Binding Affinity and Specificity of Neuromyelitis Optica Autoantibodies to Aquaporin-4 M1/M23 Isoforms and Orthogonal Arrays

• Published in: The Journal of biological chemistry Vol. 286; no. 18; pp. 16516 - 16524
• Main Authors: Crane, Jonathan M., Lam, Chiwah, Rossi, Andrea, Gupta, Tripta, Bennett, Jeffrey L., Verkman, A.S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.05.2011; American Society for Biochemistry and Molecular Biology
• Subjects: Antibodies; Antibodies, Monoclonal - immunology; Antibody Affinity; Antibody Specificity; AQP4; Aquaporin; Aquaporin 4 - genetics; Aquaporin 4 - immunology; Autoantibodies - genetics; Autoantibodies - immunology; Biophysics; Cell Line; Fluorescence; Humans; Immunoglobulin G - immunology; Membrane Biology; Microscopic Imaging; Mutation; Neuromyelitis Optica; Neuromyelitis Optica - genetics; Neuromyelitis Optica - immunology; NMO; Water Channel; NMO; Neuromyelitis Optica; Antibodies; Fluorescence; AQP4; Biophysics; Microscopic Imaging; Water Channel; Aquaporin
• DOI: 10.1074/jbc.M111.227298
• ISSN: 0021-9258

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.