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K. L. Schey, D. B. Gutierrez, A. C. Grey, Z. Wang; Characterization of Novel Fatty Acid Acylated Aquaporin 0 Products and Their Spatial Distributions in Bovine and Human Lenses. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3876.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize novel , highly abundant acylated aquaporin 0 products and to map their distributions in bovine and human lenses.
Standard proteomics methods (LC-MS/MS) were used to identify modified AQP0 peptides generated from digestion of lens membrane protein preparations. Membrane pellets were digested with trypsin, washed with 0.1% TFA, and extracted with acetonitrile/0.1% TFA to enrich for modified hydrophobic peptides. For MALDI tissue profiling and imaging, frozen lenses were sectioned equatorially and 20 micron sections were placed on conductive coated glass slides. Sections were washed with water prior to either manual spotting of MALDI matrix (profiling) or automated spotting of matrix in 200 micron spacings (imaging). MALDI mass spectra were acquired using a Bruker AutoFlexIII mass spectrometer.
Extraction of hydrophobic modified peptides enriched the sample in fatty acid acylated AQP0 peptides. The modified forms were identified as Lysine 238 and Methionine 1 modified with palmitate (C16:0) or oleate (C18:1) in both bovine and human lenses. The abundance of the oleate modification was higher than the palmitate modification. Profiling and imaging of intact AQP0 signals revealed significant abundance (up to 1:1 modified:unmodified) of the acylated AQP0 in the inner cortical/outer nuclear regions. Other lipid modifications were not observed.
Lysine palmitoylation has only been observed in one other eukaryotic protein and the two fatty acid modifications may be indicative of the lens lipid environment. The high abundance observed in the inner cortical/outer nuclear region suggests an age related mechanism. The functional consequences of the acylation have yet to be determined; however, the modified C-terminal residue lies within the calmodulin binding/regulatory region of AQP0 suggesting a potential role for this modification in modulating AQP0 permeability. In addition, these modifications may act to anchor the AQP0 cytoplasmic tails to the plasma membrane.
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