Amino- acid sequence analysis of a 38-kDa protein purified from bovine liver in our laboratory revealed > 90% similarity with a human sterol reductase, SR-1, encoded by the TM7SF2 gene,
Trang 1Cloning and expression of sterol A14-reductase from bovine liver
Rita Roberti', Anna Maria Bennati', Giovanni Galli?, Donatella Caruso2, Bruno MarasỶ, Cristina Aisa‘, Tommaso Beccari*, Maria Agnese Della Fazia* and Giuseppe Servillo*
' Department of Internal Medicine, University of Perugia, Italy; * Department of Pharmacological Sciences, University of Milan, Italy; *Department of Biochemical Sciences ‘A Rossi Fanelli’, Universita ‘La Sapienza Roma, Italy;
4 Department of Biochemical Sciences and Molecular Biotechnology, University of Perugia, Italy
Biosynthesis of cholesterol represents one of the funda-
mental cellular metabolic processes Sterol Al4-reductase
(A14-SR) is a microsomal enzyme involved in the con-
version of lanosterol to cholesterol in mammals Amino-
acid sequence analysis of a 38-kDa protein purified from
bovine liver in our laboratory revealed > 90% similarity
with a human sterol reductase, SR-1, encoded by the
TM7SF2 gene, and with the C-terminal domain of human
lamin B receptor A cDNA encoding the 38-kDa protein,
similar to human TM7SF2, was identified by analysis
of a bovine expressed sequence tag (EST) database
The cDNA was synthesized by RT-PCR, cloned, and
sequenced The cDNA encodes a 418 amino-acid poly-
peptide with nine predicted transmembrane domains The
deduced amino-acid sequence exhibits high similarity with
Al4-SR from yeasts, fungi, and plants (55-59%), sug- gesting that the bovine cDNA encodes Al4-SR Northern blot analysis of bovine tissues showed high expression of mRNA in liver and brain The polypeptide encoded by the cloned cDNA was expressed in COS-7 cells Immu- nofluorescence analysis of transfected cells revealed a distribution of the protein throughout the ER COS-7 cells expressing the protein exhibited Al4-SR activity about sevenfold higher than control cells These results demonstrate that the cloned bovine cDNA encodes A14-
SR and provide evidence that the human 7M7SF2 gene encodes Al4-SR
Keywords: sterol biosynthesis; sterol reductase; cloning; endoplasmic reticulum
Sterol Al4-reductase (Al4-SR), an essential enzyme for
sterol biosynthesis in eukaryotic cells, is an integral protein
of the ER that acts on A'“(>-ynsaturated sterols in different
organisms In mammalian cells the elimination of a
14a-methyl group from the C30 sterols, lanosterol and
24,25-dihydrolanosterol, during conversion to cholesterol
(C27A°) generates the intermediates 4,4-dimethyl-S-cho-
lesta-8,14,24-trien-3B-ol (C29A*!*™*) and 4,4-dimethyl-So-
cholesta-8,14-dien-3B-ol [1] that are transformed into
4,4-dimethyl-5o-cholesta-8,24-dien-3B-ol (C29A*?*) and
4,4-dimethyl-Sa-cholesta-8-en-3f-ol, respectively, by the
action of Al4-SR [2] The saturation of the Cl4=C15
Correspondence to R Roberti, Department of Internal Medicine,
Laboratory of Biochemistry, University of Perugia, Via del Giochetto,
06122 Perugia, Italy Fax: + 39 0755857428, Tel.: + 39 0755857426,
E-mail: roberti@unipg.it
Abbreviations: A14-SR, sterol Al4-reductase; SR-1, sterol reductase 1;
LBR, lamin B receptor; C29A81424 4,4-dimethyl-S5a-cholesta-8, 14,24-
trien-3B-ol; C29A**, 4,4-dimethyl-5e-cholesta-8,24-dien-3B-ol;
C27A° 4 5a-cholesta-8,14-dien-3B-ol; C27AŸ, 5œ-cholesta-8-en-3-ol;
C27A°, cholesterol; E-64, N-[N-(L-3-trans-carboxyrane-2-carbonyl)-L-
leucyl]-agmantine; EST, expressed sequence tag; DMEM, Dulbecco’s
modified Eagle’s medium; PVDF, poly(vinyiledene difluoride);
FITC-conjugated, fluorescein isothiocyanate-conjugated; EPT,
ethanolaminephosphotransferase
Note: the nucleotide sequence reported in this paper has been
submitted to GenBank and is available under accession number
AY03968 1
(Received 2 August 2001, revised 26 October 2001, accepted 31
October 2001)
double bond may occur at different stages of the pathway leading from C30 to C27 sterols [3,4]
Biochemical characterization, solubilization, and purifi- cation of Al4-SR from rat liver have been reported [5,6] The liver enzyme is responsive to cholesterol lowering agents, as well as to changes in diet and circadian rhythm [6] Al4-SR has been cloned from yeast [7-9] and fungi [10] Gene cloning of Al4-SR from Arabidopsis thaliana and analysis of mutants has highlighted the role of the protein in cell growth and embryonic development of the plant [11,12]
Inherited human disorders caused by defects in choles- terol biosynthesis have been identified, suggesting a major role for cholesterol and/or intermediates of biosynthesis in embryogenesis and morphogenesis [13] Among these, the Greenberg skeletal dysplasia has been hypothesized to originate from Al4-SR deficiency [14] In addition, interest
in the C29A*!*** and C29A**" sterols has been consid- erably stimulated by the finding that they play a crucial role during meiosis in mammals [15] The C29A*!4™* sterol is a positive regulator of the nuclear receptor LXRa [16] These data indicate that Al4-SR is one of the regulatory enzymes in the complex pathway of cholesterol biosynthesis
Recently the human lamin B receptor (LBR), an integral protein of the inner nuclear membrane, has been shown to exhibit Al4-SR_ activity [17] Two protein paralogues of human LBR, sharing high similarity with plant and yeast sterol reductases, have been identified These proteins, sterol reductase 1 and 2 (SR-1 and SR-2), are encoded by TM7SF2 and DHCR7 genes, respectively [18-20] SR-2 is sterol A7-reductase, a Smith—Lemli—Opitz syndrome-related
Trang 2protein [19-22], whereas no functional characterization of
the TM7SF2 gene product has been reported It has been
hypothesized that human 7M7SF2 encodes A14-SR [13,23],
but upon expression in yeast, no sterol Al4-, A7-, or A24-
reductase activities were detected [13]
We isolated a 38-kDa protein from bovine liver ER witha
high degree of identity with both human SR-1 and human
LBR A cDNA encoding this protein was identified by
bovine expressed sequence tag (EST) analysis, cloned, and
expressed as a functional Al4-SR
MATERIALS AND METHODS
Chemicals
M-MLYV reverse transcriptase, lipofectamine reagent, Dul-
becco’s modified Eagle’s medium (DMEM), and foetal
bovine serum were purchased from Gibco-BRL (Milan,
Italy) TOPO-cloning kit was from Invitrogen (Leek, the
Netherlands) RNAse inhibitor was from Ambion (Austin,
TX, USA) The Expand Long Template PCR System,
Staphylococcus aureus V8 protease, N-[N-(L-3-trans-carb-
oxyrane-2-carbonyl)-L-leucyl]-agmantine (E-64), leupeptine,
phenylmethylsulfonyl fluoride (PMSF), and thesit were all
purchased from Roche Molecular Biochemicals (Milan,
Italy) Q-Sepharose fast flow, 5a-cholestane, glucose oxi-
dase, reduced glutathione, NADPH, commercial antibod-
ies, protein A-Sepharose CL 4B, SDS/PAGE reagents, and
enhanced chemiluminescence reagents were from Sigma
(Milan, Italy) Biogel HTP was from Bio-Rad (Milan,
Italy) Poly(vinylidene difluoride) (PVDF) membranes
(Immobilon PS) were purchased from Millipore (Bedford,
MA, USA) 5o-cholesta-8,14-dien-3B-ol (C27A*") was
synthesized according to Fieser & Ourisson [24] Diacyl-
glycerol was prepared from egg yolk as described previously
[25] Other reagents were from Gibco-BRL and Sigma
Isolation of sterol A14-reductase
Bovine A14-SR was co-purified from liver ER together with
the previously reported ethanolaminephosphotransferase
(EPT) [25] Briefly, microsomes (3 mg proteinmL™') were
solubilized with 1.5% thesit in the presence of | mm NaCl
and diacylglycerol (0.3 mgmL7') The purification proce-
dure included chromatography on Biogel HTP and two
chromatographic steps on Q-Sepharose, performed at
pH 7.0 and pH 8.5, as described previously [25] The
protein preparation was concentrated and freed of lipids
as follows The sample was dialysed extensively against
distilled water and freeze-dried The residue was suspended
in a 10-mL mixture of chloroform/methanol (1 : 9, v/v) for
10 min at 37 °C The insoluble protein pellet was recovered
by centrifugation and the extraction was repeated twice The
protein pellet was vacuum dried, resuspended in 5% SDS
and adjusted to 100 mm Tris/HCl (pH 6.8), 1% SDS (w/v),
10% glycerol (v/v), and 100 mm dithiothreitol (SDS/PAGE
sample buffer)
Sequence analysis of sterol A14-reductase
A 20-ug aliquot of lipid-free protein was subjected to SDS/
PAGE, electroblotted on a PVDF membrane, and stained
with Coomassie blue The N-terminal amino-acid sequence
was determined by automated Edman degradation using a PerkinElmer model AB 476A sequencer For internal sequence determination the protein (30 1g) was subjected
to SDS/PAGE After staining the gel with Coomassie blue, the 38-kDa band was cut and equilibrated for 10 min with
100 mm Tris/HCl (pH 6.8) containing 12% (v/v) glycerol,
50 mm 2-mercaptoethanol, and 2% (w/v) SDS (buffer A) The slice was then inserted into a gel well and covered with buffer A containing 20% glycerol (v/v) Staphylococcus aureus V8 protease solution (2 ug in 10 uL of buffer A) was layered onto the top [26] The separating gel contained 15% (w/v) polyacrylamide (acrylamide/bisacrylamide
30 : 0.8, w/w) After the sample had been stacked with a 4-mA constant current, the power was turned off for 2 h
at room temperature to achieve proteolysis Fragments were separated by applying a 30-mA constant current and electroblotted on PVDF membrane Bands were excised and amino-acid sequence analysis was performed as described above
The amino-acid sequences were analysed using the BLAST search program (National Center for Biotechnology Infor- mation; http://www.ncbi.nlm.nhi.gov) [27]
Antibody production Polyclonal antibodies against Al4-SR were raised in rabbits
by multiple subcutaneous injections of a solution containing
~50 ug of lipid-free protein preparation in 0.9% NaCl mixed with an equal volume of Freund’s complete adjuvant Boost injections of 50-ug protein were performed 21 and
42 days after the initial administration The IgG fraction was purified on a protein A-Sepharose CL 4B column equilibrated with 0.1 m Tris/HCl (pH 8.0) and eluted with 0.1 m glycine buffer (pH 3.0) [28]
RT-PCR cloning of the bovine cDNA encoding sterol A14-reductase
BLASTN search of the bovine EST database was performed
to identify bovine cDNA clones homologous to human SR-1 cDNA [27] The putative bovine cDNA was used to design PCR primers for amplification of the ORF Total RNA (5 ug), purified from liver as described below, was used to synthesize first-strand cDNA using a reaction mixture containing 50 mm Tris/HCl (pH 8.3), 40 mm KCl, 6mm MgCl, | mm dithiothreitol, 40 UmL™! of RNase inhibitor, 2.5 mm dNTP, 0.2 mm oligo-dT 15—18mer, and
200 U of M-MLYV reverse transcriptase First-strand syn- thesis was performed at 42 °C for 45 min and then the enzyme was inactivated at 90 °C for 5 min Following first- strand synthesis, PCR of 4/4-SR cDNA was carried out using appropriate primers and the Expand Long Template PCR System The two primers used were the sense primer (ŠS-ATTCTAGAAGCGGAGACCATGGCCCCTCCTC AG-3’) and the antisense primer (5-ATTCTAGATAG GGTACAGGCCCTTGTGTCCCG-3’), both bearing the Xbal restriction site (underlined) PCR conditions were as follows: 4 min at 94 °C (1 cycle); 1 min at 94 °C, 1 min at
65 °C, 1 min at 68 °C (30 cycles); 5 min at 68 °C (1 cycle) The RT-PCR product was cloned into the pCR2.1 vector
by TOPO-cloning kit and bidirectionally sequenced at MWG Biotech (Mtinchen, Germany) The PCR product (1370 bp) was used as a probe for Northern blot analysis
Trang 3RNA isolation and Northern blot analysis
Total RNA was isolated from different bovine tissues (liver,
brain, lung, skeletal muscle, heart, adrenal, and testis) by
homogenizing the samples in guanidium isothiocyanate
solution (100 mg tissuemL™') followed by CsCl step
gradient centrifugation [29] RNA was denatured in
formamide, separated in denaturing agarose gel (1%
agarose/2.2 m formaldehyde), and blotted onto a nitrocel-
lulose filter The RNAs (20 pg) extracted from different
tissues were hybridized with random priming P cDNA
specific for bovine 4/4-SR [30]
Expression of sterol A14-reductase in COS-7 cells
Bovine 4/4-SR cDNA was subcloned in the Xbal site of the
eukaryotic expression vectors pCS2-myc-tag, containing the
CMV promoter [31], and modified pMT2, containing the
SV40 promoter [32], kindly provided by N S Foulkes
(IGBMC of Strasbourg, France) and F Grignani (Univer-
sity of Perugia, Italy), respectively The cDNA was subcl-
oned in pCS2-myc-tag 3’ to a sequence encoding six copies
of a 13-residue c-myc epitope COS-7 cells were grown in a
5% CO, incubator at 37 °C in DMEM supplemented with
10% foetal bovine serum and 2 mm glutamine Cells were
cultured in 10-cm Petri dishes until 50-80% confluence and
transfected for 5 h with the two plasmids (4 tug) separately,
using lipofectamine in serum-free DMEM Control cells
were transfected with empty pMT2 or pCS2-myc-tag
vectors After transfection, the medium was replaced with
complete DMEM and cells were incubated for 35 h at
37 °C Transfected cells were recovered with 0.9% NaCl
containing 1 mm EDTA, | um leupeptine, 0.1 mm PMSF,
0.3 um E-64 and then sonicated three times for 10 s The
microsomal fraction was prepared by centrifugation of the
500 g supernatant at 100 000 g for 1 h at 4 °C The pellet
was resuspended in 10 mm K-phosphate/0.05 mm EDTA
(pH 7.4) Protein concentration was determined by the
method of Bradford [33], using BSA as a standard
Microsomal proteins separated by SDS/PAGE were
blotted on PVDF membranes and incubated with poly-
clonal rabbit anti-(Al4-SR) Ig or monoclonal mouse anti-
(c-myc-tag) Ig, as indicated Peroxidase-conjugated goat
anti-(rabbit IgG) Ig or anti-(mouse IgG) Ig were used as
secondary antibodies The protein was detected by the
enhanced chemiluminescence assay
Indirect immunofluorescence
Transfected COS-7 cells, grown on coverslips, were washed
with NaCl/P; and fixed in ice-cold methanol for 10 min at
—20 °C Cells were subsequently permeabilized by treatment
with 0.1% Triton X-100 in NaCl/P; for 5 min at room
temperature, washed with NaCl/P;, blocked with 3% BSA
in NaCl/P;, and incubated for 60 min at room temperature
with rabbit anti-(Al4-SR) IgG After washing with NaCl/P;
containing 0.1% Tween-20, cells were incubated for 60 min
at room temperature with Cy3-conjugated sheep anti-
(rabbit IgG) Ig Cells transfected with the pCS2-myc-tag
vector were subsequently treated with monoclonal mouse
anti-(c-myc-tag) Ig and fluorescein isothiocyanate-conjugat-
ed (FITC-conjugated) goat anti-(mouse IgG) Ig The cells
were examined by fluorescence microscopy and the images
were acquired by using a Spot-2 cooled camera (Diagnostic Instruments)
Sterol A14-reductase assay A14-SR activity was assayed in microsomes prepared from Al4-SR cDNA-transfected COS-7 cells and from bovine liver, using 5a-cholesta-8,14-dien-3B-ol (C27A*"*) as a substrate [5] The sterol was added as a 0.3-mmM suspension
in 0.8% Tween-80, at 60 uM final concentration (13.5 Lug) to 0.5 mL of a mixture containing 0.1 mM K-phosphate buffer (pH 7.4), 0.5 mm EDTA, | mM reduced glutathione, 2 mm NADPH, 0.14 ™ glucose, and 10 U of glucose oxidase, that had been preincubated for 4 min at 37°C under N> atmosphere Incubation was carried out under N> for
30 min at 37 °C with 0.24 mg of microsomal proteins and terminated by the addition of 1 mL of 20% KOH in 50% methanol, followed by additional 30 min incubation at
37 °C After the addition of 5a-cholestane (5 wg) as an internal standard, sterols were extracted three times with
3 mL of petroleum ether and the organic phases were evaporated to dryness under nitrogen stream
The sterol extracts were acetylated with acetic anhydride- pyridine, 2 : 1 (v/v) for 1 hat 60 °C The samples were taken
to dryness and the residues were dissolved in ethyl acetate Aliquots of the samples were analysed by GC-MS in multiple ion detection mode using a Varian Saturn 2100T apparatus with a Varian CP-Sil8 CB low bleed/MS column Temperature was programmed from 150 to 300°C at
12 °C-min™! Sterol retention times were: 14.5 min, 5o-
cholestane (M* = 372); 18.2 min, cholesterol (M* = 368); 18.3 min, C27A*"* (M~ = 426); 18.5 min, 5øœ-cholesta- 8(9)-en-3B-ol (C27A®, M* = 428)
A14-SR activity was evaluated on the basis of peak area ratios between m/z 426 and m/z 372 ions (C27A*"4/5o- cholestane) or m/z 428 and m/z 372 ions (C27AŸ/5a- cholestane) at the expected retention time
RESULTS AND DISCUSSION
Isolation of sterol A14-reductase During the preparation and delipidation of a bovine liver 38-kDa protein exhibiting EPT activity [25], a protein co- migrating in SDS/PAGE was revealed by amino-acid sequence analysis The determined N-terminal sequence of the protein, APPQGSRAPLEFGGPLGAAALML, was 87% identical to residues 2-24 of human SR-1 (GenBank accession no AF096304) [18] The digestion of the 38-kDa band with S aureus V8 protease produced three major fragments with molecular masses of ~27, 19.5, and 9.5 kDa The 27- and 9.5-kDa fragments confirmed the N-terminal sequence, whereas the sequence of the 19.5-kDa fragment was AVLTTMDITHDGFGFMLAF, 95% iden- tical to residues 243-261 of human SR-1 and 440-458 of human LBR (GenBank accession no L25931) Human SR-1 has been reported to be a sterol reductase, based on similarities with sterol reductases from yeast, fungi, and plants, although its catalytic activity has not been identified [18] Moreover, human SR-1 exhibits 58% identity with the C-terminal domain (residues 197-615) of human LBR [18], which possesses Al4-SR activity [17] For this reason we hypothesized that the purified 38-kDa bovine protein is a
Trang 4
™ 1
hibri97 197 -F EVTPIRAKDLEFGGVPGVFLIMFGLPVFLFLLLLMCK - QK
Ae 8 Se SSE Sete Bella Sa Sie eee Slee S m6: Bì S m HÔI GÀ 672V 12 sikaiie sisike G3 íŒ6 Am.sE L É
TM 2
ati4ga 1 - - - MLLDMDLGVL|LỊPSLQSVYVLVFYFVYLAVAGELLPGKỆV! RịG|V LỊL
TM 3
bi4s 187 ATLTAFIFSLLLYILKALLAPASA - LAPGGNISGNI|L !I Y|DIF F LỊG hSRE1 137 ATLTAFIFSLFLIY|MKAQVAPVSA- - - - LAPGGNSGN/PI YIDIFFLIG hibri97 333 ATVFCVVLSVYLIYIMRSLKAPRND - LSPAS -ISGNAVY|DIFF I/G at14q 92 TFIFCVLVTLAL]Y|VTGRSSSNKGS- - - SLKPHVISGNL VHIDIWWFIG
™ 5
b1i4sK 22 E QL LỊY|V 6e[D|A L W Y[E E|A V L T TMÍD !ÌI HDGFÍG F ML|A[F GD T Lịa hSR-1 225 |LƒVNGFQL L|Y|V G|DỊA L W HỊE E{A VL T TMID I|THDGFIG FML|AIFGD T Lịa hlbr187 422 |LỊVN SF QL L|Y|V V|DÍA L WN|E E|A LL T TMID I|I HDGF|IG F ML|AIFGD | FIQ at14srẽ 179 |L[Y Q1 ECA LỊY|! L|D|Y F V HỊE ElY MT STWD I|I AERLIG F ML|VỊF GDL LW IỊP FỊT F|S|I|Q
sc 14sr 249 |L]VN F LQG F|Y| I F|DIG V L N|E El|G VL T MMID I|TTDGF|G F ML|AIFGD T Lịa
TM8
ati4a 229 GWWLILHNKVELTVPAIVVNCLVF GIA NIKIQ KIH | |/FIKKN- - - PKTP scl4sr 209 ARYILISVSPVELGWVKVVGILAIM SIA N|KÌQ KỊS EF|RQG - - - -KLE
bi4sr 325
hSR1 325
hlbr197 522
at14sq 276
bi4s 375
hSR-1 375
hlbr187 572
at14 329
Fig 1 Amino-acid sequence alignment of sterol A14-reductase and related sterol reductases Alignment was performed using the om1ca 2.0 program run with the default parameters Positions with consensus residues present in all sequences are boxed Positions with consensus residues present in at least three sequences are shaded Bovine A14-SR (b 14 sr); human SR-1 (h SR-1); residues 197-615 of human lamin B receptor (h Ibr197);
A thaliana A\4-SR (at 14 sr) (GenBank accession no AF256535); S cerevisiae A14-SR (sc 14 sr) (GenBank accession no $69420) For bovine AI4-SR, regions of the deduced amino-acid sequence corresponding to the N-terminal and V8 peptide sequences determined in the sequencing experiments of the protein purified from bovine liver are underlined TmpRED program (ExPASy Molecular Biology Server, http://www.expasy.ch/) was used to predict transmembrane domains, indicated by thick lines on top.
Trang 5A14-SR To verify our hypothesis, bovine cDNA encoding
the 38-kDa protein was cloned to identify the catalytic
activity of the expressed protein
Cloning of the cDNA encoding bovine sterol
A14-reductase
Bovine cDNA clones, similar to human SR-/ (TM7SF2),
were retrieved by a BLASTN search in the EST database The
putative cDNA of the bovine Al4-SR was obtained by
aligning four different clones (GenBank accession nos
BE756766, BE756734, BE754556 and AW427392) [34] The
bovine cDNA was synthesized by RT-PCR using synthetic
primers based on the EST sequences, cloned into the
pCR2.1 vector, and sequenced on both strands The cloned
cDNA was 1370 bp long and contained an ORF of
1257 bp, encoding a protein of 418 amino acids with a
calculated molecular mass of 46 751 Da
The N-terminal amino-acid sequence of the protein
purified from liver and the amino-acid sequence of the 19.5-
kDa fragment generated by S aureus V8 protease digestion
corresponded to residues 2—24 and 243-261, respectively, of
the putative protein (Fig 1) In the purified protein, the
N-terminal methionine was cleaved out, as previously
described for most eukaryotic proteins [35] Moreover, the
sequenced 27- and 19.5-kDa fragments appeared to origi-
nate from cleavage of the protein in two parts (Fig 1),
which accounted for the calculated molecular mass of
46.7 kDa Therefore, the discrepancy between the appar-
ent molecular mass of 38 kDa estimated by SDS/PAGE
and the calculated molecular mass may be due to an
aberrant electrophoretic migration, as reported for other
structurally related proteins [19,36]
The putative protein was rich in leucine (19.1%) and
highly hydrophobic, with nine predicted membrane-span-
ning domains (Fig 1) The deduced amino-acid sequence
displayed similarity to putative human SR-1 (92%), the
197-615 domain of human LBR (71%), A thaliana Al4-SR
(59%), Saccharomyces cerevisiae Al4-SR (55%) (Fig 1),
and other sterol reductases [50% and 49% similarity to
human and 4A thaliana sterol A7-reductases, respectively,
and 44% to S cerevisiae sterol A24(28)-reductase]
The EFGGx(2)G signature of sterol A24(28)-reductase
and Al4-SR and the LLxSGWWGx(2)RH signature of
sterol reductases family [37] were present at positions 12-18
and 337-348 of the deduced amino-acid sequence, respec-
tively Ergosterol biosynthesis ERG4/ERG?24 family signa-
tures, Gx(2)[LIVM][YH]Dx[FYV]xGx(2)LNPR — and
[LIVM](2)HRx(2)Rpx(3)Cx(2)K YG [38] were found at
positions 167-182 and 383-399 of the deduced amino-acid
sequence, respectively A leucine-zipper region was present
at position 139-160
The presence of signature patterns conserved from yeast
Al4-SR (ERG24 gene) and sterol A24(28)-reductase (ERG4
gene), as well as the degree of similarity with human LBR
and Al4-SR from plants and yeast, strongly suggest that the
cloned cDNA corresponds to A/4-SR
Sterol A14-reductase mRNA expression in bovine tissues
Northern blot analysis of bovine tissues was performed with
Al4-SR cDNA A single transcript of ~ 1.8 kb was detected
in different tissues High levels of mRNA expression were
found in liver and brain (Fig 2) No transcript was detected
in the heart, contrary to TM7SF2, highly expressed in the human tissue [18]
Expression of sterol A14-reductase cDNA
in transfected COS-7 cells Western blot analysis Immunoblot analysis of the ex- pressed A/4-SR cDNA was performed using a polyclonal antibody raised against the bovine liver Al4-SR The antibody recognized a single band of ~38 kDa both in Al4-SR transfected cells and in bovine liver microsomes (Fig 3) No protein was detected in cells transfected with control vector The expressed myc-tag-A14-SR was detected
by both anti-(Al4-SR) Ig and anti-(c-myc) Ig as a protein of
=~ 56 kDa, consistent with the fusion of six myc epitopes (= 9.3 kDa) at the N-terminus of the protein (Fig 3) Cellular localization The cellular localization of myc-tag- Al4-SR was examined in transiently transfected COS-7 cells Double immunofluorescence analysis of cells showed a similar labelling pattern with anti-(myc-tag) Ig and anti- (A14-SR) Ig (Fig 4) The images showed that the newly formed protein was distributed throughout the ER in the proximity of the nucleus The same localization was observed in transfected cells over-expressing Al4-SR; no label was observed in control cells These results are consistent with the known subcellular localization of the enzymes involved in cholesterol biosynthesis and with the purification of the bovine protein from the ER
Determination of Al4-SR activity To demonstrate that the cloned bovine liver cDNA encodes a protein with Al4-
SR activity, cDNA was cloned in the expression vector pMT2 and transfected into COS-7 cells Microsomes prepared from transfected cells were assayed for Al4-SR
adrenal skeletal
testis liver
E
se
xc 2 brain
:
Fig 2 Northern blot analysis of bovine tissues The RNAs (20 ug) extracted from different tissues were blotted onto a nitrocellulose filter and hybridized by *°P-labelled cDNA specific for bovine A14-SR (A) Hybridized A/4-SR transcript (arrow) (B) Nitrocellulose filter show- ing total RNA (288 and 18S rRNAs are indicated).
Trang 6A B C D kDa
Fig 3 Immunoblot analysis of bovine sterol A14-reductase expressed in
COS-7 cells Microsomal proteins were separated on a 12% (w/v) SDS
gel and transferred to PVDF membranes Lane A, bovine liver (40 pg
protein); lanes B and D, COS-7 cells transfected with myc-tag-A14-S'R
cDNA (5 ug protein); lane C, COS-7 cells transfected with 4/4-SR
cDNA (5 ug protein) Blots were probed with specific antibodies: anti-
(bovine liver Al4-SR) Ig danes A—-C) and anti-(myc-tag) Ig dane D)
Detection was performed by the enhanced chemiluminescence proce-
dure Molecular size markers are shown on the right
activity by incubation with C27A*"* sterol C27A® sterol was
undetectable at the beginning of incubation both in COS-7
cells and bovine liver microsomes Endogenous Al4-SR
activity of microsomes obtained from control COS-7 cells,
measured on the basis of C27A®* formation and C27A®*'*
disappearance, was much lower than that observed in
bovine liver microsomes (Fig 5) COS-7 cells expressing
Al4-SR cDNA exhibited Al4SR microsomal activity
sixfold to sevenfold higher than that of control cells and
comparable to that of bovine liver microsomes (Fig 5)
These results indicate that the cloned bovine cDNA encodes
a functional Al4-SR
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“control Al4SR liver
microsomes Fig 5 Sterol Al4reductase activity of transfected COS-7 cells Microsomes (0.24 mg protein), prepared from cells transfected with the empty pMT2 vector (control) or with 4/4-SR cDNA (A14-SR) and bovine liver microsomes (0.24 mg protein), were assayed for sterol Al4-reductase activity by incubation for 30 min with C27A*"* in the conditions described in Materials and methods Enzymatic activity was evaluated on the basis of peak area ratios between m/z 426 and m/z 372 ions (C27A*'*/5a-cholestane) or m/z 428 and m/z 372 ions (C27A*/Sa- cholestane) at the expected retention time At zero incubation time the C27A*'*/5a-cholestane peak area ratio determined for control cells, transfected cells, and liver microsomes was 4.23 + 0.56 Data shown are mean + SD (n = 3)
The present study describes the cloning and functional characterization of bovine Al4-SR, thus providing evi- dence that the previously cloned human T7M7SF?2 corre- sponds to A/4-SR Identification of TM7SF2 as the human gene encoding Al4-SR paves the way for studies
on molecular regulatory mechanisms of the 4/4-SR gene expression and its possible role in the metabolism of meiosis activating sterols Mutation analysis of TM7SF2 will clarify whether a defect in this gene underlies the Greenberg skeletal dysplasia
Fig 4 Cellular localization of sterol Al4-reductase (A) and (B) Immunofluorescence photomicrographs of transfected COS-7 cells expressing myc-tag-Al4-SR Cells were labelled with rabbit anti-(Al4-SR) Ig and secondary Cy3-conjugated sheep anti-(rabbit IgG) Ig (A) and then with monoclonal mouse anti-(myc-tag) Ig and secondary FITC-conjugated goat anti-(mouse IgG) Ig (B) (C) Immunoflurescence photomicro- graphs of transfected COS-7 cells expressing Al4-SR Cells were labelled with rabbit anti-(Al4-SR) Ig and secondary Cy3-conjugated sheep anti-(rabbit IgG) Ig.
Trang 7ACKNOWLEDGEMENTS
We are grateful to Prof D Barra and Prof L Binaglia for critical
reading of the manuscript and helpful suggestions Thanks are extended
to D Piobbico and A Toia for excellent technical assistance This study
was supported by grants from the University of Perugia, Italy
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