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Zhen Wang, Kevin L Schey; Qualitative and quantitative analysis of AQP5 modifications in lens fiber cells. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
Lens fiber cells have two transmembrane water channels: highly abundant, low water permeability AQP0 and low abundance, high water permeability AQP5. AQP5 was found to relocate to plasma membrane from cytoplasm during fiber cell differentiation. Membrane trafficking of AQP5 could be regulated by multiple mechanisms and phosphorylation through PKA activity has been identified as one of the mechanisms. The purpose of this study is to identify new modifications that could play a role in AQP5 membrane trafficking and to quantify the level of modifications in different lens regions.
Human or bovine lens membrane fractions were prepared by removing water-soluble fraction and urea-soluble fraction. The urea insoluble fraction was either separated by SDS-PAGE followed by in-gel digestion or directly digested by trypsin, chymotrypsin or Lys C in 50 mM Tris-HCl (pH 8.0). The peptides were extracted from the gel or membrane pellets after digestion and analyzed by LC-MS/MS. An acyl-biotin exchange experiment was done to prescreen cysteine palmitoylation. A pseudo-MRM method was used for quantifying the level of modifications in different lens regions.
Phosphorylation on T259 was detected in both bovine and human lens. The level of phosphorylation is high in the outer cortex region and dramatically decreases in the inner cortex and outer nucleus region. Phosphorylation was further decreased in the inner nucleus region. An acyl-biotin exchange experiment suggested AQP5 is palmitoylated. Palmitoylation of AQP5 was confirmed by direct detection of palmitoylation on cysteine 6. Palmitoylation was detected in the cortex region and dramatically decreased in the nucleus region.
In addition to phosphorylation, AQP5 also undergoes cysteine palmitoylation. Considering the important role of palmitoylation in membrane trafficking and protein subcellular localization, palmitoylation of cysteine 6 in AQP5 could be one of multiple mechanisms involved in AQP5 membrane trafficking.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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