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Analyses of theCYP11Bgene family in the guinea pig suggestthe existence of a primordialCYP11Bgene with aldosterone synthaseactivityHannes E. Bu¨ low1,* and Rita Bernhardt21Max-Delbru¨ck-Centrum fu¨r Molekulare Medizin, Berlin-Buch, Germany;2Universita¨t des Saarlandes, FR Biochemie, Saarbru¨cken,GermanyIn this study we describe the isolation of three genes of theCYP11B family of the guinea pig. CYP11B1 codes for thepreviously described11b-hydroxylase[Bu¨low, H.E., Mo¨bius,K., Ba¨hr, V. & Bernhardt, R. (1996) Biochem. Biophys. Res.Commun. 221, 304–312] while CYP11B2 represents thealdosterone synthase gene. As no expression for CYP11B3was detected this gene might represent a pseudogene.Transient transfection assays show higher substrate speci-ﬁcity for its proper substrate for CYP11B1 as compared toCYP11B2, which could account for the zone-speciﬁc syn-thesis of mineralocorticoids and glucocorticoids, respec-tively. Thus, CYP11B2 displayed a fourfold higher ability toperform 11b-hydroxylation of androstenedione thanCYP11B1, while this diﬀerence is diminished with the size ofthe C17 substituent of the substrate. Furthermore, analyseswith the electron transfer protein adrenodoxin indicate dif-ferential sensitivity of CYP11B1 and CYP11B2 as well as thethree hydroxylation steps catalysed by CYP11B2 to theavailability of reducing equivalents. Together, both mecha-nisms point to novel protein intrinsic modalities to achievetissue-speciﬁc production of mineralocorticoids and gluco-corticoids in the guinea pig. In addition, we conductedphylogenetic analyses. These experiments suggest that acommon CYP11B ancestor gene that possessed both11b-hydroxylase and aldosterone synthase activity under-went a gene duplication event before or shortly after themammalian radiation with subsequent independent evolu-tion of the system in diﬀerent lines. Thus, a diﬀerentialmineralocorticoid and glucocorticoid synthesis might be anexclusive achievement of mammals.Keywords: guinea pig; 11b-hydroxylase; aldosterone synth-ase; phylogeny.Higher vertebrates regulate vital processes like volume/electrolyte homeostasis and glucose/lipid metabolism bymeans of steroid hormones, namely mineralocorticoids andglucocorticoids. The biosynthesis of these steroids occursprimarily in the adrenal cortex within morphologically andfunctionally distinct zones. Accordingly, mineralocorticoidsare 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 consecutiveoxidations and dehydrogenations where all oxidative reac-tions are catalysed by enzymes of the cytochrome P450superfamily . The ﬁrst and rate-limiting step is theconversion 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 yield11-deoxycortisol or 11-deoxycorticosterone, respectively.Both compounds in turn are substrates for the cytochromeP450 enzymes of the CYP11B subfamiliy, namely the11b-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 formsaldosterone as the major mineralocorticoid by means ofan 11b-hydroxylation and an 18-hydroxylation/oxidation of11-deoxycorticosterone. Thus, the proteins of the CYP11Bsubfamily catalysing the last biosynthetic steps are the keyenzymes for the synthesis of both mineralocorticoids andglucocorticoids.From molecular cloning of the corresponding genes andanalyses of the cDNAs it became obvious that theencoded isoenzymes share a very high degree of similarityranging up to 95% on the amino acid level for humanCYP11B1 and CYP11B2 . There are, however, anumber of signiﬁcant species differences. For example,humans , mice , rats , and hamsters [6,7], possess atleast two functionally different genes with the encodedproteins exhibiting different enzymatic activities. Whileone protein modiﬁes the steroid entity predominantly inposition 11, the other one is able to hydroxylate andoxidize position 18 as well. In contrast, cows , pigs ,sheep , and frogs  apparently possess only one typeof a bipotent enzyme that is capable of catalysing thereactions at both positions 11 and 18. Nonetheless, theproduction of mineralocorticoids and glucocorticoids isstrictly zone speciﬁc in all species. It is, however, unknownCorrespondence to R. Bernhardt, Universita¨t des Saarlandes, FRBiochemie, PO Box 15 11 50, D-66041 Saarbru¨cken, Germany.Fax: + 49 681302 4739, Tel.: + 49 681302 3005,E-mail: firstname.lastname@example.org*Present address, Columbia University, College of Physicians &Surgeons, New York, NY 10032, USA.Note: a website is available athttp://www.uni-saarland.de/fak8/bernhardt.(Received 12 April 2002, revised 11 June 2002, accepted 26 June 2002)Eur. J. Biochem. 269, 3838–3846 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03076.xwhich factors convey this speciﬁcity and how these similarbut distinct systems evolved.To investigate the zone-speciﬁc synthesis of mineralocor-ticoids and glucocorticoids and the evolution of thehormonal system in more detail we chose the guinea pigas a model. The guinea pig is an interesting species becauseits taxonomical position remains controversial [12,13].These features should provide new insight into the evolutionand function of the hormonal system. We ﬁrst cloned thegenes of the CYP11B family of the guinea pig. The 11b-hydroxylase of the guinea pig showed higher substratespeciﬁcity than the aldosterone synthase. In addition, thealdosterone synthase exhibited unique properties in that18-hydroxylase activity was strongly dependent on thepresence of high levels of reducing equivalents whereas basiclevels were sufﬁcient for high 11b-hydroxylase activity ofthis enzyme. This suggests a new regulatory level inaldosterone synthesis that together with the higher substratespeciﬁcity of the 11b-hydroxylase could be crucial for thetissue-speciﬁc synthesis of steroid hormones. Phylogeneticanalyses indicate a gene duplication event of a bipotentCYP11B ancestor gene before the mammalian radiationwith subsequent distinct evolution in different clades. Thisindicates that a differential glucocorticoid and mineralocor-ticoid synthesis is an exclusive property of mammals.EXPERIMENTAL PROCEDURESGeneral proceduresMolecular biology procedures were carried out according tostandard protocols  unless stated otherwise. Chemicalsand enzymes were purchased from the highest qualitysources commercially available.Screening of a guinea pig genomic libraryA total of 1 · 106clones of a guinea pig genomic library(Stratagene, #946110) were screened under low stringencyconditions as described for Southern blots using a guineapig CYP11B1 full-length probe (1618 bp XbaIfragmentof pHBL5 . Positive clones were puriﬁed to homoge-neity and analysed by Southern blotting using variousrestriction endonucleases. Appropriate genomic fragmentswere subcloned into pBluescript SK(–) (Stratagene) andsequenced using gene-speciﬁc primers. Furthermore, tosequence parts not represented by genomic phage clonesgenomic fragments were ampliﬁed by PCR and sequenceddirectly.RNA preparationTissue was homogenized in 6Mguandinium thiocyanateand subsequently RNA was puriﬁed by centrifugationthrough a CsCl gradient . PolyA+RNA was isolated bythree rounds of afﬁnity puriﬁcation on oligodT cellulose(Stratagene).RNAse protection analysesRNAse protection analyses were carried out using aHybSpeedTMRPA Kit (Ambion) according to themanufacturer’s recommendations. Brieﬂy, speciﬁc32Plabelled RNA antisense transcripts (corresponding tonucleotides 1491–1700 in the CYP11B1 cDNA  andnucleotides 1511–1750 in the CYP11B2 cDNA; Fig. 2) werehybridized with total RNA from different tissues. Afterdigestion of the reaction mixture with RNAse A/Hprotected fragments were separated by PAGE and visual-ized by autoradiography.RACEThe cDNA for CYP11B2 of the guinea pig was ampliﬁedand cloned using a MarathonÒ cDNA Ampliﬁcation Kit(Clontech) following the supplier’s recommendations. Inbrief, after reverse transcription of 1 lg of polyA+RNAand second-strand synthesis an adapter comprising the T7promoter sequence combined with a NotIandaSmaIsitewas ligated to both ends of the cDNA pool. Using acombination of a primer complementary to the adapter(adapter primer: 5¢-CCATCCTAATACGACTCACTATAGGGC-3¢) and a gene-speciﬁc sense primer (5¢-GCCGCTCGAGTTTGAGTTAGCCAGAAACTCC-3¢, XhoIsite underlined) or antisense primer (5¢-ATACGGGCCCGACAGTGGTGTGCCTGGGAAC-3¢, Bsp120I siteunderlined), respectively, a PCR reaction was carried outwith 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 at1.4 °C per cycle), 72 °C 3 min polymerization; 10 cycles,followed by 25 cycles at 94 °C45s,58°C1minand72°C3 min with a ﬁnal extension step for 8 min at 72 °C. The5¢-RACE product was cloned directly into a TAÒ Cloningvector pCR2.1 (Invitrogen) yielding pCR2.1/HG17 while3¢-RACE products were inserted by using the XhoIandNotI sites into pBluescript SK(–) (Stratagene) givingpBSSK/3¢RACE HG17.DNA sequencingDNA sequencing was carried out using a Thermo Sequen-aseTMCycle Sequencing Kit (Amersham/USB) in combi-nation with [a-35S]dCTP followed by autoradiography withHyperﬁlmTMMP (Amersham).Southern blottingGenomic DNA was digested with the appropriate enzymes,extracted twice with phenol/chloroform and precipitatedusing EtOH and sodium acetate. After extensive washingthe DNA was redissolved in Tris/EDTA, pH 8.0 and sep-arated on a 1 · Tris/borate/EDTA, 0.9% agarose gel. Aftercapillary transfer to HybondTMnylon 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% SDS and 50 lgÆmL)1sonicated salmon spermDNA for 2 h at 65 °C. [a-32P]dCTP labelled DNA probes( 1 · 106c.p.m.ÆmL)1) were hybridized in the same solu-tion for 16 h. For low stringency hybridization the blot waswashed twice at room temperature in 2 · NaCl/Cit, 0.1%SDS for 10 min followed by two 30 min washes at 50 °Cin1 · NaCl/Cit, 0.1% SDS. Autoradiography was carried outwith HyperﬁlmTMMP (Amersham).Ó FEBS 2002 Guinea pig CYP11B genes (Eur. J. Biochem. 269) 3839Construction of expression plasmidsFor the construction of pCMV/11B2, pRc/CMV wasdigested with Bsp120I, trimmed with Pfu polymerase(Stratagene) and subsequently digested with NotI. Likewise,pCR2.1/HG17 was digested with SpeI, trimmed with Pfupolymerase and digested with NotI to release a fragmentcomprising the ORF of the guinea pig CYP11B2. Thisfragment was ligated using NotI/blunt into the eukaryoticexpression vector.Hydroxylation assaysCOS-1 cells were maintained as described previously .Transfections were carried out using LipofectAMINETM(Gibco/BRL) according to the manufacturer’s recommen-dations. One mL of transfection mix contained 2 lgoftherespective expression construct together with 1 lg pBAdx4(bovine adrenodoxin; gift of M. R. Waterman, VanderbiltUniversity, Nashville, TN, USA) and 6 lLLipofectAMINETMunless stated otherwise. Twenty-fourh after transfection cells were incubated with appropriatesubstrates for 48 h using [1,2-3H]cortisol, [14C]11-deoxy-corticosterone or [1,2–3H]androstenedione, respectively, astracers. Media were extracted and analysed by highperformance TLC as described previously .Phylogenetic analysesPhylogenetic analyses were conducted using thePHYLIPpackage (Version 3.5c, 1993) .The sequences have been submitted to GenBank underthe accession numbers AF191278, AF191279 (forCYP11B1), AF191281, AF191280 (for CYP11B2), andAF191282 (for CYP11B3).RESULTSIn a previous study we isolated an 11b-hydroxylase of theguinea pig  by screening an adrenal cDNA library with aPCR ampliﬁed orthologous probe. Upon expression, theisolated cDNA turned out to be a pure 11b-hydroxylasewith no detectable 18-hydroxylation activity suggesting theexistence of additional isoenzymes of the CYP11B subfam-ily in the guinea pig. To investigate this notion, a Southernblot was performed utilizing an exon-1-speciﬁc probe ofCYP11B1 under low stringency conditions and digesting thegenomic DNA with various restriction endonucleases thatdid not cut within exon 1. The result (Fig. 1) stronglysuggested the existence of at least three different genes asjudged from the appearance of three bands if the DNA was,e.g. digested with EcoRI/EcoRV, XbaI, or XbaI/HindIII.Although guinea pigs had been sodium depleted tostimulate the expression of a putative aldosterone synthaseas much as possible , repeated screening of the cDNAlibrary did not result in the identiﬁcation of any cDNAother than CYP11B1 (data not shown). Thus, we devisedanother strategy for the identiﬁcation of additional genes ofthe CYP11B subfamily in the guinea pig. To this end, agenomic library was screened under low stringency (seeExperimental procedures) utilizing a full-length guinea pigCYP11B1 cDNA as a probe. As opposed to a cDNAlibrary, screening of a genomic library should yield clones inrelation to their abundance in the genome rather than theirrelative abundance due to differential expression. Indeed,this approach lead to the isolation of eight genomic clonesthat were classiﬁed into three subgroups based on restrictiondigests and hybridization experiments (data not shown).One clone termed kHG13 turned out to represent theCYP11B1 gene while kHG17 and kHG15 representedclosely related genes of the CYP11B family demonstratedby similarities of > 75% at the nucleotide level. They weretentatively named CYP11B2 and CYP11B3, respectively.To clone the corresponding cDNAs, the RACE tech-nique was used. PolyA+RNA was converted into a double-stranded cDNA pool and adapters comprising the promotersequence of the T7 bacteriophage were ligated to both ends.The sequences of the T7 promoter are extremely rare ineukaryotic genomes and thus convey a high degree ofspeciﬁcity in subsequent PCR reactions. Using a primercombination of an adapter primer and gene-speciﬁc sense orantisense primers, respectively, we were able to amplify twooverlapping fragments in case of kHG17. Upon sequencingof these cDNA fragments the complete sequence of thecDNA of CYP11B2 could be deduced. It comprised2611 bp and an ORF of 1503 bp coding for a putativemitochondrial preprotein of 501 amino acids with acalculated molecular weight of 57.7 kDa (Fig. 2). AfterLeu24 a cleavage site for the matrix-associated protease waspredicted resulting in a mature mitochondrial protein of55 kDa. The deduced amino acid sequence showed 81%similarity to the guinea pig CYP11B1 and 80% similarity tothe human CYP11B2, respectively (see below). The 3¢-UTRcomprised 1079 bp with a canonical polyadenylation site16 bp upstream of the polyA tail with no indications for theexistence of alternative poly adenylation sites (Fig. 2).We next investigated the expression of the CYP11Bgenes. A Northern blot probed with a CYP11B2-speciﬁcprobe showed a single band of 2.9 kb (data not shown)which is consistent with the length of the isolated cDNA forCYP11B2 assuming a polyA tail of 200–300 adenineresidues. To see where the CYP11B genes were expressedFig. 1. Southern blot analyses with a CYP11B1 exon 1-speciﬁc probe.Fifteen micrograms of guinea pig genomic DNA was digested withthe indicated endonucleases. After transfer, membranes were probedunder low stringency conditions with an exon 1-speciﬁc probe ofCYP11B1 (nucleotides 1–141; see Experimental procedures fordetails). Sizes of fragments are indicated on the right.3840 H. E. Bu¨low and R. Bernhardt (Eur. J. Biochem. 269) Ó FEBS 2002and whether they played a role during postnatal develop-ment we used a highly sensitive RNAse protection assaywith RNAs from different tissues and developmental stages.As shown in Fig. 3, expression of both the 11b-hydroxylaseand the aldosterone synthase was exclusively in the adrenalgland. Moreover, there was no difference in expressionbetween postnatal day 1 and the adult stages suggesting thatthe genes were not differentially regulated during postnataldevelopment. With respect to kHG15 we were not able todemonstrate expression of the gene in adult tissues usingRT/PCR with various gene-speciﬁc primer combinations(data not shown). Thus, this clone might represent apseudogene of the CYP11B family or a gene that is notexpressed in adult tissues.To compare the enzymatic activities of CYP11B2 andCYP11B1, the cDNAs were cloned under the control of aviral promoter and transiently transfected into COS-1 cells.Transfected cells were incubated with different substratesand the resulting metabolites were analysed using TLC. Asseen in Fig. 4A, CYP11B2 converted 11-deoxycorticoster-one to corticosterone and both 18(OH)-corticosterone andaldosterone. These results clearly demonstrate thatCYP11B2 is the aldosterone synthase of the guinea pig asit is capable of modifying position 11 and 18 of the steroidring. In contrast, CYP11B1 produced only corticosteroneand traces of 18/19(OH)-deoxycorticosterone, conﬁrmingearlier results . Furthermore, CYP11B2 transfected cellsefﬁciently 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 alsoused androstenedione as a substrate. Under the experi-mental conditions large amounts of 11b(OH)-androstendi-one were synthesized by CYP11B2 in comparison withCYP11B1 (Fig. 4C). It is noteworthy, that CYP11B2displayed a higher enzymatic activity than CYP11B1 basedon 11b-hydroxylase activity. These differences were highestFig. 2. Sequence of the CYP11B2 cDNA of the guinea pig. The nucleotide sequence and the deduced amino acid sequence are both shown. The ORF(putative start and stop codon underlined) encodes a mitochondrial preprotein with a calculated molecular mass of 57.7 kDa. An arrowheadindicates the presumptive cleavage site for the mitochondrial matrix associated protease. Numbers on the left denote amino acids, those on the rightindicate nucleotides. A canonical polyadylation site is shown boldface.Ó FEBS 2002 Guinea pig CYP11B genes (Eur. J. Biochem. 269) 3841for androstenedione (fourfold) and lowest for 11-deoxy-cortisol (Fig. 4). This shows a higher substrate speciﬁty ofCYP11B1 which could be due to differences in the activecentre and/or the entry channel. Moreover, it could beimportant for tissue-speciﬁc synthesis of glucocorticoidsgiven the differences in expression levels of the twoenzymes.We next asked whether other accessory proteins mightcontribute to the zone-speciﬁc synthesis of steroid hor-mones. A good candidate is adrenodoxin, an iron sulfurcontaining electron donor protein that is required for thefunction of mitochondrial cytochrome P450 proteins 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 oromitted. After transfection, cells were incubated with11-deoxycorticosterone as a substrate. As shown in Fig. 5,the omission of Adx leads to a sharp decrease in the activityfor the 11b-hydroxylase, CYP11B1. Intriguingly, however,the 11b-hydroxylase activity of the aldosterone synthaseCYP11B2 was basically unaffected whereas the 18-hydroxy-lation and oxidation potential were abrogated almostcompletely. These results indicate clear structural differenceson the surface of these proteins involved either in glucocor-ticoid or in mineralocorticoid biosynthesis despite a highdegree of similarity between the two isoenzymes. Moreimportantly, these results indicate a new level of regulationfor tissue-speciﬁc aldosterone synthesis depending on theavailability of reducing equivalents.One intriguing question is how and when animalsdeveloped a hormonal system that differentially regulatedthe control of both electrolyte/volume homeostasis andglucose metabolism. Knowing when and how differentialsynthetic pathways for mineralocorticoids and glucocorti-coids developed would lead to deeper understanding ofthese important evolutionary processes. Because the guineapig’s taxonomical classiﬁcation is controversial [12,13], thisspecies is extremely interesting in terms of vertebrateevolution and might provide insight into some aspects ofthe evolution of the hormonal system.Fig. 4. Enzymatic acivities of CYP11B2. COS-1 cells were transfectedwith pBAdx4 (bovine adrenodoxin) and the expression plasmidpCMV5  (CYP11B1), pRc/CYP11B2 (CYP11B2), or pRC/CMV(mock), respectively. Twenty-four h after transfection cells wereincubatedfor48hwith5lM11-deoxycorticosterone (DOC) includ-ing 4 nCiÆmL)1[14C]DOC (A), 5 lMandrostendione including0.5 lCiÆmL)1[3H]androstendione (B), or 2.5 lM11-deoxycortisolincluding 0.5 lCiÆmL [3H]11-deoxycorticosterone (C). Subsequently,steroids were extracted and separated by TLC . In culture mediumincubated substrates served as an additional control (substrate).Positions of cold standards are denoted on the left. On the rightpercentage of total radioactivity or relative activity is given ± SD; dataare from at least two diﬀerent experiments performed in triplicate.Fig. 3. Tissue and age-speciﬁc RNAse protection assays. DiﬀerentamountsoftotalRNAfromdiﬀerenttissuesandstagesasindicatedwere hybridized in solution with CYP11B1 and CYP11B2-speciﬁcprobes. Both probes were chosen from the 3¢ untranslated regions ofthe genes where sequence divergence was maximal between the twoisoenzymes. Following RNAse digestion the probes protected a 210nucleotide fragment of CYP11B1 (corresponding to nucleotides 1491–1700 ) or a 240 nucleotide fragment for CYP11B2 (corresponding tonucleotide 1511–1750; Fig. 2), respectively. A control lane withoutRNAse (–RNAse) shows the corresponding undigested riboprobes of242 nucleotides (for CYP11B1) and 272 nucleotides (for CYP11B2). Amolecular size marker is given on the left. Diﬀerent developmentalstages are denoted on the right: P1, postnatal day 1; adult.3842 H. E. Bu¨low and R. Bernhardt (Eur. J. Biochem. 269) Ó FEBS 2002To answer the question how these genes might haveevolved we determined the complete genomic structure ofboth genes. As shown in Fig. 6, both genes exhibit thetypical organization of the family, characterized by nineexons and eight introns supporting the idea of a geneduplication event. It is, however, noteworthy that the exonsare grouped into three clusters comprising exons 1 and 2,exons 3–5 and exons 6–9, respectively. These clusters areseparated by intron 2 and intron 5, which are not onlylarger than any other intron but also show considerabledifferences in sequence similarity. For example, an align-ment  of intron sequences requires the introduction of 11and 14 gaps for intron 2 and 5, respectively, as compared to1 (intron 7) to 7 (intron 6) for the remaining introns. Finally,the 3¢ UTRs of the guinea pig CYP11B genes show only42% similarity whereas the 3¢UTR of the guinea pigCYP11B2 gene shares up to 72% identity with thecorresponding human homologue indicative of a closerelationship of CYP11B2 sequences between species.Comparing the protein sequences of the CYP11B family(Table 1) we were surprised to ﬁnd that both CYP11B1 andCYP11B2 of the guinea pig are always more closely related(or equal) to the CYP11B2 sequences of other species.Moreover, the guinea pig CYP11B2 is always more similarto CYP11B proteins of other species than CYP11B1 of theguinea pig (compare lines A and B). Together these resultssuggest that the CYP11B2 genes are the primordial genesand that a common ancestor containing both enzymaticactivities was duplicated. The resulting two genes subse-quently evolved to give both different regio-speciﬁcities anddifferential regulatory circuits.We next asked when the aforementioned gene duplicationevent might have occurred. To this end we conductedphylogenetic analyses with all known sequences of theCYP11B family of proteins. Including the sequences of theguinea pig with its highly controversial taxonomical posi-tion into this highly homologous family of proteins couldpossibly give new insights into both its taxonomicalclassiﬁcation and the evolutionary relationships within thisprotein family. Also, these analyses might indicate when thegene duplication event occurred that subsequently led toisoenzymes harbouring different enzymatic activities like,for example, those in humans or to an exclusively differen-tial regulation like seen in cattle. Amino acid sequences of 16CYP11B proteins were subjected to phylogenetic analysesusing two fundamentally different methods. The use ofvarious methods should provide an estimate of methodicalerrors. On the one hand, two distance matrix methods, theUPGMA (unweighted pairgroup method using arithmeticmean) and the neighbor joining method (reviewed in ),were used. The distance matrices for the calculation ofphylogenetic trees were produced with three differentalgorithms for amino acid exchanges, namely the Dayhoffmodel , Kimura’s model  and the categories modeldeveloped by Felsenstein . On the other hand, themaximum parsimony method as a single character statealgorithm was used to evaluate the phylogenetic relation-ships between these proteins. This approach assumes themost probable phylogeny to be the one that requires thefewest nucleotide exchanges . The frog was used as anoutgroup in all applications and the reliability of a giventopology was assessed using the bootstrapping procedure.The results are depicted in Fig. 7. No matter whichalgorithm was used, the guinea pig sequences were groupedtogether with bootstrapping probabilities of at least 98%.The maximum parsimony method placed the guinea piginto one group with rodents requiring 1349 nucleotideexchanges thus supporting monophyly of the rodents.Within the rodents the branching consistently grouped theorthologues of rat and mouse together demonstrating theclose relationship between these species. In contrast, bothdistance matrix methods showed the guinea pig togetherFig. 5. Inﬂuence of bovine adrenodoxin on enzymatic activities. COS-1cells were transfected with pBAdx4 (bovine adrenodoxin) and theexpression plasmids pCMV5  (CYP11B1), pRc/CYP11B2(CYP11B2), or pRC/CMV (mock), respectively. Twenty-four hoursafter transfection, cells were incubated for 48 h with 5 lM11-deoxy-corticosterone (DOC) including 4 nCiÆmL)1[14C]DOC. Metaboliteswere analysed by TLC. Enzymatic activity is given as relative radio-activity. White bars represent cotransfection with adrenodoxin (¼ 100)andblackbarsrepresentnocotransfectionexpressedinrelationto100 ± SD; n ¼ 9 for each data point.Fig. 6. Genomic structure of CYP11B1 and CYP11B2 of the guinea pig.Shown is the complete genomic structure of CYP11B1 and CYP11B2.Exon and intron boundaries are indicated. Scale bar represents 1000nucleotides.Table 1. Pair-wise sequence similarities of CYP11B proteins. Similarities for the CYP11B proteins were determined pair-wise using thePALIGNprogram (PcGENE, Intelligenetics) for the proteins from human (HS), mouse (MM), rat (RN) hamster (MA), guinea pig (CP), sheep (OA), cow(BT), pig (SS) and frog (RC).HSB1HSB2MMB1MMB2RNB1RNB2MAB1MAB2CPB1CPB2OAB0BTB0SSB0 RCB0CP B1 74% 75% 72% 76% 73% 76% 72% 74% – 81% 73% 73% 72% 58% ACP B2 80% 80% 75% 80% 77% 79% 76% 79% 81% – 77% 77% 77% 58% BÓ FEBS 2002 Guinea pig CYP11B genes (Eur. J. Biochem. 269) 3843with artiodactyls and primates, i.e. supported the paraphylyof the order rodentia. The bootstrapping probabilities werehowever, comparatively low (Fig. 7D) using the neighborjoining approach with 49, 52 or 68% for the categoriesmodel, the Dayhoff model or Kimura’s model, respectively.Using UPGMA the probabilities were slightly higher;between 70 and 82%. Moreover, the hamster proteins werenow assigned to their rat and mouse paralogues. Interest-ingly, Kimura’s model consistently produced the highestbootstrapping probabilities for a given topology.DISCUSSIONIn this paper, we describe the isolation and characterizationof the CYP11B genes of the guinea pig. In an earlier study we isolated a single cDNA using an orthologous probethat had been obtained using degenerated primers to screena guinea pig adrenal cDNA library. This cDNA proved tocode for the abundantly expressed 11b-hydroxylase,CYP11B1, of the guinea pig which exhibited exclusive11b-hydroxylase activity . Although cloning strategiesusing PCR-based approaches with degenerated primershave often been successful this is sometimes hampered bylarge differences in expression levels. Thus, we were unableto isolate more than one cDNA of the CYP11B family ofthe guinea pig either by PCR-based approaches or byrepeated screening of the library under low stringencyconditions, even under conditions in which sodium wasdepleted to achieve induction of the aldosterone synthasegene. The enzymatic activity of the cloned protein, however,strongly suggested the existence of additional isoenzymeswith different enzymatic activities. To test this hypothesis,we performed a Southern blot analysis using an exon 1-spe-ciﬁc probe of CYP11B1. This experiment indicated theexistence of at least two additional genes of this family in theguinea pig. To clone these genes we screened a genomiclibrary which should circumvent difﬁculties associated withlargely differing expression levels. This led to the isolation oftwo additional genes, tentatively named CYP11B2 andCYP11B3.ForCYP11B2,wewereabletoisolateacDNAwith a complete ORF. In contrast, no speciﬁc transcriptscould be detected for CYP11B3 in sensitive RT-PCRexperiments with RNA from adult animals. Thus, this genemight be a pseudogene that evolved as a consequence of asecondary gene duplication event (see below). Similarobservations have also been made in cows that have ﬁvegenes of the CYP11B family only two of which are functional. Alternatively, CYP11B3 might be a developmentallyregulated gene as in rats, in which it is expressed solely duringa few speciﬁc days of postnatal development .However, no differential expression was observed foreither CYP11B1 or CYP11B2 during postnatal develop-ment of the guinea pig using RNAse protection assays.Instead, we saw exclusive adrenal expression of both genes.This does, however, not rule out expression in other tissuesat lower levels. For example, expression of CYP11B genes inthe rat has been demonstrated in brain  and in the heart using very sensitive RT-PCR and in situ hybridizationtechniques. The physiological signiﬁcance of this low levelexpression remains unclear.To compare the catalytic activities of the guinea pigCYP11B isoenzymes we cloned the cDNAs downstream ofa cytomegalovirus promoter to drive expression in COS-1cells. This system has been proven suitable for thecharacterization of enzymes of the steroidogenic pathway. These analyses demonstrated a potent 18-hydroxyla-tion and 18-oxidation activity of CYP11B2 using varioussubstrates thus showing it to be the aldosterone synthaseof the guinea pig. It was interesting to note that CYP11B2of the guinea pig had a considerably higher enzymaticactivity in terms of 11b-hydroxylated product formedthan the guinea pig CYP11B1. These results are in contrastwith ﬁndings in other species. For example, the humanCYP11B1 has a 20-fold higher activity towards 11-deoxy-cortisol than CYP11B2 . Moreover, the activity of theguinea pig CYP11B2 compared to CYP11B1 could becorrelated to the size of the C17 substituent of thesubstrate. Thus, the differences were most pronouncedwith androstenedione and least with 11-deoxycortisol as asubstrate. These results might indicate steric hindrance inthe entry channel of the cytochrome P450 enzymes withCYP11B1 being more selective for its proper substrate.This would contribute to the zone-speciﬁc synthesis ofglucocorticoids given the vast differences in expression levelof the two genes.In a second set of experiments, we investigated thesigniﬁcance of adrenodoxin, an iron sulfur protein that isessential for electron transfer from adrenodoxin reductaseto mitochondrial cytochrome P450 enzymes . Adreno-doxin has been shown to signiﬁcantly increase enzymaticactivities of steroidogenic enzymes in transfection experi-ments . If this cotransfection was omitted, the twoguinea pig enzymes showed differential properties. The 11b-hydroxylase activity of CYP11B1 was strongly reduced(Fig. 5), whereas that of the aldosterone synthase wasbasically unaffected, while the 18-hydroxlase and oxidaseactivity was also greatly diminished. These differences canFig. 7. Phylogenetic analyses. Shown are the phylogenetic treesobtained by the maximum parsimony method (A), the UPGMAmethod (categories model) (B), and the neighbour joining method(categories model) (C). Numbers represent bootstrapping probabilitiesof 1000 replicates. The Table in D gives the bootstrapping probabilitiesfor monophyly and paraphyly of the order rodentia in detail. Pr,primates; Ar, artiodactyls; My, myomorphs; Cp, guinea pig.3844 H. E. Bu¨low and R. Bernhardt (Eur. J. Biochem. 269) Ó FEBS 2002be explained by a lower binding afﬁnity of CYP11B1 toadrenodoxin when compared with CYP11B2. CYP11B1has a tryptophan at position 366 while all other proteins ofthe CYP11B family including CYP11B2 of the guinea pighave a basic residue (arginine or lysine, respectively) at thecorresponding position. These two basic residues have beenshown in site-directed mutagenesis experiments to be ofsigniﬁcance to the electrostatic interaction of bovineCYP11A1 (cytochrome P450SCC), a closely related mito-chondrial protein with adrenodoxin . Accordingly, the11b-hydroxylase activity of CYP11B2 was hardly affectedby omission of cotransfected adrenodoxin. However, thesubsequent enzymatic reactions involving the 18-hydroxy-lation and oxidation were severely impaired. This couldindicate altered binding afﬁnities for adrenodoxin afterthe 11b-hydroxylation. In this regard it is interesting tonote that in vitro experiments with puriﬁed bovineCYP11B0 indicate a conformational change of the proteindue to rearrangement of the substrate after the ﬁrsthydroxylation step . This might account for an alteredbinding site for adrenodoxin or modiﬁed binding afﬁni-ties. Also, experiments in a reconstituted system withbovine CYP11B0 using mutant forms of adrenodoxin thathave increased electron transfer capabilities showed a shiftin the spectrum of products formed towards compoundsmodiﬁed at position 18 . Interestingly, studies with themicrosomal cytochrome P450 enzyme 17a-hydroxylase/17,20-lyase demonstrated a dependence of the more electronconsuming lyase reaction on the presence of high concen-trations of the electron donor protein . Taken together,our results with the guinea pig suggest a new regulatory levelof aldosterone synthesis by the availability of reducingequivalents. In the light of results with the bovine andhuman enzymes  this might be a more commonly usedmechanism which could be crucial for the zone-speciﬁcbiosynthesis of mineralocorticoids. In this respect it wouldbe interesting to see whether expression of adrenodoxin isdifferentially regulated.To investigate the evolution of the CYP11B genes, wecompared protein sequences of the CYP11B family. Theseanalyses showed that CYP11B2 proteins are more closelyrelated to each other than CYP11B1 proteins across species.Furthermore, the 3¢ UTR of the guinea pig CYP11B2shows considerable similarity to its human counterpartwhile exhibiting only low resemblance to its paralogue in theguinea pig. On a functional level the CYP11B1 proteins alsoshow greater differences across species. For example, thehamster CYP11B1 produces 50% of 19-hydroxylatedproduct besides the 11b-hydroxylated steroid . Moreover,as mentioned above, human CYP11B1 is a very potent 11b-hydroxylase as compared with CYP11B2 while in theguinea pig this situation is reversed. These ﬁndings suggestthat CYP11B2 genes, i.e. those encoding bipotent enzymes,are the primordial genes that underwent a subsequentduplication and, as a consequence, the CYP11B1 genesevolved independently in different species within the limitsof functional constraints.To see how this evolution might have occurred, wedetermined the complete genomic structure of the twofunctional guinea pig genes. Interestingly, we observed thegreatest differences between these closely related genes withinintron 2 and intron 5. This could indicate frequent recom-bination between the genes for which close association hasbeen shown in mice  and humans . Indeed, in humansan unequal crossover between CYP11B genes, fusing theCYP11B2 gene undercontrol of the CYP11B1 promoter andvice versa, has been demonstrated to cause glucocorticoidremediable aldosteronism, an autosomal dominant disorderleading to severe hypertension , or congenital adrenalhyperplasia . In this context it is interesting to note thatthe crucial determinants for regio-speciﬁcity have beenshown to reside in exon 5 . Moreover, analysis ofbreakpoints in patients suffering from glucocorticoid reme-diable aldosteronism indicate that important regulatoryelements are contained within intron 2 . Thus, a scenariois conceivable where recombination between the two genes inintron 2 and intron 5 eventually lead to both distinct regio-speciﬁcities and/or differential regulation.We next sought to determine when this gene duplicationevent might have occurred. To this end, we conductedphylogenetic analyses with 16 sequences of the CYP11Bfamily of proteins. To assess possible methodical problemswe used different algorithms, namely the maximumparsimony and two distance matrix methods. The maxi-mum parsimony method consistently grouped the guineapig with a bootstrapping probability of 98% into one cladewith rodents thus favouring monophyly of the orderrodentia. This is in contrast with the ﬁndings of Graur andcolleagues  who postulated paraphyly of the order. Ourresults are however, in accordance with the results ofHasegawa et al.  who questioned the paraphyly of theorder rodentia using the same data as Graur. In contrast,the distance matrix algorithms again placed the guinea pigtogether with artiodactyls and primates, thus supportingparaphyly albeit only with bootstrapping probabilitiesbetween 49 and 82%. Interestingly, the highest values wereobtained when Kimura’s model  was used to calculatethe matrices. This might reﬂect the fact that this modelassumes conservative and nonconservative changes to beequally likely which could lead to an overestimation ofconservative changes.According to Frye et al.  the ambiguity of thephylogeny as seen in our analyses with respect to the guineapig can be interpreted as insufﬁcient methodology. Forexample, the data and the algorithms might not be adequateto assign a statistically unambiguous topology if the radia-tion of species has occurred in a sufﬁciently small time frame.Thus, the guinea pig presumably branched off at a very earlytime point within the mammalian radiation irrespective ofthe branching order. Intriguingly, however, the guinea pigpossesses already two functional CYP11B genes as have allother mammals investigated so far. Theoreticallyit is possiblethat the gene duplication occurred in all lines independently.It seems, however, much more likely that the gene wasduplicated in an ancestor mammal. In this respect, it isinteresting that analyses in the frog as an amphibian gave noindications of two CYP11B genes . Thus, the differentialmineralocorticoid and glucocorticoid synthesis is presum-ably an exclusive property of mammals.ACKNOWLEDGEMENTSWe thank K. Denner, B. Bo¨ttner and members of the Bernhardt labfor helpful discussions and advice. We also thank W. 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Analyses of the CYP11B gene family in the guinea pig suggest the existence of a primordial CYP11B gene with aldosterone synthase activity Hannes. the genes of the CYP11B family of the guinea pig. The 11b-hydroxylase of the guinea pig showed higher substratespeciﬁcity than the aldosterone synthase.
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Xem thêm: Tài liệu 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 docx, Tài liệu 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 docx, Tài liệu 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 docx