title: Mutagenesis of the aquaporin 4 extracellular domains defines restricted binding patterns of pathogenic neuromyelitis optica IgG.
abstract: 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 the 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 we evaluated the effects on binding of NMO AQP4-reactive rAbs by quantitative immunofluorescence. Whereas all NMO rAbs required conserved loop C ((137)TP(138) and Val(150)) and loop E ((230)HW(231)) 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 ((148)TM(149) and His(151)) and loop E (Asn(226) and Glu(228)). Alternatively, pattern 2 NMO rAbs showed significantly reduced binding following amino acid changes in loop A ((63)EKP(65) and Asp(69)) and loop C (Val(141), His(151), and Leu(154)). Amino acid substitutions at (137)TP(138) 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.
authors: From the Departments of Neurology and.
authors: Gregory P
authors: GP
authors: Owens
authors: From the Departments of Neurology and.
authors: Alanna
authors: A
authors: Ritchie
authors: the Department III-Developmental Genetics, Max Planck Institute for Heart and Lung Research, Ludwigstrasse 43, 61231 Bad Nauheim, Germany, and.
authors: Andrea
authors: A
authors: Rossi
authors: From the Departments of Neurology and.
authors: Kristin
authors: K
authors: Schaller
authors: From the Departments of Neurology and.
authors: Scott
authors: S
authors: Wemlinger
authors: From the Departments of Neurology and.
authors: Hannah
authors: H
authors: Schumann
authors: From the Departments of Neurology and.
authors: Andrew
authors: A
authors: Shearer
authors: the Departments of Medicine and Physiology, University of California, San Francisco, San Francisco, California 94143.
authors: Alan S
authors: AS
authors: Verkman
authors: From the Departments of Neurology and Ophthalmology and Neuroscience Program, University of Colorado at Denver, Anschutz Medical Campus, Aurora, Colorado 80045, jeffrey.bennett@ucdenver.edu.
authors: Jeffrey L
authors: JL
authors: Bennett
mesh_terms: D000409:Alanine / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D051401:Aquaporin 4 / Q000737:chemistry*; D001323:Autoantibodies / Q000737:chemistry*; D016466:CHO Cells; D002469:Cell Separation; D006224:Cricetinae; D003412:Cricetulus; D004195:Disease Models, Animal; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry; D005434:Flow Cytometry; D005998:Glycine / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D016296:Mutagenesis; D009154:Mutation; D009471:Neuromyelitis Optica / Q000134:cerebrospinal fluid / Q000276:immunology*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: C496030:AQP4 protein, human; D000911:Antibodies, Monoclonal; D051401:Aquaporin 4; D001323:Autoantibodies; D000939:Epitopes; D007074:Immunoglobulin G; D000409:Alanine; D005998:Glycine
keywords: Aquaporin; Autoimmunity; Demyelination; Epitope Mapping; Monoclonal Antibody; Neuroimmunology; Neuromyelitis Optica