MDGA2 targeting polypeptide and application thereof

Abstract

The invention discloses an MDGA2 targeting polypeptide and application thereof, the MDGA2 targeting polypeptide is composed of 8-55 continuous amino acids of RPS23RG1 protein and contains QHNSX1CMR, and X1 is F or L. The invention further discloses a preparation method of the MDGA2 targeting polypeptide. The MDGA2 targeting polypeptide and the fusion protein can significantly inhibit lysosomal degradation of MDGA2 so as to improve the level of MDGA2, so that the MDGA2 targeting polypeptide and the fusion protein can be used for treating diseases (such as nervous system diseases such as autism and gastric cancer) related to too low MDGA2 activity, and have great clinical value.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to an MDGA2 targeting polypeptide and an application thereof. Background Art

[0002] Autism is called autism spectrum disorder (ASD), also known as autism, which is a type of early-onset neurological disorder in children. The main clinical symptoms are social disorders, narrow interests, repetitive stereotyped behaviors, etc. At the same time, some patients are accompanied by complications such as anxiety, depression, intellectual disabilities, epilepsy, etc., which seriously affect the development and mental health of children (Canitano et al., 2022). According to the "China Autism Education and Rehabilitation Industry Development Status Report IV" released in my country in 2022, it has become one of the primary causes of children's problems, bringing huge economic and mental burdens to families and society. However, the existing drugs cannot completely cure ASD, and can only improve the patient's symptoms to a small extent, so effective preventive measures and treatments are urgently needed to solve the health problems caused by autism.

[0003] ASD is mainly 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 the main cause of ASD (Gilbert and Man, 2017; Glessner et al., 2009; Peca and Feng, 2012). Recently, loss-of-function and missense mutations of the MDGA2 gene have been found in ASD patients (Bucan et al., 2009; Cirnigliaro et al., 2023; De Rubeis et al., 2014). The human MDGA2 gene is located on chromosome 14, and its encoded protein has 956 amino acids. As a vertebrate-specific immunoglobulin superfamily protein, the MDGA2 protein contains 6 extracellular immunoglobulin-like (Ig) domains, 1 fibronectin type III (FNIII) domain, 1 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 mainly localized to the postsynaptic membrane through the GPI anchor (Connor et al., 2016; Fertan et al., 2021). Studies have found that MDGA2 interacts with the neuroligin NLGN to jointly regulate synaptic function, and MDGA2 deficiency leads to increased excitatory synaptic activity and ASD-like behavior in mice (Connor et al., 2016; Fertan et al., 2021). Summary of the invention

[0004] The present invention aims to provide a MDGA2 targeting polypeptide.

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

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

[0007] An MDGA2 targeting peptide, which consists of 8-55 consecutive amino acids of RPS23RG1 protein and contains QHNSX 1 CMR (SEQ ID NO.31), wherein X 1 F or L.

[0008] Preferably, the MDGA2 targeting polypeptide consists 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 X 2 YFX 3 QHNSX 1 CMRX 4 TX 5 (SEQ ID NO.32), wherein X 2 For S or P, X 3 S or F, X 4 S or G, X 5 It is S or P.

[0010] Further preferably, the amino acid sequence comprises SYFSQHNSFCMRSTS (human origin, SEQ ID NO. 08) or PYFFQHNSLCMRGTP (mouse origin, SEQ ID NO. 14).

[0011] A conjugate having the above-mentioned MDGA2 targeting polypeptide and a modification portion, wherein the modification portion is selected from another polypeptide, a detectable label (to make it useful for detection) or any combination thereof, wherein the other polypeptide is selected from CPP (to improve its ability to penetrate cell membranes), a targeting portion (to make it targeted) and / or a protein tag (to facilitate its expression, detection, tracing and / or purification).

[0012] A fusion protein comprising the above-mentioned MDGA2 targeting polypeptide and another polypeptide, wherein the other polypeptide is selected from CPP (cell membrane penetrating peptide, which improves its ability to penetrate the cell membrane), a targeting portion (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 modification portion is optionally linked to the N-terminus or C-terminus of the MDGA2 targeting polypeptide 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 targeting moiety is a ligand, a receptor or an 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 has the nucleotide sequence of the above-mentioned MDGA2 targeting polypeptide or the above-mentioned fusion protein.

[0020] A vector having the above-mentioned isolated nucleic acid molecule. The vector may be a cloning vector or an expression vector. In a preferred embodiment, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a cosmid, etc. In a preferred embodiment, the vector can express the MDGA2 targeting polypeptide of the present invention or the fusion protein of the present invention in a subject (e.g., a mammal, e.g., a human).

[0021] A host cell having the above-mentioned isolated nucleic acid molecule or the above-mentioned vector. 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 polypeptide or the above-mentioned fusion protein comprises: culturing the above-mentioned host cell under suitable conditions, and recovering the MDGA2 targeting polypeptide or fusion protein from the culture of the host cell.

[0023] A pharmaceutical composition comprises the MDGA2 targeting polypeptide, the conjugate, the fusion protein, the isolated nucleic acid molecule, the vector or the 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 additional pharmaceutically active agent is a drug active in treating a neurological disease (eg, a neurodegenerative disease).

[0026] Or the additional pharmaceutically active agent is selected from omega-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] Use of the MDGA2 targeting polypeptide, the conjugate, the fusion protein, the isolated nucleic acid molecule, the vector or the host cell in preparing a drug for treating diseases associated with low MDGA2 activity, or inhibiting lysosomal degradation of MDGA2, or increasing the expression level of MDGA2.

[0028] It has been previously reported that deletion or dysregulation of MDGA2 impairs synaptic function and causes autistic-like behavioral deficits (Connor et al., 2016; Fertan et al., 2021). In particular, previous studies have found MDGA2 loss-of-function mutations in the brains of ASD patients (Bucan et al., 2009; Cirnigliaro et al., 2023; De Rubes et al., 2014). In addition, MDGA2 is associated with epilepsy and schizophrenia (Lesca et al., 2012). In addition, during the development of cancer, for example, in patients with gastric cancer, increased methylation levels of MDGA2, i.e., decreased activity, were observed (Zhao et al., 2013). Therefore, the disease associated with low MDGA2 activity is a neurological disease or cancer.

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

[0030] The cancer is preferably gastric cancer.

[0031] A method for inhibiting lysosomal degradation of MDGA2 or increasing the expression level of MDGA2 in vitro for non-diagnostic treatment purposes, characterized in that the MDGA2 targeting polypeptide, the conjugate or the fusion protein is contacted with a cell in need thereof.

[0032] Preferably, the cell is a neuronal cell or a gastric cancer cell.

[0033] The MDGA2 targeting polypeptide, fusion protein or pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, lyophilized powders), etc. In some embodiments, the MDGA2 targeting polypeptide, fusion protein or pharmaceutical composition of the present invention can be formulated into injection solutions or lyophilized powders.

[0034] In addition, the MDGA2 targeting polypeptide or fusion protein of the present invention can be present in a pharmaceutical composition in a unit dosage form for easy administration.

[0035] MDGA2 targeting polypeptide of the present invention, fusion rotein or pharmaceutical composition can be used by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, leaf sheath, endocytoplasmic reticulum groove, groin, bladder, local (such as, powder, ointment or drops), or nasal route.But, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection).Technicians should understand that route of administration and / or mode will change according to the intended purpose.In a preferred embodiment, MDGA2 targeting polypeptide of the present invention, fusion rotein or pharmaceutical composition are given by intravenous infusion or injection.

[0036] MDGA2 targeting polypeptide, fusion protein or pharmaceutical composition provided by the present invention can be used alone or in combination, and can also be used in combination with other pharmaceutically active agents (for example, medicines with the activity of treating nervous system diseases). In certain preferred embodiments, MDGA2 targeting polypeptide of the present invention or fusion protein and other medicines with the activity of treating nervous system diseases are used in combination to prevent and / or treat diseases (for example, nervous system diseases) related to the low activity of MDGA2. This other pharmaceutically active agent can be used before, simultaneously or afterwards using MDGA2 targeting polypeptide of the present invention, fusion protein or pharmaceutical composition.

[0037] The pharmaceutical composition of the present invention may include a "therapeutically effective amount" or a "preventive effective amount" of the MDGA2 targeting polypeptide or fusion protein of the present invention. A "preventive effective amount" refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease (e.g., a disease associated with too low MSGA2 activity). A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications of a patient who has already suffered from the disease. The therapeutically effective amount of the MDGA2 targeting polypeptide or fusion protein of the present invention may vary according to the following factors: the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0038] In the present invention, the dosage regimen can be adjusted to obtain the best desired response (e.g., therapeutic or preventive response). For example, the dosage can be administered once, multiple times over a period of time, or the dosage can be reduced or increased in proportion to the urgency of the treatment situation.

[0039] The typical non-limiting range of the treatment or prevention effective amount of the MDGA2 targeting polypeptide or fusion protein of the present invention is 0.001-100 mg / kg body weight, such as 0.01-50 mg / kg body weight, 0.1-25 mg / kg body weight. It should be noted that the dosage may vary with the type and severity of the symptoms to be treated. In addition, it is understood by those skilled in the art that for any particular patient, a specific dosage regimen should be adjusted over time according to the patient's needs and the doctor's professional evaluation; the dosage range given here is only for illustrative purposes, and does not limit the use or scope of the pharmaceutical composition of the present invention.

[0040] In the present invention, the subject may be a mammal, such as a human.

[0041] Definition of terms

[0042] In the present invention, unless otherwise specified, the scientific and technical terms used in the present invention have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used in the present invention are all conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0043] As used in the present invention, the term "RPS23RG1" refers to a protein expressed by the ribosomal protein S23 mRNA retrotransposon gene Rps23rg1, which is well known to those skilled in the art and described in (Yan et al., 2016; Zhao et al., 2019), and U.S. Patent Application US2010 / 0286252. In the present invention, RPS23RG1 can be from any source, for example, human or non-human, such as non-human mammalian, such as mouse (such as mouse).

[0044] As used in the present invention, the term "cell penetrating peptide (CPP)" is also called "cell penetrating peptide", "protein translocation domain (PTD), "Trojan horsepeptides" or "transduction peptide (transduction peptide)", etc., which refers to a polypeptide that can promote the cellular uptake of various molecules (e.g., various macromolecules including proteins or nucleic acids; for example, the MDGA2 targeting polypeptide of the present invention or its variant). Such polypeptides are well known in the art and are described in, for example, 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 the methods described in detail in U.S. patent application US2008 / 0234183, all of which are incorporated herein by reference.

[0045] In the present invention, examples of the CPP include but are not limited to: (i) protein derived peptides: such as sequences derived from the gene controlling antennae (Antennapedia), such as pAntp (43-58); sequences derived from HIV-1, such as 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: for example VT5; MAP; or arginine stretch, etc.; (iii) designed peptides: for example MPG; Transportan; Transportan 10; Pep-1; a peptide selected from KALA; or a peptide selected from Bulforin2, etc.

[0046] In addition, the CPP used in the conjugate of the present invention can also be selected from a polypeptide sequence having about 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 the cellular uptake of the isolated polypeptide of the present invention (or its variant) and / or promoting the isolated polypeptide of the present invention (or its variant) to pass through the blood-brain barrier.

[0047] As used in the present invention, the term "targeting moiety" refers to a domain that can guide the MDGA2 targeting polypeptide of the present invention (or its variant) to a desired location, which can be a specific tissue, a specific cell, or even a specific intracellular location (e.g., a nucleus, a ribosome, an endoplasmic reticulum, a lysosome, or a peroxisome). Those skilled in the art know how to design a corresponding targeting domain by the characteristics of the desired location.

[0048] As used in the present invention, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0049] As used in the present 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 in the present invention, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both compared sequences is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent 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 6 out of 10 positions of the two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Usually, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent 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), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0051] As used in the present invention, the term "separated" refers to that the target (for example, polypeptide) has been purified from the pollutant present in the sample, and the sample is for example the sample comprising the target obtained from a natural source. The term separated need not exclude the existence of other components that are intended to be combined with the isolate to work. For example, MDGA2 targeting polypeptide of the present invention can be described as separated, although it can be connected with a cell-penetrating peptide.

[0052] As used in the present invention, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, ionic strength enhancers, agents that maintain osmotic pressure, agents that delay absorption, diluents, adjuvants, preservatives, stabilizers, and the like. For example, pH regulators 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 that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay 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), and the like. 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, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings commonly understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug (e.g., inhibitory activity on lysosomal degradation of MDGA2), including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein) or their degradation products (e.g., lactalbumin hydrolysate), etc.

[0053] As used herein, the term "treat" refers to treating or curing a disease (eg, a nervous system disease), delaying the onset of symptoms of a disease (eg, a nervous system disease), and / or delaying the progression of a disease (eg, a nervous system disease).

[0054] As used in the present invention, the term "prevention" refers to preventing, inhibiting or delaying the occurrence of a disease (eg, a nervous system disease).

[0055] As used in the present invention, 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 nervous system disease), delay the onset of symptoms of a disease (e.g., a nervous system disease) and / or delay the development of a disease (e.g., a nervous system disease). A preventive effective amount can be an amount that is effective or sufficient to prevent, inhibit or delay the occurrence of a disease (e.g., a nervous system disease). Such an effective amount can be easily determined by a person skilled in the art or a doctor, and can be related to the intended purpose (e.g., treatment or prevention), the general health of the subject, age, sex, weight, severity of the disease to be treated, complications, mode of administration, etc. The determination of such an effective amount is fully within the capabilities of those skilled in the art.

[0056] As used in the present invention, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (e.g., a human) suffers from a disease (e.g., a nervous system disease) associated with too low MDGA2 activity, or has a risk of suffering from a disease (e.g., a nervous system disease) associated with too low MDGA2 activity.

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

[0058] 1) Ability to specifically bind to MDGA2;

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

[0060] 3) the ability to increase MDGA2 levels in a subject (optionally after conjugating the polypeptide to a CPP);

[0061] 4) the ability to improve synaptic dysfunction and / or social dysfunction and repetitive stereotyped behaviors caused by low MDGA2 levels in a subject (optionally after conjugating the polypeptide to a CPP);

[0062] 5) The ability to treat a disease associated with low MDGA2 activity (eg, a neurological disease) in a subject (optionally after conjugating the polypeptide to a CPP).

[0063] The beneficial effect of the present invention is that the MDGA2 targeting polypeptide and fusion protein of the present invention can significantly inhibit the lysosomal degradation of MDGA2, thereby increasing the level of MDGA2, and can therefore be used to treat diseases associated with low MDGA2 activity (such as autism and other neurological diseases and gastric cancer), and has great clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 a-1c: Analysis of RPS23RG1 regulating MDGA2 degradation level. Figure 1 a: Primary neurons from WT and KO mice were isolated, and cycloheximide (CHX) was added on the 9th day of in vitro culture. The protein levels of MDGA2 at different times were analyzed by WB. Figure 1 b: HEK293T cells were treated with CHX, MG132, CQ, and NH4Cl at the same time, and the protein level of MDGA2 was analyzed. Figure 1 c: Brain tissues of 1.5-month-old WT and KO mice were extracted, lysosomal components were separated, and WB was used to analyze the protein level of MDGA2 in lysosomes. Statistical analysis was performed using Image J software to analyze the grayscale of protein levels in the figures. The 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 ttest).

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

[0066] Figure 3 a-3c: Analysis of RPS23RG1 sequence. Figure 3 a: Amino acid sequence alignment of mouse RPS23RG1 and human RPS23RG1, and residues interacting with MDGA2 in structure prediction. Gray indicates identical amino acids or interacting residues. Figure 3 b: Schematic diagram of the full-length sequence of human RPS23RG1 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 indicates a truncated form lacking amino acids 57-67. Δ103-128 indicates a truncated form lacking amino acids 103-128. Figure 3c: Co-immunoprecipitation analysis of human RPS23RG1 and its different truncations with MDGA2, with IgG as a negative control.

[0067] Figure 4 a-4b: Analysis of the role of RPS23RG1 core sequence in MDGA2 degradation. Figure 4 a: Full-length RPS23RG1 and Δ103-128 truncation were overexpressed in Lyso-HA cells, and the levels of MDGA2 in lysosomes and total proteins were detected by WB by isolating lysosomes. Figure 4 b: Full-length RPS23RG1 and Δ103-128 truncation were overexpressed in HEK293T cells, and the levels of MDGA2 in the cell membrane and total protein were detected by WB. Statistical analysis: Grayscale analysis of protein levels in the figures was performed using Image J software. 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: Analysis of 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: The biotin-coupled 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 colocalization of the peptide and MDGA2 was analyzed by immunofluorescence. Scale bar: 10 μm.

[0069] Figure 6 a-6b: Analysis of RPS23RG1-based peptide regulation of MDGA2 degradation. Figure 6 a: Peptide P2 and RPS23RG1 siRNA were transfected into Lyso-HA cells. After sufficient reaction, cell lysosomes were separated and WB was used to detect the levels of MDGA2 in lysosomes and total proteins. The right side is a statistical analysis chart. Image J software was used to perform grayscale analysis of the protein levels in the chart. The results are expressed as mean ± standard error (SEM), n = 3. Figure 6b: Peptide P2 and RPS23RG1 siRNA were transfected into HeLa cells, and the colocalization of MDGA2 on the cell membrane and with LAMP1 was analyzed by immunofluorescence. The right side is a statistical analysis chart, and the colocalization was analyzed using Image J 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 a-7d: Construction and identification of Mdga2 gene knockout mice. Figure 7 a: Construction of Mdga2 knockout mice. Schematic diagram of the Mdga2 gene structure and CRISPR / Cas9 technology. The CDS sequence of RPS23RG1 starts from the first exon to the end of the 17th exon, and the second exon represents the knockout region. Figure 7 b: Genotype identification of WT and gene knockout mice. + / - and Mdga2 - / - Mouse DNA, SDS-PAGE analysis. Figure 7 c: Analysis of RNA expression levels of Mdga2 in WT and knockout mice. + / - and Mdga2 - / - The level of Mdga2 in mouse brain tissue RNA was detected by reverse transcription and quantitative PCR, and then normalized with β-actin level for comparison. n = 4. Figure 7 d: Analysis of MDGA2 protein expression levels in WT and knockout mice. + / - and Mdga2 - / - Mouse brain tissue, WB detection of MDGA2 protein level. Image J software was used to analyze the grayscale of protein levels in the figure, and the 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 RPS23RG1-based peptides in Mdga2 knockout mice. Figure 8 a: Schematic diagram of peptide injection into the tail vein of mice. 1.5-month-old mice were injected with P2 and control peptides into the tail vein for 5 consecutive days, and behavioral and biochemical tests were performed on the 7th day. Figure 8b: The brains of mice injected with P2 and control peptides were taken respectively, and biotin immunoprecipitation-immunoblotting was used to verify that the peptides passed through the blood-brain barrier, entered the brain tissue and bound to MDGA2.

[0072] Fig. 9 a-9b: Effects of RPS23RG1-based peptides on MDGA2 levels in Mdga2 knockout mice. Fig. 9 a: For mice treated with P2 or control peptide, brain tissue protein lysates were extracted and MDGA2 protein levels were detected by immunoblotting. Fig. 9 b: Protein level statistics. Image J software was used to analyze the grayscale of protein levels in the figure, with β-actin as the 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] Fig.10 a-10d: Evaluation of the therapeutic efficacy of RPS23RG1-based peptides in MDGA2-deficient ASD model mice. Fig.10 a: Mine field experiment, analyzing the total activity distance and the time spent in the mine field by mice treated with P2 or control peptide. Fig.10 b: Stereotyped grooming experiment, analyzing the frequency of repetitive stereotyped behaviors of self-grooming and jumping in the mine field in mice treated with P2 or control peptide. Fig.10 c: Mouse free social interaction experiment, analyzing the time that mice treated with P2 or control peptide spent interacting and sniffing with stranger mice during free space exploration. Fig.10 d: Three-box social experiment of mice. In the social preference test phase, the time that mice sniffed the stranger mouse (S1) and the empty cage (E) was analyzed, and the social preference index PI = T (S1-E) / T (S1 + E) was also analyzed. Then, the time that mice sniffed another stranger mouse (S2) and (S1) mouse was analyzed in the social novelty test phase. 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 DESCRIPTION

[0074] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0075] Sequence information

[0076] The information of the sequences involved in the present invention is provided in Table 1 below.

[0077] Table 1: Description of sequences

[0078]

[0079]

[0080]

[0081]

[0082] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., A Compendium of Molecular Biology Experiments, 3rd edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the conditions recommended by the product manufacturers. The reagents whose sources are not indicated in the examples are all conventional reagents in the art or commercially available reagents.

[0083] Experimental Materials and Methods

[0084] Real-time quantitative PCR (qRT-PCR)

[0085] Total RNA was extracted from mouse tissues using TRIzol reagent according to the manufacturer's instructions. After the concentration was determined by a microplate reader, the mRNA was reverse transcribed into cDNA using a kit. FastStart Universal SYBR Green Master (Roche) was used for qRT-PCR experiments. After the qRT-PCR experiment, a small amount of PCR product was analyzed by 1.5% agarose gel electrophoresis to determine whether the PCR product fragment size was correct and whether it was specifically amplified.

[0086] Protein extraction and Western blot

[0087] 1. Extraction of cell sample protein:

[0088] (1) Cell collection: discard the culture medium and wash the cells twice with PBS; discard the PBS, scrape the cells with a cell scraper and collect them in a centrifuge tube; centrifuge at 1000 rpm for 5 min, collect the cells at the bottom of the tube, and discard the supernatant.

[0089] (2) Cell lysis: Add TNEN cell lysis buffer containing cocktail protease inhibitors and phosphatase inhibitors (added just before use) to the cell mass collected in (1), shake on ice for 30 min or place in a silent mixer at 4°C for slow lysis overnight, centrifuge at 12,000 rpm at 4°C for 10 min, collect the supernatant, and measure the protein concentration.

[0090] 2. Extraction of tissue sample protein:

[0091] Fresh tissues were immediately frozen in liquid nitrogen after being taken out, and then stored in a -80°C ultra-low temperature refrigerator for later use. The tissues were minced with surgical scissors before homogenization, and then homogenized with a polytron in RIPA lysis buffer. After sufficient lysis, the tissues were centrifuged at 4°C, 12,000 rpm for 15 minutes, the supernatant was collected, and the centrifugation was repeated once to measure the protein concentration.

[0092] 3. Determination of protein concentration:

[0093] (1) Prepare BCA protein assay solution by mixing solution A and solution B at a ratio of 50:1 and set aside;

[0094] (2) Using BSA as the standard sample, prepare a protein standard curve;

[0095] (3) Protein samples were added to a 96-well plate, and TNEN / RIPA lysis buffer was used as a blank control. Three wells were set up in each group.

[0096] (4) Add 200 μl of the prepared protein assay solution to each well, mix well, and react in a 37°C incubator for 30 min;

[0097] (5) Use an ELISA instrument to measure OD562 and substitute the obtained value into the protein standard curve to calculate the protein sample concentration.

[0098] 4.Westernblot:

[0099] (1) Protein electrophoresis: Take 30-40 μg of protein sample, add 1 / 4 volume of 5× SDS loading buffer, boil at 100℃ for 3-5 min, and run electrophoresis in Tris-Glycine electrophoresis buffer using SDS-PAGE protein gel.

[0100] (2) Electrophoretic transfer of proteins: Precool the electrophoretic transfer buffer at 4°C, cut a PVDF membrane of appropriate size, soak it with methanol, and soak it in the electrophoretic transfer buffer together with filter paper for 10 minutes. At the same time, cut the gel after electrophoresis and soak it in the electrophoretic transfer buffer for 5 minutes. Then, stick the PVDF membrane on the gel, cover both sides with filter paper, remove all bubbles, and install it in the electrotransfer tank with the membrane facing the positive electrode, and perform constant current electrotransfer (300mA, 90min) at 4°C.

[0101] (3) Antigen-antibody reaction:

[0102] a. Blocking: Block with 5% skim milk powder at room temperature for 1 hour;

[0103] b. Primary antibody reaction: dilute the primary antibody in blocking solution at an appropriate ratio and incubate at 4°C overnight;

[0104] c. Secondary antibody reaction: Wash three times with PBST, 10 min each time, dilute the primary antibody in blocking solution at an appropriate ratio, and incubate at room temperature for 1 h.

[0105] (4) ECL detection:

[0106] Wash three times with PBST, 10-15 min each time; mix solution A and solution B in the ECL kit at a ratio of 1:1 (V / V), add ECL to the membrane surface in a dark room, incubate for 5 min and then expose, adjusting the exposure time according to the fluorescence intensity.

[0107] Immunoprecipitation (IP) or co-immunoprecipitation (CoIP)

[0108] (1) Collect protein samples, add 10 μl of Protein-ASepharose to the protein samples, and incubate with rotation at 4°C for 1-2 h.

[0109] (2) Centrifuge at 4°C, 5000 rpm for 2 min. Transfer the supernatant to a new EP tube, fill up to 700 ul with 0.5% TNEN lysis buffer, add 20 μl Protein-GSepharose and corresponding antibodies, and incubate at 4°C with rotation overnight.

[0110] (3) Centrifuge at 5000 rpm for 2 min at 4°C. Remove the supernatant by aspiration, add pre-cooled 1% TNEN solution, and incubate at 4°C with rotation for 8 min.

[0111] (4) Repeat step (3) three times. The last time, carefully suck up the supernatant with a microsyringe, then add 20 μL of 2× loading buffer, mix well, and boil the sample at 100°C for 5 min. The obtained sample is used for immunoblotting analysis.

[0112] Immunofluorescence

[0113] (1) Place the treated coverslips into a 24-well plate (one per well), evenly seed with cells at a certain density (5% to 10%), and culture for 24 hours.

[0114] (2) Transfer the corresponding plasmid according to the lipo2000 transfection method.

[0115] (3) 24 hours after transfection, remove the culture medium and wash the cells three times with pre-cooled PBS. Remove the PBS and fix the cells with 4% paraformaldehyde at room temperature for 10 min.

[0116] (4) 0.1% TritonX100-PBS penetration at room temperature for 5-10 minutes (the time should be strictly controlled here).

[0117] (5) Block with 3% BSA-PBS for 1 h.

[0118] (6) Incubate with different primary antibodies (1:100 dilution in 3% BSA-PBS) at 4°C overnight.

[0119] (7) Wash with PBS 4 times, 5 min each time. Let the culture plate stand still without shaking.

[0120] (8) Incubate with corresponding fluorescent secondary antibody at room temperature for 60 min.

[0121] (9) If necessary, incubate with DAPI at room temperature for 3-5 min.

[0122] (10) Wash with PBS 4 times, 5 min each time.

[0123] (11) Seal the slides at room temperature overnight. After the sealant is dry, observe and photograph under a confocal microscope.

[0124] Isolation of lysosomes

[0125] 1. Tissue (Bebo Biotechnology, lysosome extraction kit, BB-3603)

[0126] (1) Remove the mouse brain tissue, cut it into small pieces with surgical scissors, wash it twice with cold PBS, add 400 μl of cold reagent A, place it on ice for 10 min, and homogenize it with a tight Dounce homogenizer for 30-40 times;

[0127] (2) The homogenate was centrifuged at 4°C and 1000 × g for 5 min. The precipitate was discarded and the supernatant was collected.

[0128] (3) Centrifuge the supernatant at 4°C, 3000×g for 10 min. Discard the precipitate and collect the supernatant.

[0129] (4) Centrifuge the supernatant at 4°C, 5000×g for 10 min. Discard the precipitate and collect the supernatant;

[0130] (5) Centrifuge the supernatant at 4°C, 20,000-30,000 × g for 20 min. Discard the supernatant, collect the precipitate, add 400 μl of cold reagent B to the precipitate, and mix well.

[0131] (6) Centrifuge at 4°C, 20,000-30,000 × g for 20 min. Discard the supernatant, collect the precipitate, and resuspend the precipitate in Lysosome Preservation Buffer C to obtain a lysosome sample. Store in a refrigerator for later use or use directly in downstream experiments.

[0132] 2. Cells (LysoIP)

[0133] (1) Using HeLa cells stably transfected with TMEM192-3xHA, the treated cells were rinsed twice with PBS, then collected with KPBS (136 mM KCl, 10 mM KH2PO4, pH 7.25, adjusted with KOH), and a portion of the cells were lysed as the whole protein fraction;

[0134] (2) The KPBS cell suspension was homogenized slowly with a tight Dounce homogenizer for 30 seconds, and centrifuged at 1000 g for 2 min at 4°C;

[0135] (3) The supernatant was collected and incubated with KPBS pre-washed anti-HA beads on a gentle rotating shaker for 30 min;

[0136] (4) Wash the immunoprecipitate three times with KPBS and resuspend it in TNEN buffer to obtain the lysosome sample. Store it in the refrigerator for later use or use it directly in downstream experiments.

[0137] Biotin labeling of cell surface proteins

[0138] Dissolve biotin (EZ-Link Sulfo-NHS-SS-Biotin) in freshly prepared PBS / CM buffer to a final concentration of 0.5 mg / mL and place on ice for later use; remove cells from the incubator, place on ice, remove the culture medium, add an appropriate amount of pre-cooled PBS / CM buffer to wash the cells three times, remove the buffer, add biotin solution to the cell culture plate, culture for 20 min in an ice bath, remove the biotin solution, and repeat once; add 50 mM NH 4 Cl PBS / CM buffer to terminate the reaction (10min); lyse the cells with TNEN lysis buffer (containing protease inhibitors), centrifuge at 4℃, 12000rpm for 10min, transfer the supernatant to a new centrifuge tube, determine the protein concentration by BCA method, take an equal amount of protein sample, add an equal volume of 2× loading buffer, mix well, boil at 100℃ for 5min, and detect protein expression by immunoblotting analysis; add streptavidin beads to the equal amount of remaining protein sample, and precipitate biotinylated protein at 4℃ overnight; the next day, centrifuge at 4℃, 5000RPM for 2min, remove the supernatant, rinse the streptavidin beads with 1% TNEN 3 times, 10min each time, remove the residual liquid with a microinjector, add 20μL 2× loading buffer, boil at 100℃ for 5min, and determine the content of the target protein by protein immunoblotting analysis.

[0139] Plasma membrane separation

[0140] The cell membrane preparation process uses a cell membrane protein extraction kit (Phygene, PH0710). Remove the cells from the incubator, place them on ice, remove the culture medium, add an appropriate amount of pre-cooled PBS buffer to wash the cells twice, resuspend the collected cells in pre-cooled extraction reagent A and incubate for 15 minutes, then use a loose Dounce homogenizer to slowly homogenize 200 times, stop grinding when about 70% of the cells are broken, centrifuge the cell suspension at 700g for 10 minutes, collect the supernatant, and then centrifuge at 120,000g for 30 minutes to collect the membrane precipitate. Finally, resuspend the membrane protein in the precipitate and dissolve it with a lysis buffer containing protease inhibitors, centrifuge at 12,000g for 10 minutes, and collect it for the next experiment.

[0141] Protein interaction structure prediction

[0142] The researchers of the present invention selected the PDB file of MDGA2 (AF-Q7Z553-F1) suitable for the present invention in 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). The interaction between proteins was predicted by GRAMM: Docking network server (http: / / gramm.compbio.ku.edu / gramm, Vakser laboratory), and finally PYMOL was used to perform structural visualization and molecular docking analysis on the three-dimensional protein model and its interaction.

[0143] Tail vein injection

[0144] Dissolve the synthesized peptide in physiological saline, add no more than 2% dimethyl sulfoxide to help dissolve, and place the fully dissolved peptide on ice for later use. When injecting mice, make sure the mouse is comfortably placed in the mouse tube and positioned with the tail vein syringe barrel, while placing the mouse tail in the groove of the squeeze plate. Adjust the light so that the blood vessels are clearly visible, and then start the injection from the bottom of the tail. During the injection process, slowly push the peptide solution and always observe the health of the mouse. After the injection, the needle stays to ensure the normal absorption of the drug. After the needle is removed, use a cotton swab to press and stop bleeding for 1 minute. Inject at the same time for 5 consecutive days.

[0145] Animal behavior experiments

[0146] All mouse behavioral experiments were performed using Smart Video Tracking Software (Panlab, Harvard Apparatus) for data collection and analysis. Animal behavioral experiments were conducted between 9:00 a.m. and 18:00 p.m. every day, with a light intensity of 650 lux in the laboratory.

[0147] (1) Touch and stroke the mice once a day for 3 days before the start of the experiment. Gently grasp the mouse's tail and pick it up. Let the mouse stay in your hand for 30 seconds, then use a marker to draw a line on the mouse's tail. After marking the mouse, still hold it in your palm, grasp its tail, and then gently put it back into the cage.

[0148] (2) On the day of the experiment, the mice were transferred to the preparation room and allowed to adapt for 60 minutes. Before the experiment, the boxes and mazes were cleaned with 70% alcohol. After each experiment, the boxes and mazes were cleaned with 70% alcohol to remove the feces and urine excreted by the mice during the experiment and to eliminate the interference of the residual smell of the mice on the experiment.

[0149] 1. Open field test

[0150] Used to study the autonomous movement ability and anxiety behavior of mice, mainly based on the avoidance of mice to bright light and open space. In the open field test, the mice were placed in the center of the box (40cm (L) × 40cm (W) × 40cm (H)), and allowed to freely explore the maze for 10 minutes, and the total movement distance (Total distance) and the time spent in the middle area (Time in center) were recorded.

[0151] 2. Self-grooming test

[0152] The mice were placed in the center of the box (40cm (L) × 40cm (W) × 40cm (H)) and allowed to explore the maze freely for 15 minutes. The Smart3.0 video tracking system was used to record the entire process. It is generally believed that the first 5 minutes is the adaptation stage for mice to enter a new environment. Therefore, by observing the video data of the mice in the last 10 minutes, the total number of times and total time that each experimental mouse combed its own hair with its limbs were counted.

[0153] 3. Social affiliation

[0154] The experimental mouse was placed in the center of the box (40 cm (L) × 40 cm (W) × 40 cm (H)). Before the experiment, a wild-type mouse (stimulus mouse) of the same gender and age as the experimental mouse was placed in a metal cage. The experimental mouse was then placed in the box and allowed to explore freely for 10 min. The Smart3.0 video tracking system was used to record the entire process. The exploration time of the mouse in the metal cage and within 2 cm around it was recorded.

[0155] 4. Three chamber sociability test

[0156] There are two transparent partitions in the box (60 width × 40 depth × 22 height cm) that separate the left, middle and right chambers (20 × 40 cm each), and each partition has a small opening (5 × 5 cm) for access to each chamber. A fixed wire cylinder (8.5 cm in diameter) is placed in each of the left and right chambers. The test consists of three stages: adaptation, sociability test, and social novelty test. In the adaptation stage, both the left and right cylinders are empty, and the test mouse is placed in the middle and allowed to explore freely for 10 minutes. In the sociability test, a gender-matched stranger mouse (S1) is enclosed in an empty cylinder, and the mouse can explore freely for 10 minutes. The time the mouse explores S1 or another empty cylinder (E) is measured, and the social preference index (T(S1-E) / T(S1+E)) is calculated: the time exploring S1 minus the time exploring E, divided by the time exploring S1 and E). During the social novelty test, another unfamiliar mouse (S2) of the same sex was placed in the empty cylinder and allowed to explore freely for 10 min. The time the mouse explored 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, divided by the time spent exploring S2 and S1.

[0157] Materials and reagents

[0158]

[0159]

[0160]

[0161] Example 1: RPS23RG1 regulates lysosomal degradation of MDGA2

[0162] First, the researchers detected that the MDGA2 protein level was reduced in Rps23rg1 knockout mice, and then treated hippocampal neuron cultures of Rps23rg1 KO and WT mice with cycloheximide (CHX) to inhibit protein synthesis. It was found that the degradation rate of MDGA2 in Rps23rg1 KO neurons was significantly accelerated compared with that in WT controls ( Figure 1 a). In addition, treatment with the lysosomal inhibitors chloroquine (CQ) or NH4Cl blocked the degradation of MDGA2, but the proteasome inhibitor MG-132 did not. Figure 1 b). Further tissue lysosome separation experiments revealed that MDGA2 in Rps23rg1 KO mice was reduced in the brain fraction, but increased in the lysosomal fraction ( Figure 1 c), confirming that MDGA2 degradation occurs via lysosomal processing. These findings suggest that RPS23RG1 is involved in regulating the lysosomal localization and degradation of MDGA2.

[0163] Example 2: Interaction between RPS23RG1 and MDGA2 and its effect on MDGA2 degradation

[0164] 2.1: Analysis of the interaction between RPS23RG1 and MDGA2

[0165] RPS23RG1 is a typical Ib transmembrane protein. Protein-protein interaction analysis predicted that RPS23RG1 and MDGA2 interact with each other ( Figure 2 a) To further confirm, the researchers of the present invention analyzed the interaction between RPS23RG1 and MDGA2 by immunoprecipitation-immunoblotting. First, the RPS23RG1-specific antibody was used to precipitate the interacting proteins in the brain tissue of WT mice. The results are as follows Figure 2 As shown in b, RPS23RG1 showed obvious interaction with MDGA2. We then constructed a full-length Myc-tagged human RPS23RG1 plasmid and a full-length GFP-tagged MDGA2 plasmid, transfected the two plasmids into HEK293T cells simultaneously, and performed immunoprecipitation-immunoblotting analysis. The results are shown in Figure 2 As shown in c, RPS23RG1 and MDGA2 bind to each other in cells. Finally, we co-transfected RPS23RG1-Myc and MDGA2-GFP plasmids into HeLa cells and performed immunofluorescence experiments, and also detected the co-localization of the two in the cell membrane and cytoplasm ( Figure 2 d), indicating the interaction between the two.

[0166] 2.2: Determination of the core sequence of the extracellular segment of RPS23RG1

[0167] In previous studies, the researchers of the present invention respectively elaborated on the important role of the interaction between the intracellular segment and transmembrane region of RPS23RG1 and synaptic proteins in maintaining synaptic structure and synaptic transmission. In order to clarify the binding region of RPS23RG1 and MDGA2, the researchers of the present invention compared and analyzed the protein sequences of human RPS23RG1 (SEQ ID NO.01) and mouse RPS23RG1 (SEQ ID NO.02), determined the region of homologous sequences, and combined the analysis of the interacting residues of RPS23RG1 and MDGA2 in the structure prediction ( Figure 2 a), and finally identified two amino acid-rich regions in the extracellular segment 57-67 and 103-128 ( Figure 3 a). Then, expression plasmids of Myc-modified human full-length RPS23RG1 (1-173) or different truncations (Δ57-67 and Δ103-128) were constructed, see Table 2 and Figure 3 b. The coding nucleic acid sequences of the above polypeptides were respectively inserted into pCMV-Myc plasmids to obtain plasmids expressing the above polypeptides. The above plasmids were respectively transfected into HEK293T cells to overexpress the full-length and different truncated RPS23RG1, and in the above cells, MDGA2-GFP was overexpressed for immunoprecipitation-immunoblotting analysis. The results are shown in Figure 3 As shown in c, the full-length RPS23RG1 (1-173) and truncated form 57-67 both showed obvious interaction with MDGA2, while the truncated form 103-128 lacking the extracellular segment could not interact with MDGA2, suggesting that the 103-128 sequence of the extracellular segment of RPS23RG1 is the key region for interaction with MDGA2.

[0168] Table 2: Truncations of human full-length RPS23RG1

[0169]

[0170] 2.3: Effect of the core sequence of the extracellular segment of RPS23RG1 on MDGA2 degradation

[0171] In order to determine whether the interaction between the two mediates the lysosomal degradation of MDGA2. We used the lysosomal immunoprecipitation (LysoIP) method to transfect the full-length and 103-128 sequence-deficient RPS23RG1 plasmids into the TMEM192-3*HA-coupled lysosome stable cell line (HA-Lyso). After the plasmid was stably expressed, the cell lysate was collected, the lysosomal components were enriched using HA magnetic beads, and the protein levels in the lysosomes were detected by immunoblotting. The results showed that overexpression of full-length RPS23RG1 (but not RR1-Δ103-128) mediated a decrease in MDGA2 levels in lysosomes and an increase in total protein levels ( Figure 4a). In addition, the full-length and 103-128 sequence-deficient RPS23RG1 plasmids were transfected into HEK293T cells to overexpress the full-length and truncated RPS23RG1, respectively. The protein content in the membrane fraction was detected by biotin labeling and immunoprecipitation-immunoblotting experiments. It was found that overexpression of full-length RPS23RG1 could simultaneously upregulate the surface localization and total protein level of MDGA1, while the 103-128 sequence-deficient RPS23RG1 had no effect ( Figure 4 b) Taken together, these results suggest that RPS23RG1 interacts with MDGA2 through its 103-128 fragment and prevents its lysosomal degradation.

[0172] This result further confirmed that the core sequence of RPS23RG1 that interacts with MDGA2 and inhibits 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 the level of MDGA2, and thus improve neurological diseases caused by the reduction of MDGA2 (such as autism).

[0173] Example 3: Evaluation of the efficacy of peptides based on the extracellular segment of RPS23RG1 in inhibiting MDGA2 degradation in cells

[0174] In order to further determine the core action site of the RPS23RG1 extracellular segment, the researchers of the present invention artificially synthesized human RPS23RG1 extracellular segment polypeptides peptide1 (GRIWLFG; SEQ ID NO.34) and peptide2 (SYFSQHNSFCMRSTS; SEQ ID NO.08) ( Figure 5 a), and added a FITC fluorescent or biotin marker to the amino terminus of the above polypeptides, and the obtained polypeptides were respectively called P1 (SEQ ID NO.30) and P2 (SEQ ID NO.33). The above polypeptides were synthesized by Sangon Biotech Co., Ltd. The above biotin-labeled polypeptides were added to HEK293T cells and incubated for 16 hours, then the cells were lysed to extract proteins, and the polypeptides and their binding proteins were captured with biotin beads. The results are shown in Figure 5 As shown in Figure 2, peptide P2, but not P1, can bind to MDGA2. We then added FITC-labeled peptide P2 to HeLa cells and incubated them for 16 hours. We detected the co-localization of P2 and endogenous MDGA2 on the cell membrane by immunofluorescence ( Figure 5 c). These results suggest that the P2 sequence plays an important role in the binding of RPS23RG1 and MDGA2.

[0175] Subsequently, RPS23RG1 siRNA (SEQ ID NO.24-27) was transfected into HA-Lyso stable cells, and the peptide P2 was added while silencing the RPS23RG1 gene. The lysosomal immunoprecipitation (LysoIP) method was used to detect the effect of P2 on MDGA2 degradation. The results showed that knocking down RPS23RG1 led to increased lysosomal degradation of MDGA2 and decreased total cellular protein levels, while the addition of P2 could significantly inhibit its degradation and increase the overall level of MDGA2 ( Figure 6 a). RPS23RG1 siRNA and P2 were transfected into HeLa cells simultaneously. Immunofluorescence detection also showed that in cells incubated with P2, the aggregation of MDGA2 in lysosomes was significantly reduced, while the localization in the cell membrane was significantly increased ( Figure 6 b). The above experiments confirmed that polypeptide P2 can effectively inhibit the lysosomal degradation of MDGA2, stabilize its membrane localization, and increase the cellular level of MDGA2.

[0176] Example 4: Evaluation of the therapeutic efficacy of peptides based on the extracellular segment of RPS23RG1 in autism (ASD) model mice

[0177] MDGA2 is considered to be a susceptibility gene associated with ASD. MDGA2 protein deficiency can lead to increased excitatory synaptic transmission, and mice will show anxiety, social disorders and stereotyped behaviors. Therefore, Mdga2 gene knockout mice are widely used as a typical autism model.

[0178] 4.1: Construction and identification of Mdga2 knockout mice

[0179] In this example, CRISPR / Cas9 technology was used to construct Mdga2 knockout mice.

[0180] The Mdga2 gene is located on chromosome 12 of mice and contains 17 exons. First, based on the Mdga2 gene sequence on NCBI (GenBank ID: 320772), a targeted gDNA (guide DNA) was designed for exon 2 of the Mdga2 gene. Under the synergistic action of Cas9, specific site recognition and cutting were performed, resulting in gene deletion mutations and early termination of protein translation ( Figure 7 a). Obtaining Mdga2 knockout mice (Mdga2 - / - The Mdga2 knockout mice were crossed with C57BL / 6 wild-type mice to obtain offspring mice, and the Mdga2 knockout heterozygous mice (Mdga2 + / - ).

[0181] Then, the construction of Mdga2 knockout mice was verified at the DNA, RNA and protein levels. Figure 7 shown. Figure 7 b shows the genotype identification results, and the wild-type C57BL / 6 mice (abbreviated as WT mice in the present invention) and heterozygous mice (Mdga2 + / - ), knockout mice (Mdga2 - / - ) DNA, and designed primers: 5'-ccagaggcctatgaagtccg-3' (SEQ ID NO.20) and 5'-tccacagtgaaattccctcaa-3' (SEQ ID NO.21) for PCR amplification; the genotype of the PCR product was identified by 15% SDS-PAGE gel electrophoresis, among which the PCR product of wild-type mice (WT) had only one band with a length of 314 bp; the gene knockout mice (Mdga2 - / - ) PCR product has only one band with a length of 582bp; while the gene knockout heterozygous mice (Mdga2 + / - ) PCR product contained the above two bands. The results showed that KO mice were successfully constructed at the DNA level. Figure 7 c shows the RNA expression of WT and knockout mice. + / - ), knockout mice (Mdga2 - / - ) brain tissue RNA was used to detect Mdga2 levels by reverse transcription and fluorescence quantitative PCR, and then normalized with β-actin levels for comparison. - / - The mRNA level of the Rps23rg1 gene in mice is almost zero, that is, there is no transcription product of the Rps23rg1 gene, which fully proves that at the RNA level, Mdga2 - / - The mouse construct was successful. Figure 7 d shows the expression of RPS23RG1 protein in WT and knockout mice. Brain tissues of WT and knockout mice were extracted, and the MDGA2 protein level was detected by immunoprecipitation and western blot. The results showed that Mdga2 - / - There is no MDGA2 protein expression in mouse brain tissue, which fully proves that at the protein level, Mdga2 - / - The mouse was successfully constructed. In summary, Figure 7 The results showed that the Mdga2 gene knockout mice were successfully constructed.

[0182] 4.2: Evaluation of the efficacy of peptides based on the extracellular segment of RPS23RG1 in Mdga2 knockout mice

[0183] The above experimental results have shown that polypeptide P2 can directly interact with MDGA2, interfere with the lysosomal degradation level of MDGA2, and increase its overall level, which may become an effective therapy for improving neurological diseases (such as autism) caused by MDGA2 reduction. Therefore, this example further evaluates the therapeutic effect of the polypeptide based on the extracellular segment of RPS23RG1 on autism model mice.

[0184] The researchers of the present invention artificially synthesized the human RPS23RG1 extracellular segment polypeptide P2 (as shown in SEQ ID NO.33) labeled with biotin and the scrambled control polypeptide Scramble (as shown in SEQ ID NO.29) and injected them into the tail vein of 1.5-month-old Mdga2 at a dose of 4 mg / kg / d. + / - Mice or littermate control WT mice were treated for 5 consecutive days, followed by one day of rest. On the 7th day, the mice underwent behavioral testing and biochemical analysis ( Figure 8 a)

[0185] First, the brains of mice in the control group injected with scramble and the experimental group injected with P2 were lysed and then subjected to biotin immunoprecipitation and immunoblotting experiments. The protein MDGA2 was detected in the brain tissue lysates of the experimental group, indicating that the peptide successfully passed through the blood-brain barrier and entered the brain tissue and interacted with MDGA2 ( Figure 8 b). Next, we took the WT control group (scramble injection), Mdga2 + / - Control group (scramble injection) and Mdga2 + / - The brain tissue of mice in the experimental group (injected with P2) was used to detect the MDGA2 protein level by immunoblotting. The results showed that the injection of peptide P2 can effectively improve the expression of Mdga2 + / - Insufficient MDGA2 levels in mouse brain tissue Fig. 9 ab). The results of mouse behavioral testing showed that there was no difference in the movement of mice in the control group and the experimental group in the open field test and the time spent in the central area ( Fig.10 a), Fig.10 b shows that injection of P2 did not alter Mdga2 + / - However, in the free social interaction and three-box test, Mdga2 + / - Mice showed normal social interests and preferences ( Fig.10 c, 10d). The above results show that the MDGA2 targeting polypeptide of the present invention can significantly improve the + / - Social deficits in mice.

[0186] In addition to ASD, synaptic stability and homeostasis, as well as inactivation of postsynaptic density proteins, also exist in other neurological diseases. For example, it has been reported that multiple risk genes with genetic diversity were found in genomic analysis of patients with epileptic encephalopathy, including MDGA2, SHANK3 and other genes, which are also susceptibility genes for autism (Bucan et al., 2009; Cirnigliaro et al., 2023; De Rubeis et al., 2014; Lesca et al., 2012), suggesting the clinical association between the two diseases. In addition, it was found that high methylation of MDGA2 in gastric cancer cells led to gene inactivation (Zhao et al., 2013). On the contrary, high expression of MDGA2 can inhibit the proliferation of tumor cells and promote cell apoptosis, and is considered to be a new tumor suppressor in gastric cancer (Wang et al., 2016). Therefore, the polypeptide of the present invention that can target and regulate MDGA2 metabolism to increase its expression level is also suitable for treating other neurological diseases (such as epilepsy) and tumors (such as gastric cancer) caused by MDGA2 inactivation.

[0187] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention 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: It consists of 8-55 consecutive amino acids of RPS23RG1 protein and contains QHNSX1CMR (SEQ ID NO.31), wherein X1 is F or L.

2. An MDGA2 targeting polypeptide as claimed in claim 1, characterized in that: 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.

3. An MDGA2 targeting polypeptide as claimed in claim 2, characterized in that: The amino acid sequence thereof comprises SYFSQHNSFCMRSTS (SEQ ID NO. 08) or PYFFQHNSLCMRGTP (SEQ ID NO. 14).

4. A conjugate, characterized in that: It has the MDGA2 targeting polypeptide according to any one of claims 1 to 3 and a modification portion, wherein the modification portion is selected from another polypeptide, a detectable marker or any combination thereof, and the other polypeptide is selected from CPP, a targeting portion and / or a protein tag.

5. A fusion protein, characterized in that: The invention comprises the MDGA2 targeting polypeptide according to any one of claims 1 to 3 and another polypeptide, wherein the other polypeptide is selected from CPP, a targeting moiety, a protein tag or any combination thereof.

6. An isolated nucleic acid molecule, characterized in that: It has the nucleotide sequence of the MDGA2 targeting polypeptide according to any one of claims 1 to 3, or the fusion protein according to claim 5.

7. A carrier, characterized in that: It has the isolated nucleic acid molecule according to claim 6.

8. A host cell, characterized in that: It has the isolated nucleic acid molecule of claim 6 or the vector of claim 7.

9. A method for preparing the MDGA2 targeting polypeptide according to any one of claims 1 to 3 or the fusion protein according to claim 5, characterized in that: include: The host cell of claim 8 is cultured under suitable conditions, and the MDGA2 targeting polypeptide or fusion protein is recovered from the culture of the host cell.

10. A pharmaceutical composition, characterized in that: It comprises the MDGA2 targeting polypeptide according to any one of claims 1 to 3, the conjugate according to claim 4, the fusion protein according to claim 5, the isolated nucleic acid molecule according to claim 6, the vector according to claim 7 or the host cell according to claim 8, and a pharmaceutically acceptable carrier and / or excipient.

11. Use of the MDGA2 targeting polypeptide according to any one of claims 1 to 3, the conjugate according to claim 4, the fusion protein according to claim 5, the isolated nucleic acid molecule according to claim 6, the vector according to claim 7 or the host cell according to claim 8 in the preparation of a medicament for treating a disease associated with low MDGA2 activity, or inhibiting lysosomal degradation of MDGA2, or increasing the expression level of MDGA2.

12. A method for inhibiting lysosomal degradation of MDGA2 or increasing the expression level of MDGA2 in vitro for non-diagnostic therapeutic purposes, characterized in that: The MDGA2 targeting polypeptide according to any one of claims 1 to 3, the conjugate according to claim 4 or the fusion protein according to claim 5 is contacted with a cell in need thereof.

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