title: Anti-NMDA receptor encephalitis antibody binding is dependent on amino acid identity of a small region within the GluN1 amino terminal domain.
abstract: Anti-NMDA receptor (NMDAR) encephalitis is a newly identified autoimmune disorder that targets NMDARs, causing severe neurological symptoms including hallucinations, psychosis, and seizures, and may result in death (Dalmau et al., 2008). However, the exact epitope to which these antibodies bind is unknown. A clearly defined antigenic region could provide more precise testing, allow for comparison of immunogenicity between patients to explore potential clinically relevant variations, elucidate the functional effects of antibodies, and make patients' antibodies a more effective tool with which to study NMDAR function. Here, we use human CSF to explore the antigenic region of the NMDAR. We created a series of mutants within the amino terminal domain of GluN1 that change patient antibody binding in transfected cells in stereotyped ways. These mutants demonstrate that the N368/G369 region of GluN1 is crucial for the creation of immunoreactivity. Mass spectrometry experiments show that N368 is glycosylated in transfected cells and rat brain regions; however, this glycosylation is not directly required for epitope formation. Mutations of residues N368/G369 change the closed time of the receptor in single channel recordings; more frequent channel openings correlates with the degree of antibody staining, and acute antibody exposure prolongs open time of the receptor. The staining pattern of mutant receptors is similar across subgroups of patients, indicating consistent immunogenicity, although we have identified one region that has a variable role in epitope formation. These findings provide tools for detailed comparison of antibodies across patients and suggest an interaction between antibody binding and channel function.
authors.affiliation: Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Gleichman
authors.forename: Lynn A
authors.initials: LA
authors.lastname: Spruce
authors.forename: Josep
authors.initials: J
authors.lastname: Dalmau
authors.forename: Steven H
authors.initials: SH
authors.lastname: Seeholzer
authors.forename: David R
authors.initials: DR
authors.lastname: Lynch
mesh_terms: D000367:Age Factors; D060426:Anti-N-Methyl-D-Aspartate Receptor Encephalitis / Q000134:cerebrospinal fluid* / Q000235:genetics / Q000473:pathology; D000906:Antibodies / Q000134:cerebrospinal fluid* / Q000276:immunology; D001666:Binding Sites, Antibody / Q000187:drug effects / Q000235:genetics; D019921:Biotinylation; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002461:Cell Line, Transformed; D000939:Epitopes / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D018698:Glutamic Acid / Q000494:pharmacology; D005998:Glycine / Q000494:pharmacology; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation / Q000235:genetics; D018408:Patch-Clamp Techniques; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D018091:Receptors, AMPA / Q000737:chemistry* / Q000235:genetics; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000276:immunology*; D018709:Statistics, Nonparametric; D053719:Tandem Mass Spectrometry; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D000906:Antibodies; D000939:Epitopes; D018698:Glutamic Acid; D005998:Glycine; D049452:Green Fluorescent Proteins; D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D014415:Tunicamycin; C121001:NR2B NMDA receptor