Determination Of Dominant Autoimmune Epitope In Human NR1a Membrane-binding Protein And Functional Analysis Of The Monoclonal Antibody

N-methy-D-aspartate (NMDA) subtype of ionotropic glutamate receptors is proved to play a critical role in pathophysiological processes such as acute and delayed ischemic neuronal death as occurs in stroke, status epilepticus, brain trauma and brain tumour, and also contributes to the emergence of chronic neurodegenerative disorders. Because of their central involvement in the cascade leading to neuronal death following a variety of the central nervous system injuries, pharmacological NMDA receptor antagonists have been evaluated for potential clinical use for several decades. However, the therapeutic efficacy of NMDA antagonists has yet to be established, because the therapeutic ratio for most NMDA antagonists is poor, with significant adverse effects at clinically effective doses, thus limiting their utility in human. Therefore, new therapeutic strategies are needed badly in this field. Vaccine-based strategy and antibody-therapy is a new exploration: NMDA receptor can be applied as a vaccine to immunize the high-risk crowd of above-mentioned brain disorders, and the specific antibodies can be induced. When the antibodies bind to their epitopes in NMDA receptor, the excessive excitement of NMDA receptor can be inhibited. When a too long protein antigen be used as a vaccine, the potential cytotoxic T lymphocyte epitope in it can induce harmful cytotoxic response in body, but the epitope-based subunit vaccine can settle this problem effectively. Accordingly, the epitope selection is a critical work for vaccine design. In this study, two extracellular domain of NMDA receptor subunit 1a (NR1a) membrane protein was analysed, and the M3-M4 loop was considered as a more secure target in the auto-immunity intervention strategy than the extracellular N terminal. Then a dominant auto-immune dominant epitope was selected in M3-M4 loop, and the monoclonal antibody against this epitope was proved to have anti-excitotoxic effect.1.Analysis of autoimmune dominant epitope in human NR1a membrane- binding protein by bioinformaticsStudies on previous experimental data indicated that in NR1a membrane protein, M3-M4 loop can induce secure and effective immune response more easily than the extracellular N terminal during auto-immunity intervention. In order to avoid the cytotoxic response induced by the cytotoxic T lymphocyte epitopes in a too long vaccine, it is critical to select an auto-immune dominant epitope in M3-M4 loop. Now, it is possible to predict the B cell epitope in a heterogenous antigen, but due to the existence of immunotolerance, it is not easy to predict the B cell epitope in the auto-proteins. In this study, we established a method to predicte the dominant epitope in auto-proteins. This method includes seven aspects: B cell epitope, cytotoxic T lymphocyte epitope, helper T lymphocyte epitope, proteasome cleavage site, endosome/lysosome proteases cleavage site, glycosylation site and protein secondary structure. In order to test the veracity of this prediction method, we analysed about forty self-immune molecule which dominant epitopes have been defined. Our result showed that the veracity ratio of this method was above 70%, which was higher than the veracity ratio of bioinformatics epitope predictional methods in existence. Then we analysed M3-M4 loop in NR1a membrane protein using this method, and selected an auto-immune dominant epitope R(22)LRNPS.2.Studies on neuroexcitotoxicity protective effect of monoclonal antibody against auto-immune dominant epitope in human NR1a membrane-binding proteinMAb363 is a specific monoclonal antibody directly against M3-M4 loop. The dominant epitope obtained by random phage displayed dodecapeptide library screening with mAb363 was consistent with the epitope we selected by the method of bioinformatics. Previous study reported that it has neuroprotective activity in vitro. To further validate this conclusion and investigate the mechanism of neuroprotective activity of mAb363, we primarily culture the hippocampal neurons of the newborn Sprague-Dawley rats, and the intr...