Light chain variants of botulinum toxin type e

Abstract

The present disclosure provides a light chain variant of botulinum toxin type E, in which a specific lysine residue is substituted with arginine. The light chain variant of botulinum toxin type E according to the present disclosure exhibits a significantly increased in vivo half-life due to the suppression of degradation via the ubiquitin-proteasome pathway.

Description

Light chain variant of botulinum toxin type E

[0001] The present invention relates to a variant of the light chain of botulinum toxin type E (also referred to as a 'botulinum E light chain protein'). More particularly, the present invention relates to a variant of the light chain of botulinum toxin type E having an increased half-life, obtained by substituting lysine at a specific position in the light chain of botulinum toxin type E with arginine.

[0002] Protein degradation within eukaryotic cells occurs through two pathways: lysosomes and proteasomes. The lysosomal pathway, which degrades 10-20% of proteins, lacks substrate specificity and precise temporal regulation. This process primarily degrades extracellular or membrane proteins, such as cell surface proteins that are internalized by endocytosis and degraded in lysosomes. However, for proteins to be selectively degraded in eukaryotic cells, they must undergo the ubiquitin-proteasome pathway, which involves the conjugation of ubiquitin to target proteins by ubiquitin conjugation enzymes, forming polyubiquitin chains, which are then recognized and degraded by the proteasome. This process is known to degrade over 80% of eukaryotic proteins and, by regulating the degradation of most proteins within eukaryotic cells, is responsible for protein functional transitions and homeostasis.

[0003] Botulinum toxin consists of a heavy chain of approximately 100 kDa and a light chain of approximately 50 kDa, linked by disulfide bonds. These disulfide bonds play a crucial role in the biological activation and action of the toxin, but their weak binding makes them easily cleaved by other factors. The heavy chain consists of two functional terminals. The N-terminal region, a potential site, is known to form ion channels in the lipid bilayer, while the C-terminal region, a binding site, plays a crucial role in the toxin's attachment to the cell membrane and its internalization. The light chain acts as a zinc-dependent peptide intermediate. Botulinum toxin is primarily used for facial cosmetic purposes. It is injected into the skin to improve wrinkles in areas such as the glabella and forehead. It is also used in the treatment of diseases due to its beneficial effects, such as nerve paralysis.

[0004] Clinical studies using botulinum toxin type E are currently underway, and interest in developing products using it is growing. Botulinum toxin type E can produce rapid effects within a few days. However, its duration is short, lasting approximately 2 to 7 weeks, after which the skin and affected area return to their original state (Pons et al, 407; 116516, 2019). A technological solution that can deliver rapid and sustained effects is needed for both cosmetic and disease treatment.

[0005] The present inventors have conducted various studies to develop a method that can effectively increase the in vivo half-life of botulinum toxin, thereby enabling long-term effects of botulinum toxin or enabling procedures and disease treatments using small doses.

[0006] The present inventors confirmed that the light chain of botulinum toxin type E undergoes a degradation pathway mediated by the ubiquitin-proteasome, and compared the ubiquitination degradation pathway by producing various mutants. As a result, they discovered that mutants obtained by substituting lysine at specific positions (i.e., lysine at positions 3, 62, 85, 230, 281, 288, 342, 413, or 419) in the light chain of botulinum toxin type E significantly inhibited ubiquitination degradation.

[0007] Accordingly, the present invention aims to provide a light chain mutant of botulinum toxin type E obtained by substituting lysine at a specific position in the light chain of botulinum toxin type E with arginine.

[0008] In addition, the present invention aims to provide a nucleic acid composed of or including a polynucleotide sequence encoding a light chain variant of the botulinum toxin type E of the present invention.

[0009] In addition, the present invention aims to provide a vector comprising the nucleic acid.

[0010] In addition, the present invention aims to provide a cell transfected with the above vector.

[0011] In addition, the present invention aims to provide a method for increasing the half-life of a light chain of botulinum toxin type E, which comprises substituting lysine at a specific position in the light chain of botulinum toxin type E with arginine.

[0012] In addition, the present invention aims to provide a use of a light chain variant of botulinum toxin type E according to the present invention or a nucleic acid encoding the same for use in a subject requiring cosmetic treatment or procedure with botulinum toxin.

[0013] In addition, the present invention aims to provide a use of a light chain variant of botulinum toxin type E according to the present invention or a nucleic acid encoding the same for use in a subject requiring therapeutic treatment or procedure with botulinum toxin.

[0014] According to one aspect of the present invention, a light chain variant of botulinum toxin type E having an amino acid sequence of SEQ ID NO: 1 is provided, wherein lysine at position 3, 62, 85, 230, 281, 288, 342, 413 or 419 is substituted with arginine.

[0015] According to another aspect of the present invention, a nucleic acid comprising or consisting of a polynucleotide sequence encoding a light chain variant of the botulinum toxin type E is provided.

[0016] According to another aspect of the present invention, a vector is provided comprising a nucleic acid consisting of or including a polynucleotide sequence encoding a light chain variant of the botulinum toxin type E.

[0017] According to another aspect of the present invention, a cell is provided that is transfected with a vector comprising a nucleic acid comprising or consisting of a polynucleotide sequence encoding a light chain variant of the botulinum toxin type E.

[0018] According to another aspect of the present invention, a method for increasing the half-life of a light chain of a botulinum toxin type E comprising substituting lysine at positions 3, 62, 85, 230, 281, 288, 342, 413 or 419 with arginine in a light chain of a botulinum toxin type E having an amino acid sequence of SEQ ID NO: 1 is provided.

[0019] According to another aspect of the present invention, there is provided a use of a light chain variant of botulinum toxin type E according to the present invention or a nucleic acid encoding the same for use in a subject requiring cosmetic treatment or procedure with botulinum toxin.

[0020] According to another aspect of the present invention, there is provided a use of a light chain variant of botulinum toxin type E according to the present invention or a nucleic acid encoding the same for use in a subject in need of therapeutic treatment or procedure with botulinum toxin.

[0021] The light chain variant of botulinum toxin type E according to the present invention has a significantly increased in vivo half-life due to inhibition of degradation by the ubiquitin-proteasome. Therefore, the light chain variant of botulinum toxin type E according to the present invention can be useful in the production of botulinum toxin type E that can enable long-term treatment or treatment using low doses.

[0022] Figure 1 shows the results of confirming expression after inducing transfection by increasing the amount of the botulinum toxin type E light chain, which is a plasmid gene, in the HEK293T cell line.

[0023] Figure 2 shows the results of confirming the degradation regulation pathway of the light chain of botulinum toxin type E through ubiquitination analysis experiments.

[0024] Figure 3 shows the results of comparing the degree of ubiquitination of the light chain protein of wild type botulinum toxin type E and the light chain protein mutant of botulinum toxin type E.

[0025] Figure 4 shows the results comparing the degree of stabilization of the light chain of intracellular botulinum toxin type E after treatment with cycloheximide.

[0026] Figure 5 is a graph that numerically represents the results of Figure 4. (*: 0.01 < p < 0.05, **: 0.001 < p < 0.01).

[0027] The present inventors confirmed that the light chain of botulinum toxin type E undergoes a degradation pathway mediated by the ubiquitin-proteasome.

[0028] The present inventors, through site-directed mutagenesis, produced conservative amino acid substitution mutants of the light chain protein of botulinum toxin type E, i.e., mutants in which lysine at position 62 or 288 of the light chain protein of botulinum toxin type E was substituted with arginine, and confirmed the degree of degradation by ubiquitin-proteasome. As a result, it was found that the level of degradation was significantly reduced, i.e., the resistance to degradation was increased.

[0029] Furthermore, the inventors have found that variants obtained by substituting lysine at positions 3, 62, 85, 230, 281, 288, 342, 413 or 419 of the light chain protein of botulinum toxin type E with arginine exhibit improved resistance to degradation by the ubiquitin-proteasome, thereby having significantly increased in vivo half-lives.

[0030] Therefore, the variant according to the present invention can be very useful in cases where the effect of botulinum toxin needs to be sustained for a long time, or where cosmetic procedures and / or disease treatments using small doses are required.

[0031] In addition, the variant of the light chain protein of botulinum toxin type E according to the present invention can be used for all purposes for which botulinum toxin type E is conventionally known, and can be applied to related fields. Specifically, for cosmetic purposes, it can be used for improving skin wrinkles and elasticity, removing wrinkles, restoring skin, etc., and for therapeutic purposes, it can be used for treating or alleviating diseases or disorders such as upper limb stiffness (including upper limb stiffness due to stroke), strabismus, migraine, and eyelid tremors, or for relieving pain before and after surgery, treating itching, treating wounds, etc.

[0032] The present invention provides a variant of a light chain protein of botulinum toxin type E. Specifically, the present invention provides a light chain variant of botulinum toxin type E, wherein lysine at positions 3, 62, 85, 230, 281, 288, 342, 413, or 419 in the light chain of botulinum toxin type E having an amino acid sequence of SEQ ID NO: 1 is substituted with arginine.

[0033] The amino acid sequence of the light chain protein of botulinum toxin type E and the base sequence encoding it are both known; for example, the amino acid sequence of the light chain protein of botulinum toxin type E is as shown in SEQ ID NO: 1, and the base sequence encoding it is as shown in SEQ ID NO: 2.

[0034] In one embodiment, the light chain variant of botulinum toxin type E according to the present invention may be a variant consisting of the amino acid sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17 or 19.

[0035] In addition, the light chain variant of botulinum toxin type E according to the present invention may be a variant (K288R variant) consisting of the amino acid sequence of SEQ ID NO: 13.

[0036] The light chain variant of botulinum toxin type E according to the present invention can be produced by substituting lysine at position 288 in the light chain of botulinum toxin type E with arginine according to a method commonly used in the field of biotechnology.

[0037] For example, by performing a polymerase chain reaction using a gene encoding a light chain protein of botulinum toxin type E (e.g., a gene having a base sequence of SEQ ID NO: 2) as a template using a primer set of SEQ ID NOs: 31 and 32, a gene including or consisting of a polynucleotide sequence encoding a variant in which lysine at position 288 is substituted with arginine can be obtained.

[0038] As used herein, the term "gene" has substantially the same meaning as "(isolated) nucleic acid or nucleic acid molecule" or "polynucleotide" as commonly used in the art, and can be used interchangeably, as will be readily apparent to those skilled in the art.

[0039] In one embodiment, a gene encoding a variant in which lysine at position 288 in the amino acid sequence of the light chain of botulinum toxin type E represented by SEQ ID NO: 1 is substituted with arginine may be composed of the base sequence of SEQ ID NO: 14.

[0040] After producing an expression vector expressing a gene encoding a variant according to the present invention according to a conventional method used in the field of biotechnology, the expression vector can be transfected into a host cell to obtain a transfected cell, and the cell can be cultured to obtain a light chain variant protein of botulinum toxin type E according to the present invention.

[0041] Therefore, the scope of the present invention includes a vector (i.e., an expression vector) comprising a gene encoding the light chain variant of the botulinum toxin type E. The gene encoding the light chain variant of the botulinum toxin type E may be composed of the base sequence of SEQ ID NO: 14. The expression vector may be prepared using a vector commonly used in the field of biotechnology, such as pcDNA3, pCS4, pcDNA3.1, etc., as an empty vector, using an appropriate restriction enzyme. The empty vector may be labeled with Flag, etc., as needed.

[0042] Additionally, the scope of the present invention includes cells transfected with a vector (i.e., an expression vector) comprising a gene encoding the light chain variant of the botulinum toxin type E. The gene encoding the light chain variant of the botulinum toxin type E may be composed of the base sequence of SEQ ID NO: 14. Host cells include, but are not limited to, HEK293T cells, B16F10 cells, A549 cells, A2780 cells, SKOV3 cells, HeLa cells, and the like, for example.

[0043] The present invention also provides a method for increasing the half-life of a light chain of a botulinum toxin type E, comprising substituting lysine at positions 3, 62, 85, 230, 281, 288, 342, 413 or 419 in the light chain of a botulinum toxin type E having an amino acid sequence of SEQ ID NO: 1 with arginine. In the method of the present invention, the substitution is as described above.

[0044] Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention and the present invention is not limited by the examples.

[0045] Example

[0046] 1. Experimental method

[0047] (1) Production of expression vector

[0048] The polynucleotide of the light chain of botulinum toxin type E consisting of the base sequence of SEQ ID NO: 2 was cloned into the pCS4-3Flag vector (4.3 kb) (E7908, Sigma-Aldrich) using restriction enzymes StuI and XbaI, thereby constructing an expression vector of the light chain protein of wild-type botulinum toxin type E (pCS4-3Flag-Bont-E-LC WT).

[0049] An expression vector for a mutant in which lysine at position 288 of the light chain protein of botulinum toxin type E was substituted with arginine was constructed through site-directed mutagenesis using 5'-TAC TTG TAC TCT GCT AAG TTT AGA CGC TAT TTT TTT ATA ATC AG-3' (SEQ ID NO: 31) as a forward primer and 5'-AAA CTT AGC AGA GTA CAA GTA TCT AAT CCA CTA CTT AAT CC-3' (SEQ ID NO: 32) as a reverse primer.

[0050] Specifically, using the pCS4-3Flag-Bont-E-LC WT wild-type botulinum toxin type E light chain protein gene cloned above as a template, polymerase chain reaction (PCR) was performed using the above primer set under the following conditions: 95°C for 40 seconds, 62°C for 40 seconds, and 68°C for 7 minutes, for a total of 18 cycles. An expression vector for a mutant of the botulinum toxin type E light chain, i.e., pCS4-3Flag-Bont-E-LC (K288R), was constructed through mutagenesis.

[0051] (2) Transfection

[0052] Transfection of HEK293T cells (ATCC, CRL-3216) was induced using the expression vector constructed above. HEK293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin and streptomycin (Gibco, Grand Island, NY, USA) in a 5% CO2 incubator. For transfection of botulinum toxin type E light chain wild type (WT), mutant (K288R) and ubiquitin pRK5-HA-Ub, 3 μg of wild type or mutant botulinum toxin type E light chain, 3 μg of ubiquitin, 600 μl of NaCl and 54 μl of polyethylenimine reagent (PEI, Polysciences, Inc., Warrington, PA, USA) were mixed to prepare a mixture, and then incubated at room temperature for 15 min. HEK293T cells (1 × 10 6 The mixture was added to 6 ml of culture medium containing cells and cultured at 37°C for 48 hours.

[0053] (3) Immunoblotting and antibody

[0054] Immunoblotting was performed using anti-Flag antibody (MBL), anti-HA antibody (12CA5 hybridoma cell media), and anti-β-actin antibody (Santa Cruz Biotechnology). After SDS-PAGE, the blots were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore), and detected with HRP-conjugated secondary antibodies.

[0055] (4) Immunoprecipitation

[0056] Transfected cells were lysed in lysis buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 10% glycerol, 300 mM NaCl, and 1% Triton X-100) on ice for 20 minutes, and then centrifuged at 13,000 rpm for 20 minutes. The supernatant was collected, antibody (Flag antibody) was added, and the mixture was incubated overnight at 4°C. A / G PLUS agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added, and the mixture was incubated for 2 hours on a 4°C rotator to obtain only the Flag-labeled botulinum toxin type E light chain (Flag-Bont-E-LC) among the proteins expressed in the HEK293T cell line.

[0057] Antibodies, beads, and proteins were mixed with 2X sodium dodecyl sulfate (SDS) buffer, boiled at 100°C for 7 minutes to break the bonds and induce structural unfolding of the botulinum toxin light chain, and then separated by SDS-PAGE. The separated proteins were transferred to a polyvinylidene difluoride membrane, and then primary antibodies [anti-Flag (MBL), anti-HA (12CA5 hybridoma cell media), and anti-β-actin (Santa Cruz Biotechnology)] and 2% skim milk were added and reacted overnight at 4°C. After addition of anti-mouse secondary monoclonal antibody, the blot was developed onto a photosensitive film using an enhanced chemiluminescence (ECL) system.

[0058] (5) Confirmation of derived results and statistical analysis

[0059] Densitometric analysis was performed using Image J (National Institutes of Health), and Turkey was performed using GraphPad Prism version 5 (GraphPad Software). ANOVA was performed using one-way analysis to indicate significant differences.

[0060] 2. Experimental results

[0061] Agarose gel electrophoresis was performed on cells transfected with an expression vector for the light chain protein of wild-type botulinum toxin type E. Antibodies were used to specifically select only Flag-Bont-E-LC, and transfections were performed with increasing amounts of plasmid genes to confirm the exact protein size. Flag-Bont-E-LC was confirmed to have a size of approximately 51-54 kDa, and expression was confirmed to be induced in the cell line (Fig. 1).

[0062] To analyze the ubiquitination of the light chain of botulinum toxin type E, HEK293T cells were transfected with the plasmid genes pCS4-3Flag-Bont-E-LC WT and pRK5-HA-Ub. The ubiquitination degree of the light chain of botulinum toxin type E transfected into the cell line was confirmed by precipitation using an immunoprecipitation assay. As a result of treatment with MG132 reagent, the degree of ubiquitination increased, confirming that the light chain of botulinum toxin type E undergoes a degradation pathway mediated by the ubiquitin-proteasome (Fig. 2).

[0063] HEK293T cells were transfected with pCS4-3Flag-Bont-E-LC WT, pCS4-3Flag-Bont-E-LC(K288R), and pRK5-HA-Ub plasmid genes, and the degree of ubiquitination was compared using the same method as above. As a result, the degree of ubiquitination was significantly reduced in the case of mutants in which lysine at positions 62 and 288 was substituted with arginine compared to the control group (Fig. 3).

[0064] The same amount of pCS4-3Flag-Bont-E-LC WT and pCS4-3Flag-Bont-E-LC(K288R) plasmid genes were transfected into HEK293T cells, and after 24 hours, each cell medium was treated with cycloheximide (CHX) at a concentration of 100 μg / ml for 0, 6, and 12 hours, and then immunoblotting was performed (Fig. 4). The graph representing the immunoblotting results of Fig. 4 in numerical form is presented in Fig. 5.

[0065] As confirmed in FIGS. 4 and 5, when the 62nd lysine residue was replaced with arginine, the half-life of the mutant was significantly increased (i.e., 1.20 times at 6 hours) compared to the wild type, and also, when the 288th lysine residue was replaced with arginine, the half-life of the mutant was significantly increased (i.e., 1.30 times at 6 hours, 1.31 times at 12 hours) compared to the wild type.

[0066] The light chain variant of botulinum toxin type E according to the present invention exhibits improved resistance to degradation by the ubiquitin-proteasome, thereby exhibiting a significantly increased in vivo half-life. Therefore, the light chain variant of botulinum toxin type E according to the present invention can be usefully utilized in botulinum toxin-related products for use in cosmetic and / or therapeutic treatment or procedures using botulinum toxin.

Claims

1. A light chain mutant of botulinum toxin type E, comprising an amino acid sequence of sequence number 1, in which lysine at positions 3, 62, 85, 230, 281, 288, 342, 413, or 419 is substituted with arginine.

2. A light chain variant of botulinum toxin type E, wherein the light chain variant in paragraph 1 is composed of an amino acid sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17 or 19.

3. A nucleic acid comprising or including a polynucleotide sequence encoding a light chain variant of botulinum toxin type E according to paragraph 1.

4. A nucleic acid according to claim 3, wherein the polynucleotide sequence is composed of the base sequence of SEQ ID NO:

14.

5. A vector containing the nucleic acid of paragraph 4.

6. Cells transfected with the vector of clause 5.

7. A method for increasing the half-life of a light chain of botulinum toxin type E, comprising the step of replacing lysine at position 288 of the amino acid sequence of sequence number 1 with arginine.

8. A method for increasing the half-life of a light chain of botulinum toxin type E, comprising the step of replacing lysine at position 62 of the amino acid sequence of sequence number 1 with arginine.

9. Use of the light chain variant of botulinum toxin type E according to claim 1 or the nucleic acid according to claim 3 for use in a subject requiring cosmetic treatment or procedure with botulinum toxin.

10. Use of a light chain variant of botulinum toxin type E according to claim 1 or a nucleic acid according to claim 3 for use in a subject requiring therapeutic treatment or procedure with botulinum toxin.

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