JP2002335972A - Insulin receptor-associated receptor-binding protein and its application - Google Patents

Abstract

PROBLEM TO BE SOLVED: To identify a ligand bondable to an insulin receptor-associated receptor-bonding protein (IRR), and to provide an application thereof and a method for retrieving an agonist or an antagonist of the IRR ligand. SOLUTION: The protein is the one having the following properties or a modified product thereof, and having activities of bonding to the IRR: (a) containing an amino acid sequence represented by sequence number 1 (refer to the specification); and (b) having 6,135, 6,206, 6,250 or 6,321 molecular weight measured by a mass spectrometry using a Fourier transformation ion cyclotron method. The agonist or the antagonist of the IRR ligand is retrieved by bonding the protein with the insulin receptor-associated receptor in the presence of a specimen, and measuring the inhibition of the bond.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a therapeutic agent for controlling proliferation and differentiation of cells and a method for searching for such a therapeutic agent.
More specifically, the present invention relates to a therapeutic agent that controls proliferation and differentiation of cells involved in diabetes, neuropathy, kidney disorder, gastrointestinal disorder, and the like, and a method for searching for such a therapeutic agent.

[0002]

2. Description of the Related Art Many growth and differentiation factor receptors constitute a plurality of families, each of which comprises an intracellular kinase domain having a similar structure and an extracellular ligand binding domain.

[0003] In recent years, a number of novel genes, which are thought to structurally encode receptors from genetic information, have been discovered, and these are called orphan receptors because their ligands are unknown. It is inferred from the function of the family receptor and the specificity of the expression site that there are important factors regulating the bioregulatory action in the ligand.

[0004] Insulin receptor-related receptor (insulin re
ceptor-related receptor (hereinafter sometimes referred to as “IRR”) is an orphan receptor belonging to the insulin receptor family (Shier et al., J. Biol. Chem., 264: 14).
605, 1989). Insulin, insulin-like growth factor (IG
Known insulin families, such as F) -1, bind to the corresponding receptor as a ligand, and through receptor autophosphorylation, proliferation of cells expressing the receptor, expression of glucose transporter, hepatic gluconeogenesis Regulate functions such as system inhibition. Therefore, it is expected that the use of a ligand for IRR can regulate the proliferation, differentiation and function of IRR-expressing cells through binding to IRR and phosphorylation of IRR.
However, it has been reported that known insulin families, such as insulin and IGF-1, cannot cause binding to IRR and transduction of phosphorylation signals (Zhang et al., JB
iol. Chem., 267: 18320, 1992), and the identity of the ligand that specifically binds to IRR was still unknown.

[0005] The increase in insulin resistance due to changes in lifestyle such as obesity is a major cause of the recent increase in the number of type 2 diabetes patients. However, it has also been reported that if insulin resistance causes compensatory proliferation and differentiation of β-cells, which are insulin-secreting cells in the islets of Langerhans in the pancreas, diabetes does not occur (Terauchi et al.
J. Clin. Invest., 99: 861, 1997). The physiological mechanism that regulates pancreatic β-cell capacity is unknown, but elucidation of its regulators is a new perspective that aims to increase and maintain endogenous or transplanted pancreatic β-cell capacity in diabetic patients. , To provide treatment.

[0006] In recent years, findings from mice knocking out IRS-2, one of the intracellular substrates of the insulin receptor kinase, suggest that IRS-2 is essential for increasing the capacity of pancreatic β-cells in response to insulin demand. And began to strongly suggest the involvement of the insulin receptor family (Wither
s et al., Nature, 391: 900, 1998). On the other hand, it was confirmed that IRR is highly expressed in pancreatic β-cells, and that IRR kinase phosphorylates IRS-2 using artificially produced insulin receptor (IR) / IRR chimeric receptor. (Hirayam
a et al., Diabetes, 48: 1237, 1999), it is postulated that an unknown ligand (IRR ligand) that binds to IRR acts on pancreatic β-cells and induces proliferation, differentiation, and maintenance of the cells. You. Therefore, obtaining an IRR ligand and an agonist thereof provides a means and a method for regulating the proliferation and differentiation of pancreatic β cells, and if this is administered to a living body, diabetes through the proliferation and differentiation of pancreatic β cells. Is expected to be a therapeutic agent for.

It has been reported that IRR is expressed in specific localized tissues and cells such as nerves, kidneys and stomach (Reinhardt et al., Endocrinology, 133: 3, 1993, Watt.
Adv. Exp. Med. Biol., 343: 125, 1993), and are considered to be involved in the proliferation and differentiation of these cells. Therefore, by using IRR ligands and agonists to induce proliferation and differentiation of these tissues and cells, therapeutic agents for innate or acquired neurological disorders such as drugs, autoimmunity, inflammation, renal disorders, gastrointestinal disorders, etc. Is also expected to be useful.

[0008] Further, an antagonist of the IRR ligand is expected to be a therapeutic agent for diseases caused or accompanied by hyperproliferation or hyperfunction of the tissues or cells, for example, proliferative nephritis. However, as described above, since the IRR ligand has not been identified and the IRR ligand is unknown, there is no simple method for searching for its agonist or antagonist.

[0009] By the way, factors inhibiting the growth of cancer cells (Shoyab et al., Proc. Natl. Acad. Sci. USA, 87: 7912, 1
990) or as a cysteine-rich protein present in leukocyte granules (Bateman et al., Biochem. Biophys.
s. Commun., 173: 1161, 1990), and Epithelin / granulins are known. As the epithelin / granulins, a total of seven types from granulin A (epitelin 1) and B (epitelin 2) and C to G are known.

The functions and uses of the polypeptides belonging to epithelin / granulins include, as epithelins 1 and 2, tumor cell growth-inhibiting effects, epithelial cell growth-promoting effects (promotion of wound healing), keratinocyte growth-promoting effects (treatment of psoriasis with antagonists) (Shoyab et al., Proc. Natl. Acad.
Sci. USA, 87: 7912, 1990, WO91 / 15510A). In addition, epithelin / granulin precursor itself (PCDGF, GP88)
Have been reported to promote the growth of tumor cells and fibroblasts (Zhou et al., J. Biol. Chem., 268: 10863,1993, Xu
Et al., J. Biol. Chem., 273: 20078, 1998, WO 98/5260.
7). In addition, epithelin 1 was found to be 140-1 on human breast cancer cell membrane.
Binding to a 45 kDa protein (Culouscou et al., J. Biol.
Chem., 268: 10458, 1993) and that PCDGF binds to a 120 kDa protein on the membrane of the mink lung epithelial cell line (Xia).
Biophys. Res. Commun., 245: 539, 199.
8) is suggested. However, these epithelins 1
And the proteins to which PCDGF binds have not been identified, and the receptors for epithelin / granulins are not yet known.
There are no reports suggesting an association with

[0011]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above, and has as its object to provide a method for identifying an IRR ligand unknown so far, its use, and searching for an agonist or antagonist of the IRR ligand. Is a major issue.

[0012]

Means for Solving the Problems As a result of intensive studies for the above purpose, the present inventors have purified a protein having an activity that specifically binds to IRR from cell culture supernatant, and identified this protein. And succeeded in completing the present invention.

Specifically, for 76 types of cells, culture supernatants stimulated with phorbol ester were collected, concentrated, and
Surface plasmon resonance (SPR) showing R-specific binding
Screening was performed, and IRR-specific binding activity was observed in eight cell culture supernatants. Among them, rat C6 glioma cells (ATCC No. CCL-107) were cultured in large amounts, the culture supernatant was collected and roughly fractionated, and then purified by three-step reverse phase HPLC using IRR-specific binding activity as an index. went. Then, the N-terminal amino acid sequence of the purified protein was determined, the molecular weight was measured by mass spectrometry, and the corresponding protein was identified from database information. As a result, the purified protein was a protein belonging to epithelin / granulins having a molecular weight of 6135, 6206, 6250 or 6321 and consisting of 57 to 59 amino acids represented by the amino acid sequences shown in SEQ ID NOS: 3 to 7. That is, the present invention is as follows.

(1) A protein having the following properties or a variant thereof, which has an activity of binding to an insulin receptor-related receptor. (A) contains the amino acid sequence shown in SEQ ID NO: 1. (B) Molecular weight measured by mass spectrometry using Fourier transform ion cyclotron method: about 6135, 620
6, 6250 or 6321. (2) the protein of (1), which has any one of the amino acid sequences of SEQ ID NOs: 3 to 7;

(3) A pharmaceutical composition comprising, as an active ingredient, a protein that binds to an insulin receptor-related receptor or an agonist or antagonist of the protein. (4) The pharmaceutical composition according to (3), which has an action of controlling proliferation and differentiation of cells expressing an insulin receptor-related receptor. (5) The pharmaceutical composition according to (4), wherein the cells are cells involved in diabetes, neuropathy, kidney disorder, and gastrointestinal disorder. (6) The pharmaceutical composition according to (5), wherein the cells are pancreatic β cells. (7) The protein that binds to the insulin receptor-related receptor is epithelin / granulin (3) to (6).
The pharmaceutical composition of any one of the above. (8) Epithelin / granulin is a protein contained in the culture supernatant of rat glioma cells stimulated with phorbol ester and concentrated in a fraction eluted with 8 to 20% acetonitrile from a C18 reverse phase HPLC column. (7) The pharmaceutical composition according to (7). (9) The pharmaceutical composition according to (7), wherein the epithelin / granulin comprises the amino acid sequence of SEQ ID NO: 8. (10) The epithelin / granulin is SEQ ID NO: 3 to
7. The pharmaceutical composition according to (7), which is a protein having any one of the amino acid sequences of 7 or a variant having an activity of binding to an insulin receptor-related receptor.

(11) DN encoding the protein of (1)
A. (12) The DNA of (11), which encodes any of the amino acid sequences of SEQ ID NOs: 3 to 7. (13) an insulin receptor-related receptor-binding protein, wherein the binding between an insulin receptor-related receptor and a protein that binds to the receptor is performed in the presence of a test substance, and the inhibition of the binding is measured. And a method for searching for an agonist or antagonist. (14) The binding between the insulin receptor-related receptor and a protein that binds to the receptor is detected by a shift change in surface plasmon resonance, thereby measuring the inhibition of the binding. A) a method for searching for an agonist or antagonist of an insulin receptor-related receptor-binding protein.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The IRR ligand of the present invention is a protein that specifically binds to IRR.

The IRR ligand of the present invention may be purified from the culture supernatant of rat C6 glioma cells by reverse-phase HPLC or other means, for example, as described in Examples below, or may be synthesized by a peptide synthesis method. May be combined. Alternatively, a gene corresponding to the amino acid sequence may be obtained by a method well known in the art, and the gene may be used in a bacterial, yeast, insect cell, mammalian cell or the like by a gene recombination method.
IRR purified from culture supernatant of rat C6 glioma cells
The amino acid sequences shown in SEQ ID NOs: 3 to 7 have been obtained by amino acid sequencing and mass spectrometry of the ligand.

The IRR ligand of the present invention may be derived from a rat or as long as it has the property of binding to IRR.
IRR ligands derived from mammals such as human, monkey, mouse, dog, cow, rabbit, and birds, fish, and any other animals may be used. When an IRR ligand is used as a component of a pharmaceutical composition, it is preferably derived from a mammal.

In addition to the IRR ligand protein identified in the examples, the IRR ligand may have one or more amino acid sequences in SEQ ID NO: 8 or SEQ ID NOS: 3 to 7 as long as it has the property of binding to IRR. May have one or several amino acid substitutions, deletions, insertions or additions at the position (1). In the present invention, the “variant” refers to one having such amino acid substitution, deletion, insertion or addition, or one having modification.

The IRR ligand of the present invention may be one in which one or a plurality of the same or different proteins are bound in tandem as long as it has the property of binding to IRR. Further, as long as it has the property of binding to IRR, it may be a sugar chain-bound or non-linked sugar chain.

A database search was performed on the N-terminal amino acid sequence of the IRR ligand purified in the Examples described below. As a result, the amino acid sequence at positions 440 to 460 of the amino acid sequence encoded by the gene known as rat epithelin / granulin precursor was obtained. Matches the granulin D domain (Bha
Natl. Acad. Sci. USA, 89: 1715, 199.
It was estimated that the protein was equivalent to 2). Furthermore, repurification of the IRR ligand and the results of mass spectrometry by the Fourier transform ion cyclotron method (6135, 6250, 63
21 or 6206) from rat C6 cell culture supernatant
The IRR ligand is the epithelin / granulin precursor 439
The aspartic acid or isoleucine at position 440 to N
A protein consisting of 57 to 59 amino acids 439 to 497 with C-terminal lysine at position 495, aspartic acid at position 496, or alanine at position 497, and having six intramolecular Cys-Cys bonds. It became clear that there was.

Briefly describing epithelin / granulins, epithelin is a protein purified from rat kidney as a factor for suppressing the growth of cancer cells (Shoyab et al., Proc. Natl. Acad. Sci. USA, 87 : 7912,
1990), granulin is a protein purified as a cysteine-rich protein present in human leukocyte granules (Bateman et al., Biochem. Biophys. Res. Commun., 173:
1161, 1990). By epithelin / granulin cDNA cloning, the cDNA encodes a common precursor of the epithelin / granulin group and has a total of seven cysteine-rich motifs, from granulin A (epitelin 1), B (epitelin 2) to C to G. Has been found to be included in tandem. Also, amino acids 440 to 495 of rat epithelin precursor (Plowman et al., J. Biol. Chem., 2
67: 13073, 1992, WO91 / 15510A), or 438-492 (Granulin D; Bhandari et al., Endocrinology, 133: 2).
682, 1993) is also disclosed as one of the motifs. In addition, the above-mentioned document (WO91 / 15510A) discloses that CX _5-6 CX ₅ is a common structure of epithelin / granulins.
Disclosed are a structure consisting of CCX ₈ CCX ₆ CCXDXXHCCPX ₄ CX _5-6 C (SEQ ID NO: 8) and a sequence obtained by adding any two amino acids before and after this sequence. The IRR ligand purified in the examples retains the common structure represented by SEQ ID NO: 8.

From the above, other granulins / epitelins or their variants, specifically, for example, CX
_5-6 CX ₅ CCX ₈ CCX ₆ CCXDXXHCCPX ₄ CX _5-6 C is a protein of epithelin / granulins, which is assumed to bind to IRR.

In the present invention, the IRR ligand is not limited to those having the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NOs: 3 to 7, and is collectively referred to as IRR ligand including the protein that binds to IRR as described above. The DNA of the present invention is a DNA encoding such an IRR ligand.

The granulin D reported and disclosed is estimated to be a homodimer or a heterodimer (Batema
n et al., Biochem. Biophys. Res. Commun., 173: 1161, 19
90, WO 93/15195), but the IRR ligand purified in the present invention is a monomer having no intermolecular bond based on the result of mass spectrometry.

IR including rat C6 glioma cells
Determination of the active fraction when purifying the IRR ligand from the cell supernatant expressing the R ligand, or identification of granulin / epitelins or their mutants as IRR ligands can be accomplished by determining the binding activity of the sample to IRR. It can be done by measuring. The binding activity of the test sample to IRR can be measured using a surface plasmon resonance (SPR) measuring device, for example, BIACORE (Biacore). Specifically, for example, the extracellular domain of IRR is prepared as a soluble protein, which is immobilized on the surface of a BIACORE sensor chip, and the test sample is sent to the sensor chip surface at a constant flow rate through a microchannel system. I do. If the test sample contains a molecule that binds to IRR (IRR ligand), the binding between the IRR-IRR ligand molecules increases the mass of the sensor chip surface and shifts the SPR signal in proportion to the amount of binding to the resonance unit. (RU) can be detected.
IRR-specific binding means that the so-called bulk effect due to solvents etc. can be reduced by simultaneously preparing a sensor chip on which proteins other than IRR are immobilized, and subtracting the change in resonance units when a test sample is applied to this chip. It can be confirmed by removing it. When an insulin receptor (IR) protein is used as a protein other than the IRR, for example, the IRR binding activity can be measured more accurately.

The soluble IRR protein used for the search is IRR cDNA
Can be prepared in the same manner as in the production of a protein by a normal gene recombination method. For example, mouse IRR cDNA
For example, using the cDNA fragment of the kinase region of mouse IRR amplified by PCR as a probe by the method shown in Hirayama et al., Diabetes, 48: 1237, 1999, pancreatic β cell line bH
It can be obtained by cloning from a C9 or MIN6 cDNA library. Soluble as control
When using an IR protein, the IR cDNA is, for example, Whittaker
Natl. Acad. Sci. USA, 84: 5237, 1987, can be obtained from a human kidney cDNA library using a synthetic oligonucleotide as a probe.

When IRR or IR is obtained as a soluble protein, a fragment encoding an extracellular region is obtained from the above IRR cDNA or IR cDNA, and if necessary, an appropriate synthetic oligonucleotide is used as an adapter and a spacer to prepare an expression vector. The protein may be introduced, transfected into an appropriate host, cultured for an appropriate time, and then the target protein may be purified from the cell lysate or culture supernatant. Further, it is more preferable to produce a fusion protein of IRR or IR and an IgG heavy chain constant region (Fc region), alkaline phosphatase, FLAG, or the like, because purification of the target protein is simplified. As IgG cDNA, for example, as shown in Nishimura et al., Cancer Res., 47: 999, 1987, human IgG1 from human plasma leukemia cell line ARH77.
You can get a chain. For example, when preparing an IRR-Fc or IR-Fc fusion protein, DNA containing the extracellular region of mouse IRR (residues 1 to 920) or the extracellular region of human IR (residues 1 to 944)
DNA containing fragment and H chain C (constant) region (Fc region) of human IgG1
The fragment was expressed in the expression vector pEF-BOS (Mizushima et al., Nucleic A
cid Res. 18: 5322, 1990) to construct a plasmid. This is transfected into CHO cells according to a standard method, cultured in a medium containing G418, and a resistant clone is selected. Then, the culture supernatant is added to the protein A column, and the adsorbed fraction is collected, and then the target IRR-Fc fusion protein, IR-F
c fusion protein can be obtained.

The IRR ligand of the present invention specifically binds to IRR of a cell in which IRR is expressed on a cell, such as pancreatic β cell,
It has the activity of controlling the proliferation and differentiation of those cells. Therefore, it is considered that the IRR ligand, or an agonist or antagonist thereof, can be used as a therapeutic agent for these diseases by suppressing or enhancing the proliferation and differentiation of cells involved in diabetes, neuropathy, kidney disorder, and gastrointestinal disorder. Can be

As described above, the onset of type 2 diabetes requires both insulin secretion deficiency and insulin resistance, and even if the mice are insulin resistant, the mouse undergoes compensatory proliferation of β-cells. Suggest that diabetes does not develop. It has also been reported that those who do not develop diabetes, even if they are obese and have insulin resistance, have a greater number of pancreatic β-cells than diabetic patients. Conversely, it is assumed that the compensatory proliferation of β cells is insufficient in diabetic patients. Therefore, the compensatory proliferation mechanism of β-cells is artificially enhanced to promote β-cell proliferation and regeneration and / or
Or, if it is possible to induce the maintenance of survival, it is expected to be a fundamental treatment for the treatment and prevention of type 2 diabetes, especially for severe type 2 diabetes in which glycemic control is performed by insulin replacement therapy. You.

In addition, agonists and antagonists that inhibit the binding of the IRR ligand to the IRR can be searched for by using the IRR ligand. That is, by binding an IRR ligand to an IRR (protein that binds to strikethrough) in the presence of a test substance and measuring the inhibition of the binding, an agonist or antagonist of the IRR ligand can be searched. As an example of a specific method for searching for agonists and antagonists, it is possible to determine whether the biological response caused by the addition of an IRR ligand to cells expressing IRR differs depending on the presence or absence of the test substance. Viewing method or IRR labeled with radioactive or fluorescent substances
A method of measuring the inhibition of the binding between a ligand and an IRR-expressing cell or an IRR prepared from the same cell, in the presence or absence of a test substance, and the like can be mentioned. Also, IR
Modifications to biochemical reactions such as phosphorylation that occur when an IRR ligand is added to IRR prepared from the cell membrane of an R-expressing cell can also be used as an index.

Further, as described in Examples described later, the extracellular partial protein of IRR (soluble IRR) prepared by a gene recombination method or the like is immobilized on a suitable solid phase to inhibit the binding of the IRR ligand to this. It can also be used as an index. Specifically, for example, using a surface plasmon resonance (SPR) measurement device,
Prepare a sensor chip bound to soluble IRR and a sensor chip bound to nothing or bound to immunoglobulin or soluble IR. Then, before, simultaneously with, or after the IRR ligand solution, a solution of the test substance is flowed, and the IRR
By searching for a substance that suppresses a change in SPR shift specific to an IRR-binding sensor chip caused by a ligand, a small molecule agonist or antagonist can be found with high sensitivity. As described above, any method may be used as long as the method detects the modification or inhibition of the reaction induced specifically by the IRR ligand by the IRR.

The IRR ligand used as a search means is not limited to the proteins described in the Examples of the present invention as long as they are epiterin / granulin-like proteins that bind to IRR. In the present invention, an IRR ligand is an agonist thereof,
When used as a means of searching for an antagonist, it differs depending on the evaluation method, but there is generally no particular limitation on the solvent and the like,
The concentration is usually 1 pg / ml to 1 g / ml, more preferably 1 ng / ml to 1 mg
/ ml, even more preferably in a concentration range of 10 ng / ml to 10 μg / ml.

When the IRR ligand or an agonist or antagonist thereof is used as a therapeutic agent in the present invention, it is administered as an injection (intravenous, subcutaneous, intramuscular, etc.) or an oral preparation. The dosage varies depending on the condition, but is usually in the range of 1 pg to 1 g per day for an adult.

Formulation of the IRR ligand or an agonist or antagonist thereof is made into a dosage form such as an injection, an inhalant, a tablet, a granule, a powder, a capsule, a suppository and the like by a usual method. For example, when preparing an injection, a pH adjuster, a buffer, a stabilizer, a preservative, and the like are added to the main drug as necessary. IRR ligands, agonists thereof, antagonists thereof may be used in other pharmaceutical compositions such as diabetes, neuropathy,
It can be used in combination with drugs for kidney and gastrointestinal disorders.

[0037]

The present invention will be described more specifically with reference to the following examples.

[0038]

[Example 1] Purification of IRR ligand from rat C6 cell culture supernatant <1> Collection of cell culture supernatant Rat C6 cells (ATCC No. CCL-107) were collected at an initial density of 3 × 10 ⁴
Α containing 400 ml of 10% FCS (fetal calf serum) at cells / ml
The cells were suspended in -MEM nucleic acid-containing medium and cultured in a roller bottle (Falcon 3007) at 37 ° C. at a rate of 0.5 rotation / minute in a closed rotation. After culturing for 3 days, the medium was removed and 400 ml of α-MEM nucleic acid-containing medium and 100 ng / ml of TPA (phorbol 12-myristate 13-a
cetate), and the mixture was further cultured for 72 hours, and the culture supernatant was collected.

<2> Purification of IRR Ligand 1.95 ml of TFA (trifluoroacetic acid) was added to 1.95 L of the culture supernatant, and a reversed phase column (5 cm diameter × 7.6 cm length; 150 ml / YMC C4300)
A S-40 (YMC)). After washing with 300 ml of 0.1% TFA, the column was eluted with 300 ml of 20% acetonitrile-0.1% TFA.

The IRR binding activity of the 20% eluted fraction was confirmed by the measurement method described below, and then diluted with 2 volumes of 0.1% TFA.
The mixture was adsorbed on an LC column (10 mm diameter × 250 mm length; Cosmosil 5TMS-300 (Nacalai Tesque)), sufficiently washed with 0.1% TFA containing 8% acetonitrile, and then eluted with an acetonitrile concentration gradient (1.6% / min). The flow rate was 3 ml / min, and fractionation was performed in 3 ml increments. The HPLC chromatogram is shown in FIG. The IRR binding activity was mainly recovered in the fraction indicated by the thick black line (FIG. 1).

After diluting the above active fraction with twice the amount of 0.1% TFA, an HPLC column (4.6 mm diameter × 250 mm length; Cosmosil 5TMS-
300%) and 0.1% TF containing 8% acetonitrile
After sufficient washing with A, acetonitrile concentration gradient (0.8% / min)
Eluted. The flow rate was 1 ml / min, and the peak at 280 nm was visually fractionated. This HPLC chromatogram is shown in FIG.
The IRR binding activity was mainly recovered in the fraction indicated by the thick black line (FIG. 2).

After diluting the above active fraction with twice the amount of 0.1% TFA, an HPLC column (4.6 mm diameter × 250 mm length; Vydac 218TP54)
(Vydac), washed thoroughly with 0.1% TFA containing 8% acetonitrile, and eluted with an acetonitrile concentration gradient (1.6% / min). The flow rate is 1 ml / min.
The peak at 280 nm was fractionated. The HPLC chromatogram is shown in FIG. IRR binding activity was recovered mainly in the fractions indicated by the thick black lines (FIG. 3).

<3> Measurement of IRR binding activity In each of the above purification steps, IRR binding activity was measured as follows.

(1) Measurement sample 40 μg of bovine serum albumin (BSA) was added as a carrier to the test sample solution, and after freezing, the solvent was removed using Speedvac AS290A (Servant). 200 μl residue
Dissolve in 1 mM hydrochloric acid and add 200 μl of 2 × HBS (40 mM HEPES, 0.6%
NaCl, 12 mM EDTA (pH 7.5)) to obtain a measurement sample.

In the last two steps of the purification process, the amount of the lysis solution and the amount of the neutralizing solution were each 100 μl, and the amount of BSA added was 20 μg.

(2) Preparation of Soluble IRR and Soluble IR The mouse IRR cDNA was obtained by using the cDNA fragment of the kinase region of the mouse IRR amplified by PCR as a probe with the pancreatic β cell line bHC9
/ MIN6 cDNA library (Hirayama et al., Diabetes, 48, 1
237-1244, 1999).

Next, the pFas-FcII vector (Suda et al., J. E.
xp. Med., 179, 873-879, 1994, provided by Dr. Takashi Suda, Kanazawa University Cancer Institute) by replacing the extracellular region (residues 1 to 920) of mouse IRR with IRR. Human
An IRR-Fc fusion protein expression plasmid expressing a protein fused to the H chain C (constant) region (Fc region) of IgG1 was constructed. As a control, a human IR cDNA (Kaburagi et al., J. Biol. Chem., 2
68, 16610-16622, 1993, provided by Dr. Yasushi Kaburagi, Faculty of Medicine, The University of Tokyo) and the Fas portion of pFas-FcII vector
Similarly, an IR-Fc fusion protein expression plasmid was prepared by replacing the extracellular region of IR (residues 1 to 944).
These plasmids were transfected into CHO cells according to a standard method, cultured in a medium containing G418, and resistant clones were selected. Clones secreting the desired fusion protein into the supernatant were selected by Western blotting using an anti-human IgG antibody.

The confluence of these recombinant CHO cells dish 10cm culture (Falcon 3003A) (1~2 × 10 7 /
After culturing to a dish, the medium was replaced with 20 ml of HamF-12 medium (containing no serum or G418), and the supernatant was aseptically collected 48 to 72 hours later. Further, a new HamF-12 medium was added, the same culture was repeated (up to 3 cycles), and the supernatant was collected. The culture supernatant was concentrated 30 to 100 times with a regenerated cellulose membrane (PLGC0MP04) using a minitan system (Millipore), and a protein A column (Amersham Pharmacia, HiTrap ProteinA, 1 ml) was used. -
Fc and IR-Fc fusion proteins were purified.

(3) Evaluation of IRR binding activity by surface plasmon resonance (SPR) measurement method SPR measurement was performed using BIACORE2000 (Biacore). Replace each flow cell of CM5 sensor chip with E
Activated with DC / NHS (mixture of N '-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide) (flow rate 5 μl / min, 7 minutes), IRR-Fc (20 μl
g / ml, 10 mM acetate buffer (pH 3.9)), IR-Fc (20 μg / ml, 1
0 mM formate buffer (pH 3.7)) was coupled to each of the separate flow cells by the amine coupling method.

The flow rate was set to 10 μl / min, and the measurement sample was 30 μl
20 seconds before sample injection (baseline) and start of injection 16
5 seconds later (15 seconds before completion: bound1) SPR (resonance units:
RU). The chip is 10 mM Glycine-HCl (pH 2.3
5) After washing and regenerating with 10 μl and the baseline was stabilized, the next sample was measured.

For each sample, the binding value [bound1-baseline] of the IRR-Fc-immobilized flow cell and the binding value [bound1-baseline] of the IRR-Fc-immobilized flow cell were determined.
The difference between the binding value and the IR-Fc binding value was taken as the binding value of the sample.

The binding value is indicated by a circle and a numeral in the text and in FIGS.

[0053]

Example 2 Determination of N-Terminal Amino Acid Sequence of IRR Ligand The N-terminal amino acid sequence of the IRR-binding activity fraction sample purified in Example 1 was analyzed using a protein sequencer 476A (Applied Biosystems) in accordance with the manual. Therefore, it carried out by the method using the glass filter which performed the polybrene treatment. First, 45 μl of the active fraction sample of FIG. 3 was added to a glass filter treated with polybrene according to the manual method, and amino acid sequence analysis was performed.

Table 1 shows the PTH amino acids considered to correspond to amino acids derived from the sample up to the 22nd residue, and the detected amounts calculated from the peak heights for the PTH amino acid standard.

[0055]

[Table 1]

From the above results, it was found that the active fraction sample was N
The terminal amino acid sequence consists of a peptide having the amino acid sequence of SEQ ID NO: 1 at the N-terminus and an amino acid sequence of SEQ ID NO: 2
It was considered to be a mixture of peptides having terminals.
Note that Xaa in each sequence was considered to be a cysteine residue that was not detected under these conditions, or a serine or threonine residue with a low recovery rate.

Amino acid sequence homology (GENETY
Using X), a protein having homology to the above amino acid sequence was searched from the database (NBRF). As a result, the following proteins were hit as proteins having 50% or more homology.

Epithelin / granulin precursor-mouse Epithelin / granulin precursor-rat Acrogranin-mouse granulin-rat Granulin precursor-human The cells used for the purification of IRR ligand (C6 cells) are derived from rats. When the amino acid sequences of SEQ ID NOS: 1 and 2 were compared with the rat epithelin / granulin precursor (589 amino acids), the sequence of SEQ ID NO: 1 was 440-460 of the rat epithelin / granulin precursor, SEQ ID NO: 2
And the amino acid sequence at positions 439 to 460 were identical.

[0059]

[Example 3] Repurification of IRR ligand Culture supernatant of rat C6 cells cultured as in Example 1
From 4 L, IRR ligand was newly purified. From the culture supernatant, YMC C4 300A S-40, Cosmosil 5TMS-300, Vydac
Half of the active crude fraction (400 μl) fractionated stepwise in the same manner as in Example 1 using 218TP54 was added to 1.2 ml of 0.05% HFBA.
(Heptafluorobutyric acid), and then HPLC column (4.
6mm diameter x 250mm length; adsorbed on Vydac 218TP54), 8%
After sufficiently washing with 0.05% HFBA containing acetonitrile, elution was carried out with an acetonitrile concentration gradient (1% / min for 30 minutes, and thereafter 0.5% / min). The flow rate was 1 ml / min, and the peak at 280 nm was visually fractionated. FIG. 4 shows the chromatogram at this time.
The binding activity was recovered in the fractions indicated by the arrows (peak 1 and peak 2) (FIG. 4). Furthermore, peak 1 and peak 2 were each repurified under the same conditions, and 150-200 μl
Concentrated. About 1/10 amount of each of the repurified preparations (about 10 μg) was lyophilized, dissolved in 200 μl of buffer, and the binding activity was measured. Peak 1 showed 13 RU and peak 2 showed 21 RU.

FIG. 4 Peaks 1 and 2 of the purified fraction
Was used for amino acid sequence analysis. twenty two
As a result of analysis up to the residue, an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 2 was obtained from peak 1, and an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1 was obtained from peak 2.

[0061]

Example 4 Determination of molecular weight of IRR ligand by mass spectrometry A 10 μl sample of the active fraction shown in FIG.
After adding 4 μl, using a Fourier transform ion cyclotron mass spectrometer APEXII70e (Bruker Daltonics), GADT-micro electrospray ion method,
The spectrum was measured. Bovine insulin for mass calibration,
Bovine ubiquitin and chicken lysozyme were used.

As a result, m / z = 2045.6716, 1534.7963, 12
28.0888, 1023.3715 main peaks were found,
Since it corresponds to [M + 3H] ⁺³ , [M + 4H] ⁺⁴ , [M + 5H] ⁺⁵ , [M + 6H] ⁺⁶ , the molecular weight was estimated to be 6135. Combined with the results of Example 2, the major component of the active fraction in FIG. 3 is that the aspartic acid at position 439 of the rat epithelin / granulin precursor has the N-terminal and the lysine at position 495 has the C-terminal, The protein was considered to be a protein having the amino acid sequence of SEQ ID NO: 4 or a protein having the amino acid sequence of SEQ ID NO: 4 having isoleucine at position 440 at the N-terminus and aspartic acid at position 496 at the C-terminus.

DIGCDQHTSCPVGQTCCPSLKGSWACCQLPHAVCCEDR
QHCCPAGYTCNVKARTCEK (SEQ ID NO: 3) IGCDQHTSCPVGQTCCPSLKGSWACCQLPHAVCCEDRQHCCPAGYTCNVK
ARTCEKD (SEQ ID NO: 4)

Further, m / z corresponding to [M + 4H] ^{+ 4} = 1563.547
9, 1581.2989 and 1552.5515 were found, and the corresponding protein had a molecular weight of 6250 and 632, respectively.
It was estimated at 1,6206. Combined with the result of Example 2, the third
The active fraction in the figure contains rat epithelin /
It was considered that a protein having the amino acid sequence of SEQ ID NO: 5, 6, or 7, having the amino acid at position 439 or 440 of the granulin precursor at the N-terminus, was included.

DIGCDQHTSCPVGQTCCPSLKGSWACCQLPHAVCCEDR
QHCCPAGYTCNVKARTCEKD (SEQ ID NO: 5) DIGCDQHTSCPVGQTCCPSLKGSWACCQLPHAVCCEDRQHCCPAGYTCNV
KARTCEKDA (SEQ ID NO: 6) IGCDQHTSCPVGQTCCPSLKGSWACCQLPHAVCCEDRQHCCPAGYTCNVK
ARTCEKDA (SEQ ID NO: 7)

The 12 cysteine residues contained in each of the above proteins are presumed to be crosslinked in the molecule.

Further, the molecular weight was similarly measured using 10 μl each of peak 1 and peak 2 of the purified fraction in FIG. As a result, peak 1 was estimated to be a protein having the amino acid sequences of SEQ ID NOS: 5 and 6, with the main peaks being proteins having molecular weights of 6250 and 6321. Peak 2 was estimated to be a protein having an amino acid sequence of SEQ ID NO: 4 with a protein having a molecular weight of 6135 as a main peak.

[0068]

According to the present invention, pancreatic β cells, nerves,
Epiterin / granulin-like proteins can be provided as IRR ligands for controlling proliferation and differentiation of kidney and gastrointestinal cells.

Further, a method for searching for an agonist or antagonist of an IRR ligand can be provided.

[0070]

[Sequence List] SEQUENCE LISTING <110> Ajinomoto Co., Inc. <120> Insulin receptor-related receptor binding protein and its use <130> P-8911 <140> <141> 2001-06- 06 <150> JP 2000-170912 <151> 2000-06-07 <160> 8 <170> PatentIn Ver. 2.0

<210> 1 <211> 21 <212> PRT <213> Mus musculus <220> <221> UNSURE <222> (3,8,9,15,16,18) <223> Xaa = Cys or Ser or Thr <400> 1 Ile Gly Xaa Asp Gln His Thr Xaa Xaa Pro Val Gly Gln Thr Xaa Xaa 1 5 10 15 Pro Xaa Leu Lys Gly 20

<210> 2 <211> 22 <212> PRT <213> Mus musculus <220> <221> UNSURE <222> (4,9,10,16,17,19) <223> Xaa = Cys or Ser or Thr <400> 2 Asp Ile Gly Xaa Asp Gln His Thr Xaa Xaa Pro Val Gly Gln Thr Xaa 1 5 10 15 Xaa Pro Xaa Leu Lys Gly 20

<210> 3 <211> 57 <212> PRT <213> Mus musculus <400> 3 Asp Ile Gly Cys Asp Gln His Thr Ser Cys Pro Val Gly Gln Thr Cys 1 5 10 15 Cys Pro Ser Leu Lys Gly Ser Trp Ala Cys Cys Gln Leu Pro His Ala 20 25 30 Val Cys Cys Glu Asp Arg Gln His Cys Cys Pro Ala Gly Tyr Thr Cys 35 40 45 Asn Val Lys Ala Arg Thr Cys Glu Lys 50 55

<210> 4 <211> 57 <212> PRT <213> Mus musculus <400> 4 Ile Gly Cys Asp Gln His Thr Ser Cys Pro Val Gly Gln Thr Cys Cys 1 5 10 15 Pro Ser Leu Lys Gly Ser Trp Ala Cys Cys Gln Leu Pro His Ala Val 20 25 30 Cys Cys Glu Asp Arg Gln His Cys Cys Pro Ala Gly Tyr Thr Cys Asn 35 40 45 Val Lys Ala Arg Thr Cys Glu Lys Asp 50 55

<210> 5 <211> 58 <212> PRT <213> Mus musculus <400> 5 Asp Ile Gly Cys Asp Gln His Thr Ser Cys Pro Val Gly Gln Thr Cys 1 5 10 15 Cys Pro Ser Leu Lys Gly Ser Trp Ala Cys Cys Gln Leu Pro His Ala 20 25 30 Val Cys Cys Glu Asp Arg Gln His Cys Cys Pro Ala Gly Tyr Thr Cys 35 40 45 Asn Val Lys Ala Arg Thr Cys Glu Lys Asp 50 55

<210> 6 <211> 59 <212> PRT <213> Mus musculus <400> 6 Asp Ile Gly Cys Asp Gln His Thr Ser Cys Pro Val Gly Gln Thr Cys 1 5 10 15 Cys Pro Ser Leu Lys Gly Ser Trp Ala Cys Cys Gln Leu Pro His Ala 20 25 30 Val Cys Cys Glu Asp Arg Gln His Cys Cys Pro Ala Gly Tyr Thr Cys 35 40 45 Asn Val Lys Ala Arg Thr Cys Glu Lys Asp Ala 50 55

<210> 7 <211> 58 <212> PRT <213> Mus musculus <400> 7 Ile Gly Cys Asp Gln His Thr Ser Cys Pro Val Gly Gln Thr Cys Cys 1 5 10 15 Pro Ser Leu Lys Gly Ser Trp Ala Cys Cys Gln Leu Pro His Ala Val 20 25 30 Cys Cys Glu Asp Arg Gln His Cys Cys Pro Ala Gly Tyr Thr Cys Asn 35 40 45 Val Lys Ala Arg Thr Cys Glu Lys Asp Ala 50 55

<210> 8 <211> 53 <212> PRT <213> Mus musculus <220> <221> UNSURE <222> (2,3,4,5,6,7,9,10,11,12 , 13,16,17,18,19,20,21,22,23,26,27,28,29,30,31,34,36,37,42,43,44,45,47,48,49 , 50,51,52) <223> Xaa = optional amino acid <220> <221> UNSURE <222> (7,52) <223> Xaa = may be nonexsistent <400> 8 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys 20 25 30 Cys Xaa Asp Xaa Xaa His Cys Cys Pro Xaa Xaa Xaa Xaa Cys Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Cys 50

[Brief description of the drawings]

FIG. 1 shows an HPLC column (10 mm diameter × 250 mm length; Cosmosil 5TMS-3) of a fraction eluted with 20% acetonitrile in Example 1.
00) It is a figure showing an elution result and an IRR binding value of each fraction.

FIG. 2 shows a HPLC column (4.6 mm diameter × 250 mm length; Cosmosil) obtained by pooling fractions 10 and 11 in FIG. 1 in Example 1.
5TMS-300) shows the elution results and the IRR binding values of each fraction.

FIG. 3 is a diagram showing the results of elution of the HPLC column (4.6 mm diameter × 250 mm length; Vydac 218TP54) of fraction 1 in FIG. 2 and the IRR binding value of each fraction in Example 1.

FIG. 4 shows a HPLC column (4.6 mm diameter × 250 mm length; Vydac 218T) in the final purification of the active fraction in Example 3.
P54) It is the figure which showed the elution result.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 13/12 C07K 14/47 25/00 G01N 21/05 C07K 14/47 21/27 C G01N 21/05 27/62 V 21/27 33/53 D 27/62 M 33/53 33/566 C12N 15/00 ZNAA 33/566 A61K 37/02 F term (reference) 2G057 AA02 AB07 AC01 BA03 BA05 2G059 AA01 BB04 BB12 CC16 DD01 EE02 FF08 MM05 4B024 AA01 BA21 CA04 DA02 EA04 GA11 HA01 4C084 AA02 AA17 BA01 BA08 BA20 CA23 CA56 DC50 MA17 MA31 MA35 MA37 MA41 MA43 MA52 MA66 NA14 ZA012 ZA662 ZA812 ZC352 4H045 AA10 AA30 BA17 BA20 CA40 DA01

Claims (14)

[Claims] 1. A protein having the following properties or a variant thereof, which has an activity of binding to an insulin receptor-related receptor. (A) contains the amino acid sequence shown in SEQ ID NO: 1. (B) Molecular weight measured by mass spectrometry using Fourier transform ion cyclotron method: about 6135, 620
6, 6250 or 6321. 2. The protein according to claim 1, which has any one of the amino acid sequences of SEQ ID NOs: 3 to 7. 3. A pharmaceutical composition comprising, as an active ingredient, a protein that binds to an insulin receptor-related receptor or an agonist or antagonist of the protein. 4. The pharmaceutical composition according to claim 3, which has an action of controlling proliferation and differentiation of cells expressing an insulin receptor-related receptor. 5. The pharmaceutical composition according to claim 4, wherein the cells are cells involved in diabetes, neuropathy, kidney disorder, and gastrointestinal disorder. 6. The pharmaceutical composition according to claim 5, wherein said cells are pancreatic β cells. 7. The protein that binds to an insulin receptor-related receptor is epithelin / granulin.
The pharmaceutical composition according to any one of claims 1 to 6. 8. A protein in which epithelin / granulins are contained in rat glioma cell culture supernatant stimulated with phorbol ester and concentrated in a fraction eluted with 8-20% acetonitrile from a C18 reverse phase HPLC column. The pharmaceutical composition according to claim 7, wherein 9. The pharmaceutical composition according to claim 7, wherein the epithelin / granulin comprises the amino acid sequence of SEQ ID NO: 8. 10. A protein, wherein the epithelin / granulin has an amino acid sequence of any one of SEQ ID NOs: 3 to 7,
Alternatively, the pharmaceutical composition according to claim 7, which is a variant having an activity of binding to an insulin receptor-related receptor. 11. D encoding the protein according to claim 1.
NA. 12. The DNA according to claim 11, which encodes any one of the amino acid sequences of SEQ ID NOs: 3 to 7. 13. An insulin receptor-related receptor characterized by binding an insulin receptor-related receptor to a protein that binds to the receptor in the presence of a test substance, and measuring the inhibition of the binding. A method for searching for an agonist or antagonist of a binding protein. 14. The method according to claim 1, wherein the binding between the insulin receptor-related receptor and a protein that binds to the receptor is detected by a shift change in surface plasmon resonance, thereby measuring the inhibition of the binding. A method for searching for an agonist or antagonist of the insulin receptor-related receptor-binding protein according to claim 13.