title: Characterization of the binding pattern of human aquaporin-4 autoantibodies in patients with neuromyelitis optica spectrum disorders.
abstract: The discovery of a highly specific antibody against the aquaporin-4 (AQP4) water channel (AQP4-IgG) unified the spectrum of neuromyelitis optica spectrum disorders (NMOSD), which are considered to be antibody-mediated autoimmune diseases. The AQP4 water channel is located on astrocytic end-feet processes and consists of six transmembrane helical domains forming three extracellular loops A, C, and E in which defined amino acids were already proven to be critical for AQP4-IgG binding. However, the clinical relevance of these findings is unclear. Therefore, we have characterized the epitope specificity of AQP4-IgG-positive NMOSD patients.
  
  
  
  We established a cell-based flow cytometry assay for the quantitative detection of AQP4-IgG-positive serum samples. Human embryonic kidney (HEK) cells were transiently transfected with an EmGFP-tagged AQP4-M23, AQP4-M1, or six AQP4-M23 extracellular loop mutants including two mutations in loop A (serial AA substitution, insertion of a myc-tag), two in loop C (N153Q, insertion of a myc-tag), and two in loop E (H230G, insertion of a myc-tag). Fourty-seven baseline and 49 follow-up serum samples and six paired cerebrospinal fluid (CSF) baseline samples of 47 AQP4-IgG-positive Austrian NMOSD patients were then tested for their binding capability to AQP4-M1 and AQP4-M23 isoforms and these six extracellular loop mutants.
  
  
  
  Overall, we could identify two broad patterns of antibody recognition based on differential sensitivity to mutations in extracellular loop A. Pattern A was characterized by reduced binding to the two mutations in loop A, whereas pattern B had only partial or no reduced binding to these mutations. These two patterns were not associated with significant differences in demographic and clinical parameters or serum titers in this retrospective study. Interestingly, we found a change of AQP4-IgG epitope recognition pattern in seven of 20 NMOSD patients with available follow-up samples. Moreover, we found different binding patterns in five of six paired CSF versus serum samples, with a predominance of pattern A in CSF.
  
  
  
  Our study demonstrates that AQP4-IgG in sera of NMOSD patients show distinct patterns of antibody recognition. The clinical and diagnostic relevance of these findings have to be addressed in prospective studies.
authors.affiliation: Clinical Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria.
authors.forename: Friederike
authors.initials: F
authors.lastname: Tuller
authors.affiliation: Clinical Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria.
authors.forename: Hannah
authors.initials: H
authors.lastname: Holzer
authors.affiliation: Clinical Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Schanda
authors.affiliation: Department of Neurology, Karl Landsteiner Institute for Neuroimmunological and Neurodegenerative Disorders, Sozialmedizinisches Zentrum Ost Donauspital, Vienna, Austria.
authors.forename: Fahmy
authors.initials: F
authors.lastname: Aboulenein-Djamshidian
authors.affiliation: Institute of Neurology, Medical University of Vienna, Vienna, Austria.
authors.forename: Romana
authors.initials: R
authors.lastname: Höftberger
authors.affiliation: Department of Neurology, Medical University of Graz, Graz, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Khalil
authors.affiliation: Department of Neurology, Medical University of Graz, Graz, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Seifert-Held
authors.affiliation: Department of Neurology, Medical University of Vienna, Vienna, Austria.
authors.forename: Fritz
authors.initials: F
authors.lastname: Leutmezer
authors.affiliation: Clinical Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Berger
authors.affiliation: Clinical Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria. Markus.Reindl@i-med.ac.at.
authors.forename: Markus
authors.identifier: 0000-0003-2817-1402
authors.initials: M
authors.lastname: Reindl
mesh_terms: D000328:Adult; D000368:Aged; D051401:Aquaporin 4 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001323:Autoantibodies / Q000097:blood*; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D009471:Neuromyelitis Optica / Q000097:blood* / Q000276:immunology; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D000072417:Protein Domains / Q000235:genetics; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D014162:Transfection
chemical_list: C496030:AQP4 protein, human; D051401:Aquaporin 4; D001323:Autoantibodies; D000939:Epitopes; D020033:Protein Isoforms
keywords: Aquaporin-4; Autoantibodies; Epitope specificity; Flow cytometry; Neuromyelitis optica spectrum disorders