Host-Cell Type Dependent Features of Recombinant Human Aquaporin-4 Orthogonal Arrays of Particles-New Insights for Structural and Functional Studies

Francesco Pisani, Laura Simone, Maria Grazia Mola, Manuela De Bellis, Maria Mastrapasqua, Maddalena Ruggieri, Maria Trojano, Grazia Paola Nicchia, Maria Svelto, Antonio Frigeri

Research output: Contribution to journalArticlepeer-review

Abstract

The CNS plasma-membrane water channel aquaporin-4 (AQP4) is expressed as two major isoforms able to aggregate into supramolecular assemblies known as 'orthogonal arrays of particles' (OAPs). OAP subnanometric features are largely unknown mainly because a method for the expression, isolation, and crystallization of integral human OAPs has not been developed. Here, the human OAP-forming isoform M23-AQP4 was expressed in insect and mammalian cell lines and AQP4 and OAP features evaluated. Native size exclusion chromatography was employed to isolate and analyze authentically folded OAPs, and neuromyelitis optica (NMO)-specific sandwich ELISA was developed to test OAP-integrity. The results demonstrate that in insect cells most AQP4 remains intracellular and unfolded and that OAPs are largely disassembled after the detergent extraction step. In mammalian cells, AQP4 showed regular plasma membrane targeting and OAPs exhibited strong post-extraction stability. Starting from the mammalian cell expression system, we isolated authentically folded OAPs. Together these data suggest a new strategy for expressing and isolating integral recombinant human OAPs and providing new insights into the cell-type dependent OAP-assembly and post-extraction stability, potentially useful to design new approaches for structural and functional studies of OAP and for other plasma membrane proteins organized into supramolecular structures.

Original languageEnglish
JournalCells
Volume8
Issue number2
DOIs
Publication statusPublished - Feb 2 2019

Keywords

  • Animals
  • Aquaporin 4/chemistry
  • Cell Line
  • Cell Membrane/metabolism
  • Humans
  • Immunoglobulin G/metabolism
  • Insecta
  • Mammals
  • Protein Binding
  • Protein Transport
  • Rats
  • Recombinant Proteins/chemistry
  • Structure-Activity Relationship

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