JP2012075408A - Physiologically active peptide and application thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new peptide which has energy metabolism modulating activity or circulation modulating activity.SOLUTION: There are provided a peptide composed of specific amino acid sequence or pharmacologically permissible salt thereof, and energy metabolism regulator or circulation regulator which contains the peptides and the salts as active ingredients. Also a method for screening substances which promote or inhibit activity of the peptide or agonist or antagonist to a receptor of the peptide utilizing the peptide or salts thereof is provided.

Description

  The present invention relates to a physiologically active peptide, a polynucleotide encoding the peptide, a method for producing the peptide, a medicament containing the peptide, a method for screening a substance that inhibits or promotes the activity of the peptide, and an agonist for the receptor of the peptide Alternatively, the present invention relates to an antagonist screening method and the like.

  Abnormalities in regulation of energy metabolism in the body cause eating disorders such as obesity and anorexia, drinking disorders, metabolic disorders, and sleep disorders. In addition, abnormalities in blood circulation regulation in a living body cause hypertension and the like, and can lead to the onset of serious diseases such as myocardial infarction, ischemic heart disease, and cerebral infarction. Therefore, abnormalities in these regulatory functions related to maintaining the homeostasis of living organisms are not only a problem in daily life but also may lead to death, which is a serious social problem.

The hypothalamus and pituitary are tissues that produce and receive physiologically active peptides such as hormones and neurotransmitters. Biological homeostasis is regulated by physiologically active peptides that are produced and received in the hypothalamus and pituitary gland.
Examples of physiologically active peptides produced in the hypothalamus and pituitary gland include thyroid stimulating hormone releasing hormone (TRH), gonadotropin releasing hormone (GnRH), corticotropin releasing hormone (CRH), growth hormone releasing hormone (GRH), somatostatin (growth) Hormone suppressor hormone), prolactin suppressor hormone (PIH), prolactin releasing hormone (PRH), neuropeptide Y (NPY), orexin (OX), melanin-concentrating hormone (MCH), agouti-related protein (AGRP), pigment cell stimulating hormone (ARP) MSH), cocaine amphetamine-regulated transcript (CART) and the like are known.

  Examples of receptors for bioactive peptides whose expression has been confirmed in the hypothalamus and pituitary are type 1 neuromedin U receptor (NMU1R), type 2 neuromedin U receptor (NMU2R), ghrelin receptor, type 1 orexin receptor (OX1R), type 2 orexin receptor (OX2R), type 1 neuropeptide Y receptor (NPY1R), type 5 neuropeptide Y receptor (NPY5R), type 4 melanocortin receptor (MC4R), type 1 corticotropin releasing hormone receptor Body (CRHR-1), type 2 corticotropin releasing hormone receptor (CRHR-2) and the like.

  Various intracellular signal transduction reactions are caused by the interaction of such a bioactive peptide receptor and a bioactive peptide as a ligand, but the signal transduction reaction through an increase in intracellular calcium concentration is particularly prominent. is there.

  In the hypothalamus and pituitary, for example, type 1 neuromedin U receptor (NMU1R), type 2 neuromedin U receptor (NMU2R), ghrelin receptor, type 1 orexin receptor (OX1R), type 2 orexin receptor (OX2R) Bind to individual bioactive peptides that are ligands, etc., cause an increase in intracellular calcium concentration. This increase in intracellular calcium concentration becomes a second messenger, and further causes a reaction for homeostasis regulation. For example, type 1 neuromedin U receptor (NMU1R), type 2 neuromedin U receptor (NMU2R), ghrelin receptor regulates feeding responses, and type 1 orexin receptor (OX1R), type 2 orexin receptor ( OX2R) is known to regulate feeding response, arousal, and motivational response.

On the other hand, muscle contraction in myocardium, skeletal muscle, and smooth muscle is caused by the interaction between actin filaments and myosin filaments, and the interaction is triggered by an increase in intracellular calcium concentration. Therefore, a substance that causes an increase in calcium concentration in muscle cells is used as a muscle contraction agent. In particular, substances that cause an increase in calcium concentration in circulating tissues such as the myocardium and vascular smooth muscle have a circulatory regulating action such as an increase in blood pressure accompanying increased muscle contraction.
For example, physiologically active peptides such as endothelin and angiotensin II are known to cause an increase in intracellular calcium concentration by interacting with each receptor expressed in circulating tissues including the kidney, resulting in an increase in blood pressure. Yes.
The adrenal gland controls the production of carbohydrate steroids and mineral steroids, and the production and secretion of catecholamines. Through these actions, body fluid volume, electrolyte concentration, heart rate and blood pressure are controlled. Thus, the adrenal gland is one of the tissues involved in circulation regulation.

  The CCDC80 (coiled-coil domain containing 80) gene was identified as a gene that is highly expressed in undifferentiated human bone marrow stromal cells (Non-patent Document 1). This gene is highly expressed in white adipose tissue, lung, bone marrow stromal cells, cardiac fibroblasts and the like (Non-patent Document 2). The protein encoded by the human CCDC80 gene is a secreted glycoprotein consisting of 950 amino acid residues and having a molecular weight of approximately 150 kDa.

In the process of differentiation of preadipocytes 3T3-L1 cells into adipocytes, it has been reported that expression of CCDC80 gene is high immediately before differentiation and after differentiation maturation, and when this gene is knocked down, adipose differentiation is suppressed. CCDC80 is thought to control fat differentiation (Non-patent Document 3).
However, the existence of a physiologically active peptide derived from the CCDC80 protein is not known, and of course, there is no report on the function of the derived peptide.

Journal of Cellular Biochemistry, (USA), 2004, Vol. 92, No. 4, p. 733-744 Biochemical and Biophysical Research Communications, (USA), 2008, Vol. 367, No. 2, p. 370-376 The Journal of Biological Chemistry, (USA), 2009, 284, 12, p. 8136-8147

  The present invention relates to a compound useful for energy metabolism regulation or circulatory regulation such as increased feeding, suppression of feeding, increased drinking, drinking suppression, sleep induction, increased arousal, increased metabolism, or metabolic suppression, and a pharmaceutical comprising the compound The purpose is to provide. Furthermore, this invention aims at providing the screening method of the substance which adjusts the effect | action of these compounds.

  As a result of diligent studies to solve the above problems, the present inventors have found that a peptide derived from CCDC80 is secreted into the medium in rat non-myocardial primary cultured cells. A detailed analysis of the physiological activities of these peptides revealed that cells in the hypothalamus or pituitary gland, which are organs involved in the energy metabolism regulation system, and adrenal gland, kidney, heart or blood vessel cells, which are organs involved in the circulatory system. It has been found to have an activity of increasing the concentration of calcium ions in the inside. The present inventors have further studied based on these findings and have completed the present invention.

That is, the present invention is as follows.
[1] Any peptide selected from the following (a) to (d) or a pharmacologically acceptable salt thereof:
(A) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(B) a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 7 and having a length of 80 amino acids or less;
(C) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, comprising an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and includes a mammalian hypothalamus, pituitary gland, adrenal gland, A peptide having an activity to increase intracellular calcium ion concentration of cells in kidney, heart, blood vessel or brain tissue and having a length of 80 amino acids or less; and (d) represented by any one of SEQ ID NOs: 1 to 7 Activity that increases the intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel, or brain tissue of mammals And a peptide having a length of 80 amino acids or less.
[2] The following formula (I):

Wherein R ¹ is a hydrogen atom, substituted or unsubstituted alkanoyl, substituted or unsubstituted aroyl, substituted or unsubstituted heteroarylcarbonyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, Or substituted or unsubstituted heteroaryloxycarbonyl, R ² represents hydroxy, substituted or unsubstituted alkoxy, or substituted or unsubstituted amino, and A represents any one of claims 1 (a) to (d). Represents a peptide residue of the peptide described in 1.)
Or a pharmacologically acceptable salt thereof.
[3] A polynucleotide comprising a nucleotide sequence encoding the peptide according to [1].
[4] A vector comprising the polynucleotide according to [3].
[5] A transformant into which the vector according to [4] has been introduced.
[6] The method according to [1], comprising culturing the transformant according to [5], producing and accumulating the peptide according to [1] in the culture, and isolating the peptide from the culture. A method for producing the peptide according to the description.
[7] A pharmaceutical comprising any of the following peptides (a) to (d) or a pharmaceutically acceptable salt thereof:
(A) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(B) a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(C) in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, comprising an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and a mammalian hypothalamus, pituitary gland, A peptide having an activity of increasing intracellular calcium ion concentration of cells in adrenal gland, kidney, heart, blood vessel or brain tissue; and (d) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7 and 54% or more A peptide comprising an amino acid sequence having identity and having an activity of increasing intracellular calcium ion concentration in cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal.
[8] The following formula (II):

Wherein R ³ is a hydrogen atom, substituted or unsubstituted alkanoyl, substituted or unsubstituted aroyl, substituted or unsubstituted heteroarylcarbonyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, Or substituted or unsubstituted heteroaryloxycarbonyl, R ⁴ represents hydroxy, substituted or unsubstituted alkoxy, or substituted or unsubstituted amino, and B represents any of [7] (a) to (d) Represents the peptide residue of the peptide.)
Or a pharmacologically acceptable salt thereof.
[9] The medicament according to [7], which is an energy metabolism regulator.
[10] The medicament according to [7], which is a circulation regulator.
[11] The peptide of any of the following (a) to (d) or a pharmacologically acceptable salt thereof and a test substance are administered to the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain of a mammal. A step of measuring a cellular response when contacted with a cell in a tissue, compared with a cellular response when the peptide or a pharmacologically acceptable salt thereof is contacted with the cell in the absence of the test substance In the case where the cellular response is suppressed, the test substance inhibits the increase in intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of the mammal by the peptide. Intracellular calcium ion concentration of a cell in the hypothalamus, pituitary, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal by the peptide or a pharmacologically acceptable salt thereof of Screening method for a substance that inhibits the temperature:
(A) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(B) a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(C) in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, comprising an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and a mammalian hypothalamus, pituitary gland, A peptide having an activity of increasing intracellular calcium ion concentration of cells in adrenal gland, kidney, heart, blood vessel or brain tissue; and (d) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7 and 54% or more A peptide comprising an amino acid sequence having identity and having an activity of increasing intracellular calcium ion concentration in cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal.
[12] The peptide of any of the following (a) to (d) or a pharmacologically acceptable salt thereof and a test substance are administered to the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain of a mammal. A step of measuring a cellular response when contacted with a cell in a tissue, compared with a cellular response when the peptide or a pharmacologically acceptable salt thereof is contacted with the cell in the absence of the test substance When the cellular response is promoted, the test substance promotes an increase in intracellular calcium ion concentration of cells in the mammalian hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel, or brain tissue. Intracellular calcium ion concentration of a cell in the hypothalamus, pituitary, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal by the peptide or a pharmacologically acceptable salt thereof of Screening method for a substance that promotes the rise:
(A) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(B) a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(C) in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, comprising an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and a mammalian hypothalamus, pituitary gland, A peptide having an activity of increasing intracellular calcium ion concentration of cells in adrenal gland, kidney, heart, blood vessel or brain tissue; and (d) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7 and 54% or more A peptide comprising an amino acid sequence having identity and having an activity of increasing intracellular calcium ion concentration in cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal.
[13] The peptide of any of the following (a) to (d) or a pharmacologically acceptable salt thereof and a test substance are administered to the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain of a mammal. Measuring a binding amount of the peptide or a pharmacologically acceptable salt thereof to a cell in a tissue or a membrane fraction of the cell when contacted with the cell or a membrane fraction of the cell, Binding amount of the peptide or pharmacologically acceptable salt thereof when the peptide or pharmacologically acceptable salt thereof is contacted with the cell or the membrane fraction of the cell in the absence of the test substance Screening of the agonist or antagonist for the receptor of the peptide comprising the step of identifying the test substance as an agonist or antagonist for the receptor of the peptide when the amount of binding is reduced compared to Packaging method:
(A) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(B) a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(C) in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, comprising an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and a mammalian hypothalamus, pituitary gland, A peptide having an activity to increase intracellular calcium ion concentration of cells in adrenal gland, kidney, heart, blood vessel or brain tissue;
(D) an amino acid sequence having 54% or more identity with the amino acid sequence represented by any of SEQ ID NOs: 1 to 7, and a mammalian hypothalamus, pituitary, adrenal gland, kidney, heart, blood vessel, or A peptide having an activity of increasing intracellular calcium ion concentration of cells in brain tissue.
[14] An antibody that specifically recognizes any of the following peptides (a) to (d):
(A) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(B) a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 7 and having a length of 80 amino acids or less;
(C) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, comprising an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and includes a mammalian hypothalamus, pituitary gland, adrenal gland, A peptide having an activity to increase intracellular calcium ion concentration of cells in kidney, heart, blood vessel or brain tissue and having a length of 80 amino acids or less; and (d) represented by any one of SEQ ID NOs: 1 to 7 Activity that increases the intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel, or brain tissue of mammals And a peptide having a length of 80 amino acids or less.

  INDUSTRIAL APPLICABILITY According to the present invention, a novel peptide having activity to increase intracellular calcium ion concentration of cells in hypothalamus, pituitary, adrenal gland, kidney, heart, blood vessel or brain tissue, DNA encoding the peptide, and method for producing the peptide A medicament containing the peptide is provided. Since the peptide of the present invention has energy metabolism regulation activity or circulatory regulation activity, diseases of eating metabolism such as obesity and anorexia, water intake disorders, metabolic disorders, sleep disorders, etc., or myocardial infarction It is useful for the treatment of cardiovascular diseases such as ischemic heart disease and cerebral infarction. The present invention also provides a screening method for a substance that promotes or suppresses the activity of the peptide of the present invention, and an agonist or antagonist for the receptor of the peptide. Substances obtained by the screening method of the present invention include food intake regulators, water intake regulators, metabolic regulators, circulatory regulators, pressors, therapeutic agents for energy metabolic regulation diseases, therapeutic agents for cardiovascular diseases, Alternatively, it is useful as a candidate substance for an antihypertensive agent.

FIG. 1 shows an increase in intracellular calcium concentration in spleen cells of apoaequorin-expressing mice by 1 μmol / L of peptide 2 (SEQ ID NO: 2). The horizontal axis represents the time (seconds) after addition of the medium, and the vertical axis represents the relative luminescence (RLU) per second. Peptide 2 was added in 25 seconds. FIG. 2 shows an increase in intracellular calcium concentration in pituitary cells of apoaequorin-expressing mice by peptide 2 at 1 μmol / L. The horizontal axis represents the time (seconds) after addition of the medium, and the vertical axis represents the relative luminescence (RLU) per second. Peptide 2 was added in 25 seconds. FIG. 3 shows an increase in intracellular calcium concentration in cardiac cells of apoaequorin-expressing mice by 1 μmol / L of peptide 5 (SEQ ID NO: 5). The horizontal axis represents the time (seconds) after addition of the medium, and the vertical axis represents the relative luminescence (RLU) per second. Peptide 5 was added in 25 seconds. FIG. 4 shows an increase in intracellular calcium concentration in kidney cells of apoaequorin-expressing mice by 1 μmol / L of peptide 5. The horizontal axis represents the time (seconds) after addition of the medium, and the vertical axis represents the relative luminescence (RLU) per second. Peptide 5 was added in 25 seconds. FIG. 5 shows the increase in intracellular calcium concentration in hypothalamic cells of apoaequorin-expressing mice by 1 μmol / L of peptide 5. The horizontal axis represents the time (seconds) after addition of the medium, and the vertical axis represents the relative luminescence (RLU) per second. Peptide 5 was added in 25 seconds. FIG. 6 shows the increase in intracellular calcium concentration in adrenal cells of apoaequorin-expressing mice by peptide 5 of 1 μmol / L. The horizontal axis represents the time (seconds) after addition of the medium, and the vertical axis represents the relative luminescence (RLU) per second. Peptide 5 was added in 25 seconds. FIG. 7 shows an increase in intracellular calcium concentration in cardiac cells of apoaequorin-expressing mice by 1 μmol / L of peptide 7 (SEQ ID NO: 7). The horizontal axis represents the time (seconds) after addition of the medium, and the vertical axis represents the relative luminescence (RLU) per second. Peptide 7 was added in 25 seconds. FIG. 8 shows the increase in intracellular calcium concentration in aortic cells of apoaequorin-expressing mice by 1 μmol / L of peptide 7. The horizontal axis represents the time (seconds) after addition of the medium, and the vertical axis represents the relative luminescence (RLU) per second. Peptide 7 was added in 25 seconds. FIG. 9 shows the increase in intracellular calcium concentration in vena cava cells of apoaequorin-expressing mice by 1 μmol / L of peptide 7. The horizontal axis represents the time (seconds) after addition of the medium, and the vertical axis represents the relative luminescence (RLU) per second. Peptide 7 was added in 25 seconds. FIG. 10 shows the increase in intracellular calcium concentration in hypothalamic cells of apoaequorin-expressing mice by 1 μmol / L of peptide 7. The horizontal axis represents the time (seconds) after addition of the medium, and the vertical axis represents the relative luminescence (RLU) per second. Peptide 7 was added in 25 seconds. FIG. 11 shows the increase in intracellular calcium concentration in adrenal cells of apoaequorin-expressing mice by peptide 7 at 1 μmol / L. The horizontal axis represents the time (seconds) after addition of the medium, and the vertical axis represents the relative luminescence (RLU) per second. Peptide 7 was added in 25 seconds.

<1. Peptide of the present invention and pharmacologically acceptable salt thereof>
The present invention provides a peptide of any of the following (a) to (d) or a pharmacologically acceptable salt thereof. In the present specification, any of the following peptides (a) to (d) is also referred to as “the peptide of the present invention”.

(A) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(B) a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(C) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, comprising an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and a mammalian hypothalamus, pituitary gland, adrenal gland A peptide having an activity of increasing intracellular calcium ion concentration of cells in kidney, heart, blood vessel or brain tissue; and (d) 54% or more identical to the amino acid sequence represented by any of SEQ ID NOs: 1 to 7 A peptide comprising an amino acid sequence having sex and having an activity of increasing intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal.

Examples of the peptide (a) include any of the following peptides:
The amino acid sequence represented by SEQ ID NO: 1, a peptide comprising the 28th to 52nd partial amino acid sequences of the amino acid sequence of human CCDC80 (NCBI accession number: NP — 955805) (SEQ ID NO: 9), the amino acid represented by SEQ ID NO: 2 The sequence corresponds to the amino acid sequence represented by SEQ ID NO: 1 in the amino acid sequence of rat CCDC80 (NCBI accession number: NP_071988) (SEQ ID NO: 10), and the 28th to 52nd positions of the amino acid sequence of rat CCDC80. A peptide consisting of a partial amino acid sequence, the amino acid sequence represented by SEQ ID NO: 3, and the sequence corresponding to the amino acid sequence represented by SEQ ID NO: 1 in the amino acid sequence of mouse CCDC80 (NCBI accession number: NP — 08715) (SEQ ID NO: 11) And mouse CCD A peptide consisting of the 28th to 52nd partial amino acid sequence of the 80 amino acid sequence, the amino acid sequence represented by SEQ ID NO: 4, and a peptide consisting of the 54th to 75th partial amino acid sequence of the amino acid sequence of human CCDC80, SEQ ID NO: 5 A peptide corresponding to the amino acid sequence represented by SEQ ID NO: 4 in the amino acid sequence of rat CCDC80, the peptide consisting of the 54th to 75th partial amino acid sequence of the amino acid sequence of rat CCDC80, SEQ ID NO: A peptide corresponding to the amino acid sequence represented by SEQ ID NO: 4 in the amino acid sequence of mouse CCDC80, the partial amino acid sequence of positions 54 to 75 of the amino acid sequence of mouse CCDC80, The amino acid sequence represented by SEQ ID NO: 7, In 940-950 th of the amino acid sequence, 939-949 amino acid sequence represented by SEQ ID NO: 10, and a peptide consisting of 939-949 th partial amino acid sequence of the amino acid sequence represented by SEQ ID NO: 11.

  CCDC80 is a known secreted glycoprotein having a coiled-coil domain and consisting of 950 amino acid residues in humans and 949 amino acid residues in rats and mice.

  In the present specification, mammals include laboratory animals such as mice, rats, hamsters, guinea pigs, etc., domestic animals such as pigs, cows, goats, horses, sheep, minks, pets such as dogs, cats, etc. , Primates such as humans, monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees and the like, but are not limited thereto. The mammal is preferably a primate (such as a human) or a rodent (such as a rat).

Examples of the peptide (b) include the following peptides (b-1) and (b-2):
(B-1) A peptide comprising the amino acid sequence represented by SEQ ID NO: 1 and consisting of a partial sequence of the amino acid sequence represented by SEQ ID NO: 9, a peptide comprising the amino acid sequence represented by SEQ ID NO: 2, and SEQ ID NO: 10 A peptide comprising a partial sequence of the amino acid sequence represented by SEQ ID NO: 3, a peptide comprising the amino acid sequence represented by SEQ ID NO: 3 and comprising a partial sequence of the amino acid sequence represented by SEQ ID NO: 11, an amino acid represented by SEQ ID NO: 4 A peptide comprising the sequence and consisting of a partial sequence of the amino acid sequence represented by SEQ ID NO: 9, a peptide comprising the amino acid sequence represented by SEQ ID NO: 5 and consisting of a partial sequence of the amino acid sequence represented by SEQ ID NO: 10, A peptide comprising the amino acid sequence represented by SEQ ID NO: 6 and comprising a partial sequence of the amino acid sequence represented by SEQ ID NO: 11, an amino acid represented by SEQ ID NO: 7 A peptide comprising an acid sequence and consisting of a partial sequence of the amino acid sequence represented by SEQ ID NO: 9, 10, or 11; and (b-2) a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, The peptide may have an additional amino acid sequence at the N-terminal and / C-terminal of the amino acid sequence represented by SEQ ID NO: 1, and the additional amino acid sequence is a partial sequence of the amino acid sequence represented by SEQ ID NO: 9. (However, in the case of N-terminal addition, the C-terminal of the partial sequence is the 27th amino acid in the amino acid sequence represented by SEQ ID NO: 9, and in the case of C-terminal addition, the N-terminal of the partial sequence. Is the 53rd amino acid in the amino acid sequence represented by SEQ ID NO: 9) and 60% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more. Consists amino acid sequence, peptides having the hypothalamus in mammals, pituitary, adrenal, kidney, heart, an activity of increasing the intracellular calcium ion concentration in cells of the blood vessels or brain tissue;
A peptide comprising the amino acid sequence represented by SEQ ID NO: 2, wherein the peptide may have an additional amino acid sequence at the N-terminus and / C-terminus of the amino acid sequence represented by SEQ ID NO: 2, and the addition The amino acid sequence is a partial sequence of the amino acid sequence represented by SEQ ID NO: 10 (in the case of N-terminal addition, the C-terminal of the partial sequence is the 27th amino acid in the amino acid sequence represented by SEQ ID NO: 10). In the case of C-terminal addition, the N-terminal of the partial sequence is the 53rd amino acid in the amino acid sequence represented by SEQ ID NO: 10) and 60% or more, preferably 80% or more, more preferably 90 %, Most preferably 95% or more of the amino acid sequence, and the intracellular calci of cells in the mammalian hypothalamus, pituitary, adrenal gland, kidney, heart, blood vessel or brain tissue Peptides having an activity of increasing the Ion concentration;
A peptide comprising the amino acid sequence represented by SEQ ID NO: 3, wherein the peptide may have an additional amino acid sequence at the N-terminus and / C-terminus of the amino acid sequence represented by SEQ ID NO: 3, and the addition The amino acid sequence is a partial sequence of the amino acid sequence represented by SEQ ID NO: 11 (however, in the case of N-terminal addition, the C-terminal of the partial sequence is the 27th amino acid in the amino acid sequence represented by SEQ ID NO: 11). In the case of C-terminal addition, the N-terminal of the partial sequence is the 53rd amino acid in the amino acid sequence represented by SEQ ID NO: 11) and 60% or more, preferably 80% or more, more preferably 90 %, Most preferably 95% or more of the amino acid sequence, and the intracellular calci of cells in the mammalian hypothalamus, pituitary, adrenal gland, kidney, heart, blood vessel or brain tissue Peptides having an activity of increasing the Ion concentration;
A peptide comprising the amino acid sequence represented by SEQ ID NO: 4, wherein the peptide may have an additional amino acid sequence at the N-terminal and / C-terminal of the amino acid sequence represented by SEQ ID NO: 4, and the addition The amino acid sequence is a partial sequence of the amino acid sequence represented by SEQ ID NO: 9 (in the case of N-terminal addition, the C-terminal of the partial sequence is the 53rd amino acid in the amino acid sequence represented by SEQ ID NO: 9). In the case of C-terminal addition, the N-terminal of the partial sequence is the 76th amino acid in the amino acid sequence represented by SEQ ID NO: 9) and 60% or more, preferably 80% or more, more preferably 90 %, Most preferably 95% or more of the amino acid sequence, and intracellular calcium ions of cells in the mammalian hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue. Peptides having an activity of increasing the emissions concentration;
A peptide comprising the amino acid sequence represented by SEQ ID NO: 5, wherein the peptide may have an additional amino acid sequence at the N-terminal and / C-terminal of the amino acid sequence represented by SEQ ID NO: 5, and the addition The amino acid sequence is a partial sequence of the amino acid sequence represented by SEQ ID NO: 10 (however, in the case of N-terminal addition, the C-terminal of the partial sequence is the 53rd amino acid in the amino acid sequence represented by SEQ ID NO: 10). In the case of C-terminal addition, the N-terminus of the partial sequence is the 76th amino acid in the amino acid sequence represented by SEQ ID NO: 10) and 60% or more, preferably 80% or more, more preferably 90 %, Most preferably 95% or more of the amino acid sequence, and the intracellular calci of cells in the mammalian hypothalamus, pituitary, adrenal gland, kidney, heart, blood vessel or brain tissue Peptides having an activity of increasing the Ion concentration;
A peptide comprising the amino acid sequence represented by SEQ ID NO: 6, wherein the peptide may have an additional amino acid sequence at the N-terminal and / C-terminal of the amino acid sequence represented by SEQ ID NO: 6, and the addition The amino acid sequence is a partial sequence of the amino acid sequence represented by SEQ ID NO: 11 (in the case of N-terminal addition, the C-terminal of the partial sequence is the 53rd amino acid in the amino acid sequence represented by SEQ ID NO: 11). In the case of C-terminal addition, the N-terminus of the partial sequence is the 76th amino acid in the amino acid sequence represented by SEQ ID NO: 11) and 60% or more, preferably 80% or more, more preferably 90 %, Most preferably 95% or more of the amino acid sequence, and the intracellular calci of cells in the mammalian hypothalamus, pituitary, adrenal gland, kidney, heart, blood vessel or brain tissue A peptide having an activity to increase muon concentration; or a peptide comprising the amino acid sequence represented by SEQ ID NO: 7, wherein the peptide has an additional amino acid sequence at the N-terminus of the amino acid sequence represented by SEQ ID NO: 7 The additional amino acid sequence may be a partial sequence of the amino acid sequence represented by SEQ ID NO: 9, 10 or 11 (provided that the C-terminal of the partial sequence is the 939th amino acid sequence represented by SEQ ID NO: 9). The 9th amino acid, or the 938th amino acid in the amino acid sequence represented by SEQ ID NO: 10 or 11, and 60% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more. Intracellular calci of cells in the mammalian hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue consisting of amino acid sequences with identity Peptides having an activity of increasing the Ion concentration.

  As the peptide of (c) above, in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, 1 to 5 amino acid residues are substituted, deleted or added. Means that there is substitution, deletion or addition of 1 to 5 (preferably 1 to 3, more preferably 1 or 2) amino acid residues at positions in one or a plurality of amino acid sequences. Deletions or additions may occur simultaneously. In the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, examples of the amino acid sequence in which 1 to 5 amino acid residues are substituted, deleted or added include (1) any of SEQ ID NOs: 1 to 7 An amino acid sequence in which 1 to 5 (preferably 1 to 3, more preferably 1 or 2) amino acids have been deleted in the amino acid sequence represented by (2) An amino acid sequence in which 1 to 5 (preferably 1 to 3, more preferably 1 or 2) amino acids are added to the amino acid sequence represented, (3) represented by any one of SEQ ID NOs: 1 to 7 An amino acid sequence in which 1 to 5 (preferably 1 to 3, more preferably 1 or 2) amino acids in the amino acid sequence are substituted with other amino acids, or (4) the above (1) to (3) A combined amino acid sequence (in this case 1-5 is the sum of the mutated amino acid (preferably 1 to 3, more preferably 1 or 2)) and the like.

  Examples of amino acids to be substituted or added include 20 L-amino acids known as amino acids constituting proteins, namely L-alanine, L-asparagine, L-aspartic acid, L-arginine, L-glutamine, L- Glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L- Examples include, but are not limited to, valine and L-cysteine, such as tert-leucine, norleucine, norvaline, 2-aminobutanoic acid, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine. , Isoaspartic acid, isoglutamic acid, -Other amino acids such as amino adipic acid, 2-aminosuberic acid, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid, 3-hydroxyproline, 4-hydroxyproline, homoserine, and D -It may be an amino acid or a β-amino acid.

Examples of amino acid residues that can be substituted with each other are shown below. Amino acid residues contained in the same group can be substituted for each other.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine, tert-leucine B group: aspartic acid, glutamic acid, Isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid group C: asparagine, glutamine group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid group E : Proline, 3-hydroxyproline, 4-hydroxyproline F group: Serine, threonine, homoserine G group: phenylalanine, tyrosine

  Regarding the peptide of (d) above, the amino acid sequence having 54% or more identity with the amino acid sequence represented by any of SEQ ID NOs: 1 to 7 is the homology analysis program BLAST 2 Sequences [FEMS Microbiol Lett. 174, 247 (1999)] using the default conditions (program: blastp, matrix: BLOSUM62, open gap: 11 penalties, extension gap: 1 penalty, gap x_dropoff: 50, expect: 10.0, word size: 3) When alignment is performed, the identity (the number of the same amino acids between the target sequence and the sequence subjected to homology analysis of SEQ ID NOs: 1 to 7 / homology analysis of SEQ ID NOs: 1 to 7 is performed) The total number of amino acids in the sequence plus the number of gaps inserted during alignment is 54% or more (preferably 63% or more, 72% or more, 77% or more, 80% or more, 81% or more, 84% or more, 86 % Or more, 88% or more, 90% or more 92% or more, or refers to an amino acid sequence at least 95%). Although higher amino acid sequence identity is preferred, the peptide of the present invention or a pharmacologically acceptable salt thereof can be used for cells in mammalian hypothalamus, pituitary, adrenal gland, kidney, heart, blood vessel or brain tissue. The identity may be low as long as it has an activity to increase intracellular calcium ion concentration.

  The peptide of the present invention includes the following formula (I):

Wherein R ¹ is a hydrogen atom, substituted or unsubstituted alkanoyl, substituted or unsubstituted aroyl, substituted or unsubstituted heteroarylcarbonyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, Or substituted or unsubstituted heteroaryloxycarbonyl, R ² represents hydroxy, substituted or unsubstituted alkoxy, or substituted or unsubstituted amino, and A represents any of the peptides (a) to (d) above May be used.

In Formula (I), R ¹ is the N-terminal amino group of any of the peptides (a) to (d), and R ² is the C-terminal of any of the peptides (a) to (d). Each is arranged on a carboxyl group. Specifically, R ¹ is substituted with one or both hydrogen atoms in the N-terminal amino group, and R ² is substituted with a hydroxy group in the C-terminal carboxyl group.

In the definition of each group of the above formula (I), alkanoyl includes, for example, linear or branched alkanoyl having 1 to 20 carbon atoms such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl , Heptanoyl, lauroyl, eicosanoyl and the like.
Examples of the aryl moiety of aroyl and aryloxycarbonyl include aryl having 6 to 15 carbon atoms such as phenyl and naphthyl.

  Heteroarylcarbonyl and heteroaryloxycarbonyl heteroaryl moieties include, for example, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, indazolyl, benzimidazolyl, quinolyl, isoquinolyl, cinnolinyl Quinazolinyl, quinoxalinyl, naphthyridinyl and the like.

Examples of alkoxycarbonyl and the alkyl part of alkoxy include linear or branched alkyl having 1 to 20 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, decyl, dodecyl, eicosyl and the like. Can be mentioned.
Substituents in substituted alkanoyl, substituted alkoxycarbonyl and substituted alkoxy are the same or different and have 1 to 3 substituents such as hydroxy, carboxy, 3 to 8 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl And cycloaliphatic alkyl such as cyclooctyl, substituted or unsubstituted phenyl, fluorenyl and the like. Substituents of substituted phenyl are the same or different and have 1 to 3 substituents, for example, alkyl, alkoxy, hydroxy, nitro, sulfo, cyano, halogen, etc. Examples of halogen include fluorine, chlorine, bromine and iodine. Each atom is mentioned. The alkyl part of the substituted phenyl and the alkyl part of alkoxy have the same meaning as the alkyl part of alkoxycarbonyl and alkoxy.

Substituents in substituted aroyl, substituted aryloxycarbonyl, substituted heteroarylcarbonyl and substituted heteroaryloxycarbonyl are the same or different and have 1 to 3 substituents, and have the same meaning as the substituent of the above substituted phenyl.
Examples of the substituent in the substituted amino include the same or different substituents having 1 to 2 substituents, such as substituted or unsubstituted alkyl, substituted or unsubstituted aryl, and the like. Alkyl has the same meaning as the alkyl moiety such as alkoxy, and the substituent of the substituted alkyl has the same meaning as the substituent such as substituted alkoxy. Aryl is synonymous with the aryl moiety of the aroyl or aryloxycarbonyl, and the substituted aryl substituent is synonymous with the aroyl or aryloxycarbonyl substituent.

  Moreover, the functional group of the side chain of the amino acid residue constituting the peptide residues A and B may be chemically modified or protected. Examples of amino acid residues whose side chain functional groups are chemically modified or protected include aspartic acid residues or glutamic acid residues in which the side chain carboxy group is protected with a benzyl ester, and the side chain thiol group is a carboxy group. Examples include methylated cysteine residues.

  The peptide of the present invention may be modified. Examples of such modifications include lipid chain addition (aliphatic acylation (palmitoylation, myristoylation, etc.), prenylation (farnesylation, geranylgeranylation, etc.), phosphorylation (serine residue, threonine residue, tyrosine residue) Phosphorylation), acetylation, addition of sugar chain (N-glycosylation, O-glycosylation) and the like.

The peptide of the present invention contains a suitable labeling agent such as a radioisotope (eg, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, etc.), an enzyme (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase). , Malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), affinity tags (eg, biotin, etc.), etc. It may be labeled with.

  The length of the peptide of the present invention is not particularly limited as long as it has an activity to increase intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of mammals. A peptide having a desired length can be selected according to the purpose. The total length is preferably 80 amino acids or less, more preferably 60 amino acids or less, particularly preferably 40 amino acids or less (for example, 30 amino acids or less, 29 amino acids or less, 28 amino acids or less, 27 amino acids or less, 26 amino acids or less, 25 amino acids or less). 24 amino acids or less, 23 amino acids or less, 22 amino acids or less, 21 amino acids or less, 20 amino acids or less, 19 amino acids or less, 18 amino acids or less, 17 amino acids or less, 16 amino acids or less, 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 Amino acids or less) can be appropriately selected.

  Examples of the pharmacologically acceptable salt of the peptide of the present invention include acid addition salts, metal salts, organic base addition salts and the like. Examples of the acid addition salt include inorganic acid salts such as hydrochloride, sulfate and phosphate, and organic acid salts such as acetate, maleate, fumarate, tartrate and citrate. Examples of the metal salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, zinc salt and the like. Examples of organic base addition salts include primary amines such as methylamine, ethylamine and aniline, secondary amines such as dimethylamine, diethylamine, pyrrolidine, piperidine, morpholine and piperazine, trimethylamine, triethylamine, N, N-dimethylaniline and pyridine. And salts formed with tertiary amines such as ammonium salts and the like.

  The peptide of the present invention or a pharmacologically acceptable salt thereof is preferably isolated or purified. “Isolation or purification” means that an operation for removing components other than the target component has been performed. Purity of the peptide of the present invention or a pharmaceutically acceptable salt thereof isolated or purified (the peptide of the present invention or a pharmacologically acceptable salt thereof with respect to the total weight of the peptide or a pharmaceutically acceptable salt thereof) The ratio of the weight of the salt formed) is usually 50% or more, preferably 70% or more, more preferably 90% or more, and most preferably 95% or more (for example, 100%).

  The peptide of the present invention or a pharmacologically acceptable salt thereof has an activity of increasing intracellular calcium ion concentration in cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal. . Here, the brain tissue refers to a tissue included in the brain, such as cerebrum, midbrain, cerebellum, diencephalon, and medulla.

  The peptide of the present invention or a pharmacologically acceptable salt thereof has an activity of increasing the intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal. It can be confirmed by the method shown in the following (i) or (ii).

(I) Utilization of apoaequorin-expressing transgenic mice From the transgenic mouse that expresses apoaequorin expressed throughout the body (International Publication No. 02/010371 pamphlet) produced by introducing an apoaequorin gene expression vector into fertilized eggs, the thalamus Collect tissue from the lower, pituitary, adrenal, kidney, heart, blood vessel, or part of the brain. The obtained hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue is shredded, suspended and cultured in a medium containing coelenterazine, and coelenterazine is taken up into cells and aequorin (apoaequorin and coelenterazine Complex). Aequorin emits light by binding to intracellular calcium ions, so the relative luminescence of the cells per second before and after the addition of the medium containing the peptide or its pharmacologically acceptable salt is measured with a luminometer over time. And used as an index of intracellular calcium ion concentration. When the amount of relative luminescence is increased by the addition of a peptide or a pharmacologically acceptable salt thereof, the peptide or a pharmacologically acceptable salt thereof has an activity of increasing intracellular calcium ion concentration. I can confirm. In general, the intracellular concentration of calcium ions as a second messenger decreases rapidly or gradually after increasing. On the other hand, in the case of nonspecific inflow of calcium ions from the outside of the cell due to cell membrane damage, the intracellular calcium ion concentration continues to increase for a certain period of time. This time change of intracellular calcium ion concentration, that is, time change of relative luminescence level, increases the calcium ion concentration as a second messenger due to physiological action, or nonspecific calcium ion concentration due to simple cell membrane damage. It is possible to distinguish between rising.

(Ii) Use of calcium-binding fluorescent reagent Hypothalamic, pituitary, adrenal, kidney, heart, blood vessel or brain tissue collected from an animal individual, or hypothalamus, pituitary, adrenal, kidney, heart, Calcium ion binding properties of cell lines derived from cells in blood vessels or brain tissues such as Fura-2, Indo-1, and Fluo-3, whose excitation wavelength, fluorescence wavelength, or fluorescence intensity changes depending on the presence or absence of calcium ions. Are suspended in a buffer containing a fluorescent reagent and cultured, and taken up into cells. Since Fura-2 binds to calcium ions, the fluorescence excitation wavelength peak shifts from 380 nm to 340 nm. Therefore, before and after the addition of a buffer containing a peptide or a pharmacologically acceptable salt thereof, 380 nm The ratio of the fluorescence intensity when excited at a wavelength of 340 nm to the fluorescence intensity when excited at a wavelength of is measured with a fluorescence measuring device and used as an index of intracellular calcium ion concentration. When the ratio of fluorescence intensity is increased by the addition of a peptide or a pharmacologically acceptable salt thereof, the peptide or a pharmacologically acceptable salt thereof has an activity of increasing intracellular calcium ion concentration. Can be confirmed. Since Indo-1 binds to calcium ions, the fluorescence wavelength shifts from 480 nm to 400 nm. Therefore, before and after the addition of a buffer containing a peptide or a pharmaceutically acceptable salt thereof, the wavelength of 480 nm is increased. The ratio of the fluorescence intensity at a wavelength of 400 nm to the fluorescence intensity is measured with a fluorescence measuring device and used as an index of intracellular calcium ion concentration. When the ratio of fluorescence intensity is increased by the addition of a peptide or a pharmacologically acceptable salt thereof, the peptide or a pharmacologically acceptable salt thereof has an activity of increasing intracellular calcium ion concentration. Can be confirmed. Since Fluo-3 binds to calcium ions, the fluorescence intensity at a wavelength of 520 nm is greatly increased. Therefore, before and after the addition of a buffer containing a peptide or a pharmacologically acceptable salt thereof, a wavelength of 520 nm is used. The fluorescence intensity ratio is measured with a fluorescence measuring device and used as an index of intracellular calcium ion concentration. When the ratio of fluorescence intensity is increased by the addition of a peptide or a pharmacologically acceptable salt thereof, the peptide or a pharmacologically acceptable salt thereof has an activity of increasing intracellular calcium ion concentration. Can be confirmed.

  Since the peptide of the present invention and a pharmacologically acceptable salt thereof have an activity of increasing intracellular calcium concentration in mammalian adrenal, kidney, blood vessel or heart cells, in a preferred embodiment, the peptide of the present invention and The pharmacologically acceptable salt has activity (circulatory regulation activity) that regulates (enhances or inhibits) blood pressure and blood flow in mammals. The circulatory regulation activity also includes an activity to regulate (enhance or suppress) various parameters (for example, body fluid volume, electrolyte concentration in body fluid, heart rate, etc.) that vary by regulating blood pressure and blood flow.

  The fact that the peptide of the present invention or a pharmacologically acceptable salt thereof has circulatory regulation activity can be confirmed, for example, by measuring the activity of changing blood pressure by the following measurement method.

  A catheter is inserted into the external jugular vein and internal carotid artery of an animal such as a rat under anesthesia, and the arterial pressure is continuously measured by connecting the catheter of the internal carotid artery to a pressure monitor. Administer peptide or its pharmacologically acceptable salt dissolved in physiological saline from the external jugular vein, and compare the arterial pressure before and after administration of the peptide or its pharmacologically acceptable salt, When the arterial pressure fluctuates (increases or decreases) due to administration of a peptide or a pharmacologically acceptable salt thereof, the peptide or a pharmacologically acceptable salt thereof increases circulatory regulation activity (particularly increases blood pressure). Or a lowering activity).

  In addition, since the peptide of the present invention and a pharmacologically acceptable salt thereof have an activity to increase the intracellular calcium concentration of cells in the hypothalamus or pituitary gland of a mammal or brain tissue, another preferred embodiment In the above, the peptide of the present invention and a pharmacologically acceptable salt thereof have an activity (energy metabolism-regulating activity) for regulating (enhancing or suppressing) the amount of intake and water intake and energy metabolism of mammals. The energy metabolism regulation activity regulates (enhances or suppresses) various parameters (such as body weight, body fat mass, amount of exercise, awakening time, body temperature, mental activity, etc.) that change by regulating food intake, water intake, and energy metabolism. Activity).

  It can be confirmed by the following measurement method that the peptide of the present invention or a pharmacologically acceptable salt thereof has energy metabolism regulating activity.

  A catheter is inserted into the lateral ventricle of an anesthetized animal such as a rat, and the peptide dissolved in physiological saline or a pharmacologically acceptable salt thereof is transiently or appropriately applied to the lateral ventricle through the catheter. Administer multiple doses over a period, and measure body weight, water consumption, food intake, exercise, wakefulness, body fat mass, body temperature, etc. Alternatively, a catheter is inserted into the external jugular vein or the like of an animal such as a rat under anesthesia, and the peptide or pharmacologically acceptable salt thereof dissolved in physiological saline from the external jugular vein or the like is passed through the catheter, Or it administers several times in a suitable period, and measures the body weight, the amount of drinking water, the amount of intake, the amount of exercise, the awakening time, the body fat mass, the body temperature, etc. under the free behavior after the administration. When these measurement items vary as compared with the non-administered group of the peptide, it can be confirmed that the peptide or a pharmacologically acceptable salt thereof has an activity of regulating energy metabolism.

<2. Polynucleotide>
The present invention provides a polynucleotide comprising a nucleotide sequence encoding the peptide of the present invention. The polynucleotide may be DNA, RNA, or a DNA / RNA chimera, preferably DNA. The polynucleotide may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid.

  Examples of the polynucleotide containing the nucleotide sequence encoding the peptide of the present invention include genomic DNA, cDNA, synthetic DNA derived from mammals such as human or rat. Genomic DNA and cDNA containing the nucleotide sequence encoding the peptide of the present invention were prepared using Polymerase Chain Reaction (hereinafter referred to as “PCR method”) using the genomic DNA fraction prepared from the mammal and the total RNA or mRNA fraction as templates, respectively. Can also be directly amplified by Reverse Transcriptase-PCR. Alternatively, genomic DNA and cDNA containing a nucleotide sequence encoding the peptide of the present invention can be prepared by inserting genomic DNA prepared from the mammal and a fragment of total RNA or mRNA into an appropriate vector. It can also be cloned from a cDNA library by colony or plaque hybridization method or PCR method, respectively. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.

  As a polynucleotide containing the nucleotide sequence which codes the peptide of this invention, the polynucleotide containing the nucleotide sequence represented by sequence number 8 can be mentioned, for example. The nucleotide sequence represented by SEQ ID NO: 8 encodes the peptide of the present invention consisting of the amino acid sequence represented by SEQ ID NO: 1.

  The polynucleotide of the present invention is amplified by a PCR method using a synthetic DNA primer having a partial sequence of a nucleotide sequence encoding the peptide of the present invention, or a DNA incorporated into an appropriate vector is a part of the peptide of the present invention. Alternatively, it can be cloned by hybridization with nucleotides encoding the entire region or with a synthetic DNA label. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.

  The cloned DNA can be used as it is or after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon on the 5 'end side, and may have TAA, TGA or TAG as a translation termination codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

  The polynucleotide of the present invention is preferably isolated or purified. “Isolation or purification” means that an operation for removing components other than the target component has been performed. The purity of the isolated or purified polynucleotide of the present invention (ratio of the weight of the polynucleotide of the present invention to the total weight of the polynucleotide) is usually 50% or more, preferably 70% or more, more preferably 90% or more, Most preferably, it is 95% or more (for example, 100%).

<3. Vector containing the polynucleotide of the present invention>
The present invention provides a vector comprising the polynucleotide of the present invention. The vector of the present invention can be produced, for example, by operably linking a polynucleotide containing a nucleotide sequence encoding the peptide of the present invention downstream of a promoter in an appropriate expression vector. As the expression vector, an appropriate vector (plasmid vector, viral vector, retroviral vector, baculovirus vector, adenovirus vector, etc.) can be selected depending on the host to be used.

  When a prokaryotic organism such as a bacterium is used as a host cell, the recombinant vector containing the DNA encoding the peptide of the present invention can autonomously replicate in the prokaryotic organism, and at the same time, a promoter, a ribosome binding sequence, and a transcription A vector comprising a termination sequence is preferred. A gene that controls the promoter may also be included.

Examples of expression vectors include pSE420 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-30 (manufactured by Qiagen), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agric. Biol. Chem., 48 , 669 (1984)], pLSA1 (Agric. Biol. Chem., 53 , 277 (1989)), pGEL1 (Proc. Natl. Acad. Sci., USA, 82 , 4306 (1985)), pBluescript II SK (-) (Stratagene), pTrs30 [prepared from transformed E. coli strain (FERM BP-5407)], pTrs32 [prepared from transformed E. coli strain (FERM BP-5408)], pGHA2 [transformed E. coli strain ( FERM BP-400)], pGKA2 [prepared from transformed E. coli strain (FERM P-6798)], pTerm2 (Japanese Patent Laid-Open No. 3-22979), pGEX-2T (manufactured by GE Healthcare), pET (Novagen) PKK223-2 (manufactured by GE Healthcare), pMAL-c2X (manufactured by New England Biolabs), and the like.

Any promoter can be used as long as it functions in the host cell. For example, trp promoter (P _trp), lac promoter, P _L promoter, P _R promoter include promoters derived from such T7 promoter, E. coli or phage, or the like. Further, artificially designed and modified promoters such as a promoter in which two P _trp are connected in series (P _trp × 2), tac promoter, lacT7 promoter, let I promoter, and the like can also be used.

It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
In the recombinant vector of the present invention, a transcription termination sequence is not necessarily required for the expression of the DNA of the present invention, but it is preferable to place the transcription termination sequence immediately below the structural gene.

  When yeast is used as a host cell, examples of expression vectors include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like.

  Any promoter can be used as long as it can be expressed in yeast strains. For example, a glycolytic gene promoter such as hexose kinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFα1 promoter, CUP 1 promoter and the like.

When animal cells are used as a host, examples of expression vectors include pcDNA3.1 (+) (manufactured by Invitrogen), pAGE107 (Japanese Patent Laid-Open No. 3-22979, Cytotechnology, 3 , 133 (1990)), pAS3- 3 (Japanese Patent Laid-Open No. 2-227075), pCDM8 [Nature, 329 , 840 (1987)], pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochem., 101 , 1307 (1987)] and the like. .

  Any promoter can be used as long as it functions in animal cells. For example, cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter, metallothionein Examples include promoters, heat shock promoters, SRα promoters, and the like. In addition, an enhancer of human CMV IE gene may be used together with a promoter.

When using insect cells as a host, for example, Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), Bio / Technology, 6 , 47 ( 1988) and the like, the peptide can be expressed.
In other words, the recombinant gene transfer vector and the defective baculovirus genome are co-introduced into insect cells to obtain recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the peptide. Can be made.

  Examples of gene transfer vectors used in the method include pVL1392, pVL1393 (Becton Dickinson), pBlueBac4.5 (Invitrogen), and the like.

  When plant cells are used as host cells, examples of expression vectors include Ti plasmids and tobacco mosaic virus vectors.

  As the promoter, any promoter can be used as long as it can be expressed in plant cells. Examples thereof include cauliflower mosaic virus 35S promoter and rice actin 1 promoter.

<4. Transformant capable of expressing the peptide of the present invention>
A vector containing a polynucleotide containing a nucleotide sequence encoding the peptide of the present invention is known, such as lipofection method, calcium phosphate method, microinjection method, proplast fusion method, electroporation method, DEAE dextran method, gene transfer by Gene Gun, etc. A transformant capable of expressing the peptide of the present invention can be produced by introducing it into the host according to the above method.

When a prokaryotic organism such as a bacterium is used as a host cell, any method can be used as long as it introduces a vector containing the polynucleotide of the present invention into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69 , 2110 (1972)], protoplast method (JP-A 63-248394), or Gene, 17 , 107 (1982) or Molecular & General Genetics, 168 , 111 (1979) The method etc. can be mentioned.

As the host cell, Escherichia (Escherichia) genus Serratia (Serratia) genus Bacillus (Bacillus) genus Brevibacterium (Brevibacterium) genus Corynebacterium (Corynebacterium) genus-mycobacterial (Microbacterium) genus Pseudomonas (Pseudomonas ) microorganisms belonging to the genus or the like, for example, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49 , Escherichia coli W3110, Escherichia coli TB1 , Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefacines, Brevibacterium ammoniagenes, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Brevibacterium flavum ATCC14067, Brevibacterium lactofer mentum ATCC13869, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, Pseudomonas putida , Pseudomonas sp. D-0110 and the like.

When yeast is used as a host cell, any method for introducing a vector containing the polynucleotide of the present invention into the host cell can be used. For example, electroporation [Methods Enzymol., 194 , 182 ( 1990)), spheroplast method (Proc. Natl. Acad. Sci. USA, 75 , 1929 (1978)), lithium acetate method (J. Bacteriology, 153 , 163 (1983)), Proc. Natl. Acad. Sci. Examples include the methods described in USA, 75 , 1929 (1978).

As the host cell, Saccharomyces (Saccharomyces) genus Schizosaccharomyces (Schizosaccharomyces) genus Kluyveromyces (Kluyveromyces) genus Trichosporon (Trichosporon) genus Schwanniomyces (Schwanniomyces) genus Pichia (Pichia) sp., Candida (Candida ) Microorganisms belonging to the genus, such as Saccharomyces cerevisiae , Schizosaccharomyces pombe , Kluyveromyces lactis , Trichosporon pullulans , Schwanniomyces alluvius , Candida utilis and the like.

When animal cells are used as hosts, any method can be used as long as the vector containing the polynucleotide of the present invention is introduced into animal cells. For example, electroporation [Cytotechnology, 3 , 133 (1990) , Calcium phosphate method (JP-A-2-27075), lipofection method [Proc. Natl. Acad. Sci. USA, 84 , 7413 (1987)], Virology, 52, 456 (1973), and the like.

  Examples of host cells include human cells such as Namalwa cells, monkey cells, COS cells, Chinese hamster cells, CHO cells, and HBT5637 (Japanese Patent Laid-Open No. 63-299). it can.

When using insect cells as a host, for example, Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), Bio / Technology, 6 , 47 ( 1988) and the like, the peptide can be expressed.
In other words, the recombinant gene transfer vector and the defective baculovirus genome are co-introduced into insect cells to obtain recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the peptide. Can be made.

  As the baculovirus, for example, an autographa californica nuclear polyhedrosis virus, which is a virus that infects a serpentine insect, can be used.

Examples of insect cells include Sf9, Sf21 (Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992)), which is an ovary cell of Spodoptera frugiperda , and High 5 (Invitrogen), an ovary cell of Trichoplusia ni. Etc.) can be used.
Examples of a method for co-introducing the recombinant gene introduction vector and the baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the lipofection method [Proc. Natl Acad. Sci. USA, 84 , 7413 (1987)].

When a plant cell is used as a host cell, any method can be used as long as it is a method for introducing a vector containing the polynucleotide of the present invention into a plant cell. For example, Agrobacterium (Japanese Patent Laid-Open No. Sho 59-59 ). 140885, JP-A-60-70080, WO94 / 00977 pamphlet), electroporation method (JP-A-60-251887), method using particle gun (gene gun) (patent no. No. 2606856, Japanese Patent No. 2517813) and the like.

  Examples of host cells include plant cells such as tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat and barley.

<5. Method for producing peptide of the present invention>
(1) Production Method by Chemical Synthesis The peptides of the present invention are prepared, for example, by Nobuo Izumiya, Tetsuo Kato et al., “Basics and Experiments of Peptide Synthesis”, Maruzen, (1985), Saburo Aimoto et al. 4th edition, Volume 22, “Organic Synthesis IV Acids, Amino Acids and Peptides”, Maruzen, (1999), Int. J. Pept. Protein Res. 35 , 161-214 (1990), Fields, GB, Solid-Phase Known in Peptide Synthesis, Methods in Enzymology, vol. 289, Academic Press, (1997), Pennington, MW and Dunn, BM, Peptide Synthesis Protocols, Methods in Molecular Biology, vol. 35, Humana Press, (1994), etc. It can obtain by refine | purifying after the synthesis | combination by the peptide synthesis method. Specific examples of the synthesis method include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a dichloromethane method, an active ester method, a carboimidazole method, and a redox method. Moreover, the solid phase synthesis method and the liquid phase synthesis method can be applied to the synthesis. That is, the target peptide can be synthesized by condensing the amino acid constituting the peptide of the present invention and the remaining portion and removing the protecting group when the product has a protecting group.

  Furthermore, when the peptide of the present invention is one in which the side chain of the amino acid residue constituting the peptide and / or the amino terminus of the peptide and / or the carboxy terminus of the peptide are chemically modified or protected, peptide synthesis Methods conventionally known in the field of peptide synthesis chemistry, such as a method of chemical modification later, a method of peptide synthesis using chemically modified amino acids, or a method of appropriately selecting reaction conditions for final deprotection of peptide synthesis [Nobuo Izumiya et al. , "Basics and Experiments of Peptide Synthesis", Maruzen, (1985), Supervised by Yajima Haruaki, "Continued Drug Development", Volume 14, "Peptide Synthesis", Hirokawa Shoten, (1991), Japan Biochemical Society “Biochemistry Experiment Course”, Volume 1, “Protein Chemistry IV—Chemical Modification and Peptide Synthesis”, Tokyo Chemical Doujin, Ohno Motonori et al., “Biochemical Experiment Method”, 12, 13 Volume, "protein of chemical modification (top) (bottom)", Gakkai Shuppan Center, can be produced by (1981)].

  The peptide of the present invention can also be synthesized using an automatic peptide synthesizer. Peptide synthesis using a peptide synthesizer was carried out using a commercially available peptide synthesizer such as a peptide synthesizer manufactured by Shimadzu Corporation or a peptide synthesizer manufactured by Advanced ChemTech, Inc. Fmoc (9-fluorenylmethyloxycarbonyl) -amino acid or Nα-Boc (t-butyloxycarbonyl) -amino acid or the like can be used according to each synthesis program. Protected amino acids and carrier resins used as raw materials are Applied Biosystems, Inc., Shimadzu, Kokusan Chemicals, EMD Biosienses, Inc., Watanabe Chemical, Advanced It can be obtained from Chemtech, AnaSpec, Inc., Peptide Institute, Inc.

  The peptide of the present invention can be purified by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like.

(2) Production method by genetic engineering method When the peptide of the present invention consists of the above-mentioned 20 essential amino acids and has not undergone side chain, N-terminal or C-terminal modification, Molecular Cloning: A Laboratory Manual, 3rd The DNA encoding the peptide of the present invention can be expressed and produced in a host cell using the method described in edition, Cold Spring Harbor Laboratory Press (2001), etc., for example, by the following method.

  The DNA encoding the peptide of the present invention can be synthesized by designing a base sequence encoding from the amino acid sequence of the peptide of the present invention and using a DNA synthesizer. In the case where the peptide of the present invention is a partial peptide of human CCDC80 containing the amino acid sequence represented by any of SEQ ID NOs: 1 to 7, PCR using cDNA of human white adipose tissue-derived or bone marrow-derived cells as a template Can also be isolated. When the DNA encoding the peptide of the present invention is used for the production of the peptide of the present invention, the end of the region encoding the peptide of the present invention is made to be a stop codon.

Below, the manufacturing method in case the N terminal of the peptide of this invention is methionine is described.
A recombinant vector is prepared by inserting the DNA encoding the peptide of the present invention obtained above downstream of the promoter of an appropriate expression vector, and the recombinant vector is transferred to a host cell suitable for the expression vector. Introduced to obtain a transformant capable of expressing the peptide of the present invention.
Any host cell can be used as long as it can express the target gene, such as bacteria, yeast, animal cells, insect cells, and plant cells.

The peptide of the present invention can be produced by culturing the transformant in a medium, producing and accumulating the peptide of the present invention in the culture, and collecting from the culture.
In addition, the method of culturing the transformant in a medium can be performed according to a usual method used for culturing a host.

When the transformant is a transformant obtained by using a prokaryote such as E. coli or a eukaryote such as yeast as a host, as a medium for culturing the transformant, a carbon source that can be assimilated by the transformant, Either a natural medium or a synthetic medium may be used as long as it contains a nitrogen source, inorganic salts, and the like and can efficiently culture the transformant.
Any carbon source may be used as long as the transformant can assimilate, for example, glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, etc. Alcohols such as ethanol and propanol can be used.

  Examples of the nitrogen source include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.

Examples of inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
The culture is preferably performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C., and the culture time is usually preferably 16 hours to 7 days. The pH during the culture is preferably maintained at 3.0 to 9.0. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

Moreover, you may add antibiotics, such as an ampicillin and a tetracycline, to a culture medium as needed during culture | cultivation.
When culturing a microorganism transformed with a recombinant vector using an inducible promoter as a promoter, it is preferable to add an inducer to the medium as necessary. For example, when cultivating a microorganism transformed with a recombinant vector using the lac promoter, when culturing a microorganism transformed with isopropyl-β-D-thiogalactopyranoside or the like with a recombinant vector using the trp promoter. Indoleacrylic acid or the like may be added to the medium.

Examples of a medium for culturing a transformant obtained using an animal cell as a host include RPMI 1640 medium [The Journal of the American Medical Association, 199 , 519 (1967)], Eagle's minimal essential medium (MEM) [Science, 122 , 501 (1952)], Dulbecco's modified Eagle medium (Virology, 8 , 396 (1959)), 199 medium (Proceeding of the Society for the Biological Medicine, 73 , 1 (1950)) or fetal calf serum etc. And the like, and the like.

In general, the culture is preferably performed in the presence of 5% CO ₂ , the pH is preferably 6 to 8, the temperature is preferably 30 to 40 ° C., and preferably 1 to 7 days.
Moreover, you may add antibiotics, such as kanamycin and penicillin, to a culture medium as needed during culture | cultivation.
Further, according to the method described in JP-A-2-27075, the production amount can also be increased using a gene amplification system using a dihydrofolate reductase gene or the like.

As a medium for culturing a transformant obtained using insect cells as a host, commonly used TNM-FH medium (Becton Dickinson), Sf-900 II SFM medium (Invitrogen), ExCell400, ExCell405 (Both manufactured by JRH Biosciences), Grace's insect medium [Nature, 195 , 788 (1962)] and the like can be used.
In general, the culture is preferably carried out at a pH of 6 to 7 and a temperature of 25 to 30 ° C., preferably 1 to 5 days.
Moreover, you may add antibiotics, such as a gentamicin, to a culture medium as needed during culture | cultivation.

A transformant obtained using a plant cell as a host can be cultured as a cell or differentiated into a plant cell or organ. As a medium for cultivating the transformant, a commonly used Murashige and Skog medium, a white medium, or a medium in which a plant hormone such as auxin or cytokinin is added to these mediums can be used. .
In general, the culture is preferably performed at a pH of 5 to 9 and a temperature of 20 to 40 ° C., and preferably 3 to 60 days.
Moreover, you may add antibiotics, such as kanamycin and a hygromycin, to a culture medium as needed during culture | cultivation.

  As described above, a transformant derived from a microorganism, animal cell, or plant cell having a recombinant vector incorporating a DNA encoding the peptide of the present invention is cultured according to a normal culture method to produce and accumulate the peptide. The peptide can be produced by collecting the peptide from the culture.

  In order to avoid degradation in the host cell, the peptide of the present invention is produced by producing a fusion protein of an arbitrary polypeptide (hereinafter referred to as polypeptide X) and the peptide of the present invention, and then using the peptide of the present invention as a fusion protein. You may isolate and manufacture from. An expression vector for expressing the fusion protein is prepared by adding a DNA encoding a specific protease recognition sequence or methionine to the 5 ′ end of the DNA encoding the peptide of the present invention described above. The peptide X expression vector can be prepared by ligating DNA encoding polypeptide X together in frame. However, the DNA encoding methionine is added only when the peptide of the present invention does not contain methionine. As the polypeptide X, any polypeptide can be used, and examples thereof include glutathione S-transferase, maltose binding protein, DsbA, DsbC, and protein A. Examples of specific protease recognition sequences include factor Xa recognition sequence (Ile-Glu-Gly-Arg) and enterokinase recognition sequence (Asp-Asp-Asp-Asp-Lys). The expression vector for polypeptide X can be prepared by inserting a DNA encoding polypeptide X in place of the DNA encoding the peptide of the present invention in accordance with the expression vector for the peptide of the present invention described above. In addition, commercially available fusion protein expression vectors such as fusion protein expression vector pGEX-3 (manufactured by GE Healthcare) with glutathione S-transferase, fusion protein expression vectors pMAL-c2X, pMAL-p2E with maltose binding protein (New England Biolabs), DsbA fusion protein expression vector pET-39b (+) (EMD Biosciences) or the like may be used. When the peptide of the present invention is fused to the polypeptide X through a recognition sequence of a specific protease, the peptide of the present invention can be cleaved from the fusion protein by protease treatment corresponding to the recognition sequence. When the peptide of the present invention is fused to the C-terminus of polypeptide X via methionine, the peptide of the present invention is treated with cyanogen bromide according to the method described in JP-A-1-02096. Can be cleaved from the fusion protein. After treatment with protease or cyanogen bromide, the peptide of the present invention can be isolated and purified by combining gel filtration, reverse phase HPLC, affinity chromatography and the like.

Literature [J. Biol. Chem., 264 , 17619 (1989); Proc. Natl. Acad. Sci. USA, 86 , 8227 (1989); Genes Develop., 4 , 1288 (1990); In preparation of the DNA encoding the peptide of the present invention described above, the DNA encoding the signal peptide of the secreted protein is added to the 5 ′ end of the DNA according to the description of JP 94/23021 pamphlet]. The peptide of the present invention can be secreted into the culture medium by producing a recombinant vector using the DNA in the same manner as described above and introducing it into a host cell.

In the case where the N-terminus of the peptide of the present invention is not methionine, it can be produced by a method in which the above fusion protein is produced and then isolated or secreted into a medium. For example, the peptide of the present invention consisting of the amino acid sequence represented by SEQ ID NO: 1 can be produced as follows. First, a DNA consisting of the base sequence represented by SEQ ID NO: 8 and a DNA consisting of a base sequence complementary to SEQ ID NO: 8 are chemically synthesized with a DNA synthesizer, and then annealed to prepare double-stranded DNA. The double-stranded DNA and pMAL-c2X cleaved with XmnI are ligated to produce a plasmid in which the double-stranded DNA is inserted into the XmnI site of pMAL-c2X. The obtained plasmid encodes a fusion protein in which a peptide consisting of the factor Xa recognition sequence (Ile-Glu-Gly-Arg) and the amino acid sequence represented by SEQ ID NO: 1 is fused to the C-terminus of the maltose binding protein. Escherichia coli is transformed with the obtained plasmid. The obtained transformant is cultured in a medium, and the fusion protein is expressed in the cells of the transformant. After the cultured cells are isolated by centrifugation, the cells are crushed to obtain a solution containing the fusion protein. The fusion protein is isolated from the resulting solution by affinity chromatography using a column on which maltose is immobilized, and then the fusion protein is treated with factor Xa, and the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 is fused to the fusion protein. Disconnect from. The peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 can be isolated and purified by gel filtration or reverse phase HPLC.

In order to isolate and purify the peptide produced by the transformant, a normal protein isolation and purification method can be used.
For example, when the peptide of the present invention is expressed in a cell in a dissolved state, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, a Manton Gaurin homogenizer. Then, the cells are disrupted with dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylamino Anion exchange chromatography using resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), cation exchange chromatography using resin such as S-Sepharose FF (manufactured by Pharmacia) Methods such as electrophoresis, hydrophobic chromatography using resin such as butyl sepharose, phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing etc. A purified preparation can be obtained by using alone or in combination.

  When the peptide is expressed by forming an insoluble substance in the cell, the cell is similarly collected, crushed, and centrifuged to recover the peptide insoluble substance as a precipitate fraction. The recovered peptide insolubles are solubilized with a protein denaturant. The peptide is returned to a normal three-dimensional structure by diluting or dialyzing the solubilized solution and reducing the concentration of the protein denaturant in the solubilized solution. After the operation, a purified preparation of the peptide can be obtained by the same isolation and purification method as described above.

  When the peptide of the present invention is secreted extracellularly, the peptide can be recovered in the culture supernatant. That is, the culture supernatant is obtained by treating the culture by a technique such as centrifugation as described above, and a purified sample is obtained from the culture supernatant by using the same isolation and purification method as described above. Obtainable.

<6. Pharmaceutical containing the peptide of the present invention or a pharmacologically acceptable salt thereof>
The peptide of the present invention and a pharmacologically acceptable salt thereof have an activity to increase intracellular calcium concentration in mammalian adrenal, renal, vascular or cardiac cells, and thus regulate blood circulation and blood flow. Has activity. Therefore, the peptide of the present invention and a pharmacologically acceptable salt thereof are used as an active ingredient of a circulation regulator, a pressor, or a therapeutic agent for cardiovascular disease such as myocardial infarction, ischemic heart disease or cerebral infarction. be able to.

  In addition, since the peptides of the present invention and pharmacologically acceptable salts thereof have the activity of increasing intracellular calcium concentration in the hypothalamus or pituitary gland of mammals or cells in brain tissue, the amount of food intake and intake It has energy metabolism regulating activity to regulate water volume and energy metabolism. Therefore, the peptide of the present invention and a pharmacologically acceptable salt thereof are a food intake regulator, a water intake regulator, a metabolic regulator, or a treatment for diseases of the energy metabolic regulation system such as obesity, anorexia or insomnia. It can be used as an active ingredient of drugs.

  The medicament containing the peptide of the present invention or a pharmacologically acceptable salt thereof may be used as an active ingredient alone or as an active ingredient for any other treatment. It can contain as a mixture of these. In addition, these medicaments are produced by any method well known in the technical field of pharmaceutics by mixing the active ingredient together with one or more pharmaceutically acceptable carriers.

It is desirable to use the most effective route for treatment, and it is administered orally or parenterally, for example, intracerebroventricularly or intravenously.
Administration forms include tablets, powders, granules, syrups, injections and the like.
Liquid preparations such as syrups suitable for oral administration include saccharides such as water, sucrose, sorbit, fructose, glycols such as polyethylene glycol, propylene glycol, oils such as sesame oil, olive oil, soybean oil, p -It can be produced using preservatives such as hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint. Tablets, powders and granules include excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy It can be produced using a binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin.

Formulations suitable for parenteral administration preferably comprise a sterile aqueous solution containing the active compound that is isotonic with the blood of the recipient. For example, in the case of an injection, a solution for injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of salt water and a glucose solution.
Also in these parenteral agents, one kind selected from diluents, preservatives, flavors, excipients, disintegrants, lubricants, binders, surfactants, plasticizers and the like exemplified for oral agents. Or more auxiliary components can be added.

  The dose and frequency of administration of the peptide of the present invention or a pharmacologically acceptable salt thereof vary depending on the dosage form, patient age, body weight, nature or severity of the condition to be treated. 0.01 mg to 1 g, preferably 0.05 to 50 mg per adult is administered once to several times a day. In the case of parenteral administration such as intravenous administration, 0.001 to 100 mg, preferably 0.01 to 10 mg per adult is administered once to several times a day. However, the dose and the number of doses vary depending on the various conditions described above.

  In addition, the peptide can be expressed in the body by transplanting a recombinant vector that expresses the peptide of the present invention or a predetermined cell into which the recombinant vector has been introduced. The peptide of the present invention expressed in the body can be expected to have the same effect as the case where itself or a pharmacologically acceptable salt thereof is administered from outside the body. A recombinant vector that expresses the peptide and a predetermined cell into which the recombinant vector has been introduced are described in 3. above. Or 4. Can be produced by the method described in the above.

<7. Method for screening a substance that inhibits or promotes an increase in intracellular calcium ion concentration of cells in hypothalamus, pituitary, adrenal gland, kidney, heart, blood vessel or brain tissue with the peptide of the present invention>
The increase in calcium concentration in the hypothalamus and pituitary cells by the peptide of the present invention is caused by the fact that the test substance has increased feeding, suppression of feeding, increased drinking, suppression of drinking, sleep induction, increased arousal, increased metabolism, or decreased metabolism. Therefore, it can be used for screening drug candidate substances.

  A substance that inhibits the increase in intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel, or brain tissue of a mammal by the peptide of the present invention or a pharmaceutically acceptable salt thereof. (I) cells when the peptide of the present invention or a pharmacologically acceptable salt thereof and a test substance are contacted with cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue (Ii) the presence of the test substance in comparison with the cell response when the peptide of the present invention or a pharmacologically acceptable salt thereof is contacted with the same cell in the absence of the test substance. When the cellular response is suppressed, the test substance inhibits the increase in intracellular calcium ion concentration of cells in the hypothalamus, pituitary, adrenal gland, kidney, heart, blood vessel or brain tissue by the peptide of the present invention As a substance It is specified by it screened.

  Similarly, (i) the peptide of the present invention or a pharmacologically acceptable salt thereof and a test substance are brought into contact with cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue. (Ii) in comparison with the cellular response when the peptide of the present invention or a pharmacologically acceptable salt thereof is contacted with the same cell in the absence of the test substance. When the cellular response is promoted in the presence of the substance, the test substance is increased by the peptide of the present invention to increase the intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue. A substance that promotes an increase in intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel, or brain tissue by the peptide of the present invention. You can ring.

  The cellular response can be measured when the peptide of the present invention or a pharmacologically acceptable salt thereof is brought into contact with cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue. Any cell response may be used as long as it is a proper cell response, and examples thereof include an increase in intracellular calcium ion concentration.

  Cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue used in the screening method of the present invention described above are brought into contact with the peptide of the present invention or a pharmacologically acceptable salt thereof. Cell lines derived from cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal may be used as long as they show a response, or the hypothalamus, pituitary gland, collected from a mammal, The adrenal gland, kidney, heart, blood vessel or brain tissue may be shredded. The hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of a transgenic mouse (international publication 2002/010371 pamphlet) that is produced by introducing the apoaequorin gene and expresses apoaequorin is collected. Cells obtained by chopping are preferred because the intracellular calcium ion concentration can be easily measured by measuring the amount of luminescence with a luminometer in the presence of coelenterazine. The intracellular calcium ion concentration is as described in 1. above. It can be measured by the method described in (i) and (ii).

  The intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue by the peptide of the present invention obtained by the screening method of the present invention or a pharmacologically acceptable salt thereof. The substance that promotes the increase has energy metabolism regulation activity or circulatory regulation activity like the peptide of the present invention or a pharmacologically acceptable salt thereof. , Circulatory regulator, vasopressor, or therapeutic agent for disorders of energy metabolism such as eating disorders, drinking disorders, metabolic disorders, sleep disorders and cardiovascular diseases such as myocardial infarction, ischemic heart disease or cerebral infarction It is useful for drug development as a candidate substance. In addition, a substance that inhibits an increase in intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue by the peptide of the present invention or a pharmaceutically acceptable salt thereof, Since it inhibits the circulatory regulatory activity of the peptide of the present invention or a pharmacologically acceptable salt thereof, for example, the activity of increasing blood pressure, it is useful for the development of a medicament as a candidate substance for an antihypertensive agent.

<8. Method for screening agonist or antagonist for receptor of peptide of the present invention>
An agonist or antagonist for the receptor of the peptide of the present invention comprises (i) cells of the hypothalamus, pituitary, adrenal gland, kidney, heart or blood vessel, or cells in brain tissue or membrane fractions of the cells. A binding amount of the peptide or a pharmacologically acceptable salt thereof to the cell or a membrane fraction of the cell when the peptide or a pharmacologically acceptable salt thereof and a test substance are contacted, (Ii) the same cell or membrane fraction with respect to the cell or membrane fraction of the cell when the peptide of the present invention or a pharmaceutically acceptable salt thereof is contacted in the absence of a test substance Compared with the binding amount of a peptide or a pharmacologically acceptable salt thereof, if the binding amount of the peptide or a pharmacologically acceptable salt thereof decreases in the presence of the test substance, the test substance is Departure Of it is identified as candidates for agonists or antagonists for the receptor of the peptide, the possible screening.

  Cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue include a piece of tissue obtained by chopping the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue taken from a mammal, Alternatively, cell lines derived from cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel, or brain tissue can be used. The cells or the cell membrane fraction of the cells are suspended in a suitable buffer. The buffer may be any buffer as long as it does not inhibit the binding of the peptide of the present invention or a pharmacologically acceptable salt thereof to the cell or the cell membrane fraction of the cell, for example, pH 4 to 10 (preferably A pH 6-8) phosphate buffer, a Tris-HCl buffer, or the like is used. For the purpose of reducing non-specific binding, surfactants such as CHAPS, Tween-80, digitonin and deoxycholic acid, and various proteins such as bovine serum albumin and gelatin can be added to the buffer. Furthermore, protease inhibitors such as PMSF, leupeptin, E-64, and pepstatin can be added for the purpose of suppressing degradation of the peptide or ligand of the present invention by protease.

A binding experiment is carried out in the presence of 10 μL to 10 mL of the cell or a cell membrane fraction of the cell coexisting with the peptide of the present invention having a certain radioactivity labeled with a radioisotope such as ¹²⁵ I and ³ H. . The reaction is carried out at 0 to 50 ° C., preferably 4 to 37 ° C., for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and then the radioactivity remaining on the glass fiber filter is measured with a γ-counter or a liquid scintillation counter. The binding amount at this time is defined as the total binding amount (A). The same reaction is carried out under the condition where a large excess of the same unlabeled compound is added, and the binding amount at that time is defined as the nonspecific binding amount (B). The same reaction is performed under the condition where the test substance is added, and the binding amount at that time is C. The binding inhibition rate of the test substance can be determined by the following formula.

  Since the agonist for the receptor of the peptide of the present invention obtained by the screening method of the present invention has an energy metabolism regulating activity or a circulation regulating activity, like the peptide of the present invention or a pharmacologically acceptable salt thereof, Energy-regulatory diseases, myocardial infarction, ischemic heart disease, such as feeding regulators, water regulators, metabolic regulators, circulatory regulators, pressors, or eating disorders, drinking disorders, metabolic disorders, sleep disorders Or it is useful for the development of a drug as a candidate substance for a therapeutic agent for cardiovascular diseases such as cerebral infarction. In addition, since the antagonist to the receptor of the peptide of the present invention inhibits the circulation regulating activity of the peptide of the present invention or a pharmacologically acceptable salt thereof, for example, the activity to increase blood pressure, Useful for the development of medicines.

<9. Antibody specifically recognizing the peptide of the present invention>
The antibody specifically recognizing the peptide of the present invention comprises (i) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7 and (ii) any one of SEQ ID NOs: 1 to 7 contained in the peptide of the present invention. Or an amino acid sequence represented by any one of (iii) SEQ ID NOs: 1 to 54, which is 54% or more identical. It is an antibody that binds to an epitope present in an amino acid sequence having sex and can specifically recognize the peptide of the present invention.

  Here, “specifically recognize” means that the affinity of a certain antibody for a specific antigen is higher than the affinity for another antigen.

In a preferred embodiment, the antibody specifically recognizes a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1-7. The antibody binds to an epitope present in one amino acid sequence selected from SEQ ID NOs: 1 to 7. The antibody has high antigen specificity. For example, the affinity of the antibody specifically recognizing the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is represented by any one of SEQ ID NOs: 2 to 7 It is 100 times higher than the affinity for peptides consisting of amino acid sequences. Similarly, the affinity of the antibody specifically recognizing the peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 for the peptide is the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 3 to 7 The affinity of the antibody specifically recognizing the peptide consisting of the amino acid sequence represented by SEQ ID NO: 3 as compared to the affinity, which is one of SEQ ID NO: 1, 2 or 4-7, The affinity for the peptide of the antibody that specifically recognizes the peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 is 100 times or more higher than the affinity for the peptide consisting of the amino acid sequence represented by SEQ ID NO: 4. 100 or more times higher than the affinity for the peptide consisting of the amino acid sequence represented by any one of 1 to 3 or 5 to 7, represented by SEQ ID NO: 5 The affinity of the antibody specifically recognizing the peptide consisting of the amino acid sequence to the peptide is 100 compared with the affinity to the peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1-4, 6 or 7. A peptide comprising an amino acid sequence represented by any one of SEQ ID NOs: 1 to 5 or 7, wherein the affinity of the antibody specifically recognizing the peptide comprising the amino acid sequence represented by SEQ ID NO: 6 is more than doubled The affinity of the antibody specifically recognizing the peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 relative to the affinity for the peptide is represented by any one of SEQ ID NOS: 1 to 6 More than 100 times higher than the affinity for a peptide comprising an amino acid sequence. The affinity of the antibody is evaluated by IC ₅₀ (the amount or concentration of the target peptide that inhibits the maximum binding amount of the selected peptide to the antibody by 50%).

Those antibodies may be polyclonal antibodies or monoclonal antibodies. Such antibodies also include antibody fragments such as Fab, Fab ′, F (ab ′) ₂ prepared from polyclonal antibodies or monoclonal antibodies. Monoclonal antibodies include human chimeric antibodies composed of variable regions of monoclonal antibodies prepared in non-human animals and constant regions of human antibodies, and complementarity determining regions (CDRs) of monoclonal antibodies prepared in non-human animals. Also included are human CDR-grafted antibodies comprising a variable region inserted in the framework region and a constant region of a human antibody.

(1) Preparation of polyclonal antibody By using the peptide of the present invention as an antigen as an antigen, the antibody can be prepared intradermally, intravenously, intraperitoneally, intramuscularly, etc. to produce a polyclonal antibody that binds to the peptide. it can. In the case where it is difficult to produce an antibody due to problems such as solubility of the peptide, it is also possible to use an epitope obtained by modifying the peptide with a hydrophilic modifying group such as polyethylene glycol. This polyclonal antibody can specifically bind to the peptide of the present invention. In this case, it is desirable to administer an antigen peptide, which is covalently bound to a carrier protein such as keyhole, limpet, hemocyanin, bovine thyroglobulin, or ovalbumin, together with an adjuvant. The covalent bond of the peptide serving as the antigen to the carrier protein can be performed by a reaction using a cross-linking agent such as maleimide, carbodiimide, glutaraldehyde or the like. When carrying out the reaction of maleimide, a peptide having a cysteine residue added to the N-terminal or C-terminal of the amino acid sequence of the peptide to be used as an antigen is prepared by the method described in 2 above and covalently bonded via cysteine. Examples of adjuvants include Freund's complete adjuvant, aluminum hydroxide gel, pertussis vaccine, and the like. Rabbits, goats, rats, mice, hamsters and the like can be used as non-human animals to administer the antigen, and the dosage is preferably an amount containing 50 to 200 μg of the peptide serving as the antigen per animal.

  The antigen is preferably administered 3 to 10 times, for example, every 1 to 3 weeks after the first administration, until the antibody titer of the serum is sufficiently increased. The antibody titer of serum can be measured by collecting blood on 3 to 7 days after each administration, preparing serum, and using enzyme immunoassay, radioimmunoassay or the like. The enzyme immunoassay is based on the literature [Enzyme Immunoassay, Medical School (1976); Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. Is immobilized on a suitable plate with another carrier protein, (ii) blocked and washed, (iii) reacted with serum prepared from the immunized animal, washed (iv ) Reaction with an enzyme-labeled antibody against IgG of animal used for immunization and washing, (v) Reaction using a substrate that develops or emits light with the labeled enzyme, and measures the amount of color or the amount of luminescence to measure the antibody titer. It can be performed by the procedure of using as an index.

Serum is prepared by collecting blood from a non-human animal whose serum showed a sufficient antibody titer against the peptide as an antigen. This serum, that is, antiserum can be used as a polyclonal antibody, or a polyclonal antibody can be purified from the antiserum.
Methods for purifying polyclonal antibodies from antisera include, for example, centrifugation, salting out with 40-50% saturated ammonium sulfate, caprylic acid precipitation (Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, (1988)), or DEAE-Sepharose. Examples include a method of treating a column, an anion exchange column, a protein A or G-column, a chromatography using a gel filtration column, or the like alone or in combination.

(2) Production of monoclonal antibody A monoclonal antibody that binds to an epitope present in the amino acid sequence of the peptide of the present invention can be produced by the method shown below. In the case where it is difficult to produce an antibody due to problems such as solubility of the peptide, it is also possible to use an epitope obtained by modifying the peptide with a hydrophilic modifying group such as polyethylene glycol. This monoclonal antibody can specifically bind to the peptide of the present invention.

(A) Preparation of antibody-producing cells A mouse or rat is used as an animal to which an antigen is administered, and the same antigen is administered as in the production of the polyclonal antibody in (1). A mouse or rat that shows a titer can be used as a source of antibody-producing cells. Spleen cells can be used as antibody-producing cells. Antibody-producing cells can be prepared from mice or rats that have shown a sufficient antibody titer, for example, as follows.

  Three to seven days after the final administration of the antigen to the mouse or rat showing the antibody titer, the spleen is removed. The spleen is shredded in MEM, loosened with tweezers, centrifuged, the supernatant is discarded, and the precipitated splenocytes are collected. The obtained spleen cells are treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove erythrocytes, and then washed three times with MEM can be used as antibody-producing cells.

(B) Preparation of myeloma cells As myeloma cells, cell lines established from mouse or rat myeloma cells can be used. Examples of established myeloma cells include 8-azaguanine-resistant mouse (BALB / c-derived) myeloma cell line P3-X63Ag8-U1 [Curr. Topics. Microbiol. Immunol., 81 , 1 (1978); Europ. J Immunol., 6 , 511 (1976)], SP2 / 0-Ag14 [Nature, 276 , 269 (1978)], P3-X63-Ag8653 [J. Immunol., 123 , 1548 (1979)], P3-X63. -Ag8 [Nature, 256 , 495 (1975)] and the like. These cell lines consisted of 8-azaguanine medium [RPMI-1640 medium with glutamine (1.5 mmol / L), 2-mercaptoethanol (5 × 10 ⁻⁵ mol / L), gentamicin (10 μg / mL) and fetal calf. It is preferable to subculture with medium supplemented with serum (10%) (hereinafter referred to as normal medium) and medium further added with 8-azaguanine (15 μg / mL), and 3 to 4 days before cell fusion. It is preferable to culture in a normal medium. It is preferable to use 2 × 10 ⁷ or more of the cells for fusion.

(C) Production of Hybridoma A hybridoma can be produced by fusing the antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) using, for example, polyethylene glycol as follows. The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) are prepared by MEM or PBS (1.83 g disodium phosphate, 0.21 g monopotassium phosphate, 7.65 g sodium chloride, 1 liter distilled water). Wash well with pH 7.2), mix so that the number of cells is antibody-producing cells: myeloma cells = 5 to 10: 1, centrifuge at 1,200 rpm for 5 minutes, and discard the supernatant. The cell group of the obtained precipitate fraction was thoroughly loosened, and the cell group was mixed with polyethylene glycol-1000 2 g, MEM 2 mL, and dimethyl sulfoxide 0.7 mL per 10 ⁸ antibody-producing cells at 37 ° C. with stirring. Add 0.2-1 mL of the solution and add 1-2 mL of MEM several times every 1-2 minutes. After the addition, MEM is added to prepare a total volume of 50 mL. The preparation is centrifuged at 900 rpm for 5 minutes, and the supernatant is discarded.

The cells after cell fusion can be cultured, for example, as follows, and hybridomas with high antibody production can be selected. After the precipitated fraction of the cells obtained was gently loosened, discharging from a graduated pipette, hypoxanthine ⁽¹⁰ -4 mol / L) to gently HAT medium [normal medium in blowing, thymidine (1.5 × 10 ^{- 5} mol / L) and aminopterin (4 × 10 ⁻⁷ mol / L) added medium] are suspended in 100 mL. The suspension is preferably dispensed at 100 μL / well into a 96-well culture plate and cultured at 37 ° C. for 7 to 14 days in a 5% CO ₂ incubator. After culturing, a part of the culture supernatant is collected, and using the culture supernatant instead of serum, the antibody titer is measured as described in (1) above, and a hybridoma corresponding to the culture supernatant having a high antibody titer is obtained. It can be selected as a hybridoma with high antibody production.

  The hybridoma is cloned, and a hybridoma cell that stably produces a high amount of antibody can be obtained by selecting those that produce a high amount of antibody from the obtained clones of each hybridoma. Cloning can be performed, for example, by a limiting dilution method or the like, and is preferably repeated twice (the first time is HT medium (a medium obtained by removing aminopterin from HAT medium), and the second time is a normal medium). . Using the culture supernatant of each clone obtained by cloning, the above-mentioned antibody titer is measured, and a clone of a hybridoma corresponding to the culture supernatant in which a strong antibody titer is recognized is stably produced with high antibody. It can be selected as a hybridoma cell.

(D) Preparation of monoclonal antibody The monoclonal antibody can be prepared from ascites after the hybridoma selected in (c) is grown as ascites cancer in nude mice as follows. 2,6,10,14-tetramethylpentadecane (pristane) 0.5 mL is intraperitoneally administered, and the monoclonal antibody obtained in (c) is produced in 8-10 week old mice or nude mice raised for 2 weeks Preferably, 5-20 × 10 ⁶ hybridoma cells / animal are injected intraperitoneally. After 10 to 21 days, ascites is collected from the mouse in which the hybridoma has developed ascites tumor, and the solid content is removed by centrifugation at 3,000 rpm for 5 minutes. A monoclonal antibody can be purified and obtained from the obtained ascites supernatant by the same method as that used for the polyclonal antibody.

  The subclass of the antibody can be determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. The amount of peptide can be calculated from the Raleigh method or the absorbance at 280 nm.

(3) Method for Measuring Peptide of the Present Invention The antibody of the present invention can be used to immunologically detect or quantify the peptide of the present invention. Immunological detection or quantification methods include competitive methods, sandwich methods, immunohistochemistry, Western blot, agglutination (monoclonal antibody experiment manual, Kodansha Scientific, 1987; secondary biochemistry experiment course 5, immunobiochemistry Research method, Tokyo Chemical Doujin, 1986).

  In the competition method, the prepared antibody is reacted with a sample solution and a certain amount of a competitive substance (the peptide of the present invention to be measured labeled with an enzyme, biotin, radioisotope, fluorescent substance, etc.) in the sample solution. In this method, after the peptide of the present invention and the competitive substance are competitively bound to the antibody, the amount of the competitive substance bound to the antibody is measured using a label, and the peptide of the present invention is quantified from the bound amount. . For example, the antibody is immobilized on a solid phase such as a plate or a bead, the peptide of the present invention and the competitive substance are competitively bound to the antibody, the solid phase is washed, and the binding amount of the competitive substance to the antibody on the solid phase The peptide of the present invention and a competitive substance are competitively bound to an antibody, and then an immune complex is precipitated with γ-globulin and polyethylene glycol to separate it from a competitor that is not bound to the antibody. And a method for measuring the amount of a competitive substance bound to. For example, a solution of the peptide of the present invention having a concentration of 5 to 10 points is prepared, and when the solution is used as a sample solution, the binding amount of the competitor to the antibody is measured, and the peptide concentration and the competitor A calibration curve in which the amount of binding is plotted is prepared, and the binding amount of the competitor for the sample solution for quantifying the peptide of the present invention is applied to the calibration curve, whereby the peptide of the present invention in the sample solution can be quantified.

  The sandwich method is a method using two types of antibodies that specifically bind to the peptide of the present invention. For example, a sample solution is reacted with one antibody immobilized on a solid phase such as a plate or a bead, and a sample is obtained. After binding the peptide of the present invention to the antibody on the solid phase, the other antibody labeled with enzyme, biotin, radioisotope, fluorescent substance, etc. is reacted to bind to the antibody on the solid phase. Examples include a method in which the labeled antibody is further bound to the peptide of the invention, the binding amount of the labeled antibody is measured using a labeling substance, and the peptide of the invention is quantified from the binding amount. For example, a peptide solution of the present invention having a concentration of 5 to 10 points is prepared, and when this solution is used as a sample solution, the binding amount to the labeled antibody is measured, and the peptide concentration and the binding amount of the label are measured. Is prepared, and the binding amount of the labeled antibody for the sample solution for quantifying the peptide of the present invention is applied to the calibration curve, whereby the peptide of the present invention in the sample solution can be quantified.

  The enzyme immunoassay method is the above competitive method or sandwich method, wherein the competitor or antibody is labeled with an enzyme such as alkaline phosphatase or peroxidase, reacted with a reagent that develops or emits light with the labeled enzyme, and the color or amount of emitted light is determined. This is a quantitative method for measuring the binding amount of a competitor or labeled antibody. Radioimmunoassay is a quantitative method in which the competitive substance or antibody is labeled with a radioisotope in the competition method or the sandwich method, and the binding amount of the competitive substance or labeled antibody is measured from the radioactivity.

  Immunohistochemistry involves reacting antibodies labeled with enzymes, biotin, radioisotopes, fluorescent substances, gold colloids, etc., on sections prepared by freezing tissues or cells in paraffin or embedding them in paraffin. This is a method for detecting the peptide of the present invention in a tissue or cell by detecting using a labeling substance.

  In Western blotting, proteins and peptides contained in a sample are separated by SDS-polyacrylamide gel, and then the proteins and peptides are blotted from the gel onto a polyvinylidene fluoride (PVDF) membrane, nitrocellulose membrane, etc. This is a method of detecting the peptide of the present invention on a membrane by reacting an antibody labeled with an element or the like and then detecting the antibody using a labeling substance.

  In the aggregation method, particles such as latex to which an antibody is immobilized are reacted with a sample solution, and the aggregation of particles caused by binding of the antibody on the particles to the peptide of the present invention in the sample is measured by measuring the absorbance. A method for detection or quantification.

  Examples of the present invention are shown below.

Example 1: Isolation and Structure Determination of Peptide Derived from CCDC80 Rat non-myocardial primary cultured cells were prepared from Wistar rats a day of age according to a conventional method [Metabolism, 53 , 710 (2004)]. The cells on the third day of culture were cultured in RPMI medium without phenol red and serum for 6 hours, and then the medium was collected. After centrifuging the medium and adding 1/50 volume of 1 mol / L hydrochloric acid to the resulting supernatant, a sample was extracted using a Sep-Pak C18 cartridge (manufactured by Waters). . The cartridge was washed with 0.1% (v / v) trifluoroacetic acid (hereinafter abbreviated as TFA), and the sample was eluted with 60% (v / v) acetonitrile-0.1% (v / v) TFA. It was. The eluate was freeze-dried, dissolved in 60% (v / v) acetonitrile-0.1% (v / v) TFA, and equilibrated with the same solution, gel filtration HPLC column [TSKgel G2000SW _XL , 21.5 mm × 30 cm (Manufactured by Tosoh Corporation)] was separated by HPLC [HPLC pump L-2100 (manufactured by Hitachi)] at a flow rate of 1.5 ml / min, and the peptide fraction eluted at approximately 30 to 35 minutes was recovered. The lyophilized sample was dissolved in 1 mol / L acetic acid, neutralized with 1 mol / L Tris (pH 11), and reduced reaction solution (1 mmol / L EDTA, 25 mmol / L dithiothreitol, 0.5 mol / l Tris, The mixture was heated at 37 ° C. for 1 hour in pH 8.5). Next, iodoacetamide was added to a final concentration of 50 mmol / L, and the mixture was allowed to react at room temperature for 15 minutes in the dark. The reaction was quenched with glacial acetic acid and desalted using a Sepp-Pack C18 cartridge. After desalting, the lyophilized sample was dissolved in 0.1% (v / v) formic acid, 2% (v / v) acetonitrile, and reverse-phase nanospray column [Monosplay C18, 100 μm × 10 mm (manufactured by GL Sciences)] ] [Nano HPLC pump Chorus (manufactured by CTC Analytics)] with a flow rate of 200 nL / min, and a hybrid mass spectrometer (LTQ-Orbitrap-XL, manufactured by Thermofisher Scientific) ] Was carried out. The obtained tandem mass spectrum was identified on the basis of the NCBI and Swiss-Prot amino acid sequence databases using analysis software Mascot MS / MS ion search (Matrix Science). As a result, three types of peptides consisting of the amino acid sequences represented by SEQ ID NOs: 2, 5, and 7 corresponding to the partial amino acid sequence of rat CCDC80 were found.

Example 2: Preparation of peptide derived from CCDC80 Peptides having the amino acid sequences represented by SEQ ID NOs: 2, 5, and 7 found in Example 1 (hereinafter referred to as peptides 2, 5 and 7, respectively) Chemical synthesis was carried out at the request of Sigma-Genosys.

  The structure of the synthesized peptide was confirmed by MALDI mass spectrometry using a MALDI-TOF mass spectrometer Autoflex (Autoflex, manufactured by Brucker). As a matrix, a saturated solution of α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich) prepared with 50% (v / v) ACN-0.1% (v / v) TFA was used. Furthermore, the structure of the peptide was also confirmed by amino acid analysis [Anal. Biochem., 222, 19 (1994)]. The hydrolysis was performed using a picotag workstation (manufactured by Waters) in hydrochloric acid vapor at 110 ° C. for 22 hours, and the amino acid composition of the hydrolyzate was performed using an amino acid analyzer L-8500 (manufactured by Hitachi).

Example 3 Activity of Increasing Intracellular Calcium Ion Concentration of Peptide Derived from CCDC80 Utilizing each organ of apoaequorin gene transgenic mouse (hereinafter referred to as apoaequorin expressing mouse) expressing apoaequorin throughout the body Thus, it was examined whether peptides 2, 5 and 7 synthesized in Example 2 had an activity to increase the intracellular calcium ion concentration. Apoaequorin-expressing mouse cells can monitor the intracellular calcium ion concentration because apoaequorin binds to calcium ions in the presence of the luminescent substrate coelenterazine to emit light. International Publication No. 2002/010371 pamphlet describes a method for producing an apoaequorin-expressing mouse, a method for evaluating a physiologically active substance using a biological sample derived from the mouse, and a physiologically active peptide as shown below. Experimental results evaluated using organs are disclosed. That is, when angiotensin II was added to each organ obtained from apoaequorin-expressing mice at a final concentration of 1 μmol / L, strong luminescence was observed in blood vessels, uterus and adrenal gland, and bradykinin was added at a final concentration of 10 μmol / L. However, there is a description that strong luminescence was observed in blood vessels, uterus and adrenal gland, and apoaequorin-expressing mice can be used for evaluating the physiological activity of novel peptides.

Apoaequorin-expressing mice were prepared according to the method disclosed in Reference Example 4 of WO 2002/010371 pamphlet. After killing the apoaequorin-expressing mice, the thymus, hypothalamus, spleen, bone, aorta, heart, kidney, adrenal gland, pancreas, pituitary gland, uterus, medulla oblongata, and spinal cord were removed, and each organ was about 1-2 mm. Shredded to ³ size. Next, in a 5 mL tube (Rohren-Tubes; manufactured by Sarstedt, No. 55.476), 10 μmol / L coelenterazine (manufactured by Molecular Probes) solution dissolved in RPMI1640 medium was added to 50 μL. Three prepared various organ samples were added and cultured at 37 ° C. for 3 hours. After 3 hours, RPMI 1640 medium was added, and after 25 seconds, peptides 2, 5, and 7 dissolved in RPMI 1640 medium were added to a final concentration of 1 μmol / L, and a luminometer [AutoLumat LB953, The relative luminescence was measured every second immediately after the addition of the RPMI1640 medium.

  As a result, peptide 2 is the thymus, spleen (FIG. 1), pituitary gland (FIG. 2), medulla, kidney, and peptide 5 is the heart (FIG. 3), kidney (FIG. 4), hypothalamus (FIG. 5). , Adrenal gland (Fig. 6), aorta, vena cava, spleen, peptide 7 for thymus, heart (Fig. 7), aorta (Fig. 8), vena cava (Fig. 9), hypothalamus (Fig. 10), adrenal gland (Fig. 11) Luminescence was observed in the spleen, skull and medulla respectively. Based on the above results, these peptides show intracellular calcium ions in the hypothalamus or pituitary gland, which are organs involved in the energy metabolism control system, and in adrenal gland, kidney, heart, or vascular cells, which are organs involved in the circulatory system. It was found to have an activity of increasing the concentration of. Moreover, it turned out that it is a raise of the intracellular calcium ion concentration by the physiological effect from the mode of the time change of the relative light-emission quantity.

  According to the present invention, a novel physiologically active peptide having energy metabolism regulation activity or circulation regulation activity is provided. In addition, a method for screening for a substance that promotes or suppresses the activity of the peptide or an agonist or antagonist for the receptor of the peptide using the peptide is provided. The peptide, a substance that promotes or suppresses the activity of the peptide obtained by the screening method provided by the present invention, or an agonist or antagonist for the receptor of the peptide has an energy metabolism regulating activity or a circulation regulating activity. Dietary regulators, water intake regulators, metabolism improvers, circulatory regulators, pressors, or diseases of energy metabolism regulation such as eating disorders, drinking disorders, metabolic disorders, sleep disorders, or myocardial infarction, ischemic heart It can be used for drug development as a candidate substance for a therapeutic drug for diseases or circulatory diseases such as cerebral infarction.

[SEQ ID NO: 7]
The partial amino acid sequence of the 940-950th position of SEQ ID NO: 9, the 939-949th position of SEQ ID NO: 10, or the 939-949th position of SEQ ID NO: 11

Claims (14)

Any peptide selected from the following (a) to (d) or a pharmaceutically acceptable salt thereof:
(A) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(B) a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 7 and having a length of 80 amino acids or less;
(C) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, comprising an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and includes a mammalian hypothalamus, pituitary gland, adrenal gland, A peptide having an activity to increase intracellular calcium ion concentration of cells in kidney, heart, blood vessel or brain tissue and having a length of 80 amino acids or less; and (d) represented by any one of SEQ ID NOs: 1 to 7 Activity that increases the intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel, or brain tissue of mammals And a peptide having a length of 80 amino acids or less. The following formula (I):

Wherein R ¹ is a hydrogen atom, substituted or unsubstituted alkanoyl, substituted or unsubstituted aroyl, substituted or unsubstituted heteroarylcarbonyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, Or substituted or unsubstituted heteroaryloxycarbonyl, R ² represents hydroxy, substituted or unsubstituted alkoxy, or substituted or unsubstituted amino, and A represents any one of claims 1 (a) to (d). Represents a peptide residue of the peptide described in 1.)
Or a pharmacologically acceptable salt thereof.   A polynucleotide comprising a nucleotide sequence encoding the peptide of claim 1.   A vector comprising the polynucleotide according to claim 3.   A transformant into which the vector according to claim 4 has been introduced.   The peptide according to claim 1, comprising culturing the transformant according to claim 5, producing and accumulating the peptide according to claim 1 in the culture, and isolating the peptide from the culture. Manufacturing method. A medicament comprising the peptide of any of the following (a) to (d) or a pharmaceutically acceptable salt thereof:
(A) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(B) a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(C) in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, comprising an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and a mammalian hypothalamus, pituitary gland, A peptide having an activity of increasing intracellular calcium ion concentration of cells in adrenal gland, kidney, heart, blood vessel or brain tissue; and (d) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7 and 54% or more A peptide comprising an amino acid sequence having identity and having an activity of increasing intracellular calcium ion concentration in cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal. Formula (II) below:

Wherein R ³ is a hydrogen atom, substituted or unsubstituted alkanoyl, substituted or unsubstituted aroyl, substituted or unsubstituted heteroarylcarbonyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, Or substituted or unsubstituted heteroaryloxycarbonyl, R ⁴ represents hydroxy, substituted or unsubstituted alkoxy, or substituted or unsubstituted amino, and B represents any of claims 7 (a) to (d). Represents the peptide residue of the peptide.)
Or a pharmacologically acceptable salt thereof.   The medicament according to claim 7, which is an energy metabolism regulator.   The medicament according to claim 7, which is a circulation regulator. The peptide of any of the following (a) to (d) or a pharmacologically acceptable salt thereof and a test substance are administered in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal. Measuring a cellular response when contacted with a cell, comparing the cellular response when contacting the cell with the peptide or a pharmacologically acceptable salt thereof in the absence of the test substance, When the cellular response is suppressed, the test substance is a substance that inhibits the increase in intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel, or brain tissue of a mammal by the peptide. An increase in intracellular calcium ion concentration of cells in the hypothalamus, pituitary, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal by the peptide or a pharmaceutically acceptable salt thereof, Hindrance Substance of the screening methods to:
(A) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(B) a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(C) in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, comprising an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and a mammalian hypothalamus, pituitary gland, A peptide having an activity of increasing intracellular calcium ion concentration of cells in adrenal gland, kidney, heart, blood vessel or brain tissue; and (d) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7 and 54% or more A peptide comprising an amino acid sequence having identity and having an activity of increasing intracellular calcium ion concentration in cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal. The peptide of any of the following (a) to (d) or a pharmacologically acceptable salt thereof and a test substance are administered in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal. Measuring a cellular response when contacted with a cell, comparing the cellular response when contacting the cell with the peptide or a pharmacologically acceptable salt thereof in the absence of the test substance, When the cellular response is promoted, the test substance is used as a substance that promotes an increase in intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel, or brain tissue of the mammal by the peptide. An increase in intracellular calcium ion concentration of cells in the hypothalamus, pituitary, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal by the peptide or a pharmaceutically acceptable salt thereof, Prompt Substance of the screening methods to:
(A) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(B) a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(C) in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, comprising an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and a mammalian hypothalamus, pituitary gland, A peptide having an activity of increasing intracellular calcium ion concentration of cells in adrenal gland, kidney, heart, blood vessel or brain tissue; and (d) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7 and 54% or more A peptide comprising an amino acid sequence having identity and having an activity of increasing intracellular calcium ion concentration in cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal. The peptide of any of the following (a) to (d) or a pharmacologically acceptable salt thereof and a test substance are administered in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel or brain tissue of a mammal. A step of measuring the binding amount of the peptide or a pharmacologically acceptable salt thereof to the cell or the membrane fraction of the cell when contacted with the cell or the membrane fraction of the cell, the peptide or the Compared with the binding amount of the peptide or a pharmacologically acceptable salt thereof when a pharmacologically acceptable salt is brought into contact with the cell or a membrane fraction of the cell in the absence of the test substance. And a method for screening an agonist or antagonist for the receptor of the peptide, comprising identifying the test substance as an agonist or antagonist for the receptor of the peptide when the binding amount decreases :
(A) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(B) a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(C) in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, comprising an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and a mammalian hypothalamus, pituitary gland, A peptide having an activity to increase intracellular calcium ion concentration of cells in adrenal gland, kidney, heart, blood vessel or brain tissue;
(D) an amino acid sequence having 54% or more identity with the amino acid sequence represented by any of SEQ ID NOs: 1 to 7, and a mammalian hypothalamus, pituitary, adrenal gland, kidney, heart, blood vessel, or A peptide having an activity of increasing intracellular calcium ion concentration of cells in brain tissue. An antibody that specifically recognizes any of the following peptides (a) to (d):
(A) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 7;
(B) a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 7 and having a length of 80 amino acids or less;
(C) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7, comprising an amino acid sequence in which 1 to 5 amino acids are substituted, deleted or added, and includes a mammalian hypothalamus, pituitary gland, adrenal gland, A peptide having an activity to increase intracellular calcium ion concentration of cells in kidney, heart, blood vessel or brain tissue and having a length of 80 amino acids or less; and (d) represented by any one of SEQ ID NOs: 1 to 7 Activity that increases the intracellular calcium ion concentration of cells in the hypothalamus, pituitary gland, adrenal gland, kidney, heart, blood vessel, or brain tissue of mammals And a peptide having a length of 80 amino acids or less.

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