MALDI Imaging Mass Spectrometry Spatially Maps Age-Related Deamidation and Truncation of Human Lens Aquaporin-0.

The ocular lens is a transparent optical element, focusing light onto the retina for clear vision. Tight cellular packing, lack of light-scattering organelles, and a careful balance of protein and water concentration ensure lens transparency and provide focusing properties.1–3 Epithelial cells on the lens anterior surface differentiate into lens fiber cells, such that new fiber cells are continuously added to the lens cortex atop older fiber cells in the lens core, creating concentric rings that reflect cellular age.4 Lens fibers mature through a process of elongation, cellular compaction, and removal of organelles. Mature fiber cells in the core experience no cellular turnover and they are devoid of light-scattering nuclei as well as other organelles.5 Without machinery for cellular turnover or protein synthesis, long-lived cells and their contents survive for decades in the lens.6 Moreover, aging lens proteins are subject to modification over time. Some posttranslational modifications (PTMs), like phosphorylation, are programmed to occur in a specific region of the lens, while truncation accumulates in a time-dependent manner with age.7 In the lens, PTMs may alter protein function by modulating existing protein function when no new protein is produced. One essential, yet extensively modified, protein is the lens major intrinsic protein (MIP), or aquaporin-0 (AQP0). Aquaporin-0 is a dual-function water channel and adhesion molecule comprising approximately 50% of lens membrane protein.8 The central pore of each AQP0 monomer shuttles water across the plasma membrane.9 Aquaporin-0 also is involved in junction formation and cell-to-cell adhesion.10–16 Mutations or loss of AQP0 lead to cataract, highlighting the importance of this protein for lens function.17–28 Aquaporin-0 is a 28 kD protein comprised of six transmembrane domains and an intracellular C-terminal tail. The highly modified tail regulates AQP0 permeability via calmodulin binding.29–31 This region of AQP0 also binds cytoskeletal proteins, including filensin and phakinin,32 and may have a role in establishing fiber cell organization. Numerous PTMs decorate the AQP0 C-terminus over time, some of which have been functionally characterized. Phosphorylation of Ser-235 reduces the binding affinity of calmodulin, thereby boosting the permeability of the channel.31,33,34 Cleavage of the C-terminal tail removes protein–protein binding regions and may encourage AQP0 to form cell–cell junctions.7,13 More recently, fatty acid modifications were discovered on lysine 238 and on the N-terminal methionine, which may target the protein to detergent-resistant lipid raft regions of the membrane.35,36 Deamidation and isomerization of asparagine residues have been reported but have not been fully characterized.37,38 The localization of these PTMs within the lens can provide clues of the changing physiological role of AQP0 over time. Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) has been used to map the spatial localization of modified AQP0 within the lens.35,39 In MALDI IMS, proteins are detected by their mass-to-charge (m/z) value and each m/z value can be mapped across a tissue section with intensity encoded as a heat map.40 Protein modifications are characterized by specific shifts in m/z values. Previous MALDI experiments showed the localization of truncated and lipid-modified AQP0.35,41 Although informative, these molecular images captured a single time point in the complex aging process. Furthermore, the spatial distribution and accumulation of deamidated AQP0 is unknown. The aim of this study was to characterize the localization of modified AQP0 in human lens from birth through middle age using MALDI IMS. We optimized methods to image modified AQP0, revealing unprecedented images of deamidated and truncated forms of the functionally-important AQP0 C-terminal tail. Understanding the localization of these modifications in young or old fiber cells can provide clues on how AQP0 function changes during development, aging, and cataract formation.