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Feiye Guo, Ying Ding, Nora Blanca Caberoy, Feng Wang, Rui Chen, Wei Li; ABCF1 extrinsically regulates retinal pigment epithelial cell phagocytosis. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5484.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelial (RPE) phagocytosis of shed photoreceptor outer segments (POSs) is critical to maintaining retinal homeostasis and preventing retinal degeneration. However, molecular mechanisms of RPE phagocytosis are largely undefined. The purpose of this study is to identify unknown phagocytosis ligands by a new method and characterize ATP-binding cassette subfamily F member 1 (ABCF1) as a novel RPE phagocytic ligand.
Open reading frame phage display (OPD) was recently developed for unbiased identification of phagocytosis ligands. We combined OPD with next generation DNA sequencing (NGS) to systematically identify RPE phagocytosis ligands for RPE cells. Recombinant ABCF1 was expressed as the fusion protein of maltose-binding protein in bacteria and purified with amylose columns. POS vesicles were prepared from fresh bovine retina, labeled with pHrodo and used for phagocytosis assay with D407 RPE cells, primary RPE cells and RPE cups in the presence or absence of purified ABCF1. Phagocytosed POSs were analyzed by confocal microscopy. Immunohistochemistry and immunocytochemistry were performed with α-ABCF1, α-rhodopsin or α-Rab7 antibody. ABCF1-FLAG binding to shed POS, apoptotic and healthy cells was analyzed by confocal microscopy and flow cytometry.
OPD-NGS systematically identified hundreds of putative RPE phagocytosis ligands. One of the identified ligands is ABCF1. ABCF1 was independently verified to extrinsically promote phagocytosis of shed POSs by D407 RPE cell line. This finding was further corroborated with primary RPE cells and RPE cups. Internalized POS vesicles were co-localized with phagosome marker Rab7, suggesting that ABCF1-mediated engulfment is through a phagocytic pathway. ABCF1 was released from apoptotic cells. Released ABCF1 selectively bound to shed POS vesicles and apoptotic cells but not healthy cells. ABCF1 is predominantly expressed in POSs and co-localized with POS marker rhodopsin, providing geographical convenience to regulate RPE phagocytosis. Collectively, these results suggest that ABCF1 is released from and binds to shed POSs in an autocrine manner to facilitate RPE phagocytosis through a conserved pathway.
These data suggest that ABCF1 is a genuine RPE phagocytosis ligand. These findings in turn support the validity of OPD-NGS as a new approach to systematically identify RPE phagocytosis ligands in the absence of receptor information.
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