title: Identification of two major conformational aquaporin-4 epitopes for neuromyelitis optica autoantibody binding.
abstract: Neuromyelitis optica (NMO) is an autoimmune demyelinating disease characterized by the presence of anti-aquaporin-4 (AQP4) antibodies in the patient sera. We recently reported that these autoantibodies are able to bind AQP4 when organized in the supramolecular structure called the orthogonal array of particles (OAP). To map the antigenic determinants, we produced a series of AQP4 mutants based on multiple alignment sequence analysis between AQP4 and other OAP-forming AQPs. Mutations were introduced in the three extracellular loops (A, C, and E), and the binding capacity of NMO sera was tested on AQP4 mutants. Results indicate that one group of sera was able to recognize a limited portion of loop C containing the amino acid sequence (146)GVT(T/M)V(150). A second group of sera was characterized by a predominant role of loop A. Deletion of four AQP4-specific amino acids ((61)G(S/T)E(N/K)(64)) in loop A substantially affected the binding of this group of sera. However, the binding capacity was further reduced when amino acids in loop A were mutated together with those in loop E or when those in loop C were mutated in combination with loop E. Finally, a series of AQP0 mutants were produced in which the extracellular loops were progressively changed to make them identical to AQP4. Results showed that none of the mutants was able to reproduce in AQP0 the NMO-IgG epitopes, indicating that the extracellular loop sequence by itself was not sufficient to determine the rearrangement required to create the epitopes. Although our data highlight the complexity of the disease, this study identifies key immunodominant epitopes and provides direct evidence that the transition from AQP4 tetramers to AQP4-OAPs involves conformational changes of the extracellular loops.
authors.affiliation: Department of General and Environmental Physiology, Centre of Excellence in Comparative Genomics, University of Bari, I-70126 Bari, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Pisani
authors.forename: Mauro
authors.initials: M
authors.lastname: Mastrototaro
authors.forename: Andrea
authors.initials: A
authors.lastname: Rossi
authors.forename: Grazia Paola
authors.initials: GP
authors.lastname: Nicchia
authors.forename: Carla
authors.initials: C
authors.lastname: Tortorella
authors.forename: Maddalena
authors.initials: M
authors.lastname: Ruggieri
authors.forename: Maria
authors.initials: M
authors.lastname: Trojano
authors.forename: Antonio
authors.initials: A
authors.lastname: Frigeri
authors.forename: Maria
authors.initials: M
authors.lastname: Svelto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D051401:Aquaporin 4 / Q000235:genetics / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D006367:HeLa Cells; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D051379:Mice; D009471:Neuromyelitis Optica / Q000235:genetics / Q000276:immunology*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D051381:Rats; D017384:Sequence Deletion
chemical_list: C496030:AQP4 protein, human; C496031:Aqp4 protein, mouse; C496032:Aqp4 protein, rat; D051401:Aquaporin 4; D001323:Autoantibodies; D000939:Epitopes; D007074:Immunoglobulin G