rpe65a encodes retinoid isomerohydrolase RPE65a, one of three zebrafish RPE65 paralogs and the one expressed in the retinal pigment epithelium (rpe65b is extra-ocular; rpe65c is expressed in retinal Muller glia). It is an Fe(II)-dependent member of the carotenoid-oxygenase fold family that, unlike its BCO1/NinaB relatives, does not use O2: it cleaves the O-alkyl bond of membrane-stored all-trans-retinyl esters and couples the cleavage to trans-cis isomerization, releasing 11-cis-retinol (EC 3.1.1.64), which is oxidized to the 11-cis-retinal chromophore of rod and cone visual pigments. The protein cycles between a soluble cytoplasmic form and a palmitoylated, membrane-anchored form. Mammalian RPE65 additionally isomerizes lutein to meso-zeaxanthin. In zebrafish, rpe65a is expressed in the pineal gland from 16 hpf and in the RPE from 40 hpf; morpholino knockdown lowers ocular 11-cis-retinal and disrupts rod outer segments, while cone vision persists because larval cones can be supplied through an RPE65-independent, Muller-cell visual cycle.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0042574 retinal metabolic process | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000057192 (carotenoid oxygenase family: Drosophila ninaB, mouse Bco1/Bco2/Rpe65, human BCO1) asserts retinal metabolism. Rpe65a produces the 11-cis-retinol that is oxidized to the 11-cis-retinal chromophore, and rpe65a knockdown lowers 11-cis-retinal in larval eyes (PMID:17868371). Reason: The gene product performs the isomerization step that generates the cis-retinoid precursor of 11-cis-retinal; in vivo, morphants have reduced 11-cis-retinal and disorganized rod outer segments, so the zebrafish protein sits inside the clade function. Accepted as the core process. Supporting Evidence: PMID:17868371 Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11- cis -retinal levels. PMID:32238432 Zebrafish rpe65a knockdown significantly reduced but did not completely attenuate 11cRAL synthesis, supporting the presence of an alternative pathway for chromophore synthesis in zebrafish retinae file:DANRE/rpe65a/rpe65a-uniprot.txt FUNCTION: Plays important roles in the production of 11-cis retinal and in visual pigment regeneration. file:DANRE/rpe65a/rpe65a-deep-research-falcon.md only one paralog, RPE65a, is expressed in the zebrafish eye (in the RPE) |
| GO:0003834 beta-carotene 15,15'-dioxygenase activity | IBA GO_REF:0000033 | REMOVE | Summary: PAINT node PTN000830314 is a deep carotenoid-oxygenase family node seeded by BCO1 orthologs (mouse Bcmo1, rat Bco1, human BCO1, chicken BCO1); the RPE65 clade descends from it and so inherits beta-carotene 15,15'-dioxygenase activity (all-trans-beta-carotene + O2 -> 2 all-trans-retinal). Reason: The RPE65 lineage diverged from the oxygenases: its primary chemistry is O-alkyl cleavage of a retinyl ester coupled to trans-cis isomerization, with no O2 incorporation, whereas carotenoid oxygenation is the chemistry of NinaB/BCO1 (PMID:38355721). The node placement therefore over-reaches for the RPE65 subclade, which shows lineage-specific loss of the dioxygenase reaction even though it keeps the Fe(II) center. No RPE65 ortholog has been shown to cleave beta-carotene, and the zebrafish enzyme is annotated by UniProt only with EC 3.1.1.64 and the lutein isomerase reaction. Removed as an over-propagated family inference; this does not touch the BCO1 annotations. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE PSEUDO OR SUBACTIVITY LOSS Sources checked: PANTHER:PTN000830314 SUPPORTS SOURCE BUT NOT TARGET Node spans BCO1 oxygenases and the RPE65 isomerohydrolases; the dioxygenase activity is genuine for the BCO1 descendants but was lost in the RPE65 subclade, which hydrolyses retinyl esters without incorporating O2. Supporting Evidence: PMID:38355721 two homologous enzymes catalyzing distinct primary chemistry on disparate substrates—retinyl ester hydrolysis in the case of RPE65 and carotenoid oxygenation in the case of NinaB—both possess the ability to catalyze trans–cis isomerization at a C11–C12 retinoid/carotenoid double bond file:DANRE/rpe65a/rpe65a-uniprot.txt Reaction=an all-trans-retinyl ester + H2O = 11-cis-retinol + a fatty acid + H(+); Xref=Rhea:RHEA:31771 file:DANRE/rpe65a/rpe65a-uniprot.txt Name=Fe(2+); Xref=ChEBI:CHEBI:29033 |
| GO:0052885 all-trans-retinyl-ester hydrolase, 11-cis retinol forming activity | IBA GO_REF:0000033 | ACCEPT | Summary: The EC 3.1.1.64 isomerohydrolase reaction (all-trans-retinyl ester + H2O -> 11-cis-retinol + fatty acid), the defining activity of RPE65. For the IBA, PAINT node PTN002576077 is the RPE65 subclade (human, bovine, dog, mouse, rat RPE65 and a second zebrafish RPE65 paralog); ISS transfers from bovine RPE65 and the Rhea/ARBA IEA give the same term. UniProt assigns EC 3.1.1.64 to the zebrafish entry by similarity. Reason: Zebrafish Rpe65a has not itself been assayed in vitro (PMID:22512451 notes this explicitly), so the activity rests on orthology plus in vivo genetics: it is the only RPE65 paralog expressed in the RPE, and morpholino knockdown reduces 11-cis-retinal and damages rod outer segments (PMID:17868371), with residual chromophore attributed to an RPE65-independent cone pathway (PMID:32238432). The transfer within the RPE65 subclade is sound and the term is the core molecular function. Supporting Evidence: file:DANRE/rpe65a/rpe65a-uniprot.txt Reaction=an all-trans-retinyl ester + H2O = 11-cis-retinol + a fatty acid + H(+); Xref=Rhea:RHEA:31771 file:DANRE/rpe65a/rpe65a-uniprot.txt EC=3.1.1.64 PMID:17868371 Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11- cis -retinal levels. PMID:22512451 It is noteworthy that the enzymatic activity of RPE65a in zebrafish has not been studied or characterized. PMID:32238432 Zebrafish rpe65a knockdown significantly reduced but did not completely attenuate 11cRAL synthesis, supporting the presence of an alternative pathway for chromophore synthesis in zebrafish retinae |
| GO:1901827 zeaxanthin biosynthetic process | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT node PTN002576077 (RPE65 subclade) asserts zeaxanthin biosynthesis from the human RPE65 lutein-to-meso-zeaxanthin isomerase finding; UniProt lists the lutein = (3R,3'S)-zeaxanthin reaction (EC 5.3.3.22) on the zebrafish entry by similarity. Reason: The node is seeded by one human experimental annotation, which is legitimate for an IBA, and there is no zebrafish evidence either for or against a lutein isomerase side-reaction; the reaction is mechanistically related (C-C double-bond isomerization by the same Fe(II) active site) so there is no basis to argue loss in fish. It is a secondary activity relative to the visual-cycle role, so it is kept as non-core. Supporting Evidence: file:DANRE/rpe65a/rpe65a-uniprot.txt Capable of catalyzing the isomerization of lutein to meso-zeaxanthin an eye-specific carotenoid (By similarity). file:DANRE/rpe65a/rpe65a-uniprot.txt Reaction=lutein = (3R,3'S)-zeaxanthin; Xref=Rhea:RHEA:12729 |
| GO:0050251 retinol isomerase activity | IBA GO_REF:0000033 | MODIFY | Summary: GO:0050251 is defined as all-trans-retinol = 11-cis-retinol, i.e. isomerization of free retinol. PAINT placed it at the RPE65 subclade node PTN002576077 on the strength of a single ZFIN experimental annotation to the other zebrafish RPE65 paralog (Rpe65c, the Muller-cell isomerohydrolase; PMID:21676174). Reason: RPE65-family enzymes, including zebrafish Rpe65c in the source paper, act on all-trans-retinyl esters and release 11-cis-retinol; free all-trans-retinol is not their substrate (PMID:38355721: retinyl ester hydrolysis is the primary chemistry of RPE65). The isomerization the term is reaching for is the coupled isomerohydrolase reaction already captured by GO:0052885, which should replace this less accurate term; the rpe65c source data are sound but were mapped to a term whose substrate is wrong. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN002576077 SUPPORTS TRANSFER RPE65 subclade node; the isomerohydrolase function is correctly placed here. ZFIN:ZDB-GENE-081104-505 SUPPORTS SOURCE BUT NOT TARGET Zebrafish RPE65 paralog with in vitro 11-cis-retinol-forming isomerohydrolase activity from retinyl esters; the experimental result is genuine but the free-retinol isomerase term does not describe the assayed reaction. Proposed replacements: all-trans-retinyl-ester hydrolase, 11-cis retinol forming activity Supporting Evidence: PMID:38355721 two homologous enzymes catalyzing distinct primary chemistry on disparate substrates—retinyl ester hydrolysis in the case of RPE65 and carotenoid oxygenation in the case of NinaB—both possess the ability to catalyze trans–cis isomerization at a C11–C12 retinoid/carotenoid double bond file:DANRE/rpe65a/rpe65a-uniprot.txt Reaction=an all-trans-retinyl ester + H2O = 11-cis-retinol + a fatty acid + H(+); Xref=Rhea:RHEA:31771 PMID:21676174 zebrafish RPE65c, RPE65a and 13cIMH are three homologous proteins encoded by distinct genes |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location Cytoplasm (transferred from an RPE65 ortholog, A9C3R9), reflecting the soluble, unpalmitoylated form of the enzyme; no zebrafish subcellular localization has been reported, though immunostaining places the protein in RPE cells (PMID:17868371). Reason: RPE65 is a peripheral, palmitoylation-regulated protein that partitions between a soluble cytoplasmic pool and intracellular membranes, so cytoplasm is a correct location for the zebrafish ortholog even though the finer-grained site of catalysis is the ER membrane (ISS row). Accepted. Supporting Evidence: file:DANRE/rpe65a/rpe65a-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000250|UniProtKB:A9C3R9}. Cell membrane {ECO:0000250|UniProtKB:A9C3R9}; Lipid-anchor file:DANRE/rpe65a/rpe65a-uniprot.txt Attached to the membrane by a lipid anchor when palmitoylated (membrane form), soluble when unpalmitoylated. PMID:17868371 In crosssections it became clear that retinal RPE65a expression was confined to RPE cells |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Mapped from the UniProt "Cell membrane; Lipid-anchor" location, itself transferred from an RPE65 ortholog. UniProt describes a palmitoylated membrane form and a soluble form. Reason: The membrane form of RPE65 is real, but the membranes that hold its retinyl-ester substrate are intracellular (the ER membrane transferred by ISS from human RPE65), and no zebrafish data place Rpe65a at the plasma membrane. The SubCell keyword mapping is therefore a loose rendering of "membrane-associated"; kept as a non-core location rather than removed because the lipid-anchored form is genuine. Supporting Evidence: file:DANRE/rpe65a/rpe65a-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000250|UniProtKB:A9C3R9}. Cell membrane {ECO:0000250|UniProtKB:A9C3R9}; Lipid-anchor file:DANRE/rpe65a/rpe65a-uniprot.txt Attached to the membrane by a lipid anchor when palmitoylated (membrane form), soluble when unpalmitoylated. |
| GO:0016702 oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen | IEA GO_REF:0000002 | REMOVE | Summary: InterPro IPR004294 (carotenoid oxygenase family) maps to the dioxygenase parent term (two atoms of O2 incorporated into a single donor). The family signature is shared by BCO1/BCO2/NinaB, which are oxygenases, and by RPE65, which is not. Reason: Rpe65a does not incorporate molecular oxygen: its reaction is hydrolytic ester cleavage coupled to isomerization (Rhea 31771, EC 3.1.1.64), a distinct primary chemistry from the carotenoid oxygenation of its NinaB/BCO relatives (PMID:38355721). The family-level mapping is wrong for this member and there is no child of GO:0016702 that fits, so the row should be removed rather than modified; the correct activity is carried by the GO:0052885/GO:0052884 rows. Supporting Evidence: PMID:38355721 two homologous enzymes catalyzing distinct primary chemistry on disparate substrates—retinyl ester hydrolysis in the case of RPE65 and carotenoid oxygenation in the case of NinaB—both possess the ability to catalyze trans–cis isomerization at a C11–C12 retinoid/carotenoid double bond file:DANRE/rpe65a/rpe65a-uniprot.txt Reaction=an all-trans-retinyl ester + H2O = 11-cis-retinol + a fatty acid + H(+); Xref=Rhea:RHEA:31771 file:DANRE/rpe65a/rpe65a-uniprot.txt EC=3.1.1.64 |
| GO:0042572 retinol metabolic process | IEA GO_REF:0000120 | ACCEPT | Summary: Derived automatically from the GO:0052884/GO:0052885 activity rows (the reaction product is 11-cis-retinol). Reason: The enzyme produces 11-cis-retinol from stored esters, which is a retinol-metabolizing step by definition, and rpe65a knockdown reduces downstream 11-cis-retinal (PMID:17868371). Consistent with the retinal/retinoid process rows; accepted. Supporting Evidence: file:DANRE/rpe65a/rpe65a-uniprot.txt Reaction=an all-trans-retinyl ester + H2O = 11-cis-retinol + a fatty acid + H(+); Xref=Rhea:RHEA:31771 PMID:17868371 Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11- cis -retinal levels. |
| GO:0052884 all-trans-retinyl-palmitate hydrolase, 11-cis retinol forming activity | IEA GO_REF:0000116 | ACCEPT | Summary: The palmitate-specific instance of the isomerohydrolase reaction (Rhea 31775; all-trans-retinyl hexadecanoate + H2O -> 11-cis-retinol + hexadecanoate), mapped from Rhea and transferred by ISS from human RPE65. Retinyl palmitate is the predominant stored ester in the RPE. Reason: Same activity as GO:0052885 stated for the physiological substrate; UniProt records the reaction on the zebrafish entry and the in vivo requirement for 11-cis-retinal production is shown by knockdown (PMID:17868371). Accepted as core, with the caveat that no direct zebrafish enzyme assay exists (PMID:22512451). Supporting Evidence: file:DANRE/rpe65a/rpe65a-uniprot.txt Reaction=all-trans-retinyl hexadecanoate + H2O = 11-cis-retinol + hexadecanoate + H(+); Xref=Rhea:RHEA:31775 PMID:17868371 Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11- cis -retinal levels. PMID:22512451 It is noteworthy that the enzymatic activity of RPE65a in zebrafish has not been studied or characterized. |
| GO:0005737 cytoplasm | ISS GO_REF:0000024 | ACCEPT | Summary: UniProt subcellular location Cytoplasm (transferred from an RPE65 ortholog, A9C3R9), reflecting the soluble, unpalmitoylated form of the enzyme; no zebrafish subcellular localization has been reported, though immunostaining places the protein in RPE cells (PMID:17868371). Reason: RPE65 is a peripheral, palmitoylation-regulated protein that partitions between a soluble cytoplasmic pool and intracellular membranes, so cytoplasm is a correct location for the zebrafish ortholog even though the finer-grained site of catalysis is the ER membrane (ISS row). Accepted. Supporting Evidence: file:DANRE/rpe65a/rpe65a-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000250|UniProtKB:A9C3R9}. Cell membrane {ECO:0000250|UniProtKB:A9C3R9}; Lipid-anchor file:DANRE/rpe65a/rpe65a-uniprot.txt Attached to the membrane by a lipid anchor when palmitoylated (membrane form), soluble when unpalmitoylated. PMID:17868371 In crosssections it became clear that retinal RPE65a expression was confined to RPE cells |
| GO:0005886 plasma membrane | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Mapped from the UniProt "Cell membrane; Lipid-anchor" location, itself transferred from an RPE65 ortholog. UniProt describes a palmitoylated membrane form and a soluble form. Reason: The membrane form of RPE65 is real, but the membranes that hold its retinyl-ester substrate are intracellular (the ER membrane transferred by ISS from human RPE65), and no zebrafish data place Rpe65a at the plasma membrane. The SubCell keyword mapping is therefore a loose rendering of "membrane-associated"; kept as a non-core location rather than removed because the lipid-anchored form is genuine. Supporting Evidence: file:DANRE/rpe65a/rpe65a-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000250|UniProtKB:A9C3R9}. Cell membrane {ECO:0000250|UniProtKB:A9C3R9}; Lipid-anchor file:DANRE/rpe65a/rpe65a-uniprot.txt Attached to the membrane by a lipid anchor when palmitoylated (membrane form), soluble when unpalmitoylated. |
| GO:0052884 all-trans-retinyl-palmitate hydrolase, 11-cis retinol forming activity | ISS GO_REF:0000024 | ACCEPT | Summary: The palmitate-specific instance of the isomerohydrolase reaction (Rhea 31775; all-trans-retinyl hexadecanoate + H2O -> 11-cis-retinol + hexadecanoate), mapped from Rhea and transferred by ISS from human RPE65. Retinyl palmitate is the predominant stored ester in the RPE. Reason: Same activity as GO:0052885 stated for the physiological substrate; UniProt records the reaction on the zebrafish entry and the in vivo requirement for 11-cis-retinal production is shown by knockdown (PMID:17868371). Accepted as core, with the caveat that no direct zebrafish enzyme assay exists (PMID:22512451). Supporting Evidence: file:DANRE/rpe65a/rpe65a-uniprot.txt Reaction=all-trans-retinyl hexadecanoate + H2O = 11-cis-retinol + hexadecanoate + H(+); Xref=Rhea:RHEA:31775 PMID:17868371 Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11- cis -retinal levels. PMID:22512451 It is noteworthy that the enzymatic activity of RPE65a in zebrafish has not been studied or characterized. |
| GO:0052885 all-trans-retinyl-ester hydrolase, 11-cis retinol forming activity | ISS GO_REF:0000024 | ACCEPT | Summary: The EC 3.1.1.64 isomerohydrolase reaction (all-trans-retinyl ester + H2O -> 11-cis-retinol + fatty acid), the defining activity of RPE65. For the IBA, PAINT node PTN002576077 is the RPE65 subclade (human, bovine, dog, mouse, rat RPE65 and a second zebrafish RPE65 paralog); ISS transfers from bovine RPE65 and the Rhea/ARBA IEA give the same term. UniProt assigns EC 3.1.1.64 to the zebrafish entry by similarity. Reason: Zebrafish Rpe65a has not itself been assayed in vitro (PMID:22512451 notes this explicitly), so the activity rests on orthology plus in vivo genetics: it is the only RPE65 paralog expressed in the RPE, and morpholino knockdown reduces 11-cis-retinal and damages rod outer segments (PMID:17868371), with residual chromophore attributed to an RPE65-independent cone pathway (PMID:32238432). The transfer within the RPE65 subclade is sound and the term is the core molecular function. Supporting Evidence: file:DANRE/rpe65a/rpe65a-uniprot.txt Reaction=an all-trans-retinyl ester + H2O = 11-cis-retinol + a fatty acid + H(+); Xref=Rhea:RHEA:31771 file:DANRE/rpe65a/rpe65a-uniprot.txt EC=3.1.1.64 PMID:17868371 Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11- cis -retinal levels. PMID:22512451 It is noteworthy that the enzymatic activity of RPE65a in zebrafish has not been studied or characterized. PMID:32238432 Zebrafish rpe65a knockdown significantly reduced but did not completely attenuate 11cRAL synthesis, supporting the presence of an alternative pathway for chromophore synthesis in zebrafish retinae |
| GO:0001523 retinoid metabolic process | ISS GO_REF:0000024 | ACCEPT | Summary: ISS from human RPE65 for the generic retinoid metabolic process; the zebrafish morphant has reduced 11-cis-retinal and altered rod outer segments (PMID:17868371). Reason: Correct parent of the retinal/retinol process rows and directly supported by zebrafish metabolite profiling; accepted, with the specific terms GO:0042574/GO:0042572 carrying the detail. Supporting Evidence: PMID:17868371 Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11- cis -retinal levels. PMID:17868371 Cone vision of RPE65-deficient larvae remained functional as demonstrated by behavioural tests and by metabolite profiling for retinoids. file:DANRE/rpe65a/rpe65a-uniprot.txt FUNCTION: Plays important roles in the production of 11-cis retinal and in visual pigment regeneration. |
| GO:0005789 endoplasmic reticulum membrane | ISS GO_REF:0000024 | ACCEPT | Summary: ISS from human RPE65, which associates with ER/microsomal membranes of the RPE where retinyl esters are stored; UniProt describes the palmitoylated membrane form of the zebrafish protein. Reason: The substrate of the isomerohydrolase is a membrane-embedded retinyl ester (PMID:38355721), so the membrane-bound form on intracellular ER membranes is the catalytically relevant location; the curator-judged transfer from human RPE65 is consistent with this and nothing in the zebrafish data contradicts it. Accepted as the core membrane location. Supporting Evidence: file:DANRE/rpe65a/rpe65a-uniprot.txt Attached to the membrane by a lipid anchor when palmitoylated (membrane form), soluble when unpalmitoylated. PMID:38355721 two homologous enzymes catalyzing distinct primary chemistry on disparate substrates—retinyl ester hydrolysis in the case of RPE65 and carotenoid oxygenation in the case of NinaB—both possess the ability to catalyze trans–cis isomerization at a C11–C12 retinoid/carotenoid double bond |
| GO:0052885 all-trans-retinyl-ester hydrolase, 11-cis retinol forming activity | IEA GO_REF:0000120 | ACCEPT | Summary: The EC 3.1.1.64 isomerohydrolase reaction (all-trans-retinyl ester + H2O -> 11-cis-retinol + fatty acid), the defining activity of RPE65. For the IBA, PAINT node PTN002576077 is the RPE65 subclade (human, bovine, dog, mouse, rat RPE65 and a second zebrafish RPE65 paralog); ISS transfers from bovine RPE65 and the Rhea/ARBA IEA give the same term. UniProt assigns EC 3.1.1.64 to the zebrafish entry by similarity. Reason: Zebrafish Rpe65a has not itself been assayed in vitro (PMID:22512451 notes this explicitly), so the activity rests on orthology plus in vivo genetics: it is the only RPE65 paralog expressed in the RPE, and morpholino knockdown reduces 11-cis-retinal and damages rod outer segments (PMID:17868371), with residual chromophore attributed to an RPE65-independent cone pathway (PMID:32238432). The transfer within the RPE65 subclade is sound and the term is the core molecular function. Supporting Evidence: file:DANRE/rpe65a/rpe65a-uniprot.txt Reaction=an all-trans-retinyl ester + H2O = 11-cis-retinol + a fatty acid + H(+); Xref=Rhea:RHEA:31771 file:DANRE/rpe65a/rpe65a-uniprot.txt EC=3.1.1.64 PMID:17868371 Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11- cis -retinal levels. PMID:22512451 It is noteworthy that the enzymatic activity of RPE65a in zebrafish has not been studied or characterized. PMID:32238432 Zebrafish rpe65a knockdown significantly reduced but did not completely attenuate 11cRAL synthesis, supporting the presence of an alternative pathway for chromophore synthesis in zebrafish retinae |
Loading supporting content…
Download this section (compressed HTML)Q: Does zebrafish Rpe65a have measurable isomerohydrolase activity in vitro, and what is its product specificity (11-cis vs 13-cis retinol) relative to the paralogs Rpe65c and 13cIMH, given that only the paralogs have been assayed?
Q: Does Rpe65a, like mammalian RPE65, isomerize lutein to meso-zeaxanthin, and is meso-zeaxanthin present in the zebrafish eye?
Experiment: Express zebrafish rpe65a with LRAT in 293A cells and quantify 11-cis-retinol formation from all-trans-retinyl palmitate by HPLC, alongside rpe65c and chicken RPE65 as controls, to confirm the isomerohydrolase activity directly rather than by orthology.
Experiment: Generate a stable rpe65a null allele and profile ocular retinoids in dark-adapted larvae and adults to separate rod (RPE65-dependent) from cone (Muller-cell) chromophore supply without morpholino caveats.
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)