Can Phage Display Be Used as a Tool to Functionally Identify Endogenous Eat-Me Signals in Phagocytosis?

Abstract

Removal of apoptotic cells and cellular debris by phagocytosis is essential for development, tissue homeostasis and resolution of inflammation. Eat-me signals control the initiation of phagocytosis, holding a key to our understanding of phagocyte biology. Due to lack of functional cloning strategy, eat-me signals are conventionally identified and characterized on a case-by-case basis. To investigate the feasibility of functionally cloning eat-me signals by phage display, we characterize the biological behavior of T7 phages displaying two well-known eat-me signals: growth arrest-specific gene 6 (Gas6) and milk fat globule-EGF8 (MFG-E8). Gas6-phage binds to all three known Gas6 receptors, Mer, Axl and Tyro3 receptor tyrosine kinases. Gas6-phage and MFG-E8-phage are capable of binding to phagocytes and non-phagocytes. However, both phages stimulate phage uptake only in phagocytes, including macrophages, microglia and retinal pigment epithelium (RPE) cells, but not in non-phagocytes. Furthermore, functional phage selection by phagocytosis in phagocytes enriches both Gas6-phage and MFG-E8-phage, suggesting that phage display can be used as a tool to functionally identify unknown eat-me signals from phage display cDNA library.

Identification of tubby and tubby-like protein 1 as eat-me signals by phage display

“…Human ARPE19 RPE cells were cultured as previously described [8]. Rat RPE-J and human D407 RPE cell lines, gifts from Dr. G. Hoppe (Cleveland Clinic, Ohio), were cultured in Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine.…”

“…The question is whether this strategy can be used to identify unknown eat-me signals. We recently demonstrated that T7 phage displaying MEG-E8 or Gas6, two well-characterized eat-me signals, was preferentially phagocytosed and enriched by retinal pigment epithelium (RPE) cells or macrophages, but not by non-phagocytes [8], suggesting that the strategy of phagocytosis-based phage selection is feasible for functional cloning of unknown eat-me signals. However, unlike Ab libraries, cDNA repertoires with unpredictable reading frames and stop codons may not be expressed in correct frames as phage capsid fusion proteins [9].…”

Unraveling the Molecular Mystery of Retinal Pigment Epithelium Phagocytosis

“…We recently characterized the feasibility to enrich phagocytosis ligands by phage display (Caberoy et al 2009). The question is whether this approach can be used for identification of unknown RPE phagocytosis ligands.…”

Mesd extrinsically promotes phagocytosis by retinal pigment epithelial cells

“…D407 human RPE and HEK293 cell lines were previously described (Caberoy et al, 2009) and cultured in Dulbeccos's modified essential medium (DMEM) supplemented with 10% FBS and 2 mM L-glutamine (Mediatech, Manassas, VA).…”