Cholesterol Biosynthesis from Lanosterol
MOLECULAR CLONING, TISSUE DISTRIBUTION, EXPRESSION, CHROMOSOMAL LOCALIZATION, AND REGULATION OF RAT 7-DEHYDROCHOLESTEROL REDUCTASE, A SMITH-LEMLI-OPITZ SYNDROME-RELATED PROTEIN*

Soo-Han BaeDagger , Joon No LeeDagger , Barbara U. Fitzky§, Jekyung Seong, and Young-Ki PaikDagger parallel

From the Dagger  Department of Biochemistry and Bioproducts Research Center and § Institut fur Biochemische Pharmakologie A-6020 Innsbruck, Peter Mayr-Strasse 1, Austria and  Medical Research Center, Yonsei University, 134 Shinchon-dong, Sudaemoon-ku, Seoul 120-749, Korea

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The cDNA encoding the 471-amino acid rat 7-dehydrocholesterol reductase (DHCR), an enzyme that has been implicated in both cholesterol biosynthesis and developmental abnormalities (e.g. Smith-Lemli-Opitz syndrome) in mammals, has been cloned and sequenced, and the primary structure of the enzyme has been deduced. The DHCR gene was mapped to chromosome 8q2.1 by fluorescence in situ hybridization. Rat DHCR, calculated molecular mass of 54.15-kDa polypeptide, shares a close amino acid identity with mouse and human DHCRs (96 and 87%, respectively) as compared with its other related proteins (e.g. fungal sterol Delta 14-reductase) and exhibits high hydrophobicity (>68%) with 9 transmembrane domains. Five putative sterol-sensing domains were predicted to be localized in transmembrane domains 4-8, which are highly homologous to those found in 3-hydroxymethylglutaryl-CoA reductase, sterol regulatory element-binding protein cleavage-activating protein, and patched protein. The polypeptide encoded by DHCR cDNA was expressed in yeast as a 55.45-kDa myc-tagged fusion protein, which was recognized with anti-myc monoclonal antibody 9E10 and shown to possess full DHCR activity with respect to dependence on NADPH and sensitivity to DHCR inhibitors. Northern blot analysis indicates that the highest expression of DHCR mRNA was detected in liver, followed by kidney and brain. In rat brains, the highest level of mRNA encoding DHCR was detected in the midbrain, followed by the spinal cord and medulla. Feeding rats 5% cholestyramine plus 0.1% lovastatin in chow resulted in both approximately a 3-fold induction of DHCR mRNA and a 5-fold increase of the enzymic activity in the liver. When rats were fed 0.1% (w/w) AY-9944 (in chow) for 14-days, a complete inhibition of DHCR activity and a significant reduction in serum total cholesterol level were observed. However, the level of hepatic DHCR mRNA fell only slightly, suggesting that AY-9944 may act more rapidly at the protein level than at the level of transcription of the DHCR gene under these conditions.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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It has been suggested that the 19-step conversion of lanosterol to cholesterol in mammals begins with C14-demethylation and is followed by C14-reduction, C4-demethylation, C8right-arrow7 isomerization, C24-reduction, C5-desaturation, and C7-reduction (see Fig. 1) (1); this sequence has only recently been established following the determination of the precise location of C24-reduction (2). The enzymes involved in this pathway (e.g. lanosterol 14alpha -demethylase) are major targets for various lipid-lowering drugs, and mutations in the genes encoding these enzymes can lead to sterol-related genetic diseases such as Smith-Lemli-Opitz syndrome (SLOS)1 because of mutation in the 7-dehydrocholesterol reductase (DHCR) gene (3-5), and desmosterolosis following mutation in the gene encoding sterol Delta 24-reductase (6) (Fig. 1).

As the terminal enzyme in cholesterol biosynthesis in mammals, DHCR (E.C. 1.3.1.21) catalyzes the reduction of the Delta 7 double bond of sterol intermediates in the presence of NADPH under anaerobic conditions (1, 7). Studies on this enzyme have recently attracted attention because DHCR has been implicated in both cholesterol metabolism and developmental malformations in mammals (8-10). That is, a deficiency of DHCR activity because of genetic mutation in humans has recently been found to cause SLOS (3-5). SLOS is an autosomal recessive disorder with various developmental abnormalities and is characterized by elevated 7-dehydrocholesterol (DHC) in serum body fluids and tissues (11-19). The connection between cholesterol and development in mammals was established based on the unexpected role of cholesterol in the autoprocessing of sonic hedgehog protein (8-10), a morphogen that binds to patched (PTC) protein in the developing central nervous system and limbs. For example, mice mutated in sonic hedgehog show a SLOS-like phenotype (20, 21).

SLOS-like developmental malformations with elevation of DHC in animals have also been observed when DHCR activity is inhibited in vivo by either BM15.766 (16, 22, 23) or AY-9944 (24-28); these compounds have also been shown to inhibit other cholesterogenic enzymes including sterol Delta 14-reductase (29, 30) and sterol Delta 8-isomerase (31, 32), collectively termed AY-9944-sensitive cholesterogenic enzymes (7) (Fig. 1).


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Fig. 1.   Sequence of the biosynthesis of cholesterol from lanosterol (2) and some representative inhibitors blocking this pathway. The mark perp  denotes blocking of the reactions by inhibitors, and the asterisk represents AY-9944-sensitive cholesterogenic enzymes. Some of inhibitors used for this study are indicated in bold.

The gene encoding rat DHCR had not been cloned previously. In order that we might investigate the molecular regulation of DHCR gene expression and the importance of impaired DHCR activity for the development of SLOS-like phenotypes in rats (24-28), we isolated a full-length cDNA encoding rat DHCR. Work of this nature is important in order that we may understand more fully developmental cause of the SLOS phenotype and the regulation of cholesterol biosynthesis in animals.

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Materials-- A rat liver cDNA library (oligo(dT) primed and randomly primed) and rat multiple tissue Northern blot were purchased from CLONTECH (Palo Alto, CA). The following materials were purchased from the manufacturers that are indicated in parentheses: Multiple ChoiceTM Rat Brain Northern Blot (Origen); [alpha -32P]dCTP (3000 Ci/mmol) and Hyper x-ray film, rainbow prestained protein marker, ECL Western blotting detection system (Amersham Pharmacia Biotech); random-primed DNA labeling kit (Roche Molecular Biochemicals), total RNA isolation kit (Invitrogen); DNA preparation kit (Qiagen). Sterols included ergosterol, 7-dehydrocholesterol, lathosterol, and cholesterol (Steraloid). The sources of the following drugs or agents are also indicated in parentheses: AY-9944 (Wyth-Ayerst, Princeton, NJ); cholestyramine (LG Chem, Pharmaceutical Division, Seoul, Korea); lovastatinR (Dr. Y-K. Sim at Choongwae Pharmaceutical Co., Suwon, Korea). Oligonucleotide primers were synthesized by DNA synthesizer Applied Biosystem (Foster City). Yeast strain JB811 was obtained from K. Nasmyth (Vienna, Austria). Roswell Park Memorial Institute 1640 medium, fetal bovine serum, penicillin/streptomycin, Dulbecco's modified Eagle's medium, and L-glutamine were purchased from Life Technologies, Inc. Anti-myc monoclonal antibody (9E10) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents were the best grade available.

Screening and Isolation of DHCR cDNA from Rat Liver cDNA Library-- A 1.5-kb human DHCR cDNA fragment covering the entire open reading frame (ORF) (33) was labeled with [alpha -32P]dCTP with a random-primed labeling kit according to the suggested procedures for a specific activity >5 × 108 dpm/µl. The labeled fragment was denatured at 100 °C for 10 min and used as a probe to screen >3 × 106 plaques from a rat liver cDNA library according to standard protocols provided by CLONTECH. After tertiary screening, 12 positive clones were used for further analyses using the slot blot hybridization (34). After digestion of 12 clones with EcoRI, three different clones were identified; they are 7B-3-1(2.5 kb), 7B-1-1 (1.8 kb), and 7-5 (1.5 kb) (Fig. 2A), respectively.


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Fig. 2.   Schematic presentation of cDNA clones for the rat DHCR. A, sequencing strategies and restriction map for rat DHCR cDNA clone. The sites for the restriction endonucleases used to generate probes and confirm the sequence are shown. The restriction map of the cDNA (top) corresponds to the compilation of three independent clones (i.e. 7B-3-1, 7B-1-1, and 7-5) sequenced; the arrows indicate the direction of sequencing using either sense or antisense primers; the black box indicates the open reading frame, and the white boxes represent the untranslated 3' and 5' ends. B, the nucleotide sequence and translation product (bottom) are shown. The numbers on the left refer to the nucleotide sequence; numbers on the right refer to the amino acid sequence. Lowercase sequences indicate the 48 additional 5' nucleotides and the 892 additional 3' nucleotides in clone 7B-3-1. The location of the codon for the translation initiator methionine, ATG (black-down-triangle ) and the translation stop codon TGA (***) are indicated. The position of the AATAAA polyadenylation signal in the 3'-untranslated region is double-underlined.

DNA Sequencing and Analysis of DHCR cDNA-- A DNA fragment (2.5-kb size) that was isolated by EcoRI digestion of cloned DNA from 7B-3-1 (Fig. 2A) was sequenced on both strands by the dideoxy sequencing method using a Taq Dye Deoxy Terminator Cycle sequencing kit (Applied Biosystems). Sequence and structure analyses of the cloned cDNA and its predicted protein product were carried out using BLAST programs in the GenBankTM. Rat cDNA clone homologous to human DHCR (GenBankTM accession number AF034544), Mus musculus (GenBankTM accession number AF057368), and Arabidopsis (GenBankTM accession number U49398) was analyzed with the BLASTX algorithm. Alignments and hydrophobicity analysis were calculated with the Wisconsin sequence analysis package according to the algorithms of Smith and Waterman and Kyte and Doolittle, respectively. Analysis of transmembrane segments was performed by using the following prediction programs: PHD  htm at EMBL, TMpred at ISREC, SOSUI at Tokyo University, and HMMTOP prediction as described (35). Topology of rat DHCR structure was predicted by at least three of the four programs. The sequences for each protein from GenBankTM are indicated in parentheses: Chinese hamster sterol regulatory element-binding protein cleavage activation protein SCAP (280-446, GenBankTM accession number U67060); Chinese hamster 3-hydroxymethylglutaryl-CoA reductase (57-224, GenBankTM accession number L00165); mouse Niemann-Pick type C1 (617-791, GenBankTM accession number AF003348); PTC (420-589, GenBankTM accession number U46155). The alignment of these sequences with rat DHCR was performed using CLUSTALW.

Chromosomal Localization of the Rat DHCR Gene-- Two genomic EcoRI-HindIII fragments, 0.16 and 1.6 kb in length, that mainly included the coding and spacer region of 5 S rRNA, respectively, were used for fluorescence in situ hybridization analysis. The fragments were amplified by polymerase chain reaction and inserted into pUC119. The 0.16-kb fragment, which was derived from SD rats, included a 121-bp gene for mouse 5 S rRNA, which was amplified from total genomic DNA with the primers 5'-TGTCGGCGCCGCCCGCCCTC-3' and 5'-AGAGACAGAGGGGAGTCCAA-3'. One genomic DNA fragment of DHCR gene was also prepared by polymerase chain reaction using rat genomic DNA as template and the primers derived from rat genomic sequences covering the 5' end of exon 5 (5'-CATAGCAACGGGATCCA-3') and the 3' end (5'-CGTAGCCTTTCACCAAA-3') of exon 6 (2.1-kb size: 17 bp of exon 5, 2030 bp of intron, and 17 bp of exon 6) of rat gene.2 The probe was labeled by nick translation with biotin-16-dUTP (Roche Molecular Biochemicals) according to the manufacturer's protocol. Preparation of R-banded chromosomes and fluorescence in situ hybridization were performed as described (36).

Heterologous Expression of Myc-tagged Rat DHCR in Saccharomyces cerevisiae-- Rat DHCR cDNA was subcloned into c-myc-tagged yeast episomal plasmid c-myc-YEp351ADC1, and transformation of S. cerevisiae JB811 (ade2-1 leu2-3, 112 pep4-3 trp1-289 ura3-52) was performed as described (33). Cells were harvested at an A600 of 1.2 and lysed with glass beads in 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride. For the preparation of microsomes, the lysates were spun for 5 min at 500 × g (4 °C), and the supernatant was pelleted for 45 min at 100,000 × g (4 °C) as described (33). Immunoblotting with a 9E10 c-myc monoclonal antibody following SDS/polyacrylamide gel electrophoretic analysis was exactly the same as described previously (33). Protein concentrations were determined by the method of Bradford (37) using bovine serum albumin as a standard.

Animals, Diet Feeding, and Drug Treatment-- Male Sprague-Dawley rats (200-250-g body weight) were maintained on a standard rodent chow under a reverse light cycle (light 6:00 p.m-6:00 a.m.; dark 6:00 a.m.-6:00 p.m.) as described previously (30). In the diet feeding experiments, each diet-fed group (4 to 5 rats/group) was fasted for 12 h and then fed diets supplemented with various agents such as 5% cholestyramine plus 0.1% lovastatin (CL-diet) in or 0.1% AY-9944 in chow for 14 days (30). At the end of the treatment, the rats were fasted for 24 h and killed by CO2 inhalation. Blood was drawn by heart puncture and analyzed for serum lipids. The plasma serum cholesterol was determined by an automatic analyzer Hitachi 7150. Low density lipoprotein cholesterol was calculated from the method of Friedewald formulation (38).

Preparation of Microsomes and Enzyme Assays-- Rats that had been fed under various diet conditions were killed by decapitation at the midpoint of the dark period (10 p.m. for rats), and their livers were excised and processed for microsome preparation as described previously (30). A standard assay for DHCR was carried out using 300 µM substrate (ergosterol, 7-dehydrocholesterol, or lathosterol) that had been suspended in the detergent Tween 80 (70:1, detergent:sterol) and 1 to 2 mg of microsomal protein as described (7). For the in vitro inhibition experiments for both rat liver microsomes and yeast overexpressed protein, drugs or agents were dissolved in dimethyl sulfoxide (Me2SO) such that the final concentration of Me2SO was less than 0.3% (w/v) that of the incubation mixture (2). The sterol concentration in enzyme assay samples was measured in a Hewlett Packard gas chromatograph 5890 II or a Young-In GC520 gas chromatograph (Seoul, Korea) (FID, capillary column; SAC-5, 5% diphenyl, 95% dimethylsiloxane, 30 m × 0.25 mm, 0.25 µm inner diameter, flow rate 2.44 ml/min) using 5alpha -cholestane as a standard (30, 32).

RNA Preparation and Northern Blot Analysis-- For the effects of diet on DHCR gene expression, total RNA from liver of either CL-diet, AY-9944-fed, or normal diet-fed rats were prepared with total RNA isolation kit (Invitrogen) according to the manufacturer's procedures. Fifteen µg of each tissue RNA were separated on a 1.2% (w/v) agarose gel in 30% (v/v) formaldehyde and blotted. RNAs were probed with 32P-labeled rat DHCR cDNA (GenBankTM accession number AF071500). A rat beta -actin cDNA probe was used as a control. After hybridization (42 °C, 50% (v/v) formamide), blots were washed at low stringency (e.g. 0.2 × SSC (1 × SSC = 0.15 M NaCl and 0.015 M sodium citrate), 0.1% (w/v) SDS at 22 °C). Rat multiple tissue Northern blot (CLONTECH) and Multiple ChoiceTM Rat Brain Northern Blot (Origen) were probed with [32P]-labeled rat DHCR cDNA. The blots were exposed to Hyper x-ray film with intensifying screens at -70 °C for longer than 48 h. The bands that appeared in the blots were quantitatively analyzed with a Molecular Dynamics 425DE PhosphorImager using ImageQuaNT program. DHCR mRNA signals obtained from different tissues by the Northern blot hybridization were normalized to those of beta -actin mRNA expression.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Cloning and Sequence Analysis of Rat DHCR cDNA-- Screening of a rat liver cDNA library (CLONTECH) with the human cDNA probe yielded three positive clones (i.e. 7B-3-1, 7B-1-1, and 7-5) (Fig. 2A) of approximately >3.0 × 106 plaques screened. The nucleotide sequences of these clones were determined on both strands and revealed that all of these clones (e.g. clone 7B-3-1) encode the rat DHCR cDNA (Fig. 2B). The DHCR cDNA (clone 7B-3-1) is 2356 nucleotides long and consists of a 48-nucleotide 5'-untranslated region followed by a 1416-nucleotide coding region and a 892-nucleotide 3'-untranslated region. As expected, the nucleotide sequence of rat DHCR showed highest homology with mouse DHCR. That is, the coding region (ORF) of the cDNA sequence (1416 nucleotides) showed 96, 87, and 36% identity with mouse (GenBankTM accession number AF057368), human (GenBankTM accession number AF034544), and Arabidopsis thaliana (GenBankTM accession number U49398) DHCR cDNA sequences, respectively. Unlike human DHCR cDNA, which has an additional polyadenylation site, rat DHCR cDNA contains only a single polyadenylation site, similar to the mouse ortholog (3). A striking feature of the rat DHCR mRNA sequence is that it contains an exceptionally long 3'-untranslated region (892 nucleotides), indicating the polyadenylation site is quite distant from the termination codon. The deduced amino acid sequence of rat DHCR (Fig. 2B) predicts a 471-residue protein with a calculated molecular mass of 54,155 Da, quite similar to the human (475 amino acid (33)) and mouse (471 amino acids (3)) enzymes.

Structural Feature of Rat DHCR Protein-- Fig. 3A presents the alignment of rat DHCR polypeptide with other mammalian enzymes (human and mouse (3, 33)) and A. thaliana (39). At the polypeptide level, the rat DHCR ORF shows 96 and 87% identity to the mouse and human DHCR polypeptides over 471 amino acids (Table I). The relatively high degree of sequence similarity in ORF between DHCRs of different species suggests that they may be involved in fundamental physiological processes that are evolutionarily conserved between animals and plants. There is also a significant degree of similarity between the amino acid sequence of DHCR and related proteins (Table I). In particular, a relatively high similarity is seen between DHCR and fungal sterol Delta 14-reductase (47%) (39), human lamin B receptor (48%), which has been suggested as a putative sterol Delta 14-reductase (41), and fungal sterol Delta 24-reductase (43%) (42). However, although rat sterol Delta 8-isomerase is a member of AY-9944-sensitive cholesterogenic enzymes, its homology to rat DHCR is low (29%) (Table I).


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Fig. 3.   Structural feature of DHCR protein. A, homology between DHCRs of different species. Residues of rat DHCR (RDHCR) cDNA sequence (GenBankTM accession number AF071500) identical or partially identical to the corresponding residues in the human (HDHCR) (GenBankTM accession number AF034544), mouse (MDHCR, GenBankTM accession number AF057368), and Arabidopsis (cress) ((ADHCR) GenBankTM accession number U49398) DHCR cDNA sequences are marked by asterisks, colons, and single dots, respectively. The residues comprising the nine putative transmembrane (TM) domains are boxed. Potential N-glycosylation sites (single dots) on rat DHCR are indicated. B, Kyte-Doolittle hydrophobicity plot of the deduced DHCR protein sequence. Hydropathy analysis with an average window size of 19-amino acid residues plotted at one-residue intervals. On the ordinate, hydrophobicity and hydrophilicity are indicated by positive and negative numbers, respectively. Domains that appear above the central line are predicted to be hydrophobic.

                              
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Table I
Comparison of the amino acid sequence of rat DHCR with sequences of related proteins

Several structural motifs are conserved among the mouse, human, and rat proteins (Fig. 3A). They are hydrophobic regions, transmembrane domain (marked as box), and glycosylation sites (dots). The DHCR polypeptides contains a highly conserved motif, GVQEGA (residue 124-129), which is thought to represent potential N-myristoylation sites (43, 44). The deduced amino acid sequence of DHCR also contains a total of 23 serine (4.8%) and 22 threonine residues (4.7%) located throughout the backbone that may serve as putative phosphorylation sites for serine/threonine kinases as suggested (45). There are also three putative protein kinase C phosphorylation sites (i.e. 73-75 (TGR), 89-91 (TAK), and 437-439 (THR)) (46, 47) and two tyrosine kinase phosphorylation sites (i.e. 372-378 (KAIECSY) and 450-458 (KYGRDWERY)) (48, 49). The deduced amino acid sequence of rat DHCR contains four potential N-linked glycosylation sites (Asn-Xaa-(Ser/Thr) (50-52) at 8-11 (NASK), 248-251 (NLSF), 283-286 (NETW), and 403-406 (NYTG) (Fig. 3A). The first and the fourth (at 8-11 and 403-406) are positioned in relatively long loops of the DHCR that are projected into the ER lumen. The second and third are localized inside the transmembrane domain (248-251) or very close to it (283-286).

A hydropathy plot indicates that there is a large proportion (>68%) of hydrophobic amino acids throughout the polypeptide backbone (except for the region of amino acid residues 3-30 and 349-388) of the deduced amino acid sequence (Fig. 3B). More than 68% of the rat DHCR polypeptide appears to be hydrophobic, a feature shared by other cholesterogenic microsomal enzymes (Fig. 3B) (e.g. 3-hydroxymethylglutaryl-CoA reductase (53)). The existence of at least nine transmembrane alpha -helical domains is predicted by Kyte-Doolittle hydrophobicity analysis (54), in keeping with our previous observation that solubilization of membrane-bound DHCR by various nonionic detergents (e.g. CHAPS) was extremely difficult to accomplish (7).

The most intriguing feature of DHCR protein structure is that it contains a sterol-sensing domain (SSD) (Fig. 4A), which has been well reported in five other sterol-mediating regulatory proteins: sterol regulatory element-binding protein (55), sterol regulatory element-binding protein cleavage activation protein (56, 57), PTC (58), TRC8, a PTC-related protein (35), and Niemann Pick-type C1 protein (59); the SSD of rat DHCR was predicted to be localized at transmembrane segments 4-8, covering amino acid residues 177 and 358 (Fig. 4A). The alignment of the SSDs of various proteins is shown in Fig. 4B. The putative SSD region of rat DHCR (residues 177-358) showed 47% similarity and 12.6% identity to the SSD of mouse sterol regulatory element-binding protein cleavage activation protein (280-446). The significance of the similarity of rat DHCR to other sterol-related proteins is currently unknown. However, among human SLOS patients, three missense mutations have been found in this putative SSD region: A247V, V326L, and R352W (3). These mutations in human DHCR also resulted in 95-97% reduction in enzyme activity (3), suggesting that loss both of enzyme activity and sterol-sensing response may contribute to development of SLOS during embryogenesis.


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Fig. 4.   Predicted membrane topology and sterol-sensing domain of rat DHCR. A, membrane topology showing the putative sterol-sensing domain of rat DHCR, which is located between amino residues 177 and 358. B, alignment of putative SSD of various proteins. The thick lines with numbers on top represent the alignment of putative SSD of sterol regulatory element binding protein cleavage activation protein (SCAP, 280-446, TM 2-6) with those of rat DHCR (RDHCR), 3-hydroxymethylglutaryl-CoA reductase (HMGR), PTC, and Niemann-Pick type C1 (NPC1). Identical (dark shading) and similar (light shading) amino acids within the putative SSD of each protein are indicated.

Assignment of the Rat DHCR Gene to Chromosome 8q2-- DNA isolated from the rat genomic library by polymerase chain reaction was hybridized to the region of the long arm of rat chromosome 8q2 from 31 metaphase chromosome slides of 4 Sprague-Dawley rats (Fig. 5). Using replicative R-banding, we confirmed that the DHCR genome probe was localized to rat chromosome 8q2.1 (data not shown). Human and mouse DHCR genes were previously localized to chromosome 11q12-13 (5) and 7F5 (3), respectively.


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Fig. 5.   Assignment of the rat DHCR gene to chromosome 8q2.1. An idiogram of chromosome 8 summarizes the fluorescence in situ hybridization analysis using the 2.1-kb genomic DNA clone covering intron 5 of the rat DHCR gene. Each dot represents the localization of double fluorescent signals on banded chromosome 8.

Functional Analysis of the Cloned DHCR cDNA-- To examine the function of cloned rat DHCR cDNA, a control expression vector for the cDNA (i.e. c-myc-YEp351ADC1) encoding a myc-tagged DHCR protein (myc-DHCR) was transfected into JB811 S. cerevisiae cells (33). Because it is already known that the expression of a myc-tagged DHCR significantly increased DHCR activity in yeast crude microsomes (33), we designed and used a myc-DHCR expression vector for the experiment. An advantage of this tagged protein is that it enables the use of an immunoblot detection assay system without the need for a DHCR-specific antibody, which is not available at the present time. As anticipated, transfected yeast cells expressed an approximately 55.45-kDa rat DHCR protein, which was recognized with the anti-myc monoclonal antibody 9E10 (Fig. 6A). No protein was detected in the immunoblot of yeast cells transfected with control vector. Immunoblotting of expression of myc-DHCR showed excellent agreement in calculated molecular mass of DHCR (i.e. Mr 54,550) in 12% SDS/polyacrylamide gels. Rat DHCR activity in yeast crude membranes is illustrated in Fig. 6B. The overexpressed cloned myc-DHCR fusion protein showed a similar pattern of inhibition to that previously seen in the rat liver microsomal protein upon exposure to AY-9944 (IC50 = 0.243 µM) or U18666A (IC50 = 2.690 µM) when enzyme activities were determined under conditions where the activities were linear with respect to time and substrate concentration (Fig. 6C). This observation is in excellent agreement with our previous results that had been obtained with rat hepatic DHCR in which IC50 of AY-9944, for example, was estimated to be 0.25 µM (7). The overexpressed protein was not inhibited significantly by tamoxifen (a specific inhibitor of sterol Delta 8-isomerase) (60) or miconazole (a specific inhibitor of lanosterol 14alpha -demethylase) (61, 62). Furthermore, the DHCR overexpressed in yeast exhibited essentially the same properties as those of hepatic microsomes (Table II) (7). For example, the overexpressed DHCR was highly sensitive to the presence of AY-9944 (Fig. 6C) and sulfhydryl-binding agents and dependent on NADPH (i.e. NADH cannot substitute for NADPH as a proton donor) (Table II). The activity of the overexpressed DHCR protein was mildly affected by the presence of oxygen and phosphate ions but not by some metal ions (4 mM), as previously seen in the case of rat hepatic DHCR (7). Thus, these results suggest that the cloned rat hepatic DHCR cDNA indeed encodes functional DHCR protein, and there is essentially no difference in the functionality of the enzyme when it is expressed in yeast.


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Fig. 6.   Heterologous expression of a myc-tagged rat DHCR cDNA (myc-DHCR-ORF) in S. cerevisiae strain JB811 (ade2-1 leu2-3, 112 pep4-3 trp1-289 ura3-52). A, Western blotting was carried out as described (33) using 10 µg of microsomal protein that was separated on a 12% (w/v) SDS gel under reducing conditions. The blot was immunostained with 80 ng/ml 9E10 c-myc antibody and analyzed with the ECL Western blotting detection system using horseradish peroxidase-conjugated secondary antibody. The arrow indicates the migration of a c-myc immunoreactive band with a migration corresponding to 55 kDa (lane 1, mock; lane 2, rat myc-DHCR). B, time course of overexpressed rat DHCR activity. Microsomes (0.5 mg) prepared from strains transformed with the vector without cDNA (mock (open circle )) or myc-DHCR-ORF () were incubated anaerobically for the indicated times with DHC in the presence of 2 mM NADPH. Each value represents the average of duplicate assays of two separate experiments. C, inhibition of myc-DHCR by AY-9944 () (IC50 = 0.243 µM), U18666A (open circle ) (IC50 = 2.690 µM), miconazole (black-square), and tamoxifen (). Each drug was dissolved in Me2SO, the concentration of which was kept at less than 0.3% (w/v). Data shown are mean ±S.D. (n = 3). Enzyme assays were carried out for 20 min in the presence of each inhibitor as described in the legend of panel B. The absolute values of enzyme activity (nmol/min/mg of protein) at 10-3 µM of each inhibitor were 1.16 (miconazole), 1.13 (tamoxifen), 1.09 (U18666A), and 1.2 (AY-9944), respectively. The initial measurement for the inhibition of DHCR activity by each drug started at a concentration of 10-4 µM.

                              
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Table II
The properties of myc-tagged rat DHCR heterologously expressed in S. cerevisiae strain
Enzyme assays were conducted anaerobically at 37 °C using 300 µM 7-dehydrocholesterol, 0.5 mg of yeast microsomal protein, 2 mM NADPH, 25 mg of glucose, 20 units of glucose oxidase (Type V) in 0.1 M Tris-HCl buffer including 20% glycerol, 0.5 mM EDTA, and 1 mM reduced glutathione. Data represent the mean of duplicate determinations.

Tissue Distribution of DHCR mRNA-- The distribution of mRNAs encoding DHCR in different rat tissues was examined by Northern blot analysis using a rat DHCR cDNA ORF probe. The signals of DHCR mRNA on blots were normalized to those of beta -actin mRNA. As shown in Fig. 7A, the liver (100% A = 41 by PhosphorImager analysis) exhibited the highest signal, and that is followed by the kidney (13% A = 5.3) and the brain (9% A = 3.7). No signal was detected in other rat tissues (heart, testis, lung, spleen, and skeletal muscle). This result suggests that the liver is the major site of rat DHCR mRNA expression. To determine tissue distribution of DHCR mRNA in the rat brain, Multiple Choice Northern brain blot (Origen) was hybridized with rat cDNA probe in which the hybridization signal of each part of brain was normalized to that of rat beta -actin. As shown in Fig. 7B, midbrain(as 100%) showed the highest densities followed by spinal cord and medulla. It should be noted that there is a marked difference between human (33) and rat DHCR mRNA distribution. That is, the mRNA for rat DHCR is expressed highly in rat liver and moderately in brain and kidney, which corresponds well to the known tissue distribution of the enzyme activity,2 whereas ubiquitous expression of human DHCR mRNA (in organs such as the adrenal gland, liver, brain, heart, and testis) is seen (33). In particular, in humans the level of DHCR mRNA expression in the brain is very high, at almost the same as that of the liver (33), whereas in the rat the level of mRNA of liver is predominant over that of brain (almost 14-fold). The significance of this difference between the two species remains to be determined.


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Fig. 7.   Tissue-specific expression of rat DHCR mRNA. A, Northern dot blots with 2 µg of poly(A)+ RNA (rat multiple tissue Northern blot, CLONTECH) were probed with the 32P-labeled rat DHCR cDNA (>108 cpm/ng) and exposed for 16 h. The arrow indicates the migration of the 2.5-kb DHCR mRNA. The signals of DHCR mRNA bands on blots were normalized to those of rat beta -actin mRNA. B, ubiquitous expression of rat DHCR mRNA in brain. Analysis of DHCR expression in adult rat brain was performed using brain Multiple Choice Northern blot (Origen). Twenty µg of total RNA were probed with the 32P-labeled rat DHCR cDNA and exposed for 16 h. Hybridization signals (expressed as AU, absorbance unit) were quantified with a PhosphorImager. The signals of each lane on blots were first normalized to those of beta -actin as described in panel A and then compared with those of midbrain (100%). Data shown are the mean from two different blots in which variability was less than 10%.

In Vivo Regulation of the Hepatic DHCR Gene by Lipid-lowering Drugs-- To determine whether DHCR mRNA levels are regulated by feeding lipid-lowering drugs (e.g. cholestyramine, lovastatin, and AY-9944), DHCR mRNAs in liver tissue from chow-fed and CL-diet-fed rats (30, 7) were quantified by Northern blot analysis (Fig. 8). In the CL-diet-fed rats, both hepatic DHCR mRNA (expressed as an optical density of the hybridized bands in the blot by densitometric scanning) and the specific enzymic activity (expressed as nmol/min/mg of protein) were increased nearly 3-fold (i.e. in optical densities of mRNA signals, 35.8 ± 6 (control) versus 97.3 ± 8 (CL-diet), A values normalized to beta -actin signal n = 3) and 5-fold (i.e. in enzymic activities, 4.42 ± 0.7 (control) versus 21.37 ± 2.8 (CL-diet), n = 3), respectively, relative to the control rats in three independent experiments (Fig. 8). Thus, the DHCR induction by CL-diet shown here and previously seen (7) was mainly because of elevation of DHCR mRNA levels in these animals. Although AY-9944 has long been known to inhibit DHCR activity, the molecular nature of this inhibitor has not been reported. This inhibitor in fact also causes teratogenic SLOS-like phenotypes in rodents (24, 26, 28). Northern blot analysis was performed on RNA from livers of rats fed AY-9944 for 14 days. Surprisingly, the level of DHCR mRNA was not much changed in liver at this dose of AY-9944 (0.1%, w/w, approximately 7.0 mg/kg/day). However, there was a huge reduction in serum lipids (e.g. average values of 65% total cholesterol, 49% low density lipoprotein cholesterol, and 38% triglyceride of control (n = 3) (30)3 and complete inhibition of enzyme activity (to an undetectable level) in vivo. The failure of the same dose of AY-9944 to alter the level of expression of DHCR mRNA (from 35.8 to 30.1; A values normalized to beta -actin signal) suggests that the mechanism of action of this inhibitor may occur most rapidly at the protein level, as seen previously (7), than at the transcriptional level. However, we do not rule out the possibility that in the longer term (e.g. >1 month) ingestion of higher doses of AY-9944 (e.g. >75 mg/kg (27)), which have been known to cause SLOS-like phenotype in rats, may modulate the level of transcription of the DHCR gene. This point remains to be clarified.


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Fig. 8.   In vivo effects of cholesterol-lowering drugs on the expression of DHCR mRNA and DHCR enzymic activity in rat liver. Total RNA and microsomes were isolated from each liver from each of 4-5 rats in each feeding group. Northern blot analysis was carried out using 15 µg of total RNA from normal-, CL-diet-, or AY-9944-fed rats. Enzyme activity was determined using 0.5 mg of protein as described under "Experimental Procedures." Quantitation of blots was performed using a 425DE PhosphorImager with ImageQuaNT program (Molecular Dynamics). Data are shown as mean ±S.D. (n = 3) obtained from three different blots following normalization of the DHCR-specific hybridization signals to those of beta -actin. (+)CL, 5% (w/w) cholestyramine plus 0.1% (w/w) lovastatin in chow; (+)AY-9944, 0.1% (w/w) AY-9944 in chow.

In conclusion, our results on the molecular characterization of DHCR cDNA may provide many useful opportunities with regard to 1) the elucidation of the molecular regulatory mechanism for DHCR gene expression in vitro and in vivo, 2) the creation of an animal model for investigation and treatment of SLOS, 3) investigation of structure-function relationships in DHCR, and 4) other useful pharmacokinetic studies including determination of the effect of targeted mutations during investigation of treatment strategies. In particular, the elucidation of the role of sterol-sensing domains in DHCR expression may provide some clues about the connection between this enzyme and developmental malformations such as the SLOS-like phenotypes in mammals.

    ACKNOWLEDGEMENT

We thank Drs. H. Glossmann, F. Moebius, and D. J. Adams for their help and critical readings of this manuscript. We also thank Dr. Yoischi Matsuda for his help on fluorescence in situ hybridization work and Dr. W.-T. Lee and J.-H. Kim for their assistance on this project.

    FOOTNOTES

* This work was supported by grants from the Korean Ministry of Science and Technology Molecular Medicine Project 98MM-0204A03 (to Y.-K. P.) and Korea Science and Engineering Foundation through the Bioproducts Research Center at Yonsei University (9514-0401-00-12-3 (to Y.-K. P.)).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF071500.

parallel To whom correspondence should be addressed: Yonsei University, Dept. of Biochemistry, 134 Shinchon-dong, Sudaemoon-ku, Seoul, 120-749, Korea. Tel.: 82-2-361-2702; Fax: 82-2-362-9897; E-mail: paikyk{at}bubble.yonsei.ac.kr.

2 J. N. Lee and Paik, Y.-K., unpublished data.

3 S-H. Bae and Y.-K. Paik, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: SLOS, Smith-Lemli-Opitz syndrome; DHCR, 7-dehydrocholesterol reductase; kb, kilobase(s); ORF, open reading frame; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; SSD, sterol-sensing domain; AY-9944, trans1,4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride; BM15. 766, 4(2-(1-(4-chlorocinnamyl)-piperazin-4-ylethyl)-benzoic acid; CL-diet, 5% cholestyramine plus 0.1% lovastatin in normal chow; IC50, a concentration of inhibitor required for 50% inhibition; lanosterol, 4,4',14alpha -trimethyl-5alpha -cholesta-8,24-dien-3beta -ol; lathosterol, 5alpha -cholesta-7-en-3beta -ol; PTC, patched protein; tamoxifen, trans-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethylamine; U18666A, 3-beta -[2-(diethylamino)ethoxy]androst-5-en-17-one.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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