Protein Interactions in Retina Development and Function

National Eye Institute

Investigators

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Abstract

Work in this research group is aimed at elucidating the mechanisms that regulate the activities of pigment epithelium-derived factor (PEDF), an extracellular serpin with neurotrophic and antiangiogenic activities. We continued working on the characterization of a putative receptor for PEDF, PEDF-R, and demonstrated that this novel protein had binding affinity for PEDF and phospholipase activity. We compared the affinities of PEDF and other serpins to PEDF-R, engineered cells overexpressing the PEDF-R gene, constructed mutants and deletion versions of PEDF-R, and developed antibodies to this interesting protein. We determined its subcellular localization to the cell-surface and cytosol of mammalian cells using antibodies to predicted extracellular and intracellular loops. We exposed live cells to flourescein-conjugated PEDF (Fl-PEDF) and showed fluorescent staining on cell-surfaces in a dose-dependent and specific fashion. We examined the effects of PEDF on extracellular ATP synthesis activity of endothelial cells and compared them to those by angiostatin and ATP synthase inhibitors, and found that all of them inhibited significantly the extracellular ATP synthesis activity. We examined the direct binding of PEDF to highly purified yeast F1-ATP synthase with a His-tagged a-subunit. Size-exclusion ultrafiltration, Ni-NTA pull-down and Surface Plasmon Resonance assays showed that PEDF bound to F1 with similar affinities as those between PEDF and intact endothelial cells. We continued to develop and optimize a method for evaluating and quantifying chroroidal neovascularization (CNV) in rodents. CNV lesions were induced with laser and the morphology and volume of the lesions evaluated in choroid/RPE flat mounts by confocal microscopy using 3-D reconstructions. We studied the structure-function relationships of PEDF?s antiangiogenic activity. We compared the efficacy of PEDF peptides, other antiangiogenic factors and serpins using tube formation, an ex vivo chick aortic arch and the in vivo CNV assay. We found that PEDF and 34-mer, a small PEDF peptide towards the amino-end region, inhibited angiogenesis in these assays. We examined the direct binding of PEDF to hyaluronan by coprecipitation with cetylpyridinium chloride. We prepared site-specific mutants encoding modified PEDF at a putative hyaluronan binding region and found that single point alterations reduced >70% the binding affinity relative to the unmodified protein, while cumulative modifications abolished the hyaluronan-binding activity of PEDF. All the modified proteins retained binding affinity to collagen similar to that of the unmodified protein. These results showed that the homologous hyaluronan-binding motif is a functional region in this serpin. In the spatial PEDF structure it is located between helix F and strand s3A, and is distinct from the previously reported collagen- and heparin-binding regions.