September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Differential rescue of protein expression and localization of two adRP-linked PRPH2 mutants in rods is mediated by their specific binding characteristics
Author Affiliations & Notes
  • Elvir Becirovic
    Department of Pharmacology - Center for Drug Research, Ludwig-Maximilians Universitaet, Munich, Germany
  • Sybille Böhm
    Department of Pharmacology - Center for Drug Research, Ludwig-Maximilians Universitaet, Munich, Germany
  • Ong Nam Phuong Nguyen
    Department of Pharmacology - Center for Drug Research, Ludwig-Maximilians Universitaet, Munich, Germany
  • Lisa Maria Riedmayr
    Department of Pharmacology - Center for Drug Research, Ludwig-Maximilians Universitaet, Munich, Germany
  • Nadja Schroeder
    Department of Biology, Animal Physiology, University of Erlangen-Nuremberg, Erlangen, Germany
  • Andreas Giessl
    Department of Biology, Animal Physiology, University of Erlangen-Nuremberg, Erlangen, Germany
  • Stylianos Michalakis
    Department of Pharmacology - Center for Drug Research, Ludwig-Maximilians Universitaet, Munich, Germany
  • Martin Biel
    Department of Pharmacology - Center for Drug Research, Ludwig-Maximilians Universitaet, Munich, Germany
  • Footnotes
    Commercial Relationships   Elvir Becirovic, None; Sybille Böhm, None; Ong Nguyen, None; Lisa Riedmayr, None; Nadja Schroeder, None; Andreas Giessl, None; Stylianos Michalakis, None; Martin Biel, None
  • Footnotes
    Support  Deutsche Forschunggemeinschaft BE 4830/1-1
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 1170. doi:
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      Elvir Becirovic, Sybille Böhm, Ong Nam Phuong Nguyen, Lisa Maria Riedmayr, Nadja Schroeder, Andreas Giessl, Stylianos Michalakis, Martin Biel; Differential rescue of protein expression and localization of two adRP-linked PRPH2 mutants in rods is mediated by their specific binding characteristics. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1170.

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

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Abstract

Purpose : Some autosomal-dominant retinitis pigmentosa (adRP)-linked mutations in the photoreceptor outer segment (OS)-specific peripherin-2 (PRPH2) interfere with binding to wild type (WT) PRPH2, leading to protein mislocalization and reduced protein expression in rods. Using recombinant adeno-associated virus (rAAV)-mediated gene delivery in combination with FRET on isolated murine rod OS, this study addressed two questions: i) Is it possible to rescue protein expression or localization of two adRP-linked PRPH2 mutants, P210L and C214S, by a simultaneous co-delivery of WT PRPH2? ii) What is the role of protein-protein interactions between the single mutants and WT PRPH2 in this context?

Methods : WT mice (N=3) were injected on P14 with rAAV particles bearing WT+WT, WT+P210L, or WT+C214S PRPH2 constructs fused to FRET fluorophores citrine or cerulean driven by the human Rhodopsin promoter. Rescue efficiencies were analyzed via fundus photography, confocal and transmission electron microscopy, and Western blotting of injected retinas 3 weeks post injection. FRET was measured 4 weeks post injection on isolated rod OS for the WT+WT (N=17), WT+P210L (N=17), and WT+C214S (N=14) combinations. For binding curves, FRET ratio was plotted against the cerulean/citrine molar ratio. The relative maximal binding affinity given by the maximal FRET ratio (FRmax) and the relative Kd values were calculated using the FR=(FRmax+x)/(Kd+x) formula.

Results : Simultaneous co-delivery of WT PRPH2 led to an almost full rescue of protein expression and localization for the C214S mutant and to a partial rescue of these parameters for the P210L mutant. WT+WT interaction yields a Kd=0.06±0.02 and FRmax=5.66±0.42. The C214S mutant increases the Kd to 0.12±0.04 and reduces the FRmax to 2.15±0.23. The Kd was even stronger increased for the P210L mutant (0.21±0.08) explaining its lower rescue efficiency. Surprisingly, however, the FRmax of P210L was increased up to the highest levels detectable via FRET (11.35±1.31).

Conclusions : We show that differential rescue effects of WT PRPH2 on protein expression and localization of two adRP-linked PRPH2 mutants in photoreceptors is shaped by their differential binding characteristics. We also provide a proof-of principle for rAAV-based ex vivo FRET as a tool to address specific binding properties of proteins in a subcellular compartment.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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