Primary Structure of Human 11-cisRetinol Dehydrogenase and Organization and Chromosomal Localization of the Corresponding Gene

GENOMICS 36, 424 –430 (1996)ARTICLE NO. 0487

Primary Structure of Human 11-cis Retinol Dehydrogenaseand Organization and Chromosomal Localization of

the Corresponding Gene

ANDRAS SIMON,* JACOB LAGERCRANTZ,† SVETLANA BAJALICA-LAGERCRANTZ,† AND ULF ERIKSSON*,1

*Ludwig Institute for Cancer Research, Stockholm Branch, Box 240, S-171 77 Stockholm, Sweden; and †Clinical Genetics Unit,Department of Molecular Medicine, Karolinska Hospital, S-171 76 Stockholm, Sweden

Received December 13, 1995; accepted July 4, 1996

ceptor cells to all-trans retinol (Bliss, 1948; Wald andThe universal chromophore of visual pigments in Hubbard, 1949).

higher animals is 11-cis retinaldehyde. The final step 11-cis Retinaldehyde is generated in the retinal pig-in the biosynthetic pathway generating this compound ment epithelium (RPE)2 (Das and Gouras, 1988). All-is catalyzed by 11-cis retinol dehydrogenase, a mem- trans retinol, the ultimate precursor in this process, isbrane-bound enzyme abundantly expressed in the reti- accumulated by the RPE from the photoreceptor cellsnal pigment epithelium of the eye. In this work we following light exposure of the visual pigments anddemonstrate that the primary structure of human 11- from plasma retinol-binding protein present in the gen-cis retinol dehydrogenase is highly conserved with eral circulation. Following the isomerization of all-91% identity to the bovine enzyme. The gene encoding

trans retinol into 11-cis retinol via an all-trans reti-11-cis retinol dehydrogenase spans over É4.1 kb ofnylester intermediate (Bernstein and Rando, 1986;DNA and is divided into four translated exons. Analy-Bridges and Alvarez, 1988), 11-cis retinol is oxidizedsis of a panel of somatic cells hybrids and fluorescenceinto 11-cis retinaldehyde. This last metabolic step isin situ hybridization on metaphase chromosomes re-catalyzed by the stereo-specific enzyme 11-cis retinolvealed that the gene is located on chromosome 12q13–dehydrogenase (11-cis RDH) (Lion et al., 1975), in aq14. Due to the unique role of 11-cis retinol dehydroge-reaction requiring NAD/ as the cofactor. The 11-cisnase in the generation of visual pigments, it is a candi-RDH is a 32-kDa membrane-bound enzyme abundantlydate gene for involvement in hereditary eye disease.expressed by the RPE (Simon et al., 1995). This enzymeq 1996 Academic Press, Inc.

is the first identified retinol-metabolizing enzyme be-longing to the family of short-chain alcohol dehydroge-nases/reductases (SDR), a large family of enzymes thatINTRODUCTIONmetabolize a variety of substrates including steroids,prostaglandins, and fatty acids (Jornvall et al., 1995).Vitamin A derivatives play a central role in the biol-Recently, three additional retinol dehydrogenasesogy of the eye and particularly in the visual processesclosely related to 11-cis RDH have been identified (Chai(for a recent review see Saari, 1994). The universalet al., 1995a,b, 1996).chromophore of all visual pigments in higher animals

It is well known that inability to synthesize func-is 11-cis retinaldehyde, a metabolite of all-trans retinoltional visual pigments is a common cause of hereditary(vitamin A alcohol). In the photoreceptor cells of theretinal degeneration, a group of diseases collectivelyeye, 11-cis retinaldehyde is covalently complexed to theknown as retinitis pigmentosa (for a review see Berson,opsins, the protein moieties of the visual pigments.1993). The most common forms of autosomal dominantUpon light exposure, 11-cis retinaldehyde undergoesretinitis pigmentosa affect rhodopsin, the protein moi-an 11-cis to all-trans isomerization, and this change inety of the rod visual pigments. Much less is knownmolecular structure of the visual pigments initiates awith regard to the role of 11-cis retinaldehyde in theseries of events in the photoreceptor cells that eventu-biogenesis of functional visual pigments and the possi-ally lead to visual perception (Wald, 1968). All-transbility that failure to synthesize this compound may beretinaldehyde is subsequently reduced in the photore-involved in hereditary retinal degeneration.

The nucleotide sequence of the cDNA encoding human 11-cis reti-nol dehydrogenase has been deposited with the GenBank/EBI Data 2 Abbreviations used: RPE, retinal pigment epithelium; bp, base-

pair(s); DAPI, 4,6-diamino-2-phenyl-indole; HSD, hydroxysteroid de-Bank under the Accession No. U43559.1 To whom correspondence should be addressed. Telephone: /46-8-728 hydrogenase; nt, nucleotide(s); 11-cis RDH, 11-cis retinol dehydroge-

nase; SDR(s), short-chain alcohol dehydrogenase(s)/reductase(s).7109. Fax: /46-8-332812. E-mail: [email protected].

4240888-7543/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

AID Genom 4297 / 6r1c$$$361 08-26-96 23:47:27 gnma AP: Genomics

HUMAN 11-cis RETINOL DEHYDROGENASE GENE 425

FIG. 1. Primary structure of human 11-cis retinol dehydrogenase and mapping of the 5* untranslated region of the correspondingmRNA. (A) Alignment of the deduced amino acid sequence of human 11-cis RDH (h 11-cis RDH) and bovine 11-cis RDH (b 11-cis RDH;Simon et al., 1995). Amino acid residues that differ between the two enzymes are boxed. Amino acid numbering is to the right. (B) Mappingthe length of the 5* untranslated region in the mRNA encoding human 11-cis RDH by primer extension analysis using total human retinaRNA and tRNA from yeast (negative control) as the templates. A major band corresponding to a 5* untranslated region of 226 nt is visualized.

RPE cells, were carefully dissected out and total retina RNA wasTo elucidate the possible involvement of 11-cis RDHprepared using standard techniques (Chirgwin et al., 1979). A 30-in human eye disease, we have isolated and character-mer oligonucleotide 5*-AGCTGGAGCCCAAGTGACCTACAGGTG-ized the human gene encoding 11-cis RDH. The results GCA (reverse) corresponding to nucleotides 031 to 01 upstream of

showed that the gene maps to chromosome 12q13–q14, the initiation codon was labeled with [32P]ATP (Amersham Inc.) us-ing T4 polynucleotide kinase. The labeled oligonucleotide (4 1 105a locus with no previous association to human eye dis-cpm) was annealed to 40 mg of total retina RNA or to 40 mg of tRNAease.from yeast (negative control). The details of the procedure includingthe primer extension reaction with reverse transcriptase was per-

MATERIALS AND METHODS formed as recommended (Ausubel et al., 1992). The reactions wereanalyzed on a sequencing gel and the length of the formed products

Isolation of cDNA and genomic clones encoding human 11-cis RDH. was determined by a comparison with a normal sequence reactionA human eye cDNA library cloned in the lgt11 vector (Clontech Inc.) run in parallel. A phosphoimager was used to visualize the resultswas screened using bovine cDNA as the probe (Simon et al., 1995). (Fuji Bas 2000).Approximately 21 105 plaque-forming units were screened and three

The genomic organization of the gene encoding human 11-cis RDH.independent clones were isolated. The inserts of the clones wereThe exon–intron structure of the 11-cis RDH gene was establishedsubcloned into pBluescript (Stratagene). The inserts in two of theby nucleotide sequence analysis of overlapping DNA fragments ob-clones were not full length while a third clone, termed he1, containedtained from standard PCRs using the isolated genomic l clones (100a 1.6-kb insert and was selected for further characterization. Theng/reaction) as the templates. Different combinations of primers de-nucleotide sequence of the insert in clone he1 was established byrived from the nucleotide sequence of the human cDNA were used.sequence analysis of both stands (Sequenase 2.0, U. S. Biochemicals).Following analysis by agarose gel electrophoresis, suitable amplifiedA human genomic library cloned in the lFIX II vector (Stratagene)fragments were cloned into the pCR II vector (Invitrogen Inc.). Nucle-was screened using a 1153-bp 32P-labeled PCR fragment from cloneotide sequence analysis of the cloned fragments using vector specifiche1. The primers used in the generation of the PCR fragment wereprimers or internal primers allowed the identification of the exon–5*-GCTTCGGGCGCTGTAGTA (sense) and 5*-TTTAAACAATAC-intron boundaries of the gene.ACTTTTG (reverse). Approximately 1 1 106 plaque-forming units

were screened according to the instructions from the manufacturer. Southern blot analysis of somatic cell hybrids and fluorescence inSeveral positive plaques were isolated, rescreened and l DNA was situ hybridization (FISH) of human chromosomes. High-molecular-prepared from the positive clones using the glycerol step gradient weight DNA was isolated from human leukocytes, Chinese hamstermethod. Throughout this work standard molecular biology tech- cells, mouse liver cells, and hamster/human and mouse/human so-niques were used (Ausubel et al., 1992). The DNA probes used were matic hybrid cell lines, each one retaining one of the human chromo-labeled with [32P]dCTP (Amersham Inc.) using random priming, to somes in addition to the rodent genomes (NIGMS Coriell Cell Reposi-a specific activity of 5 1 108 to 1 1 109 cpm/mg DNA. tories, panel 2). Following digestion with HindIII, the DNA was frac-

tionated by agarose gel electrophoresis and transferred to a nylonPrimer extension analysis of mRNA for human 11-cis RDH. Thefilter. The blots were hybridized under stringent conditions with theposterior parts of several human eyes were kindly obtained from the

Missouri Eye Bank, Columbia, Missouri. The retinas, including the 1153-bp PCR generated fragment from clone he1.


SIMON ET AL.426

FIG. 2. Exon –intron organization of the gene encoding human 11-cis retinol dehydrogenase and a comparison with the genomic struc-tures of three related human hydroxysteroid dehydrogenases. (A) Schematic representation of the exon–intron organization of the humangene for 11-cis retinol dehydrogenase. Noncoding regions appear in gray. Initiation and termination codons are marked. The invarianttyrosine found in all SDRs is indicated by the Y in E3. The length of exons E1 and E5 were not established. (B) A comparison of thetranscripts encoding 11-cis RDH and the related 17b-, 3b-, and 11b-HSD. The segments of the transcripts encoded by the different exonsin the corresponding genes have been marked as well as the initiation and termination codons. The position of the highly conserved cofactorbinding motif G-X-X-X-G-X-G ( ), present in all four enzymes, N-N-A-G (ø), present in 11-cis RDH and 17b-HSD, and Y-X-X-X-K (º),present in all four enzymes, are marked.

Chromosome slides were prepared from lymphocyte cultures using tern obtained. The signals were visualized using a Zeiss Axiophotfluorescence microscope equipped with a cooled CCD-camera (Photo-standard procedures. l DNA from three genomic clones was mixed

and labeled with biotin-16 –dUTP (Boehringer Mannheim) by nick- metrics Nu 200/CH 250) for image capturing. The results were ana-lyzed using the SmartCapture software (Digital Scientific, Cam-translation. A centromere-specific probe for the chromosome 12 cen-

tromere (D12Z1, ATCC) was labeled with fluoro-red–dUTP (Amer- bridge).sham) by nick-translation. Preannealing of the probes, pretreatment

RESULTS AND DISCUSSIONof the slides, hybridization conditions, and signal amplification anddetection were performed as previously described (Bajalica et al.,

To explore the possible role of 11-cis RDH in heredi-1995). The chromosomes were counterstained with DAPI and thepositions of the signals were determined based on the banding pat- tary eye disease, we have as a first step determined

TABLE 1

Exon–Intron Junctions of the Gene for Human 11-cis Retinol Dehydrogenase

Exon length (bp) Donor site Intron length (bp) Acceptor site

E1 5*-UNT (nd)a CCA CAG GAG/gtab É450 tag/GCT GCC ACCE2 342 AG GAA GCA G/gta É400 cag/GG CTT TTT G

ys Glu Ala G ly Leu Phe GE3 259 C AGC CTG AG/gtg É1900 tag/G CGG GAT GT

p Ser Leu Ar g Arg Asp VaE4 164 TC ACC AAG T/gtg É250 cag/AC CTG AAA A

eu Thr Lys T yr Leu Lys ME5 221c — — —

a Not determined.b Exon sequences appear in uppercase letters while intron sequences are in lowercase letters.c Refers to the translated part of the exon; the total size of the exon was not determined.



nus of the deduced amino acid sequence of the enzymereported by these investigators differs from our pre-viously published sequence, and the length of their en-zyme was 319 amino acids instead of the 318 aminoacids found by us (Simon et al., 1995). The differenceswere due to three additional Gs in the 5* end of theisolated cDNA clone, which generated a divergent se-quence of the 30 N-terminal amino acid residues. Theremaining amino acid sequences were identical. TheFIG. 3. Chromosomal assignment of the gene for human 11-cis

retinol dehydrogenase to chromosome 12 by Southern blot analysis high evolutionary conservation along the entire poly-of somatic cell hybrids. The filter was hybridized with a PCR frag- peptides of the human and bovine enzymes, as deter-ment of the human cDNA for 11-cis RDH as the probe. Hybridization

mined by us from the cDNAs, strongly suggest that theto genomic DNA from human, hamster, and mouse and to genomicenzyme has 318 amino acids. The exon sequences ofDNA from a somatic cell hybrid cell line (GM10868), containing hu-

man chromosome 12 on a hamster background, is shown. the human gene confirmed the nucleotide sequence ofthe isolated cDNA clone (see below). It should also benoted that the N-terminal amino acid sequences of hu-the primary structure of the human enzyme and theman and bovine 11-cis RDH reported here and by Si-organization and chromosomal localization of the corre-mon et al. (1995), respectively, have a significantlysponding gene.higher homology to three recently cloned retinol dehy-A 1.6-kb cDNA clone encoding human 11-cis RDHdrogenases (Chai et al., 1995a,b, 1996) than that re-was isolated from an eye-specific human cDNA libraryported by Driessen et al. (1995) (É40% versus É6%).using bovine cDNA for this enzyme as the probe. Se-However, we cannot fully rule out that other alleles ofquence analysis identified an open reading frame en-the bovine gene might exist.coding 318 amino acid residues, the same length of the

The 11-cis RDH was the first enzyme of the SDRdeduced amino acid sequence as found for the bovinefamily of proteins to be identified as a retinoid-metabo-enzyme (Simon et al., 1995). Alignment of the humanlizing enzyme, and it appeared to be of interest to exam-and bovine amino acid sequences showed a 91% iden-ine the structure of the corresponding human gene.tity between the two enzymes (Fig. 1A). The amino acidSeveral genomic clones were isolated and the codingreplacements were scattered along the entire polypep-regions of the gene were amplified by PCR using combi-tides and were mainly conservative in nature. Thus thenations of primers derived from the cDNA sequenceoverall features of the human and bovine enzymes areincluding the 5* and 3* untranslated regions. Two over-very similar, i.e., a hydrophobic N-terminal putativelapping 1.5- and 2.7-kb PCR fragments were clonedsignal sequence, several other hydrophobic regions be-and analyzed by restriction mapping and nucleotideing putative transmembrane domains, and the highlysequencing.conserved cofactor binding and active site motifs found

The gene for human 11-cis RDH spanned over É4.1in most SDRs (cofactor binding consensus G-X-X-X-G-kb of DNA and was divided into four translated exonsX-G; active site consensus Y-X-X-X-K, Jornvall et al.,ranging from 164 to 342 bp. In addition, we found an1995).exon in the 5* untranslated region. The length of thisThe nucleotide sequence of the extreme 5* untranslateduntranslated exon and the length of the last exon wereregion (approximately 250 bp from the EcoRI cloning site)not fully established. The introns vary in length fromof the cDNA insert was identical to that of human heat-É250 bp to É1.9 kb. A schematic outline of the exon–shock protein hsp70 cDNA (Accession No. M59828). Weintron structure of the gene is shown in Fig. 2A. Sequencebelieve that the fusion of 11-cis RDH and hsp70 cDNAsanalysis of the exon–intron boundaries showed that allwas due to a cloning artifact. To estimate the length ofsplice donor and acceptor sites followed the canonical GT/the 5* untranslated region in the mRNA encoding 11-cisAG rule (Padgett et al., 1986) (Table 1). Exon 2, the firstRDH, primer extension analysis was carried out usingtranslated exon, was 342 bp in length and had 32 bp oftotal human retina RNA as the template. The results5* untranslated nucleotide sequence and 310 bp of trans-showed a major band corresponding to a length of the 5*lated sequence with the splice junction in the codon foruntranslated region to 226 nt. A minor band of 216 ntG104. Exon 3 was 259 bp in length and ended in thewas also visualized (Fig. 1B). By summarizing the lengthcodon for R190. Exon 4, the shortest exon in the gene,of the 5* untranslated region, the translated region (957was 164 bp in length and ended in the codon for Y245.nt including the stop codon), and the 3* untranslatedThe translated part of exon 5 terminated after 221 bp byregion (165 nt), we conclude that the mRNA for humana stop codon. Highly conserved structural elements like11-cis RDH is 1348 nt excluding the poly(A) tract. Thisthe cofactor binding site and the active site with the in-figure is close to the length of the mRNA correspondingvariant tyrosine residue were encoded by exons 2 and 3,to the bovine enzyme as determined by Northern blottingrespectively. One difference in the nucleotide sequences(Simon et al., 1995).of the human cDNA clone he1 and that of the exon se-Recently, Driessen et al. (1995) reported the cloning

of a cDNA encoding bovine 11-cis RDH. The N-termi- quences was noted. However, the C r T substitution in


SIMON ET AL.428

FIG. 4. Localization of the gene for human 11-cis retinol dehydrogenase by fluorescence in situ hybridization to 12q13–q14. (A) Apartial metaphase spread with specific hybridization of both chromatids of both chromosomes 12 is shown in green. Centromere-specifichybridization to chromosome 12 is shown in red. The chromosomes were counterstained with DAPI. (B) An enlargement of one chromosome12 (left) and an idiogram of chromosome 12 (right) demonstrating the localization of the human gene for 11-cis RDH to 12q13–q14.

the third base of the codon for I141 did not change the al., 1991). None of the three HSD genes have genomicstructures that are highly similar to that of 11-cis RDH,deduced amino acid sequence.

The structure of the gene for 11-cis RDH was com- an indication that the genes diverged from an ancestralgene early on during evolution. This is also demon-pared with the structures of the genes for 17b-, 3b-,

and 11b-hydroxysteroid dehydrogenase (HSD), three strated by the low amino acid sequence identity sharedbetween the HSDs and the 11-cis RDH (15–35%, un-human SDRs with known genomic structures (La-

chance et al., 1990; Luu-The et al., 1990; Tannin et published observation). However, some features of the



genomic organization of the gene for 11-cis RDH could phy have a thickening of Bruch membrane, caused bya mutation in a gene encoding an inhibitor of metallo-be found in the HSDs. A comparison of the transcripts

derived from the four genes, where the exon boundaries proteinase-3, which leads to reduced ability of the RPEto accumulate retinol and other nutrients from the cho-have been separately marked, showed that the gene

for 17b-HSD is most similar to that of 11-cis RDH (Fig. roid circulation (Weber et al., 1995; Jacobson et al.,1995). Common to the above mentioned diseases is the2B). The exon 2–exon 3 junctions in these genes were

found just upstream of a conserved motif present in fact that treatment with large doses of vitamin A willalleviate the symptoms.many, but not all SDRs (N-N-A-G, Jornvall et al., 1995).

Furthermore, the exon 3–exon 4 junction in 11-cis Nutritional studies in rats have shown that severelyretinol-deficient animals develop retinal degenerationRDH and the corresponding exon 4–exon 5 junction in

17b-HSD were located downstream and close to the (Wohlbach and Howe, 1925). Supplementation of suchanimals with RA, another metabolite of retinol in-active site. Also the apparent organization of exons 4

and 5 in 11-cis RDH and that of exons 5 and 6 in 17b- volved in gene regulation during growth and differenti-ation of many cell types (Gudas et al., 1994), does notHSD were similar. The similarities with the other

HSDs were less obvious, although an untranslated prevent or reverse the retinal degeneration phenotype(Dowling and Wald, 1960). Although, these studiesexon just upstream of the initiation codon could also

be found in 3b-HSD. have indicated that failure to form proper visual pig-ments might be the cause of the degeneration, it hasThe chromosomal assignment of the human 11-cis

RDH gene was examined by Southern blot analysis of not been possible to exclude fully the possibility thatthese effects are due to reduced synthesis of retinoicDNA from mouse and hamster somatic cell hybrids,

each retaining one of the human chromosomes. The acid in the eye. However, given the striking phenotypesof retinol-deficient animals and humans, and givenhybridization with the human 11-cis RDH cDNA as

the probe showed that the human gene was located on that humans suffering from the above-mentioned dis-eases are unable to form functional visual pigments, itchromosome 12 (Fig. 3). No hybridization to human

DNA was obtained in the somatic cell hybrids carrying seems likely that 11-cis RDH is a candidate gene forinvolvement in some form of hereditary retinal degen-other human chromosomes. The analysis also showed

a specific cross hybridization with mouse and hamster eration or allied diseases. Further experiments aimingat the identification of disease-associated alleles of thegenomic DNA, suggesting a high nucleotide sequence

similarity between the human and rodent genes for 11- 11-cis RDH gene will address this issue.cis RDH.

Using fluorescence in situ hybridization analysis, the ACKNOWLEDGMENTSchromosomal localization of the gene for 11-cis RDH

We thank Drs. Hans Jornvall and Bengt Persson for stimulatingwas established. The centromere of chromosome 12 wasdiscussions on SDRs and Dr. Ron Walkenbach at Missouri Lionsvisualized with a 12-centromere specific probe (red inEye Foundation for supplying us with human eyes. This study wasFig. 4). Specific hybridization using the l DNA genomicsupported in part by grants from the Swedish Cancer Society (to

fragments containing the gene for 11-cis RDH was ob- Magnus Nordenskiold).tained on the long arm of chromosome 12, a result con-sistent with the Southern blot analysis. Detailed analy-

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Primary Structure of Human 11-cisRetinol Dehydrogenase and Organization and Chromosomal Localization of the Corresponding Gene