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.

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.

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

Mola, Maria Grazia;
2019-01-01

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.
2019
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.
aquaporin-4
OAPs isolation
OAPs structure
neuromyelitis optica
Animals
Aquaporin 4
Cell Line
Cell Membrane
Humans
Immunoglobulin G
Insecta
Mammals
Protein Binding
Protein Transport
Rats
Recombinant Proteins
Structure-Activity Relationship
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12572/20626
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