June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
PEDF Alterations that Modify Receptor Affinity: Implications for the construction of PEDF with optimal biological effects
Author Affiliations & Notes
  • Jeanee Bullock
    Section of Protein Structure and Function, NEI-NIH, Bethesda, MD
    Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC
  • Federica Polato
    Section of Protein Structure and Function, NEI-NIH, Bethesda, MD
  • Patricia Becerra
    Section of Protein Structure and Function, NEI-NIH, Bethesda, MD
  • Footnotes
    Commercial Relationships Jeanee Bullock, None; Federica Polato, None; Patricia Becerra, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 804. doi:
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      Jeanee Bullock, Federica Polato, Patricia Becerra; PEDF Alterations that Modify Receptor Affinity: Implications for the construction of PEDF with optimal biological effects. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):804.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: A 17mer fragment within the neurotrophic 44mer fragment of pigment epithelium-derived factor (PEDF) binds to an extracellular ectodomain of the cell surface receptor PEDF-R to stimulate prosurvival activity. Recently, an alanine scan of the 17mer peptide revealed residues that play a role in the interactions with PEDF-R and in turn retinoprotective activity. The present study tests the hypothesis that these changes in the overall full length PEDF are also important for the interactions with its receptor PEDF-R and biological effects.

Methods: Full length human PEDF gene SERPINF1 was used. Mutant SERPINF1[R99A] and SERPINF1[H105A] were generated by targeting wild-type (WT) SERPINF1 cDNA by Site-Directed Mutagenesis. WT and mutant SERPINF1 cDNA were cloned into p3XFLAG-CMV™-9 expression vector. The resulting expression vectors were transfected into BHK cells. After selection with neomycin, stably transfected cells were cycled between serum and serum-free media every 24 hours. Recombinant proteins were purified from conditioned serum-free media. A purification protocol for the recombinant proteins was optimized. Western blots to detect PEDF and FLAG were performed. PEDF proteins were quantified using ELISA specific for human PEDF. Binding to P1 peptide, the ectodomain of PEDF-R, was assayed by peptide affinity chromatography.

Results: The nucleotide sequences of the cDNA for PEDF fused to three FLAG epitopes at its N-terminus in the expression vectors SERPINF1[WT], SERPINF1[R99A] and SERPINF1[H105A] were confirmed. Recombinant proteins in culture medium of transient and stably-transfected BHK cells transfected with PEDF expression plasmids migrated as expected for 3X-FLAG-PEDF protein. Highly purified recombinant proteins were obtained after cation- and anion-exchange column chromatography. They migrated in SDS-PAGE as 50kDa proteins and immunoreacted with specific PEDF and FLAG antibodies. FLAG-tagged PEDF bound to P1 in a similar fashion as unmodified PEDF, and the PEDF[H105A] version had higher affinity than wild type FLAG PEDF, while PEDF[R99A] was not detected in bound fractions of P1 resins.

Conclusions: Alterations to full length PEDF have similar effects on the affinity for its receptor PEDF-R when compared to the 17mer fragment. The results imply that single point alterations can lead to the generation of new versions of PEDF with enhanced biological activities.

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