June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Light-driven regeneration of 11-cis-retinal in the retinal pigment epithelium
Author Affiliations & Notes
  • Elliot H Choi
    Pharmacology, Case Western Reserve University, Cleveland, Ohio, United States
    Ophthalmology, Gavin Herbert Eye Institute, Irvine, California, United States
  • Jianye Zhang
    Ophthalmology, Gavin Herbert Eye Institute, Irvine, California, United States
  • Aleksander Tworak
    Ophthalmology, Gavin Herbert Eye Institute, Irvine, California, United States
  • David Salom
    Ophthalmology, Gavin Herbert Eye Institute, Irvine, California, United States
  • Grazyna Palczewska
    Medical Devices, Polgenix, Inc, Irvine, California, United States
  • Philip David Kiser
    Physiology and Biophysics, University of California, Irvine, Irvine, California, United States
  • Krzysztof Palczewski
    Ophthalmology, Gavin Herbert Eye Institute, Irvine, California, United States
  • Footnotes
    Commercial Relationships   Elliot Choi, None; Jianye Zhang, None; Aleksander Tworak, None; David Salom, None; Grazyna Palczewska, None; Philip Kiser, None; Krzysztof Palczewski, None
  • Footnotes
    Support  NIH EY009339, EY027283, EY025451 and EY019312, NIH T32GM007250 and T32GM008803, VA IK2BX002683
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 1519. doi:
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    • Get Citation

      Elliot H Choi, Jianye Zhang, Aleksander Tworak, David Salom, Grazyna Palczewska, Philip David Kiser, Krzysztof Palczewski; Light-driven regeneration of 11-cis-retinal in the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1519.

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

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Abstract

Purpose : The retina requires a continuous supply of 11-cis-retinal for activation of rod and cone photoreceptors. One source of regenerated 11-cis-retinal is through a series of enzymatic reactions referred to as the classical visual cycle. However, it remains unclear whether an additional pathway that operates upon light exposure also exists. We hypothesize that light can drive regeneration of 11-cis-retinal through a biochemical process in the retinal pigment epithelium (RPE) and contribute to the maintenance of daytime vision.

Methods : Microsomes prepared from fresh bovine RPE were illuminated with different light conditions (n = 3) to examine the isomerization of all-trans-retinal to 11-cis-retinal. Size-exclusion chromatography was then used to obtain the fractions containing the photoisomerase activity. Mass spectrometry was used to identify the photoisomerase as RGR (the retinal G protein-coupled receptor) in bovine RPE. Recombinant RGR was subsequently purified and evaluated for photoisomerase activity under different light conditions (n = 3-4). Lastly, we generated a HEK 293S GnTI- cell line stably expressing RGR for further studies.

Results : Bovine RPE had a photosensitive response resulting in the production of 11-cis-retinal, which scaled with light intensity. We found that RGR was responsible for the photoisomerization. The presence of cellular retinaldehyde-binding protein (CRALBP) strongly enhanced (640 %) the generation of 11-cis-retinal by RGR. Using recombinant RGR, we confirmed that RGR accounts for the photoisomerase activity found in the RPE. The substitution of a lysine with an alanine at position 255 in RGR abolished the photoisomerization (P<0.001). To achieve a higher yield of RGR, we established a stable mammalian expression system and optimized purification of functional RGR. Illumination of purified RGR produced 11-cis-retinal (536±121 pmol/reaction), whereas purified RGR in the dark did not. We also expressed LRAT and RPE65 in the expression system to explore the interplay between the two isomerases.

Conclusions : RGR expressed in RPE is responsible for light-driven production of 11-cis-retinal. CRALBP enhances this activity by binding the 11-cis-retinal produced by RGR and prevents its re-isomerization to all-trans-retinal. Our results indicate that the light-driven pathway works in a complementary fashion to the classical visual cycle to regenerate visual chromophore in daylight conditions.

This is a 2020 ARVO Annual Meeting abstract.

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