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Wei Li, Nora Caberoy, Gabriela Alvarado, Hui Wang, Feng Wang, Rui Chen; Global identification of RPE phagocytosis ligands by phagoligandomics. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2650. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) phagocytosis is critical to maintaining retinal homeostasis. Defects in RPE phagocytosis lead to retinal degeneration. Phagocytosis ligands, receptors and pathways were traditionally identified on a case-by-case basis with daunting technical challenges. As a result, we really do not know how many new phagocytosis ligands and pathways are yet to be identified. This study is to thoroughly identify all possible RPE phagocytosis ligands by a new approach of phagoligandomics.
We previously developed a new approach of phagocytosis-based functional cloning (PFC) to identify unknown phagocytosis ligands in the absence of receptor information. After three rounds of PFC selection with mouse eye cDNA library, all clones enriched via phagocytosis were identified by next generation DNA sequencing (NGS). Identified putative phagocytosis ligands were experimentally verified for their capability to stimulate RPE phagocytosis.
Three rounds of PFC selection resulted in ~30-fold internalization activity of cDNA library. NGS analysis identified that a total of 6,611,438 valid sequence reads, which matched to 1,382 proteins in NCBI CCDS database. Among the most active phagocytosis ligands were Tulp1 and Gas6, both of which are well-characterized ligands for MerTK phagocytic receptor. Other identified ligands included Abcf1 and Lyar, which were previously reported as putative phagocytosis ligands identified by our manual PFC screening. Moreover, we independently validated Abcf1 as a MerTK-binding protein, suggesting that Abcf1 is likely a new ligand for MerTK.
These results suggest that our PFC-NGS is a valid and biologically relevant approach of phagoligandomics for global identification of RPE phagocytosis ligands. To our knowledge, ligandomics is currently impossible to any cells, receptors and functions. PFC-NGS is the first paradigm of ligandomics for global mapping of cell-wide phagocytosis ligands, including unknown ligands. This approach will systematically unravel the mystery of RPE molecular phagocyte biology and delineate the underlying mechanisms for RPE phagocytosis dysfunction in retinal aging and disease conditions, such as age-related macular degeneration (AMD).
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