Identification of two major conformational aquaporin-4 epitopes for neuromyelitis optica autoantibody binding - PubMed (original) (raw)

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

Francesco Pisani et al. J Biol Chem. 2011.

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.

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Figures

FIGURE 1.

FIGURE 1.

NMO-IgG does not recognize non-OAP-forming M23-AQP4. AQP4 immunoblot was performed after BN-PAGE using M23-mCherry (A) and GFP-M23 (B) protein extracts. Note the presence of multiple bands only in sample extracts from M23-mCherry, indicating the expression of OAPs. Representative immunofluorescence analysis of NMO-IgG binding using M23-mCherry (C) and GFP-M23 cells (D) is shown. Expression of M23-mCherry (red, E) and GFP-M23 (green, F) in transiently transfected HeLa cells is shown. Inset in C shows high magnification of a transfected cells with dot staining. Scale bars, 1 μm and 15 μm for the others.

FIGURE 2.

FIGURE 2.

Comparison of the primary amino acid sequence of rat, mouse, and human AQP4 and AQP0 and of Cicadella AQP. Residues identical in all sequences are indicated by asterisk, and partially conserved amino acids are indicated by a double or single dot. The exposed extracellular loops are highlighted. The amino acid positions are referred to the AQP4-M1 isoform.

FIGURE 3.

FIGURE 3.

Schematic representation of mutations performed in AQP4 extracellular loops. Mutated amino acids, highlighted in red, are flanked by the mutant name and sequence. For more details see also Table 1.

FIGURE 4.

FIGURE 4.

Contribution of loop A, C, and E in the formation of NMO-IgG epitope. Immunofluorescence experiments performed on HeLa cells transfected with mutants are reported in Table 1. Anti-AQP4 antibody (AQP4) and NMO-1, NMO-2, and NMO-3 sera are shown. Scale bars, 20 μm.

FIGURE 5.

FIGURE 5.

Immunoprecipitation experiment to evaluate the loop A, C, and E contributions into the NMO-IgG epitopes. A, experiment performed with NMO-1, NMO-2, and NMO-3 sera in the AQP4 mutants described previously. Immunoprecipitated proteins were revealed with anti-AQP4 commercially available antibodies. AQP4 antibodies and multiple sclerosis (MS) sera were used as positive and negative control, respectively. B, densitometric analysis of the immunoprecipitated AQP4 described in A (n = 3).

FIGURE 6.

FIGURE 6.

Schematic model on how NMO epitopes could be associated to OAPs formation. Side and top views of an AQP4 tetramer (A) and OAPs are shown (B). A, extracellular loops interactions in the tetramer do not generate the NMO-IgG epitope. B, when AQP4 organizes in OAPs, the extracellular loops of each tetramer rearrange and create at least two different NMO-IgG epitopes.

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