Methods of extending half-life of botulinum toxin type A light chain proteins

Abstract

The invention relates to a method for prolonging the half-life period of a botulinum toxin type A light chain (BT-LC) protein, or a botulinum toxin type A light chain (AUT-BT-LC) protein with prolonged half-life period. The method comprises the step of replacing more than one lysine residue in the amino acid sequence of the botulinum toxin type A light chain (BT-LC) protein. The proteins with prolonged half-life of the present invention are useful in cosmetic and pharmaceutical compositions.

Description

Technical Field

[0001] The present invention relates to a method for extending the half-life of botulinum toxin type A by replacing one or more amino acid residues in the light chain region of the botulinum toxin type A, and also relates to a botulinum toxin type A light chain protein with extended half-life prepared by the method. Background Art

[0002] In eukaryotic cells, 80-90% of proteins are degraded through the ubiquitin-proteasome pathway (UPP). Ubiquitin is a highly conserved protein composed of 76 amino acids, which exists in almost all eukaryotic cells. Among them, the amino acid residues at positions 6, 11, 27, 29, 33, 48, and 63 are lysine (Lys, K), and positions 48 and 63 play a major role in the formation of polyubiquitin chains. The ubiquitin-proteasome pathway includes two independent continuous processes, the first of which is the process of covalently labeling substrates with multiple ubiquitin molecules, and the second is the process of degradation of ubiquitin-labeled proteins by the 26S proteasome complex. The binding of ubiquitin to the substrate occurs through an isopeptide bond between the lysine residue of the substrate molecule and the C-terminal glycine of ubiquitin, and is achieved by the formation of thioesters between ubiquitin and the enzyme through ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2, and ubiquitin ligase E3. Among them, ubiquitin-activating enzyme E1 activates ubiquitin through an ATP-dependent reaction. Ubiquitin-conjugating enzyme E2 receives activated ubiquitin from E1 at the cysteine ​​residue in the ubiquitin binding domain and transfers it to E3 ligase or directly to the substrate protein. The E3 enzyme also catalyzes the stable isopeptide bond between the lysine residue of the substrate protein and the glycine residue of ubiquitin. Another ubiquitin can be connected to the C-terminal lysine residue of ubiquitin bound to the substrate protein. By repeating this process, multiple ubiquitin molecules are connected to the substrate protein in a branched shape to form a polyubiquitin chain, which is recognized and selectively degraded by the 26S proteasome.

[0003] Botulinum toxin is a neurotoxin produced by Clostridium botulinum, a Gram-positive anaerobic bacterium found in spoiled meat and canned food. It is reported that there are eight types of neurotoxins, of which seven, A, B, C, D, E, F, and G, can cause nerve paralysis. Usually, due to the combination of heavy and light chains, its size is about 150 kDa, but it is reported that the size of the complex can be as high as 900 kDa by binding to a complex of non-toxin proteins other than botulinum toxin protein, depending on the type of neurotoxin. Botulinum toxin type A is considered to be the most lethal biological agent. In the current use of botulinum toxin, when botulinum toxin is injected into skin tissue, the nerve paralysis effect weakens within 3 to 6 months, or regular treatment is required, and repeated administration leads to the formation of antibodies against botulinum toxin in the body, so there are limitations such as reduced effect.

[0004] Therefore, the present inventors have made efforts to develop a botulinum toxin type A protein with a prolonged half-life, and as a result, have confirmed that the half-life of the botulinum toxin type A light chain protein can be prolonged by replacing specific amino acid residues with arginine, thereby completing the present invention. Summary of the invention

[0005] Technical issues

[0006] The object of the present invention is to provide a method for prolonging the half-life of botulinum toxin type A light chain protein.

[0007] Furthermore, the present invention aims to provide a botulinum toxin type A light chain protein with an extended half-life, wherein one or more lysine residues present in the amino acid sequence of the light chain region of the botulinum toxin type A are substituted.

[0008] Furthermore, the present invention aims to provide a cosmetic raw material and a pharmaceutical composition comprising a botulinum toxin type A light chain protein with an extended half-life.

[0009] Technical Solution

[0010] In order to achieve the above-mentioned object, the present invention provides a method for extending the half-life of a botulinum toxin type A light chain protein, comprising: replacing one or more lysine residues present in the amino acid sequence of the botulinum toxin type A light chain protein. Specifically, the present invention provides a method for extending the half-life of a botulinum toxin type A light chain (BT-LC) protein, comprising: replacing one or more of the lysines (lysine) bound to the C-terminal glycine (glycine) of ubiquitin in the amino acid sequence of the botulinum toxin type A light chain (BT-LC) protein with arginine (arginine). More specifically, the botulinum toxin type A light chain protein may have an amino acid sequence as shown in SEQ ID No. 1, and one or more of the lysine residues at positions 89, 212, 301, 330, and 335 from its N-terminus is replaced by arginine.

[0011] The present invention also provides a botulinum toxin type A light chain (AUT-BT-LC) protein with an extended half-life, wherein one or more of the lysine residues in the amino acid sequence of the botulinum toxin type A light chain protein that binds to the C-terminal glycine of ubiquitin are substituted with arginine. Specifically, the botulinum toxin type A light chain protein with an extended half-life may have an amino acid sequence as shown in SEQ ID No. 1, and one or more of the lysine residues at positions 89, 212, 301, 330, and 335 from its N-terminus are substituted with arginine.

[0012] Furthermore, the present invention provides a cosmetic raw material and / or a cosmetic composition comprising the botulinum toxin type A light chain protein with an extended half-life.

[0013] Furthermore, the present invention provides a pharmaceutical composition for treating facial spasm, eyelid spasm, torticollis, blepharospasm, cervical dystonia, middle pharyngeal dystonia, spasmodic dysphonia, migraine, anal pruritus or hyperhidrosis, and comprising the half-life extended botulinum toxin type A light chain protein and a pharmaceutically acceptable carrier or excipient.

[0014] Furthermore, in order to achieve the above-mentioned purpose, the present invention can provide an expression vector and / or a host cell, wherein the expression vector comprises: (a) a promoter; (b) a base sequence encoding a botulinum toxin type A light chain (BT-LC) protein; and any linker.

[0015] In the present invention, the lysine residues of the botulinum toxin type A light chain protein can be replaced by "conservative amino acids". In the present invention, "conservative amino acid" substitution refers to the substitution of one amino acid residue by another amino acid residue with a side chain having similar chemical properties (such as charge or hydrophobicity). In general, conservative amino acid substitution does not actually change the functional properties of the protein. Examples of amino acid groups with side chains of similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic hydroxyl side chains: 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) acidic side chains: aspartic acid and glutamic acid; 7) sulfur-containing side chains: cysteine ​​and methionine.

[0016] In the present invention, the lysine residue of the botulinum toxin type A light chain protein may be substituted with arginine or histidine having a basic side chain, preferably with an arginine residue.

[0017] Effects of the Invention

[0018] According to the present invention, the half-life of the botulinum toxin type A light chain protein in which one or more lysine residues in the amino acid sequence of the botulinum toxin type A light chain protein are replaced by arginine is prolonged and can remain in the body for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The structure of the mammalian expression vector for the light chain region of botulinum toxin type A is shown.

[0020] Figure 2 The figure shows the expression of the light chain region of botulinum toxin type A in HEK-293T cells. The expression was confirmed using a tagged Flag antibody.

[0021] Figure 3 The ubiquitination process of the light chain region of botulinum toxin type A confirmed by ubiquitination analysis is shown, thereby indicating that the degradation pathway of the light chain of botulinum toxin type A is the ubiquitin-proteasome pathway.

[0022] Figure 4 Shown are ubiquitin levels for light chain region substituents of botulinum toxin type A in which lysine residues are replaced with arginine, compared to wild type.

[0023] Figure 5 The graph shows the change in half-life of the light chain region of botulinum toxin type A after treatment with cycloheximide (CHX), a protein synthesis inhibitor.

[0024] Figure 6The graph shows the change in half-life of the light chain region of botulinum toxin type A.

[0025] Figure 7 The structure of the E. coli expression vector for the light chain region of botulinum toxin type A is shown.

[0026] Figure 8 The expression of the light chain region of botulinum toxin type A in E. coli is shown, with each purification step and the purity of the final product indicated, and the purification process of each step is also indicated by numbers. DETAILED DESCRIPTION

[0027] In one embodiment of the present invention, the protein is a light chain of botulinum toxin type A. In the amino acid sequence of the light chain region of botulinum toxin type A as shown in SEQ ID No. 1, one or more of the lysine residues at positions 89, 212, 301, 330, and 335 from the N-terminus are replaced by arginine residues.

[0028] In another embodiment of the present invention, the half-life extended botulinum toxin type A light chain protein obtained by substitution as described above can be used as a cosmetic raw material, or can be used to prepare a pharmaceutical composition for treating facial spasm, eyelid spasm, torticollis, blepharospasm, cervical dystonia, middle pharyngeal dystonia, spasmodic dysphonia, migraine, anal pruritus or hyperhidrosis. (Long-term stable efficacy of botulinum toxin A in facial movement disorders with no need for increasing dose. S Badarny et al. Medicine (Baltimore) (2021 Jun 25; 100 (25): e26481; Botulinum Toxin for the Treatment of Hemifacial Spasm: An Update on Clinical Studies. Nicola Tambasco et al., 2021 Dec 9; 13 (12): 881).

[0029] The present invention will be described in more detail below based on examples. The following examples are only used to illustrate the present invention, and the present invention is not limited to the following examples.

[0030] Mode for carrying out the invention

[0031] Example: Ubiquitination analysis of botulinum toxin type A light chain (BT-LC) protein and preparation of five half-life extended substitutions

[0032] Example 1: Cloning into mammalian expression vector and confirming protein expression

[0033] We commissioned gene synthesis and obtained the light chain gene of botulinum toxin type A of SEQ ID No. 1. In order to insert it into a mammalian expression vector, we prepared fragments using EcoRI and XhoI, and then spliced ​​and cloned them. The structure of the expression vector was illustrated using SnapGeneViewer software, as shown in the figure below: Figure 1 As shown ( Figure 1 In the example, the amino acid sequence of the light chain of botulinum toxin type A is shown in SEQ ID No. 1. In order to confirm whether the DNA prepared in this way is correctly expressed as protein, an anti-flag (Sigma-aldrich, F1804) antibody was used to Figure 1 The flag present in the pCS4-flag vector shown in the map was subjected to Western blotting. The results confirmed that the botulinum toxin type A light chain protein bound to the flag was well expressed ( Figure 2 ).

[0034] Example 2: In vivo ubiquitination analysis

[0035] HEK-293T cells (purchased from Abcam) were infected with plasmids encoding pCS4-flag-A-type botulinum toxin light chain WT and pMT123-HA-Ubiquitin DNA (J Biol Chem., 279 (4), 2368-2376, 2004; Cell Research, 22, 873-885, 2012; Oncogene, 22, 1273-1280, 2003; Cell, 78, 787-798, 1994). In order to confirm the degree of ubiquitination, 5 μg of pCS4-flag-A-type botulinum toxin light chain WT and 1 μg of pMT123-HA-ubiquitin DNA were co-transfected (co-transfection) into the cells. After 24 hours of transfection, the cells were treated with MG132 (purchased from Sigma-Aldrich, proteasome inhibitor, 5 μg / ml) for 4 hours, and then immunoprecipitation analysis was performed ( Figure 3 ).

[0036] The samples obtained for immunoprecipitation were lysed with lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8, 1 mM phenylmethanesulfonyl fluoride (PMSF)) and mixed with anti-flag (Sigma-Aldrich, F1804) primary antibody and incubated overnight at 4°C. Immunoprecipitates were separated using protein A / G magnetic beads (Santa Cruz Biotechnology) at 4°C for 2 hours. Then, the samples were washed twice with lysis buffer. For immunoblotting, protein samples were mixed with 2X SDS buffer, heated at 100°C for 7 minutes, and then separated by SDS-PAGE. The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and then developed by the ECL system (Western blot detection kit, ABfrontier, Seoul, Korea) using a blocking solution containing anti-Flag (Sigma-Aldrich, F3165), anti-HA (Santa Cruz Biotechnology, sc-7392), anti-β-actin (Santa Cruz Biotechnology, sc-47778) at a weight ratio of 1:1000 and anti-mouse (Peroxidase-labeled antibody to mouse IgG(H+L), KPL, 074-1806) secondary antibody.

[0037] As a result, when immunoprecipitation was performed using anti-flag (Sigma-Aldrich, F1804), ubiquitin bound to the light chain WT of pCS4-flag-A-type botulinum toxin to form polyubiquitination, followed by the detection of diffuse ubiquitin and the appearance of dark bands ( Figure 3 , lane 3).

[0038] Example 3: Preparation of Lysine (K) Residues Substituted with Arginine

[0039] Site-directed mutagenesis was used to replace lysine residues with arginine, and primers were prepared using DNA sequences that can induce specific mutations (BT-LC K89R FP 5'-GAT AAT TAT TTA AGG GGA GTTACA AAA-3'(SEQ ID No.2), RP 5'-TTT TGT AAC TCC CCTTAA ATAATT ATC-3'(SEQ IDNo.3); BT-LC K212R FP 5'-GGT GCA GGC AGA TTT GCT ACAGAT-3'(SEQ ID No.4), RP 5'-ATC TGT AGC AAA TCT GCC TGC ACC-3'(SEQ ID No.5); BT-LC K301R FP 5'-CTT AATAAAGCT AGA TCAATA GTAGGT-3'(SEQ ID No.6), RP 5'-ACC TAC TAT TGA TCT AGC TTT ATTAAG-3'(SEQ ID No.7); BT-LC K330R FP 5'-ACATCT GGA AGA TTT TCG GTA GAT-3'(SEQ IDNo.8), RP 5'-ATC TAC CGA AAA TCT TCC AGATGT-3'(SEQ IDNo.9); BT-LC K335R FP 5'-TTT TCG GTA GAT AGA TTAAAATTT GAT-3'(SEQ ID No.10), RP 5'-ATC AAA TTT TAA TCT ATC TAC CGAAAA-3' (SEQ ID No. 11)), and then PCR was performed under specific conditions to prepare plasmid DNA in which specific amino acid residues were replaced. pCS4-flag-A-type botulinum toxin light chain (BT-LC) was used as a template to prepare plasmid DNA in which lysine residues were replaced by arginine (K→R) (Table 1, Figure 6 , amino acid sequence of light chain of botulinum toxin type A: SEQ No.12~16)

[0040] Table 1

[0041]

[0042] In addition, the light chain region of botulinum toxin type A (BT-LC) having a sequence that is effectively expressed in E. coli was ligated to pET21b through a codon optimization process and used as a template to prepare a plasmid DNA in which lysine residues were substituted with arginine (K→R) (Table 2, Figure 6 , the light chain amino acid sequence of botulinum toxin type A: SEQ No. 17, nucleotide sequence: SEQ No. 18).

[0043] Table 2

[0044]

[0045] Example 4: Confirmation of A by using cycloheximide (CHX) as a protein production inhibitor The half-life of the light chain of botulinum toxin type 2 (BT-LC)

[0046] 2 μg each of pCS4-flag-Botulinum toxin type A light chain (BT-LC) WT, pCS4-flag-Botulinum toxin type A light chain (BT-LC) (K89R), pCS4-flag-Botulinum toxin type A light chain (BT-LC) (K212R), pCS4-flag-Botulinum toxin type A light chain (BT-LC) (K301R), pCS4-flag-Botulinum toxin type A light chain (BT-LC) (K330R), and pCS4-flag-Botulinum toxin type A light chain (BT-LC) (K335R) were transfected into HEK 293T cells. 48 hours after transfection, the cells were treated with cycloheximide (CHX) (Sigma-Aldrich) (100 μg / ml), a protein production inhibitor, and the half-lives were measured at 8 and 16 hours. The results confirmed that the degradation of the Botulinum toxin type A light chain (BT-LC) was inhibited ( Figure 5 ). The results confirmed that the half-life of the light chain (BT-LC) of botulinum toxin type A was prolonged by the light chain (BT-LC) of botulinum toxin type A (K212R) and the light chain (BT-LC) of botulinum toxin type A (K301R), and the results are shown in a curve graph ( Figure 6 ).

[0047] Example 5: Cloning into E. coli expression vector and confirming protein expression

[0048] In order to insert the light chain region of botulinum toxin type A that has undergone codon optimization into an E. coli expression vector, a fragment was prepared from the pCS4-flag-codon-optimized light chain DNA of botulinum toxin type A using NdeI and XhoI, and then spliced ​​and cloned with the pET21b vector. The GCT sequence inserted during this process was removed using site-directed mutagenesis, and the image was visualized using SnapGene Viewer software ( Figure 7 ). In order to express the light chain region of botulinum toxin type A, IPTG (1 mM) was used for treatment, and then expression was induced overnight at 18°C. Then, Coomasie staining was performed to confirm expression, and the results confirmed that the expression of botulinum toxin type A light chain protein was good ( Figure 8 ).

Claims

1. A method for extending the half-life of botulinum toxin type A light chain protein, characterized in that: include: In the amino acid sequence of the botulinum toxin type A light chain protein, one or more of the lysine residues that bind to the C-terminal glycine residue of ubiquitin are replaced with arginine residues.

2. The method for extending the half-life of botulinum toxin type A light chain protein according to claim 1, characterized in that: The botulinum toxin type A light chain protein has an amino acid sequence as shown in SEQ ID No. 1, and one or more of the lysine residues at positions 89, 212, 301, 330 and 335 from its N-terminus is substituted with arginine.

3. A botulinum toxin type A light chain protein with an extended half-life, characterized in that: In the amino acid sequence of the botulinum toxin type A light chain protein, one or more lysine residues that bind to the C-terminal glycine residue of ubiquitin are replaced by arginine residues.

4. The botulinum toxin type A light chain protein with extended half-life according to claim 3, characterized in that: The botulinum toxin type A light chain protein has an amino acid sequence as shown in SEQ ID No. 1, and one or more of the lysine residues at positions 89, 212, 301, 330 and 335 from its N-terminus is substituted with arginine.

5. A cosmetic raw material, characterized in that: Comprising the botulinum toxin type A light chain protein with extended half-life according to claim 4.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition is used to treat facial spasm, eyelid spasm, torticollis, blepharospasm, cervical dystonia, middle pharyngeal dystonia, spasmodic dysphonia, migraine, anal pruritus or hyperhidrosis, and comprises the half-life extended botulinum toxin type A light chain protein according to claim 4 and a pharmaceutically acceptable carrier or excipient.

7. An expression vector comprising: (a) Promoter; (b) a base sequence encoding a botulinum toxin type A light chain protein; and Any connector, The expression vector is characterized in that The promoter is operably linked to the base sequence.

8. A host cell, characterized in that Comprising the expression vector according to claim 7.

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