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Department of Pediatrics and the Metabolic Research Unit (W.A., J.W.M.M., M.S., J.T.W., W.L.M.), University of California, San Francisco, California 94143-0978; and Division of Endocrinology (R.J.A.), Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857
Address all correspondence and requests for reprints to: Prof. Walter L. Miller, Department of Pediatrics, Building MR-IV, Room 209, University of California, San Francisco, San Francisco, California 94143-0978. E-mail: wlmlab{at}itsa.ucsf.edu.
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Abstract |
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-hydroxylase and 17,20 lyase activities of a single enzyme, P450c17. To ascertain whether sequence differences in P450c17 contribute to adrenarche, we cloned the rhesus monkey cDNA from adrenal tissue and cloned the chimpanzee and baboon cDNAs from genomic DNA using an exon-trapping strategy. Using microsomes from yeast transformed with rhesus, baboon, chimp, or human P450c17, we measured the Michaelis constant and maximum velocity for the 17-hydroxylase and 17,20 lyase activities. The human and chimp enzymes differ at only two amino acids and baboon and rhesus P450c17 only at a single residue; the human/chimp enzyme differed from the baboon/rhesus enzyme by 25–27 residues (95% identity). Surprisingly, the greatest difference in enzymatic activities was a marked increase in 17-hydroxylase activity of P450c17 in the baboon, which differs from rhesus only at residue 255 [arginine (Arg) in baboon, histine (His) in rhesus]. Residue 255 is also Arg in human and chimp. Wild-type human P450c17 and its Arg255His mutant had similar 17-hydroxylase activities, but the Arg255Ala mutant had decreased 17-hydroxylase activity. These data establish that Arg255 is important for 17-hydroxylase activity and show that the evolution of adrenarche in higher primates is not determined by variations in the sequence of P450c17. |
Introduction |
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The biosynthesis of DHEA from cholesterol requires only two steroidogenic enzymes (11). The mitochondrial cholesterol side chain cleavage enzyme, P450scc, catalyzes 20-hydroxylation, 22-hydroxylation, and scission of the 20,22 carbon bond of cholesterol to yield pregnenolone. Microsomal P450c17 then catalyzes 17-hydroxylation of pregnenolone to 17-hydroxypregnenolone (17-Preg) and of progesterone to 17-OH-progesterone (17OHP), and scission of the 17,20 carbon bond of 17-Preg to yield DHEA. P450c17 also catalyzes the 16-hydroxylation of progesterone to 16-OH-progesterone (12, 13, 14), but the physiological significance of this reaction is unclear. Although P450c17 from rodents and cattle can convert 17OHP to androstenedione, human P450c17 cannot catalyze this reaction effectively (12, 14, 15). The 17,20 lyase activity of human P450c17 requires phosphorylation of serine and/or threonine residues of P450c17 (16), but the precise mechanism by which this phosphorylation promotes 17,20 lyase, but not 17-hydroxylase activity, is unknown. P450c17 requires P450 oxidoreductase (OR) as an obligatory electron donor. However, 17,20 lyase activity also requires cytochrome b5 (b5), which allosterically facilitates the interaction of P450c17 with OR (14). Without b5, there is virtually no 17,20 lyase activity (14). Expression of b5 is largely confined to the zona reticularis of the human (17, 18) and rhesus monkey (19) adrenal and very little b5 is found in the zonae fasciculata and glomerulosa.
To determine if sequence differences in P450c17 contribute to the evolution of adrenarche, we compared the sequences and enzymology of this enzyme from baboon and rhesus monkey, which lack adrenarche, to P450c17 from human beings and chimpanzees, which undergo adrenarche.
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Materials and Methods |
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). PCRs in a final volume of 50 µl contained 500 µM deoxy (d)-NTP, 2 U Taq polymerase, and 100 ng each of sense and antisense primers. The PCR conditions were 95 for 3 min, followed by 35 cycles of 95 C for 1 min, 65 C for 1 min, and 68 C for 2 min. The resulting 1.5-kb fragment was cloned into pGEM3 (Promega Corp., Madison, WI), amplified and sequenced using primers complementary to the human sequence (21). The cDNA was then reamplified from pGEM3-c17 by nested PCR using sense and antisense primers overlapping the start and stop codon and including a BamHI site at the 5' overhang and an XhoI site at the 3' overhang (Table 1). The resulting PCR product was subcloned into the expression vector pcDNA3 (Invitrogen, Carlsbad, CA).
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). The 5' sense primers included a BamHI restriction site and were complementary to the 5'UTR of the chimpanzee and baboon sequences as identified by PCR and sequencing of exon1. Direct sequencing revealed sequence identity at the C-termini of P450c17 from rhesus monkey, baboon, and chimpanzee; thus, we employed the same antisense primer including an XhoI site that was used for the rhesus monkey cDNA amplification (Table 1). The PCR conditions were 94 C for 10 sec, 15 cycles of 94 C for 10 sec, then 68 C for 3 min 45 sec (1 min + 2.5 sec per 100 bp), followed by 15 cycles of 94 C for 10 sec, then 68 C for 4 min. The resulting 6.5-kb PCR products, encompassing the full-length genes for baboon and chimpanzee P450c17, were cloned into pcDNA3 and expressed in COS-1 cells. Cells were harvested 48 h after transfection, and total RNA was subjected to RT-PCR using the conditions described above and employing the same primers as used for amplification of the full-length gene. The resulting 1.5-kb baboon and chimpanzee cDNAs were subcloned into pcDNA3 and sequenced directly; sequences were verified by comparison to the sequences identified by sequencing the individual exons.
Subcloning of rhesus monkey, baboon, and chimpanzee P450c17 cDNA into yeast expression vector
The pcDNA3 vectors containing the three ape P450c17 cDNAs were digested with Xho, blunt-ended, and digested with BamHI. This facilitated directional cloning into the yeast expression vector V10 (23), which had been previously digested with EcoRI, followed by blunt-ending and digestion with BglII. Cloning the P450c17 cDNAs into V10 destroyed the BglII and EcoRI sites and placed the P450c17 cDNA under the control of the constitutive pgk promoter, producing vectors V10-Cc17 (chimpanzee), V10-Bc17 (baboon), and V10-Rc17 (rhesus monkey).
Site-directed mutagenesis and subcloning
Human P450c17 cDNA (20) subcloned into pcDNA3 was subjected to PCR-based site-directed mutagenesis, using the forward primer 5'-CAAGGAGAAATTCCACAGTGACTCTATCACC-3' and the reverse primer 5'-GGTGATAGAGTCACTGTGAATTTCTCCTTG-3'. This changed two bases (in boldface) in codon 255 (underlined) resulting in the change Arg255His. Similarly, the forward primer 5'-CAAGGAGAAATTCGCGAGTGACTCTATCACC-3' and the reverse primer 5'-GGTGATAGAGTCACTCGCGAATTTCTCCTTG-3' were used to create the human P450c17 mutant Arg255Ala. PCR using 50 ng of plasmid DNA was performed in 50 µl of 500 µM dNTP, 1 U of pfu polymerase (Stratagene, La Jolla, CA), and 125 ng each of sense and antisense oligonucleotides. The reaction conditions were 95 C for 30 sec, followed by 18 cycles of 95 C for 30 sec, 55 C for 1 min, and 65 C for 14 min (2 min/kb DNA). PCR products were directly treated with 10 U of DpnI at 37 C for 60 min and used to transform Escherichia coli DH5 cells. The resulting cDNAs were sequenced in their entirety to confirm the mutations and to ensure that no other bases had been changed. The mutagenized human P450c17 cDNAs were then subcloned into V10 as described (11), yielding the constructs V10-hc17–255RH and V10-hc17–255RA.
Yeast transformation, yeast microsome preparation, and characterization
Saccharomyces cerevisiae strain W303B (23) was transformed by the lithium acetate procedure (24) with the yeast expression vector pYcDE2 (25) expressing human OR cDNA (14). To ensure similar levels of OR expression, one OR-expressing yeast clone was grown and subsequently transformed with V10 expressing human, chimpanzee, baboon, or rhesus monkey P450c17 cDNA or the human P450c17 mutants, Arg255His, and Arg255Ala. The transformed yeast were grown and microsomes were prepared. To control for a similar level of P450c17 expression, total yeast microsomal protein was estimated by the Bradford method (Bio-Rad Laboratories, Inc., Hercules, CA) and 10 µg aliquots from yeast transformed with P450c17 from each species were separated on a SDS-10% polyacrylamide gel and elctrotransferred to a polyvinylidene membrane (Millipore, Bedford, MA). Immunodetection used polyclonal rabbit antiserum against human P450c17 (12) and a secondary peroxidase-conjugated antibody in combination with the ECL chemiluminescent detection method (Amersham International, Arlington Heights, IL). To confirm that equivalent amounts of immunodetectable P450c17 contained equivalent amounts of heme, the microsomal P450 content was measured by difference spectroscopy. Microsomes were suspended at 1 mg/ml in 0.1 M potassium phosphate (pH 7.4), divided between two 0.1-ml cuvettes, and mixed with 2 µl ethanol (reference) or 1 mM progesterone (sample) to achieve 20 µM final concentration. P450 content was calculated from the 
A386–420 using 
= 110 mM-1 cm-1. Difference spectra demonstrated comparable P450 contents in all samples, varying between 20–30 pmol/mg total protein.
Yeast enzyme assays
The 17-hydroxylase and 16-hydroxylase activities of P450c17 were measured by preincubating yeast microsomes in 200 µl of 50 mM potassium phosphate buffer (pH 7.4) with 0.5–4 µM progesterone added in 4 µl ethanol and 20,000 cpm of [14C]-progesterone (55.4 mCi/mmol) (NEN Life Science Products, Boston, MA) for 2 min at 37 C and catalysis was initiated by adding 1 mM NADPH (Sigma, St. Louis, MO). The 17,20 lyase activity of P450c17 was measured in 200 µl of 50 mM potassium phosphate buffer (pH 7.4) with 0.5–4 µM 17-Preg added in 4 µl ethanol, 80,000 cpm of [3H]-17-Preg (21.1 Ci/mmol) (NEN Life Science Products), and purified recombinant human b5 (PanVera, Madison, WI) in 10-fold molar excess compared with the total P450 content of the microsomes. All assays were done in the linear time range of the enzymatic reaction.
Steroids were extracted from the reaction mixtures with 400 µl ethyl acetate/isooctane (1:1), concentrated by evaporation under continuous nitrogen flow, and assayed by thin layer chromatography on PE SIL G/UV silica gel plates (Whatman, Maidstone, UK) using 3:1 chloroform:ethyl acetate as the solvent system (14). The radiolabeled steroids were quantified by phosphorimager analysis on a Storm 860 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). All assays were performed in triplicate, and data are presented as means ± SD. Kinetic behavior was approximated as a Michaelis-Menten system, and data were plotted as described by Lineweaver-Burk. The Michaelis constant Km and maximum velocity Vmax were calculated from the equation Vapp = Vmax x [S]/(Km + [S]). Data fitting was carried out by LEONORA Version 1.0 for analysis of steady-state enzyme kinetics (26).
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Results |
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). The human and chimpanzee sequences were nearly identical, and the baboon and rhesus sequences were nearly identical, but the human/chimp sequences were only about 95% identical to the rhesus/baboon sequences (Table 2).
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-hydroxylase activity (12, 14). To measure the activities of P450c17, we first stably transformed yeast strain W303B with vector pYcDE2 expressing human OR cDNA. A single colony of this transformant was then propagated and transformed with vector V10 expressing P450c17 from each species. Immunoblotting confirmed indistinguishable levels of OR and P450c17 expression in the four engineered yeast strains expressing each primate P450c17 (not shown).
Human P450c17 coexpressed with OR catalyzes the 17-hydroxylation of pregnenolone to 17-Preg and of progesterone to 17OHP equally well (12, 14). The 17,20 lyase reaction of human P450c17 is less efficient than the 17-hydroxylase reaction, but it is 100 times more efficient with 17-Preg as the substrate than with 17OHP (12, 14). Thus, under physiologic conditions, most C-19 steroids are produced by conversion of 17-Preg to DHEA, whereas only trace amounts of 17OHP are converted to androstenedione. Therefore, 17-hydroxylase activity was measured as the conversion of progesterone to 17OHP and 17,20 lyase activity as the conversion of 17-Preg to DHEA (14). As 17,20 lyase activity is significantly enhanced by allosteric interaction of the P450c17/OR complex with b5 (14, 28), 17,20 lyase activity was measured in the presence of exogenously added b5.
P450c17 from baboon had considerably higher 17-hydroxylase activity than that from the other primates and there appeared to be an inverse correlation between 17-hydroxylase and 16-hydroxylase activity among the four species (Fig. 2). The ratios of 16-hydroxylase/17-hydroxylase activity in human (0.34) and chimpanzee (0.31) P450c17 were eight times higher than in baboon (0.04) and twice as high as in rhesus (0.18) (Fig. 2). Analysis of enzyme kinetics confirmed these observations (Fig. 3). Baboon P450c17 had the lowest Km and the highest Vmax (and thus the highest catalytic efficiency) for 17-hydroxylase activity and the lowest Vmax for 16-hydroxylase activity (Table 3).
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-hydroxylase activity (Fig. 4). Kinetic analysis of 17,20 lyase activity showed that baboon P450c17 had the highest Vmax, whereas there were only slight differences between 17,20 lyase activities from human, chimpanzee and rhesus (Fig. 3C, Table 3). However, there were no major differences in the 17-hydroxylase and 17,20 lyase activities of P450c17 when comparing the enzymes from the species with adrenarche to those that do not have adrenarche.
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, Table 2). Not surprisingly, the enzymatic activities of the human and chimp enzymes were very similar, but there were substantial differences in the 17-hydroxylase and 16-hydroxylase activities catalyzed by the rhesus and baboon enzymes (Fig. 2). The baboon and rhesus sequences differ only at residue 255, where the baboon, like the human and chimp, has arginine (Arg), whereas rhesus has histine (His). Thus, it appears that Arg255 influences both hydroxylation reactions. To test the potential role of Arg255 in human P450c17, we changed this residue to His and to alanine (Ala) in the human enzyme and examined its activities.
Compared with the 17-hydroxylase activity of wild-type human P450c17, the Arg255His mutant had equivalent activity (106 ± 3%) and the Arg255Ala mutant had decreased activity (73 ± 3%). The effect of these mutations on 17,20 lyase activity were similar; Arg255His had equivalent activity (109 ± 6%) and Arg255Ala had somewhat decreased activity (79 ± 8%). Kinetic analysis showed that the reduced 17-hydroxylase activity of the Arg255Ala mutant was due to a lower Vmax, whereas the reduction in 17,20 lyase activity was due to a higher Km (Fig. 5, Table 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase activity of baboon and rhesus P450c17 had similar Vmax values, but the Km for the baboon enzyme was lower, resulting in greater catalytic efficiency. The 17,20 lyase activity of baboon P450c17 was also increased, largely due to a higher Vmax compared with the other species, but the 17,20 lyase activity of baboon P450c17 was increased to a lesser degree than its 17-hydroxylase activity.
Arg255 in baboon and His255 in rhesus monkey is the only amino acid difference in the P450c17 sequence of these two primates. Modeling of human P450c17 (29) indicates that Arg255 lies near to the redox-partner binding site (Fig. 6). Arg255 is conserved in human, chimpanzee, and baboon P450c17, but the baboon enzyme differs from the human or chimpanzee enzyme by 25 amino acids, which may contribute to differences in enzymatic activity. We observed a modest loss in 17-hydroxylase and 17,20 lyase activity when Arg255 was changed to Ala in the human enzyme. Thus, Arg255 is important in modulating 17-hydroxylase activity.
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-hydroxylase activity then human or chimpanzee P450c17. The clustered amino acid differences in the region encompassing helices E through H may alter the position of steroids in the substrate-binding pocket, reducing accessibility of the 16 hydrogen to the heme iron, and thereby reducing 16-hydroxylase activity. The greater 17-hydroxylase and lower 16-hydroxylase activities in baboon and rhesus may provide increased amounts of 17-hydroxylated steroids, thereby increasing the substrate pool for the enzyme’s 17,20 lyase activity contributing to the higher serum DHEA/S concentrations in baboon and rhesus (5, 6). However, these higher concentrations of DHEA/S in baboon and rhesus may also reflect decreased activity of enzymes that metabolize DHEA, such as the 3ß- and 17ß-hydroxysteroid dehydrogenases. Thus, the recent evolutionary appearance of adrenarche in higher primates cannot be explained by differences among the primate P450c17 sequences. DHEA secretion also requires inhibition of 3ß-hydroxysteroid dehydrogenase (3ßHSD), which would otherwise convert DHEA to androstenedione. By contrast, high levels of gonadal 3ßHSD expression (30) divert DHEA to the sex steroid synthesis, reducing gonadal secretion of DHEA. Thus, adrenarche requires both the acquisition of the 17,20 lyase activity of P450c17 and a decreased 3ßHSD expression in the adrenal zona reticularis (31). Decreased amounts of 3ßHSD and increased P450c17, OR, and b5 have recently been demonstrated by immunohistochemistry and RT-PCR in the adrenal zona reticularis from children undergoing adrenarche (18, 32, 33, 34). Thus, the regulation of adrenarche is a complex event at multiple levels of regulation of adrenal steroidogenesis.
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Acknowledgments |
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Footnotes |
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Abbreviations: Ala, Alanine; Arg, arginine; b5, cytochrome b5; C-19, 19-carbon; d, deoxy; DHEA, dehydroepiandrosterone; DHEAS, sulfate ester of DHEA; DHEA/S, combination of DHEA and DHEAS; His, histine; 3ßHSD, 3ß-hydroxysteroid dehydrogenase; Km, Michaelis constant; 17OHP, 17-OH-progesterone; OR, P450 oxidoreductase; 17-Preg, 17-hydroxypregnenolone; Vmax, maximum velocity.
Received April 29, 2002.
Accepted for publication August 2, 2002.
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References |
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