Yasuhiro Mitsuuchi

Congenitally defective aldosterone biosynthesis in humans: The involvement of point mutations of the P-450C18 gene (CYP11B2) in CMO II deficient patients

The gene for steroid 18-hydroxylase (P-450C18) has been recently assigned to encode corticosterone methyl oxidases Type I and Type II which were previously postulated to catalyze the final two steps in the biosynthesis of aldosterone in humans. Molecular genetic analysis of the P-450C18 gene is three patients from three different families affected with CMO II deficiency has indicated that a point mutation of CGG----TGG (181Arg----Trp) in exon 3 and one of GTG----GCG (386Val----Ala) in exon 7 occur exclusively in the gene of the patients. Analysis of PCR products by restriction enzymes (HapII and HphI) has indicated that the patients are homozygous and the unaffected parent is heterozygous for both mutations, in accordance with the established concept that CMO II deficiency is inherited in an autosomal recessive manner. These data clearly provide the molecular genetic basis for the characteristic biochemical phenotype of CMO II clinical variants.

Alternative usage of different poly(A) addition signals for two major species of mRNA encoding human aromatase P-450.

Two cDNA clones for human placental aromatase P‐450 (P‐450AROM) have been isolated and sequenced. The insert of one clone (2894 bp) contains an open reading frame encoding a protein consisting of 503 amino acid residues together with a 49 bp 5′‐untranslated stretch and a 1336 bp 3′‐noncoding region to which a poly(A) tract is attached. Three potential poly(A) addition signals are detected in this 3′‐noncoding region. The other clone contains a shorter cDNA insert, the nucleotide sequence of which overlaps with most of the sequence of the longer cDNA insert (nucleotides 36–2355) except for one nucleotide substitution. The 3′‐noncoding region of this shorter cDNA is only 846 bp in length, but a poly(A) tract is also attached to its 3′‐terminus. Northern blot analysis of human placental RNA reveals the presence of two major mRNA species of 3.4 and 2.9 kb when probes excised from the overlapping region of these two cDNAs are employed. The 2.9 kb mRNA is not detected, however, when a fragment of the non‐overlapping region of the longer cDNA is used as a probe. It is therefore concluded that the two major species of P‐450AROM mRNA are formed as a consequence of alternative processing of precursor mRNA(s).

Molecular genetic studies on the biosynthesis of aldosterone in humans.

Corticosterone methyl oxidase Type I (CMO I) and II (CMO II) have been postulated to be the enzymes involved in the final two steps of aldosterone biosynthesis in humans. We have isolated human cDNAs for P450c11 and P450c18 as well as the corresponding genes, CYP11B1 and CYP11B2. Both protein products of these two genes as expressed in COS-7 cells exhibit steroid 11β-hydroxylase activity, but only P450c18, a product of CYP11B2, carried steroid 18-hydroxylase activity to form aldosterone. These results indicate that CYP11B2 encodes CMO, the actual catalytic function of which is retained by P450c18, a multifunctional enzyme. This conclusion is further supported by the finding that the P450c18 gene, CYP11B2, is mutated at several different loci in patients deficient in CMO I or II.

Inborn errors of aldosterone biosynthesis in humans

Corticosterone methyl oxidase (CMO) type I and type II deficiencies are inborn errors at the penultimate and ultimate steps in the biosynthesis of aldosterone in humans. Recently, steroid 18-hydroxylase (P450C18), or aldosterone synthase (P450aldo), was shown to be a multifunctional enzyme catalyzing these two steps of aldosterone biosynthesis, i.e., the conversion of corticosterone to 18-hydroxycorticosterone and the subsequent conversion of 18-hydroxycorticosterone to aldosterone. This observation suggests that CMO I and CMO II deficiencies are derived from two different mutations in the P450C18 gene (CYP11B2). To elucidate whether or not this is the case, we performed molecular genetic studies on CYP11B2 of both types of patients. Nucleotide sequence analysis has indicated that the gene of CMO I deficient patients is completely inactivated by a frameshift to form a stop codon due to a 5-bp nucleotide deletion in exon 1. Sequence analysis of CYP11B2 of CMO II deficient patients has revealed two point mutations, CGG-->TGG (Arg181-->Trp) in exon 3 and GTG-->GCG (Val386-->Ala) in exon 7. CYP11B1, the gene for steroid 11 beta-hydroxylase (P45011 beta) which was previously postulated to be the target for CMO II deficiency, is not impaired in these two types of patients. Expression studies using the corresponding mutant cDNAs have shown that CMO I deficient patients are null mutants with a complete lack of P450C18 whereas CMO II deficient patients are leaky mutants with an altered P450C18 activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The domain structure and the function of poly(ADP-ribose) synthetase

Poly(ADP-ribose) synthetase is a chromatin-bound enzyme which synthesizes a protein-bound homopolymer of ADP-ribose utilizing NAD as a substrate. The characteristic nature of this enzyme is that it requires DNA for catalytic activity. The enzyme is rich in malignant tumor cells as well as in normal tissues where cell proliferation is very rapid. The enzyme has been purified to homogeneity from calf thymus, mouse testis and human placenta. The amino acid composition of these enzymes is very similar and a monoclonal antibody as well as antisera against the calf enzyme cross-reacts with mouse, chicken and human enzymes, suggesting that the antigenic structures of poly(ADP-ribose) synthetase are highly conserved in various animal cells. The native enzyme (Mr = 120K) is cleaved by limited proteolytic digestion into three different domains (Mr = 46K, 22K, 54K), the first containing the site for DNA binding, the second containing the site for automodification and the third containing the site for NAD binding. The DNA binding domain (Mr = 46K), like the native enzyme, has the ability to preferentially suppress nick induced random transcription initiation in a HeLa cell lysate, resulting in the production of run-off RNA initiated from the correct late promoter site on truncated DNA of adenovirus 2. The native enzyme poly(ADP-ribosyl)ates RNA polymerase and some other nuclear enzymes. These results, taken together, indicate that poly(ADP-ribose) synthetase plays a critical role in regulating gene expression in various eukaryotic cells.

Biosynthesis and Degradation of Poly(ADP-Ribose) Synthetase

Poly(ADP-ribose) synthetase is a chromatin-bound enzyme which produces a protein bound homopolymer of ADP-ribose using NAD as a substrate (1). The enzyme from calf thymus has been purified to homogeneity and extensively characterized in several laboratories (2–4). Using limited proteolysis, we recently demonstrated that the enzyme (Mr = 120,000) consists of three functionally different domains, the first (Mr = 46,000) for binding of DNA, the second (Mr = 22,000) for accepting poly(ADP-ribose) and the third (Mr = 54,000) for binding of the substrate, NAD (5, 6). We also demonstrated by immunoblotting that endogenous degradation products of the enzyme were present in calf thymus (7–9). Nevertheless, detailed processes of synthesis and degradation of this enzyme in vivo are not as yet fully understood.

Molecular Structure of Poly(ADP-Ribose) Synthetase

During the past several years, it has become clear that poly(ADP-ribose) synthetase has two unique features (1, 2). One is that the enzyme requires DNA for catalytic activity and another is that the enzyme is subjected to automodification during the reaction. These two unique features will provide us with a key for clarifying the physiological function of this enzyme in vivo. In this article, we will present our recent data on molecular cloning of human poly(ADP-ribose) synthetase and will discuss the physiological functions of this enzyme on the basis of its structural characteristics.