(Received for publication, November 9, 1995; and in revised form, December 18, 1995)
From the
Four types of 17-hydroxysteroid dehydrogenases have been
identified so far. The porcine peroxisomal 17
-hydroxysteroid
dehydrogenase type IV catalyzes the oxidation of estradiol with high
preference over the reduction of estrone. A 2.9-kilobase mRNA codes for
an 80-kDa (737 amino acids) protein featuring domains which are not
present in the other 17
-hydroxysteroid dehydrogenases. The 80-kDa
protein is N terminally cleaved to a 32-kDa fragment with
17
-hydroxysteroid dehydrogenase activity. Here we show for the
first time that both the 80-kDa and the N-terminal 32 kDa (amino acids
1-323) peptides are able to perform the dehydrogenase reaction
not only with steroids at the C17 position but also with
3-hydroxyacyl-CoA. The central part of the 80-kDa protein (amino acids
324-596) catalyzes the 2-enoyl-acyl-CoA hydratase reaction with
high efficiency. The C-terminal part of the 80-kDa protein (amino acids
597-737) is similar to sterol carrier protein 2 and facilitates
the transfer of 7-dehydrocholesterol and phosphatidylcholine between
membranes in vitro. The unique multidomain structure of the
80-kDa protein allows for the catalysis of several reactions so far
thought to be performed by complexes of different enzymes.
The redox reactions at position C17 of the steroid molecule are
catalyzed by a number of different 17-hydroxysteroid
dehydrogenases
(17
-HSD)(
)(1, 2, 3) . Until
now, four human 17
-HSDs were characterized. The soluble
17
-HSD type I consisting of 327 amino acids (aa) was cloned from
human placenta and performs the oxidation of estradiol at the same
efficiency as the reduction of
estrone(4, 5, 6) . The 17
-HSD type II is
a microsomal enzyme of 387 aa that slightly prefers the oxidation over
the reduction of estrogens and androgens and is expressed at high
levels in the human placenta(7, 8) . The testes
predominantly express the microsomal 17
-HSD type III consisting of
310 aa and is responsible for the reduction of estrogens and
androgens(9) . The porcine 17
-HSD type IV inactivates
hormones very efficiently because of its 360-fold preference for
steroid oxidation (10, 11) and is the first steroid
metabolizing enzyme localized in peroxisomes(12) . The enzyme
is primarily translated as an 80-kDa protein from a 2.9-kilobase
message(13) . The post-translational modifications include an
N-terminal cleavage leading to a 32-kDa peptide(10) . A
fraction of the 32-kDa peptide is covalently linked to actin through an
(
-glutamyl)-lysine bond(14) . Recently, cloning of
the human and mouse 80-kDa 17
-HSD type IV showed a close
relationship revealing 85% amino acid similarity, the same multidomain
structure, and identical kinetic parameters of the 17
-HSD
IV(15, 16) . In contrast, the overall similarity
between sequences of four human 17
-HSD type I-IV is less than 25%.
The amino acid sequence comparison with the Swissprot and EMBL data
bases (17) revealed several interesting features of the type IV
enzyme (Fig. 1). The N-terminal part shows homologies to the
family of short chain alcohol
dehydrogenases(18, 19, 20) , especially to
the two short chain alcohol dehydrogenases domains of the
multifunctional (hydratase-dehydrogenase) enzymes of peroxisomal
-oxidation of fatty acids in Saccharomyces cerevisae(21) and Candida tropicalis(22) . The central
domain of the 17
-HSD type IV is 40 and 38% identical (Fig. 1) with the C-terminal parts of the S. cerevisae and C. tropicalis multidomain proteins, respectively. The
C-terminal extension of the 80-kDa protein shows an intriguing
similarity to the sterol carrier protein 2 (SCP2) which is assumed to
participate in the intracellular transport of sterols and
lipids(23, 24, 25, 26, 27) .
Although the SCP2 was first identified as a 13-kDa protein it is,
however, as well part of a 60-kDa fusion protein between SCP2 and a
peroxisomal 3-oxoacyl-CoA thiolase named
SCPx(28, 29, 30) . Recently, it was
demonstrated that SCP2 and SCPx are expressed from a single gene via
alternative transcription initiation from two distinct
promoters(31, 32) .
Figure 1:
Amino acid similarities of multidomain
proteins. FOX2, hydratase-dehydrogenase of S. cerevisiae; 17-HSD, porcine 80-kDa
protein; SCAD, similarity to short chain alcohol dehydrogenase
superfamily.
The 80-kDa protein reveals a
complex structure which was unknown among other 17-hydroxysteroid
dehydrogenases and enzymes of peroxisomal
-oxidation of fatty
acids. To evaluate the activities suggested by the amino acid
similarities, the functionalities of the purified porcine
17
-hydroxysteroid dehydrogenase as well as the expressed
recombinant single domains were assayed.
Figure 2:
Expression of 17-hydroxysteroid
dehydrogenase activity in HEK 293 cells transfected with pRep10-p80.
HEK 293 cells were transfected with a pRep10-p80 vector coding for the
full-length 80-kDa protein and harvested at time points after
transfection as indicated. The 17
-HSD activity was assayed with
17
-estradiol in cell homogenates as described under
``Materials and Methods.'' Open bars, control cells; dark bars, transfected cells.
To clarify if the processing of the 80-kDa
protein to its N-terminal 32-kDa fragment is necessary for the
activation of the 17-hydroxysteroid dehydrogenase Western blot
analysis was performed. HEK 293 cells transfected with plasmid coding
for the full-length or the N-terminal domain were subjected to
immunoblotting with a mouse monoclonal antibody F1 (10) recognizing both the 80- and 32-kDa forms of the enzyme (Fig. 3). The effect of in vivo processing is shown in
porcine kidney homogenates (Fig. 3, lane 2). The cells
transfected with pRep10-p80 reveal a single band at 80 kDa (lane
3), those transfected with pRep10-p32 a band at 32 kDa (lane
4). Since the transfected cells show comparable specific activity
of the 17
-HSD (Fig. 3) and no 32-kDa band is seen in cells
transfected with pRep10-p80, the 80-kDa protein is active as a
17
-hydroxysteroid dehydrogenase without cleavage to the 32-kDa
fragment.
Figure 3:
Processing of 80-kDa protein. Samples were
subjected to SDS-PAGE and immunoblotting with monoclonal antibody F1
conjugated with peroxidase. Lane 1, molecular mass standards; lane 2, porcine kidney homogenates; lane 3, HEK 293
cells transfected with pRep10-p80 vector coding for full-length 80-kDa
protein; lane 4, cells transfected with pRep10-p32 vector
coding for N-terminal 32-kDa fragment. Lane 2, 5 µg of
protein; lanes 3 and 4, 20 µg of protein.
Specific activities of the 17-HSD IV are given at the bottom.
Figure 4: Purified domains of the porcine 80-kDa protein. Domains were expressed in E. coli in pGex-2T PL2 vector using inserts coding for the N-terminal domain (amino acids 1-323), central hydratase-like domain (aa 324-596) and C-terminal SCP2-like domain (aa 597-737). The glutathione S-transferase fusion proteins were adsorbed on glutathione-agarose digested with thrombin and eluted as described under ``Materials and Methods.'' Samples (5 µg) were subjected to SDS-PAGE and stained with Coomassie Blue. Lane 1, molecular mass markers; lane 2, N-terminal domain; lane 3, central domain; lane 4, C-terminal domain.
More direct evidence on the involvement of the SCP2-related domain in the sterol and lipid transfer was obtained with the porcine recombinant peptide. Both the GST-SCP2 fusion product and the SCP2-like protein stimulate the transfer of 7-DHC and PC from donors to acceptors. The purified porcine SCP2-like peptide increases the transfer of 7-DHC and PC 147- and 158-fold over the control levels, respectively (Table 2).
Amino acid sequence comparisons suggest a relationship
between the four human 17-hydroxysteroid dehydrogenases and
bacterial proteins involved in fatty acid metabolism(35) . The
high similarity of 17
-HSD IV to the C. tropicalis or S. cerevisiae enzymes participating in the
peroxisomal
-oxidation of fatty acids even suggests a common
ancestor(17, 35, 36) . The porcine
17
-HSD IV is the first peroxisomal enzyme with proven
dehydrogenase activity against steroids and fatty acids. The K
values for 17
-estradiol (0.2-0.4
µM) and for crotonyl-CoA (31-35 µM) are
compatible with the physiological concentrations of the substrates and
are close to K
observed in other dehydrogenases
specialized in either
substrate(1, 34, 37, 38) . Recently,
a rat homologue (85% amino acid identity) of the porcine protein has
been purified (39) and cloned. (
)The isolated rat
enzyme shows activity of 3-hydroxyacyl-CoA dehydrogenase with fatty
acids, 2-methyl-branched fatty acids, and bile acid intermediates
(3-hydroxyacyl derivative of trihydroxycoprostanic acid)(39) .
It remains to be settled which substances are the preferred in vivo substrates for the 80-kDa protein.
The domains of the
multifunctional (fatty acids hydratase-dehydrogenase) FOX2 gene product
of S. cerevisiae were studied by Hiltunen et
al.(21) . The deletion of the carboxyl-terminal domain
(271 aa) resulted in a loss of hydratase activity (converting
trans-2-enoyl CoA into D-3 hydroxyacyl-CoA) but the D-specific 3-hydroxyacyl-CoA dehydrogenase activity was
retained. This pointed indirectly to the localization of the
dehydrogenase activity in the N-terminal part. In our report different
enzymatic activities have been assigned unequivocally to the individual
regions of the 80-kDa protein by analyses of the isolated domains
expressed in E. coli. All the functionalities suggested by the
amino acid similarities were observed in both the 80-kDa protein and in
its isolated recombinant domains. This excludes the possibility that
the processing into smaller peptides is a prerequisite for the release
of the activities. However, at least the processing into a 32-kDa
fragment was observed in porcine tissues. There are different cleavage
efficiencies: high in hormone target organs like uterus and breast
epithelium but low in non-target tissues such as kidney and
liver(40) . It is yet unclear if the separation of the 32-kDa
17-HSD IV from the other parts is an advantage in hormone
inactivation. Most probably the lack of the hydratase and SCP2 domains
in the vicinity of the 17
-HSD IV could reduce the competition
between steroids and fatty acids for the active center of 17
-HSD
IV.
All amino acids which were shown to be essential for the lipid
transfer activity of human SCP2 by site-directed mutagenesis are
conserved in the porcine SCP2-like domain of the 80-kDa
protein(17, 25) . The relatively low activity of
porcine SCP2 may be due to the fact that some amino acids, which may be
required for the full activity are missing at the N terminus. The high
activity obtained with the purified native protein supports the view
that the separation between hydratase and SCP2 domains is not yet
optimally set. As presumed earlier by Pfeiffer et al.(26) the SCP2 might have an important function in the
beginning of steroidogenesis by stimulating the pregnenolone synthesis.
The biological role of the C-terminal sterol transfer domain contained
within the 80-kDa protein is not known at present. The results shown in Table 1strongly suggest that the domain is not involved in the
hydratase/dehydrogenase activity of the enzyme. Because the SCP2-like
domain contains the peroxisomal targeting sequence AKI (other domains
are devoid of any targeting signals) one possibility would be to ensure
proper peroxisomal localization of the whole protein. On the other hand
this exclusive function does not explain the considerable degree of
structural and functional conservation of the domain with SCP2.
Therefore, an additional function appears to be likely. In vitro SCP2 seems not to facilitate the movement of most steroids (41) or fatty acids(42) . However, we currently cannot
exclude that 17-HSD IV also utilizes other, more hydrophobic
substrates in vivo which may require transfer from the
peroxisomal membrane to the catalytically active site, located
primarily in the peroxisomal matrix. Alternatively, at present it
cannot be ruled out that the SCP2-like domain has no direct functional
relevance with respect to the catalytic activity of the 80-kDa protein.
Additional studies are clearly necessary in order to discriminate
between these possibilities.
The advantages of the multidomain
structure of porcine 80-kDa protein (17-HSD IV + hydratase
+ SCP2) or of the SCPx (3-oxoacyl-CoA thiolase + SCP2) remain
to be established. It permits the coordination of regulation of gene
expression for functionally related but yet diverse enzymes. The
composition of the 80-kDa protein allows for the catalysis of several
processes of peroxisomal
-oxidation of fatty acids by a single
macromolecule instead of a participation of several
enzymes(21, 38, 39, 43) . Such a
concerted action might further be essential in the metabolism of
sterols and steroids.
The porcine 17-hydroxysteroid
dehydrogenase IV is the first peroxisomal enzyme known to be stimulated
by progesterone(40) . The hormone is as well responsible for
the regulation of other types of 17
-HSD(44, 45) .
The recently purified and cloned rat 80-kDa homologue responds as well
to peroxisomal proliferators such as clofibrate and WY
14,643(39, 46) . The 80-kDa protein seems to be
controlled by modulators of steroid and fatty acid metabolism. The high
level of conservation of amino acid sequence (85% identity) between
human, mouse, rat, and porcine 80-kDa proteins suggests an essential
function of this type of protein.