B2m-glu n1 blocking peptides, pharmaceutical compositions thereof, and uses thereof

Abstract

The application belongs to the field of biological medicine, and relates to a B2M-GluN1 blocking peptide, a pharmaceutical composition thereof and purposes. Specifically, the application relates to an isolated polypeptide which is a polypeptide shown in SEQ ID NO: 8 or a truncated fragment of the polypeptide shown in SEQ ID NO: 8. The isolated polypeptide of the application can effectively treat or prevent Down syndrome, Alzheimer's disease or cognitive impairment caused by Down syndrome or Alzheimer's disease, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to B2M-GluN1 blocking peptide, its pharmaceutical composition and uses. Background Technology

[0002] Down syndrome (DS), also known as trisomy 21, is one of the most common intellectual disabilities, affecting approximately 1 in 800 newborns worldwide. Individuals with Down syndrome have an extra copy or portion of chromosome 21. This increased copy number of the chromosome 21 gene leads to abnormal gene expression, ultimately causing various symptoms, including developmental delay, intellectual disability, language delay, immune and endocrine system abnormalities, and defects in the skeletal, cardiac, and digestive systems. Intellectual disability is the most prominent and severe symptom of Down syndrome. The vast majority of affected children have varying degrees of intellectual developmental impairment, with IQs typically ranging from 25 to 50 (normal range is above 90). This impairment becomes more pronounced with age, affecting language, memory, and abstract thinking. Furthermore, almost all individuals with Down syndrome develop neuropathological features similar to Alzheimer's disease (AD) after age 40, with 60% exhibiting significant Alzheimer's dementia symptoms by age 65. However, currently, effective drug treatments and interventions for the intellectual disability of individuals with Down syndrome remain lacking.

[0003] Alzheimer's disease is one of the most common neurodegenerative diseases in humans. According to the World Alzheimer's Disease Report 2018, 50 million people worldwide had Alzheimer's disease in 2018, and this number is projected to rise to 152 million by 2050. The main pathological features of the disease include amyloid deposits formed by the oligomerization of β-amyloid protein (Aβ) produced by the cleavage of amyloid precursor protein (APP) in the brain; neurofibrillary tangles (NFTs) formed by the aberrant phosphorylation of intracellular microtubule-binding protein tau; neuronal loss; and excessive neuroinflammation. The main clinical manifestations of Alzheimer's disease are memory decline and cognitive impairment, which worsens as the disease progresses, ultimately leading to the loss of all memory and the ability to live independently, until death. Given the global aging population, the continuously rising incidence of Alzheimer's disease, and the current lack of effective treatments, Alzheimer's disease has become one of the most serious threats to human health.

[0004] GluN1, a constituent subunit of the NMDA receptor, is encoded by a gene on human chromosome 9. It contains 938 amino acids and is a three-terminal transmembrane protein, comprising an N-terminal extracellular segment, a C-terminal intracellular segment, a transmembrane structure, and an extracellular loop. The NMDA receptor is considered a potential pathogenic feature of various neurological diseases, such as ischemic stroke, traumatic brain injury, Alzheimer's disease, epilepsy, mood disorders, and schizophrenia. The NMDA receptor exhibits diversity in subunit composition, biophysical and pharmacological properties, interactions, and subcellular localization. The subunit composition varies across different regions of the central nervous system during development and in disease states. Therefore, the NMDA receptor has been a hot research topic and drug target in neuropharmacology. In recent years, interest in NMDA receptor modulators as therapeutic agents has also increased significantly. These compounds will provide new tools for studying the physiology of NMDA receptor signaling, thereby revealing new therapeutic opportunities. Currently, the FDA-approved Alzheimer's disease treatment drug, memantine, is a reversible blocker of NMDA-type glutamate receptors, but its mechanism of action and mode of action are completely different from those of this invention.

[0005] β2-microglobulin (B2M) is an aging factor that has garnered increasing attention in recent years. B2M is a subunit of Major Histocompatibility Complex I (MHC-I), encoded by a gene on human chromosome 15, and contains 119 amino acids. Because B2M is not anchored to the cell membrane via a transmembrane domain, it can migrate from the MHC-I complex into the intercellular space. Normally, B2M exists as a soluble monomer, but it can aggregate and deposit under certain pathological conditions, including aging, chronic renal dysfunction, and inflammation. B2M amyloid deposition is primarily found in the bone and joint regions, ultimately leading to severe arthritis, fractures, and carpal tunnel syndrome. Furthermore, serum and plasma B2M levels are increased in many disease states. However, whether B2M has a direct or indirect impact on the development and progression of Down syndrome and Alzheimer's disease remains unreported. Summary of the Invention

[0006] Through in-depth research and creative work, the inventors discovered the role of B2M in the development of Down syndrome. Surprisingly, they found that blocking peptides that inhibit the binding of B2M to GluN1 have the potential to be used as drugs for the prevention and treatment of cognitive impairment caused by Down syndrome or Alzheimer's disease. Therefore, the following invention is provided:

[0007] One aspect of the present invention relates to an isolated polypeptide, which is the polypeptide shown in SEQ ID NO:8 or a truncated fragment of the polypeptide shown in SEQ ID NO:8; preferably, the truncated fragment comprises the polypeptide shown in SEQ ID NO:11.

[0008] EKHNYESAAEAIQAVRDNKLHAFIWDSAVLEFEASQKCDLVTTGELFFRSGFGIGMR(SEQ ID NO:8)

[0009] KLHAFIWDSAVLEFEASQ(SEQ ID NO:11)

[0010] In some embodiments of the present invention, the isolated polypeptide is a polypeptide represented by any of the sequences in SEQ ID NO:11 or SEQ ID NOs:14-32.

[0011] AVRDNKLHAFIWDSAVLEFEASQ(SEQ ID NO:14)

[0012] VRDNKLHAFIWDSAVLEFEASQ(SEQ ID NO:15)

[0013] RDNKLHAFIWDSAVLEFEASQ(SEQ ID NO:16)

[0014] DNKLHAFIWDSAVLEFEASQ(SEQ ID NO:17)

[0015] NKLHAFIWDSAVLEFEASQ(SEQ ID NO:18)

[0016] KLHAFIWDSAVLEFEASQKCDLV(SEQ ID NO:19)

[0017] KLHAFIWDSAVLEFEASQKCDL(SEQ ID NO:20)

[0018] KLHAFIWDSAVLEFEASQKCD(SEQ ID NO:21)

[0019] KLHAFIWDSAVLEFEASQKC(SEQ ID NO:22)

[0020] KLHAFIWDSAVLEFEASQK(SEQ ID NO:23)

[0021] RDNKLHAFIWDSAVLEFEASQKCD(SEQ ID NO:24)

[0022] RDNKLHAFIWDSAVLEFEASQKC(SEQ ID NO:25)

[0023] RDNKLHAFIWDSAVLEFEASQK(SEQ ID NO:26)

[0024] DNKLHAFIWDSAVLEFEASQKCD(SEQ ID NO:27)

[0025] DNKLHAFIWDSAVLEFEASQKC(SEQ ID NO:28)

[0026] DNKLHAFIWDSAVLEFEASQK(SEQ ID NO:29)

[0027] NKLHAFIWDSAVLEFEASQKCD(SEQ ID NO:30)

[0028] NKLHAFIWDSAVLEFEASQKC(SEQ ID NO:31)

[0029] NKLHAFIWDSAVLEFEASQK(SEQ ID NO:32)

[0030] Another aspect of the present invention relates to isolated polynucleotides encoding the isolated polypeptides described in any one of the present invention.

[0031] Another aspect of the invention relates to a recombinant expression vector comprising the isolated polynucleotides of the present invention.

[0032] Another aspect of the invention relates to a transformed cell comprising the recombinant expression vector of the invention.

[0033] Another aspect of the invention relates to a pharmaceutical composition comprising one or more (e.g., 2, 3, 4 or 5) isolated polypeptides as described in any one of the invention.

[0034] In some embodiments of the present invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0035] Another aspect of the invention relates to the use of the isolated polypeptide described in any one of the invention in the preparation of a medicament for the treatment or prevention of Down syndrome, Alzheimer's disease, or cognitive impairment caused by Down syndrome or Alzheimer's disease.

[0036] Another aspect of the present invention relates to the use of the isolated polypeptide described in any one of the present invention in the preparation of the following pharmaceutical products:

[0037] Drugs that reduce B2M levels in the human brain, drugs that reduce amyloid precursor protein levels in the human brain, drugs that inhibit the binding of GluN1 to B2M in the human brain, or drugs that repair synaptic damage in the human brain caused by increased B2M.

[0038] The isolated polypeptide according to any one of the present invention is used to treat or prevent Down syndrome, Alzheimer's disease, or cognitive impairment caused by Down syndrome or Alzheimer's disease.

[0039] The isolated polypeptide according to any one of the present invention is used to reduce the level of B2M in the human brain, reduce the level of amyloid precursor protein in the human brain, inhibit the binding of GluN1 to B2M in the human brain, or repair synaptic damage in the human brain caused by increased B2M.

[0040] Another aspect of the invention relates to a method for treating or preventing Down syndrome, Alzheimer's disease, or cognitive impairment caused by Down syndrome or Alzheimer's disease, comprising the step of administering to a subject in need an effective amount of the isolated polypeptide described in any one of the invention.

[0041] Another aspect of the present invention relates to a method for inhibiting the binding of GluN1 to B2M in the human brain or for repairing synaptic damage in the human brain caused by increased B2M, comprising the step of administering to a subject in need an effective amount of the isolated polypeptide described in any one of the present invention.

[0042] In some embodiments of the present invention, the method for treating or preventing Down syndrome, Alzheimer's disease, or cognitive impairment caused by Down syndrome or Alzheimer's disease, or the method for inhibiting the binding of GluN1 to B2M in the human brain or repairing synaptic damage in the human brain caused by increased B2M, wherein,

[0043] The single dose of the isolated polypeptide described in any one of the present invention is 0.1-100 mg per kilogram of body weight, preferably 5-50 mg or 5-15 mg per kilogram of body weight;

[0044] Preferably, the medication is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks, or every 3 weeks;

[0045] Preferably, the administration method is intravenous infusion or intravenous injection.

[0046] This invention is the first to discover that B2M expression is significantly elevated in the brain tissue of patients with Down syndrome, and increased B2M can impair synaptic plasticity and cognitive function. Furthermore, the inventors found a direct interaction between B2M and the extracellular domain of GluN1. Using a truncated GluN1 amino acid sequence as a blocking peptide can prevent B2M from binding to GluN1, inhibiting the damage of B2M to NMDA receptors. In vivo experiments showed that the blocking peptide can significantly inhibit the binding of B2M to GluN1 in the brain and enhance synaptic plasticity. This discovery provides a potential drug target and a novel therapeutic approach based on this target for the clinical treatment of Down syndrome.

[0047] In some embodiments of the present invention, the amino acid sequence of GluN1 is shown in SEQ ID NO:1.

[0048] The amino acid sequence (N-terminus to C-terminus) of rat GluN1 protein is as follows:

[0049] MSTMHLLTFALLFSCSFARAACDPKIVNIGAVLSTRKHEQMFREAVNQANKRHGSWKIQLNATSVTHKPNAIQMALSVCEDLISSQVYAILVSHPPTPNDHFTPTPVSYTAGFYRIPVLGLTTRMSIYSDKSIHLSFLRTVPPYSHQSSVWFEMMRVYNWNHIILLVSDDHEGRAAQKRLETLLEERESKAEKVLQFDPGTKNVTALLMEARELEARVIILSASEDDAAT VYRAAAMLNM TGSGYVWLVGEREISGNALRYAPDGIIGLQLINGKNESAHISDAVGVVAQAVHELLEKENITDPPRGCVGNTNIWKTGPLFKRVLMSSKYADGVTGRVEFNEDGDRKFANYSIMNLQNRKLVQVGIYNGTHVIPNDRKIIWPGGETEKPRGYQMSTRLKIVTIHQEPFVYVKPTMSDGTCKEEFTVNGDPVKKVICTGPNDTSPGSPRHTVPQCCYGFCIDLLIKLARTMNFTYEVHLVADGKFGTQERVNNSNKKEWNGMMGELLSGQADMIVAPLTINNERAQYIEFSKPFKYQGLTILVKKEIPRSTLDSFMQPFQSTLWLLVGLSVHVVAVMLYLLDRFSPFGRFKVNSEEEEEDALTLSSAMWFSWGVLLNSGIGEGAPRSFSARILGMVWAGFAMIIVASYTANLAAFLVLDRPEERITGINDPRLRNPSDKFIYATVKQSSVDIYFRRQVELSTMYRHMEKHNYESAAEAIQAVRDNKLHAFIWDSAVLEFEASQKCDLVTTGELFFRSGFGIGMRKDSPWKQNVSLSILKSHENGFMEDLDKTWVRYQECDSRSNAPATLTFENMAGVFMLVAGGIVAGIFLIFIEIAYKRHKDARRKQMQLAFAAVNVWRKNLQDRKSGRAEPDPKKKATFRAITSTLASSFKRRRSSKDTSTGGGRGALQNQKDTVLPRRAIEREEGQLQ LCSRHRES(SEQ ID NO:1)

[0050] In some embodiments of the present invention, the amino acid sequence of the GluN1 is shown in SEQ ID NO:2.

[0051] The amino acid sequence (N-terminus to C-terminus) of human GluN1 protein is as follows:

[0052] MSTMRLLTLALLFSCSVARAACDPKIVNIGAVLSTRKHEQMFREAVNQANKRHGSWKIQLNATSVTHKPNAIQMALSVCEDLISSQVYAILVSHPPTPNDHFTPTPVSYTAGFYRIPVLGLTTRMSIYSDKSIHLSFLRTVPPYSHQSSVWFEMMRVYSWNHIILLVSDDHEGRAAQKRLETLLEERESKAEKVLQFDPGTKNVTALLMEAKELEARVIILSASEDDAATVYRAAAMLNMTGSGYVWLVGEREISGNALRYAPDGILGLQLINGKNESAHISDAVGVVAQAVHELLEKENITDPPRGCVGNTNIWKTGPLFKRVLMSSKYADGVTGRVEFNEDGDRKFANYSIMNLQNRKLVQVGIYNGTHVIPNDRKIIWPGGETEKPRGYQMSTRLKIVTIHQEPFVYVKPTLSDGTCKEEFTVNGDPVKKVICTGPNDTSPGSPRHTVPQCCYGFCIDLLIKLARTMNFTYEVHLVADGKFGTQERVNNSNKKEWNGMMGELLSGQADMIVAPLTINNERAQYIEFSKPFKYQGLTILVKKEIPRSTLDSFMQPFQSTLWLLVGLSVHVVAVMLYLLDRFSPFGRFKVNSEEEEEDALTLSSAMWFSWGVLLNSGIGEGAPRSFSARILGMVWAGFAMIIVASYTANLAAFLVLDRPEERITGINDPRLRNPSDKFIYATVKQSSVDIYFRRQVELSTMYRHMEKHNYESAAEAIQAVRDNKLHAFIWDSAVLEFEASQKCDLVTTGELFFRSGFGIGMRKDSPWKQNVSLSILKSHENGFMEDLDKTWVRYQECDSRSNAPATLTFENMAGVFMLVAGGIVAGIFLIFIEIAYKRHKDARRKQMQLAFAAVNVWRKNLQDRKSGRAEPDPKKKATFRAITSTLASSFKRRRSSKDTSTGGGRGALQNQKDTVLPRRAIEREEGQLQLCSRHRES(SEQ ID NO:2)

[0053] The homology (similarity) between human GluN1 protein and rat GluN1 protein was 99.25%.

[0054] In this invention, unless otherwise stated, the scientific and technical terms used herein 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 herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0055] In this invention, when referring to the amino acid sequence of GluN1, it includes the full-length GluN1 and its fusion protein. However, those skilled in the art will understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of GluN1 without affecting its biological function. In one embodiment of the invention, the amino acid sequence of GluN1 is shown in SEQ ID NO:1. In one embodiment of the invention, the amino acid sequence of GluN1 is shown in SEQ ID NO:2.

[0056] In this invention, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" substance, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated substance. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the activity of the substance.

[0057] In this invention, the term "host cell" refers to the cell into which the vector is introduced, including many cell types such as 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 fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or animal cells such as human cells.

[0058] In this invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide that inhibits a protein can be inserted. For example, vectors include: plasmids; phage particles; Cosmids; 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 used as vectors include retroviruses (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.

[0059] The term "disease and / or symptom" refers to a physical condition of the subject that is related to the disease and / or symptom described in this invention.

[0060] The term "subject" can refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the pharmaceutical composition of the present invention to treat, prevent, reduce and / or alleviate the disease or condition described in the present invention.

[0061] As used herein, the term "pharmaceuticalally acceptable excipient" means 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, adjuvants, and ionic strength enhancers. 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; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0062] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing a disease (e.g., Down syndrome or Alzheimer's disease) means an amount sufficient to prevent, stop, or delay the onset of the disease (e.g., Down syndrome or Alzheimer's disease); an effective amount for treating a disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on 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 manner of administration of the drug, and other concurrent treatments, etc.

[0063] The term "blocking peptide" refers to a biological effect that can competitively bind to B2M with the full-length GluN1 protein, thereby inhibiting the B2M binding to GluN1 and damaging synaptic plasticity.

[0064] In this invention, unless otherwise specified, the concentration units μM represent μmol / L, mM represent mmol / L, and nM represent nmol / L.

[0065] In this invention, when referring to the amount of drug added to cells, unless otherwise specified, it generally refers to the final concentration of the drug after addition.

[0066] Beneficial effects of the invention

[0067] This invention provides novel drug targets for the prevention, treatment, or improvement of cognitive impairment caused by Down syndrome or Alzheimer's disease. Inhibiting or blocking β2-microglobulin activity can effectively prevent and treat Down syndrome, Alzheimer's disease, or cognitive impairment caused by Down syndrome or Alzheimer's disease. Attached Figure Description

[0068] Figures 1A to 1D B2M expression levels were increased in the fetal brain and tissues of Down syndrome mice.

[0069] in:

[0070] Figure 1A Immunoblotting results of B2M expression in fetal brain tissue of patients with Down syndrome.

[0071] Figure 1B Image J Analysis Figure 1A B2M expression levels were measured in brain tissue samples from 6 individuals in the control group and 7 individuals in the Down syndrome group. Data were statistically analyzed using Student's t-test. No significant differences were found in the expression levels within ns; P>0.05; *P<0.05; **P<0.01; ***P<0.001.

[0072] Figure 1C Immunoblotting results of B2M expression in brain tissue of Dp16 mice.

[0073] Figure 1D Image J Analysis Figure 1C B2M expression levels were measured in brain tissue samples from n=6 mice in the WT group and n=6 mice in the Dp16 group. Data were statistically analyzed using Student's t-test. No significant difference was found in ns, P>0.05; *P<0.05; **P<0.01; ***P<0.001.

[0074] Figure 2NMDAR EPSC amplitude statistics. Under in vitro conditions, WT mouse brain slices were incubated with B2M protein (10 μg / ml) and ACSF for two hours, respectively. Electrophysiological monitoring was performed after incubation, recording the NMDAR EPSC amplitude of the Scheffer collateral circuit in the hippocampus. The stimulating electrode was placed near CA3 to record pyramidal cell currents in the CA1 region. 50 μM MPTX and 20 μM CNQX were added to the perfusion fluid to block GABA, respectively. A Receptor and AMPA receptor ion channels were clamped at a voltage of +40 mV. Cell numbers were recorded in the ACSF group (n=16) and the B2M group (n=14). Data represent mean ± standard error (SEM). Statistical analysis was performed using one-way ANOVA. No significant differences were found in ns; P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0075] Figures 3A to 3D Results of co-immunoprecipitation (co-IP) experiments demonstrating the interaction between B2M and the extracellular cyclic peptide of GluN1. B2M plasmids tagged with HA were co-transfected into HEK293T cells with GluN1, N-terminal deleted GluN1, C-terminal deleted GluN1, and the extracellular cyclic peptide plasmid of GluN1, respectively. Equal volumes of protein lysis buffer were incubated overnight with IgG (control group), HA antibody, and Protein G beads for co-immunoprecipitation. Immunoblot analysis was performed the following day. (Note:)

[0076] Figure 3A Immunoprecipitation of B2M and GluN1 with anti-HA antibodies was performed.

[0077] Figure 3B Immunoprecipitation of B2M and N-terminal-deleted GluN1 with anti-HA antibody was performed.

[0078] Figure 3C Immunoprecipitation of B2M and C-terminal-deficient GluN1 with anti-HA antibody was performed.

[0079] Figure 3D Immunoprecipitation of B2M and GluN1 extracellular cyclic peptides was performed using anti-HA antibodies.

[0080] Figure 4A To further confirm the minimum region on the extracellular circular peptide of GluN1 that binds to B2M, a schematic diagram of the truncation of the extracellular circular peptide sequence of GluN1 into three short peptides with no overlapping sequences is shown.

[0081] Figure 4B The GST-pull-down assay showed that the GST fusion protein of the GluN1 extracellular cyclic peptide L2 had a stronger binding ability to B2M.

[0082] Figure 4C To further narrow down the range of amino acid sequences that exert the inhibitory effect, the inventors further divided the GluN1 extracellular cyclic peptide L2 into three segments of short peptides with non-overlapping sequences.

[0083] Figure 4D The three non-overlapping short peptides were pre-incubated with Ni-NTA Agarose in PBS solution at 4°C for 8 hours, followed by incubation with hB2M protein at 4°C overnight. Immunoblot detection and analysis were performed the next day.

[0084] Figure 4E The glutN1-P2 truncated peptide and GST-B2M fusion protein were pre-incubated in PBS at 4°C for 8 hours by rotation. Then, 800 μg of 293T cell lysate transfected with glutN1 plasmid was added and incubated overnight at 4°C. The next day, Western blot analysis was performed.

[0085] Figure 5A NMDAR EPSC amplitude statistics. Six-month-old Dp16 mice and control WT mice underwent stereotactic injection of 1 μl (1 μg / μl) of GluN1-P2 truncated peptide or non-sense peptide into the hippocampus (left and right controls for each mouse's own brain). Electrophysiological recordings were performed one day after injection. The NMDAR EPSC amplitude of the Scheffer collateral circuit in the hippocampus was recorded. The stimulating electrode was placed near CA3. Pyramidal cell currents in the CA1 region were recorded. 50 μM PTX and 20 μM CNQX were added to the perfusion fluid to block GABA, respectively. A Receptor and AMPA receptor ion channels were clamped at a voltage of +40 mV. Cell numbers recorded were WT Scrambled n=14, WTGluN1-P2 n=14, Dp16 Scrambled n=15, and Dp16GluN1-P2 n=16. Data represent mean ± standard error (SEM). Statistical analysis was performed using one-way ANOVA. No significant difference was considered (P>0.05); *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0086] Figure 5BNMDAR EPSC amplitude statistics. Three-month-old C57BL / 6 mice were stereotactically injected into the hippocampus with 1 μl (1 μg / μl) of GluN1-P2 truncated peptide or non-sense peptide (left and right controls for each mouse's own brain). One day after injection, brain slices were incubated in vitro with B2M protein (10 μg / ml) and ACSF for two hours, respectively. Electrophysiological recordings were performed after incubation. The NMDAR EPSC amplitude of the Scheffer collateral circuit in the hippocampus was recorded. The stimulating electrode was placed near CA3. Pyramidal cell currents in the CA1 region were recorded. 50 μM PTX and 20 μM CNQX were added to the perfusion fluid to block GABA, respectively. A Receptor and AMPA receptor ion channels were clamped at a voltage of +40 mV. Cell numbers recorded were WT-ACSF n=15, WT-B2M n=15, WT-B2M Scrambled n=16, and WT-B2M GluN1-P2 n=16. Data represent mean ± standard error (SEM). Statistical analysis was performed using one-way ANOVA. No significant difference was considered (n=0.05); *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0087] Figure 5C Analysis of LTP recordings in the CA1 region of brain slices. Six-month-old Dp16 mice and control WT mice were stereotactically injected into the hippocampus with 1 μl (1 μg / μl) of truncated GluN1-P2 peptide or non-sense peptide (left and right controls for each mouse). Electrophysiological recordings were performed one day after injection. The number of brain slices recorded were WT Scrambled n=9, WT GluN1-P2 n=8, Dp16 Scrambled n=8, and Dp16 GluN1-P2 n=8. Data represent mean ± standard error (SEM). Statistical analysis was performed using one-way ANOVA, ns. No significant difference was considered P>0.05; *P<0.05; **P<0.01; ***P<0.001; ***P<0.0001.

[0088] Figure 5D : Figure 5C Statistical analysis of the slope of fEPSP in the last 10 minutes of LTP recording results. The n value for each group is the same. Figure 5C .

[0089] Figure 5ELTP recording analysis results of the CA1 region of brain slices. 1 μl (1 μg / μl) of truncated GluN1-P2 peptide or non-sense peptide (left and right controls for each mouse) was injected into the hippocampus of 8-month-old 5×FAD mice via stereotactic injection. LTP recordings were performed one day after injection. A total of 5 mice were injected, and the number of brain slices recorded were 5×FAD Scrambled n=8 and 5×FAD GluN1-P2 n=10. Data represent mean ± standard error (SEM). Statistical analysis was performed using unpaired t-test, ns. No significant difference was considered P>0.05; *P<0.05; **P<0.01; ***P<0.001.

[0090] Figure 5F : Figure 5E Statistical analysis of the slope of fEPSP in the last 10 minutes of LTP recording results. The n value for each group is the same. Figure 5E . Detailed Implementation

[0091] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0092] 5×FAD mice are transgenic mouse models of Alzheimer's disease. These mice exhibit the pathological features of β-amyloid plaques characteristic of Alzheimer's disease in their brains. 5×FAD mice were purchased from Jackson Laboratory (Ellsworth, ME, USA) and have the serial number 034840-JAX.

[0093] Dp16 mice are Down syndrome model mice, purchased from The Jackson Laboratory, USA, catalog number 013530-JAX.

[0094] The WT control mice were C57BL / 6 mice, purchased from the Experimental Animal Center of Xiamen University.

[0095] Example 1: Increased expression levels of B2M in human Down syndrome fetal brain and Down syndrome mouse tissues

[0096] Brain tissue from patients with Down syndrome and non-Down syndrome control human brain tissue (obtained from Xiamen University Affiliated Women and Children's Hospital, with informed consent obtained before the brain tissue samples were stored), hippocampal tissue from Dp16 mice and WT control mice were collected. After tissue grinding and lysis with RIPA protein lysis buffer, total protein was extracted, and BCA concentration was measured to prepare the sample. Then, Western blot detection was performed (the detection antibody was from Abcam, catalog number ab75853).

[0097] The results are as follows Figures 1A to 1D As shown.

[0098] The results showed that, compared with the control group, patients with Down syndrome had significantly higher levels of total B2M protein in the cerebral cortex. Compared with the control group, B2M protein levels in the hippocampus of Dp16 mice were significantly higher.

[0099] In addition, the expression level of amyloid precursor protein (APP) encoded by chromosome 21 was found to be significantly higher in the brain tissue of patients with Down syndrome and Dp16 mice than in their respective control groups.

[0100] Example 2: B2M protein incubation damages NMDA receptor function

[0101] Under in vitro conditions, WT mouse brain slices were incubated with B2M protein (10 μg / ml) and artificial cerebrospinal fluid (ACSF) for two hours. Electrophysiological monitoring was performed after incubation, recording the NMDAR EPSC of the Scheffer collateral circuit in the hippocampus. The stimulating electrode was placed near CA3 to record the pyramidal cell current in CA1. 5 mM QX-314 was added to the electrode fluid, and 50 μM PTX and 20 μM CNQX were added to the perfusion fluid to block GABA, respectively. A The receptor and AMPA receptor ion channel have a clamping voltage of +40mV.

[0102] Experimental results are as follows Figure 2 As shown.

[0103] The results showed that, compared with the control group, B2M protein treatment significantly reduced the NMDAREPSC amplitude of pyramidal neurons in the CA1 region of the hippocampus, indicating that B2M protein treatment impairs the NMDA receptor function of hippocampal neurons.

[0104] Example 3: Interaction exists between the extracellular loop of GluN1 and B2M.

[0105] Under in vitro conditions, HA antibody (Sigma, Cat#H6908) and IgG (control) antibody (Invitrogen, Cat#02-6102) were incubated with protein lysis buffers expressing GluN1 and its truncated protein and B2M-HA, respectively, for in vitro co-precipitation experiments. Immunoblot analysis was then performed to determine if there was a direct interaction between the two in vitro. The specific procedures are as follows:

[0106] B2M plasmids tagged with HA were co-transfected into HEK293T cells with plasmids containing GluN1 (SEQ ID NO:1), GluN1 C-terminal intracellular fragment deletion, GluN1 N-terminal extracellular fragment deletion, or GluN1 extracellular circular peptide fragment, and expressed for 24 hours. Equal volumes of protein lysis buffer were incubated overnight with IgG (control group), HA antibody, and Protein G magnetic beads (for binding to the Fc region of antibody IgG, Thermo Fisher Scientific, Cat#88848) for immunoprecipitation. Immunoblot analysis was performed the following day. 3% of the immunoprecipitated protein lysis buffer was used as input (positive control).

[0107] Deletion of the C-terminal intracellular segment of rat GluN1 (99.16% homology with deletion of the C-terminal intracellular segment of human GluN1)

[0108] MSTMHLLTFALLFSCSFARAACDPKIVNIGAVLSTRKHEQMFREAVNQANKRHGSWKIQLNATSVTHKPNAIQMALSVCEDLISSQVYAILVSHPPTPNDHFTPTPVSYTAGFYRIPVLGLTTRMSIYSDKSIHLSFLRTVPPYSHQSSVWFEMMRVYNWNHIILLVSDDHEGRAAQKRLETLLEERESKAEKVLQFDPGTKNVTALLMEARELEARVIILSASEDDAAT VYRAAAMLNM TGSGYVWLVGEREISGNALR YAPDGIIGLQLINGKNESAHISDAVGVVAQAVHELLEKENITDPPRGCVGNTNIWKTGPLFKRVLMSSKYADGVTGRVEFNEDGDRKFANYSIMNLQNRKLVQVGIYNGTHVIPNDRKIIWPGGETEKPRGYQMSTRLKIVTIHQEPFVYVKPTMSDGTCKEEFTVNGDPVKKVICTGPNDTSPGSPRHTVPQCCYGFCIDLLIKLARTMNFTYEVHLVADGKFGTQERVNNSNKKEWNGMMGELLSGQADMIVAPLTINNERAQYIEFSKPFKYQGLTILVKKEIPRSTLDSFMQPFQSTLWLLVGLSVHVVAVMLYLLDRFSPFGRFKVNSEEEEEDALTLSSAMWFSWGVLLNSGIGEGAPRSFSARILGMVWAGFAMIIVASYTANLAAFLVLDRPEERITGINDPRLRNPSDKFIYATVKQSSVDIYFRRQVELSTMYRHMEKHNYESAAEAIQAVRDNKLHAFIWDSAVLEFEASQKCDLVTTGELFFRSGFGIGMRKDSPWKQNVSLSILKSHENGFMEDLDKTWVRYQECDSRSNAPATLTFENMAGVFMLVAGGIVAGIFL IFI(SEQ ID NO:3)

[0109] Rat GluN1 N-terminal extracellular domain deletion (100% homology with human GluN1 N-terminal extracellular domain deletion)

[0110] STLWLLVGLSVHVVAVMLYLLDRFSPFGRFKVNSEEEEEDALTLSSAMWFSWGVLLNSGIGEGAPRSFSARILGMVWAGFAMIIVASYTANLAAFLVLDRPEERITGINDPRLRNPSDKFIYATVKQSSVDIYFRRQVELSTMYRHMEKHNYESAAEAIQAVRDNKLHAFIWDSAVLEFEASQKCDLVTTG ELFFRSGFGIGMRKDSPWKQNVSLSILKSHENGFMEDLDKTWVRYQECDSRSNAPATLTFENMAGVFMLVAGGIVAGIFLIFIEIAYKRHKDARRKQMQLAFAAVNVWRKNLQDRKSGRAEPDPKKKATFRAITSTLASSFKRRRSSKDTSTGGGRGALQNQKDTVLPRRAIEREEGQLQLCSRHRES(SEQ ID NO:4)

[0111] Extracellular cyclic peptide of rat GluN1 cells (100% homology with human GluN1 extracellular cyclic peptide)

[0112] TANLAAFLVLDRPEERITGINDPRLRNPSDKFIYATVKQSSVDIYFRRQVELSTMYRHMEKHNYESAAEAIQAVRDNKLHAFIWDSAVLEFEASQKCDLVTTGELFFRSGFGIGMRKDSPWKQNVSLSILKSHENGFMEDLDKTWVRYQECDSRSNAPATLTF EN(SEQ ID NO:5)

[0113] B2M proteins with HA tag

[0114] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMYPYDVPDYA(SEQ ID NO:6)

[0115] The plasmid construction, cell transfection, and protein sample preparation are described below.

[0116] Plasmid construction:

[0117] (1) PCR target gene fragment (e.g., GluN1 N-terminal extracellular segment deletion, GluN1 extracellular circular peptide, GluN1 extracellular circular peptide). (2) Double digestion of PCR product and vector (pcDNA3.1(+) / myc-His A) (restrictions EcoR1 and Xba1, respectively) at 37℃ for 1 hour. (3) Separation of the digested vector by agarose gel electrophoresis. Then, gel extraction and purification were performed under UV light after staining with Gel Green and the digested plasmid DNA fragments were purified using the SanPrep column DNA gel extraction kit. (4) Ligation of the annealed oligonucleotide double strands with the digested and recovered pcDNA3.1(+) / myc-His A vector was performed using T4 DNA Ligase at 22℃ for 1 hour. (5) Transform the ligation product into E. coli DH5α competent cells, incubate on ice for 30 min, heat shock at 42℃ for 90 s, incubate on ice for 3 min, add antibiotic-free LB medium, and culture at 37℃ with shaking (250 rpm) for 45 min. Then, spread the revived E. coli DH5α bacteria on plates containing 50 μg / ml Amp / LB, incubate upright at 37℃ for 30 min, and then invert for 12-16 hours. (6) Pick single colonies and culture them in 4-6 ml of medium containing 50 μg / ml Amp / LB at 37℃ with shaking (250 rpm) for 12-16 hours. Then, extract plasmid DNA using the SanPrep column-based plasmid DNA mini-extraction kit (Shanghai Sangon Biotech Co., Ltd., Cat#HC17KA2946). (7) Send the plasmid to Xiamen Platinum Biotech Co., Ltd. for sequencing identification.

[0118] Cell transfection:

[0119] Cell transfection was performed using PEI. (1) One day before transfection, cells were seeded into 60 mm culture dishes. The cell density at the time of transfection should be 70-90%. (2) The plasmid to be transfected was Opti-MEM:PEI (1:50:5.5, w / v / v). 4 μg of plasmid was transfected into a 60 mm culture dish. After mixing, the mixture was allowed to stand at room temperature for 10 min. (3) The mixture was added dropwise to the culture medium. The mixture was gently shaken and cultured in a 37°C, 5% CO2 incubator. (4) Eight hours after transfection, the culture medium was replaced with fresh medium. Cells were collected after culturing for another 24 hours.

[0120] Protein sample preparation:

[0121] (1) Remove the culture medium from the culture dish, add pre-cooled PBS, shake well, remove the PBS, and repeat three times; (2) Add 500 μl of RIPA cell lysis buffer, scrape off the cells with a cell scraper, transfer to a pre-cooled centrifuge tube, centrifuge at 1000g for 5 min at 4℃, and collect the cells; vortex lyse at 4℃ for 30 min; (3) After lysis, centrifuge at 12000g for 10 min at 4℃, and the resulting supernatant is the required protein lysis buffer. Transfer the supernatant to a new 1.5 ml centrifuge tube; (4) Determine the protein concentration using the BCA method, and prepare the sample according to the obtained protein concentration. Protein concentration was determined using the BCA Protein Assay Kit.

[0122] Experimental results are as follows Figures 3A to 3D As shown.

[0123] The results showed that GluN1 and B2M interacted. Figure 3A ).

[0124] GluN1 is a three-terminal transmembrane protein, comprising an N-terminal extracellular segment, a C-terminal intracellular segment, a transmembrane structure, and an extracellular loop. To detect the region where B2M binds to GluN1, the inventors deleted both the C-terminal intracellular segment and the N-terminal extracellular segment of GluN1, and performed immunoprecipitation experiments with B2M. The results showed that, compared with the control group, B2M bound to the N-terminal deletion (GluN1-N terminal deletion-myc)... Figure 3B ) and C-terminal deletion of GluN1 (GluN1-Cterminal deletion-myc) Figure 3C All of them interact with each other.

[0125] Both N-terminal and C-terminal deleted GluN1 molecules contain a common extracellular cyclic peptide region. To investigate whether the GluN1 extracellular cyclic peptide binds to B2M, an immunoprecipitation assay was performed on the GluN1 extracellular cyclic peptide and B2M. The results showed that the GluN1 extracellular cyclic peptide can interact with B2M. Figure 3D ).

[0126] In summary, there is an interaction between the extracellular loop of GluN1 and B2M.

[0127] Example 4: The truncated GluN1 peptide can act as a blocking peptide, preventing GluN1 from binding to B2M and thus inhibiting the function of GluN1 by B2M. Inhibition of action

[0128] To clarify the amino acid sequence in which GluN1 interacts with B2M, such as Figure 4A As shown, the inventors truncated the extracellular loop sequence of GluN1 into three non-overlapping amino acid sequences, and then synthesized the GST fusion expression protein of these amino acid sequences.

[0129] Rat GluN1-S2 loop-L1 (100% homology with human GluN1-S2 loop-L1)

[0130] TANLAAFLVLDRPEERITGINDPRLRNPSDKFIYATVKQSSVDIYFRRQVELSTMYRHM(SEQ IDNO:7)

[0131] Rat GluN1-S2 loop-L2 (100% homology with human GluN1-S2 loop-L2)

[0132] EKHNYESAAEAIQAVRDNKLHAFIWDSAVLEFEASQKCDLVTTGELFFRSGFGIGMR(SEQ ID NO:8)

[0133] Rat GluN1-S2 loop-L3 (100% homology with human GluN1-S2 loop-L3)

[0134] KDSPWKQNVSLSILKSHENGFMEDLDKTWVRYQECDSRSNAPATLTF EN(SEQ ID NO:9)

[0135] The GST fusion proteins containing the three GluN1 extracellular loop sequences described above were incubated overnight at 4°C with 0.5 μghB2M protein at an approximately 1:1 molar ratio, and immunoblotting was performed the following day. Figure 4B As shown, GluN1-S2loop-L2 interacts more strongly with B2M.

[0136] like Figure 4C As shown, based on GluN1-S2 loop-L2, the inventors further shortened the amino acid sequence to narrow the effective binding range. The inventors truncated the GluN1-S2 loop-L2 sequence into three non-overlapping amino acid sequences (specific sequences are shown in Table 1 below), and then synthesized these small peptides. It should be noted that, considering the 100% homology between rat GluN1-S2 loop-L2 and human GluN1-S2 loop-L2, the truncated fragments GluN1-P1, GluN1-P2, and GluN1-P3 in Table 1 also show 100% homology with their corresponding truncated fragments in human GluN1-S2 loop-L2.

[0137] Table 1

[0138]

[0139]

[0140] 2.5 μg of each of the three non-overlapping short peptides were pre-incubated with Ni beads in PBS solution at 4°C for 8 hours, followed by incubation with 1 μg of B2M protein at 4°C overnight. Immunoblot analysis was performed the next day. Figure 4D As shown, B2M specifically binds to the GluN1-P2 short peptide.

[0141] To further verify whether the GluN1-P2 short peptide can competitively bind to GluN1 in vivo, 0.055 μg, 0.55 μg, 1.1 μg, and 2.2 μg mass gradients of the GluN1-P2 short peptide and approximately 5 μg of the GST-B2M fusion protein were incubated in PBS solution at 4°C for 8 hours. The GluN1 plasmid (a recombinant plasmid of rat GluN1 and pcDNA3.1) was then transformed into 293T cells. An equal mass of protein lysis buffer was added to the above system, and the cells were incubated overnight at 4°C. Immunoblotting analysis was performed the next day. 0.3% of the mass of the IP protein lysis buffer was used as an input control. Figure 4E As shown, the ability of the GluN1-P2 short peptide to bind to B2M increases with increasing mass.

[0142] In summary, the GluN1-P2 short peptide can act as a blocking peptide, preventing GluN1 from binding to B2M and thus preventing B2M from inhibiting NMDA receptor function.

[0143] Example 5: GluN1-P2 blocking peptides inhibit the binding of B2M to GluN1, thereby reducing synaptic damage.

[0144] GluN1 is an essential subunit of the NMDA receptor, and its dysfunction can severely impair synaptic plasticity. Since the GluN1-P2 blocking peptide can prevent B2M from binding to GluN1, it is necessary to investigate whether the GluN1-P2 blocking peptide can prevent B2M from damaging NMDA receptor function and thus impairing excitatory synaptic function.

[0145] Six-month-old Dp16 mice and control WT mice underwent stereotactic injection of 1 μl (1 μg / μl) of GluN1-P2 short peptide or non-sense peptide into the hippocampus (GluN1-P2 short peptide was injected into the left CA1 region and non-sense peptide into the right CA1 region of each mouse). Electrophysiological recording was performed one day after injection. The Scheffer collateral circuit NMDAR EPSC in the hippocampus was recorded. The stimulating electrode was placed near CA3 to record pyramidal cell currents in the CA1 region. 5 mM QX-314 was added to the electrode fluid, and 50 μM PTX and 20 μM CNQX were added to the perfusion fluid to block GABA, respectively. AThe receptor and AMPA receptor ion channel have a clamping voltage of +40mV.

[0146] Experimental results are as follows Figure 5A As shown in the figure. The results showed that in Dp16 mice, the NMDA EPSC amplitude was significantly increased in the GluN1-P2 truncated peptide group compared with the group injected with the insignificant peptide group, while the injection of GluN1-P2 truncated peptide had no significant effect on the NMDA EPSC amplitude in WT mice, suggesting that GluN1-P2 truncated peptide significantly improves NMDA receptor function in Dp16 mice.

[0147] Three-month-old C57BL / 6 mice were stereotactically injected into the hippocampus with 1 μl (1 μg / μl) of GluN1-P2 truncated peptide or non-sense peptide (left and right controls from each mouse's own brain). One day after injection, brain slices were incubated in vitro with B2M protein (10 μg / ml) and ACSF for two hours, respectively, followed by electrophysiological recording. The amplitude of NMDAR EPSC in the Scheffer collateral circuit of the hippocampus was recorded. The stimulating electrode was placed near CA3, and the pyramidal cell current in CA1 was recorded. 5 mM QX-314 was added to the electrode fluid, and 50 μM PTX and 20 μM CNQX were added to the perfusion fluid to block GABA, respectively. A The receptor and AMPA receptor ion channel have a clamping voltage of +40mV.

[0148] Experimental results are as follows Figure 5B As shown in the figure. The results showed that, compared with brain slices incubated with ACSF, brain slices incubated with B2M significantly reduced the NMDA EPSC amplitude in WT mice, and injection of GluN1-P2 truncated peptide significantly improved the NMDA EPSC amplitude after B2M incubation.

[0149] Six-month-old Dp16 mice and control WT mice underwent stereotactic injections of 1 μl (1 μg / μl) of truncated GluN1-P2 peptide or non-sense peptide into the hippocampus (the left CA1 region of each mouse received the GluN1-P2 peptide, and the right CA1 region received the non-sense peptide). Eight-month-old 5×FAD mice underwent the same procedure. Electrophysiological recordings were performed one day after injection. After anesthesia, the brain tissue was rapidly removed from the mice and placed in ice-cold, oxygen-saturated artificial cerebrospinal fluid (ACSF). The tissue was then transferred to a vibratory microtome for coronal sectioning, with a slice thickness of 400 μm. The brain slices were incubated at 32°C in oxygen-saturated ACSF for 1 hour, followed by incubation at room temperature for 1 hour. The recording electrodes were placed in the radiative layer of the CA1 region of the Schaffer collateral pathway, and the stimulation electrodes were placed in the CA3 region. The stimulation intensity was 30% of the maximum amplitude of the excitatory postsynaptic potential (fEPSP). After the fEPSP baseline was stabilized for 20 minutes, high-frequency stimulation (HFS) induced LTP (two stimuli, each containing 100 stimulation pulses, with a 30-second interval between each stimuli), and the recording continued for 60 minutes.

[0150] Experimental results are as follows Figures 5C to 5F As shown in the figure. The results showed that in Dp16 mice, the injection of GluN1-P2 blocking peptide significantly improved LTP in the CA1 region of the hippocampus compared to the injection of meaningless peptide, while the injection of GluN1-P2 blocking peptide had no significant effect on LTP in WT mice, suggesting that GluN1-P2 blocking peptide reverses synaptic damage in Dp16 mice. Similarly, similar results were observed in 5×FAD mice.

[0151] In summary, the GluN1-P2 blocking peptide inhibits the binding of B2M to GluN1, thereby reducing synaptic damage.

[0152] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof. SEQUENCE LISTING <110> Xiamen University <120> B2M-GluN1 blocking peptide, its pharmaceutical composition and uses <130> IDC220225 <160> 32 <170> PatentIn version 3.5 <210> 1 <211> 938 <212> PRT <213> Rattus norvegicus <400> 1 Met Ser Thr Met His Leu Leu Thr Phe Ala Leu Leu Phe Ser Cys Ser 1 5 10 15 Phe Ala Arg Ala Ala Cys Asp Pro Lys Ile Val Asn Ile Gly Ala Val 20 25 30 Leu Ser Thr Arg Lys His Glu Gln Met Phe Arg Glu Ala Val Asn Gln 35 40 45 Ala Asn Lys Arg His Gly Ser Trp Lys Ile Gln Leu Asn Ala Thr Ser 50 55 60 Val Thr His Lys Pro Asn Ala Ile Gln Met Ala Leu Ser Val Cys Glu 65 70 75 80 Asp Leu Ile Ser Ser Gln Val Tyr Ala Ile Leu Val Ser His Pro Pro 85 90 95 Thr Pro Asn Asp His Phe Thr Pro Thr Pro Val Ser Tyr Thr Ala Gly 100 105 110 Phe Tyr Arg Ile Pro Val Leu Gly Leu Thr Thr Arg Met Ser Ile Tyr 115 120 125 Ser Asp Lys Ser Ile His Leu Ser Phe Leu Arg Thr Val Pro Pro Tyr 130 135 140 Ser His Gln Ser Ser Val Trp Phe Glu Met Met Arg Val Tyr Asn Trp 145 150 155 160 Asn His Ile Ile Leu Leu Val Ser Asp Asp His Glu Gly Arg Ala Ala 165 170 175 Gln Lys Arg Leu Glu Thr Leu Leu Glu Glu Arg Glu Ser Lys Ala Glu 180 185 190 Lys Val Leu Gln Phe Asp Pro Gly Thr Lys Asn Val Thr Ala Leu Leu 195 200 205 Met Glu Ala Arg Glu Leu Glu Ala Arg Val Ile Ile Leu Ser Ala Ser 210 215 220 Glu Asp Asp Ala Ala Thr Val Tyr Arg Ala Ala Ala Met Leu Asn Met 225 230 235 240 Thr Gly Ser Gly Tyr Val Trp Leu Val Gly Glu Arg Glu Ile Ser Gly 245 250 255 Asn Ala Leu Arg Tyr Ala Pro Asp Gly Ile Ile Gly Leu Gln Leu Ile 260 265 270 Asn Gly Lys Asn Glu Ser Ala His Ile Ser Asp Ala Val Gly Val Val 275 280 285 Ala Gln Ala Val His Glu Leu Leu Glu Lys Glu Asn Ile Thr Asp Pro 290 295 300 Pro Arg Gly Cys Val Gly Asn Thr Asn Ile Trp Lys Thr Gly Pro Leu 305 310 315 320 Phe Lys Arg Val Leu Met Ser Ser Lys Tyr Ala Asp Gly Val Thr Gly 325 330 335 Arg Val Glu Phe Asn Glu Asp Gly Asp Arg Lys Phe Ala Asn Tyr Ser 340 345 350 Ile Met Asn Leu Gln Asn Arg Lys Leu Val Gln Val Gly Ile Tyr Asn 355 360 365 Gly Thr His Val Ile Pro Asn Asp Arg Lys Ile Ile Trp Pro Gly Gly 370 375 380 Glu Thr Glu Lys Pro Arg Gly Tyr Gln Met Ser Thr Arg Leu Lys Ile 385 390 395 400 Val Thr Ile His Gln Glu Pro Phe Val Tyr Val Lys Pro Thr Met Ser 405 410 415 Asp Gly Thr Cys Lys Glu Glu Phe Thr Val Asn Gly Asp Pro Val Lys 420 425 430 Lys Val Ile Cys Thr Gly Pro Asn Asp Thr Ser Pro Gly Ser Pro Arg 435 440 445 His Thr Val Pro Gln Cys Cys Tyr Gly Phe Cys Ile Asp Leu Leu Ile 450 455 460 Lys Leu Ala Arg Thr Met Asn Phe Thr Tyr Glu Val His Leu Val Ala 465 470 475 480 Asp Gly Lys Phe Gly Thr Gln Glu Arg Val Asn Asn Ser Asn Lys Lys 485 490 495 Glu Trp Asn Gly Met Met Gly Glu Leu Leu Ser Gly Gln Ala Asp Met 500 505 510 Ile Val Ala Pro Leu Thr Ile Asn Asn Glu Arg Ala Gln Tyr Ile Glu 515 520 525 Phe Ser Lys Pro Phe Lys Tyr Gln Gly Leu Thr Ile Leu Val Lys Lys 530 535 540 Glu Ile Pro Arg Ser Thr Leu Asp Ser Phe Met Gln Pro Phe Gln Ser 545 550 555 560 Thr Leu Trp Leu Leu Val Gly Leu Ser Val His Val Val Ala Val Met 565 570 575 Leu Tyr Leu Leu Asp Arg Phe Ser Pro Phe Gly Arg Phe Lys Val Asn 580 585 590 Ser Glu Glu Glu Glu Glu Asp Ala Leu Thr Leu Ser Ser Ala Met Trp 595 600 605 Phe Ser Trp Gly Val Leu Leu Asn Ser Gly Ile Gly Glu Gly Ala Pro 610 615 620 Arg Ser Phe Ser Ala Arg Ile Leu Gly Met Val Trp Ala Gly Phe Ala 625 630 635 640 Met Ile Ile Val Ala Ser Tyr Thr Ala Asn Leu Ala Ala Phe Leu Val 645 650 655 Leu Asp Arg Pro Glu Glu Arg Ile Thr Gly Ile Asn Asp Pro Arg Leu 660 665 670 Arg Asn Pro Ser Asp Lys Phe Ile Tyr Ala Thr Val Lys Gln Ser Ser 675 680 685 Val Asp Ile Tyr Phe Arg Arg Gln Val Glu Leu Ser Thr Met Tyr Arg 690 695 700 His Met Glu Lys His Asn Tyr Glu Ser Ala Ala Glu Ala Ile Gln Ala 705 710 715 720 Val Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu 725 730 735 Glu Phe Glu Ala Ser Gln Lys Cys Asp Leu Val Thr Thr Gly Glu Leu 740 745 750 Phe Phe Arg Ser Gly Phe Gly Ile Gly Met Arg Lys Asp Ser Pro Trp 755 760 765 Lys Gln Asn Val Ser Leu Ser Ile Leu Lys Ser His Glu Asn Gly Phe 770 775 780 Met Glu Asp Leu Asp Lys Thr Trp Val Arg Tyr Gln Glu Cys Asp Ser 785 790 795 800 Arg Ser Asn Ala Pro Ala Thr Leu Thr Phe Glu Asn Met Ala Gly Val 805 810 815 Phe Met Leu Val Ala Gly Gly Ile Val Ala Gly Ile Phe Leu Ile Phe 820 825 830 Ile Glu Ile Ala Tyr Lys Arg His Lys Asp Ala Arg Arg Lys Gln Met 835 840 845 Gln Leu Ala Phe Ala Ala Val Asn Val Trp Arg Lys Asn Leu Gln Asp 850 855 860 Arg Lys Ser Gly Arg Ala Glu Pro Asp Pro Lys Lys Lys Ala Thr Phe 865 870 875 880 Arg Ala Ile Thr Ser Thr Leu Ala Ser Ser Phe Lys Arg Arg Arg Ser 885 890 895 Ser Lys Asp Thr Ser Thr Gly Gly Gly Arg Gly Ala Leu Gln Asn Gln 900 905 910 Lys Asp Thr Val Leu Pro Arg Arg Ala Ile Glu Arg Glu Glu Gly Gln 915 920 925 Leu Gln Leu Cys Ser Arg His Arg Glu Ser 930 935 <210> 2 <211> 938 <212> PRT <213> Homo sapiens <400> 2 Met Ser Thr Met Arg Leu Leu Thr Leu Ala Leu Leu Phe Ser Cys Ser 1 5 10 15 Val Ala Arg Ala Ala Cys Asp Pro Lys Ile Val Asn Ile Gly Ala Val 20 25 30 Leu Ser Thr Arg Lys His Glu Gln Met Phe Arg Glu Ala Val Asn Gln 35 40 45 Ala Asn Lys Arg His Gly Ser Trp Lys Ile Gln Leu Asn Ala Thr Ser 50 55 60 Val Thr His Lys Pro Asn Ala Ile Gln Met Ala Leu Ser Val Cys Glu 65 70 75 80 Asp Leu Ile Ser Ser Gln Val Tyr Ala Ile Leu Val Ser His Pro Pro 85 90 95 Thr Pro Asn Asp His Phe Thr Pro Thr Pro Val Ser Tyr Thr Ala Gly 100 105 110 Phe Tyr Arg Ile Pro Val Leu Gly Leu Thr Thr Arg Met Ser Ile Tyr 115 120 125 Ser Asp Lys Ser Ile His Leu Ser Phe Leu Arg Thr Val Pro Pro Tyr 130 135 140 Ser His Gln Ser Ser Val Trp Phe Glu Met Arg Val Tyr Ser Trp 145 150 155 160 Asn His Ile Ile Leu Leu Val Ser Asp Asp His Glu Gly Arg Ala Ala 165 170 175 Gln Lys Arg Leu Glu Thr Leu Leu Glu Glu Arg Glu Ser Lys Ala Glu 180 185 190 Lys Val Leu Gln Phe Asp Pro Gly Thr Lys Asn Val Thr Ala Leu Leu 195 200 205 Met Glu Ala Lys Glu Leu Glu Ala Arg Val Ile Ile Leu Ser Ala Ser 210 215 220 Glu Asp Asp Ala Ala Thr Val Tyr Arg Ala Ala Ala Met Leu Asn Met 225 230 235 240 Thr Gly Ser Gly Tyr Val Trp Leu Val Gly Glu Arg Glu Ile Ser Gly 245 250 255 Asn Ala Leu Arg Tyr Ala Pro Asp Gly Ile Leu Gly Leu Gln Leu Ile 260 265 270 Asn Gly Lys Asn Glu Ser Ala His Ile Ser Asp Ala Val Gly Val Val 275 280 285 Ala Gln Ala Val His Glu Leu Leu Glu Lys Glu Asn Ile Thr Asp Pro 290 295 300 Pro Arg Gly Cys Val Gly Asn Thr Asn Ile Trp Lys Thr Gly Pro Leu 305 310 315 320 Phe Lys Arg Val Leu Met Ser Ser Lys Tyr Ala Asp Gly Val Thr Gly 325 330 335 Arg Val Glu Phe Asn Glu Asp Gly Asp Arg Lys Phe Ala Asn Tyr Ser 340 345 350 Ile Met Asn Leu Gln Asn Arg Lys Leu Val Gln Val Gly Ile Tyr Asn 355 360 365 Gly Thr His Val Ile Pro Asn Asp Arg Lys Ile Ile Trp Pro Gly Gly 370 375 380 Glu Thr Glu Lys Pro Arg Gly Tyr Gln Met Ser Thr Arg Leu Lys Ile 385 390 395 400 Val Thr Ile His Gln Glu Pro Phe Val Tyr Val Lys Pro Thr Leu Ser 405 410 415 Asp Gly Thr Cys Lys Glu Glu Phe Thr Val Asn Gly Asp Pro Val Lys 420 425 430 Lys Val Ile Cys Thr Gly Pro Asn Asp Thr Ser Pro Gly Ser Pro Arg 435 440 445 His Thr Val Pro Gln Cys Cys Tyr Gly Phe Cys Ile Asp Leu Leu Ile 450 455 460 Lys Leu Ala Arg Thr Met Asn Phe Thr Tyr Glu Val His Leu Val Ala 465 470 475 480 Asp Gly Lys Phe Gly Thr Gln Glu Arg Val Asn Asn Ser Asn Lys Lys 485 490 495 Glu Trp Asn Gly Met Met Gly Glu Leu Leu Ser Gly Gln Ala Asp Met 500 505 510 Ile Val Ala Pro Leu Thr Ile Asn Asn Glu Arg Ala Gln Tyr Ile Glu 515 520 525 Phe Ser Lys Pro Phe Lys Tyr Gln Gly Leu Thr Ile Leu Val Lys Lys 530 535 540 Glu Ile Pro Arg Ser Thr Leu Asp Ser Phe Met Gln Pro Phe Gln Ser 545 550 555 560 Thr Leu Trp Leu Leu Val Gly Leu Ser Val His Val Val Ala Val Met 565 570 575 Leu Tyr Leu Leu Asp Arg Phe Ser Pro Phe Gly Arg Phe Lys Val Asn 580 585 590 Ser Glu Glu Glu Glu Glu Asp Ala Leu Thr Leu Ser Ser Ala Met Trp 595 600 605 Phe Ser Trp Gly Val Leu Leu Asn Ser Gly Ile Gly Glu Gly Ala Pro 610 615 620 Arg Ser Phe Ser Ala Arg Ile Leu Gly Met Val Trp Ala Gly Phe Ala 625 630 635 640 Met Ile Ile Val Ala Ser Tyr Thr Ala Asn Leu Ala Ala Phe Leu Val 645 650 655 Leu Asp Arg Pro Glu Glu Arg Ile Thr Gly Ile Asn Asp Pro Arg Leu 660 665 670 Arg Asn Pro Ser Asp Lys Phe Ile Tyr Ala Thr Val Lys Gln Ser Ser 675 680 685 Val Asp Ile Tyr Phe Arg Arg Gln Val Glu Leu Ser Thr Met Tyr Arg 690 695 700 His Met Glu Lys His Asn Tyr Glu Ser Ala Ala Glu Ala Ile Gln Ala 705 710 715 720 Val Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu 725 730 735 Glu Phe Glu Ala Ser Gln Lys Cys Asp Leu Val Thr Thr Gly Glu Leu 740 745 750 Phe Phe Arg Ser Gly Phe Gly Ile Gly Met Arg Lys Asp Ser Pro Trp 755 760 765 Lys Gln Asn Val Ser Leu Ser Ile Leu Lys Ser His Glu Asn Gly Phe 770 775 780 Met Glu Asp Leu Asp Lys Thr Trp Val Arg Tyr Gln Glu Cys Asp Ser 785 790 795 800 Arg Ser Asn Ala Pro Ala Thr Leu Thr Phe Glu Asn Met Ala Gly Val 805 810 815 Phe Met Leu Val Ala Gly Gly Ile Val Ala Gly Ile Phe Leu Ile Phe 820 825 830 Ile Glu Ile Ala Tyr Lys Arg His Lys Asp Ala Arg Arg Lys Gln Met 835 840 845 Gln Leu Ala Phe Ala Ala Val Asn Val Trp Arg Lys Asn Leu Gln Asp 850 855 860 Arg Lys Ser Gly Arg Ala Glu Pro Asp Pro Lys Lys Lys Ala Thr Phe 865 870 875 880 Arg Ala Ile Thr Ser Thr Leu Ala Ser Ser Phe Lys Arg Arg Arg Ser 885 890 895 Ser Lys Asp Thr Ser Thr Gly Gly Gly Arg Gly Ala Leu Gln Asn Gln 900 905 910 Lys Asp Thr Val Leu Pro Arg Arg Ala Ile Glu Arg Glu Glu Gly Gln 915 920 925 Leu Gln Leu Cys Ser Arg His Arg Glu Ser 930 935 <210> 3 <211> 833 <212> PRT <213> Artificial Sequence <220> <223> Rat GluN1 C-terminal intracellular segment deletion <400> 3 Met Ser Thr Met His Leu Leu Thr Phe Ala Leu Leu Phe Ser Cys Ser 1 5 10 15 Phe Ala Arg Ala Ala Cys Asp Pro Lys Ile Val Asn Ile Gly Ala Val 20 25 30 Leu Ser Thr Arg Lys His Glu Gln Met Phe Arg Glu Ala Val Asn Gln 35 40 45 Ala Asn Lys Arg His Gly Ser Trp Lys Ile Gln Leu Asn Ala Thr Ser 50 55 60 Val Thr His Lys Pro Asn Ala Ile Gln Met Ala Leu Ser Val Cys Glu 65 70 75 80 Asp Leu Ile Ser Ser Gln Val Tyr Ala Ile Leu Val Ser His Pro Pro 85 90 95 Thr Pro Asn Asp His Phe Thr Pro Thr Pro Val Ser Tyr Thr Ala Gly 100 105 110 Phe Tyr Arg Ile Pro Val Leu Gly Leu Thr Thr Arg Met Ser Ile Tyr 115 120 125 Ser Asp Lys Ser Ile His Leu Ser Phe Leu Arg Thr Val Pro Pro Tyr 130 135 140 Ser His Gln Ser Ser Val Trp Phe Glu Met Met Arg Val Tyr Asn Trp 145 150 155 160 Asn His Ile Ile Leu Leu Val Ser Asp Asp His Glu Gly Arg Ala Ala 165 170 175 Gln Lys Arg Leu Glu Thr Leu Leu Glu Glu Arg Glu Ser Lys Ala Glu 180 185 190 Lys Val Leu Gln Phe Asp Pro Gly Thr Lys Asn Val Thr Ala Leu Leu 195 200 205 Met Glu Ala Arg Glu Leu Glu Ala Arg Val Ile Ile Leu Ser Ala Ser 210 215 220 Glu Asp Asp Ala Ala Thr Val Tyr Arg Ala Ala Ala Met Leu Asn Met 225 230 235 240 Thr Gly Ser Gly Tyr Val Trp Leu Val Gly Glu Arg Glu Ile Ser Gly 245 250 255 Asn Ala Leu Arg Tyr Ala Pro Asp Gly Ile Ile Gly Leu Gln Leu Ile 260 265 270 Asn Gly Lys Asn Glu Ser Ala His Ile Ser Asp Ala Val Gly Val Val 275 280 285 Ala Gln Ala Val His Glu Leu Leu Glu Lys Glu Asn Ile Thr Asp Pro 290 295 300 Pro Arg Gly Cys Val Gly Asn Thr Asn Ile Trp Lys Thr Gly Pro Leu 305 310 315 320 Phe Lys Arg Val Leu Met Ser Ser Lys Tyr Ala Asp Gly Val Thr Gly 325 330 335 Arg Val Glu Phe Asn Glu Asp Gly Asp Arg Lys Phe Ala Asn Tyr Ser 340 345 350 Ile Met Asn Leu Gln Asn Arg Lys Leu Val Gln Val Gly Ile Tyr Asn 355 360 365 Gly Thr His Val Ile Pro Asn Asp Arg Lys Ile Ile Trp Pro Gly Gly 370 375 380 Glu Thr Glu Lys Pro Arg Gly Tyr Gln Met Ser Thr Arg Leu Lys Ile 385 390 395 400 Val Thr Ile His Gln Glu Pro Phe Val Tyr Val Lys Pro Thr Met Ser 405 410 415 Asp Gly Thr Cys Lys Glu Glu Phe Thr Val Asn Gly Asp Pro Val Lys 420 425 430 Lys Val Ile Cys Thr Gly Pro Asn Asp Thr Ser Pro Gly Ser Pro Arg 435 440 445 His Thr Val Pro Gln Cys Cys Tyr Gly Phe Cys Ile Asp Leu Leu Ile 450 455 460 Lys Leu Ala Arg Thr Met Asn Phe Thr Tyr Glu Val His Leu Val Ala 465 470 475 480 Asp Gly Lys Phe Gly Thr Gln Glu Arg Val Asn Asn Ser Asn Lys Lys 485 490 495 Glu Trp Asn Gly Met Met Gly Glu Leu Leu Ser Gly Gln Ala Asp Met 500 505 510 Ile Val Ala Pro Leu Thr Ile Asn Asn Glu Arg Ala Gln Tyr Ile Glu 515 520 525 Phe Ser Lys Pro Phe Lys Tyr Gln Gly Leu Thr Ile Leu Val Lys Lys 530 535 540 Glu Ile Pro Arg Ser Thr Leu Asp Ser Phe Met Gln Pro Phe Gln Ser 545 550 555 560 Thr Leu Trp Leu Leu Val Gly Leu Ser Val His Val Val Ala Val Met 565 570 575 Leu Tyr Leu Leu Asp Arg Phe Ser Pro Phe Gly Arg Phe Lys Val Asn 580 585 590 Ser Glu Glu Glu Glu Glu Asp Ala Leu Thr Leu Ser Ser Ala Met Trp 595 600 605 Phe Ser Trp Gly Val Leu Leu Asn Ser Gly Ile Gly Glu Gly Ala Pro 610 615 620 Arg Ser Phe Ser Ala Arg Ile Leu Gly Met Val Trp Ala Gly Phe Ala 625 630 635 640 Met Ile Ile Val Ala Ser Tyr Thr Ala Asn Leu Ala Ala Phe Leu Val 645 650 655 Leu Asp Arg Pro Glu Glu Arg Ile Thr Gly Ile Asn Asp Pro Arg Leu 660 665 670 Arg Asn Pro Ser Asp Lys Phe Ile Tyr Ala Thr Val Lys Gln Ser Ser 675 680 685 Val Asp Ile Tyr Phe Arg Arg Gln Val Glu Leu Ser Thr Met Tyr Arg 690 695 700 His Met Glu Lys His Asn Tyr Glu Ser Ala Ala Glu Ala Ile Gln Ala 705 710 715 720 Val Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu 725 730 735 Glu Phe Glu Ala Ser Gln Lys Cys Asp Leu Val Thr Thr Gly Glu Leu 740 745 750 Phe Phe Arg Ser Gly Phe Gly Ile Gly Met Arg Lys Asp Ser Pro Trp 755 760 765 Lys Gln Asn Val Ser Leu Ser Ile Leu Lys Ser His Glu Asn Gly Phe 770 775 780 Met Glu Asp Leu Asp Lys Thr Trp Val Arg Tyr Gln Glu Cys Asp Ser 785 790 795 800 Arg Ser Asn Ala Pro Ala Thr Leu Thr Phe Glu Asn Met Ala Gly Val 805 810 815 Phe Met Leu Val Ala Gly Gly Ile Val Ala Gly Ile Phe Leu Ile Phe 820 825 830 Ile <210> 4 <211> 379 <212> PRT <213> Artificial Sequence <220> <223> Rat GluN1 N-terminal extracellular domain deletion <400> 4 Ser Thr Leu Trp Leu Leu Val Gly Leu Ser Val His Val Val Ala Val 1 5 10 15 Met Leu Tyr Leu Leu Asp Arg Phe Ser Pro Phe Gly Arg Phe Lys Val 20 25 30 Asn Ser Glu Glu Glu Glu Glu Asp Ala Leu Thr Leu Ser Ser Ala Met 35 40 45 Trp Phe Ser Trp Gly Val Leu Leu Asn Ser Gly Ile Gly Glu Gly Ala 50 55 60 Pro Arg Ser Phe Ser Ala Arg Ile Leu Gly Met Val Trp Ala Gly Phe 65 70 75 80 Ala Met Ile Ile Val Ala Ser Tyr Thr Ala Asn Leu Ala Ala Phe Leu 85 90 95 Val Leu Asp Arg Pro Glu Glu Arg Ile Thr Gly Ile Asn Asp Pro Arg 100 105 110 Leu Arg Asn Pro Ser Asp Lys Phe Ile Tyr Ala Thr Val Lys Gln Ser 115 120 125 Ser Val Asp Ile Tyr Phe Arg Arg Gln Val Glu Leu Ser Thr Met Tyr 130 135 140 Arg His Met Glu Lys His Asn Tyr Glu Ser Ala Ala Glu Ala Ile Gln 145 150 155 160 Ala Val Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val 165 170 175 Leu Glu Phe Glu Ala Ser Gln Lys Cys Asp Leu Val Thr Thr Gly Glu 180 185 190 Leu Phe Phe Arg Ser Gly Phe Gly Ile Gly Met Arg Lys Asp Ser Pro 195 200 205 Trp Lys Gln Asn Val Ser Leu Ser Ile Leu Lys Ser His Glu Asn Gly 210 215 220 Phe Met Glu Asp Leu Asp Lys Thr Trp Val Arg Tyr Gln Glu Cys Asp 225 230 235 240 Ser Arg Ser Asn Ala Pro Ala Thr Leu Thr Phe Glu Asn Met Ala Gly 245 250 255 Val Phe Met Leu Val Ala Gly Gly Ile Val Ala Gly Ile Phe Leu Ile 260 265 270 Phe Ile Glu Ile Ala Tyr Lys Arg His Lys Asp Ala Arg Arg Lys Gln 275 280 285 Met Gln Leu Ala Phe Ala Ala Val Asn Val Trp Arg Lys Asn Leu Gln 290 295 300 Asp Arg Lys Ser Gly Arg Ala Glu Pro Asp Pro Lys Lys Lys Ala Thr 305 310 315 320 Phe Arg Ala Ile Thr Ser Thr Leu Ala Ser Ser Phe Lys Arg Arg Arg 325 330 335 Ser Ser Lys Asp Thr Ser Thr Gly Gly Gly Arg Gly Ala Leu Gln Asn 340 345 350 Gln Lys Asp Thr Val Leu Pro Arg Arg Ala Ile Glu Arg Glu Glu Gly 355 360 365 Gln Leu Gln Leu Cys Ser Arg His Arg Glu Ser 370 375 <210> 5 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> Rat GluN1 extracellular loop peptide <400> 5 Thr Ala Asn Leu Ala Ala Phe Leu Val Leu Asp Arg Pro Glu Glu Arg 1 5 10 15 Ile Thr Gly Ile Asn Asp Pro Arg Leu Arg Asn Pro Ser Asp Lys Phe 20 25 30 Ile Tyr Ala Thr Val Lys Gln Ser Ser Val Asp Ile Tyr Phe Arg Arg 35 40 45 Gln Val Glu Leu Ser Thr Met Tyr Arg His Met Glu Lys His Asn Tyr 50 55 60 Glu Ser Ala Ala Glu Ala Ile Gln Ala Val Arg Asp Asn Lys Leu His 65 70 75 80 Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe Glu Ala Ser Gln Lys 85 90 95 Cys Asp Leu Val Thr Thr Gly Glu Leu Phe Phe Arg Ser Gly Phe Gly 100 105 110 Ile Gly Met Arg Lys Asp Ser Pro Trp Lys Gln Asn Val Ser Leu Ser 115 120 125 Ile Leu Lys Ser His Glu Asn Gly Phe Met Glu Asp Leu Asp Lys Thr 130 135 140 Trp Val Arg Tyr Gln Glu Cys Asp Ser Arg Ser Asn Ala Pro Ala Thr 145 150 155 160 Leu Thr Phe Glu Asn 165 <210> 6 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> HA‑B2M <400> 6 Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg 20 25 30 His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser 35 40 45 Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu 50 55 60 Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp 65 70 75 80 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp 85 90 95 Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile 100 105 110 Val Lys Trp Asp Arg Asp Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 115 120 125 <210> 7 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> GluN1‑S2 loop‑L1 <400> 7 Thr Ala Asn Leu Ala Ala Phe Leu Val Leu Asp Arg Pro Glu Glu Arg 1 5 10 15 Ile Thr Gly Ile Asn Asp Pro Arg Leu Arg Asn Pro Ser Asp Lys Phe 20 25 30 Ile Tyr Ala Thr Val Lys Gln Ser Ser Val Asp Ile Tyr Phe Arg Arg 35 40 45 Gln Val Glu Leu Ser Thr Met Tyr Arg His Met 50 55 <210> 8 <211> 57 <212> PRT <213> Artificial Sequence <220> <223> GluN1‑S2 loop‑L2 <400> 8 Glu Lys His Asn Tyr Glu Ser Ala Ala Glu Ala Ile Gln Ala Val Arg 1 5 10 15 Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe 20 25 30 Glu Ala Ser Gln Lys Cys Asp Leu Val Thr Thr Gly Glu Leu Phe Phe 35 40 45 Arg Ser Gly Phe Gly Ile Gly Met Arg 50 55 <210> 9 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> GluN1‑S2 loop‑L3 <400> 9 Lys Asp Ser Pro Trp Lys Gln Asn Val Ser Leu Ser Ile Leu Lys Ser 1 5 10 15 His Glu Asn Gly Phe Met Glu Asp Leu Asp Lys Thr Trp Val Arg Tyr 20 25 30 Gln Glu Cys Asp Ser Arg Ser Asn Ala Pro Ala Thr Leu Thr Phe Glu 35 40 45 Asn <210> 10 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> GluN1‑P1 <400> 10 Glu Lys His Asn Tyr Glu Ser Ala Ala Glu Ala Ile Gln Ala Val Arg 1 5 10 15 Asp Asn <210> 11 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> GluN1‑P2 <400> 11 Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe Glu Ala 1 5 10 15 Ser Gln <210> 12 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> GluN1‑P3 <400> 12 Lys Cys Asp Leu Val Thr Thr Gly Glu Leu Phe Phe Arg Ser Gly Phe 1 5 10 15 Gly Ile Gly Met Arg 20 <210> 13 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Non‑sense <400> 13 Trp Ser Phe Ala Glu Gln Asp Lys Ala Phe Ile Val His Leu Glu Ala 1 5 10 15 Leu Ser <210> 14 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 14 Ala Val Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val 1 5 10 15 Leu Glu Phe Glu Ala Ser Gln 20 <210> 15 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 15 Val Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu 1 5 10 15 Glu Phe Glu Ala Ser Gln 20 <210> 16 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 16 Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu 1 5 10 15 Phe Glu Ala Ser Gln 20 <210> 17 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 17 Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe 1 5 10 15 Glu Ala Ser Gln 20 <210> 18 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 18 Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe Glu 1 5 10 15 Ala Ser Gln <210> 19 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 19 Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe Glu Ala 1 5 10 15 Ser Gln Lys Cys Asp Leu Val 20 <210> 20 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 20 Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe Glu Ala 1 5 10 15 Ser Gln Lys Cys Asp Leu 20 <210> 21 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 21 Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe Glu Ala 1 5 10 15 Ser Gln Lys Cys Asp 20 <210> 22 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 22 Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe Glu Ala 1 5 10 15 Ser Gln Lys Cys 20 <210> 23 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 23 Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe Glu Ala 1 5 10 15 Ser Gln Lys <210> 24 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 24 Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu 1 5 10 15 Phe Glu Ala Ser Gln Lys Cys Asp 20 <210> 25 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 25 Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu 1 5 10 15 Phe Glu Ala Ser Gln Lys Cys 20 <210> 26 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 26 Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu 1 5 10 15 Phe Glu Ala Ser Gln Lys 20 <210> 27 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 27 Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe 1 5 10 15 Glu Ala Ser Gln Lys Cys Asp 20 <210> 28 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 28 Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe 1 5 10 15 Glu Ala Ser Gln Lys Cys 20 <210> 29 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 29 Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe 1 5 10 15 Glu Ala Ser Gln Lys 20 <210> 30 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 30 Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe Glu 1 5 10 15 Ala Ser Gln Lys Cys Asp 20 <210> 31 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 31 Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe Glu 1 5 10 15 Ala Ser Gln Lys Cys 20 <210> 32 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment <400> 32 Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe Glu 1 5 10 15 Ala Ser Gln Lys 20

Claims

1. An isolated polypeptide, which is the polypeptide shown in SEQ ID NO: 8 or the polypeptide shown in SEQ ID NO:

11.

2. An isolated polynucleotide encoding the isolated polypeptide of claim 1.

3. A recombinant expression vector comprising the isolated polynucleotide of claim 2.

4. A transformed cell comprising the recombinant expression vector of claim 3.

5. A pharmaceutical composition comprising one or more isolated polypeptides as described in claim 1.

6. The pharmaceutical composition according to claim 5, further comprising one or more pharmaceutically acceptable excipients.

7. Use of the polypeptide shown in SEQ ID NO: 11 in the preparation of a medicament for the treatment or prevention of Down syndrome, Alzheimer's disease, or cognitive impairment caused by Down syndrome or Alzheimer's disease.

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