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University of Freiburg, Institute of Biology I, Animal Physiology and Neurobiology, Hauptstrasse 1, D-79104 Freiburg, Germany
2To whom correspondence should be addressed. E-mail: Johannes.von.Lintig{at}biologie.uni-freiburg.de.
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ABSTRACT |
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 TOP ABSTRACT CONCLUSIONSLITERATURE CITED |
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KEY WORDS: • carotenoids • provitamin A • vitamin A • retinoids • bioconversion
The elucidation of the physiological roles played by vitamins has always been a major concern of biochemists and nutritionists. In humans, vitamin A deficiency leads in milder forms to night blindness, while longer onset can lead to corneal malformations, e.g., xerophthalmia. Besides visual defects, its deficiency affects the immune system, leads to infertility and causes severe malformations during embryogenesis. The molecular basis for these diverse effects is found in the dual role exerted by vitamin A derivatives in animal physiology. In visual systems, retinal or closely related compounds such as 3-hydroxy-retinal serve as the chromophore of the various visual pigments (rhodopsins) (1,2). In chordates, the vitamin A derivative retinoic acid (RA)2 is an important signaling molecule influencing developmental processes and cell differentiation by binding two classes of nuclear receptors which mediate the transcriptional regulation of target genes (3,4).
Vitamin A deficiency, particularly in Third World countries, is still a major problem leading to blindness and childhood mortality (5). The requirements for this vitamin can be satisfied either by animal food containing vitamin A or plant food containing provitamin A carotenoids. This phenomenon was first explained by Moore in 1930 (6). Moore described a conversion of ß-carotene to vitamin A in the small intestine, providing the first evidence that these carotenoids are the direct precursors for vitamin A in animals. Today we know that all naturally occurring vitamin A in the food chain derives from carotenoids and that the world’s population mainly relies on carotenoids from plants for its vitamin A supply.
For the conversion of ß-carotene to vitamin A a central cleavage mechanism at the 15,15' carbon double bond was first proposed by Karrer (7). In 1954 Glover proposed an eccentric cleavage reaction and a stepwise process, leading ultimately only to 1 mole vitamin A per mole carotene consumed (8). Evidence for this eccentric cleavage was provided by the observation that radioactive ß-apocarotenals were converted in mammals to vitamin A esters with the release of "small" radioactive fragments (9). The observation of eccentric or asymmetric cleavage of ß-carotene led to a controversial debate on the significance of this reaction (10,11).
A milestone in our understanding of vitamin A formation was independently achieved by James Allen Olsen and Dewitt Goodman (12,13) in 1965. Both researchers were able to show in cell-free extracts derived from rat small intestine that ß-carotene is enzymatically cleaved at the central 15,15' carbon double bond, yielding two molecules of vitamin A aldehyde (retinal). This enzymatic activity depends on molecular oxygen and since no cofactors are required the enzyme was termed ß,ß-carotene-15,15'-oxygenase. The enzyme was reported to be soluble, have a slightly alkaline pH-optimum and be inhibited by ferrous iron chelators and by sulfhydryl-binding compounds. Subsequently, the enzyme was characterized in more detail and most of what we know about the enzyme’s properties and substrate specificity was ascertained in this careful and elegant work. Thus, the way was open to purify the enzyme and to determine its molecular structure. Several endeavors were undertaken and while highly enriched enzyme fractions could be obtained (14), all attempts to purify the enzyme to homogeneity failed. Therefore, the molecular nature of this enzyme remained elusive for a long time. However, the identification of its cDNA or the corresponding gene would be of particular importance. With this tool in hand, old questions in vitamin A research could be addressed definitively on the molecular levels contributing to a mechanistic understanding, e.g., of the regulation of vitamin A homeostasis or tissue specificity of vitamin A formation, with an impact on animal physiology and human health.
Molecular identification and characterization of a ß,ß-carotene-15,15'-oxygenase
To explain how we molecularly identified a gene coding this enzyme, a short excursion to plant carotenoid metabolism must be made. In plants, in addition to the formation of carotenoids, carotenoid cleavage also occurs. Here, by eccentric oxidative cleavage of carotenoids, apocarotenoids such as saffron or ß-ionone are synthesized. Most important in the context of our research is the plant growth factor abscisic acid (ABA). In maize, several mutants are known to exert an abscisic acid defective phenotype. The ABA defective phenotype becomes visible since the maize kernels germinate directly on the corncob and, therefore, these mutants are called viviparous (vp). By analyzing the maize vp14 mutant, Zeevaart and co-workers cloned the first gene coding a carotenoid cleaving enzyme (15). The heterologously expressed and purified recombinant enzyme catalyzes the oxidative cleavage of 9 cis-epoxycarotenoids to form xanthoxin, the direct precursor of ABA. The recombinant enzyme is soluble and depends on molecular oxygen and ferrous iron. Thus, this plant enzyme has quite similar properties compared to the animal ß,ß-carotene-15,15'-oxygenase. The assortment of apocarotenals found in nature results from the large number of carotenoids (>600) and variations in the cleavage site, including retinal formation, in green algae and halobacteria (Fig. 1). This led us to infer that vitamin A (retinoid) formation in animals is just a variation of this theme and is most probably catalyzed by the same class of enzymes. If so, this should be reflected in sequence similarity.
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On the basis of the supposition that the animal ß,ß-carotene-15,15'-oxygenase shares sequence identity with the plant VP14 (9-cis neoxanthin-cleavage enzyme from Zea mais), we searched the entire animal database. We found an expressed sequence tag (EST) with weak sequence similarity to the plant VP14 from the fruit fly Drosophila melanogaster. To obtain its full-length cDNA, we performed RACE-PCR and cloned the cDNA in a bacterial expression vector. The construct was transformed into the E. coli strain capable of synthesizing and accumulating ß-carotene. Indeed, the resultant E. coli strain turned white, indicating that in the presence of the encoded enzyme retinoids are formed at the expense of ß-carotene. Overnight cultures were grown and subjected to further analyses. The lipophilic compounds were extracted and HPLC analyses were performed. The control strain transformed with the vector alone lacked the ability to cleave ß-carotene and no traces of retinoids were detectable. However, bacteria expressing the Drosophila cDNA contained significant amounts of retinoids in addition to ß-carotene. The retinoids were identified by retention time as well as cochromatography with authentic standards and by their absorption spectra. The dominant retinal isomer was the all-trans form, with 
20% of the 13-cis isomer.
The sequence analyses revealed that the insect cDNA encoded a protein of 620 amino acid residues with a calculated molecular mass of 69.9 kDa. The deduced amino acid sequence shares sequence homology to the plant carotenoid oxygenase VP14, to lignostilbene synthase from Pseudomonas paucimobilis and to several proteins of unknown function in animals, plants and cyanobacteria. These sequence similarities represent evidence that the animal carotene oxygenases belong to a large and diverse class of polyene chain oxygenase heretofore only described in plants and microorganisms.
For further analysis of the enzymatic properties of the insect enzyme, the cDNA was cloned in the expression vector pGEX-4T-1 and expressed as a fusion protein. After expression in E. coli, the protein was purified by affinity-chromatography. The purification could be achieved without the addition of detergents, indicating that the fusion-protein was soluble and not tightly associated to membranes. To test for enzymatic activity in vitro, the recombinant purified protein was incubated in the presence of ß-carotene. HPLC analyses revealed exclusively the formation of retinal. The addition of FeSO4/ascorbate in the assays led to an increase in the formation of the cleavage product while addition of EDTA inhibited the conversion of ß-carotene to retinal. These results indicate that the enzyme is soluble and the enzymatic activity of the cloned oxygenase depends on ferrous iron as has been reported for its mammalian counterpart (12,13). Since exclusively retinoids were found in the E. coli test system as well as in the in vitro assay, it must be supposed that a central cleavage of ß-carotene is catalyzed (17). Thus, the insect cDNA encodes a ß,ß-carotene-15,15'-oxygenase (BCO).
Analysis of the blind Drosophila mutant ninaB identifies BCO as the key enzyme for vitamin A formation in vivo
The completed human genome project has revealed that 60% of the genes found in the Drosophila genome possess homologues in the human genome. The identification of the insect bco cDNA may provide the key toward understanding vitamin A formation in animals. However, direct genetic evidence that the encoded proteins catalyze vitamin A formation needed for the visual chromophores in vivo was still missing. In contrast to vertebrates, in Drosophila vitamin A functions are restricted to the visual system. Therefore, a complete vitamin A deficiency leads to a blind but viable phenotype, making it feasible to mutagenize and subsequently identify genes involved in carotenoid and retinoid metabolism. Among the various blind Drosophila mutants affected in vision, one existing mutant, ninaB, manifests a phenotype which fits to a genetically caused vitamin A deficiency, but this mutant had so far not been molecularly characterized (18). The ninaB phenotype could be rescued exclusively by feeding flies retinal, indicating that a loss of the bco function might cause this phenotype. This possibility was made more plausible by the fact that the ninaB mutation has been mapped to position 87E-F in the Drosophila genome coinciding with the physical location of the bco gene.
We then analyzed whether a mutation in the bco gene is responsible for the ninaB phenotype (19). For this purpose we cloned bco cDNAs from two different mutant alleles, ninaBP315 and ninaB360d, into the expression vector pTOPO-BAD. To test directly for enzymatic activity of the encoded proteins we transformed the resultant plasmid constructs into the E. coli strain able to synthesize and accumulate ß-carotene. In contrast to the E. coli strain expressing the wild-type (wt) cDNA, the E. coli strains expressing the BCO encoded by the ninaB alleles remained yellow and subsequent HPLC analyses revealed that mutant proteins failed to catalyze retinoid formation. We then performed sequence analysis of the ninaB alleles. In summary, in both ninaB alleles amino acid sequence alterations could be detected. In the ninaB360d allele a nonsense mutation at position 41 interrupts the open reading frame, while in the ninaBP315 allele the missense mutation at position 838 leading to a Glu to Lys exchange in the encoded BCO protein is responsible for the observed loss of its enzymatic activity. Thus, the molecular analyses of the ninaB Drosophila mutants revealed that their vitamin A deficient phenotype is indeed caused by a mutation in the bco gene and identified it as the gene encoding the key enzyme for vitamin A formation.
Vertebrates have two different types of carotene oxygenases
In vertebrates, there has long been a controversy over symmetric versus asymmetric cleavage of ß-carotene in the biosynthesis of vitamin A and its derivatives (10,11). By sequence similarity to the Drosophila BCO, we and others identified cDNAs encoding its mammalian counterpart (20–23). In an independent approach, Wyss and colleagues (24) succeeded in the cloning and functional characterization of a ß,ß-carotene-15,15'-oxygenase from chicken by an approach relying on partial protein purification, determination of peptide sequences and using this information for the synthesis of oligonucleotide primers to generate a partial cDNA to screen a cDNA library derived from small intestine. By the use of the E. coli test system or by in vitro assays for enzymatic activity with the soluble purified recombinant proteins, it could be shown that these enzymes catalyze exclusively the symmetric oxidative cleavage of ß-carotene, yielding two molecules of retinal, thus demonstrating on the molecular level the existence of the symmetric cleavage pathway in the synthesis of vitamin A.
As in Drosophila, vitamin A derivatives serve as the visual chromophores in vertebrates. Furthermore, RA is an important signal molecule influencing development and cellular differentiation processes. This dual function of vitamin A in vertebrates and a more complex retinoid metabolism are reflected in a large subset of different retinoid modifying enzymes, such as various retinoid dehydrogenases as well as in a large subset of cellular and extra-cellular retinoid binding proteins. Napoli and Race (25) described that besides the formation of RA from retinal, as the initial product of symmetric ß-carotene cleavage, RA is directly formed from ß-carotene in vertebrates. In these investigations retinal was not found to be a freely diffusible intermediate in RA formation, indicating that an alternative pathway exists in vertebrates. Evidence for this alternative pathway in RA formation comes from the observation that, besides symmetric cleavage of ß-carotene, asymmetric cleavage occurs (8,9,26). This asymmetric cleavage leads to the formation of two molecules of ß-apocarotenal with different chain lengths. For RA formation, the ß-apocarotenal with the longer chain length must be shortened, yielding one molecule of RA. For this a mechanism similar to the ß-oxidation of fatty acids has been proposed (27).
In Drosophila only one family member of polyene chain oxygenase, BCO encoded by the ninaB gene, is found in the entire genome. In vertebrates, however, besides the ß,ß-carotene-15,15'-oxygenase, another protein with significant sequence identity, RPE65, has been found in the retinal pigment epithelium (RPE) (28,29). A role in retinoid metabolism of the eye has been proposed for RPE65 by mutant analysis (30). Although its exact role is still unknown, the high degree of sequence identity to BCO points to a related biochemial function. We searched mammalian EST-databases and found an EST-fragment from the mouse with significant peptide sequence similarity to both RPE65 and BCO. However, it was not identical with the mouse RPE65 or BCO and, thus, represented possibly a candidate cDNA for an enzyme catalyzing the asymmetric oxidative cleavage of carotenoids (20). To obtain its full-length cDNA, we designed up-stream primers deduced from the EST-fragment. Then, we performed RACE-PCR on a total RNA preparation and cloned it into the vector pBAD-TOPO. Sequence analyses revealed that the cDNA encoded a protein of 532 amino acids, and the deduced amino acid sequence shared 40% sequence identity with the mouse ß,ß-carotene 15,15'-oxygenase. From a commercial cDNA library, a cDNA encoding its human counterpart could also be cloned. Thus, in mammals, besides BCO and RPE65, a third type of polyene chain oxygenase [BCO-II (ß,ß-carotene-9',10'-oxygenase)] exists. For functional characterization of BCO-II of the mouse, we expressed it as a recombinant protein in E. coli and performed an in vitro test for enzymatic activity. HPLC analysis revealed that no retinoids were formed from ß-carotene. However, a compound could be detected with a UV/Vis spectrum resembling those of ß-apocarotenals. To obtain large amounts of this material for further chemical analysis, we decided to take advantage of our E. coli test system. As a control we used the BCO from mouse. While the E. coli strain expressing the BCO from mouse became white, in the E. coli strain expressing the BCO-II no such pronounced color shift occurred. However, the ß-carotene content of the E. coli strain expressing the BCO-II was significantly reduced compared to a control strain (Fig. 2). To identify the putative cleavage products, they were extracted and subjected to HPLC analyses. Besides ß-carotene, ß-apo-10'-carotenal and significant amounts of ß-apo-10'-carotenol could be detected as judged from UV/VIS spectra as well as by LC-MS determination of molecular masses. Thus, ß-apo-10'-carotenal is formed from ß-carotene. However, the second compound which should result from the oxidative cleavage of ß-carotene at the 9',10' double bond of ß-carotene, ß-ionone, was not detectable by HPLC. This could be explained either by its volatility and/or its being partitioned to the medium. Therefore, we analyzed the bacterial growth medium after solid phase extraction of lipophilic compounds by GC-MS. Indeed, in the medium of the E. coli strain expressing BCO-II significant amounts of ß-ionone could be detected. Taken together, these analyses demonstrated that BCO-II catalyzes the asymmetric cleavage of ß-carotene at the 9',10' carbon double bond, resulting in the formation of ß-apo-10'-carotenal and ß-ionone. Therefore, we have termed this enzyme ß,ß-carotene-9',10'-oxygenase (BCO-II).
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Tissue specificity of vitamin A formation in vertebrates
Since the work of Moore (6) it has been known that vitamin A formation takes place in the small intestine in mammals. Thereafter, ß-carotene oxygenase activity was also described in liver as well as in lung, kidney and brain (12,32).
To establish the tissue specific expression of the carotene oxygenases on the molecular level, we analyzed total RNA from several tissues of mice and estimated the steady-state mRNA levels of the two types of carotene oxygenases by RT-PCR. Both types of carotene oxygenase mRNAs became detectable in small intestine, liver, kidney and testis as well as in uterine tissues. Additionally, low-abundance steady-state mRNA levels of bco-II were present in spleen, brain, lung and heart (20). The expression patterns of the symmetric cleaving enzyme from the mouse could be recently verified by Northern blot analyses (21,23). In chicken, the tissue specific expression patterns of bco were analyzed by a combination of Northern blot and in situ hybridization experiments. Its mRNA was mainly localized in liver, in duodenal villi, as well as in tubular structures of the lung and the kidney (33). To analyze the expression patterns of the asymmetric cleaving enzyme in humans, we used a commercial multiple tissue mRNA blot. With a riboprobe of the human type II oxygenase cDNA, we could find a 2.2 kb message in heart, skeletal muscle and liver. By using the more sensitive RT-PCR method, the occurrence of its mRNA in humans could also be established in small intestine, liver, kidney and brain.
Interesting results were obtained by others by analyzing the expression of bco in humans (22). The authors show that bco is preferentially expressed in the retinal pigment epithelium (RPE) of the human eye and only at much lower levels in kidney, testis, liver and brain. The surprising results of all these current investigations is that the carotene oxygenase steady state mRNA levels in small intestine are quite low even though its highest enzymatic activity was reported here in mammals. This could be either explained by interspecies differences in carotene metabolism—biochemical investigations were mainly performed with the rat—or be due to the age and nutritional status of the individuals investigated. In addition, the expression of both types of carotene oxygenases in the same tissues, e.g., small intestine and liver, confirms biochemical investigations on the molecular level and explains the observation of both symmetric and asymmetric cleavage activity in homogenates of the same tissue. However, it is not yet clear whether both enzymes are expressed in the same or in different cell types of these tissues and this needs further elucidation. To sum up, these first investigations on the molecular level revealed that carotene oxygenase expression is found in a variety of different tissues. These findings suggest that vitamin A synthesis is not just restricted to small intestine and liver. Therefore, besides supplementation from the retinoid pool of the body several tissues may rely on a local vitamin A synthesis from carotenoids catalyzed via carotene oxygenase activities. This supposition is in agreement with the fact that in the circulation of many mammals significant amounts of carotenoids are present in addition to vitamin A derivatives.
Putative roles of the carotene oxygenases in vertebrate development
Retinoids play an essential role in vertebrate development. The present model in developmental biology assumes that the retinoids needed for development derive mainly from maternal, preformed vitamin A. Vertebrate reproduction demands an elevated vitamin A supply for the egg or embryo, respectively. Under laboratory conditions, the test animals are normally kept under a vitamin A fortified diet that is low in carotenoids. However, an interesting issue is the embryonic supply of vitamin A under conditions when dams are fed a diet mainly providing the provitamin. In most vertebrates including man those diets correspond to the naturally occurring situation. In addition, since molecular data were missing, it could not yet be elucidated whether the vertebrate embryo itself takes advantage of the nontoxic provitamin to partially synthesize the retinoids needed during development. With the identification of the carotene oxygenases, these issues can be now addressed on the molecular level. We used the zebrafish (Danio rerio) as a vertebrate model system to investigate the expression patterns of the carotene oxygenases during development. For this purpose we first cloned the two types of carotene oxygenases from this fish. The existence of the two different types of carotene oxygenases in zebrafish demonstrates that their occurrence is not restricted to mammals. By whole mount in situ hybridization with anti-sense RNA probes, we established their spatial and temporal expression patterns. The zebrafish bco homologue was expressed with the beginning of segmentation stages and could be detected in several structures of the head including the developing eye until embryonic day 2 (E2). In contrast, the bco-II mRNA first became detectable at E2 and its expression was restricted to the developing heart. An expression of bco-II in the human fetal heart could also be found upon analyzing a commercial multi-tissue RNA panel, indicating that it may play a role during the development of this organ. By using the anti-sense morpholino oligonucleotide technique, we analyzed the consequence of a loss of function of BCO during zebrafish development. This treatment led to malformations in the architecture of the branchial arch skeleton and the eye, thus indicating a crucial developmental role of this enzyme in zebrafish. Even though only small amounts of carotenoids, besides huge amounts of retinoids, are found in the zebrafish yolk, these studies reveal that some tissues may rely on a local vitamin A synthesis from the provitamin during development (34).
In the mouse embryo, high expression levels of bco mRNA at E7 by Northern blot analysis were reported while at later stages of development (E11-E15) its mRNA levels decrease but remain still detectable (21). In contrast, by in situ hybridization experiments, in utero bco expression could be found mainly in the maternal tissues surrounding the embryo, but it was not present in embryonic tissues in detectable levels (7.5 and 8.5 d) (23). The expression of bco in the maternal tissues surrounding the mammal embryo indicates that BCO may contribute directly to the elevated vitamin A demand of the mammalian embryo. Even these results are contradictory, and any putative role of an endogenous embryonic vitamin A synthesis should to be addressed in more detail, examining early and late stages of mouse embryogenesis.
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3 Abbreviations used: ABA, abscisic acid; BCO, ß,ß-carotene-15,15'-oxygenase; BCO-II, ß,ß-carotene-9',10'-oxygenase; EST, expressed sequence tag; GC, gas chromatography; LC, liquid chromatography; MS, mass spectroscopy; PCR, polymerase chain reaction; RA, retinoic acid; kb, kilobase (pair); RPE, retinal pigment epithelium; VP14, 9-cis neoxanthin-cleavage enzyme from Zea mais.
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