Aim: 

Neuromyelitis optica (NMO) is a devastating autoimmune demyelinating disease, associated with the presence of anti-AQP4 water channel auto-antibodies. The pathogenic effect of the autoantibody is probably due to complement activation and AQP4 internalization, contributing to astrocyte injury and breakdown of the blood brain barrier. Our recent data show that the NMO-IgG, bnding an extracellular epitope, selectively recognizes AQP4 only when this protein organizes in the plasma membrane as a supermolecular structure called orthogonal array of particles (OAPs). However, up to now, the epitope structure remains unknown.

Methods: 

In this study a site-specific mutagenesis approach was used to mutate AQP4 in the A, C and E loops, separately, and in the C and E loops, simultaneously. These mutants were transiently expressed in HeLa cells and the level of the NMO-IgG binding capacity tested by immunofluorescence (IF) and immunoprecipitation (IP) experiments. Eight AQP4 positive NMO patient sera were used individually.

Results: 

Both IF and the IP experiments demonstrated that the epitope is not linear but it is instead a conformational epitope formed by the interaction of the three extracellular loops A, C and E being the loops A and C key in the structure. Interestingly, these interactions are generated only when AQP4 tetramers associate in OAPs. Furthermore we found, among tested sera, that two classes of antibodies with different binding sites on the extracellular loops, can be identified.

Conclusion: 

Our data indicate that the NMO-IgG epitope has a very complex and unhomogeneous structure that need to be taken into account in further characterization studies.