An MDGA2-targeting peptide and its application

Abstract

This invention discloses an MDGA2-targeting polypeptide and its applications, which consists of 8-55 consecutive amino acids of the RPS23RG1 protein and contains QHNSX1CMR, where X1 is F or L. The MDGA2-targeting polypeptide and fusion protein of this invention can significantly inhibit the lysosomal degradation of MDGA2, thereby increasing MDGA2 levels. Therefore, it can be used to treat diseases associated with low MDGA2 activity (such as autism and other neurological disorders and gastric cancer), and has significant clinical value.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to an MDGA2-targeting polypeptide and its applications. Background Technology

[0002] Autism spectrum disorder (ASD) is a neurological disorder that occurs early in childhood. Clinical symptoms primarily include social impairment, restricted interests, and repetitive, stereotyped behaviors. Some patients also experience complications such as anxiety, depression, intellectual disability, and epilepsy, severely impacting children's development and mental health. However, current medications cannot completely cure ASD; they can only improve patients' symptoms to a very small extent. Therefore, effective preventive and treatment measures are urgently needed to address the health problems caused by autism.

[0003] ASD is primarily caused by gene mutations. Genetic studies have identified multiple genes involved in ASD, most of which (such as SHANK, NRXN, and NLGN) encode synaptic proteins, indicating that synaptic dysfunction is a major cause of ASD. Recently, loss-of-function and missense mutations in the MDGA2 gene have been found in ASD patients. The human MDGA2 gene is located on chromosome 14. Its encoded protein has 956 amino acids. As a vertebrate-specific immunoglobulin superfamily protein, MDGA2 contains six extracellular immunoglobulin-like (Ig) domains, one fibronectin type III (FNIII) domain, one receptor protein tyrosine phosphatase (MAM) domain, and a glycosylphosphatidylinositol (GPI) anchor. MDGA2 is highly conserved in humans and mice, widely expressed in the nervous system, and primarily localized to the postsynaptic membrane via the GPI anchor. Studies have found that MDGA2 interacts with the neural connective protein NLGN to jointly regulate synaptic function, and MDGA2 deficiency leads to increased excitatory synaptic activity and ASD-like behavior in mice. . Summary of the Invention

[0004] The purpose of this invention is to provide an MDGA2-targeting polypeptide.

[0005] Another object of the present invention is to provide the use of the above-mentioned MDGA2-targeting peptide.

[0006] The technical solution of the present invention is as follows:

[0007] An MDGA2-targeting polypeptide consisting of 8-55 consecutive amino acids of the RPS23RG1 protein and containing QHNSX1CMR (SEQ ID NO. 31), wherein X1 is F or L.

[0008] Preferably, the MDGA2-targeting polypeptide is composed of 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, or 8 consecutive amino acid residues.

[0009] In a preferred embodiment of the present invention, it comprises X2YFX3QHNSX1CMRX4TX5 (SEQ ID NO. 32), wherein X2 is S or P, X3 is S or F, X4 is S or G, and X5 is S or P.

[0010] Further preferred, its amino acid sequence contains SYFSQHNSFCMRSTS (human, SEQ ID NO. 08) or PYFFQHNSLCMRGTP (mouse, SEQ ID NO. 14).

[0011] A conjugate having the above-described MDGA2-targeting polypeptide and a modifying moiety selected from other polypeptides, detectable markers (for detection), or any combination thereof, wherein the other polypeptide is selected from CPP (to enhance its ability to penetrate cell membranes), a targeting moiety (to make it targeted), and / or a protein tag (to facilitate its expression, detection, tracing, and / or purification).

[0012] A fusion protein having the aforementioned MDGA2 targeting peptide and an additional peptide selected from CPP (cell membrane permeation peptide, enhancing its ability to penetrate cell membranes), a targeting moiety (to make it targeted), a protein tag (to facilitate its expression, detection, tracing and / or purification), or any combination thereof.

[0013] For the above conjugates and fusion proteins:

[0014] Preferably, the modified portion is optionally linked to the N-terminus or C-terminus of the MDGA2-targeting peptide via a linker.

[0015] Preferably, the CPP is a Tat-derived peptide; for example, the CPP has a sequence as shown in SEQ ID NO. 17.

[0016] Preferably, the target component is a ligand, receptor, or antibody.

[0017] Preferably, the detectable label is a fluorescent dye, such as FITC.

[0018] Preferably, the protein tag is HA, Myc, GFP, or biotin.

[0019] An isolated nucleic acid molecule having the nucleotide sequence of the aforementioned MDGA2 targeting polypeptide or the aforementioned fusion protein.

[0020] A vector having the isolated nucleic acid molecules described above. This vector can be a cloning vector or an expression vector. In a preferred embodiment, the vector of the present invention is, for example, a plasmid, granule, bacteriophage, cosmid, etc. In a preferred embodiment, the vector is capable of expressing the MDGA2 targeting peptide of the present invention or the fusion protein of the present invention in a subject (e.g., a mammal, such as a human).

[0021] A host cell having the isolated nucleic acid molecules or the aforementioned carriers. The host cell includes: prokaryotic cells such as *Escherichia coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The cell of the present invention can also be a cell line, such as 293T cells.

[0022] The method for preparing the above-mentioned MDGA2 targeting peptide or the above-mentioned fusion protein includes: culturing the above-mentioned host cells under suitable conditions, and recovering the MDGA2 targeting peptide or fusion protein from the culture of the host cells.

[0023] A pharmaceutical composition comprising the above-described MDGA2-targeting polypeptide, the above-described conjugate, the above-described fusion protein, the above-described isolated nucleic acid molecule, the above-described carrier or the above-described host cell, and a pharmaceutically acceptable carrier and / or excipient.

[0024] Preferably, the pharmaceutical composition optionally further comprises an additional pharmaceutically active agent;

[0025] The other pharmaceutically active agent is a drug that has activity in treating nervous system diseases (e.g., neurodegenerative diseases).

[0026] Alternatively, the other pharmaceutically active agent may be selected from ω-3 fatty acids, vitamin B12, intravenous immunoglobulin, hyperbaric oxygen therapy, glutamate receptor antagonists, catechol-O-methyltransferase (COMT) inhibitors, dopa decarboxylase inhibitors, or any combination thereof.

[0027] The above-mentioned MDGA2-targeting polypeptide, the above-mentioned conjugate, the above-mentioned fusion protein, the above-mentioned isolated nucleic acid molecule, the above-mentioned carrier or the above-mentioned host cell are used in the preparation of a drug for treating diseases associated with low MDGA2 activity, or inhibiting lysosomal degradation of MDGA2, or increasing MDGA2 expression levels.

[0028] It has been previously reported that deletion or dysregulation of MDGA2 can impair synaptic function and cause autism-like behavioral deficits. In particular, previous studies have found loss-of-function mutations in MDGA2 in the brains of ASD patients. Furthermore, MDGA2 is associated with epilepsy and schizophrenia. Furthermore, during cancer development, such as in gastric cancer patients, elevated MDGA2 methylation levels, i.e., decreased activity, have been observed. Therefore, the diseases associated with low MDGA2 activity are neurological disorders or cancer.

[0029] This neurological disorder is characterized by social impairment and repetitive, stereotyped behaviors, preferably autism.

[0030] The preferred cancer is stomach cancer.

[0031] A method for inhibiting lysosomal degradation of MDGA2 or increasing MDGA2 expression levels in vitro for non-diagnostic and therapeutic purposes, characterized in that: the above-mentioned MDGA2-targeting polypeptide, the above-mentioned conjugate, or the above-mentioned fusion protein is contacted with cells in need.

[0032] Preferably, the cells are neuronal cells or gastric cancer cells.

[0033] The MDGA2-targeting peptides, fusion proteins, or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, and injections (including injectable solutions and lyophilized powders). In some embodiments, the MDGA2-targeting peptides, fusion proteins, or pharmaceutical compositions of the present invention can be formulated into injectable solutions or lyophilized powders.

[0034] Furthermore, the MDGA2-targeting polypeptide or fusion protein of the present invention can be present in the pharmaceutical composition in unit dose form for ease of administration.

[0035] The MDGA2-targeting peptides, fusion proteins, or pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In a preferred embodiment, the MDGA2-targeting peptides, fusion proteins, or pharmaceutical compositions of the present invention are administered by intravenous infusion or injection.

[0036] The MDGA2-targeting peptides, fusion proteins, or pharmaceutical compositions provided by this invention can be used alone or in combination, or in combination with other pharmaceutically active agents (e.g., drugs with activity for treating neurological disorders). In some preferred embodiments, the MDGA2-targeting peptides or fusion proteins of this invention are used in combination with other drugs with activity for treating neurological disorders to prevent and / or treat diseases associated with low MDGA2 activity (e.g., neurological disorders). Such additional pharmaceutically active agents can be administered before, simultaneously with, or after the administration of the MDGA2-targeting peptides, fusion proteins, or pharmaceutical compositions of this invention.

[0037] The pharmaceutical compositions of the present invention may include a “therapeutic effective amount” or a “preventative effective amount” of the MDGA2-targeting peptide or fusion protein of the present invention. A “preventative effective amount” refers to an amount sufficient to prevent, stop, or delay the onset of a disease (e.g., a disease associated with low MSGA2 activity). A “therapeutic effective amount” refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. The therapeutic effective amount of the MDGA2-targeting peptide or fusion protein of the present invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient’s own immune system, the patient’s general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0038] In this invention, the dosing regimen can be adjusted to obtain the optimal target response (e.g., treatment or prevention). For example, it can be administered as a single dose, multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0039] The typical non-limiting range of therapeutic or preventative effective doses of the MDGA2-targeting peptides or fusion proteins of the present invention is 0.001-100 mg / kg body weight, for example 0.01-50 mg / kg body weight, 0.1-25 mg / kg body weight. It should be noted that the dosage may vary depending on the type and severity of the symptoms requiring treatment. Furthermore, those skilled in the art will understand that for any given patient, a specific dosing regimen should be adjusted over time based on the patient's needs and the physician's professional evaluation; the dosage ranges given herein are for illustrative purposes only and do not limit the use or scope of the pharmaceutical compositions of the present invention.

[0040] In this invention, the subject can be a mammal, such as a human.

[0041] Terminology Definition

[0042] In this invention, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in this invention are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0043] As used herein, the term "RPS23RG1" refers to the protein expressed by the ribosomal protein S23 mRNA repositioning gene Rps23rg1, which is well known to those skilled in the art and described herein. And US patent application US 2010 / 0286252. In this invention, RPS23RG1 can be from any source, such as human or non-human, such as non-human mammalian, such as mouse (e.g., mouse).

[0044] As used herein, the term "cell penetrating peptide (CPP)," also known as "cell-penetrating peptide," "protein translocation domain (PTD)," "Trojan horsepeptides," or "transduction peptide," refers to a polypeptide capable of promoting cellular uptake of various molecules (e.g., various macromolecules including proteins or nucleic acids; for example, the MDGA2-targeting polypeptide or variants thereof of this invention). Such polypeptides are well known in the art and described, for example, in Stewart, et al., 2008 and Chinese patent application CN101490081A (all of which are incorporated herein by reference); or can be obtained by methods known in the art, such as those described in detail in U.S. patent application US 2008 / 0234183, all of which are incorporated herein by reference.

[0045] In this invention, examples of CPPs include, but are not limited to: (i) protein-derived CPPs: such as sequences derived from genes controlling antennae (Antennapedia), e.g., pAntp (43-58); sequences derived from HIV-1, e.g., Tat-derived peptides, such as amino acid residues 37-72, 37-60, 48-60, or 49-57 from TAT; hCT (9-32); pVEC; plSL; mouse PRP (1-28); E ms (194-220); or Restricocin L3 (60-73), etc.; (ii) Model peptides: such as VT5; MAP; or arginine stretch, etc.; (iii) Designed CPPs: such as MPG; Transportan; Transportan 10; Pep-1; peptides selected from KALA; or peptides selected from Bulforin2, etc.

[0046] Furthermore, the CPP used in the conjugates of the present invention may also be selected from polypeptide sequences having approximately 60, 70, 80, 90, 95, 99% or 100% sequence identity with any polypeptide sequence as described above, as long as the polypeptide sequence still retains its biological activity, i.e., promoting cellular uptake of the isolated polypeptide (or its variants) of the present invention and / or promoting the isolated polypeptide (or its variants) of the present invention across the blood-brain barrier.

[0047] As used herein, the term "targeting portion" refers to a domain capable of directing the MDGA2 targeting peptide (or a variant thereof) of the present invention to a desired location, which may be a specific tissue, a specific cell, or even a specific intracellular location (e.g., the nucleus, ribosomes, endoplasmic reticulum, lysosomes, or peroxisomes). Those skilled in the art know how to design corresponding targeting domains based on the characteristics of the desired location.

[0048] As used in this invention, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0049] As used in this invention, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0050] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be performed using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48:443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970) algorithm in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0051] As used herein, the term "isolated" means that the target analyte (e.g., a peptide) has been purified from contaminants present in a sample, such as a sample containing the target analyte obtained from a natural source. The term "isolated" does not necessarily exclude the presence of other components intended to work in conjunction with the isolated analyte. For example, the MDGA2-targeting peptide of this invention may be described as isolated, although it may be linked to cell-penetrating peptides.

[0052] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, ionic strength enhancers, agents for maintaining osmotic pressure, agents for delaying absorption, diluents, adjuvants, preservatives, stabilizers, etc. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents for delaying absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), etc. Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which stabilize the desired activity of the active ingredient in the drug (e.g., inhibitory activity against MDGA2 lysosomal degradation), including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0053] As used in this invention, the term "treatment" means treating or curing a disease (e.g., a neurological disease), delaying the onset of symptoms of a disease (e.g., a neurological disease), and / or delaying the progression of a disease (e.g., a neurological disease).

[0054] As used in this invention, the term "prevention" means preventing, inhibiting, or delaying the occurrence of a disease (such as a neurological disease).

[0055] As used herein, the term "effective amount" refers to an amount that can effectively achieve the intended purpose. For example, a therapeutically effective amount can be an amount that is effective or sufficient to treat or cure a disease (e.g., a neurological disease), delay the onset of symptoms of a disease (e.g., a neurological disease), and / or delay the progression of a disease (e.g., a neurological disease). A preventatively effective amount can be an amount that is effective or sufficient to prevent, inhibit, or delay the occurrence of a disease (e.g., a neurological disease). Such effective amounts can be readily determined by those skilled in the art or by a physician and can be related to the intended purpose (e.g., treatment or prevention), the general health condition of the subject, age, sex, weight, severity of the disease to be treated, complications, method of administration, etc. The determination of such effective amounts is entirely within the capabilities of those skilled in the art.

[0056] As used in this invention, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) suffers from a disease associated with low MDGA2 activity (e.g., a neurological disorder), or is at risk of suffering from a disease associated with low MDGA2 activity (e.g., a neurological disorder).

[0057] As used in this invention, the biological functions of the MDGA2-targeting peptides of this invention include, but are not limited to, one or more selected from the following:

[0058] 1) The ability to specifically bind to MDGA2;

[0059] 2) The ability to inhibit MDGA2 lysosomal degradation;

[0060] 3) The ability to increase MDGA2 levels in subjects (optionally, after conjugating the peptide to CPP).

[0061] 4) The ability to improve synaptic dysfunction and / or social dysfunction and repetitive stereotyped behaviors caused by low MDGA2 levels in subjects (optionally, after conjugating the peptide to CPP).

[0062] 5) The ability to treat diseases associated with low MDGA2 activity (e.g., neurological disorders) in subjects (optionally, after conjugating the peptide to CPP).

[0063] The beneficial effects of this invention are: the MDGA2-targeting peptide and fusion protein in this invention can significantly inhibit the lysosomal degradation of MDGA2, thereby increasing the level of MDGA2. Therefore, it can be used to treat diseases associated with low MDGA2 activity (such as autism and other neurological diseases and gastric cancer), and has significant clinical value. Attached Figure Description

[0064] Figure 1 a-1c: Analysis of the regulation of MDGA2 degradation levels by RPS23RG1. Figure 1 a: Primary neurons from WT and KO mice were isolated and cultured in vitro on day 9. Actinomycin C (CHX) was added, and the protein level of MDGA2 at different time points was analyzed by Western blotting. Figure 1 b: HEK293T cells were treated with CHX, MG132, CQ and NH4Cl simultaneously, and the protein level of MDGA2 was analyzed. Figure 1 c: Brain tissue was extracted from 1.5-month-old WT and KO mice, lysosomal components were separated, and Western blotting was used to analyze the protein level of MDGA2 in lysosomes. Statistical analysis of the graphs was performed using ImageJ software to analyze the protein levels in the graphs. Results are expressed as mean ± standard error (SEM). *P<0.05, **P<0.01, ***P<0.001, “ns” indicates no significant difference (2-tailed Student's test).

[0065] Figure 2 a-2d: Evaluation of the interaction between RPS23RG1 and MDGA2. Figure 2 a: The interaction relationship and interaction sites between RPS23RG1 and MDGA2 were predicted using PYMOL molecular docking software. Figure 2 b: Brain tissue was extracted from adult WT mice, and the lysate was used for IP-WB analysis of RPS23RG1 interacting proteins. Figure 2 c: RPS23RG1 and MDGA2 were overexpressed in HEK293T cells, and their relationship was analyzed by IP-WB. Figure 2 d: Overexpression of RPS23RG1 and MDGA2 in HeLa cells, and co-localization of the two cells by immunofluorescence analysis. Scale bar: 10 μm.

[0066] Figure 3 Analysis of the a-3c:RPS23RG1 sequence. Figure 3 a: Amino acid sequence alignment of mouse and human RPS23RG1, and structural prediction of residues in which RPS23RG1 interacts with MDGA2. Gray indicates identical amino acids or interacting residues. Figure 3 b: Schematic diagram of the full-length human RPS23RG1 sequence and its truncated form. The full-length human RPS23RG1 consists of 173 amino acids. Positions 131 to 154 are the transmembrane domain (TM domain). Δ57-67 represents a truncated form with amino acids 57-67 missing. Δ103-128 represents a truncated form with amino acids 103-128 missing. Figure 3c: Immunoprecipitation analysis of human RPS23RG1 and its different truncated forms with MDGA2, with IgG as a negative control.

[0067] Figure 4 a-4b: Analysis of the role of the RPS23RG1 core sequence in MDGA2 degradation. Figure 4 a: Full-length RPS23RG1 and Δ103-128 truncated variants were overexpressed in Lyso-HA cells. The levels of MDGA2 in lysosomes and total protein were detected by Western blotting after lysosome isolation. Figure 4 b: Full-length RPS23RG1 and its truncated Δ103-128 variant were overexpressed in HEK293T cells. MDGA2 levels in the cell membrane and total protein were detected by Western blotting using biotin-labeled precipitates. Statistical analysis of the graphs was performed using ImageJ software for grayscale analysis of protein levels. Results are expressed as mean ± standard error (SEM), *P<0.05, **P<0.01 (one-way ANOVA with Tukey's multiple comparisons test).

[0068] Figure 5 a-5c: Peptide binding to MDGA2 based on RPS23RG1. Figure 5 a: Schematic diagram of the synthesis of peptides P1 / P2 based on the RPS23RG1 core sequence. Figure 5 b: Biotin-conjugated peptides were added to HEK293T cells, and the binding of P1 / P2 to MDGA2 was detected by IP-WB. Figure 5 c: FITC-conjugated P2 was added to HeLa cells, and the co-localization of the peptide with MDGA2 was analyzed by immunofluorescence. Scale bar: 10 μm.

[0069] Figure 6 a-6b: Analysis of peptide-mediated MDGA2 degradation based on RPS23RG1. Figure 6 a: Peptide P2 and RPS23RG1 siRNA were transfected into Lyso-HA cells. After sufficient reaction, lysosomes were isolated, and the levels of MDGA2 in lysosomes and total protein were detected by Western blotting. The right side shows the statistical analysis graph. ImageJ software was used to perform grayscale analysis on the protein levels in the graph. The results are expressed as mean ± standard error (SEM), n=3. Figure 6b: HeLa cells were transfected with peptide P2 and RPS23RG1 siRNA. Immunofluorescence was used to analyze the localization of MDGA2 on the cell membrane and its co-localization with LAMP1. The right side shows the statistical analysis graph. Co-localization was analyzed using ImageJ software, n=40. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA with Tukey's multiple comparisons test). Scale bar: 10μm.

[0070] Figure 7 Construction and identification of a-7d:Mdga2 gene knockout mice. Figure 7 a: Construction of Mdga2 gene knockout mice. Schematic diagram of the Mdga2 gene structure and CRISPR / Cas9 technology. The CDS sequence of RPS23RG1 extends from exon 1 to exon 17, with exon 2 representing the knockout region. Figure 7 b: Genotyping of WT and gene knockout mice. Extracts were taken from WT and Mdga2 mice respectively. + / - and Mdga2 - / - Mouse DNA, analyzed by SDS-PAGE gel chromatography. Figure 7 c: Analysis of Mdga2 RNA expression levels in WT and gene knockout mice. RNA was extracted from WT and Mdga2 mice respectively. + / - and Mdga2 - / - Mdga2 levels were detected by reverse transcription and quantitative real-time PCR of mouse brain tissue RNA, and then compared after normalization with β-actin levels. n=4. Figure 7 d: Analysis of MDGA2 protein expression levels in WT and gene knockout mice. MDGA2 was extracted from WT and Mdga2 mice respectively. + / - and Mdga2 - / - Mouse brain tissue, Western blot analysis was used to detect MDGA2 protein levels. ImageJ software was used to perform grayscale analysis of protein levels in the images. Results are expressed as mean ± standard error (SEM), n=4. **P<0.01, ***P<0.001 (one-way ANOVA with Tukey's multiple comparisons test).

[0071] Figure 8 a-8b: In vivo injection of an RPS23RG1-based peptide in Mdga2 gene knockout mice. Figure 8 a: Schematic diagram of peptide injection into mouse tail vein. 1.5-month-old mice were injected with P2 and control peptides via tail vein for 5 consecutive days, and behavioral and biochemical tests were performed on day 7. Figure 8b: The brains of mice injected with P2 and control peptides were collected respectively, and biotin immunoprecipitation-immunoblotting was used to verify that the peptides crossed the blood-brain barrier, entered the brain tissue, and bound to MDGA2.

[0072] Figure 9 a-9b: Effects of RPS23RG1-based peptides on MDGA2 levels in Mdga2 knockout mice. Figure 9 a: For mice treated with P2 or control peptides, brain tissue protein lysates were extracted, and MDGA2 protein levels were detected by immunoblotting. Figure 9 b: Protein level statistics. ImageJ software was used to perform grayscale analysis on the protein levels in the graph, with β-actin as an internal reference. The results are expressed as mean ± standard error (SEM), n=8. **P<0.05, "ns" indicates no significant difference (one-way ANOVA with Tukey's multiple comparisons test).

[0073] Figure 10 a-10d: Evaluation of the efficacy of RPS23RG1-based peptides in MDGA2-deficient ASD model mice. Figure 10 a: Mine field experiment, analyzing the total distance traveled and the time spent in the mine by mice treated with P2 or control peptides. Figure 10 b: Stereotyped grooming experiment, analyzing the frequency of repetitive stereotyped behaviors such as self-grooming and jumping in mice treated with P2 or control peptides in a mine. Figure 10 c: Mouse free socialization experiment, analyzing the time mice treated with P2 or control peptides spent interacting and sniffing with unfamiliar mice in free space exploration. Figure 10 d: In the three-box social experiment in mice, the time mice spent sniffing a strange mouse (S1) and an empty cage (E) during the social preference test phase was analyzed, along with the social preference index PI = T(S1-E) / T(S1+E). Then, the time mice spent sniffing another strange mouse (S2) and mouse (S1) during the social novelty test phase was analyzed. The social novelty preference index PI = T(S2-S1) / T(S1+S2) was also analyzed. *P<0.05, **P<0.01, ***P<0.001, “ns” indicates no significant difference (one-way ANOVA with Tukey's multiple comparisons test). Detailed Implementation

[0074] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0075] Sequence information

[0076] Information about the sequence involved in this invention is provided in Table 1 below.

[0077] Table 1: Sequence Description

[0078] SEQ ID NO. Name / Description Sequence Information 1 Human RPS23RG1 full-length protein MSPWSLGGMQKTGLASDSQSGRHPPLPAPQFLEAGGQKKDTGYFDNLKANSRNITNSMTFLTKSSNQSFMVFHNKVQATITEYKGCDFLAILDPLDPDTLPNGRIWLFGFNSYFSQHNSFCMRSTSKRVGLQGCAQMGFLVLFIMPLLILLVTTETPSSMRSTTLAHPAVLRA 2 Murine RPS23RG1 full-length protein MQSQQRNIGYFNHLKADSRNITYSMTFSTKSSNQNFIIFLNEVQAAIIGHERCDLLPVLNELHPDALPDGRIWLFGLNPYFFQHNSLCMRGTPKRIGLQGCAQVGFLVLFVMPLLIPSVTAELPGSSETTTLAHLAGATGP 3 Human Rps23rg1 gene sequence atgtccccatggagccttggagggatgcagaagacaggattggccagcgacagtcaaagtggccggcacccccctctaccagctccccagttcctggaggctgggggccaaaagaaagacacaggctactttgacaacctgaaagcgaactccagaaatatcaccaacagcatgacctttttgaccaaatccagcaaccagagctttatggttttccacaataaggttcaagcaaccatcactgaatacaaaggctgtgattttcttgccatccttgatccactggaccctgacacacttcctaatggcagaatttggctttttggcttcaactcctacttttcccagcacaattccttttgcatgagaagcacctccaaaagggttggccttcagggctgtgcccaaatgggctttcttgtactgtttatcatgccacttctgatcttgttggtgactacagagactcctagcagtatgaggtccacgacacttgcccatcctgcagtgctacgggcctga 4 Murine Rps23rg1 gene sequence atgtccccatggagccttggagggatgcagaagacaggattggccagcgacagtcaaagtggccggcacccccctctaccagctccccagttcctggaggctgggggccaaaagaaagacacaggctactttgacaacctgaaagcgaactccagaaatatcaccaacagcatgacctttttgaccaaatccagcaaccagagctttatggttttccacaataaggttcaagcaaccatcactgaatacaaaggctgtgattttcttgccatccttgatccactggaccctgacacacttcctaatggcagaatttggctttttggcttcaactcctacttttcccagcacaattccttttgcatgagaagcacctccaaaagggttggccttcagggctgtgcccaaatgggctttcttgtactgtttatcatgccacttctgatcttgttggtgactacagagactcctagcagtatgaggtccacgacacttgcccatcctgcagtgctacgggcctga atgcagagccaacagagaaacattggctactttaaccaccttaaagcggactccaggaatatcacctacagcatgaccttttcgacgaaatccagcaaccagaacttcatcattttcctcaatgaagttcaggcagccatcattgggcacgaacgctgtgatcttcttcccgttcttaatgagctgcaccctgacgcacttcctgatggcagaatttggctgtttggcctcaacccctactttttccagcacaattccctttgcatgagaggcacccccaaacggattggccttcagggctgtgcccaagtgggctttcttgtactgtttgtcatgccacttctgatcccgtcggtgactgcggagcttccgggcagttcggagaccacgacacttgcccatcttgccggcgccacgggcccctaa 5 H-S1 QHNSFCMR 6 H-S2 QHNSFCMRSTS 7 H-S3 SYFSQHNSFCMR 8 H-S4 / peptide2 SYFSQHNSFCMRSTS 9 H-S5 FNSYFSQHNSFCMRSTS 10 H-S6 FNSYFSQHNSFCMRSTSKR 11 M-S1 QHNSLCMR 12 M-S2 QHNSLCMRGTP 13 M-S3 PYFFQHNSLCMR 14 M-S4 PYFFQHNSLCMRGTP 15 M-S5 LNPYFFQHNSLCMRGTP 16 M-S6 LNPYFFQHNSLCMRGTPKR 17 CPP / Tat YARAARRAARR 18 H-S4-CPP SYFSQHNSFCMRSTSYARAARRAARR 19 Linker GGG 20 Forward primer of Mdga2 ccagaggcctatgaagtccg 21 Reverse primer of Mdga2 tccacagtgaaattccctcaa 22 Forward primer of β-actin agccatgtacgtagccatcca 23 Reverse primer of β-actin tctccggagtccatcacaatg 24 Sense strand of human RPS23RG1 siRNA-1 ccagcaaccagagcuuuautt 25 Antisense strand of human RPS23RG1 siRNA-1 auaaagcucugguugcuggtt 26 Sense strand of human RPS23RG1 siRNA-2 cacuuccuaauggcagaautt 27 Antisense strand of human RPS23RG1 siRNA-2 gagacuccuagcaguaugatt 28 MDGA2 encoding nucleic acid sequence atggatttagtgtacggtctcgtgtggctgctgacagtcctcctggagggaatctctggccaaggagtgtacgctcccccgacagttcggattgtgcactcaggattggcctgtaatatcgaggaggagcgctactccgaaagggtctataccatccgggaaggagaaactctagaattgacctgcctggttactggacatccacgcccacagatcaggtggaccaaaacagcaggaagcgcctctgacagattccaagactcaagtgtcttcaatgagactttgaggattacaaacattcagagacaccaaggaggccgatattactgtaaagcagagaatggcttggggtcaccagcaataaagtcgatccgagtggatgtgtactacttggatgatccagtagtaactgttcatcaaagtataggtgaagctaaagaacaattttactatgagagaacagtgtttctccgatgtgtggccaattccaacccccctgtgcgctatagctggagacgtggtcaggaggtattactacaaggatctgacaagggagtggagatttatgagcccttttttacccagggggaaacgaagatcttaaagctaaagaatcttcgccctcaagattatgctaactacagctgcattgcttcagtaaggaacgtgtgtaatattccagacaagatggtgtcgtttagactttccaataaaactgcttctccgtcaattaagctcttggtggatgatcctatagttgtaaatcctggagaggccataacgttagtatgtgttacaactggaggagagcctacaccctctcttacctgggtcaggtccttcgggactctgcctgaaaagattgttttgaatggagggacattgaccatacctgccatcacctctgatgacgctggtacttacagctgcattgccaataataatgtgggaaaccctgcaaaaaagtccaccaacatcattgtgagagcattaaaaaaaggacgattttggatcactcctgatccttatcacaaagatgacaacatccagattgggcgtgaggtgaaaatatcttgccaagtagaagctgttccctctgaggagttaacattcagttggtttaaaaatggtcgtccattaagaagttctgaacggatggttattacacagactgaccctgatgtgtcccccggaacaacaaacttggatatcattgacttaaaattcacagactttgggacttacacatgcgtggcctctctgaagggaggaggaatatctgatatcagtattgatgttaacatttccagcagtacagttccacccaatctgactgttccacaggaaaaatcacctttggtcaccagagaaggagacacaatagaacttcagtgtcaagtaactgggaaaccaaaaccaatcatcctttggtctagagcagataaagaagttgcaatgcctgatgggacaatgcaaatggaaagttatgatgggaccctgaggattgtgaatgtttcaagggaaatgtcaggaatgtacagatgccagaccagccagtacaatggattcaacgtgaagccaagagaagccttggtgcagctcattgtgcagtatcccccagctgtggaaccagcattcttggaaatccgtcaaggacaggacagaagtgtgaccatgagctgtagggtcctgagagcctatccaatacgagtgctgacctatgagtggcgtctgggcaataaattattacggaccggacagtttgactctcaggagtatacagagtacccactaaagagtctttccaatgaaaactatggagtttataactgcagcatcataaatgaagctggagcagggagatgcagctttcttgttacaggaaaggcctatgccccagagttctactatgacacctacaaccccgtgtggcagaaccgacaccgagtgtattcttacagtctacagtggacacaaatgaaccctgatgcagttgatcgaattgttgcataccgcttgggtattcggcaggctggacagcaacgctggtgggaacaggagattaaaataaatggaaatattcagaaaggagaactgattacatataacttgactgaacttattaaaccagaggcctatgaagtccgattgactcctctcactaaatttggtgaaggagattcaacaattcgtgtaattaaatatacagcacctgtaaatcctcatttgagggaatttcactgtggatttgaagatggtaatatttgtttgttcacccaagacgatacggataactttgactggacaaagcaaagcactgcaacaaggaacacaaaatacacccctaacacgggtcccagtgcagatcggagtggttccaaagaaggtttttatatgtacattgagacatcgcgacccagactagaaggcgaaaaggctcgacttctcagccctgtattcagcatagctcccaaaaatccatatggacctacaaatagtgcatattgtttcagtttcttctatcacatgtacgggcaacatataggggttttaaatgtatatctacgtttgaaagggcagacaacgatagagaatccgctatggtcgtcgagtgggaacaaaggacaacgatggaatgaagctcatgttaatatatatccaattacttcatttcagttaatttttgaaggcattcgaggtcctgggatagagggtgacatcgccattgatgatgtatcaattgctgaaggagaatgtgcaaaacaagacctaccaactaagaattccgtggatggtgctgttgggatcttagttcatatatggctttttccagttatcatcctcatctctatcttaagccctcgaaggtga 29 Scramble, a disordered control peptide of human RPS23RG1 extracellular domain peptide. Biotin-FNSSFHRSTSMQSCY 30 Human RPS23RG1 extracellular peptide P1 FITC / Biotin-GRIWLFGFN 31 QHNSX1CMR 32 [X2YFX3QHNSX1CMRX4TX5] 33 Human RPS23RG1 extracellular peptide P2 FITC / Biotin-SYFSQHNSFCMRSTS 34 peptide1 GRIWLFGFN

[0079] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the manufacturer's recommendations. Reagents not otherwise specified in the examples are conventional reagents in the art or commercially available reagents.

[0080] Experimental Materials and Methods

[0081] Real-time quantitative PCR (qRT-PCR)

[0082] Total RNA was extracted from mouse tissues using TRIzol reagent according to the manufacturer's instructions. After determining the concentration using a microplate reader, the mRNA was reverse transcribed into cDNA using a kit. qRT-PCR experiments were performed using a FastStart Universal SYBR Green Master (Roche). After the qRT-PCR experiments, a small amount of PCR product was analyzed by 1.5% agarose gel electrophoresis to determine if the PCR product fragment size was correct and if it was a specific amplification.

[0083] Protein extraction and Western blot

[0084] 1. Extraction of proteins from cell samples:

[0085] (1) Cell collection: Discard the culture medium and wash the cells twice with PBS; discard the PBS, scrape off the cells with a cell scraper and collect them into a centrifuge tube; centrifuge at 1000 rpm for 5 min, collect the cells at the bottom of the tube, and discard the supernatant.

[0086] (2) Cell lysis: Add TNEN cell lysis buffer containing cocktail protease inhibitor and phosphatase inhibitor (added immediately before use) to the cell clusters collected in (1), shake on ice for 30 min or lyse slowly overnight at 4°C in a silent mixer, centrifuge at 12000 rpm for 10 min at 4°C, collect the supernatant and measure the protein concentration.

[0087] 2. Protein extraction from tissue samples:

[0088] Fresh tissue was immediately frozen in liquid nitrogen after extraction and then stored in an ultra-low temperature freezer at -80°C for later use. Before homogenization, the tissue was minced with surgical scissors and then homogenized in RIPA lysis buffer using a polytron electric homogenizer. After complete lysis, the tissue was centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatant was collected. The centrifugation was repeated once, and the protein concentration was measured.

[0089] 3. Determination of protein concentration:

[0090] (1) Prepare the BCA protein assay solution by mixing solution A and solution B in a 50:1 ratio and set aside.

[0091] (2) Using BSA as the standard sample, a protein standard curve was prepared;

[0092] (3) Add protein samples to 96-well plates, with TNEN / RIPA lysis buffer as a blank control, and set up 3 wells for each group;

[0093] (4) Add 200 μl of the prepared protein assay solution to each well, mix well, and react in a constant temperature oven at 37°C for 30 min;

[0094] (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure OD562, and substitute the obtained value into the protein standard curve to calculate the protein sample concentration.

[0095] 4. Western blot:

[0096] (1) Protein electrophoresis: Take 30-40 μg of protein sample, add 1 / 4 volume of 5×SDS loading buffer, boil at 100°C for 3-5 min, use SDS-PAGE protein gel, and electrophoresis in Tris-Glycine electrophoresis buffer.

[0097] (2) Electrophoretic transfer of proteins: Pre-cool the electrophoresis transfer buffer at 4°C, cut out an appropriate size of PVDF membrane, wet it with methanol, and soak it together with filter paper in the electrophoresis transfer buffer for 10 min. At the same time, cut the electrophoresis gel and soak it in the electrophoresis transfer buffer for 5 min. Then, attach the PVDF membrane to the gel, cover both sides with filter paper, remove all air bubbles, and place it in the electrophoresis tank with the membrane facing the positive electrode. Electrophoresis is performed at a constant current of 4°C (300 mA, 90 min).

[0098] (3) Antigen-antibody reaction:

[0099] a. Sealing: 5% skim milk powder sealed at room temperature for 1 hour;

[0100] b. Primary antibody reaction: Dilute the primary antibody in the blocking buffer at an appropriate ratio and incubate overnight at 4°C;

[0101] c. Secondary antibody reaction: Wash three times with PBST for 10 min each time, dilute the primary antibody in blocking buffer at an appropriate ratio, and incubate at room temperature for 1 h.

[0102] (4) ECL detection:

[0103] Wash three times with PBST, 10-15 min each time; mix solutions A and B from the ECL kit at a 1:1 (V / V) ratio, add ECL to the membrane surface in a dark room, incubate for 5 min, and then expose to light. Adjust the exposure time according to the fluorescence intensity.

[0104] Immunoprecipitation (IP) or co-immunoprecipitation (CoIP)

[0105] (1) Collect protein samples, add 10 μl Protein-ASepharose to the protein samples, and incubate at 4℃ for 1-2 h.

[0106] (2) Centrifuge at 5000 rpm for 2 min at 4℃. Transfer the supernatant to a new EP tube, add 0.5% TNEN lysis buffer to a final volume of 700 μl, add 20 μl Protein-GSepharose and the corresponding antibody, and incubate overnight at 4℃ by rotation.

[0107] (3) Centrifuge at 5000 rpm for 2 min at 4℃, remove the supernatant, add pre-cooled 1% TNEN solution, and incubate at 4℃ for 8 min by rotation.

[0108] (4) Repeat step (3) three times. On the last time, carefully aspirate the supernatant with a microsyringe, then add 20 μL of 2× loading buffer, mix well, and boil at 100°C for 5 min. The obtained sample is used for immunoblotting analysis.

[0109] Immunofluorescence

[0110] (1) Place the treated coverslip into a 24-well plate (one per well), and evenly distribute cells at a certain density (5%~10%), and culture for 24 h.

[0111] (2) Transfect the corresponding plasmid according to the lipo2000 transfection method.

[0112] (3) 24 h after transfection, remove the culture medium and wash the cells 3 times with pre-cooled PBS. Remove the PBS and fix with 4% paraformaldehyde at room temperature for 10 min.

[0113] (4) 0.1% TritonX100-PBS, room temperature penetration for 5-10 min (the time should be strictly controlled here).

[0114] (5) Block with 3% BSA-PBS for 1 h.

[0115] (6) Incubate overnight at 4°C with different primary antibodies (1:100 diluted in 3% BSA-PBS).

[0116] (7) Wash with PBS 4 times, 5 min each time. At this time, let the culture dish stand still without shaking.

[0117] (8) Incubate with the corresponding fluorescent secondary antibody at room temperature for 60 min.

[0118] (9) If necessary, incubate DAPI at room temperature for 3-5 min.

[0119] (10) Wash with PBS 4 times, 5 min each time.

[0120] (11) Mount the slide at room temperature overnight. After the mounting medium dries, observe and photograph it under a confocal microscope.

[0121] Lysosome isolation

[0122] 1. Tissue (BioBio, Lysosome Extraction Kit, BB-3603)

[0123] (1) Remove mouse brain tissue, cut it into small pieces as much as possible with surgical scissors, wash it twice with cold PBS, add 400 μl of cold reagent A, place it on ice and let it stand for 10 min, and homogenize it 30-40 times with a tight Dounce homogenizer.

[0124] (2) The homogenate was centrifuged at 4℃ and 1000×g for 5 min. The precipitate was discarded and the supernatant was collected;

[0125] (3) Centrifuge the supernatant at 4℃ and 3000×g for 10 min. Discard the precipitate and collect the supernatant;

[0126] (4) Centrifuge the supernatant at 4℃ and 5000×g for 10 min. Discard the precipitate and collect the supernatant;

[0127] (5) Centrifuge the supernatant at 4℃ and 20000-30000×g for 20 min. Discard the supernatant, collect the precipitate, add 400 μl of cold reagent B to the precipitate, and mix well;

[0128] (6) Centrifuge at 4℃ and 20,000-30,000 × g for 20 min. Discard the supernatant, collect the precipitate, and resuspend the precipitate in lysosome preservation solution C to obtain the lysosome sample. Store in a refrigerator for later use or use directly in downstream experiments.

[0129] 2. Cells (LysoIP)

[0130] (1) Using HeLa cells stably transfected with TMEM192-3xHA, the treated cells were washed twice with PBS, and then collected with KPBS (136 mM KCl, 10 mM KH2PO4, pH 7.25, adjusted with KOH), and a portion of the cell lysate was retained as the total protein component.

[0131] (2) The KPBS cell suspension was slowly homogenized for 30 cycles using a tight-fitting Dounce homogenizer, and then centrifuged at 1000 g for 2 min at 4°C.

[0132] (3) Collect the supernatant and pre-wash the anti-HA beads with KPBS and incubate them on a gentle rotating shaker for 30 min;

[0133] (4) Wash the immunoprecipitate three times with KPBS and resuspend it in TNEN buffer to obtain the lysosome sample. Store it in a refrigerator for later use or use it directly in downstream experiments.

[0134] Cell surface protein biotin labeling

[0135] Biotin (EZ-LinkSulfo-NHS-SS-Biotin) was dissolved in freshly prepared PBS / CM buffer to a final concentration of 0.5 mg / mL and kept on ice. Cells were removed from the incubator, placed on ice, and the culture medium was aspirated. Cells were washed three times with a suitable amount of pre-chilled PBS / CM buffer, and the buffer was aspirated. Biotin solution was added to the cell culture dish, and the cells were incubated on ice for 20 min. The biotin solution was aspirated, and the process was repeated once. The reaction was terminated by adding PBS / CM buffer containing 50 mM NH4Cl (10 min). Cells were lysed with TNEN lysis buffer (containing protease inhibitors), centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was transferred to a new centrifuge tube. After determining the protein concentration by the BCA method, an equal volume of protein sample was added to an equal volume of 2× loading buffer, mixed well, and boiled at 100 °C for 5 min. Protein expression was detected by Western blotting. Streptavidin beads were added to an equal volume of the remaining protein sample. (Beads), precipitate biotinylated protein at 4℃ overnight; the next day, centrifuge at 5000RPM for 2 min at 4℃, aspirate the supernatant, wash the streptavidin beads 3 times with 1% TNEN for 10 min each time, aspirate the residual liquid with a microsyringe, add 20 μL of 2× loading buffer, boil at 100℃ for 5 min, and determine the content of the target protein by Western blotting analysis.

[0136] plasma membrane separation

[0137] Cell membrane preparation was performed using a cell membrane protein extraction kit (Phygene, PH0710). Cells were removed from the incubator, placed on ice, and the culture medium was aspirated. The cells were washed twice with pre-chilled PBS buffer. The collected cells were resuspended in pre-chilled extraction reagent A and incubated for 15 min. Then, the cells were slowly homogenized 200 times using a loose-fitting Dounce homogenizer until approximately 70% of the cells were ruptured. The cell suspension was then centrifuged at 700 g for 10 min, and the supernatant was collected. The membrane precipitate was then collected by centrifugation at 120,000 g for 30 min. Finally, the membrane proteins in the precipitate were resuspended and dissolved using lysis buffer containing protease inhibitors. The precipitate was then collected by centrifugation at 12,000 g for 10 min for further experiments.

[0138] Protein-protein interaction structure prediction

[0139] The researchers selected the MDGA2 (AF-Q7Z553-F1) PDB file, suitable for their research in this invention, from the SWISS-MODEL protein structure data. The protein structure of RPS23RG1 (C-score = -4.62) was predicted by Zhang Lab_I-TASSER (UNIVERSITY OF MICHIGAN). Protein-protein interactions were predicted using the GRAMM:Docking web server (http: / / gramm.compbio.ku.edu / gramm, Vacker Lab). Finally, PYMOL was used to visualize the three-dimensional protein model and its interactions, and to analyze molecular docking.

[0140] Tail vein injection

[0141] The synthesized peptides were dissolved in physiological saline, with no more than 2% dimethyl sulfoxide added to aid dissolution. The fully dissolved peptides were then placed on ice for later use. When injecting mice, ensure the mice were comfortably positioned in the syringe and located using the tail vein syringe barrel, while simultaneously placing the mouse's tail in the groove of the compression plate. Adjust the lighting to make the veins clearly visible, then begin injection from the base of the tail, slowly advancing the peptide solution while constantly monitoring the mouse's health. After injection, leave the needle in place to ensure proper drug absorption. After needle removal, apply pressure with a cotton swab for 1 minute to stop bleeding. Injections were repeated at the same time for 5 consecutive days.

[0142] Animal behavioral experiments

[0143] All mouse behavioral experiments were conducted using SmartVideoTracking Software (Panlab, Harvard Apparatus) for data acquisition and analysis. Animal behavioral experiments were performed daily between 9:00 am and 6:00 pm, with a laboratory light intensity of 650 lux.

[0144] (1) Three days before the start of the experiment, touch the mice once a day, touch one mouse each time, gently grasp the tail and pick up the mouse, let the mouse stay in your hand for 30 seconds, then mark the tail with a marker, after marking the mouse, still put it in your palm, grasp the tail, and then gently put it back into the cage.

[0145] (2) On the day of the experiment, the mice were transferred to the preparation room before the experiment and allowed to adapt for 60 minutes. Before the experiment began, the test chamber and maze were cleaned with 70% alcohol. After each experiment, the test chamber and maze were cleaned with 70% alcohol to remove the feces and urine excreted by the mice during the experiment and to eliminate the interference of residual odor from the mice on the experiment.

[0146] 1. Open field test

[0147] This study investigates the voluntary movement ability and anxiety behavior of mice, primarily based on their tendency to avoid bright light and open spaces. In the open field experiment, mice are placed in the center of a maze (40 cm (L) × 40 cm (W) × 40 cm (H)) and allowed to explore freely for 10 minutes. The total distance traveled and the time spent in the center are recorded.

[0148] 2. Self-grooming test

[0149] Mice were placed in the center of a maze (40 cm (L) × 40 cm (W) × 40 cm (H)) and allowed to explore freely for 15 minutes. The entire process was recorded using the Smart3.0 video tracking system. It is generally believed that the first 5 minutes are the adaptation phase for mice entering a new environment. Therefore, by observing the video data of the mice in the last 10 minutes, the total number of times and the total time each mouse groomed itself with its limbs were recorded.

[0150] 3. Social affiliation

[0151] The experimental mouse was placed in the center of a box (40 cm (L) × 40 cm (W) × 40 cm (H)). Before the experiment, a wild-type mouse of the same sex and age as the experimental mouse (the stimulation mouse) was placed in a metal cage. The experimental mouse was then placed in the box and allowed to explore freely for 10 minutes. The entire process was recorded using the Smart3.0 video tracking system. The exploration time of the mouse within the metal cage and its surrounding 2 cm area was recorded.

[0152] 4. Three-chamber sociability test

[0153] The test chamber (60cm wide × 40cm deep × 22cm high) contains two transparent partitions that separate the left, middle, and right chambers (each 20cm × 40cm). Each partition has a small opening (5cm × 5cm) for access to each chamber. A fixed wire cylinder (8.5cm in diameter) is placed in each of the left and right chambers. The test consists of three phases: adaptation, social skills test, and social novelty test. In the adaptation phase, both cylinders are empty, and the test mouse is placed in the middle and allowed to explore freely for 10 minutes. In the social skills test, a sex-matched unfamiliar mouse (S1) is confined in an empty cylinder and allowed to explore freely for 10 minutes. The time spent exploring S1 or the other empty cylinder (E) is measured, and the social preference index (T(S1-E) / T(S1+E)): the time spent exploring S1 minus the time spent exploring E, then divided by the time spent exploring both S1 and E). During the social novelty test, another sex-matched unfamiliar mouse (S2) was placed in an empty tube and allowed to explore freely for 10 minutes. The time spent exploring S1 or S2 was measured, and the social novelty preference index (T(S2-S1) / T(S2+S1)) was calculated: the time spent exploring S2 minus the time spent exploring S1, and then divided by the time spent exploring S2 and S1.

[0154] Materials and reagents

[0155]

[0156] Example 1: RPS23RG1 regulates lysosomal degradation of MDGA2

[0157] First, the researchers of this invention detected a decrease in MDGA2 protein levels in Rps23rg1 gene knockout mice. Therefore, they treated hippocampal neuronal cultures from Rps23rg1 KO and WT mice with actinomycin (CHX) to inhibit protein synthesis. They found that, compared to the WT control, the degradation rate of MDGA2 in Rps23rg1 KO neurons was significantly accelerated. Figure 1a). Furthermore, treatment with the lysosomal inhibitor chloroquine (CQ) or NH4Cl can prevent the degradation of MDGA2, but the proteasome inhibitor MG-132 cannot prevent the degradation of MDGA2. Figure 1 b). Further lysosomal isolation experiments revealed that MDGA2 was reduced in the brain components of Rps23rg1 KO mice, but increased in the lysosomal components. Figure 1 (c) This confirms that MDGA2 degradation occurs via a lysosomal process. These findings suggest that RPS23RG1 is involved in regulating the lysosomal localization and degradation of MDGA2.

[0158] Example 2: Interaction between RPS23RG1 and MDGA2 and its effect on MDGA2 degradation

[0159] 2.1: Interaction Analysis between RPS23RG1 and MDGA2

[0160] RPS23RG1 is a typical Class Ib transmembrane protein. Protein-protein interaction analysis predicted an interaction between RPS23RG1 and MDGA2. Figure 2 a). To further confirm this, the researchers of this invention analyzed the interaction between RPS23RG1 and MDGA2 using immunoprecipitation-immunoblotting. First, the interacting proteins were precipitated in WT mouse brain tissue using an RPS23RG1-specific antibody, and the results are as follows: Figure 2 As shown in b, RPS23RG1 exhibits a significant interaction with MDGA2. Next, we constructed a full-length human RPS23RG1 plasmid with a Myc tag and a full-length MDGA2 plasmid with a GFP tag. Both plasmids were simultaneously transfected into HEK293T cells, and immunoprecipitation-Western blotting analysis was performed. The results are shown in Figure b. Figure 2 As shown in c, RPS23RG1 and MDGA2 bind to each other in cells. Finally, we co-transformed the RPS23RG1-Myc and MDGA2-GFP plasmids into HeLa cells and performed immunofluorescence experiments, which also detected the co-localization of the two in the cell membrane and cytoplasm. Figure 2 d) indicates the interaction between the two.

[0161] 2.2: Determination of the core sequence of the extracellular segment of RPS23RG1

[0162] In previous studies, the researchers of this invention elucidated the important roles of the interaction between the intracellular segment and transmembrane region of RPS23RG1 and synaptic proteins in maintaining synaptic structure and synaptic transmission. To clarify the binding region between RPS23RG1 and MDGA2, the researchers of this invention performed a comparative analysis of the protein sequences of human RPS23RG1 (SEQ ID NO. 01) and mouse RPS23RG1 (SEQ ID NO. 02), identifying homologous sequence regions. This was combined with analysis of the interacting residues of RPS23RG1 and MDGA2 in structural prediction. Figure 2 a), ultimately identifying two amino acid enrichment regions in the extracellular region: 57-67 and 103-128. Figure 3 a). Next, expression plasmids of Myc-modified human full-length RPS23RG1 (1-173) or different truncated versions (Δ57-67 and Δ103-128) were constructed, see Table 2 and... Figure 3 b. The coding nucleic acid sequences of the above-mentioned peptides were inserted into the pCMV-Myc plasmid to obtain plasmids expressing each peptide. These plasmids were then transfected into HEK293T cells to overexpress full-length and truncated RPS23RG1. Furthermore, MDGA2-GFP was overexpressed in these cells for immunoprecipitation-Western blotting analysis. Results are as follows: Figure 3 As shown in Figure c, both the full-length RPS23RG1 (1-173) and the truncated variants 57-67 exhibit significant interactions with MDGA2, while the truncated variants 103-128, which lack the extracellular segment, do not interact with MDGA2. This suggests that the 103-128 sequence of the RPS23RG1 extracellular segment is a key region for interaction with MDGA2.

[0163] Table 2: Truncated human full-length RPS23RG1

[0164] name describe Sequence information 1-173 Human RPS23RG1 full-length sequence SEQID NO:1 Δ57-67 Human RPS23RG1 with amino acid residues missing from positions 57-67 Missing 57-67 human source RPS23RG1 Δ103-128 Human RPS23RG1 with amino acid residues missing from positions 103-128 Missing RPS23RG1 (103-128)

[0165] 2.3: Effect of the extracellular core sequence of RPS23RG1 on MDGA2 degradation

[0166] To determine whether their interaction mediates lysosomal degradation of MDGA2, we used lysosomal immunoprecipitation (LysoIP) to transfect full-length and 103-128-deleted RPS23RG1 plasmids into a stable TMEM192-3*HA-coupled lysosome cell line (HA-Lyso). After stable plasmid expression, cell lysates were collected, and lysosomal components were enriched using HA magnetic bead precipitation. Protein levels in lysosomes were then detected by Western blotting. The results showed that overexpression of full-length RPS23RG1 (but not RR1-Δ103-128) mediated a decrease in MDGA2 levels and an increase in total protein levels in lysosomes. Figure 4a). Additionally, full-length and truncated RPS23RG1 plasmids (with the 103-128 sequence deletion) were transfected into HEK293T cells to overexpress full-length and truncated RPS23RG1, respectively. Biotin labeling and immunoprecipitation-Western blotting assays were used to detect protein content in membrane components. The results showed that overexpression of full-length RPS23RG1 simultaneously upregulated the surface localization of MDGA1 and total protein levels, while the deletion of the 103-128 sequence had no effect. Figure 4 (b) In summary, these results indicate that RPS23RG1 interacts with MDGA2 through its 103-128 fragment and prevents its lysosomal degradation.

[0167] This result further confirms that the core sequence of RPS23RG1 that interacts with MDGA2 to inhibit its lysosomal degradation is 103-128 amino acids (human RPS23RG1 protein). The RPS23RG1 fragment containing this core sequence can interact with MDGA2 to inhibit its degradation, increase MDGA2 levels, and thus improve neurological disorders (such as autism) caused by MDGA2 reduction.

[0168] Example 3: Evaluation of the therapeutic effect of peptides based on the extracellular domain of RPS23RG1 in inhibiting MDGA2 degradation in cells.

[0169] To further determine the core action site of the RPS23RG1 extracellular domain, the researchers of this invention artificially synthesized human RPS23RG1 extracellular domain peptides peptide1 (GRIWLFG; SEQ ID NO. 34) and peptide2 (SYFSQHNSFCMRSTS; SEQ ID NO. 08). Figure 5 a) A FITC fluorescent or biotin label was added to the amino terminus of the above peptides, and the resulting peptides were named P1 (SEQ ID NO. 30) and P2 (SEQ ID NO. 33), respectively. All the above peptides were synthesized by Sangon Biotech. The biotin-labeled peptides were added to HEK293T cells and incubated for 16 hours. The cells were then lysed to extract the proteins, and the peptides and their binding proteins were captured using biotin beads. The results are as follows: Figure 5 As shown in b, peptide P2, but not P1, can bind to MDGA2. Subsequently, we added FITC-labeled peptide P2 to HeLa cells and incubated them for 16 hours. Immunofluorescence was used to detect the co-localization of P2 and endogenous MDGA2 on the cell membrane. Figure 5 c). This indicates that the P2 sequence plays an important role in the binding of RPS23RG1 and MDGA2.

[0170] Subsequently, RPS23RG1 siRNA (SEQ ID NO. 24-27) was transfected into HA-Lyso stable cells to silence the RPS23RG1 gene. Peptide P2 was added simultaneously, and the effect of P2 on MDGA2 degradation was detected using lysosomal immunoprecipitation (LysoIP). The results showed that knockdown of RPS23RG1 led to increased lysosomal degradation of MDGA2 and a decrease in total cellular protein levels, while the addition of P2 significantly inhibited its degradation and increased the overall MDGA2 level. Figure 6 a). Simultaneous transfection of HeLa cells with RPS23RG1 siRNA and P2 revealed, by immunofluorescence assay, that in cells incubated with P2, MDGA2 accumulation in lysosomes was significantly reduced, while its localization in the cell membrane was significantly increased. Figure 6 b). The above experiments confirm that peptide P2 can effectively inhibit the lysosomal degradation of MDGA2, stabilize its membrane localization, and increase the cellular level of MDGA2.

[0171] Example 4: Evaluation of the therapeutic effect of peptides based on the extracellular domain of RPS23RG1 in autism (ASD) model mice.

[0172] MDGA2 is considered a susceptibility gene associated with ASD. Insufficient MDGA2 protein leads to increased excitatory synaptic transmission, and mice exhibit anxiety, social impairment, and stereotyped behaviors. Therefore, Mdga2 knockout mice are widely used as a typical autism model.

[0173] 4.1: Construction and Identification of Mdga2 Gene Knockout Mice

[0174] In this embodiment, Mdga2 gene knockout mice were constructed using CRISPR / Cas9 technology.

[0175] The Mdga2 gene is located on mouse chromosome 12 and contains 17 exons. Based on the Mdga2 gene sequence (GenBank ID: 320772) on NCBI, a targeted gDNA (guide DNA) was designed for exon 2 of the Mdga2 gene. With the synergy of Cas9, this gDNA specifically identifies and cuts the gene at specific sites, leading to a deletion mutation and premature termination of protein translation. Figure 7 a). Obtain Mdga2 gene knockout mice (Mdga2 - / - Mdga2 gene knockout mice were crossed with C57BL / 6 wild-type mice to obtain offspring mice, and Mdga2 gene knockout heterozygous mice were obtained through genotyping. + / - ).

[0176] Then, the successful construction of the Mdga2 gene knockout mouse was verified at the DNA, RNA, and protein levels, with the results as follows: Figure 7 As shown. Figure 7 b shows the genotype identification results, extracted from wild-type C57BL / 6 mice (hereinafter referred to as WT mice) and heterozygous mice (Mdga2 mice) from the same litter. + / - ), gene knockout mice (Mdga2) - / - DNA was collected, and primers 5'-ccagaggcctatgaagtccg-3' (SEQ ID NO. 20) and 5'-tccacagtgaaattccctcaa-3' (SEQ ID NO. 21) were designed for PCR amplification. Genotyping of the PCR products was performed by 15% SDS-PAGE gel electrophoresis. The wild-type mouse (WT) PCR product showed only one band, 314 bp in length; the gene knockout mouse (Mdga2)... - / - The PCR product showed only one band, 582 bp in length; while the gene knockout heterozygous mouse (Mdga2) + / - The PCR product contained the two bands mentioned above. The results indicate that, at the DNA level, KO mice were successfully constructed. Figure 7 c shows the RNA expression levels of wild-type mice (WT) and gene knockout mice, extracted from wild-type mice (WT) and heterozygous mice (Mdga2). + / - ), gene knockout mice (Mdga2) - / - RNA from brain tissue was analyzed, and Mdga2 levels were detected by reverse transcription and quantitative real-time PCR. The results were then compared after normalization with β-actin levels. The results showed that Mdga2... - / - The mRNA level of the Rps23rg1 gene in mice is almost zero, meaning there is no transcript of the Rps23rg1 gene. This strongly suggests that at the RNA level, Mdga2... - / - The mouse model was successfully constructed. Figure 7 The image shows the expression of RPS23RG1 protein in WT and gene knockout mice. Brain tissues from WT and gene knockout mice were extracted, and MDGA2 protein levels were detected by immunoprecipitation and Western blot. The results showed that MDGA2... - / - The absence of MDGA2 protein expression in mouse brain tissue strongly suggests that, at the protein level, MDGA2... - / - The mouse model was successfully constructed. In summary, Figure 7 The results showed that the Mdga2 gene knockout mouse was successfully constructed.

[0177] 4.2: Evaluation of the therapeutic effect of RPS23RG1 extracellular peptides in Mdga2 gene knockout mice

[0178] The above experimental results show that peptide P2 can directly interact with MDGA2, intervening in the lysosomal degradation level of MDGA2 and upregulating its overall level, potentially becoming an effective therapy for improving neurological disorders (such as autism) caused by MDGA2 reduction. Therefore, this embodiment further evaluates the therapeutic effect of peptides based on the extracellular domain of RPS23RG1 on autism model mice.

[0179] The researchers of this invention artificially synthesized biotin-labeled human RPS23RG1 extracellular peptide P2 (as shown in SEQ ID NO. 33) and disordered control peptide Scramble (as shown in SEQ ID NO. 29), and administered them via tail vein injection at a dose of 4 mg / kg / day to 1.5-month-old Mdga2 pharmacies. + / - In mice or littermate control WT mice, treatment was administered for 5 consecutive days followed by a 1-day rest period. On day 7, behavioral and biochemical analyses were performed on the mice. Figure 8 a)

[0180] First, after lysing the brains of mice in the control group injected with scramble and the experimental group injected with P2, biotin immunoprecipitation and Western blotting experiments were performed. The protein MDGA2 in the lysed brain tissue of the experimental group could be detected, indicating that the peptide successfully crossed the blood-brain barrier and entered the brain tissue and interacted with MDGA2. Figure 8 b). Next, we took the WT control group (injected with scramble) and Mdga2 respectively. + / - Control group (injected scramble) and Mdga2 + / - The brain tissue of mice in the experimental group (injected with P2) was analyzed by Western blotting to detect MDGA2 protein levels. The results showed that injection of peptide P2 effectively improved MDGA2 levels. + / - Insufficient MDGA2 levels in mouse brain tissue ( Figure 9 ab). Behavioral testing of mice showed no difference in movement patterns or time spent in the central region between the control and experimental groups during the open field experiment. Figure 10 a) Figure 10 b shows that P2 injection did not change Mdga2. + / - Stereotypical self-grooming behaviors in mice. However, in free socialization and three-box experiments, Mdga2 mice treated with P2 showed... + / - The mice exhibited normal social interests and preferences. Figure 10 c, 10d). The above results indicate that the MDGA2-targeting peptide of the present invention can significantly improve Mdga2. + / - Social deficits in mice.

[0181] Besides ASD, synaptic stability and homeostasis, as well as the inactivation of postsynaptic density proteins, are also present in other neurological disorders. For example, previous genomic analyses of patients with epileptic encephalopathy have identified multiple risk genes exhibiting genetic diversity, including MDGA2, SHANK3, and several other genes that are also susceptibility genes for autism. This suggests a clinical link between the two diseases. Furthermore, high methylation of MDGA2 was found to lead to gene inactivation in gastric cancer cells. Conversely, high expression of MDGA2 can inhibit tumor cell proliferation and promote apoptosis, and is considered a novel tumor suppressor in the development of gastric cancer. Therefore, the peptides of the present invention that can target and regulate MDGA2 metabolism to increase its expression level are also applicable to the treatment of other neurological diseases (e.g., epilepsy) and tumors (e.g., gastric cancer) caused by MDGA2 inactivation.

[0182] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. An MDGA2-targeting polypeptide, characterized in that: Its amino acid sequence is SYFSQHNSFCMRSTS (SEQ ID NO.08).

2. A conjugate, characterized in that: It consists of the MDGA2-targeting peptide as described in claim 1 and a modified portion, wherein the modified portion is a detectable marker.

3. An isolated nucleic acid molecule, characterized in that: It encodes the MDGA2-targeting polypeptide of claim 1.

4. A carrier, characterized in that: It has the isolated nucleic acid molecule as described in claim 3.

5. A host cell, characterized in that: It has the isolated nucleic acid molecule as described in claim 3 or the vector as described in claim 4, wherein the host cell is not a plant cell.

6. The method for preparing the MDGA2-targeting polypeptide according to claim 1, characterized in that: include: The host cell of claim 5 is cultured under suitable conditions, and the MDGA2 targeting peptide is recovered from the culture of the host cell.

7. A pharmaceutical composition, characterized in that: It comprises the MDGA2 targeting peptide of claim 1, the conjugate of claim 2, the isolated nucleic acid molecule of claim 3, the vector of claim 4 or the host cell of claim 5, and a pharmaceutically acceptable carrier and / or excipient.

8. Use of the MDGA2-targeting peptide of claim 1 in the preparation of a medicament for the treatment of autism.

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