Mutagenesis of the Aquaporin 4 Extracellular Domains Defines Restricted Binding Patterns of Pathogenic Neuromyelitis Optica IgG [Immunology]

March 19th, 2015 by Owens, G. P., Ritchie, A., Rossi, A., Schaller, K., Wemlinger, S., Schumann, H., Shearer, A., Verkman, A. S., Bennett, J. L.

Neuromyelitis optica immunoglobulin G (NMO-IgG) binds to aquaporin-4 (AQP4) water channels in the central nervous system leading to immune-mediated injury. We have previously demonstrated that a high proportion of CSF plasma cells of NMO patients produce antibody to extracellular domains of the AQP4 protein and that recombinant IgG (rAb) derived from these cells recapitulate pathogenic features of disease. We performed a comprehensive mutational analysis of the three extracellular loops of the M23 isoform of human AQP4 using both serial and single point mutations and evaluated the effects on binding of NMO AQP4-reactive rAbs by quantitative immunofluorescence. Whereas all NMO rAbs required conserved loop C (137TP138 and 150V) and loop E (230HW231) amino acids for binding, two broad patterns of NMO-IgG recognition could be distinguished based on differential sensitivity to loop A amino acid changes. Pattern 1 NMO rAbs were insensitive to loop A mutations and could be further discriminated by differential sensitivity to amino acid changes in loop C (148TM149 and 151H) and loop E (226N and 228E). Alternatively, Pattern 2 NMO rAbs showed significantly reduced binding following amino acid changes in loop A (63EKP65 and 69D) and loop C (141V, 151H, and 154L). Amino acid substitutions at 137TP138 altered loop C conformation and abolished the binding of all NMO rAbs and NMO-IgG, indicating the global importance of loop C conformation to the recognition of AQP4 by pathogenic NMO Abs. The generation of human NMO rAbs has allowed the first high resolution mapping of extracellular loop amino acids critical for NMO-IgG binding and identified regions of AQP4 extracellular structure that may represent prime targets for drug therapy.