JP4024282B2 - Novel polypeptide ESDN, method for producing the same, cDNA encoding ESDN, vector comprising the cDNA, host cell transformed with the vector, antibody of ESDN, and pharmaceutical composition containing ESDN or antibody - Google Patents

Description

  The present invention relates to a novel polypeptide vascular endothelial cell / smooth muscle cell-derived neuropilin-like molecule (hereinafter abbreviated as “ESDN”), its production method, and ESDN. Encoding cDNA, vector comprising the cDNA, host cell transformed with the vector, ESDN antibody, ESDN or pharmaceutical composition containing antibody, method for measuring ESDN, reagent for measuring ESDN and ESDN Related to the screening method.

Since the cloning of vascular endothelial growth factor (VEGF), elucidation of the complex extracellular signaling network that governs angiogenesis has been revolutionary. The study of knockout mice of this ligand and tyrosine kinases such as Flk-1 / VEGFR2 and Flt-1 / VEGFR1 was the first important point regarding the first step of angiogenesis and development.
VEGF receptor-2 (VEGFR2) is necessary for endothelial cell formation, and this gene deficiency leads to a lack of blood cell groups or organized blood vessels leading to embryonic death on day 9.5. However, from the report that initial hematopoiesis and endothelial progenitor cells are actually produced in the absence of VEGFR2 by analysis of initial markers using RT-PCR, VEGFR2 is not essential for the formation of hematoblasts, It suggests that it is necessary for the subsequent increase of the strain.
VEGF receptor-1 (VEGFR1) knockout mice also die at 9.5 days of development, but their endothelial cell differentiation is not directly affected. Instead, the migration of interstitial hematoblasts is thought to be lacking, and congested endothelial progenitor cells lead to severe tissue destruction of the vasculature.
VEGF knockout mice also die with a significant decrease at 9.5 days of development, but do not lack endothelial cell differentiation. That is, it is essentially the same as the VEGFR1 knockout mouse, but shows a milder phenotype. A striking feature of this gene is that even a heterozygous gene deletion results in embryonic lethality on day 11.5. This indicates that there is a remarkably stringent dose dependence on VEGF during embryonic life.
Other receptor tyrosine kinases are also specifically expressed in vascular endothelial cells such as Tie-2 and its ligand angiopoietin-1, and are involved in the later stages of angiogenesis, angiogenesis steps. While disruption of these genes does not affect angiogenesis, it disrupts vascular remodeling and leads to embryonic death at 10.5 days of development. They play a role in the interaction between endothelial cells and the vascular smooth muscle cells (VSMC) or mesenchymal cells that surround them. In addition to these vascular specific systems, PDGF-BB or TGF-β and their receptors also play a similar role between endothelial cells and the cells surrounding them. Identification of the relationship of neuropilin-1 (NP-1) to the vascular system introduced a new aspect of the field. NP-1 was cloned as an antigen recognized by a monoclonal antibody that specifically recognizes the initially developed nervous system.
The identification of semaphorin 3A (semaphorin 3A) as a receptor has attracted a great deal of attention in axon formation. The search for other VEGF receptors led to the discovery of NP-1, which is also a receptor for VEGFR2. And it not only increases binding to a single VEGF isoform, but also promotes endothelial cell migration and possibly mitosis as well. Before this was identified, it was shown that chimeric mice that forcedly expressed NP-1 formed enlarged vessels. NP-1 knockout mice exhibited low vascularity in the central nervous system and also exhibited large vascular malformations. On the other hand, the presence or absence of vascular abnormality has not been reported in sema 3A knockout mice, but it is interesting that the phenotype exhibits a thin myocardium observed in NP-1 forced expression mice.
The Eph / ephrin system is another example and first became famous in neuroscience. However, to date it has been identified as the only surface marker that distinguishes arteries and veins. They also show expression in surrounding mesenchymal cells, which are contacts with endothelial cells. In addition to molecules with these roles, we assumed that many more extracellular signaling molecules exist in the vascular field. And I thought that elucidation of this could contribute to further understanding of this complex system.

  Conventionally, when obtaining a specific polypeptide or cDNA encoding the same, the target biological activity is confirmed in tissue or cell culture medium, and then the gene is cloned through isolation and purification of the polypeptide. In general, a method of expressing and cloning a gene using its biological activity as an index has been used. However, in vivo bioactive polypeptides often have a variety of biological activities, and as a result of cloning a gene using one activity as an indicator, it may be the same as a known polypeptide. Increasing cases are found later. In addition, there are many factors that are produced only in trace amounts or expressed only under special physiological conditions, which makes isolation, purification, and confirmation of biological activity difficult.

  The present inventors have studied the cloning of a gene for a growth / differentiation factor that works in the hematopoietic system and the immune system. And most of secreted proteins such as growth differentiation factors (for example, various cytokines) and membrane proteins such as receptors thereof (hereinafter collectively referred to as secreted proteins etc.) have a signal peptide at the N-terminus. Focusing on the fact that it has a sequence called, we have intensively studied a method for efficiently and selectively cloning a gene encoding a signal peptide. As a result, a method (signal sequence strap (SST) method) was found that can easily search for the presence or absence of a signal peptide using animal cells (see Patent No. 3,229,590). Furthermore, based on the same concept, a method (yeast SST method) for isolating a gene encoding a signal peptide more easily and easily using yeast was also developed (US Patent No. 5,536,637).

The present inventors have provided a novel novel factor (polypeptide) that plays an important role in pathologic smooth muscle after restenosis and arteriosclerosis after PTCA in the cardiovascular region, in particular secreted proteins and membranes with secretory signals. In order to find out by focusing on the protein, we conducted intensive studies.
The present inventors performed newly modified SST screening on vascular cells and succeeded in cloning a novel transmembrane protein. The protein ESDN of the present invention is a novel type I transmembrane protein having a characteristic domain like neuropilin.

  As will be described in detail later, the ESDN protein of the present invention is expressed in coronary artery cells and smooth muscle cells, and also expressed in the vascular smooth muscle of balloon-injured arteries and the media of the common carotid artery. It is considered useful for the treatment of restenosis after PTCA and arteriosclerosis in the region.

  The cDNA sequence provided by the present invention is identified as a human ESDN clone represented by SEQ ID NO: 2, and information obtained using a yeast SST method from a cDNA library prepared from coronary artery primary cultured endothelial cells and smooth muscle cells is obtained. Originally isolated. The human ESDN clone shown in SEQ ID NO: 3 is a full-length cDNA containing a complete cDNA sequence encoding a secreted protein (herein expressed as a human ESDN protein).

  The cDNA sequence provided by the present invention was identified as a mouse ESDN clone represented by SEQ ID NO: 6, and was isolated from a mouse cDNA library based on information obtained using the yeast SST method. The mouse ESDN clone shown in SEQ ID NO: 5 is a full-length cDNA containing a complete cDNA sequence encoding a secreted protein (herein expressed as mouse ESDN protein).

  The cDNA sequence represented by SEQ ID NO: 9 provided by the present invention was identified as a rat ESDN clone and was isolated from a rat cDNA library based on information obtained using the yeast SST method. The mouse ESDN clone shown in SEQ ID NO: 8 is a full-length cDNA containing a complete cDNA sequence encoding a secreted protein (herein expressed as mouse ESDN protein).

  For known nucleic acid sequences registered in the nucleic acid sequence database by GenBank and BLASTN, FASTA and UNIGENE using NCBI, etc., and for amino acid sequences of known polypeptides registered in the amino acid sequence database As a result of searching with BLASTP, Fly Database, SwissProt, etc., there was no sequence that matches the rat ESDN which is the polypeptide of the present invention and the nucleic acid sequence encoding them. From this, it was found that the polypeptide of the present invention is a novel secreted protein.

The present invention
1. A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 5, or 8 in a substantially pure form, or a polypeptide comprising a homologue thereof, a fragment thereof or a homologue thereof.
2. The polypeptide according to item 1 above, comprising the amino acid sequence represented by SEQ ID NO: 2, 5 or 8;
3. CDNA encoding the polypeptide described in the preceding item 1 or 2,
4). The cDNA according to the aforementioned item 3, which has the base sequence represented by SEQ ID NO: 3, 6 or 9, or a fragment which selectively hybridizes to the sequence.
5). A replication or expression vector comprising the cDNA according to item 3 or 4;
6). A host cell transformed with the replication or expression vector of paragraph 5;
7). The method for producing the polypeptide according to item 1 or 2, characterized by culturing the host cell according to item 6 under conditions for expressing the polypeptide according to item 1 or 2.
8). A polyclonal or monoclonal antibody of the polypeptide described in item 1 or 2,
9. A pharmaceutical composition comprising the polypeptide according to item 1 or 2, or the antibody according to item 8, and a pharmaceutically acceptable excipient and / or carrier,
10. A pharmaceutical composition effective for the treatment of restenosis after PTCA, comprising the polypeptide according to item 1 or 2, or the antibody according to item 8 above and a pharmaceutically acceptable excipient and / or carrier. Composition,
11. 10. A pharmaceutical composition effective for the treatment of arteriosclerosis according to item 10 above, comprising the polypeptide according to item 1 or 2, or the antibody according to item 8 above, and a pharmaceutically acceptable excipient and / or carrier. Composition,
12 A method for measuring the polypeptide according to 1 or 2 above,
13. The method for immunochemical measurement of a polypeptide according to item 1 or 2, wherein the antibody according to item 8 is used,
14 A reagent for detecting the polypeptide according to item 1 or 2, wherein the method according to item 12 or 13 is used,
15. A reagent for examining restenosis after PTCA, characterized by using the method according to item 12 or 13 above,
16. 16. A reagent for examining arteriosclerosis, which comprises using the method according to item 12 or 13, and The present invention relates to a method for screening a compound having activity as an antagonist or agonist for the polypeptide, characterized by using the polypeptide described in the preceding item 1 or 2.

The inventors of the present invention have confirmed that the expression of the polypeptide is enhanced in the synthetic trait than the contractile trait of vascular smooth muscle. Therefore, it is considered that the polypeptide is useful for treatment of restenosis after PTCA and arteriosclerosis in the circulatory region.
In addition, the polypeptide of the present invention exhibited a cell growth inhibitory action in the experiment, and since it has a structure similar to VEGF, it may have the following actions.
"Cytokine activity and cell proliferation / differentiation activity"
Since the protein of the present invention suppresses cell proliferation in a forced expression system, it may exhibit cytokine activity and cell proliferation (induction or inhibition) / differentiation activity (induction or inhibition), or other cytokines may be present in a cell population. It is thought to induce or suppress production.
"Immune stimulation / suppression activity"
The protein of the present invention is also considered to exhibit immunostimulatory activity and immunosuppressive activity. For example, by controlling (stimulating or suppressing) the growth and proliferation of either T lymphocytes and / or B lymphocytes, and also affecting the cytotoxic activity of NK cells and other populations. It appears to be effective in the treatment of immune deficiencies and diseases. In particular, it is considered to be effective in suppressing lymphatic cancer metastasis caused by tumor angiogenesis.
"Ischemic angiogenesis inhibitory activity"
The protein of the present invention is considered to suppress retinopathy, which is a complication of diabetes. VEGF is known to have a strong angiogenic effect upon induction of ischemia. Angiogenesis of diabetic retinopathy is also ischemic angiogenesis that occurs after the formation of a retinal avascular region. It is considered that the protein of the present invention may act as a novel VEGF receptor to suppress blood vessel growth due to the structural similarity with neuropilin. This is considered to be effective in diabetic retinopathy.

  The hybridizing cDNA includes a complementary sequence of the above sequence. Hybridization is preferably performed under stringent conditions.

  A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, 5, or 8 in substantially pure form is generally 90% or more, eg, 95, 98, or 99% of the polypeptide as produced. It means a polypeptide consisting of the amino acid sequence shown by 2, 5 or 8.

  The homologue of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, 5 or 8 is generally a region of at least 20, preferably at least 30, for example 40, 60, 80 or 100 consecutive amino acids and at least 70 %, Preferably at least 80 or 90%, more preferably 95% or more homologous, and such homologues are hereinafter described as polypeptides of the invention.

  Furthermore, a fragment of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, 5 or 8 or a homologous fragment thereof is at least 10 amino acids, preferably at least 15 amino acids, for example 20, 25, 30, 40, 50 Or means the 60 amino acid part.

  The cDNA that selectively hybridizes to the cDNA consisting of the base sequence shown in SEQ ID NO: 3, 6 or 9 is generally at least 20, preferably at least 30, for example 40, 60, 80 or 100 consecutive. The nucleotide sequence region is at least 70%, preferably at least 80 or 90%, more preferably 95% or more homologous, and such cDNA is hereinafter described as the cDNA of the present invention.

  The fragment of cDNA consisting of the base sequence shown in SEQ ID NO: 3, 6 or 9 means a portion of at least 10 bases, preferably at least 15 bases, such as 20, 25, 30 or 40 bases. Included in the cDNA of the invention.

  Furthermore, the present invention includes a replication or expression vector comprising the cDNA of the present invention. Examples of the vector include a plasmid, virus, or phage vector comprising an ori region and, if necessary, a promoter for expression of the cDNA, a promoter control factor, and the like. The vector may contain one or more selective marker genes, such as an ampicillin resistance gene. The vector can be used in vitro, for example, for production of RNA corresponding to cDNA and transformation of host cells.

  Furthermore, the present invention includes a host cell transformed with a vector for replicating or expressing the cDNA of the present invention comprising the nucleotide sequence represented by SEQ ID NO: 3, 6 or 9 or a cDNA comprising the open reading frame thereof. Is also included. Examples of the cell include bacteria, yeast, insect cells, and mammalian cells.

  Furthermore, the present invention includes a method for producing the polypeptide of the present invention, which comprises culturing the host cell of the present invention under conditions for expressing the polypeptide of the present invention. Culturing is preferably performed under conditions where the polypeptide of the present invention is expressed and produced from a host cell.

  Antisense RNA can also be produced by inserting the cDNA of the present invention into the antisense region of the vector as described above. Such antisense RNA can also be used to control the level of a polypeptide of the invention in a cell.

  The present invention also includes monoclonal or polyclonal antibodies of the polypeptides of the present invention. Furthermore, a method for producing a monoclonal or polyclonal antibody of the polypeptide of the present invention is also included. A monoclonal antibody can be produced by a conventional hybridoma technique using the peptide of the present invention or a fragment thereof as an antigen. Polyclonal antibodies can be produced by a conventional method in which a host animal (eg, rat, rabbit, etc.) is inoculated with the polypeptide of the present invention and immune serum is collected.

  The present invention also includes a pharmaceutical composition containing the polypeptide of the present invention, its antibody, and a pharmaceutically acceptable excipient and / or carrier.

  As the polypeptide of the present invention, in addition to the amino acid sequence shown by SEQ ID NO: 2, 5 or 8, a part thereof is deleted (for example, a part essential for expression of biological activity in SEQ ID NO: 2). A mature polypeptide consisting of only amino acids), a part of which is substituted with other amino acids (for example, those substituted with amino acids having similar physical properties), and other amino acids are added or inserted into the polypeptide of the present invention. Also included.

  As is well known, 1 to 6 types of codons encoding one amino acid are known (for example, 1 type for Met and 6 types for Leu). Therefore, the base sequence of cDNA can be changed without changing the amino acid sequence of the polypeptide.

  The cDNA of the present invention includes all nucleotide sequences encoding the polypeptide represented by SEQ ID NO: 2, 5, or 8. Polypeptide productivity may be improved by changing the base sequence.

  The cDNA specified by SEQ ID NO: 3, 6 or 9 is one embodiment of the base sequence group encoding the polypeptide shown by SEQ ID NO: 2, 5 or 8, and represents the natural type sequence.

  Preparation of cDNA consisting of the base sequence represented by SEQ ID NO: 3, 6 or 9 is performed according to the following method.

First, an outline of the yeast SST method (described in US Pat. No. 5,536,637) will be described.
In order for yeasts such as Saccharomyces cerevisiae to use sucrose or raffinose as an energy or carbon source, invertase must be secreted into the medium (invertase converts raffinose into sucrose and melibiose, sucrose Is an enzyme that breaks down into fructose and glucose.) It is also known that many known mammalian signal peptides can secrete yeast invertase.

  Based on these findings, the present method was developed as a method for screening a novel signal peptide that enables secretion of yeast invertase from a mammalian cDNA library using the growth of yeast on raffinose medium as an indicator.

  A non-secreted invertase gene SUC2 (GENBANK accession No. V01311) from which the translation start point ATG was deleted was incorporated into a yeast expression vector to prepare a yeast SST vector pSUC2. The expression vector incorporates an expression promoter (ADH promoter) and terminator (ADH terminator) derived from the AAH5 plasmid (Gammerer, Methods in Enzymol. 101, 192-201, 1983). Is TRP1, ColE1 ori as the E. coli replication origin, and ampicillin resistance gene is incorporated into the E. coli drug resistance marker. A mammalian cDNA was incorporated upstream of the SUC2 gene to prepare a yeast SST cDNA library. This library was transformed into yeast lacking secreted invertase.

  When the incorporated mammalian cDNA encodes a signal peptide, it is considered to have a secretory effect on invertase expressed in yeast, and as a result, growth on a raffinose medium is possible. Thus, yeast was cultured from the colonies that emerged, plasmids were prepared, and the nucleotide sequence of the insert cDNA was determined, thereby making it possible to search for new signal peptides quickly and easily.

Preparation of yeast SST cDNA library
(1) mRNA is isolated from a target cell, and a double-stranded cDNA is synthesized using a random primer linked with a specific restriction enzyme (enzyme I) site,
(2) Ligating an adapter containing a specific restriction enzyme (enzyme II) site different from enzyme I, digesting with enzyme I, fractionating at an appropriate size,
(3) A step of ligating and transforming a cDNA fragment obtained upstream of the invertase gene from which the signal peptide in the yeast expression vector has been deleted.

  Each step will be described in detail. In step (1), after stimulation with an appropriate stimulant as necessary from the target mammalian organ or cell line, a known method (hereinafter, known methods are not particularly described). Molecular Cloning (Sambrook, J., Fritsch, EF and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1989) or Current Protocol in Molecular Biology (edited by FM Ausubel et al., John Wiley & Sons, Inc.) The isolation of mRNA is carried out according to the method described in the publication.

  An example of a target tissue is a mouse fetal heart. Double-stranded cDNA synthesis using random primers is performed by a known method.

  The restriction enzyme (enzyme I) site connected to the adapter and the restriction enzyme (enzyme II) site used in the next step (2) may be any as long as they are different from each other. Preferably, XhoI is used as enzyme I and EcoRI is used as enzyme II.

  In step (2), the ends are blunted with T4 DNA polymerase, the enzyme II adapter is ligated, digested with enzyme I, and 300-800 bp cDNA is fractionated by agarose electrophoresis (AGE). The enzyme II may be anything as long as it is different from the enzyme I as described above.

  Step (3) is a step of transforming E. coli by incorporating the cDNA fragment obtained in (2) upstream of the invertase gene from which the signal peptide linked to the yeast expression plasmid vector has been deleted. Here, various plasmid vectors for yeast expression are known. For example, YEp24 that functions also in E. coli is used, and the above-described plasmid pSUC2 is preferably used.

  Many host E. coli strains for transformation are already known, and are preferably DH10B competent cells. The transformation method may be any known method, but is preferably performed by electroporation. The transformant is cultured by a known method to obtain a cDNA library for yeast SST.

  In this cDNA library, not all cloned cDNA fragments have been introduced, and not all gene fragments encoding unknown (new) signal peptides. Therefore, it is necessary to screen a gene fragment encoding an unknown signal peptide from the library.

  Introducing the cDNA library into a yeast without a gene, Saccharomycs cerevisiae (for example, YT455 strain) or a strain in which the invertase gene is artificially deleted (can be prepared according to a known method), A fragment having a sequence encoding a signal peptide is screened. Transformation of yeast is performed by a known method such as the lithium acetate method. After growing the transformant in a selective medium, the transformant is transferred to a medium containing raffinose as a carbon source, viable colonies are selected, and the plasmid is recovered. The fact that yeast grew using raffinose as a carbon source indicates that a signal peptide of some secreted protein was incorporated into the library.

  Next, the nucleotide sequence of the isolated positive clone is determined, and for cDNA that has been revealed to encode an unknown protein, a full-length clone is isolated using it as a probe, and the full-length nucleotide sequence is determined. Can do. These operations are all performed by methods known to those skilled in the art.

  A cDNA encoding the protein of the present invention present in a mammal or a cDNA encoding a homologue and a subset of the protein of the present invention when part, preferably all, of the base sequence represented by SEQ ID NO: 3, 6 or 9 is determined Obtainable. By synthesizing an oligonucleotide having an appropriate base sequence and using it to PCR from a cDNA library or mRNA derived from a mammal, or by hybridizing with a fragment of an appropriate base sequence as a probe, other mammalian cDNA live From the library or the genomic library, cDNAs encoding the other mammalian proteins can be obtained.

If the cDNA thus obtained contains the nucleotide sequence of the cDNA fragment obtained by SST (or a homologous sequence thereof), it means that the signal peptide is encoded. It is clear that it is almost full length (the signal peptide is encoded at the 5 ′ end of the open reading frame of the cDNA because it exists at the N-terminus of the protein without exception).
Furthermore, according to a known method, the full length may be confirmed by Northern analysis using the cDNA as a probe. The size of the mRNA obtained from the hybridized band is compared with the size of the cDNA. If the size is almost the same, the cDNA is considered to be almost full length.

  The present invention provides both full-length as well as mature forms of the disclosed proteins. The full-length forms of these proteins are identified by the translated amino acid sequence of the base sequence shown in SEQ ID NO: 2, 5 or 8. These mature proteins can be obtained by expressing the full-length DNA represented by SEQ ID NO: 3, 6 or 9 disclosed in an appropriate mammalian cell or other host cell in an appropriate mammalian cell or other host cell. . The sequence of the mature protein can be predicted from the full-length amino acid sequence (see FIG. 1).

  Once the base sequence represented by SEQ ID NO: 3, 6 or 9 is determined, thereafter, by chemical synthesis, or by chemically synthesizing a fragment of the base sequence and hybridizing it with a probe, cDNA can be obtained. Furthermore, a necessary amount of the target cDNA can be obtained by introducing a vector cDNA containing this cDNA into an appropriate host and propagating it.

As a method for obtaining the polypeptide of the present invention,
(1) A method for purification and isolation from living organisms or cultured cells,
(2) a method of peptide synthesis, or (3) a method of production using genetic recombination technology,
Industrially, the method described in (3) is preferable.

  Examples of the expression system (host-vector system) for producing a polypeptide using a gene recombination technique include bacterial, yeast, insect cell and mammalian cell expression systems.

  For example, when expressed in E. coli, an initiation codon (ATG) is added to the 5 ′ end of the cDNA encoding the mature protein portion, and the resulting cDNA is converted into an appropriate promoter (eg, trp promoter, lac promoter, λPL An expression vector is prepared by connecting downstream of a promoter, T7 promoter, etc.) and inserting into a vector that functions in E. coli (eg, pBR322, pUC18, pUC19, etc.).

  Next, E. coli transformed with this expression vector (for example, E. Coli DH1, E. Coli JM109, E. Coli HB101 strain, etc.) is cultured in an appropriate medium, and the desired polypeptide is obtained from the cells. Obtainable. In addition, if a bacterial signal peptide (for example, a pelB signal peptide) is used, the target polypeptide can be secreted into the periplasm. In addition, fusion proteins with other polypeptides can be produced.

  In addition, when expressed in mammalian cells, for example, the base sequence represented by SEQ ID NO: 3, 6 or 9 is used as an appropriate vector (for example, retrovirus vector, papilloma virus vector, vaccinia virus vector, SV40 vector, etc.) An expression vector is prepared by inserting it downstream of an appropriate promoter (for example, SV40 promoter, LTR promoter, metallothionein promoter, etc.). Next, an appropriate mammalian cell (for example, monkey COS-1 cell, COS-7 cell, Chinese hamster CHO cell, mouse L cell, etc.) is transformed with the obtained expression vector, and the transformant is transformed into an appropriate medium. The protein of the present invention, which is a secreted protein, is expressed as a desired polypeptide in the cell supernatant. Furthermore, a fusion protein can also be produced by ligation with a cDNA fragment encoding the constant region (Fc portion) of another polypeptide, such as an antibody. The polypeptide obtained as described above can be isolated and purified by a general biochemical method.

  The effects or biological activities described for the protein of the present invention can be attributed to administration or use of the protein, or administration or use of cDNA encoding the protein (eg, gene therapy (including regenerative medicine) and cDNA introduction). A suitable vector).

  In addition, the polypeptide can be quantified in a living body using a polyclonal antibody or a monoclonal antibody of the polypeptide, and can be used for studying the relationship between the polypeptide and a disease or for diagnosing a disease. Polyclonal antibodies and monoclonal antibodies can be prepared by known methods using the polypeptide or a fragment thereof as an antigen.

  Further, by using the polypeptide, for example, an affinity column can be prepared, and a known or unknown protein (ligand) that binds to the polypeptide can be identified and purified, or gene cloning thereof can be performed.

  Identification of a molecule that interacts with the polypeptide using the polypeptide, for example, by the West-Western method, or using the cDNA (preferably cDNA encoding the polypeptide), for example, by the yeast two-hybrid method. Gene cloning can also be performed.

  Furthermore, by using the present polypeptide, it is possible to screen for the present polypeptide receptor agonists, antagonists, inhibitors between receptor and signaling molecules, and the like.

Screening can be performed, for example, by the following method. That is, a) a reaction mixture comprising a polypeptide of the present invention, a compound to be screened, and cells under conditions in which the cells are normally stimulated by the peptide (the reaction mixture in the cells as the cells proliferate) And a peptide other than the peptide for effectively observing the function of the peptide); then b) measuring the degree of cell proliferation and the compound is an effective antagonist or agonist Decide whether or not.

The cDNA of the present invention is not only an important and indispensable template for producing the polypeptide of the present invention, which is expected to be very useful, but also diagnosis and treatment of genetic diseases (treatment of gene deficiency or antisense). DNA (RNA) can be used for treatment by stopping the expression of a polypeptide.
Further, genomic DNA can be separated using the cDNA of the present invention as a probe.

[Application to pharmaceutical products]
In order to adapt to the above-mentioned diseases, the polypeptide of the present invention or the antibody against the polypeptide of the present invention is usually administered systemically or locally, generally in an oral or parenteral form. Oral administration, intravenous administration, and intracerebroventricular administration are preferable.

  The dose varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but it is usually administered orally once to several times a day in the range of 100 μg to 100 mg per adult. Alternatively, it is administered parenterally once or several times a day in the range of 10 μg to 100 mg per adult per person.

  Of course, as described above, the dose varies depending on various conditions, so that a dose smaller than the above dose may be sufficient or may be necessary beyond the range.

  When the compound of the present invention is administered, it is used as a solid composition, liquid composition and other compositions for oral administration, injections for parenteral administration, external preparations, suppositories and the like.

  Solid compositions for oral administration include tablets, pills, capsules, powders, granules and the like. Capsules include soft capsules and hard capsules.

  In such solid compositions, one or more active substances are present in at least one inert diluent (eg lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, Acid magnesium aluminate etc.). The composition is prepared in accordance with conventional methods, and additives other than inert diluents, such as lubricants (magnesium stearate, etc.), disintegrants (calcium fibrin glycolate, etc.), stabilizers (human serum albumin, lactose, etc.) ), A solubilizing agent (arginine, aspartic acid, etc.) may be contained.

  Tablets or pills may be coated with a gastric or enteric film such as sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc., if necessary, or may be coated with two or more layers. Also included are capsules of absorbable substances such as gelatin.

  Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, etc., and commonly used inert diluents (eg, purified water, Ethanol etc.) may be included. Such a composition may contain an auxiliary agent such as a wetting agent and a suspending agent, a sweetening agent, a flavoring agent, a fragrance and a preservative in addition to the inert diluent.

  Other compositions for oral administration include sprays containing one or more active substances and formulated by methods known per se. In addition to the inert diluent, this composition contains a stabilizer that provides isotonicity with a stabilizer such as sodium bisulfite, and an isotonic agent such as sodium chloride, sodium citrate or citric acid. May be. The production method of the spray is described in detail in, for example, US Pat. Nos. 2,868,691 and 3,095,355.

  The injection for parenteral administration according to the present invention includes sterile aqueous or non-aqueous solutions, suspensions, and emulsions. As an aqueous or non-aqueous solution or suspension, one or more active substances are mixed with at least one inert diluent. Examples of the aqueous diluent include distilled water for injection and physiological saline. Examples of the non-aqueous diluent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, alcohols such as ethanol, polysorbate 80 (registered trademark), and the like.

  Such a composition further includes adjuvants such as preservatives, wetting agents, emulsifiers, dispersants, stabilizers (eg, human serum albumin, lactose, etc.), and solubilizing agents (eg, arginine, aspartic acid, etc.). May be included.

  The present invention will be described more specifically with reference to examples of the clone ESDN of the present invention, but these do not limit the scope of the present invention.

Example 1: Screening by SST Human coronary artery primary cultured endothelial cells (hCAEC) and smooth muscle cells (hCASMC) (Clontech), and their co-culture (mixed hCAEC and hCASMC of the same number of cells, EGM-2-MV (commodity) Name), purchased from BioWhittaker) for 2 days) and a cDNA library was constructed (see Japanese Patent Application No. 10-119731). Next, as a result of screening by the yeast SST method (see US Pat. No. 5,536,637), an ESDN having a CUB domain was obtained from a cDNA library using co-culture as a source.

Example 2: Full-length cDNA cloning of human, mouse and rat 5'-3 'using a Marathon cDNA amplification kit (trade name, purchased from CLONTECH) to obtain full-length cDNA -5'-, 3'-terminal cDNA was cloned using RACE (Rapid Amplification of cDNA End) method. At the same time, primers were designed based on human base sequence information, and rat and mouse ESDNs were also isolated by RT-PCR.
Mouse primer 5'-CTGCCTCACTACTCTCCTCTCTC-3 '(SEQ ID NO: 10)
5′-CTGCTTTATTCCTTTCCACCAACCTG-3 ′ (SEQ ID NO: 11)
Rat primer 5'-TGGCTGGTCATGGTCTCACTACTTCTC-3 '(SEQ ID NO: 12)
5′-TGTGCTTTAAAACGATGCTTTG-3 ′ (SEQ ID NO: 13)
As a result, it was revealed that ESDN has high homology among human, mouse, and rat, but no 5 ′ terminal sequence including the translation start point ATG can be found in any species. It was. This may be due to the high GC content of the 5 ′ end region of ESDN. Therefore, screening was performed using a mouse phage genomic library (Lambda FIXII library (trade name, purchased from Strategene)). As a result, two positive clones containing the translation start point (Met) were obtained. Therefore, a 5'-side primer containing this Met was prepared, and an antisense primer containing a downstream exon (not included in the genomic clone)
Sense primer: 5′-GCACTATGCGGGCGGATTGC-3 ′ (SEQ ID NO: 14)
antisense primer: 5′-GAGATGTAAGGGTTCCACTCTCAGG-3 ′ (SEQ ID NO: 15)
As a result of RT-PCR, human and rat counterparts were obtained. From the obtained amino acid sequence, human and rodent showed 84 to 5% homology, and mouse and rat showed 92% homology. The alignment of human, rat and mouse ESDN is shown in FIG.
From the results of the motif search, it was found that ESDN is a type I transmembrane protein having one CUB domain and one factor V / VIII homology domain outside the cell. Moreover, the structural similarity with neuropilin (having two CUBs, two FV / VIII domains, and one MAM domain) can be pointed out from this structural feature. In other regions, there is a highly homologous region in which horseshoe crab Factor C, and Coch, which is one of the genes responsible for hearing loss in humans, are conserved. This region is new in Coch because it is a part where all four mutations discovered so far in DFNA9 hereditary deafness are localized, and it is also found in living organisms such as horseshoe crabs. It is considered a domain structure. It is shown in FIG. 2 and FIG.

Example 3: Expression of human ESDN protein using mammalian cells and identification with anti-peptide antibody The original V5 epitope of the expression vector for mammalian cells was replaced with a FLAG tag, and pEF6V5-His was obtained. The product was subcloned into (trade name, purchased from Invitrogen). These expression vectors were introduced into cells by Cellphect (trade name, purchased from Amersham Life Science) or Lipofectamine (trade name, purchased from Life Technologies, Inc.) into 293T cells and COS7 cells. . The target expressed protein was confirmed by preparing a cell lysate and then Western blotting with various antibodies.
anti-FLAG M2 monoclonal antibody (trade name, purchased from Sigma)
GlyGluArgIleArgIleLysPheGlyAspGlyAspIleGluAspSerAsp (SEQ ID NO: 16) was used as a rabbit anti-CUB polyclonal antibody GlnAspLysIlePheGlnGlyAsnLysAspLysAgV Obtained (Sawady Technology).
Western blotting followed the protocol of ECL (trade name, purchased from Amersham Life Science) Renaissance (NEN Life Science). As a result, bands that were not observed in the cell lysate into which only the expression vector was introduced were detected at around 127, 106, and 93 kDa (shown in FIG. 6).

Example 4: Analysis of signal sequence From the hydrophobicity plot (shown in Fig. 4) of the obtained amino acid sequence, it was found that the signal sequence of ESDN was atypical longer than the normal sequence. Therefore, COS7 cells introduced with human full-length cDNA were fixed with 4% paraformaldehyde, and then reacted with a primary antibody for 30 minutes at room temperature. Next, the secondary antibody, Texas red anti-mouse IgG (trade name, purchased from Vector Laboratories) or FITC anti-rabbit IgG (FITC-anti-rabbit IgG) (trade name, The reaction was carried out using a Bio-Rad confocal laser scanning microscope (trade name, model MRC-1024). As a result, it was found that the protein was expressed on the cell surface (shown in FIG. 7).
Next, an attempt was made to confirm the cleavage site of the signal sequence.
MESDN-Ex (Edns) with His-tag added to the C-terminus of the extracellular region of mouse ESDN and human CD5 signal sequence: MetProMetGlySerLeuGlnProLeuThlaLeuTyrLeuLeuGlyMetLeuGalLuS 600-603 (1993)). After introduction into 293T cells, the target protein secreted into the culture supernatant was detected by Western blotting using “His-probe H-15 polyclonal antibody” (trade name, purchased from Santa Cruz). As a result, proteins of the same size were confirmed from both constructs of mESDN-Ex (Edns) and mESDN-Ex (CD5) (shown in FIG. 5). The signal sequences of human CD5 and mouse ESDN are smaller for 39 amino acids (about 4 kDa) human CD5, but the size of the obtained protein is the same, so that the same cleavage site was predicted (shown in FIG. 8). .)

Example 5: Southern zooblot analysis of ESDN protein Using [ ³² P] dCTP-labeled human ESDN as a probe, mammals (mouse, rat, rabbit, cow, human), Xenopus, fly ( Fly, Southern zooblot analysis in yeast seeds was performed (washed at 37 ° C., 1 × SSC). As a result, a strong band was confirmed in mammals, and it was revealed that ESDN was highly conserved in mammals. On the other hand, a weak band was confirmed in Xenopus. In Drosophila (fly) and yeast (yeast), no band was confirmed (shown in FIG. 9A).

Example 6: Gene locus of human ESDN In humans, radiation hybrid map D3S1603-D3S1271, from information of two (stSG29921, sts-D29024) independent sequence-tagged sites (STS), It became clear that there is a locus in the vicinity of D3S1552-D3S1603 (site that seems to correspond to chromosome 3q11.2 in cytogenetic map). In order to confirm the results obtained from this database, genomic southern hybridization was performed using the mouse lines A9 (Neo3) and A9 (Neo12) (JCRB Cell Bank). . As a result, the mouse ESDN was confirmed in both A9 (Neo3) and A9 (Neo12) because the human ESDN probe can also recognize mouse ESDN. On the other hand, human ESDN was detected only in A9 (Neo3). From this, it became clear that human ESDN has a locus at a site which seems to correspond to chromosome 3q11.2 (shown in FIG. 9B).

Example 7: Analysis of the expression site of ESDN mRNA Northern analysis of cultured human cells was performed by extracting total RNA from hCAEC, hCASMC and their co-cultures with TRIzol (trade name, purchased from Life Technologies). did. For Northern analysis of rat tissues / cells, total RNA was extracted with TRIzol LS (for whole blood) and TRIzol (for other tissues and cultured cells). Next, poly (A) RNA was purified by “Oligotex ™ -dT30 Super” (trade name, purchased from Roche Molecular Biochemicals). Next, [ ³² P] dCTP-labeled human, rat ESDN or GAPDH cDNA was used as a probe for Northern blot analysis (washed at 65 ° C., 0.2 × SSC). As a result, strong expression was observed in hCASMC cells (6.4, 3 kb). On the other hand, hCAEC cells showed weaker expression than hCASMC. In addition, the change of the expression of ESDN mRNA by co-culture was not recognized (shown in FIG. 10A).
As a result of Northern analysis of rat tissues / cells, it was found that ESDN mRNA was not confirmed from whole blood cells and its expression was considerably low in the liver (shown in FIG. 10B).

Example 8: Expression of ESDN protein using human coronary artery smooth muscle cells hCASMC was cultured in serum-starved DMEM / 2mM glutamine for 48 hours. Next, the medium was replaced with a medium containing PDGF-BB, AT-II, and FCS (trade name, Sigma, purchased from Life Technologies) at designated concentrations, and stimulation was performed. Total RNA was prepared by Trizol (trade name, purchased from Life Technologies), and cDNA synthesis was performed by SuperScript Preamplification System (trade name, First Strand cDNA Synthesis: Life Technologies). Purchased). MRNA was measured by real-time quantitative RT-PCR (PE Applied Biosystems Prism Model 7700 Sequence Detection System). The sequences of the forward and reverse primers are as follows.
ESDN forward: 5′-CCCACGCAAGGTGATGGATG-3 ′ (SEQ ID NO: 19)
ESDN reverse: 5′-CAAGAATCAGAATCTTCAATGTCAAAG-3 ′ (SEQ ID NO: 20)
ESDN probe: 5 '-(6-FAM) -CCTGGAGATGGAACCCTTACATCCATAAAC- (TAMRA) -3' (SEQ ID NO: 21)
Although these sequences are based on humans, they were confirmed to be applicable to rodents.
The sequence of human GAPDH is as follows.
human GAPDH forward: 5′-GAAGGTGAAGGTCGGATC-3 ′ (SEQ ID NO: 22)
human GAPDH reverse: 5′-GAAGATGGTGATGGGGATTC-3 ′ (SEQ ID NO: 23)
human GAPDH probe: 5 '-(VIC) -CAAGCTTCCCGTTCTCAGCC- (TAMRA) -3' (SEQ ID NO: 24)
“TaqMan Rodent GAPDH Control Reagents” (PE biosystems) was used for the quantification of rat GAPDH. The mRNA levels of ESDN and GAPDH mean copy number. Therefore, the previous expression level was normalized by GAPDH. This was accomplished by a standard curve generated from serial dilutions of the standard. As a standard, a known amount of plasmid obtained by subcloning ESDN or GAPDH into pBlueScript SK (-) (trade name, purchased from Stratagene) was prepared.
As a result, the expression of ESDN increased in a concentration-dependent manner upon stimulation with PDGF-BB, but no increase in expression was observed upon stimulation with AT-II. In addition, the increase in expression of ESDN was also observed in the FCS stimulation in a concentration-dependent manner, but the amount was smaller than that in the PDGF-BB stimulation (shown in FIG. 11).

Example 9: Expression of ESDN protein in rat common carotid artery The carotid artery was collected 0, 5, and 14 days after balloon injury (n = 5, day 0, 5, n = 4, day 14). Total RNA was prepared by Trizol (trade name, purchased from Life Technologies), and cDNA synthesis was performed using "SuperScript Preamplification System" (trade name, First Strand cDNA Synthesis: purchased from Life Technologies). Rat ESDN and GAPDH mRNA were measured by “real-time quantitative RT-PCR” (PE Applied Biosystems Prism Model 7700 Sequence Detection System). As a result, a trend of increasing ESDN was observed on the 5th day, and a significant increase of 30% was observed on the 14th day. Next, the expression of this ESDN was analyzed immunohistologically (shown in FIG. 12).
Treated or untreated rats were anesthetized and the tissues were perfused with physiological saline previously cooled to 4 ° C. And the local perfusion part was fixed with 4% paraformaldehyde. The carotid artery was carefully collected and embedded with “Tissue-Tek OCT Compound” (trade name, purchased from Sakura Finetechnical) placed on a dry ice-ethanol bath. The tissue was sliced to a thickness of 4 μm, and immunohistochemical analysis was performed by the “avidin-biotin-alkaline phosphatase complex” (trade name, purchased from Vector Laboratories) method. Alkaline phosphatase was developed with “Vector Red” (trade name, purchased from Vector Laboratories). Sections were counterstained with methyl green. Rabbit anti-peptide polyclonal antibody (primary antibody) was used at a concentration of 5 to 10 μg / ml. As a negative control, normal rabbit IgG (DAKO) was used at the same concentration. As a result, although the media of the aorta and common carotid artery where vascular smooth muscle exists are stained, the central nervous system such as the brain and spinal cord (arrows) and peripheral nerves such as the vagus nerve (arrowhead) are strongly stained (Shown in FIG. 13). A, B, E and F were stained with anti-CUB antibody. C, D, G and H were stained with the same concentration of rabbit anti-IgG antibody as the primary antibody (control). M and N in E and F indicate the vascular media and intima, respectively.

Example 10: Measurement of BrdU incorporation using 293T cells Full-length ESDN and its deletion mutants (hESDN-ΔEC, hESDN-ΔCy) were expressed using the expression vector “QIAfilter Plasmid Midi Kit” (trade name, QIAGEN) (Purchased more). Then, for further purification, extraction was performed twice with phenol / CIAA, and CIAA extraction was performed once. 293T cells were transfected using CellPhect (trade name, purchased from Amersham Life Science). After 12 hours, the medium was replaced with fresh DMEM / 10% FCS and incubated for 2 hours. Next, the cells were once collected with trypsin, and then replated on two 96-well plates. One plate was uptaked after 2 hours of BrdU incorporation for 24 hours. BrdU incorporation was measured by “Cell Proliferation ELISA, BrdU” (colorimetric) (trade name, purchased from Roche Diagnostics). On the other plate, the number of cells for 2 hours after seeding was measured by “Premix WST-1 assay kit” (trade name, purchased from TaKaRa Biomedicals). As a result, it was revealed that BrdU incorporation was significantly suppressed in cells in which full-length ESDN was expressed. On the other hand, those lacking the extracellular domain attenuated this effect. Moreover, it was confirmed that this effect was completely lost in the case of lacking the intracellular domain (shown in FIG. 14).

Represents alignment of human, mouse and rat ESDN. Represents the domain structure in ESDN, neuropilin and Coch. FIG. 4 represents alignment of LCCL portions of human, mouse, rat ESDN, rim lass factor C, rat Lgl-1, human Coch, mouse Coch and rat Coch. Represents the hydrophobicity of human and mouse ESDN. The structure of an expression vector is represented. FIG. 6 represents Western blot analysis of human full-length ESDN. It shows how ESDN is expressed on the cell membrane surface. Fig. 4 represents Western blot analysis of cleaved signal sequences. A represents Southern blotting using human ESDN cDNA. B indicates that human ESDN is in chromosome 3. A and B show Northern blotting analysis with cDNA of ESDN using RNA of human carotid artery cells and RNA of each organ of rat. In human smooth muscle cells, the appearance of ESDN mRNA induced by PDGF or FCS stimulation is shown. FIG. 5 shows that the expression of ESDN mRNA is increased by damaging the rat carotid artery with a balloon. FIG. 5 shows that the expression of ESDN is increased by damaging the rat carotid artery with a balloon. It shows that BrdU incorporation is suppressed in ESDN overexpressing cells.

Claims (7)

  A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, 5 or 8.   A cDNA encoding the polypeptide of claim 1.   The cDNA according to claim 2, comprising the base sequence represented by SEQ ID NO: 3, 6 or 9.   A replication or expression vector comprising the cDNA of claim 3.   A host cell transformed with the replication or expression vector according to claim 4.   The method for producing a polypeptide according to claim 1, wherein the host cell according to claim 5 is cultured under conditions for expressing the polypeptide according to claim 1.   A polyclonal or monoclonal antibody of the polypeptide of claim 1.

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