Role of Protein Interactions in Retina Development and Function

National Eye Institute

Investigators

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Abstract

We completed studies on the interactions of PEDF with hyaluronan. Surface plasmon resonance (SPR) and affinity column chromatography were performed to investigate the affinity of PEDF for hyaluronan from several sources. Biophysical binding parameters of the interactions were determined by SPR with human recombinant PEDF protein immobilized in sensor chips. Sensitivity to ionic strength was determined by hyaluronan-affinity column chromatography and SPR in the presence of NaCl. PEDF was chemically modified with fluorescein, and its binding affinities for hyaluronan and heparin were compared. PEDF binding competition assays between hyaluronan and heparin were performed. We found that PEDF chemically modified with fluorescein retained the capacity for interacting with HA but lacked heparin affinity, suggesting a distinct HA-binding region(s) on PEDF. We concluded that the HA-binding activity of PEDF may contribute to deposition in the extracellular matrix and to its reported antitumor/antimetastatic effects. [unreadable] [unreadable] We completed the study on the upregulation of PEDF by dexamethasone in trabecular meshwork (in collaboration with Terete Borras). To further confirm previous results we performed ELISA to determine the levels of PEDF in effluents from organotypic cultures of anterior segments from human eyes. The results demonstrated that PEDF was secreted to the extracellular milieu of the trabecular meshwork and that its levels increased specifically and progressively over perfusion time with dexamethasone.[unreadable] [unreadable] To continue examining the implications of the interactions between PEDF and ectopic ATP synthase, bovine retina endothelial cells (BRECs) were assayed for extracellular ATP production in the presence of PEDF and other inhibitors of F1 ATP synthase. We found that PEDF inhibited the extracellular ATP synthesis activity of BRECs. [unreadable] [unreadable] We continued working on the characterization of PEDF-R protein, a receptor for PEDF with binding affinity for PEDF and phospholipase activity. Several peptides were designed from L4, a putative PEDF-R extracellular loop, and were chemically synthesized. The peptides were termed according to their exon mapping on the PEDF-R gene. To analyze protein-protein interactions we developed ligand blot assays with peptides immobilized on nitrocellulose membranes using fluoresceinated PEDF protein as ligand. We performed real-time binding analyses by SPR using recombinant human PEDF immobilized on sensor chips. We found that one peptide from a central region of L4 specifically bound PEDF with high affinity, and that their interactions were partially sensitive to increasing NaCl concentrations. We obtained structural models of PEDF-R and PEDF to envision the ligand-receptor interface.[unreadable] [unreadable] To explore the signaling pathways triggered by PEDF-R, retina R28 cells precursors were differentiated to have neuron-like phenotype. Western blots of R28 membrane fractions were performed and PEDF-R protein was immunodetected. PLA2 activity in-vitro was assayed in these fractions. We investigated the effects of PEDF on the PLA activity of PEDF-R. We also performed assays to determine activation of Akt in R28 cells by PEDF and whether this activation can prevent cell death.