Identification of Two Major Conformational Aquaporin-4 Epitopes for Neuromyelitis Optica Autoantibody Binding (1)
Neuromyelitis optica (NMO) is an autoimmune demyelinating disease characterized by the presence of anti-aquaporin-4 (AQP4) antibodies in the patient sera. Autoantibodies against AQP4 (IgG) are found in about 75% of NMO patients, together with other autoantibodies for other proteins, including anti-myelin oligodendrocyte glycoprotein, anti-myelin basic protein, anti-S100 calcium-binding protein B (S100β), anti-cleavage polyadenylation specificity factor (CPSF-73), and anti-RING finger protein 141 (RNF-141). Anti-AQP4 IgG may act through the activation of multiple potentially neuropathogenic mechanisms contributing to injury to astrocytes and to the breakdown of the blood-brain barrier.
Cells expressing AQP4-M23 with an N-terminal GFP tag (GFP-M23) and AQP4-M23 with a C-terminal mCherry tag (M23-mCherry) were subjected to BN-PAGE analysis. The presence of several distinct bands, likely corresponding to AQP4-OAPs of different sizes, was found only in M23-mCherry-expressing cells, whereas GFP-M23 showed no significant levels of higher order structures. Immunofluorescence experiments showed that the NMO sera labeled M23-mCherry transfected cells with a typical punctated staining, but no staining was observed in the GFP-M23 cells.
An alignment analysis of AQP0 and AQPcic and human, rat, and mouse AQP4 primary amino acid sequences was performed. An amino acid sequence specific for AQP4 in the first extracellular loop (loop A, amino acids 60–68) between residues 61 and 64. This sequence was absent in AQP0 and in AQPcic as well as in all other human AQPs. Other differences were found within loops C (amino acids from 137 to 156) and E (amino acids from 225 to 230).
A series of rat AQP4 mutants where AQP4 specific extracellular sequences in loops A, C, and E were replaced with the corresponding sequences of AQP0 was generated.
Immunofluorescence experiments showed nearly complete absence of a fluorescence signal for the NMO-1 and NMO-3 sera and a faint reduction for the NMO-2 sera. A strong reduction of the staining was observed for NMO-2, and a complete absence of the staining for NMO-3 sera was found in G146–150 mutants. No staining alteration was found for NMO-1 sera. The mutant G152–157 showed a weak reduction of the fluorescence signal for NMO-1 and NMO-3 but not for NMO-2 sera. The contribution of the E loop was evaluated using the mutant W227–228. Using W227–228, a slight reduction of fluorescence signal was observed for NMO-1 and -2, although for NMO-3 it was found to be more pronounced.
Immunoprecipitation experiments showed that Similar results were obtained when the human sequence Gly61–Lys64 was deleted or when the two conserved AQP4 amino acids Gly61 and Glu63 were both mutated in lysine. These results indicate a central role of the additional sequence in the AQP4 loop A in the NMO-binding site. All these results were confirmed in the corresponding immunofluorescence experiments.
From the immunofluorescence and immunoprecipitation results, a group of sera (NMO-2) was able to recognize a specific portion of loop C (CA) including G146–150 region corresponds to the tip of the loop C. All the other mutants did not significantly affect the binding capacity of the NMO-2 group, suggesting the NMO-2 epitope is generated by pair associations, among tetramers, of loop C. The deletion of the four AQP4-specific amino acids (Δ61–64) substantially affected the binding of this NMO-1 group of sera. Graber et al. have recently tried to create an NMO animal model by injecting AQP4 linear peptides of the loop E (206–231) and C (272–291). These animals possessed high antibody titers able to bind the M1 and M23 isoforms, but they failed to induce any specific NMO symptoms.
