Rat Intestinal β-Carotene Dioxygenase Activity Is Located Primarily in the Cytosol of Mature Jejunal Enterocytes

260

225

We have identified from mouse the first mammalian ␤-carotene 15,15-dioxygenase (␤-CD), a crucial enzyme in development and metabolism that governs the de novo entry of vitamin A from plant-derived precursors. ␤-CD is related to the retinal pigment epithelium-expressed protein RPE65 and belongs to a diverse family that includes the plant 9-cis-epoxycarotenoid dioxygenase and bacterial lignostilbene dioxygenases. ␤-CD expression in Escherichia coli cells engineered to produce ␤-carotene led to the accumulation of all-trans-retinal at the expense of ␤-carotene, confirming that ␤-CD catalyzed the central cleavage of this vitamin A precursor. Purified recombinant ␤-CD protein cleaves ␤-carotene in vitro with a V max of 36 pmol of retinal/mg of enzyme/ min and a K m of 6 M. Non-provitamin A carotenoids were also cleaved, although with much lower activity. By Northern analysis, a 2.4-kilobase (kb) message was observed in liver, kidney, small intestine, and testis, tissues important in retinoid/carotenoid metabolism. This message encoded a 63-kDa cytosolic protein expressed in these tissues. A shorter transcript of 1.8 kb was found in testis and skin. Developmentally, the 2.4-kb mRNA was abundant at embryonic day 7, with lower expression at embryonic days 11, 13, and 15, suggesting a critical role for this enzyme in gastrulation. Identification of ␤-CD in an accessible model organism will create new opportunities to study vitamin A metabolism.In vertebrates, vitamin A in its various oxidative and isomeric forms is essential for embryonic development (1), pattern formation (2, 3), and vision (4). Retinoic acid, through its interaction with the nuclear retinoic acid receptor and retinoid X receptor, profoundly affects cell differentiation and development. Because animals are unable to synthesize vitamin A de novo from endogenous isoprenoid precursors, they must instead derive it from cleavage of ␤-carotene and certain other carotenoids with an unsubstituted ␤-ring (e.g. ␥-and ␣-carotenes, ␤-zeacarotene, and ␤-cryptoxanthin). It is generally accepted that central cleavage of ␤-carotene by a putative dioxygenase gives rise to two molecules of all-trans-retinal, whereas eccentric cleavage with subsequent processing leading to a single molecule of retinoic acid from an apocarotenal is quantitatively far less important (5). ␤-Carotene cleavage activity is reported highest in the intestinal mucosa, but is found at high activity levels in liver, kidney, lung, and fat tissues, among other sites. However, an inability to purify the protein catalyzing this reaction has hindered thorough investigation of this crucial first step in vitamin A metabolism.Because of a loose similarity between the mammalian protein RPE65 and the neoxanthin cleavage enzymes of plants, our laboratories have considered the hypothesis that the putative ␤-CD 1 would belong to an emerging family of carotenoidcleaving dioxygenases known mainly from examples in plants (6), but with members also in bacteria and Metazoa. The first described representative was a ba...

Section: Discussionmentioning

confidence: 99%

Identification, Expression, and Substrate Specificity of a Mammalian β-Carotene 15,15′-Dioxygenase

Redmond

Gentleman

Duncan

et al. 2001

260

225

“…For this reason, NinaD cannot be the only missing component, and other components need to be identified. Mammalian BCO activity also may be restricted to particular cell types, as the majority of the intestinal activity comes from jejunal enterocytes (39). Thus it is possible that mammals also retain cellular specializations supporting ␤-carotene metabolism in a limited number of cell types.…”

Section: Discussionmentioning

confidence: 99%

Drosophila NinaB and NinaD Act Outside of Retina to Produce Rhodopsin Chromophore

Gu¹,

Yang²,

Mitchell

et al. 2004

The Drosophila ninaB gene encodes a ␤,␤-carotene-15,15 -oxygenase responsible for the centric cleavage of ␤-carotene that produces the retinal chromophore of rhodopsin. The ninaD gene encodes a membrane receptor required for efficient use of ␤-carotene. Despite their importance to the synthesis of visual pigment, we show that these genes are not active in the retina. Mosaic analysis shows that ninaB and ninaD are not required in the retina, and exclusive retinal expression of either gene, or both genes simultaneously, does not support rhodopsin biogenesis. In contrast, neuron-specific expression of ninaB and ninaD allows for rhodopsin biogenesis. Additional directed expression studies failed to identify other tissues supporting ninaB activity in rhodopsin biogenesis. These results show that nonretinal sites of NinaB ␤,␤-carotene-15,15 -oxygenase activity, likely neurons of the central nervous system, are essential for production of the visual chromophore. Retinal or another C 20 retinoid, not members of the ␤-carotene family of C 40 carotenoids, are supplied to photoreceptors for rhodopsin biogenesis.Mutants in eight Drosophila genes, designated ninaA through ninaH, are characterized by reduced rhodopsin levels in photoreceptors and altered electroretinograms (1). The opsin protein component of rhodopsin is coded by the ninaE gene (2, 3). The low rhodopsin phenotypes observed in other nina mutants are caused by deficits in the post-translational rhodopsin maturation process. For example, ninaA encodes a molecular chaperone required for movement of newly synthesized rhodopsin from the endoplasmic reticulum to the photosensitive rhabdomeric membranes (4, 5). In the case of ninaB and ninaD, defective rhodopsin production is the result of a failure to generate the chromophore of rhodopsin, 3-OH retinal (6).Animal species usually obtain retinals in their food. Plants and microorganisms produce C 40 carotenoids such as ␤-carotene and zeaxanthin, which animals metabolize to C 20 retinoids. In Drosophila, the ninaD gene encodes a membrane "scavenger" receptor proposed to mediate the cellular uptake of carotenoids (7). The ninaB gene encodes a ␤,␤-carotene-15,15Ј-oxygenase (BCO) 1 responsible for the centric cleavage of ␤-carotene to form retinal (8). This enzyme was originally named as a ␤-carotene dioxygenase. It has now been renamed BCO (9) in light of recent data showing related enzymes act as monooxygenases (10).In vertebrates, vitamin A is essential for development and differentiation processes as well as its role in vision. The availability of vitamin A for metabolic processes is governed by multiple factors, including dietary absorption, transport, metabolism, and storage (11). A human BCO is expressed in the retinal pigment epithelium and also in the kidney, intestine, liver, brain, stomach, and testis (12, 13), suggesting that the processing of dietary carotenoids occurs in a variety of vertebrate tissues. Additional studies show that centric cleavage of ␤-carotene plays a major role in the processing of carote...

“…This may be attributed to species differences between the mouse and the rat (and other mammalian species) because the published studies of tissue activity levels have not employed mouse small intestine as a source of enzyme activity. Moreover, the literature indicates that carotene 15,15Ј-dioxygenase activity is limited to only the proximal portion of the small intestine, including the duodenum and part of jejunum (8,15,40) and is present only in mature functional enterocytes within these anatomic regions (18). Because we were concerned about the possible rapid degradation of intestinal RNA, as a source of intestinal total RNA we employed the entire small intestine without first separating the mucosal lining.…”

Section: Discussionmentioning

confidence: 99%

Expression and Characterization of a Murine Enzyme Able to Cleave β-Carotene

Paik¹,

During²,

Harrison³

et al. 2001

145

Because animals are not able to synthesize retinoids de novo, ultimately they must derive them from dietary provitamin A carotenoids through a process known as carotene cleavage. The enzyme responsible for catalyzing carotene cleavage (␤-carotene 15,15-dioxygenase) has been characterized primarily in rat intestinal scrapings. Using a recently reported cDNA sequence for a carotene cleavage enzyme from Drosophila, we identified a cDNA encoding a mouse homolog of this enzyme. When the cDNA was expressed in either Escherichia coli or Chinese hamster ovary cells, expression conferred upon bacterial and Chinese hamster ovary cell homogenates the ability to cleave ␤-carotene to retinal. Several lines of evidence obtained upon kinetic analyses of the recombinant enzyme suggested that carotene cleavage enzyme interacts with other proteins present within cell or tissue homogenates. This was confirmed by pulldown experiments upon incubation of recombinant enzyme with tissue 12,000 ؋ g supernatants. Matrix-assisted laser desorption ionization-mass spectrometry analysis of pulled-down proteins indicates that an atypical testis-specific isoform of lactate dehydrogenase associates with recombinant carotene cleavage enzyme. mRNA transcripts for the carotene cleavage enzyme were detected by reverse transcription-polymerase chain reaction in mouse testes, liver, kidney, and intestine. In situ hybridization studies demonstrated that carotene cleavage enzyme is expressed prominently in maternal tissue surrounding the embryo but not in embryonic tissues at 7.5 and 8.5 days postcoitus. This work offers new insights for understanding the biochemistry of carotene cleavage to retinoids.