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Shadi K. Safuri, Ido Perlman; Bestrophin Modulates Phagocytosis of Photoreceptors Outer Segments by Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1771.
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© ARVO (1962-2015); The Authors (2016-present)
Best vitelliform macular dystrophy (Best Disease) result from specific mutations in the bestrophin-1 gene, which is localized to the retinal pigment epithelium (RPE) cells. Despite established molecular functions, the physiological role played by the bestrophin-1 protein (Best1) in RPE cells and the mechanism of Best Disease are yet unknown. Lipofuscin accumulation suggests involvement of photoreceptors outer segments (POS) in disease pathogenesis. Accordingly, we tested the effects of Best1 mutations on the phagocytosis process of POS by RPE cells.
Different Best1 mutations associated with distinct phenotypes were generated using site-directed mutagenesis. Verified vectors were cloned into pBABE-puro retroviral vectors and subsequently used to transduce ARPE19 cells, thus generating RPE cell lines stably expressing different variants of Best1. Monolayers of these lines were challenged with FITC labeled photoreceptor outer segment (POS) according to different protocols. Trypan blue was used to discriminate between bound and engulfed POS.
Phagocytosis of POS by RPE lines displayed saturation characteristics. Following a saturating pulse of POS, RPE lines expressing Best Disease associated mutants (R218S or E300D) displayed enhanced binding and engulfment compared to wild-type (wt). In contrast, V86M, associated with autosomal dominant vitreoretinochoroidopathy (ADVIRC), resulted in reduction in these processes. To better characterize this modulation, curves were fitted to a Michaelis-Menten model. Vmax and Km were increased in Best, but both were decreased in ADVIRC, compared to wt. Interestingly, the line expressing R47H mutant associated with adult-onset vitelliform dystrophy (AVMD) had Vmax and Km values intermediate between Best and wt variants.
Best1 mutations modulate phagocytosis in a phenotype-dependent manner. The decreased affinity for POS observed in Best Disease and AVMD, and decreased maximal phagocytosis capability observed in ADVIRC, may underlie the mechanism of lipofuscin accumulation in RPE cells, characteristic of these phenotypes.
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