Structure of the gene for the human 17␤-hydroxysteroid
dehydrogenase type IV
* Vincent Dolez,
Johannes Ch. Gloeckner,
Yvan de Launoit,
UMR 319, CNRS-Institut Pasteur de Lille, Institut de Biologie de Lille, 1 rue Calmette, BP 447, 59021 Lille Cedex, France
GSF-National Research Center for Environment and Health, Institute of Mammalian Genetics, Ingolstaedter Landstr. 1,
D-85764 Neuherberg, Germany
Received: 4 June 1998 / Accepted: 12 August 1998
Abstract. The 17␤-hydroxysteroid dehydrogenase type IV (17␤-
HSD IV) is a multifunctional enzyme that is localized in the per-
oxisomes. The N-terminal part has dehydrogenase activity, the
central part has hydratase activity, and the carboxy-terminal part is
responsible for sterol transport. Recent observations of mutations
in the human 17␤-HSD IV cDNA leading to a severe peroxisomal
disorder motivated us to define the genomic organization of this
gene mapped to Chromosome (Chr) 5q2. We show here that this
gene consist of 24 exons and 23 introns with classical intron-exon
junctions spanning more than 100 kbp. By mapping the regulatory
region of this gene, we have shown that the first 400 bp upstream
of the transcription start site are sufficient to activate transcription.
The data presented here will permit sequence analysis of patients
with peroxisomal disorders.
Several 17␤-hydroxysteroid dehydrogenases (17␤-HSDs) catalyze
the redox reactions at position C17 of steroid molecule at the final
steps in androgen and estrogen biosynthesis and modulate the bio-
logical function of steroid hormones. Until now, four different
human 17␤-HSDs have been characterized (Penning 1997). Fur-
ther orthologs of the 17␤-HSDs, types V and VI, have been de-
scribed in human and rat (Biswas and Russell 1997; Zhang et al.
1995) and await further characterization. 17␤-HSD I and III par-
ticipate in the synthesis and 17␤-HSD II and IV in the inactivation
We have recently identified a cDNA of the human 17␤-HSD
IV (gene name HSD17B4) that catalyzes the oxidation of the
steroid hormones ⌬5-androstene-3␤,17␤-diol and 17␤-estradi
ol (Adamski et al. 1995). The cDNAs of HSD17B4 genes were
cloned from human, rat, mouse, pig, chicken, and guinea pig and
code for 80 kDa three domain multifunctional enzymes (Adamski
et al. 1995; Caira et al. 1998; Corton et al. 1996; Kobayashi et al.
1997; Leenders et al. 1994a; Normand et al. 1995; Qin et al. 1997).
Using immunogold electron microscopy, we have previously
shown that the 17␤-HSD IV protein is the first steroid metaboliz-
ing enzyme located in peroxisomes (Markus et al. 1995). The
N-terminal part has activities of 17␤-estradiol dehydrogenase and
-specific 3-hydroxyacyl CoA dehydrogenase; the central part
-specific 2-enoyl CoA hydratase activity with straight and
2-methylbranched 2-enoyl-CoAs and bile acids; and the C-
terminus acts as sterol carrier protein 2 (Dieuaide-Noubhani et al.
1997b; Leenders et al. 1996; Qin et al. 1997). Because of the
multitude of inherited activities, the enzyme was also termed mul-
tifunctional protein 2 (MFP2) (Jiang et al. 1997). Through its
substrate specificity it can be distinguished from the
multifunctional protein 1 (MFP1; Osumi et al. 1985). MFP1 and
MFP2 participate in separate pathways and cannot compensate for
each other (Dieuaide-Noubhani et al. 1997a)
The unraveling of the genomic organization became of special
interest after the observation that mutations in the human
HSD17B4 gene led to a severe peroxisomal disorder (Novikov et
al. 1997; Suzuki et al. 1997; van Grunsven et al. 1998). To un-
derstand the molecular basis of this inherited disease, we have
recently mapped the gene to Chr 5q2 (Leenders, 1996) and now
present the characterization of its genomic organization and its
Materials and methods
Screening and characterization of 17␤-HSD IV clones.
dash DNA libraries from human NB4 lymphoblastic cells and human nor-
mal blood were screened (10
recombinant phage plaques) with a randomly
P-dCTP (Amersham, Little Chalfont, UK)-labeled probe (1 × 10
cpm/ml) corresponding to the full length of the 17␤-HSD IV cDNA pre-
viously cloned (Adamski et al. 1995). In parallel, we screened a P1 (male
ICRFP 700) and a PAC (female RPCI6 709) library obtained from the
Resource Center, Primary Database of the German Human Genome Project
at the Max-Planck-Institute for Molecular Genetics (Berlin, Germany) with
a 1.4 kb EcoRI fragment of the human 17␤-HSD IV cDNA. For the cloning
of the promoter region the human genomic NB4 library was screened with
a 200-bp fragment located in the most 5Ј-region (starting at the first ATG)
of the human 17␤-HSD IV cDNA.
After hybridization, the filters were washed and exposed to a Kodak
X-OMAT film for 12 h at −80°C as described (Adamski et al. 1995).
Positive phages were isolated and the inserts excised by different endo-
nuclease digestion. The inserts were subcloned into the polylinkers of
(Stratagene, La Jolla, Calif.), PCRII (InVitrogen,
San Diego, Calif.), or pGem-3Zf (Promega, Charbonnie`res, France). Syn-
thetic oligonucleotides, SP6, T7, or T3 vector primers, and modified T7
DNA polymerase were used to sequence both strands of the subcloned
insert DNA by the dideoxy chain termination method with [
(Pharmacia, Piscataway, N.J.). Sequences were also confirmed with an
Applied Biosystem 370A automatic system with fluorescent dye-labeled
cDNA sequence-specific primers and a Taq dye-primer sequencing kit
(Applied Biosystem, Foster City, Calif.).
The sizes of the introns were determined either by sequencing or by
long-range PCR using the Elongase system (Gibco, Eggenstein, Germany).
For PCR, specific primers were derived from known exon sequences lead-
ing to exon-spanning products. PCR was performed with an initial hot start,
10 cycles (93°C for 1 min, 62°C for 30 s, 68°C for 15 min), followed by
* Present address: University Clinic Charite´, Humboldt University Berlin,
Institute of Pharmacology and Toxicology, 10098 Berlin, Germany.
Correspondence to: J. Adamski
The nucleotide sequence data reported in this paper have been submitted to
GenBank and have been assigned the accession numbers: AF057720–
Mammalian Genome 9, 1036–1041 (1998).
© Springer-Verlag New York Inc. 1998