The expression of a functional cDNA encoding the chicken cytochrome P-450arom (aromatase) that catalyzes the formation of estrogen from androgen

A complementary DNA (cDNA) copy of the aromatase P-450 has been isolated from a chicken ovary library using as probe a partial cDNA believed to encode the human placental aromatase. The predicted amino acid sequence of the chicken aromatase cDNA possesses regions of homology to that of its human counterpart, but only limited homology to other cytochrome P-450 enzymes. The introduction of the cDNA clone into COS-1 cells results in the production of high levels of aromatase activity. The chicken enzyme is targeted to the appropriate subcellular fraction in the transfected COS cells, and the apparent Km of the chicken aromatase activity, measured in microsomes prepared from the transfected cells, is similar to that of the enzyme prepared from chicken ovary microsomes. These findings establish that the cDNA clone encodes chicken ovarian aromatase and demonstrate that this protein can catalyze the three successive oxidation reactions necessary to form estrogen from androgen.     

Cloning and expression of a cDNA for human cytochrome P-450aldo as related to primary aldosteronism

A cDNA clone encoding human aldosterone synthase cytochrome P-450 (P-450aldo) has been isolated from a cDNA library derived from human adrenal tumor of a patient suffering from primary aldosteronism. The insert of the clone contains an open reading frame encoding a protein of 503 amino acid residues together with a 3 bp 5'-untranslated region and a 1424 bp 3'-untranslated region to which a poly(A) tract is attached. The nucleotide sequence of P-450aldo cDNA is 93% identical to that of P-450(11) beta cDNA. Catalytic functions of these two P-450s expressed in COS-7 cells are very similar in that both enzymes catalyze the formation of corticosterone and 18-hydroxy-11-deoxycorticosterone using 11-deoxycorticosterone as a substrate. However, they are distinctly different from each other in that P-450aldo preferentially catalyzes the conversion of 11-deoxycorticosterone to aldosterone via corticosterone and 18-hydroxycorticosterone while P-450(11)beta substantially fails to catalyze the reaction to form aldosterone. These results suggest that P-450aldo is a variant of P-450(11)beta, but this enzyme is a different gene product possibly playing a major role in the synthesis of aldosterone at least in a patient suffering from primary aldosteronism.     

Molecular cloning and sequence analysis of cDNA containing the entire coding region for human fetal liver cytochrome P-450

From a human fetal liver cDNA library, a cDNA clone (lambda HFL33) containing the entire coding region for a form of cytochrome P-450 related to P-450 HFLa was obtained. The clone was 1,971 bp long and had an open reading frame of 1,509 nucleotides coding for a 503 amino acid polypeptide. The nucleotide and the deduced amino acid sequences of lambda HFL33 were very similar to but clearly distinct from those of NF25 and HLp cDNAs, which code for forms of cytochrome P-450 in adult human liver. The deduced N-terminal amino acid sequence of the HFL33 protein was identical to that of P-450 HFLa.     

Characterization of a cDNA for the unexpressed form of cytochrome P-450g from the (-g) rat and differentiation of its mRNA from that of the (+g) phenotype using specific oligoprobes

Our laboratory recently isolated a cDNA for cytochrome P-450g (IIC13), a male-specific, highly polymorphic P-450 isozyme, from livers of the high phenotype (+g) of Sprague-Dawley rats [McClellan-Green et al. (1989) Biochemistry 28, 5832-5839]. Hybridization studies using a specific oligonucleotide probe for P-450 (+g) indicated that equivalent amounts of P-450g mRNA were present in livers of both the high and low phenotypes (+g and -g) of male Sprague-Dawley, Fischer (inbred -g), or ACI (inbred +g) rats. In the present study, we isolated one full-length and one nearly full-length cDNA clone coding for the unexpressed form of cytochrome P-450g from a cDNA library constructed from mRNA from a (-g) male Sprague-Dawley rat. The longest cDNA had an open reading frame of 1473 nucleotides which coded for a 490 amino acid polypeptide of Mr 55,839. Although the 5'-noncoding leader sequence and the 3'-noncoding region were unchanged, the coding sequence of the (-g) phenotype differed from that of the cDNA isolated from the (+g) phenotype by nine bases changes. These base changes would result in seven amino acid differences between the protein sequences for the two phenotypes. Two specific oligonucleotide probes for (+) P-450g and (-) P-450g containing three base differences between the (+g) and (-g) sequences hybridized differentially to mRNA from the (+g) and (-g) phenotypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of aromatase in estrogen-producing cells

Human adipose stromal cells in monolayer culture aromatize androstenedione to estrone. The rate of aromatization is stimulated 20- to 30-fold by glucocorticoids when fetal calf serum is present in the culture medium and by dibutyryl cyclic AMP in the absence of serum. The action of dibutyryl cyclic AMP to stimulate aromatase activity is potentiated markedly by phorbol esters and inhibited by growth factors, such as EGF. In order to investigate the mechanisms underlying this multifactorial regulation, we have prepared polyclonal and monoclonal antibodies specific for aromatase cytochrome P-450. By use of these antibodies it was demonstrated that the action of these various factors to regulate aromatase activity was caused by alterations in the rate of synthesis of aromatase cytochrome P-450, whereas the synthesis of the reductase component of the aromatase enzyme complex was relatively unaffected. The changes in the rate of synthesis of aromatase cytochrome P-450 were, in turn, reflective of changes in the levels of translatable mRNA specific for this protein. In order to analyze the levels of aromatase cytochrome P-450 mRNA directly, we have isolated a cloned cDNA insert complementary to the mRNA encoding aromatase cytochrome P-450, by screening a lambda gt 11 human placental cDNA library utilizing the polyclonal anti-aromatase P-450 IgG. Use of this cDNA probe in Northern analysis of RNA extracted from human adipose stromal cells revealed that the changes in translatable mRNA resulting from incubation of the cells with the various regulatory factors were due to changes in the absolute levels of mRNA encoding this protein.     

Molecular cloning and sequence analysis of cDNA encoding human cholesterol 7 alpha-hydroxylase

A complete cDNA clone encoding human cholesterol 7 alpha-hydroxylase has been isolated using a rat P-450ch7 alpha cDNA insert [(1989) FEBS Lett. 257, 97-100] as a probe and totally sequenced. The cDNA contained 1512-base pair open reading frame encoding 504 amino acid residues (Mr 57,630), 39-base pair 5'-untranslated region 1322-base pair 3'-ultranslated region including 20 nucleotides of poly A tail in the total length of 2873 base pairs. The deduced amino acid sequence showed 82% similarity to rat P-450ch7 alpha. Unique amino acid residues were observed in putative binding domains for heme and steroid which are highly conserved in most steroidogenic P-450s.     

Characterization of the cytochrome P-450IID subfamily in bovine liver. Nucleotide sequences and microheterogeneity.

To elucidate the molecular mechanisms underlying drug detoxification, the structures of the members of the microsomal cytochrome P-450IID subfamily were analyzed by isolating, mapping and sequencing cytochrome P-450IID (CYP2D) cDNA clones from bovine liver. The screening was performed under nonstringent conditions so that most of the P-450IID subfamily members could be obtained. 114 of the 147 positive clones were classified into four groups on the basis of their restriction-enzyme maps. The maps of the four groups were highly similar, however, the clones of one group contained an insertion of approximately 500 bp in the coding region. Analysis of partial nucleotide sequences of several representative clones from each group showed that the bovine P-450IID subfamily in liver consisted of several, not many, highly similar members, differing by less than 7% in their nucleotide sequences. The location of the insertion found in the minor group corresponded to intron 7 and the GT/AG rule was found at the exon/intron boundary, suggesting that intron 7 was retained in this group. The complete nucleotide sequences of two clones from the major group were examined to determine the structures of the P-450IID subfamily in bovine liver. A full-length cDNA clone (1615 bp) and a partial cDNA clone (1538 bp) contained open reading frames encoding 500 and 487 amino acid residues, respectively. The partial clone lacked the nucleotide sequence corresponding to the first 13 N-terminal amino acid residues. The deduced amino acid sequences of the two clones were 98% similar, and 80% and 68% similar to those from human CYP2D6 and rat CYP2D1, respectively. Comparisons of the amino acid sequences of the P-450IID subfamily members showed the highly conserved C-terminal region of their molecules and the high similarity between the members in one species, especially in cattle and man.     

Isolation of a full-length cDNA encoding mouse aromatase P450

A full-length cDNA clone for aromatase P450 has been isolated from a pregnant mouse ovarian cDNA library. The insert of this clone (2394 bp) contains a 1509-bp open reading frame encoding 503 amino acid residues together with a 46-bp 5'-untranslated stretch and an 839-bp 3'-untranslated region to which a poly(A) tract is attached. Northern blot analysis of ovarian RNA from pregnant mice reveals a major mRNA band of 2.5 kb with a minor band of 2.1 kb. Comparison of mouse aromatase P450 with that of rat, human, and chicken shows 91, 81, and 69% identity in the nucleotide sequence and 92, 79, and 69% identity in the deduced amino acid sequence, respectively. The membrane-spanning domain of mouse aromatase P450 is estimated to be an extremely hydrophobic segment located within the N-terminal region of the molecule. Furthermore, a highly conserved heme-binding domain is noticed.     

Immunologic identification of the aromatase enzyme system in human endometrium

Confluent human endometrial stromal cells were cultured in medium with no hormone or supplemented with medroxyprogesterone acetate (MPA), estradiol (E2), and porcine relaxin (RLX) for 5 days. These stromal cells were then labeled with [35S]methionine for 3 h. The radioactive proteins in the particulate fraction of cell homogenate were extracted by detergent and incubated with antisera to purified placental aromatase cytochrome P-450 (P-450arom) and NADPH-cytochrome P-450 reductase to isolate the radio-labeled aromatase enzyme components. Analysis of the radio-labeled protein, isolated by antibody to the cytochrome P-450arom from different preparations (P45FBIII or R-8-2) showed a major band at molecular weight 54k on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The intensity of 54k band was stronger in hormone treated stromal cells than that of control in parallel with the increase of aromatase activity. The radio-labeled protein isolated by anti-NADPH cytochrome P-450 reductase, REDFBIV, showed a major band at the molecular weight 73k on SDS-PAGE with comparable intensity in control and hormone treated samples. Thus, the apparent molecular weights of endometrial cytochrome P-450arom and cytochrome P-450 reductase were identical to placental aromatase enzyme system. When a secretory endometrium and a decidua were labeled with [35S]methionine, the cytochrome P-450arom was detected only in the decidua. NADPH cytochrome P-450 reductase was detected both in the endometrium and the decidua. These results show that antisera to placental aromatase enzyme system cross reacts with the endometrial aromatase enzyme components. The synthesis of cytochrome P-450arom was stimulated by MPA, E2 and RLX while the synthesis of the NADPH-cytochrome P-450 reductase aromatase component was not affected by the hormone.     

Molecular cloning and sequence analysis of guinea-pig NADPH-cytochrome P-450 oxidoreductase [corrected]

A cDNA clone coding for cytochrome P-450 oxidoreductase was isolated from a guinea-pig liver cDNA library. The cDNA, MSr2, contained a complete coding region of 678 amino acids. The amino acid sequence of the guinea-pig cytochrome P-450 oxidoreductase showed approx. 90% identities with those of rat, human, rabbit, pig enzymes indicating conservation of primary structure of the enzyme during evolutionary divergence of species. The high conservation of acidic residues of the enzyme sustained the importance of them to maintain its function [corrected].     

Use of molecular probes to study regulation of aromatase cytochrome P-450.

Aromatase, an enzyme complex localized in the endoplasmic reticulum of estrogen-producing cells, is composed of NADPH-cytochrome P-450 reductase, and aromatase cytochrome P-450 (cytochrome P-450AROM). To define the molecular mechanisms involved in the multifactorial regulation of cytochrome P-450AROM in estrogen-producing cells, we have isolated a cDNA specific for human cytochrome P-450AROM and have used this cDNA to isolate the human cytochrome P-450AROM gene. The cDNA sequence encodes a polypeptide of 503 amino acids and contains--near the carboxy-terminus, a region of high homology with the putative heme-binding regions of other P-450 cytochromes. COS1 cells transfected with an expression plasmid containing the cytochrome P-450AROM cDNA had the capacity to aromatize testosterone, androstenedione and 16 alpha-hydroxyandrostenedione, suggesting that a single polypeptide catalyzes all steps of the aromatization reaction using either of the three major C19-substrates. The human cytochrome P-450AROM gene is greater than 52 kb in size and consists of 10 exons and 9 introns. Hormonally induced changes in aromatase activity of human ovarian granulosa and adipose stromal cells are associated with comparable changes in cytochrome P-450AROM gene expression and synthesis, whereas the reductase component is only modestly affected. Studies are in progress to define the molecular mechanisms involved in the regulation of cytochrome P-450AROM gene expression in estrogen-producing cells.     

Mouse pulmonary cytochrome P-450 naphthalene hydroxylase: cDNA cloning, sequence, and expression in Saccharomyces cerevisiae.

We have isolated a cDNA clone, Nah-2, encoding the cytochrome P-450Nah (naphthalene hydroxylase) from a mouse lung lambda ZAP cDNA library using anti-cytochrome P-450Nah IgG as a probe. This same antibody selectively blocked [Nagata, K., Martin, B.M., Gillette, J.R., & Sasame, H.A. (1990) Drug Metab. Dispos. 18, 557-564] the cytochrome P-450 in mouse lung microsomes that catalyzed the conversion of naphthalene to (1R,2S)-naphthalene 1,2-oxide, which has been postulated as a causative agent in the naphthalene-induced tissue-specific necrosis of Clara cells in mouse lung. The toxic effect is seen in mouse and not in rat. The cDNA encodes a polypeptide of 491 amino acids with a molecular mass of 50 kDa. Northern blot analysis with an Nah-2-specific probe revealed that the mRNA is expressed in a species- and tissue-specific manner, present only in mouse lung and liver and not in that of rat. The mRNA encoding Nah-2 is constitutively expressed and is not induced by either phenobarbital, pyrazole, pregnenolone 16 alpha-carbonitrile, or 3-methylcholanthrene. Comparative amino acid sequence analyses with other documented members of the P-450 gene superfamily revealed that this encoded protein is in the IIF subfamily. To analyze its substrate specificity, the cDNA was inserted into the vector, pAAH5, and expressed in the Saccharomyces cerevisiae strain, AH22. The presence of cytochrome P-450Nah in the microsomes isolated from transformed cells and analyzed by Western blot was confirmed by immunocomplexing product with anti-cytochrome P450Nah IgG. Furthermore, activity toward naphthalene in the microsomes from the transformed cells established that this clone encodes a naphthalene hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aromatase isozyme in goldfish brain and stereochemistry of the aromatization [Poster 40]

Aromatase activity of goldfish brain synaptosomes was not suppressed by a mouse anti-human placental aromatase cytochrome P-450 monoclonal antibody. A rabbit antiserum to human placental aromatase cytochrome P-450 did not show a significant suppression of the goldfish brain activity when compared to the placental aromatase. However, the stereomechanism of 1,2-hydrogen elimination during the brain aromatization was determined to be stereospecific 1ß, 2ß-elimination which was identical to that of human placentas and ovaries.     

Nucleotide sequence of a full-length cDNA coding for 3-methylcholanthrene-induced rat liver cytochrome P-450MC

We constructed a full-length cDNA coding for 3-methylcholanthrene-inducible rat liver cytochrome P-450MC by the method of Okayama and Berg. The isolated clone pAU157 contained the cDNA insert of 2.7 kb in length. Sequence analysis of the cDNA insert revealed that the amino acid sequence of cytochrome P-450MC was composed of 523 amino acid residues, including the initial 22 N-terminal amino acids whose sequence was determined with the purified protein. The primary structure was found to contain two highly conserved regions as pointed out from comparisons of the reported amino acid sequences of cytochrome P-450 species. The predicted molecular weight of the apoprotein was 59,300 daltons. Therefore, we concluded that the amino acid sequence determined here is for cytochrome P-450MC, probably corresponding to cytochrome P-450c.     

Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.