Abstract
Purpose:
Pigment epithelium-derived factor (PEDF) protects the retina by blocking pathogenic angiogenesis, and by protecting the neural retina against degeneration. Two biologically active PEDF-derived peptides have been discovered, an antiangiogenic 34mer and a neurotrophic 44mer, which bind distinct receptors; not yet elucidated. Our lab has identified a PEDF receptor, PEDF-R and mapped its ligand binding domain to amino acid positions 203-232 of the human PEDF-R. However, the region on PEDF that binds PEDF-R has not been identified. Therefore, this study is designed to map the region on PEDF that binds PEDF-R to exert biological activity.
Methods:
Peptides designed from the human PEDF sequence, 34mer (44-77), 44mer (78-121), 17mer (98-104) and alanine scan of 17mer peptides, and from human PEDF-R, P1 (210-249) were chemically synthesized and purified (Biosynthesis, Inc.). Binding was determined by ligand blotting, pull-down, surface plasmon resonance (SPR), and fluorescence anisotropy. The structure of P1 was predicted in an ab initio model, and the resultant structure was then used in a global docking search using the Rosetta software. Retina survival assays were performed with rat retina precursor R28 cells induced to cell death by serum starvation, and measuring apoptosis by TUNEL staining.
Results:
The 44mer peptide bound specifically to P1 peptide by ligand blot, SPR, and anisotropy assays; whereas, the 34mer did not bind. The 44mer, and not the 34mer, bound to full-length PEDF-R in a pull-down assay. The affinity of the 44mer:P1 interaction calculated from ligand blot data was KD=70±7.7 nM compared to 9.1±1.1 nM for that of PEDF:P1. The 17mer bound P1 with a KD=410±80 nM. Alanine scanning of the 17mer demonstrated that R99 of 17mer was critical for P1 binding, and that 17mer[H105A] variant bound P1 more tightly than the unmodified peptide. Molecular modeling revealed that P1 can fold into three alpha helices and dock in the 44mer region of the PEDF protein. Like PEDF, the 44mer peptide, protected R28 cells from apoptosis induced by serum deprivation, but the 34mer had no neuroprotective effect.
Conclusions:
Our results identify a novel PEDF-R binding site on PEDF in its neurotrophic region. Further, these data support the role of PEDF-R as a neurotrophic receptor for PEDF.
Keywords: 615 neuroprotection •
659 protein structure/function •
449 cell survival