Cloning and tissue distribution of a novel human cytochrome p450 of the CYP3A subfamily, CYP3A43

On the basis of the detection of an expressed sequence tag ('EST') similar to the human cytochrome P450 3A4 cDNA, we have identified a novel member of the human cytochrome P450 3A subfamily. The coding region is 1512-bp long and shares 84, 83, and 82% sequence identity on the cDNA level with CYP3A4, 3A5, and 3A7, respectively, with a corresponding amino acid identity of 76, 76, and 71%. Quantitative real time based mRNA analysis revealed CYP3A43 expression levels at about 0.1% of CYP3A4 and 2% of CYP3A5 in the liver, with significant expression in 70% of the livers examined. Gene specific PCR of cDNA from extrahepatic tissues showed, with the exception of the testis, only low levels of CYP3A43 expression. The CYP3A43 cDNA was heterologously expressed in yeast, COS-1 cells, mouse hepatic H2.35 cells and in human embryonic kidney (HEK) 293 cells, but in contrast to CYP3A4 which was formed in all cell types, no detectable CYP3A43 protein was produced. This indicates a nonfunctional protein or specific conditions required for proper folding. It is concluded that CYP3A43 mRNA is expressed mainly in liver and testis and that the protein would not contribute significantly to human drug metabolism.     

Molecular cloning of a cDNA encoding a member of a novel cytochrome P450 family in the mollusc Lymnaea stagnalis.

We isolated a cDNA encoding a cytochrome P450 from the mollusc Lymnaea stagnalis. The mRNA is 2.1 nucleotides long and contains an open reading frame encoding a protein of 545 amino acids. A conserved home-binding domain, characteristic of cytochrome P450 proteins, is present in the deduced amino acid sequence. The Lymnaea cytochrome P450 protein shares less than 40% positional identity with any known member of the cytochrome P450 superfamily, and therefore, represents a separate family, which we propose to name CYP10. The CYP10 mRNA is shown to be uniquely and abundantly expressed in the female gonadotropic hormone producing dorsal bodies of L. stagnalis.     

棉酚和烟碱对棉铃虫的生长和细胞色素P-450单加氧酶活性的影响

以人工饲料添加法测定了 0 5%的棉酚和烟碱对棉铃虫的生长和细胞色素P 4 50单加氧酶 (简称P 4 50酶系 )活性的影响。研究结果显示 ,在测定浓度下 ,高龄棉铃虫短期取食含棉酚和烟碱的人工饲料后 ,对幼虫的生长没有显著影响 ,由此表明 ,棉铃虫对其主要寄主植物中的次生物质棉酚和烟碱具有很好的适应能力。与此同时 ,棉铃虫中肠微粒体P 4 50酶系的蛋白组成和酶活性发生了不同的变化 ,有升有降 ,有的没有变化。棉铃虫可能通过调整P 4 50酶系的各种蛋白含量和酶的活力水平 ,来适应对植物次生物质的代谢解毒的需要。另外 ,棉铃虫取食棉酚和烟碱后 ,细胞色素B5含量均显著提高 ,而细胞色素P 4 50含量均显著降低 ,细胞色素B5在棉铃虫对棉酚和烟碱的解毒代谢中可能发挥着更为重要的作用     

A novel cytochrome P450 gene (CYP4G25) of the silkmoth Antheraea yamamai: cloning and expression pattern in pharate first instar larvae in relation to diapause

A new cytochrome P450 gene, CYP4G25, was identified as a differentially expressed gene between the diapausing and post-diapausing pharate first instar larvae of the wild silkmoth Antheraea yamamai, using subtractive cDNA hybridization. The cDNA sequence of CYP4G25 has an open reading frame of 1674 nucleotides encoding 557 amino acid residues. Sequence analysis of the putative CYP4G25 protein disclosed the motif FXXGXRXCXG that is essential for heme binding in P450 cytochromes. Hybridization in situ demonstrated predominant expression of CYP4G25 in the integument of pharate first instar larvae. Northern blotting analysis showed an intensive signal after the initiation of diapause and no or weak expression throughout the periods of pre-diapause and post-diapause, including larval development. These results indicate that CYP4G25 is strongly associated with diapause in pharate first instar larvae.     

棉铃虫细胞色素P450 cDNA片段的克隆与序列分析

以 5龄实验室敏感品系棉铃虫Helicoverpaarmigera的总RNA为模板 ,采用简并性引物 ,利用反转录 -多聚酶链式反应 (RT PCR)扩增出了 2个新的长度分别为 2 3 7bp和 2 40bp的cDNA片段。序列分析表明 ,2 3 7bp的cDNA片段与棉铃虫P45 0CYP4家族有较高的相似性 ,最高达 73 %;而 2 40bp的cDNA片段与CYP6家族有较高的相似性 ,最高达 49%。     

棉铃虫P450基因CYP9A12酵母表达产物对拟除虫菊酯的代谢作用

细胞色素P450氧化酶解毒代谢作用增强是棉铃虫Helicoverpa armigera对拟除虫菊酯类杀虫剂产生抗性的主要原因,棉铃虫细胞色素P450氧化酶基因CYP9A12的组成型过量表达与拟除虫菊酯抗性相关。为了进一步明确棉铃虫细胞色素P450氧化酶基因CYP9A12与拟除虫菊酯类杀虫剂抗性的关系,采用酿酒酵母Saccharomyces cerevisiae表达系统异源表达了CYP9A12基因,检测了该基因的酵母表达产物对溴氰菊酯、氟氯氰菊酯、甲氰菊酯和联苯菊酯4种药剂的离体代谢作用。结果表明：含有CYP9A12外源基因的重组酵母细胞裂解液对溴氰菊酯、氟氯氰菊酯和联苯菊酯的代谢率分别为8.58,5.85和3.94 pmol/min·mg protein,而没有检测到对甲氰菊酯的代谢。本研究表明了CYP9A12具有代谢多种拟除虫菊酯的能力,也为CYP9A12参与拟除虫菊酯的解毒代谢提供了直接证据。     

Soluble and membrane-bound forms of cytochrome b5 are the products of a single gene in chicken

In order to determine the relationship of the soluble cytochrome b5 found in erythrocytes to the membrane-bound form found in other tissues, a cDNA clone encoding cytochrome b5 in chicken erythrocytes was isolated by using mixed oligonucleotides based on a partial amino acid sequence of the protein. Complete nucleotide sequence identity between the erythrocyte cDNA and the sequence of a cDNA clone of the liver protein suggests that they are transcribed from the same gene. The isolation and structural analysis of genomic clones was also consistent with the presence of only one cytochrome b5 gene in chicken. These results suggest that the formation of soluble erythrocyte cytochrome b5 occurs by proteolytic processing of the membrane-bound form. Thus, previous reports indicating that the carboxyl terminal amino acid residue of the erythrocyte form differs from the corresponding residue of the membrane-bound form may suggest the existence of a novel post-translational modification.     

Characterization of cytochrome b(5) in the ascidian Polyandrocarpa misakiensis and budding-specific expression

A cDNA for cytochrome b(5) was cloned from a cDNA library of buds of the ascidian, Polyandrocarpa misakiensis, by a hybridization method involving a digoxigenin-labeled cDNA probe of human soluble cytochrome b(5). The nucleotide sequence of the cDNA for the ascidian cytochrome b(5) (Pmb5) consisted of about 1,800 base pairs including 5'- and 3'-noncoding regions, and a coding sequence of 405 base pairs. The amino acid sequence of 135 residues deduced from the coding nucleotide sequence exhibited 54% identity and 76% similarity to chicken cytochrome b(5). A highly conserved amino acid sequence was observed in the amino-terminal domain of 96 residues containing two heme-binding histidine residues. The putative soluble form of the recombinant Pmb5 expressed in Escherichia coli was purified to homogeneity by column chromatographies on an anion-exchanger and gel filtration. The purified Pmb5 showed the typical absorption spectrum of cytochrome b(5) with an asymmetric peak at 556 nm and a shoulder at 560 nm upon reduction with NADH and NADH-cytochrome b(5) reductase. The low temperature spectrum of the dithionite-reduced form of the protein contained the split peaks at 551 and 555 nm, this spectrum being very similar to that of mammalian liver cytochrome b(5). Expression of Pmb5 in the ascidian was examined immunohistochemically with a monoclonal antibody against the Pmb5. Apparently high level expression of Pmb5 was found in the developing buds, but the levels of cytochrome b(5) in the parents and juvenile adults were very low. This is the first report on the characterization of Pmb5, and the increased expression of Pmb5 in the ascidian.     

Expression of the Csp protein family upon cold shock and production of tetracycline in Streptomyces aureofaciens

The biosynthesis of the steroidal molting hormone, 20-hydroxyecdysone, of arthropods involves a series of cytochrome P450-catalyzed hydroxylations. None of the many sequences of insect cytochromes P450, known to date, is related to ecdysteroid pathways. Here, we report the cloning and sequencing of a full-length cDNA of a new cytochrome P450, classified as CYP6H1, from malpighian tubules of the locust, Locusta migratoria. The 1854 bp DNA contained an open reading frame coding for a protein of 542 amino acids, a 5'-leader sequence and a 3'-untranslated region containing a polyadenylation signal and a poly(A) tail. The encoded protein had been isolated as an ecdysone-binding cytochrome P450 from microsomes of the same tissue in previous work. The closest homolog of CYP6H1 was CYP6A2 from Drosophila with 42.1% identity. According to Northern analysis, CYP6H1 is predominantly expressed at larval instars and in malpighian tubules. Evidence is presented for a functional assignment of CYP6H1 to microsomal ecdysone 20-hydroxylase of the locust.     

Cloning and expression analysis of a new member of the cytochrome P450, CYP2A15 from the Chinese hamster, encoding testosterone 7alpha-hydroxylase.

We cloned a new cytochrome P450 cDNA encoding testosterone 7alpha-hydroxylase in the Chinese hamster, designated CYP2A15 which shares significant amino acid sequence homology with members of the CYP2A subfamily. The CYP2A15 cDNA was isolated by screening a liver cDNA library and the sequence contains an open reading frame of 1482 nucleotides encoding a polypeptide of 493 amino acids with a calculated molecular mass of 56,295 Da. This is flanked by a 5'-untranslated region of 2 bp and a 3' untranslated region of 191 bp including the poly(A) tail. We determined the catalytic activity of CYP2A15 using microsomes obtained by transient expression of its cDNA in transfected COS-7 cells. The heterologously expressed CYP2A15 was found to hydroxylate testosterone at position 7alpha in a reconstituted system. RT-PCR experiments revealed that the mRNA of CYP2A15 was expressed in liver, but not detected in kidney, lung, or small intestine. The expression of CYP2A15 mRNA was slightly induced by treatment with either rifampicin or 3-methylcholanthrene.     

Induction of the mRNA for CYP6B2, a pyrethroid inducible cytochrome p450, in Helicoverpa armigera (Hubner) by dietary monoterpenes

A cDNA clone encoding a cytochrome P450 from H. armigera was used to examine the expression of two homologous cytochrome P450 mRNAs, one of which, CYP6B2, is probably involved in pyrethroid metabolism. The mRNA for CYP6B2 in particular can be induced up to tenfold by peppermint oil and the monoterpene α-pinene as well as to a smaller extent by menthol, butylated hydroxyanisole, and piperonyl butoxide. Both mRNAs are present in the major larval tissues, midgut, fat body, and cuticle, although only the mRNAs in the midgut and fat body are inducible by peppermint oil. The induction is a rapid process occurring within a period of 4 h with a similarly rapid rate of decrease in the absence of the inducing compounds. During normal development both mRNAs are absent from eggs and increase during the larval stage, reaching a maximum during mid fifth instar. Both mRNAs then decrease to undetectable levels during pupation and remain undetectable in the adult stage. © 1997 Wiley-Liss, Inc.     

棉铃虫细胞色素P450 CYP9A12基因5′-上游区的克隆及序列分析

细胞色素P450基因CYP9A12的过量表达已被证实与棉铃虫Helicoverpa armigera对拟除虫菊酯的抗性相关。为探明棉铃虫CYP9A12基因的表达调控机理,根据棉铃虫CYP9A12基因cDNA全长的5′-末端核苷酸序列,采用基因组步移方法,获得CYP9A12的5′-上游区序列（总长为3 575 bp）。与cDNA序列进行比对,表明在起始密码子上游3 bp处有一长为2 124 bp的内含子。利用NNPP分析软件预测出转录起始位点,与根据CYP9A12全长cDNA序列推测的结果是一致的。TFSEARCH 1.3软件分析转录因子结合位点的结果显示,该序列不仅包含启动子的核心结构序列——TATA-box和CAAT-box,亦包含多个转录因子结合位点,如GATA-1,CdxA,Dfd等。本研究结果为深入研究棉铃虫CYP9A12的表达调控机制及其参与杀虫剂抗性的分子机理奠定了一定基础。     

棉铃虫HaKettin1基因的全长cDNA克隆、序列分析和表达谱

【目的】为了克隆棉铃虫Helicoverpa armigera编码肌肉蛋白Kettin基因的全长cDNA序列以及鉴定该基因在棉铃虫发育周期内的表达模式。【方法】利用兼并引物,通过分段RT-PCR和5′-和3′-RACE的方法克隆全长cDNA序列。利用半定量RT-PCR进行表达谱分析。【结果】编码棉铃虫Kettin蛋白的基因HaKettin1全长cDNA序列为13 805 bp,包含一个13 365 bp的开放阅读框,编码4 454个氨基酸,蛋白分子量约为504.3 kD。组织表达结果显示HaKettin1基因在棉铃虫的整个生育周期都有表达,幼虫期的表达尤为显著。【结论】HaKettin1与家蚕的Kettin蛋白具有90％的同源性,表明鳞翅目昆虫的Kettin蛋白之间具有很高的保守性。表达谱结果显示HaKettin1基因在棉铃虫的整个发育过程中都发挥重要作用。     

Role of cytochrome b5 in catalysis by cytochrome P450 2B4

Cytochrome b5 has been shown to stimulate, inhibit or have no effect on catalysis by P450 cytochromes. Its action is known to depend on the isozyme of cytochrome P450, the substrate, and experimental conditions. Cytochrome P450 2B4 (CYP 2B4) has been used in our laboratory as a model isozyme to study the role of cytochrome b5 in cytochrome P450 catalysis using two substrates, methoxyflurane and benzphetamine. One substrate is the volatile anesthetic, methoxyflurane, whose metabolism is consistently markedly stimulated by cytochrome b5. The other is benzphetamine, whose metabolism is minimally modified by cytochrome b5. Determination of the stoichiometry of the metabolism of both substrates showed that the amount of product formed is the net result of the simultaneous stimulatory and inhibitory actions of cytochrome b5 on catalysis. Site-directed mutagenesis studies revealed that both cytochrome b5 and cytochrome P450 reductase interact with cytochrome P450 on its proximal surface on overlapping but non-identical binding sites. Comparison of the rate of reduction of oxyferrous CYP 2B4 and the rate of substrate oxidation by cyt b5 and reductase with stopped-flow spectrophotometric and rapid chemical quench experiments has demonstrated that although cytochrome b5 and reductase reduce oxyferrous CYP 2B4 at the same rate, substrate oxidation proceeds more slowly in the presence of the reductase.     

The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P45017A1 activities

Male pigs are routinely castrated to prevent the accumulation of testicular 16-androstene steroids, in particular 5α-androst-16-en-3-one (5α-androstenone), which contribute to an off-odour and off-flavour known as boar taint. Cytochrome P450C17 (CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids from pregnenolone by the andien-β synthase reaction or the synthesis of the glucocorticoid and sex steroids via 17α-hydroxylase and C17,20 lyase pathways respectively. We have expressed CYP17A1, along with cytochrome P450 reductase (POR), cytochrome b5 reductase (CYB5R3) and cytochrome b5 (CYB5) in HEK-293FT cells to investigate the importance of the two forms of porcine CYB5, CYB5A and CYB5B, in both the andien-β synthase as well as the 17α-hydroxylase and C17,20 lyase reactions. Increasing the ratio of CYB5A to CYP17A1 caused a decrease in 17α-hydroxylase (p < 0.013), a transient increase in C17,20 lyase, and an increase in andien-β synthase activity (p < 0.0001). Increasing the ratio of CYB5B to CYP17A1 also decreased 17α-hydroxylase, but did not affect the andien-β synthase activity; however, the C17,20 lyase, was significantly increased. These results demonstrate the differential effects of two forms of CYB5 on the three activities of porcine CYP17A1 and show that CYB5B does not stimulate the andien-β synthase activity of CYP17A1.