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D. Maiguel, N. Caberoy, Y. Kim, W. Li; Identification of Phagocytic Protein Ligands for Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1656.
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Trabecular meshwork (TM) cells play a major role in regulating aqueous humor outflow. They are phagocytically active and may prevent the drainage pathways clogging by removing cellular debris and pigment through phagocytosis. Although TM cell phagocytosis has been well-documented at cellular level with latex beads, its molecular mechanism is poorly defined with no identified phagocytic ligand. The purpose of this study is to identify and characterize phagocytic ligands in TM cells.
A T7 bacteriophage display cDNA library has been generated from mouse eye, and a novel functional cloning strategy of phage display has been developed. Phage clones with phagocytic activities were enriched with human trabecular meshwork TM-3 cell line with 4 rounds of selection. Individual phage clones with phagocytic activity were isolated and identified by DNA sequencing. Active domains of identified phagocytic ligands were further analyzed by deletion and mutation analysis. Their phagocytic activities were further validated by non-phage-based phagocytosis assays.
Four rounds of phage selection resulted in 50-fold increase in phagocytic activity. After screening a total of 96 phage clones, we isolated a handful of proteins with at least 20-fold higher phagocytic activity vs. control phage. One of the proteins identified was Max protein with 35-fold increase in its phagocytic activity. Max protein is a transcription factor that plays a role in cell proliferation. The phagocytic activity of these identified proteins is currently under characterization, including the determination of their minimal phagocytic domains.
The innovative functional cloning strategy of phage display described in this study provides a powerful tool to investigate phagocytic mechanisms in TM cells. This will advance our knowledge of TM cell phagocytosis from the current cellular level to the molecular level, and may lead to the identification of phagocytic ligands, receptors and signal pathways that play critical roles in glaucoma pathogenesis.
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