Kyuichiro Okuda

Molecular Cloning and Expression of Rabbit Sterol 12α-Hydroxylase

Sterol 12α-hydroxylase is an important enzyme in bile acid biosynthesis, responsible for the balance between formation of cholic acid and chenodeoxycholic acid. The enzyme has been purified to apparent homogeneity from rabbit liver (Ishida, H., Noshiro, M., Okuda, K., and Coon, M. J. (1992) J. Biol. Chem.267, 21319-21323), and we here describe the cloning and sequencing of a cDNA coding for this enzyme. After tryptic digestion of purified protein in a polyacrylamide gel, eight different peptides were isolated and sequenced. Using oligonucleotides deduced from the amino acid sequences, clones were isolated from a rabbit liver cDNA library. In addition to several overlapping clones, one full-length clone was obtained that coded for a polypeptide of 500 amino acids, corresponding to a molecular mass of 57 kDa. All of the eight peptides and the reported NH2-terminal amino acid sequence were matched against the sequence. The peptide sequence showed a 39% similarity with human prostacyclin synthase (CYP8) and 31% similarity with the rate-limiting enzyme in over-all synthesis of bile acids, the cholesterol 7α-hydroxylase (CYP7) of the rabbit. The similarity with most other sterol cytochrome P-450 hydroxylases was less. Thus, this species of cytochrome P-450 should belong to a group of its own, here denoted CYP12. Transfection of COS cells with the coding part of the cDNA resulted in a significant expression of sterol 12α-hydroxylase activity toward 7α-hydroxy-4-cholesten-3-one. Northern blotting showed that the enzyme was exclusively expressed in the liver. The major mRNA fraction in rabbit liver had a size of approximately 2.9 kilobases, and those found in rat and human liver were about 2.5 and 4.5 kilobases, respectively. Fasting of rats and mice led to a severalfold increase in both enzyme activity and mRNA levels. In contrast, starvation of rabbits had little or no stimulatory effect on enzyme activity and mRNA levels.

Expression of 25-Hydroxyvitamin D3-24-Hydroxylase mRNA in Cultured Human Keratinocytes

Abstract It is well documented that 1α,25-dihydroxyvitamin D3 (1α,25[OH]2D3), the most active vitamin D metabolite, inhibits epidermal keratinocyte proliferation and promotes differentiation. 1α,25(OH)2D3 can be produced in keratinocytes from 25-hydroxyvitamin D3 by the enzyme 25-hydroxyvitamin D3-1α-hydroxylase (1-OHase). Hydroxylation of 1α,25(OH)2D3 by 25-hydroxyvitamin D3-24-hydroxylase (24-OHase), the first step in the catabolic pathway of 1α,25(OH)2D3 could significantly reduce the intracellular concentration of 1α,25(OH)2D3. Therefore, the expression of 24-OHase could have a critical regulatory role in 1α,25(OH)2D3-dependent gene expression. As a first step to examine this possibility, the steady state level of 24-OHase mRNA in cultured human keratinocytes (CHK) was investigated. 24-OHase mRNA was not detected in control CHK. 1α,25(OH)2D3 caused a dose- and time-dependent increase in 24-OHase mRNA level. The highest accumulation of 24-OHase mRNA was observed in CHK treated with 0.1-1 μM 1α,25(OH)2D3. The level of 24-OHase mRNA reached a plateau 12-24 hr after 1α,25(OH)2D3 treatment. 1β,25-dihydroxyvitamin D3, the stereoisomer of 1α,25(OH)2D3, failed to induce 24-OHase mRNA expression significantly. In addition to 24-OHase mRNA, a 1.0-kb mRNA hybridized strongly with both rat and human 24-OHase cDNA probes. The origin of this 1.0-kb message is unknown at present, however, it was regulated by 1α,25(OH)2D3. These results demonstrate that 1α,25(OH)2D3 up-regulates the expression of 24-OHase mRNA, and this may be an important first step in the initiation of catabolism of 1α,25(OH)2D3 in human keratinocytes.

delta 4-3-Oxosteroid 5 beta-reductase. Structure and function.

delta 4-3-Oxosteroid 5 beta-reductase catalysing reduction of delta 4-3-oxosteroids to give A/B cis-conformation was intraperitoneally injected into BALB/c strain mice with Ribi adjuvant. Monoclonal antibody specific for this enzyme was prepared from their spleen cells. Using this monoclonal antibody as a probe the enzyme was further purified using reversed phase liquid chromatography to determine amino-acid sequence protein-chemically. Attempts to determine the N-terminal amino acid failed, indicating that the N-terminal amino acid is blocked. The protein was therefore subjected to digestion with lysyl endopeptidase after alkylating with iodoacetate. The peptides thus formed were isolated and purified by reversed-phase high-performance liquid chromatography and their amino-acid sequences were determined. Using antibodies and oligonucleotides as probes a cDNA which contained a 978 bp long open reading frame encoding 326 amino-acid residues (Mr 37376) was isolated from rat liver cDNA libraries and the entire sequence of the protein was deciphered from its nucleotide sequence. The COS cells transfected with this cDNA revealed a versatile activity to reduce varied kinds of delta 4-3-oxosteroids, i.e. 7 alpha-hydroxy-4-cholesten-3-one, androstenedione and cortisone as postulated by Okuda and Okuda (1984, J. Biol. Chem. 259, 7519-7524) and Furuebisu et al. (1987, Biochim. Biophys. Acta 912, 110-114. With a newly established immunoblotting assay method several tissues and organs were surveyed and it was found that the enzyme exists only in the liver and there is an apparent difference between sexes as to the content of this enzyme. However, there was little if any difference in the amount of mRNAs between both sexes, which may indicates that the sexual difference of rat liver cytosol 5 beta-reductase is due to a posttranslational modification and/or degradation.

Assay of vitamin D derivatives and purification of vitamin D hydroxylases.

Publisher Summary Vitamin D 3 is hydroxylated at position 25 by liver microsomal and mitochondrial vitamin D 3 25-hydroxylases (25-hydroxylase). The microsomal enzyme purified from male rats is the same as CYP2C11, a male-specific P450. The mitochondrial enzyme is identical with 5 β -cholestane-3 α ,7 α ,12 α -triol (an intermediate in the conversion of cholesterol to cholic acid) 27-hydroxylase, and is categorized as CYP27. The product, 25-hydroxyvitamin D 3 (25-OH-D 3 ), is transported to the kidney through the bloodstream, where it is hydroxylated at the 1 α -position to give 1 α ,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ] in calcium or 1,25-(OH) 2 D 3 depleted animals. Both 25-and 24-hydroxylases are purified and their cDNA clones are isolated. These hydroxylases are members of the P450 superfamily that are b -type hemoproteins functioning as terminal monooxygenase in the oxygenation of various organic compounds. This chapter describes the assay and purification of both 25- and 24-hydroxylases.

The Enzymes Responsible for Metabolizing Vitamin D

Vitamin D is a generic name for the antirachitic agents capable of curing rickets of which vitamin D3 is the major constituent. Vitamin D3 is formed in the skin from 7-dehydrocholesterol, a precursor of cholesterol, by the action of ultraviolet light (1,2). It is then transported to the liver and is hydroxylated at position 25 by vitamin D3 25-hydroxylase (25-hydroxylase) to 25-hydroxyvitamin D3 [25(OH)D3]. The 25(OH)D3 formed is again transported to the kidney through the blood stream. In the kidney 25(OH)D3 is further hydroxylated at position la by 25-hydroxyvitamin D3 1α-hydroxylase (lα-hydroxylase) to 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] which is now considered the hormonal form of the vitamin. It plays a central role in the physiology of calcium homeostasis (2,3). In some calcium statuses, another metabolite of vitamin D3, 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3], is formed from 25(OH)D3 by 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase), existing in proximal convoluted tubules of the kidney (4). The two kidney hydroxylases are induced in the reciprocal calcium statuses. lα-Hydroxylase activity is enhanced in a calcium- and/or 1,25(OH)2D3depleted status, whereas that of 24-hydroxylase is enhanced in the replete status (4,5).

MOLECULAR CLONING OF VITAMIN D3 HYDROXYLASES

Publisher Summary Vitamin D 3 is hydroxylated to 25-hydroxyvitamin D 3 by a vitamin D 3 25-hydroxylase (25-hydroxylase) located in liver mitochondria or endoplasmic reticulum. 25-hydroxyvitamin D 3 is then transported to the kidney through the bloodstream. In kidney mitochondria, it is hydroxylated at the 1 α -position by a 25-hydroxyvitamin D 3 1 α -hydroxylase to 1 α ,25-dihydroxyvitamin D 3 in calcium-depleted animals or hydroxylated at the 24 R -position to 24 R ,25-dihydroxyvitamin D 3 by a 25-hydroxyvitamin D 3 24-hydroxylase (24-hydroxylase) in animals that are calcium or 1,25-dihydroxyvitamin D 3 repleted. Both 25- and 24-hydroxylases are purified and their cDNA clones are isolated. These hydroxylases constitute novel families in the P450 super family, that is, CYP27 for 25-hydroxylase and CYP24 for 24-hydroxylase. This chapter discusses the molecular cloning of these hydroxylases. Screening of cDNA libraries is performed by nucleic acid hybridization or by an immunological detection method. Construction of cDNA libraries, isolation of cDNA clones, and nucleotide sequencing are performed according to established methods.