Analysing Human Aquaporin-4 Purified from P. pastoris in the Context of Neuromyelitis Optica Spectrum Disorder

Abstract

Aquaporin-4 (AQP4) is the predominant water channel in the central nervous system (CNS), controlling water homeostasis in the brain, spinal cord, and optic nerve. Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune disease in which antibodies attack the optic nerves and spinal cord. In the majority of cases, NMOSD is associated with anti-AQP4 autoantibodies (NMO-IgGs). NMO-IgGs bind preferentially to higher order oligomers of AQP4 tetramers, known as orthogonal arrays of particles (OAPs). Solubilisation and purification of membrane proteins (MPs) is challenging due to their large hydrophobic domains that lie within biological membranes. The styrene-maleic acid (SMA) co-polymer can form nanodisc-like lipid particles (SMALPs) from biological membranes, and can encapsulate MPs within a native annulus of lipid. Recently, mass photometry has been developed as an analytical technique for label-free measurement of the mass of single particles in solution. Mass photometry requires samples of low concentration (~10 nM) and low heterogeneity, making it applicable to the analysis of purified MP-SMALPs. This thesis presents recombinant expression of human AQP4 in Pichia pastoris, solubilisation of AQP4 with SMA, and purification of the AQP4-SMALPs for analysis with mass photometry amongst other biophysical characterisation techniques. This work showed that AQP4-SMALPs exist as a distribution of different nanodisc sizes after SMA solubilisation. These SMALPs of increasing size were hypothesised to contain different tetrameric assemblies of AQP4. When analysed by dot blotting, NMO-IgGs preferentially targeted purified AQP4-SMALPs predicted to contain large arrays of AQP4. Blue-native PAGE identified that hAQP4 was able to form higher-order oligomers in P. pastoris and, using a biochemical assay, hAQP4 was found to be palmitoylated by P. pastoris. This may represent a possible mechanism of OAP regulation in this host. These results suggest that AQP4 tetramers and OAPs can be captured and characterised in SMALPs from the P. pastoris membrane. P. pastoris can replicate features of hAQP4 that are observed in mammalian expression systems. Therefore, this could be the basis for a future model in understanding AQP4 oligomerisation in the context of NMOSD.