Abstract
Purpose :
To investigate cytoplasmic aquaporin-5 (AQP5) trafficking to the plasma membrane in the cortex of the bovine lens by identifying AQP5 protein-protein interactions (PPIs) and by characterizing the subcellular localization of AQP5 and phosphorylated AQP5 (pAQP5).
Methods :
Membrane fractions from homogenates of bovine lens cortical fiber cells were isolated by differential centrifugation. AQP5 co-immunoprecipitation was performed on membrane fractions following detergent solubilization. Eluted proteins from AQP5 co-immunoprecipitation were separated by SDS-PAGE and identified by in-gel digestion and LC-MS/MS analysis on a Velos LTQ ion trap mass spectrometer with a nanoelectrospray ionization source. Tandem mass spectra were searched against a bovine protein database and interpreted manually. The subcellular localization of AQP5 in fixed bovine lenses was determined by immunohistochemistry using an AQP5 antibody and fluorescent organelle markers. Bovine lens tissue cryosections were imaged with confocal microscopy. The subcellular localization of pAQP5 in fixed bovine lenses was determined analogously using an AQP5 pT259 antibody.
Results :
LC-MS/MS analyses revealed several trafficking-related proteins enriched with AQP5 including plectin, filamin B, and peripherin. AQP5 immunolabeling showed intracellular, diffuse, and generally punctate staining in epithelial and cortical fiber cells. Additionally, AQP5 immunolabeling was localized to nuclei and prominent, spheroid structures in the cytoplasm. Cortical AQP5 pT259 immunolabeling was similar to, but less prominent than, AQP5 immunolabeling. AQP5 and AQP5 pT259 immunolabeling on the plasma membrane increased with fiber cell age. Intracellular NBD C6-ceramide and calnexin labeling is similar to, but less prominent, than AQP5 staining. Preliminary Rab 5 immunolabeling is punctate and diffuse.
Conclusions :
Preliminary results indicate potential trafficking-related AQP5 PPIs in the bovine lens cortex. AQP5 spatiotemporal expression in the bovine lens appears to be primarily localized to the nucleus, Golgi apparatus, endoplasmic reticulum, and intracellular vesicles. AQP5 pT259 spatiotemporal expression in the bovine lens is similar, but reduced, relative to unmodified AQP5 expression. Together, AQP5 PPIs, spatiotemporal mapping, and phosphorylation are expected to reveal potential trafficking mechanisms of lenticular AQP5.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.