Closing the Gap: Identification of Human 3-Ketosteroid Reductase, the Last Unknown Enzyme of Mammalian Cholesterol Biosynthesis
Zrinka Marijanovic,
Daniela Laubner,
Gabriele Möller,
Christian Gege,
Bettina Husen,
Jerzy Adamski and
Rainer Breitling
GSF-National Research Center for Environment and Health, Institute of Experimental Genetics (Z.M., D.L., G.M., J.A.), 85764 Neuherberg, Germany; Jenapharm GmbH & Co. KG (C.G.), 07745 Jena, Germany; Deutsches Primatenzentrum (B.H.), 37077 Göttingen, Germany; and Department of Biology (R.B.), San Diego State University, San Diego, California 92182
Address all correspondence and requests for reprints to: Jerzy Adamski, GSF-National Research Center for Environment and Health, Institute of Experimental Genetics, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany. E-mail: adamski{at}gsf.de.
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ABSTRACT
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The protein encoded by the HSD17B7 gene was originally described as a prolactin receptor-associated protein and as 17ß-hydroxysteroid dehydrogenase (HSD) type 7. Its ability to synthesize 17ß-estradiol in vitro has been reported previously. However, we demonstrate that HSD17B7 is the ortholog of the yeast 3-ketosteroid reductase Erg27p and converts zymosterone to zymosterol in vitro, using reduced nicotinamide adenine dinucleotide phosphate as cofactor. Expression of human and murine HSD17B7 in an Erg27p-deficient yeast strain complements the 3-ketosteroid reductase deficiency of the cells and restores growth on sterol-deficient medium. A fusion of HSD17B7 with green fluorescent protein is located in the endoplasmic reticulum, the site of postsqualene cholesterogenesis. Further critical evidence for a role of HSD17B7 in cholesterol metabolism is provided by the observation that its murine ortholog is a member of the same highly distinct embryonic synexpression group as hydroxymethyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme of sterol biogenesis, and is specifically expressed in tissues that are involved in the pathogenesis of congenital cholesterol-deficiency disorders. We conclude that HSD17B7 participates in postsqualene cholesterol biosynthesis, thus completing the molecular cloning of all genes of this central metabolic pathway. In its function as the 3-ketosteroid reductase of cholesterol biosynthesis, HSD17B7 is a novel candidate for inborn errors of cholesterol metabolism.
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INTRODUCTION
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CHOLESTEROL BIOSYNTHESIS IS a complex multistep reaction: three two-carbon units [acetyl-coenzyme A (CoA)] from fatty-acid ß-oxidation (or other metabolic pathways) are combined to form hydroxymethyl-glutaryl-CoA (HMG-CoA), which is then converted to mevalonate by the main rate-controlling enzyme of the pathway HMG-CoA reductase. Further reactions transform the six-carbon molecule mevalonate to isopentenyl diphosphate, the five-carbon precursor of all isoprenoids. Sequential polymerization of five-carbon units yields squalene, which is the first intermediate unique to sterol biosynthesis. Thirteen further reactions, catalyzed by eleven enzymes convert squalene to cholesterol by cyclization followed by a series of demethylations, isomerizations, dehydrogenations, and reductions (Fig. 1
and Table 1
). The molecular characterization of this pathway has mainly relied on the identification of yeast mutants that are defective in ergosterol biosynthesis and therefore show changed sensitivity to antibiotics (reviewed in Ref. 1). By 1999, the last unidentified gene of yeast ergosterol biosynthesis (ERG27) had been cloned (2). Surprisingly, until very recently comparatively little was known about the postsqualene part of cholesterol biosynthesis in mammals. Only in 1995 the first enzymesrat squalene epoxidase, lanosterol synthase, and lanosterol 14
-demethylasewere molecularly cloned (3, 4, 5). Interest in the molecular aspects of postsqualene cholesterol biosynthesis was dramatically stimulated when, in 1998, three groups reported the cloning of human 7-dehydrocholesterol reductase and confirmed previous predictions (6, 7) that the gene is mutated in children with Smith-Lemli-Opitz syndrome, a severe developmental malformation syndrome (8, 9, 10). This was the first identification of a metabolic disorder leading to a complex morphogenetic defect, including characteristic craniofacial dysmorphisms, holoprosencephaly, and syn- and polydactyly. Together with the slightly earlier report that developmental morphogens of the hedgehog family are posttranslationally modified by attachment of a C-terminal cholesterol moiety (11), this suggested a close link between cholesterol biosynthesis and embryonic signaling pathways reviewed in (12, 13). Since then, further inborn errors of cholesterol metabolism have been genetically characterized in quick succession: X-linked chondrodysplasia punctata (14), CHILD (chondrodysplasia with ichithyosis and limb defects) syndrome (15), desmosterolosis (16), and lathosterolosis (17), whereas two others have been suggested by biochemical measurements [Greenberg dysplasia (18); Antley-Bixler syndrome (19), Table 1
, and reviewed in Refs. 20, 21, 22 ]. All of these disorders are associated with developmental malformations. By now, all but one of the enzymes of the postsqualene part of mammalian cholesterol biosynthesis have been cloned, and 7 out of 10 have been implicated in severe congenital malformation syndromes.
Here we report the molecular characterization and embryonic expression pattern of the last enzyme of mammalian cholesterol biosynthesis that had not been cloned previously. 3-Ketosteroid reductase takes part in the multistep C-4 demethylation reaction that also involves
4-methyl oxidase and 3ß-hydroxysteroid dehydrogenase (1). Breitling et al. (23) predicted 3-ketosteroid reductase function for a human protein (HSD17B7) by sequence similarity to its yeast ortholog ERG27 and phyletic profiling. The refined phylogenetic analysis in this work strengthens this hypothesis. This enzyme previously had been identified as a prolactin receptor-associated protein in rat (24) and as an estradiol-producing 17ß-hydroxysteroid dehydrogenase type 7 in mouse (25). Here we demonstrate 3-ketosteroid reductase activity of HSD17B7 by enzymatic measurements and by complementation in an ERG27-deficient yeast strain. As predicted for all enzymes of the post-squalene pathway, HSD17B7 is localized in the endoplasmic reticulum. A detailed mRNA in situ hybridization analysis demonstrated that in mouse embryos HSD7B7 was expressed in a highly specific pattern during early development. The expression pattern closely corresponds to that of HMG-CoA reductase, the rate-limiting enzyme of cholesterogenesis, and was predominant in areas that are commonly affected in the developmental malformation syndromes associated with cholesterol deficiency.
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RESULTS
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Orthology of HSD17B7 and Erg27p
The initial indication that HSD17B7 might be the 3-ketosteroid reductase of cholesterol biogenesis came from our observation that the protein shows considerable similarity with the yeast 3-ketosteroid reductase Erg27p (23). However, although the similarity to Erg27p is higher than to any other short-chain alcohol dehydrogenase, the phylogenetic distance between the two species is very large and leads to a low absolute level of conservation (Saccharomyces cerevisiae Erg27p and human HSD17B7 share only 22% identity and 43% similarity distributed over the whole length of the protein). This makes the establishment of a convincing orthology relationship between the two proteins difficult and is crucially dependent on the inclusion of as many diverse sequences as possible. The previous phylogenetic analysis (23) included only five orthologs of HSD17B7/Erg27p, from human, mouse, rat, S. cerevisiae, and Schizosaccharomyces pombe. We now identified additional ERG27 orthologs from two further fungi (Neurospora crassa and Phanerochaete chrysosporium) as well as in two fish genomes (Takifugu rubripes (two isozymes) and Tetraodon nigroviridis). Further orthologs have recently been reported from rabbit (26) and marmoset monkey (27). In addition to these proteins, we also included the two closest relatives of HSD17B7 (WWOX and PAN2) from the completed genomes of human, as well as Drosophila, mosquito, and Caenorhabditis elegans proteins. Both maximum parsimony and neighbor joining analysis of the data set yield identical phylogenetic trees with very high bootstrap support (Fig. 2
). In both trees, Erg27p orthologs from yeasts and HSD17B7 orthologs from animals form monophyletic groups. Both closest relatives of HSD17B7 from the human genome have affinities to the fly proteins in the analysis; none of these proteins shows a closer relationship to any of the members of the Erg27p/HSD17B7 group, confirming the monophyly of the latter.

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Fig. 2. Neighbor Joining Tree of HSD17B7 and ERG27 Proteins and Their Closest Relatives
Bootstrap support in 1000 pseudoreplicates is indicated at the branches (top number, maximum parsimony; bottom number, neighbor joining). GenBank identification numbers: agCP3205 21292610; agCP6069 21298770; CG3842 7290709; CG7221 20129347; DC2.5p 17559170; E04F6.15.p 17532805; Erg27p S. pombe 19112232, S. cerevisiae 6323129; HSD17B7 human 7705421, marmoset monkey 8050859, rabbit 13383374, mouse 6754250, rat 8393576; PAN2 10039619; WWOX 8927391. Neurospora Erg27p is a hypothetical protein (NCU0599.1) from contig 3.343. A fragment of Phanerochaete Erg27p has been translated from scaffold 4 (beginning at nucleotide 293369). The fugu fish HSD17B7-1 and -2 are predicted proteins 1184 (from scaffold 107) and 9983 (scaffold 194). The Tetraodon sequence has been extracted from the whole-genome shotgun contig 3892.1.
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The results of the phylogenetic analysis at the single-amino acid level are corroborated by the presence of two characteristic secondary structure elements in Erg27p and HSD17B7, an hydrophobic C-terminal helix and a central insertion (28) (published as supplemental data Fig. 1 on The Endocrine Societys Journals Online web site at http://mend.endojournals.org.). We found that both elements are present in the newly identified HSD17B7 orthologs but not in other related members of the short-chain dehydrogenase reductase family, to which these proteins belong.
Another important argument for the functional conservation between ERG27 and HSD17B7 came from the phyletic profile of the proteins (23). Members of the ERG27/HSD17B7 group were detected in mammals and yeast, but not in the cholesterol auxotrophic invertebrates Drosophila and C. elegans. This indicates that a HSD17B7 homolog was present in the common ancestor of yeast, human, fly, and worm and was specifically lost in those organisms that also lost squalene epoxidase and the ability for cholesterol de novo biosynthesis. In addition, we have now found that ERG27/HSD17B7 orthologs are absent from the complete genomes of the mosquito Anopheles gambiae and the sea squirt Ciona intestinalis, both of which lack squalene epoxidase and are therefore unable to produce cholesterol de novo. These data confirm the initial observation that ERG27/HSD17B7-orthologs are only found in sterol synthesizing organisms (23).
HSD17B7 Has 3-Ketosteroid Reductase Activity
The next step was to test whether HSD17B7 indeed possesses 3-ketosteroid reductase activity and accepts zymosterone as a substrate. We expressed recombinant human HSD17B7, truncated HSD17B7, HSD17B1, HSD17B5, and murine Hsd17b7 as GST fusion proteins and tested the enzymes for the ability to convert zymosterone to zymosterol (Fig. 3
). Only GST-HSD17B7 and GST-Hsd17b7 showed measurable 3-ketosteroid reductase activity with zymosterone that was absent in reactions with GST alone, GST-HSD17B1, GST-HSD17B5 or when the cofactor reduced nicotinamide adenine dinucleotide phosphate (NADPH) was left out. The fusion protein with truncated HSD17B7 lacking the C-terminal 127 amino acids of HSD17B7 including the membrane-associated helix was as well inactive. All enzymes accept the fusion protein with truncated HSD17B7 were active on 17ß-estradiol (HSD17B1, HSD17B7, Hsd17b7) and androstenedione (HSD17B5) (data not shown). The results indicate that the conversion of zymosterone to zymosterol by HSD17B7 and Hsd17b7 is indeed specific.

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Fig. 3. HSD17B7 Converts Zymosterone to Zymosterol
Recombinant GST-fusion proteins of murine Hsd17b7 and human HSD17B7, truncated HSD17B7, HSD17B1, HSD17B5, and GST were incubated with zymosterone for 90 min at 37 C as indicated. Steroid extracts were separated by TLC using toluol:ethylacetate (80:20) as the mobile phase and detected by H2SO4/EtOH staining. Z0, Zymosterone; Z1, zymosterol; arrow indicates direction of flow.
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Yeast Deficient in Erg27p Is Complemented by HSD17B7 Expression
To test whether HSD17B7 is functionally equivalent to Erg27p we performed a yeast complementation assay in the ERG27-deficient yeast strain SDG110 (2). Two yeast transformants from each transformation selection plate were verified by PCR (not shown) and plated on complementation selection plates. The plates were left at 30 C for 3 wk and checked for colony formation. Both human HSD17B7 and mouse Hsd17b7, as well as Erg27p, complemented the gene disruption in SDG110 and restored growth in cholesterol-deficient medium (Fig. 4
). No complementation was seen with a truncated HSD17B7 or with two other human hydroxysteroid dehydrogenases (HSD17B1 and HSD17B5). However, in human HSD17B7 transformants, colony formation was much slower than in ERG27 transformants. Western blots showed that all human proteins, except HSD17B5, were very poorly expressed in the SDG110 cells (data not shown), indicating that the slower growth of HSD17B7 transformants was probably caused by low enzyme levels and not by lack of specific activity.

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Fig. 4. Complementation of Erg27-Deficient Yeast Strain
SDG110 yeast were transformed with pYES2.1 constructs encoding the proteins indicated. Transformants were plated in serial dilutions (1:1, 1:10, 1:100, 1:1000) and incubated 3 wk on complementation selection plates in the absence of cholesterol. Only transformants expressing native yeast Erg27p, human HSD17B7, and murine Hsd17b7 were able to complement the ERG27 deficiency of SDG110. HSD17B7tr, Truncated form of human HSD17B7 lacking the last 127 amino acids.
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HSD17B7 Is Localized in the Endoplasmic Reticulum
In yeast, Erg27p is part of larger enzyme complex in the endoplasmic reticulum (ER), and it is expected that the corresponding reactions in humans also occur in the ER membrane (29). Therefore, we checked the cellular localization of HSD17B7 using fusions with green fluorescent protein (GFP). Human HeLa and murine NIH3T3 cells were transfected with plasmids coding for conspecific HSD17B7 fused N-terminally to GFP, and colocalization with ER-Tracker Blue-White was examined by confocal microscopy. Both human and mouse HSD17B7 were found to be localized in the ER (Fig. 5
, AF). C-terminal truncation, which removes the predicted membrane-associated helix of HSD17B7, led to uniform mislocalization of the protein to cytosol and nucleus (Fig. 5G
). Cytosolic localization was as well observed when the fluorescent label was fused to the C terminus of HSD17B7 (data not shown), indicating that the C-terminal part of HSD17B7 is essential for its proper targeting/anchoring to the ER.

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Fig. 5. Localization of N-Terminal GFP Fusions of Human and Murine HSD17B7 in the ER of HeLa and NIH3T3 Cells
HeLa and NIH3T3 were transfected with human HSD17B7 (AC) and mouse Hsd17b7 (DF) fused N-terminally to GFP. Twenty-four hours after transfection with the fusion construct, the cells were incubated with ER-Tracker Blue-White DPX and examined by confocal microscopy. A and D, Fluorescence of GFP-HSD17B7. B and E, ER-Tracker fluorescence. C and F, Overlay of both fluorescence signals. G, Deletion of the C terminus of HSD17B7 leads to uniform mislocalization to the cytosol and nucleus of HeLa cells. Colocalization is represented in light blue. ER, Endoplasmic reticulum.
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Expression Analysis during Embryonic Development
If Hsd17b7 is involved in cholesterol biosynthesis, it should be a member of the same synexpression group as other cholesterogenic genes, that means it should show the same expression pattern in different tissues and the same transcriptional responses to changing conditions. In a recent microarray analysis of gene expression in fifty human primary fibroblast cell cultures, HSD17B7 was a member of a comprehensive cluster of canonical sterol regulatory element binding protein target genes, including low-density lipoprotein receptor, HMG-CoA reductase, squalene epoxidase, lanosterol synthase, and about 20 other genes of lipid and sterol metabolism (30). We reevaluated a subset of data by Chang et al. (30) to illustrate the coregulation of HSD17B7 (see Fig. 2 in supplemental data). The selection and order of genes in this figure is the result of an automatic computation (Pearson correlation). The fact that all genes that are present in duplicates (HSD17B7, FADS2, HMGCS1) are nearest neighbors indicates the excellent reproducibility of the data in this experiment. To extend these observations to different tissues and because the most complex localized changes in cholesterol biosynthesis requirements occur during embryogenesis (31), we compared the expression patterns of Hsd17b7 and Hmgcr, which codes for the rate-limiting enzyme of cholesterol biosynthesis, during several stages of development in the mouse embryo [embryonic day (E) 10.514.5]. Using mRNA whole mount in situ hybridization and whole mount immunostaining we found both genes to be expressed in the same complex pattern over the whole period investigated (Figs. 6
and 7
).

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Fig. 6. Coexpression of Hsd17b7 and Hmgcr in the Mouse Embryo at E10.5-E12.5
A and B, Both Hsd17b7 and Hmgcr show moderate ubiquitous expression, but specific high expression is seen in the trigeminal ganglion (red arrowheads), the dorsal root ganglia (white arrowhead) and the distal tip of the forelimb buds (asterisks). C and D, Both genes are detected at the anterior margin of the second branchial arch and the posterior margin of the third branchial arch (anterior, up; posterior, down). II, Second branchial arch. III, Third branchial arch. E and F, by E11.5, the expression of both genes in the dorsal root ganglia (arrowheads) and in the neural tube (asterisks) is strongly increased including the transient ganglionic crest (Frorieps ganglia; G and H, arrowheads). Dynamic expression of Hsd17b7 and Hmgcr could be observed during limb development (I and J). At E11.5, expression is found in the autopodal region of the limb mesenchyme but becomes restricted to the interdigital zones by E12.5 (K and L).
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Fig. 7. Expression Domains of Hsd17b7 and Hmgcr in the Face (E13.5)
A, Immunostaining using an antibody against HSD17B7 detects the protein in the hair follicles of the vibrissae. B, In situ hybridization; the very characteristic pattern spares the centers of the follicles and is identical to that of Hsd17b7 mRNA at the same stage.
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The analysis of the expression of Hsd17b7 and Hmgcr at E10.5 is shown in Fig. 6
, AD. High expression levels of both genes are found in the ganglion of the trigeminal nerve and the dorsal root ganglia that begin to appear around E10.5 rostrally, in addition to a weak ubiquitous staining. The figures also show increased expression of the genes in the distal tip of the emerging limb buds. An even more characteristic similarity in the expression pattern is seen in the second and third branchial arches (Fig. 6
, C and D), where Hsd17b7 and Hmgcr are expressed at the anterior margin of the second and the posterior margin of the third branchial arch. The same pattern is observed for several other cholesterogenic enzymes e.g. Nsdhl and Dhcr7 (32).
By E11.5, expression of both genes in the dorsal root ganglia is found along the complete body axis and is strongly increased compared with E10.5. At this stage very high expression is also seen in the neural tube (Fig. 6
, E and F) and the tail bud (data not shown). Hsd17b7 as well as Hmgcr are furthermore coexpressed in the transient ganglionic crest (Frorieps ganglia; Fig. 6
, G and H) and the cranial ganglia (data not shown). The expression in the various ganglia persists at least until E14.5 (not shown).
During limb development, both genes show dynamic expression beginning in the autopodal region where homogeneous expression is seen up to E12.5. From then on the mRNAs become more and more restricted to the interdigital regions (Fig. 6
, K and L). Especially at later stages (around E13.5-E14.5), this pattern resembles that of apoptotic markers like p53 or Bax1 (not shown).
Another striking case of coexpression occurs around E13.5 in the hair follicles of the vibrissae. Figure 7A
shows the facial region of a mouse embryo after immunostaining using a peptide antibody directed against HSD17B7. The enzyme is detectable in circles around the follicle center. The same pattern is observed after mRNA in situ hybridization for Hsd17b7 (data not shown) as well as Hmgcr (Fig. 7B
). This comparison also shows that the expression seen by in situ hybridization is indeed specific and correlates with enzyme level.
Because Hsd17b7 was originally described as an estrogenic enzyme, we were also interested in comparing its expression to that of the estrogenic 17ß-hydroxysteroid dehydrogenase type 1 (Hsd17b1). In contrast to Hsd17b7, which shows a distinct and dynamic pattern of expression identical to that of cholesterogenic genes, mRNA in situ hybridization of Hsd17b1 did not show any specific expression pattern at the same developmental stages (data not shown).
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DISCUSSION
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In this study, we present experimental evidence that HSD17B7 is the 3-ketosteroid reductase of cholesterol biogenesis in mammals. This corroborates speculations by Breitling et al. (23) that were based on a computational analysis, including phyletic profiling, molecular modeling, and in silico Northern blots.
Our extended phylogenetic analysis using maximum parsimony and neighbor joining algorithms confirmed the hypothesis that HSD17B7 is the vertebrate ortholog of Erg27p and showed that orthologs are absent from C. elegans, Drosophila, and Ciona intestinalis, indicating that the gene was selectively lost in cholesterol-auxotroph organisms. HSD17B7 and the yeast 3-ketosteroid reductase Erg27p are associated by the presence of two conserved secondary structure elements (see Fig. 1 in supplemental data) that are shared derived characters (synapomorphies) of the group and are predicted to be involved in substrate recognition and membrane association, respectively.
To test our computational prediction that HSD17B7 is the 3-ketosteroid reductase of cholesterogenesis, we first of all determined its enzymatic activity in vivo and in vitro. Recombinant HSD17B7 protein catalyzed the conversion of zymosterone to zymosterol with NADPH as a cofactor. Neither HSD17B1 (17ß-hydroxysteroid dehydrogenase) nor HSD17B5 (3
- hydroxysteroid dehydrogenase and 17ß-hydroxysteroid dehydrogenase) catalyzed the reduction of zymosterone. The results of the in vitro tests were verified by a yeast complementation assay. Expression of HSD17B7 in an ERG27-deficient yeast strain rescued the defect and demonstrated that HSD17B7 can act as a zymosterone reductase under physiological conditions in vivo. The rescue effect was specific to HSD17B7 and was not observed with other hydroxysteroid dehydrogenases (HSD17B1 and HSD17B5).
The yeast ortholog of HSD17B7, Erg27p, is localized in a multiprotein complex in the endoplasmic reticulum (29). Correspondingly, we found that GFP-fusion constructs of human and murine HSD17B7 colocalized with an endoplasmic reticulum marker. The localization, as well as activity, was specifically dependent on the presence of an intact and accessible C terminus. This agrees well with our prediction that the C terminus contains the membrane interaction domain of HSD17B7 (see Fig. 1 in supplemental data) (23, 33, 34, 35, 36). Present UniGene information on HSD17B7 reveals two gene copies as cluster Hs.187579 on chromosome 1q23 and as cluster Hs.380900 on chromosome 10p11.2. Although both genes have identical structure consisting of nine exons, the copy on chromosome 10p11.2 features a premature stop codon in exon six. The corresponding protein would be missing the membrane-associated helix and therefore be inactive as enzyme.
Phylogeny, subcellular localization, and activity measurements can only provide circumstantial evidence regarding the physiological function of an enzyme. To establish the involvement of HSD17B7 in cholesterogenesis we made use of a recent observation by Laubner et al. (32), who showed that cholesterogenic genes are members of a highly distinct synexpression group in the mouse embryo. And indeed the expression pattern of HSD17B7 during mouse embryogenesis faithfully copies that of HMG-CoA reductase and many other cholesterogenic enzymes. Expression is prominently restricted to those tissues that have been implicated in the pathogenesis of cholesterol deficiency disorders, such as neural crest derivatives, hair follicles and the emerging limbs. Our observation shows that HSD17B7 is closely coregulated with cholesterogenesis in a wide variety of tissues over a range of rapidly changing conditions. This again is in the accordance with the pattern of cholesterogenic genes coregulated with HSD17B7 in human primary fibroblast cultures upon serum starvation (30).
How do these data fit in with the fact that HSD17B7 was originally described as an estradiol-producing enzyme and is abundantly expressed in the ovaries of pregnant mice (25)? In part, the expression in the ovary may be explained by the increased local demand for cholesterol as steroid hormone precursor, but up-regulation of HSD17B7 in corpora lutea during pregnancy persists much longer than up-regulation of HMG-CoA reductase (37, 38, 39) indicating an interesting acquisition of dual functionality by HSD17B7, at least in rodents. In primates, such as human and marmoset monkeys, the strongest expression is seen in hepatic cells, a classical estradiol inactivating tissue (23, 27, 28). This preferential expression in liver agrees well with a role of HSD17B7 in cholesterol biosynthesis.
HSD17B7 is not the only 17ß-hydroxysteroid dehydrogenase that was originally identified based on its function in organ-specific hormone metabolism and later turned out to be predominantly involved in another metabolic pathway. As reported recently (40, 41) the HSD17B7 reveals further activity as 3ß-hydroxysteroid dehydrogenase in that it can convert dihydrotestosterone to 3ß-androstane-diol and progesterone to 4-pregnen-3ß-ol-20-one.
In another example, the HSD17B4 was first identified as the estradiol-inactivating dehydrogenase from porcine endometrium (42, 43), whereas later experiments (44, 45, 46, 47), identification of human mutants (48, 49) and a recent targeted gene disruption in the mouse (50) have conclusively determined that its main function is in peroxisomal ß-oxidation and bile acid metabolism (multifunctional protein type 2 activity). The presence of 3-ketosteroid reductase and 17ß-hydroxysteroid dehydrogenase in a single enzyme is not unique to HSD17B7. For example, HSD17B5 has dual activity as a 3
- and 17ß-hydroxysteroid dehydrogenase within the same active center (51, 52). Flexibility in substrate preference seems to be a general feature of 17ß-hydroxysteroid dehydrogenases (reviewed in Refs. 53 and 54). Interestingly, so far mutations in only two human 17ß-hydroxysteroid dehydrogenases have been identified that cause monogenic diseases like male pseudohermaphroditism and D-specific multifunctional protein type 2 deficiency, i.e. HSD17B3 (55) and HSD17B4 (48), respectively. Phenotypes of mutations in other hydroxysteroid metabolizing enzymes might be lethal and therefore not observed yet. Definitely a functional deletion of further 17ß-hydroxysteroid dehydrogenases under controlled conditions like short interfering RNA or Cre/Lox technologies (56, 57) would be instrumental in understanding their metabolic role.
Identification of the 3-ketosteroid reductase completes the molecular inventory of cholesterol biosynthesis in mammals. Given that both upstream and downstream reactions have been implicated in severe inborn errors of cholesterol biosynthesis (CHILD syndrome and X-linked chondrodysplasia punctata), hypomorphism in HSD17B7 should have similar consequences making it a new candidate gene for this important class of congenital malformation syndromes.
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MATERIALS AND METHODS
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Sequences
Genome draft sequences for the fugu fish Takifugu rubripes (Fugu version 3.0), the sea squirt Ciona intestinalis and the whiterot fungus Phanerochaete chrysosporium (version 020216) were obtained from the DOE Joint Genome Institute (http://www.jgi.doe.gov). Genomic sequences for the freshwater puffer fish Tetraodon nigroviridis (assembly version 6) are from Genoscope (http://www.genoscope.cns.fr/externe/tetraodon). The unfinished genome of the filamentous fungus Neurospora crassa (assembly 3) was accessed at the Whitehead Institute Center for Genome Research (http://www-genome.wi.mit.edu/annotation/fungi/neurospora). All other sequences are available at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov).
Phylogenetic Analysis
Protein sequences of human HSD17B7 and its homologs were identified by BLAST searches and aligned using ClustalW (58). After removal of positions containing gaps, phylogenetic trees were reconstructed using maximum parsimony or neighbor joining as implemented in the Phylip package (59). The stability of the resulting topology was assessed by bootstrap analysis using 1000 pseudoreplicates.
Enzymic Activity Assay
For activity measurements cDNA of human HSD17B7, truncated HSD17B7, HSD17B1, HSD17B5, and murine Hsd17b7 were PCR-amplified (for primer sequences, see Table 2
) and cloned into a modified pGEX vector (Amersham Biosciences, Freiburg, Germany) (44). The truncated form of HSD17B7 was shortened by 127 amino acids at the C terminus and did not contain the membrane-associated helix. The proteins were expressed as fusion proteins with glutathione-S-transferase (GST) in Escherichia coli BL21 DE3 Codon Plus (Stratagene, Amsterdam, The Netherlands) after standard protocols.
Zymosterone was synthesized as follows: zymosterol was oxidized with pyridinium chlorochromate in dichloromethane at room temperature to give zymosterone in 65% yield. The enzymatic reaction contained 880 µl buffer [10 mM KPi, 0.05% BSA, and 1 mM EDTA (pH 8.0)], 100 µl bacterial lysate (4.5 nmol enzyme), 10 µl substrate (5 mM zymosterone in dimethylsulfoxide) and 10 µl NADPH (5 mg/ml). The incubation was carried out for 90 min at 37 C. The steroids contained in the reaction were then extracted using reverse phase chromatography on RP18 columns (Phenomenex, Aschaffenburg, Germany) and eluted with two times 200 µl methanol (MetOH):chloroform 1:1 and once with 200 µl chloroform. After evaporation of the solvent the steroids were solubilized in chloroform, separated on Kieselgel 60 F254 thin-layer chromatography plates (Merck, Darmstadt, Germany) using toluene:ethylacetate (80:20) as the mobile phase and detected by spraying with 30% H2SO4 in ethanol (EtOH) and development at 135 C. Substrate and metabolites were identified by comparison with reference steroids. Because zymosterone was not available as a radioactive substance, the maximum velocity and Michaelis constant were not determined.
Complementation Analysis of ERG27-Deficient Yeasts
For the assay complete cDNAs of yeast ERG25, ERG27, human HSD17B7, truncated HSD17B7, HSD17B1 and HSD17B5, and mouse Hsd17b7 were amplified by PCR (for primer sequences see Table 2
) and cloned into the vector pYES2.1/V5/His (Invitrogen, La Jolla, CA) according to the manufacturers protocol. Yeast SDG110 (MATa upc2 ade2 his3 ura352 erg27
:HIS3) (courtesy of Dr. M. Bard, Purdue University, Indianapolis, IN) were grown in YPDA medium containing 30 µg/ml cholesterol (Sigma-Aldrich, Taufkirchen, Germany; solubilized in EtOH:Tween 20 = 1:1) to an OD600 of approximately 3. Transformation was done with the BIO 101 kit (BIO 101 Inc., Carlsbad, CA) according to the manufacturers protocol. After transformation the entire reaction mixture from each transformant was plated on transformation selection plates [synthetic complete (SC) medium -HIS (histidine)/-URA (uracil), 30 µg/ml cholesterol and 2% glucose] and incubated at 30 C for 3 d until colonies formed. Two colonies from each plate were resuspended in 20 µl of sterile water and checked for the presence of plasmid by PCR. Positive yeast clones were resuspended in 50 µl of sterile water and serially diluted (1:1, 1:10, 1:100, 1:1000). 5 µl of each dilution was plated on the complementation selection plate (SC medium -HIS/-URA/-cholesterol, 2% galactose, 2% raffinose). The plates were incubated at 30 C up to 3 wk.
Subcellular Localization
Human HSD17B7, truncated human HSD17B7, and mouse Hsd17b7 cDNAs were amplified by PCR (for primer sequences see Table 2
) and cloned into the pEGFP-C1 vector (CLONTECH, Heidelberg, Germany), respectively. The human cell line HeLa was maintained in RPMI 1640 medium/10% fetal bovine serum (Invitrogen, Karlsruhe, Germany) and the murine cell line NIH3T3 was cultivated in DMEM/10% fetal bovine serum (Invitrogen). HeLa cells were grown to 50% confluency and transfected with human HSD17B7 and truncated human HSD17B7 subcloned in the vector pEGFP-C1 using FuGENE 6 Transfection Reagent (Roche, Mannheim, Germany) according to the manufacturers protocol. This results in expression of a fusion protein with enhanced GFP (excitation 488 nm, emission 507 nm). NIH3T3 cells were transfected the same way with mouse Hsd17b7 subcloned in pEGFP-C1 vector. Twenty-four hours after transfection, cells were incubated with 300 nM of ER-Tracker Blue-White DPX (Molecular Probes, Eugene, OR; peak fluorescence: 374 nm, emission: 430640 nm) and examined with a LSM 410 confocal microscope (Zeiss, Jena, Germany) using a x63 water objective.
Preparation of mRNA Probes for in Situ Hybridization Experiments
Probes for murine HMG-CoA reductase (Hmgcr) and Hsd17b7 were PCR-cloned from cDNA of NIH3T3 cells. cDNA derived from mouse placenta was used for PCR amplification and subcloning of Hsd17b1 (for primers see Table 2
). Digoxigenin-labeled RNA probes were synthesized from the linearized template using the digoxigenin RNA labeling kit (Roche, Mannheim, Germany). RNA probes were purified (RNEasy Mini kit, QIAGEN, Hilden, Germany) and denatured at 80 C for 3 min.
mRNA Whole Mount in Situ Hybridization
C3HeB/FeJ wild-type mouse embryos were dissected in PBS at E10.5-E14.5 and fixed over night in freshly prepared 4% paraformaldehyde in PBS at 4 C. Embryos were washed twice with PBS on ice, dehydrated through a MetOH series, and stored at -20 C. In situ hybridization was done according to Spörle and Schughart (60). Rehydrated embryos were bleached with 6% hydrogen peroxide for 30 min up to 1 h depending on the age, and the heads were perforated using a thin needle to avoid unspecific precipitation of staining reagentin the ventricles. Proteinase K treatment (10 µg/ml) was done as follows: E10.52 min, E11.54 min, E12.56 min, E13.5/E14.510 min incubation at room temperature. Precipitating BM Purple AP substrate (Roche, Mannheim, Germany) was used for staining.
HSD17B7 Antibodies
Polyclonal antipeptide antibodies reactive against HSD17B7 were generated by immunization of rabbits (Pineda Antikörper-Service, Berlin, Germany). The synthetic peptide (NH2-C)KMDLDEDTAEKFYQK(-CONH2) corresponding to conserved amino acids 301315 of human HSD17B7 coupled to keyhole limpet hemocyanine was used as antigen. The antiserum was purified by affinity chromatography.
Whole Mount Immunostaining
After dissection embryos were fixed overnight in 4% paraformaldehyd in PBS and bleached for 2 h at 4 C by adding 1 vol 30% H2O2. The embryos were washed twice in 100% MetOH and stored in 100% MetOH at -20 C. For immunostaining the embryos were rehydrated through series of 75%50%25% MetOH in PBS. After two washing steps in PBS, the embryos were washed twice in PBSMT (2% instant skim milk powder; 0.1% Triton X-100 in PBS) for 1 h at room temperature, followed by incubation with anti-HSD17B7 antibody in PBSMT overnight at 4 C. Embryos were washed twice in PBSMT for 2 h at 4 C followed by 3 times for 1 h in PBSMT at room temperature. Embryos were incubated with secondary antibody (goat-antirabbit-FAB2-HRP conjugate; 1:200; Promega, Mannheim, Germany) in PBSMT over night at 4 C followed by 5 washing steps in PBSMT and a final 20 min washing in PBT (PBS + 0.1% Tween 20). After the embryos were incubated with 0.3 mg/ml diaminobenzidine in PBT for 2040 min, 30% H2O2 was added in 1 µl steps until structures became visible. Staining was stopped by rinsing in PBT.
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ACKNOWLEDGMENTS
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We would like to thank Dr. Almuth Einspanier for her help in preparation of anti HSD17B7 antibodies, Dr. Josef-Karl Gerber for his advice on yeast cultivation, Christina Fürthner for her help with whole mount immunostaining, and Marion Schieweg for expert technical assistance. We wish to thank Dr. Peter Hutzler for his advice on confocal microscopy. We thank Dr. M. Bard (Purdue University, Indianapolis, IN) for kindly providing the SDG110 yeast strain.
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FOOTNOTES
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This work was supported by the Bundesministerium fuer Bildung und Forschung and Deutsche Forschungsgemeinschaft (DFG) grants (to J.A.) and in part by DFG Grant EI333/6-3 (to A.E.).
Z.M. and D.L. contributed equally to this work
Current address for B.H.: Solvay Pharmaceuticals GmbH, 30173 Hannover, Germany.
Abbreviations: CHILD, Chondrodysplasia with ichthyosis and limb defects; CoA, coenzyme A; Erg27p, yeast 3-ketosteroid reductase; ERG27, gene of yeast 3-ketosteroid reductase; EtOH, ethanol; GFP, green fluorescent protein; GST, glutathione-S-transferase; HIS, histidine; HMG, hydroxymethyl-glutaryl; HSD, hydroxysteroid dehydrogenase; HSD17B7, gene of human HSD17B7; Hsd17b7, gene of mouse HSD17B7; HMG-CoA, hydroxymethyl-glutaryl-CoA reductase; Hmgcr, gene of mouse HMG-CoA; MetOH, methanol; NADPH, reduced nicotinamide adenine dinucleotide phosphate; URA, uracil.
Received for publication December 23, 2002.
Accepted for publication June 16, 2003.
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