Yoshihiko Ohyama

Cloning and expression of cDNA encoding 25-hydroxyvitamin D3 24-hydroxylase

A cDNA encoding 25‐hydroxyvitamin D3 24‐hydroxylase (P450cc24) was isolated from a rat kidney cDNA library using specific antibodies to the enzyme. The isolated cDNA wax 3.2 kbp long and contained a 1542‐bp open reading frame encoding 514 amino acids. The deduced amino acid sequence contained a presequence typical or mitochondrial enzymes in the N‐terminal region. The amino acid sequence shows less than 30% similarity to those of any other cytochrome P450s so far reported and, therefore, P450cc24 constitutes a novel family or P450, COS‐7 cells transfected with the cDNA produced a protein that was reactive with the antibodies and catalyzed NADPH‐dependent 24‐hydroxylation of 25‐hydroxy‐vitamin D3 in the presence or adrenodoxin and NADPH‐adrenodoxin reductase. Using the cDNA as a probe we demonstrated that the increase of 24‐hydroxylation activity caused by administration or vitamin D3 into rats was accompanied by an increase of the mRNA.

Studies on the Transcriptional Regulation of Cholesterol 24-Hydroxylase (CYP46A1) MARKED INSENSITIVITY TOWARD DIFFERENT REGULATORY AXES

Mammalian CNS contains a disproportionally large and remarkably stable pool of cholesterol. Despite an efficient recycling there is some requirement for elimination of brain cholesterol. Conversion of cholesterol into 24S-hydroxycholesterol by the cholesterol 24-hydroxylase (CYP46A1) is the quantitatively most important mechanism. Based on the protein expression and plasma levels of 24S-hydroxycholesterol, CYP46A1 activity appears to be highly stable in adults. Here we have made a structural and functional characterization of the promoter of the human CYP46A1 gene. No canonical TATA or CAAT boxes were found in the promoter region. Moreover this region had a high GC content, a feature often found in genes considered to have a largely housekeeping function. A broad spectrum of regulatory axes using a variety of promoter constructs did not result in a significant transcriptional regulation. Oxidative stress caused a significant increase in transcriptional activity. The possibility of a substrate-dependent transcriptional regulation was explored in vivo in a sterol-deficient mouse model (Dhcr24 null) in which almost all cholesterol had been replaced with desmosterol, which is not a substrate for CYP46A1. Compared with heterozygous littermates there was no statistically significant difference in the mRNA levels of Cyp46a1. During the first 2 weeks of life in the wild-type mouse, however, a significant increase of Cyp46a1 mRNA levels was found, in parallel with an increase in 24S-hydroxycholesterol level and a reduction of cholesterol synthesis. The failure to demonstrate a significant transcriptional regulation under most conditions is discussed in relation to the turnover of brain and neuronal cholesterol.

Functional Assessment of Two Vitamin D-responsive Elements in the Rat 25-Hydroxyvitamin D3 24-Hydroxylase Gene

Two vitamin D-responsive elements (VDRE-1 and VDRE-2) were recently identified in the 5′-upstream region of the rat 25-hydroxyvitamin D3 24-hydroxylase gene at −151/−137 and −259/−245, respectively. We studied the transcriptional regulation of this gene by vitamin D by means of mutational analysis. Introducing mutations into VDRE-1 and VDRE-2 in the native promoter −291/+9 reduced vitamin D-dependent chloramphenicol acetyltransferase activity by 86 and 41%, respectively. Mutation of the direct repeat −169/−155 located at 3 base pairs upstream of VDRE-1 also caused 50% decrease of chloramphenicol acetyltransferase activity. Connection of the element −169/−155 to VDRE-1 enhanced the vitamin D responsiveness of VDRE-1 5-fold through the heterologous β-globin promoter. The fragment −291/−102 containing the two VDREs showed two shifted bands in the presence of the vitamin D receptor and retinoid X receptor in gel retardation analysis, and the appearance of the slower migrating band indicates that two sets of receptor complexes bind to this fragment simultaneously. These results demonstrate that VDRE-1 is a stronger mediator of vitamin D function than VDRE-2 due to the presence of the accessory element −169/−155 located adjacent to VDRE-1, although VDRE-2 exhibits a smaller dissociation constant for the vitamin D receptor-retinoid X receptor complex than VDRE-1.

Recognition of Formamidopyrimidine by Escherichia coli and Mammalian Thymine Glycol Glycosylases DISTINCTIVE PAIRED BASE EFFECTS AND BIOLOGICAL AND MECHANISTIC IMPLICATIONS

The activity of prokaryotic and mammalian thymine glycol (Tg) glycosylases including Escherichia coliendonuclease III (Endo III) and endonuclease VIII (Endo VIII) and mouse Endo III homologue (mNth1) for formamidopyrimidine (Fapy) has been investigated using defined oligonucleotide substrates. 2,6-Diamino-4-hydroxy-5-N-methylformamidopyrimidine, a methylated Fapy derived from guanine, was site specifically incorporated in the oligonucleotide. The substrates containing Fapy:N pairs (N = A, G, C, T) as well as a Tg:A pair, a physiological substrate of Endo III, Endo VIII, and mNth1, were treated by the enzymes and nicked products were quantified by gel electrophoresis. The activity of Endo III and Endo VIII for Fapy varied markedly depending on the paired base, being the highest with G (activity relative to Tg = 0.55 (Endo III) and 0.41 (Endo VIII)) and the lowest with C (0.05 (Endo III) and 0.06 (Endo VIII)). In contrast, mNth1 recognized all Fapy pairs equally well and the activity was comparable to Tg. The results obtained in the nicking assay were further substantiated by the analysis of the Schiff base intermediate using NaBH4trapping assays. These results indicate that Escherichia coli and mammalian Tg glycosylases have a potential activity to recognize Fapy. However, as demonstrated for Fapy:C pairs, their distinctive activities implicate unequal participation in the repair of Fapy lesions in cells.

Characterization of rat and human CYP2J enzymes as Vitamin D 25-hydroxylases

vitamin D is 25-hydroxylated in the liver, before being activated by 1alpha-hydroxylation in the kidney. Recently, the rat cytochrome P450 2J3 (CYP2J3) has been identified as a principal vitamin D 25-hydroxylase in the rat [Yamasaki T, Izumi S, Ide H, Ohyama Y. Identification of a novel rat microsomal vitamin D3 25-hydroxylase. J Biol Chem 2004;279(22):22848-56]. In this study, we examine whether human CYP2J2 that exhibits 73% amino acid homology to rat CYP2J3 has similar catalytic properties. Recombinant human CYP2J2 was overexpressed in Escherichia coli, purified, and assayed for vitamin D 25-hydroxylation activity. We found significant 25-hydroxylation activity toward vitamin D3 (turnover number, 0.087 min(-1)), vitamin D2 (0.16 min(-1)), and 1alpha-hydroxyvitamin D3 (2.2 min(-1)). Interestingly, human CYP2J2 hydroxylated vitamin D2, an exogenous vitamin D, at a higher rate than it did vitamin D3, an endogenous vitamin D, whereas, rat CYP2J3 hydroxylated vitamin D3 (1.4 min(-1)) more efficiently than vitamin D2 (0.86 min(-1)). Our study demonstrated that human CYP2J2 exhibits 25-hydroxylation activity as well as rat CYP2J3, although the activity of human CYP2J2 is weaker than rat CYP2J3. CYP2J2 and CYP2J3 exhibit distinct preferences toward vitamin D3 and D2.

Unique property of liver mitochondrial P450 to catalyze the two physiologically important reactions involved in both cholesterol catabolism and vitamin D activation.

The cDNA for vitamin D 25‐hydroxylase in rat liver mitochondria was transfected in COS cells in order to confirm our previous postulation that both 5β‐cholestane‐3α,7α,12α‐triol 27‐hydroxylation and vitamin D 25‐hydroxylation are catalyzed by a common enzyme. As a result it was found that both enzyme activities could be reconstituted from the solubilized extract of mitochondria of these cells. NADPH. NADPH‐adrenodoxin reductase and adrenodoxin, giving unequivocal evidence that the two enzyme activities are catalyzed by a common enzyme.

Enzymatic Repair of 5-Formyluracil I. EXCISION OF 5-FORMYLURACIL SITE-SPECIFICALLY INCORPORATED INTO OLIGONUCLEOTIDE SUBSTRATES BY AlkA PROTEIN (Escherichia coli 3-METHYLADENINE DNA GLYCOSYLASE II)

5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation. We have previously shown that fU is a potentially mutagenic lesion due to its elevated frequency to mispair with guanine. Therefore, fU can exist in DNA as a correctly paired fU:A form or an incorrectly paired fU:G form. In this work, fU was site-specifically incorporated opposite A in oligonucleotide substrates to delineate the cellular repair mechanism of fU paired with A. The repair activity for fU was induced inEscherichia coli upon exposure toN-methyl-N′-nitro-N-nitrosoguanidine, and the induction was dependent on the alkA gene, suggesting that AlkA (3-methyladenine DNA glycosylase II) was responsible for the observed activity. Activity assay and determination of kinetic parameters using purified AlkA and defined oligonucleotide substrates containing fU, 5-hydroxymethyluracil (hU), or 7-methylguanine (7mG) revealed that fU was recognized by AlkA with an efficiency comparable to that of 7mG, a good substrate for AlkA, whereas hU, another major thymine methyl oxidation products, was not a substrate. 1H and 13C NMR chemical shifts of 5-formyl-2′-deoxyuridine indicated that the 5-formyl group caused base C-6 and sugar C-1′ to be electron deficient, which was shown to result in destabilization of the N-glycosidic bond. These features are common in other good substrates for AlkA and are suggested to play key roles in the differential recognition of fU, hU, and intact thymine. Three mammalian repair enzymes for alkylated and oxidized bases cloned so far (MPG, Nth1, and OGG1) did not recognize fU, implying that the mammalian repair activity for fU resided on a yet unidentified protein. In the accompanying paper (Terato, H., Masaoka, A., Kobayashi, M., Fukushima, S., Ohyama, Y., Yoshida, M., and Ide, H.,J. Biol. Chem. 274, 25144–25150), possible repair mechanisms for fU mispaired with G are reported.

Effects of a Guanine-derived Formamidopyrimidine Lesion on DNA Replication TRANSLESION DNA SYNTHESIS, NUCLEOTIDE INSERTION, AND EXTENSION KINETICS

2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3′-5′ exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (f ins =V max/K m for insertion) and extension (f ext =V max/K m for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative f ins = 1) ≫ dTMP (2.4 × 10−4) ≈ dAMP (8.1 × 10−5) > dGMP (4.5 × 10−7)), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative f ext = 1) > mFapyG:T (4.6 × 10−3) ≫ mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.

Purification of 25-hydroxyvitamin D3 24-hydroxylase from rat kidney mitochondria

25‐Hydroxyvitamin D3 24‐hydroxylase was purified to an electrophoretically homogeneous state (M r=53000) from kidney mitochondria of female rats treated with vitamin D3.

DIFFERENTIAL TIME COURSE OF INDUCTION OF 1α,25-DIHYDROXYVITAMIN D3-24-HYDROXYLASE mRNA EXPRESSION IN RATS BY 1α,25-DIHYDROXYVITAMIN D3 AND ITS ANALOGS

In order to investigate the in vivo mechanisms of target gene activation by vitamin D3 analogs, we compared the effects of two vitamin D3 analogs, 22-oxa-1alpha,25-(OH)2D3 (OCT) and 2beta (3-hydroxypropoxy) -1alpha,25-(OH)2D3 (ED-71) with that of 1alpha,25-(OH)2D3 on 1alpha,25-(OH)2D3 -24-hydroxylase[24(OH)ase] mRNA expression in the kidney and intestine of normal rats. In these experiments, all three compounds induced 24(OH)ase mRNA, but the time course of induction for each respective treatment was clearly different. OCT caused the most rapid onset of increased 24(OH)ase mRNA expression and its subsequent return to pre-injection levels. In marked contrast, ED-71 was the slowest to increase expression which was prolonged over that observed with the other compounds tested. These differences probably relate to the pharmacokinetic properties of these analogs, which are mainly generated by the affinity of analogs for the vitamin D-binding protein(DBP).