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NIH Research Festival

September 16 – 18, 2015

Alterations on pigment epithelium-derived factor (PEDF) that modify receptor affinity

Wednesday, September 16, 2015 – Poster Session I
3:30 – 5:00 p.m.

FAES Terrace

NEI

MOLBIO-5

Authors

  • JL Bullock
  • F Polato
  • V Marigo
  • SP Becerra

Abstract

PEDF, a protective protein of the eye, is a multifunctional secreted serpin with neurotrophic properties. Our lab previously identified a cell surface receptor, PEDF-R, with high binding affinity for PEDF. A neurotrophic peptide from positions 98-114 of PEDF binds to an extracellular ectodomain of PEDF-R to stimulate prosurvival activity. An alanine scan of the 17-mer peptide revealed that R99 is critical for receptor binding and prosurvival activity, while an H105A alteration enhances these activities. The present study tests the hypothesis that these changes in the overall full-length PEDF are also important for the interactions with PEDF-R and biological effects. We created full-length constructs containing single point mutations to change each R99 and H105 to alanine in human PEDF fused to a 3X-FLAG tag and transfected them into BHK cells. Conditioned serum-free media containing the recombinant proteins were harvested and purified by a two-step ion-exchange column chromatography. Circular dichroism spectra indicated that the three recombinant PEDF versions share similar fold. Peptide affinity chromatography revealed higher binding of FLAG-PEDF[H105A] than FLAG-PEDF to P1, a peptide from the PEDF-R ectodomain. P1 binding of FLAG-PEDF[R99A] was undetectable. Photoreceptor survival assays in vivo showed that FLAG-PEDF and FLAG-PEDF[H105A] decreased the number of TUNEL-positive nuclei in the rd1 mouse model of retina degeneration, with FLAG-PEDF[H105A] being more effective than the unmodified version. The results demonstrate that the R99 in PEDF plays a critical role in binding the receptor, while alteration at H105 to alanine generates PEDF molecules with enhanced survival activities.

Scientific Focus Area: Molecular Biology and Biochemistry

This page was last updated on Friday, March 26, 2021

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