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N. B. Caberoy, Y. Zhou, W. Li; Characterization of Eat-Me Signals in RPE Cell Phagocytosis by Phage Display. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1869.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) cell phagocytosis is essential to remove shed photoreceptor outer segments (POS) and maintains viability and excitability of photoreceptors. There is no approach to systematically identify "eat-me" signals or recognition ligands controlling the initiation of RPE phagocytosis. A limited number of known eat-me signals were identified on a case-by-case basis with daunting challenges. The purpose of this study is to establish the feasibility of using phage display technology to functionally clone eat-me signals by characterizing the biological behavior of phages in RPE phagocytosis.
Two phage clones were constructed to display the well-characterized eat-me signals of growth arrest-specific gene 6 (Gas6) and milk fat globule-EGF8 (MFG-E8). The expression of both proteins on phage surface was characterized by phage binding assay. Their stimulation on RPE phagocytosis was analyzed by phage phagocytosis assay. Gas6-phage and MFG-E8-phage were diluted with control phage and enriched by multiple rounds of phagocytosis selection. Phage enrichment was characterized by plaque assay and PCR analysis.
Binding study showed that both Gas6 and MFG-E8 were expressed on phage surface. Gas6-phage bound to all three known Gas6 receptors, including Mer, Axl and Tryo3 receptor tyrosine kinases. Gas6-phage and MFG-E8-phage were capable of binding to various phagocytes and non-phagocytes. However, both eat-me signals stimulated phage uptake only in professional phagocytes, including ARPE19, macrophage and microglial cell lines, but not in non-phagocytes. Furthermore, functional phage selection by phagocytosis in RPE cells substantially enriched both Gas6-phage and MFG-E8-phage,
These data demonstrated that Gas6-phage and MFG-E8-phage are specifically enriched by phagocytosis selection, suggesting that phage display can be used as a powerful tool to functionally identify unknown eat-me signals from phage display cDNA library.
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