KR101955885B1 - A method for extending half-life of PDGFA - Google Patents

Abstract

The present invention relates to a method for increasing the half-life of PDGFA or a PDGFA having an increased half-life, including substituting at least one lysine residue present in the amino acid sequence of PDGFA, wherein the PDGFA substituted with the lysine residue of the present invention It remains for a long time and has excellent therapeutic effect.

Description

A method for increasing PDGFA half-life time

The present invention relates to a method for increasing the half-life of a protein or (poly) peptide by substituting at least one amino acid residue of the protein or (poly) peptide. Further, the present invention relates to a protein or (poly) peptide having an increased half-life produced by such a method.

Intracellular proteolysis occurs through two pathways: lysosomes and proteasomes. The lysosomal pathway, which degrades 10-20% of the protein, lacks substrate specificity and elaborate temporal regulation. In other words, it is a process of decomposing mostly extracellular or membrane proteins as the cell surface proteins incorporated into cells by endocytosis are degraded in lysosomes. However, in order for proteins to be selectively degraded in eukaryotic cells, a process in which ubiquitin is bound to a target protein by a ubiquitin-binding enzyme to form a polyubiquitin chain, which is recognized and degraded by proteasome, that is, ubiquitin- The ubiquitin-proteasome pathway (UPP). More than 80% to 90% of eukaryotic proteins are degraded through this process, and the ubiquitin-proteasome pathway regulates protein degradation and protein homeostasis by regulating protein degradation in eukaryotic cells.

Ubiquitin is a highly conserved 76 amino acid protein that is present in almost all eukaryotic cells, of which 6, 11, 27, 29, 33, 48 and 63 amino acid residues are lysine (Lysine, Lys, K) 48 and 63 play a major role in the formation of the poly ubiquitin chain. The ubiquitination of ubiquitin involves a series of enzymes (E1, E2, E3), which are degraded by the ATP-dependent protease complex 26S proteasome. The ubiquitin-proteasome pathway involves two separate, sequential processes, the first of which is the process of labeling multiple ubiquitin molecules with a covalent bond to the substrate, and the second is that the protein labeled by ubiquitin is a 26S pro It is a process that is decomposed by a teasome complex. The binding of ubiquitin and substrate takes place via an isopeptide bond between the lysine residue of the substrate molecule and the glycine at the C-terminal of ubiquitin, and the ubiquitin-activating enzyme E1, the ubiquitin-binding enzyme E2 and the ubiquitin ligase E3 The formation of thiol esters between ubiquitin and enzymes. E1 (ubiquitin-activating enzyme) activates ubiquitin by ATP-dependent reaction. E2 (ubiquitin-conjugating enzyme) takes ubiquitin activated from E1 to cysteine residue in ubiquitin-conjugated domain and transfers it to E3 ligase or directly to substrate protein. The E3 enzyme also catalyzes stable isopeptide binding between the lysine residue of the substrate protein and the glycine residue of ubiquitin. Another ubiquitin may be linked to the C-terminal lysine residue of the ubiquitin bound to the substrate protein. When this process is repeated and several ubiquitin molecules are ligated to the substrate protein in a branched form to form a polyubiquitin chain, 26S proteasome and is selectively degraded.

On the other hand, various kinds of proteins and (poly) peptides having a therapeutic effect in vivo are known. Such a protein or (poly) peptide having a therapeutic effect in vivo can be used as a therapeutic agent for the treatment of diseases such as growth hormone releasing hormone (GHRH), growth hormone releasing peptide, interferons, interferon-α or interferon-β, interferon receptors, colony stimulating factors (CSFs), glucagon-like peptides, interleukins, interleukin receptors, Human interleukin binding proteins, cytokine binding proteins, G-protein-coupled receptors, human growth hormone (hGH), and the like. Macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A (prot ein A), allergy inhibitor, cell necrosis glycoproteins, G-protein-coupled receptor, immunotoxin, lymphotoxin, tumor necrosis Tumor necrosis factor, tumor suppressors, metastasis growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin, Apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietins, hemoglobin, thrombin, and the like. Thrombin receptor activating peptide, thrombomodulin, factor VII, factor VIIa, factor VIII, factor IX, factor IX, Factor XIII, A plasminogen activating factor, a urokinase, a streptokinase, a hirudin, a protein C, a C-reactive protein, a renin inhibitor renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor, , Epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone stimulating protein, calcitonin, insulin, art Atriopeptin, a cartilage inducing factor, a fibrin-binding peptide, elcatonin, a connective tissue activating factor, A tissue factor pathway inhibitor, a follicle stimulating hormone, a luteinizing hormone, a luteinizing hormone releasing hormone, a nerve growth factor, a parathyroid hormone parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecytokinin, cholesterol, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin lactoferrin, myostatin, receptors, receptor antagonists, cell surface antigens, ns, virus derived vaccine antigens, monoclonal antibodies, polyclonal antibodies, and antibody fragments.

Platelet-derived growth factor (PDGF) is one of the growth factors that regulates cell growth and division and is involved in angiogenesis and plays a role in platelet-derived growth factor subunit A (PDGFA ) And platelet derived growth factor subunit B (PDGFB) functions (Biochim. Biophys. Acta., 989 (1): 110, 1989; EMBO J., 11 (12) : 42514259, 1992). PDGF is synthesized, stored in alpha granules of platelets, secreted when platelets become active, and is also produced in cells such as muscle cells, active macrophages and epidermal cells (Curr Pharm Des., 19 (19) : 3384-3390, 2013).

The present invention aims to provide a method for increasing the half-life of a protein.

The present invention also aims to provide a protein having one or more lysine residues substituted in the amino acid sequence, wherein the protein has an increased half-life.

It is also an object of the present invention to provide a pharmaceutical composition comprising a protein having an increased half-life.

In order to achieve the above object, the present invention provides a method for increasing the half-life of a protein, including replacing one or more lysine residues present in the amino acid sequence of the protein.

In the present invention, lysine residues of proteins may be substituted with conservative amino acids. In the present invention, "conservative amino acid substitution" means that an amino acid residue is substituted by another amino acid residue having a similar side chain, e.g., a charge or hydrophobic chemical character. In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. Examples of amino acid groups having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chain: serine and threonine; 3) Amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) Basic side chains: lysine, arginine and histidine; 6) Acid side chains: aspartate and glutamate 7) Sulfur-containing side chains: include cysteine and methionine.

In the present invention, the lysine residue of the protein may be substituted with arginine or histidine containing a basic side chain, preferably arginine residue.

According to the present invention, a protein in which at least one lysine residue present in the amino acid sequence of a protein is substituted with arginine may have an increased half-life and may remain in the body for a long time.

Figure 1 shows the structure of a PDGFA expression vector.
Fig. 2 shows the PDGFA gene size and the PCR result.
Figure 3 shows the expression of the protein through plasmid of PDGFA in HEK-293T cells.
Figure 4 shows the degradation pathway of PDGFA through ubiquitination assay.
Figure 5 shows the degree of ubiquitination of a PDGFA substituent in which the lysine residue was replaced with arginine as compared to the wild type.
Figure 6 shows the half-life change of PDGFA after treatment with cycloheximide (CHX), a protein synthesis inhibitor.
Figure 7 shows the results for effects such as JAK-STAT, PI3K-AKT and MAPK / ERK signal induction.

In one embodiment of the invention, the protein is PDGFA. At least one of the 160, 165, and 206 lysine residues from the N-terminus in the amino acid sequence of PDGFA represented by SEQ ID NO: 1 is substituted with an arginine residue. Accordingly, there is provided a platelet-derived growth factor with increased half-life and a pharmaceutical and / or cosmetic composition for restoring cellular growth, angiogenesis, chronic ulcer and bone loss.

In the present invention, site-directed mutagenesis was used to replace the lysine residue present in the amino acid sequence of the protein with the arginine (R) residue. In this method, a primer is prepared using a DNA sequence that induces a specific mutation, and then a PCR is carried out under specific conditions to prepare a plasmid DNA in which a specific amino acid residue is substituted.

In the present invention, the target protein was transfected into the cell line by immunoprecipitation analysis and precipitated to confirm the degree of ubiquitination. As a result of treatment with the MG132 (proteasome inhibitor) reagent, the degree of ubiquitination was increased, - proteasome degradation pathway.

In the present invention, the pharmaceutical composition may be delivered into the body by various routes including oral, transcutaneous, subcutaneous, intravenous or intramuscular administration and may be administered as a syringe formulation . In addition, the pharmaceutical composition of the present invention can be formulated according to methods well known to those skilled in the art, such as rapid release, delayed release or slow release after administration according to the method. The formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, Aerosol, soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders, and the like. Suitable carriers, excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, mannitol, xylitol, erythritol, maltitol, carbohydrates, Gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate, and mineral oil. . In addition, the formulations can additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives, and the like. have.

In the present invention, the singular forms include plural forms unless expressly stated otherwise. Furthermore, terms such as "comprising" in the present invention are interpreted to have a similar meaning to "including". In the present invention, " physiologically active (poly) peptide or protein " means a (poly) peptide or protein which exhibits biological activity useful when administered to a mammal including a human.

Hereinafter, the present invention will be described in more detail based on examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: Analysis of ubiquitination of PDGFA protein and confirmation of half-life increase and confirmation of intracellular signaling

1. Cloning into expression vector and confirmation of protein expression

(1) Expression vector cloning

The PDGFA DNA amplification product by polymerase chain reaction and pcDNA3-myc (5.6 kb) were ligated with restriction enzymes BamHI and XhoI, and then ligated and cloned (Fig. 1, amino acid sequence of PDGFA: SEQ No.1). The results were confirmed by agarose gel electrophoresis after restriction enzyme digestion (Fig. 2). In addition, the underlined and bolded parts on the nucleotide sequence of FIG. 1 are part of the primer set used for confirmation of the cloned site through polymerase chain reaction, and the results are shown in the agarose gel electrophoresis (Fig. 2). Polymerase chain reaction conditions were as follows; The initial denaturation was carried out at 94 ° C for 3 minutes, followed by repeated cycles of 25 cycles of denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, and extension at 72 ° C for 45 seconds, And then reacted at 72 ° C for 10 minutes. In order to confirm whether the DNA thus prepared properly expressed in the protein, myc present in the pcDNA3-myc vector shown in the map of Fig. 1 was Western blotted using an anti-myc (9E10, Santa Cruz Biotechnology, sc- Lt; / RTI > expression. PDGFA bound to myc was well expressed and quantitatively loaded through a blot identified with actin (Fig. 3).

(2) Substitution of lysine (K) residue

The lysine residue was replaced with arginine (R) using site-directed mutagenesis and the primers (PDGFA K160R FP 5'-GAATACGTCAGGAGGAAGCCAAAATTA-3 '(SEQ ID NO: SEQ ID No. 2), RP 5'-TAATTTTGGCTTCCTCCTGACGTATTC-3 '(SEQ ID No. 3); PDGFA K165R FP 5'-AAGCCAAAATTAAGAGAAGTCCAGGTG-3' (SEQ ID No. 4); RP 5'-CACCTGGACTTCTCTTAATTTTGGCTT-3 '(SEQ. 5 '); PDGFA K206R FP 5'-AAACGGAAAAGAAGAAGGTTAAAACCC-3' (SEQ No. 6) and RP 5'-GGGTTTTAACCTTCTTCTTTTCCGTTT-3 '(SEQ No. 7) were prepared and PCR was performed to obtain plasmids substituted with specific amino acid residues Three plasmid DNAs were prepared using pcDNA3-myc-PDGFA as a template and lysine residues substituted with arginine (K → R) (Table 1).

Lysine (K) residue site Lysine (K) is replaced by Arginine (R) -derived PDGFA construct 160 pcDNA3-myc-PDGFA (K160R) 165 pcDNA3-myc-PDGFA (K165R) 206 pcDNA3-myc-PDGFA (K206R)

2. In vivo ubiquitination assay

78, Cells, 78, 237-2736, 2004; Oncogene, 22, 12731280, 2003; Cell, 78, 787-798, 1994) was used to infect HEK 293T cells (ATCC, CRL-3216). To confirm the ubiquitination process, 3 μg of pcDNA3-myc-PDGFA WT and 1 μg of pMT123-HA-ubiquitin DNA were co-transfected into the cells. After 24 hours, MG132 (protease inhibitor, 5 μg / , Sigma-Aldrich) for 6 hours, followed by immunoprecipitation analysis (FIG. 4). In order to compare the degree of ubiquitination between WT and the substituent, pcDNA3-myc-PDGFA WT, pcDNA3-myc-PDGFA substituent (K160R), pcDNA3-myc-PDGFA substituent (K165R) and pcDNA3-myc-PDGFA substituent 3 μg was co-transfected with HEK 293T cells (ATCC, CRL-3216) together with 1 μg of pMT123-HA-ubiquitin DNA and subjected to immunoprecipitation analysis after 24 hours (FIG.

Protein samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride) and then incubated with anti-myc (9E10) (Santa Cruz Biotechnology, sc-40) and incubated overnight at 4 ° C. Immunoprecipitates were separated by reaction with protein A / G beads (Santa Cruz Biotechnology) at 4 ° C. for 2 hours. The protein samples were mixed with 2X SDS buffer, heated at 100 ° C for 7 minutes, and separated by SDS-PAGE. The separated proteins were separated by polyvinylidene difluoride (PVDF) ) (Santa Cruz Biotechnology, sc-7392) and anti-beta-actin (Santa Cruz Biotechnology, sc-47778 ) In a weight ratio of 1: 1,000 and anti-mouse (Peroxidase-1 Abeled antibody to mouse IgG (H + L), KPL, 074-1806) secondary antibody was developed with an ECL system (ABfrontier, Seoul, Korea) sc-40), pcDNA3-myc-PDGFA WT was bound to ubiquitin and poly ubiquitination was formed, so that smear ubiquitin was detected and the band became darker (Fig. 4, lanes 3 and 4 In addition, when MG132 (protease inhibitor, 5 / / ml) was treated for 6 hours, the formation of ubiquitin was increased by the increase of polyubiquitination (Fig. 4, lane 4). These results suggest that PDGFA is bound to ubiquitin and polyubiquitinated through the ubiquitin-proteasome system. In the case of pcDNA3-myc-PDGFA substituent (K160R), pcDNA3-myc-PDGFA substituent (K165R) and pcDNA3-myc-PDGFA substituent (K206R) This indicates that the ubiquitin was not bound to these substituents and thus the ubiquitin was little detected (Fig. 5, lanes 3 to 5).

3. PDGF by the protein synthesis inhibitor cycloheximide (CHX) A Half-life of

transfection of HEK 293T cells with 3 ㎍ each of pcDNA3-myc-PDGFA WT, pcDNA3-myc-PDGFA substitution (K160R), pcDNA3-myc-PDGFA substitution (K165R), and pcDNA3- . After 48 hours of transfection, the protein production inhibitor cycloheximide (CHX) (Sigma-Aldrich) (100 ug / ml) was treated and incubated at 30, 60 and 90 min and 15, 30 and 60 min The half-life was measured. As a result, it was confirmed that decomposition of human PDGF and PDGFB was inhibited (Fig. 6). It can be seen that the human PDGFA is decomposed after 60 minutes, whereas the human PDGFA substituent (K160R), PDGFA substituent (K165R) and PDGFA substituent (K206R) are not decomposed for more than 90 minutes, 6).

4. Intracellular PDGF A And PDGF A  Identification of signaling by substituents

PDGF has been reported to activate Erk1 / 2 by Ras-Raf-MEK1 / 2-Erk signaling as a growth factor (Journal of Cell Science 117, 4619-4628, 2004).

This example confirmed the signaling process by PDGF A and PDGF A substituents in the cells. HeLa cells were transfected with 3 각 each of pcDNA3-myc-PDGFA WT, pcDNA3-myc-PDGFA substitution (K160R), pcDNA3-myc-PDGFA substituent (K165R) and pcDNA3-myc-PDGFA substituent (K206R) . After 2 days of infection, proteins were extracted from the cells, quantified, and Western blotting was performed to confirm intracellular signal transduction. To this end, the proteins isolated from HeLa cells infected with pcDNA3-myc-PDGFA WT, pcDNA3-myc-PDGFA substituent (K160R), pcDNA3-myc-PDGFA substituent (K165R) and pcDNA3-myc- PDGFA substituent (K206R) (9E10, Santa Cruz Biotechnology, sc-40), anti-STAT3 (Santa Cruz Biotechnology, sc-21876), anti-phospho-STAT3 (Cell signaling 9131S), anti-AKT (H-136, Santa Cruz Biotechnology, sc-8312), anti-phospho-AKT (S473, cell signaling 9271S), anti-Erk1 / 2 (9B3, Abfrontier LF-MAO134) Blocking solution containing anti-phospho-Erk1 / 2 (Thr202 / Tyr204, Abfrontier LF-PA0090) and anti-β-actin (Santa Cruz Biotechnology, sc-47778) at a weight ratio of 1: 1000 to 1: 3000, (ECL) using a secondary antibody with anti-rabbit (goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology, sc- System (Western blot detection kit, ABf rontier, Seoul, Korea). As a result, the pcDNA3-myc-PDGFA substituent (K160R), the pcDNA3-myc-PDGFA substituent (K165R) and the pcDNA3-myc-PDGFA substituent (K206R) STAT3 signaling (Fig. 7).

According to the present invention, a PDGFA with an increased half-life is provided. Accordingly, the present invention relates to PDGFA which can be used as a therapeutic agent, and can be usefully used in the pharmaceutical industry and the beauty industry.

&Lt; 110 > UbiProtein. Corp <120> A method for extending half-life of PDGFA <130> UBPRN17P-0003 <160> 7 <170> KoPatentin 3.0 <210> 1 <211> 211 <212> PRT <213> Artificial Sequence <220> <223> Platelet-derived growth factor subunit A <400> 1 Met Arg Thr Leu Ala Cys Leu Leu Leu Leu Gly Cys Gly Tyr Leu Ala   1 5 10 15 His Val Leu Ala Glu Glu Ala Glu Ile Pro Arg Glu Val Ile Glu Arg              20 25 30 Leu Ala Arg Ser Ser Gln Ile His Ser Ile Arg Asp Leu Gln Arg Leu Leu          35 40 45 Glu Ile Asp Ser Val Gly Ser Glu Asp Ser Leu Asp Thr Ser Leu Arg      50 55 60 Ala His Gly Val His Ala Thr Lys His Val Pro Glu Lys Arg Pro Leu  65 70 75 80 Pro Ile Arg Arg Lys Arg Ser Ile Glu Glu Ala Val Pro Ala Val Cys                  85 90 95 Lys Thr Arg Thr Val Ile Tyr Glu Ile Pro Arg Ser Gln Val Asp Pro             100 105 110 Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val Glu Val Lys Arg         115 120 125 Cys Thr Gly Cys Cys Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg     130 135 140 Val His His Arg Ser Val Lys Val Ala Lys Val Glu Tyr Val Arg Lys 145 150 155 160 Lys Pro Lys Leu Lys Glu Val Gln Val Arg Leu Glu Glu His Leu Glu                 165 170 175 Cys Ala Cys Ala Thr Thr Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp             180 185 190 Thr Gly Arg Pro Arg Glu Ser Gly Lys Lys Arg Lys Arg Lys Arg Leu         195 200 205 Lys Pro Thr     210 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 gaatacgtca ggaggaagcc aaaatta 27 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 taattttggc ttcctcctga cgtattc 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 aagccaaaat taagagaagt ccaggtg 27 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 cacctggact tctcttaatt ttggctt 27 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 aaacggaaaa gaagaaggtt aaaaccc 27 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 gggttttaac cttcttcttt tccgttt 27

Claims (6)

Wherein any one of the lysine residues at positions 160, 165 and 206 from the N-terminus of the platelet-derived growth factor subunit A (PDGFA) having the amino acid sequence of SEQ ID NO: 1 is substituted with arginine PDGFA having an increased half-life. delete A cosmetic composition comprising the PDGFA of claim 1. (a) a promoter; And (b) an expression vector comprising a base sequence encoding the PDGFA of claim 1, wherein the promoter and the base sequence are operatively linked. A host cell comprising the expression vector of claim 4.


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