RPE65 Operates in the Vertebrate Visual Cycle by Stereospecifically Binding All-<i>trans</i>-Retinyl Esters

Gollapalli, Deviprasad R.; Maiti, Pranab; Rando, Robert R.

doi:10.1021/bi035227w

Cited by 69 publications

(70 citation statements)

References 33 publications

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“…We did not observe any effect of all-trans-retinol addition on the localization of LRAT, which remained ER-associated. Of note is that the T-REx-293 cells we used for these experiments do not express detectable levels of RPE65, a protein proposed in some studies to be a retinyl ester-binding/transport protein (42,43), yet we were still able to detect retinosome formation. The N Terminus of LRAT Is Not Conserved in All VertebratesAnalysis of the N-terminal domains of the known and putative vertebrate homologs of LRAT showed a low degree of conservation.…”

Section: The Putative C-terminal Transmembrane Domain Is Necessary Fomentioning

confidence: 80%

“…The RPE65 protein is currently the only protein known to associate with retinyl esters (42,43). It has been reported that RPE65 is involved in the isomerization reaction that produces 11-cis-retinol and that retinyl esters are the preferred substrates for this reaction (51)(52)(53).…”

Section: Discussionmentioning

confidence: 99%

“…The NCEHF domain found in several other unrelated proteins is also shown. Gly 35 and Ser 195 (N-and C-terminal residues of mouse tLRAT, respectively) and Ile 42 and Asp 128 (mutated to N-linked glycosylation acceptor sites) are shown in boldface. activity assays (Fig.…”

Section: Lrat Is An Endoplasmic Reticulum (Er)-localized Mem-mentioning

confidence: 99%

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Topology and Membrane Association of Lecithin: Retinol Acyltransferase

Moise

Golczak

Imanishi

et al. 2007

Journal of Biological Chemistry

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Fatty acid retinyl esters are the storage form of vitamin A (alltrans-retinol) and serve as metabolic intermediates in the formation of the visual chromophore 11-cis-retinal. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester formation, acts by transferring an acyl group from the sn-1 position of phosphatidylcholine to retinol. To define the membrane association and localization of LRAT, we produced an LRAT-specific monoclonal antibody, which we used to study enzyme partition under different experimental conditions. Furthermore, we examined the membrane topology of LRAT through an N-linked glycosylation scanning approach and protease protection assays. We show that LRAT is localized to the membrane of the endoplasmic reticulum (ER) and assumes a single membrane-spanning topology with an N-terminal cytoplasmic/C-terminal luminal orientation. In eukaryotic cells, the C-terminal transmembrane domain is essential for the activity and ER membrane targeting of LRAT. In contrast, the N-terminal hydrophobic region is not required for ER membrane targeting or enzymatic activity, and its amino acid sequence is not conserved in other species examined. We present experimental evidence of the topology and subcellular localization of LRAT, a critical enzyme in vitamin A metabolism.The metabolism of vitamin A (all-trans-retinol) leads to the formation of all-trans-retinoic acid and 11-cis-retinal derivatives that play crucial roles in such processes as development, immunity, and vision (1-6). Recent studies have highlighted the importance of lecithin:retinol acyltransferase (LRAT) 3 in the absorption and retention of retinol in intracellular stores (7). LRAT catalyzes the synthesis of retinyl esters, thereby drawing retinol from the circulation to storage depots such as the lipid droplets of hepatic stellate cells and the retinosome structures found in the retinal pigment epithelium (RPE) (8, 9). Mutations in the LRAT gene lead to early-onset severe retinal dystrophy (10). Disruption of the Lrat gene in Lrat Ϫ/Ϫ mice produces a severe impairment in retinol uptake and storage capacity. As a result, Lrat Ϫ/Ϫ mice are blind (11) and more susceptible to vitamin A deficiency than their wild-type (WT) counterparts (12). Lrat Ϫ/Ϫ mice possess only trace amounts of retinyl esters in most tissues (11, 12), yet, surprisingly, the levels of retinyl esters in their adipose tissue are elevated compared with those in WT mice (12). Adipose stores of retinyl esters could depend on the activity of acyl-CoA:retinol acyltransferase (12).LRAT plays a pivotal role in determining retinol availability, which in turn affects the levels of all-trans-retinoic acid, a potent regulator of the expression of many genes via retinoic acid receptors (13). Expression of LRAT in the liver is influenced by multiple factors, including vitamin A status (14), retinoic acid (15), other synthetic retinoic acid receptor activators (16), and peroxisome proliferator-activated receptor ␤/␦ agonists (17). Thus, regulation of LRAT activi...

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Section: The Putative C-terminal Transmembrane Domain Is Necessary Fomentioning

confidence: 80%

Section: Discussionmentioning

confidence: 99%

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Topology and Membrane Association of Lecithin: Retinol Acyltransferase

Moise

Golczak

Imanishi

et al. 2007

Journal of Biological Chemistry

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“…The accumulation of retinyl esters is clearly dependent on LRAT activity (Figure 2), and these esters accumulate in Rpe65−/− mice. RPE65 was also proposed to be a retinyl esterbinding protein (27,44,45), hinting of its possible role in this process. Most insightful was the observation made by Pepperberg et al that, in normal RPE, there is a substantial exchange of all-trans-retinol with the blood circulation, whereas in Rpe65−/−mice, despite the presence of abnormally high molar levels of RPE retinyl esters, the outward movement is inhibited (43).…”

Section: Accumulation Of All-trans-retinyl Esters In the Rpementioning

confidence: 99%

Aberrant Metabolites in Mouse Models of Congenital Blinding Diseases: Formation and Storage of Retinyl Esters

Maeda

Imanishi

et al. 2006

Biochemistry

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Regeneration of the visual chromophore, 11-cis-retinal, is a critical step in restoring photoreceptors to their dark-adapted conditions. This regeneration process, called the retinoid cycle, takes place in the photoreceptor outer segments and the retinal pigment epithelium (RPE). Disabling mutations in nearly all of the retinoid cycle genes are linked to human conditions that cause congenital or progressive defects in vision. Several mouse models with disrupted genes related to this cycle contain abnormal fatty acid retinyl ester levels in the RPE. To investigate the mechanisms of retinyl ester accumulation, we generated single or double knockout mice lacking retinoid cycle genes. Alltrans-retinyl esters accumulated in mice lacking RPE65, but they are reduced in double knockout mice also lacking opsin, suggesting a connection between visual pigment regeneration and the retinoid cycle. Only Rdh5-deficient mice accumulate cis-retinyl esters, regardless of the simultaneous disruption of RPE65, opsin, and prRDH. 13-cis-Retinoids are produced at higher levels when the flow of retinoid through the cycle was increased, and these esters are stored in specific structures called retinosomes. Most importantly, retinylamine, a specific and effective inhibitor of the 11-cisretinol formation, also inhibits the production of 13-cis-retinyl esters. The data presented here support the idea that 13-cis-retinyl esters are formed through an aberrant enzymatic isomerization process.Vitamin A transformations in the eye are essential for vision. Absorption of light by the vitamin A-derived chromophore of visual pigments, 11-cis-retinal, leads to its photoisomerization to all-trans-retinal and the initiation of the signal transduction cascade (1-4). Through a chain of reactions, all-trans-retinal is recycled enzymatically back to 11-cis-retinal (5-8). These retinoid cycle reactions (Figure 1) take place in the outer segments of photoreceptor cells and in the adjacent retinal pigment epithelium (RPE). 1 Characterization of the knockout mice lacking genes involved in the retinoid cycle provides important insight into the flow of retinoids in the eye.11-cis-Retinol dehydrogenase (RDH), also known as RDH5, catalyzes the final oxidation reaction of 11-cis-retinol in the RPE (9, 10). Although RDH5 is responsible for the majority of 11-cis-RDH activity, RDH11 and other RDHs also have a measurable role in regenerating the visual pigment by complementing RDH5 in RPE cells (11). Disruption of the gene encoding RDH5 causes fundus albipunctatus in humans (12,13). Fundus albipunctatus is an autosomal recessive form of congenital stationary night blindness characterized by the appearance of numerous small white dots located in the RPE, a delayed course of dark adaptation, and † This research was supported by NIH Grant EY09339. Lipid droplet-like organelles known as retinosomes were characterized as specific sites of alltrans-retinyl ester accumulation in the RPE (19,20). All-trans-retinyl esters accumulate in the RPE of wild-type mice as the...

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“…This is partly because the putative IMH (3), or alternate isomerase (5), has yet to be identified at the molecular level, and partly due to uncertainties in understanding the mechanism of isomerization (5,17,18). The precise role of RPE65 in isomerization has been particularly perplexing, although its absolute necessity is demonstrated by the Rpe65-deficient mouse (14).…”

mentioning

confidence: 99%

Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle

Redmond

Poliakov

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

361

357

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RPE65 is essential for isomerization of vitamin A to the visual chromophore. Mutations in RPE65 cause early-onset blindness, and Rpe65-deficient mice lack 11-cis-retinal but overaccumulate alltrans-retinyl esters in the retinal pigment epithelium (RPE). RPE65 is proposed to be a substrate chaperone but may have an enzymatic role because it is closely related to carotenoid oxygenases. We hypothesize that, by analogy with other carotenoid oxygenases, the predicted iron-coordinating residues of RPE65 are essential for retinoid isomerization. To clarify RPE65's role in isomerization, we reconstituted a robust minimal visual cycle in 293-F cells. Only cells transfected with RPE65 constructs produced 11-cisretinoids, but coexpression with lecithin:retinol acyltransferase was needed for high-level production. Accumulation was significant, amounting to >2 nmol of 11-cis-retinol per culture. Transfection with constructs harboring mutations in residues of RPE65 homologous to those required for interlinked enzymatic activity and iron coordination in related enzymes abolish this isomerization. Iron chelation also abolished isomerization activity. Mutating cysteines implicated in palmitoylation of RPE65 had generally little effect on isomerization activity. Mutations associated with Leber congenital amaurosis͞early-onset blindness cause partial to total loss of isomerization activity in direct relation to their clinical effects. These findings establish a catalytic role, in conjunction with lecithin:retinol acyltransferase, for RPE65 in synthesis of 11-cisretinol, and its identity as the isomerohydrolase.11-cis-retinoids ͉ Leber congenital amaurosis ͉ retinal pigment epithelium R egeneration of 11-cis-retinal, the chromophore of all visual pigments (opsins), occurs by a process in the retinal pigment epithelium (RPE) termed the visual cycle that involves isomerization of all-trans-retinyl esters to 11-cis-retinol. In outline, lecithin:retinol acyltransferase (LRAT) (1, 2) esterifies incoming all-trans-retinol to all-trans-retinyl esters, the substrate for the putative isomerohydrolase (IMH) (3, 4). IMH is postulated to perform a concerted hydrolysis and isomerization yielding 11-cis-retinol, which is trapped by cellular retinaldehyde-binding protein (CRALBP) and then oxidized to 11-cis-retinal by 11-cis-retinol dehydrogenase͞RDH5 (11cisRDH). An alternate mechanism proposes generation of 11-cis-retinol via a retinyl carbocation intermediate (5). Mutations of RDH5, RPE65, LRAT, and CRALBP in the human (6 -12) and cognate disruptions in the mouse (13-16) result in mild to severe blindness due to derangement of RPE retinoid metabolism. CRALBP and RDH5 disruptions do not block the visual cycle but reduce its efficiency (13, 16). Although mutation or loss of LRAT and RPE65 each block the visual cycle, they do so differently. With LRAT loss, no vitamin A accumulates in the RPE, obviating retinoid metabolism (15). With RPE65 loss, all-trans-retinyl ester accumulates to very high levels, but 11-cis-retinoids are not formed (14).Howev...

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RPE65 Operates in the Vertebrate Visual Cycle by Stereospecifically Binding All-trans-Retinyl Esters

Cited by 69 publications

References 33 publications

Topology and Membrane Association of Lecithin: Retinol Acyltransferase

Topology and Membrane Association of Lecithin: Retinol Acyltransferase

Aberrant Metabolites in Mouse Models of Congenital Blinding Diseases: Formation and Storage of Retinyl Esters

Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle

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