title: Role of the H-bond between L53 and T56 for Aquaporin-4 epitope in Neuromyelitis Optica.
abstract: Aquaporin-4 (AQP4) is the CNS water channel organized into well-ordered protein aggregates called Orthogonal Arrays of Particles (OAPs). Neuromyelitis Optica (NMO) is an autoimmune disease caused by anti-OAP autoantibodies (AQP4-IgG). Molecular Dynamics (MD) simulations have identified an H-bond between L53 and T56 as the key for AQP4 epitope and therefore of potential interest for drug design in NMO field. In the present study, we have experimentally tested this MD-prediction using the classic mutagenesis approach. We substituted T56 with V56 and tested this mutant for AQP4 aggregates and AQP4-IgG binding. gSTED super-resolution microscopy showed that the mutation does not affect AQP4 aggregate dimension; immunofluorescence and cytofluorimetric analysis demonstrated its unaltered AQP4-IgG binding, therefore invalidating the MD-prediction. We later investigated whether AQP4, expressed in Sf9 insect and HEK-293F cells, is able to correctly aggregate before and after the purification steps usually applied to obtain AQP4 crystal. The results demonstrated that AQP4-IgG recognizes AQP4 expressed in Sf9 and HEK-293F cells by immunofluorescence even though BN-PAGE analysis showed that AQP4 forms smaller aggregates when expressed in insect cells compared to mammalian cell lines. Notably, after AQP4 purification, from both insect and HEK-293F cells, no aggregates are detectable by BN-PAGE and AQP4-IgG binding is impaired in sandwich ELISA assays. All together these results indicate that 1) the MD prediction under analysis is not supported by experimental data and 2) the procedure to obtain AQP4 crystals might affect its native architecture and, as a consequence, MD simulations. In conclusion, given the complex nature of the AQP4 epitope, MD might not be the suitable for molecular medicine advances in NMO.
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Centre of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Pisani
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Centre of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, Italy; IRCCS "Casa Sollievo della Sofferenza", Research Hospital, San Giovanni Rotondo, Foggia, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Simone
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Centre of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, Italy.
authors.forename: Concetta Domenica
authors.initials: CD
authors.lastname: Gargano
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Centre of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: De Bellis
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Centre of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Cibelli
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Centre of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, Italy.
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Mola
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Centre of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, Italy.
authors.forename: Giacomo
authors.initials: G
authors.lastname: Catacchio
authors.affiliation: School of Medicine, Basic Medical Sciences, Neuroscience and Sense Organs, University of Bari "Aldo Moro", Bari, Italy; Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY, USA.
authors.forename: Antonio
authors.initials: A
authors.lastname: Frigeri
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Centre of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, Italy; Institute of Biomembranes and Bioenergetics, National Research Council, Bari, Italy; National Institute of Biostructures and Biosystems (INBB), Rome, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Svelto
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutics and Centre of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, Italy; Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY, USA. Electronic address: graziapaola.nicchia@uniba.it.
authors.forename: Grazia Paola
authors.initials: GP
authors.lastname: Nicchia
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D051401:Aquaporin 4 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006860:Hydrogen Bonding; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009471:Neuromyelitis Optica / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D051401:Aquaporin 4; D000939:Epitopes; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D008239:Lysine
keywords: AQP4-IgG; Aquaporin-4; Molecular dynamics; NMO; OAPs