3P-HSD - The Journal of Biological Chemistry

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3@-Hydroxysteroid Dehydrogenase/A6-A4 Isomerase (3P-HSD) Family. THE EXCLUSIVE .... Moreover, we have recently demonstrated that congenital adrenal hyperplasia due to classical 3P-HSD deficiency results from mutation(s) in the ...
VOl. 268, No. 26, Issue of September 15,PP. 19659-19668,1993 Printed in U.S A .

T H EJOURNAL OF BIOLOGICAL CHEMISTRY

0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure and Tissue-specific Expression of a Novel Member of the Rat 3@-HydroxysteroidDehydrogenase/A6-A4 Isomerase (3P-HSD) Family THE EXCLUSIVE 3P-HSD GENEEXPRESSEDIN

THE SKIN* (Received for publication, May 11, 1993, and in revised form, June 1, 1993)

Jacques SimardS, JacquesCouetP, Francine Durocherll, Yvan Labrie, Rocio Sanchezl), Nathalie Breton,Carl Turgeon, and Fernand Labrie From the Medical Research Council Group in Molecular Endocrinology, CHUL Research Center and Lava1 University, Qdbec G1 V 4G2, Canada

Structures of cDNA clones encoding three members in thetransformation of all 5-pregnen-3P-01and 5-androstenof the rat 38-hydroxysteroid dehydrogenase/An-A4 iso3P-01 steroids intothe corresponding A‘-3-ketosteroids, merase (38-HSD)family were characterized. To searchnamely progesterone as well as all the precursors of androgens, for potential new types of 3@-HSD, rattypes I and I1 estrogens, glucocorticoids, and mineralocorticoids. In addi38-HSD cDNAs were used as probes to screen a rat tion, 3P-HSD is responsible for the interconversion of 38genomic DNA library. Among the clones isolated, one hydroxy- and3-keto-5a-androstane steroids. In mammals, encodes a novelpredictedrat3B-HSDisoenzyme, chronologically designated type IV. The corresponding the 3P-HSD isoenzymes are expressed not only in classical full-length cDNA was thereafter isolated by selective steroidogenic tissues, namely, the adrenal cortex, ovary, testis, andplacenta (1-7), but also in several peripheral tissues polymerasechainreactionamplificationfromrat ovary and day-15 placenta cDNA libraries. The rat including the skin, liver, adipose tissue, kidney, epididymis, type IV 3B-HSD cDNA encodes a predicted 372-amino lung, mammary gland, prostate, and brain (5, 8-21), where acidproteinof 41,854 daltons,whichshares 90.9, they areinvolved in intracrineformation of sex steroids acting 87.9, and 78.8%sequence identity with rat types I, 11, locally (22). and I11 proteins, respectively. Ribonuclease protection The structures and tissue-specific expression of two types assay reveals that type IV 38-HSD is the sole 3&HSD of human 3p-HSD cDNA clones encoding the types I and I1 mRNA species detectable in the skin and represents 3P-HSD isoenzymes of 372 and 371 amino acids, respectively, the predominant species in the placenta while being I also detectable in the ovary and, to a lower degree, in were recently characterized (5,23-25). Human type 3P-HSD theadrenalgland.Transientexpressionoftype IV is the predominant 3P-HSD gene expressed in the placenta cDNA inSW-13 cells indicates 3O-HSD activity similar and peripheraltissues, such as theskin, while the human type to that of rat typeI 38-HSD. The presence of multipleI1 is the almost exclusive mRNA species observed in the 3P-HSD genes should permit differential and tissue- adrenals and gonads (5). The type I protein possesses higher specific regulation ofthis rate-limiting enzymatic ac- 3P-HSD activity than type I1 (5). The structure of the corretivity essential inthebiosynthesisof all classes of sponding genes consists of 4 exons and 3 introns included steroid hormones in both classical steroidogenic and within a genomic DNA fragment of about 7.8 kilobases (26intracrine peripheraltissues. 28). Moreover, we have recently demonstrated that congenital adrenal hyperplasia due to classical 3P-HSD deficiency results from mutation(s) in the type I1 3P-HSD gene, while the The membrane-bound enzyme 38-hydroxysteroid dehydro- finding of a normal type I 3P-HSD gene provides the basis genase/A6-A4isomerase (3P-HSD)’ catalyzes an essential step for the well recognized intact peripheral steroidogenesis in these patients (29, 30). * This work wassupported in part by the Medical Research Council Multiple 3P-HSDs have also been characterized in the rat of Canada, Socihtk d‘Investissement R&D Andros Inc., and Endorecherche. The costs of publication of this article were defrayed in and mouse (4, 12, 15). The structure of three types of rat 38part by the payment of page charges. This article must therefore be HSD cDNAs which all encode a 372-amino acid protein has hereby marked “advertisement” in accordance with 18U.S.C. Section been elucidated (4, 12, 14, 31, 32). The predicted rat types I 1734 solelyto indicate this fact. and I1 3P-HSD proteins expressed in theadrenal, ovary, testis, The nucleotide sequencefs)reported in thispaperh m been submitted kidney, and adipose tissue share 93.8% homology. The much totheGenBankTM/EMBLDataBankwith accession numberfs) lower activity of expressed rat type I1 compared to rat type I L17138. $ Recipient of a Medical Research Council scholarship. To whom BB-HSD is due to a change of four residues involved in a correspondence should be addressed MRC Group in Molecular En- putativemembrane-spanning domain as revealed by sitedocrinology,CHUL Research Center, 2705 Laurier Blvd., QukbecG1V directed mutagenesis (33). The male liver-specific type I11 4G2, Canada. Tel.: 418-654-2296;Fax: 418-654-2735. isoform (12,13) does not display the expected 3P-HSD activity Recipient of a Medical Research Council studentship. with As-hydroxysteroid precursors but is rather a 3-ketosterll Recipient of a studentship from the Fonds pour la Formation de Chercheur et L’Aide a la Recherche. oid reductase using NADPH as cofactor, which catalyzes the 11 Recipient of a studentship from the Ministbre de YEnseignement conversion of 3-keto-saturated steroids such asdihydrotestosSuphrieur et de la Science du Qubbec. terone and dihydroprogesterone into their corresponding 3PThe abbreviations used are: 3P-HSD, 3P-hydroxysteroid dehydrogenase/A’-A‘ isomerase; PCR, polymerase chain reaction; M-MLV, hydroxy metabolite (34). The aim of the present study was to Moloney murine leukemia virus; DHEA, dehydroepiandrosterone; investigate the potential existence of a new type of 3P-HSD PREG, pregnenolone. isoenzyme, which could be the predominant species expressed

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Structure and Expression of a Novel Rat 3p-HSD Isoenzyme

in the placenta andthe skin, in analogy to thesituation found in the human (1,5, 8, 23).

directly sequenced in both orientationsas described below. DNA Sequencing and Computer Analyses-Synthetic oligonucleotides, as well as T7 and T3 vector primers and modified T7 DNA polymerase were used to sequence both strands of double-stranded EXPERIMENTALPROCEDURES plasmid DNA using the dideoxy chain termination method as shown Animal Tissues-Adult male and female Sprague-Dawley rats in Fig. 1 (37, 38). Direct sequencing of PCR products was performed (CrLCD(SD)Br) were obtained from Charles River Inc. (St. Con- from single-strand DNA produced from both strands by an asymstant, Canada). The tissue distributionof 3P-HSD mRNA was studied metric amplification. After selective precipitation, one-third of the in sex-specific organs such as the ovary, placenta, and uterus in asymmetric PCR products was then used for each sequencing reacfemales and testis in male animals. In addition, 3P-HSD mRNA tion. Sequencing was performed using the limiting PCR primer or species distribution was investigated in the female adrenal,skin, internal sequence-specific primers as illustrated in Fig. 1F. Three p1 kidney, and intra-abdominal fat aswell as in male liver. of the reaction mixtures were incubated at 80 "C for 3 min and Materials-All restriction endonucleases, T4polynucleotide kinase, resolved on a polyacrylamide-urea sequencing gel. The dried gel was T, DNA ligase, T7 sequencing kit, Transprobe T kit, and oligo(dT)- exposed to Kodak X-Omat AR films overnight. DNA sequences were cellulose were purchased from Pharmacia LKB Technology Inc. The analyzed with the Microgenie 7.0 software (Beckman Instruments). RPA I1 RNase protection assay kit was obtained from Ambion Putative membrane-spanning protein segments were determined by (Austin, TX). The placenta plasmid cDNA library was constructed the programs SOAP (39), HELIXMEM (40), and RAOARGOS (41) with the Superscript cDNA synthesis system and integrated in the (PC/GENE software, release 6.01, IntelliGenetics Inc./Genofit SA, plasmid vector pSport 1 both obtained from Life Technologies Inc. Mountain View, CA). using the Superscript M-MLV reverse transcriptase. Bluescript KS Transient Expression of Rat Types I and IV 30-HSD cDNAs-A and SK vectors were obtained from Stratagene (La Jolla, CA). The full-length cDNA insert corresponding to the rat ovary type IV 38Quiagen plasmid kit was purchased from Quiagen Corp. (Chatsworth, HSD clone was PCR-isolated using primers 1and 2 modified in order CA) The rat genomic DNA library constructed in the X-charon 4 to contain a HindIII restrictionsite sequence at their5' termini. This phage vector as well astherat ovary Xgtll cDNA library were fragment was then subcloned into the unique HindIII site of the obtained from Clontech Laboratories Inc. (Palo Alto, CA). PCR RNA pCMV vector (kindly provided by Dr. Michael B. Mathews, Cold Kit as well as Taq DNA polymerase were obtained from Perkin- Spring Harbor Laboratories), downstream from the cytomegalovirus Elmer Cetus. Synthetic oligonucleotides were synthesized in our (CMV) promoter, to produce the recombinant plasmid pCMV-type laboratory using a Biosearch DNA synthetizer. Total RNA was IV 38-HSD. The insert was sequenced in both orientations, amplified, extracted from rat tissues using the Applied Biosystems model 340 and subsequently purified using the Quiagen plasmid kit. Human automated nucleic acid extractor (Foster City, CA). Nitrocellulose SW-13 adrenal cortex adenocarcinoma cells (American Type Culture (Hybond C) and nylon membranes (Hybond N), [ c Y - ~ ' P ] ~ C(3000 TP Collection, Rockville, MD) were obtained at their 22-25th passage (800 Ci/mmol), [-y-32P]ATP(>lo00 Ci/mmol), and grown in L-15medium containing 5% fetal bovine serum suppleCi/mmol, [cY-~'P]UTP [y-36S]ATP(>lo00 Ci/mmol), and the Megaprime DNA labeling kit mented with 2 mM L-glutamine, 1 mM sodium pyruvate, 15 mM were obtained from Amersham Corp. [7-3H]Pregnenolone (25 Ci/ HEPES, 100 IU of penicillin/ml, and 100 pg of streptomycin sulfate/ mmol) and [1,2,6,7-3H]dehydroepiandrosterone (DHEA, 89 Ci/mmol) ml. SW-13 cells were plated at an initial density of lo' cells/cm* in were obtained from Du Pont-New England Nuclear. All other re- FalconT-75 flasks and the medium was changed 24 h later, immediagents were ofanalytical grade and purchased from ICN Biochemicals ately before the transfections. The pCMV type I or pCMV-type IV Inc. (Cleveland OH) and Bio-Rad. 3P-HSD plasmid was introduced into SW-13 cells by the calcium Isolation of Genomic and cDNA Clones-A rat genomic DNA library phosphate precipitation procedure as described elsewhere (42). Mock constructed in the X-charon 4 phage vector was screened with types transfections were carried out with pCMV alone while transfection I and I1 rat 3P-HSD cDNAs labeled with [32P]dCTPby the random efficiency was monitored by co-transfection of the plasmid with primer method (35). Prehybridization and hybridization were per- pXGH5 plasmid (43). pXGH5 constitutively expresses growth horformed as previously described (26, 28). Among the five clones iso- mone which is secreted into the culture medium. The media were lated, one encodes a novel predicted rat 3P-HSD isoenzyme which collected 72 h after transfection and stored at -20 "C until assayed has been designated type IV. EcoRI restriction fragments were sub- for growth hormone by radioimmunoassay while cells were harvested cloned into Bluescript SK Vector, and plasmid DNA was prepared by scraping with a rubber policeman and resuspended at a concentraby alkaline lysis (36). From the partial genomic sequence, the two tion of 10 X 10' cells/ml in buffer containing 50 mM phosphateexonic primers: 1, 5'-CCAGGGTCACCGTGCATTAACCCCACT-3'buffered saline, pH 7.4, 1 mM EDTA, and 20% glycerol. Cells were then sonicated. To measure 30-HSD activity, the indicated concenfrom -56 to -30 (see Table I) and 2, 5"GACAAATATTTATTGTTCCTTCTATT-3' from +1442 to +1417 were synthesized tration of protein from cell homogenates was incubated for the in order to isolate the entire cDNA from a rat ovary Xgtll cDNA indicated time periods at 37 "C in 50 mM phosphate-buffered saline library. The sequence and nucleotide positions of the primers used (pH 7.5), 20% glycerol,containing 1mM NAD+ in the presence of the for PCR amplification of type IV 38-HSD cDNA fragments arelisted indicated concentrations of [1,2,6,7-3H]DHEAor [7-3H]-PREG.The in Table I and their position is further illustrated in Fig. 1. The rat enzymatic reaction was stopped by adding 4 volumes of diethylether ovarian type IVcDNAwas amplified using the indicated set of and chilling the incubation mixture in adry ice-ethanol bath. Steroids primers (1and 2) in a 100-p1 volumecontaining 10 mM Tris (pH8.3), were analyzed by thin-layer chromatography (4, 5, 33,34). The 50 mM KCl, 1 mM MgC12, 50 p~ dNTP, 0.25 p M of each primer, and substrates and newly formed steroids were identified by co-migration 0.25 pg of rat ovary Xgtll cDNA. The reaction mixture was covered on each TLC plate of the appropriate purified labeled steroids. The with mineral oil, and DNA templates were denatured at 100 "C for corresponding areas were quantitated with a digital autoradiograph, 10 min and then cooled down to 72 "C prior to the addition of Taq Berthold (Wildbrod, Germany). K,,, and V,, values were calculated polymerase. PCR reactions were carried out using a Perkin-Elmer using ENZFITTER software (Biosoft, Cambridge, UK) aspreviously Cetus thermal cycler (model 480) with a temperature cycle consisting described (34, 44) after normalization for the amount of translated of 1 min of denaturation at 95 "C, 1 min of annealing a t 60 "C, and types I and IV 3P-HSD proteins in each cell homogenate preparation 1.5 min of extension at 72 "C. After 40 cycles, the primers were as measured by immunoblot analysis as previously described (5, 33, removed by selective DNA precipitation. The fragment was subcloned 34). Messenger RNA Purification and Ribonuclease Protection Assayinto Bluescript SK Vector and the plasmid insert was sequenced in both orientations (Fig. 1F).In parallel, a pSPORT 1plasmid cDNA Total RNA was extracted with an Applied Biosystems model 340 library from rat placenta poly(A+)RNA obtained at day 15 of gesta- automated nucleic acid extractor according to thesupplier's protocol. tion was constructed following the supplier's protocol. This library Total RNA samples were solubilized in sterile water, ethanol-precipwas used to purify placental type IV 3P-HSD cDNA fragments by itated, resuspended in sterile water, and quantitated by absorbance PCR (Fig. IC). Furthermore, type IV 3P-HSD cDNA fragments were at 260 nm. Poly(A+) RNA was purified by two cycles of chromatogisolated directly from 2 pgof rat day-13 placenta or skin poly(A+) raphy on oligo(dT)-cellulose columns as previously described (45). From the genomic type IV 3P-HSD clone, a 195-nucleotide fragRNA using reverse transcriptase and PCR amplification under the same conditions described above and as illustrated in Fig. 1, D and ment was isolated by PCR amplification using the following set of E. The Perkin-Elmer Cetus RNA PCR Kit was used for this purpose. primers: 5'-GGAATTCACAAAGAATCCTCTCAATGATCTG-3', The PCR products (1/30) were then subjected to a subsequent 40- and 5"GTCTGGTAGGATCTACAGTT-3'. The PCR product was cycle asymmetric amplification under the same conditions, except for purified and the blunt ends subcloned in the SmaI restriction site of the use of 3 nM limiting primer. All of these PCR products were then the Bluescript SK vector. The plasmid was linearized with the HindIII

Structure and Expression of a Novel Rat 3p-HSD Isoenzyme

19661

TABLEI Primer sequences

ce

Nucleotide position

Primer 1 2 3 4 5 6 7 8 9 10 11 12 13 14

’Positions

(5’+ 3‘)”

-56 -+ -30 +1442 -+ +1417 +177 +150 +742 -+ +713 +150 -+ +177 +69 -+ +96 +339 -+ +366 +1125 -+ +1106 +366 + +346 +693 -+ +714 +lo75 -+ +lo96 +lo19 -+ +lo39 +1416 + +1392 -30 -+ -56

5’-CCAGGGTCACCGTGCATTAACCCCACT-3’ 5’-GACAAATATTTATTGTTCCTTCTATT-3’ 5’-CAACACTGTCACCTTGATGCTTGTCCCT-3’ 5’-TTTGTGACTTCTTGGGGTCTCGAAGG-3’

--.)

5’-AGGGACAAGCATCAAGGTGACAGTGTTG-3’ 5’-GGTGCAGGAGAAAGATCTGAAGGAGGTC-3’

5’-CCAAGCTAGTGTGCCAGCCTTCATCTAC-3’ 5’-CCATTACTGAGACTTTGTGT-3’ 5’-GTAGATGAAGGCTGGCACACT-3’

5’-GGCACATATTCTGGCTGCCAGG-3’ 5’-ACACTAGTGGAGCAGCACAGGG-3’

5’-ATGAGCCACTTGTCAGCTGGG-3’ 5’-GGGCTTCACACAAAGCAAGCAGACTTT-3’ 5’-AGTGGGGTTAATGCACGGTGACCCTGG-3’

are relative to first nucleotide of the translation initiation codon labeled +l.

FIG. 1. Restriction endonuclease map of rat type IV 3D-HSD cDNA, selective PCR amplification and sequencing strategies. In panel A, the coding region is represented by the black box while the flanking 5‘- and 3’-noncoding regions are shown as open boxes. Panels B and C show the various type IV 3P-HSD cDNA fragments obtained by direct PCR amplification using the indicated sets of primers from a rat ovary Xgtll cDNA library and a rat placenta pSPORT I plasmid cDNA library. Panels D and E show partial type IV cDNA fragments obtained from rat placenta andskin poly(A+)RNA by reverse transcriptase-PCR using M-MLV reverse transcriptase. The location and the directionality of the oligonucleotide primer binding sites used for PCR are shown by numbered arrowheads. The primer sequences are listed in Table I. Panel F shows the direction and extent of sequencing using synthetic oligonucleotide primers (solid circles). A scale in base pairs is shown below where 0 corresponds to the ATG initiation codon.

1)

1498bD

4 PCR Rat Ovary t g t l l cDNA

1 b A d 3 798 bo

1)

*

5)

6)

1293 bD

7,

1

5



42 1104 bD

3

42 Rat

976 bD

7) 6)

42

1374 bD

298DD

PCR

Placenta pSPORT I cDNA

48

1104bO

-2 RT PCR Rat Placenta

49

10,

433 bD 21- 1 11)

6)

298 DD

36800

4 RTPCRRat Skin

49

3-1

. . . . ”. . . ... 1 2 3 -

c

f

Sequencing Strategy

t__.





” ,

0

200

400

enzyme and the TB RNA polymerase enzyme was used for probe synthesis as previously described (4,12,13). Thetype IV cRNA probe contains 82 nucleotides from the vector, the 7 nucleotides of the EcoRI site (underlined nucleotides) plus 38 nucleotides in the intron 2 downstream of the 157 nucleotides of rat type IV 3P-HSD cDNA (from nucleotide corresponding to +146 to +302 in the cDNA). The total length of the type IV cRNA probe was 284 nucleotides. Types I, 11, or I11 cRNA probe preparations have been described elsewhere (4, 12, 13). Hybridization of 10 pg of poly(A+)RNA from various rat tissues with the specific cRNA probe for each of the four types of 3PHSD was processed using the RPA I1 kit following supplier’s protocol. After resolution of the protected fragments on a polyacrylamide-urea sequencing gel, signals were detected using a PhosphorImager model 440E (Molecular Dynamics, Sunnyvale, CA), and resultsare expressed in arbitrary units after removal of the background value, where a value of 100,000 has been arbitrarily set for the signal corresponding to protected rat type I 3P-HSD mRNA transcript in the adrenals. Primer Extension Analysis-To map the 5”terminal nucleotide of rat 3P-HSD type IV cDNA, 3 X 10’ cpm 32P-5’-end-labeledsynthetic oligonucleotide 14 (Table I), which corresponds to nucleotides -30 to -56 specific for the type IV transcript, was hybridized to 10 pg of rat day-13 placenta and ovary poly(A+) RNA overnight at 42 “C. The DNA-RNA hybrid was then extracted with a phenol-chloroform solution, ethanol-precipitated, and then extended using 200 units of

.

I

600

.

b

BOO

.

I

1000

.

I

1200

.

I

,

1400

Superscript M-MLV reverse transcriptase at 42 “C for 90 min. The reaction products were again extracted with a phenol-chloroform solution and ethanol-precipitated before analysis on a denaturing polyacrylamide-urea sequencing gel alongside a control sequencing reaction. RESULTS

In order to identify a possible new type of rat 3P-HSD isoenzyme and to overcome the problems observed using a cDNA library and secondary to a relative abundance of multiple 3P-HSD mRNA species, we have first screened a rat genomic DNA library with 32P-labeledrat types I and I1 3pHSD cDNA probes. Among the clones isolated, only one included in a genomic DNA fragment of about 12 kilobase pairs, encodes a novel predicted rat 3P-HSD isoenzyme, chronologically designated type IV. The structure of the corresponding full- length cDNA encoding the type IV 3p-HSD protein was thereafter established by selective PCR amplification from rat ovary and day-15 placenta cDNA libraries using two oligonucleotides selected from the partial sequence of the genomic clone. As illustrated in Fig. 1, a PCR product of 1498 base pairs (including the two primers 1 and 2) was

Structure and Expression of a Novel Rat 3p-HSD Isoenzyme

19662

-40 -1 CCAGGGTCACCGTGCATTAACCCCACTCCCACTGTGATCTGCTTCCTGTGTTGACC

60

30

90

ATG CCT GGG TGG AGCTGC CTG GTG ACT GGA GCA GGA TTT GGG TTG GGC CAG AGGATC GTC CAG TTGTTG GTG CAG GAG AAA GAT CTG AAG Met Pro Gly TrpSer Cys Leu Val Thr Gly Ala Gly Gly Phe Leu GlyGln Arg Ile Val Gln Leu Leu Val Gln Glu Lys Asp Leu LyS 10

20

30

120

150

180

GAG GTC AGG GTC CTG GAC AAG GTC TTC AGA CCA GAA ACC AGA GAA GAA TTC TTC AAC CTA GGG ACA AGC ATC AAG GTG ACATTG GTG GAA Glu Val Arg Val Leu Asp Lys Val Phe Arg Pro Glu Thr Arg Glu Glu Phe Phe Asn Leu GlyThr Ser Ile Lys Val Thr Val Leu GlU 40

50

60

210

240

270

GGA GAC ATT CTT GAC ACC CAG TGC CTG AGG AGA GCG TGC CAG GGC ATC TCT GTT GTC ATC CAC ACT GCC GCC CTC ATTGAT GTC ACA GGT Gln Gly Ile Ser Val Val Ile H i s Thr Ala Ala Leu Ile Asp Thr Val Gly Gly Asp Ile Leu Asp Thr Gln Cys Leu Arg Arg Ala Cys 70

80

90

330

300

GTC AAT CCC AGG CAG ACC ATC CTA GAT GTC AAT CTG AAA GGT ACC CAG AAT CTA TTG GAG GCC TGT GTC CAA GCT AGT GTG CCA GCC TTC Val Asn Pro ArgGln Thr Ile Leu Asp Val Asn Leu Lys Thr GlyGln Asn Leu LeuGlu Ala Cys Val Gln Ala Ser Val Pro AlaPhe 100

110

120

390

420

450

ATC TAC TGC AGC ACA GTT GAC GTT GCA GGG CCC AAC TCC TAC AAG AAG ATC ATC CTG AAC GGC CAT GAG GAA GAG CAT CAT GAA AGC ACA Ile Tyr Cys SerThr Val Asp Val Ala Gly Pro Asn Ser Tyr Lys Lys Ile Ile LeuAsn Gly H i s Glu Glu GluH i s H i s Glu Ser Thr 130

140

150

480

510

540

TGG TCA AAT CCA TAC CCA TAC AGC AAA AAG ATG GCC GAG AAG GCA GTG CTG GCC AAT GCA GGGAGC ATC CTGAAA AAT GGT GGC ACA TTG Lys Ala Val Leu Ala Ala Asn Ser Gly Ile Leu LYS Asn Gly GlY Thr Leu Trp Ser Asn Pro Tyr Pro Tyr Ser Lys Lys Met Ala Glu 160

170

180

570

600

630

CAT

ACTTGT GCC TTA AGG CCC ATG TAC ATT TAT GGG GAG AGAAGT CCA TTC CTT TCT GTC ATG ATA CTT GCG GCC CTC AAA AAT AAGGGT H i s Thr cys Ala Leu ArgPro Met Tyr IleTyr Gly G1U ArgSer Pro Phe Leu Ser Val Met Ile Leu Ala Ala Leu Lys LysAsn Gly 190

200

210

660

690

720

TAT GTA GGC AAT GTGGCC TGG GCA CAT ATT CTGGCT GCC AGG GGC CTT ATT CTG AATGTT ACT GGC AAA TTC TCC ATA GCC AAT CCA GTG i s Ile Leu Ala Ala Arg GlY Leu Ile Leu Asn Val Thr Gly Lys Phe Ser Ile Ala Asn Pro ValTyr Val Gly Asn Val Ala Trp HAla 220

230

240

840

870

900

CGA GAC CCC AAG AAG TCA CAA Arg Asp Pro Lys Lys Ser Gln

TAC TGG CTT GCC TTC CTG CTG GAA ATT ACC CTG AGC AAG GAA TGG GGC CTC CACCTT GAT TCC AGT TGG AGC CTT CCT CTG CCC CTG CTC P r o Leu Pro Leu Leu Tyr Trp Leu Ala Phe Leu LeuGlu Ile Thr Leu Ser Lys Glu Trp Gly Leu H i s Leu Asp Ser Ser Trp Ser Leu 300 280 290 930

960

GTG AGC TTC TTT CTG CAT CCA GTG TAC AAC TAT AGG Val Ser Phe Phe Leu H i s Pro Val Tyr AsnTyr Arg 310

1020

TAC AAG AAA GCT CAG AGA GAT CTG GGCTAT GAG Tyr Lys Lys Ala Gln Arg Asp Leu Gly Tyr Glu 340

990

CCATCC TTT AAC CGC CAC TTG GTC ACA CTG TCA AAT AGC TTC AAG ACT TTC TCC Pro Ser Phe Asn ArgH i s Leu Val Thr Leu Ser Asn Ser Lys Phe Thr Phe Ser 320

330

1050

1080

CCA CTT GTC AGC TGG GAG GAA GCC AAG CAGAAA ACC TCG GAG TGG ATC GGG ACA CTA Pro Leu Val Ser Trp Glu Glu LysAla Gln Lys Thr Ser GlU Trp Ile Gly Thr Leu 360 350

1185 1110 1145 GTG GAG CAG CAC AGG GAG ACAGACTTG ACA AAG TCT CAG TAA TGGGAAGAGGGCAGGGACATGGTCTGGGTGTTTACTGGATCCTCCAGCAAGGACAGACATATA Val Glu Gln H i s Arg Glu Thr Leu Asp Thr LYS Ser Gln End 370

1343

1383

1423

AGATACACAGCTTCCTAAGCACCTTTTGTACCTCAGAGCTCTCTCCATTCTTTTCTCTCCCCATCCAAAGCATGTCTAACCTTCAG~GTCTGCTTGCTTTGTGAAGCCCAATAGAA

GGAACAATAAATATTTGTC

1442

FIG. 2. Nucleotide and predicted amino acid sequences of rat type IV 3B-HSD cDNA. Nucleotides are numbered on the top of the sequence while amino acids are numbered below the sequences. Nucleotides at positions 5’ of the ATG initiation codon are given negutiue numbers. The nucleotides corresponding to theputative polyadenylation signal AATAAA are underlined with a solid line.

first obtained after amplification using DNA from a rat ovary Xgtll cDNA library. This fragment was subcloned into Bluescript SK vector and the plasmid insert was sequenced in both orientations as illustratedin Fig. 1F.Thereafter, several overlapping partial PCR products corresponding to the rat type IV 3B-HSD were characterized from a rat ovary Xgtll cDNA library (Fig. 1B) aswell as from a rat placenta pSPORT I cDNA library (Fig. IC) using the same procedure. Furthermore, we have also determined by direct sequencing the nucleotide sequence of several PCR products generated from rat day-13 placenta (Fig. 1D) or skin (Fig. 1E)poly(A+) RNA

after a M-MLV reverse transcriptase reaction. The sequence of all amplified PCR fragments was identical to that of the overlapping rat type IV 3P-HSD sequence. The nucleotide sequence of rat type IV 3P-HSD cDNA as well as the corresponding deduced amino acid sequence are illustrated in Fig. 2. The first in-frame ATG codon is designated as position 1. The cDNA sequence of type IV 3P-HSD includes an open reading frame of 1119 nucleotides. The nucleotide sequence of the coding region of rat type IV shares 94.3,93.1, and 84.7% identity with that of rat types I, 11, and I11 38-HSD cDNAs (4, 12), respectively, while the same

Structure and Expression ofRat a Novel

P O

190 170 150 -

ACGT

220

I I )

130 120

-

-. -.

-

-

100 90 80 -

...

110

70

-

60

-

I. i

r-?

br

FIG. 3. Primer extensionanalysis. 10 pg of poly(A+)RNA from were hybridized to the3zP-5’rat day-13 placenta (P)and ovary (0) end-labeled oligonucleotide corresponding to nucleotides -30 to -56 of the noncoding strand of rat 3P-HSD type IV and extended with M-MLV reverse transcriptase. Lanes ACGT, control sequence.

sequence shows 89.7,90.2, and 89.6%homology with the overlapping mouse types I, 11, and I11 3P-HSD cDNA sequences (15). Furthermore, the nucleotide sequence of the coding region of rat type IV 3P-HSD cDNA shares 78.1,78.0, 79.3, and 77.0% identity with that of human types I (23) and I1 (5), macaque (3), and bovine (46) 3P-HSD cDNA clones, respectively. The longest type IV 3P-HSD cDNA fragment characterized includes in its 3’-untranslated region, a sequence of 309 nucleotides before the expected polyadenylation consensus signal AATAAA (47). This 3“untranslatedregion shares only 58-62% homology with the overlapping regions of rat types I, 11, and I11 (excluding the additional downstream non-homologous 420-nucleotide segment in the 3”untranslated region of type I11 cDNA). In order to locate the 5’-end of the rat type IV 3P-HSD mRNA species, a 32P-5’-end-labeled27-mer oligonucleotide specific to nucleotides -30 to -56 of the noncoding strand of rat type IV 3P-HSD cDNA was hybridized to 10 pg of rat day-13 placenta or ovary poly(A+) RNA and

3p-HSD Isoenzyme

19663

extended with M-MLV reverse transcriptase (Fig. 3). The predominant 214 base pairs long extension product shown in Fig. 3 suggests that the 5’-non-coding sequence contains about 243 nucleotides, thus suggesting that approximately 187 nucleotides are missing inthe5”untranslated region upstream to thesequence of primer 1. It can be seen in Fig. 2 that the sequence G A C C m C containing the first in-frame initiating codon of rat type IV 3P-HSD cDNA is identical with that in rat types I, 11, and I11 38-HSD cDNAs (4, 12). This sequence corresponds to the consensus sequence C A / & C m G needed for optimal initiation by eukaryotic ribosomes, with the knowledge that as long as a purine is located at position -3, deviations from the rest of the consensus sequence surrounding the AUG codon only slightly impair initiation of translation (48). The rattype IV 3P-HSD cDNA thus predicts a 41,854-dalton protein with 372 amino acid residues (excluding the first Met). The deduced amino acid sequence of rat type IV 3P-HSD shares 90.9,87.9, and 78.8% identity with that of rat types I, 11, and I11 3P-HSD proteins, respectively, thus having 34,45, and 79 non-identical residues with types I, 11, and I11 3P-HSD proteins (Fig. 4). Furthermore, the percentage of identity of the rat type IV 3P-HSD amino acid sequence with that of human types I and 11, macaque, bovine and mouse types I, 11, and I11 38-HSD proteins is 73.2,73.4,75.0, 76.1 86.8,84.9, and 86.8%, respectively (Fig. 4). Comparative analysis of the deduced 3P-HSD proteins illustrated in Fig. 4 indicates that 186 residues (50.0%) are conserved in all eleven amino acid sequences. Conservative changes are found at 46 additional positions, thus leading to an overall similarity of62.4% across the eleven different sequences from five species, thus showing that this enzyme family is well conserved throughout the course of evolution. The 38-HSD enzyme is a membrane-bound protein located in the endoplasmic reticulum and in mitochondrial membranes (24). The hydropathy profiles of rat type IV and other rat type 3P-HSD proteins are quite superimposable (data not shown). It is of interest tonote that computer analysis of the rat type IV 3P-HSD protein performed according to Klein et al. (39) predicts two membrane-spanning domains extending from residues 75-91 and 284-305 (Fig. 5). These two transmembrane segments are also predicted using two other algorithms (40,41), aspreviously reported for human types I and 11, macaque, bovine, and rat types I and I11 3P-HSD proteins (1).We have recently provided strong evidence by site-directed mutagenesis that the absence of the putative membrane-spanning segment between residues 75-91 in rat type I1 3P-HSD explains the much lower activity of rat type I1 compared to that of rat type I (33). It can also be seen that analysis of the rattype IV 3P-HSD protein performed according to Eisenberg et al. (40) predicts a membrane spanning segment extending from 3 to 23 in contrast toother rat types. This segment is, however, as deduced for human type I, macaque, and bovine 3P-HSD proteins (1). The predicted rat type IV 3P-HSD protein contains three potential sites of N-glycosylation (49) at the Asn residues located at positions 169, 212, and 268 (excluding the first Met). These sites are also present in rat types I and I1 38HSD proteins, while only those located at residues 169 and 268 are predicted in the rat type I11 protein. It is of interest to mention that only about one-third of the potential glycosylation sites in eukaryotic proteins are in fact glycosylated (49). To demonstrate that rattype IV 3P-HSD cDNA encodes a protein able to catalyze both A5-3B-hydroxysteroiddehydrogenation and A5-A4-isomerizationandto assess potential

Structure and Expression of a Novel Rat 3p-HSD Isoenzyme 20 30 GGFLGQRIVQ LLVQEKDLKE . . . . . . . . I R . . . K..E... . . . . . . . . . . . L. . . . . . R . . . E . . E . . . T......... R . . .E..E... A . . . . . . . .G IC E....Q. A . . . . . . . . . . . . V . . . . I K Y . . . . . E .Q . 10

R A TI V HUMAN I HUMAN II MACAQUE BOVINE MOUSE I M O U S E II M O U S E FII RAT I RAT II RAT Ill

R A TI V HUMAN I HUMAN II MACAQUE BOVINE MOUSE I MOUSE II MOUSE Ill RAT I R A T II R A T Ill

R A TI V HUMAN I HUMAN II MACAQUE BOVINE MOUSE I M O U S E II M O U S E Ill RAT I RAT II RAT Ill

R A T IV HUMAN I HUMAN II MACAQUE BOVINE MOUSE t MOUSE II M O U S E Ill RAT I R A T I1 RAT Ill

RATIV HUMAN I HUMAN II MACAQUE BOVINE MOUSE I M O U S E II M O U S E Ill RAT I R A T II R A T Ill

R A TI V HUMAN I HUMAN II MACAQUE BOVINE MOUSE I M O U S E II M O U S E Ill R A T. .I . R A T II R A T Ill

RATIv HUMAN I H U M A N 11 MACAQUE BOVINE MOUSE 1 MOUSE 11 MOUSE (11 RAT I R A T II R A T 111

PGWSCLVTGA T.........

......... ........ ...

"""""

"""""

"""""

40 VRVLDKVFRP I . . . . .A . G . 1 . A . . .A , . . I . . . . . A... I......... ..A

50 ETREEPBNLG .L....SK.Q .L....SK.Q .L....SK.Q .V....SK.Q

. . . . . . . . .K . . . S K . Q

"""""

"""""

60 TSIKVTVLEG NKT.L . . . . . NRT.L . . . . . NKT.L.. . . . S K . .L . L . . . .KT. . . . . . . """""

. . . . . . . . . . . . . . . . . .I Q . . . . . . . . P . I . . . . . . . K . . . . . Q . . . . . . . . . . . . . . . . . . . . . . . . . . . .V . . . . I RH . . . . . E. Q . . .A . . . . . . . . . K . . . S K . Q . K A . . . Y . . . . . . . . . . . . . . . . V . . . . I R H . . . . . E . Q . . . A. . . . . . . . . K . . . S K . Q . K A . . . M . . . . . . . . . . . . . . . . . . . . . . . M . . . . . E . Q. . . . .Y R T . S . K E K . . L S K . Q . X A . . . . . R . 80 70 90 DILDTQCLRR ACQGISVVIE TAALIDVTGV C I . ..I.. EPT.K. . . .DV..I.. L P T . K . . . . D V . . . . . . . C I . . .r . . C I . . . .~ . EPT.K. DV . . . . . . . . . E...KG . . . . T . . . . . . . S V . . . R N A v. . . . . . A..... ..........

.... .... .... ... .... . . . . . . Y... . . . . . . . . . . . . . .A . Y . . . . . . . . . . . . . . . . .A . Y . . . . . . . . . . . . .

"""""

. .V.A.P . . .

"""""

....M.. I . .

.. .. ...

100 NPRQTILDVN TE.ES.YN.. TH.ES.MN.. TE.ES.YN.. V..E..HN.. I.........

110 LKGTQNLLIA V....L.... V....L.... V....L.... V....L....

120 CVQASVPArI v..

V....L..D.

. .E... . . . .

....... . . . . . . . v.. . . . . . . .v . . . . . . . . .v . . ."""_. . . . . . . . . . . . . . . . . . . . . . .I . . . . . . . . . . .I. . . . . . I . . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . V . . . S E . L . . . . . . . . . . . . . . .I . . . . . E . . . . . . . ..SVW.TSR. L . . . . . . . . . . . . . . . . . . . G I B . . . . . . .

"""""

"""""

. . . A L . I A . ~L . . . . . . . . .

180 SILKNGGTLE WN. . . . . . .Y WN. . . . D..Y W T . . . . . . .Y W A . . . . . . .Y . M . . . . . . .N

140 150 130 YCSTVDVAGP NSYKKIILNG EEEEEELSTW . . . . PL.N.. . T . S I E . . . . . . . .E . . Q . . PL.N.. . T . S I E . . . . . . . .E . . Q . . . T . . L E . . . . . . . . E . . Q . . . . . .P L . N . . ET..IE . . . . . . .RE..QD. R . . . . . . .A . . . . . . . v... . . . Q N . . . . . T . . S . . . . . . E.V . . . . . . .c..... BS.S . . . . . . PS.S . . . . . . . . . . D.V . . . . .D..R....

170 160 SNPYPYSKKY ALKAVLAANG P A . . . E...L . . . . . . . . . . P T . . . . . . .L . . . . . . . . . . PA. . . . . . .L . . . . . . . . . . G... . s. . . . . . . L . D. . . . . . . . . . . . . . . . . . .D . . . . . . . . . . . . . . . . . . .D . . . . . . . . . . . . . . . . . . .DA.. . . . R . . . . . . . . . . .

190 200 210 TCALRPMYIY GERSPFLSVM ILAALKNKGI . . . . . . . . . . . G . R . . . A.SN E . . N . N . . . . . . . . T . . . . . GG . . . . A S . N E . . N . N . . . . . . . . . . . . . . GG. . A S .NE..N.N.. . . . . . . . . . . . .G . . . . .AY MEG..N.N.. . . . . . . . . . . . . . . . . I T N A .IR . . . . . . . . . . . . . . c.. . . . . . LI.NI . I M . . . B . . . . . . . . . . c.. . . . . Q . . .N T . I K . . . . . F .

220 230 240 LNVTGKFSIA NPVYVGNVAW AHILAARGLR .SSV....TV . . . . . . . . . . . . . . . L.A.Q . S S V . . . . T V . . . . . . . . . . . . . . . L.A.. .SSV....TV . . . . . . . . . . . . . . . L.A.. .TNHC...RV . . . . . . . . . . . . . . . L.A.. . . . . .E.... . . . . . . . . . . .c... . . . .RSF...NT. . . . . . . . . . . . . . . . . . . . . .RGG....T. . . . . . . . . . . . . . . . . . . . .

....

....

......

. . . . . . .Q

.H .M .

......Q .......... .......... .......... .......... . . . ....... . . . . . . . . . . . . . . . . . . . . . . . . .T . . . . R . . . . . . . . . . . . . . . . . . . . S . S T G . . . . . . . . ET . . .D R . . . . R . . . . . . . . . . . . R . . . . . . . . . . . . . . . . . . .F .

.

..

.......... .......... .......... .......... .......... .......... . . . . . . . . . . . . . G Q . . . R I . I n . . . . . .v . . . . . . . . . v . . . . . . . . . . . . . . . . . . . . . . . . . L P T . . ..I.QII.T. V N R . . . . N S .I K R E A T . . . . . . . . . . . A . . . . . . . . . . . .

250 260 290 300 270 280 DPKKSQNVQG QTYYISDDTP HQSYDDLNYT LSKEWGLHLD SSWSLPLPLL YWLATLLEIV . . . .A P S I R . . . . . . . . . . . . . . . . N. . . . . . . . Y . . R . . . R . . F . . S . M . . I C . . . . . . . . . .A P S . R . . . . . . N...I . . . . P..R.. .R T.M IG....V . . . . .A P S . . . . . . . . . . . . . . . . . . N...I . . . . P . . C . . .R . . . . .A . M . . I C . . . .V . . . . .V P . 1 . . . . . . . . . . . . . . . . . .rc.. .RY...IS.Q . . . . .T S I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R P N A . . . . . . . . . . . . . . . . . T. . . . . . P.I.. E . . . . . . . .T. T..Is.. T C P . . . . . . .v . N....P.I.. E . . . . . . . . . . . . . . . . . . . .P C . N . R . Y . . V . I . . . . . . . . . T . .......... c. .R.. . . . . . . . . . . . . . . . . . . T. . . . . . . . I . . . . . . . . . . . . . . . . . . . . c. . . . . . . . R . . . . . . . . . . . . . . . . . . . . T . . . E...SI.. . . . . . .TC.. T.

.....

.......... ..........

..........

......... ... ...... .......... ........ ...... .......... ..........

310 SPBLEPVYNY ..L.R.I.T. ..L.S.I.S. . . L . S . . . s. ..L.S.I.K. L . R . . . R. ..L.S.I.R. ..L.S.I.R. . .L.R.F . .L.R.F . . . . .L.R.F . . .

..

370 EQHRETLDTK DR.K.. .IS. DR.K. . X S . DR.K.. .KS. K. .K.. .K.. . . . . . I . .. .

.

..

..

.......... ........

360 350 320 340 330 RPSTNRELVT LSNSKTTTSY KKAQRDLGYL PLVSWLEAKQ XTSEWIGTLV . . P.. ..I.. . . . . v..... A.K . . Y. . . . . . . ..v..v.s.. Q . P . . . . T . . . . . .V . . . . . . . . . . . . A . K . . Y . . . . . . . . . v . . v . s . . ..v..v.s.. Q . P . . . . T . . . . . .V . . . . . . . . . . . . A . K . . Y N . C . . . . . . . . . . . V . . . . . . . . . . . . . . . . . Y T . . . . . . . . K....S.. . . L. . . . . I. . . . . T..... I. . . . N...... 1.P . . . . . . . . . G.T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.P....... .TA.T..... .......... .......... . . P . . C . . . . . . . . . . . . . . . . . . . . . . .v . . . . . . . . . . . . . . . . . . . . . .P..C.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .P...FM.. IL..V..I... . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.......

.......

.......... ..........

........

SQ

T. T. T. IE

c.

............ ............ ............ ............ ............

FIG. 4. Comparison of the predicted amino acid sequence of rat type I V 3&HSD protein with that of human type I (23,25), human type I1 (5),macaque (3), bovine (46), m o u e types I, 11,and I11 (15), as well asrat types I (4, 31), I1 (4, 14),and I11 (12) 3&HSD proteins. Amino acid sequences are designated by the single-letter code. Residues common to rat type IV 3p-HSD are designated by a dot (. ). The noncharacterized NH2-terminal sequence in mouse type I1 3p-HSD is indicated by a dotted line ( - - - - -).

Structure and Expression of a Novel Rat

3P-HSD Isoenzyme

19665

........4

-

RAT TYPE II

mLZz9

RAT TYPE 111

0

100

50

150

200

250

300

350

AMINO ACID NUMBER

FIG.5. Comparison of the predicted membrane-spanning domains of deduced rat types I, 11,111, and I V 38-HSD proteins according to Klein et al. (39) (open boxes), Eisenberg et al. (40) (hatched boxes), and Rao and Argos (41)(closed boxes). A

,

I

p

1 / [PREG] (pM)

30

properties of the expressed rat types I and I V 3D-HSD proteins. Theenzymatic reaction was performedduring 60 min at 37 "C using 50 pg of homogenate protein prepared in the presence of 20% glycerol from SW-13 cells transfected with pCMV-type I 30-HSD (open circles) or pCMV-typeIV3p-HSD (close circles) plasmids. Panels A and B, Michaelis-Menten plot of velocity ( V , in picomoleslminlpg of normalized total protein) against [PREGI (panel A ) or [DHEA] (panel B ) in micromolar. Panels C and DI data are displayed as LineweaverBurk plot. The amounts of translated typesI and IV 3fl-HSD proteins were equivalent in each corresponding cell homogenate preparation as measured by immunoblot analysis. The results are presented as the mean f S.E. of the mean (n = 3). When the standard errorof the mean overlaps with the symbol used, only the symbol is illustrated.

20

B

8

1 I [DHEA] (pM)

FIG.7. Comparison of thekinetic

10

5 * o

I

T h e lminl

I

FIG.6. Comparison of the relative activities of rat types I and I V 3&HSD recombinant proteins. Time courseof enzymatic conversion of [3H]PREG into [3H]progesterone (PROG) ( A )and [3H] DHEA into[3H]-A4-dione( B ) in homogenates from SW-13 cells transfected with 40 pg of pCMV, pCMV type I, or type IV 3P-HSD plasmid. Incubations with 40 nM of [3H]PREGor 11 nM of [3H] DHEA were carried out for 10, 30, and 60 min in the presence of 1 mM NAD+ using 20 pg of proteinhomogenates.Theresults are presented as the mean f S.E. of the mean (n = 3). functional difference@) between types I and IV 3P-HSD, plasmids which contained either the rat type I or IV 3P-HSD cDNA insert driven by the CMV promoter derived from pCMV were transiently expressed in human SW-13 adrenal cortex adenocarcinoma cells. TransfectioninSW-13 cells with pCMV-type IV 3P-HSD resulted in the biosynthesis of a single 42-kDa protein that cross-reactswith polyclonal antibodies raised against purified human placental 3P-HSD protein used as a positive control, while no signal was detected in mock-transfected SW-13 cells that aredevoid of significant endogenous 3P-HSD activity (data notshown). In uitro incubation with homogenates from SW-13 cells transfected with pCMV type I 3P-HSD or pCMV type IV 38HSD plasmids in the presence of 1 mM NAD' and 3H-labeled substrates shows that the rat type IV 3P-HSD isoenzyme possesses a genuine 3P-HSD/A5-A4-isomeraseactivity similar to thatof the rat type I 38-HSD protein (Fig. 6). In addition to being involved in the irreversible dehydrogenation and

isomerization of A5-3P-hydroxysteroidprecursors, the type IV SB-HSD isoenzyme is also able to catalyze the interconversion of 3P-hydroxy- and 3-keto-5a-androstane steroids (data not shown), as previously demonstrated for rat types I and I1 (4, 34) and human types I and I1 (5,25) 3P-HSD proteins. As illustrated in Fig. 7, the rat type IV 3D-HSD isoenzyme shows a similar affinity for both [3H]pregnenolone (PREG) and [3H]dehydroepiandrosterone(DHEA),showing& values of 0.65 p M and 0.71 pM, respectively. Similar results were obtained using homogenates from SW-13 cells transfected with pCMV-type I 3@-HSD,with K,,, values of 0.43 PM and 0.16 p~ using as labeled substrates PREG and DHEA, respectively. It can also be seen in Fig. 7 that rattypes I and IV possess an equivalent relative specificity for PREG with respective relative specific activity ( Vmax)/Km ratio values of 0.14 and 0.15 l/min. mg protein/ml (when normalized for the estimated amount of translated 42-kDa types I and IV 3PHSD proteinsmeasured by immunoblot analysis). The similar relative specificity of the rat type I 3P-HSD isoenzyme compared to type IV 3P-HSD is also confirmed using [3H]DHEA as substratewith respective Vm,,/Km ratio values of 0.61 and 0.30 llmin-mg proteinlml (Fig. 7, B and D). To determine the tissue-specific expression of the rattype IV 3@-HSDgene compared to that of rat types I, 11, and 111 3P-HSD genes, we have studied the distribution of these four 3@-HSDmRNA species in rat tissues using the ribonuclease protection assay which permits to discriminate accurately a few base mismatches occurring after annealing of specific rat 3P-HSD cRNA probes with the different 3P-HSD mRNA species. As illustrated in Fig. 8, the type 1V 3B-HSD mRNA population was detectable in 10 pg of poly(A+)RNA extracted from rat placenta, ovary, andadrenalas revealed by the

Structure and Expression of a Novel Rat 3P-HSD Isoenzyme

19666

Placenta AdrenalTestis Ovary Probe 1&11 111

I

IV

II

111

- 1

I

IV

II

111

I

IV

II

111

I

IV

I

I

II

111

I)

I

1-

!I ;'

IV

1

I

4

157

!i

t

:

I !

!

j !

io i

1

I!! .

.

FIG.8. Ribonuclease protectionanalysis of the distributionof rat typesI, II, IIand I, IV 38-HSD mRNA species in classical steroidogenic tissuesof the rat. Samples of 10 pg of poly(A+) RNA from rat ovary, testis, adrenal, and day-15 placenta were hybridized to the types I, 11, 111, or IV cRNA probes, digested with ribonuclease A and TI, and protected fragments were resolved on a denaturing polyacrylamide-urea sequencing gel. The longest protected fragment (223 nucleotides for types I and 11; 250 nucleotides for type 111; 157 nucleotides for type IV) corresponds to thehomologous RNA species protected by the respective cRNA probe. Blots were exposed for 3 days before quantitation with PhosphorImager model 440E. TABLE I1 Tissue-specific expressionof types I, II, III, and IV 38-HSD mRNA species in ratclassical steroidogenictissues and peripheral tissues Relative 3b-HSD mRNA levels were directly quantitated with a PhosphorImager model 440E following the RNase protection analysis illustrated inFigs. 8 and 9 where a value of 100,000 has been arbitrary set for the signal corresponding to rat type I mRNA level in the adrenal. Tissue

I Adrenal 100,000 Ova7 72,904 Testis 7,720 Male liver ND Placenta 3,028 Kidney 246 Skin ND Fat 12 15 Uterus 'ND, not detectable.

Types

I1

58,593 40,121 10,393 ND 2,252 118 ND 28 10

I11 ND" ND ND 41,590 ND ND ND ND ND

N 94 2,255 ND ND 3,541 ND 117 ND ND

presence of the expected full-length protected fragment of 157 nucleotides using the specific type IV cRNA probe. Under the conditions used, no signal was detectable in rat testis. It can also be seen that, the type I 38-HSD mRNA population was

the predominant species in the rat ovary and adrenal gland as previously observed (4, 12, 32) (Fig. 8, Table 11). On the other hand, the type IV transcript corresponds to the predominant 3P-HSD species in poly(A+)RNA extracted from day15 placenta, although types I and I1 3P-HSD mRNAs were also relatively abundant at this stage of gestation (Fig. 8, Table 11). We have next examined the distribution of the rattype IV 38-HSD mRNA compared to thethree other types in several peripheral tissues. It can be seen in Fig. 9 that the rat type IV mRNA population corresponds to the sole detectable 3PHSD species inthe skin. In contrastto types I and I1 3P-HSD mRNAs, it was not possible to detect type IV 3B-HSD mRNA in poly(A+)RNA extracted from kidney,fat tissue and uterus. As expected (12,13), rat type I11 mRNA wasthe sole 3B-HSD species detectable in the male liver, while it was not detectable in all other tissues tested (Figs. 8 and 9, Table 11). DISCUSSION

The rat type IV mRNA thus represents the sole 3P-HSD mRNA species detectable in the skin and the major 3j3-HSD mRNA species present in the placenta. By analogy with our recent demonstration that the human type I 38-HSD is the

3p-HSD Isoenzyme

Structure and Expression of a Novel Rat Skin Kidney Liver

19667

Male Probe

-

ldrll 111 IV -

347 e

306

1

I

I1

111

IV

I

I1

1

111

I

IV I

I1

111

IV

I

I1

111

I

IV I

I

I1

111

IV 1

w

.-. ”

4

250

4

223

4

157

!

.

f

FIG. 9. Ribonuclease protection analysis of the distribution of rat types I, 11, III, and IV 3B-HSD mRNA species in rat peripheral tissues. Samples of 10 pg of poly(A+) RNA from male liver, female kidney, female skin, and female abdominal fat tissue and female uterus were hybridized to the types I, 11,111, or IV cRNA probes and digested with ribonucleases A and T1,while the protected fragments were resolved on a denaturing polyacrylamide-urea sequencing gel. The longest protected fragment (223 nucleotides for types I and 11; 250 nucleotides for type 111; 157 nucleotides for type IV) corresponds to the homologous RNA species protected by the respective cRNA probe. Blots were exposed for 3 days before quantitation with PhosphorImager model 440E.

sole mRNA species detectable in theskin and in the placenta kidney, provides a molecular explanation for our preliminary (5,8), it is tempting to speculate that rattype IV and human results demonstrating the opposite regulation of 3P-HSD actype I 3P-HSD genes have evolved from a common ancestor tivity in these two tissues by pituitary and steroid hormones gene. In this regard, it could be suggested that both human (51). type I and rat type IV 38-HSD genes have conserved some The expression of this specific 3O-HSD isoenzyme in the specific cis-acting elements in their promoter region involved skin is in agreement with the demonstration of3/3-HSD/ in a tissue-specific transcriptional controlcommon to the skin isomerase activity in sebaceous glands in human skin during and the placenta. Moreover, we have recently observed that embryonic development and in developed skin (10, 11, 52). rat types I, 11, and IV 3P-HSD mRNA levels show marked Microdissected sebaceous glands have also been shown to and specific changes during gestation (50). The type IV 38- possess 3P-HSD activity (52). Recently, immunohistochemiHSD mRNA population was always the predominant 3P-HSD cal localization of 3P-HSD using antibodies raised against mRNA species detectable in the placenta, especially during human type I 3@-HSD,shows that 30-HSD immunoreactivity the 2nd week of gestation, when it was an order of magnitude is located in sebaceous glands, the highest level of expression more abundant than types I and I1 3j3-HSD mRNA species being observed in the most differentiated sebocytes (8, 9). (50). These results well support the predominant involvement The physiologic importance of 3P-HSD in the skin is supof type IV 3P-HSD in placental steroidogenesis. ported by the observation that DHEA can stimulatesebaceous The presence of multiple 3P-HSD isoenzymes offers the gland secretion in humans (53, 54) and by the finding of a unique possibility of tissue- and/or cell-specific expression close correlation between 3P-HSD activity and in vivo sebum and regulation of this enzymatic activity that plays an essen- secretion rate in humans (10). The observation that androsttial role in the biosynthesis of all hormonal steroids in clas- 5-ene-3/3,17/3-diol (A5-diol) is a potent stimulator of sebum sical as well as in peripheral intracrine steroidogenic tissues. secretion in the rat(55) is also in agreement with the role of In thisregard, the present findingshowing that rattype IV is 3P-HSD in lipogenesis in thesebaceous gland. the sole detectable 3B-HSD gene expressed in theskin, while The recent observation of the peripheral conversion of only the types I and I1 3P-HSD genes are expressed in the PREG into DHEA and AS-diol, primarily resulting from a

19668

Structure and Expression of a Novel Rat 3p-HSD Isoenzyme

17a-hydroxylase/l7,20 lyase activity in nonclassical steroidogenic tissues including the rat stomach and intestine (56), combined with the ubiquitous distribution of 17B-hydroxysteroid dehydrogenase activity in rat tissues (57), further supports the physiological relevance of the involvement of the type IV 3P-HSD isoenzyme in the intracrinesynthesis of androgens in the rat skin from these C-19 A5-hydroxysteroid precursors. Such data indicate that the precursor used for synthesis of active steroids in extragonadal tissues can also be PREG in addition to DHEA. The availability of rat type IV 3P-HSD cDNA thus offers the opportunity of investigating the specific regulation of its expression in the rat skin, which appears as a good experimental model to better understand the role of 3P-HSD in androgen biosynthesis locally formed in the skin. The existence of multiple 3P-HSDs was also described in the mouse (15, 58). It has been demonstrated by RNase protection assay that mouse type I 3P-HSD is specifically expressed in the adrenal gland, ovary and testis, while types I1 and I11 3P-HSD are bothexpressed in kidney and liver (15). It could be thus speculated that on the basis of tissue-specific expression, that mouse type I ( E ) , rat types I and I1 (4, 12, 32), bovine ovary (46), macaque ovary (3), and humantype I1 (5) 3P-HSD have evolvedfrom a common duplicated ancestor gene. In agreement with this hypothesis, it interesting to note that deduced mouse type I 3P-HSD protein shares 87.9 and 86.3% sequence identity with that of rat types I and I1 3PHSD proteins, respectively, while having only 83.3% identity with that of deduced mouse type I11 3P-HSD protein. More recently, the more distantly related mouse kidney-specific type IV 3P-HSD cDNA was characterized (58). This cDNA appears to encode an enzyme showing similar peculiar activity as previously described for the male liver-specific type I11 member of the rat 3P-HSD family (35, 58). This observation thus suggests that this mouse type IV has evolved from an ancestor gene common to that of rat type I11 gene, which diverged from other rodenttypes after agene duplication that took place earlier in the evolution of mammals as revealed by phylogenetic analysis (59). On the basis of the available literature, it is impossible to delineate which type(s) among the mouse 3P-HSD gene(s) are evolutionary and/or functionally homologous to rat type IV 3P-HSD, the latterbeing most likely related to human type I 3P-HSD. Acknowledgments-We thank Dr. Dimcho Bachavarov, Carole Dumas, Carolle Samson, and Dominique Paradis for skillful technical assistance. REFERENCES 1. Labrie, F., Simard, J., Luu-The, V., BBlanger, A., and Pelletier, G. (1992) J. Steroid Bcochem. Mol. Biol. 4 3 , 805-826 2. Pelletier, G., Dupont, E., Simard, J., Luu-The, V., BBlanger, A., and Labrie, F. (1992) J. Steroid Biochem. Mol. Biol. 43,451-467 3. Simard J., Melner, M. H., Breton, N., Low, K. G., Zhao, H. F., Periman, L. M:, and Labrie, F. (1991) Mol. Cell. Endocrinol. 7 5 , 101-110 4. Zhao, H. F., Labrie, C., Simard, J., de Launoit, Y., Trudel, C., Martel, C., RhBaume, E., Dupont, E., Luu-The, V., Pelletier, G., and Labrie, F. (1991) J. Biol. Chem. 266,583-593 5. Rheaume, E., Lachance, Y., Zhao, H. F., Breton, N., de Launoit, Y., Trudel, C., Luu-The, V., Simard, J., and Labrie, F. (1991) Mol. Endocrinol. 5 , 1147-1157 V., andPelletier, G. (1992) J. Clin. 6. Dupont, E., Labrie,F.,Luu-The, Endocrinol. & Metab. 74,994-998 7. Riley, S. C., Dupont, E., Walton, J. C., Luu-The, V., Labrie, F., Pelletier, G., and Challis, J. R. G. (1992) J. Clin. Endocrinol. & Metab. 7 5 , 956961 8. Dumont, M., Luu-The, V., Dupont, E., Pelletier, G., and Lahrie, F. (1992) J. Inuest. Dermatol. 9 9 , 415-421 9. Sawaya, M. E., and Penney, N. S. (1991) J. Cutaneous Pathol. 19,309-314 10. Simpson, N. B., Cunliffe, W. J., and Hodgins, M. B. (1983) J. Inuest. Dermatol. 8 1 , 139-144 11. Sharp, F. (1978) Histochem. J. 10,517-528 12. Zhao, H. F., RhBaume, E., Trudel, C., Couet, J., Labrie, F., and Simard, J.

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