Báo cáo Y học: Analyses of the CYP11B gene family in the guinea pig suggest the existence of a primordial CYP11B gene with aldosterone synthase activity

Eur. J. Biochem. 269, 3838–3846 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03076.x Analyses of the CYP11B gene family in the guinea pig suggest the existence of a primordial CYP11B gene with aldosterone synthase activity Hannes E. Bu¨low1,* and Rita Bernhardt2 1Max-Delbruck-Centrum fur Molekulare Medizin, Berlin-Buch, Germany; 2Universitat des Saarlandes, FR Biochemie, Saarbrucken, Germany In this study we describe the isolation of three genes of the CYP11B family of the guinea pig. CYP11B1 codes for the previouslydescribed11b-hydroxylase[Bulow,H.E.,Mobius, K., Bahr, V. & Bernhardt, R. (1996) Biochem. Biophys. Res. Commun. 221, 304–312] while CYP11B2 represents the aldosterone synthase gene. As no expression for CYP11B3 was detected this gene might represent a pseudogene. Transient transfection assays show higher substrate speci-ficity for its proper substrate for CYP11B1 as compared to CYP11B2, which could account for the zone-specific syn-thesis of mineralocorticoids and glucocorticoids, respec-tively.Thus,CYP11B2displayedafourfoldhigherabilityto perform 11b-hydroxylation of androstenedione than CYP11B1,whilethisdifferenceisdiminishedwiththesizeof the C17 substituent of the substrate. Furthermore, analyses with the electron transfer protein adrenodoxin indicate dif-ferentialsensitivityofCYP11B1andCYP11B2aswellasthe Higher vertebrates regulate vital processes like volume/ electrolyte homeostasis and glucose/lipid metabolism by means of steroid hormones, namely mineralocorticoids and glucocorticoids. The biosynthesis of these steroids occurs primarily in the adrenal cortex within morphologically and functionally distinct zones. Accordingly, mineralocorticoids are produced by the outer zona glomerulosa while gluco-corticoids are formed in the two inner layers of the cortex, the zonae fasciculata/reticularis. Originating from cholester-ol they are synthesized by a number of consecutive oxidations and dehydrogenations where all oxidative reac-tions are catalysed by enzymes of the cytochrome P450 superfamily [2]. The first and rate-limiting step is the conversion of cholesterol to pregnenolone by the mitochon-drial cytochrome P450 side-chain cleavage enzyme (P450scc, CYP11A1). Subsequently, pregnenolone is dehydroge-nated and oxidized in position 17 and/or 21 to yield 11-deoxycortisol or 11-deoxycorticosterone, respectively. Correspondence to R. Bernhardt, Universitat des Saarlandes, FR Biochemie, PO Box 15 11 50, D-66041 Saarbrucken, Germany. Fax: + 49 681302 4739, Tel.: + 49 681302 3005, E-mail: ritabern@mx.uni-saarland.de *Present address, Columbia University, College of Physicians & Surgeons, New York, NY 10032, USA. Note: a website is available at http://www.uni-saarland.de/fak8/bernhardt. (Received 12 April 2002, revised 11 June 2002, accepted 26 June 2002) three hydroxylation steps catalysed by CYP11B2 to the availability of reducing equivalents. Together, both mecha-nisms point to novel protein intrinsic modalities to achieve tissue-specific production of mineralocorticoids and gluco-corticoids in the guinea pig. In addition, we conducted phylogenetic analyses. These experiments suggest that a common CYP11B ancestor gene that possessed both 11b-hydroxylase and aldosterone synthase activity under-went a gene duplication event before or shortly after the mammalian radiation with subsequent independent evolu-tion of the system in different lines. Thus, a differential mineralocorticoid and glucocorticoid synthesis might be an exclusive achievement of mammals. Keywords: guinea pig; 11b-hydroxylase; aldosterone synth-ase; phylogeny. Both compounds in turn are substrates for the cytochrome P450 enzymes of the CYP11B subfamiliy, namely the 11b-hydroxylase (CYP11B1) and the aldosterone synthase (CYP11B2). While CYP11B1 hydroxylates 11-deoxycorti-sol in position 11 to give cortisol as the major glucocorti-coid, the closely related aldosterone synthase forms aldosterone as the major mineralocorticoid by means of an11b-hydroxylationandan18-hydroxylation/oxidationof 11-deoxycorticosterone. Thus, the proteins of the CYP11B subfamily catalysing the last biosynthetic steps are the key enzymes for the synthesis of both mineralocorticoids and glucocorticoids. From molecular cloning of the corresponding genes and analyses of the cDNAs it became obvious that the encoded isoenzymes share a very high degree of similarity ranging up to 95% on the amino acid level for human CYP11B1 and CYP11B2 [3]. There are, however, a number of significant species differences. For example, humans [3], mice [4], rats [5], and hamsters [6,7], possess at least two functionally different genes with the encoded proteins exhibiting different enzymatic activities. While one protein modifies the steroid entity predominantly in position 11, the other one is able to hydroxylate and oxidize position 18 as well. In contrast, cows [8], pigs [9], sheep [10], and frogs [11] apparently possess only one type of a bipotent enzyme that is capable of catalysing the reactions at both positions 11 and 18. Nonetheless, the production of mineralocorticoids and glucocorticoids is strictly zone specific in all species. It is, however, unknown Ó FEBS 2002 which factors convey this specificity and how these similar but distinct systems evolved. To investigate the zone-specific synthesis of mineralocor-ticoids and glucocorticoids and the evolution of the hormonal system in more detail we chose the guinea pig as a model. The guinea pig is an interesting species because its taxonomical position remains controversial [12,13]. Thesefeaturesshouldprovidenewinsightintotheevolution and function of the hormonal system. We first cloned the genes of the CYP11B family of the guinea pig. The 11b-hydroxylase of the guinea pig showed higher substrate specificity than the aldosterone synthase. In addition, the aldosterone synthase exhibited unique properties in that 18-hydroxylase activity was strongly dependent on the presenceofhighlevelsofreducingequivalentswhereasbasic levels were sufficient for high 11b-hydroxylase activity of this enzyme. This suggests a new regulatory level in aldosterone synthesis thattogetherwiththe highersubstrate specificity of the 11b-hydroxylase could be crucial for the tissue-specific synthesis of steroid hormones. Phylogenetic analyses indicate a gene duplication event of a bipotent CYP11B ancestor gene before the mammalian radiation with subsequent distinct evolution in different clades. This indicates that a differential glucocorticoid and mineralocor-ticoid synthesis is an exclusive property of mammals. EXPERIMENTAL PROCEDURES General procedures Molecularbiologyprocedureswerecarriedoutaccordingto standard protocols [14] unless stated otherwise. Chemicals and enzymes were purchased from the highest quality sources commercially available. Screening of a guinea pig genomic library A total of 1 · 106 clones of a guinea pig genomic library (Stratagene, #946110) were screened under low stringency conditions as described for Southern blots using a guinea pig CYP11B1 full-length probe (1618 bp XbaI fragment of pHBL5 [1]. Positive clones were purified to homoge-neity and analysed by Southern blotting using various restriction endonucleases. Appropriate genomic fragments were subcloned into pBluescript SK(–) (Stratagene) and sequenced using gene-specific primers. Furthermore, to sequence parts not represented by genomic phage clones genomic fragments were amplified by PCR and sequenced directly. RNA preparation Tissue was homogenized in 6 M guandinium thiocyanate and subsequently RNA was purified by centrifugation througha CsClgradient[14]. PolyA+ RNAwas isolatedby three rounds of affinity purification on oligodT cellulose (Stratagene). RNAse protection analyses RNAse protection analyses were carried out using a HybSpeedTM RPA Kit (Ambion) according to the Guinea pig CYP11B genes (Eur. J. Biochem. 269) 3839 manufacturer’s recommendations. Briefly, specific 32P labelled RNA antisense transcripts (corresponding to nucleotides 1491–1700 in the CYP11B1 cDNA [1] and nucleotides1511–1750intheCYP11B2cDNA;Fig. 2)were hybridized with total RNA from different tissues. After digestion of the reaction mixture with RNAse A/H protected fragments were separated by PAGE and visual-ized by autoradiography. RACE The cDNA for CYP11B2 of the guinea pig was amplified and cloned using a MarathonÒ cDNA Amplification Kit (Clontech) following the supplier’s recommendations. In brief, after reverse transcription of 1 lg of polyA+ RNA and second-strand synthesis an adapter comprising the T7 promoter sequence combined with a NotI and a SmaI site was ligated to both ends of the cDNA pool. Using a combination of a primer complementary to the adapter (adapter primer: 5¢-CCATCCTAATACGACTCACTA TAGGGC-3¢) and a gene-specific sense primer (5¢-GCCG CTCGAGTTTGAGTTAGCCAGAAACTCC-3¢, XhoI site underlined) or antisense primer (5¢-ATACGGGCCC GACAGTGGTGTGCCTGGGAAC-3¢, Bsp120I site underlined), respectively, a PCR reaction was carried out with KlenTaqTM (Clontech) under the following conditions: 94 °C 2 min initial denaturation, 94 °C 45 s denaturation, 72 °C 1 min annealing (annealing temperature reduced at 1.4 °C per cycle), 72 °C 3 min polymerization; 10 cycles, followed by 25 cycles at 94 °C 45 s, 58 °C 1 min and 72 °C 3 min with a final extension step for 8 min at 72 °C. The 5¢-RACE product was cloned directly into a TAÒ Cloning vector pCR2.1 (Invitrogen) yielding pCR2.1/HG17 while 3¢-RACE products were inserted by using the XhoI and NotI sites into pBluescript SK(–) (Stratagene) giving pBSSK/3¢RACE HG17. DNA sequencing DNA sequencing was carried out using a Thermo Sequen-aseTM Cycle Sequencing Kit (Amersham/USB) in combi-nation with [a-35S]dCTP followed by autoradiography with HyperfilmTM MP (Amersham). Southern blotting Genomic DNA was digested with the appropriate enzymes, extracted twice with phenol/chloroform and precipitated using EtOHand sodium acetate. After extensive washing the DNA was redissolved in Tris/EDTA, pH8.0 and sep-aratedona1 · Tris/borate/EDTA,0.9%agarosegel.After capillary transfer to HybondTM nylon membranes (Amer-sham) nucleic acids were UV cross-linked (0.24 JÆcm)2). Prehybridization was performed in 5 · NaCl/Cit, 5 · Den-hardt’s,0.5%SDSand50 lgÆmL)1 sonicatedsalmonsperm DNA for 2 h at 65 °C. [a-32P]dCTP labelled DNA probes ( 1 · 106 c.p.m.ÆmL)1) were hybridized in the same solu-tion for 16 h. For low stringency hybridization the blot was washed twice at room temperature in 2 · NaCl/Cit, 0.1% SDS for 10 min followed by two 30 min washes at 50 °C in 1 · NaCl/Cit,0.1%SDS.Autoradiographywascarriedout with HyperfilmTM MP (Amersham). 3840 H. E. Bulow and R. Bernhardt (Eur. J. Biochem. 269) Ó FEBS 2002 Construction of expression plasmids For the construction of pCMV/11B2, pRc/CMV was digested with Bsp120I, trimmed with Pfu polymerase (Stratagene) and subsequently digested with NotI. Likewise, pCR2.1/HG17 was digested with SpeI, trimmed with Pfu polymerase and digested with NotI to release a fragment comprising the ORF of the guinea pig CYP11B2. This fragment was ligated using NotI/blunt into the eukaryotic expression vector. Hydroxylation assays COS-1 cells were maintained as described previously [15]. Transfections were carried out using LipofectAMINETM (Gibco/BRL) according to the manufacturer’s recommen-dations. One mL of transfection mix contained 2 lg of the respective expression construct together with 1 lg pBAdx4 (bovine adrenodoxin; gift of M. R. Waterman, Vanderbilt University, Nashville, TN, USA) and 6 lL LipofectAMINETM unless stated otherwise. Twenty-four h after transfection cells were incubated with appropriate substrates for 48 h using [1,2-3H]cortisol, [14C]11-deoxy-corticosterone or [1,2–3H]androstenedione, respectively, as tracers. Media were extracted and analysed by high performance TLC as described previously [16]. Phylogenetic analyses Phylogenetic analyses were conducted using the PHYLIP package (Version 3.5c, 1993) [17]. The sequences have been submitted to GenBank under the accession numbers AF191278, AF191279 (for CYP11B1), AF191281, AF191280 (for CYP11B2), and AF191282 (for CYP11B3). RESULTS In a previous study we isolated an 11b-hydroxylase of the guinea pig [1] by screening an adrenal cDNA library with a PCR amplified orthologous probe. Upon expression, the isolated cDNA turned out to be a pure 11b-hydroxylase with no detectable 18-hydroxylation activity suggesting the existence of additional isoenzymes of the CYP11B subfam-ily in the guinea pig. To investigate this notion, a Southern blot was performed utilizing an exon-1-specific probe of CYP11B1underlowstringencyconditionsanddigestingthe genomic DNA with various restriction endonucleases that did not cut within exon 1. The result (Fig. 1) strongly suggested the existence of at least three different genes as judged from theappearanceofthreebandsiftheDNAwas, e.g. digested with EcoRI/EcoRV, XbaI, or XbaI/HindIII. Although guinea pigs had been sodium depleted to stimulate the expression of a putative aldosterone synthase as much as possible [18], repeated screening of the cDNA library did not result in the identification of any cDNA other than CYP11B1 (data not shown). Thus, we devised another strategy for the identification of additional genes of the CYP11B subfamily in the guinea pig. To this end, a genomic library was screened under low stringency (see Experimental procedures) utilizing a full-length guinea pig CYP11B1 cDNA as a probe. As opposed to a cDNA library, screening ofa genomic library shouldyieldclones in Fig. 1. Southern blot analyses with a CYP11B1 exon 1-specific probe. Fifteen micrograms of guinea pig genomic DNA was digested with the indicated endonucleases. After transfer, membranes were probed under low stringency conditions with an exon 1-specific probe of CYP11B1 (nucleotides 1–141; see Experimental procedures for details). Sizes of fragments are indicated on the right. relation to their abundance in the genome rather than their relative abundance due to differential expression. Indeed, this approach lead to the isolation of eight genomic clones thatwereclassifiedintothreesubgroupsbasedonrestriction digests and hybridization experiments (data not shown). One clone termed kHG13 turned out to represent the CYP11B1 gene while kHG17 and kHG15 represented closely related genes of the CYP11B family demonstrated by similarities of >75% at the nucleotide level. They were tentatively named CYP11B2 and CYP11B3, respectively. To clone the corresponding cDNAs, the RACE tech-niquewasused.PolyA+ RNAwasconvertedintoadouble-strandedcDNApoolandadapterscomprisingthepromoter sequence of the T7 bacteriophage were ligated to both ends. The sequences of the T7 promoter are extremely rare in eukaryotic genomes and thus convey a high degree of specificity in subsequent PCR reactions. Using a primer combinationofanadapterprimerandgene-specificsenseor antisense primers, respectively, we were able to amplify two overlapping fragments in case of kHG17. Upon sequencing of these cDNA fragments the complete sequence of the cDNA of CYP11B2 could be deduced. It comprised 2611 bp and an ORF of 1503 bp coding for a putative mitochondrial preprotein of 501 amino acids with a calculated molecular weight of 57.7 kDa (Fig. 2). After Leu24 a cleavage site for the matrix-associated protease was predicted resulting in a mature mitochondrial protein of 55 kDa. The deduced amino acid sequence showed 81% similarity to the guinea pig CYP11B1 and 80% similarity to the human CYP11B2, respectively (see below). The 3¢-UTR comprised 1079 bp with a canonical polyadenylation site 16 bp upstream of the polyA tail with no indications for the existence of alternative poly adenylation sites (Fig. 2). We next investigated the expression of the CYP11B genes. A Northern blot probed with a CYP11B2-specific probe showed a single band of 2.9 kb (data not shown) which is consistent with the length of the isolated cDNA for CYP11B2 assuming a polyA tail of 200–300 adenine residues. To see where the CYP11B genes were expressed Ó FEBS 2002 Guinea pig CYP11B genes (Eur. J. Biochem. 269) 3841 Fig. 2. SequenceoftheCYP11B2cDNAoftheguineapig. Thenucleotidesequenceandthededucedaminoacidsequencearebothshown.TheORF (putative start and stop codon underlined) encodes a mitochondrial preprotein with a calculated molecular mass of 57.7 kDa. An arrowhead indicatesthepresumptivecleavagesiteforthemitochondrialmatrixassociatedprotease.Numbersontheleftdenoteaminoacids,thoseontheright indicate nucleotides. A canonical polyadylation site is shown boldface. and whether they played a role during postnatal develop-ment we used a highly sensitive RNAse protection assay withRNAsfromdifferenttissuesanddevelopmentalstages. As shown in Fig. 3, expression of both the 11b-hydroxylase and the aldosterone synthase was exclusively in the adrenal gland. Moreover, there was no difference in expression betweenpostnatalday1andtheadultstagessuggestingthat the genes were not differentially regulated during postnatal development. With respect to kHG15 we were not able to demonstrate expression of the gene in adult tissues using RT/PCR with various gene-specific primer combinations (data not shown). Thus, this clone might represent a pseudogene of the CYP11B family or a gene that is not expressed in adult tissues. To compare the enzymatic activities of CYP11B2 and CYP11B1, the cDNAs were cloned under the control of a viral promoter and transiently transfected into COS-1 cells. Transfected cells were incubated with different substrates and the resulting metabolites were analysed using TLC. As seen in Fig. 4A, CYP11B2 converted 11-deoxycorticoster-one to corticosterone and both 18(OH)-corticosterone and aldosterone. These results clearly demonstrate that CYP11B2 is the aldosterone synthase of the guinea pig as it is capable of modifying position 11 and 18 of the steroid ring. In contrast, CYP11B1 produced only corticosterone and traces of 18/19(OH)-deoxycorticosterone, confirming earlier results [1]. Furthermore, CYP11B2 transfected cells efficiently converted 11-deoxycortisol to cortisol and fur-ther to 18(OH)-cortisol (Fig. 4B). As 11b(OH)-androsten-edione is the major C19 steroid in the guinea pig, we also used androstenedione as a substrate. Under the experi-mental conditions large amounts of 11b(OH)-androstendi-one were synthesized by CYP11B2 in comparison with CYP11B1 (Fig. 4C). It is noteworthy, that CYP11B2 displayed a higher enzymatic activity than CYP11B1 based on 11b-hydroxylase activity. These differences were highest 3842 H. E. Bulow and R. Bernhardt (Eur. J. Biochem. 269) Fig. 3. Tissue and age-specific RNAse protection assays. Different amounts of total RNA from different tissues and stages as indicated were hybridized in solution with CYP11B1 and CYP11B2-specific probes. Both probes were chosen from the 3¢ untranslated regions of the genes where sequence divergence was maximal between the two isoenzymes. Following RNAse digestion the probes protected a 210 nucleotide fragment of CYP11B1 (corresponding to nucleotides 1491– 1700 [1]) or a 240 nucleotide fragment for CYP11B2 (corresponding to nucleotide 1511–1750; Fig. 2), respectively. A control lane without RNAse (–RNAse) shows the corresponding undigested riboprobes of 242 nucleotides (for CYP11B1) and 272 nucleotides (for CYP11B2). A molecular size marker is given on the left. Different developmental stages are denoted on the right: P1, postnatal day 1; adult. for androstenedione (fourfold) and lowest for 11-deoxy-cortisol (Fig. 4). This shows a higher substrate specifity of CYP11B1 which could be due to differences in the active centre and/or the entry channel. Moreover, it could be important for tissue-specific synthesis of glucocorticoids given the differences in expression levels of the two enzymes. We next asked whether other accessory proteins might contribute to the zone-specific synthesis of steroid hor-mones. A good candidate is adrenodoxin, an iron sulfur containing electron donor protein that is required for the function of mitochondrial cytochrome P450 proteins [19] and has been shown to interact directly with the cyto-chromes. To test its significance we carried out an experi-ment where adrenodoxin was either cotransfected or omitted. After transfection, cells were incubated with 11-deoxycorticosterone as a substrate. As shown in Fig. 5, the omission of Adx leads to a sharp decrease in the activity for the 11b-hydroxylase, CYP11B1. Intriguingly, however, the 11b-hydroxylase activity of the aldosterone synthase CYP11B2wasbasicallyunaffectedwhereasthe18-hydroxy-lation and oxidation potential were abrogated almost completely.Theseresultsindicateclearstructuraldifferences on the surface of these proteins involved either in glucocor-ticoid or in mineralocorticoid biosynthesis despite a high degree of similarity between the two isoenzymes. More Ó FEBS 2002 Fig. 4. Enzymatic acivities of CYP11B2. COS-1 cells were transfected with pBAdx4 (bovine adrenodoxin) and the expression plasmid pCMV5 [1] (CYP11B1), pRc/CYP11B2 (CYP11B2), or pRC/CMV (mock), respectively. Twenty-four h after transfection cells were incubated for 48 h with 5 lM 11-deoxycorticosterone (DOC) includ-ing 4 nCiÆmL)1 [14C]DOC (A), 5 lM androstendione including 0.5 lCiÆmL)1 [3H]androstendione (B), or 2.5 lM 11-deoxycortisol including 0.5 lCiÆmL [3H]11-deoxycorticosterone (C). Subsequently, steroids were extracted and separated by TLC [16]. In culture medium incubated substrates served as an additional control (substrate). Positions of cold standards are denoted on the left. On the right percentageoftotalradioactivityorrelativeactivityisgiven±SD;data are from at least two different experiments performed in triplicate. importantly, these results indicate a new level of regulation for tissue-specific aldosterone synthesis depending on the availability of reducing equivalents. One intriguing question is how and when animals developed a hormonal system that differentially regulated the control of both electrolyte/volume homeostasis and glucose metabolism. Knowing when and how differential synthetic pathways for mineralocorticoids and glucocorti-coids developed would lead to deeper understanding of these important evolutionary processes. Because the guinea pig’s taxonomical classification is controversial [12,13], this species is extremely interesting in terms of vertebrate evolution and might provide insight into some aspects of the evolution of the hormonal system. ... - tailieumienphi.vn