Neuromyelitis optica (NMO) is an inflammatory autoimmune demyelinating disease of the central nervous system (CNS). NMO autoantibodies (NMO-IgG) recognize the glial water channel Aquaporin-4 which exists as two major isoforms differing in the length of the N terminus, the shorter AQP4-M23 and the longer AQP4-M1. Both isoforms form tetramers, which further aggregate in the plasma membrane to form typical Orthogonal Arrays of Particles (OAPs). We recently demonstrated that NMO-IgG epitope is 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. In this study we map the NMO-IgG antigenic determinants in the OAP extracellular surface. To identify the AQP4-OAP extracellular epitope for NMO IgG, we generated a series of AQP4 mutants, based on multi-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 by immunofluorecence and immunoprecipitation. Results indicate that one group of sera was able to recognize a limited portion of loop C containing the amino acid sequence G146VTTV150. It is likely that this conformational epitope is generated by surface associations of the C loops among different tetramers. A second group of sera was characterized by a predominant role of the loop A. Deletion of four AQP4-specific amino acids (G61SEN64) 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 suggesting that loop C and E contribute together with loop A to generate the conformational epitope. Our data indicate that the NMO-IgG autoantibodies have a polyclonal origin and that the three AQP4 extracellular loops (A, C, and E) participate in the formation of the NMO-IgG epitope. This study identifies two major key immunodominant conformational epitopes and provides crucial information for the generation of a NMO disease model.

Identification of two major conformational AQP4 epitopes for neuromyelitis optica autoantibodies binding

PISANI, FRANCESCO;NICCHIA, GRAZIA PAOLA;SVELTO, Maria;A. FRIGERI
2010

Abstract

Neuromyelitis optica (NMO) is an inflammatory autoimmune demyelinating disease of the central nervous system (CNS). NMO autoantibodies (NMO-IgG) recognize the glial water channel Aquaporin-4 which exists as two major isoforms differing in the length of the N terminus, the shorter AQP4-M23 and the longer AQP4-M1. Both isoforms form tetramers, which further aggregate in the plasma membrane to form typical Orthogonal Arrays of Particles (OAPs). We recently demonstrated that NMO-IgG epitope is 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. In this study we map the NMO-IgG antigenic determinants in the OAP extracellular surface. To identify the AQP4-OAP extracellular epitope for NMO IgG, we generated a series of AQP4 mutants, based on multi-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 by immunofluorecence and immunoprecipitation. Results indicate that one group of sera was able to recognize a limited portion of loop C containing the amino acid sequence G146VTTV150. It is likely that this conformational epitope is generated by surface associations of the C loops among different tetramers. A second group of sera was characterized by a predominant role of the loop A. Deletion of four AQP4-specific amino acids (G61SEN64) 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 suggesting that loop C and E contribute together with loop A to generate the conformational epitope. Our data indicate that the NMO-IgG autoantibodies have a polyclonal origin and that the three AQP4 extracellular loops (A, C, and E) participate in the formation of the NMO-IgG epitope. This study identifies two major key immunodominant conformational epitopes and provides crucial information for the generation of a NMO disease model.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11586/37339
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