WO2018135738A1 - COMPOSITION FOR INHIBITING MYOSTATIN ACTIVITY, CONTAINING MATRIX gla PROTEIN AS ACTIVE INGREDIENT - Google Patents

Abstract

The present invention relates to a composition for inhibiting myostatin activity, containing a matrix gla protein as an active ingredient. More specifically, it is confirmed that a composition containing a matrix gla protein as an active ingredient promotes the differentiation of muscle satellite cells by increasing the expression of genes associated with the differentiation of the muscle and inhibits myostatin activity by inhibiting the binding of myostatin, which induces muscle wasting, with ACVRIIB, and thus the composition containing a matrix gla protein as an active ingredient can be effectively usable as an agent for modulating myostatin activity and an agent for treating muscle diseases such as myostatin-induced muscle wasting.

Description

Composition for inhibiting myostatin activity containing matrix gla protein as an active ingredient

The present invention relates to a composition for inhibiting the activity of myostatin to induce muscle loss.

Diseases that cause muscle weakness include muscular dystrophy and cardiac atrophy that progress with aging, muscular atrophy caused by imbalances in protein metabolism or decreased muscle use, starvation, and wasting diseases.

Muscular dystrophy refers to a decrease in muscle strength due to a decrease in muscle mass during aging, and not only a decrease in muscle mass, but also a change in the type of muscle fibers. This myotropia is induced by a decrease or growth of growth hormone, a change in physiological activity, a change in metabolism, an increase in the amount of sex hormone or fat or catabolic cytokines, and a balance change in protein synthesis and differentiation.

Reduction of muscle mass, the biggest hallmark of muscular dystrophy, is known to be due to the decrease of satellite cell activity. Satellite cells are small mononuclear cells located between the basement membrane and the muscles of myofibrils, which are activated by stimulation such as injury or movement, and proliferate into myoblasts. When differentiation progresses, they fuse with other cells to form multinucleated myofiber.

Accordingly, the decrease in the activity of the satellite cells, the ability to regenerate damaged muscle or the response to the differentiation signal is reduced, resulting in a decrease in muscle formation.

Muscular dystrophy is caused by malnutrition or long-term muscle inactivity, resulting in a breakdown in the balance of normal protein synthesis and degradation.

Appropriate exercise and drug treatment methods are progressing as a treatment method for such sarcopenia. Exercise may increase skeletal muscle protein synthesis in the short term, but is inadequate for long-term treatments, and testosterone or anabolic steroids may be used for medications, but this induces masculinity in women and prostate symptoms in men. Appears.

Recently, stem cell therapy that separates satellite cells, differentiates them in vitro, and introduces them into the body, and directly activates satellite cells in the body to promote muscle differentiation and maintain or strengthen muscles, such as muscle weakness treatment methods such as myotropenia. However, in order to treat muscle weakness-related diseases, there is a need for a more fundamental and no side effect treatment method and development of a substance capable of promoting differentiation of myoblasts.

The present invention is to provide a composition containing the matrix gla protein as an active ingredient in order to inhibit the activity of myostatin to induce muscle loss to be used as a therapeutic agent for muscle diseases related to myostatin.

The present invention provides a composition for inhibiting myostatin activity comprising a matrix gla protein (MGP) consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating muscle diseases, comprising as an active ingredient a matrix gla protein consisting of the amino acid sequence represented by SEQ ID NO: 1.

The present invention comprises the steps of treating a candidate with a cell; And Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, in the myostatin amino acid sequence represented by SEQ ID NO: 2 in cells treated with the candidates Pro76, Thr77, Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 to provide a myostatin inhibitor screening method comprising the step of identifying the binding level of any one or more amino acids and candidates selected from the group consisting of. Can be.

In addition, the present invention comprises the steps of treating the candidate material to the cell; And Arg49, Pro46, Gln48, Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1, and Phe43 in the matrix gla protein amino acid sequence represented by SEQ ID NO: 1 in cells treated with the candidates. Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, Pro76, Thr77 in the myostatin amino acid sequence represented by SEQ. , Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 may provide a matrix gla protein and myostatin binding promoter screening method comprising the step of confirming the increase in the binding level of any one or more amino acids selected from the group consisting of have.

The present invention comprises a matrix gla protein (MGP) consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient, the matrix gla protein is Arg49, Pro46, Gln48, in the amino acid sequence represented by SEQ ID NO: 1, Leu20, Val22, Phe27, Trp29, Trp31 in the amino acid sequence of myostatin, wherein any one or more amino acids selected from the group consisting of Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1 and Phe43 are represented by SEQ ID NO: 2 At least one amino acid selected from the group consisting of Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, Pro76, Thr77, Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101. It can provide a reagent composition for inhibiting myostatin activity by binding to inhibit myostatin activity.

According to the present invention, a composition containing the matrix gla protein as an active ingredient increases the expression of genes related to muscle differentiation, thereby promoting the differentiation of muscle satellite cells and inhibiting the binding of myostatin and ACVRIIB to induce muscle reduction. As it has been found to inhibit the activity, the composition containing the matrix gla protein as an active ingredient can be effectively used as a therapeutic agent for muscle diseases such as myostatin activity modulators and muscle loss induced by myostatin.

FIG. 1 shows MGP expression in C2C12 cells grown in differentiation medium over time. FIG. 1 (A) shows RT-PCR results confirming MGP mRNA levels, and FIG. 1 (B) shows Western MGP protein levels. As a result of the blot, MGP shRNA-injected C2C12 cells after differentiation and cultured for 2 days after the result of confirming the effect of reducing MGP expression, Figure 1 (C) is the result of RT-PCR confirming the MGP mRNA level, Figure 1 (D ) Is a Western blot result, Figure 1 (E) is a result of confirming myotube cell formation in C2C12 cells with reduced MGP expression, Figure 1 (F) is a result of confirming the fusion index in C2C12 cells with reduced MGP expression, 1 (G) is an immunocytochemical result confirming MGP protein expression.

Figure 2 shows the results of confirming the expression of MGP protein in the cytoplasm of myotubes.

Figure 3 is a result of confirming the extracellular matrix (ECM) and myogenic marker gene expression in C2C12 cells (MGPkd) reduced MGP expression in order to confirm the effect of MGP expression on the root canal cell formation, Figure 3 (A) Is the result of confirming the RNA and protein expression levels of the myogenic marker gene MYOG, Figure 3 (B) is the result of confirming the RNA and protein expression levels of the myogenic marker gene MYOD, Figures 3 (C) and 3 (D) This is the result of confirming the expression of the extracellular matrix (ECM) genes COL1α1 and FMOD in cells with reduced MGP expression during the differentiation period (day 2).

Figure 4 is a result of confirming the MGP expression level in the cells suppressed FMOD, COL1α1, or MSTN gene, Figure 4 (A) is a result of confirming the MGP expression level in the FMOD reduced expression cells (FMODkd), Figure 4 (B) is a result of confirming the MGP expression level in the COL1α1 expression reduced cells (COL1α1kd), Figure 4 (C) is a result of confirming the MGP expression level in the MSTN expression reduced cells (MSTNkd), Figure 4 (D ) And (E) are immunochemical analyzes confirming the expression level of MSTN, a muscle development negative regulator, in cells with reduced MGP expression and in normal cells.

5 is a result of confirming the correlation between MGP and MSTN and MGP and FMOD in in vitro conditions by complex immunoprecipitation and Western blot analysis, Figure 5 (A) is confirmed in normal cells, Figure 5 (B) 5 is a result of confirming the structure of the produced MGP, Figure 5 (D) is a result of confirming the correlation between MGP and MSTN by performing In silico experiments 5 (E) shows the results of confirming the effect on the receptor ACVRIIB that binds to MSTN.

The present invention can provide a composition for inhibiting myostatin activity containing a matrix gla protein (MGP) consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

 The matrix gla protein may inhibit myostatin activity by inhibiting the binding of myostatin and its receptor, activin receptor type IIB.

More specifically, the matrix gla protein is any one selected from the group consisting of Arg49, Pro46, Gln48, Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1 and Phe43 in the amino acid sequence represented by SEQ ID NO: 1 The above amino acids are Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, Pro76, Thr77, Lys78 in the amino acid sequence of myostatin represented by SEQ ID NO: 2. Myostatin activity can be regulated by binding to any one or more amino acids selected from the group consisting of Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 and inhibiting the binding of myostatin and its receptor, ACVRIIB.

More preferably, the matrix gla protein amino acid Arg49 binds to the amino acid Leu20 of myostatin, the matrix gla protein amino acid Arg38 binds to any one of the amino acids Tyr55, Pro76, Thr77, Lys78 or Met79 of myostatin, and the matrix gla Protein amino acid Arg37 binds to either amino acid Met79 or Ala100 of myostatin, the matrix gla protein amino acid Gln48 binds to amino acid Phe27 of myostatin, and the matrix gla protein amino acid Gln24 is either amino acid Arg67 or Gly68 of myostatin The matrix gla protein amino acid Leu4 binds to any one of amino acids Trp29 or Trp31 of myostatin, and the matrix gla protein amino acid Met26 binds to any of amino acids Gly68, Ser69, Ala70, Gly71 or Cys73 of myostatin Combine, The matrix gla protein amino acid Met1 binds to any one of amino acids Tyr86, Asn88 or Gly89 of myostatin, and the matrix gla protein amino acid Phe43 binds to any of amino acids Ala100 or Met101 of myostatin, and the matrix gla protein amino acid Pro46 Is bound to any one of amino acids Val22 or Phe27 of myostatin, the matrix gla protein amino acid Ser45 binds to any of amino acids Ala40, Asn41 or Tyr42 of myostatin, and the matrix gla protein amino acid Ser25 is amino acid Ala70 of myostatin And the matrix gla protein amino acid Thr42 can modulate myostatin activity by binding to either of amino acids Pro76 or Met79 of myostatin.

The composition may be selected from the group consisting of a pharmaceutical composition and health food.

Myostatin (MSTN) of the present invention is one of transforming growth factor-β (TGF-β) superfamily as an inhibitor of autocrine / paracrine which is very important for muscle growth It's working. Reportedly, the absence of myostatin expression in mice significantly increased the mass of skeletal muscle, resulting in muscular dystrophy and hyperplasia.

Recent reports have shown that the action of myostatin binds directly to activin receptor type IIB (ACVRIIB) and induces muscle loss.

The activin receptor type 2B is a protein encoded by the ACVR2B gene and is involved in the activin signaling mechanism. Signal transduction by activin is known to be involved in the production, secretion of follicle stimulating hormone (FSH), regulation of the menstrual cycle, and in response to proliferation, differentiation and apoptosis of cells. Works.

The matrix gla protein (MGP) of the present invention is another protein of the extracellular matrix (ECM), which is vitamin K2-dependent and includes a gla (γ-carboxyglutamate) domain. It has high affinity binding, serves as an inhibitor of blood vessel mineralization and plays an important role in bone tissue.

According to one embodiment of the present invention, the matrix gla protein was found to inhibit MSTN and ACVRIIB binding. A protein-protein correlation study showed that the overall energy score of MSTN and ACVRIIB binding was -56.99. As shown in 5d, the binding efficiency was reduced to -25.08 in the presence of MGP, and the amino acid residues included in MSTN and ACVRIIB binding were significantly reduced in the presence of MGP as shown in FIGS. 5E and 2.

From the above results, it was confirmed that MGP binds to MSTN and thereby inhibits MSTN and ACVRIIB binding to regulate MSTN activity.

According to another embodiment of the present invention, in order to confirm the effect of MSTN expression regulation by MGP, as a result of confirming the mRNA and protein levels of MSTN in MGP reduced cells (MGPkd), MGP reduced cells as shown in Figure 4d The decrease in MSTN expression was confirmed, and as a result of immunostaining using the MSTN antibody, MSTN expression was reduced in cells with reduced MGP as shown in FIG. 4E.

Accordingly, the present invention can provide a pharmaceutical composition for preventing or treating muscle diseases containing the matrix gla protein consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

More specifically, the pharmaceutical composition for preventing or treating muscle diseases, the matrix gla protein is Arg49, Pro46, Gln48, Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1 in the amino acid sequence represented by SEQ ID NO: 1 And at least one amino acid selected from the group consisting of Phe43 is Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, in the amino acid sequence of myostatin represented by SEQ ID NO: 2. Myostatin is inhibited by binding to one or more amino acids selected from the group consisting of Gly71, Cys73, Pro76, Thr77, Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 to inhibit the binding of myostatin and its receptor ACVRIIB. It can suppress induced muscle loss.

More preferably, the matrix gla protein amino acid Arg49 binds to the amino acid Leu20 of myostatin, the matrix gla protein amino acid Arg38 binds to any one of the amino acids Tyr55, Pro76, Thr77, Lys78 or Met79 of myostatin, and the matrix gla Protein amino acid Arg37 binds to either amino acid Met79 or Ala100 of myostatin, the matrix gla protein amino acid Gln48 binds to amino acid Phe27 of myostatin, and the matrix gla protein amino acid Gln24 is either amino acid Arg67 or Gly68 of myostatin The matrix gla protein amino acid Leu4 binds to any one of amino acids Trp29 or Trp31 of myostatin, and the matrix gla protein amino acid Met26 binds to any of amino acids Gly68, Ser69, Ala70, Gly71 or Cys73 of myostatin Combine, The matrix gla protein amino acid Met1 binds to any one of amino acids Tyr86, Asn88 or Gly89 of myostatin, and the matrix gla protein amino acid Phe43 binds to any of amino acids Ala100 or Met101 of myostatin, and the matrix gla protein amino acid Pro46 Is bound to any one of amino acids Val22 or Phe27 of myostatin, the matrix gla protein amino acid Ser45 binds to any of amino acids Ala40, Asn41 or Tyr42 of myostatin, and the matrix gla protein amino acid Ser25 is amino acid Ala70 of myostatin And the matrix gla protein amino acid Thr42 may bind to any one of amino acids Pro76 or Met79 of myostatin.

In addition, the matrix gla protein may promote the differentiation of muscle satellite cells by increasing the expression of the muscle differentiation genes MYOD, MYOG, Col1α1 and FMOD.

The muscular diseases include muscle dystrophy, rigid spine syndrome, muscle-eye-brain disease, amyotrophic lateral sclerosis, and Charco-Marie- It may be selected from the group consisting of Charcot-Marie-Tooth disease, chronic inflammatory neuropathy and distal myopathy.

The present invention comprises the steps of treating a candidate with a cell; And Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, in the myostatin amino acid sequence represented by SEQ ID NO: 2 in cells treated with the candidates Pro76, Thr77, Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 to provide a myostatin inhibitor screening method comprising the step of identifying the binding level of any one or more amino acids and candidates selected from the group consisting of. Can be.

The screening method may further comprise the step of identifying the reduced level of binding of myostatin and its receptor activin receptor type IIB in combination with the myostatin candidate.

The binding level between the matrix gla protein and the myostatin receptor is reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), immunoprecipitation, immunocytochemistry , Western blotting and flow cytometry (FACS) can be identified by any one selected from the group consisting of.

The myostatin activity inhibitor may be selected from the group consisting of a therapeutic agent for muscle disease, a meat enhancer and an anabolic agent for livestock.

The present invention can provide a pharmaceutical composition for preventing or treating muscle diseases, which contains a myostatin activity inhibitor selected by the screening method as an active ingredient.

In addition, the present invention comprises the steps of treating the candidate material to cells isolated from mammals; And Arg49, Pro46, Gln48, Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1, and Phe43 in the matrix gla protein amino acid sequence represented by SEQ ID NO: 1 in cells treated with the candidates. Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, Pro76, Thr77 in the myostatin amino acid sequence represented by SEQ. , Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 may provide a matrix gla protein and myostatin binding promoter screening method comprising the step of confirming the increase in the binding level of any one or more amino acids selected from the group consisting of have.

In addition, the present invention contains a matrix gla protein (MGP) consisting of an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient, the matrix gla protein is Arg49, Pro46, in the amino acid sequence represented by SEQ ID NO: 1; Any one or more amino acids selected from the group consisting of Gln48, Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1 and Phe43 are represented in SEQ ID NO: 2 by Leu20, Val22, Phe27, Trp29 At least one selected from the group consisting of, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, Pro76, Thr77, Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 It is possible to provide a reagent composition for inhibiting myostatin activity by binding to amino acids to inhibit myostatin activity.

In one embodiment of the invention, the pharmaceutical composition is any one selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or solutions according to conventional methods Formulations of

In another embodiment of the invention, suitable carriers, excipients, disintegrants, sweeteners, coatings, swelling agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatics, diluents, conventionally used in the manufacture of the pharmaceutical compositions, It may further comprise one or more additives selected from the group consisting of dispersants, surfactants, binders and lubricants.

Specifically, the carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules. And the like, and such solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the composition. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used. Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. Witsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used as the base material of the suppository.

According to one embodiment of the invention the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, nasal, inhaled, topical, rectal, oral, intraocular or intradermal Via the route can be administered to the subject in a conventional manner.

The preferred dosage of the matrix gla protein may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg. Administration may be administered once a day or divided into several times, thereby not limiting the scope of the invention.

In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.

In addition, the health food is used with other food or food additives in addition to the matrix gla protein (MGP), and may be appropriately used according to conventional methods. The mixed amount of the active ingredient can be suitably determined depending on the purpose of use thereof, for example, prophylactic, health or therapeutic treatment.

The effective dose of the compound contained in the health food may be used in accordance with the effective dose of the therapeutic agent, but may be less than the above range in the case of long-term intake for health and hygiene purposes or health control purposes It is evident that the component can be used in an amount above the range because there is no problem in terms of safety.

There is no particular limitation on the kind of the health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, Drinks, alcoholic drinks, vitamin complexes, etc. are mentioned.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Experimental Example 1 Cell Culture

C2C12 cells from mouse myoblasts were purchased from Korean Cell Line Bank (KCLB), 10% fetal bovine serum (FBS; HyClone Laboratories) and 1% penicillin / streptomycin (P / S) (Invitrogen, Carlsbad, Incubated in DMEM (Dulbecco's modified Eagle's medium; HyClone Laboratories, Logan, UT) medium containing 37%, 5% CO ₂ conditions.

To induce differentiation, C2C12 cells were grown to 70% confluence, replaced with DMEM differentiation medium containing 2% fetal bovine serum and 1% penicillin / streptomycin and grown for 1 to 5 days, and the differentiation medium was replaced every other day. .

Experimental Example 2 RNA Extraction and Real-Time RT-PCR Analysis

Trizol ™ reagent (Invitrogen) according to the manufacturer's instructions using a, and stored in C2C12 cells Total RNA was extracted diethyl fatigue from -80 ℃ condition until use carbonate (diethylpyrocarbonate) in treated H ₂ O a.

Oligo (dT) 20 primer (Bioneer, Daejeon, Korea) was prepared from 20 μl of the reaction mixture containing total RNA (1 μg), and reverse transcription was performed at 42 ° C. for 50 minutes and 72 ° C. for 15 minutes. PCR was performed using a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) using the cDNA (2 μl) and each gene specific primer (10 pmoles).

Power SYBR® Green PCR Master Mix (Applied Bio systems) was used as a fluorescence source, and Primer 3 software (http://frodo.wi.mit.edu) and the US National Biotechnology Information Center's base information list were used. Primer was prepared.

Gene expression analysis was performed and fold differences were confirmed using GAPDH as a control, and the primer sequences used are shown in Table 1 below.

Species Gene Product size (bp) Tm (℃) Sequence direction Mouse GAPDH 155 55 5'-tgctggtgctgagtatgtcg-3 ' Forward direction 5'-caagcagttggtggtacagg-3 ' Reverse FMOD 155 59 5'-tgcagaagatccctcctgtc-3 ' Forward direction 5'-cttgatctcgttcccatcca-3 ' Reverse MSTN 163 59 5'-acgctaccacggaaacaatc-3 ' Forward direction 5'-ggagtcttgacgggtctgag-3 ' Reverse MYOG 185 59 5'-tccagtacattgagcgccta-3 ' Forward direction 5'-caaatgatctcctgggttgg-3 ' Reverse MYOD 213 59 5'-aggagcacgcacacttctct-3 ' Forward direction 5'-tctcgaaggcctcattcact-3 ' Reverse MYL2 177 59 5'-aaagaggctccaggtccaat-3 ' Forward direction 5'-cctctctgcttgtgtggtca-3 ' Reverse COL1α1 244 59 5'-ctttgcttcccagatgtcct-3 ' Forward direction 5'-ccccatcatctccattcttg-3 ' Reverse MGP 174 59 5'-acaggagaaatgccaaca-3 ' Forward direction 5'-ggttgtaggcagcgttgt-3 ' Reverse

Experimental Example 3 Immunocytochemistry

C2C12 cells were incubated for 2 or 4 days in differentiation medium in cover glass-bottom dishes and stained with MGP protein.

First, cells were washed with PBS, fixed with 4% formaldehyde, permeated with 0.2% Triton X-100 (Sigma Aldrich) and incubated with Image-iTTM FX signal amplifier.

Thereafter, the cells were blocked with PBS containing 5% goat serum for 1 hour and then incubated with MGP antibody (rabbit polyclonal IgG MGP, 1:50; Proteintech) overnight at 4 ° C. wet conditions.

It was then washed three times with PBS and a secondary antibody (1: 100; Alexa Fluor 488 goat anti-rabbit, Molecular Probes, Eugene, OR, USA) was applied for 1 hour at room temperature.

Samples were washed with PBS and counterstained with nuclei using 4'6'-diamino-2-phenylindole (DAPI; Sigma-Aldrich) and photographed with a fluorescence microscope (Nikon) equipped with a digital camera.

Experimental Example 4 Western Blot Analysis

Western blots were performed with protein samples of cells differentiated at different times.

The cells were washed with ice-cold PBS and lysed with RIPA buffer containing protease inhibitor cocktail (Thermo Scientific, NH, USA), followed by centrifugation at 4 ° C and 12,000 rpm for 10 minutes to separate whole protein and supernatant. Were collected and analyzed for protein by Bradford method.

40 μg of a dye containing β-mercaptoethanol (Sigma-Aldrich) and a protein were mixed and heated at 95 ° C. for 5 minutes, followed by electrophoresis on 15% SDS-PAGE, and a PVDF membrane (Immobilon®-p, Millipore, Billerica MA). , USA).

Blots were blocked for 1 hour with 3% BSA and incubated with primary antibody at 4 ° C. overnight. The membrane was then washed with TBST and the peroxidase labeled secondary antibody (Santa Cruz) was treated for 1 hour at room temperature. After washing with TBST again, Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific) was treated and exposed to a luminescent image analyzer (LAS-4000 mini; Fujifilm, Tokyo) to detect immune response proteins.

Experimental Example 5 Gene Expression Reduction and Fusion Index Confirmation

According to each manufacturer's instructions, mixed vectors or shRNAs for specific genes (shMGP, shFMOD, shMSTN) were transfected into C2C12 cells.

C2C12 cells grown to 30% confluence were transfected overnight with transfection reagent (Santa Cruz Biotechnology, Calif., USA) treated with 1 ng of gene specific shRNA and mixed vector (control), respectively, and the cells were 80% confluent. Transfected cells were treated with 2 μg / ml puromycin (Sigma Life Sciences).

To induce expression reduction with siRNA, 100 mM control or Col1α1 siRNA were transfected into C2C12 cells for 5 hours and then cultured in differentiation medium for 2 days.

To confirm the efficiency of expression reduction, RT-PCR and Western blots were performed to confirm mRNA and protein expression levels.

The percent difference in Col1α1 gene expression between wild-type and reduced expression cells was used to confirm the transfection efficiency of shRNA / siRNA knock down constructs.

In addition, the melting index was confirmed in MGPkd and MGPwt cells by the reported method (Brigitle et al., 1998 and Velic et al., 2012).

First, stain the cell nuclei with Giemsa G250 (Sigma Aldrich) and randomly obtain images of three different sites, count the number of nuclei in the root canal cells and the total number of nuclei in each image in each image, Fusion index was calculated by expressing the number of nuclei contained in the root canal cells as a percentage.

Experimental Example 6 Co-Immunoprecipitation

To confirm protein-protein interactions between MGP, FMOD and MSTN, Co-Immunoprecipitation was performed.

First, samples were obtained from normal C2C12 cells differentiated with 2% fetal bovine serum (FBS), C2C12 cells transfected with MGP shRNA, and C2C12 cells transfected with a complex vector.

Briefly in cells washed with PBS were denatured buffer (20 mM Tris HCl, pH 8), 137 mM NaCl, 1% Nonidet P-40, 2 mM EDTA and HaltTM protease inhibitor (Thermo Scientific) 100: It was added in 1 ratio.

Cells were collected with a cell scraper and incubated for 30 minutes with gentle shaking every 10 minutes on ice, followed by centrifugation at 12,000 rpm for 10 minutes at 4 ° C.

300 μg of total protein and 20 μl of Protein A agarose (Santa Cruz Biotechnology) were incubated and then incubated at 4 ° C. at 900 rcf for 3 to 4 hours.

After removing the supernatant, the pellet was mixed with FMOD or MSTN antibody (Santa Cruz), incubated overnight, and centrifuged at 12,000 rpm for 10 minutes at 4 ° C. to remove the supernatant.

200 μl of undenatured buffer was added to the sample obtained by the above procedure, washed twice by centrifugation at 12,000 rpm for 5 minutes, the supernatant was removed, and western blot of MGP protein was performed.

Experimental Example 7 Protein Structure Prediction and Interaction Study

The crystal structure of MSTN (pdb id: 3HH2) was retrieved from Protein Data Bank (PDB) and modeled with Modeller 9v14 using the crystal structure of human activin receptor type II kinase domain (pdb id: 2QLU) as a template. Five ACVRIIB models were made and the best model was selected based on the dope score.

On the other hand, since the MGP structure of the protein data bank was not available, ab initio was obtained using the amino acid sequence retrieved from the UniProt database (P19788). The structure of MGP was fabricated with I-TASSER using a combination of folding and mowing methods.

After that, the binding between MSTN and ACVRIIB, its receptor, was examined, and it was confirmed whether MGP could block the interaction of MSTN and ACVRIIB on in silico .

Protein-protein interactions between ACVRIIB and MSTN were confirmed using the PatchDock server (http://bioinfo3d.cs.tau.ac.il/PatchDock/) in the presence or absence of MGP.

The results obtained from PatchDock were further purified and recorded based on protein-protein interaction energies using the Fire Dock algorithm.

Composite structures with low interaction energies were selected as the final model for analysis.

Example 1 Confirmation of Increased MGP Expression During Muscle Differentiation

Expression patterns of MGP according to muscle differentiation induction for 0 to 5 days were confirmed.

As a result, as shown in FIG. 1A, MGP mRNA expression was confirmed to be 8-fold higher on the second day of muscle differentiation induction, and similar results were observed in the Western blot pattern of FIG. 1B. In addition, referring to Figure 2 MGP protein expression was confirmed in the cytoplasm of the myotubes.

To confirm the role of MGP in muscle differentiation, MGP shRNAs were transfected into C2C12 cells and RT-PCR and Western blot analysis was performed.

As a result, MGP expression was reduced by 60% in the transfected cells as shown in Figs. 1c and 1d, and it was confirmed that myotube formation was reduced as shown in Figs. 1e and 1f by decreasing MGP expression. As a result of confirming the effect of reducing cell formation, as shown in FIG. 1G, more myotubes were found in normal cells (MGPwt) than in cells with reduced MGP (MGPkd).

From the above results, it was confirmed that MGP plays a role in regulating myotube cell formation during differentiation period.

Example 2 Confirmation of MGP Effect on Extracellular Matrix and Myogenic Marker Gene Expression

In order to confirm the role of MGP in the development of muscle tissue, the effects of myogenic markers (MYOD and MYOG) and Col1α1 gene expression according to MGP expression reduction were examined.

MYOD and MYOG are known to regulate the progression of muscle stem cell differentiation in the course of muscle tissue development. As a result of confirming the relationship with MGP, mRNA and protein of MYOD and MYOG in MGP reduced cells (MGPkd) as shown in FIGS. 3A and 3B It was confirmed that all levels were significantly reduced.

In addition, mRNA and protein expression levels of Col1α1, an important extracellular matrix gene, were also reduced, as shown in FIG. Indicated. On the other hand, Western blot analysis showed high expression of FMOD protein in cells with reduced MGP expression.

From these results, MGP was found to regulate the expression of genes that induce muscle differentiation.

On the other hand, as it is known that FMOD plays an important role in the regulation of MSTN during the muscle tissue development process, MGP expression pattern was confirmed in C2C12 cells that inhibited the expression of Col1α1 and FMOD.

Example 3 Confirmation of ECM Gene Knockdown Effect on MGP Expression

As the cross-linking mechanism between MGP and ECM genes during muscle tissue development is considered to be likely to indicate the effect of the gene expression, the role of MGP in regulating ECM gene expression was identified.

To confirm the potential for interaction, C2C12 cells were transfected with shRNA / siRNA (shFMOD, siCol1α, or shMSTN).

As a result, MGP expression was increased in FMODkd, Col1α1kd and MSTNkd cells as compared to the wild type as shown in FIGS. 4A to 4C.

From the above results, it was confirmed that FMOD, Col1α1, and MSTN interacted in the same way, and to understand the mechanism regulating muscle tissue development process, MSTN expression was confirmed in cells with reduced MGP.

Example 4 Confirmation of the Effect of MGP on MSTN Expression During Muscle Differentiation

To understand the regulation of MSTN expression by MGP and its correlation with FMOD, MSTN expression was confirmed in cells with reduced MGP.

For comparison with MGP wild type cells, RT-PCR and Western blot analysis were performed to confirm mRNA and protein levels of MSTN in MGP reduced cells (MGPkd).

As a result, as shown in FIG. 3d, it was confirmed that MSTN expression was decreased in cells in which MGP was reduced.

From the above results, it was confirmed that MGP is a gene regulating MSTN expression. As a result of performing immunostaining using MSTN antibody for further confirmation of the result, MSTN expression was decreased in cells with reduced MGP as shown in FIG. 4E. I could confirm that.

Example 5 Confirmation of Interaction of MGP

In recent studies, the present inventors confirmed that FMST interacts with MSTN and thus, MSTN expression is regulated by preventing binding of MSTN and ACVRIIB, a receptor. Accordingly, in order to confirm the correlation between MGP, FMOD, and MSTN, Western blot was performed using MGP antibody on a sample of FMOD or MSTN precipitated after complex immunoprecipitation.

As a result, a strong interaction between MGP and FMOD and MGP and MSTN protein was confirmed as shown in FIG. After confirming the interaction between the MGP, FMOD and MSTN protein in the normal cells as described above, complex immunoprecipitation was performed even in normal cells (MGPwt) and MGP reduced cells (MGPkd).

As a result, as shown in FIG. 5B, weak interactions were observed in cells with reduced MGP.

In silico analysis was also performed to confirm the interaction between MGP, FMOD and MSTN. The MGP structure of the protein information bank (PDB) has no applicability, and thus an MGP structure was produced as shown in FIG. 5C.

The protein-protein correlation study confirmed that the overall energy score of MSTN and ACVRIIB binding was -56.99. However, in the presence of MGP as shown in FIG. 5D, the binding efficiency was reduced to -25.08.

From these results, it was confirmed that MGP inhibits the binding of MSTN and ACVRIIB.

In addition, as shown in Figure 5e and Table 2 it can be seen that the amino acid residues included in the MSTN and ACVRIIB bond in the presence of MGP is greatly reduced.

From the above results, it was confirmed that MGP can disrupt MSTN and ACVRIIB binding.

Complex Residues involved MGP-MSTN MGP-MSTN Hydrogen bonding Hydrophobic interaction MGP MSTN MGP MSTN NIL R49, P46, Q48, L4, S45, E24, M26, S25, R38, T42, R37, M1F43 L20, V22, P27, W29, W31, A40, N41, Y42, Y55, R67, G68, S69, A70, G71, C73, P76, T77, K78, M79, Y86, N88, G89, P100, A100, M101

Complex Residues involved ACVRIIB - MSTN -MGP ACVRIIB-MSTN Hydrogen bonding Hydrophobic interaction ACVRIIB MSTN ACVRIIB MSTN NIL P167, G168, P170 Y55, H57 ACVRIIB-MGP Hydrogen bonding Hydrophobic interaction ACVRIIB MGP ACVRIIB MGP G191, N194, D283, E285 Y29, V85, R81, Y86 P167, G168, E190, G191, A192, I193, N194, F195, Q196, R202, D283, E285 P6, L7, L10, Y29, I31, P33, F34, R81, Y82, Y86, R94

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (17)

Composition for inhibiting myostatin activity containing a matrix gla protein (MGP) consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient. The composition for inhibiting myostatin activity according to claim 1, wherein the matrix gla protein inhibits the binding of myostatin and its receptor, activin receptor type IIB. The method according to claim 1, wherein the matrix gla protein is any one selected from the group consisting of Arg49, Pro46, Gln48, Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1 and Phe43 in the amino acid sequence represented by SEQ ID NO: Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, Pro76, Thr77, in the amino acid sequence of myostatin represented by SEQ ID NO: 2 Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 bind to any one or more amino acids selected from the group consisting of inhibiting myostatin activity, characterized in that the composition for inhibiting myostatin activity. The method of claim 3, wherein the matrix gla protein amino acid Arg49 binds to amino acid Leu20 of myostatin, and the matrix gla protein amino acid Arg38 binds to any one of amino acids Tyr55, Pro76, Thr77, Lys78 or Met79 of myostatin, Matrix gla protein amino acid Arg37 binds to either amino acid Met79 or Ala100 of myostatin, the matrix gla protein amino acid Gln48 binds to amino acid Phe27 of myostatin, and the matrix gla protein amino acid Gln24 is amino acid Arg67 or Gly68 of myostatin The matrix gla protein amino acid Leu4 binds to any one of amino acids Trp29 or Trp31 of myostatin, and the matrix gla protein amino acid Met26 is any of amino acids Gly68, Ser69, Ala70, Gly71 or Cys73 of myostatin Combined with one The matrix gla protein amino acid Met1 binds to any one of amino acids Tyr86, Asn88 or Gly89 of myostatin, and the matrix gla protein amino acid Phe43 binds to any of amino acids Ala100 or Met101 of myostatin, and the matrix gla protein amino acid Pro46 binds to any of amino acids Val22 or Phe27 of myostatin, the matrix gla protein amino acid Ser45 binds to any of amino acids Ala40, Asn41 or Tyr42 of myostatin, and the matrix gla protein amino acid Ser25 is an amino acid of myostatin Ala70 and the matrix gla protein amino acid Thr42 is a composition for inhibiting myostatin activity, characterized in that binding to any one of amino acids Pro76 or Met79 of myostatin. The composition for inhibiting myostatin activity according to claim 1, wherein the composition is selected from the group consisting of pharmaceutical compositions and health foods. A pharmaceutical composition for preventing or treating muscle diseases comprising the matrix gla protein consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient. The method according to claim 6, wherein the matrix gla protein is any selected from the group consisting of Arg49, Pro46, Gln48, Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1 and Phe43 in the amino acid sequence represented by SEQ ID NO: Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, Pro76, Thr77, in the amino acid sequence of myostatin represented by SEQ ID NO: 2 Inhibiting myostatin-induced muscle loss by inhibiting the binding of myostatin and its receptor ACVRIIB by binding to any one or more amino acids selected from the group consisting of Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 Pharmaceutical composition for preventing or treating muscle diseases, characterized in that. The method of claim 7, wherein the matrix gla protein amino acid Arg49 binds to amino acid Leu20 of myostatin, and the matrix gla protein amino acid Arg38 binds to any one of amino acids Tyr55, Pro76, Thr77, Lys78 or Met79 of myostatin, Matrix gla protein amino acid Arg37 binds to either amino acid Met79 or Ala100 of myostatin, the matrix gla protein amino acid Gln48 binds to amino acid Phe27 of myostatin, and the matrix gla protein amino acid Gln24 is amino acid Arg67 or Gly68 of myostatin The matrix gla protein amino acid Leu4 binds to any one of amino acids Trp29 or Trp31 of myostatin, and the matrix gla protein amino acid Met26 is any of amino acids Gly68, Ser69, Ala70, Gly71 or Cys73 of myostatin Combined with one The matrix gla protein amino acid Met1 binds to any one of amino acids Tyr86, Asn88 or Gly89 of myostatin, and the matrix gla protein amino acid Phe43 binds to any of amino acids Ala100 or Met101 of myostatin, and the matrix gla protein amino acid Pro46 binds to any of amino acids Val22 or Phe27 of myostatin, the matrix gla protein amino acid Ser45 binds to any of amino acids Ala40, Asn41 or Tyr42 of myostatin, and the matrix gla protein amino acid Ser25 is an amino acid of myostatin Ala70 and the matrix gla protein amino acid Thr42 is combined with any one of amino acids Pro76 or Met79 of myostatin pharmaceutical composition for preventing or treating muscle diseases. The pharmaceutical composition for preventing or treating muscle diseases according to claim 6, wherein the matrix gla protein promotes differentiation of muscle satellite cells by increasing expression of the muscle differentiation genes MYOD, MYOG, Col1α1 and FMOD. The method of claim 6, wherein the muscle disease is muscular dystrophy, rigid spine syndrome, muscle-eye-brain disease, amyotrophic lateral sclerosis , Charcot-Marie-Tooth disease (Charcot-Marie-Tooth disease), chronic inflammatory neuropathy (chronic inflammatory neuropathy) and terminal myopathy (distal myopathy) pharmaceutical composition for preventing or treating muscle diseases, characterized in that selected from the group. Treating the candidate with cells; And Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, Pro76 in the myostatin amino acid sequence represented by SEQ ID NO: 2 in cells treated with the candidates , Thr77, Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 Myostatin inhibitor screening method comprising the step of identifying the binding level of any one or more amino acids selected from the group consisting of. The method according to claim 11, wherein the screening method further comprises the step of identifying the reduced level of binding of the myostatin and activin receptor type IIB (myostatin and its receptor) in combination with the myostatin. Myostatin activity inhibitor screening method. The method according to claim 11, wherein the binding level between the myostatin amino acid and the candidate substance is Reverse Transcription-Polymerase chain Reaction (RT-PCR), enzyme immunoassay (ELISA), immunoprecipitation, immune cells A method for screening a myostatin activity inhibitor, characterized in that it is identified by any one selected from the group consisting of chemistry (Immunocytochemistry), Western blotting and flow cytometry (FACS). The method of claim 11, wherein the myostatin activity inhibitor is selected from the group consisting of a therapeutic agent for muscle disease, a meat enhancer and an anabolic agent of livestock. A pharmaceutical composition for preventing or treating muscle diseases, comprising a myostatin activity inhibitor selected by the screening method of any one of claims 11 to 13 as an active ingredient. Treating the candidate with cells isolated from the mammal; And The candidate material is selected from the group consisting of Arg49, Pro46, Gln48, Leu4, Ser45, Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1 and Phe43 in the matrix gla protein amino acid sequence represented by SEQ ID NO: 1 in cells treated with the candidates Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, Pro76, Thr77, in any one or more amino acids and the myostatin amino acid sequence represented by SEQ ID NO: 2 A method for screening a matrix gla protein and a myostatin binding promoter comprising confirming an increase in binding level of any one or more amino acids selected from the group consisting of Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101. Matrix gla protein (MGP) consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient, the matrix gla protein is Arg49, Pro46, Gln48, Leu4, Ser45 in the amino acid sequence represented by SEQ ID NO: 1 Any one or more amino acids selected from the group consisting of Glu24, Met26, Ser25, Arg38, Thr42, Arg37, Met1, and Phe43 are Leu20, Val22, Phe27, Trp29, Trp31, Ala40, Myo in combination with any one or more amino acids selected from the group consisting of Asn41, Tyr42, Tyr55, Arg67, Gly68, Ser69, Ala70, Gly71, Cys73, Pro76, Thr77, Lys78, Met79, Tyr86, Asn88, Gly89, Pro100, Ala100 and Met101 A reagent composition for inhibiting myostatin activity that inhibits statin activity.

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