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Rajendra K. Gangalum, Zhe Jing, Suraj P. Bhat; A free C-terminus is Essential for the Secretion of αB-crystallin via Exosomes in Human Adult Retinal Pigment Epithelial Cells in Culture. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3339.
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© ARVO (1962-2015); The Authors (2016-present)
αB-crystallin (αB) is a small heat shock protein that is known to protect cells in culture against stress. It is also a component of drusen associated with age-related macular degeneration (AMD). We have recently shown that this protein is secreted out of the human ARPE19 cells in culture via microvesicles (exosomes). These studies were undertaken to understand the molecular events associated with the secretion of αB from these cells.
Two hybrid proteins were made: αB-GFP (with tGFP attached in-frame on the c-terminus of αB) and tGFP-αB (with tGFP attached in frame on the N-terminus of αB). Stable ARPE19 cell lines that express hybrid αB/tGFP (turbo Green Fluorescent Protein) proteins under the control of the homologous αB promoter were generated. Time-lapse Confocal microscopy was used to follow secretion of the recombinant molecules from ARPE cells. Sucrose density gradients, immunoblotting and Confocal fluorescent microscopy were used to follow the distribution of hybrid proteins in the cells.
Out of the two hybrid proteins only GFP-αB (GFP on the N-terminus of αB) is secreted out of the RPE cells. The other recombinant molecule αB-GFP is not found in the medium. Further analyses shows that GFP-αB is found in the exosomes; it can be followed by time-lapse Confocal imaging exiting the cell into the medium. The GFP-αB protein seems to localize (in the perinuclear Golgi) and distribute ‘normally’ in the RPE cells. On the other hand αB-GFP aggregates are seen in the cell at non-specific sites (as also assessed by sucrose density gradient fractionation) and are unable to exit the cell.
These data indicate that a free C-terminus is important for the secretion of αB-crystallin out of ARPE cells via exosomes.
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