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S. P. Bhat, R. K. Gangalum, Z. Jing; The Small Heat Shock Protein B-Crystallin Is Secreted From Retinal Pigment Epithelial Cells ARPE in Culture. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4183.
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Elevated expression of the small heat shock protein, B-crystallin (B) has been associated with cancer, cardiomyopathies and a large number of neurodegenerative disorders including, Alzheimer’s, Parkinson’s, Alexander’s diseases, multiple sclerosis and retinal degenerations. Interestingly, the expression of this protein in retinal pigment epithelium (RPE) and its presence in ‘drusen’ has been implicated in age-related macular degeneration. In this investigation we have explored the status of B in ARPE cells in culture with a focus on mechanistic understanding of how B becomes associated with the ‘drusen’?
The association of B with Golgi membranes was studied by sucrose density gradient fractionation and confocal microscopy. Protein transport was studied using various inhibitors. Exosomes were isolated from the culture medium by differential centrifugation, characterized by AChE assay and immuno-electron microscopy. Optiprep gradients were used to isolate lipid rafts from ARPE cells and characterized by the presence of established raft markers.
Using synchronized cultures we show that B is a Golgi -associated protein in ARPE. We demonstrate that B is secreted out of ARPE and that this secretion is not inhibited by classical protein transport inhibitors such as Brefeldin and Monnensin, and glycosylation inhibitor, Tunicamycin and microtubule disrupting agent, Nocodazole. Examination of the culture medium reveals that B is associated with exosomes and multivesicular bodies (MVB). Interestingly the secretion of this protein is inhibited by methyl β-cyclodextrin (MBC) suggesting lipid raft dependent exocytosis of vesicles containing B.
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