April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Mechanism for RPE65 Mutation-Induced Vision Impairment
Author Affiliations & Notes
  • Olga Nikolaeva
    Department of Physiology, Oklahoma Univ Health Science Ctr, Oklahoma City, Oklahoma
  • Gennadiy P. Moiseyev
    Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, Oklahoma
  • Yusuke Takahashi
    Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, Oklahoma
  • Jian-Xing Ma
    Medicine, Physiology,
    Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, Oklahoma
  • Footnotes
    Commercial Relationships  Olga Nikolaeva, None; Gennadiy P. Moiseyev, None; Yusuke Takahashi, None; Jian-Xing Ma, None
  • Footnotes
    Support  NIH grants EY018659, EY012231, EY019309, P20RR024215 and ADA
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 3644. doi:
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      Olga Nikolaeva, Gennadiy P. Moiseyev, Yusuke Takahashi, Jian-Xing Ma; Mechanism for RPE65 Mutation-Induced Vision Impairment. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3644.

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

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Purpose: : RPE65 is isomerohydrolase, a key enzyme in the visual cycle. Point mutations in the RPE65 gene were found in patients with retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). The mechanism by which these mutations lead to impaired vision is unknown. The purpose of this study is to reveal how patients’ mutations of RPE65 affect its enzymatic activity and substrate binding.

Methods: : Recombinant human RPE65 (hRPE65) with a His-tag and its mutants A132T (RP), A132V, A132L, and E102K (LCA) were generated by site-directed mutagenesis. Wt hRPE65 and the mutants were expressed in 293-LRAT cells by the adenovirus system and purified using affinity chromatography. The subcellular fractionation was performed using FractPrepTM kit. Catalytic activities of hRPE65 and the mutants were quantified by HPLC. The binding affinity of all-trans retinyl ester towards wt hRPE65 and its mutants (A132T and E102K) was measured by monitoring quenching of RPE65 tryptophan emission.

Results: : Expression levels of the mutants were found similar to that of wt hRPE65. The wt hRPE65 showed a high stability with a half-life more than 10h. The mutants have reduced half-lives, suggesting that the mutations decrease protein stability. The subcellular fractionation revealed that considerable amount of wt and A132T were located in membrane fractions whereas the other mutants were predominantly found in the cytoskeleton fraction. These results suggest that mutations lead to disruption of hRPE65-membrane association. Wt hRPE65 generated significant amount of 11-cis retinol. The A132T produced 5-fold less 11-cis retinol than wt hRPE65 at a similar expression level, whereas the other mutants had no detectable catalytic activity. The wt hRPE65 demonstrated atRE-binding affinity with Kd=2.6 nM whereas A132T had lower binding affinity with Kd=9.0 nM. No atRE binding was detected for E102K mutant.

Conclusions: : A132T mutations decreased stability and membrane association of RPE65. Together with the lower substrate binding affinity, it leads to an impaired catalytic activity and in turn, debilitates the visual cycle. The lack of activity of the E102K mutant corresponds to the absence of a detectable substrate binding that halts the visual cycle.

Keywords: protein structure/function • retinoids/retinoid binding proteins • protein purification and characterization 

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