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. 2013 Jan;9(1):30-6.
doi: 10.1038/nchembio.1114. Epub 2012 Nov 11.

Identification of DES1 as a vitamin A isomerase in Müller glial cells of the retina

Joanna J Kaylor  1 , Quan Yuan, Jeremy Cook, Shanta Sarfare, Jacob Makshanoff, Anh Miu, Anita Kim, Paul Kim, Samer Habib, C Nathaniel Roybal, Tongzhou Xu, Steven Nusinowitz, Gabriel H Travis
Affiliations

Identification of DES1 as a vitamin A isomerase in Müller glial cells of the retina

Joanna J Kaylor et al. Nat Chem Biol. 2013 Jan.

Abstract

Absorption of a light particle by an opsin-pigment causes photoisomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the resulting apo-opsin requires chemical re-isomerization of the photobleached chromophore. This is carried out by a multistep enzyme pathway called the visual cycle. Accumulating evidence suggests the existence of an alternative visual cycle for regenerating opsins in daylight. Here we identified dihydroceramide desaturase-1 (DES1) as a retinol isomerase and an excellent candidate for isomerase-2 in this alternative pathway. DES1 is expressed in retinal Müller cells, where it coimmunoprecipitates with cellular retinaldehyde binding protein (CRALBP). Adenoviral gene therapy with DES1 partially rescued the biochemical and physiological phenotypes in Rpe65(-/-) mice lacking isomerohydrolase (isomerase-1). Knockdown of DES1 expression by RNA interference concordantly reduced isomerase-2 activity in cultured Müller cells. Purified DES1 had very high isomerase-2 activity in the presence of appropriate cofactors, suggesting that DES1 by itself is sufficient for isomerase activity.

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Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Visual cycle in RPE cells
The light sensitive protein in rod photoreceptors is rhodopsin, located within the membranous outer-segment. Rhodopsin contains the visual chromophore, 11-cis-retinaldehyde (11-cis-RAL). Absorption of a photon (hv) induces 11-cis to all-trans isomerization of the chromophore, converting rhodopsin into active metarhodopsin II. Metarhodopsin II decays to yield apo-opsin and free all-trans-retinaldehyde (all-trans-RAL). The all-trans-RAL is reduced by NADPH-dependent retinol dehydrogenase type-8 (RDH8) to all-trans-retinol (all-trans-ROL). The all-trans-ROL is released by the outer segment into the IPM where it binds IRBP. The all-trans-ROL is taken up by RPE apical processes where it is esterified to a fatty acid by lecithin:retinol acyl transferase (LRAT) utilizing phosphatidylcholine (PC) as an acyl-donor. The resulting all-trans-retinyl esters (all-trans-RE’s) are the substrate for isomerase-1 (Rpe65), which catalyzes both hydrolysis of the ester and isomerization of all-trans-ROL to 11-cis-retinol (11-cis-ROL). The final catalytic step in the visual cycle is oxidation of 11-cis-ROL to 11-cis-RAL by retinol dehydrogenase type-5 (RDH5). 11-cis-RAL is released by the RPE into the IPM where it binds IRBP and is subsequently taken up by a rod outer segment where it recombines with an apo-opsin to form a new rhodopsin pigment molecule.
Figure 2
Figure 2. Immunohistochemical and immunoblot analysis showing DES1 expression in the retina and RPE
(a) Sections from a six-month-old 129/Sv mouse retina were reacted with antibodies against mouse DES1 (green) or CRALBP (red). Nuclei were counter-stained with DAPI (blue). The merged image shows overlapping expression of DES1 and CRALBP (yellow). Morphologic features of the retina are shown to the right of the Nomarski image. RPE, retinal pigment epithelium; IPM, interphotoreceptor matrix; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Note that DES1- and CRALBP-containing apical processes of Müller cells extend into the IPM, which also contains photoreceptor outer segments and the apical processes of RPE cells. The Müller-cell endfeet contact the vitreous within the GCL. Scale bar shows 20 μm. (b) Immunoblots showing DES1 in mouse retina and RPE (left), and chicken retina, RPE and primary cultured Müller cells (right).
Figure 3
Figure 3. Isomerase-2 activities of human membrane desaturases
DES1, DES2, FADS1, FADS2 and SCD were expressed in 293T cells. Cell homogenates were assayed for synthesis of 11-cis-ROL (a), 9,13-di-cis-ROL (b), 9-cis-ROL (c) and 13-cis-ROL (d), using all-trans-ROL as substrate. Activities are expressed as pmoles per minute per mg-total protein (n=3; s.e.m.).
Figure 4
Figure 4. Levels of visual chromophore and ERG amplitudes in rpe65 −/− mice after intravitreal injection of ad-DES1 or ad-RFP
(a) Normal-phase chromatogram showing elution of retinaldehyde oximes (ROX) and retinols in extracts of rpe65 −/− retinas following intravitreal injection of ad-DES1 (red tracing) or ad-RFP (gray tracing). Peaks containing syn- and anti- oximes for all-trans-RAL and 9-cis-RAL are labeled. Note the elevated 9-cis-RAL (syn- and anti-9-cis-ROX). 11-cis-RAL oximes were undetectable in these mice that received no supplemental all-trans-ROL. (b) Levels of 11-cis-RAL and 9-cis-RAL in extracts of rpe65 −/− retinas following intravitreal injection of ad-DES1 (black bars) or ad-RFP (gray bars). Mice in this experiment received 5.0 nmoles of intravitreal all-trans-ROL supplementation. Background levels of 9-cis-RAL and 11-cis-RAL in ad-RFP-injected rpe65 −/− eyes were from thermal isomerization and oxidation of the added all-trans-ROL substrate (n=9; s.e.m.). (c) Representative dark-adapted ERGs recorded to the brightest flash (5.791 cd-s/m2) modulated at 10 Hz in rpe65 −/− mice after intravitreal injection of ad-DES1 (black tracing) or ad-RFP (gray tracing). Shown are the first 200 msec of the responses. Tracings are vertically displaced for clarity (d) Interocular differences in Fourier-derived ERG responses between eyes injected with ad-DES1 versus ad-RFP. Positive interocular amplitude differences (values > 0) indicate that the eye receiving the ad-DES1 injection performed better than the ad-RFP eye (n=9; s.d.; * p<0.05, ** p<0.01).
Figure 5
Figure 5. Co-immunoprecipitation of CRALBP with DES1 and effects of CRALBP on isomerase-2 activity
(a) Immunoblots of protein samples detected with antibodies against chicken DES1 or CRALBP. Samples include the starting homogenates (load sample); unbound samples after incubation with immobilized DES1 or CRALBP antibody (flow through); unbound samples after the first (wash 1) or final (wash 4) wash of the column with wash/lysis buffer; unbound samples after washing with Conditioning buffer (conditioning buffer); and eluted proteins after washing with elution buffer (eluate). Homogenates were from chicken retinas (first and second rows of blots) or 293T-cells expressing CRALBP (third row of blots). (b) Isomerase-2 activities of 293T-D cell homogenates that stably express DES1 minus or plus transient transfection with CRALBP. Figures show synthesis rates of 11-cis-ROL, 9,13-di-cis-ROL, 9-cis-ROL, and 13-cis-ROL expressed as specific activities (pmol/min/mg-total protein) (n=3; s.e.m.).
Figure 6
Figure 6. Proposed alternate visual cycle in Müller cells
Cone opsins use 11-cis-RAL or 9-cis-RAL (9/11-cis-RAL) as visual chromophore. Absorption of a photon by a cone opsin isomerizes the 9/11-cis-RAL to all-trans-RAL, as in rods. After reduction by RDH8 in the cone OS, the all-trans-ROL is released into the IPM and taken up by a Müller cell. Here, the all-trans-ROL is isomerized by DES1 to 11-cis-ROL, which is bound to CRALBP, and to 9-cis-ROL and 13-cis-ROL, which are not. CRALBP interacts with DES1 and protects 11-cis-ROL from further isomerization. Interaction with negatively charged phospholipids causes holo-CRALBP to release its 11-cis-ROL ligand into the IPM, where it binds IRBP and subsequently is taken up by a cone outer segment. In contrast, the 9-cis-ROL diffuses directly into the IPM without binding to CRALBP. In the cone OS, 9-cis- and 11-cis-ROL (9/11-cis-ROL) are oxidized by an unknown NADP+-dependent retinol dehydrogenase to 9/11-cis-RAL, which combine with apo-opsin to form a new opsin pigment. Simultaneous reduction of all-trans-RAL and oxidation of 9/11-cis-ROL in the cone OS provides a self-renewing supply of NADPH/NADP+ cofactors. Müller cells express RDH10, which non-specifically oxidizes retinol isomers to their cognate aldehydes. The resulting 9/11-cis-RAL may be utilized as chromophore by rod or cone apo-opsins. This is the likely source of iso-rhodopsin in rpe65 −/− retinas.
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References

    1. Crouch R, Purvin V, Nakanishi K, Ebrey T. Isorhodopsin II: artificial photosensitive pigment formed from 9,13-dicis retinal. Proceedings of the National Academy of Sciences of the United States of America. 1975;72:1538–42. - PMC - PubMed
    1. Jin M, Li S, Moghrabi WN, Sun H, Travis GH. Rpe65 is the retinoid isomerase in bovine retinal pigment epithelium. Cell. 2005;122:449–59. - PMC - PubMed
    1. Redmond TM, et al. Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle. Proc Natl Acad Sci U S A. 2005;102:13658–63. - PMC - PubMed
    1. Goldstein EB. Cone pigment regeneration in the isolated frog retina. Vision Res. 1970;10:1065–8. - PubMed
    1. Hood DC, Hock PA. Recovery of cone receptor activity in the frog’s isolated retina. Vision Research. 1973;13:1943–51. - PubMed

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