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Nora B. Caberoy, Yixiong Zhou, Gabriela Alvarado, Wei Li; Systematic Mapping of RPE Phagocytosis Pathways. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3702.
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© ARVO (1962-2015); The Authors (2016-present)
RPE phagocytosis plays an important role in maintaining retinal homeostasis. Defects in RPE phagocytosis lead to retinal degeneration, and dysfunction in RPE phagocytosis may contribute to age-related macular degeneration. However, daunting challenge to define unknown phagocytosis pathways has hindered our understanding of the physiological and pathological roles of RPE phagocytosis. This study is to systematically identify unknown RPE phagocytosis ligands and receptor-specific phagocytosis ligands.
An innovative strategy of phagocytosis-based functional cloning was developed to identify RPE phagocytosis ligands in the absence of receptor information. Furthermore, a novel dual functional cloning technique was developed by combining phagocytosis-based functional cloning with receptor-based affinity cloning to identify receptor-specific RPE phagocytosis ligands. Identified ligands were expressed as recombinant proteins, purified and independently verified by assays for RPE phagocytosis, receptor binding and receptor activation with intracellular signal cascades.
Phagocytosis-based functional cloning identified tubby and Tulp1 as novel phagocytosis ligands for RPE cells. Both proteins were characterized as bridging molecules for MerTK, a well-known phagocytic receptor. Phagocytic receptor-binding domain (PRBD) of tubby and Tulp1 was mapped to their N-terminal K/R(X)1-2KKK motif(s). PRBP was essential for MerTK binding and receptor phosphorylation induced by tubby and Tulp1. Phagocytosis prey-binding domain (PPBD) of tubby and Tulp1 was mapped to their highly conserved C-terminal 44 amino acids, deletion of which cause retinal degeneration. Removal of either PRBD or PPBD disrupted the bridging function of tubby and Tulp1, abolishing their stimulation of RPE phagocytosis. Dual functional cloning identified galectin-3 (Gal-3) as new MerTK-specific phagocytosis ligand. Similar to tubby and Tulp1, Gal-3 was also characterized as a new bridging molecule for MerTK to facilitate RPE phagocytosis.
These results identified three new phagocytosis ligands for MerTK. These studies demonstrated that phagocytosis-based functional cloning is a valid technology for unbiased identification of RPE phagocytosis ligands in the absence of receptor information and that dual functional cloning is valid for systematic identification of receptor-specific RPE phagocytosis ligands. These technologies will improve our capability to systematically map RPE phagocytosis ligands, receptors and signaling pathways, and open a new chapter for RPE molecular phagocyte biology and exploration of their therapeutic potentials.
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