ³ H] estrone-sulfate conjugates of various conjugate compounds in order to determine which of the sulfate conjugates, glucuronic acid conjugates, and glutathionic acid conjugates is acceptable. The effect on was investigated.
As a result, among the examined conjugate compounds shown in FIGS. 10A and 10B, estrone-sulfate conjugate, dehydroepiandrosterone-sulfate conjugate, p-nitrophenol-sulfate conjugate (P-nitrophenyl sulfate), β-estradiol-sulfate conjugate (β-estradiol sulfate), 4-methylumbelliferone-sulfate conjugate (4-methlumbelliferyl sulfate), and minoxidil-sulfate conjugate (significant) A cis-inhibitory effect was observed. However, no significant cis-inhibiting effect was observed in other compounds including the glucuronic acid conjugate and glutathionic acid conjugate shown in FIGS. 10A and 10B.

The figure which shows the comparison of the amino acid sequence of human OAT7, human OAT1, rat OAT2, human OAT3, and human OAT4. Expected transmembrane sites are indicated by a line. The predicted glycosylation site is indicated by *, and the expected C-kinase-dependent phosphorylation site is indicated by #. Phylogenetic tree of molecular evolution constructed based on the amino acid sequences of human OAT7, human OAT1, rat OAT2, human OAT2, human OAT3, human OAT4 and similar organic cation transporters human OCT1, human OCT2, and human OCT3 FIG. The figure which shows the exon-intron construction | assembly of a human OAT7 chromosome gene. The photograph which showed the result of having analyzed the expression of OAT7 gene mRNA in each human organ tissue by Northern blotting. A: Adult tissue and placenta. B: Fetal tissue. A: Photograph showing the results of Western blot analysis for human liver protein with anti-OAT7 antibody. B to E: Photographs showing the results of immunohistochemical analysis of OAT7 with anti-OAT7 antibody in human liver. B. Reacted without adding anti-OAT7 antibody. No staining is observed. C. Slightly enlarged image of the stained image with anti-OAT7 antibody. Staining is seen throughout the liver. D. Strongly magnified image of an image stained with an anti-OAT7 antibody. Staining is seen in the basolateral membrane of hepatocytes. E. Absorption experiment with antigenic peptide. The staining detected with D disappeared, indicating the specificity of the staining. A: [ ³ H] estrone-sulfate conjugate ([ ³ H] estrone sulfate) and [ ³ H] dehydroepiandrosterone-sulfate conjugate ([ ³ H] dehydroepiandrosterone sulfate) by oocytes injected with human OAT7 gene cRNA ) Diagram showing results of uptake experiment. B: Diagram showing the time course of [ ³ H] estrone-sulfate conjugate uptake by oocytes injected with human OAT7 gene cRNA. C: Human OAT7 ^{[3 H]} by the oocytes injected with gene cRNA estrone - shows the results of examining the effect of salt added in the sulfate conjugate uptake experiments. Substrate estrone-sulfate conjugates and dehydroepiandrosterone-sulfate conjugates in [ ³ H] estrone-sulfate conjugate and [ ³ H] dehydroepiandrosterone-sulfate conjugate uptake experiments by oocytes injected with human OAT7 gene cRNA The figure which shows the result of having investigated the influence of the density | concentration of coalescence. The figure which shows the result of having investigated the influence of various organic anions and organic cation addition to a system in the [< ³ > H] estrone-sulfate conjugate uptake | capture experiment by the oocyte which injected human OAT7 gene cRNA. The figure which shows the result of having investigated the influence of the organic anion addition considered to be transported in various hepatocytes to the system in the [< ³ > H] estrone-sulfate conjugate uptake experiment by the oocyte which inject | poured human OAT7 gene cRNA. . The figure which shows the result of having investigated the influence of various conjugate compounds addition to a system in the [< ³ > H] estrone-sulfate conjugate uptake experiment by the oocyte which inject | poured human OAT7 gene cRNA.

Claims (24)

A protein selected from the following (A) and (B), which has an ability to selectively transport a sulfate conjugate.
(A) Protein comprising the amino acid sequence represented by SEQ ID NO: 1 (B) Protein comprising the amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1   The protein according to claim 1, which is derived from a mammal.   The protein according to claim 2, which is derived from human.   The protein according to any one of claims 1 to 3, which is derived from an organ, a tissue, or a cultured cell.   The gene which codes the protein in any one of Claims 1-4. A gene comprising a DNA selected from the following (a) and (b), which encodes a protein having an ability to selectively transport a sulfate conjugate.
(A) DNA consisting of the base sequence represented by SEQ ID NO: 2.
(B) DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence represented by SEQ ID NO: 2.   The gene according to claim 5 or 6, which is derived from a mammal.   The gene according to claim 7, which is derived from human.   The gene according to any one of claims 5 to 8, which is derived from an organ, tissue or cultured cell.   A vector comprising the gene according to claim 5 or a gene encoding a protein in the gene.   The vector according to claim 10, which is an expression plasmid.   A transformant transformed with the vector according to claim 10 or 11.   A nucleotide comprising a partial sequence of 14 or more consecutive bases in the base sequence represented by SEQ ID NO: 2 or a complementary sequence thereof.   The nucleotide according to claim 13, which is used as a probe for detecting a gene encoding a protein having an ability to selectively transport a sulfate conjugate.   14. The nucleotide according to claim 13, which is used for modulating the expression of a gene encoding a protein having the ability to selectively transport sulfate conjugates.   The antibody with respect to the protein in any one of Claims 1-4.   A method for detecting the action of a test substance as a substrate on the ability of the protein according to claim 1 to selectively transport a sulfate conjugate of the protein.   A method for detecting the action of a test substance as an inhibitor on the ability to selectively transport a sulfate conjugate of the protein using the protein according to claim 1.   A method for detecting a drug-drug interaction in mass transport through the protein using the protein according to claim 1.   Using the protein according to any one of claims 1 to 4, a specific antibody thereof, a function-promoting substance or a function-inhibiting substance, by modulating the ability to selectively transport a sulfate conjugate of the protein, a drug A method of altering kinetics.   By using the protein according to any one of claims 1 to 4, a specific antibody thereof, a function promoting substance or a function suppressing substance thereof, and modulating the ability of the protein to selectively transport a sulfate conjugate, a toxic substance Or a method of altering the pharmacokinetics of a foreign substance.   Using the protein according to any one of claims 1 to 4, a specific antibody thereof, a function-promoting substance or a function-inhibiting substance, by modulating the ability of the protein to selectively transport a sulfate conjugate, a steroid A method of altering the pharmacokinetics of hormones.   A method for reducing side effects of a drug and a toxic effect of a foreign substance by the method according to claim 21.   Using the protein according to any one of claims 1 to 4, a specific antibody thereof, a function promoting substance or a function suppressing substance thereof, by modulating the ability of the protein to selectively transport a sulfate conjugate, liver A method for protecting cells from drugs and foreign substances.

Images