Identification of two major conformational aquaporin-4 epitopes for neuromyelitis optica autoantibody binding.

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.

Aquaporin-4 orthogonal arrays of particles are the target for neuromyelitis optica autoantibodies.

Neuromyelitis optica (NMO) is an inflammatory autoimmune demyelinating disease of the central nervous system (CNS) which in autoantibodies produced by patients with NMO (NMO-IgG) recognize a glial water channel protein, Aquaporin-4 (AQP4) expressed as two major isoforms, M1- and M23-AQP4, in which the plasma membrane form orthogonal arrays of particles (OAPs). AQP4-M23 is the OAP-forming isoform, whereas AQP4-M1 alone is unable to form OAPs. The function of AQP4 organization into OAPs in normal physiology is unknown; however, alteration in OAP assemblies is reported for several CNS pathological states. In this study, we demonstrate that in the CNS, NMO-IgG is able to pull down both M1- and M23-AQP4 but experiments performed using cells selectively transfected with M1- or M23-AQP4 and native tissues show NMO-IgG epitope to be intrinsic in AQP4 assemblies into OAPs. Other OAP-forming water-channel proteins, such as the lens Aquaporin-0 and the insect Aquaporin-cic, were not recognized by NMO-IgG, indicating an epitope characteristic of AQP4-OAPs. Finally, water transport measurements show that NMO-IgG treatment does not significantly affect AQP4 function. In conclusion, our results suggest for the first time that OAP assemblies are required for NMO-IgG to recognize AQP4.

Aquaporin-4 expression is severely reduced in human sarcoglycanopathies and dysferlinopathies.

Aquaporin-4 (AQP4) is the major water channel expressed in fast-twitch skeletal muscle fibers. AQP4 is reduced in Duchenne and Becker Muscular Dystrophies, but not in caveolinopathies, thus suggesting an interaction with dystrophin or with members of the dystrophin-glycoprotein complex (DGC) rather than a nonspecific effect due to muscle membrane damage. To establish the role of sarcoglycans in AQP4 decrease occurring in muscular dystrophy, AQP4 expression was analyzed in muscle biopsies from patients affected by Limb Girdle Muscular Dystrophies (LGMDs) 2C-F genetically confirmed. In all the LGMD 2C-F (2alpha-, 1beta-, 2gamma-, 1delta-deficiency), AQP4 was severely decreased. This effect was associated to a marked reduction in alpha1-syntrophin levels. In control muscle AQP4 did not show a direct interaction with any of the four sarcoglycans but, it co-immunoprecipitated with alpha1-syntrophin, indicating that this modular protein may link AQP4 levels with the DGC complex. To determine whether AQP4 expression could be affected in other LGMDs due to the defect of a membrane protein not associated to the dystrophin complex, we examined AQP4 expression in 6 patients affected by dysferlin deficiency genetically confirmed. All the patients displayed a reduction of the water channel, and AQP4 expression appeared to correlate with the severity of the muscle histopathological lesions. However, differently from what observed in the sarcoglycans, alpha1-syntrophin expression was normal or just slightly reduced. These results seem to indicate an additional mechanism of regulation of AQP4 levels in muscle cells. In accordance with a specific effect of membrane muscle disorders, AQP4 protein levels were not changed in 3 mitochondrial and 3 metabolic myopathies. In conclusion, AQP4 expression and membrane localization are markedly reduced in LGMD 2B-2F. The role of AQP4 in the degenerative mechanism occurring in these diseases will be the object of our future research.