Abstract
Purpose :
The water channel function of Aquaporin-0 (AQP0) can be altered by the plasma membrane lipid environment and age-related modifications may alter the protein structure. The purpose of this work is to use Native MS and Hydrogen-Deuterium Exchange (HDX) MS to examine AQP0 structure and lipid binding as a function of fiber cell age.
Methods :
Bovine and rabbit AQP0 were purified by anion exchange chromatography from lenses dissected into cortex and nucleus regions. Native MS samples were concentrated and buffer exchanged into C8E4 detergent in 200 mM ammonium acetate. Samples were introduced into a Q Exactive plus EMR mass spectrometer using nanoelectrospray ionization. AQP0 complexes were dissociated by collision induced dissociation (CID) or by surface induced dissociation (SID). AQP0 was prepared for HDX experiments by placing purified AQP0 in 1% octylglucoside into D2O for varying times. Pepsin digestion was performed offline or on-line via a pepsin column. HDX analysis was performed on a Waters Xevo G2-XS instrument and data were analyzed using Waters DynamX software.
Results :
Intact AQP0 tetramers were detected in protein solubilized in C8E4 detergent. Dissociation of the tetramer produced monomers and trimers, confirming the parent signal identity as the AQP0 tetramer. We observed non-phosphorylated, singly phosphorylated and doubly phosphorylated forms of tetrameric AQP0. CID spectra indicated that the tetramer is composed of only non-phosphorylated and singly-phosphorylated monomers. Also, we observed truncated forms of AQP0 depending on the region of the lens from which the protein was isolated. Lipidated AQP0 signals were also detected; consistent with previous reports of covalently bound lipids. An additional series of signals, attributed to endogenous non-covalent lipid binding, was observed with the primary lipid bound identified as a C16:0 phosphatidylcholine lipid.
HDX analysis of purified AQP0 revealed solvent exposed regions that include intracellular and extracellular loops as well as the C-terminal tail. Such data can be used as a basis for examining AQP0-protein interaction sites and AQP0 conformational changes.
Conclusions :
Our results indicate that Native and HDX MS are useful tools to study AQP0 quaternary structure, endogenous lipid binding, surface into exposed regions, and protein-protein interactions of this integral membrane protein.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.