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Molecular cloning and functional expression of a cDNA for mouse squalene synthase
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Biochi~ic~a et Biophysica AEta
ELSEVIER
Biochimica et Biophysica Acta 1260 (1995) 49-54
Molecular cloning and functional expression of a cDNA for mouse squalene synthase Takayuki Inoue 1, Takashi Osumi, Shingo Hata * Department of Life Science, Faculty of Science, Himeji Institute of Technology, Kamigori, Ako-gun, Hyogo 678-12, Japan Received 24 June 1994
Abstract
Using a probe obtained by PCR amplification, a full-length cDNA encoding squalene synthase was isolated from a mouse liver cDNA library. Its nucleotide sequence had an open reading frame for a 416 amino acid polypeptide (calculated molecular mass, 48 kDa). In vitro transcription of the cDNAfollowed by in vitro translation produced a protein of the expected size. The deduced amino acid sequence was 93%, 88% and 46% identical to those of the rat, human and budding yeast squalene synthases, respectively. Blotting analyses showed that the mRNA is 1.6 kb in size and that less than two copies of the gene are present in the mouse genome. To establish the enzyme activity, the entire coding region was subcloned into an expression plasmid so that it was in frame with the N-terminal region of fl-galactosidase. Escherichia coli, which was transformed with the recombinant plasmid, expressed high activity of converting farnesyl diphosphate into squalene. Keywords: Squalene synthase; Cholesterol biosynthesis; cDNA cloning; Recombinant enzyme; (Mouse liver)
I. Introduction
Squalene synthase (farnesyl diphosphate:farnesyl diphosphate farnesyl transferase, EC 2.5.1.21) is an integral microsomal enzyme. It condenses two molecules of farnesyl diphosphate (FPP) into squalene, via an intermediate, presqualene diphosphate [1,2]. Since FPP is metabolized to a number of isoprenoid compounds, squalene synthase is the first enzyme specific for cholesterol biosynthesis. Therefore, the reaction represents a potentially important control point for balancing sterol synthesis and other isoprenoid syntheses. In cultured fibroblasts, the level of squalene synthase activity dramatically varies
Abbreviations: FPP, farnesyl diphosphate; PCR, polymerase chain reaction; SSC, standard saline citrate; TLC, thin-layer chromatography. The nucleotide sequence data reported in this paper have been submined to the DDBJ/EMBL/GenBank Databases under the accession number D29016. * Corresponding author. Present address: Laboratory of Applied Botany, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan. Fax: +81 75 7536146. 1 Present address: Department of Gene Sciences, Faculty of Science, Hiroshima University, Higashi-Hiroshima 724, Japan. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSD10167-4781(94)00178-2
according to the demand for sterol synthesis [3]. In rat liver, the level is also altered by feeding inhibitors of hydroxymethylglutaryl-CoA reductase [4]. Little is known, however, about the mechanisms of these observed phenomena. It had been very hard to isolate squalene synthase gene until highly purified enzyme preparations were obtained from yeast [5] and rat [4]. Nevertheless, cloning has been achieved from budding yeast [6,7], fission yeast [8], rat [9] and man [8,10,11], successively, in a short period. The mouse is the most suitable animal for gene manipulation experiments [12]. Using transgenic mice, control mechanisms of lipid metabolism and the causes of metabolic diseases have been elucidated. For example, atherosclerosis research greatly advanced with overexpression of the apolipoprotein E gene [13] or targeted disruption of the gene [14,15]. It may be possible to generate recombinant animals with altered abilities of sterol synthesis. As a first step to understand the complex regulation of cholesterol synthesis in mouse, we isolated and characterized a cDNA for mouse squalene synthase. We also established the enzymatic activity by expressing a recombinant protein in Escherichia coli.
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2. Materials and methods
2.4. In vitro transcription / translation
2.1. Polymerase chain reaction (PCR)
pMMSS-2 was linearized by digestion with XhoI. mRNA was transcribed with T7 RNA polymerase (Bethesda Research Laboratories) by the standard method [17]. About 1 /xg of this material was translated with 5 /xl of a rabbit reticulocyte lysate (Amersham) in the presence of [35S]methionine (4 /zCi) at 30°C for 1 h. The protein products were electrophoresed in a SDS/10% polyacrylamide gel and then detected with a Bio-imaging analyzer, BAS 2000 (Fuji).
Two oligonucleotide primers were synthesized. H042 (5'-GCCTCGAGGATGGAGTTCGTGAAGTGT-3') and H041 (5'-CGAATTCAGTGT£CTCTCTGGACA-3') were designed to be specific for the sense 5' end and antisense 3' end, respectively, of the coding region of rat squalene synthase cDNA [9]. They had XhoI and EcoRI sites for the cloning of the PCR products. The 50 /zl reaction mixture comprised 30 ng of random hexamer-primed cDNA of mouse F9 cells [16], 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgC12, 0.01% gelatin, 0.2 mM each of dTFP, dCTP, dGTP and dATP, 1.5 units of Takara Taq DNA polymerase (Takara), and 1 /xM each of the primers. The PCR conditions were 94°C for 1 min, 45°C for 2 min, and 72°C for 3 min, for 40 cycles. The amplified products (1.3 kb in size) were digested with XhoI and EcoRI, and then ligated into XhoI-EcoRI digested pBluescript II K S ( - ) (Stratagene). Partial sequencing of the resulting plasmid showed that the subclone is highly homologous to the rat squalene synthase gene. Its insert was cut out by digestion with XhoI and EcoRI, and then used as a probe for library screening. 2.2. Screening of a cDNA library
An oligo(dT)-primed AZAP cDNA library containing liver cDNA inserts of B6CBA mouse (C57 Black/6 X CBA) (Stratagene) was plated with E. coli BB4, and then blotted onto Hybond-N + membranes (Amersham; 5 . 1 0 4 plaques/sheet). After radiolabeling of the probe using a Megaprime labeling kit (Amersham) and [32p]dCTP, hybridization was carried out at 65°C for 24 h in 6 x SSPE (1 X SSPE = 0.18 M NaC1, 0.01 M sodium phosphate (pH 7.7), 1 mM EDTA), 5 X Denhardt's reagent, 0.5% SDS, and 100 /zg/ml salmon sperm DNA [17]. The membranes were initially washed with 1 × SSC (1 × SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) and 0.1% SDS at room temperature once for 15 min, and then with 0.1 X SSC and 0.1% SDS at 60°C four times for 15 min each. 2.3. Subcloning and DNA sequencing
The longest cDNA insert (1.6 kb in size) of positive clones was cut out with EcoRI and then ligated into the EcoRI site of pBluescript II. The resulting plasmid, designated as pMMSS-2, was digested with exonuclease III (Promega), and then an overlapping series of deletions was prepared. The nucleotide sequence was determined on both strands by the dideoxy chain termination method [18] using [32p]dCTP and Sequenase (US Biochemical). Sequence data were analyzed with GENETYX software (Software Development).
2.5. Northern and Southern analysis
30 /xg of mouse liver total RNA (Clontech) was fractionated by electrophoresis in a 1.2% agarose gel containing 2.2 M formaldehyde and then blotted onto a HybondN ÷ membrane. 4/xg per each lane of mouse chromosomal DNA preparations of B A L B / c mouse and F9 cells (gifts from Dr. T. Tsukamoto of this department) was digested with BamHI, EcoRI or HindlII, separated by 0.8% agarose gel electrophoresis, and then blotted onto a membrane. The probes used were fragments of pMMSS-2, i.e., an EcoRIPvulI fragment ( ~ 1.3 kb in size) containing the entire coding sequence of the cDNA was used for Northern hybridization, and an RsaI-EcoRI fragment ( ~ 420 bp in size) containing the 3' end of the coding region and the 3'-untranslated region was used for the Southern hybridization. The probes were radiolabelled with [32p]dCTP. The membranes were hybridized and washed as described above. 2.6. Construction of an expression vector of mouse squalene synthase
The coding region of mouse squalene synthase was reamplified by PCR using the H042 and H041 primers, and pMMSS-2 as a template. The amplified fragment was digested with XhoI and EcoRI, and then inserted between the XhoI and EcoRI sites of pBluescript II. The resulting plasmid was designated as pXE-7. 2. 7. Preparation of the enzyme
Prokaryotic expression of mouse squalene synthase was carried out based on the method of McKenzie et al. [9] with some modifications. E. coli XL-1 Blue cells were transformed with pXE-7, pBluescript II KS( - ) without an insert was also introduced into cells as a negative control. A single colony of the transformant was inoculated into 25 ml of TB medium in a 100 ml Erlenmeyer flask. The bacteria were grown at 37°C with shaking for 3 h to an optical density of 0.3 at 600 nm. IPTG, 0.5 mM final concentration, was added to induce the lacZ promoter, and then the bacteriawere regrown at 37°C for an additional 3 h. They were collected by centrifugation, and one-fourth of
T. Inoue et al. / Biochimica et Biophysica Acta 1260 (1995) 49-54
them (0.1 g in wet weight) was suspended in 0.5 ml of 0.1 M Tris-HC1 (pH 8.0), 50 mM EDTA and 1 m g / m l lysozyme, and then incubated at 30°C for 10 min. After freezing at - 8 0 ° C and thawing on ice, the cells were disrupted by ultrasonication and then centrifuged at 9000 × g for 3 min. The resulting superuatant was passed through a Sephadex G-50 column (Pharmacia), which had been equilibrated with 100 mM Tris-HC1 (pH 7.6) and 0.2 mM dithiothreitol. The protein concentration was determined by the method of Bradford [19] and adjusted to 1.7 mg/ml.
2.8. Assaying of the enzyme activity The reaction mixture (final volume, 100 /.d) contained [laC]FPP (American Radiolabeled Chemicals; 125 nCi, 11.5 /xM), 3 mM NADPH, 5 mM MgC12, 0.1 mM dithiothreitol, 10 mM KF, 50 mM Tris-HCl (pH 7.6), and the enzyme (85 tzg protein). It was incubated at 37°C for 1 h. After the incubation, 50 ng each of squalene and famesol was added as carriers with the aid of 10/xl of ethanol, and then 10 /xl of the reaction mixture was applied to a silica gel G TLC plate (Merck). The sample was separated with ethylacetate-benzene (1:4, v / v ) , and the radioactivity of each spot was determined with a Bio-imaging analyzer.
51
1 CGCTCCCAGCAGTCGCAAGGATGGAGTTCGTCAAGTGTCTAGGCCACCCGC4~GGAGTTCT M E F V K C L G H P E E F Y 14 61 ATAACCTGCTGCGAI-[CCC-CATGGGAGGC~GGAAI-TFTATACCCAAGATGGACCAGG N L L R F R M G G R R N F I P K M D O D 34 121 ACTCACTCAGCAGCAC-CTTGAAGACCTGCTACAAGTATCTCAATCAGACCAGTCGCAGCT S L S S S L K T C Y K Y L N O T S R S F 54 181 TTGCCGCGGI-[ATCCAGGCC-CTGGAT@CATACC£K;ACC-CCATATGTGTGI-FCTACC A A V I 0 A L D G D I R H A I C V F Y L 74 241 TC431-FCTCCGAGCCCTGGATACAGTGGAGGATGACATGAGCATCAGTGTGGA~GA V L R A L D T V E D D M S I S V E K K I 94 301 TCCCACTGCTGTGTAACTTCCACAC-lIFCCTCTATGACCCAGAGTGGCCK~I-FCACTGAGA P L L C N F H T F L Y D P E W R F T E S I14 361 GCAAGGAGAAGGACCCV~CAAGTGCTGGAGGACTTCCCCACGATCTCCCTGGAGT IFAGAA K E K D R 0 V L E D F P T I S L E F R N 134 421 AI-IT ' GGCTGAGAAATATCAAACAGTGATCCC~TGACATCTGCCACCAGAT~TGT~ L A E K Y O T V I D D 1 C H 0 M G C G M 154 481 TGGCAGAA1-FTGTAGACAAGGATGTGACCTCCAAACAGGACTGGGACAAGTACTGCCACT A E F V D K D V T S K 0 D W D K Y C H Y 174 541 ACGT-FGCTGC£K;TGGT~I-FGGCClI-fCTCGTCTATTCTCTGCCTCAGAGITTGAAG V A G L V G i G L S R L F S A S E F E D 194 601 ACCCCATAG1-FGGTGAAGACATAGAGTGTGCCAACTCAATGGGTCTG-IFCCTGCAGAV / ~A P I V G E D I E C A N S M G L F L 0 K T 214 661 CAAATATCA-IFCGTGATTATCTGGAAGACCAACAGGAAGGAAGGAAGt i I IGC-CCTCAGG N I I R D Y L E D 0 0 E G R K F W P 0 E 234 721 AGGTGTGGGGCAGATACAF 'IAAGAAGF 'IGCPk~GACTTTGCTAAGCCAGAGAACGTAGATG V W G R Y I K K L E D F A K P E N V D V254 781 TGGCCGTGCAGTGC-G [l AATGAACTCATAACCAACACCCTACAGCACATCCCTGACGTCC A V 0 C L N E L I T N T L 0 H I P D V L274 841 TCACCTACCTGTCAAGGCTCCC-C¢V~CCAGAGTGTGTTTV / kCTTCTGTGCTATTCCACAC-G T Y L S R L R N 0 S V F N F C A I P 0 V 294
901 TAATGGCCA'I-FGCCACACTGGCTGCCTGTTACAATAACCAGCAGGTAI-FCAAAGGAGTAG M A I
A T
L A A C Y N N 0
0 V F K G V V 314
961 TGAAGAI-FCGGAAGGGC-CAAGCAGTCACCCTCA TGATGGATGCCACCAACATC-CCTGCOG K I R K G 0 A V T L M M D A T N M P A V 334 1021 TCAAAGCTATCATATACCAGTACATAGAAGAGAI]-I'ATCACCGGATCCCCAACTCAGACC K A I I Y 0 Y I E E I Y H R I P N S D P354
3. Results and discussion
3.1. Preparation of a probe for library screening Since mouse is a species very close to rat, we presumed that the 5' and 3' ends of the coding region of the squalene synthase gene might be conserved. Based on this idea, a PCR experiment involving primers specific to the rat cDNA [9] and a mouse F9 cDNA library [16] as a template was carried out. Analysis of the PCR products by agarose gel electrophoresis showed a major product of the expected size, 1.3 kb (data not shown). Subcloning and partial sequencing of the amplified product revealed that its nucleotide sequence is closely related to that of the rat squalene synthase cDNA [9]. Therefore, we concluded that the PCR product encoded mouse squalene synthase.
108t CATCATCAAC-CAAAACCAAC,-CAGGTCATCTCCAAGATCAGGACACAGAACC'FICCC/kACT S S S K T K 0 V I S K I R T 0 N L P N C 374 1141 GCCAGCTCATCTCCCGAAC-CCACTACTCGCCCAlqqACCTGTCATTTATCATC'CTC T-FGG 0 L I S R S H Y S P I Y L S F I M L L A 394 1201 CTC-CCCTGAGCT~GTACCTGAC-CACCCTGTCCCAGGTCACAGAAGACTATGTC CAGA A L S W O Y L S T L S 0 V T E D Y V 0 R 414 1261 GAGAACACTGA]{l IGTTTAC-CCGGAAGTGGAAG1-FCCCGTGGAGTGGG1111 ICCTTI-r E H * (416) t 321 CCTCCAGCTGGATI-FTGACTTCCCI-IGr; i i I CCTCCTACTCTAAAATCTTTGGGAGAAC 1381 TGAGTGTGGGACCTITA~CT~GGATGCCTTGCCCTCAC.-CAGCC TGGT 1441 C-CTGGCT~CI-IGGI-rCCTCTGCCTCTTGTAGCCACTGGCAGCGTGCCGACTGCTGCA 1501 CTTGTGAGGCCACGTGTGATGGTCACAAGAGCCTAGTGAACCTC-C-CTAGAATGCTGAI-IG 1561 GATFFATTTAATTTGAAACAGCC13TGAA TACCTATGACAATAGAAAATGAAAC.-CAAAAA 1621 A A A A A A ~
Fig. 1. Nucleotide and deduced amino acid sequences of the mouse squalene synthase cDNA clone, pMMSS-2 was sequenced in its entirety. Nucleotide positions (beginning at the 5' end) are indicated on the left, and amino acid positions (numbered from the putative translation initiation codon) are indicated on the right. A stop codon (TGA) at the end of the translated sequence is marked with an asterisk.
3.2. Isolation and sequencing of a cDNA clone for mouse squalene synthase Next, we screened a mouse liver cDNA library using the PCR product as a probe. About 60 positive spots were detected for 9- 105 clones. Six recombinant phages were picked at random and the longest cDNA insert among them was subcloned into pBluescript II. The resulting plasmid, designated as pMMSS-2, had an open reading frame for a 416 amino acid polypeptide (Fig. 1). Through analogy with the enzymes of rat and man [8-11], we think the ATG codon at nucleotide position 21 is the transla-
tional initiation codon, though no in-frame stop codon was found upstream. The calculated molecular mass of the polypeptide is 48 126. 3.3. In vitro transcription and translation In order to check the validity of the predicted molecular mass, in vitro transcription followed by in vitro translation was carried out using XhoI-cut pMMSS-2 as an initial material. As shown in Fig. 2, a rabbit reticulocyte lysate
T. Inoue et a l . / Biochimica et Biophysica Acta 1260 (1995) 49-54
52
1
49
2
3.6. Southern blot analysis
kDa I ~I L
~i i
To obtain information about the copy number of the mouse squalene synthase gene, Southern blot analysis was carried out using genomic DNA preparations of B A L B / c mouse and F9 cells. The probe, which contained the C-terminus of the coding region and the 3'-noncoding region, detected two bands in each lane (Fig. 5). Therefore, it can be said that the copy number is less than two. Further study is necessary to clarify whether or not the mouse squalene synthase gene is a single copy.
3. 7. Establishment of the enzyme activity
Fig. 2. In vitro transcription and translation analysis. The translation mixture contained the pMMSS-2-derived R N A template (lane 1) or no R N A (lane 2). The products radiolabeled with [35S]methionine were
separated by 10% SDS-polyacrylamide gel electrophoresis.
We constructed an expression vector by inserting a PCR-amplified coding region of pMMSS-2 into pBluescript II. The resulting plasmid, designated as pXE-7, had a nucleotide mismatch compared to pMMSS-2, since the H042 primer had the 5'-terminal sequence of the rat squalene synthase gene. Nevertheless, the deduced amino acid 49
Mouse Rut Human Yeast
NEFVKC~G-~E~iFYNLLRFRMH~RRNFIPKM~!~DSL~SS~!KTOYKY~N~ ~EFVKC~:G-~E~iFYNLLRFRM6~RRNFIPKM~iRN~L~NS~KTOYKY~DQ ~EFVKC~G-H~iE~FYNLVRFRIO~KRKVMPKM~QD~L~HS~iKTGYKY~NQ MGKLL~AL~V~IMKAALKLKF-CRTPLFSIY~Q-ST~PY~:LHOFHLLNL
produced a major protein of 49 kDa depending on the pMMSS-2-derived RNA template. The observed molecular mass of the in vitro expressed protein is fairly consistent.
Mouse Rat Human Yeast
~8RHFAAVI~A~DGDIKHAICV~,LV~LBi~D#S{~HVEKKIP~CN ~HR~tV|i~A~D~DI~HAVCV~II~MBIV~N~liHVEK~IP~RN ~iSR~Yl]I~,I~A~D~EM~AVCI~V~L~L~Ti!SVEK~VP~HN ~6~F~IRE~HPEL~NCVTLF~ilgI~JIL~I~S!~iHHDL[ID~RH
3.4. Comparison of mouse squalene synthase with the enzymes of other species
Mouse Rat Human Yeast
~TF~YDPE~RF--TESKE{D~E~PT!I%~RNLAEK~T~DD|~C ~flTF~iYEPE~RF--TESKE~H~V~IE~PTISLE~IRN~AEK~T~AD~IC ~SF~iYQPDWRP---MESKE~D~E~PT~:SL~RN~AEKY~T~ADIC ~EKL:LLTKNSFDHNAPDV~DRAV~TBi~HSIiLI~HK~KPE~E~KE~!T
147 147 147 148
Mouse Rat Human Yeast
HOJ~fiC~MAEFVDKD.......VTSKQDW~K~O~¥~h@~V~II~S~FSASE HR~C~i~EFLNKD ...... VTSKQDW~K~II~,~%~I~!~H~FSASE RR~I~II~EFLDKH ...... VTSEQEW~K~"II{~!I~!~S~$SASE EK~N~{DYILDENYNLNOL~TVHDY~V¥~¥V{~iDH~TRL!IVIAK
191 192 191 198
Mouse Rat Human Yeast
~EDPIVHEDIECAN8MHL~L~KTNI~:~B?LHO~E~K~QH~GR~IK gEDPIVSEDTECANS{i~'tK|~L~E~Q~J{FQ~SKYVK
241 241
~NESLYSNEQLYESM~I[%L~I~I~RI~YN~LVDa~S~M~ilWS~YAP
248
Mouse Rat Human Yeast
K~E~t,A~VDVAVQOL§IE~IT~TLQ~IP~LT~SRLRN~iV~N~OA~ K~E~iVK~.~VDVAVK~LNE~ITNALO~IP~I~SRLRN~$V~N~:I K~OD~A~#IDLAV~%NE~ITNALHHIP~I~SRLRN~SV~N~ ~K~iMK~:NEQLGLDfflKH~VLNALSHVI~L~ASIHE~ITFQFOAI~I
291 291 291 298
Mouse
Rat Human Yeast
P~MAI~CYXHiQQ~FK~iV~iKiI{LK.GQAVTLIMMDA-TNMPA~KAIIY~Q ~%?/~&I!~TI,/tACY~NHQ~FK~V~[I~I[~AVT~MMDA-~NMPA~KAIIYQ ~CYNN~Q~FK~A~AVT~MMDA-~!NMPA~KAIIYQ P~VI~AI~i~LVF~NRE~LH~N~i~I~TTCY~ILKSR~LRGC~EIFDMY
340 340 340 348
Mouse Rat Human Yeast
YIEBIYHRI PNSB~HSS~TKQV~SK IRTt~NLPNCIILISRSHYSPIYLS-E YIEEIYHRVPNS~SAS~KQLt~NI:RTi~SLPNCQLISRSHYS~IYLS-~ YMF,E{YHRIPDSD~SSSI~TRQI|~T ~RTQNLPNC~LI SRSHYS~IYLS-~ LRD-{KSKLAVQD~NFL~LNI~|~K IEQFMEEMY~DKLPPNVK~NETPI ~
389 389 389 397
Mouse Rat Humau Yeast
. . . . . . . . . . . . . . . . . . I MLLAALSWQY~ST-~QVTED~VQR EH ......................... IMLLAALSWQY~ST-LHQVTED~VQR- EH . . . . . . . . . . VMLLAALSWQY~TT-~$QVTEDYVQTOEH LKVKEI~HRYDD ELVPTQQEEEYKFNMV~S IIL$~LLG FIYI YTLMRA
416 416 417 444
Fig. 3 shows protein comparison of the rat, mouse, human and budding yeast squalene synthase sequences. Highly conserved segments A, B and C [9] of known squalene synthases corresponded to amino acids 171-186, 208-228, and 283-301, respectively, of the mouse enzyme. These segments were suggested to represent the crucial regions for the active site [8,9]. The amino acid residues in the segments were almost completely conserved among the reported squalene synthases. The overall amino acid sequence of the mouse enzyme was 93%, 88%, and 46% identical to those of the rat, human and budding yeast enzymes, respectively.
3.5. Northern blot analysis On Northem blot analysis, a single band corresponding to ~ 1.6 kb in size was detected for mouse liver (Fig. 4). This is in contrast to the results for human squalene synthase, for which two or three distinct transcripts were observed [8,10,11], but is similar to in the cases of budding yeast [7,8] and fission yeast [8], for which a single band was detected. The pattern of rat squalene synthase transcripts has not been reported. The reason for the interspecies discrepancy is not yet known.
49 49
48 99 99 99 98
acid sequence alignmemt of squalene synthase polypeptidcs from rat, mouse, man and yeast. P~mino acids identicalthroughout are shaded. Numbers indicate amino acid residues in the sequences. Fig. 3. A m i n o
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T. Inoue et al. / Biochimica et Biophysica Acta 1260 (1995) 49-54
1
2
SF--
Sq-
1.
6
k b
FOH--
Fig. 4. Northern blot analysis of the mouse squalene synthase transcript. A 30 /xg sample of mouse liver total RNA was electrophoresed through denaturing 1.2% agarose, transferred to a Hybond-N + membrane, and probed with the 32P-labeled 1.3 kb EcoRI-PvulI fragment of pMMSS-2, which contained the entire coding sequence. The size of the detected band was estimated using a poly(A)-tailed RNA ladder as molecular size markers.
1
2
3
4
5
6
O--
Fig. 6. Prokaryotic expression of mouse squalene synthase activity by the recombinant plasmid, pXE-7. Cell-free extracts of E. coli transfected with pXE-7 (lane 1) or pBluescript II (lane 2) were incubated with [14C]FPP as described under Materials and methods. The reaction products were separated by TLC and detected with a Bio-imaging analyzer. The positions of squalene (Sq), farnesol (FOH), and the chromatographic origin (O) and solvent front (SF) are indicated. Unconverted FPP remained at the origin.
(kb)
n
19. u
7.
7
6.
2
4°
3
--
3.
5
-
2.
7
--
W
Fig. 5. Southern blot analysis of the mouse squalene synthase gene. Samples of genomic DNAs from B A L B / c mouse (lanes 1-3) and F9 cells (lanes 4-6) were digested with BamHI (lanes 1 and 4), EcoRl (lanes 2 and 5), or HindlII (lanes 3 and 6), subjected to electrophoresis through 0.8% agarose, transferred to Hybond-N ÷ membranes, and then probed with the 420 bp RsaI-EcoRI fragment of pMMSS-2, which contained the C-terminal of the coding region and the 3'- untranslated region. The )t-EcoT14I digest served as molecular markers.
sequence of pXE-7 was exactly the same as that of p M M S S - 2 . Partial s e q u e n c i n g of p X E - 7 showed a correct open reading frame for expression of a chimeric protein c o m p o s e d of the 26 a m i n o acid N-terminal end of /3galactosidase, followed by the entire mouse squalene synthase (data not shown). E. coli is u n a b l e to synthesize sterols, hence it lacks squalene synthase. Biochemical assaying of cell-free extracts of p X E - 7 - t r a n s f o r m e d E. coli established the efficient c o n v e r s i o n of F P P to squalene (Fig. 6, lane 1). A n apparent specific activity of 87 p m o l / m g protein per m i n was observed. On the other hand, the activity of E. coli transformed with vector only was negligible (Fig. 6, lane 2). Therefore, the c D N A for m o u s e squalene synthase e n c o d e d a functional e n z y m e . Farnesol is likely to be generated from F P P through the action of e n d o g e n e o u s phosphatases in E. coli. 3.8. P e r s p e c t i v e s
The c D N A c l o n i n g of m o u s e squalene synthase will lead to the isolation of m o u s e g e n o m i c D N A clones for the e n z y m e . A n a l y s i s of the promotor region and its trans-
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T. Inoue et al. /Biochimica et Biophysica Acta 1260 (1995) 49-54
acting factors may allow elucidation of the regulatory mechanism of the gene expression. Since squalene synthase is the first committed enzyme in cholesterol biosynthesis, it will also be feasible to generate a recombinant mouse with abnormal cholesterol synthesis. This attempt may give new insights into the regulation of cholesterol synthesis among a number of isoprenoid biosyntheses. Specific inhibitors of squalene synthase have attracted much attention as cholesterol-lowering drugs without side effects [20-22]. The functional expression of squalene synthase in E. coli may be a convenient system for screening novel inhibitors of the enzyme.
Acknowledgements We wish to thank Y. Yabusaki, T. Sakaki and Y. Shigeri for the retrieval of literature and the valuable discussions. We also thank T. Tsukamoto for the gifts of mouse chromosomal DNAs. This work was supported in part by research grants from the Ministry of Education, Science and Culture of Japan.
References [1] Rilling, H.C. and Epstein, W.W. (1969) J. Am. Chem. Soc. 19, 1041-1042. [2] Poulter, C.D. and Rilling, H.C. (1981) in Biosynthesis of Isoprenoid Compounds (Porter, J.W. and Spurgeon, S.L., eds.), Vol. 1, pp. 413-442, John Wiley and Sons, New York. [3] Faust, J.R., Goldstein, J.L. and Brown, M.S. (1979) Proc. Natl. Acad. Sci. USA 76, 5018-5022. [4] Shechter, I., Klinger, E., Rucker, M.L., Engstrom, R.G., Spirito, J.A., Islam, M.A., Boettcher, B.R. and Weinstein, D.B. (1992) J. Biol. Chem. 267, 8628-8635. [5] Sasiak, K. and Rilling, H.C. (1988) Arch. Biochem. Biophys. 260, 622-627.
[6] Jennings, S.M., Tsay, Y.H., Fisch, T.M. and Robinson, G.W. (1991) Proc. Natl. Acad. Sci. USA 88, 6038-6042. [7] Fegueur, M., Richard, L., Charles, A.D. and Karst, F. (1991) Curr. Genet. 20, 365-372. [8] Robinson, G.W., Tsay, Y.H., Kienzle, B.K., Smith-Monroy, C.A. and Bishop, R.W. (1993) Mol. Cell. Biol. 13, 2706- 2717. [9] McKenzie, T.L., Jiang, G., Straubhaar, J.R., Conrad, D.G. and Shechter, I. (1992) J. Biol. Chem. 267, 21368-21374. [10] Jiang, G., McKenzie, T.L., Conrad, D.G. and Shechter, I. (1993) J. Biol. Chem. 268, 12818-12824. [11] Summers, C., Karst, F. and Charles, A.D. (1993) Gene 136, 185-192. [12] Joyner, A.L. (1993) Gene Targeting: A Practical Approach, IRL Press, Oxford. [13] Shimano, H., Yamada, N., Katsuki, M., Shimada, M., Gotoda, T., Harada, K., Murase, T., Fukazawa, C., Takaku, F. and Yazaki, Y. (1992) Proc. Natl. Acad. Sci. USA 89, 1750-1754. [14] Plump, A.S., Smith, J.D., Hayek, T., Aalto-Setala, K., Walsh, A., Verstuyfl, J.G., Rubin, E.M. and Breslow, J.L. (1992) Cell 71, 343-353. [15] Zhang, S.H., Reddick, R.L., Piedrahita, J.A. and Maeda, N. (1992) Science 258, 468-471. [16] Hata, S, Tsukamoto, T., Osumi, T., Hashimoto, J. and Suzuka, I. (1992) Biochem. Biophys. Res. Commun. 184, 576-581. [17] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor. [18] Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. [19] Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. [20] Baxter, A., Fitzgerald, B.J., Hutson, J.L., McCarthy, A.D., Motteram, J.M., Ross, B.C., Sapra, M., Snowden, M.A., Watson, N.S., Williams, R.J. and Wright, C. (1992) J. Biol. Chem. 267, 1170511708. [21] Bergstrom, J.D., Kurtz, M.M., Rew, D.J., Amend, A.M., Karkas, J.D., Bostedor, R.G., Bansal, V.S., Dufresne, C., VanMiddlesworth, F.L., Hensens, O.D., Liesch, J.M., Zink, D.L., Wilson, K.E., Onishi, J., Milligan, J.A., Bills, G., Kaplan, L., Omstead, M.N., Jenkins, R.G., Huang, L., Meinz, M.S., Quinn, L., Burg, R.W., Kong, Y.L., Mochales, S., Mojena, M., Martin, I., Pelaez, F., Diez, M.T. and Alberts, A.W. (1993) Proc. Natl. Acad. Sci. USA 90, 80- 84. [22] Hasumi, K., Tachikawa, K., Sakai, K., Murakawa, S., Yoshikawa, N., Kumazawa, S. and Endo, A. (1993) J. Antibiot. 46, 689-691.
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Biochi~ic~a et Biophysica AEta
ELSEVIER
Biochimica et Biophysica Acta 1260 (1995) 49-54
Molecular cloning and functional expression of a cDNA for mouse squalene synthase Takayuki Inoue 1, Takashi Osumi, Shingo Hata * Department of Life Science, Faculty of Science, Himeji Institute of Technology, Kamigori, Ako-gun, Hyogo 678-12, Japan Received 24 June 1994
Abstract
Using a probe obtained by PCR amplification, a full-length cDNA encoding squalene synthase was isolated from a mouse liver cDNA library. Its nucleotide sequence had an open reading frame for a 416 amino acid polypeptide (calculated molecular mass, 48 kDa). In vitro transcription of the cDNAfollowed by in vitro translation produced a protein of the expected size. The deduced amino acid sequence was 93%, 88% and 46% identical to those of the rat, human and budding yeast squalene synthases, respectively. Blotting analyses showed that the mRNA is 1.6 kb in size and that less than two copies of the gene are present in the mouse genome. To establish the enzyme activity, the entire coding region was subcloned into an expression plasmid so that it was in frame with the N-terminal region of fl-galactosidase. Escherichia coli, which was transformed with the recombinant plasmid, expressed high activity of converting farnesyl diphosphate into squalene. Keywords: Squalene synthase; Cholesterol biosynthesis; cDNA cloning; Recombinant enzyme; (Mouse liver)
I. Introduction
Squalene synthase (farnesyl diphosphate:farnesyl diphosphate farnesyl transferase, EC 2.5.1.21) is an integral microsomal enzyme. It condenses two molecules of farnesyl diphosphate (FPP) into squalene, via an intermediate, presqualene diphosphate [1,2]. Since FPP is metabolized to a number of isoprenoid compounds, squalene synthase is the first enzyme specific for cholesterol biosynthesis. Therefore, the reaction represents a potentially important control point for balancing sterol synthesis and other isoprenoid syntheses. In cultured fibroblasts, the level of squalene synthase activity dramatically varies
Abbreviations: FPP, farnesyl diphosphate; PCR, polymerase chain reaction; SSC, standard saline citrate; TLC, thin-layer chromatography. The nucleotide sequence data reported in this paper have been submined to the DDBJ/EMBL/GenBank Databases under the accession number D29016. * Corresponding author. Present address: Laboratory of Applied Botany, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan. Fax: +81 75 7536146. 1 Present address: Department of Gene Sciences, Faculty of Science, Hiroshima University, Higashi-Hiroshima 724, Japan. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSD10167-4781(94)00178-2
according to the demand for sterol synthesis [3]. In rat liver, the level is also altered by feeding inhibitors of hydroxymethylglutaryl-CoA reductase [4]. Little is known, however, about the mechanisms of these observed phenomena. It had been very hard to isolate squalene synthase gene until highly purified enzyme preparations were obtained from yeast [5] and rat [4]. Nevertheless, cloning has been achieved from budding yeast [6,7], fission yeast [8], rat [9] and man [8,10,11], successively, in a short period. The mouse is the most suitable animal for gene manipulation experiments [12]. Using transgenic mice, control mechanisms of lipid metabolism and the causes of metabolic diseases have been elucidated. For example, atherosclerosis research greatly advanced with overexpression of the apolipoprotein E gene [13] or targeted disruption of the gene [14,15]. It may be possible to generate recombinant animals with altered abilities of sterol synthesis. As a first step to understand the complex regulation of cholesterol synthesis in mouse, we isolated and characterized a cDNA for mouse squalene synthase. We also established the enzymatic activity by expressing a recombinant protein in Escherichia coli.
50
T. Inoue et aL / Biochimica et BiophysicaActa 1260 (1995) 49-54
2. Materials and methods
2.4. In vitro transcription / translation
2.1. Polymerase chain reaction (PCR)
pMMSS-2 was linearized by digestion with XhoI. mRNA was transcribed with T7 RNA polymerase (Bethesda Research Laboratories) by the standard method [17]. About 1 /xg of this material was translated with 5 /xl of a rabbit reticulocyte lysate (Amersham) in the presence of [35S]methionine (4 /zCi) at 30°C for 1 h. The protein products were electrophoresed in a SDS/10% polyacrylamide gel and then detected with a Bio-imaging analyzer, BAS 2000 (Fuji).
Two oligonucleotide primers were synthesized. H042 (5'-GCCTCGAGGATGGAGTTCGTGAAGTGT-3') and H041 (5'-CGAATTCAGTGT£CTCTCTGGACA-3') were designed to be specific for the sense 5' end and antisense 3' end, respectively, of the coding region of rat squalene synthase cDNA [9]. They had XhoI and EcoRI sites for the cloning of the PCR products. The 50 /zl reaction mixture comprised 30 ng of random hexamer-primed cDNA of mouse F9 cells [16], 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgC12, 0.01% gelatin, 0.2 mM each of dTFP, dCTP, dGTP and dATP, 1.5 units of Takara Taq DNA polymerase (Takara), and 1 /xM each of the primers. The PCR conditions were 94°C for 1 min, 45°C for 2 min, and 72°C for 3 min, for 40 cycles. The amplified products (1.3 kb in size) were digested with XhoI and EcoRI, and then ligated into XhoI-EcoRI digested pBluescript II K S ( - ) (Stratagene). Partial sequencing of the resulting plasmid showed that the subclone is highly homologous to the rat squalene synthase gene. Its insert was cut out by digestion with XhoI and EcoRI, and then used as a probe for library screening. 2.2. Screening of a cDNA library
An oligo(dT)-primed AZAP cDNA library containing liver cDNA inserts of B6CBA mouse (C57 Black/6 X CBA) (Stratagene) was plated with E. coli BB4, and then blotted onto Hybond-N + membranes (Amersham; 5 . 1 0 4 plaques/sheet). After radiolabeling of the probe using a Megaprime labeling kit (Amersham) and [32p]dCTP, hybridization was carried out at 65°C for 24 h in 6 x SSPE (1 X SSPE = 0.18 M NaC1, 0.01 M sodium phosphate (pH 7.7), 1 mM EDTA), 5 X Denhardt's reagent, 0.5% SDS, and 100 /zg/ml salmon sperm DNA [17]. The membranes were initially washed with 1 × SSC (1 × SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) and 0.1% SDS at room temperature once for 15 min, and then with 0.1 X SSC and 0.1% SDS at 60°C four times for 15 min each. 2.3. Subcloning and DNA sequencing
The longest cDNA insert (1.6 kb in size) of positive clones was cut out with EcoRI and then ligated into the EcoRI site of pBluescript II. The resulting plasmid, designated as pMMSS-2, was digested with exonuclease III (Promega), and then an overlapping series of deletions was prepared. The nucleotide sequence was determined on both strands by the dideoxy chain termination method [18] using [32p]dCTP and Sequenase (US Biochemical). Sequence data were analyzed with GENETYX software (Software Development).
2.5. Northern and Southern analysis
30 /xg of mouse liver total RNA (Clontech) was fractionated by electrophoresis in a 1.2% agarose gel containing 2.2 M formaldehyde and then blotted onto a HybondN ÷ membrane. 4/xg per each lane of mouse chromosomal DNA preparations of B A L B / c mouse and F9 cells (gifts from Dr. T. Tsukamoto of this department) was digested with BamHI, EcoRI or HindlII, separated by 0.8% agarose gel electrophoresis, and then blotted onto a membrane. The probes used were fragments of pMMSS-2, i.e., an EcoRIPvulI fragment ( ~ 1.3 kb in size) containing the entire coding sequence of the cDNA was used for Northern hybridization, and an RsaI-EcoRI fragment ( ~ 420 bp in size) containing the 3' end of the coding region and the 3'-untranslated region was used for the Southern hybridization. The probes were radiolabelled with [32p]dCTP. The membranes were hybridized and washed as described above. 2.6. Construction of an expression vector of mouse squalene synthase
The coding region of mouse squalene synthase was reamplified by PCR using the H042 and H041 primers, and pMMSS-2 as a template. The amplified fragment was digested with XhoI and EcoRI, and then inserted between the XhoI and EcoRI sites of pBluescript II. The resulting plasmid was designated as pXE-7. 2. 7. Preparation of the enzyme
Prokaryotic expression of mouse squalene synthase was carried out based on the method of McKenzie et al. [9] with some modifications. E. coli XL-1 Blue cells were transformed with pXE-7, pBluescript II KS( - ) without an insert was also introduced into cells as a negative control. A single colony of the transformant was inoculated into 25 ml of TB medium in a 100 ml Erlenmeyer flask. The bacteria were grown at 37°C with shaking for 3 h to an optical density of 0.3 at 600 nm. IPTG, 0.5 mM final concentration, was added to induce the lacZ promoter, and then the bacteriawere regrown at 37°C for an additional 3 h. They were collected by centrifugation, and one-fourth of
T. Inoue et al. / Biochimica et Biophysica Acta 1260 (1995) 49-54
them (0.1 g in wet weight) was suspended in 0.5 ml of 0.1 M Tris-HC1 (pH 8.0), 50 mM EDTA and 1 m g / m l lysozyme, and then incubated at 30°C for 10 min. After freezing at - 8 0 ° C and thawing on ice, the cells were disrupted by ultrasonication and then centrifuged at 9000 × g for 3 min. The resulting superuatant was passed through a Sephadex G-50 column (Pharmacia), which had been equilibrated with 100 mM Tris-HC1 (pH 7.6) and 0.2 mM dithiothreitol. The protein concentration was determined by the method of Bradford [19] and adjusted to 1.7 mg/ml.
2.8. Assaying of the enzyme activity The reaction mixture (final volume, 100 /.d) contained [laC]FPP (American Radiolabeled Chemicals; 125 nCi, 11.5 /xM), 3 mM NADPH, 5 mM MgC12, 0.1 mM dithiothreitol, 10 mM KF, 50 mM Tris-HCl (pH 7.6), and the enzyme (85 tzg protein). It was incubated at 37°C for 1 h. After the incubation, 50 ng each of squalene and famesol was added as carriers with the aid of 10/xl of ethanol, and then 10 /xl of the reaction mixture was applied to a silica gel G TLC plate (Merck). The sample was separated with ethylacetate-benzene (1:4, v / v ) , and the radioactivity of each spot was determined with a Bio-imaging analyzer.
51
1 CGCTCCCAGCAGTCGCAAGGATGGAGTTCGTCAAGTGTCTAGGCCACCCGC4~GGAGTTCT M E F V K C L G H P E E F Y 14 61 ATAACCTGCTGCGAI-[CCC-CATGGGAGGC~GGAAI-TFTATACCCAAGATGGACCAGG N L L R F R M G G R R N F I P K M D O D 34 121 ACTCACTCAGCAGCAC-CTTGAAGACCTGCTACAAGTATCTCAATCAGACCAGTCGCAGCT S L S S S L K T C Y K Y L N O T S R S F 54 181 TTGCCGCGGI-[ATCCAGGCC-CTGGAT@CATACC£K;ACC-CCATATGTGTGI-FCTACC A A V I 0 A L D G D I R H A I C V F Y L 74 241 TC431-FCTCCGAGCCCTGGATACAGTGGAGGATGACATGAGCATCAGTGTGGA~GA V L R A L D T V E D D M S I S V E K K I 94 301 TCCCACTGCTGTGTAACTTCCACAC-lIFCCTCTATGACCCAGAGTGGCCK~I-FCACTGAGA P L L C N F H T F L Y D P E W R F T E S I14 361 GCAAGGAGAAGGACCCV~CAAGTGCTGGAGGACTTCCCCACGATCTCCCTGGAGT IFAGAA K E K D R 0 V L E D F P T I S L E F R N 134 421 AI-IT ' GGCTGAGAAATATCAAACAGTGATCCC~TGACATCTGCCACCAGAT~TGT~ L A E K Y O T V I D D 1 C H 0 M G C G M 154 481 TGGCAGAA1-FTGTAGACAAGGATGTGACCTCCAAACAGGACTGGGACAAGTACTGCCACT A E F V D K D V T S K 0 D W D K Y C H Y 174 541 ACGT-FGCTGC£K;TGGT~I-FGGCClI-fCTCGTCTATTCTCTGCCTCAGAGITTGAAG V A G L V G i G L S R L F S A S E F E D 194 601 ACCCCATAG1-FGGTGAAGACATAGAGTGTGCCAACTCAATGGGTCTG-IFCCTGCAGAV / ~A P I V G E D I E C A N S M G L F L 0 K T 214 661 CAAATATCA-IFCGTGATTATCTGGAAGACCAACAGGAAGGAAGGAAGt i I IGC-CCTCAGG N I I R D Y L E D 0 0 E G R K F W P 0 E 234 721 AGGTGTGGGGCAGATACAF 'IAAGAAGF 'IGCPk~GACTTTGCTAAGCCAGAGAACGTAGATG V W G R Y I K K L E D F A K P E N V D V254 781 TGGCCGTGCAGTGC-G [l AATGAACTCATAACCAACACCCTACAGCACATCCCTGACGTCC A V 0 C L N E L I T N T L 0 H I P D V L274 841 TCACCTACCTGTCAAGGCTCCC-C¢V~CCAGAGTGTGTTTV / kCTTCTGTGCTATTCCACAC-G T Y L S R L R N 0 S V F N F C A I P 0 V 294
901 TAATGGCCA'I-FGCCACACTGGCTGCCTGTTACAATAACCAGCAGGTAI-FCAAAGGAGTAG M A I
A T
L A A C Y N N 0
0 V F K G V V 314
961 TGAAGAI-FCGGAAGGGC-CAAGCAGTCACCCTCA TGATGGATGCCACCAACATC-CCTGCOG K I R K G 0 A V T L M M D A T N M P A V 334 1021 TCAAAGCTATCATATACCAGTACATAGAAGAGAI]-I'ATCACCGGATCCCCAACTCAGACC K A I I Y 0 Y I E E I Y H R I P N S D P354
3. Results and discussion
3.1. Preparation of a probe for library screening Since mouse is a species very close to rat, we presumed that the 5' and 3' ends of the coding region of the squalene synthase gene might be conserved. Based on this idea, a PCR experiment involving primers specific to the rat cDNA [9] and a mouse F9 cDNA library [16] as a template was carried out. Analysis of the PCR products by agarose gel electrophoresis showed a major product of the expected size, 1.3 kb (data not shown). Subcloning and partial sequencing of the amplified product revealed that its nucleotide sequence is closely related to that of the rat squalene synthase cDNA [9]. Therefore, we concluded that the PCR product encoded mouse squalene synthase.
108t CATCATCAAC-CAAAACCAAC,-CAGGTCATCTCCAAGATCAGGACACAGAACC'FICCC/kACT S S S K T K 0 V I S K I R T 0 N L P N C 374 1141 GCCAGCTCATCTCCCGAAC-CCACTACTCGCCCAlqqACCTGTCATTTATCATC'CTC T-FGG 0 L I S R S H Y S P I Y L S F I M L L A 394 1201 CTC-CCCTGAGCT~GTACCTGAC-CACCCTGTCCCAGGTCACAGAAGACTATGTC CAGA A L S W O Y L S T L S 0 V T E D Y V 0 R 414 1261 GAGAACACTGA]{l IGTTTAC-CCGGAAGTGGAAG1-FCCCGTGGAGTGGG1111 ICCTTI-r E H * (416) t 321 CCTCCAGCTGGATI-FTGACTTCCCI-IGr; i i I CCTCCTACTCTAAAATCTTTGGGAGAAC 1381 TGAGTGTGGGACCTITA~CT~GGATGCCTTGCCCTCAC.-CAGCC TGGT 1441 C-CTGGCT~CI-IGGI-rCCTCTGCCTCTTGTAGCCACTGGCAGCGTGCCGACTGCTGCA 1501 CTTGTGAGGCCACGTGTGATGGTCACAAGAGCCTAGTGAACCTC-C-CTAGAATGCTGAI-IG 1561 GATFFATTTAATTTGAAACAGCC13TGAA TACCTATGACAATAGAAAATGAAAC.-CAAAAA 1621 A A A A A A ~
Fig. 1. Nucleotide and deduced amino acid sequences of the mouse squalene synthase cDNA clone, pMMSS-2 was sequenced in its entirety. Nucleotide positions (beginning at the 5' end) are indicated on the left, and amino acid positions (numbered from the putative translation initiation codon) are indicated on the right. A stop codon (TGA) at the end of the translated sequence is marked with an asterisk.
3.2. Isolation and sequencing of a cDNA clone for mouse squalene synthase Next, we screened a mouse liver cDNA library using the PCR product as a probe. About 60 positive spots were detected for 9- 105 clones. Six recombinant phages were picked at random and the longest cDNA insert among them was subcloned into pBluescript II. The resulting plasmid, designated as pMMSS-2, had an open reading frame for a 416 amino acid polypeptide (Fig. 1). Through analogy with the enzymes of rat and man [8-11], we think the ATG codon at nucleotide position 21 is the transla-
tional initiation codon, though no in-frame stop codon was found upstream. The calculated molecular mass of the polypeptide is 48 126. 3.3. In vitro transcription and translation In order to check the validity of the predicted molecular mass, in vitro transcription followed by in vitro translation was carried out using XhoI-cut pMMSS-2 as an initial material. As shown in Fig. 2, a rabbit reticulocyte lysate
T. Inoue et a l . / Biochimica et Biophysica Acta 1260 (1995) 49-54
52
1
49
2
3.6. Southern blot analysis
kDa I ~I L
~i i
To obtain information about the copy number of the mouse squalene synthase gene, Southern blot analysis was carried out using genomic DNA preparations of B A L B / c mouse and F9 cells. The probe, which contained the C-terminus of the coding region and the 3'-noncoding region, detected two bands in each lane (Fig. 5). Therefore, it can be said that the copy number is less than two. Further study is necessary to clarify whether or not the mouse squalene synthase gene is a single copy.
3. 7. Establishment of the enzyme activity
Fig. 2. In vitro transcription and translation analysis. The translation mixture contained the pMMSS-2-derived R N A template (lane 1) or no R N A (lane 2). The products radiolabeled with [35S]methionine were
separated by 10% SDS-polyacrylamide gel electrophoresis.
We constructed an expression vector by inserting a PCR-amplified coding region of pMMSS-2 into pBluescript II. The resulting plasmid, designated as pXE-7, had a nucleotide mismatch compared to pMMSS-2, since the H042 primer had the 5'-terminal sequence of the rat squalene synthase gene. Nevertheless, the deduced amino acid 49
Mouse Rut Human Yeast
NEFVKC~G-~E~iFYNLLRFRMH~RRNFIPKM~!~DSL~SS~!KTOYKY~N~ ~EFVKC~:G-~E~iFYNLLRFRM6~RRNFIPKM~iRN~L~NS~KTOYKY~DQ ~EFVKC~G-H~iE~FYNLVRFRIO~KRKVMPKM~QD~L~HS~iKTGYKY~NQ MGKLL~AL~V~IMKAALKLKF-CRTPLFSIY~Q-ST~PY~:LHOFHLLNL
produced a major protein of 49 kDa depending on the pMMSS-2-derived RNA template. The observed molecular mass of the in vitro expressed protein is fairly consistent.
Mouse Rat Human Yeast
~8RHFAAVI~A~DGDIKHAICV~,LV~LBi~D#S{~HVEKKIP~CN ~HR~tV|i~A~D~DI~HAVCV~II~MBIV~N~liHVEK~IP~RN ~iSR~Yl]I~,I~A~D~EM~AVCI~V~L~L~Ti!SVEK~VP~HN ~6~F~IRE~HPEL~NCVTLF~ilgI~JIL~I~S!~iHHDL[ID~RH
3.4. Comparison of mouse squalene synthase with the enzymes of other species
Mouse Rat Human Yeast
~TF~YDPE~RF--TESKE{D~E~PT!I%~RNLAEK~T~DD|~C ~flTF~iYEPE~RF--TESKE~H~V~IE~PTISLE~IRN~AEK~T~AD~IC ~SF~iYQPDWRP---MESKE~D~E~PT~:SL~RN~AEKY~T~ADIC ~EKL:LLTKNSFDHNAPDV~DRAV~TBi~HSIiLI~HK~KPE~E~KE~!T
147 147 147 148
Mouse Rat Human Yeast
HOJ~fiC~MAEFVDKD.......VTSKQDW~K~O~¥~h@~V~II~S~FSASE HR~C~i~EFLNKD ...... VTSKQDW~K~II~,~%~I~!~H~FSASE RR~I~II~EFLDKH ...... VTSEQEW~K~"II{~!I~!~S~$SASE EK~N~{DYILDENYNLNOL~TVHDY~V¥~¥V{~iDH~TRL!IVIAK
191 192 191 198
Mouse Rat Human Yeast
~EDPIVHEDIECAN8MHL~L~KTNI~:~B?LHO~E~K~QH~GR~IK gEDPIVSEDTECANS{i~'tK|~L~E~Q~J{FQ~SKYVK
241 241
~NESLYSNEQLYESM~I[%L~I~I~RI~YN~LVDa~S~M~ilWS~YAP
248
Mouse Rat Human Yeast
K~E~t,A~VDVAVQOL§IE~IT~TLQ~IP~LT~SRLRN~iV~N~OA~ K~E~iVK~.~VDVAVK~LNE~ITNALO~IP~I~SRLRN~$V~N~:I K~OD~A~#IDLAV~%NE~ITNALHHIP~I~SRLRN~SV~N~ ~K~iMK~:NEQLGLDfflKH~VLNALSHVI~L~ASIHE~ITFQFOAI~I
291 291 291 298
Mouse
Rat Human Yeast
P~MAI~CYXHiQQ~FK~iV~iKiI{LK.GQAVTLIMMDA-TNMPA~KAIIY~Q ~%?/~&I!~TI,/tACY~NHQ~FK~V~[I~I[~AVT~MMDA-~NMPA~KAIIYQ ~CYNN~Q~FK~A~AVT~MMDA-~!NMPA~KAIIYQ P~VI~AI~i~LVF~NRE~LH~N~i~I~TTCY~ILKSR~LRGC~EIFDMY
340 340 340 348
Mouse Rat Human Yeast
YIEBIYHRI PNSB~HSS~TKQV~SK IRTt~NLPNCIILISRSHYSPIYLS-E YIEEIYHRVPNS~SAS~KQLt~NI:RTi~SLPNCQLISRSHYS~IYLS-~ YMF,E{YHRIPDSD~SSSI~TRQI|~T ~RTQNLPNC~LI SRSHYS~IYLS-~ LRD-{KSKLAVQD~NFL~LNI~|~K IEQFMEEMY~DKLPPNVK~NETPI ~
389 389 389 397
Mouse Rat Humau Yeast
. . . . . . . . . . . . . . . . . . I MLLAALSWQY~ST-~QVTED~VQR EH ......................... IMLLAALSWQY~ST-LHQVTED~VQR- EH . . . . . . . . . . VMLLAALSWQY~TT-~$QVTEDYVQTOEH LKVKEI~HRYDD ELVPTQQEEEYKFNMV~S IIL$~LLG FIYI YTLMRA
416 416 417 444
Fig. 3 shows protein comparison of the rat, mouse, human and budding yeast squalene synthase sequences. Highly conserved segments A, B and C [9] of known squalene synthases corresponded to amino acids 171-186, 208-228, and 283-301, respectively, of the mouse enzyme. These segments were suggested to represent the crucial regions for the active site [8,9]. The amino acid residues in the segments were almost completely conserved among the reported squalene synthases. The overall amino acid sequence of the mouse enzyme was 93%, 88%, and 46% identical to those of the rat, human and budding yeast enzymes, respectively.
3.5. Northern blot analysis On Northem blot analysis, a single band corresponding to ~ 1.6 kb in size was detected for mouse liver (Fig. 4). This is in contrast to the results for human squalene synthase, for which two or three distinct transcripts were observed [8,10,11], but is similar to in the cases of budding yeast [7,8] and fission yeast [8], for which a single band was detected. The pattern of rat squalene synthase transcripts has not been reported. The reason for the interspecies discrepancy is not yet known.
49 49
48 99 99 99 98
acid sequence alignmemt of squalene synthase polypeptidcs from rat, mouse, man and yeast. P~mino acids identicalthroughout are shaded. Numbers indicate amino acid residues in the sequences. Fig. 3. A m i n o
53
T. Inoue et al. / Biochimica et Biophysica Acta 1260 (1995) 49-54
1
2
SF--
Sq-
1.
6
k b
FOH--
Fig. 4. Northern blot analysis of the mouse squalene synthase transcript. A 30 /xg sample of mouse liver total RNA was electrophoresed through denaturing 1.2% agarose, transferred to a Hybond-N + membrane, and probed with the 32P-labeled 1.3 kb EcoRI-PvulI fragment of pMMSS-2, which contained the entire coding sequence. The size of the detected band was estimated using a poly(A)-tailed RNA ladder as molecular size markers.
1
2
3
4
5
6
O--
Fig. 6. Prokaryotic expression of mouse squalene synthase activity by the recombinant plasmid, pXE-7. Cell-free extracts of E. coli transfected with pXE-7 (lane 1) or pBluescript II (lane 2) were incubated with [14C]FPP as described under Materials and methods. The reaction products were separated by TLC and detected with a Bio-imaging analyzer. The positions of squalene (Sq), farnesol (FOH), and the chromatographic origin (O) and solvent front (SF) are indicated. Unconverted FPP remained at the origin.
(kb)
n
19. u
7.
7
6.
2
4°
3
--
3.
5
-
2.
7
--
W
Fig. 5. Southern blot analysis of the mouse squalene synthase gene. Samples of genomic DNAs from B A L B / c mouse (lanes 1-3) and F9 cells (lanes 4-6) were digested with BamHI (lanes 1 and 4), EcoRl (lanes 2 and 5), or HindlII (lanes 3 and 6), subjected to electrophoresis through 0.8% agarose, transferred to Hybond-N ÷ membranes, and then probed with the 420 bp RsaI-EcoRI fragment of pMMSS-2, which contained the C-terminal of the coding region and the 3'- untranslated region. The )t-EcoT14I digest served as molecular markers.
sequence of pXE-7 was exactly the same as that of p M M S S - 2 . Partial s e q u e n c i n g of p X E - 7 showed a correct open reading frame for expression of a chimeric protein c o m p o s e d of the 26 a m i n o acid N-terminal end of /3galactosidase, followed by the entire mouse squalene synthase (data not shown). E. coli is u n a b l e to synthesize sterols, hence it lacks squalene synthase. Biochemical assaying of cell-free extracts of p X E - 7 - t r a n s f o r m e d E. coli established the efficient c o n v e r s i o n of F P P to squalene (Fig. 6, lane 1). A n apparent specific activity of 87 p m o l / m g protein per m i n was observed. On the other hand, the activity of E. coli transformed with vector only was negligible (Fig. 6, lane 2). Therefore, the c D N A for m o u s e squalene synthase e n c o d e d a functional e n z y m e . Farnesol is likely to be generated from F P P through the action of e n d o g e n e o u s phosphatases in E. coli. 3.8. P e r s p e c t i v e s
The c D N A c l o n i n g of m o u s e squalene synthase will lead to the isolation of m o u s e g e n o m i c D N A clones for the e n z y m e . A n a l y s i s of the promotor region and its trans-
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acting factors may allow elucidation of the regulatory mechanism of the gene expression. Since squalene synthase is the first committed enzyme in cholesterol biosynthesis, it will also be feasible to generate a recombinant mouse with abnormal cholesterol synthesis. This attempt may give new insights into the regulation of cholesterol synthesis among a number of isoprenoid biosyntheses. Specific inhibitors of squalene synthase have attracted much attention as cholesterol-lowering drugs without side effects [20-22]. The functional expression of squalene synthase in E. coli may be a convenient system for screening novel inhibitors of the enzyme.
Acknowledgements We wish to thank Y. Yabusaki, T. Sakaki and Y. Shigeri for the retrieval of literature and the valuable discussions. We also thank T. Tsukamoto for the gifts of mouse chromosomal DNAs. This work was supported in part by research grants from the Ministry of Education, Science and Culture of Japan.
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