WO2004061106A1 - Novel mammal-origin protein negatively regulating the expression of antigen presentation-associated molecule such as b7-2 molecule or mhc class ii molecule on cell surface and utilization of the same - Google Patents

Abstract

Human c-MIR cloned presently is a novel protein originating in a mammal such as a human homolog having an immune suppression function which is a molecule being similar in function to viral proteins, for example, K3 and K5 proteins of Kaposi’s sarcoma-associated herpes virus. By analyzing the functions, it is observed that c-MIR has an immune suppression effect of inhibiting the expression of B7-2 molecule and MHC class II molecule, which are antigen presentation-associated molecules, on the cell surface. Because of having this immune suppression effect, c-MIR is considered as being a factor controlling the expression of an antigen presentation-associated molecule in a human immune system and, therefore, expected as usable in developing a new immune suppression method, inhibiting rejections in cell and organ transplantation, treating autoimmune diseases, developing new drugs and so on and thus highly useful in medicine and industries.

Description

 Specification

Novel mammalian-derived proteins that negatively regulate cell surface expression of antigen-presentation-related molecules such as B7-2 molecules or MHC class II molecules and their uses

 The present invention relates to a novel mammal-derived protein that negatively regulates cell surface expression of an antigen-presentation-related molecule such as a B7-2 molecule or an MHC class II molecule, and its use. Background art

 The virus infects humans and cleverly evades immunological elimination, causing persistent and latent infections. This phenomenon is often observed in herpes virus and hepatitis virus, but recently, (1) proteins expressed by the virus have been expressed on the cell surface of antigen-presentation-related molecules (MHC etc.) that are important for immune recognition. (2) This indicates that the virus evades virus recognition by cytotoxic T lymphocyte (CTL) and natural killer cell (NK), and the virus can avoid immunological elimination. (For example, see Reference 1: Immunity, 2000 Sep: 13 (3): 365-374 and Reference 2: Journal of Virology, 2000 Jun: 74 (ll): 5300-5309).

More specifically, the K3 and K5 proteins (also called “MIR1” and “MIR2”) of lipopositive sarcoma-associated herpes virus (KSHV) and mouse γ-herpes virus -68 (MHV-68), respectively. Viral proteins, such as the Κ3 protein, function as キ 3 ubiquitin ligase and release MHC class I molecules on the cell surface. It has been found that the present is controlled negatively. These viral proteins share a catalytic site for ligase activity called the BKS-PHD / LAP zinc finger domain, and ubiquitinate MHC class I molecules via this domain. This ubiquitination induces rapid end-site lysis and degradation of MHC class I molecules. By virtue of the K3 and K5 proteins (immunosuppressive action), the lipopositive sarcoma-associated virus suppresses the expression of MHC class I molecules on the cell surface, thereby circumventing immunological elimination.

 As described above, the myelopositive sarcoma-associated virus and the like have an immunosuppressive mechanism by the viral proteins such as the K3 and Κ5 proteins. However, the present inventors have analyzed the functions of the Κ3 and Κ5 proteins. As we proceeded, we thought that the genes encoding these viral proteins were derived from the mammalian genome, and that genes encoding functionally similar molecules of these viral proteins might also be present in the mammalian genome, especially in the human genome.

 If a human homologue having such an immunosuppressive function can be found, it will not only help to elucidate the immunoregulatory mechanism of humans, but also, for example, develop a method to efficiently and artificially suppress immunosuppression. It was also considered that this method could be applied to the suppression of rejection during cell and organ transplantation, and the treatment of autoimmune diseases.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel mammalian-derived protein such as a human homologue having an immunosuppressive function, which is a molecule similar in function to a viral protein such as the Κ3 and Κ5 proteins. The purpose is to provide headlines and how to use them. Disclosure of the invention In view of the above problems, the present inventors searched for a human homolog of the K3 and K5 proteins in a human genome, and as a result, identified a plurality of genes and proteins as human homologs. As a result of further detailed functional analysis, several proteins were found to have immunosuppressive effects on cell surface expression of antigen presentation-related molecules (分子 7-2 costimulatory molecules and MHC class II molecules) that are important for immune recognition. As a result, they have found that the functions are similar to the above-mentioned proteins # 3 and # 5, and have completed the present invention.

 That is, the present invention includes the following A) to A) as medically or industrially useful methods and substances.

 Ii) A mammal-derived protein described in (a) or (b) below.

 (a) a protein comprising any one of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16;

 () In any of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16, one or several amino acids are substituted, deleted, or inserted. A protein comprising an amino acid sequence with or without an amino acid, and which negatively regulates the expression of antigen-presentation-related molecules on the cell surface.

 B) The protein according to A) above, which negatively regulates the expression of a B7-2 molecule, which is an antigen-presentation-related molecule, on the cell surface.

 C) The protein of B) above, which ubiquitinates the B7-2 molecule.

 D) The protein according to any one of A) to C) above, which negatively controls the expression of MHC class II molecules on the cell surface, which are molecules related to antigen presentation.

E) The protein according to any one of A) to D) above, wherein the protein is human or Is a mouse-derived E3 ubiquitin ligase protein.

 F) An antibody against the protein according to any one of A) to E). G) A gene encoding the protein according to any one of A) to E).

 H) The gene of G) above, wherein SEQ ID NOs: 1, 3, 5, 7, 9, 1

A gene having any one of the nucleotide sequences of 1, 13 and 15 as an open reading frame region.

 I) Under stringent conditions with a DNA consisting of a nucleotide sequence complementary to a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15 A gene encoding a protein that hybridizes with and negatively regulates the expression of antigen-presentation-related molecules on the cell surface.

 J) A recombinant expression vector containing any one of the above genes G) to 1).

K) A transformant into which any one of the above genes G) to 1) has been introduced.

L) A gene detection instrument using, as a probe, at least a part of the nucleotide sequence of any of the above genes G) to 1) or a complementary sequence thereof.

 M) The protein according to any one of A) to E) above, or G

Or an immunosuppressant comprising the gene of H).

 N) A method for screening a substance which regulates the action of a protein according to any one of the above A) to E).

 O) A method for screening a substance having an action of negatively controlling the expression of an antigen-presentation-related molecule on the cell surface, similarly to the protein according to any one of the above A) to E).

P) The disk described in M) or N) above, wherein any one of the following substances (1) to (3) and one of the following substances (4) to (7) are used: Reading method.

 (1) The protein according to any one of the above A) to E)

 (2) A partial protein of the protein according to any one of the above (A) to (E), which comprises at least a transmembrane region in the amino acid sequence of the full-length protein.

 (3) A variant of the protein of (1) or the partial protein of (2)

(4) B7-2 molecule or MHC class II molecule

 (5) B7-2 molecule or variant of MHC class II molecule

 (6) A partial protein of the B7-2 molecule or MHC class II molecule, which contains at least the transmembrane region or its intracellular region in the amino acid sequence of the full-length protein

 (7) A variant of the partial protein of (6) above

 Q) A therapeutic drug for an immunological disease obtained by using the screening method according to any one of the above N) to P).

 The present invention relates to a novel mammal-derived protein that negatively regulates the expression of B7-2 molecule or MHC class II molecule on the cell surface and its use.As described above, the development of a new immunosuppression method, cell, It can be used for suppressing rejection during organ transplantation, treating autoimmune diseases, developing new drugs, and has various other usefulness.

Further objects, features, and advantages of the present invention will be made clear by the description below. Also, the advantages of the present invention will become apparent in the following description with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 (a) shows the amino acid sequence of the protein c-MIR of the present invention, and FIG. 1 (b) shows whether c-MIR has the effect of suppressing the expression of antigen-presentation-related molecules. Fig. 2 shows the results of an investigation using the c-MIR method.Fig. 2 is a flow cytometry using c-MIR constitutively expressing cells to determine whether c-MIR has the effect of suppressing the expression of antigen presentation-related molecules. FIG. 6 is a diagram showing the results of an examination by the method.

 Figures 3 (a) to 3 (d) show how c-MIR suppresses the expression of B7-2 molecule. It is a figure showing the result of examination.

 FIGS. 4 (a) and 4 (b) show the results of examining whether the B7-2 molecule is rapidly endocytosed by c-MIR.

 Figure 5 shows the results of using a chimeric protein to determine whether the transmembrane region and cytoplasmic region of the B7-2 molecule are sufficient for c-MIR-mediated downregulation of the B7-2 molecule. It is.

 FIGS. 6 (a) to 6 (c) show the results of examining whether the BKS-PHD / LAP domain of c-MIR has E3 ubiquitin ligase activity.

 FIGS. 7 (a) to 7 (c) show the results of examining whether ubiquitination of B7-2 molecule is essential for down-regulation of B7-2 molecule via c-MIR. .

 Figures 8 (a)-(d) show whether the intermolecular interaction between c-MIR and the B7-2 molecule is essential for ubiquitination of the B7-2 molecule and its down-regulation. It is a figure showing a result.

FIG. 9 is a diagram illustrating the molecular mechanism of c-MIR. FIG. 10 shows the amino acid sequence of the protein HSPC240 of the present invention.

 FIG. 11 is a diagram showing the results of examining whether HSPC240 has an effect of suppressing the expression of antigen-presentation-related molecules by flow cytometry.

 FIGS. 12 (a) and (b) are diagrams showing the results of suppression of the expression of MHC class II molecule and B7-2 molecule by c-MIR, which suppresses the function of antigen presentation. BEST MODE FOR CARRYING OUT THE INVENTION

 The following will describe one embodiment of the present invention. Note that the present invention is not limited to this.

 (I) Structure of the protein of the present invention and its gene

 The present inventor has conducted studies on the existence of the human homologues of the K3 and K5 proteins of KSHV. Six genes' proteins were identified as similar human homologs.

SEQ ID NOs: 1, 3, 5, 7, 9, and 11 show the base sequences of the six genes c-MIR, HSPC240, 43171, XP-055276, CAD28529, and 20445, respectively, in their open form. It is shown with each amino acid sequence encoded by the reading frame (ORF) region. The amino acid sequences of the proteins encoded by the above six genes are shown in SEQ ID NOs: 2, 4, 6, 8, 10, and 12, respectively. The amino acid sequences of c-MIR and HSP C240 are also shown in FIGS. 1 (a) and 10 respectively. In FIG. 1 (a), “TM1” is a region determined to be the first transmembrane region of c-MIR, and “TM2” is a region determined to be the second transmembrane region. BKS-PHD / LAP Jin Tough The inger motif is located on the amino terminal side of the first transmembrane region. For the above HSPC240, its BKS-PHD / LAP zinc finger motif is underlined in FIG.

 As a result of conducting a detailed analysis on the above c-MIR, as described in detail in the examples below, the following knowledge was obtained regarding its action and properties.

 (1) c-MIR ubiquitinates B7-2 costimulatory molecule (B7-2 molecule), one of the antigen presentation-related molecules, and negatively regulates its expression on the cell surface.

 (2) The ubiquitination of the B7-2 molecule causes rapid endcytosis and degradation in the lysosome of the B7-2 molecule. That is, c-MIR ubiquitinates the B7-2 molecule, thereby inducing rapid endosite lysis and degradation of the B7-2 molecule.

 (3) c-MIR binds to the transmembrane region of the B7-2 molecule or its nearby intracellular region via its transmembrane region. This binding is essential for ubiquitination of the B7-2 molecule.

 (4) c-MIR also negatively regulates cell surface expression of MHC class II molecules, other antigen presentation-related molecules.

 (5) c-MIR functions as an E3 ubiquitin ligase like the K3 and K5 proteins, and is a catalytic site whose BKS-PHD / LAP zinc finger domain has ligase activity.

Thus, c-MIR has an effect of negatively regulating the expression of B7-2 molecules and MHC class II molecules on the cell surface, in other words, an immunosuppressive effect of suppressing the expression of these antigen presentation-related molecules. Admitted. Therefore, c-MIR is considered to be a regulator that regulates the expression of antigen-presentation-related molecules in the human immune system by such an immunosuppressive effect. As for the above-mentioned HSPC240, which is a structural analog of c-MIR, the effect of negatively controlling the expression of the B7-2 molecule on the cell surface, in other words, suppressing the expression of antigen-presentation-related molecules, was also described as a result of the functional analysis. An immunosuppressive effect was observed. Similarly, it is highly probable that the remaining four molecules that are structurally similar to c-MIR also have the effect of suppressing the expression of antigen-presentation-related molecules. It can be said to be a regulator that suppresses expression and regulates antigen presentation and immune recognition in the human immune system by such an immunosuppressive action.

 Furthermore, the present inventors cloned the mouse homologues of c-MIR and HSPC240, and analyzed their functions. SEQ ID NOS: 13 and 15 show the respective base sequences of the above mouse homologues of c-MIR and HSPC240, together with the respective amino acid sequences encoded by their open reading frame (ORF) regions. The amino acid sequences of the proteins respectively encoded by the above two genes are shown in SEQ ID NOs: 14 and 16.

In the mouse homologue of the above c-MIR, the gene was introduced into A20 cells, and the expression of MHC class I molecules and MHC class II molecules was examined. As a result, specific suppression of MHC class II molecules was observed. In addition, the above mouse homologue of HSPC240 was transfected into A20.2J cells, and the expression of B7-2 molecule, MHC class I molecule, and MHC class II molecule was examined. Suppression was observed. These results suggest that the mouse homologues of c-MIR and HSPC240 also have a function to specifically suppress the expression of any of the antigen-presentation-related molecules, similar to the human gene. . Furthermore, it was also clarified that the specific suppression of MHC class II molecules and B7-2 molecules by c-MIR strongly suppressed the antigen presenting function. Therefore, it is suggested that c-MIR may suppress the hyperimmune reaction. These are considered to give an interesting perspective on the origin of virus immune evasion molecules, and it is thought that elucidation of these physiological functions will reveal new immune control mechanisms.

 In addition, the protein of the present invention is not limited to the above eight molecules, and may be a mutant protein partially modified. That is, the protein of the present invention includes not only (a) a protein consisting of any of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 10, 12, 14, or 16, but also (B) in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 10, 12, 14, or 16, one or several amino acids are replaced or deleted. It also includes a protein comprising a lost, inserted, and Z or added amino acid sequence, and which negatively regulates cell surface expression of an antigen presenting-related molecule.

The above-mentioned “one or several amino acids are substituted, deleted, inserted, and Z or added” means that substitution, deletion, or insertion is performed by a known method for producing a mutant protein such as site-directed mutagenesis. , And Z or the number of amino acids that can be added (preferably 10 or less, more preferably 7 or less, and still more preferably 5 or less) are substituted, deleted, inserted, and / or added. means. As described above, the protein of (b) is a mutant protein of the protein of (a), and the “mutation” referred to herein is mainly a mutation artificially introduced by a known method for producing a mutant protein. However, it may be a protein obtained by isolating and purifying a similar naturally occurring mutant protein. Further, the protein according to the present invention may include an additional polypeptide. Examples of the case where such a polypeptide is added include a case where the protein of the present invention is labeled with an epitope by His, Myc, Flag, or the like.

 The gene of the present invention encodes the protein of the above (a) or (b), and may be any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 or 15. It includes a gene having a nucleotide sequence as an open reading frame region, and a modified gene obtained by modifying a part of the nucleotide sequence.

 The “gene” of the present invention includes DNA and RNA. D

NA includes, for example, cDNA and genomic DNA obtained by cloning and chemical synthesis techniques or a combination thereof. The DNA / RNA may be double-stranded or single-stranded, and the single-stranded DNA / RNA may be a coding strand serving as a sense strand or an anti-coding strand serving as an antisense strand (antisense The strand can be used as a probe or as an antisense drug). Further, siRNA obtained by using a part of the gene sequence of the present invention is also useful.

Further, the "gene" of the present invention includes, in addition to the sequence encoding the protein of (a) or (b), a sequence of an untranslated region (UTR) or a vector sequence (including an expression vector sequence). It may be something. Furthermore, the gene according to the present invention is limited to a gene having the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15 as an ORF region. Not the base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 11, 13 or 15. And DNA that hybridizes under stringent conditions. The above-mentioned "stringent conditions" means that at least 90% identity, preferably at least 95% identity, and most preferably at least 97% identity between sequences. Means that hybridization occurs only when present in

 The above hybridization can be performed by a conventionally known method such as the method described in J. Sambrook et al. Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory (1989). . Generally, the higher the temperature and the lower the salt concentration, the higher the stringency (the less likely it is to hybridize).

 (II) Method of using the protein of the present invention and its gene (utility) The B7-2 molecule assists in the presentation of antigen to T cells on the surface of antigen presenting cells (dendritic cells, B cells, etc.) It functions as a modulator of immune synapse formation between cells and antigen presenting cells. Immune synapse formation activates T cells. Signaling by the B7-2 molecule has also been implicated in the progression of autoimmune diseases.

The protein of the present invention has an immunosuppressive effect of suppressing the expression of antigen-presentation-related molecules such as the B7-2 molecule, and is considered to be a regulator that regulates antigen presentation important for immune recognition in the human immune system. Thus, the protein of the present invention and the gene thereof are useful not only for elucidating the mechanism of human immune regulation but also for various medical and industrial uses. For example, it can be used for the development of an efficient and artificial method of immunosuppression as an immunosuppressant, and is also used in the suppression of rejection during cell and organ transplantation and the treatment of autoimmune diseases. The protein and gene of the invention are applicable. Further, the protein of the present invention and the mammalian homologue of the gene thereof are useful in experiments for examining the immunosuppressive effect using model animals, and such mammalian homologues are also included in the present invention. For example, it is useful for experiments in which a rejection reaction model is prepared using small animals such as mice and the immunosuppressive effect is examined in vivo. In fact, the present inventors have cloned the mouse homologues of c-MIR and HSPC240 and analyzed their functions.As described above, the expression of the B7-2 molecule or MHC class II molecule was similar to that of the human gene as described above. The function of specifically suppressing was observed.

 Further, the protein of the present invention and its gene are useful for screening candidate molecules (drug discovery targets) of drugs. For example, (1) a method for screening a substance that regulates the action of the protein of the present invention;

(2) Similar to the protein of the present invention, it can be used for a method for screening a substance having an action of negatively controlling the expression of antigen-presenting-related molecules such as B7-2 molecule or MHC class II molecule on the cell surface, etc. It is. The substance (1) that regulates the action of the protein of the present invention is, for example, a substance that inhibits the binding of the protein of the present invention to a target molecule (such as a B7-2 molecule or MHC class II molecule). Substances that inhibit the immunosuppressive action of the protein of the present invention, or, on the other hand, substances that enhance the immunosuppressive action of the protein of the present invention. Such a substance can be used as a therapeutic or diagnostic agent for an immune disease, and a screening method thereof is also included in the present invention.

As the screening method of the present invention, various conventionally known methods for examining the presence or absence of bonding or dissociation between substances can be applied, and are not particularly limited. For example, the c-MIR binds to the transmembrane region of the B7-2 molecule or a nearby intracellular region via the transmembrane region. Since it is indispensable for ubiquitination of the B7-2 molecule (see Examples below), any of the following (1) to (3) and the following (4) are used in the in vitro reaction system (ceU-free system). ) To (7), and a screening method in which a molecule or the like that inhibits the binding of both molecules is detected from candidate molecules by ELISA or the like.

 (1) The protein of the present invention such as the above c-MIR

 (2) A partial protein of the protein of the present invention, which comprises at least a transmembrane region in the amino acid sequence of the full-length protein

(3) A variant of the protein of (1) or the partial protein of (2)

 (4) B7-2 molecule or MHC class II molecule

 (5) B7-2 molecule or variant of MHC class II molecule

 (6) A partial protein of the B7-2 molecule or MHC class II molecule, which contains at least the transmembrane region or its intracellular region in the amino acid sequence of the full-length protein

 (7) A variant of the partial protein of (1) above

 The above-mentioned “variant (of protein)” means that one or several (preferably 7 or less, more preferably 5 or less, and more preferably 3 or less) amino acids of the protein are substituted. , Deletion, insertion, and / or addition, when the protein is labeled with a tag such as His or Myc, or when the protein is a fluorescent protein (such as GFP luciferase) or another protein. The term is used to include the case where the protein is modified by phosphorylation, sugar chain binding, or the like.

Of course, the screening method of the present invention is not limited to the above method. Instead, screening may be performed in cells using cultured cells, etc., instead of screening in a cell-free system.

 In addition, (1) the transmembrane region of the protein of the present invention, such as c-MIR, or the transmembrane region of the B7-2 molecule or MHC class II molecule or the intracellular region in the vicinity thereof is fixed to the column and bound And (2) immunoprecipitation-immunoblotting methods to search for substances that inhibit the binding of c-MIR to B7-2 molecules or MHC class II molecules. Various conventionally known methods for examining the presence or absence of binding and the presence or absence of dissociation can be applied to the screening method of the present invention.

 Furthermore, in the screening method of the present invention, screening may be performed using proteins other than human, for example, mouse homologs, rat homologs, and homologs of other organisms.

 (I I I) Recombinant expression vector etc. of the present invention

 The recombinant expression vector of the present invention contains the gene of the present invention that encodes the protein of (a) or (b), and includes, for example, SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 1. A recombinant expression vector into which cDNA having any one of the nucleotide sequences shown in 3, and 15 has been inserted. Plasmid, phage, cosmid, or the like can be used for production of the recombinant expression vector, but is not particularly limited.

The transformant of the present invention is a transformant into which the gene of the present invention encoding the protein of (a) or (b) has been introduced. Here, "the gene has been introduced" means that the gene can be expressed in a target cell (host cell) by a known genetic engineering technique (gene manipulation technique). In addition, the above “transformants” include not only cells, tissues and organs but also animals It is a meaning including. The target animals are not particularly limited, but examples include mammals such as porcupines, pigs, higgins, goats, rabbits, dogs, cats, monoremots, nomsters, mice, and rats. You. In particular, rodents such as mice and rats are widely used as experimental animals and pathological model animals, among which a large number of inbred strains have been produced, fertilized egg culture, in vitro fertilization, etc. Mice with the above technology are preferred as experimental animals and pathological model animals.Knockout mice and the like are used for further analysis of the function of the above-mentioned protein c-MIR and its homologue, and diseases involving these proteins. It is useful for the development of diagnostic methods and the development of treatment methods.

 The gene detection device of the present invention uses at least a part of the nucleotide sequence of the gene of the present invention or its complementary sequence as a probe. The gene detection device can be used for detecting and measuring the expression pattern of the gene of the present invention under various conditions. Examples of the gene detection device of the present invention include a DNA chip in which the above-described probe that specifically hybridizes with the gene of the present invention is immobilized on a substrate (carrier).

 The antibody of the present invention is an antibody obtained as a polyclonal antibody or a monoclonal antibody by a known method using the protein of (a) or (b) or a partial peptide thereof as an antigen. The antibody of the present invention can be used for detection and measurement of the protein of the present invention, and may be used for diagnosis, treatment, and the like.

 (IV) Method for obtaining genes, proteins, etc. according to the present invention

 [IV-1: Gene acquisition method]

The method for obtaining the gene according to the present invention is not particularly limited, and each of the above methods can be obtained by various methods based on the above-described sequence information and the like. A DNA fragment containing the gene sequence can be isolated and cloned. For example, a probe that specifically hybridizes with a partial sequence of each of the above-mentioned cDNA sequences may be prepared, and a human or mouse genomic DNA library or cDNA library may be screened. As such a probe, any probe having any sequence and length may be used as long as it specifically hybridizes to at least a part of each cDNA sequence or its complementary sequence. In addition, each step in the above-described screening may be performed under a condition generally used.

 The clones obtained by the above screening can be analyzed in more detail by constructing a restriction map and determining their nucleotide sequences (sequencing). By these analyses, it can be easily confirmed whether a DNA fragment containing the gene sequence according to the present invention has been obtained.

 In addition, the sequence of the above-mentioned probe is selected from regions that are well conserved between human c-MIR and its analogous molecules (for example, BKS-PHD / LAP zinc finger domain). By screening genomic DNA (or cDNA) libraries of other organisms, there is a high possibility that genes encoding homologous molecules and related molecules having the same function as the above c-MIR can be isolated and cloned.

As a method for obtaining the gene according to the present invention, there is a method using amplification means such as PCR in addition to the above-mentioned screen-Jung method. For example, among the above cDNA sequences, primers are respectively prepared from the sequences on the 5, 5 'and 3' side (or their complementary sequences), and human or mouse genomic DNA ( Or cDNA), etc., and perform PCR, etc., to amplify the DNA region sandwiched between both primers, thereby obtaining the residue of the present invention. A large amount of DNA fragments containing genes can be obtained.

 [IV-2: Method for obtaining protein]

 The method for obtaining the protein according to the present invention is not particularly limited. For example, a recombinant expression vector into which each of the above cDNA sequences is introduced is prepared, and microorganisms such as Escherichia coli and yeast are prepared by a well-known method. Alternatively, the protein according to the present invention can be easily obtained by expressing the protein encoded by the cDNA and purifying the transformant by incorporating it into an animal cell or the like as a transformant.

 When a foreign gene is introduced into a host in this way, there are various expression vectors and hosts that incorporate a promoter that functions in the host for the expression of the foreign gene. You just have to choose one. The method for removing the produced protein varies depending on the host used and the properties of the protein, but the target protein can be relatively easily purified by using a tag or the like.

The method for producing a mutant protein is not particularly limited. For example, site-directed mutagenesis (Hashimoto-Gotoli, Gene 152, 271-275 (1995), etc.) Using a well-known mutant protein production method such as a method for producing a mutant protein by introducing a point mutation into the cDNA, or a method for producing a mutant strain by inserting a transposon, one or more nucleotide sequences in each of the above cDNAs are used. Can be produced by modifying the base such that it is substituted, deleted, inserted, and / or added. In addition, a commercially available kit (for example, QuikChange Site-Directed Mutagenesis Kit manufactured by Stratagene) may be used to prepare the mutant protein. Many examples of the mutant protein produced as described above having the same activity and function as the wild type are already known.On the other hand, a mutant protein is introduced by introducing a mutation so as to cause an abnormality in its activity and function. Many have already been made. For example, the mutant protein mZnc-MIR (a protein in which a mutation was introduced into the BKS-PHD / LAP zinc finger region of c-MIR) prepared in a later example could not ubiquitinate a target molecule.

 [IV-3: Gene detection instrument]

 The gene detection device uses a partial base sequence of the gene of the present invention or its complementary sequence as a probe. An example is a DNA chip in which an oligonucleotide (probe) is immobilized on a base (carrier). Here, the term “DNA chip” mainly refers to a synthetic DNA chip using a synthesized oligonucleotide as a probe, but also includes an attached DNA microarray using cDNA such as a PCR product as a probe. It shall be.

 The sequence used as a probe can be determined by a conventionally known method for identifying a characteristic sequence from a cDNA sequence. For example, SAGE: Serial Analysis oi Gene Expression method (Science 276: 1268, 1997; Cell 88) : 243, 1997; Science 270: 484, 1995; Nature 389: 300, 1997; U.S. Patent No. 5,695,937).

 It is to be noted that a known method may be employed for manufacturing the DNA chip. For example

When a synthetic oligonucleotide is used as the oligonucleotide, the oligonucleotide may be synthesized on a substrate by a combination of a photolithography technique and a solid-phase DNA synthesis technique. On the other hand, when cDNA is used as the oligonucleotide, the base is determined using an array machine. Just stick it on the board.

 In addition, similar to a general DNA chip, a perfect match probe (oligonucleotide) and a mismatch probe mono-substituted in the perfect match probe are arranged to further improve the detection accuracy of the gene. Is also good. Furthermore, in order to detect different genes in parallel, a DNA chip may be configured by immobilizing multiple types of oligonucleotides on the same substrate.

 [IV-4: Recombinant expression vector and transformant]

 The recombinant expression vector contains the gene of the present invention. The specific type of the vector is not particularly limited, and a vector that can be expressed in the host cell may be appropriately selected. That is, a promoter sequence is appropriately selected according to the type of the host cell to ensure that the gene is expressed.

A vector obtained by incorporating the gene of the present invention into various plasmids or the like may be used as an expression vector. Various markers may be used to confirm whether or not the gene of the present invention has been introduced into a host cell, and whether or not the gene has been reliably expressed in the host cell. For example, a gene deleted in a host cell is used as a marker, and a plasmid or the like containing this marker and the gene of the present invention is introduced into the host cell as an expression vector. Thus, the introduction of the gene of the present invention can be confirmed from the expression of the marker gene. Alternatively, the protein according to the present invention may be expressed as a fusion protein.For example, the protein according to the present invention may be used as a GFP fusion protein using, as a marker, green fluorescent protein GFP (Green Fluorescent Protein) derived from O jellyfish. It may be expressed. The host cell is not particularly limited, and conventionally known various cells can be suitably used. Specifically, cells derived from human or mouse, for example, bacteria such as Escherichia coli, yeast 1¾: (Saccharomyces cerevisiae fission yeast Sc zosaccnaromvc es pombe), A Caenorhabditis elegans Oocytes of the African amphitheater (Xenopas laevis), cultured cells of various mammals (rats, magpies, pigs, monkeys, etc.), or cultured cells of insects such as Drosophila melanogaster and Bombyx mori The method for introducing the above-described expression vector into a host cell, that is, the method for transformation is not particularly limited, and includes the electroporation method and the calcium phosphate method. Conventionally known methods such as the ribosome method and the DEAE dextran method can be suitably used.

 [IV-5: Antibody]

 Antibodies use the protein of the present invention or a partial peptide thereof as an antigen.

An antibody obtained as a polyclonal antibody or a monoclonal antibody by a known method. Known methods include, for example, the literature (I Antibodies by Harlow et al .: A laboratory manual (Cold Spring Harbor Laboratory, New York (1988)), and Iwasaki et al., “Monoclonal antibody hybridoma and ELISA, Kodansha ( 1991) ””. The antibody thus produced is effective for detecting the protein of the present invention.

 Hereinafter, the results of functional analysis performed on the protein of the present invention, c-MIR, and HSPC240 will be described with reference to the drawings.

[Example 1: Kaposi's sarcoma-associated herpesvirus (KSHV) K3,5 protein White functionIdentification of c-MIR as a structural homolog)

 The human homologues of KSHV K3 and K5 proteins were searched from the database of Celle Lagenomics. As a result, no sequence with high homology to the K3 and K5 proteins was found at the level of the entire amino acid sequence, but one clone, hCP36279, was found to contain the K3 and K5 proteins, secondary structure, and BKS-PHD / LAP zinc. The location of the finger and the relevant jinta finger were common. Since the amino-terminal sequence of hCP36279 was lacking in the database, the full-length amino acid sequence of hCP36279 was obtained from the NK cell line YTS by performing ACE analysis using the cap-trapping method. Were determined. The sequence is shown in FIG. 1 (a) (the sequence is also shown in SEQ ID NO: 2 in the sequence listing;). In the figure, the underlined TM1 and TM2 are transmembrane regions predicted by the PHDhtm program, and the other underlined are amino acid residues important for the BKS-PHD / LAP zinc finger structure. By RT-PCR analysis, this transcript was also found in BJAB cells and 293T cells.

 The present inventors have named this novel protein "cellular MIR (c-MIR)". In order to examine the functional similarity between this c-MIR and the K3 and Κ5 proteins, the His-tagged c-MIR was transiently expressed in BJAB cells by electroporation, and important for immunorecognition. The expression of antigen-presentation-related molecules on the cell surface was analyzed by two-color flow cytometry. The result is shown in Fig. 1 (b).

In the figure, MHC I, ICAM-1, and B7-2 are antigen presentation-related molecules, and are the results of examining the surface expression levels of MHC class I molecules, ICAM-1 molecules, and B7-2 molecules, respectively. In the above experiment, the GFP-c-MIR vector was transfused into BJAB cells, and 24 hours after introduction, the surface expression of each of the above molecules was performed. Levels and GFP expression levels were analyzed by flow cytometry. The vertical axis of the figure shows the surface expression levels of MHC class I molecules, ICAM-1 molecules, and B7-2 molecules, and the horizontal axis shows the GFP expression levels.

 As shown in the figure, as the expression of c-MIR increased, the expression of the B7-2 molecule on the cell surface was negatively regulated (ie, its expression was suppressed), but the MHC class I molecule and ICAM-1 were suppressed. The expression of the molecule was not negatively regulated. Similar results were obtained when c-MIR with a Flag tag was expressed and examined (data not shown).

 Furthermore, for detailed functional analysis of c-MIR, BJAB cells were modified to constitutively overexpress c-MIR (this cell is hereinafter referred to as “c-MIR cell”), and various antibodies were used. The expression of molecules related to antigen presentation (immune recognition) on the cell surface was examined by flow cytometry. The results are shown in Fig. 2.In the figure, ICAM-1, B7-2, Classl, and Class II (DR) are antigen-presenting-related molecules, and are ICAM-1 molecule, B7-2 molecule, MHC class I molecule, and MHC molecule, respectively. It is the result of examining the surface expression level of class II (HLA-DR) molecules. In the figure, c-MIR is the result of examining the above c-MIR cells that expressed c-MIR by gene transfer, and Cont was the result of control BJAB cells that did not express such c-MIR. Merge is a superposition of both results (the results for c-MIR cells are outlined).

As shown in the figure, the expression of B7-2 molecule and MHC class II molecule on the cell surface was significantly negatively regulated, while MHC class I molecule and ICAM-1 molecule were negatively regulated. Not controlled. Thus, the novel protein c-MIR was found to have an effect of specifically suppressing the expression of B7-2 molecules and MHC class II molecules. Was called.

 [Example 2: The above c-MIR induces rapid endocytosis and lysosomal degradation of B7-2 molecule]

To examine how the c-MIR suppresses the expression of the B7-2 molecule, we examined protein synthesis, degradation, and translocation of the B7-2 molecule. Therefore, BJAB cells (Cont) and c-MIR cells (c-MIR) were pulse-labeled with 35 g methionine and ³⁵ S cysteine for 30 minutes, and then followed for a specified period (Oh, lh, 3h, 6h). did. After each period, the cells were lysed and immunoprecipitated with anti-B7-2 antibody (upper panel) or anti-MHC I antibody (lower panel). The results are shown in Fig. 3 (a).

 Due to the higher degree of glycosylation of the B7-2 molecule, the mature B7-2 molecule migrated more slowly than the immature one. On the other hand, mature MHC I molecules did not migrate more slowly than immature ones, presumably due to the lower degree of glycosylation. Also, as shown in the figure, the amount of immature B7-2 molecule observed in both control cells (Cont) and c-MIR cells (c-MIR) at the start of tracking (0 o'clock) was almost the same. However, in control cells (Cont), the amount of mature B7-2 molecules did not decrease substantially until 6 hours, whereas in c-MIR cells (c-MIR), maturation was already completed after 1 hour. The amount of the B7-2 molecule was quite small. This result indicates that c-MIR induces rapid degradation of B7-2 molecule.

Rapid degradation of the target molecule was also observed in BJAB cells expressing KSHV K3 and K5 proteins, and this degradation has been reported to occur in lysosomes. Therefore, we examined whether the degradation of the B7-2 molecule induced by c-MIR occurs in lysosomes. In the experiment, c-MIR cells were After treatment with afilomycin Al for 2 to 4 hours, immunoblotting analysis was performed with an anti-B7'2 antibody (upper panel) or an anti-actin antibody (lower panel). The result is shown in Fig. 3 (b). Treatment of cells with bafilomycin A1 increases the pH in lysosomes by inhibiting vacuolar H ⁺ -ATPase. As a result, as shown in the figure, the expression of B7-2 protein was stably increased.

 Furthermore, after treatment with bafilomycin A1, pulse-chase analysis was performed in the same manner as in the experiment in Fig. 3 (a), and the result that bafilomycin Al inhibits the decomposition of B7-2 molecule was obtained. (See Fig. 3 (c): "Baf ilo +" in the figure was treated with bafilomycin Al, while ": Bafilo_" was treated with DMSO as a control.) These results indicate that c-MIR targets B7-2 molecules and degrades them in lysosomes.

Next, the intracellular kinetics of the B7-2 molecule was examined by a digestion experiment with Endo-H (endo-j3-N-acetylglucosamine nidase H). When the B7-2 molecule is modified with a darican, it becomes resistant to Endo-H digestion ', indicating that the B7-2 molecule has reached at least the intermediate Golgi. In the experiments, BJAB cells (Cont) or c-MIR cells (c-MIR) were pulse-labeled with ³⁵ S-methionine and ³⁵ S-cysteine for 30 minutes and chased for 15 to 30 minutes. After each period, the cells were lysed with a lysis buffer containing 0.1% SDS and immunoprecipitated with an anti-B7-2 antibody. Thereafter, each sample was digested with Endo-H (+) or without (1) and separated by SDS-PAGE. The result is shown in Fig. 3 (d). In the figure, ★ indicates Endo-H resistant B7-2 molecule. As shown in the figure, after 30 minutes, the amount of the Endo-H resistant B7-2 molecule was almost the same in the control cells and c-MIR cells, and the intracellular kinetics of the B7-2 molecule was B7-2. The experimental results show that the molecule is not affected until it reaches the intermediate Golgi. From the above experimental results, it was considered that c-MIR might enhance endcytosis of B7-2 molecule. To confirm this possibility, we used TACS with a fluorescence-activated cell sorter (FACS) -based endoscope and the cis angle 军 i ^ ". The results are shown in Fig. 4 (a).

 In the above experiment, BJAB cells (Cont) and c-MIR cells (c'MIR) were stained with PE-conjugated anti-B7-2 antibody at 4 ° C, washed with PBS to remove unbound antibody, and then washed with PBS. Incubated for 10 or 30 minutes at ° C. After the incubation, the antibody not internalized into the cells was removed with an acidic solution, and the internalized fluorescent signal was measured with a flow cytometer. These results are shown in the panels labeled “10” and “30” in the figure. To determine the background level of fluorescence, after staining with a PE-conjugated anti-B7-2 antibody, the antibody on the cell surface was removed with an acidic solution, and the background level was measured with a flow cytometer. The results are shown in the panel marked “0” in the same figure, as well as in the “10” and “30” panels in bold and f springs. The panel labeled “Pre” shows the cell surface expression level of the B7-2 molecule before removing the antibody on the cell surface.

As shown in the figure, after incubation for 10 minutes, a remarkable fluorescent signal was observed inside the c-MIR cells. On the other hand, no signal was observed in the control cells. After 30 minutes of incubation, no increase in the fluorescent signal into the cells was observed in the c-MIR cells, and no signal into the cells was still observed in the control cells. These results indicate that c-MIR-induced rapid end-site lysis of the B7-2 molecule occurs within 10 minutes. Also, a similar When experiments were performed on MHC class I molecules (MHC I), c-MIR cells did not enhance MHC I endocytosis (data not shown).

 In addition, c-MIR cells and control cells were reacted with FITC-labeled anti-B7-2 antibody at 37 ° C for 2 hours to confirm the rapid endogenesis of B7-2 molecules induced by c-MIR. After that, the localization of the B7-2 molecule was examined using a confocal microscope. The result is shown in Fig. 4 (b). As shown in the figure, B7-2 molecule was observed in c-MI • R cells, but not in control cells. These results indicate that c-MIR induces rapid endocytosis of the B7-2 molecule.

 [Example 3: Transmembrane domain and cytoplasmic domain of B7-2 molecule are related to target specificity of c-MIR]

 The present inventors have analyzed the function of the K3 and K5 proteins of KSHV by analyzing the CD8 chimera protein to find that the transmembrane region and the cytoplasmic region of the target molecule are down-regulated through the Κ3 and Κ5 proteins. (Expression suppression) was previously shown to be sufficient. Therefore, in order to examine whether the same region of the Β7-2 molecule is sufficient for down-regulation of the Β7-2 molecule via c-MIR, CD8 chimeric proteins were prepared for the HLA-Α2 and Β7-2 molecules ( These chimeric proteins are called “CD8 / A2” and “CD8 / B7-2,” respectively. CD8 / A2 and CD8 / B7-2 each contain a transmembrane region and a cytoplasmic region of the HLA-A2 molecule 7-2 molecule at the carboxyl terminus of the extracellular region of CD8α. These CD8 chimeric proteins were expressed in control BJAB cells or c-MIR cells, and the expression of CD8 on the cell surface was examined by flow cytometry. Figure 5 shows the results.

Since CD8 is not expressed on the surface of BJAB cells, Good detection of down regulation of the target molecule. As shown in FIG. 5, all CD8 chimeric proteins were efficiently expressed in control BJAB cells. On the other hand, the CD8 / A2 chimeric protein was sufficiently expressed in c-MIR cells, but the CD8 / B7-2 chimeric protein was not sufficiently expressed. This result indicates that the transmembrane domain and cytoplasmic domain of the B7-2 molecule are involved in c-MIR target specificity.

 [Example 4: c-MIR functions as E3 ubiquitin ligase] As described above, for the K3 and K5 proteins of KSHV, BKS-PHD / LAP zinc finger is a functional domain of Ε3 ubiquitin ligase. It is shown that there is. Therefore, in order to examine whether BKS-PHD / LAP jintafinger of c-MIR has Ε3 ubiquitin ligase activity, the BKS-PHD / LAP zinc finger (amino acid residues 8 to 57) in K3 protein was A chimeric protein was prepared in which the chimeric protein was substituted with that of c-MIR (amino acid residues at positions 78 to 137) (this chimeric protein is hereinafter referred to as "Zn MIR-K3"). Then, it was examined whether this chimeric protein had ligase activity against MHC I, a target molecule of K3, and suppressed its expression on the cell surface. In addition, a chimeric protein was also prepared in which a mutation was introduced into the BKS-PHD / LAP zinc finger region of c-MIR, and this was replaced with the BKS-PHD / LAP zinc finger of K3. (These mutations are called K3J.) The above mutations are those in which four cysteines (80, 83, 123, and 125) at the zinc (Zn) binding site are substituted with serine. The protein was expressed in A7 cells, and the surface expression of MHC I was examined by a two-color flow cytometry method, and the results are shown in Fig. 6 (a).

As shown in the figure, the Zn MIR-K3 protein sufficiently reduced the surface expression of MHC I. It was regulated. _{Since the} mZn MIR-K3 protein could not down-regulate MHC I surface expression, this down-regulation can be said to be BKS-PHD / LAP domain-dependent. This result indicates that c-MIR has E3 ubiquitin ligase activity.

 To confirm this further, an in vitro auto-ubiquitination analysis was performed. Specifically, the wild-type or mutant BKS-PHD / LAP domain of c-MIR is fused to the carboxyl terminus of GST, and the purified GST fusion protein is purified from ATP, free ubiquitin, El, E2 (UbcH5a). The mixture was used for self-ubiquitination analysis. The result is shown in Fig. 6 (b). As shown in the figure, in the GST fusion protein containing the wild-type BKS-PHD / LAP domain (c-MIR in the figure), auto-ubiquitination (ub-GST) was induced by the anti-ubiquitin antibody (α-Ubi). Although clearly observed, self-ubiquitination was observed in GST alone (GST in the figure) and in a GST fusion protein containing the mutant BKS-PHD / LAP domain (mZnc-MIR in the figure). Did not. This result indicates that the BKS-PHD / LAP domain of c-MIR has E3 ubiquitin ligase activity.

Furthermore, in order to examine whether the B7-2 molecule is ubiquitinated by c-MIR, HA-labeled ubiquitin and wild-type (wt) c-MIR (or a mutant obtained by modifying the BKS-PHD / LAP domain) were used. c-MIR (mZn c-MIE)) and B7-2 molecule were co-expressed. Each cell lysate was immunoprecipitated with an anti-B7-2 antibody, and then subjected to immunoblot analysis using an anti-HA antibody (upper panel) or an anti-B7-2 antibody (middle panel). The result is shown in Fig. 6 (c). As shown in the figure, when wild-type c-MIR (c-MIR in the figure) was expressed, the results of plotting the anti-HA antibody and the anti-B7-2 antibody were ubiquitinated. B7-2 (ub -B7-2) was clearly shown, but the wild-type c-MIR was not expressed (Cont in the figure) and the mutant c-MIR was expressed ( In the figure, mubicin-MIR) did not show ubiquitination. In addition, in order to confirm the expression levels of c-MIR and mZn c-MIR in each cell lysate, the results of blotting with an anti-His antibody are shown in the lower panel.

 As a result, it was shown that c-MIR functions as an E3 ubiquitin ligase for the B7-2 molecule via its BKS-PHD / LAP jintafine guard domain.

 [Example 5: Ubiquitination is essential for c-MIR-mediated downregulation of B7-2 molecule]

 So far, c-MIR has been shown to be (1) a regulator (modulator) of cell surface expression of B7-2 molecule, and (2) a novel E3 ubiquitin ligase for B7-2 molecule. However, it is unclear whether ubiquitination is essential for down-regulating cell surface expression of the B7-2 molecule. To confirm this point, the wild-type c-MIR (wt-c-MIR) and the mZn c_MIR mutant that failed to ubiquitinate the B7-2 molecule were transiently transferred to B JAB cells by electoral poration. The B7-2 molecule was expressed on the cell surface by two-color flow cytometry. The result is shown in Fig. 7 (a). As shown in the figure, mZnc′MIR did not suppress the expression of the B7-2 molecule.

Next, the following experiment was performed using the CD8 chimeric protein. The K5 protein of KSHV is known to ubiquitinate lysine residues located in the cytoplasm of the target molecule. Therefore, in order to examine whether the same applies to c-MIR, the CD8 / B7-2 chimeric protein was located in the cytoplasmic region of the B7-2 molecule. All the lysine residues were modified to arginine by PCR-based mutagenesis, and the amino terminus was labeled with an Epitope with a Flag tag (this chimeric protein was called “B7-KR”). Furthermore, the CD8 / B7-2 chimeric protein without the above modification was also labeled with a Epitope with a Flag tag (this chimeric protein is referred to as “wt-B7”).

 The B7-KR or wt-B7 was co-expressed in 293T cells together with the HA-tagged ubiquitin c-MIR, and subjected to the same experiment as in FIG. 6 (c). The result is shown in Fig. 7 (b). As shown in the figure, as a result of block analysis using anti-HA antibody and anti-Flag antibody, ubiquitination (ub-Flag-CD8 chimera in the figure) was observed in wt-B7 via c-MIR. However, ubiquitination via c-MIR was not observed in B7-KR.

 To investigate the relationship between ubiquitination of the B7-2 molecule and its suppression (down-regulation), wt-B7 and B7-KR were expressed in BJAB cells (control cells) and c-MIR cells, and CD8 Were compared by the flow cytometry method. The result is shown in Fig. 7 (c). As shown in the figure, the expression of wt-B7 was suppressed in c-MIR cells, but the expression of B7-KR was not suppressed in c-MIR cells. This result indicates that ubiquitination of lysine residues in the cytoplasmic region of the B7-2 molecule is required for its downregulation.

 [Example 6: Specific ubiquitination and down regulation of B7-2 molecule through intermolecular interaction]

Since E3 ubiquitin ligase is thought to function through binding to the target molecule, next, the intermolecular interaction between c-MIR and B7-2 was examined. CD8-B7 and c-MIR, which are the same molecules as wt-B7 used in the experiment in Fig. 7 (b), were After singly or co-expressed in cells, the cell lysate was immunoprecipitated with an anti-Flag antibody and subjected to immunoblot analysis with an anti-V5 antibody recognizing c-MIR. The results are shown in the upper panel of Fig. 8 (a).

 As shown in the figure, only when c-MIR and CD8-B7 were co-expressed, a specific band corresponding to c-MIR was detected, so the molecular interaction between c-MIR and B7-2 Was confirmed. In addition, the expression level of the CD8 chimeric protein and the expression level of c-MIR were confirmed using an anti-Flag antibody (center panel) and an anti-V5 antibody (lower panel), respectively.

 By the way, it has been reported that the transmembrane domain of KSHV K3 and K5 proteins is involved in specific targeting through binding to the transmembrane domain of the target molecule. If the binding between c-MIR and the target molecule is the same, it is considered that the mZnc-MIR mutant has the ability to bind to the target molecule because it has a wild-type transmembrane region. Can be To confirm this hypothesis, mZnc-MIR was subjected to the same experiment as in FIG. 8 (a). The results are shown in Fig. 8 (b). As shown in the figure, a specific band corresponding to mZnc-MIR was detected only when mZnc-MIR and CD8-B7 were co-occurred and expressed. In both experiments shown in Figs. 8 (a) and (b), the conjugate of mZn c-MIR and CD8-B7 was more easily detected than the conjugate of c-MIR and CD8-B7, but this was wild. This is thought to be due to the instability of the c-MIR conjugate of the type.

In order to investigate whether the specific downregulation of the B7-2 molecule by c-MIR was due to an intermolecular interaction, an experiment was performed using the CD8 / A2 chimeric protein. This is because the expression of the CD8 / A2 chimeric protein on the cell surface was not down-regulated in c-MIR cells (see FIG. 5). CD8 / A2 was modified to have a flag epitope tag at its amino terminus (this key Mela protein is called "CD8-A2"). Then, CD8-A2 or CD8-B7 was co-expressed with mZnc-MIR in 293 T cells, and intermolecular interactions were examined by immunoprecipitation one-shot immunoblotting. In this experiment, mZn c-MIR was used in this experiment, because the detection of c-MIR conjugate was easy. In this experiment, cell lysates of CD8-A2 expressing cells and CD8-B7 expressing cells were immunoprecipitated with anti-HA antibody or anti-Flag antibody, and then immobilized with anti-V5 antibody (upper) or anti-Flag antibody (middle). Analysis was performed. The result is shown in Fig. 8 (c). The results of the immunoblot analysis of the lysates of both cells with anti-V5 antibody are also shown in the lower row.

 As shown in the figure, CD8-B7 and mZnc-MIR co-precipitated, but CD8-A

2 and mZn c-MIR did not co-sediment. This result indicates that intermolecular interaction is essential for specific downregulation of B7-2 molecule via c-MIR. '

 Next, in order to examine the relationship between intermolecular interaction and ubiquitination, the above CD8-A2 and CD8-B7 were subjected to the same experiment as in FIG. 7 (b). The result is shown in Fig. 8 (d). As shown in the figure, sufficient ubiquitination (ub-CD8 chimera) was observed in CD8-B7 as compared to CD8-A2. This result indicates that intermolecular interactions are essential for c-MIR-mediated ubiquitination and subsequent down-regulation of target molecules.

Fig. 9 shows the molecular mechanism of c-MIR considered from the above experimental results. As shown in the figure, c-MIR binds to B7-2 molecule through a transmembrane site. Then, from the E2 ubiquitin converting enzyme (E2) that binds to the PHD / LAP domain (PHD), ubiquitin (Ubi) is converted to each lysine residue in the intracellular region of the B7-2 molecule. (K) and B7-2 molecule is ubiquitinated. The ubiquitinated B7-2 molecule undergoes endcytosis from the cell surface, is carried to lysosomes, and is degraded.

 [Example 7: Functional and structurally similar molecule of the above c-MIR HSPC240] A search was conducted for a novel human E3 ubiquitin ligase homologous to the above c-MIR. Genes and proteins were identified. FIG. 10 shows the amino acid sequence of HSPC240 (the same sequence is shown in SEQ ID NO: 4 in the sequence listing). In the figure, the underline indicates the BKS-PHD / LAP zinc finger motif. It was examined whether this HSPC240 suppresses the expression of antigen presentation-related molecules. Specifically, HSPC240 is transiently expressed in BJAB cells, and cell surface of MHC class I molecule (MHC I), MHC class II (MHC II / DR) molecule, ICAM-1 molecule, B7-2 molecule Was examined by flow cytometry. The results are shown in FIG. 'As shown in the figure, the expression of the B7-2 molecule was significantly suppressed by the expression of HSPC240, but the expression of each of the MHC I, MHC II, and ICAM-1 molecules was not suppressed. This result indicates that HSPC240 specifically suppresses the expression of B7-2 molecule on the cell surface and has the same effect as c-MIR in suppressing the expression of antigen presentation-related molecules. .

 [Example 8: Suppression of expression of MHC class II molecule and B7-2 molecule by c-MIR strongly suppresses antigen presentation function]

Create a c-MIR expressing A20.2J cells, OVA _{323. 339} specific I'Ad restricted Thl clo ne, using 42-6A, OVA or OVA _{323. 339} antigen presentation function for peptide of IL-2 Production was examined as an index. As a result of using OVA in Fig. 12 (a), Fig. 1

2 (b) shows the results using the OVA323-339 peptide. The dotted line shows the results for control cells, and the solid line shows the results for cells overexpressing c-MIR. Also, OVA was measured 24 hours after OVA, and peptide 6 hours after IL-2 was measured in the culture supernatant by ELISA. As a result I apparent from sea urchin, OVA or OVA ₃₂₃ - ₃₃₉ antigen presenting function for peptide significant suppression was observed. Industrial potential

 The present invention relates to a novel mammal-derived protein that negatively regulates the expression of B7-2 molecule or MHC class II molecule on the cell surface and its use.As described above, the development of a new immunosuppression method, cell, It can be used for suppressing rejection during organ transplantation, treating autoimmune diseases, developing new drugs, and has various other uses. Therefore, the present invention is considered to contribute to the development of medical care widely in various pharmaceutical industries and the like.

Claims

Range of request  1. A mammal-derived protein described in the following (a) or (b):  (a) a protein comprising any one of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16;  (b) In any of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16, one or several amino acids are substituted, deleted, 揷A protein comprising an amino acid sequence added and / or added thereto and negatively controlling the expression of a molecule related to antigen presentation on the cell surface.  2. The protein according to claim 1, which negatively controls the expression of the B7-2 molecule, which is an antigen-presentation-related molecule, on the cell surface.  3. The protein according to claim 2, which ubiquitinates the B7-2 molecule.  4. The protein according to any one of claims 1 to 3, which negatively regulates the expression of an MHC class II molecule, which is an antigen presentation-related molecule, on the cell surface. 5. The protein according to any one of claims 1 to 4, which is E3 ubiquitin ligase derived from human or mouse.  6. An antibody against the protein according to any one of claims 1 to 5. 7. A gene encoding the protein according to any one of claims 1 to 5.  8. The gene according to claim 7, which has any one of the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, or 15 as an open reading frame region. 9. Under stringent conditions, a DNA consisting of the nucleotide sequence shown in 1, 3, 5, 7, 9, 11, 11, 13 or 15 and a DNA consisting of a complementary nucleotide sequence under stringent conditions Hybridizes and displays antigen-presenting-related molecules on the cell surface A gene encoding a protein that negatively regulates expression. 10. Recombinant kakigen beta containing the gene according to any one of claims 7 to 9 ¹ to ₀ 1 1. A transformant _Q into which the gene according to any one of claims 7 to 9 has been introduced.  1 2. A gene detection instrument using at least a part of the nucleotide sequence of the gene according to any one of claims 7 to 9 or a complementary sequence thereof as a probe.  1 3. An immunosuppressant comprising the protein according to any one of claims 1 to 5, or the gene according to any one of claims 7 to 9.  1 4. A method for screening a substance that regulates the action of a protein according to any one of claims 1 to 5.  1 5. A method for screening a substance having an action of negatively controlling the expression of an antigen-presenting-related molecule on the cell surface, similarly to the protein according to any one of claims 1 to 5. +  1 6. A method according to claims 14 or 15, characterized in that any one of the following substances (1) to (3) and one of the following substances (4) to (7) are used. Screening method.  (1) The protein according to any one of claims 1 to 5  (2) A partial protein of the protein according to any one of claims 1 to 5, wherein the partial protein comprises at least a transmembrane region in the amino acid sequence of the full-length protein. (3) A variant of the protein of (1) or the partial protein of (2) (4) B7-2 molecule or MHC class II molecule  (5) B7-2 molecule or variant of MHC class II molecule  (6) A partial protein of the B7-2 molecule or MHC class II molecule, which contains at least the transmembrane region or its intracellular region in the amino acid sequence of the full-length protein  (7) A variant of the partial protein of (6) above  1 7. A therapeutic drug for an immunological disease obtained by using the screening method according to any one of claims 14 to 16.