Role of Protein Interactions in Retina Development and Function

National Eye Institute

Investigators

Linked publications & trials

Abstract

We continued working on the characterization of PEDF-R protein, a receptor for PEDF with binding affinity for PEDF and phospholipase activity. We constructed several plasmids with deletion versions of PEDF-R and a point mutation at a putative phospholipase A active site, and expressed them heterologously. The resulting recombinant polypeptides were assayed for binding to PEDF and for phospholipase activity. The C-terminal truncated PEDF-R polypeptides exhibited phospholipase A activity and had binding affinity for PEDF. PEDF-R with an altered amino acid at the putative phospholipase A active site bound PEDF but did not exhibit phospholipase A activity. Synthetic peptides were designed from a putative extracellular loop. PEDF binding assays were performed and showed that a peptide from the central region of the loop had binding affinity for PEDF.[unreadable] [unreadable] Immunohistochemistry of bovine retina and RPE showed distribution of PEDF-R the RPE, in the inner segments of photoreceptors and inner layers of the retina. Surface Plasmon Resonance experiments showed that bovine retina plasma membrane proteins contained a PEDF binding component that can be captured with antibodies to PEDF-R. The PEDF-R protein was also immunodetected in westerns of plasma membranes of retina, retinal pigment epithelium, and R28, RGC-5 and ARPE-19 cells. PEDF-R derived from ARPE-19 cells was an active phospholipase enzyme, which PEDF stimulated to release fatty acids. We genetically engineered ARPE-19, retina R28 and retinal ganglion cell RCG-5 lines to overexpress or silence the PEDF-R expression. Plasma membrane extracts from genetically modified cells overexpressing PEDF-R transcripts had higher phospholipase activity than those from untransfected cells. In contrast, transfection of silencing vectors lowered the levels of PEDF-R transcripts and those for plasma membrane phospholipase activity. The effects of these modified cell lines on oxidative-stress-induced apoptosis cell model were also studied using a label-free real-time assay using electronic cell sensor technology system. The results showed a differential behavior in the uptake of the oxidative agent and the response in cell kinetics upon PEDF addition. Living retina R28 and RCG-5 cells were also monitored by the electronic cell sensor system and cell viability assays. Cells were induced to apoptosis by serum starvation. Treatments with PEDF of increased the electronic index and viability of the cells. [unreadable] [unreadable] We continued examining the interactions between PEDF and ectopic ATP synthase. Plasma membrane fractions from human microvascular endothelial cell (HMVEC) and bovine retina contained a component with affinity for PEDF on SPR surface chips, which were captured with specific antibodies to beta subunit of F1 ATP synthase. Direct binding to highly purified yeast F1-ATPase with a His-tagged beta-subunit showed PEDF binding affinities similar to those with intact cells. PEDF competed with angiostatin K1-5 for F1 binding. PEDF inhibited significantly the extracellular ATP synthesis activity of HMVEC endothelial cells. [unreadable] [unreadable] We completed studies on the interactions of PEDF with hyaluronan. The hyaluronan-binding region was examined by chemical modification and site-directed mutagenesis. In the spatial PEDF structure the hyaluronan-binding motifs are located in areas distinct from previously reported neurotrophic and antiangiogenic active regions, and from the collagen-binding site. [unreadable] [unreadable] The regulation of PEDF by dexamethasone in trabecular meshwork continued to be examined (in collaboration with Terete Borras). We investigated the effect of DEX on metalloproteinases by gelatin solution assays and zymography. While dexamethasone can increase levels of PEDF transcripts and PEDF protein, it decreased the gelatinolytic activities of trabecular meshwork in anterior segments organs and cell cultures.