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K.L. Schey, L.E. Ball, R.K. Crouch, D.L. Garland; The Distribution of Phosphorylated and Posttranslationally Modified Aquaporin 0 Within the Normal Human Lens . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4483.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The goal of the present study is to map the spatial distribution of posttranslationally modified forms of the most abundant lens membrane protein, Aquaporin 0 (AQP0, MIP), in normal human lenses. Efforts were concentrated on the C-terminus, a putative regulatory domain that is subject to many posttranslational modifications. Methods: Human lenses, ages 34, 35, and 38, were dissected concentrically into 3 or 4 sections and the membrane protein from each section was isolated by centrifugation. The lens membranes were digested with trypsin and the C-terminal peptides released from AQP0 were isolated from the membrane pellet by centrifugation. C-terminal peptides were separated by RP-HPLC, quantified by ESI-MS, and sequenced by tandem mass spectrometry. Results: Novel posttranslational modifications identified include phosphorylation at Ser 229 and Ser 231, backbone cleavage at residues 249, 260, 261, and 262, and isomerization/ racemization of L-Asp 243 to D-iso-Asp. The most abundant sites of backbone cleavage between residues 239-263 occurred after asparagine residues 246 and 259 and were observed in the outer most lens sections. Although the levels of truncation products and D-iso-Asp 243 increased as a function of fiber cell age, the level of phosphorylation was highest in the inner cortex. In the outer cortex, cortex, and nuclear sections of the 34 year old lens, the levels of phosphorylation were 6.4, 15.1 ± 1.8, and 7.0 ± 1.1% for Ser235, and 1.0, 8.9 ± 1.7, and 8.0 ± 3.5% (n=3) for residues 229-233, respectively. Conclusions: Although the role of C-terminal phosphorylation of AQP0 is not known, these data suggest that AQP0 may be regulated spatially within the lens. Elucidation of the exact sites and extent of age-related posttranslational modifications of AQP0 will provide insight into potential mechanisms involved in protein truncation and protein repair.
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