The Study of Lens AQP0-Protein and -Lipid Interactions using Advanced Mass Spectrometry Methods

Abstract

The ocular lens is an avascular tissue that generates an internal microcirculation system that delivers nutrients, regulates lens homeostasis, and is fundamental for lens transparency and lens function. Lens membrane aquaporins are water permeable channel proteins that participate in the generation and regulation of the lens microcirculation system (MCS). The most abundant aquaporin in the lens is aquaporin-0 (AQP0) which, in addition to being a water channel, functions as a cell-adhesion molecule. Given the important role of AQP0 in lens transparency and cataract development, understanding how proteins regulate AQP0 structure and function is critical to understanding its role in the MCS. Furthermore, the lipid composition surrounding integral membrane proteins has been shown to affect membrane protein structure, function, and stability, yet prior to this work, interactions with native lens lipids had not been elucidated for AQP0. This dissertation focused on characterizing full-length AQP0-protein and AQP0-lipid interactions using crosslinking-mass spectrometry (XL-MS), hydrogen-deuterium exchange mass spectrometry (HDX-MS) and native mass spectrometry (nMS). Through XL-MS, specific regions of interaction were elucidated for several AQP0 interacting partners including phakinin, α-crystallin, connexin-46, and connexin-50 and, two new interacting partners, vimentin and connexin-46, were identified. Through nMS, a variety of endogenous lens lipids, i.e., phosphatidylcholines (PCs) and sphingomyelins (SMs) were found to differentially bind AQP0 in a regionally dependent manner (lens cortex vs nucleus) suggesting that the native lipid environment surrounding AQP0 regulates its function differentially throughout the lens. Since both proteins and lipids regulate AQP0, the specific lens proteins and lipids found in this work to interact with AQP0 can inform us on how these interactions impact AQP0 structure and function in the context of the MCS. My dissertation research advances the lens field by demonstrating how and where proteins and lipids interact with AQP0 in the lens and provides a framework for the development of therapeutics and/or practices that could help delay or prevent the onset of cataracts. Furthermore, the mass spectrometry techniques, methods, and sample preparation workflows established in my studies of AQP0 could be of particular use to membrane protein structural biologists.