Cloning and Expression of a cDNA Encoding Bovine ... - CiteSeerX
Cloning and Expression of a cDNA Encoding Bovine Retinal Pigment Epithelial 11-cis Retinol Dehydrogenase Carola A. G. G. Driessen,* Bert P. M. Janssen,* HuubJ. Winkens* Anke H. M. van Vugt,* Ton L. M. de Leeuw,* and Jacques J. M. Janssen\
Purpose. Identification of a 32-kd protein in the bovine retinal pigment epithelium. Methods. A bovine retinal pigment epithelium cDNA library was constructed in the bacteriophage XZAP Express. A monoclonal antibody, designated 21-C3/AV, was used to isolate the cDNA encoding the 21-C3/AV antigen. A positive full-length clone, designated 21-C3RDH/ CD, was sequenced. Northern blot analysis was used to determine the length of the mRNA and the tissue expression pattern. The entire open reading frame of clone 21-C3RDH/CD was used to isolate a recombinant baculovirus clone and expressed in Spodoptera frugiperda insect cells. Enzymatic activity toward 11-cis retinaldehyde was investigated. Results. The complete nucleotide sequence of 21-C3RDH/CD was obtained. The deduced amino acid sequence reveals homology with short-chain alcohol dehydrogenases. Northern blot analysis detected a 1.2-kb transcript. Although the monoclonal antibody used to isolate 21-C3RDH/CD also reacts with other ocular and nonocular tissues, the authors were unable to demonstrate any reactivity with RNA samples isolated from different (non)ocular tissues. Recombinant baculovirus-infected insect cells synthesized the 21-C3/AV antigen. This protein showed 1 1-cis retinol dehydrogenase activity. Conclusions. Homology to the human D-beta-hydroxybutyrate dehydrogenase precursor and other alcohol dehydrogenases shows that 21-C3RDH/CD encodes a short-chain alcohol dehydrogenase. Furthermore, tissue specificity and molecular weight of the antigen suggest that 21-C3RDH/CD encodes the bovine retinal pigment epithelial 11-cis retinol dehydrogenase. Direct proof came from experiments in which we used the baculovirus-based expression system for in vitro synthesis of the protein encoded by 21-C3RDH/CD . Protein extracts obtained from recombinant baculovirus-infected insect cells were found capable of reducing 11-cis retinaldehyde. Invest Ophthalmol Vis Sci. 1995; 36:1988-1996.
Xn the past, retinal pigment epithelium (RPE) research has focused on elucidation of the function of the RPE in vision. These studies have shown that the RPE fulfills several important functions in this process, including phagocytosis of photoreceptor outer segments, metabolite exchange, and rhodopsin regeneration.1 2 The latter process involves reuse of endogenous retinoids. One of the retinoids, 11-cis retinaldehyde, serves as the chromophore for visual pigments present in the outer segments of rod and cone photo-
Frwm the * Institute of Ophthalmology and tlw.^ Department of Biochemistry, University of Nijmegen, The. Netherlands. Submitted for publication September 27, 1994; revised May I, 1995; accepted May 12, 1995. Proprietary interest categoiy: N. Helmut requests: Carola A. C. G. Driessen, Institute of Ophthalmology, University of Nijmegen, l>. 0. Box 9101, 6500 I IB Nijmegen, The Netherlands.
1988
receptor cells. Photon absorbance by 11-cis retinaldehyde and its subsequent isomerization to 3\\-trans retinaldehyde is the primary event in phototransduction. The RPE is responsible for the regeneration of 11-cis retinaldehyde. 3 On isomerization, zW-trans retinaldehyde is reduced to all-/raras retinol, which is bound to the interphotoreceptor retinoid binding protein and transported to the RPE. In the RPE, all-/rans retinol is isomerized to 11-cis retinol. The formation of 11-cis retinaldehyde from 11-cis retinol is catalyzed by an 11cis retinol specific dehydrogenase. The latter enzyme was reported to be an oxidoreductase that preferentially reacts with 11-cis retinol. 4 Finally, 11-cis retinaldehyde bound to interphotoreceptor retinoid binding protein is transported to the rod and cone outer segments to regenerate the visual pigments. Recently, retinol dehydrogenase was purified partially from bovine
Investigative Ophthalmology & Visual Science, September 199:5, Vol. 3f>, No. 10 Copyright © Association for Research in Vision and Ophthalmology
Retinol Dehydrogenase
RPE.' The RPE retinol dehydrogenase was reported to have an apparent molecular weight of 33 kd. Until today, the primary structure of retinol dehydrogenase has been unknown. To characterize RPE proteins, we recently isolated a broad panel of monoclonal antibodies (mAbs) recognizing different RPE antigens.'3 Monoclonal antibodies against five types of antigens were identified. One of these, 21-C3/AV, recognizes an antigen with an apparent molecular weight of 32 kd. Immunohistochemical analysis shows that 21-C3/AV exhibits strong reactivity with RPE. The mAb 21-C3/AV was used to screen a bovine RPE cDNA library. In this article, we report on the isolation of a cDNA encoding a 32-kd RPE protein. The deduced amino acid sequence reveals significant homology with the amino acid sequence of members of the superfamily of short-chain alcohol dehydrogenases. The largest homology was found with the human D-beta-hydroxybutyrate dehydrogenase precursor protein.7 MATERIALS AND METHODS All experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The monoclonal antibody used in this study was isolated and characterized as described.6 RNA Isolation and cDNA Library Construction Bovine eyes were obtained from a local slaughterhouse. Within 3 hours of eye enucleation, the cornea, vitreous, and retina were removed. Eyecups were rinsed in 15 mM phosphate-buffered saline, pH 7.4, containing 0.25 M sucrose and 2 mM ethylenediaminetetraacetic acid (EDTA). Retinal pigment epithelial cells were removed by careful brushing in the same buffer from which EDTA was omitted. Total cellular RPE RNA was prepared by a LiCl-urea method.8 Poly(A+) RNA was isolated using a biotinylated oligo(dT) primer (Promega, Madison, WI). An expression cDNA library was constructed using 5 fj,g of poly(A+) RNA and the \ZAP Express vector in accordance with the manufacturer's instructions (Stratagene, La Jolla, CA). Plated on Escherichia coli XL1Blue, a library consisting of 8.7 X 105 recombinant clones was obtained. Immunoscreening of a Retinal Pigment Epithelial cDNA Library The cDNA library was plated on E. coli XLl-Blue. Plates were incubated for 3 % hours at 42°C. Immunoscreening was performed as described by Sambrook et al9 with minor adjustments. In brief, nitrocellulose filters impregnated with 10 mM isopropyl-l-thio-/3-Dgalactopyranoside were used to induce expression of hybrid proteins (3 hours at 37°C). To obtain duplicate
1989
filters, this procedure was repeated once. After blocking in radioimmunoassay buffer (0.3% gelatine, 0.9% NaCl, 10 mM Tris; pH 7.5, 1% Triton X-100, 0.5% desoxycholate, 0.1 mM PMSF) for 30 minutes, the filters were incubated with I2>I labeled mAb 21C3/AV in the same buffer (12 hours at room temperature). Affinity-purified mAb was radiolabeled using chloramine T and Na125I to a specific activity of 3.2 /iCi///g.10 The filters were subjected to autoradiography using an Amersham Hyperfilm-MP (Amersham, Buckinghamshire, UK) at — 70°C with two intensifying screens. Positive clones were excised in vivo by use of the ExAssist helper phage. Subclones were generated in the phagemid vector pBK-CMV (Stratagene). DNA Sequencing Sequencing was performed by the dideoxy chain termination method" using Sequenase (US Biochemical, Cleveland, OH). DNA sequences were determined in both directions. Universal T3 and T7 primers were used, as were several specific internal sequencing primers (Pharmacia, Woerden, The Netherlands). Computer analysis of nucleotide sequences and alignment of deduced amino acid sequences were accomplished using the University of Wisconsin Genetics Computer Group Software (version 7.3.1). Northern Blot Hybridization Total RNA was isolated from nonocular tissues (heart, liver, spleen, lung, kidney, small intestine) and ocular tissues (lens capsule, RPE, iris, retina, cornea epithelium, ciliary body). RNA samples were prepared by the LiCl-urea method.8 Poly(A+) RNA from kidney and RPE was isolated as described above. Per sample, 10 /ig of total RNA or 2 //g poly(A+) RNA was electrophoresed on a 0.9% agarose-formaldehyde gel and transferred to 0.2 //m nitrocellulose (Schleicher and Schuell, Dassel, Germany). The 1210 bp EcoRI/XhoIinsert of 21-C3RDH/CD was labeled with 32P by a random priming method12 and used as a probe to hybridize the blot in 1% bovine serum albumin, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA, and 0.5 M NaP; pH 7.2, overnight at 60°C. After hybridization, the blot was washed in a solution containing 0.5% SDS, 1 mM EDTA, and 50 mM NaPi pH 7.2, at 60°C for 30 minutes. Autoradiography was accomplished by exposure to a Kodak X-Omat film at —80°C using two intensifying screens (Kodak, Odijk, The Netherlands). To verify the amount and integrity of the RNA on the filters, the same filters were hybridized with a mouse actin probe or an rRNA probe. Construction of pAcJAC3/RDH To use the baculovirus-based in vitro expression system for in vitro expression of the protein encoded by 21-C3RDH/CD, we constructed the transfer vector
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Investigative Ophthalmology & Visual Science, September 1995, Vol. 36, No. 10
pAcJAC3/RDH. In brief, a multiple cloning site containing sequences recognized by the restriction enzymes BamH I, Nco I, Kpn I, Bgl II, Stu I, Not I, Nhe I, and Spe I was engineered into the BamH I position of the baculovirus transfer vector pAcDZlIH using two complementary DNA oligonucleotides 5'-GATCCCCATGGTACCAGATCTAGGCCTGCGGCCGCTAGCACTAGT-3' and 5'-GATCACTAGTGCTAGCGGCCGCAGGCCTAGATCTGGTACCATGGG-3'. The resultant transfer vector was designated pAcJAC3. The 21-C3RDH/CD cDNA fragment was cloned into pAcJAC3 as a BamH I Not I DNA-fragment to generate pAcJAC3/RDH. Both sites are derived from the multiple cloning site of the pBK-CMV phagmid vector and flank the cDNA fragment cloned into the Xho I and EcoR I sites of the pBK-CMV multiple cloning site (see above). Transcription of the inserted cDNA in pAcJAC3/RDH is controlled by the baculovirus-derived polyhedrin promoter. We used pAcJAC3/RDH to isolate the recombinant AcNPV (Autographa californica nuclear polyhedrosis virus) clone designated AcNPV.RDH as described previously.14
Cell Culture Baculovirus propagation, transfection, isolation of recombinant virus and Sf9 insect cell culture were performed as described.15 Cells were cultured at 27°C in TNMFH medium supplemented with 10% (vol/vol) fetal calf serum, 0.1% (wt/vol) pluronic F-68, 50 fig/ ml streptomycin, and 50 U/ml penicillin. Suspension cultures of 150 ml each were used for 21-C3/RDH biosynthesis. Viral infections were performed using 15 ml of a diluted virus stock solution to give a multiplicity of infection of 5. Cells used for both Western blot analysis and measurement of 1 l-cis retinol dehydrogenase activity were harvested 3 days after infection. Immunoblot Analysis Proteins were separated on a 12.5% SDS-polyacrylamide gel,"' transferred to nitrocellulose (Schleicher and Schuell BA85, pore size 85), and probed with the mAb 21-C3/AV. The mAb 21-C3/AV has been described6 and was used in a dilution of 1:1000. Specifically bound antibodies were detected through an incubation with rabbit anti-mouse immunoglobulin G conjugated to peroxidase (1:500) and a subsequent incubation with the peroxidase substrate 4-chloro-lnaphthol (0.3% in phosphate-buffered saline, 5% methanol, 0.01% H2O2). Assay for 1 l-ci*-retinol Dehydrogenase Activity At 3 days after infection, cells were harvested by centrifugation (4000g 10 minutes, 4°C). The 11-ds retinol dehydrogenase activity was measured from whole cell extracts. Briefly, cell pellets were resuspended in extraction buffer
(5 mM Tris HC1,10 mM reduced /3-nicotinamide-adenine dinucleotide (NADH), 20% glycerol, 0.15 M NaCl, and 20 mM dodecyl-/?-l-maltoside (DoM)) at a cell density of 20 X lO15 Sf9 cells per milliliter. Glycerol and NADH were added to the extraction buffer to stabilize the expressed 11-ds retinol dehydrogenase.' Samples were incubated for 30 minutes at 4°C and centrifuged (100,000g; 30 minutes, 4°C). All subsequent manipulations were performed under dim red light (Schott-Jena [Mainz, Germany], RG 645 cut-off filter). We used 50-/il aliquots, 1 X 10" infected cells, of the supernatant to mix with 50 \A of the 1 1-ds retinol dehydrogenase substrate solution (0.2 M acetate buffer, 2.5% methanol, 1.0 mM NADH, 10 nmol 11-ds retinaldehyde, pH 5.0). Under these conditions, 11-ds retinol dehydrogenase reduces 11-ds retinaldehyde to l i ds retinol. Samples were incubated at 37°C. At 1-, 5-, and 10-minute intervals, 25-//1 samples were taken. The 11-ds retinaldehyde decrease was measured through the addition of 150 fi\ thiobarbituric and 150 fA thiourea according to Futterman and Saslow.17 Samples were incubated for 30 minutes at 25°C and centrifuged (10,000g 5 minutes, 25°C). Color development was measured at 530 nm. For a positive control, we used bovine RPE cells isolated as described. 6 Whole RPE cell extracts were prepared as described above (40 X 106 cells/ml). For a negative control, we used Sf9 cells infected with wildtype AcNPV.
RESULTS Isolation of cDNA Clones With 21-C3/AV We successfully generated a bovine RPE cDNA library consisting of 8.7 X 105 independent clones. To determine the level of contaminating-retina, mRNA-derived cDNA clones, we used a bovine opsin cDNA probe to screen 100,000 recombinant clones of this RPE-specific \ZAP Express cDNA library. The complete 3' untranslated region of the opsin gene and a small part of the coding region were encoded by this probe. Hybridization showed that only a small percentage (0.1%) of the clones screened are actually opsin cDNA clones. Hence, the mRNA samples used to construct this library were minimally contaminated with mRNA derived from the neurosensory retina. Having tested the specificity of the library, we used the iodinated mAb 21-C3/AV for expression screening of clones from the amplified library. After two rounds of screening, 16 positive cDNA clones were selected. From each of these clones, the phagemid pBK-CMV was rescued by in vivo excision. Plasmid DNA was isolated and used in Southern blot experiments. These experiments showed that 13 of the 16 clones cross-hybridized. Four
Retinol Dehydrogenase
1991
1 34 10S 1 159 19 213 37
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found from the first ATG start codon at position 105 to the stop codon TAA at position 1062. The first ATG codon is situated in a good context for translation initiation according to Kozak's rules.1" '' An AATAAA polyadenylation signal is found 16 nucleotides upstream from the poly (A) tail. From the nucleotide sequence of 21-C3RDH/CD, we deduced the amino acid sequence, designated 21C3RDH, of the encoded polypeptide (Fig. 1). This conceptual translation yielded a protein sequence consisting of 319 amino acid residues. The cumulative molecular weight of the amino acid sequence was 34,301 daltons, which is in good agreement with the previously published apparent molecular weight of the antigen recognized by 21-C3/AV.(> On the N-terminus of 21-C3RDH, no signal peptide sequence was detected.
Homology With Human D-beta-hydroxybutyrate Dehydrogenase Precursor Using both the nucleotide sequence and the deduced 807 CAG GCC CTC TAT GGG GAG GCC TTC CTC ACC AAA TAC CTG AGA GTG CAG CAA CGT amino acid sequence, we searched for homologous 235 Gin Ala Leu Tyr Gly Glu Ala Phe Leu Thr Lys Tyr Leu Arg Val Gin Gin Arg sequences in the EMBL (release 39.0), PIR (release 861 ATC ATG AAC ATG ATC TGT GAT CCG GAC CTG GCC AAG GTG AGC AGG TGC CTG GAG 253 H e Met Asn Met lie Cys Asp Pro Asp Leu Ala Lys Val Ser Arg Cys Leu Glu 29.0), and SWISS-PROT (release 41.0) data bases us915 CAT GCC CTA ACT GCC CGT CAC CCC AGA ACC CGC TAC AGC CCA GGC TGG GAT GCC ing the FASTA program.20 The DNA sequence was not 271 His Ala Leu Thr Ala Arg His Pro Arg Thr Arg Tyr Ser Pro Gly Trp Asp Ala homologous to any nucleotide sequence in the data 969 AAG CTG CTC TGG TTG CCA GCC TCC TAC TTG CCA GCC AGG CTG GTG GAT GCT GTG 289 Lys Leu Leu Trp Leu Pro Ala Ser Tyr Leu Pro Ala Arg Leu Val Asp Ala Val base. The amino acid sequence showed 39% identity 1023 CTC GCC TGG GTC CTT CCC AAG CCT GCC CAG ACA GTC TAC TAA ATCCAGCCCTCCAGC in 287 amino acid residues with human D-beta-hydro307 Leu Ala Trp Val Leu Pro Lys Pro Ala Gin Thr Val Tyr . xybutyrate dehydrogenase precursor7 and 33% iden1080 AAAAGATGGTTGTTCAAGGCAAGGACTCTGATTTATTCTGTCCCCTGACCCTGGTACTGCCTGGTGTGTGG 1151 CATAAAACAGTCACTCAATAAATGTATTATTCAAAACAAAAAAAAAAAAAA tity in 210 amino acid residues with human placental 17-beta-hydroxysteroid dehydrogenase21 (Fig. 2). FIGURE l. The nucleotide sequence of the 21-C3RDH/CD cDNA {upper) and deduced amino acid sequence {lower) When conserved amino acid substitutions are taken into consideration, the degree of homology with huof bovine 21-C3RDH. The initiator codon (ATG) and the man D-beta-hydroxybutyrate dehydrogenase precurtermination codon (TAA) are indicated by circles. Undersor increases to 72%. lined nucleotides in the 3'-untranslated region represent a possible polyadenylation signal. Nucleotide and amino acid sequences are numbered on the left. Bold numbers were used for the amino acid sequence. This sequence is deposited in GenBank under the accession number L36533.
clones were found to contain a 1.2-kb insert. The apparent molecular weight of the antigen recognized by 21-C3/AV is 32 kd. Therefore, assuming that the antigen was not glycosylated, we expected a full-length cDNA to have a minimal length of 1.0 kb. Because the lengths of the largest inserts were equivalent in all four clones and, judging from their length, could contain the entire coding sequence, we used one of these clones, 21-C3RDH/CD, for sequence analysis. DNA and Deduced Protein Sequence of 21-C3RDH/CD The complete nucleotide sequence of the cDNA 21C3RDH/CD is shown in Figure 1. Sequence analysis showed 21-C3RDH/CD to contain a 1210-bp cDNA insert. An open reading frame consisting of 957 bp is
Secondary Structure Predictions The hydropathy profile of 21-C3RDH does not indicate hydrophobic stretches sufficiently long to span a phospholipid bilayer (Fig. 3). However, amino acid residues in the region 130 to 200 displayed significant hydrophobic features. A similar hydrophilicity profile pattern was obtained using an algorithm for antigenicity (result not shown). Northern Blot Analysis Northern blot analysis was used to determine the size and tissue expression pattern of 21-C3RDH mRNA. A Northern blot containing 10 //g total RNA from bovine RPE, heart, liver, spleen, lung, kidney, and small intestine was probed with the cDNA 21-C3RDH/CD. A single transcript of approximately 1.2 kb was detected in RPE (Fig. 4, panel A). No transcripts were detected in other tissues even after long exposure. Because immunohistochemical analysis showed that 21-C3/AV reacts with smooth muscle tissue in the
1992
Investigative Ophthalmology & Visual Science, September 1995, Vol 36, No. 10
bovine kidney cortex, we performed Northern blot analysis with 2 //g poly (A') RNA from bovine RPE and kidney tissue. No transcript in kidney was detected (Fig. 4, panel B). To determine the expression pattern of 2110 bdh hu
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FIGURE 3. Hydropathy analysis of 21-O3RDH. The predicted amino acid sequence of 21-C3RDH was analyzed according to Kyte and Doolittle 1 ' using a window of nine residues.
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FIGURE 2. Alignment of 21-C3RDH (rdh bo) with human Dbeta-hydroxybutyrate dehydrogenase precursor ( b d h h u ) and human placenta! 17-beta-hydroxysteroid dehydrogenase ( d h b l h ) . The alignment was obtained using the FASTA program and the SWISS-PROT data base. Vertical bars denote matches, and colons denote substitutions. Residues in the short-chain alcohol dehydrogenases family that are conserved in at least 15 of 20 aligned sequences"' are underlined. The location of a residue that is usually an isoleucine in shortchain alcohol dehydrogenases is indicated by the asterisk. The location of absolutely conserved residues are denoted by vertical airows. Residue positions of b d h h u are indicated by higher numbers. Residue positions of dbhl h are indicated by lower numbers. For residue positions r d h b o see Figure 1.
t FIGURE 4. Northern blot analysis of 21-C3RDH. Panel A: Northern blot containing 10 /xg total RNA from retinal pigment epithelium (RPE) (1), heart (2), liver (3), spleen (4), lung (5), kidney (6), small intestine (7). Panel B: Northern blot containing 2 fig poly(A') RNA from kidney (1) and RPE (2). Panel C: Northern blot containing 10 j/g total RNA from lens capsule (1), RPE (2), iris (3), retina (4), cornea epithelium (5), ciliary body (6). As a control, filters were rehybridized to a mouse actin cDNA probe or a rRNA probe (lanes beneath each panel).
Retinol Dehydrogenase
1993
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~ **
20FICURE 5. Immunoblot analysis of 21-C3/AV antigen isolated from bovine retinal pigment epithelial cells. The immunoblot was screened with 21-C3/AV. Soluble retinal pigment epithelial proteins (lane 1), proteins isolated from the bovine retinal pigment epithelium in the presence of 1% Triton X-100 (lane 2) and in the presence of 1% DoM (lane 3). ence of 1% Triton X-100 or 1% DoM. Homogenized cells were centrifuged (SOfiOOg, 20 minutes, 4°C), and the supernatant was analyzed for the presence of the 21-C3/AV antigen by immunoblotting (Fig. 5). No antigen was detected in protein samples derived from RPE cell homogenates without the use of detergents (Fig. 5, lane 1). However, in the samples obtained with both types of detergent, the antigen was clearly detectable (Fig. 5, lanes 2 and 3). Additional experiments showed that the antigen recognized by 21-C3/ AV accumulates in the microsome fraction if subcellular fractions are prepared from the RPE (data not shown). Expression of Recombinant 21-C3RDH in Sf9 Cells The entire 21-C3RDH/CD cDNA coding sequence was inserted downstream from the baculovirus polyhedrin promoter in pAcJAC3, yielding the baculovirus transfer vector pAcJAC3/RDH. Cotransfection of Sf9 cells with pAcJAC3/RDH and wild-type AcNPV DNA yielded a recombinant AcNPV clone that expressed the protein encoded by 21-C3RDH/CD (Fig. 6). Protein samples derived from recombinant virus-infected Sf9 cells were analyzed by immunoblot, using the mAb 21-C3/AV (Fig. 6, panel A). The immunoblot revealed a protein band of approximately 32 kd (lane 3), which was not present in mock-infected cells (lane 1) or wildtype AcNPV infected cells (lane 2). This band comigrated with the 21-C3/AV antigen recognized in a Triton X-100 protein extract obtained from bovine RPE cells (lane 4). Functional Analysis of 21-C3RDH/CD Encoded 1 l-cis Retinol Dehydrogenase An assay to investigate the 11-eis retinol dehydrogenase activity of 21-C3RDH was performed using pro-
tein samples obtained from AcNPV.RDH infected Sf9 cells (3 days after infection) (Fig. 6, panel B). Retinol dehydrogenase activity was assayed by measuring the initial velocity of 1 l-cis retinaldehyde reduction to 11cis retinol in the presence of NADH at pH 5.0. The decrease of the 1 l-cis retinaldehyde was measured as a function of time over a 10-minute period. Whole cell extracts derived from 1 X 10(i AcNPV.RDH-infected cells reduced 7.0 nmol 1 l-cis retinal to 1 l-cis retinol within 5 minutes (Fig. 6, panel B). For a positive control, 2 X 10b RPE cells were found to reduce 4.7 nmol 1 l-cis retinaldehyde to 1 l-cis retinol within the same period. For a negative control, wild-type AcNPV-infected cells were used. In these assays, an initial velocity of only 0.7 nmol 1 l-cis retinaldehyde for 5 minutes was measured. DISCUSSION In our laboratory, two animal models were developed to resemble acute anterior uveitis and sympathetic ophthalmia in humans. Retinal pigment epithelial protein fractions were responsible for the ocular pathologic processes.22'23 To characterize these uveitogenic proteins further, we isolated a broad panel of mAb-recognizing RPE antigens. One of these mAbs
1 2
3 4
94. 67433020-
FIGURE 6.
Immunoblot and functional analysis of 21-C3RDH protein expression by recombinant baculovirus-infected Sf9 cells. Panel A; Immunoblot blot screened with 21-C3/AV. Sf9-derived cellular proteins (lane 1), wild-type AcNPV-infected Sf9 cells (lane 2), AcNPV.RDH-infected Sf9 cells expressing 11-os retinol dehydrogenase (lane 3), and native bovine retinal pigment epithelium-derived 1 l-cis retinol dehydrogenase (lane 4). Panel B: 1 l-cis retinaldehyde reduction by native bovine retinal pigment epithelial retinol dehydrogenase (open circles, 2 X 106 retinal pigment epithelial cells/reaction mixture), wild-type AcNPV-infected Sf9 cells (closed triangles, 1 X 10b cells/reaction mixture), and AcNPV.RDH-infected Sf9 cells (open triangles, 1 X 10ti cells/ reaction mixture).
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Investigative Ophthalmology & Visual Science, September 1995, Vol. 36, No. 10
was used to screen a bovine RPE cDNA library. Antibody selection resulted in 16 independently isolated clones. From these 16 clones, 13 were found to encode the same protein. Hence, the immunoscreening procedure proved to be highly selective for clones encoding the 21-C3/AV antigen. The cDNA sequence and deduced amino acid sequence of 21-C3RDH/CD were used to search for homologous sequences in the EMBL, PIR, and SWISS-PROT data libraries. Homology was found among a number of proteins thought to be members of the short-chain, nonmetalloenzyme dehydrogenase family. Members of the short-chain alcohol dehydrogenase family are composed of approximately 250 to 300 amino acid residues and do not contain catalytic metal atoms.24 The initially termed long-chain alcohol dehydrogenases, or classical alcohol dehydrogenases, are between 350 and 375 amino acid residues long. Numerous members of this family use zinc as a catalytic atom. 25 A second characteristic feature of the members of the classical dehydrogenase family is the location of the catalytic domain near the N-terminus. The coenzyme-binding domain is located in the C-terminal part of the protein. This is reversed in members of the short-chain alcohol dehydrogenase family. Here, the nucleotide-binding domain is close to the N-terminus, and the catalytic domain is found in the C-terminal part of the protein. No reaction mechanism is known for any member of the shortchain alcohol dehydrogenase family. An alignment of 21-C3RDH with human D-betahydroxybutyrate dehydrogenase precursor 7 yielded the highest homology score. The latter enzyme is located at the inner face of the mitochondrial inner membrane. The D-beta-hydroxybutyrate dehydrogenase precursor protein (34.5 kd) is synthesized on free polysomes and posttranslationally imported into the mitochondria. 26 The first 46 amino acid residues of the precursor protein function as a leader peptide. Based on the consensus cleavage site Arg-X-Tyrl (Ser/ Ala) previously reported for mitochondrial proteins, 27 the precursor protein is most likely cleaved between Tyr-46 and Ala-47 (for numbering, see Fig. 2). The leader sequence is cleaved by a matrix protease yielding a 31.5-kd protein. D-beta-hydroxybutyrate dehydrogenase catalyses the oxidation of D-beta-hydroxybutyrate and the reduction of acetoacetate. In a pairwise alignment, the members of the shortchain alcohol dehydrogenase family are approximately 25% identical to one another. 24 This is in good agreement with the homology existing between 21C3RDH and the human D-beta-hydroxybutyrate dehydrogenase, which is found to be 39% identical. The homology observed with human placental 17-beta-hydroxysteroid dehydrogenase 21 was 33%. The latter alignment was used to compare the 21-C3RDH amino acid sequence to the aligned sequences of 20 members
of the short-chain alcohol dehydrogenases, which included this human placental dehydrogenase. 24 Sequence alignment between the members of the shortchain alcohol dehydrogenase family revealed 6 strictly conserved amino acid residues and 19 conserved residues in at least 15 of 20 aligned sequences. In 21C3RDH all these strictly conserved amino acids (Gly36, Gly-42, Asp-83, Gly-157, Tyr-176, and Lys-180; for numbering see Fig. 1) are present. Of the highly conserved amino acid residues, 16 of 19 also are present in 21-C3/RDH, including residues Gly-40 and Ser-164. The coenzyme-binding site was assigned to the N-terminal site of the short-chain alcohol dehydrogenases.28 The glycine residues located at the N-terminus of 21-C3RDH (Gly-36, Gly-40 and Gly-42) are highly conserved in short-chain alcohol dehydrogenases. The spacing of these residues resembles the spacing expected for glycine residues that are part of the nicotinamide-adenine dinucleotide coenzyme-binding domain of alcohol dehydrogenases. 28 ' 29 Three putative active site residues—Ser-164, Tyr-176, and Lys-180— are conserved in 21-C3RDH. Hence, the organization of the coenzyme-binding domain and the active site domain of 21-C3RDH is characteristic for members of this superfamily of alcohol dehydrogenases. These observations suggest that 21-C3RDH encodes ll-cis retinol dehydrogenase. The most recent report on a RPE retinol dehydrogenase was by Suzuki and coworkers.5 They reported on the isolation of 11-ds retinol dehydrogenase from bovine RPE. A partially purified sample containing two proteins was obtained. The apparent molecular weights of these two proteins were 33 kd and 66 kd. Further analysis showed that the 33-kd protein exhibited retinol dehydrogenase activity. The antigen, which is recognized by the mAb 21-C3/AV, has an apparent molecular weight of 32 kd.b Hence, the molecular weight of retinol dehydrogenase and the 21C3/AV antigen are comparable, and both agree with the cumulative molecular weight of the protein encoded by 21-C3RDH/CD. Additionally, the 21-C3/AV antigen can be solubilized only with the help of detergents. Previous studies30 have shown that the ll-cis retinol dehydrogenase is associated with the microsomal membranes. Northern-blot analysis with 10 fig total RNA from different ocular and nonocular tissues showed that the 21-C3RDH gene is only transcribed in the RPE. No transcripts were detected in any of the tissues tested. Subsequently, we performed Northern blot analysis with 2 fig poly(A+) RNA from both RPE and kidney tissue. Although immunohistochemical analysis showed that 21-C3/AV reacts with smooth muscle tissue in the bovine kidney cortex,*' we were unable to detect any transcripts in kidney mRNA. However, 21C3/AV exhibited reactivity not only with RPE but also
Retinol Dehydrogenase
with the striated eye muscle and smooth muscle tissue of small arteries.0 Probably these reactions are caused by cross-reacting antigens, most likely other dehydrogenases. For example, mitochondria are abundant organelles in striated muscle tissue. Hence, a cross-reaction can occur with D-beta-hydroxybutyrate dehydrogenase if 21-C3/AV recognizes an identical epitope on both enzymes. In the alignment between both proteins, there are sufficiently long identical sequences to support this idea. Interestingly, the human D-betahydroxybutyrate gene transcript was present in all forms of muscle tissue, including smooth muscle tissue.7 In addition to muscle tissue, 21-C3/AV stains several ocular epithelial cells.6 Epithelial tissues are known to convert retinol to retinoic acid,31 including different types of ocular epithelial cells.32 Vertebrate alcohol dehydrogenases classes I and IV are expressed in these epithelial tissues. Probably 21-C3/AV also recognizes an epitope on these enzymes. All the evidence that 21-C3RDH/CD encodes one of the two key enzymes of the visual cycle has been indirect. Therefore, we used 21-C3RDH/CD for in vitro expression of the protein it encodes. Recently, biosynthesis of two dehydrogenases using the baculovirus-based expression system were published.3334 Hence, 21-C3RDH/CD was used to isolate recombinant AcNPV clones. One of these clones was used to infect Sf9 cells, and the expressed protein was assayed for 11-ds retinol dehydrogenase activity. Immunoblot analysis showed that a protein of 32 kd was produced by recombinant virus-infected cells that was recognized by 21-C3/AV. This protein was not detected in samples obtained from mock or wild-type AcNPV-infected cells. We used recombinant AcNPV-infected cells in 11-ds retinol dehydrogenase assays as described.45 These experiments showed that the protein encoded by 21-C3RDH/CD reduces 11-ds retinaldehyde. Based on observations of apparent molecular weight, homology with short-chain alcohol dehydrogenases, tissue specificity, and 11-cis retinol dehydrogenase activity measured from the expressed protein, we conclude that 21-C3RDH/CD encodes bovine 11-ds retinol dehydrogenase. Acknowledgments The authors thank Petra H. M. Bovee-Geurts (Department of Biochemistry, University of Nijmegen, The Netherlands) for her assistance with the retinol dehydrogenase assays. They also thank Dr. W. J. de Grip (Department of Biochemistry, University of Nijmegen) for helpful discussions and for providing 11-cis retinaldehyde and dodecyl-/?-l-maltoside.
NOTE ADDED IN PROOF While this manuscript was under review, Simon et al3(i reported on the cloning of bovine retinal pigment
1995
epithelial-specific 11-cis retinol dehydrogenase. The two cDNA sequences and their deduced protein sequences do not agree completely. The work by Simon et al describes a cDNA sequence encoding 318 amino acid residues, whereas we report on a protein sequence consisting of 319 amino acids. Furthermore, the first 29 amino acids do not match between the two sequences. This difference is caused by three additional guanines, present at positions 108, 177, and 192. In the sequence published by Simon et al, only one guanine is present at these positions. Both sequences align as a result of the last additional guanine. The presence of the first additional guanine immediately after the ATG start codon places the initiator codon in an optimum context for translation initiation.18 '"' The additional guanines also explain the difference in length between the two protein sequences. Key Words baculovirus, cDNA library, immunoscreening, monoclonal antibody, retinal pigment epithelium, retinol dehydrogenase References 1. Saari JC. Enzymes and proteins in the mammalian visual cycle. Prog Ret Res. 1990:360-381. 2. Bok D. The retinal pigment epithelium: A versatile partner in vision. / Cell Sri. 1993; 17:189-195. 3. Bernstein PS, Law WC, Rando RR. Isomerization of all-trans-retinoids to 11-cis-retinoids. Proc Natl Acad Sri USA. 1987;84:1849-1853. 4. Lion F, RotmansJP, Daemen FJM, Bonting SL. Stereospecificity of ocular retinol dehydrogenases and the visual cycle. Biochem Biophys Ada. 1975;384:283-292. 5. Suzuki Y, Ishiguro S, Tamai M. Identification and immunohistochemisty of retinol dehydrogenase from bovine retinal pigment epithelium. Biochem Biophys Ada. 1993; 1163:201-208. 6. Janssen JJM, Janssen BPM, Van Vugt AHM. Characterization of monoclonal antibodies recognizing retinal pigment epithelial antigens. Invest Ophthalmol Vis Sci. 1994;35:189-198. 7. Marks AR, MclntyreJO, Duncan TM, Erdjument—Bromage H, Tempst P, Fleischer S. Molecular cloning and characterization of (R)-3-hydroxybutyrate dehydrogenase from human heart. J Biol Chem. 1992; 267:15459-15463. 8. Auffray C, Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNA's from total myeloma tumor RNA. Eur J Biochem. 1980; 107:303-314. 9. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press; 1989:12.16-12. 10. Hunter WM, Greenwood FC. Preparation of iodine131 labelled human growth hormone of high specific activity. Nature. 1962; 194:495-496. 11. Sanger F, Nicklen S, Coulson A. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sd USA. 1977;74:5463-5467.
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12. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983;132:6-13. 13. Zuidema D, Schouten A, Usmany M, et al. Expression of cauliflower mosaic virus gene I in insect cells using a novel polyhedrin-based baculovirus expression vector. JGen Virol. 1990; 71:2201-2209. 14. Janssen JJM, Mulder WR, DeCaluwe GLJ, Vlak JM, DeGrip WJ. In vitro expression of bovine opsin using recombinant baculovirus: The role of glutamic acid (134) in biosynthesis and glycosylation of opsin. Biochim Biophys Ada. 1991; 1089:68-76. 15. Summers MD, Smith GE. A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Exp Station Bull. 1987:1555. 16. Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature. 1970;227:680-685. 17. Futterman S, Saslaw LD. The estimation of vitamin A aldehyde with thiobarbituric acid. / Biol Chem. 1961;236:1652-1657. 18. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eucaryotic ribosomes. Cell. 1986; 44:298-292. 19. Kozak M. An analysis of vertebrate mRNA sequences: Intimations of translation control. / Cell Biol. 1991; 115:887-903. 20. Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sri USA. 1988; 85:2444-2448. 21. Peltoketo H, Isomaa V, Maentausta O, Vihko R. Complete amino acid sequence of human placental 17beta-hydroxysteroid dehydrogenase deduced from cDNA. FEBSLett. 1988; 239:73-77. 22. Broekhuyse RM, Kuhlmann ED, Winkens HJ. Experimental autoimmune anterior uveitis (EAAU): II: Dose-dependent induction and adoptive transfer using a melanin-bound antigen of the retinal pigment epithelium. Exp Eye Res. 1992;55:401-411. 23. Broekhuyse RM, Kuhlmann ED, Winkens HJ. Experimental autoimmune anterior uveitis (EAAU): III: Induction by immunization with purified uveal and skin melanins. Exp Eye Res. 1993;56:575-583. 24. Persson B, Krook M, Jornvall H. Characteristics of short-chain alcohol dehydrogenases and related enzymes. EurJBiochem. 1991;200:537-543. 25. Borras T, Persson B, Jornvall H. Eye lens zeta-crystallin relationships to the family of 'long-chain' alcohol/
26.
27. 28.
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31.
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polyol dehydrogenases: Protein trimming and conservation of stable parts. Biochemistry. 1989; 28:61336139. Kante A, Cherkaoui-Malki M, Bailly A, Adami P, Latruffe N. Expression genique de la D-/?-hydroxybutyrate deshydrogenase: II: Incorporation de la pre-BDH dans les mitochondries. C R Soc Biol. 1987; 181:389394. Gavel Y, von Heijne G. A conserved cleavage-site motif in chloroplast transit peptides. FEBS Lett. 1990; 261: 455-458. Jornvall H, von Bahr-Lindstrom H, Jany KD, Ulmer W, Ffoschle M. Extended superfamily of short alcoholpolyol-sugar dehydrogenases: Structural similarities between glucose and ribitol dehydrogenases. FEBS Lett. 1984; 165:190-196. Wierenga RK, Maeyer De MCH, Hoi WGJ. Interaction of pyrophosphate moieties with a-helixes in dinucleotide binding proteins. Biochemistry. 1985; 24:13461357. Zimmerman WF, Lion F, Daemen FJM, Bonting SL. Biochemical aspects of the visual process: XXX: Distribution of stereospecific dehydrogenase activities in subcellular fractions of the bovine retina and pigment epithelium. Exp Eye Res. 1975;21:325-332. Satre MA, Zgombic-Knight M, Duester G. The complete structure of human class IV alcohol dehydrogenase (retinol dehydrogenase) determined from the ADH7 gene. J Biol Chem. 1994;269:15606-15612. Julia P, Farres J, Pares X. Ocular alcohol dehydrogenase in the rat: Regional distribution and kinetics of the ADH-1 isoenzyme with retinol and retinal. Exp Eye Res. 1986; 42:305-314. Breton R, Yang F, Jin J, Li B, Labrie F, Lin S. Human 17/3-hydroxysteroid dehydrogenase overproduction using a baculovirus expression system and characterization. / Steroid Biochem Mol Biol. 1994;50:275-282. Nash WE, Mercer RW, Blanco G, Strickler RC, Mason JI, Thomas JL. Over-expression of human type I (placental) 3/?-hydroxy-5-ene steroid dehydrogenase/isomerase in insect cells infected with recombinant baculovirus. / Steroid Biochem Mol Biol. 1994;50:235-240. Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982;157:105-132. Simon A, Hellman U, Wernstedt C, Eriksson U. The retinal pigment epithelial-specific 11-as retinol dehydrogenase belongs to the family of short chain dehydrogenases. /Biol Chem. 1995;270:1107-1112.
Purpose. Identification of a 32-kd protein in the bovine retinal pigment epithelium. Methods. A bovine retinal pigment epithelium cDNA library was constructed in the bacteriophage XZAP Express. A monoclonal antibody, designated 21-C3/AV, was used to isolate the cDNA encoding the 21-C3/AV antigen. A positive full-length clone, designated 21-C3RDH/ CD, was sequenced. Northern blot analysis was used to determine the length of the mRNA and the tissue expression pattern. The entire open reading frame of clone 21-C3RDH/CD was used to isolate a recombinant baculovirus clone and expressed in Spodoptera frugiperda insect cells. Enzymatic activity toward 11-cis retinaldehyde was investigated. Results. The complete nucleotide sequence of 21-C3RDH/CD was obtained. The deduced amino acid sequence reveals homology with short-chain alcohol dehydrogenases. Northern blot analysis detected a 1.2-kb transcript. Although the monoclonal antibody used to isolate 21-C3RDH/CD also reacts with other ocular and nonocular tissues, the authors were unable to demonstrate any reactivity with RNA samples isolated from different (non)ocular tissues. Recombinant baculovirus-infected insect cells synthesized the 21-C3/AV antigen. This protein showed 1 1-cis retinol dehydrogenase activity. Conclusions. Homology to the human D-beta-hydroxybutyrate dehydrogenase precursor and other alcohol dehydrogenases shows that 21-C3RDH/CD encodes a short-chain alcohol dehydrogenase. Furthermore, tissue specificity and molecular weight of the antigen suggest that 21-C3RDH/CD encodes the bovine retinal pigment epithelial 11-cis retinol dehydrogenase. Direct proof came from experiments in which we used the baculovirus-based expression system for in vitro synthesis of the protein encoded by 21-C3RDH/CD . Protein extracts obtained from recombinant baculovirus-infected insect cells were found capable of reducing 11-cis retinaldehyde. Invest Ophthalmol Vis Sci. 1995; 36:1988-1996.
Xn the past, retinal pigment epithelium (RPE) research has focused on elucidation of the function of the RPE in vision. These studies have shown that the RPE fulfills several important functions in this process, including phagocytosis of photoreceptor outer segments, metabolite exchange, and rhodopsin regeneration.1 2 The latter process involves reuse of endogenous retinoids. One of the retinoids, 11-cis retinaldehyde, serves as the chromophore for visual pigments present in the outer segments of rod and cone photo-
Frwm the * Institute of Ophthalmology and tlw.^ Department of Biochemistry, University of Nijmegen, The. Netherlands. Submitted for publication September 27, 1994; revised May I, 1995; accepted May 12, 1995. Proprietary interest categoiy: N. Helmut requests: Carola A. C. G. Driessen, Institute of Ophthalmology, University of Nijmegen, l>. 0. Box 9101, 6500 I IB Nijmegen, The Netherlands.
1988
receptor cells. Photon absorbance by 11-cis retinaldehyde and its subsequent isomerization to 3\\-trans retinaldehyde is the primary event in phototransduction. The RPE is responsible for the regeneration of 11-cis retinaldehyde. 3 On isomerization, zW-trans retinaldehyde is reduced to all-/raras retinol, which is bound to the interphotoreceptor retinoid binding protein and transported to the RPE. In the RPE, all-/rans retinol is isomerized to 11-cis retinol. The formation of 11-cis retinaldehyde from 11-cis retinol is catalyzed by an 11cis retinol specific dehydrogenase. The latter enzyme was reported to be an oxidoreductase that preferentially reacts with 11-cis retinol. 4 Finally, 11-cis retinaldehyde bound to interphotoreceptor retinoid binding protein is transported to the rod and cone outer segments to regenerate the visual pigments. Recently, retinol dehydrogenase was purified partially from bovine
Investigative Ophthalmology & Visual Science, September 199:5, Vol. 3f>, No. 10 Copyright © Association for Research in Vision and Ophthalmology
Retinol Dehydrogenase
RPE.' The RPE retinol dehydrogenase was reported to have an apparent molecular weight of 33 kd. Until today, the primary structure of retinol dehydrogenase has been unknown. To characterize RPE proteins, we recently isolated a broad panel of monoclonal antibodies (mAbs) recognizing different RPE antigens.'3 Monoclonal antibodies against five types of antigens were identified. One of these, 21-C3/AV, recognizes an antigen with an apparent molecular weight of 32 kd. Immunohistochemical analysis shows that 21-C3/AV exhibits strong reactivity with RPE. The mAb 21-C3/AV was used to screen a bovine RPE cDNA library. In this article, we report on the isolation of a cDNA encoding a 32-kd RPE protein. The deduced amino acid sequence reveals significant homology with the amino acid sequence of members of the superfamily of short-chain alcohol dehydrogenases. The largest homology was found with the human D-beta-hydroxybutyrate dehydrogenase precursor protein.7 MATERIALS AND METHODS All experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The monoclonal antibody used in this study was isolated and characterized as described.6 RNA Isolation and cDNA Library Construction Bovine eyes were obtained from a local slaughterhouse. Within 3 hours of eye enucleation, the cornea, vitreous, and retina were removed. Eyecups were rinsed in 15 mM phosphate-buffered saline, pH 7.4, containing 0.25 M sucrose and 2 mM ethylenediaminetetraacetic acid (EDTA). Retinal pigment epithelial cells were removed by careful brushing in the same buffer from which EDTA was omitted. Total cellular RPE RNA was prepared by a LiCl-urea method.8 Poly(A+) RNA was isolated using a biotinylated oligo(dT) primer (Promega, Madison, WI). An expression cDNA library was constructed using 5 fj,g of poly(A+) RNA and the \ZAP Express vector in accordance with the manufacturer's instructions (Stratagene, La Jolla, CA). Plated on Escherichia coli XL1Blue, a library consisting of 8.7 X 105 recombinant clones was obtained. Immunoscreening of a Retinal Pigment Epithelial cDNA Library The cDNA library was plated on E. coli XLl-Blue. Plates were incubated for 3 % hours at 42°C. Immunoscreening was performed as described by Sambrook et al9 with minor adjustments. In brief, nitrocellulose filters impregnated with 10 mM isopropyl-l-thio-/3-Dgalactopyranoside were used to induce expression of hybrid proteins (3 hours at 37°C). To obtain duplicate
1989
filters, this procedure was repeated once. After blocking in radioimmunoassay buffer (0.3% gelatine, 0.9% NaCl, 10 mM Tris; pH 7.5, 1% Triton X-100, 0.5% desoxycholate, 0.1 mM PMSF) for 30 minutes, the filters were incubated with I2>I labeled mAb 21C3/AV in the same buffer (12 hours at room temperature). Affinity-purified mAb was radiolabeled using chloramine T and Na125I to a specific activity of 3.2 /iCi///g.10 The filters were subjected to autoradiography using an Amersham Hyperfilm-MP (Amersham, Buckinghamshire, UK) at — 70°C with two intensifying screens. Positive clones were excised in vivo by use of the ExAssist helper phage. Subclones were generated in the phagemid vector pBK-CMV (Stratagene). DNA Sequencing Sequencing was performed by the dideoxy chain termination method" using Sequenase (US Biochemical, Cleveland, OH). DNA sequences were determined in both directions. Universal T3 and T7 primers were used, as were several specific internal sequencing primers (Pharmacia, Woerden, The Netherlands). Computer analysis of nucleotide sequences and alignment of deduced amino acid sequences were accomplished using the University of Wisconsin Genetics Computer Group Software (version 7.3.1). Northern Blot Hybridization Total RNA was isolated from nonocular tissues (heart, liver, spleen, lung, kidney, small intestine) and ocular tissues (lens capsule, RPE, iris, retina, cornea epithelium, ciliary body). RNA samples were prepared by the LiCl-urea method.8 Poly(A+) RNA from kidney and RPE was isolated as described above. Per sample, 10 /ig of total RNA or 2 //g poly(A+) RNA was electrophoresed on a 0.9% agarose-formaldehyde gel and transferred to 0.2 //m nitrocellulose (Schleicher and Schuell, Dassel, Germany). The 1210 bp EcoRI/XhoIinsert of 21-C3RDH/CD was labeled with 32P by a random priming method12 and used as a probe to hybridize the blot in 1% bovine serum albumin, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA, and 0.5 M NaP; pH 7.2, overnight at 60°C. After hybridization, the blot was washed in a solution containing 0.5% SDS, 1 mM EDTA, and 50 mM NaPi pH 7.2, at 60°C for 30 minutes. Autoradiography was accomplished by exposure to a Kodak X-Omat film at —80°C using two intensifying screens (Kodak, Odijk, The Netherlands). To verify the amount and integrity of the RNA on the filters, the same filters were hybridized with a mouse actin probe or an rRNA probe. Construction of pAcJAC3/RDH To use the baculovirus-based in vitro expression system for in vitro expression of the protein encoded by 21-C3RDH/CD, we constructed the transfer vector
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Investigative Ophthalmology & Visual Science, September 1995, Vol. 36, No. 10
pAcJAC3/RDH. In brief, a multiple cloning site containing sequences recognized by the restriction enzymes BamH I, Nco I, Kpn I, Bgl II, Stu I, Not I, Nhe I, and Spe I was engineered into the BamH I position of the baculovirus transfer vector pAcDZlIH using two complementary DNA oligonucleotides 5'-GATCCCCATGGTACCAGATCTAGGCCTGCGGCCGCTAGCACTAGT-3' and 5'-GATCACTAGTGCTAGCGGCCGCAGGCCTAGATCTGGTACCATGGG-3'. The resultant transfer vector was designated pAcJAC3. The 21-C3RDH/CD cDNA fragment was cloned into pAcJAC3 as a BamH I Not I DNA-fragment to generate pAcJAC3/RDH. Both sites are derived from the multiple cloning site of the pBK-CMV phagmid vector and flank the cDNA fragment cloned into the Xho I and EcoR I sites of the pBK-CMV multiple cloning site (see above). Transcription of the inserted cDNA in pAcJAC3/RDH is controlled by the baculovirus-derived polyhedrin promoter. We used pAcJAC3/RDH to isolate the recombinant AcNPV (Autographa californica nuclear polyhedrosis virus) clone designated AcNPV.RDH as described previously.14
Cell Culture Baculovirus propagation, transfection, isolation of recombinant virus and Sf9 insect cell culture were performed as described.15 Cells were cultured at 27°C in TNMFH medium supplemented with 10% (vol/vol) fetal calf serum, 0.1% (wt/vol) pluronic F-68, 50 fig/ ml streptomycin, and 50 U/ml penicillin. Suspension cultures of 150 ml each were used for 21-C3/RDH biosynthesis. Viral infections were performed using 15 ml of a diluted virus stock solution to give a multiplicity of infection of 5. Cells used for both Western blot analysis and measurement of 1 l-cis retinol dehydrogenase activity were harvested 3 days after infection. Immunoblot Analysis Proteins were separated on a 12.5% SDS-polyacrylamide gel,"' transferred to nitrocellulose (Schleicher and Schuell BA85, pore size 85), and probed with the mAb 21-C3/AV. The mAb 21-C3/AV has been described6 and was used in a dilution of 1:1000. Specifically bound antibodies were detected through an incubation with rabbit anti-mouse immunoglobulin G conjugated to peroxidase (1:500) and a subsequent incubation with the peroxidase substrate 4-chloro-lnaphthol (0.3% in phosphate-buffered saline, 5% methanol, 0.01% H2O2). Assay for 1 l-ci*-retinol Dehydrogenase Activity At 3 days after infection, cells were harvested by centrifugation (4000g 10 minutes, 4°C). The 11-ds retinol dehydrogenase activity was measured from whole cell extracts. Briefly, cell pellets were resuspended in extraction buffer
(5 mM Tris HC1,10 mM reduced /3-nicotinamide-adenine dinucleotide (NADH), 20% glycerol, 0.15 M NaCl, and 20 mM dodecyl-/?-l-maltoside (DoM)) at a cell density of 20 X lO15 Sf9 cells per milliliter. Glycerol and NADH were added to the extraction buffer to stabilize the expressed 11-ds retinol dehydrogenase.' Samples were incubated for 30 minutes at 4°C and centrifuged (100,000g; 30 minutes, 4°C). All subsequent manipulations were performed under dim red light (Schott-Jena [Mainz, Germany], RG 645 cut-off filter). We used 50-/il aliquots, 1 X 10" infected cells, of the supernatant to mix with 50 \A of the 1 1-ds retinol dehydrogenase substrate solution (0.2 M acetate buffer, 2.5% methanol, 1.0 mM NADH, 10 nmol 11-ds retinaldehyde, pH 5.0). Under these conditions, 11-ds retinol dehydrogenase reduces 11-ds retinaldehyde to l i ds retinol. Samples were incubated at 37°C. At 1-, 5-, and 10-minute intervals, 25-//1 samples were taken. The 11-ds retinaldehyde decrease was measured through the addition of 150 fi\ thiobarbituric and 150 fA thiourea according to Futterman and Saslow.17 Samples were incubated for 30 minutes at 25°C and centrifuged (10,000g 5 minutes, 25°C). Color development was measured at 530 nm. For a positive control, we used bovine RPE cells isolated as described. 6 Whole RPE cell extracts were prepared as described above (40 X 106 cells/ml). For a negative control, we used Sf9 cells infected with wildtype AcNPV.
RESULTS Isolation of cDNA Clones With 21-C3/AV We successfully generated a bovine RPE cDNA library consisting of 8.7 X 105 independent clones. To determine the level of contaminating-retina, mRNA-derived cDNA clones, we used a bovine opsin cDNA probe to screen 100,000 recombinant clones of this RPE-specific \ZAP Express cDNA library. The complete 3' untranslated region of the opsin gene and a small part of the coding region were encoded by this probe. Hybridization showed that only a small percentage (0.1%) of the clones screened are actually opsin cDNA clones. Hence, the mRNA samples used to construct this library were minimally contaminated with mRNA derived from the neurosensory retina. Having tested the specificity of the library, we used the iodinated mAb 21-C3/AV for expression screening of clones from the amplified library. After two rounds of screening, 16 positive cDNA clones were selected. From each of these clones, the phagemid pBK-CMV was rescued by in vivo excision. Plasmid DNA was isolated and used in Southern blot experiments. These experiments showed that 13 of the 16 clones cross-hybridized. Four
Retinol Dehydrogenase
1991
1 34 10S 1 159 19 213 37
GAAGACCTGGACCACAAATTTCTCGAGCAAAAC AAAAGGAACAGCTTCAGGTGCTATAGTACCTGCCAACAGCTTTCCCCTGAGGAGCTCACCTGGGCTCCAGC ATG GTG GCT GCC TCT GCT GCT GGG TGT CTT GCT CTG GGC AGC ACT GTG GTT GCT Met Val Ala Ala Ser Ala Ala Gly Cys Leu Ala Leu Gly Ser Thr Val Val Ala GAG GGA CCG GCA GTG CCT GGC CAG CCA GCG ATG GCC TTT ATC TTC ATC ACC GGC Gin Gly Pro Ala Val Pro Gty Gin Pro Ala Net Ala Phe lie Phe lie Thr Gly TGT GAC TCG GGC TTT GGG CGG CTC CTT GCT CTG AGG CTG GAC CAG AGA GGC TTC Cys Asp Ser Gly Phe Gly Arg Leu Leu Ala Leu Arg Leu Asp Gin Arg Gly Phe
267 55 321 73 375 91 429 109 483 127 537 US 591 163 645 181 699 199 753 217
CGA Arg GCC Ala CGG Arg CTG Leu CGG Arg ACC Thr ACC Thr TTT Phe GTA Val GAA Glu
GTA CTG Val Leu TCC TCC Ser Ser CAG GCA Gin Ala GTG AAT Val Asn GAG GAC Glu Asp CTC GCC Leu Ala AGT GTC Ser Val GGC CTG Gly Leu CGG GTC Arg Val ACT TTG Thr Leu
GCC AGC Ala Ser CGC CTC Arg Leu GTC AAG Val Lys AAT GCT Asn Ala TTC CAG Phe Gin CTG CTG Leu Leu CTT GGC Leu Gly GAG GCC Glu Ala TCT ATC Ser lie GAG GAC Glu Asp
TGC Cys CAC His TGG Trp GGT Gly CGG Arg CCC Pro CGT Arg TTC Phe GTG Val ACC Thr
CTG ACA CCC Leu Thr Pro ACC ACC CTG Thr Thr Leu GTG GAA ACG Val Glu Thr GTG GCT GGC Val Ala Gly GTG CTG AAT Val Leu Asn CTG CTG CTG Leu Leu Leu CTG GCA GCC Leu Ala Ala TCT GAC AGC Ser Asp Ser GAA CCT GGC Glu Pro Gly CTG CAG GCT Leu Gin Ala
TCG GGG Ser Gly CTG GAT Leu Asp CAT GTT His Val ATC ATT lie H e GTG AAC Val Asn CAG GCC Gin Ala AAT GGA Asn Gly CTG AGG Leu Arg TTC TTC Phe Phe TGC TGG Cys Trp
GCG Ala GTC Val GGG Gly GGT Gly ACG Thr CGG Arg GGG Gly CGA Arg CGA Arg GCA Ala
GAG Glu ACA Thr GAA Glu CCC Pro CTG Leu GGC Gly GGC Gly GAT Asp ACC Thr CGG Arg
GAC Asp GAT Asp GCA Ala ACC Thr GGT Gly CGA Arg TAC Tyr GTG Val CCT Pro CTG Leu
CTC Leu CCC Pro GGG Gly CCA Pro CCC Pro GTG Val TGC Cys GCT Ala GTG Val CCT Pro
CAG Gin CAG Gin CTT Leu TGG Trp ATC He ATC He GTC Val CCT Pro ACA Thr CCA Pro
CGG Arg AGC Ser TTT Phe CAG Gin GGG Gly AAC Asn TCC Ser TTT Phe AAC Asn GCC Ala
GTC Val ATC lie GGT Gly ACG Thr GTC Val ATC lie AAG Lys GGG Gly CTG Leu ACA Thr
found from the first ATG start codon at position 105 to the stop codon TAA at position 1062. The first ATG codon is situated in a good context for translation initiation according to Kozak's rules.1" '' An AATAAA polyadenylation signal is found 16 nucleotides upstream from the poly (A) tail. From the nucleotide sequence of 21-C3RDH/CD, we deduced the amino acid sequence, designated 21C3RDH, of the encoded polypeptide (Fig. 1). This conceptual translation yielded a protein sequence consisting of 319 amino acid residues. The cumulative molecular weight of the amino acid sequence was 34,301 daltons, which is in good agreement with the previously published apparent molecular weight of the antigen recognized by 21-C3/AV.(> On the N-terminus of 21-C3RDH, no signal peptide sequence was detected.
Homology With Human D-beta-hydroxybutyrate Dehydrogenase Precursor Using both the nucleotide sequence and the deduced 807 CAG GCC CTC TAT GGG GAG GCC TTC CTC ACC AAA TAC CTG AGA GTG CAG CAA CGT amino acid sequence, we searched for homologous 235 Gin Ala Leu Tyr Gly Glu Ala Phe Leu Thr Lys Tyr Leu Arg Val Gin Gin Arg sequences in the EMBL (release 39.0), PIR (release 861 ATC ATG AAC ATG ATC TGT GAT CCG GAC CTG GCC AAG GTG AGC AGG TGC CTG GAG 253 H e Met Asn Met lie Cys Asp Pro Asp Leu Ala Lys Val Ser Arg Cys Leu Glu 29.0), and SWISS-PROT (release 41.0) data bases us915 CAT GCC CTA ACT GCC CGT CAC CCC AGA ACC CGC TAC AGC CCA GGC TGG GAT GCC ing the FASTA program.20 The DNA sequence was not 271 His Ala Leu Thr Ala Arg His Pro Arg Thr Arg Tyr Ser Pro Gly Trp Asp Ala homologous to any nucleotide sequence in the data 969 AAG CTG CTC TGG TTG CCA GCC TCC TAC TTG CCA GCC AGG CTG GTG GAT GCT GTG 289 Lys Leu Leu Trp Leu Pro Ala Ser Tyr Leu Pro Ala Arg Leu Val Asp Ala Val base. The amino acid sequence showed 39% identity 1023 CTC GCC TGG GTC CTT CCC AAG CCT GCC CAG ACA GTC TAC TAA ATCCAGCCCTCCAGC in 287 amino acid residues with human D-beta-hydro307 Leu Ala Trp Val Leu Pro Lys Pro Ala Gin Thr Val Tyr . xybutyrate dehydrogenase precursor7 and 33% iden1080 AAAAGATGGTTGTTCAAGGCAAGGACTCTGATTTATTCTGTCCCCTGACCCTGGTACTGCCTGGTGTGTGG 1151 CATAAAACAGTCACTCAATAAATGTATTATTCAAAACAAAAAAAAAAAAAA tity in 210 amino acid residues with human placental 17-beta-hydroxysteroid dehydrogenase21 (Fig. 2). FIGURE l. The nucleotide sequence of the 21-C3RDH/CD cDNA {upper) and deduced amino acid sequence {lower) When conserved amino acid substitutions are taken into consideration, the degree of homology with huof bovine 21-C3RDH. The initiator codon (ATG) and the man D-beta-hydroxybutyrate dehydrogenase precurtermination codon (TAA) are indicated by circles. Undersor increases to 72%. lined nucleotides in the 3'-untranslated region represent a possible polyadenylation signal. Nucleotide and amino acid sequences are numbered on the left. Bold numbers were used for the amino acid sequence. This sequence is deposited in GenBank under the accession number L36533.
clones were found to contain a 1.2-kb insert. The apparent molecular weight of the antigen recognized by 21-C3/AV is 32 kd. Therefore, assuming that the antigen was not glycosylated, we expected a full-length cDNA to have a minimal length of 1.0 kb. Because the lengths of the largest inserts were equivalent in all four clones and, judging from their length, could contain the entire coding sequence, we used one of these clones, 21-C3RDH/CD, for sequence analysis. DNA and Deduced Protein Sequence of 21-C3RDH/CD The complete nucleotide sequence of the cDNA 21C3RDH/CD is shown in Figure 1. Sequence analysis showed 21-C3RDH/CD to contain a 1210-bp cDNA insert. An open reading frame consisting of 957 bp is
Secondary Structure Predictions The hydropathy profile of 21-C3RDH does not indicate hydrophobic stretches sufficiently long to span a phospholipid bilayer (Fig. 3). However, amino acid residues in the region 130 to 200 displayed significant hydrophobic features. A similar hydrophilicity profile pattern was obtained using an algorithm for antigenicity (result not shown). Northern Blot Analysis Northern blot analysis was used to determine the size and tissue expression pattern of 21-C3RDH mRNA. A Northern blot containing 10 //g total RNA from bovine RPE, heart, liver, spleen, lung, kidney, and small intestine was probed with the cDNA 21-C3RDH/CD. A single transcript of approximately 1.2 kb was detected in RPE (Fig. 4, panel A). No transcripts were detected in other tissues even after long exposure. Because immunohistochemical analysis showed that 21-C3/AV reacts with smooth muscle tissue in the
1992
Investigative Ophthalmology & Visual Science, September 1995, Vol 36, No. 10
bovine kidney cortex, we performed Northern blot analysis with 2 //g poly (A') RNA from bovine RPE and kidney tissue. No transcript in kidney was detected (Fig. 4, panel B). To determine the expression pattern of 2110 bdh hu
20
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FIGURE 3. Hydropathy analysis of 21-O3RDH. The predicted amino acid sequence of 21-C3RDH was analyzed according to Kyte and Doolittle 1 ' using a window of nine residues.
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C3RDH mRNA within the eye, we also hybridized a Northern blot containing 10 //g total RNA from bovine lens capsule, RPE, iris, retina, cornea epithelium, and ciliary body. Again, only in the RPE was a single transcript detected (Fig. 4, panel C).
rdh bo EAGLFGLfflNAGVAGUGPTPWBTREDFQRVlNVMTLGPIGVTLALLPLLL-QARGRVIN ~
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bdh hu ISWLGRMA-NPARSPYCITKFGVEAFSDCLRYEMYPLGVKVSVVEPGNFIMTSLYN-ISS l.T.tTl.l . . . I I . . I I I . . M l . . M . . i. i i.Ti. I I I I i i.. i I M ' I I I ' I " • • I I • • II I • • II I • • I I " • I ' l l ' l l ' l l l l I I" I rdh_bo lTSVLGRU-AMG-GGYCVSKFGLQAFSNSLRADVAPFGVRySlVEPGFF--RTPVTN-dhbi h TGSWSLNGLPFN-DVYCASKFALEGLCESLAVLLLPFGVHLSLiECSPV--HTAFMEKV 150 160 170 180 190 200
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Solubillzation of the 21-C3/AV Antigen Retinal pigment epithelial cells were isolated, lysed, and homogenized in phosphate-buffered saline (Potter-Elvehjem tube) without detergent and in the pres-
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FIGURE 2. Alignment of 21-C3RDH (rdh bo) with human Dbeta-hydroxybutyrate dehydrogenase precursor ( b d h h u ) and human placenta! 17-beta-hydroxysteroid dehydrogenase ( d h b l h ) . The alignment was obtained using the FASTA program and the SWISS-PROT data base. Vertical bars denote matches, and colons denote substitutions. Residues in the short-chain alcohol dehydrogenases family that are conserved in at least 15 of 20 aligned sequences"' are underlined. The location of a residue that is usually an isoleucine in shortchain alcohol dehydrogenases is indicated by the asterisk. The location of absolutely conserved residues are denoted by vertical airows. Residue positions of b d h h u are indicated by higher numbers. Residue positions of dbhl h are indicated by lower numbers. For residue positions r d h b o see Figure 1.
t FIGURE 4. Northern blot analysis of 21-C3RDH. Panel A: Northern blot containing 10 /xg total RNA from retinal pigment epithelium (RPE) (1), heart (2), liver (3), spleen (4), lung (5), kidney (6), small intestine (7). Panel B: Northern blot containing 2 fig poly(A') RNA from kidney (1) and RPE (2). Panel C: Northern blot containing 10 j/g total RNA from lens capsule (1), RPE (2), iris (3), retina (4), cornea epithelium (5), ciliary body (6). As a control, filters were rehybridized to a mouse actin cDNA probe or a rRNA probe (lanes beneath each panel).
Retinol Dehydrogenase
1993
1 2 3 94. 674330-
~ **
20FICURE 5. Immunoblot analysis of 21-C3/AV antigen isolated from bovine retinal pigment epithelial cells. The immunoblot was screened with 21-C3/AV. Soluble retinal pigment epithelial proteins (lane 1), proteins isolated from the bovine retinal pigment epithelium in the presence of 1% Triton X-100 (lane 2) and in the presence of 1% DoM (lane 3). ence of 1% Triton X-100 or 1% DoM. Homogenized cells were centrifuged (SOfiOOg, 20 minutes, 4°C), and the supernatant was analyzed for the presence of the 21-C3/AV antigen by immunoblotting (Fig. 5). No antigen was detected in protein samples derived from RPE cell homogenates without the use of detergents (Fig. 5, lane 1). However, in the samples obtained with both types of detergent, the antigen was clearly detectable (Fig. 5, lanes 2 and 3). Additional experiments showed that the antigen recognized by 21-C3/ AV accumulates in the microsome fraction if subcellular fractions are prepared from the RPE (data not shown). Expression of Recombinant 21-C3RDH in Sf9 Cells The entire 21-C3RDH/CD cDNA coding sequence was inserted downstream from the baculovirus polyhedrin promoter in pAcJAC3, yielding the baculovirus transfer vector pAcJAC3/RDH. Cotransfection of Sf9 cells with pAcJAC3/RDH and wild-type AcNPV DNA yielded a recombinant AcNPV clone that expressed the protein encoded by 21-C3RDH/CD (Fig. 6). Protein samples derived from recombinant virus-infected Sf9 cells were analyzed by immunoblot, using the mAb 21-C3/AV (Fig. 6, panel A). The immunoblot revealed a protein band of approximately 32 kd (lane 3), which was not present in mock-infected cells (lane 1) or wildtype AcNPV infected cells (lane 2). This band comigrated with the 21-C3/AV antigen recognized in a Triton X-100 protein extract obtained from bovine RPE cells (lane 4). Functional Analysis of 21-C3RDH/CD Encoded 1 l-cis Retinol Dehydrogenase An assay to investigate the 11-eis retinol dehydrogenase activity of 21-C3RDH was performed using pro-
tein samples obtained from AcNPV.RDH infected Sf9 cells (3 days after infection) (Fig. 6, panel B). Retinol dehydrogenase activity was assayed by measuring the initial velocity of 1 l-cis retinaldehyde reduction to 11cis retinol in the presence of NADH at pH 5.0. The decrease of the 1 l-cis retinaldehyde was measured as a function of time over a 10-minute period. Whole cell extracts derived from 1 X 10(i AcNPV.RDH-infected cells reduced 7.0 nmol 1 l-cis retinal to 1 l-cis retinol within 5 minutes (Fig. 6, panel B). For a positive control, 2 X 10b RPE cells were found to reduce 4.7 nmol 1 l-cis retinaldehyde to 1 l-cis retinol within the same period. For a negative control, wild-type AcNPV-infected cells were used. In these assays, an initial velocity of only 0.7 nmol 1 l-cis retinaldehyde for 5 minutes was measured. DISCUSSION In our laboratory, two animal models were developed to resemble acute anterior uveitis and sympathetic ophthalmia in humans. Retinal pigment epithelial protein fractions were responsible for the ocular pathologic processes.22'23 To characterize these uveitogenic proteins further, we isolated a broad panel of mAb-recognizing RPE antigens. One of these mAbs
1 2
3 4
94. 67433020-
FIGURE 6.
Immunoblot and functional analysis of 21-C3RDH protein expression by recombinant baculovirus-infected Sf9 cells. Panel A; Immunoblot blot screened with 21-C3/AV. Sf9-derived cellular proteins (lane 1), wild-type AcNPV-infected Sf9 cells (lane 2), AcNPV.RDH-infected Sf9 cells expressing 11-os retinol dehydrogenase (lane 3), and native bovine retinal pigment epithelium-derived 1 l-cis retinol dehydrogenase (lane 4). Panel B: 1 l-cis retinaldehyde reduction by native bovine retinal pigment epithelial retinol dehydrogenase (open circles, 2 X 106 retinal pigment epithelial cells/reaction mixture), wild-type AcNPV-infected Sf9 cells (closed triangles, 1 X 10b cells/reaction mixture), and AcNPV.RDH-infected Sf9 cells (open triangles, 1 X 10ti cells/ reaction mixture).
1994
Investigative Ophthalmology & Visual Science, September 1995, Vol. 36, No. 10
was used to screen a bovine RPE cDNA library. Antibody selection resulted in 16 independently isolated clones. From these 16 clones, 13 were found to encode the same protein. Hence, the immunoscreening procedure proved to be highly selective for clones encoding the 21-C3/AV antigen. The cDNA sequence and deduced amino acid sequence of 21-C3RDH/CD were used to search for homologous sequences in the EMBL, PIR, and SWISS-PROT data libraries. Homology was found among a number of proteins thought to be members of the short-chain, nonmetalloenzyme dehydrogenase family. Members of the short-chain alcohol dehydrogenase family are composed of approximately 250 to 300 amino acid residues and do not contain catalytic metal atoms.24 The initially termed long-chain alcohol dehydrogenases, or classical alcohol dehydrogenases, are between 350 and 375 amino acid residues long. Numerous members of this family use zinc as a catalytic atom. 25 A second characteristic feature of the members of the classical dehydrogenase family is the location of the catalytic domain near the N-terminus. The coenzyme-binding domain is located in the C-terminal part of the protein. This is reversed in members of the short-chain alcohol dehydrogenase family. Here, the nucleotide-binding domain is close to the N-terminus, and the catalytic domain is found in the C-terminal part of the protein. No reaction mechanism is known for any member of the shortchain alcohol dehydrogenase family. An alignment of 21-C3RDH with human D-betahydroxybutyrate dehydrogenase precursor 7 yielded the highest homology score. The latter enzyme is located at the inner face of the mitochondrial inner membrane. The D-beta-hydroxybutyrate dehydrogenase precursor protein (34.5 kd) is synthesized on free polysomes and posttranslationally imported into the mitochondria. 26 The first 46 amino acid residues of the precursor protein function as a leader peptide. Based on the consensus cleavage site Arg-X-Tyrl (Ser/ Ala) previously reported for mitochondrial proteins, 27 the precursor protein is most likely cleaved between Tyr-46 and Ala-47 (for numbering, see Fig. 2). The leader sequence is cleaved by a matrix protease yielding a 31.5-kd protein. D-beta-hydroxybutyrate dehydrogenase catalyses the oxidation of D-beta-hydroxybutyrate and the reduction of acetoacetate. In a pairwise alignment, the members of the shortchain alcohol dehydrogenase family are approximately 25% identical to one another. 24 This is in good agreement with the homology existing between 21C3RDH and the human D-beta-hydroxybutyrate dehydrogenase, which is found to be 39% identical. The homology observed with human placental 17-beta-hydroxysteroid dehydrogenase 21 was 33%. The latter alignment was used to compare the 21-C3RDH amino acid sequence to the aligned sequences of 20 members
of the short-chain alcohol dehydrogenases, which included this human placental dehydrogenase. 24 Sequence alignment between the members of the shortchain alcohol dehydrogenase family revealed 6 strictly conserved amino acid residues and 19 conserved residues in at least 15 of 20 aligned sequences. In 21C3RDH all these strictly conserved amino acids (Gly36, Gly-42, Asp-83, Gly-157, Tyr-176, and Lys-180; for numbering see Fig. 1) are present. Of the highly conserved amino acid residues, 16 of 19 also are present in 21-C3/RDH, including residues Gly-40 and Ser-164. The coenzyme-binding site was assigned to the N-terminal site of the short-chain alcohol dehydrogenases.28 The glycine residues located at the N-terminus of 21-C3RDH (Gly-36, Gly-40 and Gly-42) are highly conserved in short-chain alcohol dehydrogenases. The spacing of these residues resembles the spacing expected for glycine residues that are part of the nicotinamide-adenine dinucleotide coenzyme-binding domain of alcohol dehydrogenases. 28 ' 29 Three putative active site residues—Ser-164, Tyr-176, and Lys-180— are conserved in 21-C3RDH. Hence, the organization of the coenzyme-binding domain and the active site domain of 21-C3RDH is characteristic for members of this superfamily of alcohol dehydrogenases. These observations suggest that 21-C3RDH encodes ll-cis retinol dehydrogenase. The most recent report on a RPE retinol dehydrogenase was by Suzuki and coworkers.5 They reported on the isolation of 11-ds retinol dehydrogenase from bovine RPE. A partially purified sample containing two proteins was obtained. The apparent molecular weights of these two proteins were 33 kd and 66 kd. Further analysis showed that the 33-kd protein exhibited retinol dehydrogenase activity. The antigen, which is recognized by the mAb 21-C3/AV, has an apparent molecular weight of 32 kd.b Hence, the molecular weight of retinol dehydrogenase and the 21C3/AV antigen are comparable, and both agree with the cumulative molecular weight of the protein encoded by 21-C3RDH/CD. Additionally, the 21-C3/AV antigen can be solubilized only with the help of detergents. Previous studies30 have shown that the ll-cis retinol dehydrogenase is associated with the microsomal membranes. Northern-blot analysis with 10 fig total RNA from different ocular and nonocular tissues showed that the 21-C3RDH gene is only transcribed in the RPE. No transcripts were detected in any of the tissues tested. Subsequently, we performed Northern blot analysis with 2 fig poly(A+) RNA from both RPE and kidney tissue. Although immunohistochemical analysis showed that 21-C3/AV reacts with smooth muscle tissue in the bovine kidney cortex,*' we were unable to detect any transcripts in kidney mRNA. However, 21C3/AV exhibited reactivity not only with RPE but also
Retinol Dehydrogenase
with the striated eye muscle and smooth muscle tissue of small arteries.0 Probably these reactions are caused by cross-reacting antigens, most likely other dehydrogenases. For example, mitochondria are abundant organelles in striated muscle tissue. Hence, a cross-reaction can occur with D-beta-hydroxybutyrate dehydrogenase if 21-C3/AV recognizes an identical epitope on both enzymes. In the alignment between both proteins, there are sufficiently long identical sequences to support this idea. Interestingly, the human D-betahydroxybutyrate gene transcript was present in all forms of muscle tissue, including smooth muscle tissue.7 In addition to muscle tissue, 21-C3/AV stains several ocular epithelial cells.6 Epithelial tissues are known to convert retinol to retinoic acid,31 including different types of ocular epithelial cells.32 Vertebrate alcohol dehydrogenases classes I and IV are expressed in these epithelial tissues. Probably 21-C3/AV also recognizes an epitope on these enzymes. All the evidence that 21-C3RDH/CD encodes one of the two key enzymes of the visual cycle has been indirect. Therefore, we used 21-C3RDH/CD for in vitro expression of the protein it encodes. Recently, biosynthesis of two dehydrogenases using the baculovirus-based expression system were published.3334 Hence, 21-C3RDH/CD was used to isolate recombinant AcNPV clones. One of these clones was used to infect Sf9 cells, and the expressed protein was assayed for 11-ds retinol dehydrogenase activity. Immunoblot analysis showed that a protein of 32 kd was produced by recombinant virus-infected cells that was recognized by 21-C3/AV. This protein was not detected in samples obtained from mock or wild-type AcNPV-infected cells. We used recombinant AcNPV-infected cells in 11-ds retinol dehydrogenase assays as described.45 These experiments showed that the protein encoded by 21-C3RDH/CD reduces 11-ds retinaldehyde. Based on observations of apparent molecular weight, homology with short-chain alcohol dehydrogenases, tissue specificity, and 11-cis retinol dehydrogenase activity measured from the expressed protein, we conclude that 21-C3RDH/CD encodes bovine 11-ds retinol dehydrogenase. Acknowledgments The authors thank Petra H. M. Bovee-Geurts (Department of Biochemistry, University of Nijmegen, The Netherlands) for her assistance with the retinol dehydrogenase assays. They also thank Dr. W. J. de Grip (Department of Biochemistry, University of Nijmegen) for helpful discussions and for providing 11-cis retinaldehyde and dodecyl-/?-l-maltoside.
NOTE ADDED IN PROOF While this manuscript was under review, Simon et al3(i reported on the cloning of bovine retinal pigment
1995
epithelial-specific 11-cis retinol dehydrogenase. The two cDNA sequences and their deduced protein sequences do not agree completely. The work by Simon et al describes a cDNA sequence encoding 318 amino acid residues, whereas we report on a protein sequence consisting of 319 amino acids. Furthermore, the first 29 amino acids do not match between the two sequences. This difference is caused by three additional guanines, present at positions 108, 177, and 192. In the sequence published by Simon et al, only one guanine is present at these positions. Both sequences align as a result of the last additional guanine. The presence of the first additional guanine immediately after the ATG start codon places the initiator codon in an optimum context for translation initiation.18 '"' The additional guanines also explain the difference in length between the two protein sequences. Key Words baculovirus, cDNA library, immunoscreening, monoclonal antibody, retinal pigment epithelium, retinol dehydrogenase References 1. Saari JC. Enzymes and proteins in the mammalian visual cycle. Prog Ret Res. 1990:360-381. 2. Bok D. The retinal pigment epithelium: A versatile partner in vision. / Cell Sri. 1993; 17:189-195. 3. Bernstein PS, Law WC, Rando RR. Isomerization of all-trans-retinoids to 11-cis-retinoids. Proc Natl Acad Sri USA. 1987;84:1849-1853. 4. Lion F, RotmansJP, Daemen FJM, Bonting SL. Stereospecificity of ocular retinol dehydrogenases and the visual cycle. Biochem Biophys Ada. 1975;384:283-292. 5. Suzuki Y, Ishiguro S, Tamai M. Identification and immunohistochemisty of retinol dehydrogenase from bovine retinal pigment epithelium. Biochem Biophys Ada. 1993; 1163:201-208. 6. Janssen JJM, Janssen BPM, Van Vugt AHM. Characterization of monoclonal antibodies recognizing retinal pigment epithelial antigens. Invest Ophthalmol Vis Sci. 1994;35:189-198. 7. Marks AR, MclntyreJO, Duncan TM, Erdjument—Bromage H, Tempst P, Fleischer S. Molecular cloning and characterization of (R)-3-hydroxybutyrate dehydrogenase from human heart. J Biol Chem. 1992; 267:15459-15463. 8. Auffray C, Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNA's from total myeloma tumor RNA. Eur J Biochem. 1980; 107:303-314. 9. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press; 1989:12.16-12. 10. Hunter WM, Greenwood FC. Preparation of iodine131 labelled human growth hormone of high specific activity. Nature. 1962; 194:495-496. 11. Sanger F, Nicklen S, Coulson A. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sd USA. 1977;74:5463-5467.
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Investigative Ophthalmology & Visual Science, September 1995, Vol. 36, No. 10
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