title: Binding affinity and specificity of neuromyelitis optica autoantibodies to aquaporin-4 M1/M23 isoforms and orthogonal arrays.
abstract: Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for the neuroinflammatory disease neuromyelitis optica (NMO). We measured the binding of NMO autoantibodies to AQP4 in human astrocyte-derived U87MG cells expressing M1 and/or M23 AQP4, or M23 mutants that do not form orthogonal array of particles (OAPs). Binding affinity was quantified by two-color fluorescence ratio imaging of cells stained with NMO serum or a recombinant monoclonal NMO autoantibody (NMO-rAb), together with a C terminus anti-AQP4 antibody. NMO-rAb titrations showed binding with dissociation constants down to 44 ± 7 nm. Different NMO-rAbs and NMO patient sera showed a wide variation in NMO-IgG binding to M1 versus M23 AQP4. Differences in binding affinity rather than stoichiometry accounted for M1 versus M23 binding specificity, with consistently greater affinity of NMO-IgG binding to M23 than M1 AQP4. Binding and OAP measurements in cells expressing different M1:M23 ratios or AQP4 mutants indicated that the differential binding of NMO-IgG to M1 versus M23 was due to OAP assembly rather than to differences in the M1 versus M23 N termini. Purified Fab fragments of NMO-IgG showed similar patterns of AQP4 isoform binding, indicating that structural changes in the AQP4 epitope upon array assembly, and not bivalent cross-linking of whole IgG, result in the greater binding affinity to OAPs. Our study establishes a quantitative assay of NMO-IgG binding to AQP4 and indicates remarkable, OAP-dependent heterogeneity in NMO autoantibody binding specificity.
authors: Department of Medicine, University of California, San Francisco, California 94143, USA.
authors: Jonathan M
authors: JM
authors: Crane
authors: Chiwah
authors: C
authors: Lam
authors: Andrea
authors: A
authors: Rossi
authors: Tripta
authors: T
authors: Gupta
authors: Jeffrey L
authors: JL
authors: Bennett
authors: A S
authors: AS
authors: Verkman
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D000918:Antibody Specificity; D051401:Aquaporin 4 / Q000235:genetics / Q000276:immunology*; D001323:Autoantibodies / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D009154:Mutation; D009471:Neuromyelitis Optica / Q000235:genetics / Q000276:immunology*
chemical_list: C496030:AQP4 protein, human; D000911:Antibodies, Monoclonal; D051401:Aquaporin 4; D001323:Autoantibodies; D007074:Immunoglobulin G