We continued working on the characterization of PEDF-R protein, a receptor for PEDF with binding affinity for PEDF and phospholipase activity. Optimized conditions for expression of soluble PEDF-R and C-terminal truncated polypeptide PEDF-R4 were obtained. Several peptides were designed from PEDF-R, and were chemically synthesized. Binding to PEDF was assayed by ligand blot and surface plasmon resonance. One peptide region from the PEDF-R extracellular loop contained PEDF binding affinity. In collaboration with Grzegorz Piszczek (NHLBI/DIR/BBC) we analyzed the secondary structures of the peptides and found that this peptide region contained alpha-helical structure in phosphate buffer pH 7.4, but not in water. We designed smaller peptides designed from this region and assayed the synthetic peptides for PEDF binding to identify the smallest region from PEDF-R with affinity for PEDF. We obtained structural models of PEDF-R and PEDF to envision the ligand-receptor interface. To study PEDF-R in retina cells we performed western blots, immunocytochemistry and enzymatic assays. We detected PEDF-R variants in retina R28 cell membrane fractions, which exhibited phospholipase A activity in-vitro. We investigated the effects of PEDF on the PLA activity of PEDF-R. Using real-time cell impedance monitoring and cell viability assays we studied the survival effects of PEDF on R28 cells. We also examined the effects of the PEDF-binding peptides derived from PEDF-R as potential blocking agents for the PEDF-mediated survival effects on R28 cells. Vectors were used to silence the PEDF-R gene and PEDF-mediated survival assays were performed in ARPE-19 cells. We mapped epitopes of two commercial antibodies for PEDF-R and found that these antibodies recognize regions of the PEDF-binding site of PEDF-R and a flanking region. The stability of PEDF-R derived from the plasma membranes of retina cells was investigated. We found that additions of the PEDF ligand during the extraction process stabilized the PLA activity of the enzyme. We also investigated the stability of the recombinant human PEDF-R and its C-terminal truncated forms, using several potential stabilizing agents, such as detergents, chaperones, or PEDF ligand. In addition to these, we found that bacterial cell-free extracts increased the stability of the PEDF-R enzyme for at least two weeks. We also pre-incubated the PEDF-R-containing subcellular fractions from retina cells with bacterial cell-free extracts and tested to enzyme stability. The PLA activity of these recombinant proteins was stimulated upon addition of PEDF. BHK cells harboring expression vectors for PEDF with point mutations were cultured. Their culturing media contained secreted recombinant altered PEDF proteins that were harvested to develop purification protocols for each altered protein. The alterations included critical amino acids for PEDF binding to heparin, hyaluronan, collagen, and another for PEDF fused to a FLAG tag. To continue examining the implications of the interactions between PEDF and ectopic ATP synthase, we searched for cells that respond to the proapoptotic activity of PEDF and contain cell-surface ATP synthase. The effects of PEDF were tested on breast tumor MDA-MB-231-BR and 4TI-BR cells and prostate tumor LNCaP cells using real-time cell impedence monitoring systems and cell viability assays. We investigated the presence of F1/F0 ATP synthase on the cell surface using biochemical and immunocytochemistry methodologies. We assayed extracellular ATP production in the presence of PEDF and other inhibitors of F1 ATP synthase and found that PEDF inhibited the extracellular ATP synthesis activity of LNCaP cells. We also performed endothelial tube formation assays and tested the antiangiogenic effects of PEDF in the presence and absence of ATP.
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