Title: Discovery and Characterization of a Second Mammalian Thiol Dioxygenase, Cysteamine Dioxygenase
Abstract: There are only two known thiol dioxygenase activities in mammals, and they are ascribed to the enzymes cysteine dioxygenase (CDO) and cysteamine (2-aminoethanethiol) dioxygenase (ADO). Although many studies have been dedicated to CDO, resulting in the identification of its gene and even characterization of the tertiary structure of the protein, relatively little is known about cysteamine dioxygenase. The failure to identify the gene for this protein has significantly hampered our understanding of the metabolism of cysteamine, a product of the constitutive degradation of coenzyme A, and the synthesis of taurine, the final product of cysteamine oxidation and the second most abundant amino acid in mammalian tissues. In this study we identified a hypothetical murine protein homolog of CDO (hereafter called ADO) that is encoded by the gene Gm237 and belongs to the DUF1637 protein family. When expressed as a recombinant protein, ADO exhibited significant cysteamine dioxygenase activity in vitro. The reaction was highly specific for cysteamine; cysteine was not oxidized by the enzyme, and structurally related compounds were not competitive inhibitors of the reaction. When overexpressed in HepG2/C3A cells, ADO increased the production of hypotaurine from cysteamine. Similarly, when endogenous expression of the human ADO ortholog C10orf22 in HepG2/C3A cells was reduced by RNA-mediated interference, hypotaurine production decreased. Western blots of murine tissues with an antibody developed against ADO showed that the protein is ubiquitously expressed with the highest levels in brain, heart, and skeletal muscle. Overall, these data suggest that ADO is responsible for endogenous cysteamine dioxygenase activity. There are only two known thiol dioxygenase activities in mammals, and they are ascribed to the enzymes cysteine dioxygenase (CDO) and cysteamine (2-aminoethanethiol) dioxygenase (ADO). Although many studies have been dedicated to CDO, resulting in the identification of its gene and even characterization of the tertiary structure of the protein, relatively little is known about cysteamine dioxygenase. The failure to identify the gene for this protein has significantly hampered our understanding of the metabolism of cysteamine, a product of the constitutive degradation of coenzyme A, and the synthesis of taurine, the final product of cysteamine oxidation and the second most abundant amino acid in mammalian tissues. In this study we identified a hypothetical murine protein homolog of CDO (hereafter called ADO) that is encoded by the gene Gm237 and belongs to the DUF1637 protein family. When expressed as a recombinant protein, ADO exhibited significant cysteamine dioxygenase activity in vitro. The reaction was highly specific for cysteamine; cysteine was not oxidized by the enzyme, and structurally related compounds were not competitive inhibitors of the reaction. When overexpressed in HepG2/C3A cells, ADO increased the production of hypotaurine from cysteamine. Similarly, when endogenous expression of the human ADO ortholog C10orf22 in HepG2/C3A cells was reduced by RNA-mediated interference, hypotaurine production decreased. Western blots of murine tissues with an antibody developed against ADO showed that the protein is ubiquitously expressed with the highest levels in brain, heart, and skeletal muscle. Overall, these data suggest that ADO is responsible for endogenous cysteamine dioxygenase activity. There are many different processes in mammalian cells that result in the oxidation of thiol groups. Because of their reactivity, free sulfhydryl groups are highly susceptible to oxidation that results in the formation of disulfides, sulfenates, sulfinates, and sulfonates. Many of these reactions occur nonenzymatically, principally as a consequence of adventitious free radicals arising from aerobic respiration. Nevertheless, there are a small number of thiol oxidation reactions that are known to occur directly via enzymatic catalysis. The enzymes that catalyze these reactions show a high degree of substrate specificity and confer to cells the advantage of being able to precisely regulate the level of a particular reduced thiol. One interesting subset of the enzymes capable of specifically oxidizing free sulfhydryl groups are the thiol dioxygenases. In mammals this family comprises only two known proteins: cysteine dioxygenase (CDO, 3The abbreviations used are:CDOcysteine dioxygenaseADO2-aminoethanethiol dioxygenaseSSAsulfosalicylic acidsiRNAsmall interfering RNA duplexesMES2-(N-morpholino)ethanesulfonic acidORFopen reading frameWTwild type. 3The abbreviations used are:CDOcysteine dioxygenaseADO2-aminoethanethiol dioxygenaseSSAsulfosalicylic acidsiRNAsmall interfering RNA duplexesMES2-(N-morpholino)ethanesulfonic acidORFopen reading frameWTwild type. EC 1.13.11.20) and cysteamine dioxygenase (EC 1.13.11.19). CDO adds two atoms of oxygen to free cysteine to yield cysteine sulfinic acid, whereas cysteamine dioxygenase adds two atoms of oxygen to free cysteamine (2-aminoethanethiol) to form hypotaurine (Fig. 1). The activities for these two proteins were first reported in mammalian tissues almost 40 years ago (1Ewetz L. Sorbo B. Biochim. Biophys. Acta. 1966; 128: 296-305Crossref PubMed Scopus (57) Google Scholar, 2Cavallini D. Scandurra R. Demarco C. J. Biol. Chem. 1963; 238: 2999-3005Abstract Full Text PDF PubMed Google Scholar). Since that time, however, progress in our understanding of the two enzymes has been markedly unequal. Indeed, CDO has been the focus of much dedicated research, whereas cysteamine dioxygenase has received comparatively little attention. cysteine dioxygenase 2-aminoethanethiol dioxygenase sulfosalicylic acid small interfering RNA duplexes 2-(N-morpholino)ethanesulfonic acid open reading frame wild type. cysteine dioxygenase 2-aminoethanethiol dioxygenase sulfosalicylic acid small interfering RNA duplexes 2-(N-morpholino)ethanesulfonic acid open reading frame wild type. The CDO gene has been cloned from a diverse array of species ranging from bacteria to mammals (3Hirschberger L.L. Daval S. Stover P.J. Stipanuk M.H. Gene (Amst.). 2001; 277: 153-161Crossref PubMed Scopus (27) Google Scholar, 4Hosokawa Y. Matsumoto A. Oka J. Itakura H. Yamaguchi K. Biochem. Biophys. Res. Commun. 1990; 168: 473-478Crossref PubMed Scopus (44) Google Scholar, 5McCann K.P. Akbari M.T. Williams A.C. Ramsden D.B. Biochim. Biophys. Acta. 1994; 1209: 107-110Crossref PubMed Scopus (23) Google Scholar, 6Dominy J.E. Simmons Jr., C.R. Karplus P.A. Gehring A.M. Stipanuk M.H. J. Bacteriol. 2006; 188: 5561-5569Crossref PubMed Scopus (77) Google Scholar). Studies with purified recombinant protein have allowed the kinetic constants of the enzyme to be determined, have established that there is a strict substrate specificity for cysteine, and have also verified that CDO requires a ferrous iron cofactor for activity (6Dominy J.E. Simmons Jr., C.R. Karplus P.A. Gehring A.M. Stipanuk M.H. J. Bacteriol. 2006; 188: 5561-5569Crossref PubMed Scopus (77) Google Scholar, 7Chai S.C. Jerkins A.A. Banik J.J. Shalev I. Pinkham J.L. Uden P.C. Maroney M.J. J. Biol. Chem. 2005; 280: 9865-9869Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 8Simmons C.R. Hirschberger L.L. Machi M.S. Stipanuk M.H. Protein Expression Purif. 2006; 47: 74-81Crossref PubMed Scopus (48) Google Scholar). In terms of the larger physiological role of CDO, it has become generally accepted that CDO plays an important part in the homeostatic regulation of steady-state free cysteine levels as well as in the provision of important oxidized metabolites of cysteine such as sulfate, hypotaurine, and taurine (9Stipanuk M.H. Annu. Rev. Nutr. 2004; 24: 539-577Crossref PubMed Scopus (735) Google Scholar, 10Dominy J.E. Hwang J. Stipanuk M.H. Am. J. Physiol. Endocrinol. Metab. 2007; 293: E62-E69Crossref PubMed Scopus (53) Google Scholar). It has also been shown that CDO protein half-life is highly regulated by ubiquitination and degradation in response to cysteine levels both in cultured cells (11Stipanuk M.H. Hirschberger L.L. Londono M.P. Cresenzi C.L. Yu A.F. Am. J. Physiol. Endocrinol. Metab. 2004; 286: 439-448Crossref PubMed Scopus (41) Google Scholar) and in living mammals (12Dominy J.E. Hirschberger Jr., L.L. Coloso R.M. Stipanuk M.H. Biochem. J. 2006; 394: 267-273Crossref PubMed Scopus (66) Google Scholar). A major advancement in our understanding of CDO structure and function has come from the recent determination of atomic resolution crystal structures of the protein (13McCoy J.G. Bailey L.J. Bitto E. Bingman C.A. Aceti D.J. Fox B.G. Phillips Jr., G.N. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3084-3089Crossref PubMed Scopus (155) Google Scholar, 14Simmons C.R. Liu Q. Huang Q. Hao Q. Begley T.P. Karplus P.A. Stipanuk M.H. J. Biol. Chem. 2006; 281: 18723-18733Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 15Ye S. Wu X. Wei L. Tang D. Sun P. Bartlam M. Rao Z. J. Biol. Chem. 2007; 282: 3391-3402Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). These studies confirmed that CDO is a member of the cupin superfamily, a superfamily that encompasses many functionally diverse proteins including auxin-binding protein, mannose-6-phosphate isomerase, and hydroxyanthranilate dioxygenase. Members of this family possess a β-sandwich central domain with a jelly roll topology and two primary consensus sequence motifs designated as cupin motif 1 (GX5HXHX3,4EX6G) and cupin motif 2 (GX5PXGX2HX3N) separated by an intermotif distance of 15–50 amino acids (16Dunwell J.M. Culham A. Carter C.E. Sosa-Aguirre C.R. Goodenough P.W. Trends Biochem. Sci. 2001; 26: 740-746Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Many cupin proteins are capable of binding a transition metal, which often is a required cofactor in the biological activity of the protein, and do so using the residues highlighted in bold as ligands. CDO, however, is unusual among the cupins because it is missing the conserved glutamate of cupin motif 1 (Fig. 2A). CDO instead binds its iron co-factor using only the three histidine residues of its cupin motifs. This departure from other canonical cupin proteins, in conjunction with other variants in primary amino acid sequence such as the number of residues making up the intermotif distance and poor conservation of cupin motif 2, suggest that CDO may constitute a separate evolutionary clade within the cupin superfamily (16Dunwell J.M. Culham A. Carter C.E. Sosa-Aguirre C.R. Goodenough P.W. Trends Biochem. Sci. 2001; 26: 740-746Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). In contrast to the panoply of research conducted on CDO, little is known about cysteamine dioxygenase. The substrate for this protein, cysteamine, is constitutively produced by all tissues as a consequence of the degradation of coenzyme A, an acyl carrier group required for many metabolic processes. The gene for cysteamine dioxygenase has never been identified, and efforts to purify the protein from various tissues have resulted in conflicting reports about its molecular weight, specific activity, and cofactor requirements (2Cavallini D. Scandurra R. Demarco C. J. Biol. Chem. 1963; 238: 2999-3005Abstract Full Text PDF PubMed Google Scholar, 17Rotilio G. Federici G. Calabrese L. Costa M. Cavallini D. J. Biol. Chem. 1970; 245: 6235-6236Abstract Full Text PDF PubMed Google Scholar, 18Richerson R.B. Ziegler D.M. Methods Enzymol. 1987; 143: 410-415Crossref PubMed Scopus (8) Google Scholar). Nevertheless, recent work has shown that many tissues are capable of converting cysteamine to hypotaurine, and cysteamine dioxygenase could, therefore, be a quantitatively important contributor to the synthesis of hypotaurine and taurine in vivo (19Coloso R.M. Hirschberger L.L. Dominy J.E. Lee J.I. Stipanuk M.H. Adv. Exp. Med. Biol. 2006; 583: 25-36Crossref PubMed Scopus (43) Google Scholar). There is also evidence that cyst(e)amine could serve as an endogenous regulator of immune system activity (20Berruyer C. Martin F.M. Castellano R. Macone A. Malergue F. Garrido-Urbani S. Millet V. Imbert J. Dupre S. Pitari G. Naquet P. Galland F. Mol. Cell. Biol. 2004; 24: 7214-7224Crossref PubMed Scopus (138) Google Scholar, 21Berruyer C. Pouyet L. Millet V. Martin F.M. LeGoffic A. Canonici A. Garcia S. Bagnis C. Naquet P. Galland F. J. Exp. Med. 2006; 203: 2817-2827Crossref PubMed Scopus (107) Google Scholar) as well as a potential therapeutic agent for the treatment of Huntington disease (30Bailey C.D. Johnson G.V. Neurobiol. Aging. 2006; 27: 871-879Crossref PubMed Scopus (60) Google Scholar, 31Dedeoglu A. Kubilus J.K. Jeitner T.M. Matson S.A. Bogdanov M. Kowall N.W. Matson W.R. Cooper A.J. Ratan R.R. Beal M.F. Hersch S.M. Ferrante R.J. J. Neurosci. 2002; 22: 8942-8950Crossref PubMed Google Scholar, 32Lesort M. Lee M. Tucholski J. Johnson G.V. J. Biol. Chem. 2003; 278: 3825-3830Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). For these reasons, understanding the pathways by which cysteamine is metabolized is a physiologically important endeavor. Given that both CDO and cysteamine dioxygenase use a thiol substrate and catalyze a similar reaction, one might reasonably hypothesize that they may share a phylogenetic connection. Although there have been no previous reports of a closely related homolog to CDO in mammals, a PSI-BLAST search (22Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59441) Google Scholar) using murine CDO as a query sequence revealed that there is a hypothetical murine cupin protein of unknown function encoded by the gene Gm237 (GI:88984114, protein accession number Q6PDY2, hereafter referred to as protein ADO for 2-aminoethanethiol dioxygenase) that shares a low yet significant degree of overall identity (14.2%; e-value = 7 × 10–5 after 3 PSI-BLAST iterations) to CDO (Fig. 2A). ADO, like other cupins, contains two conserved cupin motifs but, like CDO, is missing the highly conserved glutamate residue found in motif 1 of many other metal binding cupins. In this report we sought to test whether ADO is a thiol dioxygenase with specificity for cysteamine. The protein was cloned, heterologously expressed, purified, and tested for its ability to catalyze the dioxygenation of cysteamine and several of its structural analogs. These data in combination with overexpression and RNA-mediated interference studies in a mammalian cell culture system and Western blot analysis of murine tissues suggest that ADO is in fact a proficient cysteamine dioxygenase expressed in many mammalian tissues. Cloning of ADO and Construction of Expression Plasmids— cDNA for ADO was prepared from an I.M.A.G.E. Consortium mouse cDNA clone (designation 5721417) obtained from ATCC. The construct, which contained cDNA for the mature mRNA of ADO cloned into the NotI/NotI regions of the pYX-ASC vector, was digested with NotI and subjected to agarose gel electrophoresis to verify the appropriate size of the predicted mRNA insert (3849 bp). The sequence of the 771-bp putative open reading frame (ORF) contained within the cDNA was verified before subsequent cloning work. For the purpose of producing recombinant protein in bacterial expression systems, the ORF of ADO was cloned into the pET SUMO expression vector (Invitrogen) and pQE30 expression vector (Qiagen). Cloning into the pET SUMO construct was done using the Champion pET SUMO protein expression system kit (Invitrogen) as per the manufacturer's instructions. The primers used for this procedure were 5′-ATGCCCCGCGACAACATGGC-3′ (forward primer) and 5′-TCAAGGTAGGACCTTGGGGC-3′ (reverse primer). A point mutation, hypothesized to ablate enzyme activity, was also prepared from the wild-type pET SUMO ADO construct in which His-95 was converted to an alanine. The mutation was introduced using a QuikChange II site-directed mutagenesis kit (Stratagene) and the primer set 5′-CGTGCATCCCGCTGGCCGACCACCCGGGCA-3′ (forward)/5′-TGCCCGGGTGGTCGGCCAGCGGGATGCACG-3′ (reverse) with the mutated His-95 codon underlined. Both wild-type and H95A pET SUMO expression plasmids were sequence verified and transformed into Escherichia coli BL21(DE3) competent cells (Novagen) for protein production. For insertion into the pQE30 vector, ADO ORF was first amplified via PCR using the following primers (introduced restriction sites are underlined, and the name is indicated in parentheses): 5′-GCGGATCCATGCCCCGCGACAACATGGC-3′ (BamHI) and 5′-CCAAGCTTTCAAGGTAGGACCTTGGGGCC-3′ (HindIII). The amplicon was then digested with BamHI and HindIII and inserted by T4 DNA ligase into pQE30 linearized by BamHI/HindIII double digestion. Sequence verified plasmid was transformed into E. coli M15 competent cells (Qiagen) for protein expression. For the production of epitope-tagged protein in mammalian cell culture, ADO was subcloned into the pCMV-3 × FLAG vector (Sigma). The ORF was first amplified using the forward primer 5′-CCAAGCTTATGCCCCGCGACAACATGGCC-3′ to create a HindIII restriction site (underlined) and the reverse primer 5′-CCGGATCCTCAAGGTAGGACCTTGGGGCC-3′ to introduce a BamHI restriction site. The amplicon was then digested with BamHI and HindIII and inserted by T4 DNA ligase into pCMV-3 × FLAG plasmid that was linearized by BamHI/HindIII double digestion. An H95A mutant of the wild-type pCMV-3 × FLAG ADO construct was also generated by using the QuikChange II site-directed mutagenesis kit and the same primer set used to mutate the pET SUMO ADO construct. Recombinant Protein Expression and Purification for Enzyme Activity Assays—Enzyme generated from the pET SUMO expression system was used for activity assays. With this expression system, ADO contained an N-terminal SUMO tag that substantially enhanced the solubility of the protein, which otherwise exhibited a tendency to aggregate and precipitate out of solution. A typical protein preparation was performed from 4 liters of bacterial culture. For protein expression, cells were grown in 2× Luria broth (LB) media at 37 °C containing 50 μgml–1 kanamycin. The cells were cultured to an A600 of ∼0.6 at which point expression of ADO was induced with 1 mm isopropyl-β-d-thiogalactopyranoside. Post-induction the cells were allowed to grow for an additional 3 h and were then harvested by centrifugation and stored at –20 °C until further processing was done. To lyse cells a total of 60 ml of lysis buffer (final pH 8.0) containing 20 mm Tris, 5 mm imidazole, 0.1% Tween, 250 mm NaCl, and one tablet of Complete protease inhibitor (Roche Applied Science) was added to the frozen cell pellets, which were then incubated in a 37 °C water bath for 10 min to thaw. After thawing, cells were resuspended and then lysed by sonication with an Ultrasonic Sonicator (Misonix). The lysate was then centrifuged for 30 min at 30,000 × g to remove cellular debris. The supernatant was filtered with a 0.2-μm syringe filter and applied onto a 5-ml HisTrap HP column (GE Healthcare) at a flow rate of 1 ml min–1. A stepwise gradient of increasing imidazole concentration was generated by using two buffers, 20 mm Tris (final pH 8.0), 5 mm imidazole, 0.1% Tween and 250 mm NaCl (IMAC Buffer A) and 20 mm Tris (final pH 8.0), 500 mm imidazole, 0.1% Tween, and 250 mm NaCl (IMAC Buffer B). The enzyme separated into 2 distinct peaks, one requiring 50 mm imidazole (10% IMAC Buffer A) for elution and the second eluting with 100 mm imidazole (20% IMAC Buffer B). Although initial tests showed similar cysteamine dioxygenase activities for the two peaks, only protein from the first peak was used in this study. The pooled fractions from peak 1 were concentrated to <2 ml and applied to a Superdex 200 16/60 gel filtration column (GE Healthcare). The elution volume of the peak fractions containing ADO from this step was consistent with a molecular weight for a single ADO monomer. The homogeneously purified ADO peak fractions were then pooled, concentrated, and analyzed for purity on a NuPAGE 4–12% SDS-PAGE gel (Invitrogen). Wild-type and H95A ADO proteins were purified in an identical fashion and exhibited similar chromatographic profiles. The relative purity for each construct after the 2-step purification was estimated to be >95% and is shown in (Fig. 3). A typical preparation of ADO yielded ∼2–3 mg of purified protein per liter of bacterial culture. Enzyme Activity Assays—In the standard assay conditions for cysteamine dioxygenase activity, 0–3 μm recombinant ADO was incubated in the presence of 50 mm Tris borate buffer and 8.0 mm cysteamine HCl (Sigma) in a total volume of 400 μl (final pH 8.0). Before the addition of enzyme, the reaction mixture was preheated to 37 °C in microcentrifuge tubes. Reactions were initiated by the addition of enzyme, and tubes were placed in an Eppendorf thermomixer (Brinkmann Instruments) for 3 min with shaking at 900 rpm. The reactions were stopped with the addition of 200 μl of 5% w/v sulfosalicylic acid (SSA). Hypotaurine production was measured by derivatization of sample with o-phthaldialdehyde and detection of derivatized products by high performance liquid chromatography coupled with fluorescence detection as previously described (19Coloso R.M. Hirschberger L.L. Dominy J.E. Lee J.I. Stipanuk M.H. Adv. Exp. Med. Biol. 2006; 583: 25-36Crossref PubMed Scopus (43) Google Scholar). Under our standard assay conditions we found that a small amount (<10%) of hypotaurine was spontaneously oxidized to taurine. For this reason taurine levels were also quantified and included in calculations of enzyme activity values. Deviations from the standard assay conditions, such as those used for the determination of pH optima and linearity of rate of product formation with time, are noted in the figure legends. Blanks were conducted for all of the experimental conditions tested and consisted of reactions wherein enzyme was left out. Mammalian Transfection Studies—HepG2/C3A hepatoma cells cultured in Dulbecco's modified Eagle's medium were seeded into 6-well tissue culture plates 24 h before transfection. Cells at a confluency of ∼95% were transfected with empty pCMV-3 × FLAG vector (4 μg), vector with wild-type ADO insert (2, 3, or 4 μg), or vector with H95A mutant ADO insert (4 μg) using Lipofectamine 2000 reagent (Invitrogen). To some cultures cystamine was added at a final concentration of 0.5 mm 6 h after transfection. Cystamine is a disulfide form of cysteamine that is efficiently taken up by cells and reduced to cysteamine intracellularly (23Qiu L. Zhang M. Sturm R.A. Gardiner B. Tonks I. Kay G. Parsons P.G. J. Investig. Dermatol. 2000; 114: 21-27Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 24Sharma R. Kodavanti U.P. Smith L.L. Mehendale H.M. Int. J. Biochem. Cell Biol. 1995; 27: 655-664Crossref PubMed Scopus (13) Google Scholar). Cells were harvested 30 h after transfection for Western blot analysis and measurement of intracellular hypotaurine/taurine content by HPLC. For Western blot analysis, cells were washed with ice-cold phosphate-buffered saline and then harvested into TNESV lysis solution, which consisted of 50 mm Tris (final pH 7.5), 1% (v/v) Nonidet P-40, 2 mm EDTA, 150 mm NaCl, and 10 mm sodium orthovanadate supplemented with mammalian protease inhibitor mixture (Sigma). The contents of each well were scraped into a 1.5-ml Eppendorf tube and centrifuged at ∼14,000 × g for 20 min at 4 °C. The cleared lysate was frozen and stored at –80 °C until Western blot analysis with anti-FLAG M2 monoclonal antibody (Sigma) was performed. For hypotaurine analysis cells were washed twice with ice-cold phosphate-buffered saline and harvested in 5% SSA. Acid extracts were centrifuged for 15 min at 16,000 × g, and hypotaurine analysis of the acid supernatant was carried out using the same HPLC protocol used for activity assays. Pellets from the acid extracts were neutralized overnight by the addition of 1 n NaOH and then quantified for protein content via the bicinchonic acid assay kit (Pierce). Protein levels were used for the normalization of cellular hypotaurine content. Metal Analysis of Recombinant ADO Protein—The metal content of purified recombinant wild-type and H95A ADO proteins was assayed by plasma emission spectroscopy using Cornell University's United States Plant, Soil, and Nutrition Laboratory services and graphite furnace atomic absorption spectrometry using Cornell University's Metabolic Mass Spectrometry Laboratory services. Small Interfering RNA Duplexes (siRNA) Studies—Three siRNAs designed to knock down the expression of C10orf22, the human ADO homolog expressed in HepG2/C3A cells, were obtained from Ambion. The sense sequences for these siRNAs (5′ → 3′), which target exon 1 of C10orf22, are as follows: GCUGAAUGUGUUAUGCUUC (siRNA #1), GGAACUUUAAUGUUCCCGA (siRNA #2), and GCAAAUACUUGGUGAGUGA (siRNA #3). A pre-designed siRNA (Silencer® Negative Control #1, Ambion) with limited sequence similarity with human genes was also used in this study as a negative control. Knockdown of C10orf22 expression required three iterative transfections, with each successive transfection separated by 24 h. For each round of transfection, Lipofectamine 2000 was used to deliver 40 pmol of the appropriate siRNA duplex to each well except for mock-transfected groups, which received vehicle only. HepG2/C3A cells were initially seeded into 6-well culture plates 24 h before the first round of transfection with siRNAs. At the time of the first transfection, cells were ∼30% confluent. Six hours after the last round of transfection, cystamine was added to each well at a final concentration of 0.5 mm, and cells were harvested 24 h later for Western blot and amino acid analysis as described above in the “Mammalian Transfection Studies” under “Experimental Procedures.” Antibody Generation and Western Blot Procedures—The pQE30-ADO construct, which produced ADO protein fused with a simple His6 N-terminal tag, was used for antibody production only. E. coli strain M15 (Qiagen) transformed with the pQE30-ADO construct were grown in LB medium containing 100 μg/ml carbenicillin at 37 °C with shaking at 250 rpm until an A600 of ∼0.6 was reached. Expression of recombinant protein was induced by the addition of isopropyl-β-d-thiogalactopyranoside at a final concentration of 1 mm. Cells were grown for an additional 3 h at 37°Cand then harvested by centrifugation at 6,000 × g for 15 min. Cells were resuspended in 50 mm K2HPO4 (final pH 7.6), 500 mm NaCl, 5 mm imidazole, 1× Complete Protease Inhibitor mixture, and 0.02% (v/v) Tween 20 and then lysed by sonication. Lysate was centrifuged at 20,000 × g for 20 min, and the supernatant was clarified by filtration through a 0.45-μm membrane. Clarified supernatant was loaded onto a gravity column packed with a 1-ml nickel-nitrilotriacetic acid resin and then washed with 14 column volumes of 50 mm K2HPO4 (final pH 7.6), 500 mm NaCl, 5 mm imidazole, 1× Complete Protease Inhibitor mixture followed by 4 column volumes of 50 mm K2HPO4 (final pH 7.6), 500 mm NaCl, and 15 mm imidazole. Protein was finally eluted in 50 mm K2HPO4 (final pH 7.6), 250 mm NaCl, and 300 mm imidazole and dialyzed at 4 °C in a 10-kDa molecular weight cutoff dialysis cassette (Pierce) overnight against a solution containing 50 mm K2HPO4 (final pH 7.6) and 250 mm NaCl. Dialyzed protein was concentrated using a 10-kDa molecular weight cutoff Centricon centrifugal filter unit (Millipore), and protein purity was assessed by SDS-PAGE and Coomassie Blue staining. With prolonged storage at 4 °C, pQE30-ADO protein had a tendency to aggregate and precipitate out of solution. Although this was not a problem for the production of antibody, it precluded the use of the fusion protein for enzyme activity assays. Purified protein was sent to Pacific Immunology, Inc. (Ramona, CA) for polyclonal antibody production in New Zealand White rabbits. Immune sera generated from the rabbits were screened against pre-immune sera to verify the generation of specific antibodies. After this screen, the IgG fraction from the immune sera was purified by column chromatography using Protein A-coupled agarose (Affi-Gel Protein A MAPS II system, Bio-Rad). To evaluate the expression of ADO protein in mouse, various tissues were harvested from adult male C57/Bl6 mice and homogenized (10–20% w/v) in ice-cold TNESV lysis buffer with a Polytron homogenizer (Brinkmann Instruments). Homogenates were then centrifuged at 16,000 × g for 30 min at 4 °C to pellet insoluble debris. The supernatants from these centrifuged samples were collected and stored at –80 °C until Western blot analysis could be performed. Statistical Methods—Data were analyzed by one-way analysis of variance followed by comparison of means with Tukey's multiple comparison test. Differences were accepted as significant at p ≤ 0.05. Each value reported in graphs is presented as the mean ± S.D. Purified ADO Shows Cysteamine Dioxygenase Activity—Recombinant wild-type (WT) ADO was initially tested for cysteamine dioxygenase activity simply by adding it to a buffered solution containing cysteamine and then measuring the formation of hypotaurine at various pH values (Fig. 4). This procedure departed in two notable ways from that described in much of the earlier literature on the measurement of cysteamine dioxygenase activity in tissues or purified protein (2Cavallini D. Scandurra R. Demarco C. J. Biol. Chem. 1963; 238: 2999-3005Abstract Full Text PDF PubMed Google Scholar, 17Rotilio G. Federici G. Calabrese L. Costa M. Cavallini D. J. Biol. Chem. 1970; 245: 6235-6236Abstract Full Text PDF PubMed Google Scholar, 18Richerson R.B. Ziegler D.M. Methods Enzymol. 1987; 143: 410-415Crossref PubMed Scopus (8) Google Scholar). First, most of these earlier studies relied upon the measurement of oxygen consumption as a surrogate marker f