From the University of Freiburg, Instiute
of Biology I, Animal Physiology and Neurobiology, Hauptstrasse 1, D-79104 Freiburg, Germany and the ¶ University of Hohenheim,
Institut für Lebensmittelchemie, Garbenstrasse 28, D-70599 Stuttgart, Germany
Received for publication, December 20, 2000, and in revised form, January 26, 2001
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ABSTRACT |
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In vertebrates, symmetric
versus asymmetric cleavage of Vitamin A and its analogs have a variety of physiological
functions. Retinal or related compounds such as 3-hydroxyretinal serve
as the chromophores of various visual pigments (rhodopsins) throughout
the animal kingdom (1, 2). In vertebrates, the vitamin A
derivative retinoic acid (RA)1 additionally exerts effects
in development and cell differentiation by binding to specific nuclear
receptors involved in the regulation of gene transcription
(3-5). The key step in the formation of vitamin A is the oxidative cleavage of -carotene in the
biosynthesis of vitamin A and its derivatives has been controversially
discussed. Recently we have been able to identify a cDNA encoding a
metazoan
,
-carotene-15,15'-dioxygenase from the fruit fly
Drosophila melanogaster. This enzyme catalyzes the key step
in vitamin A biosynthesis, symmetrically cleaving
-carotene to give
two molecules of retinal. Mutations in the corresponding gene are known
to lead to a blind, vitamin A-deficient phenotype. Orthologs of this
enzyme have very recently been found also in vertebrates and
molecularly characterized. Here we report the identification of a
cDNA from mouse encoding a second type of carotene dioxygenase
catalyzing exclusively the asymmetric oxidative cleavage of
-carotene at the 9',10' double bond of
-carotene and resulting in
the formation of
-apo-10'-carotenal and
-ionone, a substance
known as a floral scent from roses, for example. Besides
-carotene,
lycopene is also oxidatively cleaved by the enzyme. The deduced amino
acid sequence shares significant sequence identity with the
,
-carotene-15,15'-dioxygenases, and the two enzyme types have
several conserved motifs. To establish its occurrence in different
vertebrates, we then attempted and succeeded in cloning cDNAs
encoding this new type of carotene dioxygenase from human and zebrafish
as well. As regards their possible role, the apocarotenals formed
by this enzyme may be the precursors for the biosynthesis of retinoic
acid or exert unknown physiological effects. Thus, in contrast to
Drosophila, in vertebrates both symmetric and asymmetric
cleavage pathways exist for carotenes, revealing a greater complexity
of carotene metabolism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carotene by enzymes encoded in the genome of the animal. The properties of these enzymes
have been a subject of controversy because both symmetric and
asymmetric cleavage of
-carotene was reported in crude
extracts (6-8) (Fig. 1). Thanks to its
sequence similarity to a plant carotenoid-cleaving enzyme, VP14
(9-cis neoxanthin cleavage enzyme from Zea
mais) (9), we have been able to clone from a metazoan (the
fruit fly Drosophila melanogaster) a cDNA
encoding a
,
-carotene-15,15'-dioxygenase (
-diox) catalyzing
exclusively the symmetric cleavage of
-carotene to give two
molecules of retinal (10). Orthologs of this enzyme have since been
cloned and characterized from the chicken and the mouse by others (11,
12). The enzymes belong to a widespread and diverse class of polyene
chain dioxygenases previously described in bacteria and plants (10,
13). Besides the
-diox, in vertebrates another putative polyene
chain dioxygenase, RPE65, is found (14, 15). A function in retinoid
metabolism for this protein was proposed by mutant analysis, but its
biochemical function is still unknown (16).
View larger version (14K):
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Fig. 1.
Schematic overview of mammalian
-carotene/retinoid metabolism. Solid
arrows, vitamin A formation by the symmetric cleavage pathway. The
retinal formed can be further metabolized to give retinol and
retinylesters (storage) or can be oxidized to give retinoic acid.
Broken arrows,
-apo-carotenal (8', 10', 12') formation by
the asymmetric cleavage of
-carotene. For retinoic acid formation
the
-apocarotenals have to be shortened by a mechanism similar to
-oxidation of fatty acids (for further details see the
Introduction).
In contrast, in Drosophila only one representative of this
class of enzymes is found, and mutations in the corresponding gene (ninaB) cause a blind, vitamin A-deficient phenotype (17),
demonstrating that vitamin A is formed exclusively by the symmetric
cleavage of the provitamin. Besides this blindness, no other defect
becomes manifest; so here vitamin A functions are restricted to the
visual system. In vertebrates, vitamin A formation and metabolism are probably more complex, considering the multiple vitamin A effects in
development and cell differentiation exerted by its metabolite, RA.
This inference is strengthened by the fact that besides the formation of RA from retinal, as the initial product of symmetric -carotene cleavage, direct formation of RA from
-carotene has been described in vertebrates (18). In these investigations retinal was
not found to be an intermediate in RA formation, indicating that an
alternative pathway for RA formation is present in vertebrates. Biochemical evidence for this alternative pathway in RA formation comes
from the observation that, besides symmetric cleavage of
-carotene,
asymmetric cleavage occurs (7, 19, 20). This asymmetric cleavage leads
to the formation of two molecules of
-apocarotenal with different
chain lengths. For RA formation, the
-apocarotenal with the longer
chain length must be shortened, yielding one molecule of RA. For this,
a mechanism similar to the
-oxidation of fatty acids has been
proposed (21). Furthermore, in vertebrates several physiological
effects are caused by
-carotene (22).
-carotene itself or
metabolites derived from it by alternative oxidative cleavage reactions
are most likely responsible. Therefore, vertebrate
-carotene
metabolism and especially RA formation could well be more complex and
not just a matter of producing retinal and converting it to retinoic acid.
Taking the mouse as a model for vertebrate -carotene metabolism, we
searched EST libraries for additional putative polyene chain
dioxygenases. Here we report on the cloning and biochemical characterization of a carotene dioxygenase catalyzing the asymmetric cleavage of
-carotene. We were able to identify the cleavage products as
-apo-10'-carotenal (C27) and
-ionone
(C13) from
-carotene (C40). In addition,
this new type of metazoan polyene chain dioxygenase catalyzes the
oxidative cleavage of lycopene, resulting in the formation of
apolycopenals. The existence of two different types of carotene
dioxygenases in vertebrates was verified by cloning the corresponding
cDNAs from man and the zebrafish. Thus, asymmetric cleavage of
-carotene exists in vertebrates and may provide a precursor for RA
formation and/or may exert until now unknown physiological functions.
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EXPERIMENTAL PROCEDURES |
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Sequence Comparison and Phylogenetic Tree Analysis-- Vector NTI suite 6.0 (InforMax Inc., Oxford, UK) was used.
Chemicals--
The following chemicals were used:
-ionone (Roth, Karlsruhe, Germany),
-apo-12'-carotenal (BASF,
Ludwigshafen, Germany), and
-apo-8'-carotenal (Sigma).
Preparation of Total RNA from Different Tissues of Mice-- For the experiments 7-week-old BALB/c mice (male and female) were sacrified, and different tissues (colon, small intestine, stomach, spleen, brain, liver, heart, kidney, lung, and testis) were dissected by hand and frozen immediately in liquid nitrogen. 50-100 mg of each tissue was homogenized with a pestle in a mortar with liquid nitrogen, and total RNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany). The concentrations of the isolated total RNA were determined spectrophotometrically.
Cloning of cDNAs Encoding -Diox-homologous Proteins
from the Mouse--
For cloning of full-length cDNAs encoding
putative mouse
-carotene dioxygenases, RACE-PCRs were performed
using a 5'/3' RACE kit (Roche Molecular Biochemicals). Reverse
transcription was carried out using 500 ng of total RNA isolated from
liver, an oligo(dT) anchor primer, and Superscript reverse
transcriptase (Life Technologies, Inc.). For PCR the Expand PCR system
(Roche Molecular Biochemicals), an anchor primer, and a specific
up-primer, 5'-ATGGAGATAATATTTGGCCAG-3' for
-diox and
5'-ATGTTGGGACCGAAGCAAAGC-3' for
-diox-II, were used. The PCR
products were ligated into the vector pBAD-TOPO (Invitrogen),
resulting in the plasmids pDiox and pDiox-II.
Tissue-specific Expression of -Carotene Dioxygenases in the
Mouse--
With total RNA (100 ng) isolated from different tissues,
RT-PCR was performed as has been described (23). The following sets of
primers were used:
-diox, up, 5'-ATGGAGATAATATTTGGCCAG-3', and down,
5'-AACTCAGACACCACGATTC-3';
-diox-II, up,
5'-ATGTTGGGACCGAAGCAAAGC-3', and down, 5'-TGTGCTCATGTAGTAATCACC-3'. As
a control for the intactness of the individual RNA samples, the
mRNA of
-actin was analyzed using the following primers: up,
5'-CCAACCGTGAAAAGATGACCC-3'; down, 5'-CAGCAATGCCTGGGTACATGG-3'.
Determination of the Enzymatic Activity in Vitro--
For
heterologous expression of the -diox-II the plasmid pDiox-II was
transformed in the Escherichia coli strain XL1-blue (Stratagene Inc., La Jolla, CA). The bacterial culture was grown at
28 °C until it reached an A600 of 1.0. Then,
L-(+)-arabinose was added to a final concentration of 0.8%
(w/v), and the bacteria were cultivated for an additional 3 h.
After being harvesting, the bacteria were broken with a French press in
a buffer containing 50 mM Tricine/KOH (pH 7.6), 100 mM NaCl, and 1 mM dithiothreitol. The crude
extract was centrifuged at 20,000 × g for 20 min. The supernatant was dialyzed against the same buffer for 1 h at
4 °C. Enzymatic activity was determined in crude extracts (100 µg of total protein) as described (24) by adding
-carotene in micelles
of Tween 40 with a final concentration of 300 µM
-carotene and 0.2% Tween 40 in the assay. Then the lipophilic
compounds were extracted and subjected to HPLC analysis as described
(10).
HPLC Analysis of -Carotene- and Lycopene-accumulating E. coli
Strains Expressing the Two Different
-Carotene Dioxygenases from the
Mouse--
The plasmids pDiox and pDiox-II were transformed into the
appropriate E. coli strain. Growing conditions and analysis
of the carotenes and their cleavage products were as previously
described (10).
Mass Spectroscopy of the Cleavage Products by LC-MS and
GC-MS--
The E. coli strains were cultivated overnight,
and the bacteria were harvested by centrifugation. For solid phase
extraction an SPME syringe (100 µm PDMS, Supelco, Deisenhofen,
Germany) was incubated in the supernatant for 15 min. Then the
compounds absorbed to the solid phase were subjected directly to GC-MS
(GC: Hewlett-Packard 6890; MS: Hewlett-Packard 5973 (70 eV), Waldbronn,
Germany) with a temperature program starting at 100 °C and
increasing 6 °C/min to 300 °C. A DB-1 column (30 m × 0.25 mm × 0.25 µm film thickness, J & W, Folsom, Canada) was used
with helium as the carrier gas. For LC-MS analysis the bacterial pellet
was extracted in the presence of hydroxylamine as previously described
(10). LC-MS was run on an HP1100 HPLC module system (Hewlett-Packard),
coupled to a Micromass (Manchester, UK) VG platform II quadrupole mass
spectrometer equipped with an APcI (atmospheric pressure chemical
ionization) interface. UV absorbance was monitored with a diode array
detector. MS parameters (APcI+ mode) were as follows:
source temperature, 120 °C; APcI probe temperature, 350 °C;
corona, 3.2 kV; high voltage lens, 0.5 kV; cone voltage, 30 V. The
system was operated in full scan mode (m/z
250-1000). For data acquisition and processing, MassLynx 3.2 software
was used. For peak separation, a Nucleosil RP-C18 column (5 µm;
250 × 4.6 mm) from Bischoff (Leonberg, Germany) was used and kept
at 25 °C. The mobile phases consisted of a mixture of acetonitrile
and methanol at 85:15 (v/v) (A) and isopropanol (B); the gradient (% A
(min)) was as follows: 100 (8), 70 (10), 70 (25), 100 (28), 100 (32). The flow rate was 1 ml/min, and the injection volume was
20 µl.
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RESULTS |
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Cloning of a cDNA Encoding a New Type of Carotene
Dioxygenase--
For the cloning of the cDNA encoding the new type
of carotene dioxygenase, we searched mouse EST data bases and found an
EST fragment (AW044715) with significant peptide sequence similarity to
the RPE65 and the recently characterized -dioxes from
Drosophila, the chicken, and the mouse (10-12). However, it
was not identical with the mouse RPE65 and
-diox and thus
represented a new heretofore unknown representative of this class of
polyene chain dioxygenases. To obtain a full-length cDNA, we
designed upstream primers deduced from the EST fragment. Then we
performed RACE-PCR on a total RNA preparation derived from the liver of
a 7-week-old BALB/c male mouse. The PCR product was cloned into the
vector pBAD-TOPO, and sequence analyses were carried out. The cDNA
encoded a protein of 532 amino acids. Sequence comparison revealed that
the deduced amino acid sequence shared 39% sequence identity with the
mouse
,
-carotene-15,15'-dioxygenase (Fig.
2). Several highly conserved stretches of
amino acids and six conserved histidines probably involved in binding
the cofactor Fe2+ were found (10), indicating that the
encoded proteins belong to the same class of enzymes. Thus, in the
mouse, besides the
-diox and RPE65, a third type of polyene chain
dioxygenase (
-diox-II) exists.
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The New Type of Carotene Dioxygenase Catalyzes the Asymmetric
Cleavage of -Carotene, Resulting in the Formation of
-Apo-10'-carotenal and
-Ionone--
For functional
characterization of
-diox-II, we expressed it as a recombinant
protein in E. coli and performed an in vitro test
for enzymatic activity under the conditions described for
-diox
(24). HPLC analysis revealed that no retinoids were formed from
-carotene. However, a compound with a retention time of 4.6 min was
detected (Fig. 3A). In the
presence of hydroxylamine during extraction, the retention time of this
compound shifted from 4.6 to 16 min, indicating that the compound has
an aldehyde group from which the corresponding oxime can be formed
(Fig. 3B). The increase of the putative
-carotene
cleavage product catalyzed by the new type of
-carotene dioxygenase
was linear up to 2 h of incubation time. The UV-visible absorbance
spectra of the compounds resembled those of
-apocarotenals and
-apocarotenaloximes, respectively (Fig. 3C). However,
they were not identical with
-apo-8'-carotenal/oxime and
-apo-12'-carotenal/oxime, as judged by comparing the spectra of
reference substances in stock in our laboratory. The spectra resembled
the spectra of
-apo-10'-carotenal (424 nm) and
-apo-10'-carotenaloxime (435 nm) as found in the literature (25). A
definite identification of the compounds would require further
investigations. However, the turnover rates and, therefore, the amounts
of cleavage product formed were quite low in vitro, as
already observed for the
-dioxes (10-12). To obtain large amounts
of this substance for further chemical analysis, we decided to take
advantage of an E. coli test system already successfully
used to characterize the
-diox from Drosophila (10). This
test system offered the advantage of combining
-carotene
biosynthesis and further metabolism by carotene dioxygenases in one
organism. In the case of retinoid formation catalyzed by
-diox,
-carotene cleavage became visible by a color shift of the bacteria.
As a control we used the
-diox from the mouse. Whereas the E. coli strain expressing the
-diox from the mouse became white,
in the E. coli strain expressing the
-diox-II no such
pronounced color shift occurred (Fig. 4). However, the
-carotene content of the E. coli strain
expressing the
-diox-II was significantly reduced compared with a
control strain (22.8 pmol/mg of dry weight versus 60.9 pmol/mg of dry weight of the E. coli control strain). To
identify the putative cleavage products, we extracted and subjected
them to HPLC analyses as has been described (10). Besides
-carotene,
six peaks were detected, which were assigned to two classes of
compounds by their UV-visible absorbance spectra (Fig.
5). The first class (peaks 2, 5, and 6)
showed an absorbance maximum at 424 nm, identical with the putative
-apo-10'-carotenaloxime already found in the in vitro
tests (Figs. 3C and 5, B and C). The
second class of compounds (peaks 1, 3, and 4) had an absorbance maximum
at 386 nm and a UV-visible spectrum resembling that of
-apo-10'-carotenol (25) (Fig. 5, B and D). The
occurrence of compounds with the same absorbance spectra but different
retention times could be due to the stereoisomeric composition of the
products formed and/or, in the case of the aldehydes, due to the
syn or anti configuration of the oximes formed.
Depending on the induction time, first the putative
-apo-10'-carotenal and then the putative
-apo-10'-carotenol became detectable, indicating that the aldehyde is converted to the
corresponding alcohol in E. coli (data not shown). The
conversion of retinal to the corresponding alcohol retinol in E. coli was already found by expressing the
-diox from
Drosophila or from the mouse as shown here (Fig.
5A). To positively identify the putative
-apo-10'-carotenal formed, we converted it to the corresponding
-apo-10'-carotenaloxime and subjected it to LC-MS analyses. Because the system was operated in the APcI+ mode, quasimolecular
ions generally appeared as [M + H]+ signals.
-apo-10'-carotenaloxime was identified by its quasimolecular ion at
m/z 392 [M + H]+, which is the base
peak of the spectrum. The even-numbered [M + H]+ mass
signal clearly proved the presence of a nitrogen in the compound and
thus established the transformation of the aldehyde group into the
corresponding oxime. Fragmentation of the polyene chain, yielding
characteristic daughter ions, was not observed. Additionally, the
characteristic UV-visible spectrum, showing maxima at 405 (shoulder),
424, and 446 nm, is in accordance with the chromophoric system of
-apo-10'-carotenaloxime and consistent with spectroscopic data
reported previously (25).
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Thus, from -carotene
-apo-10'-carotenal is formed.
However, the second compound that should result from the oxidative
cleavage of
-carotene at the 9',10' double bond of
-carotene,
-ionone, was not detectable by HPLC. This could be explained by its
volatility and/or its partitioning to the medium. Therefore, we
analyzed the bacterial growth medium after solid phase extraction of
lipophilic compounds by GC-MS. In the medium of the E. coli
strain expressing
-diox-II, significant amounts of
-ionone
(identical in its retention time and mass spectra to a
-ionone
standard) was detected, and these were not found in the medium of the
E. coli control strain. Taken together, the analyses
demonstrated that
-diox-II catalyzes the asymmetric cleavage of
-carotene at the 9',10' carbon double bond, resulting in the
formation of
-apo-10'-carotenal and
-ionone. Therefore, we have
termed this enzyme
,
-carotene-9',10'-dioxygenase.
To test whether the enzyme catalyzes the oxidative cleavage of
carotenes different from -carotene, we transformed it into an
E. coli strain able to synthesize and accumulate lycopene
(Fig. 4). The experiment was performed as described above. In this
strain significant amounts of putative apolycopenals became detectable. This was shown by converting the aldehydes to the corresponding oximes
(data not shown). Therefore, the new type of carotene dioxygenase catalyzes the oxidative cleavage of lycopene in the E. coli
test system as well, resulting in the formation of apolycopenals
tentatively identified by their UV-visible spectra.
Cloning of cDNAs Encoding the New Type of Carotene Dioxygenase
from Man and the Zebrafish--
To verify the existence of the second
type of dioxygenase, -diox-II, in other metazoan organisms, we
searched for EST fragments with sequence identity in the data base. We
found EST fragments from man and the zebrafish. Then we cloned and
sequenced the corresponding full-length cDNAs. The cDNA cloned
from total RNA derived from human liver encoded a protein of 556 amino
acids, whereas the cDNA isolated from the zebrafish encoded a
protein of 549 amino acids. The deduced amino acid sequences shared 72 and 49% sequence identity, respectively, to the mouse
-diox-II. We
performed a phylogenetic tree calculation based on a sequence distance
method and utilized a neighbor-joining algorithm (26) with the deduced amino acid sequences of the metazoan polyene chain dioxygenases and the
plant VP14. As shown in Fig. 6, in
vertebrates three groups of polyene chain dioxygenases are found: the
two different
-carotene dioxygenases and RPE65. The sequence
analysis revealed that the three vertebrate polyene chain dioxygenases
most likely emerged from a common ancestor. In contrast, in
Drosophila and Caenorhabditis elegans, only one
type of dioxygenase was found in the entire genome. As judged by the
E. coli test system, the C. elegans dioxygenase catalyzes the symmetric cleavage of
-carotene to form retinal. Therefore, the occurrence of additional genes encoding this class of
enzymes, the
-diox-II and the RPE65, is apparently related to
vertebrate carotene/retinoid metabolism.
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Tissue-specific Expression of the New Type of Carotene
Dioxygenase--
We analyzed total RNA from several tissues of
7-week-old BALB/c mice (male and female) and estimated the steady-state
mRNA levels of the two types of carotene dioxygenases by RT-PCR
analyses. RT-PCR products of both types of carotene dioxygenase
mRNAs became detectable in small intestine, liver, kidney, and
testis, whereas low abundance steady-state mRNA of the new type of
carotene dioxygenase was additionally present in spleen, brain, lung,
and heart (Fig. 7). The intactness of the
RNA preparations was verified by analyzing the -actin mRNA. By
omitting the reverse transcriptase in the assays, it was shown that the
RT-PCR products derived from mRNA and not from DNA contaminations.
By using a multiple tissue mRNA blot
(CLONTECH), analyzed with a riboprobe of the human
cDNA, we were able to find a 2.2-kilobase pair message in heart and liver for the new type of carotene dioxygenase, whereas a transcript of
2.4 kilobase pairs for the
-diox was found mainly in kidney (data not shown).
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DISCUSSION |
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Here, we report on the cloning, characterization, and
tissue-specific expression of a new type of carotene dioxygenase from mouse catalyzing the asymmetric cleavage of -carotene. By expressing the enzyme in a
-carotene-synthesizing E. coli strain,
-apocarotenal formation at the expense of
-carotene was shown.
The cleavage products formed were identified by their absorbance
spectra, by the conversion of the aldehyde to the corresponding oxime,
and by LC-MS or GC-MS as being
-apo-10'-carotenal and
-ionone.
In vitro, the enzyme catalyzed the same reaction as in the
E. coli test system. Thus, the characterized enzyme
catalyzed the oxidative cleavage at the 9'-10' double bond in the
polyene backbone of its substrate,
-carotene.
Besides the overall sequence identity to the -diox previously
described (10-12), there is a distinct conserved pattern of histidine
residues, which has been proposed to be involved in the binding of the
cofactor Fe2+ (12). Thus, including RPE65, three different
representatives of the polyene chain dioxygenase family are found in
vertebrates. Although the biochemical function of the RPE65 protein
remains to be elucidated, we show that besides symmetrical cleavage of
-carotene, asymmetric cleavage also occurs, positively resolving the
controversial debate on the significance of this reaction. The analysis
of the tissue-specific expression showed that mRNAs for both
enzymes are found together in several tissues, e.g. small intestine and liver. These findings verify biochemical results on the
molecular level that both symmetric and asymmetric cleavage of
-carotene can be found in the same tissue. The expression patterns
in the mouse and man were not consistent. This could be either because
of interspecies differences in carotene metabolism or reflect
differences in the age and nutritional status of the individuals
investigated, thus possibly presenting an additional factor to explain
the conflicting results obtained in several investigations. In earlier
studies conducted with tissue homogenates, a variety of
-apocarotenals of different chain length resulting from asymmetric
-carotene cleavage were found. Therefore, the term random cleavage
was used for this reaction by several authors. Here we show that the
characterized enzyme does not catalyze such side reactions; instead it
is specific for the 9',10' double bond. The formation of
-apocarotenals different from
-apo-10'-carotenal found in
vitro may be caused by further metabolism of the primary cleavage
product or by additional, yet unknown, carotene dioxygenases. However,
the in vitro activity of the metazoan polyene chain
dioxygenases is difficult to obtain, and
-apocarotenal formation
from
-carotene by nonenzymatic degradation has been reported in an
aqueous environment (27).
After the molecular identification of a cDNA encoding this new type
of carotene dioxygenase, the question arose as to the physiological
relevance in vertebrate carotene metabolism. Sharma et al.
(28) showed in rats and chickens that -apocarotenals can be
bioactive precursors for RA formation. After absorption of these
compounds, the corresponding acid is first formed and then shortened to
yield retinoic acid. The same study also showed that only small
proportions of
-apocarotenals are attacked by the
-diox to give
retinal. This possibility could be of importance considering the
co-expression of both dioxygenases in several tissues shown here.
Napoli and Race (18) showed that several tissues are able to synthesize
RA and that retinal, the primary product of the symmetric cleavage of
-carotene, was not detected as an intermediate. By analyzing RA
formation from
-apocarotenals, a mechanism similar to
-oxidation
of fatty acids was proposed by Wang et al. (21). In their
study, RA formation from
-apocarotenals was ensured by giving
citral, a potent inhibitor of retinalaldehyde dehydrogenases that
catalyze the oxidation of retinal to RA. Therefore, the asymmetric
cleavage reaction most likely represents the first step in an
alternative pathway in the formation of RA and may contribute to RA
homeostasis of the body, certain tissues, or cells. The second product
resulting from asymmetric cleavage,
-ionone, is known as a scent
compound in plants. This short chain compound is volatile, and a
putative physiological role in animals remains to be investigated.
In Drosophila vitamin A is exclusively formed by the
symmetric cleavage reaction (17). In vertebrates the two different carotene dioxygenases and the RPE65 protein are found. Sequence comparison indicated that the vertebrate dioxygenases arose from a
common ancestor. In contrast to Drosophila, in vertebrates
RA plays an important role in development and cell differentiation. Thus, the existence of different -carotene dioxygenases could be
related to the emergence of RA effects. By in situ
hybridization in zebrafish embryos, high steady-state mRNA levels
of the zebrafish homologue of the
-diox were found before
gastrulation. The zebrafish homologue to the
-carotene-9',10'-dioxygenase could only be detected after
organogenesis.2 The
finding of high steady-state mRNA levels of the
-diox at early
times in development has been reported recently for the mouse (12).
This indicates that retinoid formation from
-carotene catalyzed by
the symmetric oxidative cleavage reaction may contribute to the
retinoid homeostasis of the embryo. Therefore, besides maternal,
preformed vitamin A, de novo biosynthesis from the
provitamin seems to be an important source for retinoids during
development. However, the asymmetric cleavage reaction may contribute
to RA formation in certain tissues during later stages of development. In this context, the observed expression of the
-diox-II in brain and lung could be of relevance. In cell differentiation processes in
the nervous system, RA plays an important role (29). In a ferret model,
under certain conditions such as exposure to cigarette smoke,
-carotene toxicity on lung has been reported (22). Asymmetric cleavage of
-carotene was discussed as being involved in these toxic
effects (for review see Ref. 30). Furthermore, RA formation from
-carotene has been found in vitro in the testis, small
intestine, liver, and kidney (18). Here, we show that in all these
tissues mRNAs encoding the two different types of carotene
dioxygenases are found. This indicates that besides the small intestine
and liver, several tissues may contribute to their own RA homeostasis by endogenous retinoid formation from
-carotene, until now an underestimated, unappreciated feature in retinoid homeostasis.
As judged in an E. coli test system, the enzyme was also
able to catalyze the oxidative cleavage of lycopene. This indicates with respect to substrate specificity that the polyene chain backbone of carotenes plays an important role, whereas the ionone ring structures of -carotene seem to be of marginal relevance. This result was also obtained upon analyzing the mouse
-diox (12). Favorable effects of lycopene on human health have been reported (31).
Lycopene is accumulated primarily in liver but also in intestine,
prostate, and testis, tissues in which both
-diox and
-diox-II
mRNAs are expressed. The cleavage of lycopene and the formation of
apolycopenals are indicative of a putative role in vertebrate
physiology. In vertebrates, several nuclear receptors with unknown
ligands exist, i.e. orphan receptors. Besides being a
putative precursor for RA formation in the case of
-carotene cleavage, the compounds formed by the asymmetric cleavage reaction of
-carotene and/or lycopene may represent putative ligands for these receptors.
Taken together, the data presented here led to the molecular
identification of an enzyme, ,
-carotene-9',10'-dioxgenase, catalyzing the asymmetric cleavage of
-carotene. Thus, besides the
symmetric cleavage of
-carotene, a second enzymatic activity is
present in vertebrates. The molecular identification of enzymes involved in the cleavage of
-carotene will open new avenues of research on the impact of metabolites derived from carotenes in animal
physiology and human health.
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ACKNOWLEDGEMENTS |
---|
We thank Beate Ziser for skillful technical assistance and Randy Cassada for helpful discussion and for correcting the English version of the manuscript.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ290392, AJ290393, AJ290390, and AJ290391.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: Inst. of Biology
I, Animal Physiology and Neurobiology, University of Freiburg, Hauptstrasse 1, D-79104 Freiburg, Germany. Tel.: 49 761 203 2539; Fax:
49 761 203 2921; E-mail: lintig@uni-freiburg.de.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M011510200
2 J. M. Lampert and J. v. Lintig, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
RA, retinoic acid;
-diox,
,
-carotene-15,15'-dioxygenase;
EST, expressed sequence
tag;
RACE, rapid amplification of cDNA ends;
PCR, polymerase chain
reaction;
RT-PCR, reverse transcription-PCR;
-diox-II,
,
-carotene-9',10'-dioxygenase;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
HPLC, high performance liquid chromatography;
LC, liquid chromatography;
MS, mass spectrometry;
GC, gas chromatography;
APcI, atmospheric pressure
chemical ionization;
RPE, retinal pigment epithelium.
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