Avian and Canine Aldehyde Oxidases
2006; Elsevier BV; Volume: 281; Issue: 28 Linguagem: Inglês
10.1074/jbc.m600850200
ISSN1083-351X
AutoresMineko Terao, Mami Kurosaki, Maria Monica Barzago, Emanuela Varasano, Andrea Boldetti, Antonio Bastone, Maddalena Fratelli, Enrico Garattini,
Tópico(s)Redox biology and oxidative stress
ResumoAldehyde oxidases are molybdo-flavoenzymes structurally related to xanthine oxidoreductase. They catalyze the oxidation of aldehydes or N-heterocycles of physiological, pharmacological, and toxicological relevance. Rodents are characterized by four aldehyde oxidases as follows: AOX1 and aldehyde oxidase homologs 1-3 (AOH1, AOH2, and AOH3). Humans synthesize a single functional aldehyde oxidase, AOX1. Here we define the structure and the characteristics of the aldehyde oxidase genes and proteins in chicken and dog. The avian genome contains two aldehyde oxidase genes, AOX1 and AOH, mapping to chromosome 7. AOX1 and AOH are structurally very similar and code for proteins whose sequence was deduced from the corresponding cDNAs. AOX1 is the ortholog of the same gene in mammals, whereas AOH represents the likely ancestor of rodent AOH1, AOH2, and AOH3. The dog genome is endowed with two structurally conserved and active aldehyde oxidases clustering on chromosome 37. Cloning of the corresponding cDNAs and tissue distribution studies demonstrate that they are the orthologs of rodent AOH2 and AOH3. The vestiges of dog AOX1 and AOH1 are recognizable upstream of AOH2 and AOH3 on the same chromosome. Comparison of the complement and the structure of the aldehyde oxidase and xanthine oxidoreductase genes in vertebrates and other animal species indicates that they evolved through a series of duplication and inactivation events. Purification of the chicken AOX1 protein to homogeneity from kidney demonstrates that the enzyme possesses retinaldehyde oxidase activity. Unlike humans and most other mammals, dog and chicken are devoid of liver aldehyde oxidase activity. Aldehyde oxidases are molybdo-flavoenzymes structurally related to xanthine oxidoreductase. They catalyze the oxidation of aldehydes or N-heterocycles of physiological, pharmacological, and toxicological relevance. Rodents are characterized by four aldehyde oxidases as follows: AOX1 and aldehyde oxidase homologs 1-3 (AOH1, AOH2, and AOH3). Humans synthesize a single functional aldehyde oxidase, AOX1. Here we define the structure and the characteristics of the aldehyde oxidase genes and proteins in chicken and dog. The avian genome contains two aldehyde oxidase genes, AOX1 and AOH, mapping to chromosome 7. AOX1 and AOH are structurally very similar and code for proteins whose sequence was deduced from the corresponding cDNAs. AOX1 is the ortholog of the same gene in mammals, whereas AOH represents the likely ancestor of rodent AOH1, AOH2, and AOH3. The dog genome is endowed with two structurally conserved and active aldehyde oxidases clustering on chromosome 37. Cloning of the corresponding cDNAs and tissue distribution studies demonstrate that they are the orthologs of rodent AOH2 and AOH3. The vestiges of dog AOX1 and AOH1 are recognizable upstream of AOH2 and AOH3 on the same chromosome. Comparison of the complement and the structure of the aldehyde oxidase and xanthine oxidoreductase genes in vertebrates and other animal species indicates that they evolved through a series of duplication and inactivation events. Purification of the chicken AOX1 protein to homogeneity from kidney demonstrates that the enzyme possesses retinaldehyde oxidase activity. Unlike humans and most other mammals, dog and chicken are devoid of liver aldehyde oxidase activity. Molybdo-flavoenzymes (MOFEs) 4The abbreviations used are: MOFEs, molybdo-flavoenzymes; XOR, xanthine oxidoreductase; RAL, retinaldehyde; MoCo, molybdenum cofactor; AOH, aldehyde oxidase homolog; RACE, rapid amplification of cDNA ends; RT, reverse transcription; nt, nucleotide; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; LOC, locus. constitute a small family of homodimeric oxidoreductases characterized by conserved structures (1Garattini E. Mendel R. Romao M.J. Wright R. Terao M. Biochem. J. 2003; 372: 15-32Crossref PubMed Scopus (203) Google Scholar). Until a few years ago, it was believed that the family of mammalian MOFEs consisted of only two members, i.e. xanthine oxidoreductase (XOR) (2Enroth C. Eger B.T. Okamoto K. Nishino T. Nishino T. Pai E.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10723-10728Crossref PubMed Scopus (593) Google Scholar, 3Okamoto K. Matsumoto K. Hille R. Eger B.T. Pai E.F. Nishino T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7931-7936Crossref PubMed Scopus (250) Google Scholar, 4Cazzaniga 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) and the aldehyde oxidase AOX1 5The nomenclature adopted in this study is as follows. AOX1 refers to the first identified MOFE with aldehyde oxidase activity and is the product of the gene originally annotated as AOX1 in the human and mouse sections of the NCBI data base. AOH1 and AOH2 refer to the proteins originally identified in mice as the aldehyde oxidase homologs 1 and 2 (27Terao 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). In the protein section of the NCBI data base, the two proteins are also annotated as AOX3 (accession number NP_076106) and AOX4 (accession number NP_076120). AOH3 refers to the last member of the MOFE family identified (25Kurosaki M. Terao M. Barzago M.M. Bastone A. Bernardinello D. Salmona M. Garattini E. J. Biol. Chem. 2004; 279: 50482-50498Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The protein is annotated as aldehyde oxidase 3-like 1 (Aox3l1, accession number NP_001008419) in the protein section of the NCBI database. Aldehyde oxidase is used as a general term and refers to any member of the MOFE subgroup. (5Calzi 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, 6Wright 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, 7Demontis S. Kurosaki M. Saccone S. Motta S. Garattini E. Terao M. Biochim. Biophys. Acta. 1999; 1489: 207-222Crossref PubMed Scopus (17) Google Scholar, 8Kurosaki M. Demontis S. Barzago M.M. Garattini E. Terao M. Biochem. J. 1999; 341: 71-80Crossref PubMed Scopus (57) Google Scholar). XOR has been extensively studied and is the key enzyme in the catabolism of purines, oxidizing hypoxanthine to xanthine and xanthine to uric acid (9Avis P.G. Bergel F. Bray R.C. Shooter K.V. Nature. 1954; 173: 1230-1231Crossref PubMed Scopus (18) Google Scholar, 10Godber B.L. Schwarz G. Mendel R.R. Lowe D.J. Bray R.C. Eisenthal R. Harrison R. Biochem. J. 2005; 388: 501-508Crossref PubMed Scopus (23) Google Scholar, 11Martin H.M. Hancock J.T. Salisbury V. Harrison R. Infect. Immun. 2004; 72: 4933-4939Crossref PubMed Scopus (141) Google Scholar, 12Harrison R. Drug. Metab. Rev. 2004; 36: 363-375Crossref PubMed Scopus (165) Google Scholar, 13Falciani F. Terao M. Goldwurm S. Ronchi A. Gatti A. Minoia C. Li Calzi M. Salmona M. Cazzaniga G. Garattini E. Biochem. J. 1994; 298: 69-77Crossref PubMed Scopus (27) Google Scholar, 14Falciani F. Ghezzi P. Terao M. Cazzaniga G. Garattini E. Biochem. J. 1992; 285: 1001-1008Crossref PubMed Scopus (49) Google Scholar). This function is conserved throughout evolution, as the enzyme is present from bacteria to man (1Garattini E. Mendel R. Romao M.J. Wright R. Terao M. Biochem. J. 2003; 372: 15-32Crossref PubMed Scopus (203) Google Scholar). In mammals, the protein also plays an important role in milk secretion (15Vorbach C. Scriven A. Capecchi M.R. Genes Dev. 2002; 16: 3223-3235Crossref PubMed Scopus (168) Google Scholar, 16McManaman J.L. Palmer C.A. Wright R.M. Neville M.C. J. Physiol. (Lond.). 2002; 545: 567-579Crossref Scopus (112) Google Scholar, 17Kurosaki M. Zanotta S. Li Calzi M. Garattini E. Terao M. Biochem. J. 1996; 319: 801-810Crossref PubMed Scopus (43) Google Scholar) and kidney development (18Rovira II, O.T. Starost M.F. Liu C. Finkel T. Circ. Res. 2004; 95: 1118-1124Crossref PubMed Scopus (85) Google Scholar). The function of AOX1 is ill-defined, and the enzyme lacks a recognized physiological substrate. AOX1 metabolizes N-heterocyclic compounds and aldehydes of pharmacological and toxicological relevance (19Fabre G. Fabre I. Matherly L.H. Cano J.P. Goldman I.D. J. Biol. Chem. 1984; 259: 5066-5072Abstract Full Text PDF PubMed Google Scholar, 20Beedham C. Miceli J.J. Obach R.S. J. Clin. Psychopharmacol. 2003; 23: 229-232PubMed Google Scholar, 21Beedham C. Pharm. World Sci. 1997; 19: 255-263Crossref PubMed Scopus (78) Google Scholar, 22Beedham C. Peet C.F. Panoutsopoulos G.I. Carter H. Smith J.A. Prog. Brain Res. 1995; 106: 345-353Crossref PubMed Scopus (34) Google Scholar). XOR and AOX1 are the products of two genes mapping on distinct chromosomes in rodents and different arms of chromosome 2 in humans (4Cazzaniga 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, 7Demontis S. Kurosaki M. Saccone S. Motta S. Garattini E. Terao M. Biochim. Biophys. Acta. 1999; 1489: 207-222Crossref PubMed Scopus (17) Google Scholar, 23Xu P. Huecksteadt T.P. Hoidal J.R. Genomics. 1996; 34: 173-180Crossref PubMed Scopus (86) Google Scholar, 24Terao M. Kurosaki M. Demontis S. Zanotta S. Garattini E. Biochem. J. 1998; 332: 383-393Crossref PubMed Scopus (56) Google Scholar). Recently, we demonstrated that the family of mammalian MOFEs is larger than originally anticipated (25Kurosaki M. Terao M. Barzago M.M. Bastone A. Bernardinello D. Salmona M. Garattini E. J. Biol. Chem. 2004; 279: 50482-50498Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 26Vila R. Kurosaki M. Barzago M.M. Kolek M. Bastone A. Colombo L. Salmona M. Terao M. Garattini E. J. Biol. Chem. 2004; 279: 8668-8883Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 27Terao 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, 28Terao 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). Mice and rats are endowed with three extra MOFEs structurally and biochemically more similar to AOX1 than to XOR. We named these proteins aldehyde oxidase homologs 1-3 (AOH1, AOH2, and AOH3). In rodents, AOH1 is synthesized predominantly in liver and lung, the only two organs that express significant amounts of AOX1 as well (28Terao 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). AOH2 was originally identified in the keratinized epithelia of the stomach, esophagus, and skin (28Terao 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), although the richest source of the enzyme is the Harderian gland, 6M. Terao and E. Garattini, unpublished observations. a specialized structure present in the orbital cavity of various types of animals (29Buzzell G.R. Microsc. Res. Tech. 1996; 34: 2-5Crossref PubMed Scopus (40) Google Scholar). The tissue and cell distribution of AOH3 is also peculiar, as the enzyme is selectively expressed in nasal mucosa (25Kurosaki M. Terao M. Barzago M.M. Bastone A. Bernardinello D. Salmona M. Garattini E. J. Biol. Chem. 2004; 279: 50482-50498Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Given the recent identification of AOH1, AOH2, and AOH3, the corresponding physiological substrates and homeostatic roles are unknown. The mouse AOX1, AOH1, AOH2, and AOH3 genes have strictly conserved exon structures and cluster in a small chromosomal region (aldehyde oxidase gene cluster) (25Kurosaki M. Terao M. Barzago M.M. Bastone A. Bernardinello D. Salmona M. Garattini E. J. Biol. Chem. 2004; 279: 50482-50498Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 27Terao 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). A similar arrangement of the four orthologous genes is present in rat (25Kurosaki M. Terao M. Barzago M.M. Bastone A. Bernardinello D. Salmona M. Garattini E. J. Biol. Chem. 2004; 279: 50482-50498Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). A striking conservation of exon structure is also evident when the AOX1, AOH1, AOH2, and AOH3 genes are compared with the mouse and rat XOR orthologs (1Garattini E. Mendel R. Romao M.J. Wright R. Terao M. Biochem. J. 2003; 372: 15-32Crossref PubMed Scopus (203) Google Scholar, 25Kurosaki M. Terao M. Barzago M.M. Bastone A. Bernardinello D. Salmona M. Garattini E. J. Biol. Chem. 2004; 279: 50482-50498Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 27Terao 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). Based on these as well as other observations, we proposed that all MOFE genes arose through one or more duplication events from a single ancestor with structural similarity to XOR (1Garattini E. Mendel R. Romao M.J. Wright R. Terao M. Biochem. J. 2003; 372: 15-32Crossref PubMed Scopus (203) Google Scholar). Duplications of MOFE genes are not a peculiarity of rodents, as they are also observed in plants and insects (30Seo M. Peeters A.J. Koiwai H. Oritani T. Marion-Poll A. Zeevaart J.A. Koornneef M. Kamiya Y. Koshiba T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12908-12913Crossref PubMed Scopus (320) Google Scholar, 31Ori N. Eshed Y. Pinto P. Paran I. Zamir D. Fluhr R. J. Biol. Chem. 1997; 272: 1019-1025Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 32Misra S. Crosby M.A. Mungall C.J. Matthews B.B. Campbell K.S. Hradecky P. Huang Y. Kaminker J.S. Millburn G.H. Prochnik S.E. Smith C.D. Tupy J.L. Whitfied E.J. Bayraktaroglu L. Berman B.P. Bettencourt B.R. Celniker S.E. de Grey A.D. Drysdale R.A. Harris N.L. Richter J. Russo S. Schroeder A.J. Shu S.Q. Stapleton M. Yamada C. Ashburner M. Gelbart W.M. Rubin G.M. Lewis S.E. Genome Biol. 2002; 3 (RESEARCH0083)Crossref Google Scholar). The availability of the complete sequence of an ever increasing number of genomes provides a unique opportunity to determine the number and the structure of MOFE genes in different animal species. In this study, we describe the cloning and sequencing of the avian and canine cDNAs encoding aldehyde oxidase and paralogous proteins. In addition, we reconstruct the exon structures of MOFE genes in the sequenced genomes of other vertebrates. Purification of chicken AOX1 demonstrates that the enzyme is capable of metabolizing a physiological substrate like retinaldehyde (RAL). Our results contribute to the elucidation of the biology and evolution of MOFEs. Purification of Chicken Kidney AOX1 Protein, Electrophoresis, and Western Blot Analysis—Unless otherwise stated, all the purification steps were carried out at 4 °C. Male chicken kidneys (75 g) were isolated and homogenized in 3 volumes of 100 mm sodium phosphate buffer, pH 7.5, with a mechanical Turrax homogenizer. 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. Solid ammonium sulfate was added to the supernatant (40% w/v). The precipitate was collected by centrifugation at 100,000 × g, resuspended in 50 mm Tris-HCl, pH 7.5, and dialyzed overnight against the same buffer. The sample was applied batchwise to benzamidine-Sepharose (Amersham Biosciences) equilibrated in 100 mm Tris-HCl, containing 100 mm NaCl, pH 7.5, and rolled overnight. After extensive washing of the phase in loading buffer, AOX1 was eluted in the same buffer containing 10 mm benzamidine. The eluate was concentrated using Centricon YM-100 ultrafiltration devices and diluted (1:10 v/v) in 100 mm Tris-HCl, pH 7.4. The material was applied to a 5:5 fast protein liquid chromatography Mono Q column (Amersham Biosciences) equilibrated in 100 mm Tris-HCl, pH 7.4. The AOX1 protein was eluted at 0.5 ml/min with a linear gradient from 0 to 1 m NaCl in 100 mm Tris-HCl, pH 7.5. The purification of AOX1 was monitored by RAL oxidizing activity and quantitative Western blot analysis (25Kurosaki M. Terao M. Barzago M.M. Bastone A. Bernardinello D. Salmona M. Garattini E. J. Biol. Chem. 2004; 279: 50482-50498Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) using an anti-bovine AOX1 antibody described previously (5Calzi 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), according to a chemiluminescence-based protocol (ECL, Amersham Biosciences). The antibodies do not cross-react with bovine XOR. 7E. Garattini and M. Terao, unpublished results. Chemiluminescent signals corresponding to AOX1 bands were quantitated with a scanning densitometer (Hoefer Scientific Instruments, San Francisco). The total amount of AOX1 immunoreactive protein in the various experimental samples is expressed in arbitrary units and is calculated on the basis of the intensity of the Western blot signal in OD multiplied by the total volume of each purification step. One arbitrary unit of immunoreactive protein corresponds to 1.0 OD of the specific AOX1 band in each experimental sample (25Kurosaki M. Terao M. Barzago M.M. Bastone A. Bernardinello D. Salmona M. Garattini E. J. Biol. Chem. 2004; 279: 50482-50498Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The anti-rat XOR antibodies have been described (13Falciani F. Terao M. Goldwurm S. Ronchi A. Gatti A. Minoia C. Li Calzi M. Salmona M. Cazzaniga G. Garattini E. Biochem. J. 1994; 298: 69-77Crossref PubMed Scopus (27) Google Scholar). SDS-PAGE was performed following standard techniques (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Proteins were measured according to the Bradford method with a commercially available kit (Bio-Rad). Determination of the Chicken and Dog Cross-reactivity Profiles of Anti-bovine AOX1 Antibodies—The spectrum of crossreactivity of the anti-bovine AOX1 antibodies against chicken, dog, and mouse MOFEs was determined on extracts of COS-7 transfected with chicken AOX1, AOH, and XOR as well as dog AOH2, AOH3, and XOR full-length cDNAs. The complete coding regions of the various cDNAs were cloned in the pCMVβ plasmid expression vector (Clontech). COS-7 cells were cultured and transfected with cationic liposomes, as described previously (27Terao 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). Determination of Retinaldehyde Oxidase Activity in Tissue Cytosolic Extracts—RAL oxidase activity was measured in chicken liver, kidney, and heart, C57/Bl and DBA/2 mouse liver, as well as dog liver and kidney (26Vila R. Kurosaki M. Barzago M.M. Kolek M. Bastone A. Colombo L. Salmona M. Terao M. Garattini E. J. Biol. Chem. 2004; 279: 8668-8883Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Organs were dissected, frozen, and stored at -80 °C until processed for the determination of RAL activity. Organs were homogenized in 3 volumes (w/v) of 10 mm potassium phosphate, pH 7.4. Samples were ultracentrifuged at 100,000 × g for 45 min. Supernatants were collected and passed on PD10 (Amersham Biosciences) columns to eliminate endogenous NAD+. Desalted samples were incubated in 100 μl of 10mm potassium phosphate, pH 7.4, containing all-trans-retinaldehyde (Sigma) for 10 min. The reaction was stopped by addition of 100 μlof n-butanol/methanol (95:5 v/v) containing 0.005% w/v of butylated hydroxytoluene (Sigma) and was vortexed. The organic phase was separated, and an aliquot (20 μl) was loaded onto RP-18 reverse phase HPLC columns (Waters), using a Beckman apparatus equipped with a UV-visible detector (Beckman Instruments, Palo Alto, CA). The retention times of all-trans-retinoic acid and all-trans-RAL were determined using authentic standards of the two compounds (Sigma). The amounts of retinoic acid were determined by integrating the area of the specific chromatographic peak and comparing it to an appropriate calibration curve. The enzymatic activity equivalent to the oxidation of 1 nmol of RAL to retinoic acid/min is defined as 1 unit. Characterization of the Purified Chicken AOX1 Protein by Mass Spectrometry—MALDI-mass spectrometric and electrospray ionization tandem mass spectrometric analyses of chicken AOX1 tryptic peptides were performed according to standard protocols following in gel tryptic digestion (25Kurosaki M. Terao M. Barzago M.M. Bastone A. Bernardinello D. Salmona M. Garattini E. J. Biol. Chem. 2004; 279: 50482-50498Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Briefly, the Coomassie-stained gel slice corresponding to purified AOX1 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 of trypsin (Promega, Madison, WI). Peptides were extracted twice in 50% acetonitrile, 5% formic acid. The combined extracts were lyophilized and redissolved in 0.5% formic acid and desalted using ZipTip (Millipore, Bedford, MA). Peptides were eluted in 50% acetonitrile, 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, 0.1% trifluoroacetic acid 1:3 (v/v). Mass mapping of tryptic peptides was performed with a Bruker Reflex III MALDI-TOF mass spectrometer (Bruker, Bremen, Germany). Data generated were processed with the Mascot program (25Kurosaki M. Terao M. Barzago M.M. Bastone A. Bernardinello D. Salmona M. Garattini E. J. Biol. Chem. 2004; 279: 50482-50498Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) allowing a mass tolerance of ≤0.1 Da. cDNA Cloning, Nucleotide Sequencing, and Determination of the Intron/Exon Structure of the Corresponding Genes—The chicken AOX1 cDNA was isolated as three overlapping fragments (corresponding to exons 3-17, 17-31, and 31-35) by RT-PCR from kidney RNA. The couples of oligonucleotides used as primers are as follows: 5′-CAGGAACTAAGTATGGCTGTGGAG3-′ (nt 130-153 of the chicken AOX1 cDNA); 5′-ATCTTAGCATGAGCTCTGGAACTAG-3′ (complementary to nt 1855-1879); 5′-AATGTAGAACTGAGTCAGTCTCCC-3′ (nt 1713-1736); 5′-CAACCTCTGAACAAGCAGTTCCAT-3′ (complementary to nt 3499-3522); 5′-ACGATGCAAATATGGACTGGGAGAA-3′ (nt 3442-3466); and 5′-CTGTTCAGGTCTCATGCATTCTGG-3′ (complementary to nt 4012-4035). The chicken AOH cDNA was isolated as three overlapping fragments (corresponding to exons 3-16, 15-27, and 26-35) by RT-PCR from Harderian gland RNA. The couples of oligonucleotides used as primers are as follows: 5′-GTGGTGCATGCACTGTGATGTTGT-3′ (nt 211-234 of the chicken AOH cDNA); 5′-CTTTGTAGCACTCCCAAAGCACTC-3′ (complementary to nt 1712-1735); 5′-ACAGTGGAATGATCAGATGCTGAGT-3′ (nt 1511-1535); 5′-TGAGTGACTAGTACAGACCCATCTA-3′ (complementary to nt 3148-3172); 5′-CATGTACAGAGGAGTTAACCGGAC-3′ (nt 2909-2932); and 5′-GGATATATCAATGGCCCACGGCTT-3′ (complementary to nt 4041-4064). The dog AOH2 cDNA was isolated as three overlapping fragments (corresponding to exons 1-15, 14-26, and 25-35) by RT-PCR from lacrimal gland RNA. The couples of oligonucleotides used as primers are as follows: 5′-GGTATGATGGCTTCTGTTCCCAAT-3′ (nt 15-38 of the dog AOH2 cDNA); 5′-TATTCAGTCCTCGCCTCACTTTGA-3′ (complementary to nt 1606-1629); 5′-CATTGTCAATGCTGGCATGAGTGT-3′ (nt 1340-1363); 5′-CCCCTCTTCTTCCAGTAGTTCTTT-3′ (complementary to nt 3005-3028); 5′-TACATAACTGCTGTGGCATCTCAG-3′ (nt 2814-2837); and 5′-GGATCAAGACACACGGATAGACCA-3′ (complementary to nt 4008-4031). The dog AOH3 cDNA was isolated as three overlapping fragments (corresponding to exons 1-15, 14-26, and 25-34) by RT-PCR from nasal mucosa RNA. The couples of oligonucleotides used as primers are as follows: 5′-ACAATGCCTTGCCCATCGAAATCC-3′ (nt 136-159 of the dog AOH3 cDNA); 5′-CACCAGAGTCCTCTTGAATTCCAC-3′ (complementary to nt 1681-1704); 5′-AGGAAGGCACAGGCACTATTGAGG-3′ (nt 1496-1519); 5′-CCAACTGAAAACTTCATGGGGACG-3′ (complementary to nt 3177-3200); 5′-ATTTGGCTTCCCACAAGGAACCCT-3′ (nt 2916-2939); and 5′-CATCTCTGTGAACCGATCTGCACA-3′ (complementary to nt 4093-4116). The appropriate DNA fragments were subcloned into the pCR2.1 plasmid using the TA cloning kit (Invitrogen) and sequenced according to the Sanger dideoxy chain termination method, using double-stranded DNA as template and T7 DNA polymerase (Amersham Biosciences). Oligodeoxynucleotide primers were custom synthesized by Sigma. Computer analysis of the DNA sequences was performed using the GeneWorks sequence analysis system (Intelligenetics, San Diego, CA). The nucleotide and protein sequences of the full-length chicken AOX1 and AOH, as well as dog AOH2 and AOH3 cDNAs were compared with the corresponding genomic sequences present in the NCBI public data bases. This resulted in the determination of the exon/intron structure of the corresponding genes. Determination of the 5′ and 3′ Ends of the Chicken and Dog Transcripts—Total RNA was extracted from chicken kidney (AOX1), chicken Harderian glands (AOH), dog lacrimal glands (AOH2), and dog nasal mucosa (AOH3). The poly(A+) fraction of the RNA was selected according to standard procedures (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). 5′-rapid amplification of cDNA ends (RACE) was performed with the commercially available Marathon cDNA amplification kit (Clontech), according to the nested PCR protocol included, using the primers indicated as follows: chicken AOX1 specific primer (SP1), 5′-TACTTCCAACACCTTCCACTGTGG-3′ (complementary to nt 277-300 of the cDNA), and nested primer (SP2), 5′-CTGTGGTGACTGCCATACCATACA-3′ (complementary to nt 259-282); chicken AOH SP1, 5′-CTTTGTAGCACTCCCAAAGCACTC-3′ (complementary to nt 1712-1735), and SP2, 5′-CCTGCTGACAACATGTCCTC-3′ (complementary to nt 1129-1148); dog AOH2 SP1, 5′-GTCACTGGATAGTGGTGGATCTTC3-′ (complementary to nt 221-244), and SP2, 5′-GGATAGTGGTGGATCTTCTTGGTC-3′ (complementary to nt 215-238); and dog AOH3 SP1, 5′-TCCTGTGAGGCGTAAGTTCTTTCG-3′ (complementary to nt 241-264). This 5′-RACE reaction did not require a nested protocol. The 3′-RACE was conducted as above with the following primers: chicken AOX1 SP1, 5′-TTTGCACTGAACAGCCCTCTGACT-3′ (nt 3906-3929 of the cDNA), and SP2, 5′-TGAACAAATACGAGCAGCCTGCATA-3′ (nt 3932-3956); chicken AOH SP1, 5′-GCCCAGATACATACAAGATCCCTG-3′ (nt 3742-3765), and SP2, 5′-CGGATTCGTATGGCCTGTGATGAT-3′ (nt 3975-3998); dog AOH2 SP1, 5′-GGGTGAATCTGGAATGTTCTTGGG-3′ (nt 3812-3835), and SP2, 5′-ATCTGGAATGTTCTTGGGATCCTC-3′ (nt 3818-3841); dog AOH3 SP1, 5′-TGAAGAGCCCAGCAACGCCAGAAT-3′ (nt 4055-4078), and SP2, 5′-CAGCAACGCCAGAATGGATTCGAA 3′ (nt 4064-4087). PCR products were subcloned in pCR2.1 and multiple clones were sequenced. Phylogenetic Analysis—Multiple sequence alignment was performed using the ClustalW program with default settings (Protein Gap Open Penalty, 10.0; Protein Gap Extension Penalty, 0.2; Protein matrix, Gonnet) (34Chenna R. Sugawara H. Koike T. Lopez R. Gibson T.J. Higgins D.G. Thompson J.D. Nucleic Acids Res. 2003; 31: 3497-3500Crossref PubMed Scopus (4081) Google Scholar). The multiple alignment was then used to produce a true phylogenetic tree, in the Phylip type output format, always with the ClustalW program that is based upon the neighbor-joining method of Saitou and Nei (35Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425PubMed Google Scholar). The tree was then drawn using the Phylodendron software package. The alignment shown in supplemental Fig. 3 was drawn with Color INteractive Editor for Multiple Alignments (66Attwood T.K. Beck M.E. Bleasby A.J. Degtyarenko K. Michie A.D. Parry-Smith D.J. Nucleic Acids Res. 1997; 25: 212-217Crossref PubMed Scopus (47) Google Scholar). The Complement of Avian MOFEs Consists of XOR and Two Proteins of the Aldehyde Oxidase Type, AOX1 and AOH—Chicken XOR is the only avian MOFE for which primary structural information is available (36Sato A. Nishino T. Noda K. Amaya Y. Nishino T. J. Biol. Chem. 1995; 270: 2818-2826Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). We interrogated the genome of Gallus gallus present in GenBank™ for the presence of other MOFE genes showing structural similarity with mouse aldehyde oxidases, and we identified two potential genetic loci. Partial reconstruction of the exon structure of the genes permitted the design of specific primers that were used for the cloning of tw
Referência(s)