cDNA Cloning and Expression of Oxysterol-binding Protein, an ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 264, No.28, Issue of October 5,pp. 16798-16803,1989 Printed in U.S.A.

0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

cDNA Cloning and Expressionof Oxysterol-binding Protein, an Oligomer with a Potential Leucine Zipper* (Received for publication, May 10, 1989)

Paul A. Dawson$#ll,Neale D. Ridgway#$(I, Clive A. Slaughter**, Michael S . Brown$#, and Joseph L. Goldstein$# From the Departmentsof $Molecular Genetics and §Znternal Medicine and the **HowardHughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235

Feedback repression of the genes encoding the low density lipoprotein receptor and several enzymes of the cholesterol biosynthetic pathway is mediated by 25-hydroxycholestero1 and other oxysterols. In this study, we have cloned a rabbit cDNA encoding an oxysterol-binding protein that may play a role in this regulation. The predicted amino acid sequence revealed a protein of 809 amino acids with two distinctive features: 1) a glycine- and alanine-richregion (63%of 80 residues) at theNH2 terminus, and 2) a 35residue leucine zipper motif that may mediate the previously observed oligomerization of the protein. When transfected into simian COS cells, the rabbit cDNA produced a protein that exhibited thesame affinity and specificity for sterols as the previously purified hamsterliver protein. Immunoblotting analysis showed that the rabbit cDNA encodes both the 96- and 101kilodalton forms of the oxysterol-bindingprotein that were previously observed. The availability of an expressible cDNA for theoxysterol-binding protein should help elucidate its role in sterol metabolism.

Cellular cholesterol homeostasis is achieved through regulation of the uptake, synthesis, and esterification of cholesterol (1).When sterols accumulate in cells, cholesterol synthesis is reduced through a decline in theactivities of several enzymes in the cholesterol biosynthetic pathway, cholesterol uptake is reduced througha reduction in the number of receptors for low density lipoprotein (LDL),’ and cholesterol esterification is enhancedthrough the activation of acylCoAcholesteryl acyltransferase. These regulatory events can be triggered by the addition to cells of cholesterol contained within LDL or by the addition of oxygenated sterols such as 25-hydroxycholesterol and 7-ketocholesterol added in solvents (2, 3). Cholesterol itself is not active when added to

* This work was supported in part by National Institutes of Health Grant HL 20948 and the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. The nucleotide sequencefs) reported in thispaperhas been submitted totheGenBankTM/EMBLData Bank with accession numberfsl J05056. T Recipient of Postdoctoral Fellowship HL 07524 from the National Institutes of Health. 1) Recipient of a Postdoctoral Fellowship from the Medical Research Council of Canada. The abbreviations used are: LDL, low density lipoprotein; HMGCoA, 3-hydroxy-3-methylglutaryl-CoA, kb, kilobase(s); SDS, sodium dodecyl sulfate; PCR, polymerase chain reaction; HPLC, high pressure liquid chromatography; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

cells in solvents, raising the possibility that the physiologic regulatory agent is an oxygenated sterol formed from LDL cholesterol within the cell. Oxygenated sterols carryout these regulatory actions at the transcriptional and post-transcriptional levels (4-8). They repress transcription of the genes for at least two enzymes in the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase and HMG-CoA reductase, and also of the gene for the LDL receptor. Transcriptional regulation is attributable, at least inpart, toa regulatory DNA sequence designated sterol regulatory element 1 in the 5’flanking region of each of the three genes (5-8). At the posttranscriptional level, oxygenated sterols act by enhancing the degradation of HMG-CoA reductase. They also activate acylCoA:cholesteryl acyltransferase through a mechanism that is independent of gene transcription andprotein synthesis (9). The proteins that mediate the actions of 25-hydroxycholesterol are unknown, but one candidate has been identified. Taylor and Kandutsch (10) described a protein that binds oxygenated sterols invitro roughly inproportion to their ability to repress cholesterol synthesis in vivo. We recently purified this oxysterol-binding protein 40,000-fold to homogeneity from hamster liver cytosol (11).The purified preparation containedtwo proteins with apparent M, of 96,000 and 101,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. An antipeptide antibody prepared against a peptide from one of the proteins reacted with both proteins, indicating that they were closely related. On gel filtration, these proteins formed an oligomer that had an apparent MI of280,000 (11). We hypothesized that the oligomer represented a trimer or a dimer that behaved anomalously on gel filtration, perhaps owing to an elongated shape. In this paper, we report the cDNA cloning and expression of the oxysterol-binding protein from rabbit liver. The deduced amino acid sequence reveals an apparentleucine zipper, a recently recognized structural motif that promotes oligomerization, especially in proteins that bind to DNA (12). EXPERIMENTALPROCEDURES

General Methods-Standard molecular biology techniques were used (13, 14). cDNA clones were inserted into M13 vectors and sequenced by the dideoxy chain termination method (15) using the M13 universal sequencing primer (16). Sequencing reactions were performed using a modified bacteriophage T7 DNA polymerase (17) with 35S-labelednucleotides or fluorescently labeled universal primer on an Applied Biosystems Model 370A DNA Sequencer. Poly(A)+ RNA was isolated by the guanidinium thiocyanate/CsCl centrifugation procedure (18) and oligo(dT)-cellulose chromatography (14). Northern blot hybridization of RNA was carried out asdescribed (19) using single-stranded 32P-labeledprobes. 25-[3H]Hydroxycholesterol (87 Ci/mmol) was obtained from Du Pont-New EnglandNuclear. All unlabeled steroids were obtained from Steraloids except for 1,25-

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cDNA Cloning of Oxysterol-binding Protein

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dihydroxyvitamin DB,which was kindly provided by Hoffmann-La sterol-binding protein in a PCR with 10 pl of the eluted phage. The reaction mixture was boiled, after which PCR was carried out sequenRoche. tially for 1.5 min a t 95 "C and for 5 min at 60 "C with Tag1 polymerase Amino Acid Sequenceof Peptides from Oxysterol-bindingProteinApproximately 400 pmol of oxysterol-binding protein was purified for 35 cycles. The sizes of the PCR products were determined by from Syrian hamster liver (11) and transferred from a 7.5% SDS- electrophoresis on a 1%agarose gel, and clones containing the largest polyacrylamide gel to nitrocellulose paper for solid-phase tryptic inserts were plaque-purified for further characterization (14). Construction of Expression Vector for Oxysterol-binding Proteindigestion (20). Peptides released from the paper were separated by reverse-phase HPLC on an Applied Biosystems Model 130A HPLC A cDNA containing the entire coding region of the rabbit oxysterolsystem using a Brownlee RP-300 CS column (2.1 X 100 mm). Sepa- binding protein mRNAwas constructed by joining the 5' EcoRI ration was performed in 0.1% (v/v) trifluoroacetic acid with a gradient fragment (nucleotides 1-1485) from X clone H9 and the 3' EcoRIDraI fragment (nucleotides 1486-2818) from X clone H20. To derive of 0-50% (v/v) acetonitrile over 120 min at a flow rate of 50 pl/min. Peaks were collected and brought to 1%(v/v) Lubrol and 0.1% (w/v) expression of the oxysterol-binding protein cDNA, mammalian ammonium acetate before being applied to a second HPLC gradient. expression vector pCMV2 (26) was used (kindly provided by David Separations were performed in 0.1% (w/v) ammonium acetate with a W. Russell, University of Texas Southwestern Medical Center at gradient of 0-50% (v/v) .acetonitrile over 60 min with the above Dallas). This vector contains the promoter-enhancer region of the column (20). Peaks from this second separation were collected on 1- major immediate early gene of the human cytomegalovirus, a syncm Whatman GF/C discs. Peptides were reduced, cysteine residues thetic polylinker sequence, the transcription termination and polywere alkylated (21), and the peptides were sequenced on an Applied adenylation region of the human growth hormone gene, and theSV40 DNA replication origin from plasmid pcD (26). Plasmid pOSBP was Biosystems Model 470A Sequencer (Table I). cDNA Libraries-Double-stranded cDNA libraries were con- constructed by ligating the rabbit oxysterol-binding protein cDNA described above into EcoRI-Smal-digested pCMV2. The plasmid was structed from Syrian hamster liver and New Zealand White rabbit liver poly(A)+ RNA (22) using a cDNA synthesis kit from Bethesda characterized by restriction mapping, grown in mass culture, and Research Laboratories. For the hamster liver cDNA library, first- banded in CsC12density gradients (14) prior to transfection. strand synthesis was primed with oligo(dT). After second-strand DNA Transfection and 25-Hydroxycholesterol Binding-COS-MG synthesis, the cDNA was methylated with EcoRI methylase and S- cells were grown in monolayer at 37 "C in a 5% CO, incubator in adenosylmethionine and ligated to EcoRI linkers. An aliquot (150 ng) medium A (Dulbecco's modified Eagle's medium supplemented with of the hamster liver cDNA was ligated to 250 ng of EcoRI-cleaved 10% (v/v) fetal calf serum, 20 mM Hepes, pH 7.4, 100 units/ml pUC13. The ligated cDNA was used to transform DH5 (maximum penicillin, and 100 pg/ml streptomycin). For experiments, cells were efficiency) cells (Bethesda Research Laboratories). Following ampli- seeded on day 0 at 6 X lo5 cells/lOO-mm dish in medium A. On day fication in mass culture (1 liter), the library was isolated and linear- 1, the cells were transfected with 6.6 pg of pOSBP or parent plasmid ized with SalI. Plasmids containing inserts greater than 2 kb were pCMV2/dish by the DEAE-dextran method as described previously size-selected on a0.7% agarose gel. The isolated plasmid was religated (27). On day 2, the cells were washed with Dulbecco's phosphateand used to transform D!H5 cells. Approximately 3 X lo6 colonies buffered saline, and themedium was replaced with Dulbecco's modiwere screened. Replicate filters were hybridized in 10% (v/v) form- fied Eagle's medium (minus Hepes) containing 10% (v/v) calf lipoamide containing 1.5 X 10' cpm/ml 32P-labeledoligonucleotide probe protein-deficient serum (28). On day 3, the cells were harvested and (see below). Filters were washed in 2 X SSC (I X SSC = 150 mM disrupted by repeated aspiration through a 25-gauge needle, after NaCl, 15 mM sodium citrate, pH 7) and 1%(w/v) SDS a t 55 "C.After which the extract was centrifuged at lo5 X g for 1 h at 4 "C. The a second round of colony hybridization, eight positive clones were resulting cytosol was assayed for 25-[3H]hydroxycholesterolbinding identified. Restriction mapping and dideoxy sequencing of the insert- using a modification of the dextran/charcoal assay (11). Each assay plasmid junctions indicated that all eight clones contained an iden- contained, in a finalvolume of 50 pl, 10 mM Tris-C1, pH 7.4, 200 mM tical fragment consisting of the 3' 2.2 kilobases of the mRNA for the KCl, 10% (v/v) glycerol, 1 mM disodium EDTA, 2% (w/v) polyvinyl oxysterol-binding protein. Additional screening of the pUC13 hamster alcohol, 5 mM dithiothreitol, 0.6 mM phenylmethanesulfonyl fluoride, liver cDNA library and a hamster liver X g t l O library (23) yielded no 50 p M leupeptin, 0.5 pg/ml pepstatin A, 0.5 pg/ml aprotinin, 10 pg of additional clones. cytosolic protein, and 1 p1 of ethanol containing the indicated conRandom hexamer- and oligo(dT)-primed rabbit liver cDNA was centration of 25-[3H]hydroxycholesterol(190 dpm/fmol) in the absynthesized and ligated to EcoRI linkers as described above. (The sence or presence of unlabeled steroid. Each reaction was incubated library was kindly provided by Robert Kowal (University of Texas for 12-18 h a t 4 "C.The 25-[3H]hydroxycholesterolwas then removed Southwestern Medical Center at Dallas).) cDNAs greater than 2 kb by absorption to dextran-coated charcoal (11). in size were isolated on a 0.7% agarose gel and ligated into EcoRIAntibodies and Zmmunoblotting-A synthetic peptide (obtained cleaved XgtlO DNA. After in vitro packaging with a XDNA packaging from MilliGen/Biosearch, San Rafael, CA) corresponding to amino extract (Stratagene),phage were plated out onhost strain Escherichia acids 737-755 of the rabbit oxysterol-binding protein was coupled to coli C600 hfl- cells. Approximately 1.5 X loe and 5 X lo5plaques were tuberculin-purified protein derivative (Statens Seruminstitut, Copenscreened from the random hexamer- and oligo(dT)-primed libraries, hagen, Denmark) using glutaraldehyde (29). Two New Zealand White respectively. Replicate filters were hybridized in 50% (v/v) formamide rabbits were immunized with 200 pg of coupled peptide in Freund's containing 1 X lo6 cpm/ml single-stranded M13 probe derived from the 5'-end of hamster oxysterol-binding protein clone pOSBP-28. complete adjuvant. Rabbit serum was assayed for anti-oxysterolPositive clones were characterized initially by PCR (see below) and binding proteinantibody by immunoblot analysis using partially then plaque-purified. Plate lysate DNA (24) from each clone was purified hamster liver oxysterol-binding protein (11).Immunoblotsubcloned into pUC18 and M13 vectors for restriction mapping and ting was performed as described (11) using antibody purified by protein-A Sepharose chromatography (30). Hamster liver oxysterolDNA sequencing. Polymerase Chain Reaction-The polymerase chain reaction was binding protein was partially purified for immunoblotting studies by ammonium sulfate fractionation followed by chromatography on Qused to derive an unambiguous probe for cDNA screens and to characterize hybridizing X clones. For probe synthesis, double- Sepharose and heparin-agarose (11). stranded hamster liver cDNA was used as a template for PCR (25) RESULTS as described previously (23). Oligonucleotide primers were based on the NH,- and COOH-terminal sequences of peptide 11 (Fig. 1).The The oxysterol-binding protein was purified from hamster two primers included all degenerate codons (Fig. 1).PCR primer-1 was end-labeled with [y3"P]ATP and T4kinase. Translation of the liver as previously described (ll),tryptic peptides were isowere obtained nucleotide sequence between the primers gave the expected amino lated by HPLC, and11peptidesequences acid sequence (Fig. 1).A 44-mer oligonucleotide corresponding to this (Table I). To prepare a unique probe for screening of cDNA sequence was synthesized and used to screen the hamster liver cDNA libraries, we used the polymerase chain reaction to obtain a library. To characterize the X g t l O clones that hybridized with the unique sequence for the DNA encoding peptide 11(Fig. 1). A probe, the plaque area was picked and eluted in 0.5 ml of 100 mM family of degenerate primers corresponding to the NH,- and NaCl, 8 mM MgSO,, 50 mM Tris-C1,pH 7.5, and 0.01% (w/v) gelatin. to doubleA primer of26 nucleotides (left arm) or 24 nucleotides (right arm) COOH-terminalends of peptide 11 were hybridized the corresponding to the X g t l O sequences flankingboth sides of the stranded cDNA preparedfromhamsterliver.Oneof 32P. Following PCR, the unique EcoRI site was used in combination with an oligonucleotide primerswas5"end-labeledwith corresponding to the cDNA sequence of the hamster or rabbit oxy- amplified productswere subjected to electrophoresis,and the

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fragment of the expected size was isolated and sequenced by the procedure of Maxam and Gilbert (31). The DNA sequence of the amplified product corresponded to the amino acid sequence of peptide 11. We prepared an oligonucleotide corresponding to thissequence and used it toscreen an oligo(dT)primed cDNA library prepared from hamster liver. Eight positive clones were obtained, and all were found to contain the same cDNA insert of 2.2 kb. Extensive further screening of the hamster liver library failed to yield additional clones. For this reason, we turned to screening cDNA libraries prepared from rabbit liver. TABLE I Sequence of tryptic peptidesfrom hamster oxysterol-binding protein: comparison with sequence deduced from rabbit cDNA The sequences shown were obtained from tryptic peptides isolated from the purified hamster oxysterol-binding protein as described under "Experimental Procedures." The corresponding rabbit sequences deduced from the cDNA cloning (Fig. 3) are identical to the hamster data except where indicated. Asterisks denote ambiguous residues in the protein sequencing. Hamster peptide

1

2 3 175-182

4 5 6 7

Amino acid position in rabbit cDNA sequence

Amino acid sequence

380-397 GSNISGASSDISLDEQYK VVGPGPAAIAAPGGGGAGPPAVGGGGG 9-35 V 281-290 FLMLAQT*SK V H VTALELAK EYWECKEKQD 792-801 220-232 VEDLST*NDLIAK C 327-342 ATVLPANTPGSAGSGK

H S 8 9

10

494-511 437-450

SKPFNPLLGETFELDRLE PMPVNFNEPL*MLQ S ENSLEQLCYVAAFTVSSYGTT

469-489

S 11

692-714

AENMYYFSELALTLNAWEGGTAP

PCR Primer-1 ~ZP-OCI[G(

4 ATG

[Ala Clu Asn

Met

TAC TAC TTC TCA GAG CTT

GCC

CTC

ACT CTC MC GC]

cooH

Tyr Tyr Phe Ser Glu Leu Ala Leu Thr Leu Arn xla Trp Clu Cly Gly+

HZ

CGI ACC CT; CCN CC f

PCR Primer-2

FIG. 1. Generation of cDNA probefrom amino acid sequence of peptide l l of hamster oxysterol-binding protein. The bored nucleotide sequence was generated by PCR using primer1 and primer-2 as described under "Experimental Procedures."

FIG. 2. Restriction map and sequencing strategy for rabbit oxysterol-binding protein. The structure of the oxysterol-binding protein mRNA is shown at thetop, with the coding region denoted as ahatched box and the5'- and 3"untranslated regions shown as solid lines. Restriction sites used to obtain subclones for DNA sequencing are indicated above the mRNA. Beloware shown the cDNAs obtained from the random hexamer-primed library (XH20, XH9, and XH1) and the oligo(dT)-primed library (XdT3). The arrows indicate the direction and extent of sequencing.

h dT3

Random hexamer-primed cDNA libraries and oligo(dT)primed rabbit liver libraries were screened with a probe derived from the 5'-end of the hamster sequence. Fig. 2 shows four of the clones isolated from the rabbit liver cDNA libraries and outlines the sequencing strategy. Fig. 3 shows the sequence of the rabbit cDNA. The first methionine codon occurred at nucleotide 75of the cDNA sequence, and this was followed by an open reading frame encoding a protein of 809 amino acids. This was followed by a 3"untranslated region of 1380 nucleotides, which did not extend to the poly(A) tail. In the proposed cDNA structure, the putative 5'-untranslated region comprised 74 nucleotides. The cDNA did not contain an in-frame terminator codon upstream of the proposed initiator methionine. The NH2 terminus of the protein is blocked; and therefore, the NH2terminal protein sequence is unknown. We believe that the first methionine in the cDNA is the initiator methionine for two reasons. 1) Transfection of this cDNA into COS-M6 cells leads to theproduction of an active oxysterol-binding protein of the appropriate molecular weight (discussed below); and 2) primer extension analysisof rabbit liver poly(A)+ RNA using an oligonucleotide primer corresponding to nucleotides 77106 yielded a major product of 124 nucleotides (data not shown). This result suggests that the 5'-untranslated region extends only 18 nucleotides beyond the sequence shown in Fig. 3, and it is therefore unlikely to contain any additional methionine codons. Definitive delineation of the 5'-untranslated region and the NH2 terminus of the protein will not be available, however, until genomic clones are obtained. The cDNA for the oxysterol-binding protein hybridized to a mRNA of 4.6 kb in the liver and brain (Fig. 4). A much smaller amount of hybridizing mRNA was observed in the kidney. We believe that the additional length of the mRNA as compared to thelength of the cDNA represents additional 3"untranslated sequence. The cDNA contained sequences encoding all 11of the peptides that hadbeen sequenced from the hamster oxysterol-binding protein(Table I). The few conservative changes in sequence were attributed to species differences between hamster and rabbit. The coding region of the oxysterol-binding protein cDNA was inserted into an expression plasmid and introduced into COS-M6 cells by transfection. The oxysterol-binding protein was visualized in cytosolic extracts by immunoblotting with an antipeptide antibody (Fig. 5). Lanes 1 and 4 are control lanes showing the oxysterol-binding protein that had been partially purified from hamster liver cytosol. The protein showed its characteristic closely spaced doublet at approximately 96 and 101 kDa. Lane 2 contains cytosol from COSM6 cells that were transfected with the parent plasmid that did not contain the cDNA insert. Lane 3 contains cytosol


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FIG. 3. Nucleotide and predicted amino acid sequences of rabbit oxysterol-binding pr0tei.n. Nucleotides are numbered on the right; amino acids are numbered on the left. Residue 1 is the putative initiatormethionine. Amino acid sequences corresponding tothe tryptic peptides isolated from the rabbit oxysterol-binding protein are shown in Table I. A putative leucine zipper is underlined. The5'-untranslatedand coding regions were sequenced on both strands. Approximately 75% of the 3'untranslated regionwas sequenced on both strands.

2711 2834

2954 a74

3194 3314 3434

sn

3671 3194

Be4

from COS-M6 cells that were transfected with a plasmid containing the coding regionof the oxysterol-binding protein cDNA. This cytosol contained a closely spaced doublet of proteins that appeared to have molecular weightsidentical to the oxysterol-binding protein from hamster liver. The cytosol from the cells transfected with the oxysterolbinding protein cDNA bound 25-[3H]hydroxycholesterolwith high affinity (Fig. 6B). Cytosol from cells transfected with the vector alone showed only a low level of 25-[3H]hydroxycholesterol binding (Fig. 6A). The protein in the transfected cells bound 25-[3H]hydroxycholesterol with approximately the same affinity ( K d = 10 n.M) as the purified liver protein (& = 8 nM) (11). To examine the binding specificity of the oxysterol-binding protein encoded by the cDNA, we obtained cytosol from transfected COS-M6 cells and incubated it with 25-['HH]hydroxycholesterol in the presence of varying amounts of unlabeled competitor steroids (Fig. 7). As expected, unlabeled 25hydroxycholesterol com.peted effectively for the 25-[3HJ hydroxycholesterol binding. 20-Hydroxycholesterolhad a lower affinity (Fig. 7A),and 7-ketocholesterol was even lower (Fig. 7B). Pregnenolone, p-sitosterol, 1,25-dihydroxyvitamin Ds,

and dexamethasone were all inactive competitors (Fig. 7 , A and B). These binding specificities are identical to thespecificities previously ascribed to the oxysterol-binding protein (11,32). DISCUSSION

This paper reports the cloning and expression of an oxysterol-binding protein from rabbit liver. The cDNA produces a protein that has the same molecularweight and binding properties as the oxysterol-binding protein previously described in mouse and hamster liver cytosol (10, ll, 32). The most striking feature of the predicted amino acid sequence is the apparent "leucine zipper" motif. A leucine zipper is defined by the presence of at least 4 leucine residues spaced precisely 7 residues apart, which places them on the same face of an a-helix (12, 33). This motif was first recognized by Landschulz et al. (12) in several proteins that are believed t o regulate gene transcription, including C/EBP, yeast GCN4, and oncogenes fos, jun, and myc. The characteristic feature of all of these proteins is that they form dimers in solution, either homodimers or heterodimers. Dimerization is dependent upon the interaction between twoleucine zipper


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