(Received for publication, June 16, 1994; and in revised form, November 7, 1994)
From the
We have isolated and partially characterized a 32-kDa
membrane-associated protein (p32), which forms a complex with p63, an
abundant membrane protein in bovine retinal pigment epithelium. The
sequence of a cDNA clone for p32 revealed an open reading frame
encoding 318 amino acid residues. Several hydrophobic regions could be
identified, suggesting that p32 is an integral membrane protein. A
search of data bases identified p32 as a member of the superfamily of
short chain alcohol dehydrogenases. Transcripts for p32 were
specifically expressed in retinal pigment epithelium. Overexpression of
p32 in Cos cells produced a membrane-bound stereospecific 11-cis retinol dehydrogenase, active in the presence of NAD as cofactor but not in the presence of NADP. We propose that p32
is the stereospecific 11-cis retinol dehydrogenase, which
catalyzes the final step in the biosynthesis of 11-cis retinaldehyde, the universal chromophore of visual pigments.
Under normal physiological conditions, most cells obtain all-trans retinol as their major source of retinoid. The major physiologically active derivatives of retinol, retinoic acid in non-ocular tissues(1) , and 11-cis retinaldehyde for ocular tissues (2) are then generated by specific cellular mechanisms. None of these mechanisms have been fully defined at the molecular level, and several of the enzymes involved have only been characterized as enzymatic activities(3, 4, 5, 6) .
Regarding retinoid metabolism, the polarized retinal pigment
epithelial (RPE) ()cells of the eye are unusual. These cells
obtain all-trans retinol from two different pathways. Retinol,
bound to plasma retinol-binding protein(7) , is accumulated
from the circulation through the basolateral plasma membrane, in order
to support the general need of retinoids for synthesis of visual
pigments. Furthermore, all-trans retinol, which has been
generated in the photoreceptor cells after light exposure of the visual
pigments, is taken up through the apical plasma membrane for
regeneration of 11-cis retinaldehyde. At present, it is not
known whether similar mechanisms are used with regard to cellular
retinol uptake through the basolateral and the apical plasma membranes.
However, available data show that membrane receptors for
retinol-binding protein are expressed by RPE
cells(8, 9) , and retinol uptake by the apical plasma
membrane is presumably mediated by the interstitial retinol-binding
protein located in the space between the RPE and the
photoreceptors(10, 11, 12) .
Regardless of the origin of all-trans retinol, the synthesis and apical release of 11-cis retinaldehyde is the major pathway for retinol accumulated in the RPE. This pathway involves a specific isomerase, which converts all-trans retinylesters into 11-cis retinol(13) . In the final step of this pathway, 11-cis retinol is oxidized to 11-cis retinaldehyde by a stereospecific 11-cis retinol dehydrogenase(3, 4, 5, 6, 14) .
In RPE cells, a major 63-kDa protein (p63) has been implicated as a component of the membrane receptor for retinol-binding protein(15) . To get further information on the role of this protein in retinol uptake and metabolism, we have identified interacting proteins, including a 32-kDa protein (p32) that belongs to the family of short chain alcohol dehydrogenases. Enzymatic analyses showed that p32 is a stereospecific 11-cis retinol dehydrogenase.
In order to isolate p32 for structural studies, RPE membranes were solubilized in PBS containing 1% CHAPS as above and then incubated with mAb A52 Ig coupled to CNBr-activated Sepharose 4B beads (Pharmacia Biotechnology, Inc.) in a Bio-Rad poly prep column by end-over-end rotation at 4 °C. After a 2-h incubation, the beads were allowed to settle, and the column was quickly washed with 5 column volumes of PBS containing 1% CHAPS. Bound proteins were then eluted with 50 mM triethanolamine, pH 11.2, containing 1% CHAPS. The pH of the eluate was quickly adjusted to 8.0 by the addition of 1 M Tris-HCl buffer, pH 8.0, containing 1% CHAPS. The eluted fractions were subjected to SDS-PAGE, and the separated proteins were subsequently visualized by Coomassie Blue staining.
To
carry out the PCR amplifications, first-strand cDNA was synthesized by
standard procedures using avian myeloblastosis virus reverse
transcriptase (Amersham). Twenty micrograms of total RNA from isolated
RPE cells were used, and the reaction was primed with a (dT) oligonucleotide. Aliquots corresponding to 2 µg of total RNA
were used in each subsequent PCR reaction. The PCR amplifications were
performed using Taq polymerase (Amersham) and a final
concentration of 0.2 µM of the oligonucleotide mixtures in
a 100-µl reaction. After 30 cycles (2 min at 95 °C, 1 min at 55
°C, and 2 min at 72 °C), aliquots of the reactions were
analyzed on 4% Nusieve GTG agarose gel (FMC Bioproducts) containing 5
µg/ml of ethidium bromide. The amplified products using
OM1-OM2 (61 bp) and OM3-OM2 (330 bp) were digested with EcoRI, gel-purified, and cloned into EcoRI-cut pBS
vector (Stratagene). The
P-labeled 330-bp fragment was
used to screen a RPE-specific
ZAP II cDNA library as described
previously(19) . Five positive
clones were isolated, and
the inserts were subcloned into pBluescript by in vivo excision as recommended (Stratagene). Clone p
321 contained an
insert of 1.1 kb, and both strands were fully sequenced using Sequenase
(U. S. Biochemical Corp.) with T3, T7, or M13 universal primers or with
internal primers. Standard molecular biology techniques were
used(24) . DNA probes were labeled, using random priming, to a
specific activity of 5
10
to 1
10
cpm/µg DNA.
Antisera to p32 were generated by injecting rabbits with p32 (amino acid residues 19-318) expressed as a fusion protein with glutathione S-transferase. The bacterial expression vector pGEX-2T was used, and induction and purification of the glutathione S-transferase fusion protein was as recommended by the supplier (Pharmacia). Each rabbit received a subcutaneous injection of 75 µg of fusion protein emulsified in Freund's complete adjuvant. The rabbits were boostered with 50 µg of fusion protein emulsified in Freund's incomplete adjuvant every 2nd week. Blood was collected every 2nd week. The immune rabbit sera were passed over a column containing the glutathione S-transferase fusion protein immobilized on CNBr-activated Sepharose beads. Bound Ig was eluted with 0.1 M sodium citrate buffer, pH 3.0, containing 0.5 M NaCl. To remove Ig to the glutathione S-transferase portion of the fusion protein, the eluted Ig was similarly incubated with glutathione S-transferase-coupled Sepharose beads, and the unbound Ig fraction was used. For immunoblot analysis of the overexpressed protein, the Ig was used at a concentration of 1 µg/ml. The details of the immunoblotting procedure were as described previously(15) .
11-cis retinol was
synthesized from 11-cis retinaldehyde (a kind gift from Dr. R.
Crouch, NEI) using sodium borohydride (21) and stored under
argon at -80 °C. HPLC analyses verified that 11-cis retinaldehyde was quantitatively reduced to 11-cis retinol. All manipulations with the retinoids were done in subdued
light. The enzymatic properties of p32, expressed in Cos cells, were
assayed using conditions as described previously (22) for the
study of 11-cis retinol dehydrogenase activity in microsomal
fractions of RPE cells. The final concentration of 11-cis retinol in the incubations was 100 µM. In some
reactions, all-trans retinol (Sigma) was included to a final
concentration of 100 µM. Twenty micrograms of total
membrane protein from Cos cells expressing p32 or from control cells
were used in each reaction. After a 30-min incubation at 37 °C,
with or without the addition of NAD or NADP, the
reaction mixtures were extracted once with n-hexane. The
organic phases were carefully removed and dried under a stream of
argon. The dried organic phases were then separately dissolved in
ethanol, and aliquots were analyzed on a normal phase silica HPLC
column (Chromasil KR5-Sil-250
4.6 mm) developed with n-hexane containing 4% dioxane (23) at 1 ml/min. The
effluent was monitored at 330 nm. Under the conditions specified here,
11-cis retinaldehyde and 11-cis retinol eluted at 7.0
and 22.5 min, respectively. All-trans retinaldehyde and
all-trans retinol eluted at 8 and 23.0 min, respectively.
Figure 1: p63 forms a complex with a 32-kDa protein. A, fractions of the radiolabeled proteins from detergent-solubilized RPE membranes were directly analyzed by SDS-PAGE (lane a) or subjected to indirect immunoprecipitation (lanes b-f). The autoradiogram shows the radioactive profiles after immunoprecipitation with mAb A52 against p63 (lane b), an unrelated mAb (lane c), two polyclonal rabbit antisera against highly purified p63 (lanes d and e), and preimmune rabbit antiserum (lane f). B, SDS-PAGE analysis of RPE membrane proteins solubilized in PBS containing 1% CHAPS (lane a). Solubilized RPE membrane proteins were passed over an immunoaffinity column containing the mAb A52. Bound proteins were eluted at high pH, and an aliquot was subjected to SDS-PAGE analysis (lane b). The protein profiles were visualized by staining with Coomassie Brilliant Blue. The presence of p32 in the eluted fraction from the immunoaffinity column is indicated by the arrow. Molecular weight standards are marked at the right of each panel.
In order to isolate p32, we took advantage of the fact that p32 specifically interacts with p63. Thus, detergent-solubilized RPE membrane proteins were passed over an immunoaffinity column containing A52 Ig. After a quick washing procedure, bound proteins were eluted at high pH in a CHAPS-containing buffer. SDS-PAGE analysis and Coomassie staining of the eluted fractions revealed that p63 was specifically retained and eluted from the immunoaffinity column (Fig. 1B, lane b). In addition, a weakly stained band corresponding to p32 was observed in the eluate from the A52 column. A comparison of the total protein profile of solubilized RPE membranes and the eluted fraction from the A52 column indicate that p32 is not quantitatively retained on the A52 column (Fig. 1B). Nevertheless, the appearance of p32 in the eluted fraction from the A52 column, but not in the eluted fraction from the column containing an unrelated Ig, suggests a specific interaction with p63 (data not shown). This result is consistent with the previous immunoprecipitation data and shows that p32 is complexed to p63 and that it is retained on the immunoaffinity column due to this complex formation.
Partial amino acid sequence was generated from p32 that was isolated by SDS-PAGE of eluted fractions from the A52 immunoaffinity column. Five of the identified peptides were subjected to amino acid sequence analysis (Table 1).
Figure 2: Generation of a DNA probe for p32 by PCR amplification. Agarose gel electrophoresis of PCR reactions employing degenerate oligonucleotide mixtures deduced from partial amino acid sequence determination of p32. a, 61-bp amplified fragment using oligonucleotide mixtures OM1 and OM3, both derived from peptide 321. b, 330-bp amplified fragment using oligonucleotide mixtures OM2 (derived from peptide p323) and OM3 (derived from peptide p321) (see Table 1).
We screened a RPE-specific ZAP-II cDNA library with the 330-bp
fragment as the probe. Five independent
clones were isolated from
approximately 200,000 clones and subcloned by in vivo excision. The cDNA clone p
321 contained the longest insert
(
1.1 kb), and this clone was selected for further studies. Both
strands of p
321 were fully sequenced, and the insert was 1122 bp
long, excluding the linkers used to prepare the cDNA library. The
insert contained one long open reading frame encoding 318 amino acid
residues with a calculated mass of 35,041 Da (Fig. 3). The first
methionine lies in a good context according to the rules for
translational initiation and is likely to be the initiation
codon(25) . This suggestion is strengthened by the fact that in vitro translation of synthetic mRNA transcribed from
p
321 gives rise to a M
32,000 protein in
SDS-PAGE analysis (data not shown). However, there is no stop codon
in-frame in the upstream 35-bp 5`-untranslated region of the cDNA. The
130-bp 3`-untranslated region ends with a poly(A) tract, and a putative
polyadenylation signal could be identified in the upstream sequence (bp
1094-1099) (Fig. 3).
Figure 3:
Nucleotide sequence of p321 and the
deduced amino acid sequence of p32. The nucleotides are numbered on the left and the amino acid residues on the right. Amino acid 1 is the initiation methionine. The
partial amino acid sequences previously determined from peptides
isolated from trypsin-digested p32 are underlined (see Table 1).
The deduced amino acid sequence of
p321 and the amino acid sequences of the five tryptic peptides (Table 1) differ in only three positions out of the 62 residues
available for a comparison. All three differences are found in the
peptide p321, but the nucleotide sequence in this region of a second
cDNA clone (p
324) is identical to that of p
321. This suggests
that the amino acid sequence determination of peptide p321 was probably
incorrect, although we cannot exclude that the differences are due to
allelic variation. These data demonstrate that p
321 contains the
complete coding region of p32.
In the deduced amino acid sequence, the first 18 residues are hydrophobic and have the characteristics of a classical signal sequence. However, a consensus site for signal peptidase cleavage cannot be clearly identified(26) . The region between residues 132 and 152 are also hydrophobic, and there is a relatively long hydrophobic stretch near the C terminus of the protein (residues 289-310). Thus, p32 displays several hydrophobic regions, which are potential membrane-spanning segments. In light of the homology to the family of short chain alcohol dehydrogenases (see below), it is likely that the central hydrophobic region of p32 (residues 132-152) is not used as a membrane anchor. Instead, both the N- and C-terminal regions are potential membrane-anchoring domains.
A consensus site for N-linked glycosylation (amino acid residues N-I-T) could be found in the deduced amino acid sequence at position 160-162 (Fig. 3).
Figure 4:
Amino acid sequence alignments of p32 and
some related proteins belonging to the family of short chain alcohol
dehydrogenases. The deduced amino acid sequence of p32 and of the
related proteins, the D--hydroxybutyrate dehydrogenase (BDH)(28) , the 3-oxoacyl[acyl carrier
protein]reductase from E. coli (FABG)(29) , and the human estradiol 17
-dehydrogenase (EDH) (30, 31) were
aligned. Amino acid residues conserved in all four sequences are boxed, and the invariant tyrosine residue is marked (open
triangle).
Figure 5:
Expression of transcripts corresponding to
p32. Total RNA isolated from RPE cells, liver, kidney, adrenal gland,
lung, testis, brain, and muscle were fractionated on a 1% denaturing
agarose gel using 20 µg of RNA/lane. The RNA was transferred to a
nylon membrane and probed with P-labeled insert of
p
321. A 1.4-kb mRNA is visualized in RPE. The migration of the 18
and 28 S ribosomal RNAs are indicated at the right.
Figure 6:
Expression of p32 in Cos cells and
enzymatic properties of the recombinant protein. A,
immunoblotting analyses of p32 expression in microsomal fractions from
cells transfected with a p32 expression vector or from control cells.
Ten micrograms of total microsomal protein was analyzed in each lane. B-D, HPLC analyses of products formed after incubations
of microsomal fractions from p32 expressing Cos cells with 11-cis retinol (11-cis Rol) as the substrate. The incubations were
carried out in the presence of NAD (B) or in
the presence of NADP (C). As control, an incubation of a
microsomal fraction from control cells was carried out in the presence
of NAD
(D). All-trans retinaldehyde
(at RAl) formation in B was caused by isomerization of the
11-cis compound (11-cis RAl) during the incubation and the
subsequent extraction procedures (see ``Results'' for
details).
To investigate the enzymatic properties
of p32, we incubated microsomal fractions from transfected cells and
from control cells with different combinations of 11-cis retinol, all-trans retinol, and the cofactors
(NAD or NADP). Analyses by HPLC showed that microsomal
fractions from transfected cells expressed an 11-cis retinol
dehydrogenase that was active in the presence of NAD
,
as indicated by the formation of 11-cis retinaldehyde (Fig. 6B). A second peak in the chromatogram coeluted
with all-trans retinaldehyde. Control incubations with
11-cis retinaldehyde, in the absence of cellular membranes,
showed that under the conditions used, a significant amount of the
11-cis retinaldehyde isomerized to all-trans retinaldehyde (data not shown). We conclude that all-trans retinaldehyde is generated during the incubation and extraction
procedures and not during the enzymatic reaction. No enzymatic activity
was detected with NADP as the cofactor (Fig. 6C).
Control cells, not expressing p32, lack the ability to oxidize
11-cis retinol into 11-cis retinaldehyde under the
same conditions (Fig. 6D). Incubations with
all-trans retinol verified the stereospecificity of the
enzyme, as no significant formation of all-trans retinaldehyde
was detected (data not shown). These data show the stereospecificity of
this enzyme and the requirement for NAD
as the
cofactor.
In this article, we have described a novel RPE-specific protein, p32, which forms a complex with p63 of the RPE. The primary structure of p32 has all the critical features of a functional SCAD, including a putative cofactor binding site and essential residues involved in the catalytic mechanism, namely the almost invariant tyrosine-containing sequence motif Y-X-X-X-K(27) .
A major metabolic step in retinoid metabolism in RPE cells is the conversion of 11-cis retinol to 11-cis retinaldehyde, the universal chromophore of all visual pigments in higher animals. After its synthesis in the RPE, 11-cis retinaldehyde is transported to the photoreceptor cells and covalently attached to the opsins to form the visual pigments. The enzyme responsible for synthesis of 11-cis retinaldehyde is the stereospecific11-cis retinol dehydrogenase(3, 5, 6, 14) . We can demonstrate that p32 is such a stereospecific 11-cis retinol dehydrogenase. The identification and molecular cloning of this enzyme is important for further studies of the molecular and cellular mechanisms involved in synthesis and regeneration of visual pigments. Furthermore, the role of this enzyme in various pathological conditions of the retina can be investigated.
The identification of the 11-cis retinol dehydrogenase as a member of the SCAD superfamily is interesting from several points of view. SCADs are either cytosolic or membrane-bound enzymes that utilize a large number of substrates, including steroids and prostaglandins, but this is the first report that a membrane-bound retinol dehydrogenase belonging to the SCAD superfamily has been identified. Because the SCADs are oxidoreductases, i.e. depending on whether the cofactor is reduced or oxidized, these enzymes will oxidize or reduce their substrates, respectively. Thus, it would not be surprising to find that other retinol dehydrogenases belong to the SCAD superfamily. For example, the enzyme that reduces all-trans retinal to all-trans retinol in the photoreceptors after bleaching of the visual pigments may also be a SCAD(32) . In several non-ocular tissues, all-trans retinol is oxidized to all-trans retinal for further synthesis to retinoic acid. It is generally accepted that the first step in this metabolic pathway is carried out by members of the medium chain alcohol dehydrogenases (33, 34, 35) . An appealing possibility is that oxidation of all-trans retinol in such tissues could also be carried out by membrane-bound members of the SCAD family. The identification of novel retinol dehydrogenases in non-ocular tissues would be important for further understanding of the regulation of retinoic acid biosynthesis and action during embryonic development and during cellular growth and differentiation.
Like many other epithelial cells, RPE cells are polarized. Intracellular sorting of a membrane-bound 11-cis retinol dehydrogenase, to apically located cellular compartments, might occur in order to facilitate regeneration of the visual pigments. If so, then p63 with a putative function in cellular retinol uptake may be sorted along with p32 to apical compartments. The complex formation of p63 and 11-cis retinol dehydrogenase may be an indication that cellular uptake and metabolism of retinoids are coupled events in the RPE. Further experiments along these lines, including a detailed localization study of p63 and the 11-cis retinol dehydrogenase, might shed new light on the processes of general retinol uptake in RPE cells and chromophore regeneration during the visual cycle.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X82262[GenBank].