Eur. J. Biochem. 229, 249-256 (1995) 0 FEBS 1995

Frog cytochrome P-450 (llP,aldo), a single enzyme involved in the final steps of glucocorticoid and mineralocorticoid biosynthesis Yasuki NONAKA', Hiroshi TAKEMORI', Sunil Krishna HALDER', Tiejun SUN', Miho OHTA3, Osamu HATAN04, Akira TAKAKUSU4 and Mitsuhiro OKAMOTO' ' Department of Basic Laboratory Sciences, Osaka University Medical School, Japan

Department of Molecular Physiological Chemistry, Osaka University Medical School, Japan Laboratory of Nutrition, Koshien College, Nishinomiya, Japan

* Department of Anatomy, Nara Medical University, Shijo-cho, Japan

(Received 25 November 1994) - ETB 94 1812/4

A cDNA for cytochrome P-450(11P,aldo) was cloned from a library of bullfrog interrenal tissue (tissue corresponding to the mammalian adrenal gland). The 1919-bp cDNA encoded a protein of 517 amino acids. Its amino acid sequence was highly similar to the sequences of bovine P-450(11P, and rat P-450(11p,aldo) when P-450(11B) family enzymes reported to date were examined. The enzyme ex- pressed in COS7 cells had the 11P-hydroxylation, 18-hydroxylation activities and aldosterone synthetic activity. Northern-blot and immunoblot analyses suggested that a single P-450(1 lP, enzyme was ex- pressed in bullfrog interrenal tissue. These results suggest that a single enzyme catalyzes the final steps of glucocorticoid and mineralocorticoid biosynthesis in bullfrog interrenal tissue as in bovine adrenal gland. A phylogenetic tree of CYPllB genes suggests that the frog enzyme diverged at an earlier evolu- tionary time from other vertebrate enzymes. Immunohistochemical and in situ hybridization studies indi- cated that steroidogenic cells existed in the outer region of interrenal tissue more densely than in the inner region, whereas some medullary cells made clusters like islets. Most of the cells were diffusely distributed in the tissue.

Keywords. Frog; cytochrome P-450(11a) ; CYPll B ; aldosterone synthase; expression.

The final steps of biosynthesis of glucocorticoid and minera- locorticoid from l l -deoxycorticosterone or l l -deoxycortisol oc- cur in adrenocortical mitochondria of vertebrates [l -31. Previ- ous studies have shown that these steps are catalyzed by a single molecule of cytochrome P-450(11P, in bovine adrenal cortex. Thus, all three P-450(11/3) isofoms cloned to date were shown to have not only 1 la-hydroxylation and 18-hydroxylation activi- ties for 11-deoxycorticosterone, but also aldosterone synthetic activity [4-71. In contrast to cattle, two P-450(11P)-related enzymes possessing distinct activities have been isolated from rat [8, 91, mouse [lo], and human [ll] adrenal glands. The CYPllBI product named P-450(1 lp) is responsible for 1 lp-hy- droxylation and 18-hydroxylation of 11 -deoxycorticosterone, but not responsible for aldosterone synthesis [12]. The CYPllB2 product, named P-450(11p,aldo) [8], P-450(aldo) [13], P- 450(AS) [lo] or P-450(~18) [14], produces aldosterone from 11 -deoxycorticosterone in addition to the 1 lp-hydroxylation and 18-hydroxylation products 1121.

Correspondence to M. Okamoto, Department of Molecular Physio- logical Chemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Japan 565

Abbreviations. P-450, cytochrome P-450; P-450(1 la), cytochrome P-450(1 la), i.e. steroid 11P-hydroxylase; F450(11/3,aldo), cytochrome P-450(1 lp,aldo), i.e. aldosterone synthase; CYPllB, P-450(11a) gene ; P-450,, side-chain-cleaving P-450 ; CYPllA, P-450,, gene.

Enzyme. Steroid 1 la-hydroxylase and aldosterone synthase (EC 1.14.15.4).

Note. The novel nucleotide sequence data reported here have been submitted to the GenBankEMBL data banks and are available under accession number D10984.

Earlier, endocrinological studies were reported indicating that aldosterone was not found in fish, except lungfish, and that aldosterone was present in all tetrapoda from amphibia to the more advanced groups. Frog interrenal tissue actively secretes aldosterone, but the nature of the enzyme responsible for the aldosterone production has yet to be determined. Moreover, frog interrenal tissue, unlike mammalian adrenal glands, seems not to be distinctly divided into cortical and medullary portions; a clear division of frog adrenocortical zones has not been estab- lished. Therefore, to identify steroidogenic cells in the latter tis- sue would be an interesting subject of research. To solve these problems and to investigate the molecular evolutionary process of 1 1P-hydroxylation and aldosterone production further, we attempted to isolate cDNA clones of bullfrog CYPIIB.

MATERIALS AND METHODS Cloning and sequencing of bullfrog P-450( llj3,aldo). In-

terrenal tissues from twenty frogs (Rana catesbeiana)were care- fully separated from adjoining kidneys, and immediately frozen in liquid nitrogen. Total RNA (167 pg) was extracted from the tissues (3.6 g) and poly(A)-rich RNA (30 pg) was purified by an oligo(dT)-Sepharose column. The latter was used to construct a cDNA library of bacteriophage AgtlO, and the library was screened using a '*P-labeled bovine pcP-450(1lP)-2 BglII-SphI fragment (1.3 kbp) [9]. Twenty-one positive clones were isolated out of lo4 phages. Inserts were subcloned into the EcoRI site of the pUC 118 plasmid (Takara Shuzo). The Kilo-Sequence defe- tion kit (Takara Shuzo) was used to obtain deleted inserts for sequencing.

250 Nonaka et al. (Em J. Biochem. 229)

Northern and Southern-blot analyses. The poly(A)-rich RNA prepared from frog interrenal tissue or the genomic DNA prepared from frog liver was size-fractionated by electrophoresis according to the methods described previously [ 151. The nucleic acids were transferred to nylon membranes (Hybond-Nf , Amersham). The bovine P-450(11p) cDNA fragment was used as a probe for Southem-blot analysis, and frog P-450(11P,aldo)- (1-1) cDNA was used for Northern-blot analysis.

Immunization with frog P-450(11/3,aldo) protein. A pro- tein antigen for immunization was expressed in Escherichia coli according to the method reported in [16]. A fragment from the BalI site (nucleotide 343 in Fig. 2 ) up to the 3’-terminus (BamHI site in the vector) was prepared, and it was inserted into the SmaVBamHI site of pT7-7 [17]. The plasmid was used for ex- pression. The expressed protein was purified by SDSPAGE. Af- ter electrophoresis, the protein was stained with Coomassie bril- liant blue and destained. The gel band corresponding to the pro- tein was excised and minced. The protein was electroeluted in SDS/electrophoresis buffer (3 g/1 Tris base, 14.4 gA glycine, 1 g/1 SDS). The partial amino acid sequence of the protein confirmed that the sequence began from position R116 of the enzyme. This protein was used for immunization in rabbits without further purification.

Expression of frog P-450(11p,aldo) in COS7 cells. Expres- sion of the cDNA and assays of enzyme activities were per- formed as described before [12]. A clone 1-2 fragment was di- gested by EcoRI and Sac1 (position 1624) to produce a fragment containing the entire coding region. The blunt-ended fragment was inserted into the SmaI site in the multicloning site of pSVL (Pharmacia), and the constructed plasmid was named pSV-frog- P-450(1 lP,aldo)-(l-2). COS7 cells were transfected with the plasmid (50 pg) in the presence or absence of bovine adreno- doxin-expressed plasmid (pSVADX, 10 pg) by electroporation, and the cells were incubated for 24 h in Dulbecco’s modified Eagle’s medium containing 10 % fetal bovine serum. 11-Deoxy- corticosterone (100 nmol/lO ml medium) was added, and the cells were further incubated for 24 h. The medium was recov- ered for the assay of steroids, and the cells were harvested for immunoblot analysis. The steroids were extracted from 5 ml me- dium after adding 5 nmol cortisol. The products were separated by reverse-phase HPLC (S-5 120A ODS column, YMC Co., Kyoto, Japan) with 60% (by vol.) methanol/water at a flow rate of 0.5 mumin, and were measured with an ultraviolet detector (SPD-6AV, Shimadzu, Kyoto, Japan) at 240 nm.

Immunoblot analysis. Mitochondria1 fraction of COS7 cells was prepared by the method reported in [6]. The blotted mem- brane (Immobilon, Millipore) was incubated with primary anti- body (1 : 500 dilution) and peroxidase-conjugated anti-(rabbit- IgG) antibody (1 :300 dilution) (Organon Teknika Co.). The immunostaining kit HRP-1000 (Konica) was used for develop- ment.

Immunohistochemical analysis. Bullfrog interrenal tissue with adjoining kidney was cut into small pieces, and fixed in 4 % paraformaldehyde/NaCl/PL (phosphate-buffered saline, 8 g/l NaCI, 0.2 g/l KCl, 0.2 gA KHzPO,, 1.15 g/1 Na2HP04, pH 7.2) immediately. Serial frozen sections (4 pm) were stained immu- nohistochemically according to the method reported in [6]. The antiserum against bovine tyrosine hydroxylase [18] was a ener- ous gift from Drs Ikuko Nagatsu and Toshiharu Nagatsu fFujita Health University School of Medicine, Aichi, Japan). The anti- body against bovine adrenodoxin reductase was described before [19]. The primary antibody was diluted 500 times for use, and the second antibody-conjugated peroxidase against rabbit IgG was diluted (1 : 900). Control sections were stained by a similar method using pre-immune rabbit serum instead of anti-(bullfrog

Fig. 1. Southern-blot and Northern-blot analyses of bullfrog P- 450(11/?,aldo). Genomic DNA (10 pg) of frog liver was digested by EcoRI (lane l) , BamHI (lane 2), or HindIII (lane 3). The DNA frag- ments were separated by electrophoresis in 0.7% agarose gel. The blot was hybridized with bovine P-450(11/$-2 cDNA. Marker positions (AZfindIII, kbp) are shown on the left. In lane A, poly(A)-rich RNA prepared from frog interrenal tissue (20 pg) was size-fractionated by electrophoresis using a 1 .O% agarose/formaldehyde gel. The blot was hybridized with bullfrog P-450(1 lj?,aldo)-(l-l) cDNA. The positions corresponding to 18s and 28s are shown on the right.

P-450) serum. 3-3’-Diaminobenzidine and H,O, were used for peroxidase substrates.

Analysis by in situ hybridization. The sectioned specimens (6 pm) were hybridized at 50°C for 18 h with digoxigenin-la- beled antisense, or sense, RNA probe according to the method described before [20]. The full-length cDNA was inserted into pSPT19 (Boehringer Mannheim). The digoxigenin-labeled probes were made by transcribing from SP6 and T7 promoters of the vector, and cleaved to a size of less than 1000 nucleotides by alkaline hydrolysis in NaHCO, at 60°C. An alkaline phos- phatase system (Boehringer Mannheim) was used to detect the presence of message.

RESULTS

Southern and Northern-blot analyses. Southem-blot analysis of bullfrog genomic DNA was performed using a bovine P- 450(11p) cDNA as a probe. AS shown in Fig. 1, each lane had a single band, suggesting that frog may have a single CYPIIB. The size of the fragments obtained by digestion with BamHI or HindIII (lanes 2 and 3) was smaller than that of the RNA tran- script (about 1900 bases; see below) and no other hybridized band was found in each lane, indicating that only a part of the DNA fragments of the frog P-450(11p) gene was detected by the bovine probe.

Northern-blot analysis of RNA of interrenal tissues showed a single band of transcript having a size of about 1.9 kb (Fig. 1, lane A). This suggests that only one kind of P-450(11p) enzyme is expressed in the frog interrenal tissue.

Cloning of bullfrog P-450(11m cDNA. Twenty-one positively hybridized clones were isolated from a cDNA library of bullfrog interrenal tissues. Partial sequencing revealed that four clones contained almost full-length inserts. Three inserts out of the four clones were completely sequenced. Clone 1-1 has the longest

25 1

-121

-1

120 40

240 80

360 120

480 160

588 720 240

840 280

558 1080 360

1200 400

1320 440

1440 480

1560 51T

1680

1730

Fig. 2. Nucleotide and deduced amino acid sequence of bullfrog P-450(11/l,aldo). The numbering of nucleotides and amino acids starts at the first methionine codon. The polyadenylation signal (AATAAA) is underlined (1705- 1710) and the stop position is indicated (***). The molecular mass of the protein is M, 59535, and that of the putative mature protein beginning at G46 is M, 54489.

insert (1919 bp). Clones 1-2 and 2-6 had sequences identical to that of clone 1-1 except that they lacked 24 bp and 216 bp at their 5'-terminal regions, respectively. The other eight positive clones were shorter than clone 1-1, but all had sequences iden- tical to clone 1-1, when they were both sequenced for about 300-400 bp from the 5'- and 3'-termini. From these results, we concluded that only one kind of P-450(1 lp) was present in our cDNA library.

Fig. 2 shows the nucleotide sequence of the frog P-450(11p) and its deduced amino acid sequence. The cDNA (1919 bp) was composed of a 189-bp 5'-noncoding region, a 1551-bp open reading frame, a 167-bp 3'-noncoding region, and a 9-bp poly(A) tail. The 5'-noncoding region was long compared with those of other animal species. In the open reading frame, two methionines exist at position 1 and position 15 as possible translation initiation sites. According to the optimal context for initiation [CC(;)CCKCC] [21], the methionine at position 1 is the more plausible initiation site.

Enzymic activities of bullfrog P-450(11g) expressed in COS7 cells. Immunoblot analysis (Fig. 3) suggested that the apparent molecular mass (Mr 51 000) of the frog P-450(11P, expressed in COS7 cells was the same as that in the mitochondria of the interrenal tissue. It should be noted that the enzyme present in the adrenal mitochondria was immunostained as a single band, suggesting that it is a single enzyme.

Steroids produced from 1 1-deoxycorticosterone by P- 450(118) expressed in COS7 cells are shown in Table 1. The enzyme catalyzes 1 lp-hydroxylation, 18-hydroxylation, and aldosterone synthesis at considerable rates, and is thus named P-450(11p,aldo) in this study. It should be noted that relative amounts of the main products, corticosterone, 18-hydroxycorti- costerone, and aldosterone, were very similar to those produced by bovine P-450(11p)-3 [7].

Fig. 3. Immunoblot analysis of mitochondria prepared from frog P-45O(ll/l,aldo)-expressed COS7 cells. Lane 1, mitochondria from frog interrenal tissue (3 pg protein); lane 2, mitochondria of cDNA- transfected COS7 cells (20 pg protein). The origin, the position of migra- tion of P-450(11/3,aldo), and the front are indcated (*). The positions of prestained size markers ( M , X lo-'. BioRad) are also shown (4).

Cotransfection with a vector containing bovine adrenodoxin cDNA (10 pg) had little effect on the enzymic activities of frog P-450( 1 lP,aldo). Cotransfection with more adrenodoxin cDNA (50 pg) could only slightly increase the aldosterone production (data not shown). It has been reported that the activity of bovine P-450,,, expressed in COS cells was enhanced 3.6 times by co- transfection with bovine adrenodoxin cDNA [22]. In the case of expression of rat P-450( 11p> and P -450(1 lP,aldo) cDNAs, the cotransfection with bovine adrenodoxin cDNA (10 pg) enhanced

252 Nonaka et al. ( E m J. Biochem. 229)

Table 1. The enzymic activities of bullfrog P-450(11/?,aldo) and the effect of cotransfection with bovine adrenodoxin cDNA. The extracted products were analyzed by the reverse-phase HPLC. B, 18(OH)B, and Aldo denote corticosterone, 18-hydroxycorticosterone, and aldosterone, respectively. The results represent means i- standard deviations (sigma, n = 3). Numbers in parentheses are amounts of steroids compared to cortico- sterone (%). The activity of bovine P-450(118)-3 [7] is shown for comparison.

Plasmid for P-450(11p) Plasmid for adrenodoxin Activities yielding products

B 18(OH)B Aldo

nmol '24 h-' '10 ml medium-'

Frog Frog Bovine

+ + -

1.0 t 0.3 (16) 0.62 t 0.09 (10)

6.2 -+ 0.2 (100) 6.1 -+ 0.9 (100)

2.7 t 0.1 (44) 2.6 ? 0.5 (43)

10.4 (100) 4.9 (47) 1.8 (17)

Fig. 4. Expression of P-450(11/?,aldo) adrenodoxin reductase, and tyrosine hydroxylase in bullfrog interrenal tissue. (A - C) Serial sections (4 pm) of bullfrog interrenal tissue were immunohistochemically stained using specific antisera: (A) Anti-bullfrog P-450(1 I/3,aldo) antibody; (B) anti-adrenodoxin reductase antibody; (C) anti-tyrosine hydroxylase antibody were used. Most cells of the outer region of interrenal tissue were stained with anti-P-450(1 1/3,aldo) antibody (A) or anti-adrenodoxin reductase antibody (B). Medullary cells were stained by anti-tyrosine hydroxylase antibody (C). Renal cells were not stained. (D, E) Bullfrog P-450(11/3,aldo) was detected by in situ hybridization using: (D) Frog antisense RNA probe; (E) frog sense RNA probe. The outer cells of interrenal tissue located in the middle of the picture contained the message hybridized with the antisense probe (D). The renal cells in the upper side did not contain the message. Both interrenal and renal cells did not react towards the sense probe (E). €3, S, and CV in D indicate kidney, sinusoid, and central vein, respectively. Bar, 200 pm.

steroidogenic activities 2.5-9 times [12]. Comparing the results obtained for the frog enzyme with previous reports, it was con- cluded that frog P-450(1 lj?,aldo) has less affinity toward exoge- nously added bovine adrenodoxin than the intrinsic ferredoxin in COS7 cells.

Immunohistochemistry and in situ hybridization of frog P-450(11/3,aldo). Previous histochemical studies reported that in contrast to mammalian adrenal glands no clear separation between cortical and medullary portions was observed in frog interrenal tissue [18]. In Fig. 4, P-450(11j?,aldo), adrenodoxin reductase, and tyrosine hydroxylase (one of the catecholamine- synthesizing enzymes and a marker enzyme of adrenal medul- lary cells) in interrenal tissue were immunostained using specific antibodies. The specific antibodies against P-450(1 lb,aldo) and adrenodoxin reductase reacted with the cells located in the outer region much more strongly than those in the inner region. In contrast, the antibody against tyrosine hydroxylase stained the

cells that were diffusely distributed in the interrend-tissue-like islets. In situ hybridization studies (Fig. 4) showed that more message of P-450(11b,aldo) existed in the outer region of in- terrenal tissue. It was concluded, therefore, that the cells in the outer region may synthesize steroid hormones more actively than those in the inner region.

DISCUSSION

From our cDNA library of bullfrog interrenal tissue (adrenal gland), essentially only one kind of P-450(11B) cDNA was cloned. Southern and Northem-blot analyses (Fig. I), partial se- quencing of positive clones, and immunoblot analysis (Fig. 3) strongly suggested that a single P-450(11p,aldo) enzyme exists in the interrenal tissue.

The frog P-450( 1 Ib,aldo) enzyme produced considerable amounts of aldosterone and corticosterone (Table 1). The ratio

Nonaka et al. ( E m J. Biochern. 229) 253

H O

I 611

2 4 0

320

400

480

Fig.5. Comparison of deduced amino acid sequences of cytochrome P-450(11) family enzymes. The amino acid sequences fo frog P-450(11BO) (this study), bovine P-450(11BO) [4], rat P-450(11BI) [9], rat P-450(11B2) [8], rat P-450(11B3) [29], mouse P-450(11Bl), and mouse P-450(11B2) [lo], human P-450(11Bl), human P-450(11B2) [ll], rainbow trout P-450(11A) [30], human P-450(11A) [31], porcine P-450(11A) [32], bovine P-450(11A) [33] and rat P-450(11A) [34] were aligned. The numbering is according to bullfrog P-450(11BO). Gaps are indicated as (*). A possible processing site is also indicated (V). Similar regions around the processing site are shown by solid and broken overlines. Highly similar regions are indicated by overlines with numbers. The regions 1-4 have been named oxygen-binding region, Ozols' region, aromatic region, and heme-binding region, respectively. Aldosterone synthases so far determined have conserved G305 and L318 residues which are indicated (0). Identities listed in Table 2 were computed for the sequences between amino acid residues 58-516 (0). Amino acids aligned to gaps were ignored for calculations.

of aldosterone to corticosterone was 0.16, and this value was very similar to that obtained by the bovine enzyme. The reported ratio of the secretion rates of aldosterone to corticosterone in bullfrog (R. catesbeiana) was 0.043 under the physiological con- ditions and the ratio varied from 0.01 1-0.25 under the various experimental conditions such as hypophysectomy, adrenocorti- cotropic hormone administration, and renin administration [23]. Thus, the production ratio of aldosterone to corticosterone ob- tained by the frog enzyme expressed in COS cells seemed to be within a reasonable range.

Immunohistochemical and in situ hybridization studies showed that there exists an active steroidogenic region in bull- frog interrenal tissue (Fig. 4). In bullfrog interrenal tissue, the presence of inactive cells in the subcapsular region during au- tumn and winter has been reported [23], suggesting that zone differentiation may occur in the tissue. The present results ob- tained using frogs during the spring and the summer showed that the cells in the outer region contained much more P- 450(1 lj?,aldo) transcript, P-450(11/3,aldo) protein and adreno- doxin reductase protein than the inner cells. These results agreed

254 Nonaka et al. (Eur: J. Biochern. 229)

Table 2. Identities of amino acids between frog P-450(11BO) and other cytochromes P-450(11B) and P-450(11A). The sequence align- ments were made as shown in Fig. 5. Sequences between residues 58-516, marked in Fig. 5, (0) were compared.

Species P-450 Identity

Bovine Rat Mouse Human Human Trout Mouse Rat Human Porcine Rat Bovine

11BO 11B2 11B2 11B2 11B1 11A IlBl IlBl 11A 11A 11A 11A

%

52.0 50.8 50.1 49.3 48.9 48.7 47.1 46.5 45.0 44.0 44.0 43.9

well with the previous report showing that angiotensin I1 bound only with the outer cells of frog interrenal tissue [24]. Thus, the outer cells expressing P-450( 1 lP,aldo) may produce aldosterone more actively under the control of angiotensin I1 than the inner cells.

The amino acid sequence of bullfrog P-450(11P,aldo) and those of other animal P-450(11p) enzymes are shown in Fig. 5. To avoid confusion, the following nomenclature for the CYPIl family genes and their products is used in this discussion. CYPllB and P-450(11B) are used for all genes and enzymes of the P-450(11p) family, respectively. P-450(11BO) is used when the enzyme is thought to act in both glucocorticoid and minera- locorticoid synthesis under the physiological conditions of rele- vant animals, its gene being named CYPIIBO. Thus, bullfrog P-450(1 lP,aldo) and bovine P-450(11p) are named P-450(11BO) and their genes are named CYPllBO in this study. P-450(11Bl) (CYPIIBI product) and P-450(11B2) (CYPIlB2 product) are used for two P-450(11/3) isozymes of relevant animals, such as rat, mouse, and human; the former has no aldosterone synthetic activity, and the latter is aldosterone synthase. P-450(11A) in- stead of P-450,,, is used for the CYPllA product.

Since the amino acid sequence of the N-terminal portion of bullfrog P-450(11BO) differs considerably from those of other animal P-450(11B) enzymes, we tried to align the frog protein sequence with others so that the amino acid identity around the cleavage sites of these precursor peptides is maximal. Two puta- tive processing sites were considered for the frog enzyme pre- cursor. One was between D45 and G46, shown by an arrowhead in Fig. 5, because the sequence HXARXXG (indicated by a solid line) seemed to be conserved among P-450(11B) enzymes. The other putative processing site was between L20 and D21, be- cause the sequences CLXRXRXLXTT (broken lines) seemed to be conserved. If the processing occurred at the latter site, the sequence alignment of the mature frog protein around the N- terminal region would be difficult.

The amino acid identities between the frog enzyme and the other enzymes ranged from 52.0% for bovine P-450(11BO) to 43.9% for bovine P-450(11A), as shown in Table 2. Interest- ingly, the amino acid sequence of trout P-450(11A) was more similar to that of frog P-450(11BO) than those of mouse and rat P-450(11Bl) enzymes according to this analysis. However, to establish a molecular evolutionary relationship between these molecules, a comparison should be made of their nucleotide se- quences instead of the amino acid sequences, and both synony-

A FROG 1180 BOVINE1180

MOUSE 1182 CYPl l

<RAT11 82 TROUT 11A

BOVINE 11A PORClNEllA

HUMAN11A LRATIIA

8 0 0.1 0.2 0 3 0.4

K:

B

118

CYPl1-

11 1

50 TlME(Mg) 4o

0 10 20

BOVINE 1180 [HUMAN11 81

HUMAN 1182 MOUSE 1 I81 RAT1181 RAT1183 MOUSE1182

I-TROUTllA BOVINE 11A

PORCINE 1 1 A HUMAN 11A

+RAT 11A I I 0 0.1 0.2 a 3

K: Fig. 6. Phylogenetic trees of the CYPll gene family. Phylogenetic trees were constructed by the method described by Li [35], based on the cor- rected differences of (A) amino acid substitutiondsite (c) and (B) syn- onymous substitutiondsite (G). Each difference was corrected by the equation -(3/4) In [l-(4/3) difference] [27]. The scale at the top of B was drawn by postulating that synonymous substitutions occur at the rate of 5.37X10-9 . site-' . y-' [27]. The lengths of the branches in- terrupted by short parallel lines could not be estimated by the present method. The two phylogenetic trees for the CYPllA family differed from each other (A and B). The phylogenetic tree based on synonymous sub- stitutions (B) should reflect the molecular evolution more correctly, be- cause synonymous substitutions are not influenced by the selective pres- sure during evolution and occur at a uniform speed during the evolution- ary time.

mous substitutions and amino acid substitutions should be taken into consideration [25].

Phylogenetic trees shown in Fig. 6 were constructed based on the corrected difference matrices of amino acid substitutions (Fig. 6A) and of synonymous substitutions (Fig. 6B) [25]. The overall features of the two figures were essentially identical, in- dicating that the two results reflected a single evolutionary trait of CYPIIB. As expected, the CYPll family was divided into the CYPllA and CYPllB families [26]. Thus, the frog CYPllBO and the trout CYPllA clearly belong to a different lineage.

It is interesting to note that the CYPllB family could not be grouped on the basis of the enzymic activities of their products, like 11P-hydroxylaselaldosterone synthase [P-450(1 lBO)], al- dosterone synthase [P-450(11B2)], and l lp-hydroxylase [P- 450(11Bl)]. Instead, each animal species seems to have its own cluster of the two enzymes. The CYPIIB family genes were first divided into frog and mammalian clusters. The mammalian cluster was then divided into a bovine-human lineage and a

Nonaka et al. (Eur J. Biochem. 229) 255

rodent lineage. The rodent lineage was further divided into CYPllBl and CYPllB2 lines, each of which consists of rat and mouse genes. In the CYPllBl line, CYPlIBl and CYPllB3 fur- ther diverged only in the rat lineage.

Assuming that synonymous substitutions are not influenced by the selective pressure during evolution and occur at a uniform speed during the evolutionary time [27], the phylogenetic tree constructed by this method could provide information on the time of divergence of the two genes (Fig. 6B) . From these phy- logenetic analyses, we can surmise that: (a) frog P-450(11BO) diverged from the other P-450(1 I B ) genes at the earliest time; (b) rat P-450(11Bl) and P-450(11B3) diverged very recently, about six million years ago; (c) the divergence time of rat and mouse P-450(11Bl) genes was 23 million years ago and that of rat and mouse P-450(11B2) genes was 19 million years ago. These times coincide well with the reported time of divergence of the two species (17 million years ago) [28]. Taking into con- sideration the previous report that a rodent ancestor diverged from the other mammals 75 million years ago [28], these results imply that a common ancestor of rat and mouse already had the two distinct genes, CYPllBl and CYPllB2, for some time, when the two species diverged.

The phylogenetic tree also suggests that human P-450( 1 I B I ) and P-450(11B2) diverged very recently. In this regard, the following observations must be added. First, the difference of synonymous substitutions in a range from the first to fourth ex- ons (0.02-0.1) was considerably lower than that from the eigth to ninth exons (0.2-0.23). Secondly, it has been reported that unequal cross-over involving these two genes occurred in the cases of glucocorticoid-suppressible hyperaldosteronism [36, 371 and gene conversion might occur in the cases of corticoste- rone methyloxidase I1 deficiency [38], suggesting that the human genes seemed to have evolved in parallel by intergenic recombination. Therefore, it may be concluded that the gene conversion occurred very recently between the two genes.

Overall consideration of the phylogeny indicates that an ancestor gene of P-450(11B) diverged into CYPllBl and CYPllB2 after the amphibians diverged, and probably before mammalian divergence, because the mammals whose genes were determined to date possess more than two CYPllB genes. The two related CYPllB genes in mammals have evolved in a different way in each animal species.

The authors thank Drs Ikuko Nagatsu and Toshiharu Nagatsu (Fujita Health University School of Medicine, Aichi, Japan) for generously pro- viding antibody against bovine tyrosine hydroxylase. The authors also thank Koji Wada (student at Osaka University Medical School) for his help in preparing antibody against frog P-450(11@,aldo). Part of this study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan, and from the Ministry of Health and Welfare, Japan.

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