Abstract
The cytoprotective effects of pigment epithelium-derived factor (PEDF) require interactions between an as of a yet undefined region with a distinct ectodomain on the PEDF receptor (PEDF-R). Here we characterized the area in PEDF that interacts with PEDF-R to promote photoreceptor survival. Molecular docking studies suggested that the ligand binding site of PEDF-R interacts with the neurotrophic region of PEDF (44-mer, positions 78-121). Binding assays demonstrated that PEDF-R bound the 44-mer peptide. Moreover, peptide P1 from the PEDF-R ectodomain had affinity for the 44-mer and a shorter fragment within it, 17-mer (positions 98-114). Single residue substitutions to alanine along the 17-mer sequence were designed and tested for binding and biological activity. Altered 17-mer[R99A] did not bind to the P1 peptide, whereas 17-mer[H105A] had higher affinity than the unmodified 17-mer. Peptides 17-mer, 17-mer[H105A], and 44-mer exhibited cytoprotective effects in cultured retina R28 cells. Intravitreal injections of these peptides and PEDF in the rd1 mouse model of retinal degeneration decreased the numbers of dying photoreceptors, 17-mer[H105A] being most effective. The blocking peptide P1 hindered their protective effects both in retina cells and in vivo. Thus, in addition to demonstrating that the region composed of positions 98-114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects, the findings reveal distinct small PEDF fragments with neurotrophic effects on photoreceptors.
Highlights
Pigment epithelium-derived factor (PEDF) interacts with its receptor PEDF-R to exert cytoprotection
The model predicts that the interaction between PEDF and the P1 fragment is centered around Glu101, with a combination of backbone and side chain atoms providing contacts and that likely the first six residues of the 44-mer do not affect binding
The results suggest that P1 peptide folds into ␣ helical structure and binds to a cleft in PEDF that contains the neurotrophic 44-mer, with helix C of PEDF being in closer proximity to the P1 peptide region of PEDF-R
Summary
Pigment epithelium-derived factor (PEDF) interacts with its receptor PEDF-R to exert cytoprotection. In addition to demonstrating that the region composed of positions 98 –114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects, the findings reveal distinct small PEDF fragments with neurotrophic effects on photoreceptors. We have mapped the ligand-binding domain (LBD) to an extracellular loop of the human PEDF-R (positions 203–232) and shown that peptides derived from this ectodomain act as soluble receptor fragments that block both PEDF/PEDF-R interactions and PEDF-mediating survival action [23]. The data support the pharmacotherapeutic potential of PEDF as a neuroprotectant in retinal degeneration
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