Cloning, characterization, and expression analysis of a putative 17 beta-hydroxysteroid dehydrogenase 11 in the abalone, Haliotis diversicolor supertexta

Article abstract of DOI:10.1016/j.jsbmb.2011.12.013

The 17-beta-hydroxysteroid dehydrogenases (17β-HSDs) are key enzymes for sex steroid biosynthesis. To date, relatively little is known about the presence and function of 17β-HSDs in marine gastropods. In the present study, a cDNA sequence encoding putative 17β-HSD type 11 (17β-HSD-11) was identified in marine abalone (Haliotis diversicolor supertexta). The full-length cDNA contains 1058 bp, including an open reading frame (ORF) of 900 bp that encodes a protein of 299 amino acids. Comparative structural analysis revealed that abalone 17β-HSD-11 shares relatively high homology with other 17b-HSD-11 hormologues, and a lesser degree of amino acid identity with other forms of 17b-HSD, especially in the functional domains, including the cofactor binding domain (TGxxxGxG) and catalytic site (YxxSK). Phylogenetic analysis showed that abalone 17β-HSD-11 belongs to the short-chain dehydrogenase/reductase (SDR) family. Functional analysis following transient transfection of the ORF into human embryonic kidney-293 (HEK-293) cells indicated that abalone 17β-HSD-11 has the ability to convert 5α-androstane-3α,17β-diol (3α-diol) to androsterone (A) and testosterone (T) to androstenedione (4A). Expression analysis in vivo demonstrated that abalone 17β-HSD-11 is differentially expressed during three stages (non-reproductive, reproductive, and post-reproductive). Taken together, these results indicate that ab-17β-HSD-11 is an SDR family member with a potential role in steroid regulation during the reproductive stage.

Full text of DOI:10.1016/j.jsbmb.2011.12.013

Journal of Steroid Biochemistry & Molecular Biology 130 (2012) 57–63
Contents lists available at SciVerse ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology
journal homepage: www.elsevier.com/locate/jsbmb
Cloning, characterization, and expression analysis of a putative 17
beta-hydroxysteroid dehydrogenase 11 in the abalone, Haliotis diversicolor
supertexta
Hong-ning Zhaia,b, Jin Zhou , Zhong-hua Caib,∗
b
a
School of Life Sciences, Tsinghua University, Beijing, PR China
Ocean Science and Technology Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2011
Received in revised form
The 17-beta-hydroxysteroid dehydrogenases (17␤-HSDs) are key enzymes for sex steroid biosynthesis.
To date, relatively little is known about the presence and function of 17␤-HSDs in marine gastropods. In
the present study, a cDNA sequence encoding putative 17␤-HSD type 11 (17␤-HSD-11) was identified in
marine abalone (Haliotis diversicolor supertexta). The full-length cDNA contains 1058 bp, including an open
reading frame (ORF) of 900 bp that encodes a protein of 299 amino acids. Comparative structural analysis
revealed that abalone 17␤-HSD-11 shares relatively high homology with other 17b-HSD-11 hormologues,
and a lesser degree of amino acid identity with other forms of 17b-HSD, especially in the functional
domains, including the cofactor binding domain (TGxxxGxG) and catalytic site (YxxSK). Phylogenetic
analysis showed that abalone 17␤-HSD-11 belongs to the short-chain dehydrogenase/reductase (SDR)
family. Functional analysis following transient transfection of the ORF into human embryonic kidney-293
(HEK-293) cells indicated that abalone 17␤-HSD-11 has the ability to convert 5␣-androstane-3␣,17␤-
diol (3␣-diol) to androsterone (A) and testosterone (T) to androstenedione (4A). Expression analysis
in vivo demonstrated that abalone 17␤-HSD-11 is differentially expressed during three stages (non-
reproductive, reproductive, and post-reproductive). Taken together, these results indicate that ab-17␤-
HSD-11 is an SDR family member with a potential role in steroid regulation during the reproductive stage.
1
7 December 2011
Accepted 19 December 2011
Keywords:
Abalone
1
7␤-HSD-11
Cloning
Function characterization
Steroid processing
©
2011 Elsevier Ltd. All rights reserved.
1
. Introduction
7-beta-hydroxysteroid dehydrogenases (17␤-HSDs) are
family. At present, 15 types of 17␤-HSDs have been identified
in vertebrates, and they have been numbered according to the
order of their discovery [5]. Typical 17␤-HSDs share four relatively
conserved motifs, consisting of the cofactor binding domain
TGxxxGxG, the catalytically active sites YxxSK, the structural sta-
bilization domain NAG, and the PGxxxT domain that determines
the reaction direction [6–8]. Substrate preferences of 17␤-HSDs
are various and not restricted to steroids, so different types of
17␤-HSDs might possess various physiological functions in the
steroid metabolism-related signaling pathways [9,10].
1
remarkably multifunctional enzymes that mediate the metabolism
of sex steroids. The enzymes catalyze oxidation and reduction at
position C17 of C18- and C19-steroids in which hydroxyl groups
are transformed into keto groups and vice versa. In these steroids
hydroxy-forms are more active, whereas keto-forms are relatively
inactive [1–4]. Almost all 17␤-HSDs belong to the superfamily of
short-chain dehydrogenases/reductases (SDRs) except 17␤-HSD
type 5, which is a member of the aldoketo-reductase (AKR) protein
It is well known that 17␤-HSDs play a pivotal role in the
steroidogenic pathways in mammalian species. In recent years,
1
7␤-HSDs have also been found to participate in biological func-
tions that are important for steroid metabolism in aquatic fish, such
as the zebrafish and Japanese eel [11,12]. Increasing numbers (or
isoforms) of the 17␤-HSD family are being isolated and identified
because of their diversity [13], but, to the best of our knowledge,
relatively little information is known about 17␤-HSDs in mollusks.
Porte et al. reviewed the presence of 17␤-HSD activity in mollusks,
including bivalves, cephalopods, and gastropods [14]. Given the
lack of knowledge of mollusk endocrinology, Porte et al. deemed it
Abbreviations: 17␤-HSD-11, 17␤-hydroxysteroid dehydrogenase 11; SDRs,
short-chain dehydrogenases/reductases; E1, estrone; E2, estradiol; 3␣-diol, 5␣-
androstane-3␣,17␤-diol; A, androsterone; T, testosterone; 4A, androstenedione;
DMSO, dimethyl sulfoxide; ORF, open reading frame; HEK-293, human embryonic
kidney 293 cells; qPCR, real-time quantitative PCR.
∗ Corresponding author at: Room 102, Building B, Campus of Tsinghua University,
University Town, Shenzhen City, Guangdong Province, PR China.
Tel.: +86 755 26036291; fax: +86 755 26036291.
E-mail address: caizh@sz.tsinghua.edu.cn (Z.-h. Cai).
0
960-0760/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jsbmb.2011.12.013

5
8
H.-n. Zhai et al. / Journal of Steroid Biochemistry & Molecular Biology 130 (2012) 57–63
is necessary to study endocrine-related genes, such as 17␤-HSDs.
They summarized some of the steroid-related genes in mollusks,
such as 17␤-HSD types 3, 5, and 9. However, the other members
of the 17␤-HSD family (for example, type 11) have not been func-
tionally characterized and their genes have not been cloned. In our
previous work, we demonstrated that 17␤-HSD type 12 partici-
pates in steroid-mediated physiological processes in abalones (a
marine mollusk) [15]. We now focus on 17␤-HSD type 11 (17␤-
HSD-11) in this work, to assess its molecular characteristics and
biological function in mollusks.
amplification was performed in a 20-␮L reaction volume contain-
ing 2.0 ␮L of 10× PCR buffer, 1.0 ␮L of MgCl2 (25 mM), 1.0 ␮L of
dNTPs (2.5 mM), 1.0 ␮L of each primer (10 mM), 0.1 ␮L of ExTaq
polymerase (5 U mL ) (Takara, Dalian, China), and 1 ␮L of cDNA
mixture, with DEPC-treated water making up the volume to 20 ␮L.
The touch-down PCR procedure was performed as follows: 1 cycle
−
1
◦
◦
◦
◦
◦
of 94 C for 5 min, then 10 cycles of 94 C for 30 s, 67 C for 30 s
◦
(−0.7 C per cycle), 72 C for 1 min, then 26 cycles of 94 C for 30 s,
◦
◦
◦
60 C for 30 s, 72 C for 1 min, followed by 1 cycle of 72 C for 6 min.
The PCR product was purified and subcloned into pMD18-T vec-
tor (Takara, Dalian, China), and then transformed into the E. coli
M15 competent cells. The plasmid of positive clones was purified
and sequenced by an ABI3730 Automated Sequencer (ABI, USA)
1
7␤-HSD-11 was initially discovered by Li et al. when search-
ing for a novel enzyme in glucocorticoid metabolism, as it is
homologous to other 17␤-HSDs and has the ability to trans-
form 17␤-hydroxysteroid hormones [16]. Chai et al. found that
the enzyme is similar to human retinal short-chain dehydroge-
nase/reductase (retSDR2) but cannot catalyze the metabolism of
retinoids, although the enzyme can bind to them [17]. 17␤-HSD-
ꢀ
ꢀ
using primer F2 (5 -CTG TGA CGT CAC TTC CAC CGA-3 ) and M13R
primer. By compiling the overlapping sequence of the PCR prod-
uct and the known sequence, the complete 17␤-HSD-11 sequence
was obtained. Subsequently, another PCR was carried out to verify
the 17␤-HSD-11 sequence using a new cDNA template prepared
1
1 can catalyze the conversion from 5␣-androstane-3␣,17␤-diol
ꢀ ꢀ
from the primers F3 (5 -ATA TGA AAG CTT CTA TTG GA-3 ) and R4
(
3␣-diol) to androsterone (A), and transforms testosterone (T)
ꢀ
ꢀ
ꢀ
ꢀ
into androstenedione (4A) as well [17,18]. Recent investigations
demonstrated that 17␤-HSD-11 can be up-regulated significantly
by peroxisome proliferator-activated receptor (PPAR␣) agonists in
mouse intestine, indicating that this enzyme is also involved in
the lipid metabolic pathways of mouse [19–21]. 17␤-HSD-11 has a
broad tissue distribution in humans as it was detected in the pan-
creas, kidney, liver, and lung as well as steroidogenic cells [17], and
is also found in an androgen-dependent prostate cancer cell line
(5 -GTA TCA GTC ACT CTT TGC GT-3 ) located in the 5 - and 3 -UTR,
respectively. This verified sequence was denoted as ab-17␤-HSD-
11. All primers applied in this work were synthesized by Invitrogen
Corporation, Shanghai Representative Office.
2.3. Sequence analysis
The sequence similarities between ab-17␤-HSD-11 and
other known genes were analyzed using the BLAST program
(http://www.ncbi.nim.nih.gov/blast). The Expert Protein Analysis
System (http://www.expasy.org/) was used to analyze the pro-
tein sequence, including determination of the primary structure
and prediction of the second structure and subcellular localiza-
tion. Protein multiple alignments were performed using ClustalX
1.83 software [25]. Phylogenetic trees were constructed using
MEGA4.1 software with 1000 bootstrap trials by the neighbor-
joining method [26].
[
22,23].
Although quite a few studies concerning 17␤-HSD-11 in ver-
tebrates have been published, little investigation of 17␤-HSD-11
in invertebrates, especially marine mollusks, has been launched.
Whether 17␤-HSD-11 exists in these species and plays the same
or similar roles as its counterpart in vertebrates remains unclear.
In this study, we screened a putative cDNA of 17␤-HSD-11 from
an abalone digestive gland cDNA library by data mining the EST
database. The ability of the enzyme to catalyze the transformation
of steroid substrates was then assessed by transiently transfecting
the candidate gene (i.e., 17␤-HSD-11) into HEK-293 cells. The aims
of this study were to identify the sequence and characterize the
catalytic activities of ab-17␤-HSD-11 and, consequently, provide
evidence for further investigation of the biological functions of the
target gene in the steroidogenic pathway in abalone.
2.4. Construction of ab-17ˇ-HSD-11 expression vector in
pFLAG-cmv2
The ab-17␤-HSD-11 expression vector was constructed in
ꢀ
pFLAG-cmv2. Briefly, specific primers F5 (5 -ATG CGG CCG CTA TGA
ꢀ
ꢀ
AAG CTT CTA TTG GA-3 ) and R6 (5 -GAC CCG GGT CAG TCA CTC TTT
GCG T-3 ), containing a NotI and a SmaI restriction site (underlined),
ꢀ
2
. Materials and methods
respectively, were used to amplify ab-17␤-HSD-11. PCR was per-
◦
formed for 30 cycles, with denaturation for 30 s at 94 C, annealing
2.1. Construction of the abalone digestive gland cDNA library
◦
◦
for 30 s at 53 C, and elongation for 90 s at 72 C. The PCR product
was gel-purified, digested with NotI and SmaI restriction endonu-
cleases, and then subcloned into the pFLAG-CMV2 vector. Then, the
constructed plasmid was transformed into E. coli DH5␣ competent
cells. The positive clones were screened by PCR using primers F5
and R6. The integrity of the construct, pFLAG-cmv2-ab-17␤-HSD-
The full-length cDNA library from abalone (Haliotis diversi-
color supertexta) digestive gland was constructed and 6000 EST
sequences were obtained by random sequencing using universal
primers described in a previous study [24]. A 369-base pair (bp)
fragment that shared high sequence similarity with the 17␤-HSD-
1
1, was verified by sequencing the inserted DNA fragment using
1
1 gene of other animals was screened through a BLAST search
ꢀ
pFLAG-cmv2 universal primers F7 (5 -TTC CAA AAT GTC GTA ATA
AC-3 ) and R8 (5 -ATT ATA GAA GGA CAC CTA GTC-3 ).
using 17␤-HSD-11 genes from other animals, and this sequence
was selected for further study.
ꢀ
ꢀ
ꢀ
2.5. Transient transfection of pFLAG-cmv2-ab-17ˇ-HSD-11
2.2. Cloning the full-length cDNA of 17ˇ-HSD11
The plasmid pFLAG-cmv2-ab-17␤-HSD-11 was purified using
The candidate sequence of 17␤-HSD-11 was analyzed by
the Plasmid DNA Maxi Purification Kit (free-endotoxin) (OMEGA,
Bio-Tek, USA). HEK-293 cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM; Invitrogen, USA) containing 10% (v/v) fetal
ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), and the
ꢀ
results indicated that the sequence contains the 5 end coding
region, which has a methionine at the start of the ORF preceded
bovine serum (FBS) (Invitrogen, USA) and penicillin/streptomycin
ꢀ
◦
by a short UTR. A forward primer F1 (5 -CCA CCC GCT AGA CAA
at 37 C with 5% CO . Cells were seeded into 24-well culture plates
2
ꢀ
ꢀ
5
GAT-3 ) was then designed to obtain the 3 end of 17␤-HSD-11
(0.8 × 10 /well) 36 h prior to transfection and transiently trans-
using anchored PCR with the universal reverse primer M13R. PCR
fected with the construct pFLAG-cmv2-ab-17␤-HSD-11 or empty

H.-n. Zhai et al. / Journal of Steroid Biochemistry & Molecular Biology 130 (2012) 57–63
59
vector using LipofectamineTM 2000 (Invitrogen, Carlsbad, USA),
in liquid nitrogen. Total RNA was extracted using Trizol reagent
(Invitrogen, USA), mRNA was purified by mRNA purification kit
(BBI, Canada), and then cDNA was synthesized by MMLV (Takara,
Dalian, China).
according to the manufacturers’ protocol (0.8 ␮g DNA and 2.0 ␮L
TM
Lipofectamine
2000 were transfected per well). Twenty-four
hours after transfection, six substrates (3a-diol, A, T, 4A, estrone
E1), or estradiol (E2)) at concentrations of 5 ␮g/mL were added
(
qPCR assays were performed to determine the expression of
ab-17␤-HSD-11 in the different reproductive stages using stan-
dard protocols on a 7300 RT-PCR System (Applied Biosystems, USA)
with a 96-well reaction plate. Briefly, each 25-␮L DNA amplifica-
tion reaction contained 12.5 ␮L 2× SYBR Green Premix Ex Taq with
ROX dye I (Takara, Dalian, China), 1.0 ␮L diluted cDNA, and 0.5 ␮L
10 ␮M solutions of each forward and reverse primer, with PCR-
into ab-17␤-HSD-11-transfected cells, respectively. These steroid
substrates were firstly dissolved in DMSO, then the desired con-
centration (5 ␮g/mL) was obtained by serial dilution. The culture
media from each well were collected after 23 h and were analyzed
for precursor and product steroids by TLC or HPLC. At the same time,
mock-transfected cells were used as controls in the same assay.
Each experiment was performed with three replicates.
ꢀ
grade water to make up the volume. Primers (rt-F: 5 -CCA CCC GCT
ꢀ
ꢀ
ꢀ
AGA CAA GAT-3 and rt-R: 5 -CCA ATA CGA CGG ACC TCA-3 ) for
amplification of the 170-bp gene-specific product were designed
according to the target gene sequence. The RT-PCR assay was done
2.6. Detection of steroid substrates by TLC and HPLC
◦
◦
under the following conditions: 30 s at 95 C; then 5 s at 95 C, 31 s
at 60 C for 40 cycles; 15 s at 95 C, 1 min at 60 C, and 15 s at 95 C
as the final dissociation stage. Beta-actin, with a pair of primers
Steroid metabolites were extracted from cull culture media and
analyzed using thin layer chromatography (TLC) and high per-
formance liquid chromatography (HPLC). Briefly, the supernatant
◦
◦
◦
◦
ꢀ
ꢀ
ab-actin-F (5 -GTC TTT CCC TCC ATC GTC GGA C-3 ) and ab-actin-
from each treatment was centrifuged at 13,800 × g for 10 min at
ꢀ
ꢀ
R (5 -GTC CCA GTT GGT GAC GAT TCC G-3 ), from H. diversicolor
supertexta was used as an internal control to calibrate the cDNA
template [15]. In addition, the expression of ab-17␤-HSD-11 in the
non-reproductive stage was chosen as the calibrator for normaliz-
ing different samples. Data obtained from triplicate runs for target
cDNA amplification were averaged.
◦
4
C to precipitate the residual cells and other impurities. Then each
of the supernatant fluids was extracted using two volumes of a
mixture (9:1, v/v) of methyl-tert-butyl ether and methanol. After
vortexing thoroughly (1 min) and allowing to stand (5 min), the
upper organic phase was carefully transferred into another clean
Eppendorf tube. Subsequently, the extractant was evaporated by
nitrogen flow at ambient temperature prior to further analysis.
TLC analysis was performed according to a previously published
procedure [27], with some modifications. Briefly, each sample
was dissolved in 50 ␮L chloroform/methanol (2:1, v/v), and 10-␮L
aliquots were spotted on 10 × 20 cm high silica gel GF pre-coated
TLC plates (Jiangyou Gel Co., Ltd., Yantai, China) together with
2.8. Statistical analysis
The HPLC data were analyzed by one-way analysis of variance
(ANOVA) and Tukey’s multiple comparisons using SPSS 16.0. The
RT-PCR data analysis was executed using SDS 2.0 software (ABI,
−
ꢀꢀCt
USA) according to the 2
method [28]. All values are presented
0.5 ␮g of each of the carrier steroids (3a-diol and A). The plates
as mean ± SEM, and P < 0.01 was considered statistically significant.
were developed in chloroform/toluene/acetone (50:80:20, v/v/v)
for 1 h and air-dried. Steroids were visualized by spraying with 5%
3. Results
◦
sulfuric acid in ethanol and heating the plate at 105 C for 9 min.
For separation by HPLC, each sample was dissolved in a 250-␮L
mixture of acetonitrile–water (7:3, v/v) and 10 ␮L were injected
into a LC-2010 HPLC system equipped with a double-pump unit
3
.1. cDNA cloning and sequence analysis of ab-17ˇ-HSD 11 gene
By aligning the known gene fragment and the amplified
(
LC-20AT), a controller (CBM-20A), an autosampler (SIL-20A), a UV
fragment of 689 bp, we acquired a 1058-bp sequence, which repre-
sented the full sequence of the putative abalone 17␤-HSD-11. The
sequence was named ab-17␤-HSD-11 and deposited at GenBank
with the accession number ADV02385. Ab-17␤-HSD-11 contained
an open reading frame of 900 bp encoding 299 amino acid residues,
a short 5 -UTR of 31 bp, and a 158-bp 3 -UTR with a canonical
polyadenylation signal sequence, AATAAA, located 9 bp upstream
of the poly A tail. The molecular weight of the deduced protein is
detector (SPD-20A), and a 5-␮m C18 Shim-pack VP-ODS column
(
4.6 mm × 150 mm) (Shimadzu, Japan). The samples were sepa-
◦
rated at 25 C and a flow rate of 1 mL/min with a program that
involved linear gradients of 65–70% acetonitrile in water over
2
min, 70–80% acetonitrile over 3 min, followed by 80–70% acetoni-
ꢀ
ꢀ
trile over 2 min. The UV detection wavelength was set at 230 nm.
The retention times in minutes were as follows: E2 2.52, E1 2.80, T
2
.84, and 4A 3.11. Steroids were identified by comparison with the
3
1
3.45 kDa with an estimated pI of 6.97. The deduced peptide ab-
7␤-HSD-11 includes a catalytic domain and a variable C-terminal
standards. The data were measured in triplicate and activities were
calculated as the percentage of substrate converted into product.
region. The catalytic domain contains a cofactor binding site motif
TGAGHGIG) (43–50), a putative catalytic site (YSASK) (186–190),
(
2.7. Expression analysis of ab-17ˇ-HSD-11 in the reproductive
and a typical SDR conserved domain (NNVG) (120–123).
stage
BLAST analysis of type 11 members revealed 44%, 43%, 44%,
4
3%, 47%, and 53% identity with Home sapiens (NP 057329), Pongo
Healthy adult abalones with an average shell length of
6.0 ± 3.0 mm were procured from a local hatchery (Longgang
abelii (NP 001127691), Bos Taurus (NP 001039751), Mus muscu-
lus (NP 444492), Xenopus (Silurana) tropicalis (NP 001011304), and
Saccoglossus kowalevskii (XP 002732320) respectively.
5
District, Shenzhen City, China) and used for the experiments.
Their tissue samples (digestive glands and gonads) were col-
lected from three different stages (non-reproductive, reproductive,
and post-reproductive). The reproductive stage is defined as
when the abalone is in the process of spawning, and the post-
reproductive stage refers to the seventh day after spawning; the
non-reproductive stage refers to the period when adult male and
female abalone were collected around 3 weeks prior to spawn-
ing, and was used as a control. Tissue samples from three males
or females in each stage were pooled together and homogenized
3.2. Comparison of the peptide sequence of ab-17ˇ-HSD-11 with
those of various other species
BLAST analysis revealed that the deduced peptide sequence
of ab-17␤-HSD-11 shares a high degree of sequence similarity
(39–50%) and identity (21–35%) with other 17␤-HSD family mem-
bers, and especially with other 17␤-HSD-11s. The highest similarity
(71%) and identity (53%) are shared with S. kowalevskii (an acorn

6
0
H.-n. Zhai et al. / Journal of Steroid Biochemistry & Molecular Biology 130 (2012) 57–63
Fig. 1. Phylogenetic analysis of 17␤-HSDs (from type 1 to type 14) of various species using MEGA4.1 and based on ClustalX sequence alignment. The phylogenetic tree
revealed the evolutionary relationship among the 17␤-HSDs. Bootstrap values, marked on each branch, represent the number of times out of 1000 that two organisms
grouped together during bootstrap analysis. Branch length is proportional to the distance between each protein. The type 11 family is highlighted with solid spheres. Type 2
1
1␤-HSD, which belongs to the SDR family, was selected as an outgroup.

H.-n. Zhai et al. / Journal of Steroid Biochemistry & Molecular Biology 130 (2012) 57–63
61
Fig. 3. Quantitative PCR analysis of ab-17␤-HSD-11 mRNA expression in various tis-
sues at different reproductive stages. Each column represents an average expression
level of three replications. Different tissues: md, digestive gland of male abalone;
mg, gonad of male abalone; fd, digestive gland of female abalone; fg, gonad of female
abalone. Asterisks indicate significant difference (**P < 0.01) using one-way ANOVA
by SPSS16.0.
3
1.5% at 23 h (Fig. 2B). We also conducted the reverse reactions
(i.e., from A to 3␣-diol or from 4A to T); unlike the forward reac-
tions, HEK-293 cells transfected with ab-17␤-HSD-11 exhibited
no obvious catalytic activity for reduction reactions (i.e., ab-17␤-
HSD-11 could not effectively transform A to 3␣-diol or 4A to T)
Fig. 2. Enzymatic activity assay of ab-17␤-HSD-11 expressed in HEK-293 cells. (A)
By thin layer chromatography, conversion of 3␣-diol to androsterone. 17␤-11, ab-
(Fig. 2A and B). It is important also to mention that we did not find
1
7␤-HSD-11 transfected into HEK-293 cells; control, empty vector transfected into
any catalytic activity of ab-17␤-HSD-11 towards E1/E2, although
such reactions have been observed for mammalian 17␤-HSD-11
HEK-293 cells; Std, standard steroids; 3a-diol, 3␣-diol; and A, androsterone. (B) By
high performance liquid chromatography, conversion of testosterone, androstene-
dione, estradiol, or estrone. T, testosterone; 4A, androstenedione; E1, estrone; and
E2, estradiol. Results are represented as mean ± SE of three independent measure-
ments.
(Fig. 2B).
3.4. Relative mRNA expression of ab-17ˇ-HSD-11
worm) 17␤-HSD-11, followed by Xenopus (Silurana) tropicalis (47%
identity), and other mammalian species (42–44% identity). Multi-
ple alignments indicated that 17␤-HSD-11 sequences are highly
conserved in the catalytic domain and the functionally important
motifs from the SDR family such as the cofactor-binding domain
To gain further insight into the putative functions of ab-17␤-
HSD 11 in vivo, its expression pattern was investigated by real-time
PCR at different reproductive stages. The results showed that ab-
17␤-HSD-11 is expressed in both the digestive gland and the gonad
tissues (Fig. 3). In both males and females, the relative expression
levels of ab-17␤-HSD-11 in the digestive glands were not signif-
icantly different at different reproductive stages. However, in the
gonads, the expression of the ab-17␤-HSD-11 gene was different
at various reproductive stages. Interestingly, in gonad tissues the
expression of ab-17␤-HSD-11 decreased significantly during the
reproductive period, in contrast to the non-reproductive and post-
reproductive periods, but subsequently began to recover during the
post-reproductive stage (Fig. 3).
(
TG×××G×G), the catalytic site (Y××SK), and the structural stabi-
lization domain (NNAG) (Supplemental file). Based on alignment
of the amino acid sequences of published 17␤-HSDs, a phyloge-
netic tree was constructed using the neighbor-joining algorithm
of MEGA 4.1 software. The phylogenetic relationship is shown in
Fig. 1. The results indicate that 17␤-HSDs are clustered into several
clades. 17␤-HSD types 1, 2, 6, and 9 form clade 1; types 3, 11, 12,
and 13 cluster as clade 2; types 4, 8, 10, and 14 are grouped in clade
3
; and types 5 and 7 assemble as clade 4. ab-17␤-HSD-11 is located
in clade 2: in this clade, types 3 and 12 share a sister branch with
types 11 and 13. 17␤-HSD types 11 and 13 are intertwined in the
phylogenetic tree, which means that they possess an unresolved
evolutionary relationship.
4
. Discussion
In this study, we cloned 17␤-HSD-11 from abalone, which con-
tains several typical SDR conserved motifs consisting of a cofactor
binding domain (TG×××G×G), catalytic activity site (Y××SK), and
structural stability motif (NNAG). BLAST analysis revealed that the
deduced peptide sequence of ab-17␤-HSD-11 shared a high degree
of sequence similarity with other 17␤-HSD family members, and
especially with other 17␤-HSD-11s. To further explore whether the
putative abalone 17␤-HSD-11 is indeed a 17␤-HSD-11 homologue,
an evolutionary relationship analysis was performed. By collect-
ing some representative members of all published 17␤-HSD types
(from type 1 to type 14), we constructed a phylogenetic tree. Based
on analysis of the phylogenetic tree, the 17␤-HSDs were clustered
3.3. Activity of ab-17ˇ-HSD-11
To further investigate the activity of ab-17␤-HSD-11 in pro-
cessing steroid hormones, a functional analysis was performed by
transiently transfecting the ORF of the candidate gene into HEK-
2
93 cells. As illustrated in Fig. 2, ab-17␤-HSD-11 predominantly
catalyzes oxidative reactions converting 3␣-diol and T to A and 4A,
respectively. After 23 h ab-17␤-HSD-11 had converted 53.4% of 3␣-
diol to A (Fig. 2A), whereas the conversion rate of T to 4A was about

6
2
H.-n. Zhai et al. / Journal of Steroid Biochemistry & Molecular Biology 130 (2012) 57–63
into several clades. ab-17␤-HSD-11 was located in the same branch
as other type 11 17␤-HSDs in H. sapiens, P. abelii, Macaca fascicu-
laris, B. Taurus, M. musculus, Rattus norvegicus, Taeniopygia guttata,
Xenopus (Silurana) tropicalis, and S. kowalevskii (Fig. 1). In this
branch, the closest relationship was between ab-17␤-HSD-11 and
S. kowalevskii 17␤-HSD-11, followed by the amphibian and then the
mammalian species. This result is consistent with the designation
of ab-17␤-HSD-11 as a member of the 17␤-HSD type 11 family.
It deserves mention that the evolutionary relationship between
type 11 and type 13 17␤-HSDs was somewhat perplexing since they
were intertwined in the phylogenetic tree (Fig. 1). Based on the
similarity analysis, 17␤-HSD-11 shared high similarity with type
gland tissue, we speculate that expression in the digestive gland
is independent of reproductive state. In contrast, it is notewor-
thy that in gonadal tissue, the level of ab-17␤-HSD-11 during
reproduction in abalones was demonstrably lower than that in the
non-reproductive and post-reproductive stages. Since changes in
17␤-HSD activity correlate with the reproductive cycles of animals
[36], as a consequence, variations in expression of ab-17␤-HSD-
11 suggest its potential role in regulating abalones’ reproductive
cycle.Concerning the biological activities of 17␤-HSD-11 that are
well established, we believe it plays an essential and flexible
role in regulating the actions of the steroid hormones, especially
androgens. It is well recognized that 17␤-HSD-11 can efficiently
reduce circulating levels of DHT and T, the most potent andro-
gens, by catalyzing the transformations of DHT to 3␣-diol and
T to 4A [4,37,38]. Thus, it seems that, in the process of andro-
gen metabolism, 17␤-HSD-11 functions as an essential regulator
modulating the concentrations of DHT and T, to buffer or mitigate
the detrimental impact resulting from excessive active androgens.
Therefore, it is reasonable to speculate that ab-17␤-HSD-11 like-
wise functions as a modulator to regulate active steroids, although
there is no definitive evidence regarding the presence of DHT or
T in abalone at present. In addition, it is not difficult to under-
stand why, in the gonad tissues of abalone during reproduction,
the expression of ab-17␤-HSD-11 significantly declines because at
this stage there is a high demand for active steroids (e.g., DHT,
T, or E2) to fulfill relevant physiological roles, which requires
tempering of the catabolism of active steroids by ab-17b-HSD-
11.
1
3 17␤-HSD. Additionally, it is well known that, in humans, 17␤-
HSD types 11 and 13 are located on chromosomal position 4q22.1
9]. Consequently, we can speculate that the two genes originated
[
from a common ancestor and possess different roles through gene
duplication and functional divergence. As the biological function
of 17␤-HSD-13 still remains to be characterized [9,29], we cannot
rule out the possibility that types 11 and 13 have the same function
although this is subject to proof. In order to determine the exact
relationship between the two types of 17␤-HSDs, other analyses
such as Bayesian and likelihood-based methods could be conducted
in the future.
To evaluate the functionality of abalone 17␤-HSD-11, its cat-
alytic activity was measured by monitoring the biotransformation
of steroids in HEK-293 cells transfected with ab-17␤-HSD-11. In
the present work, we characterized the conversion of candidate
steroids by using TLC and HPLC. Due to its convenience, sensitiv-
ity, and low cost, HPLC detection proved to be a very practical tool
for detecting low quantities of steroid metabolites [18,30–33]. In
view of the fact that 3␣-diol and A are difficult to separate through
HPLC because of the high similarities of their absorption spectra
and retention times, we used TLC to assess the biotransformation
of these substrates. The results clearly demonstrate ab-17␤-HSD-
The reasons why there are significant differences between male
and female gonads in the expression mode of ab-17␤-HSD-11
remain unknown. According to the available data (Fig. 3), we notice
that, in both male and female abalone gonads, the expression levels
of the target gene follow the same trend of high-low-high during
the three reproductive stages. However, in male gonads the expres-
sion peak of ab-17␤-HSD-11 appears at the post-reproductive stage
whereas, in female gonads, it emerges during the non-reproductive
phase. Unraveling this conundrum will require us to acquire more
knowledge of the role of this gene in abalones’ physiological activ-
ities.
1
1 can transform 3␣-diol and T to A and 4A, respectively (Fig. 2).
To our knowledge, this is the first study to demonstrate the cat-
alytic activity of 17␤-HSD-11 in converting androgenic hormones
in marine mollusks. The results indicate that ab-17␤-HSD-11 is
indeed a steroid metabolic enzyme that might be involved in differ-
ent steroidogenic pathways, depending on its spatial and temporal
expression patterns.
A point that should be noted is that neither E1 nor E2 can be
transformed by ab-17␤-HSD-11, whereas earlier reports showed
that 17␤-HSD-11 in humans has slight dehydrogenase activity at
the 17␤ position of estradiol [16,18]. We speculate that a difference
in three-dimensional structure is possibly the reason for this conse-
quence because we noticed that in ab-17␤-HSD-11 the amino acid
at position 226 is leucine (L) rather than the corresponding pheny-
lalanine (F) that is present in the other homologous sequences
5
. Conclusions
In summary, based on nucleotide and amino acid sequences,
we have cloned a 17␤-HSD-11 homologue from H. diversicolor
supertexta. Initial studies were also conducted to characterize the
biological activity of ab-17b-HSD-11. Currently available results
show that the functional motifs of ab-17␤-HSD-11 are endowed
with high conservation and an evolutionary relationship with
their counterparts in other species. These features indicate that
the putative ab-17␤-HSD-11 is a potential member of the 17␤-
HSD subfamily. The conversion capacity (i.e., from 3␣-diol and
T to A and 4A, respectively) was verified by transiently trans-
fecting the target gene in HEK-293 cells. Moreover, by exploring
the expression pattern of ab-17␤-HSD-11 in vivo, we suggest that
in abalone 17␤-HSD-11 probably performs an essential function
during the steroid-mediated reproductive process. However, this
work is preliminary and incomplete; the exact role and subcellu-
lar localization of ab-17␤-HSD-11 in abalone needs to be further
clarified.
(
Supplemental file). Blanchard et al. [34] pointed out that, in mam-
malian 17␤-HSD-12, the bulky amino acid F adjoins the active site
and controls the entry of estrogenic substrates, whereas the amino
acid L is responsible for the entry of androgenic substrates. Different
stereochemical configurations of 17␤-HSD-12 create differential
substrate specificities for androgens and estrogens. In accord with
this opinion, we speculate that in ab-17␤-HSD-11 it is the absence
of F at site 226 that results in the loss of catalytic activity towards
estrogens. However, this tentative proposal needs to be proven by
further experiments.
In order to procure more information about the physiologi-
cal function of ab-17␤-HSD-11, we examined mRNA expression
levels in vivo during different reproductive stages (non-, during-
,
and post-reproduction). The results show that ab-17␤-HSD-11
is expressed in the digestive glands and gonads, which is consis-
tent with published reports [9,17,35]. As there were no obvious
differences in expression levels of ab-17␤-HSD-11 in digestive
Conflict of interest
None declared.

H.-n. Zhai et al. / Journal of Steroid Biochemistry & Molecular Biology 130 (2012) 57–63
63
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