Identification of Two Major Conformational Aquaporin-4 Epitopes for Neuromyelitis Optica Autoantibody Binding

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. NMO2 is a devastating autoimmune demyelinating disease, affecting primarily young women, and is associated with NMOIgG antibodies detectable in the patient serum (1-6). Immunofluorescence using AQP4 null mice and AQP4-transfected cells (2) has amply demonstrated that the target of these autoantibodies is aquaporin-4 (AQP4), a water channel protein abundantly expressed in astrocyte end-foot near capillaries (7,8). 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 (S100␤), anti-cleavage polyadenylation specificity factor (CPSF-73), and anti-RING finger protein 141 (RNF-141) (2, 9 -12). These other autoantibodies bind extra-or intracellular antigens liberated from dead cells and do not seem to have a pathogenic role in the NMO pathology (13). 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. These mechanisms include AQP4 internalization, an alteration of water and glutamate homeostasis, activation of antibody-dependent, cell-mediated cytotoxicity, and...

Identification of a Point Mutation Impairing the Binding between Aquaporin-4 and Neuromyelitis Optica Autoantibodies

“…40 NMO sera samples were used. 21 of these sera were categorized, as reported (10), and called NMO-1 (sera: 2, 3, 5, 8, 15, 17, and 19), NMO-2 (sera: 1, 4, 10, 13, 14, 18, and 21), and NMO-3 (sera: 6, 7, 9, 11, 12, 16, and 20), based on the epitope recognized (Table 3). NMO-1 were those sera showing a predominant role of loop A (residues 61-64), NMO-2 of the tip of the loop C (residues 146 -150), and NMO-3 had an equal role as loop A and the tip of the loop.…”

“…Mutagenesis-The cloning of human AQP4-M23 and AQP0 coding DNA sequences (CDSs) into pTarget Expression Vector (Promega) and human AQP4-M23 into pmCherry-N1 (Clontech) was performed as previously reported (10). AQP0 CDS cloned into pTarget was used for AQP4-AQP0 chimera construction.…”

“…Purification Blue Native-PAGE-Blue Native-PAGE was performed as previously reported (10). Briefly, cells were lysed into 7-10 volumes of BN buffer (BN buffer: 1% Triton X-100, 12 mM NaCl, 500 mM 6-aminohexanoic acid, 20 mM BisTris, pH 7.0, 2 mM EDTA, 10% glycerol), with added protease inhibitor mixture (Roche Diagnostic Gmbh, Mannheim, Germany) on ice vortexing every 10 min for 1 h and centrifuged at 22,000 ϫ g for 30 min at 4°C.…”

“…Moreover, AQP4 epitopes may differ between patients. In particular, there are two major conformational extracellular epitopes, generated by OAP-specific inter-tetrameric interaction of extracellular loops A, C, and E (10). Mutations in loop A, or loop C, combined with loop E, reduce NMO-IgG binding, suggesting a key role of OAP-specific interactions at the level of extracellular loops (10).…”

“…In particular, there are two major conformational extracellular epitopes, generated by OAP-specific inter-tetrameric interaction of extracellular loops A, C, and E (10). Mutations in loop A, or loop C, combined with loop E, reduce NMO-IgG binding, suggesting a key role of OAP-specific interactions at the level of extracellular loops (10). It has been reported that the interaction between NMO-IgG and AQP4-OAPs induces pathogenic mechanisms in NMO by complement-dependent cytotoxicity (CDC), and/or by antibody-dependent cellular cytotoxicity (11)(12)(13)(14)(15).…”