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H. Cai, Y. Roh, C. Cai, L. V. Del Priore; Bruch’s Membrane (BM) Aging Induces Down Regulation of CD46 in Retinal Pigment Epithelium: Implications for AMD. Invest. Ophthalmol. Vis. Sci. 2010;51(13):460.
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© ARVO (1962-2015); The Authors (2016-present)
CD46, a complement regulatory protein located on the basal surface of the retinal pigment epithelium, is in the correct anatomic location to connect the complement system to Bruch’s membrane changes, and thus may play a role in the pathogenesis of age-related macular degeneration (AMD).Herein we determine the effects of BM aging on CD46 gene expression and post-translational modifications, and the role of these changes in regulating RPE function.
Human ARPE19 cells were cultured to confluence either on regular Petri dish or human Bruch’s membrane explants harvested from young (age < 50) or older (age > 70) human eye bank eyes. Confluent ARPE monolayers were harvested and protein lysates were prepared using standard techniques. Expression levels of CD46 protein were determined by densitometry of western blotting images. To study post-translation modifications, immuno-precipitates of ARPE19 cell (cultured on young or older BM) were prepared using anti-CD46 monoclonal antibodies. The immuno-precipitated samples were subjected to western blot analysis using 8-12% SDS-PAGE and transferred to a nitrocellulose membrane. Sulfo- or phospho-tyrosine specific antibodies were used to detect sulfo-or phospho-tyrosine modified CD46. Phosphatase inhibitor sodium orthovanadate (Na3VO4) were added during the tissue culture.
The levels of CD46 protein expression in ARPE19 were 2.0 -fold higher in RPE cultured on young compared to older human Bruch’s membrane. Tyrosine-sulfated CD46 was present in RPE cultured on both young and older BM. However the levels are 1.5 fold higher in RPE cells cultured on young BM. Tyrosine-phosphorylated CD46 was undetectable in RPE cultured on a Petri dish or human BM. When phosphatase inhibitor sodium orthovanadate was added into RPE culture trace amounts of tyrosine-phosphorylated CD46 could be observed.
The results suggest that BM aging affect CD46 expression, with a down regulation of CD46 as a function of increasing BM age. In addition, preliminary evidence suggests that post translation modification of tyrosine residues within CD46 may also be affected by BM age. Since CD46 plays a role in inhibition of complement-mediated damage, down regulation of CD46 would be expected to render the RPE more susceptible to complement mediated damage.
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