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K. L. Schey, D. B. Gutierrez, Z. Wang, A. C. Grey, D. Garland; Spatial Distributions of Aquaporin 0 and MP20 Modifications in Human Lenses. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3462.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the location and extent of modification to lens membrane proteins, AQP0 and MP20, in human lenses with age.
AQP0 and MP20 tryptic peptides were generated from digestion of lens membrane protein preparations from manually dissected lens regions of young (18-23y) and older (51-60y) lenses. Quantitative proteomics methods (LC-MS/MS) with isotope labeled internal standards (AQUA peptides) were used for absolute quantitation of AQP0 and MP20 phosphorylation levels in each lens region. In addition, MALDI tissue profiling and imaging were carried out on frozen lens sections after water washes to determine the extent of AQP0 truncation and fatty acid modification to AQP0. On tissue Lys-C digestion allowed distinction between N- and C-terminal fatty acid modification.
A peak in phosphorylation for both APQ0 and MP20 was observed in the outer cortical region (average r/a = 0.83-0.90) in all lenses. At an r/a of approximately 0.76 the level of AQP0 S235 phosphorylation decreased in older lenses and was significantly different than the levels in younger lenses. A similar pattern was observed for MP20 S170 phosphorylation. MALDI imaging of AQP0 revealed extensive truncation starting at the inner cortical region in older human lenses. MALDI profiling of on tissue digested AQP0 revealed little fatty acid modification in the outer cortex, but up to 50% modification in the nuclear region (r/a = 0.55-0.6). Moreover, the results indicated that a majority of the fatty acid modification is positioned at the N-terminus.
The significant decrease in AQP0 phosphorylation in older lenses occurs in the region identified as a "barrier" region. Thus, the regulation of AQP0 permeability by AQP0 phosphorylation, which affects calmodulin binding, may play a role in establishing a permeability barrier in older lenses. The functional consequence of fatty acid modification has yet to be determined; however, the major N-terminal site could be involved in protein trafficking or anchoring.
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