Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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,
Conclusions: :
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.
Keywords: retinal pigment epithelium • phagocytosis and killing