From the Laboratory of Retinal Cell and Molecular
Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892 and the ¶ Department of Cell Biology and Molecular Genetics,
University of Maryland, College Park, Maryland 20742
Received for publication, October 3, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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We have identified from mouse the first mammalian
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
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
-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 Vmax
of 36 pmol of retinal/mg of enzyme/min and a Km of
6 µM. 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
-CD1 would belong to an
emerging family of carotenoid-cleaving dioxygenases known mainly from
examples in plants (6), but with members also in bacteria and Metazoa.
The first described representative was a bacterial lignostilbene
,
-dioxygenase, which cleaves the central carbon-carbon double
bond of lignostilbene, a bicyclic lignin model compound, into two
molecules of vanillin (7). The initial plant representative of this
family is maize VP14, the neoxanthin cleavage enzyme of the abscisic
acid pathway (8) that is a 9-cis-epoxycarotenoid dioxygenase
(9). The essential similarity of these disparate reactions is shown in
Fig. 1.
View larger version (17K):
[in a new window]
Fig. 1.
Signature reactions of three members of a
diverse dioxygenase enzyme family. Arrows indicate the
double bond cleaved. Upper panel, lignostilbene
,
-dioxygenase (LSD) (39); middle
panel, 9-cis-epoxycarotenoid dioxygenase (VP14)
(8); lower panel,
-CD (this work).
The original animal representative of the family is RPE65, a protein
restricted in its expression to the retinal pigment epithelium (10,
11). Although the specific biochemical function of RPE65 is not yet
known, it is required for the all-trans- to
11-cis-isomerization reaction that regenerates the
11-cis-retinal chromophore of rhodopsin in the visual cycle
of the retina (12). A thematic feature of the eukaryotic members of the
family therefore appears to be an interaction with carotenoids or
carotenoid derivatives to cleave or otherwise alter conjugated
carbon-carbon double bonds. We used iterative searching of the genomic
and EST data bases to find genes encoding polypeptides similar in
sequence to RPE65. We describe here the characterization of one such
RPE65 homolog from mouse, detail the developmental and tissue
specificity of its expression, and define its enzymatic activity and
substrate specificity. The protein does indeed cleave -carotene to
produce all-trans-retinal, is related, although not closely,
to a
-CD recently identified from Drosophila melanogaster
(13), and is of the same family as the recently described chicken
-carotene 15,15'-dioxygenase (14). Identification of the mouse
-CD protein and its gene provides the requisite tools for study of
early steps of mammalian carotenoid metabolism in an accessible model
system and gives additional insights into an ancient family of
retinoid/carotenoid-metabolizing enzymes.
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EXPERIMENTAL PROCEDURES |
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Identification of a cDNA Encoding Mouse -CD--
A number
of cDNAs encoding polypeptides similar in sequence to RPE65,
VP14, and lignostilbene
,
-dioxygenase were detected by
iterative tBLASTn (15) searches of the data base of expressed sequence
tags (dbEST) and GenBankTM non-redundant Data Banks.
Of several IMAGE Consortium ESTs examined, AW044715 (Incyte Genomics,
St. Louis, MO) appeared, by comparison with RPE65, to be of sufficient
length to specify the full-length polypeptide and was sequenced.
Alignment of the deduced polypeptide with other metazoan dioxygenases
and RPE65 proteins was done with ClustalW Version 1.74 using a gap
opening penalty of 40 and a gap extension penalty of 0.1; the matrix
used was the Blosum series.
Escherichia coli Expression of the Prospective Mouse
-CD--
The open reading frame of the AW044715 clone was amplified
using the forward primer 5'-ATG GAG ATA ATA TTT GGC CAG-3' and the
reverse primer 5'-GCT CTA GAT TAA AGA CTT GAG CCA CCA TG-3'. An
amplification product of the expected length was cloned into the
expression vector pBAD/TOPO (Invitrogen, Carlsbad, CA) following the
manufacturer's protocol to make the recombinant clone pBAD/
-CD. The
resultant recombinant clones were isolated and sequenced to verify
orientation and sequence. Expression of
-CD was induced as follows.
Overnight cultures of E. coli containing pBAD/
-CD were
grown in LB broth supplemented with 100 µg/ml ampicillin (LB/ampicillin). These cultures were used to inoculate 5-ml
LB/ampicillin cultures. At mid-log phase, expression of
-CD was
induced with serial dilutions of L-arabinose. Control
cultures received distilled H2O in lieu of arabinose.
Cultures were incubated for an additional 4 h, and aliquots were
harvested by centrifugation. Accumulation of the expressed
-CD
protein was assayed by SDS-polyacrylamide gel electrophoresis (16)
followed by immunoblotting (17) with detection using a specific
antiserum to a peptide sequence within mouse
-CD (see below).
Assay of Cleavage Activity of Mouse -CD Expressed in E. coli--
The pBAD/
-CD expression construct was transformed into
competent cells prepared as described (18) from a variety of
carotenoid-accumulating strains of E. coli. Strains of
E. coli that produce and accumulate lycopene and
-carotene have been described (19, 20). Strains that produce
-
and
-carotenes were constructed by inserting an
Arabidopsis mono-
-cyclase (21) or a lettuce
di-
-cyclase cDNA2 into
a unique site in the plasmid that confers the ability to produce
lycopene. Culture tubes containing 5 ml of LB broth (with 150 µg/ml
ampicillin and 30 µg/ml chloramphenicol) were each inoculated with a
single fresh colony and shaken overnight at 28 °C in darkness. One
ml of the overnight cultures was used to inoculate 50 ml of LB broth
(with ampicillin and chloramphenicol) in 250-ml flasks. Arabinose was
added to a final concentration of 0.2%, and the cultures were grown at
28 °C in darkness with shaking for 48 h. Cells were harvested
by centrifugation, and the pellets were extracted immediately or frozen
at
20 °C for up to 1 week before extraction. Cultures of E. coli with cells transformed with the cloning vector containing an
irrelevant insert (pBAD/lacZ) served as controls. Carotenoids and retinoids were extracted from the bacterial pellets by
the method of von Lintig and Vogt (13). Recombinant pBAD/
-CD protein, containing a His tag fusion, was purified using His-bind resin
(Novagen, Madison, WI) from arabinose-induced cultures solubilized with
B-PER reagent (Pierce).
HPLC Analysis of -Carotene and Retinal--
HPLC analysis of
hexane extracts of the bacterial cultures was carried out on a Waters
system consisting of a Model 600E pump, Model 717 autosampler, and
Model 996 photodiode array detector. Separation was achieved by
isocratic elution on a LiChrosphere Si-60 column (5 µm, 3.2 × 250 mm) with a mobile phase consisting of 6% 1,4-dioxane in hexane at
a flow rate of 1 ml/min. Data acquisition and processing were done with
Millennium32 Chromatography Manager Version 3.05 software (Waters). All solvents were HPLC-grade (Aldrich). Standards of
-carotene, lycopene, and all-trans-retinal were obtained
from Sigma.
Kinetic Analysis of Mouse -CD Enzymatic Activity--
The
His-tagged recombinant pBAD/
-CD protein was prepared for assay by
dialysis in the presence of B-PER detergent extract of untransformed
TOP10 (Invitrogen) E. coli cells (100 µl of a 5×
concentration in B-PER of an overnight culture in LB broth per 10 µg
of recombinant protein) against a buffer containing 0.1 M
Tricine-KOH (pH 8.0), 0.1 M NaCl, 0.5% Triton X-100, 5 mM (tris(2-carboxyethyl)-phosphine hydrochloride)
(Pierce), 10 µM FeSO4, and one Complete
EDTA-free protease inhibitor mixture tablet (Roche Molecular
Biochemicals) per 25 ml. The standard assay was carried out in this
buffer (without the protease inhibitor) in a reaction volume of 0.4 ml
at 37 °C for 2 h with 10 µg of recombinant enzyme using a
substrate concentration range of 0-30 µM. The
concentration of retinal was measured by HPLC as described above. Under
these conditions, enzymatic activity was linear with enzyme
concentration up to 35 µg/ml and with time up to 3 h.
Antiserum to Mouse -CD and Immunoblotting--
A multiple
antigenic peptide (22) with the sequence NYIRKIDPQTLETLEK,
corresponding to amino acids 142-157 of mouse
-CD, was synthesized
(Princeton Biomolecules, Langhorne, PA) and used to immunize rabbits.
Serum was harvested and stored at
20 °C. This antibody was used at
a final dilution of 1:5000 in 3% bovine serum albumin and 0.05% Tween
20 in phosphate-buffered saline (TPBS). The secondary antibody was
alkaline phosphatase-conjugated goat anti-rabbit IgG (Life
Technologies, Inc.) used at a dilution of 1:3000 in 3% bovine serum
albumin and TPBS. Immunoreactive bands were detected using the
one-component 5-bromo-4-chloro-3-indolyl phosphate/p-nitro
blue tetrazolium phosphatase substrate solution (Kirkegaard & Perry
Laboratories, Inc., Gaithersburg, MD).
RNA Gel Blot Analysis of Mouse -CD mRNA
Expression--
The open reading frame of mouse
-CD was amplified
using the forward primer 5'-CGG AAT
TCC ATG GAG ATA ATA TTT GGC CAG-3' and the reverse primer
5'-GCT CTA GAT TAA AGA CTT GAG CCA
CCA TG-3' in a reaction employing the high fidelity DNA polymerase Pfu Turbo (Stratagene, La Jolla, CA). The forward and
reverse primers contain restriction sites (underlined) for
EcoRI and XbaI, respectively. Following double
digestion with these enzymes and subsequent phenol/chloroform
extraction, the resultant fragment was directionally cloned into
pBluescript II SK
digested with the same enzymes. To
generate template for riboprobe transcription reactions, the
pBluescript/
-CD clone was digested with EcoRV, and the
3.9-kilobase pair band containing the 3'-end of the
-CD
cDNA followed by an inverted T3 promoter site was separated by
agarose electrophoresis and purified by binding to Qiaex resin (QIAGEN
Inc., Valencia, CA). The purified linearized template was used in a
Strip E-Z (Ambion Inc., Austin, TX) in vitro runoff
transcription reaction to produce [
-32P]UTP-labeled
antisense riboprobe. The labeled riboprobe was used in RNA
hybridizations (NorthernMax system, Ambion Inc.) of mouse multiple
tissue (Origene, Gaithersburg, MD) and mouse embryonic development
stage (CLONTECH, Palo Alto, CA) Northern blots.
Hybridization and washing followed the manufacturer's protocols
(Ambion Inc.). Processed blots were exposed to Hyperfilm MP
autoradiographic film (Amersham Pharmacia Biotech, Little Chalfont,
Buckinghamshire, United Kingdom).
Subcellular Fractionation of Mouse Tissues--
Freshly
dissected kidney, liver, lung, testis, and small intestine were
homogenized in 10 volumes of PBS containing one Complete protease
inhibitor mixture tablet per 25 ml. The homogenate was centrifuged for
10 min at 1000 × g, and the low speed supernatant was
recentrifuged for 15 min at 30,000 × g. The resulting
supernatant was then centrifuged at 55,000 rpm for 30 min in a Beckman
TL-100 tabletop ultracentrifuge using a TLA-100.2 fixed angle rotor. The supernatant was reserved, and the pellet was resuspended in 0.2 volume of homogenizing buffer. These samples were analyzed by
SDS-polyacrylamide gel electrophoresis and immunoblotting using the
conditions described above.
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RESULTS |
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Identification and Characterization of a cDNA Encoding Mouse
-CD--
Iterative tBLASTn searches of the dbEST division of
the GenBankTM/EBI Data Bank using bovine RPE65 (accession
number L11356) as the query sequence identified an EST (AW044715)
showing 3'-similarity to RPE65, the maize epoxycarotenoid cleavage
enzyme VP14, and the bacterial lignostilbene
,
-dioxygenase. The
2120-base pair cDNA (deposited in the GenBankTM/EBI
Data Bank under accession number AF271298) contains an open reading
frame that is 1698 base pairs in length, encoding a protein 566 amino
acids in length with a calculated molecular mass of 63,859 Da. The
deduced polypeptide is without an obvious signal peptide, predicted
transmembrane domains, or potential sites for N-linked glycosylation.
Alignment of the deduced mouse polypeptide with chicken -CD
(GenBankTM/EBI accession number AJ271386),
Drosophila
-CD (accession number AB041507), and the
predicted product of a human cDNA of unknown function (accession
number AK001592) showed 70, 30, and 85% identities, respectively (Fig.
2). The human cDNA thus appears to be
an ortholog of mouse
-CD, and both are related to chicken
-CD.
The mouse polypeptide has only 37% amino acid identity to mouse RPE65.
Ten residues, including 4 histidine residues and 6 acidic residues, are
absolutely conserved in these sequences, in all RPE65 proteins (Fig.
2), and in the plant 9-cis-epoxycarotenoid dioxygenases (8).
In addition, there is a particularly well conserved region (consensus
sequence EDDGVVLSXVVS) at residues 469-480 of mouse
-CD that may be considered a family signature sequence. Besides the
mouse sequence characterized herein, its presumptive human ortholog,
and the chicken and Drosophila
-CD proteins enumerated
above, ESTs encoding presumptive orthologs from zebrafish and pig were
noted as well as more distant family members from a variety of species.
Because of the accessibility of the mouse as a model organism for
further studies, we chose to specifically characterize the mouse
gene.
|
Expression of Mouse -CD in E. coli--
Agar plate cultures of
E. coli producing
-carotene,
-carotene, and lycopene
and transformed with the pBAD/mouse
-CD expression construct showed
a distinct bleaching of the color when induced with
L-arabinose (Fig.
3A). Cultures of bacteria
containing pBAD/
-CD were induced with a range of concentrations of
L-arabinose in excess of 0.0002% (w/v) and analyzed by
SDS-polyacrylamide gel electrophoresis and immunoblotting. A protein of
the expected size of ~65 kDa was observed that was immunoreactive
with the antiserum raised against the mouse
-CD peptide epitope
(Fig. 3B). The relevant His-tagged pBAD/mouse
-CD fusion
protein was purified using histidine affinity chromatography (data not
shown).
|
In Vivo and in Vitro Activity of Mouse -CD--
HPLC analysis
of hexane extracts of
-carotene-accumulating E. coli
yielded the expected
-carotene peak (Fig.
4A). Expression of mouse
-CD in such cultures resulted in the appearance of
all-trans-retinal with a concomitant loss of
-carotene
(Fig. 4B). The spectra of both the
-carotene and
all-trans-retinal peaks were analyzed (see insets
in Fig. 4, A and B) and exhibited the expected
shapes and absorbance maxima.
-Carotene-,
-carotene-,
-carotene-, and lycopene-accumulating cultures, quantitated by HPLC
analysis, showed 2.14, 1.87, 1.23, and 1.50 pmol/mg of bacterial
protein, respectively, in cultures containing the empty vector. In
contrast, cultures containing the mouse
-CD insert had no detectable
-carotene. However,
-carotene,
-carotene, and lycopene were
present in such cultures at levels of 3.2, 57.9 and 65.6 fmol/mg of
bacterial protein, respectively.
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Purified recombinant -CD protein cleaves
-carotene in
vitro with a Vmax of 36 pmol of retinal/mg
of enzyme/min and a Km of 6 µM. At
levels of lycopene concentrations comparable to the Km for
-carotene, no acyclic retinal was
detected. However, trace amounts were seen at 2.5-3 times the
-carotene Km.
Expression of Mouse -CD in Tissues--
Multiple tissue RNA gel
blots, analyzed with riboprobe, showed substantial steady-state
mRNA levels in several of the tissues known to be active in vitamin
A metabolism (Fig. 5A). The
major message, seen in liver, kidney, testis, and small intestine, was 2.4 kb. Although the 2.4-kb message was in low abundance in the small
intestine, a tissue known to contain strong
-CD activity, it is
likely that this was due to dilution of the mRNA contribution of
the mucosa by that of the outer muscular layers of the gut. Smaller
messages were also seen in testis and skin; in particular, a 1.8-kb
message was quite abundant in testis. Analysis of a mouse developmental
blot showed a strong signal for the 2.4-kb message at 7 days
post-conception, which declined as the developmental age increased
(Fig. 5B). In immunoblots, a band of immunoreactivity of the
expected size of 63 kDa was detected in 30,000 × g and 100,000 × g supernatant fractions of liver, kidney,
testis, and small intestine, but not in the 100,000 × g pellet fractions (data not shown).
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DISCUSSION |
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In this report, we have described the catalytic activity and
tissue-specific expression pattern of a mouse -CD. The protein clearly belongs to an extended family of dioxygenases that interact with carotenoids and other polyenes and that include the plant neoxanthin cleavage enzymes, the bacterial lignostilbene dioxygenases, and the vertebrate protein RPE65. The activity of mouse
-CD
seen with alternative carotenoid substrates (Fig. 3) indicates that the
specificity of the catalytic activity is centered more in the polyene
chain of the substrate than at the end groups. This observation is in
accord with the activities previously reported for crude tissue
preparations (23-25). Therefore, a more accurate and inclusive
designation for the family as a whole may be the polyene chain
dioxygenase family.
The conserved histidines and acidic residues of the various polyene dioxygenase family members are likely to be involved in coordinating the iron required for activity in these enzymes (9, 26). The signature sequence, a run of acidic residues connected through a glycine to a hydrophobic tail, could be involved in electron transfer processes during catalysis. Although the precise enzymatic activity of RPE65 has not yet been determined, RPE65 is capable of binding ferrous iron3 and thus shares this characteristic as well as the signature sequence with other family members. The resemblance of RPE65 to the other dioxygenase family members and the fact that its absence prevents the retinal pigment epithelium-specific isomerization of all-trans- to 11-cis-retinol in the visual cycle (12) suggest a direct catalytic participation in the visual cycle.
Although -CD activity in partially purified preparations from
various sources has been localized both to cytosolic and membrane fractions, the overwhelming consensus is that it is a cytosolic protein
(23-25, 27), as we also have found. However, an important aspect of
the carotenoids and their derivatives as substrates is their
lipophilicity (including a tendency to partition in membranes) and
generally insoluble nature in aqueous solution. Interestingly, none of
the polyene dioxygenase family members, including mouse
-CD, are
predicted to be integral membrane proteins. However, the plant
9-cis-epoxycarotenoid cleavage enzymes are associated with
chloroplast membranes (9). The only metazoan member of the family whose
biochemical properties have been examined in this respect is RPE65,
which is a non-integral, microsomal membrane-associated protein, but
can be separated from retinal pigment epithelium membranes by mild
detergent or high salt extraction (11). Furthermore, RPE65 will
specifically associate with phospholipid liposomes (28). It is possible
that mouse
-CD, despite its cytosolic nature, will also interact
with membranes in the context of its physiological mechanism.
The tissue distribution of mouse -CD was, in general, as expected,
although the level in the small intestine was somewhat weak. However,
the
-CD activity of this tissue is concentrated in mature
enterocytes (and is highest in the jejunum) (29), which constitute a
relatively small proportion of the total tissue used to provide the RNA
for this blot. Thus, one possibility is that the weak signal in this
tissue is due to dilution of the RNA with that from other cell types.
Another possibility is that there is a tissue-specific
-CD in small
intestine, in addition to the one described here. The testis also is
active in the uptake and metabolism of carotenoids, as
-carotene and
lycopene have been repeatedly demonstrated in testis (30-32). Cleavage
of
-carotene has been shown to be an important source of retinoic
acid in vitro in testis as well as in small intestine,
liver, kidney, and lung (33), and the high expression of
-CD in
testis noted here is consistent with this finding. Napoli and Race (33)
have suggested that cleavage of
-carotene may be an important source
of retinoic acid in target tissues such as testis and may play an
under-appreciated role in retinoid homeostasis.
Mouse -CD has a low level of activity toward carotenoids other than
-carotene, including lycopene, observable in in vivo assays. As to its distribution, lycopene is accumulated primarily in
liver, but also in intestine, prostate, and testis (34), and appears to
have several biological activities (35). For example, in a recent
study, lycopene, but not its acycloretinoid derivative, was effective
in stimulating gap junction communication (36). Therefore, in this
case, the function of the dioxygenase with respect to lycopene might be
to terminate its activity.
The developmental expression of mouse -CD is particularly
intriguing, given its early elevated expression at embryonic day 7 and
much reduced expression thereafter. At embryonic day 7, with the embryo
proceeding into gastrulation, a number of retinoids and
retinoid-interacting proteins are being expressed (37). Analysis of
endogenous retinoids in the mouse embryo (37) shows that
all-trans-retinal is the first retinoid seen (at embryonic day 6.5), whereas both all-trans-retinoic acid and
all-trans-retinol do not appear until embryonic day 7.5. In
the mouse embryo, mRNAs of two dehydrogenases involved in retinoic
acid production, alcohol dehydrogenase IV and aldehyde
dehydrogenase I, are up-regulated between embryonic days 6.5 and
7.5 (37, 38). Although the early accumulation of
all-trans-retinal may be due to low level activity of
alcohol dehydrogenase IV on maternally supplied
all-trans-retinol, there may also be a contribution from the
cleavage of
-carotene. Ulven et al. (37) suggested this
possibility, an interpretation supported by our finding of high
-CD
mRNA expression at embryonic day 7. Thus, the activity of
-CD
could serve to produce all-trans-retinal from
-carotene,
a nontoxic provitamin source of retinoids in the early embryo.
The identification of mouse -CD establishes the existence in mammals
of an ancient family of carotenoid-metabolizing enzymes. These enzymes,
giving a means by which animals can cleave or modify these
plant-derived compounds as required, provide access to retinoids for
animals and facilitate their panoply of developmental and physiological
effects. We can surmise that the adoption by animals of hormonally and
otherwise physiologically active carotenoid metabolites (retinoids) was
initially facilitated by such enzymes evolved from an ancient common ancestor.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Rizwan Bhatti for help in the
purification of the recombinant pBAD/-CD protein.
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FOOTNOTES |
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* 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) AF271298.
§ To whom correspondence should be addressed: NEI-LRCMB, NIH, Bldg. 6, Rm. 339, 6 Center Dr. MSC 2740, Bethesda, MD 20892-2740. Tel.: 301-496-0439; Fax: 301-402-1883; E-mail: redmond@helix.nih.gov.
Published, JBC Papers in Press, November 22, 2000, DOI 10.1074/jbc.M009030200
2 F. X. Cunningham, Jr. and E. Gantt, manuscript in preparation.
3 S. Gentleman, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
-CD,
-carotene
15,15'-dioxygenase;
EST, expressed sequence tag;
HPLC, high
performance liquid chromatography;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
kb, kilobase(s).
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Morriss-Kay, G. M., and Ward, S. J. (1999) Int. Rev. Cytol. 188, 73-131[Medline] [Order article via Infotrieve] |
2. | Chen, Y., Huang, L., Russo, A. F., and Solursh, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10056-10059[Abstract] |
3. | Chen, Y., Huang, L., and Solursh, M. (1994) Dev. Biol. 161, 70-76[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Saari, J. C.
(2000)
Invest. Ophthalmol. Visual Sci.
41,
337-348 |
5. | Nagao, A., During, A., Hoshino, C., Terao, J., and Olson, J. A. (1996) Arch. Biochem. Biophys. 328, 57-63[CrossRef][Medline] [Order article via Infotrieve] |
6. | Cunningham, F. X., Jr., and Gannt, E. (1998) Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 557-583[CrossRef] |
7. | Kamoda, S., and Saburi, Y. (1993) Biosci. Biotechnol. Biochem. 57, 926-930[Medline] [Order article via Infotrieve] |
8. |
Tan, B. C.,
Schwartz, S. H.,
Zeevaart, J. A.,
and McCarty, D. R.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12235-12240 |
9. |
Schwartz, S. H.,
Tan, B. C.,
Gage, D. A.,
Zeevaart, J. A.,
and McCarty, D. R.
(1997)
Science
276,
1872-1874 |
10. |
Hamel, C. P.,
Tsilou, E.,
Pfeffer, B. A.,
Hooks, J. J.,
Detrick, B.,
and Redmond, T. M.
(1993)
J. Biol. Chem.
268,
15751-15757 |
11. | Hamel, C. P., Tsilou, E., Harris, E., Pfeffer, B. A., Hooks, J. J., Detrick, B., and Redmond, T. M. (1993) J. Neurosci. Res. 34, 414-425[Medline] [Order article via Infotrieve] |
12. | Redmond, T. M., Yu, S., Lee, E., Bok, D., Hamasaki, D., Chen, N., Goletz, P., Ma, J. X., Crouch, R. K., and Pfeifer, K. (1998) Nat. Genet. 20, 344-351[CrossRef][Medline] [Order article via Infotrieve] |
13. |
von Lintig, J.,
and Vogt, K.
(2000)
J. Biol. Chem.
275,
11915-11920 |
14. | Wyss, A., Wirtz, G., Woggon, W., Brugger, R., Wyss, M., Friedlein, A., Bachmann, H., and Hunziker, W. (2000) Biochem. Biophys. Res. Commun. 271, 334-336[CrossRef][Medline] [Order article via Infotrieve] |
15. | Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410[CrossRef][Medline] [Order article via Infotrieve] |
16. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
17. | Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract] |
18. | Chung, C. T., Niemela, S. L., and Miller, R. H. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2172-2175[Abstract] |
19. |
Sun, Z.,
Gantt, E.,
and Cunningham, F. X., Jr.
(1996)
J. Biol. Chem.
271,
24349-24352 |
20. |
Cunningham, F. X., Jr.,
Pogson, B.,
Sun, Z.,
McDonald, K. A.,
DellaPenna, D.,
and Gantt, E.
(1996)
Plant Cell
8,
1613-1626 |
21. |
Cunningham, F. X., Jr.,
Sun, Z.,
Chamovitz, D.,
Hirschberg, J.,
and Gantt, E.
(1994)
Plant Cell
6,
1107-1121 |
22. | Tam, J. P. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5409-5413[Abstract] |
23. | Lakshmanan, M. R., Pope, J. L., and Olson, J. A. (1968) Biochem. Biophys. Res. Commun. 33, 347-352[Medline] [Order article via Infotrieve] |
24. | Olson, J. A. (1983) in Biosynthesis of Isoprenoid Compounds (Porter, J. W. , and Spurgeon, S. L., eds), Vol. 2 , pp. 371-412, John Wiley & Sons, Inc., New York |
25. | Singh, H., and Cama, H. R. (1974) Biochim. Biophys. Acta 370, 49-61[Medline] [Order article via Infotrieve] |
26. | Lakshman, M. R., and Okoh, C. (1993) Methods Enzymol. 214, 256-269[Medline] [Order article via Infotrieve] |
27. | Wolf, G. (1995) Nutr. Rev. 53, 134-137[Medline] [Order article via Infotrieve] |
28. | Tsilou, E., Hamel, C. P., Yu, S., and Redmond, T. M. (1997) Arch. Biochem. Biophys. 346, 21-27[CrossRef][Medline] [Order article via Infotrieve] |
29. | Duszka, C., Grolier, P., Azim, E. M., Alexandre-Gouabau, M. C., Borel, P., and Azais-Braesco, V. (1996) J. Nutr. 126, 2550-2556[Medline] [Order article via Infotrieve] |
30. | Yamanushi, T., and Igarashi, O. (1995) J. Nutr. Sci. Vitaminol. 41, 169-177[Medline] [Order article via Infotrieve] |
31. | Kerti, A., and Bardos, L. (1999) Acta Vet. Hung. 47, 95-101[Medline] [Order article via Infotrieve] |
32. | Stahl, W., Schwarz, W., Sundquist, A. R., and Sies, H. (1992) Arch. Biochem. Biophys. 294, 173-177[Medline] [Order article via Infotrieve] |
33. |
Napoli, J. L.,
and Race, K. R.
(1988)
J. Biol. Chem.
263,
17372-17377 |
34. | Froescheis, O., Moalli, S., Liechti, H., and Bausch, J. (2000) J. Chromatogr. Biomed. Appl. 739, 291-299[CrossRef] |
35. | Clinton, S. K. (1998) Nutr. Rev. 56, 35-51[Medline] [Order article via Infotrieve] |
36. | Stahl, W., von Laar, J., Martin, H. D., Emmerich, T., and Sies, H. (2000) Arch. Biochem. Biophys. 373, 271-274[CrossRef][Medline] [Order article via Infotrieve] |
37. | Ulven, S. M., Gundersen, T. E., Weedon, M. S., Landaas, V. O., Sakhi, A. K., Fromm, S. H., Geronimo, B. A., Moskaug, J. O., and Blomhoff, R. (2000) Dev. Biol. 220, 379-391[CrossRef][Medline] [Order article via Infotrieve] |
38. | Ang, H. L., and Duester, G. (1997) Dev. Dyn. 208, 536-543[CrossRef][Medline] [Order article via Infotrieve] |
39. | Kamoda, S., and Saburi, Y. (1993) Biosci. Biotechnol. Biochem. 57, 931-934[Medline] [Order article via Infotrieve] |