JP2005168338A - New protein calphogrin, gene encoding the same, medicine, and target substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new protein which directly and specifically acts on a route for hydrolyzing PPi with IPP, a carbohydrate metabolism route in which PGM participates, or a calcium signal for transferring cyclophilin and calcineurin, to provide a gene encoding the new protein, and to provide a medicine and a target substance each containing the new protein as an active ingredient. <P>SOLUTION: The new protein calphogrin, the gene encoding the same, and the medicine and the target substance each containing the calphogrin as an active ingredient. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel protein calfoggrin, a gene encoding the same, a drug containing the same as an active ingredient, and a target substance.

  There are a number of therapeutic drugs that cause changes in human blood sugar, including those for diseases such as diabetes. It is due to an indirect mechanism that acts to suppress blood sugar, and in diabetes, the sensitivity to therapeutic drugs by the indirect mechanism is largely reduced in diabetes. Therefore, the usefulness of current blood glucose regulators is limited.

  Hydrolysis of inorganic pyrophosphate (hereinafter also referred to as PPi) to phosphoric acid (2Pi) by inorganic pyrophosphatase (hereinafter also referred to as IPP) leads to the release of many PPi such as cellular nucleic acids, proteins and polysaccharide synthesis. It is essential for the progress of the synthesis reaction. Phosphoglucomutase (hereinafter also referred to as PGM) is a central enzyme that has an action of interconverting glucose-1-phosphate and glucose-6-phosphate, and switches between gluconeogenesis and glycolysis in cells. It is a polysaccharide metabolic enzyme that is the main component of bacterial cell membranes.

  Therefore, it is considered that inhibitors of these enzymes can be used as antibacterial agents and antibiotics having a bacterial destruction effect. In addition, if a substance that acts directly and specifically on the sugar metabolic pathways involved in PGM is developed, various applications such as diet foods in addition to blood glucose regulators such as antidiabetic drugs can be considered. In addition, if a substance that directly activates IPP and PGM is developed, the substance can be a target substance for drug development that controls many metabolisms, biosynthesis, and cell division because of its core role.

  On the other hand, the calcium signal transmitted by cyclophilin and calcineurin is the central information pathway of cells that cause T lymphocyte proliferation and neuroplastic changes, and the drugs targeted here include immunosuppressants FK506 and cyclosporine, As side effects of this immunosuppressive agent, there are toxicity to the kidney and nerve and diabetes-inducing action, and a novel calcium signal modulator is required.

  Therefore, the present invention provides a novel protein that directly and specifically acts on the hydrolysis pathway of PPi by IPP, the sugar metabolic pathway involving PGM, or the calcium signal transmitted by cyclophilin and calcineurin, It is an object of the present invention to provide an encoded gene, a drug containing the novel protein as an active ingredient, and a target substance.

  In this situation, the present inventors have conducted intensive studies. As a result, the existence of a novel protein human calfoggrin, which is a defined protein predicted from the human genome, and that human calfoggrin activates IPP and activates PGM. It has been found that it has an action and a linkage to calcium signal (promotion of calcium uptake of microsomes prepared from rat muscle tissue), and the present invention has been completed.

That is, the present invention (1) is a protein of the following (a) or (b), a fragment of the following protein (a) or (b), and at least one of the biological activities of the protein of (a): Or an analog having at least one biological activity of the protein of (a) obtained by screening.
(A) human calfogulin having the amino acid sequence of SEQ ID NO: 1 (b) consisting of an amino acid sequence in which 1 to 30 amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and the protein of (a) Recombinant protein having at least one of the biological activities of

The present invention (2) is a protein of the following (a) or (b), a fragment of the protein of (a) or (b) below, and at least one of the biological activities of the protein of (a): Or a polynucleotide encoding an analog having at least one biological activity of the protein (a) obtained by screening.
(A) human calfogulin having the amino acid sequence of SEQ ID NO: 1 (b) consisting of an amino acid sequence in which 1 to 30 amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and the protein of (a) Recombinant protein having at least one of the biological activities of

  In addition, the present invention (3) provides a gene having the nucleotide sequence of SEQ ID NO: 2 encoding human calfoggrin.

  Moreover, this invention (4) provides the chemical | medical agent which uses the protein, fragment | piece, or analog of (a) or (b) as described in this invention (1) as an active ingredient.

  Moreover, this invention (5) provides the target substance which uses the protein, fragment | piece, or analog of (a) or (b) as described in this invention (1) as an active ingredient.

  The present invention encodes a novel protein that directly and specifically acts on the hydrolysis pathway of PPi by IPP, the sugar metabolic pathway involving PGM, or the calcium signal transmitted by cyclophilin and calcineurin. It is possible to provide a gene, a drug containing the novel protein as an active ingredient, and a target substance.

  The human calfogulin according to the present invention is a defined protein predicted from the human genome and cDNA, as cDNA is deciphered in Nagase et al. (Literature name; DNA Res. 7 (2): 143-150, 2000). . Calfogrin protein is obtained by the method described in this document, and the full length of the cDNA having the nucleotide sequence represented by SEQ ID NO: 2 is expressed in E. coli using an E. coli expression vector and purified by a known method. You can earn by doing. The recombinant protein is obtained by genetic recombination of the cDNA having the base sequence represented by SEQ ID NO: 2, and the amino acid of SEQ ID NO: 3 obtained by expressing the gene having the base sequence represented by SEQ ID NO: 4 Examples thereof include mouse calfogulin having the sequence and rat calfogulin having the amino acid sequence of SEQ ID NO: 5 obtained by expressing the gene having the base sequence represented by SEQ ID NO: 6. Analogues include not only proteins obtained by screening from mammals such as rats, rabbits, cows, monkeys, but also calfogrins obtained by screening from a wide range of organisms such as reptiles, amphibians, birds, plants, fungi, and bacteria. A yeast Ede1p protein having the amino acid sequence of SEQ ID NO: 7 obtained by expressing the gene of the base sequence represented by SEQ ID NO: 8, and the gene of the base sequence represented by SEQ ID NO: 10 And Escherichia coli MukB protein having the amino acid sequence of SEQ ID NO: 9 obtained in this manner. Here, one of the biological activities of the calfogulin protein indicates any one of IPP activation, PGM activation, and linkage to calcium signal shown in the following examples.

  (A) human calfogulin having the amino acid sequence of SEQ ID NO: 1 according to the present invention, (b) consisting of an amino acid sequence in which 1 to 30 amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and A recombinant protein having at least one of the biological activities of the protein of (a), a fragment thereof having at least one of the biological activities of the protein of (a), or an organism of the protein of (a) obtained by screening Analogues with at least one of the pharmacological activities include cell metabolism promoters, antibacterial agents, antibiotics, blood glucose regulators, diet food drugs, immunosuppressants, cell division, in addition to blood glucose regulators such as antidiabetics Various applications such as inhibitors and anticancer agents are conceivable. In addition, since human calfoggrin is linked with a calcium signal, the human calfoggrin of (a), the recombinant protein of (b), the fragment, or the analog according to the present invention controls phosphorus / sugar metabolism. In addition to becoming a target substance for drug development, it has the potential to be used as a target substance that promotes drug development in many calcium signal regions. Such target substances include cell metabolism promoters, antibacterial agents, antibiotics, blood glucose regulators, diet food drugs, immunosuppressants, cell division inhibitors, anticancer agents, etc., which have phosphorus metabolism, sugar metabolism, calcium signal as the active site It is thought to provide an effective principle for developing new drugs.

  When the human calfoggrin of (a), the recombinant protein of (b), the fragment or the analog according to the present invention is used as a drug, parenteral by injection, rapid infusion, nasopharyngeal absorption, skin absorption Or orally. Pharmaceutically acceptable carrier preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable organic esters such as ethyl oleate. An occlusive dressing carrier can be used to increase skin permeability and enhance absorption. Liquid dosage forms for oral administration generally can include liposomal solutions including liquid dosage forms. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups and elixirs containing inert diluents commonly used in the art, such as purified water. In addition to inert diluents, such compositions may also contain wetting agents, emulsifying agents and suspending agents, and sweetening, flavoring and flavoring agents.

  It is also possible for the medicament of the present invention to contain an adjuvant. Adjuvants are roughly divided into several groups based on their composition.

These groups include oil adjuvants (eg, Freund's complete and incomplete adjuvants), inorganic salts (eg, AlK (SO ₄ ) ₂ , AlNH ₄ (SO ₄ ), silica, alum, Al (OH) ₃ , Ca ₃ (PO ₄ ) ₂ , kaolin and carbon), polynucleotides (eg, poly IC and poly AU acids), and certain natural materials (eg, wax D from Mycobacterium tuberculosis, and Corynebacterium parvum, Bordetella pertussis And substances found in Brucella members).

  The drug of the present invention can be used for animals such as domestic animals and pets in addition to humans.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, this is merely an example and does not limit the present invention.

<Protein expression, E. coli lysate preparation, and phosphorylation experiment>
A clone was obtained by connecting the full-length cDNA of human calfoggrin protein and the cDNA of E. coli PGM to pDEST14 vector, pDEST17 vector and pET-DEST42 vector (all manufactured by Invitrogen), which are E. coli expression vectors. In addition, a clone was obtained by connecting the full-length cDNA of E. coli IPP to the pCA24N vector (gene registration number AB052891). E. coli strains BL21-AI and BL21-SI (Invitrogen) were transformed with these clones and selected with LB medium and LBON medium containing the corresponding antibiotics ampicillin and chloramphenicol. These cells were grown with vigorous agitation at 37 ° C. in 5 ml of LB and LBON medium until the mid-log growth phase was reached. 0.2% arabinose and 0.3M NaCl were added to the resulting medium to induce protein expression, respectively. After induction of expression of E. coli introduced with human calfoggrin cDNA for 2 hours, the cells were collected by centrifugation, the collected cells were resuspended, and sonicated on an ice bath for 1 minute using a microprobe. The phosphorylation reaction was initiated by the addition of 25 μl of a pH 7.5 phosphorylation mixture containing 50 mM Tris-HCl, 10 mM MgCl ₂ , 20 μCi [ ³² P] ATP, and 30 μg lysis protein. After incubation at 25 ° C. for 60 minutes, the phosphorylation reaction was stopped with 5 μl of 6XSDS loading buffer, 10 μg of phosphorylated protein was separated by SDS-PAGE using 12% gel, transferred to PVDF, auto Detected by radiography. FIG. 1 (A) shows the results of autoradiography of phosphorylation reaction of lysates of E. coli expressing human calfoggrin cDNA and E. coli (negative control) introduced with an empty vector into which no gene has been incorporated. Furthermore, pBluescript vector (manufactured by Stratagene) incorporating the human calfoggrin gene and E. coli introduced with an empty vector not incorporating the gene were separately cultured in LB medium containing ampicillin. The culture results are shown in FIG. The double transformant Escherichia coli by the PGM plasmid and the IPP plasmid used in FIG. 2 (B) and FIG. 3 (B) were selected in a medium containing ampicillin and chloramphenicol.

<Protein purification, Western blot, and antibody production>
After inducing protein expression on 400 ml of medium, the cells were collected by centrifugation and the pellet was washed with ₂₀ mM Na ₂ HPO ₄ -NaH ₂ PO ₄ , 0.5 M NaCl, and 10 mM at the same temperature as ice. Resuspended in 10 ml of a pH 7.4 solution containing imidazole. The cell suspension was sonicated for 40 minutes with a microprobe on an ice bath. The cell lysate was centrifuged at 12000 × g for 15 minutes at 4 ° C. The supernatant was collected and filtered through a 0.45 μm filter to remove cell debris. An expressed (His) _6- labeled protein (human calfoggrin, PGM, PGM and IPP) obtained from the filtered supernatant was immobilized on a nickel column (manufactured by Amersham, production number 17-1880-01), And a PD-10 desalting column (manufactured by Amersham) according to the purification method suggested by the manufacturer, and purified by affinity chromatography. 3 μg of the purified protein was electrophoresed and detected by CBB staining. The results are shown in FIGS. 2 (A) and 2 (B). Antigen detection by Western blotting using an anti-V5 antibody (manufactured by Invitrogen) and an anti-calfoggrin protein antibody (prepared using purified human calfoggrin protein) is the detection of the antigen shown by Invitrogen. The following conditions were followed according to the method. Using recombinant human calfoglin with V5 epitope and human calfoglin of human cerebellum, anti-V5 antibody was used at 1: 7000 dilution, anti-calfogulin protein antibody (anti-calfogulin rabbit polyclonal anti-antibody against full-length recombinant protein) (Made from serum) was performed as a 1: 4000 dilution. The results of Western blotting are shown in FIG. Furthermore, 30 μg of recombinant calfoggrin protein having V5 epitope was phosphorylated with 250 μl of a solution containing 300 μg of 300 μCi [ ³² P] ATP and 300 μg of a homogenized rat cerebellum homogenous solution, and 200 μl of the phosphorylated solution was subjected to affinity chromatography using a nickel column. Purified by the method. The reaction solution before purification and the purified calfoggrin protein were detected by performing autoradiography in the same manner as in the phosphorylation reaction experiment or by performing the Western blotting experiment in the same manner. The results are shown in FIG.

<Measurement of PGM activity>
Activity measurements coupled to the reduction of glucose-6-phosphate dehydrogenase and NADP ⁺ were performed. A reaction mixture containing 20 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM glucose-1-phosphate, 0.25 mM NADP ⁺ , and 0.1 mM glucose-1,6-diphosphate was prepared at pH 7.5. The reaction was initiated by adding 1 unit of glucose-6-phosphate dehydrogenase to a predetermined amount (eg 5 μg) of cell lysate or a predetermined amount (eg 0.15 μg) of purified PGM. The reduction reaction of NADP ⁺ was measured every 30 seconds for 20 minutes by measuring the absorbance at 340 nm with a spectrophotometer. Independent experiments were performed three times and the average value was adopted. FIG. 3 (A) shows the measurement results of the increase in PGM activity of the E. coli lysate expressing human calfoggrin. Furthermore, using purified PGM or IPP and PGM earned by E. coli expressing PGM alone, or E. coli expressing both IPP and PGM, PGM activity in the presence and absence of PPi as well Measurements were made. The results are shown in FIG.

<Measurement of IPP activity and total amount of cellular phosphate>
A 0.1 ml reaction mixture at pH 8.0 containing 20 mM Tris-HCl, 10 mM MgSO ₄ , 60 mM KCl, 2 mM PPi, and a predetermined amount of IPP or cell lysate was prepared. The reaction was started by adding 4 μl of 50 mM PPi. After incubation at 30 ° C. for 30 minutes, the reaction was stopped by adding 4 μl of 1M citric acid. In order to determine the hydrolysis rate of PPi, the release rate of Pi was measured by a colorimetric method according to the method of Heinonen et al. (Anal Biochem 113 (2), 313-317). The reaction mixture with citric acid added before PPi was used for the blank test. In the IPP activation experiment with human calfoggrin, 30 ng of IPP protein purified from yeast and Escherichia coli (both manufactured by Sigma) was incubated with 300 ng of human calfoggrin purified by affinity chromatography at 25 ° C. for 10 minutes. Then, IPP activity measurement was performed for 30 minutes. The average value was adopted after four independent experiments. The results of this IPP activity measurement are shown in FIG. In addition, three independent experiments were performed using a lysate of E. coli cells into which cDNA of human calfoggrin was introduced, and an average value was adopted. The result of this IPP activity measurement is shown in FIG. In an IPP inhibition experiment with Ca ²⁺ , a reaction mixture containing 2 mM MgSO ₄ instead of 10 mM MgSO ₄ was used. The average value was adopted after four independent experiments. FIG. 5 (A) shows the results of an IPP inhibition experiment using calcium ions Ca ²⁺ . In order to extract all the cellular phosphate, 1.2 ml of 1M HCl solution was added to a pellet-like dried product of a medium in which Escherichia coli expressing human calfogulin was cultured in a microcentrifuge tube, and the tube was rotated. Incubated with rotation. After 24 hours, each sample was centrifuged briefly, and then the concentration of phosphate in the supernatant was measured to determine the total amount of phosphate. The total amount of cellular phosphate is shown in FIG.

<Income of soluble protein fraction of rat>
To prepare the soluble protein fraction, 5 g of rat skeletal muscle was minced and then cooled to 50 ml of a solution containing 25 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, pH 7.5. The minced muscle was subjected to a homogenizer operation for 5 minutes three times for 5 minutes. The resulting homogenate was centrifuged at 5000 × g for 10 minutes at 4 ° C. The supernatant was taken out and further centrifuged at 12000 × g for 10 minutes, and 25 μg and 85 μg of protein obtained from the supernatant were used for measuring the activity of PGM and IPP, respectively. In each case, three independent experiments were performed and the average value was adopted. The amount of human calfogrin added was 0.3 μg. The results of measuring the activity of PGM and IPP are shown in FIGS. 6 (B) and 5 (B).

<RT-PCR>
RT-PCR using mouse calfoggrin mRNA was performed using the primers of SEQ ID NOs: 11 and 12, and RT-PCR using PGM mRNA was performed using the primers of SEQ ID NOs: 13 and 14. 5 ng of cDNA from each organ was used as a template. Amplification was performed under the following conditions. Denaturation is performed at 94 ° C. for 1 minute, denaturation is performed at 94 ° C. for 45 seconds, annealing is performed at 68 ° C. for 1 minute, complementary strand synthesis with primers at 72 ° C. for 1 minute is performed 30 cycles, and final complementary strand synthesis is performed at 72 ° C. For 5 minutes. After amplification, the reaction mixture was electrophoresed on a 1.2% agarose / ethidium bromide gel. The products of mouse calfoggrin and PGM obtained by RT-PCR were 738 bp and 886 bp. The results are shown in FIG.

<In situ hybridization>
In situ hybridization experiments were performed according to the method of Ingi et al. (Eur J Neurosci 15 (5), 929-936, 2002). Freshly cut mouse brain tissue was frozen in a plastic container placed on dry ice / ethanol. A frozen slice of 10 μm thickness was affixed onto a glass slide coated with gelatin. Prior to hybridization, the fragment was fixed with freshly prepared 4% depolymerized paraformaldehyde and then dissolved in 0.9% aqueous sodium chloride solution adjusted to pH 8.0. Acetylated with 0.5% acetic anhydride in 0.1 M triethanolamine and defatted. Hybridization was performed on 50% formaldehyde, 10 mM dithiothreitol, 600 mM NaCl, 1 × Denhards, 1 mM EDTA, 0.2 mg / ml yeast on each tissue slide placed in a sealed box of saturated humidity. A probe of 1 × 10 ⁶ cpm was placed in 100 μl of a hybridization buffer containing tRNA and 10% dextran, and the cover glass was covered at 56 ° C. After overnight incubation, the slides were washed with 2 XSSC (1 XSSC contains 150 mM NaCl, 15 mM sodium citrate, adjusted to pH 7.0) at 37 ° C. and washed with 10 μg / ml RNase A. The resultant was treated with 2XSSC and then washed 3 times with 2XSSC. After washing, the fragments were sequentially immersed in 30% ethanol, 70% ethanol, and 95% ethanol, air-dried, and exposed to Bio Max MR film (manufactured by Kodak) at room temperature for 7 to 14 days. RNA probe labeled with ³⁵ S was synthesized from [ ³⁵ S] UTP and T7 RNA polymerase or T3 RNA polymerase from calfogrin cDNA incorporated into pBluescript vector according to the method of MAXIscript (Ambion). Prepared as a cRNA transcript. The synthesized RNA probe was purified using a Sephadex G50 column (Amersham). In situ hybridization experiments were performed in the presence and absence of a 100-fold amount of unlabeled RNA probe for the purpose of distinguishing non-specific staining. The results are shown in FIG.

<Experiment of the two-hybrid method>
The two-hybrid experiment was carried out in a somewhat modified way from Cagney et al. (Methods Enzymol 328, 3-14, 2000). A DNA-binding domain vector pOBD2 and a Gal4 activation domain vector pOAD were used in the experiment. Yeast strain PJ69-4a, which contains 28 yeast genes bound to the Gal4 activation domain and expresses the respective fusion proteins, was used to screen for interacting proteins. The method of introducing pOBD2-human calfogulin (or pOBD2-E. Coli PGM) into 28 PJ69-4a containing each yeast gene was performed according to the instructions shown in the ScEasyComp transformation kit (Invitrogen). The yeast suspension was plated on a synthetic plate not containing tryptophan and leucine and incubated at 30 ° C. for 7 days. After the colonies grew, the colonies were picked and then reseeded on a synthetic plate containing 3-amino-1,2,4-triazole and no tryptophan, leucine, and histidine to detect two-hybrid activity. 3-Amino-1,2,4-triazole concentrations of 100 mM and 3 mM, respectively, were used to sort out calfogulin and PGM. The plates were recorded for viability of the transformants after 7-21 days at 30 ° C. under the conditions for sorting. A transformant containing an empty pOBD2 vector into which no gene was integrated or a transformant having only pOBD2-calphoglin (or E. coli pgm) and no yeast gene was used as a negative control in each two-hybrid measurement experiment. The result of the two-hybrid measurement experiment is shown in FIG. In FIG. 9, A row 1 and a row 1 are cmd1 protein, A row 2 and a row 2 are cmk2 protein, A row 3 and a row 3 are cmp2 protein, A row 4 And a row 4 column are cph1 protein, A row 5 column and a row 5 column are cof1 protein, A row 6 column and a row 6 column are nuf1 protein, B row 1 column and b row 1 column are myo2 protein. B row 2 and b row 2 are myo3 protein, B row 3 and b row 3 are myo4 protein, B row 4 and b row 4 are myo5 protein, B row 5 and b Row 5 is pgm2 protein, B row 6 and b row 6 are ils1 protein, C row 1 and c row 1 are vas1 protein, C row 2 and c row 2 are hyp2 proteins, C row 3 column and c row 3 column are ipp1 protein, C row 4 column and c row 4 column are ynk1 protein, C row 5 column and c row 5 column are pst2 protein, C row 6 column and c row 6 Column sod1 protein, D row 1 and d row 1 columns are uba1 protein, D row 2 and d rows 2 columns are vps13 proteins, D rows 3 and d rows 3 columns are she4 proteins, D rows 4 columns And d row 4 column are hch1 protein, D row 5 column and d row 5 column are hul5 protein, D row 6 column and d row 6 column are mlc1 protein, E row 1 column and e row 1 column are uso1 protein. E row 2 and e row 2 are myo1 protein, E row 3 and e row 3 are rad50 proteins, E row 4 and e rows 4 are pgm1 proteins, respectively, in the Gal4 activation domain. The result of a two-hybrid measurement when expressed as a fused yeast protein is shown.

< ⁴⁵ Ca ²⁺ binding experiment>
Using purified proteins of human calfogrin, E. coli IPP (Sigma), yeast IPP (Sigma), and rabbit PGM, Maruyama et al. (J. Biochem (Tokyo) 95 (2), 511-519, 1984) According to the method of ⁴⁵ Ca ²⁺ binding experiment was conducted. 30 μg of these proteins were separated by SDS-PAGE using a 12% gel and transferred to a Protran nitrocellulose membrane (Schleicher & Schuell). Thereafter, the membrane was immersed in a solution containing 10 mM imidazole-hydrochloric acid, 60 mM KCl, 5 mM MgCl ₂ and pH 6.8. Thereafter, the membrane was incubated at 25 ° C. for 10 minutes in a buffer solution having the same composition as the above solution containing 1 μCi / ml of ⁴⁵ Ca ²⁺ . The membrane was rinsed with distilled water for 5 minutes, then air-dried, and developed by exposure to X-OMAT film (Kodak) at room temperature for 1 to 5 days. The results of the ⁴⁵ Ca ²⁺ binding experiment are shown in FIG.

< ⁴⁵ Ca ²⁺ uptake experiment>
Microsomal fractions were prepared from rat muscle tissue according to the method of Ellet et al. (Biochim Biophys Acta 282 (1), 174-179, 1972). 25 μg microsomes, 3 μg human calfoggrin, and 0.3 μg yeast IPP were added to 20 mM MOPS, 100 mM KCl, 20 mM NaCl, 5 mM MgCl ₂ , 3 mM ATP, 5 mM NaN ₃ , 2 mM PPi 20 μM CaCl ₂ , 1.2 μCi ⁴⁵ CaCl. ₂ and incubated at 37 ° C. in 1 ml of pH 7.2 solution. After a predetermined time, the amount of ⁴⁵ Ca ²⁺ accumulated by microsomes was measured by rapid filtration according to the method of Fulceri et al. (Biochem J 289 (Pt 1), 299-306, 1993), and ⁴⁵ Ca ²⁺ uptake was determined. The amount was determined. Three independent experiments were performed and the average value was adopted. The result of the ⁴⁵ Ca ²⁺ uptake experiment is shown in FIG.

  Humans present in the human cerebellum at the position of 80 kd, which is the same as the molecular weight 80 kd of recombinant calfoggrin expressed from cDNA in E. coli, detected in the left and middle stages of FIG. 2 (A). Calfogrin was detected. As shown in FIG. 1C, it was found that calfogrin has an action of inhibiting the growth of Escherichia coli and can be used as an antibacterial agent and antibiotic.

  The results of the two-hybrid method in FIG. 9 indicate that human calfoggrin and yeast IPP interact, and that E. coli PGM and yeast IPP interact. Human calfoggrin and yeast IPP and E. coli It was suggested that PGM forms a complex. The association of these three different biological proteins indicates that this signaling pathway has surprising conservation in biological evolution and suggests that it is a universal function in a wide range of existing species of cells. doing.

  From the result of FIG. 4 (A), the activity of IPP is improved 2.8 times and 2.3 times in yeast and Escherichia coli by adding purified human calfoggrin, respectively. It was found to activate E. coli IPP. In addition, from the result of FIG. 4B, when the E. coli lysate expressing the human calfoggrin gene was used, the IPP activity was improved by 2.5 times compared to E. coli not expressing calfoggrin. It was found that human calfoggrin activates yeast / E. Coli IPP. In FIG. 6 (A), in the Escherichia coli lysate expressing the human calfoggrin gene giving the result of FIG. 4 (B), the intracellular Pi concentration was 1.7 times, and yeast by human calfoggrin was used. / Results consistent with activation of E. coli IPP were obtained. In addition, from the results shown in FIG. 5 (B), the IPP activity was improved by a factor of 2.0 when a physiological mammalian protein group in which purified human calfoggrin was added to the soluble protein fraction of rat skeletal muscle was used. It was found that human calfoggrin activates yeast / E. Coli IPP.

  From the result of FIG. 3 (B), the PGM enzyme activity in the case of expressing PGM alone in E. coli and the PGM enzyme activity in the case of using the protein purified by expressing both IPP and PGM are compared with IPP. It was found that the activity of PGM improved as the PPi concentration increased in the case of using the protein purified by coexpressing PGM. This indicates that PPM activity is improved by increasing the concentration of PPi and activating IPP, and PGM activation occurs through the activation of IPP by human calfogulin. It is shown that. Fig. 2 (B) shows the results of CBB confirming that PGM, IPP and PGM are expressed in E. coli expressing PGM alone and in E. coli expressing both IPP and PGM, respectively. is there.

Two findings have shown that human calfoggrin ultimately activates PGM. (1) From FIG. 1 (A), the signal intensity of 55 kd derived from PGM is increased by expressing the human calfogulin gene or recombinant human calfogulin gene in E. coli cells. It was found to promote phosphorylation. As shown in FIG. 3 (A), since the enzymatic activity of PGM in E. coli is increased by expressing the human calfoggrin gene in E. coli cells, human calfoggrin promotes phosphorylation of PGM, thereby It is thought to activate.
(2) From FIG. 6 (B), since the activity of PGM is increased in a physiological mammalian protein group system in which purified human calfoggrin is added to the soluble protein fraction of rat skeletal muscle, Was found to activate.

The following four findings suggested the linkage between human calfoggrin and calcium signals.
(1) It was found that one calcium signal protein group (consisting of the following 26 proteins) extracted from the analysis data of the whole intracellular protein complex of yeast described in Table 1 contained a calfogulin analog. did. Table 1 shows the database of Gavin et al. (Nature 415 (6868), p141-147) and Ho et al. (Nature 415 (6868), p180-183) as a database of yeast protein complexes covering all yeast proteins. This is the result of extracting 14 groups of yeast proteins containing PGM using PGM as a probe and extracting 1 group of yeast proteins containing calfoggrin analogs from these.
(2) From the results of FIG. 10 (A), it was found that the human calfoggrin C-terminal partial protein (about 1 μg) having a molecular weight of 35 kd, and purified proteins of E. coli IPP and yeast IPP bind to ⁴⁵ Ca ²⁺ . .
(3) From the results of FIG. 5 (A), it was found that Ca ²⁺ inhibits the activities of E. coli IPP and yeast IPP, and that the activities of E. coli IPP and yeast IPP are improved by human calfogulin. As shown in FIG. 1 (B), the 35 kd human calfogulin C-terminal partial protein is phosphorylated by human cerebellar protein as in the detection result by Western blotting using the anti-V5 antibody shown in the right column. Since a strong signal is shown, it is suggested that, together with the result of FIG. 10 (A), this human calfoggrin C-terminal partial protein may regulate the activities of calfoggrin and IPP in the presence of Ca ^2+. It was done.
(4) From the results of FIG. 10 (B), it was found that human calfoggrin promotes Ca ²⁺ uptake of rat skeletal muscle endoplasmic reticulum.

  As shown in FIG. 7, the mouse brain, heart, lung, liver, spleen, kidney, skeletal muscle were obtained by RT-PCR using mouse calfoggrin mRNA and RT-PCR using PGM mRNA. It was found that mouse calfoggrin and PGM were present in the testis, ovary, small intestine and embryo. As shown in FIG. 8, it was found that mouse calfogulin is present in high concentrations in mouse brain, particularly in hippocampal neurons and cerebellar granule cells. When the concentration of the unlabeled probe is high, the entire staining disappears, so that mouse calfoggrin mRNA is thought to exist evenly in the mouse brain.

The mechanism by which human calfogrin directly activates IPP has been shown to increase the number of biosynthetic reactions via PPi degradation and activate the metabolism of the entire cell. As a result, it directly affects the metabolic function that is the core of the cell, which may lead to the development of drugs with special effects. The mechanism by which human calfogrin increases PGM activity through a complex provides an action site that governs cellular phosphorus and sugar metabolism and its drug model. This mechanism for the activation of PGM by human calfogulin is a central mechanism for the regulation of phosphorus and sugar metabolism that is universally present in cells, in order to develop drugs or drugs that govern a wide range of synthesis of nucleic acids, proteins, polysaccharides, etc. Can be a target substance. It is considered that human calfogrin forms a functional complex with two core enzymes that govern the metabolism of phosphorus and glucose, IPP and PGM, as follows. First, human calfogulin forms a complex with IPP, and then a complex of human calfogulin and IPP forms a complex with PGM. The mechanism of such complex formation and activation of IPP and PGM is shown in FIG. It can be said that this human calfoggrin-IPP-PGM complex exhibits the most direct and most effective principle for regulation of phosphorus / sugar metabolism and cell activation in the living body. Furthermore, from the examples, the mechanism by which human calfoggrin is linked to the calcium signal is localized in the intracellular calcium signal protein group (Table 1), Ca ²⁺ binding and regulation by it (FIG. 10 (A) and FIG. 5 (A)), and promotion of Ca ²⁺ uptake by the endoplasmic reticulum (FIG. 10B). Human calfoggrin has the potential to promote the development of many drugs in the calcium signal region in conjunction with the regulation of phosphorus and sugar metabolism.

It is a figure which shows the result of the autoradiography of the phosphorylation reaction of the melt | dissolution solution of Escherichia coli which expressed the full-length cDNA of human calfogulin. It is a figure which shows the detection result by the autoradiography and the western blotting using the phosphorylation reaction liquid obtained by phosphorylating the recombinant human calfogulin which has a V5 epitope by the effect | action of a human cerebellum protein. It is a figure which shows the result of having culture | cultivated separately the colon_bacillus | E._coli which introduce | transduced the empty vector which has not integrated the gene, and colon_bacillus | E._coli which introduce | transduced the pBluescript vector which integrated the human calfogulin gene. It is a figure which shows the result of having detected the human calfogulin which has a V5 epitope by the CBB dyeing | staining and Western blotting, and the detection result of the human cerebellum calfoggrin by Western blotting. It is a figure which shows the result of having electrophoresed purified PGM or purified PGM and IPP, and having carried out CBB dyeing | staining. It is a figure which shows the result of having measured PGM activity using the solution of colon_bacillus | E._coli which expressed human calfogulin, and colon_bacillus | E._coli which is not expressing. PGM activity was measured in the presence and absence of PPi using purified PGM or IPP and PGM obtained by E. coli expressing PGM alone or E. coli expressing both IPP and PGM. It is a figure which shows a result. It is a figure which shows the result of having performed IPP activity measurement using yeast IPP and colon_bacillus | E._coli IPP, and 300 ng of purified human calfoggrin. It is a figure which shows the result of an IPP activity measurement using the melt | dissolution solution of colon_bacillus | E._coli which introduce | transduced cDNA of human calfogrin. It is a figure which shows the result of the inhibition experiment of IPP by Ca ^<2+ >. It is a figure which shows the result of an IPP activity measurement using the soluble protein fraction of a rat. It is a figure which shows the total amount of the intracellular phosphate in colon_bacillus | E._coli which expressed human calfogulin. It is a figure which shows the result of a PGM activity measurement using the soluble protein fraction of a rat. It is a figure which shows the result of having separated the product of RT-PCR method using mRNA of each organ of a mouse | mouth by electrophoresis. It is a figure which shows the result of the in situ hybridization experiment using the brain tissue of a rat. It is a figure which shows the result of a two-hybrid measurement experiment using the yeast calcium signal protein group which contains human calfogulin and E. coli PGM as bait protein, and IPP as prey protein. It is a figure which shows the result of ⁴⁵ Ca2 ⁺ binding experiment using human calfogulin, E. coli IPP, yeast IPP, and rabbit PGM. It is a figure which shows the result of ⁴⁵ Ca2 ⁺ uptake ^| capture experiment of the microsome prepared from the rat muscle tissue using human calfogulin and yeast IPP. It is a figure which shows the mechanism in which human calfoggrin activates IPP and then IPP activation by human calfoggrin activates PGM.

  SEQ ID NOs: 1 and 2 show the amino acid sequence and cDNA base sequence of human calfogulin, SEQ ID NOs: 3 and 4 show the amino acid sequence of mouse calfogulin and cDNA base sequence, and SEQ ID NOs: 5 and 6 show rat The amino acid sequence of calfogrin and the base sequence of cDNA are shown. SEQ ID NOs: 7 and 8 show the amino acid sequence of the yeast Ede1p protein and the base sequence of cDNA. SEQ ID NOs: 9 and 10 show the amino acid sequence of the E. coli MukB protein and the cDNA. A base sequence is shown and sequence number 11-14 shows the base sequence of a primer.

Claims (5)

A protein of the following (a) or (b), a fragment of the following protein (a) or (b), the fragment having at least one biological activity of the protein of (a), or obtained by screening: An analog having at least one of the biological activities of the protein of (a).
(A) human calfogulin having the amino acid sequence of SEQ ID NO: 1 (b) consisting of an amino acid sequence in which 1 to 30 amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and the protein of (a) Recombinant protein having at least one of the biological activities of A protein of the following (a) or (b), a fragment of the protein of the following (a) or (b), which has at least one biological activity of the protein of (a), or obtained by screening A polynucleotide encoding an analog having at least one of the biological activities of the protein of (a).
(A) human calfogulin having the amino acid sequence of SEQ ID NO: 1 (b) consisting of an amino acid sequence in which 1 to 30 amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and the protein of (a) Recombinant protein having at least one of the biological activities of   A gene having the nucleotide sequence of SEQ ID NO: 2 encoding human calfoggrin.   A drug comprising the protein, fragment or analog of (a) or (b) according to claim 1 as an active ingredient.   A target substance comprising the protein, fragment or analog of (a) or (b) according to claim 1 as an active ingredient.