The Aldehyde Oxidase Gene Cluster in Mice and Rats
2004; Elsevier BV; Volume: 279; Issue: 48 Linguagem: Inglês
10.1074/jbc.m408734200
ISSN1083-351X
AutoresMami Kurosaki, Mineko Terao, Maria Monica Barzago, Antonio Bastone, Davide Bernardinello, Mario Salmona, Enrico Garattini,
Tópico(s)Alcohol Consumption and Health Effects
ResumoMammalian molybdo-flavoenzymes are oxidases requiring FAD and molybdopterin (molybdenum cofactor) for their catalytic activity. This family of proteins was thought to consist of four members, xanthine oxidoreductase, aldehyde oxidase 1 (AOX1), and the aldehyde oxidase homologues 1 and 2 (AOH1 and AOH2, respectively). Whereas the first two enzymes are present in humans and various other mammalian species, the last two proteins have been described only in mice. Here, we report on the identification, in both mice and rats, of a novel molybdo-flavoenzyme, AOH3. In addition, we have cloned the cDNAs coding for rat AOH1 and AOH2, demonstrating that this animal species has the same complement of molybdo-flavoproteins as the mouse. The AOH3 cDNA is characterized by remarkable similarity to AOX1, AOH1, AOH2, and xanthine oxidoreductase cDNAs. Mouse AOH3 is selectively expressed in Bowman's glands of the olfactory mucosa, although small amounts of the corresponding mRNA are present also in the skin. In the former location, two alternatively spliced forms of the AOH3 transcript with different 3′-untranslated regions were identified. The general properties of AOH3 were determined by purification of mouse AOH3 from the olfactory mucosa. The enzyme possesses aldehyde oxidase activity and oxidizes, albeit with low efficiency, exogenous substrates that are recognized by AOH1 and AOX1. The Aoh3 gene maps to mouse chromosome 1 band c1 and rat chromosome 7 in close proximity to the Aox1, Aoh1, and Aoh2 loci and has an exon/intron structure almost identical to that of the other molybdo-flavoenzyme genes in the two species. Mammalian molybdo-flavoenzymes are oxidases requiring FAD and molybdopterin (molybdenum cofactor) for their catalytic activity. This family of proteins was thought to consist of four members, xanthine oxidoreductase, aldehyde oxidase 1 (AOX1), and the aldehyde oxidase homologues 1 and 2 (AOH1 and AOH2, respectively). Whereas the first two enzymes are present in humans and various other mammalian species, the last two proteins have been described only in mice. Here, we report on the identification, in both mice and rats, of a novel molybdo-flavoenzyme, AOH3. In addition, we have cloned the cDNAs coding for rat AOH1 and AOH2, demonstrating that this animal species has the same complement of molybdo-flavoproteins as the mouse. The AOH3 cDNA is characterized by remarkable similarity to AOX1, AOH1, AOH2, and xanthine oxidoreductase cDNAs. Mouse AOH3 is selectively expressed in Bowman's glands of the olfactory mucosa, although small amounts of the corresponding mRNA are present also in the skin. In the former location, two alternatively spliced forms of the AOH3 transcript with different 3′-untranslated regions were identified. The general properties of AOH3 were determined by purification of mouse AOH3 from the olfactory mucosa. The enzyme possesses aldehyde oxidase activity and oxidizes, albeit with low efficiency, exogenous substrates that are recognized by AOH1 and AOX1. The Aoh3 gene maps to mouse chromosome 1 band c1 and rat chromosome 7 in close proximity to the Aox1, Aoh1, and Aoh2 loci and has an exon/intron structure almost identical to that of the other molybdo-flavoenzyme genes in the two species. Molybdo-flavoenzymes are a subgroup of molybdo-proteins requiring molybdopterin (molybdenum cofactor) and a flavin cofactor for their catalytic activity (1Garattini E. Mendel R. Romao M.J. Wright R. Terao M. Biochem. J. 2003; 372: 15-32Crossref PubMed Scopus (203) Google Scholar, 2Schindelin H. Kisker C. Rajagopalan K.V. Adv. 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In mammals, four distinct molybdo-flavoenzymes have been identified: xanthine oxidoreductase (XOR) 1The abbreviations used are: XOR, xanthine oxidoreductase; AOX1, aldehyde oxidase 1; AOH1, AOH2, and AOH3, aldehyde oxidase homologues 1–3, respectively; RT, reverse transcription; RACE, rapid amplification of cDNA ends; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; EST, expressed sequence tag; 3′-UTR, 3′-untranslated region. (7Xu P. Huecksteadt T.P. Harrison R. Hoidal J.R. Biochem. Biophys. Res. Commun. 1994; 199: 998-1004Crossref PubMed Scopus (57) Google Scholar, 8Saksela M. Raivio K.O. Biochem. J. 1996; 315: 235-239Crossref PubMed Scopus (36) Google Scholar, 9Tsuchida S. Yamada R. Ikemoto S. Tagawa M. J. Vet. Med. Sci. 2001; 63: 353-355Crossref PubMed Scopus (5) Google Scholar, 10Ichida K. Amaya Y. Noda K. Minoshima S. Hosoya T. Sakai O. Shimizu N. Nishino T. Gene (Amst.). 1993; 133: 279-284Crossref PubMed Scopus (132) Google Scholar, 11Terao M. Cazzaniga G. 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Chem. 2001; 276: 46347-46363Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Whereas XOR and AOX1 have been isolated and characterized in humans (8Saksela M. Raivio K.O. Biochem. J. 1996; 315: 235-239Crossref PubMed Scopus (36) Google Scholar, 12Wright R.M. Vaitaitis G.M. Wilson C.M. Repine T.B. Terada L.S. Repine J.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10690-10694Crossref PubMed Scopus (80) Google Scholar, 16Terao M. Kurosaki M. Demontis S. Zanotta S. Garattini E. Biochem. J. 1998; 332: 383-393Crossref PubMed Scopus (56) Google Scholar), rodents (17Kurosaki M. Demontis S. Barzago M.M. Garattini E. Terao M. Biochem. J. 1999; 341: 71-80Crossref PubMed Scopus (57) Google Scholar, 18Cazzaniga G. Terao M. Lo Schiavo P. Galbiati F. Segalla F. Seldin M.F. Garattini E. Genomics. 1994; 23: 390-402Crossref PubMed Scopus (50) Google Scholar, 19Wright R.M. Clayton D.A. Riley M.G. McManaman J.L. Repine J.E. J. Biol. Chem. 1999; 274: 3878-3886Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), and bovines (13Calzi M.L. Raviolo C. Ghibaudi E. de Gioia L. Salmona M. Cazzaniga G. Kurosaki M. Terao M. Garattini E. J. Biol. Chem. 1995; 270: 31037-31045Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 20Berglund L. Rasmussen J.T. Andersen M.D. Rasmussen M.S. Petersen T.E. J. Dairy Sci. 1996; 79: 198-204Abstract Full Text PDF PubMed Scopus (59) Google Scholar, 21Terao M. Kurosaki M. Zanotta S. Garattini E. Biochem. Soc. Trans. 1997; 25: 791-796Crossref PubMed Scopus (23) Google Scholar), AOH1 and AOH2 have been identified only in mice (14Terao M. Kurosaki M. Saltini G. Demontis S. Marini M. Salmona M. Garattini E. J. Biol. Chem. 2000; 275: 30690-30700Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15Terao M. Kurosaki M. Marini M. Vanoni M.A. Saltini G. Bonetto V. Bastone A. Federico C. Saccone S. Fanelli R. Salmona M. Garattini E. J. Biol. Chem. 2001; 276: 46347-46363Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Mammalian molybdo-flavoenzymes are cytosolic proteins consisting of dimers of two identical subunits (1Garattini E. Mendel R. Romao M.J. Wright R. Terao M. Biochem. J. 2003; 372: 15-32Crossref PubMed Scopus (203) Google Scholar). The amino acid sequence of the ∼150-kDa subunit of all members of the family is highly conserved, suggesting a related evolutionary origin from a common ancestor protein (1Garattini E. Mendel R. Romao M.J. Wright R. Terao M. Biochem. J. 2003; 372: 15-32Crossref PubMed Scopus (203) Google Scholar). XOR, AOX1, AOH1, and AOH2 are the products of distinct genes that maintain an almost superimposing structural organization (15Terao M. Kurosaki M. Marini M. Vanoni M.A. Saltini G. Bonetto V. Bastone A. Federico C. Saccone S. Fanelli R. Salmona M. Garattini E. J. Biol. Chem. 2001; 276: 46347-46363Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). This suggests that the extant members of the mammalian molybdo-flavoenzyme family arose from one or more gene duplication events (1Garattini E. Mendel R. Romao M.J. Wright R. Terao M. Biochem. J. 2003; 372: 15-32Crossref PubMed Scopus (203) Google Scholar). In mice, Xor maps to chromosome 17 (18Cazzaniga G. Terao M. Lo Schiavo P. Galbiati F. Segalla F. Seldin M.F. Garattini E. Genomics. 1994; 23: 390-402Crossref PubMed Scopus (50) Google Scholar), whereas Aox1, Aoh1, and Aoh2 constitute an aldehyde oxidase gene cluster on chromosome 1 band c1 (15Terao M. Kurosaki M. Marini M. Vanoni M.A. Saltini G. Bonetto V. Bastone A. Federico C. Saccone S. Fanelli R. Salmona M. Garattini E. J. Biol. Chem. 2001; 276: 46347-46363Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). XOR is a relatively ubiquitous enzyme, being present in many tissues and cell types, although it is synthesized in high amounts in the intestinal tract, liver, and lactating mammary gland (22Harrison R. Free Radic. Biol. Med. 2002; 33: 774-797Crossref PubMed Scopus (657) Google Scholar). XOR catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid (23Harrison R. Drug Metab. Rev. 2004; 36: 363-375Crossref PubMed Scopus (165) Google Scholar), representing a key enzyme in the catabolism of purines (22Harrison R. Free Radic. Biol. Med. 2002; 33: 774-797Crossref PubMed Scopus (657) Google Scholar, 23Harrison R. Drug Metab. Rev. 2004; 36: 363-375Crossref PubMed Scopus (165) Google Scholar). However, the protein may have other tissue- or cell-specific functions, as suggested by the phenotype observed in the Xor knockout mouse (24Vorbach C. Scriven A. Capecchi M.R. Genes Dev. 2002; 16: 3223-3235Crossref PubMed Scopus (168) Google Scholar). Much less is known about the physiological role and substrates of the other members of the family. AOX1 and AOH1 are predominantly expressed in the liver and lung and metabolize retinaldehyde into retinoic acid (14Terao M. Kurosaki M. Saltini G. Demontis S. Marini M. Salmona M. Garattini E. J. Biol. Chem. 2000; 275: 30690-30700Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15Terao M. Kurosaki M. Marini M. Vanoni M.A. Saltini G. Bonetto V. Bastone A. Federico C. Saccone S. Fanelli R. Salmona M. Garattini E. J. Biol. Chem. 2001; 276: 46347-46363Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 17Kurosaki M. Demontis S. Barzago M.M. Garattini E. Terao M. Biochem. J. 1999; 341: 71-80Crossref PubMed Scopus (57) Google Scholar, 25Ambroziak W. Izaguirre G. Pietruszko R. J. Biol. Chem. 1999; 274: 33366-33373Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 26Huang D.Y. Furukawa A. Ichikawa Y. Arch. Biochem. Biophys. 1999; 364: 264-272Crossref PubMed Scopus (51) Google Scholar, 27Tomita S. Tsujita M. Ichikawa Y. FEBS Lett. 1993; 336: 272-274Crossref PubMed Scopus (44) Google Scholar), which is the active metabolite of vitamin A, a known morphogen (28Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2606) Google Scholar, 29Kastner P. Mark M. Chambon P. Cell. 1995; 83: 859-869Abstract Full Text PDF PubMed Scopus (940) Google Scholar) and a key regulator of many tissues and cell types in the adult animal. Thus, AOX1 and AOH1 may be of relevance to the development of vertebrates and may control the homeostasis of certain types of tissues in adults. Virtually nothing is known about the substrate specificity of AOH2. In fact, the enzyme is difficult to purify, as it is present in relatively low quantities only in keratinized epithelia (14Terao M. Kurosaki M. Saltini G. Demontis S. Marini M. Salmona M. Garattini E. J. Biol. Chem. 2000; 275: 30690-30700Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15Terao M. Kurosaki M. Marini M. Vanoni M.A. Saltini G. Bonetto V. Bastone A. Federico C. Saccone S. Fanelli R. Salmona M. Garattini E. J. Biol. Chem. 2001; 276: 46347-46363Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). AOX1 is an enzyme of pharmacological and toxicological importance. In fact, this aldehyde oxidase metabolizes numerous xenobiotics, including drugs such as zaleplon (30Lake B.G. Ball S.E. Kao J. Renwick A.B. Price R.J. Scatina J.A. Xenobiotica. 2002; 32: 835-847Crossref PubMed Scopus (80) Google Scholar) and 6-mercaptopurine (31Rooseboom M. Commandeur J.N. Vermeulen N.P. Pharmacol. Rev. 2004; 56: 53-102Crossref PubMed Scopus (438) Google Scholar) and pollutants such as nitropolycyclic hydro carbons (32Tatsumi K. Kitamura S. Narai N. Cancer Res. 1986; 46: 1089-1093PubMed Google Scholar). The enzyme has also been implicated in the hepatotoxicity of ethanol in humans and other mammals, as AOX1 oxidizes the toxic metabolite acetaldehyde into acetic acid (33Shaw S. Jayatilleke E. Biochem. J. 1990; 268: 579-583Crossref PubMed Scopus (98) Google Scholar). We proposed that AOH1 may play a similar role in the mouse; however, studies conducted on purified mouse AOX1 and AOH1 indicate that acetaldehyde is a relatively poor substrate for the two enzymes (34Vila R. Kurosaki M. Barzago M.M. Kolek M. Bastone A. Colombo L. Salmona M. Terao M. Garattini E. J. Biol. Chem. 2004; 279: 8668-8683Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In addition, results obtained in mouse strains characterized by the synthesis of remarkably different levels of both AOH1 and AOX1 show the same level of acetaldehyde metabolism in vivo (34Vila R. Kurosaki M. Barzago M.M. Kolek M. Bastone A. Colombo L. Salmona M. Terao M. Garattini E. J. Biol. Chem. 2004; 279: 8668-8683Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In this study, we demonstrate that the family of mammalian molybdo-flavoenzymes is larger than originally believed. In fact, we have identified and isolated mouse AOH3, a novel molybdo-flavoenzyme characterized by high similarity to AOX1, AOH1, AOH2, and, to a lesser extent, XOR. The protein is endowed with aldehyde oxidase activity and is selectively expressed in the epithelial mucosa at the level of Bowman's glands. The corresponding cDNAs have been cloned and sequenced in both the mouse and rat. Isolation of the mouse and rat AOH3 cDNAs proved instrumental in the definition of the exon/intron structure of the corresponding genes, which are located on chromosomes 1 and 9, respectively. Finally, we have also cloned and sequenced the cDNAs of the mouse AOH1 and AOH2 orthologous proteins in rats, demonstrating that the two molybdo-enzymes are coded for by genes that cluster with Aox1 and Aoh3 on chromosome 9. These last results demonstrate that the presence of multiple forms of aldehyde oxidases in mammals is not a peculiarity of the mouse. Cloning and Sequencing of cDNAs Coding for Mouse AOH3 and Rat AOH1, AOH2, and AOH3—Total RNA was extracted from mouse olfactory mucosa, and the poly(A+) fraction of the RNA was selected according to standard protocols (14Terao M. Kurosaki M. Saltini G. Demontis S. Marini M. Salmona M. Garattini E. J. Biol. Chem. 2000; 275: 30690-30700Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15Terao M. Kurosaki M. Marini M. Vanoni M.A. Saltini G. Bonetto V. Bastone A. Federico C. Saccone S. Fanelli R. Salmona M. Garattini E. J. Biol. Chem. 2001; 276: 46347-46363Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The full-length mouse AOH3 cDNA expressed in olfactory mucosa was isolated as two overlapping fragments, A (nucleotides 72–2332) and B (nucleotides 2247–4425), by reverse transcription followed by nested PCR amplifications. The oligonucleotides used for these experiments were as follows: primary amplimers, 5′-ATCCGAGCCAAGAGGCCAAACTCA-3′ (nucleotides 62–85) and 5′-CCTTTCCTTTCCCTCCATCTGCAGCA-3′ (complementary to nucleotides 4410–4435); and nested primers, 5′-AGAGGCCAAACTCACCAGAGCC-3′ (nucleotides 72–93), 5′-CTTCAGCTACCTGATCCACGTTCT-3′ (complementary to nucleotides 2309–2332), 5′-GCACAATTCGTTCCTGTGCCCTGAA-3′ (nucleotides 2247–2271), and 5′-CCCTCCATCTGCAGCAACATTTAC-3′ (complementary to nucleotides 4402–4425). The amplified DNA fragments (pAOH3-A and pAOH3-B) were subcloned into pBluescript (Stratagene, La Jolla, CA) with the AT cloning kit (Invitrogen) and sequenced. The mouse AOH3 cDNA was also isolated from skin poly(A+) RNA in a similar way, and its nucleotide sequence was determined. Rat AOH1, AOH2, and AOH3 cDNAs were isolated from liver, skin, and olfactory mucosa poly(A+) RNAs, respectively, by reverse transcription (RT)-PCR techniques using sense strand amplimers coding for the first eight N-terminal amino acids and antisense amplimers corresponding to the seven C-terminal amino acids and the stop codon. The resulting amplified cDNA fragments representing the entire coding regions of each protein were subcloned in pBluescript and sequenced. Determination of the 5′- and 3′-Ends of the Mouse AOH3 Transcripts—The 5′- and 3′-rapid amplification of cDNA ends (RACE) experiments for the mouse AOH3 cDNAs were performed with the commercially available Marathon cDNA amplification kit (Clontech, Palo Alto, CA) according to the nested PCR protocol and using the following amplimers: SP1, 5′-CGGATCGTGTTGTGACACCATCACTG-3′ (complementary to nucleotides 263–288 of the mouse AOH3 cDNA); and NP1, 5′-TGTGACACCATCACTGTGCAGGC-3′ (complementary to nucleotides 256–278 of the mouse AOH3 cDNA). 3′-RACE of the mouse AOH3 cDNAs was conducted as described above with the following amplimers: SP2, 5′-AAGAGACATAGCGGAGGACTTCACAG-3′ (nucleotides 3990–4015); and NP2, 5′-GACTTCACAGTGAAGAGCCCAGCA-3′ (nucleotides 4006–4029). The resulting PCR products were subcloned in pBluescript, and multiple clones were sequenced to determine the 5′- and 3′-ends of the mouse AOH3 transcript. In Situ Hybridization Experiments—The plasmid pAOH3-A was linearized with HindIII and used as template for the synthesis of anti-sense riboprobe employing T7 RNA polymerase (Stratagene) in the presence of [35S]thio-UTP as described (14Terao M. Kurosaki M. Saltini G. Demontis S. Marini M. Salmona M. Garattini E. J. Biol. Chem. 2000; 275: 30690-30700Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Mouse tissues were fixed in 4% (w/v) paraformaldehyde overnight, embedded in paraffin, sectioned to 5-μm thickness, and mounted on gelatin-coated slides. The conditions for the pretreatment of slides, hybridization, washing, and detection by the nuclear track emulsion technique were as described in a previous report (14Terao M. Kurosaki M. Saltini G. Demontis S. Marini M. Salmona M. Garattini E. J. Biol. Chem. 2000; 275: 30690-30700Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). At the end of the in situ hybridization procedure, tissue sections were stained with hematoxylin/eosin and photographed under a microscope. Purification of Mouse Olfactory Mucosa AOH3 Protein, Electrophoresis, and Western Blot Analysis—Unless otherwise stated, all purification steps were carried out at 4 °C. Male mouse olfactory mucosa was dissected and homogenized in 3 volumes of 100 mm sodium phosphate buffer (pH 7.5) containing 0.1% Triton X-100 with an Ultraturrax homogenizer (Ika, Stanten, Germany). Homogenates were centrifuged at 100,000 × g for 45 min to obtain cytosolic extracts. Extracts were heated at 55 °C for 10 min and centrifuged at 15,000 × g to remove precipitated proteins. An equal volume of saturated ammonium sulfate was added to the supernatant, and the precipitate was collected by centrifugation at 100,000 × g and resuspended in 50 mm Tris-HCl (pH 7.5). The solution was passed through a Sephadex PD-10 column (Amersham Biosciences AB, Uppsala, Sweden) to eliminate the residual ammonium sulfate. The eluate (3.5 ml) was applied to a Mono Q 5/5 fast protein liquid chromatography column (Amersham Biosciences AB) equilibrated in 50 mm Tris-HCl (pH 7.4). The AOH3 protein was eluted at 0.5 ml/min with a linear gradient (30 ml) of 0–1 m NaCl in 50 mm Tris-HCl (pH 7.5). The purification of AOH3 was monitored by quantitative Western blot analysis (34Vila R. Kurosaki M. Barzago M.M. Kolek M. Bastone A. Colombo L. Salmona M. Terao M. Garattini E. J. Biol. Chem. 2004; 279: 8668-8683Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). A specific rabbit anti-AOH3 polyclonal antibody raised against a synthetic peptide of the protein (SGRIKALDIE, amino acids 868–877) was used for Western blot analysis, which was carried out following a chemiluminescence-based protocol as described (14Terao M. Kurosaki M. Saltini G. Demontis S. Marini M. Salmona M. Garattini E. J. Biol. Chem. 2000; 275: 30690-30700Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15Terao M. Kurosaki M. Marini M. Vanoni M.A. Saltini G. Bonetto V. Bastone A. Federico C. Saccone S. Fanelli R. Salmona M. Garattini E. J. Biol. Chem. 2001; 276: 46347-46363Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 34Vila R. Kurosaki M. Barzago M.M. Kolek M. Bastone A. Colombo L. Salmona M. Terao M. Garattini E. J. Biol. Chem. 2004; 279: 8668-8683Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). This antibody is monospecific and does not cross-react with purified mouse AOX1 and AOH1 and recombinant mouse XOR, AOX1, AOH1, and AOH2 transiently expressed in human epithelial kidney HEK293 cells (data not shown). For quantitative Western blot analysis, an equivalent volume (10 μl) of protein solution, at each purification step, was loaded on the same gel and processed for analysis. Chemiluminescent signals corresponding to AOH3 bands were quantitated with a scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA). The total amount of AOH3 immunoreactive protein in the various experimental samples is expressed in arbitrary units and was calculated on the basis of the intensity of the Western blot signal in absorbance multiplied by the total volume of each purification step. One arbitrary unit of immunoreactive protein corresponds to 1.0 A unit of the specific AOH3 band in each experimental sample. Zymographic analysis of aldehyde-oxidizing activity was performed following electrophoresis on cellulose acetate plates. Plates were overlaid with 1.2% agarose containing 0.3 mm phenazine methosulfate (Sigma), 0.9 mm 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma), and the selected enzyme substrate at 10 mm (14Terao M. Kurosaki M. Saltini G. Demontis S. Marini M. Salmona M. Garattini E. J. Biol. Chem. 2000; 275: 30690-30700Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 34Vila R. Kurosaki M. Barzago M.M. Kolek M. Bastone A. Colombo L. Salmona M. Terao M. Garattini E. J. Biol. Chem. 2004; 279: 8668-8683Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). SDS-PAGE was performed according to standard techniques (14Terao M. Kurosaki M. Saltini G. Demontis S. Marini M. Salmona M. Garattini E. J. Biol. Chem. 2000; 275: 30690-30700Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15Terao M. Kurosaki M. Marini M. Vanoni M.A. Saltini G. Bonetto V. Bastone A. Federico C. Saccone S. Fanelli R. Salmona M. Garattini E. J. Biol. Chem. 2001; 276: 46347-46363Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 34Vila R. Kurosaki M. Barzago M.M. Kolek M. Bastone A. Colombo L. Salmona M. Terao M. Garattini E. J. Biol. Chem. 2004; 279: 8668-8683Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Proteins were measured according to the Bradford method with a commercially available kit (Bio-Rad). Characterization of the Purified AOH3 Protein by Mass Spectrometry—Matrix-assisted laser desorption ionization (MALDI) mass spectrometric and electrospray ionization tandem mass spectrometric analyses of AOH3 tryptic peptides were performed according to standard protocols following in-gel tryptic digestion (34Vila R. Kurosaki M. Barzago M.M. Kolek M. Bastone A. Colombo L. Salmona M. Terao M. Garattini E. J. Biol. Chem. 2004; 279: 8668-8683Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Briefly, the Coomassie Blue-stained gel slice corresponding to purified AOH3 was incubated with 10 mm dithiothreitol in 100 mm ammonium bicarbonate at 56 °C for 30 min to reduce disulfide bridges. Thiol groups were alkylated upon reaction with 55 mm iodoacetamide in 100 mm ammonium bicarbonate at room temperature in the dark for 20 min. Tryptic digestion was carried out overnight at 37 °C in 50 mm ammonium bicarbonate and 12.5 ng/μl trypsin (Promega, Madison, WI). Peptides were extracted twice in 50% acetonitrile and 5% formic acid. The combined extracts were lyophilized, redissolved in 0.5% formic acid, and desalted using ZipTip (Millipore Corp.). Peptides were eluted in 50% acetonitrile and 0.5% formic acid. The eluate was mixed 1:1 (v/v) with a saturated matrix solution of α-cyano-4-hydroxycinnamic acid in acetonitrile and 0.1% trifluoroacetic acid (1:3, v/v). Mass mapping of tryptic peptides was performed with a Bruker Reflex III MALDI-TOF mass spectrometer. The data generated were processed with the Mascot program (www.matrixscience.com/) (34Vila R. Kurosaki M. Barzago M.M. Kolek M. Bastone A. Colombo L. Salmona M. Terao M. Garattini E. J. Biol. Chem. 2004; 279: 8668-8683Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), allowing a mass tolerance of ≤0.1 Da. De novo sequence analysis was carried out via collision-induced dissociation on an API 3000 electrospray mass spectrometer (Applied Biosystems, San Diego, CA). The data were confirmed by comparison of the experimental and theoretical collision-induced dissociation spectra of the tryptic peptides derived from the AOH3 protein sequence derived from the corresponding cDNA. DNA Sequencing and Determination of the Exon/Intron Structure of the Mouse Aoh3 and Rat Aoh1, Aoh2, and Aoh3 Genes—Appropriate DNA fragments were subcloned into the pBluescript plasmid vector and sequenced according to the Sanger dideoxy chain termination method using double-stranded DNA as template and T7 DNA polymerase (Amersham Biosciences AB) or Sequenase (U. S. Biochemical Corp.). Oligodeoxynucleotide primers were custom-synthesized by M-Medical srl (Florence, Italy). Computer analysis of the DNA sequences was performed using the GeneWorks sequence analysis system (Intelligenetics, San Diego). Comparison of the nucleotide and protein sequences of the full-length cDNAs corresponding to mouse AOH3 and rat AOH1, AOH2, and AOH3 with the complete mouse and rat genomic sequences present in the NCBI Database resulted in the determination of the exon/intron structure of the corresponding genes. Cloning and Characterization of the Mouse cDNA Coding for the Novel Molybdo-flavoprotein AOH3—In silico analysis of the NCBI Database for nucleotide sequences of the mouse genome showing similarity to the AOX1, AOH1, and AOH2 cDNAs resulted in the identification of exons coding for a potential and novel molybdo-flavoprotein, which we named AOH3. The identified exon sequences were used for the design of appropriate oligonucleotides that allowed the amplification of overlapping cDNA fragments of mouse AOH3 by RT-PCR from olfactory mucosa and, subsequently, from skin RNA. The first source of AOH3 RNA was selected on the basis of a preliminary search in the mouse expressed sequence tag (EST) data base. Fig. 1 illustrates the nucleotide sequence of the full-length AOH3 cDNA isolated from mouse olfactory mucosa. The 5′-untranslated region is relatively short (93 nucleotides) and contains a stretch of DNA around the putative first methionine codon (GCCATGC) that is similar to the canonical ribosome-binding consensus sequence (RCCATGG) (35Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4172) Google Scholar). The 3′-untranslated region is much longer and is characterized by the absence of a canonical polyadenylation signal. As this portion of the cDNA was obtained by 3′-RACE, the lack of a polyadenylation signal is likely to be the result of the synthesis of an incomplete 3′ terminus. In fact, a typical polyadenylation consensus sequence (AATAAA) is present in the mouse genome (NCBI accession no. NT_039170), 215 bases downstream of the nucleotide corresponding to the last base of our AOH3 cDNA. More important, two ESTs present in the NCBI Database and corresponding to the 3′-untranslated region of the AOH3 transcript (BQ71452 and BQ898169) extend to the above-mentioned polyadenylation site. The 3′-sequence of the olfactory mucosa AOH3 cDNA is different from that of the corresponding mouse skin cDNA and is the result of a tissue-specific splicing event (see Table III).Table IIIExon/intron organization of the mouse Aoh3 gene Exon sequences are in uppercase letters, and intron sequences are in lowercase letters. The positions of nucleotides clo
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