Vitus Oberhauser

CMO1 Deficiency Abolishes Vitamin A Production from β-Carotene and Alters Lipid Metabolism in Mice

Carotenoids are currently investigated regarding their potential to lower the risk of chronic disease and to combat vitamin A deficiency in humans. These plant-derived compounds must be cleaved and metabolically converted by intrinsic carotenoid oxygenases to support the panoply of vitamin A-dependent physiological processes. Two different carotenoid-cleaving enzymes were identified in mammals, the classical carotenoid-15,15′-oxygenase (CMO1) and a putative carotenoid-9′,10′-oxygenase (CMO2). To analyze the role of CMO1 in mammalian physiology, here we disrupted the corresponding gene by targeted homologous recombination in mice. On a diet providing β-carotene as major vitamin A precursor, vitamin A levels fell dramatically in several tissues examined. Instead, this mouse mutant accumulated the provitamin in large quantities (e.g. as seen by an orange coloring of adipose tissues). Besides impairments in β-carotene metabolism, CMO1 deficiency more generally interfered with lipid homeostasis. Even on a vitamin A-sufficient chow, CMO1-/- mice developed a fatty liver and displayed altered serum lipid levels with elevated serum unesterified fatty acids. Additionally, this mouse mutant was more susceptible to high fat diet-induced impairments in fatty acid metabolism. Quantitative reverse transcription-PCR analysis revealed that the expression of peroxisome proliferator-activated receptor γ-regulated marker genes related to adipogenesis was elevated in visceral adipose tissues. Thus, our study identifies CMO1 as the key enzyme for vitamin A production and provides evidence for a role of carotenoids as more general regulators of lipid metabolism.

Two common single nucleotide polymorphisms in the gene encoding β-carotene 15,15′-monoxygenase alter β-carotene metabolism in female volunteers

The key enzyme responsible for β‐carotene conversion into retinal is β‐carotene 15,15′‐monoxygenase (BCMO1). Since it has been reported that the conversion of β‐carotene into vitamin A is highly variable in up to 45% of healthy individuals, we hypothesized that genetic polymorphisms in the BCMO1 gene could contribute to the occurrence of the poor converter phenotype. Here we describe the screening of the total open reading frame of the BCMO1 coding region that led to the identification of two common nonsynonymous single nucleotide polymorphisms (R267S: rs12934922; A379V: rs7501331) with variant allele frequencies of 42 and 24%, respectively. In vitro biochemical characterization of the recombinant 267S + 379V double mutant revealed a reduced catalytic activity of BCMO1 by 57% (P<0.001). Assessment of the responsiveness to a pharmacological dose of β‐carotene in female volunteers confirmed that carriers of both the 379V and 267S + 379V variant alleles had a reduced ability to convert β‐carotene, as indicated through reduced retinyl palmitate:β‐carotene ratios in the triglyceride‐rich lipoprotein fraction [−32% (P=0.005) and −69% (P=0.001), respectively] and increased fasting β‐carotene concentrations [+160% (P=0.025) and +240% (P=0.041), respectively]. Our data show that there is genetic variability in β‐carotene metabolism and may provide an explanation for the molecular basis of the poor converter phenotype within the population.—Leung, W. C., Hessel, S., Méplan, C., Flint, J., Oberhauser, V., Tourniaire, F., Hesketh, J. E., vonLintig, J., Lietz, G. Two common single nucleotide polymorphisms in the gene encoding β‐carotene 15,15′‐monoxygenase alter β‐carotene metabolism in female volunteers. FASEB J. 23, 1041–1053 (2009)

RBP4 Disrupts Vitamin A Uptake Homeostasis in a STRA6-Deficient Animal Model for Matthew-Wood Syndrome

The cellular uptake of vitamin A from its RBP4-bound circulating form (holo-RBP4) is a homeostatic process that evidently depends on the multidomain membrane protein STRA6. In humans, mutations in STRA6 are associated with Matthew-Wood syndrome, manifested by multisystem developmental malformations. Here we addressed the metabolic basis of this inherited disease. STRA6-dependent transfer of retinol from RBP4 into cultured NIH 3T3 fibroblasts was enhanced by lecithin:retinol acyltransferase (LRAT). The retinol transfer was bidirectional, strongly suggesting that STRA6 acts as a retinol channel/transporter. Loss-of-function analysis in zebrafish embryos revealed that Stra6 deficiency caused vitamin A deprivation of the developing eyes. We provide evidence that, in the absence of Stra6, holo-Rbp4 provokes nonspecific vitamin A excess in several embryonic tissues, impairing retinoic acid receptor signaling and gene regulation. These fatal consequences of Stra6 deficiency, including craniofacial and cardiac defects and microphthalmia, were largely alleviated by reducing embryonic Rbp4 levels by morpholino oligonucleotide or pharmacological treatments.

The retinal G protein-coupled receptor (RGR) enhances isomerohydrolase activity independent of light.

Rod and cone visual pigments use 11-cis-retinal, a vitamin A derivative, as their chromophore. Light isomerizes 11-cis- into all-trans-retinal, triggering a conformational transition of the opsin molecule that initiates phototransduction. After bleaching all-trans-retinal leaves the opsin, and light sensitivity must be restored by regeneration of 11-cis-retinal. Under bright light conditions the retinal G protein-coupled receptor (RGR) was reported to support this regeneration by acting as a photoisomerase in a proposed photic visual cycle. We analyzed the contribution of RGR to rhodopsin regeneration under different light regimes and show that regeneration, during light exposure and in darkness, is slowed about 3-fold in Rgr-/- mice. These findings are not in line with the proposed function of RGR as a photoisomerase. Instead, RGR, independent of light, accelerates the conversion of retinyl esters to 11-cis-retinal by positively modulating isomerohydrolase activity, a key step in the “classical” visual cycle. Furthermore, we find that light accelerates rhodopsin regeneration, independent of RGR.

NinaB combines carotenoid oxygenase and retinoid isomerase activity in a single polypeptide

In animals, successful production of the visual chromophore (11-cis-retinal or derivatives thereof such as 11-cis-3-hydroxy-retinal) is essential for photoreceptor cell function and survival. These carotenoid-derived compounds must combine with a protein moiety (the opsin) to establish functional visual pigments. Evidence from cell culture systems has implicated that the retinal pigment epithelium protein of 65 kDa (RPE65) is the long-sought all-trans to 11-cis retinoid isomerase. RPE65 is structurally related to nonheme iron oxygenases that catalyze the conversion of carotenoids into retinoids. In vertebrate genomes, two carotenoid oxygenases and RPE65 are encoded, whereas in insect genomes only a single representative of this protein family, named NinaB (denoting neither inactivation nor afterpotential mutant B), is encoded. We here cloned and functionally characterized the ninaB gene from the great wax moth Galleria mellonella. We show that the recombinant purified enzyme combines isomerase and oxygenase (isomerooxygenase) activity in a single polypeptide. From kinetics and isomeric composition of cleavage products of asymmetrical carotenoid substrates, we propose a model for the spatial arrangement between substrate and enzyme. In Drosophila, we show that carotenoid-isomerooxygenase activity of NinaB is more generally found in insects, and we provide physiological evidence that carotenoids such as 11-cis-retinal can promote visual pigment biogenesis in the dark. Our study demonstrates that trans/cis isomerase activity can be intrinsic to this class of proteins and establishes these enzymes as key components for both invertebrate and vertebrate vision.

Evidence for RPE65‐independent vision in the cone‐dominated zebrafish retina

An enzyme‐based cyclic pathway for trans to cis isomerization of the chromophore of visual pigments (11‐cis‐retinal) is intrinsic to vertebrate cone and rod vision. This process, called the visual cycle, is mostly characterized in rod‐dominated retinas and essentially depends on RPE65, an all‐trans to 11‐cis‐retinoid isomerase. Here we analysed the role of RPE65 in zebrafish, a species with a cone‐dominated retina. We cloned zebrafish RPE65 and showed that its expression coincided with photoreceptor development. Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11‐cis‐retinal levels. Cone vision of RPE65‐deficient larvae remained functional as demonstrated by behavioural tests and by metabolite profiling for retinoids. Furthermore, all‐trans retinylamine, a potent inhibitor of the rod visual cycle, reduced 11‐cis‐retinal levels of control larvae to a similar extent but showed no additive effects in RPE65‐deficient larvae. Thus, our study of zebrafish provides in vivo evidence for the existence of an RPE65‐independent pathway for the regeneration of 11‐cis‐retinal for cone vision.

Sequestration of Retinyl Esters Is Essential for Retinoid Signaling in the Zebrafish Embryo

For vertebrate development, vitamin A (all-trans retinol) is required in quantitative different amounts and spatiotemporal distribution for the production of retinoic acid, a nuclear hormone receptor ligand, and 11-cis retinal, the chromophore of visual pigments. We show here for zebrafish that embryonic retinoid homeostasis essentially depends on the activity of a leci-thin:retinol acyltransferase (Lratb). During embryogenesis, lratb is expressed in mostly non-overlapping domains opposite to retinal dehydrogenase 2 (raldh2), the key enzyme for retinoic acid synthesis. Blocking retinyl ester formation by a targeted knock down of Lratb results in significantly increased retinoic acid levels, which lead to severe embryonic patterning defects. Thus, we provide evidence that a balanced competition between Lratb and Raldh2 for yolk vitamin A defines embryonic compartments either for retinyl ester or retinoic acid synthesis. This homeostatic mechanism dynamically adjusts embryonic retinoic acid levels for gene regulation, concomitantly sequestering excess yolk vitamin A in the form of retinyl esters for the establishment of larval vision later during development.

NinaB Is Essential for Drosophila Vision but Induces Retinal Degeneration in Opsin-deficient Photoreceptors

In animals, visual pigments are essential for photoreceptor function and survival. These G-protein-coupled receptors consist of a protein moiety (opsin) and a covalently bound 11-cis-retinylidene chromophore. The chromophore is derived from dietary carotenoids by oxidative cleavage and trans-to-cis isomerization of double bonds. In vertebrates, the necessary chemical transformations are catalyzed by two distinct but structurally related enzymes, the carotenoid oxygenase β-carotenoid-15,15′-monooxygenase and the retinoid isomerase RPE65 (retinal pigment epithelium protein of 65 kDa). Recently, we provided biochemical evidence that these reactions in insects are catalyzed by a single enzyme family member named NinaB. Here we show that in the fly pathway, carotenoids are mandatory precursors of the chromophore. After chromophore formation, the retinoid-binding protein Pinta acts downstream of NinaB and is required to supply photoreceptors with chromophore. Like ninaE encoding the opsin, ninaB expression is eye-dependent and is activated as a downstream target of the eyeless/pax6 and sine oculis master control genes for eye development. The requirement for coordinated synthesis of chromophore and opsin is evidenced by analysis of ninaE mutants. Retinal degeneration in opsin-deficient photoreceptors is caused by the chromophore and can be prevented by restricting its supply as seen in an opsin and chromophore-deficient double mutant. Thus, our study identifies NinaB as a key component for visual pigment production and provides evidence that chromophore in opsin-deficient photoreceptors can elicit retinal degeneration.

The role of 11-cis-retinyl esters in vertebrate cone vision

A cycle of cis‐to‐trans isomerization of the chromophore is intrinsic to vertebrate vision where rod and cone photoreceptors mediate dim‐ and bright‐light vision, respectively. Daylight illumination can greatly exceed the rate at which the photoproduct can be recycled back to the chromophore by the canonical visual cycle. Thus, an additional supply pathway(s) must exist to sustain cone‐dependent vision. Two‐photon microscopy revealed that the eyes of the zebrafish (Danio rerio) contain high levels of 11‐cis‐retinyl esters (11‐REs) within the retinal pigment epithelium. HPLC analyses demonstrate that 11‐REs are bleached by bright light and regenerated in the dark. Pharmacologic treatment with all‐trans‐retinylamine (Ret‐NH2), a potent and specific inhibitor of the trans‐to‐cis reisomerization reaction of the canonical visual cycle, impeded the regeneration of 11‐REs. Intervention with 11‐cis‐retinol restored the regeneration of 11‐REs in the presence of all‐trans‐Ret‐NH2. We used the XOPS:mCFP transgenic zebrafish line with a functional cone‐only retina to directly demonstrate that this 11‐RE cycle is critical to maintain vision under bright‐light conditions. Thus, our analyses reveal that a dark‐generated pool of 11‐REs helps to supply photoreceptors with the chromophore under the varying light conditions present in natural environments—Babino, D., Perkins, B. D., Kindermann, A., Oberhauser, V., von Lintig, J., The role of 11‐cis‐retinyl esters in vertebrate cone vision. FASEB J. 29, 216–226 (2015). www.fasebj.org

SRP-35, a newly identified protein of the skeletal muscle sarcoplasmic reticulum, is a retinol dehydrogenase

In the present study we provide evidence that SRP-35, a protein we identified in rabbit skeletal muscle sarcoplasmic reticulum, is an all-trans-retinol dehydrogenase. Analysis of the primary structure and tryptic digestion revealed that its N-terminus encompasses a short hydrophobic sequence bound to the sarcoplasmic reticulum membrane, whereas its C-terminal catalytic domain faces the myoplasm. SRP-35 is also expressed in liver and adipocytes, where it appears in the post-microsomal supernatant; however, in skeletal muscle, SRP-35 is enriched in the longitudinal sarcoplasmic reticulum. Sequence comparison predicts that SRP-35 is a short-chain dehydrogenase/reductase belonging to the DHRS7C [dehydrogenase/reductase (short-chain dehydrogenase/reductase family) member 7C] subfamily. Retinol is the substrate of SRP-35, since its transient overexpression leads to an increased production of all-trans-retinaldehyde. Transfection of C2C12 myotubes with a fusion protein encoding SRP-35-EYFP (enhanced yellow fluorescent protein) causes a decrease of the maximal Ca²⁺ released via RyR (ryanodine receptor) activation induced by KCl or 4-chloro-m-chresol. The latter result could be mimicked by the addition of retinoic acid to the C2C12 cell tissue culture medium, a treatment which caused a significant reduction of RyR1 expression. We propose that in skeletal muscle SRP-35 is involved in the generation of all-trans-retinaldehyde and may play an important role in the generation of intracellular signals linking Ca2+ release (i.e. muscle activity) to metabolism.