From the Department of Neurobiology,
Brain
Research Institute and ** Jules Stein Eye Institute, School of Medicine,
University of California, Los Angeles, California 90095 and the
Department of § Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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The enzyme responsible for conversion of
all-trans-retinol into retinyl esters, the lecithin retinol
acyltransferase (LRAT) has been characterized at the molecular level.
The cDNA coding for this protein was cloned and its amino acid
sequence deduced. LRAT is composed of a polypeptide of 230 amino acid
residues with a calculated mass of 25.3 kDa. Tissue distribution
analysis by Northern blot showed expression of a 5.0-kilobase
transcript in the human retinal pigment epithelium as well as in other
tissues that are known for their high LRAT activity and vitamin A
processing. Affinity labeling experiments using specific compounds with
high affinity for LRAT and monospecific polyclonal antibodies raised in
rabbits against two peptide sequences for LRAT confirmed the molecular
mass of LRAT as a 25-kDa protein. High performance liquid chromatography analysis of the reaction product formed by HEK-293 cells
transfected with LRAT cDNA confirmed the ability of the transfected
cells to convert [3H]all-trans-retinol into
authentic [3H]all-trans-retinyl palmitate as
chemically determined.
Vitamin A (retinol) is the substrate for the biosynthesis of
several functional retinoids (retinol derivatives) which are essential
for important biological processes such as, vision, reproduction, and
development (for review, see Ref. 1). The small intestinal epithelium
is the first site of interaction and processing of retinol after
absorption from the diet. Within this epithelium, the final processing
step involves the esterification of retinol with long chain fatty
acids, primarily palmitic, and incorporation of the retinyl esters into
the hydrophobic core of chylomicrons which are secreted into the lymph
and ultimately enter the blood via the thoracic and other lymphatic
ducts (2). Chylomicron remnants are taken up by the liver and the
retinyl esters are stored until needed. Following de-esterification by the liver, retinol is complexed with a 21-kDa retinol-binding protein
(RBP)1 and secreted into the
circulation as a complex with transthyretin whereby it is distributed
to other tissues (3). Both proteins are thought to transport and
protect retinol from oxidation and/or isomerization during the
distribution process. In the eye, and more specifically in the retinal
pigment epithelium (RPE), this retinol complex interacts with the basal
membrane of RPE cells probably via an RBP receptor (4, 5), where
retinol is delivered into the cytoplasm to initiate a unique and highly
specialized process for the RPE, the visual cycle (for review, see
Refs. 6-8). During this important process, the retinol bound to a
cellular retinol-binding protein (CRBP), is trans-esterified
by an enzyme named lecithin retinol acyltransferase (LRAT) (9-11) that
transfers an acyl group from lecithin to retinol to generate
all-trans-retinyl esters. All-trans-retinyl
esters generated by the activity of LRAT are not only presumptive
storage forms of vitamin A, but they are also substrates for an
isomerohydrolase which transforms the esters into an intermediate
11-cis-retinol (12, 13). Due to a membrane-associated
alcohol dehydrogenase (14, 15), 11-cis-retinol is then
oxidized and converted into 11-cis-retinaldehyde which is
the chromophore for rhodopsin and the cone photopigments. In the RPE,
the esterification process of retinol by LRAT is critical for the
continuation of the visual cycle since it supplies the processed
substrate for the isomerization reaction. Although LRAT has been
solubilized, partially purified and substantially studied with respect
to its kinetic properties and substrate specificity (16, 17), it has
never been characterized at the molecular level. An ordered ping-pong
bi-bi mechanism in which lecithin first acylates the enzyme describes
the kinetic mechanism of action of LRAT (17). The effects of potent
competitive reversible and irreversible specific inhibitors for its
enzymatic activity have been described (18, 19). One of these has been
used as a probe to determine the putative mass of LRAT. Using
[3H]all-trans-retinyl In this study, we report on the biochemical properties of LRAT and the
generation of specific polyclonal antibodies. Additionally, we describe
its primary structure and verify its authenticity by the expression of
LRAT in transformed mammalian cell lines.
Materials--
Frozen bovine eye cups devoid of retinas were
purchased from J. A. and W. L. Lawson Co., Lincoln, NE.
[11,12-3H2]All-trans-retinol
(specific activity 31.4 Ci/mmol) was obtained from NEN Life Science
Products Inc. L- Preparation of Retinal Pigment Epithelium Membranes--
The
procedure for preparation of bovine retinal pigment epithelium
membranes is described elsewhere (18). Prior to use, the membranes were
irradiated with UV light (365 nm) on ice for 5 min in order to destroy
endogenous retinoids (13).
Partial Purification of LRAT--
The method used for the
partial purification of LRAT was substantially the same as previously
reported (17). Bovine retinal pigment epithelial membranes were
solubilized in 20 mM Tris-HCl, pH 9.0, 2 mM
DTT, 1 mM EDTA, and 1% Triton X-100. After thorough mixing
for 1 h at 4 °C, the material was centrifuged at 4 °C at 10,500 × g for 1 h. The supernatant was applied
to a DEAE-Sepharose column using a linear gradient of buffers A and B
(buffer A, 20 mM Tris-HCl, pH 9.0, 1 mM EDTA, 2 mM DTT, and 0.1% Triton X-100; buffer B, buffer A plus 1 M NaCl). The active fractions were pooled and applied to a
thiopropyl-Sepharose 6B column and eluted using a linear gradient of
buffers C and D (buffer C, 50 mM Tris-HCl, pH 9.0, 0.5 M NaCl, 1 mM EDTA, and 0.1% Triton X-100;
buffer D, buffer C plus 20 mM DTT). The active fractions
were pooled and applied to a Mono-Q column on an Pharmacia Biotech Inc.
Superose 6LCC-500 FPLC system at 4 °C and eluted using a linear
gradient of buffers A and B. Enzyme partially purified in this way was stored at Synthesis of N-Boc-L-Biocytinyl-11-aminoundecane
Chloromethyl Ketone (BACMK)--
To a chilled solution of
11-aminoundecane chloromethyl ketone hydrochloride (95 mg, 0.35 mmol),
Hunig's base (0.12 ml, 0.45 mmol), and
N-boc-L-biocytin (199 mg, 0.42 mmol) in
N,N-dimethylformamide (10 ml) was added
1-ethyl-3-C3-dimethylaminopropyl carbodiimide (96 mg, 0.50 mmol) in one
portion. The mixture was stirred overnight, then poured into EtOAc (150 ml). The organic phase was washed with 3.5% HCl (25 ml), saturated
NaCl at 25 °C (25 ml), dried over MgSO4, filtered, and
concentrated under vacuum to provide the desired product as a white
crystalline material in 74% yield. TLC (silica) RF = 0.37 (MeOH:CH2Cl2 = 80:20. 1H NMR
(Me2SO-d6, 500 MHz): 7.2 (1H, d,
j = 5.5 Hz), 6.8 (1H, d, j = 6.5 Hz), 6.38 (1H, s), 6.33 (1H,
s), 4.69-4.60 (2H, m), 4.54 (2H, s), 4.45 (1H, m), 3.36 (1H, m), 3.00 (1H, m), 2.45 (2H, t, j = 6.5 Hz), 2.31 (2H, t, J = 6.5 Hz),
1.61-1.115 (41H, m).
Inhibition of LRAT by BACMK--
A solution of
L- Affinity Labeling of LRAT and Peptide Analysis--
Partially
purified enzyme was preincubated as described earlier with cholesterol
chloroacetate to block nonspecific sites potentially labeled with BACMK
(20). The affinity labeling agent BACMK (25 µM) in a
final volume of 200 µl was incubated with pretreated enzyme solution
at room temperature for 15 min. The labeled protein was precipitated by
adding chilled acetone and analyzed by 12% SDS-polyacrylamide gel
electrophoresis gel. The labeled product was detected by an ECL Western
blotting assay (Amersham). The putative LRAT band was extracted from
the gel, digested with trypsin, and peptide analysis was performed by
the Harvard Microsequencing Facility.
RNA Isolation from Human RPE Cells--
RPE cells were dissected
from explants of human eyes collected within 4 h (adult) and less
than 1 h (fetal) postmortem. The poly(A)+ RNA fraction
was extracted from RPE cells by using the Fast-Track System (Invitrogen).
Isolation and Characterization of Human LRAT
cDNAs--
Based on peptide sequences of the putative bovine LRAT
protein corresponding to the N-terminal region and an internal peptide sequence (see "Results"), the following pair of degenerate primers were designed: 5'-ATGAARAAYCCNATGCTNGARGC-3' (forward) and
5'-DATNCCRTARTGNGTNARRTG-3' (reverse). RT-PCR experiments
were performed on poly(A)+ RNA from human RPE cells,
according to the vendor's recommendations in the RNA-PCR kit
(Perkin-Elmer). 200 ng of poly(A)+ RNA was reverse
transcribed in a reaction mixture containing a final concentration of 5 mM MgCl2, 1 × PCR buffer, 1 mM dNTPs, 1 unit/µl of RNase inhibitor, 2.5 units/µl of
reverse transcriptase, and 2.5 µM of degenerate reverse
primer. The total 20-µl reaction was overlaid with 50 µl of mineral
oil and incubated at 42 °C for 15 min. The enzymatic activity was
inactivated at 94 °C for 5 min. For PCR amplification, 78 µl of a
mixture containing a final concentration of 2.5 µM
degenerate forward primer, 2 mM MgCl2, 1 × PCR buffer, 65.5 µl of sterilized water, and 2.5 units/100 µl of
AmpliTaq DNA polymerase were added to the 20-µl reverse transcription
reaction. Amplification was performed at 94 °C, 1 min; 60 °C, 1 min; and 72 °C, 2 min for 35 cycles, followed by 10 min incubation
at 72 °C. By using the degenerate primers described above, a 189-bp
fragment was amplified. A human fetal RPE cDNA library was
constructed in the Uni-ZAP XR vector (Stratagene) by using
poly(A)+ RNA from cultured fetal human RPE cells obtained
from aborted fetus of 15-24 weeks gestation and maintained and
harvested as described earlier (24). All of the DNA inserts were
unidirectionally placed between the unique EcoRI and
XhoI restriction sites of the pBluescript vector. The
cDNA library was screened with the 189-bp DNA fragment. Five
positive cDNA clones out of 2.5 × 105 plaques
were further characterized by sequencing both strands with the dideoxy
chain termination method, using the Sequenase 2.0 system (U. S. Biochemical Corp.).
Northern Blot Analysis--
Two µg of poly(A)+ RNA
from human fetal and adult RPE cells were electrophoretically separated
in a 1.2% agarose-formaldehyde gel and blotted onto a nylon membrane.
Filter membranes containing 2 µg of poly(A)+ RNA from
several human tissues at adult and fetal stages
(CLONTECH) were also included in the analysis. All
the filter membranes were hybridized at 42 °C overnight in the
presence of 50% formamide with an LRAT probe labeled with
[ Expression of LRAT in Human Embryonic Kidney-293 and 293T Cells
(HEK-293 and HEK 293T)--
A ~1.0-kilobase EcoRI/DNA
fragment encoding for LRAT, which included 92 bp of 5'-UTR, 690 bp of
coding sequence, and 254 bp of 3'-UTR was subcloned into the
EcoRI site of the polylinker of the pcDNA3 vector
(Invitrogen) where expression is driven by the human cytomegalovirus
promoter. HEK-293 and a similar cell line HEK-293T cells (which carry
the large T antigen from SV40 and therefore exhibit higher expression
due to amplification of vectors such as the pcDNA3) were grown in
100-mm plates to 80% confluence. Transfection experiments were
performed using LipofectAMINE (Life Technologies, Inc.) and 30 µg of
DNA for LRAT/pcDNA3 construct or empty vector. Cells were collected
24, 48, and 72 h after transfection.
Polyclonal Anti-LRAT Antiserum and Western Blot
Analysis--
Two peptide sequences GAAGKDKGRNSFYETSS and
HLDESLQKKALLNEEVARRAE corresponding to positions 28-44 and 126-146 of
the LRAT polypeptide described in this work were selected by their
antigenicity and accessibility (25, 26). Rabbits were immunized with a mixture of both peptides. Polyclonal antisera were generated by contract with Alpha Diagnostics International. Native human RPE cells
stored at LRAT Activity Assay--
Unless otherwise mentioned, all
procedures were performed under dim red light with samples kept on ice.
28 pmol of
[11,12-3H2]all-trans-retinol
(solvent dried under N2 stream and retinol dissolved in 5 µl of 10% BSA) were added to 100 µl of an HEK 293 membrane
suspension (0.38 mg of protein, 100 mM Tris-HCl, pH 8.5, 0.28 µM final retinol concentration, 0.5% final BSA
concentration). The membranes were incubated at room temperature, and
50-µl aliquots of the reaction mixture were taken out for analysis
after 15 and 60 min, respectively. The reactions were quenched with
methanol (500 µl/sample), 100 µl of H2O was added, and
500 µl of hexane (containing butylated hydroxytoluene at 1 mg/ml) was
used for extraction of retinoids. The isomeric retinols were analyzed
on a 5-µm PVA-Sil column (250 × 4.00 mm, YMC) and the eluant
was 7% dioxane in hexane at a flow rate of 1.5 ml/min. Retinyl esters were separated on a 5-µl Maxisil column (250 × 4.00 mm,
Phenomenex) with 0.4% ether (preservative free) in hexane at 0.8 ml/min. The isomers were identified through co-elution of standard
mixtures of isomeric retinols (monitored at 325 nm) and retinyl esters (313 nm) prepared as described previously (28). Radioactivity was
counted with an in-line Bertold LB 506-C HPLC radioactive monitor
interfaced with an IBM 386 computer. Unless indicated otherwise, the
amounts of retinyl esters obtained are given as % of total retinoids.
Units of enzymatic activity are expressed as nanomole of ester formed
min Identification of the Reaction Product Formed Upon Addition of
[3H]All-trans-retinol to HEK 293 Membranes--
After
transfection with LRAT cDNA, 50 µl of HEK-293 cells membranes
(0.62 mg of protein) were incubated with 1 µCi of
[11,12-3H]all-trans-retinol in 100 mM Tris-HCl, pH 8.0. After 1 h the sample was
extracted as described above and subjected to HPLC under conditions
designed to separate retinyl esters from retinols (5-µm PVA-Sil
column (250 × 4.00 mm, YMC), 7% dioxane in hexane, 1.5 ml/min
detection of added standards at 325 nm). The peak shown by previous
experiments to co-elute with all-trans-retinyl esters (Sigma) was collected, spiked with cold all-trans-retinyl
esters (Sigma), and subjected to isomerization with I2
(0.1% in hexane, 15 min, room temperature) as described before (29).
The reaction products were analyzed by HPLC under conditions optimized
for the separation of retinyl ester isomers (5-µm Maxisil column
(250 × 4.00 mm, Phenomenex), 0.4% ether (preservative free) in
hexane, 0.8 ml/min; detection of added standards was at 313 nm).
Inhibition of LRAT by BACMK--
Previous studies have
demonstrated that RBA is an active-site directed affinity labeling
agent of LRAT (20). Other hydrophobic halomethyl ketones (Scheme
1) such as dodecylchloromethyl ketone and
dodecylbromomethyl ketone, are also potent affinity labeling agents of
this enzyme.2 In order to
identify the labeled protein without resorting to radioactive
synthesis, a biocytinyl-containing dodecylchloromethyl ketone analog,
BACMK (Scheme 1), was prepared and tested as an inactivator of
LRAT.
As shown in Fig. 1A, the rate
of conversion of retinol into retinyl esters by 0.52 mg of partially
purified enzyme was gradually reduced when incubated for a fixed time
period of 10 min with increasing concentrations of BACMK. The same
amounts of enzyme were used to determine the remaining LRAT activity by
a fixed concentration of 2 µM BACMK at several time
intervals (Fig. 1B). This graph clearly demonstrates a
time-dependent mode of LRAT inactivation by BACMK. After a
10-min incubation, 2 µM BACMK was able to inhibit around
45% of the LRAT activity as opposed to the activity of cells in the
absence of BACMK. These data suggested that the inhibitory effect of
BACMK for LRAT is as potent as the previously described RBA.
Labeling of LRAT with BACMK--
Since LRAT has been recalcitrant
to complete purification, it was of interest to identify the enzyme, or
a component thereof, using BACMK as a probe. Since the partially
purified enzyme preparation still contains undesirable proteins, it was
important to first eliminate the possibility that proteins other than
LRAT would react with the affinity labeling agent BACMK. To this end,
partially purified LRAT was first incubated with cholesterol
chloroacetate to block extraneous nucleophilic proteins (probably thiol
dependent) other than LRAT. Cholesterol chloroacetate does not inhibit
LRAT, and has been used to block any non-LRAT related nucleophiles
prior to treatment with RBA (20). This pretreatment dramatically
simplifies the labeling pattern when using affinity labeling reagents.
Fig. 2 shows the ECL labeling patterns of
partially purified LRAT when using a concentration of 25 µM BACMK. Lane 1, shows a major labeled product of approximately 25 kDa molecular mass. A 35-kDa protein also
appeared to be labeled in this procedure. Lane 2 shows the labeling of partially purified LRAT with 10 µM RBA,
followed by treatment with BACMK. Interestingly, when the partially
purified LRAT was first passed through a streptavidin column to remove endogenously biotinylated proteins, and afterward treated with BACMK, a
clear sharp band of 25 kDa was identified after ECL detection, lane 3.
The band in Fig. 2, lane 3, was subsequently extracted from
the gel and subjected to protein sequencing. The N terminus was not
blocked, and proved to posses the following sequence:
MKNPMLEAVSLVLEKLLFISYFKF representing the first 24 amino acids. After
enzymatic degradation of the 25-kDa product with trypsin, an internal
peptide sequence was determined to be HLTHYGIYLGDNR. Neither peptide
sequences were found homologous to any known protein previously
deposited into the GenBank sequence data base.
Molecular Cloning of LRAT--
A 189-bp DNA fragment encoding a
portion of the putative LRAT was generated by RT-PCR of
poly(A)+ RNA from human RPE cells and used as a probe to
screen a human RPE cDNA library. Five cDNA clones with sizes of
1942 bp (clone-A), 2521 bp (clone-B), 855 bp (clone-C), 481 bp (clone
D), and 824 bp (clone E) were isolated. Further characterization was
performed on these clones by sequence analysis. The overlapping
nucleotide sequence of clones A and B spanned 2718 bp (Fig.
3, panel A). This sequence
contained 92 nucleotides of 5'-untranslated region (UTR) and an open
reading frame of 690 bp encoding for a deduced 230-amino acid
polypeptide (Fig. 3, panel B) with a calculated mass of 25.3 kDa. The 3'-UTR contained 1936 nucleotides which included a poly(A)
tail of 20 residues (GenBank accession number AF071510). The methionine
initiation codon ATG at nucleotides (+1 to +3) is presumed to initiate
the transcription process of the LRAT protein and is in reasonable
agreement with consensus sequences for the translation initiation site
of eukaryotic mRNAs (30). The amino acid sequence predicts a
potential glycosylation site, N-X-T at position 21-23.
Whether this glycosylation process occurs in vivo has not
been experimentally determined. Collectively, clones C, D, and E
spanned part of the coding sequence starting at position 392, with
respect to the first nucleotide of the ATG initiation codon through the
1335 position on the 3'-UTR. An atypical polyadenylation signal ATTAAA
was localized 20 nucleotides upstream from the poly(A) tail. Other
polyadenylation signals were also found at positions 1487-1492 and
1839-1844. The entire nucleotide sequence and amino acid sequence
encoding for LRAT reported in this study was compared with sequences
deposited in the GenBank data base. The only high homology observed was
with partial nucleotide sequences of two unidentified EST clones with
accession numbers AA243120 and W90617 deposited in GenBank by the IMAGE
Consortium which, respectively, mapped at positions 1-177 and
1446-1850 in the LRAT nucleotide sequence.
Northern Blot Analysis--
The cDNA clone B was labeled with
[ Western Blot Analysis of the LRAT Protein--
The specificity of
a polyclonal antiserum raised in rabbits against a mixture of two LRAT
peptides was tested by Western blot analysis (Fig.
5). As shown in lane 1, this
antiserum reacted specifically with a single protein band of about
25-26 kDa in human RPE cells. The same band was also observed in
HEK-293T cells transfected with LRAT cDNA (lane 4, arrowhead). In contrast, this product was clearly absent in cells
that were nontransfected or transfected with the empty plasmid
(lanes 2 and 3, respectively). This result was in
agreement with the molecular mass for LRAT obtained by affinity
labeling experiments and the calculated mass of the amino acid
polypeptide deduced from the cDNA sequence. The antiserum also
cross-reacted nonspecifically with a HEK-293T kidney protein which is
not present in RPE.
Identification of the Reaction Product Formed by HEK-293 Cells
Transfected with LRAT cDNA following Addition of
[3H]All-trans-retinol--
Upon addition of
[3H]all-trans-retinol, membranes from HEK-293
cells transfected with LRAT cDNA formed a product that eluted between 2 and 3 min after loading (Fig.
6, peak 1). This elution time
is characteristic for all-trans-retinyl palmitate (Sigma) under the conditions described under "Experimental Procedures." The
radioactive peak also coeluted with authentic, nonradioactive all-trans-retinyl palmitate. The only other product found in
the same analysis was [3H]all-trans-retinol
(Fig. 6, peak 2) that had not yet been converted by the
membranes into retinyl palmitate after 1 h incubation. The
substance suspected to be all-trans-retinyl palmitate (peak 1) was collected in subsequent runs with higher amounts of material, spiked with cold all-trans-retinyl palmitate (Sigma) and
subjected to I2-catalyzed isomerization.
Spectrophotometric analysis at 313 nm of the I2-catalyzed
isomerization reaction in hexane lead to an isomeric mixture containing predominately trans and 13-cis, as well as a
small amount of 9-cis and traces of
9,13-cis-retinyl palmitate (Fig.
7A). This finding was in good
accordance with the results described earlier for the
I2-catalyzed isomerization of all-trans-retinyl
palmitate in heptane (29). The reaction product formed by
LRAT-transfected cell membranes following addition of
[3H]all-trans-retinol coeluted with
all-trans-retinyl palmitate (Fig. 7B). In the
same manner, the I2-catalyzed isomerization products of
this radioactive entity yielded an isomeric mixture identical to that
of the added cold all-trans-retinyl palmitate (Fig.
7C). The two additional peaks eluting between
13-cis and 9,13-cis-retinyl palmitate and after
all-trans-retinyl palmitate are probably
13-cis-retinyl stearate and all-trans-retinyl
stearate, respectively (28). Therefore, added radioactive
all-trans-retinol can be transformed into
all-trans-retinyl ester in an apparently LRAT-dependent process.
Analysis of All-trans-retinyl Ester Formation by Membranes from
HEK-293 Cells Transfected with LRAT cDNA--
The previously
described experiments showed that transfected HEK-293 cell membranes
exhibit LRAT activity. The relative amounts of retinyl ester formed (as
percent of total retinoids) was analyzed for membranes from
nontransfected HEK-293 cells, cells transfected with empty plasmid, and
cells transfected with LRAT plasmid. The precise amount of specific
LRAT protein contained in each sample analyzed is unknown, therefore
only relative enzymatic activity is reported. Each group of transfected
cells collected at 24, 48, and 72 h after transfection showed
similar levels of activity, therefore average data are presented.
Nontransfected cells showed a considerably lower apparent LRAT
activity. After 15 min and 1 h incubation time, 22.5 and 38.1% of
retinyl ester was generated, respectively, by these cells. Cells
transfected with empty plasmid showed no LRAT activity within the first
15 min, probably due to a competitive expression of endogenous cell
proteins and exogenous proteins such as neomycin induced by the vector
itself. However, after 1-h reaction time, these cells generated 21.4%
of retinyl ester. Interestingly, LRAT-transfected cells formed 87.1%
of retinyl ester within the first 15 min, and 91.6% within 1 h.
Apparent LRAT activity was about 4 times higher in cells transfected
with LRAT plasmid than in cells transfected with empty plasmid (at 1-h
reaction time). Compared with nontransfected cells, the
LRAT-transfected cells showed an increase in apparent LRAT activity of
4-fold after 15 min, and 2-fold after 1 h.
The relatively high ester synthetase activity in nontransfected cells
was surprising given the fact that HEK-293 cells did not have
detectable transcripts for LRAT by RT-PCR and Northern blot analysis
and expression of LRAT message was only induced in cells transfected
with LRAT cDNA (data not shown). One possible explanation for this
finding is that the retinyl ester forming activity was not due to LRAT,
but rather to the activity of other retinyl ester-forming enzymes. To
test this hypothesis, HEK-293 membranes were sedimented in 1 ml of
buffer (100 mM Tris-HCl, pH 8.0, 100,000 × g, 30 min, 4 °C) to remove soluble, cytosolic retinyl
ester synthetase activity. Washed membranes were tested for LRAT
activity as described above. No retinyl ester formation was observed
after a 15-min reaction time in either nontransfected cells or cells
transfected with empty plasmid. After 1 h incubation, washed
membranes of nontransfected cells formed 7% retinyl esters, whereas
cells transfected with empty plasmid generated 3% retinyl esters. In
contrast, LRAT-transfected cells generated 53% retinyl esters during
the same incubation time. Combined, these data suggest that specific
LRAT activity is present only in the LRAT transfected cells and absent
in nontransfected cells and cells transfected with empty plasmid.
In the present study, we describe for the first time the deduced
amino acid sequence of LRAT, an essential enzyme in the processing of
vitamin A (all-trans-retinol) into
11-cis-retinoids in the RPE and for transport and processing
of retinoids in other tissues. LRAT has been exceedingly difficult to
purify to homogeneity and consequently its primary structure and
molecular identification has been lacking. As is typical of many
membrane-bound enzymes, enzyme denaturation occurs as purification
proceeds. Therefore, indirect biochemical means were sought to clearly
identify this important enzyme as an initial step toward its further
molecular characterization.
Affinity labeling experiments were performed by using the LRAT specific
affinity labeling agent dodecylchloromethyl ketone coupled to biocytin
(BACMK) to facilitate the localization of LRAT by ECL. Initial
experiments showed that LRAT activity was clearly inhibited by BACMK in
a concentration-dependent manner, operating in the similar
concentration range found with the active site-directed affinity
labeling agent RBA (20). In addition, BACMK displayed a
time-dependent mode of inhibition, suggesting an
irreversible inhibitory effect on LRAT due to covalent binding. This
observation was supported by the fact that LRAT activity could not be
restored after removing excess BACMK. Previously, radioactive RBA has
been shown to label a protein with an apparent mass of 25 kDa (20). In
agreement with this report, ECL staining of the BACMK affinity labeled
product also showed a 25-kDa protein as the labeled entity. The fact
that the same polypeptide band was shown to be labeled by both BACMK
and RBA affinity labeling agents provided stronger evidence that the
LRAT protein was identified and has a mass of 25 kDa.
Additional evidence for the identification of LRAT was supported by the
sequence analysis of cDNA clones obtained from a human RPE cDNA
library. Encompassing nucleotide sequences of the two major clones
indicated an open reading frame of 690 bp encoding for a 230-amino acid
polypeptide with a calculated mass of 25.3 kDa, in close agreement with
the protein size obtained by the affinity labeling procedures. In
addition, the sequences of both bovine N-terminal and internal peptide
data from the biochemical characterization were present within the
human LRAT polypeptide, confirming the correlation of both peptides
with the deduced amino acid sequence from the cDNAs. Amino acid
variations were seen particularly in the N-terminal peptide residues
which are probably due to species differences since the peptide
sequences were derived from bovine tissue. A hydropathy analysis (31)
of the LRAT amino acid sequence suggests 2 possible transmembrane
regions within the protein at positions 9-31 and 195-222
(http://ulrec3.unil.ch/software/TMPRED). Thus, within the endoplasmic
reticulum where the protein is presumed to be located, the N- and
C-terminal would be found in the same lumenal (extracellular)
orientation. It is therefore hypothesized that the active site for LRAT
would be localized somewhere in the putative cytosolic loop formed by
residues 31-195 which includes cysteine residues at position 161, 168, and 182.
A nucleotide and amino acid comparison with sequences deposited in
GenBank showed no homology with any previously described protein.
Surprisingly, when LRAT was compared with sequences encoding for other
closely related acyltransferases such as the 60-kDa soluble protein
lecithin:cholesterol acyltransferase (32, 33) low homology was found
and no obvious sequence to account for a possible common active site
was identified. Although it is known that both acyltransferases remove
acyl groups from lecithin to esterify their respective substrates,
retinol for LRAT and cholesterol for lecithin:cholesterol
acyltransferase, it is possible that each process occurs in a
mechanistically different manner. In fact, LRAT has regiospecificity
for acyl groups at position sn-1, whereas
lecithin:cholesterol acyltransferase possesses regiospecificity for
acyl groups at position sn-2 of membrane phospholipids (11, 22, 34, 35).
Northern blot analysis suggested a major 5.0-kb mRNA for LRAT. The
overlapping sequences of the two major cDNA clones contain 2718 bp
of this message. The nucleotide sequence required to encompass the
remainder of the 5.0-kb message remains to be resolved. The existence
of alternative polyadenylation sites in the 3'-UTR or in the initiation
of transcription sites at the 5'-UTR could account for the variation in
the size of the observed species in the Northern blots. Our current
analysis of genomic clones for the gene characterization of LRAT will
eventually help to elucidate these issues. Interestingly, Fig. 4 also
shows an apparent higher level of expression of LRAT RNA transcripts in
adult tissues compared with fetal stages. If the levels of
mRNA in these tissues have a direct correlation with higher protein
synthesis and therefore higher enzymatic activity, it is possible that
an increased demand for esterification is required at the adult stage
of development.
In summary, evidence that the deduced amino acid sequence corresponds
to LRAT is provided by several techniques. Western blot detection of a
25-kDa protein in native RPE cells and LRAT-transfected cells is in
agreement with data obtained by affinity labeling of the protein. The
ability of HEK-293 cells transfected with LRAT cDNA to convert
retinol into retinyl palmitate (Figs. 6 and 7) provides additional
compelling evidence. Although it is known that, physiologically, LRAT
uses as a substrate retinol bound to CRBP for the formation of retinyl
esters, our experiments were performed using retinol bound to BSA, a
method whose efficacy has been well established in earlier studies (12,
13, 16). Chemical proof on the identification of
all-trans-retinyl esters by the LRAT-transfected cells comes
from chemical transformation. The expected retinyl ester stereoisomers
were generated by I2 isomerization of the major elution
product. Combined, these data strongly suggest that the observed
activity is due to LRAT. Moreover, the antibodies will serve as useful
reagents for the subcellular localization of this protein by
immunohistochemistry in multiple tissues and offer the opportunity for
co-precipitation of associated components such as the isomerohydrolase
which acts downstream of retinyl ester synthesis.
INTRODUCTION
Top
Abstract
Introduction
References
-bromoacetate (RBA)
which binds specifically to LRAT among partially purified proteins from
bovine RPE, a product of about 25 kDa was labeled (20). In contrast,
while using radiation inactivation analysis on intact microsomal
membranes from rat liver the activity of LRAT yielded a target size
averaging 52-56 kDa (21). In addition to RPE and liver, LRAT activity
has also been reported in several other tissues such as, testis,
intestine, and pancreas (11, 22, 23), suggesting a role in essential biological processes. Because of the physiological relevance of LRAT,
particularly in the metabolism and storage of vitamin A, its molecular
characterization is of vital importance.
EXPERIMENTAL PROCEDURES
-Dipalmitoylphosphatidylcholine, bovine
serum albumin (BSA), dithiothreitol (DTT), and
all-trans-retinyl palmitate were from Sigma. Triton X-100
was from Calbiochem Corp. HPLC grade solvents were from J. T. Baker. The ECL-Western blotting kit was from Amersham. Microsequencing
was performed by the Harvard Microsequencing Facility.
Boc-11-aminoundecanoic acid was obtained from Bachem. Diazomethane was
generated from Diazald (Aldrich). Cholesterol chloroacetate was from
Aldrich. Other organic reagents, unless otherwise stated, were
purchased from Fluka Chemical. Proton nuclear magnetic resonance
(1H NMR) spectroscopy was recorded on a Varian VRX 500S
Spectrometer operating at a proton frequency of 499.843 MHx. Dimethyl
sulfoxide (Me2SO-d6) was used as the
1H NMR solvent. The residual proton absorption of the
deuterated solvent was used as the internal standard. All chemicals and
solvents purchased were of the highest purity available.
80 °C and was quite stable (specific activity 0.216 nmol
min
1 mg
1). Table
I provides quantitative information on
the purification scheme used here. Protein concentrations were
determined by the Bio-Rad Dc Protein Assay kit.
Partial purification of LRAT from bovine RPE
1 mg
1.
-dipalmitoylphosphatidylcholine (400 µM)
and [11,12-3H,3H]all-trans-retinol
(0.9 mM) (NEN Life Science Products Inc.) was dried under
nitrogen and dissolved with 2.5% BSA and vortexed vigorously. The
reaction mixture was prepared containing partially purified LRAT
protein (0.52 mg), 0.1 M Tris-HCl, pH 8.0, and 2 mM DTT. Solutions of BACMK in Me2SO (0, 5, 10, 20, 50, 100, and 200 µM, respectively) were added to the
reaction mixtures in a final volume of 250 µl. After 10 min
incubation at 37 °C, LRAT activity assays were performed. The
reaction mixture for time course inhibition of LRAT by BACMK was
prepared as follows: partially purified LRAT protein (0.52 mg), 0.1 M Tris-HCl, pH 8.0, 2 mM DTT, and BACMK (2 µM) in a final volume of 250 µl. Inhibition was
measured at 0, 10, 20, 30, 40, and 60 min by taking 10-µl aliquots of
the incubation mixture and adding them to the assay mixture and the
conversion of retinol to retinyl palmitate was analyzed as describe in
the LRAT activity assays section.
-32P]dCTP (Amersham) by nick translation. Following
hybridization, the filters were washed with 2 × SSC, 0.1% SDS
twice at room temperature for 15 min, followed by high stringency
washes in 0.1 × SSC, 0.1% SDS at 50 °C for two rounds of 30 min. Filters were exposed to x-ray film using an intensifying screen at
80 °C.
80 °C after collection were analyzed. For the purpose of
higher protein expression, HEK-293T cells were used in this experiment.
In addition to non-transfected HEK-293T cells, cells transfected with
empty plasmid and cells transfected with LRAT cDNA were collected
24 h after transfection and included in the analysis. 10 µg of
microsomal protein from each sample in buffer containing 1%
2-mercaptoethanol was boiled for 2 min and loaded onto a 5%
SDS-polyacrylamide gel electrophoresis. Blot analysis was performed on
nitrocellulose filters according to Towbin et al. (27) using
antiserum diluted to 1:1000 for the identification of LRAT. Protein
bands were detected by the ECL system.
1 at 37 °C.
RESULTS
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Scheme 1.
Structures of affinity labeling agents of
LRAT.
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Fig. 1.
Effect of BACMK on LRAT activity.
A, the concentration dependent inhibition of the LRAT by
BACMK. Data points are mean values from duplicate assays. Units on the
ordinate are percent retinyl ester formation from total
retinoids after 10 min of incubation. B, time course for the
inhibition of LRAT by BACMK. Triplicate assays were performed in all
cases. The means of these values are shown in the figure (standard
deviation ± 5%). Control experiments were also performed by
incubating LRAT for 0, 10, 20, 30, 40, and 60 min in the absence of
BACMK.
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Fig. 2.
Labeling of LRAT by BACMK. Lane
1, LRAT protein labeled with BACMK (25 µM);
lane 2, LRAT protein labeled with BACMK (25 µM) and RBA (10 µM). M,
indicates molecular weight markers in kDa. Lane 3, shows the
eluate of partially purified LRAT protein labeled with BACMK (25 µM), after passage through a streptavidin affinity column
to remove endogenously biotinylated proteins.
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Fig. 3.
Analysis of human LRAT cDNAs, nucleotide
sequence, and predicted primary structure of LRAT. A,
schematic representation of the human RPE cDNA clones A and B
spanning 2718 nucleotides encoding for LRAT protein. The hatched
box represents the open reading frame of LRAT. The position of
each cDNA clone with respect to the open reading frame is
indicated. B, 5'-UTR nucleotide sequence is indicated
as position. The 1+ position represents the first nucleotide of
the start codon ATG. The deduced amino acid sequence shown
under the nucleotide sequence is indicated by the
one-letter code. A potential N-linked
glycosylation site is indicated by an asterisk. The 3'-UTR
contains a consensus polyadenylation signal at positions 1487-1492 and
1839-1844 and an atypical signal shown in bold is found 21 nucleotides upstream from the poly(A) tail. Peptide sequences obtained
by microsequencing are underlined.
-32P]dCTP by nick translation and used as a probe to
analyze the tissue distribution of RNA species encoding for LRAT in
different human tissues (Fig. 4). A major
RNA transcript of 5.0 kb was observed in several tissues, particularly
in tissues known for their high vitamin A processing activity. In fetal
tissues, the specific message was expressed in RPE, liver, and barely
in brain. In adult tissues, the highest level of expression was
observed in testis and liver, followed by the RPE, small intestine,
prostate, pancreas, colon, and low expression in brain. When using
clone B as a probe, additional smaller messages which could represent
polyadenylation variants, were also detected in these tissues and
others such as adult skeletal muscle, spleen, thymus, and uterus. A
470-bp DNA fragment corresponding to position 1996-2465 near the end
of the LRAT 3'-UTR nucleotide sequence hybridized exclusively with the
5.0-kb message (data not shown), eliminating the lower molecular weight
bands and suggesting that they represent polyadenylation variants.
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Fig. 4.
Northern blot analysis of LRAT in human
tissues. 2 µg/lane of poly(A)+ RNA from several
human tissues at adult and fetal stages was loaded. The size of RNA
markers is indicated in kilobases. The two panels on the
left side of the markers contain poly(A)+ RNA
from fetal tissues, except for the adult RPE sample. Panels on the
right side of the markers contain poly(A)+ RNA
from adult tissues.
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Fig. 5.
Western blot analysis of human LRAT
protein. 10 µg of microsomal protein was loaded in each lane.
Lane 1, human RPE cells; lane 2, nontransfected
HEK-293T cells; lane 3, cells transfected with empty vector;
lane 4, cells transfected with LRAT cDNA. A rabbit
polyclonal antiserum against a mixture of two peptides was used for the
detection. Molecular weight markers in kilodaltons are indicated on the
right.
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Fig. 6.
HPLC chromatogram of the reaction products
formed by LRAT-transfected cells upon addition of
[3H]all-trans-retinol. After 1 h reaction time, the cells were extracted with hexane and the reaction
products analyzed by HPLC optimized for the separation of retinyl
esters and retinols (5-µm PVA-Sil column (250 × 4.00 mm, YMC),
7% dioxane in hexane, 1.5 ml/min). 1, retinyl ester;
2, all-trans-retinol unconverted.
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Fig. 7.
HPLC chromatogram of the reaction products
formed by LRAT-transfected cells upon addition of
[3H]all-trans-retinol and the
corresponding I2-catalyzed isomerization products. The
retinoids were separated under conditions optimized for the separation
of retinyl ester isomers (5 µm Maxisil column (250 × 4.00 mm,
Phenomenex), 0.4% ether (preservative free) in hexane, 0.8 ml/min;
A, detection at 313 nm; B and C,
detection of 3H by radioactivity monitor). A,
I2-catalyzed isomerization products of added cold
all-trans-retinyl ester; B, reaction product
formed by LRAT-transfected cells upon addition of
[3H]all-trans-retinol; C,
I2-catalyzed isomerization products of B. 1, 13-cis-retinyl palmitate; 2, 9,13-cis-retinyl palmitate; 3, 9-cis-retinyl palmitate; 4,
all-trans-retinyl palmitate; 5, 13-cis-retinyl stearate; 6,
all-trans-retinyl stearate.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Dr. William O'Day for the culture and dissection of RPE cells, Alice Van Dyke for expert photographic assistance, and Jane Hu for transfection of HEK-293 cells. We also thank the Lions Eye Bank of Arizona for providing human adult eyes used in this study.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants EY00444, EY00331 (to D. B.), and EY04096 (to R. R.) and by a Center Grant from the National Retinitis Pigmentosa Foundation Fighting Blindness Inc.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) AF071510.
¶ To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, 250 Longwood Ave., Harvard Medical School, Boston, MA 02115. Tel.: 617-432-1794; Fax: 617-432-0471; E-mail: rando{at}warren.med.harvard.edu.
Dolly Green Professor of Ophthalmology at UCLA and a Research
to Prevent Blindness Senior Scientific Investigator. To whom correspondence should be addressed: Jules Stein Eye Institute, 100 Stein Plaza, Rm. B-182, UCLA School of Medicine, Los Angeles, CA 90095. Tel.: 310-825-6737; Fax: 310-794-2144; E-mail:
bok{at}jsei.ucla.edu.
The abbreviations used are:
CRBP, cellular
retinol-binding protein; LRAT, lecithin retinol acyltransferase; RBA, all-trans-retinyl -bromoacetate; BACMK, N-boc-L-biocytinyl-11-aminoundecane
chloromethyl ketone; RT-PCR, reverse transcription-polymerase chain
reaction; HEK-293, human embryonic kidney 293 cells; HEK-293T, human
embryonic kidney 293T cells; RPE, retinal pigment epithelium; ECL, enhanced chemiluminescence; bp base pair(s), UTR, untranslated region; BSA, bovine serum albumin; DTT, dithiothreitol; HPLC, high performance
liquid chromatography.
2 Y.-H. Lim, I. Hubacek, and R. R. Rando, unpublished data.
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REFERENCES |
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