Role of the H-bond between L53 and T56 for Aquaporin-4 epitope in Neuromyelitis Optica

Francesco Pisani, Laura Simone, Concetta Domenica Gargano, Manuela De Bellis, Antonio Cibelli, Maria Grazia Mola, Giacomo Catacchio, Antonio Frigeri, Maria Svelto, Grazia Paola Nicchia

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)368-376
Number of pages9
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1859
Issue number3
DOIs
Publication statusPublished - Mar 1 2017

Fingerprint

Aquaporin 4
Neuromyelitis Optica
Epitopes
Molecular Dynamics Simulation
Molecular dynamics
Immunoglobulin G
Insects
Purification
Fluorescent Antibody Technique
Molecular Medicine
Mutagenesis
Aquaporins
Crystals
Drug Design

Keywords

  • AQP4-IgG
  • Aquaporin-4
  • Molecular dynamics
  • NMO
  • OAPs

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

Cite this

Role of the H-bond between L53 and T56 for Aquaporin-4 epitope in Neuromyelitis Optica. / Pisani, Francesco; Simone, Laura; Gargano, Concetta Domenica; De Bellis, Manuela; Cibelli, Antonio; Mola, Maria Grazia; Catacchio, Giacomo; Frigeri, Antonio; Svelto, Maria; Nicchia, Grazia Paola.

In: Biochimica et Biophysica Acta - Biomembranes, Vol. 1859, No. 3, 01.03.2017, p. 368-376.

Research output: Contribution to journalArticle

Pisani, F, Simone, L, Gargano, CD, De Bellis, M, Cibelli, A, Mola, MG, Catacchio, G, Frigeri, A, Svelto, M & Nicchia, GP 2017, 'Role of the H-bond between L53 and T56 for Aquaporin-4 epitope in Neuromyelitis Optica', Biochimica et Biophysica Acta - Biomembranes, vol. 1859, no. 3, pp. 368-376. https://doi.org/10.1016/j.bbamem.2016.12.018
Pisani, Francesco ; Simone, Laura ; Gargano, Concetta Domenica ; De Bellis, Manuela ; Cibelli, Antonio ; Mola, Maria Grazia ; Catacchio, Giacomo ; Frigeri, Antonio ; Svelto, Maria ; Nicchia, Grazia Paola. / Role of the H-bond between L53 and T56 for Aquaporin-4 epitope in Neuromyelitis Optica. In: Biochimica et Biophysica Acta - Biomembranes. 2017 ; Vol. 1859, No. 3. pp. 368-376.
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AU - De Bellis, Manuela

AU - Cibelli, Antonio

AU - Mola, Maria Grazia

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