June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
The Putative Iron-Binding Histidines of ELOVL1 are Essential for Inhibiting 11-cis-Retinol Synthesis Catalyzed by RPE65 Isomerase
Author Affiliations & Notes
  • Minghao Jin
    Neuroscience Center of Excellence & Department of Ophthalmology, LSU School of Medicine, New Orleans, Louisiana, United States
  • Lauren N Gottschalk
    Neuroscience Center of Excellence, LSU School of Medicine, New Orleans, Louisiana, United States
  • Songhua Li
    Neuroscience Center of Excellence, LSU School of Medicine, New Orleans, Louisiana, United States
  • Footnotes
    Commercial Relationships   Minghao Jin, None; Lauren Gottschalk, None; Songhua Li, None
  • Footnotes
    Support  NIH Grant EY028572
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 4976. doi:
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      Minghao Jin, Lauren N Gottschalk, Songhua Li; The Putative Iron-Binding Histidines of ELOVL1 are Essential for Inhibiting 11-cis-Retinol Synthesis Catalyzed by RPE65 Isomerase. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4976.

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

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Abstract

Purpose : We have previously identified elongation of very long-chain fatty acids-like1 (ELOVL1) as an inhibitor of 11-cis-retinol synthesis catalyzed by RPE65, an iron-dependent retinoid isomerase in the visual cycle. ELOVL1 has a histidine-rich motif that is conserved in all ELOVL family members and is thought to bind iron. The purpose of this study was to investigate whether the putative iron-binding His residues are important for ELOVL1 to inhibit synthesis of 11-cis-retinol.

Methods : Using site-directed mutagenesis kit and pRK-ELOVL1 plasmid, we generated three mutant ELOVL1 constructs with a H141R, H144R, or H145R substitution. These ELOVL1 cDNAs were individually co-expressed at different levels with RPE65 in the 293T-LC cells stably expressing LRAT. Expression levels and subcellular distribution patterns of proteins were analyzed by immunoblot and immunohistochemical analyses. Activities of RPE65 and LRAT were determined by measuring the synthesis of 11-cis-retinol or all-trans retinyl esters from all-trans-retinol substrate incubated with the cells for different times. Retinoids were analyzed by high performance liquid chromatography.

Results : Immunoblot and immunochemical analyses showed that the His to Arg substitutions did not significantly alter expression levels and subcellular distribution patterns of ELOVL1 and RPE65 in the cells. Synthesis of 11-cis-retinol, but not all-trans retinyl esters, were strongly inhibited in the 293T-LC cells co-transfected with RPE65 and wild-type ELOVL1, as compared to the cells cotransfected with RPE65 and pRK mock vector. Similar to wild-type ELOVL1, all ELOVL1 mutants did not exhibit significant effects on the synthesis of all-trans retinyl esters. However, the His to Arg substitution at any one of the three positions almost completely abolished the inhibitory effect of ELOVL1 on the synthesis of 11-cis-retinol, although the expression levels of the ELOVL1 mutants were similar to that of wild-type ELOVL1 in the cells.

Conclusions : ELOVL1 inhibited synthesis of 11-cis-retinol, but not all-trans retinyl ester. Substitution of any histidines in the putative iron-binding motif resulted in complete loss of inhibitory activity of ELOVL1 on the synthesis of 11-cis-retinol. These data suggest that competing with RPE65 for iron cofactor is a mechanism by which ELOVL1 inhibits synthesis of 11-cis-retinol.

This is a 2020 ARVO Annual Meeting abstract.

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