The ocular lens is a transparent optical element that focuses light onto the retina for clear vision. The bulk of the lens is composed of elongated, anuclear fiber cells, which are continuously synthesized throughout life and added to older, mature fiber cells in the center of the lens. Mature fiber cells experience no cellular turnover, and the lens contains very long-lived proteins that become modified over time. Changes in protein abundance, solubility, post-translational modifications (PTMs), and protein-protein interactions can modulate protein function during lens development, differentiation, and aging, especially in regions of the lens where no new protein can be synthesized. By spatially characterizing proteins in different regions of the lens, we can gain insight into programmed and age-related modifications that may modulate protein function and therefore affect lens health or cataract development.
One critical lens protein is Aquaporin-0 (AQP0), a water channel essential for lens transparency. Here, we used MALDI IMS to characterize several AQP0 PTMs across a wide age range of human lenses. Our results shed light on the recently-discovered fatty acid acylation of AQP0, confirming it is an irreversible modification that remains on the protein during age-related C-terminal truncation. This lipid modification was detected in 2-month and 4-month human lenses, suggesting fatty acid acylation is programmed in young fiber cells. We performed in situ digestion and MALDI IMS to generate AQP0 C-terminal peptide fragments, enabling imaging of deamidated peptide forms for the first time. These unprecedented images revealed rapid deamidation of AQP0 in very young fiber cells, followed by truncation at labile asparagine residues. In a separate study, we investigated changes to a very narrow region of the human lens outer cortex that contains the morphologically-distinct remodeling zone. Using laser capture microdissection (LCM) and quantitative proteomics, our results revealed significant changes between the remodeling zone and surrounding regions. Notably, the vimentin intermediate filament to beaded filament switch occurs at the remodeling zone. Changes to other intermediate filament interacting proteins (IFAPs) were also observed. Together, these experiments advance the lens field by providing additional information on the spatial distribution of lens proteins correlated with development and age.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-01142016-152655 |
| Date | 29 February 2016 |
| Creators | Wenke, Jamie Lyn |
| Contributors | Charles Sanders, Richard Caprioli, Kevin Schey, Hassane Mchaourab, Rebecca Sappington |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-01142016-152655/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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