A steroidogenic pathway for sulfonated steroids: The metabolism of pregnenolone sulfate

Article abstract of DOI:10.1016/j.jsbmb.2014.07.005

In many tissues sulfonated steroids exceed the concentration of free steroids and recently they were also shown to fulfill important physiological functions. While it was previously demonstrated that cholesterol sulfate (CS) is converted by CYP11A1 to pregnenolone sulfate (PregS), further conversion of PregS has not been studied in detail. To investigate whether a steroidogenic pathway for sulfonated steroids exists similar to the one described for free steroids, we examined the interaction of PregS with CYP17A1 in a reconstituted in-vitro system. Difference spectroscopy revealed a K_d-value of 74.8 ± 4.2 μM for the CYP17A1-PregS complex, which is 2.5-fold higher compared to the CYP17A1-pregnenolone (Preg) complex. Mass spectrometry experiments proved for the first time that PregS is hydroxylated by CYP17A1 at position C17, identically to pregnenolone. A higher K_m- and a lower k_cat-value for CYP17A1 using PregS compared with Preg were observed, indicating a 40% reduced catalytic efficiency when using the sulfonated steroid. Furthermore, we analyzed whether the presence of cytochrome b₅(b₅) has an influence on the CYP17A1 dependent conversion of PregS, as was demonstrated for Preg. Interestingly, with 17OH-PregS no scission of the 17,20-carbon-carbon bond occurs, when b₅is added to the reconstituted in-vitro system, while b₅promotes the formation of DHEA from 17OH-Preg. When using human SOAT-HEK293 cells expressing CYP17A1 and CPR, we could confirm that PregS is metabolized to 17OH-PregS, strengthening the potential physiological meaning of a pathway for sulfonated steroids.

Full text of DOI:10.1016/j.jsbmb.2014.07.005

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SBMB 4224 1–10
Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry & Molecular Biology
journal homepage: www.elsevier.com/locate/jsbmb
1
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A steroidogenic pathway for sulfonated steroids: The metabolism of
pregnenolone sulfate
a
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Q1
J. Neunzig , A. Sánchez-Guijo , A. Mosa , M.F. Hartmann , J. Geyer , S.A. Wudy ,
a,
R. Bernhardt *
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8
a
Department of Biochemistry, Faculty of Technical and Natural Sciences III, Saarland University, 66123 Saarbrücken, Germany
Steroid Research & Mass Spectrometry Unit, Division of Pediatric Endocrinology & Diabetology, Center of Child and Adolescent Medicine,
Justus-Liebig University, 35392 Giessen, Germany
Institute of Pharmacology and Toxicology, Justus-Liebig University of Giessen, 35392 Giessen, Germany
b
c
A R T I C L E I N F O
A B S T R A C T
Article history:
In many tissues sulfonated steroids exceed the concentration of free steroids and recently they were also
shown to fulfill important physiological functions. While it was previously demonstrated that cholesterol
sulfate (CS) is converted by CYP11A1 to pregnenolone sulfate (PregS), further conversion of PregS has not
been studied in detail. To investigate whether a steroidogenic pathway for sulfonated steroids exists
similar to the one described for free steroids, we examined the interaction of PregS with CYP17A1 in a
Received 7 May 2014
Received in revised form 11 July 2014
Accepted 14 July 2014
Available online xxx
reconstituted in-vitro system. Difference spectroscopy revealed a K
CYP17A1–PregS complex, which is 2.5-fold higher compared to the CYP17A1–pregnenolone (Preg)
complex. Mass spectrometry experiments proved for the first time that PregS is hydroxylated by
CYP17A1 at position C17, identically to pregnenolone. A higher K - and a lower kcat-value for
m
CYP17A1 using PregS compared with Preg were observed, indicating a 40% reduced catalytic efficiency
d
-value of 74.8 ꢀ 4.2
mM for the
Keywords:
CYP17A1
Steroidogenesis
Sulfonated steroids
Pregnenolone sulfate
Mass spectrometry
when using the sulfonated steroid. Furthermore, we analyzed whether the presence of cytochrome
b
5
(b
Interestingly, with 17OH-PregS no scission of the 17,20-carbon–carbon bond occurs, when b
the reconstituted in-vitro system, while b promotes the formation of DHEA from 17OH-Preg. When using
human SOAT-HEK293 cells expressing CYP17A1 and CPR, we could confirm that PregS is metabolized to
7OH-PregS, strengthening the potential physiological meaning of a pathway for sulfonated steroids.
5
) has an influence on the CYP17A1 dependent conversion of PregS, as was demonstrated for Preg.
5
is added to
5
1
ã 2014 Published by Elsevier Ltd.
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1
. Introduction
unconjugated steroids [4–6]. Dehydroepiandrosterone (DHEA) for
example, one of the most abundant steroid in humans, occurs to 99%
in its sulfonated form [7], reaching concentrations of up to 10 mM in
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CYP11A1 initiates steroid hormone biosynthesis (Fig. 1) through
a side-chain cleavage reaction on cholesterol yielding pregnenolone
young adults [8]. Steroid sulfates are generated by sulfonation of
free steroids by three different sulfotransferases (SULT1E1, SULT2B1,
SULT2A1). These enzymes are widely distributed in mammalian
organism, as NCBI EST profiles indicate. Sulfonation of unconjugated
steroids seems to contribute to the modulation of the genomic
action of steroids. For instance, SULT1E1 is highly active in cultured
normal breast epithelial cells compared to tumor cell lines [9]. Since
it is known that increased exposure of estrogens increases breast
cancer development, sulfonation of estrogens might be a crucial
mechanism to impede the danger of excessive estrogenic exposure
[9]. In addition, alterations of steroid sulfonation have a severe
impact on the development in mammals. DHEA is mainly produced
in the adrenals and the gonads. In the gonads DHEA is further
metabolized to sex hormones, but in the adrenals, due to the
presence of SULT2A1, most of the DHEA is sulfonated. In case of
Q2 26
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[1,2] which represents the precursor of all steroid hormones. In a
series of reactions where six different cytochromes P450 (CYPs) and
three hydroxysteroid dehydrogenases (HSD) participate, miner-
alocorticoids, glucocorticoids and sex hormones are formed.
In order to induce a biological response, steroid hormones
interact with their corresponding receptor and thus e.g., regulation
of blood pressure, provision of carbon hydrates or development of
secondary sexual characteristics take place in mammalian organ-
isms. Interestingly, sulfonated steroids or sulfated steroids [3] often
circulate in mammals in considerably higher concentrations than
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*
Corresponding author. Tel.: +49 681 302 3005; fax: +49 681 302 4739.
E-mail address: ritabern@mx.uni-saarland.de (R. Bernhardt).
http://dx.doi.org/10.1016/j.jsbmb.2014.07.005
960-0760/ã 2014 Published by Elsevier Ltd.
0
Please cite this article in press as: J. Neunzig, et al., A steroidogenic pathway for sulfonated steroids: The metabolism of pregnenolone sulfate, J.
Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.07.005

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Fig. 1. Steroid hormone biosynthesis involving six cytochromes P450 and three hydroxysteroid dehydrogenases.
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altered DHEA sulfonation, unconjugated DHEA accumulates,
yielding to an excess of androgens and thus, virilization occurs
in women [10].
showed that sulfonated steroid metabolites are involved in many
different processes in mammalian organisms. Cholesterol sulfate
(CS) possesses a stabilizing function in cell membranes; it is
involved in the regulation of serine proteases and, moreover, in the
differentiation of keratinocytes [18]. Recently, DHEAS was shown
to induce a non-classical signaling pathway in spermatogenic cells
[19]. Pregnenolone sulfate (PregS), on the other hand, represents a
reservoir for pregnenolone (Preg), the precursor of mineralo-, and
glucocorticoids, as well as sex hormones and it is described to act
as neurosteroid modulating a big variety of ion channels, trans-
porters, and enzymes [15]. For example, PregS was demonstrated to
inhibitGABA-receptors[20], whichplayacrucialroleintheneuronal
On the other hand, excess of sulfonated steroids can lead to
diseases, like the X-linked ichthyosis, which is caused by the
accumulation of cholesterol sulfate in the epidermis [11].
Cell uptake of steroid sulfates depends on transporters of the
SLC or SLCO family [12–14] as their hydrophilicity caused by the
sulfate moiety hinders a passive passage. These transporters are
expressed in many tissues, like liver, ovary, adrenal gland and
hippocampus [15].
Sulfonated steroids have been regarded for a long time either as
an end-product of xenobiotic metabolism designated for renal
clearance [16] or as an inactive reservoir for unconjugated, active
steroids [17]. Although the knowledge about the physiological role
of steroid sulfates is still scarce, investigations about their
biological function increased in the last decade. Recent studies
network and to modulate N-methyl-D-aspartate (NMDA)-receptors.
These receptors, which are essential for neuronal development and
synapse formation, are heterodimers formed by several subunits
(NR1, NR2A–D, NR3A–B). PregS modulates these receptors by
enhancing the generation of NR1/NR2A and NR1/NR2B receptors
Please cite this article in press as: J. Neunzig, et al., A steroidogenic pathway for sulfonated steroids: The metabolism of pregnenolone sulfate, J.
Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.07.005

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and inhibiting the formation of NR1/NR2C and NR1/NR2D receptors
21]. Further studies revealed that PregS promotes NMDA-receptor
insertion in the cell surface [22] and thus, enhances the function of
NMDA-receptors.
Moreover, PregS seems to play an essential role in reproduction
as extremely increased concentrations during birth, pregnancy and
parturition indicate [15].
It was demonstrated that PregS can be formed from CS through
a side-chain cleavage reaction catalyzed by CYP11A1 [23] and
seems to be metabolized to further sulfonated steroids as Korte
et al. showed using tissue from human adrenals [24]. These
findings inevitably lead to the question, whether a steroidogenic
pathway for sulfonated steroids exists similar to the one
described for free steroids. Trying to elucidate this question,
we decided to investigate whether PregS serves as substrate for
CYP17A1, a microsomal cytochrome P450 involved in the steroid
hormone biosynthesis. CYP17A1 catalyzes the hydroxylation at
position C17 of Preg or progesterone (Prog) yielding 17OH-Preg
and 17OH-Prog, respectively, and subsequently catalyzes a
was from Carbolution (Saarbrücken, Germany). Glucose-6-phos-
phate and glucose-6-phosphate dehydrogenase were purchased
from Roche (Basel, Switzerland). Protino Ni-NTA was obtained
from Macherey-Nagel (Düren, Germany). 17OH-PregS was synthe-
sized in the Steroid Research & Mass Spectrometry Unit, Division
of Pediatric Endocrinology & Diabetology, Center of Child and
Adolescent Medicine, Justus-Liebig University. All other chemicals
were of highest purity available.
[
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3. Experimental
3.1. Construction of recombinant expression plasmids in E. coli
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The plasmid pET-17b was utilized to express bovine CYP17A1,
b
5
and CPR in E. coli. Each cDNA was cloned via the restriction sites
NdeI and BamHI into the vector. The cDNAs of b and CPR were
5
obtained through amplification from a cDNA library of bovine liver
(Zyagen, California, USA). CYP17A1 cDNA was kindly provided by
Prof. M. Waterman (Vanderbilt University, Nashville, USA). The
amino acid sequence of CYP17A1 is lacking its N-terminal
hydrophobic anchor [29] and is extended at the C-terminus by a
hexa-histidine-tag to facilitate purification. The CPR amino acid
sequence is extended at the C-terminus by three glycines and six
histidines and at the N-terminus has a lack of the first 27 amino
17,20-lyase reaction yielding dehydroepiandrosterone (DHEA)
and androstenedione (andro) [25] (Fig. 2). In human and bovidae
families (sheep, goat, bovine, bison), CYP17A1 exhibits lyase
activity only toward delta5-steroids, like Preg and 17OH-Preg
[
26]. In these mammalian species CYP17A1 hydroxylates Prog at
position C17 but 17OH-Prog is not further converted to
androstenedione. Compared to the 17-hydroxylase activity, the
5
acids [30]. Cytochrome b is extended at the C-terminus by a hexa-
histidine-tag. For human cell culture studies, the plasmid
pVITRO1-neo-mcs (Invivogen) was used to express CYP17A1 and
CPR. The cDNAs of CYP17A1 and CPR were cloned via the restriction
sites AgeI and BsrGI (CYP17A1) and BspEI and AvrII (CPR) into the
vector. Both cDNAs were obtained from a cDNA library of bovine
adrenal (CYP17A1) and bovine liver (CPR).
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17,20-lyase reaction using 17OH-Preg as substrate is weak, but it
is strongly enhanced in the presence of cytochrome b (b ) [27] or
5
5
through phosphorylation of CYP17A1 at position 258 of its amino
acid chain [28].
Here, we investigated the reaction of CYP17A1 with PregS as
substrate in
a reconstituted in-vitro system, consisting of
154
recombinantly expressed and purified CYP17A1 and its electron
transfer partner CPR, and were able to demonstrate for the first
time its conversion to 17OH-PregS, but not to DHEAS.
3.2. Protein expression and purification
155
Bovine CYP17A1 was co-expressed with chaperones GroEL and
GroES similar to CYP21 expression [31]. As host E. coli strain
C43DE3 was used. The expression was performed in 2 L baffled
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09
2. Materials
1
flaskscontaining 400 mlTB medium supplemented with 100
mg/ml
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Steroids were obtained from Sigma–Aldrich (Taufkirchen,
Germany) or from C/D/N Isotopes Inc. (Quebec, Canada).
,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC), kanamycin
ampicillin and 50 g/ml kanamycin. The expression culture was
inoculated from an overnight culture and grown at 37 C and
120 rpm. Protein expression was induced at OD600 = 0.6 with 1 mM
m
ꢁ
1
sulfate, arabinose, magnesium chloride, sodium hydroxide, sulfur
trioxide triethylamine, sulfatase from Helix pomatia Type H-1 and
HPLC-grade acetonitrile were from Sigma–Aldrich (Taufkirchen,
Germany). Yeast extract, technical was from Becton, Dickinson and
Company (Heidelberg, Germany). Pepton, pancreatically digested
and Na-acetate were from Merck (Darmstadt, Germany). LCMS
grade water and ammonium hydroxide were purchased from
Fluka (Taufkirchen, Germany). Methanol, pyridine and ethanol
were obtained from Merck (Darmstadt, Germany). SepPak C18
IPTG, 4 mg/ml arabinose, 1 mM
d
-ALA and 50
m
g/ml ampicillin.
ꢁ
Afterwards, temperature was decreased to 26 C and the cells
incubated at 95 rpm for 48 h. Cells were harvested and sonicated as
described elsewhere [32]. The purification was performed as
described for CYP11B1 [33].
Bovine CPR was expressed in E. coli strain C43DE3 using similar
conditions as described for bovine CYP17A1 with slight modifi-
cations: 2 L baffled flasks containing 300 ml TB medium were used
and after induction of protein expression temperature was reduced
ꢁ
(
360 mg) columns were from Waters Corporation (Milford, MA,
to 30 C and incubated at 100 rpm for 30 h. Cells were harvested
USA). DMEM/F12 cell culture media and fetal calf serum were from
Gibco by Life Technologies (California, USA). Imidazole and
ampicillin were from CarlRoth (Karlsruhe, Germany). NADPH
and sonicated as described elsewhere [32]. CPR was purified via
IMAC as described elsewhere [33] with 40 mM imidazole in the
washing buffer and 200 mM imidazole in the elution buffer.
Fig. 2. Reaction catalyzed by CYP17A1. Pregnolone is hydroxylated at position C17 yielding 17OH-pregnenolone, and subsequently the 17,20-carbon–carbon bond scission
takes place yielding DHEA.
Please cite this article in press as: J. Neunzig, et al., A steroidogenic pathway for sulfonated steroids: The metabolism of pregnenolone sulfate, J.
Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.07.005

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ꢁ
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Bovine b
5
was expressed according to Mulrooney et al. [34]
incubated for 70 h, 37 C and 5% CO
2
. Subsequently, samples of
using E. coli strain C43DE3. Purification was done as described for
bovine CPR.
1 ml/well were collected and prepared for LC/MS-analysis.
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3
.6. Sample analysis by liquid chromatography-tandem mass
spectroscopy (LC-MS/MS)
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.3. UV–vis spectroscopy
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The protein concentration of the CPR was determined using a
The studies were performed using a triple quadrupole mass
spectrometer (TSQ, Quantum Ultra, Thermo Fisher Scientific,
Dreieich, Germany) with electrospray ionization (ESI) in negative
mode. Mass spectrometric parameters for sulfonated steroids
were as described elsewhere [41]. For the in-house synthesized
compound, 17OH-PregS, the collision energy and tube lens values
were calculated, being similar to other sulfonated steroids. The
selected quantifier transition was 411 ! 97. The collision energy
was 32 eV and the tube lens value was 180 V. A C18 reverse phase
column was used for the chromatographic method (Hypersil Gold
ꢂ1
ꢂ1
molar extinction coefficient e585 = 2.4 mM cm [35]. Cytochrome
5
b concentration was calculated by using a difference extinction
ꢂ1
ꢂ1
coefficient of 185 mM cm
for the absorbance change at
4
24–409 nm [34]. CYP17A1 concentration was calculated from a
reduced carbon-monoxide difference spectroscopy according to
ꢂ1
Omura and Sato [36] with
e
448 = 91 mM cm . Binding of Preg and
PregS was investigated using difference spectroscopy, which was
carried out in tandem cuvettes according to Schenkman [37]. Preg
and PregS were dissolved in DMSO. The buffer utilized was
composed of 50 mM HEPES (pH 7.4), 20% glycerol, and 100
m
M
column 50 ꢃ 2.1 mm, 5
mm, Thermo Fisher Scientific, Dreieich,
dilauroyl phosphatidylcholin (DLPC). Difference spectra were
recorded from 370 to 450 nm. To determine the dissociation
Germany), mounted within a HPLC system (Agilent 1200SL,
Waldbronn, Germany). Different concentrations of pure water
and pure methanol (MeOH) were applied to achieve the
separation of the compounds (Table 1). The retention time for
both the enzymatically obtained 17OH-PregS and for the
synthetic 17OH-PregS were the same.
d
constant(K ),thevaluesfromthreetitrationswereaveragedandthe
resulting plots were fitted with hyperbolic regression.
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.4. Enzyme activity assay
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3
.7. Sample preparation and calibrations
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In-vitro substrate conversion assays were done as described
elsewhere [38] with slight modifications. The conversion buffer
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The samples were always diluted due to the high concen-
(
1
50 mM HEPES, pH 7.4, 20% glycerol, 100 M DLPC) contained
m
trations of the analytes, and of the presence of buffer components
that may affect the ionization process. At least two dilutions had
to be used for each sample, as the concentrations of PregS and
m
M
CYP17A1, 1 CPR, 1 mM MgCl , 5 mM glucose-6-
m
M
2
phosphate, 1 U glucose-6-phosphate dehydrogenase for NADPH
regeneration and varying concentrations of Preg or PregS as
substrate. When substrate conversion assays were performed to
17OH-PregS after enzymatic conversion were very different (high
concentrations of PregS when compared to 17OH-PregS concen-
trations). The dilutions were made in a mixture containing
MeOH, water and ammonia to facilitate dissolution and to
improve the ionization of the analytes in negative mode (90.00%
5 5
investigate the influence of b , 4 mM of b were added to the
conversion buffer. After starting the reaction with 1 mM NADPH
ꢁ
at 37 C for 40, 50 and 60 min, the conversion was stopped in a
boiling water bath for 5 min. Preg and the resulting product
7OH-Preg had to be converted to the corresponding 3-one-4-en
2 3
MeOH, 9.95% H O and 0.05% NH ). In case a precipitate was
1
observed, the samples were centrifuged and the clear superna-
tant was analyzed. The dilution was usually performed in glass
vials which were first vortexed and later shaked for 30 min to
allow equilibration with the internal standards. The calibration
points for the calibration curve were prepared adding the same
amount of buffer and internal standards (IS). d6DHEAS was used
as IS for 17OH-PregS and d4PregS for PregS. Calibrations showed
values always higher than 0.99 for R . Data analysis was
performed with Thermo Xcalibur 2.1 software (Thermo Fisher
Scientific, Dreieich, Germany).
form by cholesterol oxidase, to be detectable at 240 nm during
HPLC analysis. Therefore, cholesterol oxidase was added to the
ꢁ
boiled reaction mixture and incubated for 40 min at 37 C
according to Yamato [39]. Cortisol was added as internal standard.
The reaction was stopped by addition of one reaction volume of
ethylacetate. Extraction of steroids was performed twice with
ethylacetate and the ethylacetate phase was evaporated. The
steroids were resuspended in 20% acetonitrile for subsequent
HPLC analysis. Steroids were separated on a Jasco reversed phase
2
HPLC system LC2000 using
C18 reversed phase column (Macherey-Nagel) with an acetoni-
a
4 mm ꢃ 125 mm Nucleodur
276
trile/water gradient and a flow rate of 1 ml/min. Detection of the
3.8. Determination of kinetic parameters
ꢁ
steroids was performed at 240 nm within 30 min at 40 C. For
277
278
analysis of sulfonated steroids mass spectrometry and liquid
chromatography were utilized.
m
K and kcat-values were determined by plotting the substrate
conversion velocities versus the corresponding substrate concen-
trations and by using Michaelis–Menten kinetics (hyperbolic fit)
for Preg or Hill kinetics (sigmoidal fit) for PregS utilizing the
program OriginPro 8.6G.
2
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.5. Substrate conversion in SOAT-HEK293 cells
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For substrate conversion studies, 6-well plates were coated
Table 1
LC table-flows and gradients.
with poly-D-lysine for better attachment of the cells as described
5
elsewhere [40]. 3 ꢃ10 cells/well were plated and maintained in
DMEM/F-12 (Invitrogen) medium with 10% fetal calf serum
Start
.00
Seconds
Flow
% H O
% MeOH
2
(
Sigma),
L-glutamine (4 mM), penicillin (100 units/ml) and tetra-
0
60
60
20
80
60
30
30
0.45
0.50
0.50
0.50
0.50
0.50
0.45
70
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40
5
40
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70
30
45
60
95
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cycline (1
6 h at 37 C and 5% CO
m
g/ml) (for SOAT expression). The cells were grown for
1
.00
ꢁ
1
2
until they reached a confluency of 70%.
2.00
2.33
3.67
Afterwards, they were transiently transfected with a plasmid
containing the cDNAs of CYP17A1 and CPR with the Effectene
4
5
.67
.17
ꢁ
Transfection Kit of Qiagen. After 5 h of incubation (37 C and
5
% CO2), PregS (10 and 20 mM) was added and the cells were further
Please cite this article in press as: J. Neunzig, et al., A steroidogenic pathway for sulfonated steroids: The metabolism of pregnenolone sulfate, J.
Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.07.005

G Model
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J. Neunzig et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
5
Fig. 3. SDS-polyacrylamide gel electrophoresis. (A) Lane 1: supernatant; lane 2: CYP17A1 after purification via IMAC; lane 3: CYP17A1 after purification via IMAC and DEAE-
sepharose; lane 4: CYP17A1 after purification via IMAC, DEAE-sepharose and SP-sepharose; lane M: marker. (B) Lane M: marker; lane 1: supernatant; lane 2: b
5
after IMAC
purification; lane 3: CPR after IMAC purification.
2
82
307
08
3
.9. Synthesis and purification of 17OH-PregS
(70:30 v/v) and loaded onto a Sep-Pak column after activation.
3
A gradient of MeOH was then applied (0–100%) and the fractions
corresponding to 40–50% of MeOH were collected, discarding the
rest. Those fractions were free of disulfate component, showing a
clear single peak in the same chromatographic conditions
described before.
The fractions collected after purification step were evaporated
under nitrogen flow and weighed. A standard working solution was
then prepared (250 mg/mL in MeOH).
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
83
84
85
86
87
88
89
90
91
92
93 Q4
94
95
96
97
98
99
00
01
02
03
04
05
06
309
310
311
312
313
314
315
316
317
318
319
320
321
The method is based on Dusza et al. [42], with some
modifications. Briefly, 1 mg of 17OH-Preg and 0.85 mg of triethyl-
amine sulfur trioxide were mixed in 100 l of anhydrous pyridine,
in a 2 ml glass vial. The mixture was shaked at low speed during 3 h
at room temperature, and consecutively evaporated. The residue
m
was redissolved in 500 ml of ethanol. Then, 500 ml of NaOH 0.2 M in
methanol were added, and a white precipitate was observed. After
evaporation, the precipitate was washed twice with methanol.
Several HPLC-MS/MS analyses were performed to assess the
purity of the reaction product. An aliquot of the solution was
dissolved in a mixture of water:MeOH (1:1 v/v) and analyzed in
positive full scan mode as previously described [41]. The product
was free of 17OH-Preg, as no peak was present at 4.2 min, whereas
a peak with m/z 297.2 at 0.75 min was observed. This mass-to-
In order to calculate the purity of the final solution, two aliquots,
ꢁ
of 20
ml and 40
m
l, were treated with 75 units of sulfatase at 37 C
during 6 h. The reaction products were then quantified using the
aforementioned procedure described in Galuska et al. [41] for
17OH-Preg quantification. The analysis in negative mode proved
that they were free of 17OH-PregS. The calculated purity was 90%.
322
charge ratio value is identical to that of 17OH-Preg, and it is due to
4. Results and discussion
+
(
M ꢂ H
2
O ꢂ H
2
SO
4
+ H) .
323
324
325
326
327
328
329
330
The same chromatographic method was applied to ESI negative
The levels of sulfonated steroids exceed in many species and
tissues considerably the levels of the corresponding unconjugated
steroids. In human, the plasma level of PregS varies significantly
during life. The highest level is reached after birth with
mode. The m/z value of the peak at 0.75 min was 411, molecular ion.
A minority peak at 0.18 min was then found in negative mode. This
peak corresponded to the disulfate fraction of 17OH-Preg, with m/z
ꢂ2
of 245, (M ꢂ 2H) . Sulfonation of the hydroxyl group in position
concentrations between 2–3 mM, depending on the gender, and
3
beta is favored over the sterically hindered 17alpha position.
Next, the mixture was purified with a solid phase extraction
afterwards rapidly decreases in the first year up to concentrations
of about 30–50 nM [43]. During and after adrenarche, the PregS
level in plasma again increases, achieving concentrations of
step. The precipitate was re-dissolved in a water:MeOH solution
5
Fig. 4. Spectra of purified recombinant CYP17A1 (A), CPR (B) and b (C). (A) Full spectrum of purified CYP17A1 recorded from 700 nm to 260 nm. The inset shows the
CO-difference spectrum from 500 nm to 400 nm. (B) Spectrum of purified CPR recorded from 600 nm to 300 nm. (C) Spectrum of purified b
and difference spectrum (reduced–oxidized) (solid line).
5
in its oxidized form (dotted line)
Please cite this article in press as: J. Neunzig, et al., A steroidogenic pathway for sulfonated steroids: The metabolism of pregnenolone sulfate, J.
Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.07.005

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6
J. Neunzig et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
Table 2
5
Amount of heterologously expressed CPR, b and CYP17A1.
Yield of purified protein (mg/l)
CPR
34
22
73
b
5
CYP17A1
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
1
30–140 nM [43]. Although PregS represents a steroid hormone
displaying high abundance in human and other mammalian
organism [44] and exceeding its unconjugated form (0.9–6 nM
[45]) by several folds, its influence on steroid hormone biosynthe-
sis is poorly investigated. Moreover, it was already demonstrated
that CS is converted to PregS through a CYP11A1 dependent
reaction [23] leading necessarily to the issue whether also other
sulfonated steroids are converted by enzymes involved in the
steroid hormone biosynthesis, equally to free steroids. Korte et al.
Q10
Fig. 6. Difference spectroscopy of CYP17A1 titrated with 150
mM PregS (red) and
[24] suggested that CS is converted to PregS and afterwards to
1
50 M 17OH-PregS (black). (For interpretation of the references to colour in this
m
dehydroepiandrosterone sulfate (DHEAS), based on their observa-
tion that the fetal plasma contains very high levels of PregS and
DHEAS. However, experiments to support this assumption were
not performed in this early study. The conversion of PregS to
sulfonated products was demonstrated by Jaffe et al. [46] and
Lamont et al. [47] in the early 70 s, but, they did not identify the
enzyme involved in this reaction. Therefore, it was the aim of our
study to investigate, whether CYP17A1 is able to convert PregS.
figure legend, the reader is referred to the web version of this article.)
365
366
367
368
369
conversion experiments with CYP17A1 suggested an undisturbed
catalytic function of CPR.
The UV–vis spectrum categorizes the expressed CYP17A1 as a
low-spin protein with the characteristic cytochrome
P450 absorption spectrum: the Q bands at 569 nm (
a-band)
3
70
371
72
and 541 nm ( -band), the Soret band at 418 nm, the UV band at
b
3
49
4.1. Expression and purification of CPR, b
5
and CYP17A1
360 nm and the protein-band at 278 nm (Fig. 4A). Moreover, the
enzyme displays a typical CO-difference spectrum, with a major
peak at 450 nm and a minor peak at 420 nm (Fig. 4A inset).
Spectral analysis of CPR also reveals its typical absorption
spectrum: the enzyme shows its major peaks at 360 nm,
380 nm, and at 453 nm, indicating its air-stable semiquinone
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
50
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
The three proteins necessary to perform in-vitro conversions of
51
PregS were expressed in bacteria and purified from there.
For expression of the three proteins, E. coli C43DE3 were chosen
as host, as this strain was described to display high expression
yields for membrane-bound cytochrome P450’s [33,38,48] and
other membrane-bound proteins [49].
52
53
54
(FMNH
peak at 412 nm in its oxidized state, as well as a major peak at
424 nm and minor peak at 409 nm in the difference
(reduced–oxidized) spectrum [34] (Fig. 4C).
The amounts of CPR, b and CYP17A1, respectively, obtained
ꢄ
5
, FAD) form [50] (Fig. 4B). Cytochrome b exhibits a major
55
56
CPR and b
5
were purified by IMAC NTA, whereas CYP17A1 was
a
57
purified at first by IMAC NTA followed by an anion exchange DEAE-
sepharose and, in a last step, by an ion exchange SP-sepharose. The
purity of the proteins was determined via SDS-gel electrophoresis
58 Q5
59
5
after purification are listed in Table 2.
60
(
Fig. 3), in which a single band was obtained for CYP17A1 at 45 kDa
5
Using E. coli C43DE3 as host, the expression yield of b is similar
61
and for b at 17 kDa. CPR showed a main band at 70 kDa and two
5
to literature data [51], that of CYP17A1 achieved 73 mg/l, which is a
great improvement compared to published data [29]. The
expression yield of bovine CPR described here for the first time
was 34 mg/l.
62
unknown bands located above and below the band corresponding
to CPR. Further experiments, like the cytochrome c test (data not
shown), spectral characterization (Fig. 4B) and substrate
63
64
Fig. 5. Determination of substrate binding affinity to CYP17A1. The enzyme (1 mM)
was titrated with increasing concentrations of Preg (dotted line) and PregS (solid
line) dissolved in DMSO. The absorbance changes were plotted against the substrate
concentration and fitted as described; R : 0.99; n = 3.
Fig. 7. Merged chromatogram of the analytes obtained from a conversion sample,
as detected by ID-LC-MS/MS (peak at 1.66 min is d6-DHEAS, peak at 1.91 min is
17OH-PregS whereas peak at 2.28 min is PregS).
2
Please cite this article in press as: J. Neunzig, et al., A steroidogenic pathway for sulfonated steroids: The metabolism of pregnenolone sulfate, J.
Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.07.005

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J. Neunzig et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
7
4
4
4
4
4
4
4
12
13
14
15
16
17
18
steroids [41]. In Fig. 7 a merged chromatogram for PregS, 17OH-
PregS and DHEAS is displayed (detection by multiple reaction
monitoring). All three steroid sulfates are baseline-resolved.
Fig. 8 shows the product-ion mass spectra of 17OH-PregS
(
molecular ion with m/z value of 411). This methodological
development facilitates the analytical basis for the PregS metabo-
lism in our reconstituted in-vitro system.
4
4
19
20
4
.4. CYP17A1 dependent 17OH-pregnenolone and 17OH-pregnenolone
sulfate conversion
4
4
4
4
4
4
21
22
23
24
25
26
Since we could clearly demonstrate that PregS efficiently
interacts with CYP17A1, the next step was to evaluate whether the
sulfonated steroid can be converted by CYP17A1. In mammalian
steroidogenesis, CYP17A1 catalyzes the conversion of Preg to
1
7OH-Preg and DHEA. In our reconstituted in-vitro system nearly
no lyase reaction and thus very little DHEA formation could be
observed, because of the absence of b . Using PregS, we could
Fig. 8. Product-ion mass spectra of the molecular ion of 17OH-PregS (collision
energy 32 eV). The predominant fragment ion at m/z 96.9 is due to the
ꢂ
427
28
hydrogensulfate anion (HSO
4
).
5
4
demonstrate that it serves as a substrate for CYP17A1 and is
converted to a considerable amount to 17OH-PregS. The kinetic
3
3
88
89
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
4.2. Determination of dissociation constants of CYP17A1 with
pregnenolone and pregnenolone sulfate
parameters were determined to be K = 30.00 ꢀ 3.31
m
m
M and
ꢂ1
k
cat = 0.60 ꢀ 0.02 (min ) for 17OH-Preg formation using Preg as
ꢂ1
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
substrate and K = 36.72 ꢀ 3.22
m
M and kcat = 0.44 ꢀ 0.03 (min
)
In order to investigate the binding affinity of Preg and PregS
with CYP17A1 in detail, we performed spectroscopic studies to
m
for 17OH-PregS formation when PregS was utilized as substrate
(Fig. 9). Comparing the curve shapes of the kinetics of
CYP17A1 with Preg or with PregS a significant difference can be
observed: in contrast to the hyperbolic shape of CYP17A1 with Preg
(Fig. 9A), the kinetics of CYP17A1 with PregS display a sigmoidal
form (Fig. 9B). When considering the surface of the
CYP17A1 crystal structure [52], several positively charged regions
that might act as potential interaction sites with the negatively
charged sulfate moiety of PregS are found. This could lead to a
decreased conversion rate at low substrate concentrations until
these unspecific binding sites are saturated and could thus explain
the sigmoidal character of the kinetics of CYP17A1 with PregS.
In Table 3, the kinetic parameters are summarized, revealing a
catalytic efficiency kcat/Km for CYP17A1 toward Preg of 20.0
determine dissociation constants (K
-value for CYP17A1 and Preg of 28.9 ꢀ 3.1
M (Fig. 5), indicating a higher
d
). The experiments revealed a
K
d
mM
and for
CYP17A1 and PregS of 74.8 ꢀ 4.2
m
affinity of CYP17A1 toward the unconjugated pregnenolone.
Moreover, we aimed to compare the affinity of CYP17A1 toward
PregS and its product 17OH-PregS. As the spectral change of
CYP17A1 in the presence of 17OH-PregS was too low to determine
the K
presence of 150
contrast to PregS, which induces a type I shift typical for substrates,
7OH-PregS does not, or only to a very low extend, influence the
spectral properties of CYP17A1. This is an indication that
7OH-PregS might not be in a binding position suitable for further
conversion.
d
-value, we compared the spectral change of CYP17A1 in the
m
M PregS and 150 M 17OH-PregS (Fig. 6). In
m
1
1
ꢂ1
ꢂ1
(min mM ), which is two-fold higher compared to the catalytic
ꢂ1
ꢂ1
m
efficiency toward PregS with kcat/K = 11.9 (min mM ).
4
4
06
07
Consequently, the nearly 2-fold increased catalytic efficiency of
CYP17A1 converting Preg in comparison with PregS seems to be due
toahigheraffinity,astheK -andK -valuesindicateandtoanhigher
d m
kcat-value. The Kd-value for CYP17A1 and PregS is about
2.5-fold higher compared with Preg (Fig. 5). Thus, PregS possesses
less affinity to CYP17A1. By favoring Preg as a substrate,
CYP17A1 might be able to compensate for the lower concentration
4.3. Sample analysis by liquid chromatography-tandem mass
spectrometry (LC-MS/MS)
4
51
4
4
4
4
08
09
10
11
452
453
454
455
In order to analyze the conversion of sulfonated steroids, we
established a new LC-MS/MS method. This is the first time, to our
knowledge, that 17OH-PregS has been quantified by LC-MS/MS,
which represents the technique of choice to analyze sulfonated
2
Fig. 9. (A) Kinetics of Preg to 17OH-Preg conversion catalyzed by CYP17A1 using CPR as redox partner; R : 0.99. (B) Kinetics of PregS to 17OH-PregS conversion catalyzed by
2
CYP17A1 utilizing CPR as redox partner; R : 0.98.
Please cite this article in press as: J. Neunzig, et al., A steroidogenic pathway for sulfonated steroids: The metabolism of pregnenolone sulfate, J.
Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.07.005

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J. Neunzig et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
Table 3
Kinetic parameters of CYP17A1 metabolizing Preg and PregS, respectively.
Pregnenolone
Pregnenolone sulfate
(mM)
ꢂ1
ꢂ1
ꢂ1
ꢂ1
ꢂ1
ꢂ1
K
m
(mM)
k
cat (min
)
k
cat/K
m
(min mM
)
K
m
k
cat (min
)
k
cat/K
m
(min mM
)
0
.030 ꢀ 0.003
0.60 ꢀ 0.02
20.00
0.036 ꢀ 0.003
0.44 ꢀ 0.03
11.98
4
4
4
56
57
58
496
497
498
of Preg in the plasma. However, since concentrations of Preg and
PregSintissuesexpressingCYP17A1areunknown,thephysiological
importance of this observation is not clear yet.
and shown in Fig. 6, difference spectroscopy performed with
CYP17A1 in the presence of 17OH-PregS showed that this
metabolite might not to be capable to enter in the active site of
CYP17A1, as nearly no spectral change of CYP17A1 was induced.
This means that DHEAS seems to be produced solely by
sulfonation of DHEA catalyzed by sulfotransferases, whereas
PregS and 17OH-PregS can either be synthesized by sulfonation of
unconjugated Preg and 17OH-Preg or by further conversion of CS
or PregS.
4
99
500
01
4
59
4.5. Effect of b
5
on CYP17A1 dependent substrate conversions
5
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486 Q6
487
488
489
490
491
492
493
494
495
502
503
504
The first reaction step of CYP17A1 consists of a 17-hydroxyl-
ation of Preg and is followed by a 17,20-carbon–carbon bond
scission yielding DHEA, as shown in Fig. 2. The augmentation by
several folds of the lyase of CYP17A1 in the presence of b
reported [53,54]. It is postulated that the effect of b on this second
reaction step of CYP17A1 is based on an allosteric interaction of b
5
is well
505
5
4.6. CYP17A1 dependent substrate conversion in SOAT-HEK293 cells
5
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
with CYP17A1, enhancing the alignment of the iron–oxygen
complex onto the C20 rather than the C17 atom of the steroid,
hence augmenting the lyase reaction [27]. Further studies are
necessary to confirm this hypothesis. Very recently, Scott and
co-workers [55] demonstrated that structural changes occur at the
proximal surface of CYP17A1 depending on the substrate binding.
In reverse, they claim that binding of b to the proximal surface of
5
CYP17A1 induces changes in the active site of CYP17A1, leading to
an enhanced lyase activity.
To investigate whether the results concerning the conversion
of PregS observed in our reconstituted in-vitro system may
possess potential physiological meaning, we performed conver-
sion experiments in SOAT-HEK293 cells. This cell line exhibits an
integrative gene encoding for the human sodium-dependent
organic anion transporter (SOAT), which was shown to display
high specificity toward PregS [40]. SOAT-HEK293 cells were
transiently transfected with a plasmid containing genes encoding
for CYP17A1 and its electron transfer partner, CPR. LC-MS/MS
analysis after three days of incubation with PregS could clearly
confirm the formation of reasonable amounts of 17OH-PregS. As
For this reason, we investigated the effect of
b
5
on
CYP17A1 dependent conversion of Preg and PregS. In our
reconstituted in-vitro system, the lyase reaction was nearly absent
shown in Fig. 11, 2.30 ꢀ 0.30
mM 17OH-PregS was synthesized
when 20 M PregS was used as substrate. Also in this case, no
without addition of b
.08 ꢀ 0.02 M DHEA is formed. In contrast, in the presence of b
the CYP17A1 dependent conversion of Preg to DHEA is strongly
M DHEA, thus augmenting the
5
. Using 30
m
M Preg as substrate for CYP17A1,
m
0
m
5
DHEAS could be detected, confirming our in-vitro results, where no
17,20-lyase activity could be attributed to CYP17A1 using PregS as
increased, forming 1.02 ꢀ 0.07
m
substrate. In contrast, when using 20
5.30 ꢀ 0.35 M 17OH-Preg, as well as 0.86 ꢀ 0.2
formed (Fig. 11).
The utilized cell line exhibits RNA expression of b
m
M Preg as substrate,
lyase activity about 10-fold. The relatively low lyase activity is in
accordance with previous studies of Barnes et al. [56] demonstrat-
ing that the formation of DHEA is time-depending and starts after
accumulation of the intermediate 17OH-Preg. Studies using
m
mM DHEA are
5
and
MAPK14 as “The Human Protein Atlas” indicates. MAPK14
(p38alpha) is a kinase that phosphorylates CYP17A1 at position
258 of the amino acid chain [58]. This post-translational
modification of CYP17A1 is described to enhance the lyase reaction
17OH-Preg instead of Preg as substrate also show higher levels
of DHEA formation [57] (Fig. 10).
To our surprise, the conversion of PregS was not influenced by
addition of b
5
. Whether or not b
5
was present, no scission of the
of CYP17A1, similar to the effect of b
5
[28]. The presence of these
17,20-carbon–carbon bond took place and, consequently, no
two proteins (b , MAPK14) obviously supports the formation to
5
DHEAS was formed. It seems that the sulfate moiety hinders
the alignment of the iron–oxygen complex onto the C20 atom of
17OH-Preg and DHEA using Preg as substrate. However, the lyase
reaction of CYP17A1 does not take place in these cells with PregS as
substrate. In cell culture as well as in the reconstituted in-vitro
system 17OH-PregS represents the end product of the
CYP17A1 catalyzed reaction using PregS as substrate.
17OH-PregS, possibly due to the increased size of the molecule
and/or to the negative charge of the sulfate, which finally prevents
the lyase reaction of CYP17A1 on 17OH-PregS. As discussed before
Fig. 11. Conversion of 20 mM PregS (black) and 20 mM Preg (grey) in
SOAT-HEK293 cells. The product formation analyzed by LC-MS/MS (black) and
HPLC (grey) is represented as mean ꢀ standard deviation of five (black) and three
(grey) individual experiments.
Fig. 10. Comparison of DHEA and DHEAS formation by CYP17A1 in absence and in
the presence of b using Preg and PregS, respectively, as substrate (n = 5).
5
Please cite this article in press as: J. Neunzig, et al., A steroidogenic pathway for sulfonated steroids: The metabolism of pregnenolone sulfate, J.
Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.07.005

G Model
SBMB 4224 1–10
J. Neunzig et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx
9
5
36
5
. Summary
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5
89
90
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
In this work we could clearly demonstrate that PregS serves as
591
1833 (2013) 3064–3075.
a substrate for CYP17A1, being converted to 17OH-PregS.
Summarizing, we demonstrated for the first time that CYP17A1,
which is involved in the steroid hormone biosynthesis, can
convert sulfonated steroids in a similar manner as free steroids
indicating a potential alternative steroidogenic pathway for
sulfonated steroids. As already described, this pathway is
initiated by the side-chain cleavage of CS by CYP11A1, yielding
[20] M.D. Majewska, J.M. Mienville, S. Vicini, Neurosteroid pregnenolone sulfate
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5
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antagonizes electrophysiological responses to GABA in neurons, Neurosci. Lett.
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[
D
5
94
[22] E. Kostakis, C. Smith, M.K. Jang, S.C. Martin, K.G. Richards, et al., The neuroactive
595
596
steroid pregnenolone sulfate stimulates trafficking of functional
N-methyl-D-aspartate receptors to the cell surface via a noncanonical, G-
2+
PregS [23]. Subsequently, PregS is metabolized in
a
protein, and Ca -dependent mechanism, Mol. Pharmacol. 84 (2013) 261–274.
[23] R.C. Tuckey, Side-chain cleavage of cholesterol sulfate by ovarian mitochon-
dria, J. Steroid Biochem. Mol. Biol. 37 (1990) 121–127.
CYP17A1 dependent hydroxylation reaction to 17OH-PregS, but
not to DHEAS. At this point the steroidogenic pathway for
sulfonated steroids differs from the one for free steroids yielding
5
97
[
24] K. Korte, P.G. Hemsell, J.I. Mason, Sterol sulfate metabolism in the adrenals of
the human fetus, anencephalic newborn, and adult, J. Clin. Endocrinol. Metab.
55 (1982) 671–675.
598
99
5
DHEA in large quantities. The kcat- and K
-value were determined for CYP17A1 with PregS. Moreover, we
showed that b does not enhance the 17,20-lyase activity of
m
-values, as well as the
[
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