KR20210063744A - Composition comprising polypeptide originating from amyloid precursor protein - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a food composition comprising a polypeptide derived from amyloid precursor protein (APP), wherein the peptide comprising the amino acid sequence of SEQ ID NO: 1 and consisting of 9 to 30 amino acid sequences inhibits the binding of MDGA1 and APP protein, thereby being able to be usefully used for preventing, treating or alleviating brain and nervous system diseases.

Description

Composition comprising polypeptide originating from amyloid precursor protein

It relates to a composition comprising a polypeptide derived from APP.

Through the recently developed Genome-wide Association Study (GWAS), various disease-associated genes have been identified from samples taken from patients with mental disorders. In particular, proteins present in the brain are affected by various neurological disorders such as autism, attention-deficit/hyperactivity disorder, ADHD, schizophrenia, mental retardation, and emotional disorder. Facts that are related to

These cranial nervous system diseases are often caused by irreversible pathological changes caused by damaged nerve cells, so treatment is not easy and the range of sequelae is wide. According to the Global Burden of Disease report by the World Health Organization (WHO), neurological disorders in the neuropsychiatric category and neurological sequelae due to disorders and injuries in other categories accounted for 6.3%. occupies a high disease burden index of These figures represent a higher disease burden than AIDS, malignancy and ischemic heart disease.

Under this technical background, various studies for preventing or treating diseases of the cranial nervous system are being conducted (Korean Patent Registration No. 10-1130220), but the situation is still insufficient.

One aspect is to include the amino acid sequence of SEQ ID NO: 1, and to provide a pharmaceutical composition for preventing or treating brain nervous system diseases comprising a peptide consisting of 9 to 30 amino acid sequences as an active ingredient.

Another aspect is to include the amino acid sequence of SEQ ID NO: 1, to provide a food composition for preventing or improving the brain nervous system disease comprising a peptide consisting of 9 to 30 amino acid sequence as an active ingredient.

Another aspect is to include the amino acid sequence of SEQ ID NO: 1, and to provide a method for preventing or treating a brain nervous system disease, comprising administering to an individual a peptide consisting of 9 to 30 amino acid sequence.

One aspect includes the amino acid sequence of SEQ ID NO: 1, and provides a pharmaceutical composition for preventing or treating a brain nervous system disease comprising a peptide consisting of 9 to 30 amino acid sequence as an active ingredient.

The "peptide" may include the amino acid sequence of SEQ ID NO: 1. In addition, the peptide may be composed of an arbitrary integer of 9 to 30 amino acid sequences. For example, the integer may be 9, 17, or 24. The peptide may include the amino acid sequence of SEQ ID NO: 2. In addition, the peptide may have a sequence of 17 to 25 amino acids. The peptide may be derived from amyloid precursor protein (APP). According to one embodiment, the peptide inhibits the binding of the MDGA1 (MAM Domain Containing Glycosylphosphatidylinositol Anchor 1) protein and the APP protein to prevent, treat, or improve brain nervous system diseases. The inhibition may be to inhibit binding between specific domains of each of MDGA1 and APP protein. The specific domain may be a MAM domain of MDGA1 and an extension domain of an APP protein.

In particular, it was known that the MDGA1 is highly correlated with schizophrenia and bipolar disorder through existing genome-wide sequencing (Kahler et al., 2008; Li et al., 2011), and the APP is Alzheimer's Since association with autism as well as dementia has been recently reported (Sokol et al., 2019), the peptide can be usefully used to treat these diseases.

The synthesis of the peptide may be performed according to a method generally used in peptide chemistry. General synthetic methods are described in "Peptide Synthesis", Interscience, New York, 1966; "The Proteins", Vol. 2, Academic Press Inc., New York, 1976; "Peptide Synthesis (Peptide Gosei)", Maruzen, 1975; "Fundamentals and Experiments of Peptide Synthesis (Peptide Gosei no Kiso to Jikken)", Maruzen, 1985; and "The sequence of Development of Pharmaceuticals (Zoku Iyakuhin no Kaihatsu)", Vol. 14, Peptide Synthesis (Peptide Gosei), Hirokawa Shoten, 1991, and international publications such as International Publication No. WO 99/67288. In addition, the peptide may be synthesized by known genetic engineering methods. For example, the vector into which the DNA encoding the peptide is inserted may be introduced into a suitable host cell to produce transformed cells, and thus produced in the transformed cells. The peptide can be obtained by collecting the peptides. In addition, the peptide according to one embodiment may be prepared in the form of a fusion protein cleaved using an appropriate protease. They may be used in the composition as such.

In the method for preparing the fusion protein, the polynucleotide encoding the peptide may be ligated to a frame with a polynucleotide encoding another protein or peptide, which is expressed in a host. (expression) can be inserted into an expression vector. Techniques known in the art can be used for this purpose. For the peptide fused with the peptide, FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6x His residues consisting of 6 histidines (His), 10x His, influenza hemagglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragments, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, alpha-tubulin (alpha- tubulin) fragment, a B-tag, and a known peptide such as a Protein C fragment can be used. In addition, to make a fusion protein, the peptide is glutathione-S-transferase (GST), influenza hemagglutinin (HA), immunoglobulin constant regions, beta-galactosidase (beta-galactosidase), or It is possible to link maltose-binding protein (MBP) and the like.

Accordingly, in another embodiment, a polynucleotide encoding the peptide, a recombinant vector containing the polynucleotide, or a recombinant cell containing the recombinant vector may be provided.

The term “vector” refers to a means for expressing a target gene in a host cell. For example, such vectors include viral vectors such as plasmid vectors, cosmid vectors or bacteriophage vectors, adenoviral vectors, retroviral vectors or adeno-associated viral vectors. Vectors that can be used as the recombinant vector are plasmids often used in the art (eg, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14 , pGEX series, pET series and pUC19, etc.), phage (eg, λgt4λB, λ-Charon, λΔz1 and M13, etc.) or viruses (eg, SV40, etc.).

In the recombinant vector, the polynucleotide encoding the protein complex may be operably linked to a promoter. The term “operatively linked” refers to a functional linkage between a nucleotide expression control sequence (eg, a promoter sequence) and another nucleotide sequence. Such regulatory sequences may be "operably linked" to regulate the transcription and/or translation of other nucleotide sequences.

The recombinant vector is typically constructed as a vector for cloning or a vector for expression. The expression vector may be a conventional one used in the art to express foreign proteins in plants, animals, or microorganisms. The recombinant vector can be constructed through various methods known in the art.

The recombinant vector can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector used is an expression vector and a prokaryotic cell is a host, the recombinant vector is a strong promoter capable of propagating transcription (eg, pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. In the case of a eukaryotic cell as a host, the recombinant vector is an origin of replication that operates in eukaryotic cells, including, but not limited to, the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication, or the BBV origin of replication. it's not going to be In addition, a promoter derived from the genome of a mammalian cell (eg, a metallotionine promoter) or a promoter derived from a mammalian virus (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter or tk promoter of HSV) may be used, and may have a polyadenylation sequence as a transcription termination sequence.

The recombinant cell may be obtained by introducing the recombinant vector into an appropriate host cell. The host cell is a cell capable of cloning or expressing the recombinant vector stably and continuously, and any host cell known in the art may be used. For example, prokaryotic cells include E. coliJM109, E. coliBL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis. Bacillus sp. strains such as, Salmonella typhimurium, Serratia marcesens or various enterobacteriaceae and strains such as Pseudomonas. For example, in the case of transformation into eukaryotic cells, as a host cell, yeast (Saccharomyce cerevisiae), insect cell, plant cell or animal cell such as Sp2/0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines and the like can be used.

The peptide has improved chemical stability, improved pharmacokinetic or pharmacological profile (half-life, absorption, potency, potency, etc.), altered specificity (e.g., broad spectrum of biological activity), reduced antigenicity, transport to the target site, etc. It may include specific modifications to obtain.

For example, the peptide may have a protecting group attached to its N- or C-terminus. In one embodiment, the N-terminus of the peptide is an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group group), a stearyl group, a butoxycarbonyl group, an aryloxycarbonyl group, and polyethylene glycol (PEG) may be bonded to any one protecting group selected from the group consisting of; ; and/or the C-terminus of the peptide may be bonded to any one protecting group selected from the group consisting of an amino group (-NH2), a tertiary alkyl group, and an azide (-NHNH2). .

In addition, the peptide is modified at a specific position, such as pegylation, sialylation, glycosylation, deglycosylation, amino acid insertion, deletion, or substitution, dextran conjugation, carboxymethyl-dextran (CM-dex) Conjugation, diethylaminoethyl-dextran (DEAE-dex) may be modified one or more selected from the group selected from conjugation and cyclization, galactose or to improve affinity for receptor-mediated endocytosis Mannose may be conjugated. The peptide may optionally further include a targeting sequence, a tag, a labeled residue, an amino acid sequence prepared for the purpose of increasing half-life or peptide stability.

The brain nervous system disease may include a degenerative brain disease or a mental disease.

The term “degenerative brain disease” refers to a neurodegenerative disease, which may refer to a disease caused by deterioration of the structure or function of brain tissue or brain cells as aging progresses. The degenerative brain diseases include stroke, cognitive dysfunction, Alzheimer's disease, Dementia with Lewy bodies, frontotemporal dementia, Parkinson's disease, Creutzfeldt-Jacob. Disease (Creutzfeldt-Jakob disase; CJD), Huntington's disease (Huntington's disase), multiple sclerosis (multiple sclerosis), Gillian-Barre syndrome (Gillain-Barre Syndrome; GBS) may be selected from the group consisting of.

The mental disorder may include any mental disorder associated with MDGA1 or APP. Specifically, examples of the mental disorder include bipolar disorder, depression, hyperactivity, attention deficit disorder, autism, post-traumatic stress disorder (PTSD), anxiety disorder, sleep disorder, panic disorder, intellectual disorder, memory loss, drug addiction, schizophrenia , may be selected from the group consisting of obsessive compulsive disorder, grandiose delusion, personality disorder, alcoholism, and bipolar disorder.

As used herein, the term "preventing" or "prevention" refers to preventing a disease, e.g., a disease in an individual who may be predisposed to the disease, condition or disorder but has not yet experienced or exhibited the pathology or signs of the disease; To prevent a condition or disorder.

The term “treating” or “treatment” refers to inhibiting a disease, e.g., inhibiting a disease, condition or disorder in an individual experiencing or exhibiting the pathology or symptom of the disease, condition or disorder, i.e., pathology and/or or preventing the further occurrence of the sign, or ameliorating the disease, e.g., ameliorating the disease, condition or disorder in an individual experiencing or exhibiting the pathology or symptom of the disease, condition or disorder, i.e., pathology and/or or reversing the symptoms, such as reducing disease severity.

The composition may include a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" refers to an inorganic or organic acid addition salt of a compound. The pharmaceutically acceptable salt may be a salt that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound. The inorganic acid salt may be hydrochloride, bromate, phosphate, sulfate, or disulfate. The organic acid salt is formate, acetate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, besylate, camsylate, edicyl salt, trichloroacetic acid, trifluoro Acetate, benzoate, gluconate, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, galacturonic acid, embonate, glutamate, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or aspartate. The metal salt may be a calcium salt, a sodium salt, a magnesium salt, a strontium salt, or a potassium salt.

The pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier may be meant to include excipients, diluents or adjuvants. The carrier may be a stabilizing agent for offsetting the physical and chemical instability of the amino acid sequence. The carrier may be a protein, carbohydrate, cyclodextrin, surfactant, polyhydroxylated alcohol, antioxidant, chelating agent or inorganic salt. The protein may be albumin or gelatin. The carbohydrate may include glucose, sucrose, lactose, lactulose, sorbitol, maltose, dextran, glycerol, mannitol, xylitol, erythritol, maltitol, trehalose, or starch. The cyclodextrin may consist of 6, 7 and 8 sugars, and may be substituted with dimethyl, hydroxypropyl or sulfobutyl ether. The surfactant may be one or more selected from polysorbate 20, poloxamer 407, and poloxamer 188. The polyhydroxylated alcohol includes, but is not limited to, polyethylene glycol PEG 400, 600, 1000, 1500, 4000, 6000, 8000. The antioxidant may be butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, ascorbic acid, platinum, catalase, sodium thiosulfate or sodium sulfite. The chelating agent may include iron, copper, calcium, manganese, and zinc components, and may be disodium ethylenediaminetetraacetic acid (DSEDTA), citric acid, or thioglycolic acid. The carrier may be appropriately selected alone or in combination according to formulation by those skilled in the art.

The pharmaceutical composition may be prepared in any formulation according to a conventional method, respectively. In the pharmaceutical composition, the solid preparation for oral administration may be a tablet, pill, powder, granule, or capsule. The solid formulation may further include an excipient. The excipient may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, the solid formulation may further include a lubricant such as magnesium stearate or talc. In the pharmaceutical composition, the oral liquid formulation may be a suspension, an oral solution, an emulsion, or a syrup. The liquid formulation may contain water or liquid paraffin. The liquid formulation may include an excipient, a wetting agent, a sweetening agent, a flavoring agent, or a preservative. In the pharmaceutical composition, the preparation for parenteral administration may be a sterile aqueous solution, non-aqueous solution, suspension, emulsion, freeze-dried or suppository. Non-aqueous solvents or suspending agents may include vegetable oils or esters. The vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil. The ester may be, for example, ethyl oleate. The base of the suppository may be witepsol, macrogol, tween 61, cacao butter, laurin, or glycerogelatin.

The composition may be conveniently administered orally, but may be administered parenterally for reasons such as low absorption of peptides, sensitivity to enzymes, rapid clearance, or chemical instability. Accordingly, the composition according to an embodiment is an intravenous injection, a subcutaneous injection, an intramuscular injection, an intraarterial injection, an intraperitoneal injection, an intradermal injection. ), or may be administered by intraosseous infusion, and may be a lyophilized powder to be reconstituted immediately before administration. The administration may also occur by nasal, pulmonary, buccal, sternal, rectal, vaginal, ocular or transdermal routes. The injection site and depth can be appropriately determined by those skilled in the art in the thigh, abdomen, or arm, etc. in order to obtain appropriate bioavailability. The pharmaceutical composition may be administered systemically or locally, alone or in combination with other pharmaceutically active compounds.

The composition may include a polymer for desirable parenteral delivery. The polymer is biocompatible and has reasonable mechanical strength, and may be appropriately selected by those skilled in the art. The polymer may be a biodegradable polymer or a non-biodegradable polymer, and the biodegradable polymer may be a synthetic polymer or a natural polymer. The synthetic polymer may be a polyamide, polyanhydride, polyester, phosphorous-based polymer, polyacetal, poly(cyano acrylate), polydihydropyran, polyorthoester, or polyurethane. The natural polymer may be a polysaccharide or a protein. The non-biodegradable polymer may include a cellulose derivative, silicone, ethyl vinyl acetate, polyvinyl pyrrolidine, poloxamer or poloxamine. The above polymers may be used alone or in combination depending on the purpose.

The composition may utilize a parenteral delivery system for desired parenteral delivery. The parenteral delivery system may be one or more selected from the group consisting of microspheres, implants, liposomes, nanoparticles, hydrogels, microemulsions, nanoemulsions, and pumps. The microsphere system may use any one of spray drying, double emulsion, phase separation-coacervation, emulsion-desolvation method, emulsion-heat denaturation, and spray freeze-drying.

The pharmaceutical composition may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level depends on the patient's type, severity, drug activity, and drug Sensitivity, administration time, administration route and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field may be determined. For example, the dosage of the pharmaceutical composition is about 0.001 mg/kg to about 100 mg/kg, 0.005 mg/kg to about 100 mg/kg, 0.01 mg/kg to about 100 mg/kg, 0.05 for an adult. mg/kg to about 50 mg/kg, 0.1 mg/kg to about 50 mg/kg, 0.1 mg/kg to about 30 mg/kg, about 0.5 mg/kg to about 10 mg/kg, or about 0.5 mg/kg to about 1 mg/kg. Also, the administration is once a day, twice a day, three times a day, four times a day, six times a day, or once a week, once every two weeks, once every three weeks, or It may be administered once every 4 weeks or once a year. However, since it may increase or decrease depending on the route of administration, the severity of obesity, sex, weight, age, etc., the dosage is not limited in any way.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors, and this can be easily determined by a person skilled in the art.

Another aspect includes the amino acid sequence of SEQ ID NO: 1, and provides a food composition for preventing or improving brain nervous system disease comprising a peptide consisting of 9 to 30 amino acid sequence as an active ingredient.

Among the terms or elements mentioned in the description of the pharmaceutical composition, the same as those already mentioned are the same as described above.

As used herein, the term “improvement” refers to any action that at least reduces a parameter associated with an abnormal condition, for example, the severity of a symptom. In this case, the food composition may be used simultaneously with or separately from a drug for treatment before or after the onset of the disease in order to improve the brain nervous system disease.

The food composition may include additional optional ingredients in addition to containing the peptide as an active ingredient. For example, the food composition may contain various flavoring agents such as beverages or natural carbohydrates as additional ingredients. Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; or polysaccharides such as conventional sugars such as dextrin and cyclodextrin, or sugar alcohols such as xylitol, sorbitol, and erythritol. In addition, the composition may advantageously use flavoring agents such as natural flavoring agents (taumatine, stevia extract (eg rebaudioside A, glycyrrhizin, etc.) or synthetic flavoring agents (saccharin, aspartame, etc.) The ratio of the natural carbohydrate can be appropriately determined by the selection of a person skilled in the art.

In addition, the food composition includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof , organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and may contain at least one selected from the group consisting of carbonation agents used in carbonated beverages. These components may be used independently or in combination. The proportion of these additives can also be appropriately selected by those skilled in the art.

The food composition may be used in combination with other foods or food ingredients by adding the active ingredient to food as it is, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (for prevention or improvement). In general, in the production of food or beverage, the composition of the present invention is added in an amount of 60% by weight or less, preferably 40% by weight or less, based on the raw material. However, in the case of long-term ingestion for health and hygiene or health control, the amount may be less than or equal to the above range.

Another aspect includes the amino acid sequence of SEQ ID NO: 1, and provides a method for preventing or treating a brain nervous system disease, comprising administering to a subject a peptide consisting of 9 to 30 amino acid sequence.

Among the terms or elements mentioned in the description of the pharmaceutical composition or food composition, the same as those already mentioned are the same as described above.

As used herein, the term "subject" or "patient" means any animal, including mammals, e.g., mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses or primates and humans. do.

The peptide according to one aspect may be usefully used to prevent, improve, or treat various neurological diseases in which association with MDGA1 and APP protein has been reported by inhibiting the binding of MDGA1 and APP protein. In particular, it may exhibit an excellent effect on diseases associated with MDGA1 or APP protein in the brain.

1 is a result of analyzing the MDGA1 expression pattern in hippocampal neuronal culture cells using the MDGA1 antibody JK030 and antibodies against various synaptic marker proteins.
2 is a result of confirming a portion of the sequence of the APP protein using mass spectrometry.
3 is a result of confirming the size of the APP protein using Coomassie staining.
Figure 4 is the result of the total ion chromatogram (Total ion chromatogram) and the extracted ion chromatogram (extracted ion chromatogram) for a specific APP sequence.
5 is a surface labeling analysis result confirming whether MDGA1 and MDGA2 (MAM Domain Containing Glycosylphosphatidylinositol Anchor 2) bind to APP, APLP1, APLP2 and Nlgn2.
6 is a graph quantifying the results of surface labeling analysis confirming whether MDGA1 and MDGA2 bind to APP, APLP1, APLP2 and Nlgn2.
7 shows various deletion constructs for MDGA1 and MDGA2 and their binding to APP protein.
8 is a result of measuring the binding affinity between the MAM domain of MDGA1 and the APP protein.
9 is a surface labeling analysis result confirming whether the extension domain of APP binds to MDGA1, MDGA1 Ig1-3 domain, MDGA1 Ig1-6 domain, MDGA1 FNIII domain, MDGA1 MAM domain, MDGA2 or GABAbla.
10 is a graph quantifying the results of surface labeling analysis confirming whether the extension domain of APP binds to MDGA1, MDGA1 Ig1-3 domain, MDGA1 Ig1-6 domain, MDGA1 FNIII domain, MDGA1 MAM domain, MDGA2 or GABAbla.
11 shows various deletion constructs of the APP protein.
12 shows a portion of the sequence of the APP extension domain. A red box indicates a sequence that exists only in APP.
13 is a surface labeling analysis result confirming whether MDGA1 binds to various domains of APP.
14 is a graph quantifying the results of surface labeling analysis confirming whether MDGA1 binds to various domains of APP.
15 is a surface labeling analysis result to confirm whether the binding of MDGA1 and APP is calcium-dependent.
16 is a graph quantifying the results of surface labeling analysis to determine whether the binding of MDGA1 and APP is calcium-dependent.
17 is an immunoblotting result confirming whether each protein binds in HEK293T cells, P 14 brain synaptosome, and P70 brain synaptosome.
18 is a schematic diagram of a synthetic peptide design for determining the minimum unit sequence binding to MDGA1 among the amino acid sequences of the APP extension domain.
19 is a surface labeling analysis result confirming whether the 17mer, 9mer, 8mer, 7mer peptides designed from APP inhibit the binding of MDGA1 and APP.
20 is a graph quantifying the results of surface labeling analysis confirming whether the 17mer, 9mer, 8mer, and 7mer peptides designed from APP inhibit the binding of MDGA1 and APP.
21 is a confocal microscopy image of the structural change of inhibitory synapses using marker proteins after infection with several lentiviruses in neural culture cells.
22 shows the effect of knockdown of the APP protein on the inhibitory synapse quantitatively by using a confocal microscope image analyzing the structural change of the inhibitory synapse using a marker protein after infection with several lentiviruses in neural culture cells. This is the graph shown.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these examples.

Example 1. Identification of MDGA1 protein localization in inhibitory synapses

The expression distribution and location of MDGA1 protein in neurons were analyzed using antibodies against MDGA1 protein and various synaptic marker proteins.

Specifically, the immunostaining experiment was performed 12 days after the preparation of the neural culture cells (DIV 12). The neuronal culture cell specimens were fixed with 4% (w/v) paraformaldehyde/4% (w/v) sucrose at room temperature. Thereafter, the sample was permeabilized with a 0.2% Triton X-100 solution in phosphate-buffered saline (PBS). After washing twice with PBS, immersion in blocking solution (DMEM, 20 mM HEPES-NaOH pH 7.4, 4% bovin serum albumin (BSA) and 1% horse serum) for 30 minutes put After that, VGLUT1 (Synaptic Systems, RRID: AB_887880), GAD67 (clone 1G10.2; Millipore, RRID: AB_2278725), and mouse monoclonal anti-MAP2 (clone HM-2; Sigma) were treated with MDGA1 antibody at room temperature for 1 hour. reacted while Thereafter, secondary antibodies corresponding to the corresponding antibodies were reacted at room temperature for 1 hour. Each image was acquired by photographing with a confocal microscope LSM700.

As a result, as shown in FIG. 1 , the position of the MDGA1 antibody seemed to coincide with the position of the inhibitory synaptic marker protein (GAD67) antibody, but did not match the position of the excitatory synaptic marker protein (VGLUT1) antibody. In addition, it was confirmed that the MDGA1 protein was mainly expressed in the nerve cell body and dendrites.

Example 2. Confirmation of interaction between MDGA1 protein and APP protein

2-1. mass spectrometry

In order to identify a protein that additionally binds to MDGA1, the MDGA1 recombinant protein to be used for mass spectrometry was analyzed.

Specifically, MDGA1 recombinant protein and an IgC Empty vector were synthesized as a negative control in HEK293T cells. For each reaction, 10 μg of recombinant protein was used, and 2 mg of P14 mouse P2 lysate was reacted at 4° C. overnight. After washing with PBS three times, electrophoresis was performed on an 8% polyacrylamide gel containing sodium dodecyl sulfate (SDS). Then, Coomassie staining was performed to confirm a band. After that, it was cut according to the protein size and the sample was prep.

As a result, as shown in FIG. 2 , TTSTATTTTTTESVEEVVR and VESLEQEAANER were identified as sequences of the APP protein, and as shown in FIG. 3 , it was found that the APP protein was mainly detected in a band with a size of 100 kDa.

2-2. Total ion chromatogram and extracted ion chromatogram analysis

Ion chromatography is a method of moving a liquid sample and passing a mixture dissolved during the movement through a separation tube made of ion exchange resin to detect the elution state of the sample component with a conductivity detector or an engineered detector. Through this experiment, each sample cut according to the size of each protein was analyzed individually. If there is a protein that interacts with the MDGA1 recombinant protein, it will be included in the sample. Therefore, if a relatively large amount of protein is detected in the MDGA1 recombinant protein sample compared to the control, it may mean that the protein directly or indirectly interacts with the MDGA1 recombinant protein. Total ion chromatogram and extracted ion chromatogram analysis were performed for this purpose.

As a result, as shown in FIG. 4, the results for TTSTATTTTTTESVEEVVR and VESLEQEAANER, which are the APP protein sequences derived as a result of the analysis, are shown, and the results of measuring the concentration of the APP protein component in a chromatogram are also shown.

2-3. Surface Labeling Analysis

It was attempted to identify a protein to which MDGA1 protein selectively binds through surface labeling analysis using HEK293T cells.

Specifically, APP, APLP1, APLP2 expression vectors to which HA epitope is attached were transfected into HEK293T cell line. After 48 hours, the recombinant MDGA1 protein was contacted with the transfected HEK293T cell line at a concentration of 10 μg/mL. After reacting at 37 °C for 2 hours, it was fixed with 4% paraformaldehyde solution at room temperature. After that, it was reacted with a blocking solution (DMEM, 20 mM HEPES-NaOH pH 7.4, 4% BSA and 1% horse serum) at room temperature for 30 minutes. Then, it was additionally stained with human IgG Cy3 antibody at room temperature for 1 hour, and permeabilized with 0.2% Triton X-100 in PBS at room temperature for 10 minutes. The HA-ms (SC7392) antibody was reacted for 1 hour to confirm the expression of APP, APLP1, and APLP2 proteins in the HEK293T cell line. After washing twice with PBS, the reaction was performed with the ms-FITC antibody for 1 hour, and then the sample was mounted. Each image was acquired by shooting with a confocal microscope LSM700.

As a result, as shown in FIGS. 5 and 6 , it was confirmed that MDGA1 selectively binds to APP, APLP2 and Nlgn2, and MDGA2 selectively binds to Nlgn2.

Example 3. Identification of domains of MDGA1 protein binding to APP protein

3-1. Confirmation of binding affinity between the MAM domain of MDGA1 and the APP protein

7 shows a deletion construct of MDGA. Scatchard analysis was performed to determine which domain of MDGA1 binds to APP.

Specifically, 48 well HEK293T cells were transfected with HA-attached APP and two days later, Ig-tagged with 800 nM, 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM or 0 nM. Recombinant MDGA1 was contacted. It was then incubated at 37 °C for 2 h. It was fixed with 4% paraformaldehyde at room temperature and blocked with a blocking solution (DMEM, 20 mM HEPES-NaOH pH 7.4, 4% BSA and 1% horse serum) at room temperature for 30 minutes. HRP-IgG antibody was contacted on a 1:50,000 basis and then incubated for 1 hour at room temperature. After washing 3 times with PBS, 3,3',5,5'-tetramethylbenzidine (3,3',5,5'- tetramethylbenzidine) and peroxidase (peroxidase) were mixed at a ratio of 9: 1, 150 μL was added, and incubated for 15 minutes. After stopping the reaction with the same amount of 0.5 M sulfuric acid, it was measured in the 450 nm wavelength range.

As a result, as shown in FIG. 8 , it was confirmed that the Kd value between APP and MDGA1 was 41.3 ± 3.6 nM.

3-2. Surface Labeling Analysis

Surface labeling analysis was performed to determine the domain of the MDGA1 protein that binds to the APP recombinant protein.

Specifically, the MDGA1 protein consists of six Ig domains, an fnIII domain, and a MAM domain. The deletion construct was cloned into the pDisplay vector, optionally divided into an Ig1-3 domain, an Ig4-6 domain, an fnIII domain, and a MAM domain. After transfection of the prepared MDGA1 deletion construct into HEK293T cells, respectively, in _{DMEM medium containing 20 mM HEPES pH 7.4, 2 mM CaCl 2} , and 2 mM MgCl ₂ , recombinant IgC-tagged APP extended (Extended ) protein was contacted to each of the above HEK293T cells at a concentration of 10 μg/mL. Incubated at 37 °C for 2 h and fixed with 4% paraformaldehyde at room temperature. Blocking was performed with a blocking solution (DMEM, 20 mM HEPES-NaOH pH 7.4, 4% BSA and 1% horse serum) at room temperature for 30 min. Staining was performed with human IgG Cys antibody at a ratio of 1:500 at room temperature for 1 hour. After that, it was permeated with 0.2% Triton X-100 in PBS at room temperature for 10 minutes. To stain APP, APLP1, and APLP2 attached to HEK293T cells, staining was performed at 1:400 HA-ms (SC7392) for 1 hour. After washing twice with PBS, ms-FITC was stained 1:150 for 1 hour and mounted. Each image was taken with a confocal microscope LSM700.

As a result, as shown in FIGS. 9 and 10 , it was confirmed that the APP protein was bound to the MDGA1 protein, and in particular, selectively bound only to the MDGA1 MAM domain. This indicates that only the MAM domain is required for the MDGA1 protein to bind to the APP protein.

Example 4. Identification of domains of APP protein binding to MDGA1 protein

11 shows the deletion construct of APP. In addition, FIG. 12 shows in detail the sequence of the extension domain of APP, and the sequence present only in APP is indicated by a red box. It was attempted to determine which of the various domains of APP binds to the MDGA1 protein.

Specifically, APP includes an E1 domain including GFLD and CuBD, a flexible domain including Ex and ACID, and an E2 domain. After constructing deletion constructs for each domain, it was cloned into the pDisplay vector. Each of the prepared APP deletion constructs was transfected into HEK293T cells. After 48 hours, in DMEM medium containing 20 mM HEPES pH 7.4, 2 mM CaCl ₂ , and 2 mM MgCl ₂ , recombinant IgC-tagged MDGA1 protein was contacted to each of the HEK293T cells at a concentration of 10 μg/mL. Incubated at 37 °C for 2 h and fixed with 4% paraformaldehyde at room temperature. Blocking was performed with a blocking solution (DMEM, 20 mM HEPES-NaOH pH 7.4, 4% BSA and 1% horse serum) at room temperature for 30 min. Staining was performed for 1 hour at room temperature with human IgG Cys antibody at 1:500. After that, it was permeabilized with 0.2% Triton X-100 in PBS at room temperature for 10 min. To stain APP, APLP1, and APLP2 attached to HEK293T cells, 1:400 HA-ms (SC7392) was used for 1 hour. After that, it was washed twice with PBS, stained with ms-FITC at a ratio of 1:150 for 1 hour, and then mounted. Each image was taken with a confocal microscope LSM700.

As a result, as shown in FIGS. 13 and 14 , it was confirmed that the MDGA1 protein selectively binds only to the extension domain of APP. This indicates that only the extension domain is required for the APP protein to bind to the MDGA1 protein.

Example 5. Ca upon binding of APP and MDGA1 proteins ²⁺ Dependency Check

EGTA, which removes calcium by chelating, was used to determine whether calcium was required for binding between APP protein and MDGA1 protein.

Specifically, the APP constructs were transfected into HEK293T cells, respectively. After 48 hours, in DMEM medium containing 20 mM HEPES pH 7.4, 2 mM CaCl ₂ , and 2 mM MgCl ₂ , recombinant IgC-tagged MDGA1 protein at a concentration of 10 μg/mL was contacted with the HEK293T cells. In this process ^{, EGTA, a Ca 2+} chelator, was co-treated with 10 mM. Incubated at 37 °C for 2 hours and fixed with 4% paraformaldehyde at room temperature. Blocking was performed with a blocking solution (DMEM, 20 mM HEPES-NaOH pH 7.4, 4% BSA and 1% horse serum) at room temperature for 30 min. Staining was performed with human IgG Cys antibody at 1:500 for 1 hour at room temperature. After that, it was permeabilized with 0.2% Triton X-100 in PBS at room temperature for 10 min. To stain APP, APLP1, and APLP2 attached to HEK293T cells, 1:400 HA-ms (SC7392) was used for 1 hour. After washing twice with PBS, ms-FITC was stained with 1:150 for 1 hour, and then mounted. Each image was taken with a confocal microscope LSM700.

As a result, as shown in FIGS. 15 and 16 , it was confirmed that the binding of APP and MDGA1 was selectively inhibited by the addition of EGTA chelating ^{Ca 2+ .} From these results, it was found that the APP protein and the MDGA1 protein bind depending on ^{Ca 2+ .}

Example 6. Confirmation of APP and MDGA1 complex formation in the brain

A variety of neurological disorders are caused by problems with APP or MDGA1. Therefore, it was attempted to confirm whether APP and MDGA1 proteins actually bind in brain tissue.

Specifically, since MDGA1 protein is mainly expressed in the hippocampus of the actual mouse brain, synaptosomal fractions prep was performed in P70 and P14 mice. First, the recombinant Ig-tagged MDGA1 protein and the negative control IgC empty vector were reacted with 10 μg of sepharose A beads for 2 hours. Sepharose A beads bound to each protein were reacted overnight with 1 mg synaptosome sections prepared in advance. After washing twice with PBS, it was loaded on an 8% gel. Thereafter, blotting was performed with antibodies to APP (22C11), APLP1 (Cat 12305-2-AP), APLP2 (Cat15041-1-AP), and NL2 (SYSY129202).

As a result, as shown in FIG. 17 , it was confirmed that the binding of APP and MDGA1 protein also occurred in brain tissue. Specifically, FIGS. 17A and 17B show that in HEK293T cells, MDGA1 selectively binds to APP and Nlgn2, and MDGA2 does not bind to APP, but selectively binds only to Nlgn2. 17C and D show that MDGA1 protein binds weakly to APP protein in P14 brain synaptosome specimens, and both MDGA1 and MDGA2 bind strongly to Nlgn2 protein. 17E and F show that MDGA1 protein forms a complex with APP, APLP2, and Nlgn2 proteins in P70 brain synaptosome specimens.

Example 7. Identification of peptides selectively inhibiting APP and MDGA1 protein binding

18 shows the sequence of the APP extension domain. On the other hand, if the binding of APP and MDGA1 protein is selectively inhibited, it may have an effective effect on brain nervous system diseases. Therefore, it was attempted to identify a peptide capable of inhibiting the binding of APP and MDGA1 proteins among the sequences.

Specifically, as shown in Figure 18, the APP extension domain was divided into 17mer (DDSDVWWGGADTDYADG), 9mer (DDSDVWWGG), 8mer (ADTDYADG), and 7mer (SEDKVVE) in detail, and purified to 70% purity. APP constructs were transfected into HEK293T cells, respectively. After 48 hours, in DMEM medium containing 20 mM HEPES pH 7.4, 2 mM CaCl ₂ , and 2 mM MgCl ₂ , recombinant IgC-tagged MDGA1 protein was mixed at a concentration of 10 μg/mL and the purified peptides were mixed together in a concentration-dependent manner. The mixture was incubated at 4 °C for 2 hours. Thereafter, the mixed reagents were contacted with the HEK293T cells. Incubated at 37 °C for 2 hours and fixed with 4% paraformaldehyde at room temperature. Blocking was performed with a blocking solution (DMEM, 20 mM HEPES-NaOH pH 7.4, 4% BSA and 1% horse serum) at room temperature for 30 min. Staining was performed with human IgG Cys antibody at 1:500 for 1 hour at room temperature. After that, it was permeabilized with 0.2% Triton X-100 in PBS at room temperature for 10 min. To stain APP, APLP1, and APLP2 attached to HEK293T cells, 1:400 HA-ms (SC7392) was used for 1 hour. After that, it was washed twice with PBS, stained with ms-FITC 1:150 for 1 hour, and then mounted. Each image was taken with a confocal microscope LSM700.

As a result, as shown in FIG. 19 , it was confirmed that when the 17 mer or 9 mer shown in FIG. 18 was treated, the binding of APP and MDGA1 protein was effectively inhibited. In addition, as shown in FIG. 20 , as a result of quantitatively confirming whether the binding was inhibited, it was found that the 17 mer had a very good effect of inhibiting the binding of APP and MDGA1 proteins.

Example 8. Confirmation of effect of APP protein on inhibitory synaptic structure

The purpose of this study was to determine the effect of the APP protein on the inhibitory synapses through neural culture cells.

Specifically, L315 alone (control), shAPP, and shAPLP2 lentivirus were contacted to hippocampal neurons (DIV4) cultured for 4 days. Staining was performed at 12 days of culture (DIV 12) and fixed with 4% (w/v) paraformaldehyde and 4% (w/v) sucrose at room temperature. It was then permeabilized with 0.2% Triton X-100 in PBS. After washing twice with PBS, blocking solution (DMEM, 20 mM HEPES-NaOH pH 7.4, 4% BSA and 1% horse serum) was blocked for 30 minutes. After that, GAD67 (clone 1G10.2; Millipore, RRID: AB_2278725) was stained for 1 hour at room temperature at 1:100. Then, it was stained with Cys-ms at a ratio of 1:500. Each image was taken with a confocal microscope LSM700.

As a result, as shown in Figure 21, it was possible to observe the structural change of the inhibitory synapse, as a result of quantitative analysis, as shown in Figure 22, when the APP is knocked down (Knockdown), the inhibitory synapse effectively found that it could be suppressed.

<110> Daegu Gyeongbuk Institute of Science and Technology <120> Composition comprising polypeptide originating from amyloid precursor protein <130> PN129566KR <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> originating from amyloid precursor protein <400> 1 Asp Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp 1 5 10 15 Gly <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> originating from amyloid precursor protein <400> 2 Asp Asp Ser Asp Val Trp Trp Gly Gly 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> originating from amyloid precursor protein <400> 3 Ala Asp Thr Asp Tyr Ala Asp Gly 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> originating from amyloid precursor protein <400> 4 Ser Glu Asp Lys Val Val Glu 1 5

Claims (10)

A pharmaceutical composition for preventing or treating neurological diseases comprising the amino acid sequence of SEQ ID NO: 1 and comprising a peptide consisting of 9 to 30 amino acid sequences as an active ingredient. The pharmaceutical composition according to claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 2 and consists of 17 to 25 amino acid sequences. The method according to claim 1, wherein the N-terminal of the peptide is an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group ), stearyl group (stearyl group), butoxycarbonyl group (butoxycarbonyl group), aryloxycarbonyl group (allyloxycarbonyl group) and polyethylene glycol (polyethylene glycol; PEG) any one selected from the group consisting of a protecting group bonded to the pharmaceutical enemy composition. The method according to claim 1, wherein the C-terminal of the peptide is an amino group (amino group, -NH ₂ ), a tertiary alkyl group (tertiary alkyl group) and azide (azide, -NHNH ₂ ) any one protecting group selected from the group consisting of combined pharmaceutical composition. The pharmaceutical composition according to claim 1, wherein the brain nervous system disease is a degenerative brain disease or a mental disease. The method according to claim 5, wherein the degenerative brain disease is stroke, cognitive dysfunction, Alzheimer's disease, Dementia with Lewy bodies, frontotemporal dementia, Parkinson's disease , Creutzfeldt-Jakob disease (CJD), Huntington's disease (Huntington's disase), multiple sclerosis (multiple sclerosis), Gillian-Barre syndrome (Gillain-Barre Syndrome; GBS) selected from the group consisting of a pharmaceutical composition. 6. The method of claim 5, wherein the mental disorder is bipolar disorder, depression, hyperactivity, attention deficit, autism, post-traumatic stress disorder (PTSD), anxiety disorder, sleep disorder, panic disorder, intellectual disorder, memory loss, drug addiction, schizophrenia , Obsessive compulsive disorder, megalomaniac, personality disorder, alcoholism, and a pharmaceutical composition selected from the group consisting of bipolar disorder. The composition of claim 1, wherein the peptide inhibits the binding of amyloid precursor protein (APP) and MDGA1 (MAM Domain Containing Glycosylphosphatidylinositol Anchor 1). A food composition for preventing or improving brain nervous system diseases, comprising the amino acid sequence of SEQ ID NO: 1, and comprising a peptide consisting of 9 to 30 amino acid sequences as an active ingredient. The food composition of claim 9, wherein the brain nervous system disease is a degenerative brain disease or a mental disease.

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