(Received for publication, August 25, 1994; and in revised form, October 26, 1994)
From the
Squalene epoxidase (SE) (EC 1.14.99.7) catalyzes the first
oxygenation step in sterol biosynthesis and is suggested to be one of
the rate-limiting enzymes in this pathway. Rat SE cDNA was isolated by
selecting yeast transformants expressing rat cDNA in the presence of
terbinafine, an inhibitor specific for fungal SE. The expression of rat
SE in the isolated terbinafine-resistant clone was confirmed by its
survival in the presence of either terbinafine or an inhibitor specific
for mammalian SE, NB-598, but not in the presence of both terbinafine
and NB-598. Rat SE polypeptide deduced from the nucleotide sequence
contains 573 amino acids, and its molecular weight is 63,950 Da. The
amino acid sequence reveals one potential transmembrane domain, a
hydrophobic segment (Leu to Tyr
) in the
NH
-terminal region. This region also contains a
1-
A-
2 motif, which is the consensus sequence for an FAD
binding domain, suggesting that SE is a flavoenzyme. This deduced rat
SE sequence is 30.2% identical to the ERG 1 gene, which
encodes SE from an allylamine-resistant Saccharomycescerevisiae mutant. Expression of a full-length rat SE
protein in Escherichia coli confirms this polypeptide as a
functional SE. This is the first report of the molecular cloning of
mammalian SE.
Cholesterol biosynthesis is a complex biological
process(1, 2) , and it provides a therapeutic target
for the reduction of low density lipoprotein cholesterol, a key risk
factor in coronary heart disease (3) . Recent studies have
revealed a post-mevalonate pathway of cholesterol biosynthesis that
plays an important role in the supply of obligate precursors for
dolichol, ubiquinone and isopentenyl tRNA synthesis, and for
polyprenylation of p21(4) and small
GTP-binding proteins(5) , which function in the regulation of
normal cellular processes and in oncogenesis(1) . As a result,
three enzymes which are located beyond branch point in this pathway,
namely squalene synthase, squalene epoxidase (SE), (
)and
oxidosqualene cyclase, have evoked considerable interest as potential
targets for the design of hypercholesterolemic
chemotherapeutics(6, 7) . SE appears to be an
important rate-limiting enzyme in this cascade, as it has an extremely
low specific activity compared to 3-hydroxy-3-methylglutaryl-coenzyme A
reductase or squalene synthase (8, 9) . Furthermore,
the addition of exogenous cholesterol to human renal cancer cells
results in the accumulation of squalene(10) .
SE (EC 1.14.99.7) catalyzes the first oxygenation step in the sterol biosynthetic pathway, the conversion of squalene to 2,3(S)-oxidosqualene. It has been reported, however, that not only 2,3(S)-oxidosqualene but also 2,3(S);22(S),23-squalene diepoxide is accumulated in liver homogenates (11, 12) and in cultured mammalian cells (13) in the presence of an oxidosqualene cyclase inhibitor. 2,3(S);22(S),23-Squalene diepoxide is cyclized in rat liver homogenates to 24(S),25-epoxylanosterol(14) , which is then converted to 24(S),25-epoxycholesterol. 24(S),25-Epoxycholesterol was identified as one of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase repressor activities in the cultured cells(15) . Consequently, it would be interesting to know whether SE is involved in the synthesis of such oxysterols via squalene dioxide formation(16) .
SE is believed to be a microsomal membrane-bound protein. Rat SE requires FAD, NADPH-cytochrome P-450 reductase (EC 1.6.2.4), NADPH, and a supernatant protein factor for its activity(17, 18, 19, 20) . Triton X-100 can be substituted for the supernatant protein factor(19) . The fact that this enzyme is not inhibited by CO(17, 21) , as well as the product analysis of a porphyrin mutant of Saccharomyces cerevisiae(22, 23) , indicates that SE is not a cytochrome P-450 isozyme. It is the only non-cytochrome P-450 enzyme that epoxidizes a double bond in an alkyl chain(24) . Although much less is known about fungal SE, the ERG 1 gene recently cloned from an allylamine-resistant S. cerevisiae mutant was shown to encode a 55,190-Da SE protein consisting of 496 amino acids(25) . In this report, we describe the molecular cloning of rat SE by a new inhibitor selection method, its structure, and its expression in prokaryotic cells.
For immunoblot analysis, the antibody was visualized with horseradish peroxidase-conjugated goat anti-rabbit IgG (Life Technologies, Inc.), using Renaissance Western blot chemiluminescence reagent (DuPont NEN) according to the instructions of the manufacturer. Gels were calibrated with prestained low range molecular weight markers (Bio-Rad).
Figure 1: Strategy for rat SE cDNA cloning. Tb stands for terbinafine.
Figure 2:
Sensitivities of S. pombe transformants for inhibitors of SE. Transformants were grown on
leucine-deficient MMA plates supplemented with nothing (Control), terbinafine (+Tb), NB598
(+NB), and both terbinafine and NB598
(+Tb+NB). Terbinafine and NB 598
concentration was 12.5 µg/ml.
Figure 3:
Comparison of the amino acid sequences of
the predicted rat liver and yeast SE. Amino acid residues are numbered
on the left. R and Y stand for rat and yeast (S. cerevisiae) SE, respectively. Residues identical in rat
and yeast SE are boxed. The hydrophobic region
Leu-Tyr
, which is predicted to be a
membrane associated domain, is underlined with a singleline. The putative FAD binding domain is underlined with a doubleline.
The hydropathy plot, using a standard algorithm of
Kyte and Doolittle (30) , revealed one possible transmembrane
domain in the NH-terminal portion
(Leu
-Tyr
) of the protein. This domain
consists of 70% hydrophobic residues, which could be sufficient to
anchor the protein in the membrane of the endoplasmic
reticulum(31, 32) .
As shown in Fig. 4,
there is a 1-
A-
2 motif (33) in both rat SE
(Val
-Glu
) and S. cerevisiae SE (Ala
-Glu
) that demonstrates
significant homology with the FAD binding domains of human erythrocyte
glutathione reductase (33) and Pseudomonas fluorescensp-hydroxybenzoic acid hydroxylase(34) . The
presence of a consensus FAD binding domain sequence is consistent with
data suggesting that rat SE requires exogenous FAD for its activity (19) and strongly suggests that SE is directly associated with
FAD.
Figure 4:
Putative FAD binding site sequences of
SE. The highly conserved 1-
A-
2 motifs of the yeast and
rat SE and two flavoenzymes are depicted. Completely conserved amino
acid residues are boxed with boldlines,
hydrophobic residues in which conservative changes are tolerated are boxed with lightlines.
Figure 5:
Identification of SE activity of
recombinant rat SE and immunoblotting analysis of recombinant and
native rat SE. A, cell extracts were prepared as described
under ``Experimental Procedures.'' Partially purified rat
liver SE and cell extracts were incubated with
[C]squalene for 30 min at 37 °C in the
presence of 0.2 unit of NADPH-cytochrome c reductase, 1 mM NADPH, 0.01 mM FAD, and 0.3% Triton X-100. Total
nonsaponifiable substances were applied to thin-layer chromatography
plates, which were developed with 0.5% ethyl acetate/benzene and
detected autoradiographically. The migration of standard compounds on
the plate is indicated by arrows. The samples shown are: lanes1 and 2, S and P fractions,
respectively, from control vector pET3a-transformed E. coli; lanes3 and 4, S and P fractions,
respectively, from rat SE expression vector pETRSE-transformed E.
coli. B, 20 µg of fractions, described in panel
A, were separated on a 10% SDS-polyacrylamide gel in the same
lanes as in panel A. They were blotted electrophoretically
onto nitrocellulose membrane and analyzed immunochemically with
anti-recombinant rat SE antiserum as mentioned under
``Experimental Procedures.'' C, 30 µg of
partially purified SE fraction from rat liver (lane1) and crude extract from rat liver microsomes (lane2) were analyzed immunochemically as in panelB. The samples were prepared as described
previously(28) .
In order to confirm the expression of rat
SE protein in the transformants, we performed an immunoassay using an
antiserum against the purified recombinant
Glu-His
fragment of rat SE expressed
in E. coli. Although this fragment excluded the plausible
membrane binding domain, it did contain the SE activity. As shown in Fig. 5B, a 64-kDa immunoreactive protein was present in
both the S and P fractions of a pETRSE containing transformant (B, lanes 3 and 4). We also detected a
truncated protein in the P fraction and small fragments in the S
fraction. In spite of the existence of the immunoreactive protein, the
P fraction did not show any SE activity (Fig. 5A, lane 4). This might be caused by insufficient renaturation by
dialysis after solubilization of particulates with urea buffer.
Only a single immunoreactive protein, with relative molecular mass of 64 kDa, was detected in rat liver microsomes (Fig. 5C, lane2). Although the partially purified SE fraction from rat liver also contained an immunoreactive protein of 64 kDa, two other immunoreactive proteins of 57 and 51 kDa were detected. These results suggest that the molecular mass of rat SE is 64 kDa, and that a 51-kDa protein previously identified as rat SE (28) is most likely a truncated form of the enzyme produced by proteolysis during the purification procedure.
Both the recombinant rat SE and the partially purified SE from rat liver lost activity when Triton X-100 was absent during purification. Recombinant rat SE behaves as a multimeric aggregate during gel filtration in the absence of Triton X-100. Enzymatic activation by Triton X-100 at a concentration greater than its critical micelle concentration, in the presence of 1 mM DTT, suggests that the monomer is the active form of the enzyme. However, there are a number of alternative explanations for the Triton X-100 activation, such as a conformational change of the enzyme, an increased rate of enzyme diffusion or association of enzyme and substrate, and effects on enzyme kinetic parameters.
Until now mammalian liver SE had not been purified, due to its poor stability, and little is known of its structure or properties. Our new cloning strategy using a species-specific inhibitor of the enzyme demonstrates the utility of this trans-complementation cloning method, and has enabled us to produce the first purified SE. The availability of this cloned DNA will now permit detailed studies on the structure and properties of rat SE, and should also aid in the development of more effective inhibitors of SE. In addition, more precise studies of the regulation of the enzyme will now be feasible. Although it has been suggested that SE is regulated by sterols(8, 9) , the mechanism of this regulation is not known. The availability of a rat SE clone, and antibodies to the SE protein, will allow us to determine whether this regulation by sterols occurs at the level of mRNA synthesis, SE translation, or increased SE activity. Finally, we should now be able to determine if SE is involved in oxysterol formation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D37920[GenBank].