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Neuromyelitis optica 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 Orthogonal Array of Particles (OAP). To map the antigenic determinants, we produced a series of AQP4 mutants based on multi-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 G146VTTV150. A second group of sera was characterized by a predominant role of the loop A. Deletion of four AQP4-specific amino acids (G61 S/TEN/K64) in loop A substantially affected the binding of this group of sera. However, 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, 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 loops sequence by itself is 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 provide direct evidence that the transition from AQP4 tetramers to AQP4-OAPs involves conformational changes of the extracellular loops.

Read More: Identification of two major conformational AQP4 epitopes for neuromyelitis optica autoantibodies binding.