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Carla O'Neale, Hayes McDonald, Irene Zagol-Ikapitte, Sophie R. Harvey, Vicki H. Wysocki, Kevin L Schey; Studies of Native AQP0 Structure and Protein Interactions. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2064.
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© ARVO (1962-2015); The Authors (2016-present)
The lens microcirculation system is required to maintain transparency; however, how this system is established and maintained as a function of age is not well understood. Aquaporin-0 (AQP0) plays important roles in lens fiber cell adhesion and water permeability. The purpose of this study is to structurally characterize AQP0 through Native mass spectrometry (Native MS) and to develop a method to analyze AQP0-protein interactions via hydrogen deuterium exchange-mass spectrometry (HDX-MS) using AQP0-calmodulin interactions as a model.
Bovine lens membranes were solubilized in 1% octylglucoside and AQP0 was purified via anion exchange chromatography. HDX-MS: After incubation in D2O, the HDX process was quenched, proteins were digested using pepsin, and resulting peptides separated and analyzed via liquid-chromatography mass spectrometry on a Waters Xevo G2-XS QTof. Data were analyzed using Waters PLGS and DynamX software. Native MS: Purified AQP0 was buffer exchanged into C8E4 detergent in 200 mM ammonium acetate, introduced into a Thermo Exactive Plus EMR Orbitrap or a Thermo Q Exactive Plus UHMR mass spectrometer using nanoelectrospray ionization and dissociated by collision induced dissociation (CID) or by surface induced dissociation (SID).
HDX-MS: After optimization, significant sequence coverage was obtained for both AQP0 (75%) and calmodulin (90%) that includes coverage of the C-terminal tail of AQP0. As expected, AQP0 loop and C-terminal tail peptides displayed greater deuterium incorporation relative to transmembrane regions. Native MS: Multiple AQP0 proteoforms of intact tetrameric AQP0 were identified in isolated AQP0 fractions including phosphorylated and lipidated AQP0. Chromatographic separation allowed enrichment of un-, mono-, and di-phosphorylated forms.
HDX-MS methodology was optimized to yield good sequence coverage for lens membrane protein AQP0 which sets the stage for examining AQP0-protein interactions. Deuterium uptake data suggest AQP0 loop and C-terminal tail regions to be dynamic and solvent exposed whereas transmembrane regions remain largely solvent protected. Multiple proteoforms of tetrameric AQP0 were identified via Native MS which will allow us to compare unphosphorylated and phosphorylated AQP0 to understand the effects of phosphorylation on AQP0 function. Native MS data show di-phosphorylation to be the highest degree of phosphorylation of intact tetrameric AQP0.
This is a 2021 ARVO Annual Meeting abstract.
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