PEDF is a member of the serpin superfamily. It has high affinity for pigment epithelium-derived factor receptor (PEDF-R), a patatin-like phospholipase domain-containing 2 (PNPLA2) protein identified in the retina. PEDF-R is a membrane-linked phospholipase, and PEDF binding stimulates its enzymatic phospholipase A2 activity, which catalyzes the cleavage of fatty acids from phospholipids. A study on the identification of epitopes of an antibody to PEDF-R was completed. We mapped the epitopes using recombinant PEDF-R polypeptides fragments and synthetic peptides, and we identified peptides that specifically block its immunoreactivity. We continued working on the characterization of PEDF-R. His-tagged PEDF-R, and His-tagged C-terminally and internally truncated PEDF-R polypeptides (PEDFR203-232, PEDFR4 and PEDFR4203-232) were prepared with an in vitro protein expression system. We performed His-tag pull-down assays and found that His-tagged PEDF-R and His-tagged PEDF-R4 bound fluorescein conjugated PEDF, but His-tagged PEDFR203-232 and His-tagged PEDF-R4193-232 did not. We also performed cell surface biotinylation of retina R28 cells using a membrane impermeable, thiol cleavable, amine reactive sulfo-N-hydroxysuccinimide-SS-biotin reagent. Membrane proteins were isolated and subsequently the biotinylated proteins were fractionated using strepavidin-affinity resins. Western blot of the biotinylated proteins detected PEDF-R among the membrane R28 proteins, demonstrating that PEDF-R was present in the surface of R28 cells. Furthermore, we tested the capacity of PEDF-R siRNAs to decrease the expression of PEDF-R in R28 cells. RT-PCR was performed and revealed a significant decrease in the PEDF-R transcript levels in cells transfected with the PEDF-R siRNA. Western blots of membrane protein fractions were performed and showed that the PEDF-R protein levels decreased with the silencing vectors in the membrane of R28 cells. We also evaluated cell survival of serum-starved R28 cells using intracellular ATP as a biomarker for live cells. We found that PEDF did not increase the viability in cells in which PEDF-R had been silenced relative to the untransfected or scrambled siRNA-transfected cells. Using TUNNEL staining, we evaluated the apoptosis in R28 cells and found that PEDF did not decrease the TUNNEL positive nuclei number when the PEDF-R expression had been silenced relative to untransfected or scrambled siRNA-transfected cells. PEDF can induce the expression of Bcl2 in R28 cells. Bcl2 expression levels were also measured by RT-PCR in untransfected and siRNA-transfected cells to silence PEDF-R, and without and with PEDF in the presence of PEDF-R derived peptides as blocking peptides. PEDF was assayed for axon growth and survival of retinal ganglion cells, in collaboration with Dr. Yuqin Yin. PEDF protected retina cells against death and promoted axon growth of retinal ganglion cells, and a PEDF-binding peptide corresponding to the PEDF binding region of PEDF-R was an effective blocking agent for the PEDF-mediated axon growth activities. A study to identify PEDF isoforms was completed. Recombinant PEDF proteins were purified by cation- and anion-exchange column chromatography, and subjected to SDS-polyacrylamide gel electrophoresis, isoelectric focusing, deglycosylation, heparin affinity chromatography, and limited proteolysis. Cell viability, real-time electrical impedance of cells, and wound healing assays were performed using bladder and breast cancer cell lines, and retinal R28, and ARPE-19 cells. We found two distinct PEDF protein peaks after anion-exchange column chromatography: PEDF-1 eluting with lower ionic strength than PEDF-2. PEDF-1 protein had higher pI value and lower apparent molecular weight than PEDF-2 protein. Both PEDF forms were glycosylated, bound to heparin, and had identical patterns by limited proteolysis. However, PEDF-2 emerged as being highly potent in lowering cell viability in all tumor cell lines tested, and in inhibiting tumor and ARPE-19 cell migration. In contrast, PEDF-1 minimally affected tumor cell viability and cell migration but protected R28 cells against death caused by serum starvation. We concluded that the identification of two distinct biochemical forms of PEDF varying in overall charge and with distinct biological effects on tumor cell viability and migration may explain the multimodal functionality of the PEDF protein. We also completed a study on the inhibition of tumor cell surface ATP synthesis by PEDF. Incubation of urinary bladder carcinoma cell lines in media containing recombinant PEDF protein for 48-96 h dramatically decreased cell viability in a concentration-dependent fashion as monitored by real-time cell impedance with a microelectronic system, microscopic imaging and biomarkers of live cells. Intact tumor cells exhibited cell surface ATP synthesis activity, which was inhibited by piceatannol, a specific inhibitor of F1/F0-ATP synthase. Immunoblotting revealed that the subunit of F1-ATP synthase was present in plasma membrane fractions of these cells. Pre-incubation of tumor cells with PEDF inhibited the activity of cell surface ATP synthase in a concentration-dependent fashion. The PEDF-derived peptide 34-mer decreased tumor cell viability and inhibited extracellular ATP synthesis to the same extent as full-length PEDF. Moreover, ATP additions attenuated both the PEDF-mediated decrease in tumor cell viability and the inhibition of endothelial cell tube formation. The findings imply that PEDF is a novel inhibitor of tumor cell surface ATP synthase activity that exhibits a cytotoxic effect on tumor cells, and that the structural determinants for these properties are within the peptide region 34-mer of the PEDF polypeptide. Two biologically active regions have been identified in PEDF: an antiangiogenic 34-mer peptide and a neurotrophic 44-mer peptide. We began studies toward mapping the region in PEDF that binds to PEDF-R. The structure of a peptide P1 containing the PEDF binding region of PEDF-R was predicted in an ab initio model, and the resultant structure was then used in a global docking search using Rosetta, with a PEDF structural model. Evaluation of P1 by circular dichroism, indicated that P1 is composed of -helical structure. Peptides from PEDF neurotrophic and antiangiogenic regions, and P1 were chemically synthesized and purified. Binding was determined by ligand blotting of PEDF-R peptides immobilized on nitrocellulose membrane with FITC labeled PEDF ligands. Ligand blot of immobilized P1 to Fl-PEDF, FITC-conjugated-34-mer and FITC-44-mer revealed that PEDF bound to soluble and immobilized P1, and that the neuroprotective 44-mer, but not the antiangiogenic 34-mer bound to P1. Surface plasmon resonance with P1 sensor chips showed that only the 44-mer bound to P1 in real time like PEDF. Pull-down experiments with in vitro synthesized His-tagged PEDF-R revealed that the 44-mer and not the 34-mer bound to PEDF-R polypeptides. A smaller peptide derived from the central region of the 44-mer, namely a 17-mer, shares neurotrophic activity with PEDF. We performed ligand blot of P1 to FITC-17-mer peptide and found that the smaller 17-mer peptide is sufficient for binding PEDF-R. We designed a series of peptides from the 17-mer by alanine scanning in which one by one residue was substituted with an alanine. We observed that some residues of 17-mer were more critical for binding P1, and others enhanced P1 binding when altered to alanine. The results imply that the neuroprotective region of PEDF binds to PEDF-R and support the role of PEDF-R as a neurotrophic receptor.
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