Neuroprotection Against Excitotoxicity By Autoimmune To M3M4 Loop Segments In The Primary Subunit Of Human NMDA Receptor

Excitotoxicity, associated with stroke and neurodegeneration, is triggered primarily by massive Ca2+ influx arising from overactivation of glutamate receptor channels of the N-Methyl-D-aspartate (NMDA) subtype. Although approaches against excitotoxicity with antagonists or blockers of NMDA receptor have been testified to be effective in animal models, successful therapy in humans was limited by the severe side effects due to the relative wide space window and the most narrow time window. So alternative approaches to antagonize NMDA receptor are waited be found quickly. In this study, we mainly investigate the neuroprotective effects against excitotoxicity by autoimmune to M3M4 loop segments in the primary subunit (NR1a) of human NMDA receptor. Our work mainly includes:1. Epitope analysis of agonist-binding sites of NRlaPhysicochemical properties and antigenicity of two agonist-binding segments of NRla were analyzed through bioinformatics: polypeptide P1 containing 151 amino acid residues preceding the first transmembrane domain of the human NR1a, peptide P2 with 144 residues following the third transmembrane domain. Four parameters including Hopp-Woods and Kyte hydrophilicity,Janin accessibility, Karplus-Schulz flexibility, and Welling antigenicity were used to determine the antigenic sites, and Prosite programme and Chou-Fasman method were employed to analyze their related sequence motif and the secondary structures. Finally, comparison of the comprehensive predictions with some of the available experimental information was made. Compared with P1, P2 may be of higher antigenicity. The antigenic sites in P2 were dipersed rather uniformly, including or nearing receptor-activating sites: they are FLVLDRPEERI, RLRNPSDKFIYAT, YRHMEKHNYES, RDNKLHAFIW, ASQKCDLVTT, KDSPWKQNVS, ILKSHENGF. P2 may be more easily used as a molecular target than P1 in immunization intervention to control the activation of NR.2. Prokaryotic expression, purification and identification of the M3M4 Loop in the primary subuint of NRlaObtain a cDNA fragment encoding M3M4 loop in human NR1 by RT-PCR using the total RNA extracted from a human brain glioma as template. Redesign optimally the PCR primer according to a computer-aided high expression model for the foreign gene in pBV220 vector. The prediction of RNA secondary structure and the calculation of the codon adaptation were respectively finished through RNAfold and Goldkey software. Use EcoR I and BamH I to digest the PCR product and the vector pBV220, link the above two products, construct the expression system: pBV-NR1L3.Transform competent DH5a with pBV-NR1L3, use Sangers method to sequence the pBV-NR1L3. Induce pBV-NR1L3 plasmid to express the objective protein by shifting the culture temperature from 37℃ to 42℃. Confirm optimal induction time and expression form through SDS-PAGE and confirm the percentage of expressed protein accounting for total bacteria protein by gel scanning. Purify the recombinant protein by preparative SDS-PAGE. Elution protein from gel by electrophoresis.After the protein was resumed, the pure powder was obtained by frost-dryness method.Characterize the expression product by a series of identifications, such as apparent molecular weight, immunoreactivity and peptide mass fingerprinting.3. Epitope analysis in the M3M4 loop of NR1aTo determine the B cell epitope of a monoclonal antibody against the M3M4 loop of NMDAR1, a random phage displayed dodecapeptide library was screened with the monoclonal antibody MAB363 against the M3M4 loop of NMDAR1. After four rounds of biopanning, the peptide sequences of positive phage clones were determined and analyzed by DNA sequencing, ELISA and competitive inhibition assay. A positve clone was found (clone 1: VHTNPSTWQPIL). The binding between clone 1 and MAB363 were competitively inhibited by the recombined M3M4 loop expressed by E.Coli DH5 a ; The binding between M3M4 and MAB363 could be competitively inhibited by one of solid-synthesized epitope peptides: RLRNPSKD. There was a same sequence among them: NPS. Deleted with NPS, the peptide could not inhibit the binding of MAB363 to M3M4. These results demonstrate that NPS in M3M4 loop is the B cell epitope recognized by MAB363, which may be important for developing a practical immunization strategy against excitotoxic brain injury.4.1mmunogenicity of the recombinant M3M4 loop peptide of NRla, neuroprotective effects of the antiserum against excitotoxicity and the mechanisms(1) To explore the immunogenicity of M3M4 loop recombinant peptide vaccine, M3M4 recombinant peptide was injected subcutaneously to immunize Balb/C mice (H-2d) , CD4/CD8 T lymphocytes subsets, the cytokine levels in serum and the titer of specific humoral response were detected with FACS, indirect ELISA and ELISA separately. Results: compared with control mice, the percentage of CD4+ and CD8+ T lymphocytes were increased significantly, Thl and Th2 cytokines levels —TNF- α ,IFN- γ ,IL-4,IL-6 were significant increased significantly in serum. Titer of specific antibody against M3M4 was above 1:1000. Specific antibody against epitope(NPS) could also be detected in serum. Conclusion: M3M4 recombinated peptide antigen has immunogenicity and could induce Thl and Th2 types of immune response, specific antibodies against M3M4 and epitope(NPS)could be detected.(2) neuroprotective effects of the mouse antiserum against excitotoxicityTrypan blue dye staining and terminal deoxynucleotidyl transferase-mediated biotimylated deoxyuridine triphosphate nickel end labeling (TUNEL) method were used to investigate if the humoral immune response targeting the NR1 subunit of NMDA receptor has neuroprotective activity, At high concentration of glutamate (500 μ mol/L) the positive antiserum could protect 16% of neurons from necrosis, and at low concentration of glutamate (50 μ mol/L) the positive antiserum could protect 13% of neurons from apoptosis . Serum against M3M4 loop of NR1 could protect cultured hippocampal neurons from excitotoxicity, which suggests humoral autoimmune response targeting the NRl subunit of NMDA receptor might have neuroprotective activity.(3) Glutamate-neuroexcitotoxicity protective study of monoclonal antibody MAB363The epitope recognized by monoclonal antibody MAB363 against human NRl located near the important functional region of NRl. To study if MAB363 may protect neurons from excitotoxicity, We cultured primary hippocampal neurons from newborn rats and made the glutamate excitotoxicity model, examined the ratio of surviving neuron ( Trypan blue dye staining ) , LDH assay and apoptosis (TUNEL)to study the protective effects of MAB363. MAB363(0.6 μ M) could protect cultured hippocampal neurons from neuroexcitotoxicity inducedby 500 μ M glutamate with higher survival rate than control by 41%, LDH leaking decreased by 25%. Epitope peptide could block the protection of MAB363, which suggests that the epitope of MAB363 is specific target of antiexcitotoxicity. (4) The mechanisms of the neuroprotective effects of monoclonal antibody against NR1aa.Relationship between glutamate-induced Ca2+ influx and protective effect of MAB363Fluo-3 and confocal laser microscopy for measurement of intracellular calcium levels were used in this study. Pretreatment of the cultured neurons with MAB363 and MK-801(10μ mol/L) caused a marked reduction in the calcium signals generated by subsequent glutamate stimulation. Which suggest that Ca2+ influx plays a major role in protecion associated with glutamate toxicity.b.Fourier Transform Infrared Spectroscopic Investigation of excitotoxicity-protective mechanism of MAB363Cultured hippocampus neurons treated by glutamate or/and NMDAR antagonist (MAB363 and MK-801) were studied by FTIR spectroscopy. Spectroscopic differences were observed between glutamate-treated and drug-treated samples. Curve-fitting of the deconvoluted amide I band revealed the difference of protein second structure between MAB363-protected neurons and glutamate-treated neurons, by which we presume that MAB363 protects neurons against excitotoxicity by influencing the protein function. This study provides some clues for investigating excitotoxicity-protective mechanism of MAB363.In conclusion, we predicted M3M4 loop of NR1a has a certain amount of antigenic sites through bioinformatics and may be used as a molecular target in immunization intervention to control the pathological activation of NMDA receptor. We have successfully constructed the prokaryotic expression system pBV-NRlL3. The obtained gene was highly expressed with pBV220 vector by redesigning PCR primer. The result of gel scanning showed that the M3M4 loop peptide accounted for 29% of total bacteria protein. The expressed product was purified and identified. The purity was up to 95% and the results of identification were the same as those anticipated. A random phage displayed dodecapeptide library was screened with the monoclonal antibody MAB363 against the M3M4 loop of human NRla. From ELISA and competitive inhibition assay we obtained the epitope of MAB363, which may be a dominating epitope for it is identical with the result of bioinformatics. M3M4 recombinated peptide antigen has immunogenicity, antiserum and MAB363 against M3M4 loop of NR1 could protect cultured hippocampal neurons from excitotoxicity, the protective mechanism may related with decreased Ca2+ influx due to the second structure change of the target protein on neuron.