MOLECULAR CLONING AND SEQUENCE ANALYSIS OF THREE NEW FULL‐LENGTH cDNAs OF CYTOCHROME P450 (CYP6N3v1‐v3)*FROM AEDES ALBOPICTUS**

Abstract The three new full‐length cDNA sequences including the complete 5′‐and 3′‐ untranslated regions (UTR) coding for cytochrome P450s from Aedes albopictus have been obtained. The P450 proteins deduced from the nucleotide sequences shared 58.6% ‐ 62.4% amino acid identity with CYP6N1 and CYP6N2 from Anopheles gambiae, and 99% with each other. The three new complete sequences have been submitted and named as CYP6N3v1, CYP6N3v2 and CYP6N3v3 by the P450 Nomenclature Committee. The original cDNAs were obtained by rapid amplification of cDNA ends (RACE) approach with several pairs of gene specific primers based on the cDNA fragment previously obtained from deltamethrin‐resistant strain of Ae. albopictus. Further analysis showed that the three new sequences are present in both resistant strain and susceptible strain and might be effectively translated. In addition, the 5′‐ and 3′‐UTRs were compared between the CYP6N3vl‐v3 and other known insect P450s. The multiplicity of trans‐lational control of insect P450 genes was discussed.     

cytochrome p450 superfamily in Arabidopsis thaliana: isolation of cDNAs,Differential Expression,and RFLP mapping of multiple cytochromes P450

We have isolated multiple cDNAs encoding cytochromes P450 (P450s) from Arabidopsis thaliana employing a PCR strategy. Degenerate oligonucleotide primers were designed from amino acid sequences conserved between two plant P450s, CYP71A1 and CYP73A2, including the heme-binding site and the proline-rich motif found in the N-terminal region, and 11 putative P450 fragments were amplified from first-strand cDNA from 7-day-old Arabidopsis as a template. With these PCR fragments as hybridization probes, 13 full-length and 3 partial cDNAs encoding different P450s have been isolated from an Arabidopsis cDNA library. These P450s have been assigned to either one of the established subfamilies: CYP71B, CYP73A, and CYP83A; or novel subfamilies: CYP76C, CYP83B, and CYP91A. The primary protein structures predicted from the cDNA sequences revealed that the regions around both the heme-binding site and the proline-rich motif were highly conserved among all these P450s. The N-terminal structures of the predicted P450 proteins suggested that these Arabidopsis P450s were located at the endoplasmic reticulum membrane. The loci of four P450 genes were determined by RFLP mapping. One of the clones, CYP71B2, was located at a position very close to the ga4 and gai mutations. RNA blot analysis showed expression patterns unique to each of the P450s in terms of tissue specificity and responsiveness to wounding and light/dark cycle, implicating involvement of these P450s in diverse metabolic processes.     

温肾咳喘片主要成分对HepG2细胞中CYP相关基因表达的影响

观察温肾咳喘片组方中5种主要单体成分甘草酸、厚朴酚、和厚朴酚、蛇床子素和 欧前胡素对细胞色素P450(cytochrome P450, CYP) 1A2,2D6,2E1和3A4基因表达的影 响. 采用实时荧光定量PCR技术检测HepG2细胞中药物处理后各CYP mRNA的表达.厚朴酚 、和厚朴酚、蛇床子素和欧前胡素在不同浓度均能明显的诱导CYP2E1和CYP3A4,同时欧 前胡素也能诱导CYP1A2的表达,而甘草酸、厚朴酚、和厚朴酚、蛇床子素和欧前胡素在 不同浓度对CYP2D6的表达均具有较弱的抑制作用.甘草酸、厚朴酚、和厚朴酚、蛇床子 素和欧前胡素能明显影响CYP1A2、2D6、2E1或3A4的表达.此研究为中西药物代谢性相互 作用及毒理学的研究提供实验依据.     

Use of real-time gene-specific polymerase chain reaction to measure RNA expression of three family members of rat cytochrome P450 4A.

Exposure of rats to peroxisome proliferators induces members of the cytochrome P450 4A (CYP4A) family. In rats, the CYP4A family consists of four related genes, CYP4A1, CYP4A2, CYP4A3, and CYP4A8. We are specifically interested in examining CYP4A1, CYP4A2, and CYP4A3, each of which is expressed in a tissue-dependent and sex-dependent manner. While CYP4A1 is sufficiently different from the other two members to enable relatively easy specific quantitation, the close similarity between CYP4A2 and CYP4A3 makes quantitative discrimination difficult. We have combined a fluorescent real-time PCR assay (TaqMan) with the sequence-specific mismatch amplification mutation assay (MAMA) to allow us to carry out specific quantitation of all three members of this family. The assay is designed such that a single fluorescent TaqMan(R) probe binds to all three gene products, while specificity is conferred by sequence-specific primers. This specific MAMA technique takes advantage of the ability of Taq polymerase to distinguish between the two cDNAs based on mismatches at the 3' end of a PCR primer. In the 84-base PCR product used for this assay, there is only a single-base difference between CYP4A2 and CYP4A3. Despite this similarity, there is at least a 1000-fold discrimination between the two sequences, using CYP4A2 or CYP4A3 specific standards. Analysis of rat liver RNA from both sexes demonstrates that this discrimination is also achieved in complex RNA mixtures. This technique should be broadly applicable to other areas of research such as allelic discrimination, detecting mutational hotspots in tumors, and discrimination among closely related members of other gene families.     

用低G／C％含量引物通过PCR扩增家蝇细胞色素P—450 cDNA

根据昆虫细胞色素P-450基因的多型性和遗传多态性,以苯巴比妥钠诱导、室内饲养的杀虫剂敏感种群雌性家蝇Musca omestica vicina Macquart为材料,提取总RNA,以0ligo(dT)-纤维素亲和层析分离出总mRNA;以此为模板反转录合成总cDNA。再以总cDNA为模板,以P-450CYP6A1cDNA序列为参考设计一对低G/C%含量引物,进行PCR扩增,获得1.5kb左右的预期目的片段。     

Cloning of CYP9G2 from the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae).

Cytochrome P450s constitute a superfamily of hemoproteins, important in the metabolism of endogenous and xenobiotic compounds. The full-length cDNA of a novel cytochrome P450, CYP9G2, was isolated from a cDNA library. The cDNA is 2143 bp in length and contains an open reading frame from 50 to 1615 bp, encoding a protein of 521 amino acid residues. The putative P450 protein contains a highly hydrophobic N terminus and a P450 protein signature motif, FG/S*G*R*C*G***A/G, known as the important ligand for heme binding, analysis of the NH2-terminal sequence indicated that CYP9G2 is a microsomal P450. Using polymerase chain reaction with primers specific to CYP9G2, the genomic structure of CYP9G2 was analyzed, and it was found that the gene contains seven introns and eight exons within the coding region, all the sequences of the exon-intron junctions are consistent with the AG-GT rule. Multiple alignment indicated that CYP9G2 is most similar to CYP9E2 from the Blattella germanica (42.7% identity), it is also similar to the insect P450s in family 9, including CYP9L1 from Anopheles gambiae (38.7%) and CYP9A1 from Heliothis virescens (39.5%).     

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.     

Physiological, numerical and molecular characterization of alkyl ether-utilizing rhodococci

Twenty-seven Gram-positive strains were characterized physiologically and numerically and classified them into four groups according to their specific activities for utilization of linear alkyl ethers (AEs), cyclic AEs, monoalkoxybenzenes and 1,4-diethoxybenzene. The comparative analysis of the 16S ribosomal RNA gene and 16S-23S intergenic spacer region showed that they belonged to the genera Rhodococcus and Gordonia. Alkyl ether-utilizing rhodococci appeared to involve various and diverse cytochromes P450 of the families CYP116 (25 positive strains from 27), CYP153 (5/27), CYP249 (1/27) and a new family P450RR1 (27/27). The presence of P450RR1 was strongly related to the specific activity for utilization of 2-methoxyphenol and 2-ethoxyphenol. In addition, 26 of 27 strains contained multiple alkB genes coding for probable non-haem iron containing alkane monooxygenases and hydroxylases. Similar DNA fragments coding for a tetrahydrofuran monooxygenase A subunit (ThmA) were found in all cyclic AE-utilizing strains and nearly identical DNA fragments coding for likely orthologues of a propane monooxygenase A subunit (PrmA) in all linear AE-utilizing strains. The substrate availability in the degradation of aryl AEs, cyclic AEs and linear AEs agreed with the molecular probing of the respective genes encoding cytochrome P450RR1, ThmA and PrmA.     

白纹伊蚊细胞色素P450 CYP6家族基因多样性的研究(英文)

根据已获得的白纹伊蚊CYP6家族某成员cDNA序列片段AEDR ,设计基因特异性引物 ,以白纹伊蚊总RNA为模板 ,进行cDNA末端快速扩增 ,扩增产物经T -A克隆、测序。结果显示 :通过 5’ RACE获得 1个非全长cDNA序列 (GZS331 ) ,其与CYP6N1、CYP6N2的同源性分别为 59 8%和 59 1 % ,与CYP6N3v1 -v3同源性最高 ,达 83 9% - 84 3% ;通过 3’ RACE获得 6个非全长cDNA序列 ,其中来自抗性株的GZG0 33序列与CYP6N3v1 -v3的同源性达 98 2 % - 99 1 % ,而其余 3’ RACE克隆与CYP6N3v1 -v3的同源性则达 84 3% - 85 6%。上述所有非全长cDNA序列均与哺乳动物CYP3A1以及夜蛾CYP9A1有较高的同源性 ,分别为 2 3% - 36 1 %和 2 7 6% - 34 1 %。用PC/GENE软件所绘制的系统树显示出与同源性分析相一致的结果。所得非全长cDNA序列上报国际P450命名委员会进行统一的命名 ,并对蚊虫中细胞色素P450基因多样性及其形成原因进行了分析     

A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes

The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.     

A retinoic acid binding cytochrome P450: CYP120A1 from Synechocystis sp. PCC 6803

At least 35 cytochrome P450 (P450, CYP) or cytochrome P450-like genes have been identified in 10 cyanobacterial genomes yet none have been functionally characterized. CYP110 and CYP120 represent the two largest cyanobacterial P450 families with 16 and four members, respectively, identified to date. The Synechocystis sp. PCC 6803 CYP120A1 protein sequence shares high degrees of conservation with CYP120A2 from Trichodesmium erythraeum IMS101 and CYP120B1 and CYP120C1 from Nostoc punctiforme PCC 73102. In this communication, we report the cloning, expression, purification, and characterization of CYP120A1 from Synechocystis. Homology modeling predictions of the three-dimensional structure of CYP120A1 coupled with in silico screening for potential substrates and experimental spectroscopic analyses have identified retinoic acid as a compound binding with high affinity to this P450's catalytic site. These characterizations of Synechocystis CYP120A1 lay the initial foundations for understanding the basic role of cytochrome P450s in cyanobacteria and related organisms.     

De novo sequence analysis of cytochrome P450 1–3 genes expressed in ostrich liver with highest expression of CYP2G19

The cytochrome P450 (CYP) 1–3 families are involved in xenobiotic metabolism, and are expressed primarily in the liver. Ostriches (Struthio camelus) are members of Palaeognathae with the earliest divergence from other bird lineages. An understanding of genes coding for ostrich xenobiotic metabolizing enzyme contributes to knowledge regarding the xenobiotic metabolisms of other Palaeognathae birds. We investigated CYP1–3 genes expressed in female ostrich liver using a next-generation sequencer. We detected 10 CYP genes: CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2W2, CYP2AC1, CYP2AC2, CYP2AF1, and CYP3A37. We compared the gene expression levels of CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2AF1, and CYP3A37 in ostrich liver and determined that CYP2G19 exhibited the highest expression level. The mRNA expression level of CYP2G19 was approximately 2–10 times higher than those of other CYP genes. The other CYP genes displayed similar expression levels. Our results suggest that CYP2G19, which has not been a focus of previous bird studies, has an important role in ostrich xenobiotic metabolism.     

Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli

Drug oxidation activities of 12 recombinant human cytochrome P450s (P450) coexpressed with human NADPH-P450 reductase (NPR) in bacterial membranes (P450/NPR membranes) were determined and compared with those of other recombinant systems and those of human liver microsomes. Addition of exogenous membrane-bound NPR to the P450/NPR membranes enhanced the catalytic activities of CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5. Enhancement of activities of CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, and CYP2E1 in membranes was not observed after the addition of NPR (4 molar excess to each P450). Exogenous purified human cytochrome b5 (b5) further enhanced catalytic activities of CYP2A6, CYP2B6, CYP2C8, CYP2E1, CYP3A4, and CYP3A5/NPR membranes. Catalytic activities of CYP2C9 and CYP2C19 were enhanced by addition of b5 in reconstituted systems but not in the P450/NPR membranes. Apo b5 (devoid of heme) enhanced catalytic activities when added to both membrane and reconstituted systems, except for CYP2E1/NPR membranes and the reconstituted system containing purified CYP2E1 and NPR. Catalytic activities in P450/NPR membranes fortified with b5 were roughly similar to those measured with microsomes of insect cells coexpressing P450 with NPR (and b5) and/or human liver microsomes, based on equivalent P450 contents. These results suggest that interactions of P450 and NPR coexpressed in membranes or mixed in reconstituted systems appear to be different in some human CYP2 family enzymes, possibly due to a conformational role of b5. P450/NPR membrane systems containing b5 are useful models for prediction of the rates for liver microsomal P450-dependent drug oxidations.     

The cytochrome P450 gene superfamily in Drosophila melanogaster: annotation, intron-exon organization and phylogeny

The cytochrome P450 gene superfamily is represented by 90 sequences in the Drosophila melanogaster genome. Of these 90 P450 sequences, 83 code for apparently functional genes whereas seven are apparent pseudogenes. More than half of the genes belong to only two families, CYP4 and CYP6. The CYP6 family is insect specific whereas the CYP4 family includes sequences from vertebrates. There are eight genes coding for mitochondrial P450s as deduced from their homology to CYP12A1 from the house fly. The genetic map of the distribution of D. melanogaster P450 genes shows (a) the absence of P450 genes on the chromosome 4 and Y, (b) more than half of the P450 genes are found on chromosome 2, and (c) the largest cluster contains nine genes. Sequence alignments were used to draw phylogenetic trees and to analyze the intron-exon organization of each functional P450 gene. Only five P450 genes are intronless. We found 57 unique intron positions, of which 23 were phase zero, 19 were phase one and 15 were phase two. There was a relatively good correlation between intron conservation and phylogenetic relationship between members of the P450 subfamilies. Although the function of many P450 proteins from vertebrates, fungi, plants and bacteria is known, only a single P450 from D. melanogaster, CYP6A2, has been functionally characterized. Gene organization appears to be a useful tool in the study of the regulation, the physiological role and the function of these P450s.     

Functional Interactions between Cytochromes P450 1A2 and 2B4 Require Both Enzymes to Reside in the Same Phospholipid Vesicle: EVIDENCE FOR PHYSICAL COMPLEX FORMATION*

Previous studies have shown that the combined presence of two cytochrome P450 enzymes (P450s) can affect the function of both enzymes, results that are consistent with the formation of heteromeric P450·P450 complexes. The goal of this study was to provide direct evidence for a physical interaction between P450 1A2 (CYP1A2) and P450 2B4 (CYP2B4), by determining if the interactions required both enzymes to reside in the same lipid vesicles. When NADPH-cytochrome P450 reductase (CPR) and a single P450 were incorporated into separate vesicles, extremely slow reduction rates were observed, demonstrating that the enzymes were anchored in the vesicles. Next, several reconstituted systems were prepared: 1) CPR·CYP1A2, 2) CPR·CYP2B4, 3) a mixture of CPR·CYP1A2 vesicles with CPR·CYP2B4 vesicles, and 4) CPR·CYP1A2·CYP2B4 in the same vesicles (ternary system). When in the ternary system, CYP2B4-mediated metabolism was significantly inhibited, and CYP1A2 activities were stimulated by the presence of the alternate P450. In contrast, P450s in separate vesicles were unable to interact. These data demonstrate that P450s must be in the same vesicles to alter metabolism. Additional evidence for a physical interaction among CPR, CYP1A2, and CYP2B4 was provided by cross-linking with bis(sulfosuccinimidyl) suberate. The results showed that after cross-linking, antibody to CYP1A2 was able to co-immunoprecipitate CYP2B4 but only when both proteins were in the same phospholipid vesicles. These results clearly demonstrate that the alterations in P450 function require both P450s to be present in the same vesicles and support a mechanism whereby P450s form a physical complex in the membrane.