Molecular cloning, refined chromosomal mapping and structural analysis of the human gene encoding aldehyde oxidase (AOX1), a candidate for the ALS2 gene

111

We report the cloning of the AOH1 and AOH2 genes, which encode two novel mammalian molybdo-flavoproteins. We have purified the AOH1 protein to homogeneity in its catalytically active form from mouse liver. Twenty tryptic peptides, identified or directly sequenced by mass spectrometry, confirm the primary structure of the polypeptide deduced from the AOH1 gene. The enzyme contains one molecule of FAD, one atom of molybdenum, and four atoms of iron per subunit and shows spectroscopic features similar to those of the prototypic molybdo-flavoprotein xanthine oxidoreductase. The AOH1 and AOH2 genes are 98 and 60 kilobases long, respectively, and consist of 35 coding exons. The AOH1 gene has the potential to transcribe an extra leader non-coding exon, which is located downstream of exon 26, and is transcribed in the opposite orientation relative to all the other exons. AOH1 and AOH2 map to chromosome 1 in close proximity to each other and to the aldehyde oxidase gene, forming a molybdo-flavoenzyme gene cluster. Conservation in the position of exon/ intron junctions among the mouse AOH1, AOH2, aldehyde oxidase, and xanthine oxidoreductase loci indicates that these genes are derived from the duplication of an ancestral precursor.

Section: Thaliana C Elegans or D Melanogaster Aos Is The Results Omentioning

confidence: 99%

Purification of the Aldehyde Oxidase Homolog 1 (AOH1) Protein and Cloning of the AOH1 and Aldehyde Oxidase Homolog 2 (AOH2) Genes

Terao¹,

Kurosaki²,

Marini³

et al. 2001

111

“…This region contains the functional mouse proximal promoter identified previously and 700 bp of additional upstream DNA (21,22). Luciferase fusion constructs were confirmed by DNA sequence analysis as described (19). Fluorescence sequence analysis was performed using the dideoxynucleotide chain termination system from PerkinElmer Life Sciences.…”

Section: Methodsmentioning

confidence: 99%

“…Mouse XOR Promoter Cloning, Plasmid Construction, and Mutagenesis-An in-frame fusion to the luciferase translational start site of the luciferase expression vector pGL3-Basic (Promega Corp.) was constructed exactly as indicated previously (19). Briefly, upstream DNA to be cloned into pGL3-Basic was amplified by PCR, combining sequences from the mouse XOR cDNA and chromosomal locus (20,21).…”

Section: Methodsmentioning

confidence: 99%

Stress Activation of Mammary Epithelial Cell Xanthine Oxidoreductase Is Mediated by p38 MAPK and CCAAT/Enhancer-binding Protein-β

Seymour

Roberts

Fini

et al. 2006

Xanthine oxidoreductase (XOR) catalyzes the formation of uric acid from xanthine and hypoxanthine and is recognized as a source of reactive oxygen and nitrogen species. Unexpectedly, XOR was found to play an essential role in milk secretion in the differentiating mammary gland, where it is an integral component of the milk fat globule. XOR gene expression in both mammary glands and differentiating mammary epithelial cells in culture is regulated by the lactogenic hormones prolactin and cortisol. Expression in mammary epithelial cells is also regulated by inflammatory cytokines and induced by cycloheximide. Cycloheximide was found to stimulate XOR gene expression in differentiating HC11 mouse mammary epithelial cells. Activation of XOR gene expression by both cycloheximide and inflammatory cytokines suggested that XOR may be regulated by stress-activated protein kinases, the MAPKs. We demonstrate here that XOR was induced in HC11 cells by low dose cycloheximide and that expression was blocked by inhibitors of p38 MAPK. Accumulation of phospho-p38 was stimulated by low dose cycloheximide. Low dose cycloheximide stress promoted phosphorylation and nuclear accumulation of the CCAAT/enhancer-binding protein-␤ (C/EBP␤) transcription factor, which was blocked by inhibition of p38. Furthermore, C/EBP␤ was found to activate the mouse XOR promoter, and XOR promoter-C/EBP␤ protein complexes were induced by low dose cycloheximide stress. These data demonstrate, for the first time, that mouse mammary epithelial cell XOR is regulated by p38 MAPK. They identify an essential function of the C/EBP␤ transcription factor in mouse XOR expression and suggest a potential role for p38 MAPK activation of C/EBP␤ in mammary epithelial cells.

“…Northern Blot Analysis and Quantitation-Northern blots were run using formaldehyde-agarose gels and 5 g of poly(A) ϩ RNA (6,29). Hybridization probes were isolated from the clones pMID, p3ЈRATRACE, and p5ЈRATRACE by PCR amplification and agarose gel electrophoresis.…”

Section: G Of Poly(a)mentioning

confidence: 99%

“…High stringency hybridization and washing were conducted as described (6,29). Following hybridization and autoradiography, each 4,500-nt band was cut from the hybridization filter and counted by liquid scintillation counting for quantitation.…”

Section: G Of Poly(a)mentioning

confidence: 99%

cDNA Cloning, Sequencing, and Characterization of Male and Female Rat Liver Aldehyde Oxidase (rAOX1)

Wright

Clayton

Riley

et al. 1999

Molecular characterization of male and female rat liver aldehyde oxidase is reported. As described for the mouse liver, male and female rat liver expressed kinetically distinct forms of aldehyde oxidase. Our data suggest that the two forms arise as a result of differences in redox state and are most simply explained by expression of a single gene encoding aldehyde oxidase in rats. In support of this argument we have sequenced cDNAs from male and female rat liver. We examined mRNA expression by Northern blot analysis with RNA from males and females, from several tissues, and following androgen induction. Purified rat liver enzyme from males or females revealed a single 150-kDa species consistent with cDNA sequence analysis. Both male and female forms were reactive to the same carboxyl-terminal directed antisera. K m(app) values obtained in crude extracts of male or female rat liver and post-benzamidine-purified aldehyde oxidase differed substantially from each other but could be interconverted by chemical reduction with dithiothreitol or oxidation with 4,4-dithiodipyridine. Our data indicate that a single gene is most likely expressed in male or female rat liver and that the kinetic differences between male and female rat liver aldehyde oxidases are sensitive to redox manipulation. Aldehyde oxidase (AOX)1 is a member of the molybdenum cofactor containing enzymes. Native AOX is routinely prepared as a homodimer of 300 kDa. Each 150-kDa subunit contains two iron-sulfur centers, an FAD, and the molybdenum-pterin cofactor (MoCo) (1-3). AOX (EC 1.2.3.1) catalyzes the oxidation of a wide range of aldehydic compounds, purines, quinoliniums, and numerous pharmacologic agents. While substrate specificity for AOX is very broad, and wide species variation in substrate specificity exists, the general catalytic reaction takes the form of hydroxyl transfer from water to an aldehyde creating the cognate acid. For example, conversion of benzaldehyde to benzoic acid is a very efficient reaction for AOX from most species. Conversion of N-1-methyl nicotinamide (NMN) to the 2-or -4-pyridone has been used as a standard definition of AOXs, although it is usually a kinetically less efficient reaction than benzaldehyde oxidation.AOX is of interest both for its role in drug metabolism and as a source of the reactive oxygen species (ROS), hydrogen peroxide, and the superoxide anion, that have been related to numerous human pathologies. The human gene encoding AOX has been linked to a rare form of amyotrophic lateral sclerosis, although it is unknown if AOX encodes the amyotrophic lateral sclerosis locus itself (4 -6). ROS are generated from AOX in an oxidative half-reaction following reduction of the enzyme by substrate. The FeS, FAD, and MoCo cofactors comprise an internal electron transfer chain in which electrons are passed from the active site molybdenum center to the FeS centers and finally to FAD. Partial reduction of oxygen at the the reduced FAD site produces ROS. Unlike the related enzyme xanthine dehydrogenase (XDH), AOX does no...