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Deepika Arora, Chun-hong Xia, Jing Zeng, Zoey Huang, Mei Li, Xiaohua Gong; Characterization of Periaxin in the lens and cataract formation. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2968.
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© ARVO (1962-2015); The Authors (2016-present)
Periaxin (Prx) is suggested to function as a scaffold protein that plays a role in lens membrane organization and cell adhesive interactions. However, the mechanism by which Prx regulates cell adhesion and signaling in lens fiber cells is unclear. Also, the regulation of Prx gene expression and distribution during lens development is not well characterized. Our project aims to understand its mechanistic function in the lens by identifying Prx associated proteins, determining the functional domains of Prx protein, characterizing its expression and distribution during lens development and investigating its role in cataract formation in mouse models.
Lenses from wild-type 129SvJ (129) mice, C57BL/6J (B6) mice and different connexin knockout mice were prepared for biochemical analyses including western blotting, immunoprecipitation and mass-spectrometry. Wild-type and mutant mouse Prx genes were cloned and tagged with fluorescent protein gene in expression plasmids that were transfected into HEK293 cells. Stably transfected cells were isolated and grown on various extracellular matrix proteins such as fibronectin, collagen and gelatin to elucidate Prx localization by using confocal imaging analysis.
Western blotting results showed that Prx expression level was higher in 129 mouse lenses than in B6 mouse lenses. Mass-spectrometry data indicated the co-presence of Prx and other cytoskeleton proteins. Confocal imaging data revealed that Prx was profoundly localized in the membrane but not in the nucleus of 129 mouse lens while it appeared in both membrane and nucleus of B6 mouse lens. Transfected Prx proteins were localized in nucleus, cytosol and membrane of HEK293 stable cell clones.
Differences in Prx expression and distribution were observed between 129 and B6 mouse strains. Higher expression level of Prx in 129 lenses may be related to its targeting to the membrane rather than in the nucleus. Prx in the nucleus undergoes degradation during fiber cell denucleation in B6 lenses. Identification of Prx associated proteins at the lens cell membrane will be important to elucidate the function of Prx during lens formation. Prx redistribution between membrane and nucleus may influence cataract progression of Gja3 connexin knockout mice in 129 and B6 strains.
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