Proteome remodeling in the differentiating fiber cells of the human lens

Abstract

Purpose: The remodeling zone of the human lens cortex has been observed by confocal fluorescence and transmission electron microscopy at a distance of approximately 100 μm from the lens capsule. Although the fiber cells in this region undergo extensive membrane remodeling, the underlying mechanisms remain unknown. Our goal was to elucidate changes in protein abundance in four distinct regions of the human lens cortex: differentiating zone, remodeling zone, transition zone, and inner cortex.

Methods: Human lenses were fixed in ethanol/acetic acid and paraffin-embedded. Sections were placed on glass slides, dewaxed with xylenes, and stained with fluorescent wheat germ agglutinin to visualize membranes. Fluorescence microscopy and distance measurements were used in conjunction with laser capture microdissection (LCM) to capture ~2x10⁶ µm² cells from each region. The tissue was rinsed to remove soluble proteins and prepared for membrane protein analysis. The final insoluble tissue pellets were digested with trypsin and analyzed using LC-MS/MS. Searches of MS/MS spectra against a human proteome database were used to identify proteins associated with each region. Three lenses were analyzed (21, 22, and 27 years of age), with three replicates for each sample. Normalized spectral counts were used to quantitate protein abundance changes between zones.

Results: Analysis of the membrane preparation revealed differences in cytoskeletal proteins between different zones of the lens outer cortex. A dramatic decrease in vimentin was observed beyond the differentiating zone, approximately 100 μm into the lens cortex. Conversely, intermediate filament proteins filensin and phakinin were more abundant in the remodeling zone, transition zone, and inner cortex. Changes in actin and crystallin proteins were also detected. These results suggest significant cytoskeletal changes are correlated with the remodeling zone.

Conclusions: LCM and LC-MS/MS were used to quantify protein changes in distinct morphological regions of the lens outer cortex. Our results suggest intermediate filament protein expression is altered near the remodeling zone. Future work will involve targeted multiple reaction monitoring (MRM) of specific lens proteins for more accurate quantitation of protein changes in the remodeling zone. Immunohistochemistry will also be used to corroborate our proteomics data.

Keywords: 663 proteomics • 500 differentiation • 657 protein modifications-post translational