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S. Locatelli-Hoops, L. Notari, S. P. Becerra; Identification of Structural Determinants on PEDF-R Responsible for Binding PEDF. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5768. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Pigment Epithelium-derived Factor-Receptor (PEDF-R) is a cell-surface transmembrane protein present in the retina that displays phospholipase A (PLA) activity. Its ligand, PEDF, binds with high affinity to PEDF-R and stimulates its PLA activity. PEDF, a neurotrophic and antiangiogenic factor, has a biological active site located in the negatively charged side of its 3-D structure. This study aims at identifying structural determinants on PEDF-R important for the PEDF receptor-ligand interaction.
PEDF protein was obtained by recombinant expression in BHK cells and purified from the media by cation- and anion-exchange column chromatography. Peptides were designed from PEDF-R and chemically synthesized by a commercial source. Protein-peptide interactions were analyzed by Surface Plasmon Resonance (SPR) using a PEDF surface sensor chip. Purified PEDF was immobilized on surface sensor chips by amine coupling. Controls were sensor chips without PEDF.
Peptide regions were designed from the longest extracellular loop of PEDF-R and were termed according to their exon mapping on the PEDF-R gene. Sequence analyses of peptides E4a, E4b, E5, E5+6a, and E7a revealed that E4b, E5 and E5+6a were positively charged (basic), while the remaining had an acidic nature. All peptides were soluble in aqueous solutions. SPR spectroscopy of the synthetic peptides showed that only E4b and E5+6a bound to PEDF. Sensograms showed that they associated and dissociated from the PEDF surface in a specific fashion and with high and medium affinities, respectively. Acidic peptides did not bind to PEDF. Addition of NaCl to the SPR running buffer decreased the sensogram response of E4b and E5+6a. Sensograms of combinations of E4b and E5+6a showed an additive rather than a synergistic effect on the binding to PEDF.
We have identified two basic peptide regions of PEDF-R as structural determinants for PEDF binding. Electrostatic forces play a role in these interactions. These results suggest that a negatively charged area in PEDF would interact with these basic PEDF-R regions and constitute an apparent receptor binding site on the ligand.
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