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

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

doi:10.3390/cells8020119

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.

Highlights

Aquaporin-4 (AQP4) is the main astrocytic cell-membrane water channel of the central nervous system (CNS) where it contributes to the maintenance of water-ion homeostasis, to the functionality of the blood-brain-barrier and seems to play a role in the glymphatic pathway [1,2,3,4]
The OAP-forming human M23-AQP4 was expressed in Sf9 insect cells, in human embryonic kidney HEK-FS cells and in astrocyte DI TNC1 cells
Confocal analysis of stack section showed a weak intracellular staining in mammalian cells compared to that observed in insect cells (Figure 1a)

Summary

Introduction

Aquaporin-4 (AQP4) is the main astrocytic cell-membrane water channel of the central nervous system (CNS) where it contributes to the maintenance of water-ion homeostasis, to the functionality of the blood-brain-barrier and seems to play a role in the glymphatic pathway [1,2,3,4]. AQP4 monomers, similar to those of other aquaporins are organized in tetramers, but AQP4 heterotetramers are able to further aggregate in supramolecular assemblies, known as orthogonal arrays of particles (OAPs). OAP organization confers specific AQP4 localization [10,11,12,13], tissue-distribution [14,15] and is involved in some pathological processes [16,17,18,19]. The physiological role of OAPs is unclear and little is known about the molecular basis of OAP assembly and stability. OAP assembly appears to be a plasma membrane process that involves not yet well-identified cell-membrane-specific factors [22]. OAPs have been visualized and analyzed at nanometrical resolution with super-resolution microscopy approaches [6,10,23,24], sub-nanometrical resolution is required to deeply investigate the molecular properties of OAPs

Methods

Results

Conclusion