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Originally published In Press as doi:10.1074/jbc.M703089200 on June 20, 2007

J. Biol. Chem., Vol. 282, Issue 35, 25189-25198, August 31, 2007
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Discovery and Characterization of a Second Mammalian Thiol Dioxygenase, Cysteamine Dioxygenase*

John E. Dominy, Jr1, Chad R. Simmons, Lawrence L. Hirschberger, Jesse Hwang, Relicardo M. Coloso, and Martha H. Stipanuk2

From the Department of Nutritional Sciences, Cornell University, Ithaca, New York 14853

Received for publication, April 12, 2007 , and in revised form, June 19, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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,3 EC 1.13.11.20 [EC] ) and cysteamine dioxygenase (EC 1.13.11.19 [EC] ). 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 (1, 2). 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.


Figure 1
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  		FIGURE 1.
A comparison of the thiol dioxygenase reactions catalyzed by cysteine dioxygenase and cysteamine dioxygenase.

 
The CDO gene has been cloned from a diverse array of species ranging from bacteria to mammals (3–6). 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 (6–8). 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 (9, 10). 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 (11) and in living mammals (12).

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 (13–15). 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 beta-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 (16). 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 (16).


Figure 2
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  		FIGURE 2.
A, a sequence alignment of murine (Mus musculus) CDO and murine ADO (Q6PDY2) proteins. Both proteins possess motifs indicative of cupin proteins, with predicted metal binding histidine residues indicated by asterisks. Unlike most metal binding cupin proteins, both CDO and ADO are missing a conserved glutamate residue ({blacksquare}) in cupin motif 1 that is normally used by other cupin members for metal coordination. In all known CDOs, this glutamate is replaced by a cysteine or glycine (6), whereas in ADO it is a glycine or valine. B, a multiple sequence alignment of ADO homologs from representative species in three kingdoms. Homologs were identified by a BLASTP search. The sequence alignment was prepared using AlignX software (Vector NTI, Invitrogen) and the Blosum62mt scoring matrix. Accession numbers for the proteins shown in the alignment are as follows: Homo sapiens, NP_116193; Macaca mulatta, XP_001092839; M. musculus, Q6PDY2; Rattus norvegicus, XP_001080411.1; Danio rerio, AAH65461.1; Xenopus laevis, AAH82884.1; Drosophila melanogaster, NP_648176.1; Schistosoma japonicum, AAW27563.1; Strongylocentrotus purpuratus, XP_001183066.1; Dictyostelium discoideum, XP_644380.1; Leishmania major, CAJ02170.1; Trypanosoma brucei, AAX78835.1; Medicago truncatula, ABD28522.1; Arabidopsis thaliana, NP_191426.1; Oryza sativa, BAB03364.1.

 
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 (2, 17, 18). 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 (19). There is also evidence that cyst(e)amine could serve as an endogenous regulator of immune system activity (20, 21) as well as a potential therapeutic agent for the treatment of Huntington disease (30–32). 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 (22) 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 x 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 x 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 x FLAG plasmid that was linearized by BamHI/HindIII double digestion. An H95A mutant of the wild-type pCMV-3 x 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 2x 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-beta-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 x 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.


Figure 3
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  		FIGURE 3.
A SDS-PAGE gel demonstrating the relative purity of the homogeneously purified wild-type and H95A mutant SUMO-tagged ADO constructs. Protein was visualized by Coomassie Blue staining.

 
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 (19). 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 x 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 (23, 24). 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 x 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 x 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-beta-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 x g for 15 min. Cells were resuspended in 50 mM K2HPO4 (final pH 7.6), 500 mM NaCl, 5 mM imidazole, 1x Complete Protease Inhibitor mixture, and 0.02% (v/v) Tween 20 and then lysed by sonication. Lysate was centrifuged at 20,000 x 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, 1x 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 x 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (2, 17, 18). First, most of these earlier studies relied upon the measurement of oxygen consumption as a surrogate marker for hypotaurine production from cysteamine. Second, all of these studies added an exogenous electron-donating cofactor-like compound such as sulfide, methylene blue, or hydroxylamine to attain activity. Recent work from our laboratory, however, demonstrated that directly measuring hypotaurine production by HPLC is a sensitive and accurate means for assessing cysteamine dioxygenase activity (19). Using this approach we observed that exogenous electron-donating cofactors are not required for cysteamine dioxygenase activity in crude tissue extracts (19).

When purified WT ADO protein was incubated with 8 mM cysteamine, it showed substantial amounts of cysteamine dioxygenase activity over a broad range of pH values above 7.0 in three different buffer solutions (Fig. 4A). The highest amount of activity was observed in 50 mM Tris borate buffer, with activity plateauing between pH 8.0 and 10.0 (Fig. 4A). In this buffer system at pH 8.0 and 9.1, the production of hypotaurine was linear with time over the course of at least 6 min (Fig. 4, B and C). Although the specific activity was 18% higher at pH 9.1 than at pH 8.0, we chose to pursue further characterization of protein kinetics at pH 8.0 because it is closer to the physiologically relevant pH of the cytosol (pH {approx} 7.3).

Incubating WT ADO with various concentrations of cysteamine at pH 8.0 showed that the formation of product displayed Michaelis-Menten-like kinetics with an estimated Km of 3.8 mM,a Vmax of 2300 nmol·mg–1·min–1, and a kcat of 1.6 s–1 (Fig. 4D). As a point of comparison, these kinetic parameters are very similar to those for the activity of CDO on its substrate cysteine (6, 8). Although the two enzymes show similar catalytic profiles, they do not share the same substrate specificities; WT ADO is specific for the dioxygenation of cysteamine just as CDO is specific for the dioxygenation of cysteine. Cysteine sulfinic acid was not produced when WT ADO was incubated in the presence of 8 mM cysteine (data not shown). Also, cysteine was not a very effective inhibitor of cysteamine dioxygenase activity. Even when used at a molar ratio that was 6 times higher than cysteamine, cysteine produced only a 35% inhibition of activity (Fig. 4E). Additionally, the structurally related molecule 2-mercaptoethanol, which differs from cysteamine by the substitution of the amine group with a hydroxyl group, was not an effective inhibitor of activity (Fig. 4F). Collectively, these data suggest that WT ADO does not indiscriminately oxidize thiol-containing compounds but, instead, shows a high degree of specificity for the dioxygenation of cysteamine.


Figure 4
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  		FIGURE 4.
Kinetic analyses of purified wild-type ADO protein. A, a pH profile of cysteamine dioxygenase activity for ADO assayed in 50 mM Tris borate, Tris phosphate, or MES-Tris-glycine buffers adjusted to the indicated pH. Cysteamine concentration used in this set of assays was 8 mM, and the total reaction time was 3 min. B and C, an evaluation of product formation linearity over time when ADO was incubated with 8mM cysteamine in 50 mM Tris borate buffer at either pH 8.0 (B) or pH 9.1 (C). D, the dependence of cysteamine dioxygenase activity on cysteamine concentration at pH 8.0. Cysteamine dioxygenase activity for ADO protein displayed Michaelis-Menten kinetics with a Km= 3.8 mM and a Vmax = 2300 nmol·mg–1·min–1 as determined by hyperbolic regression. E and F, inhibitory effects of different concentrations of cysteine (E) and 2-mercaptoethanol (F) on cysteamine dioxygenase activity. For these inhibition experiments the concentration of cysteamine used was 2.5 mM. Assays were conducted in 50 mM Tris borate buffer (pH 8.0) for 3 min.

 
Two additional approaches were taken to show that the oxidation of cysteamine to hypotaurine was a specific biological activity of the protein in vitro. In the first approach, the protein was denatured before assaying by boiling for 5 min. This caused a complete elimination of cysteamine dioxygenase activity (data not shown). In the second approach we evaluated activity of the purified H95A mutant. Because this mutated protein lacks an important putative metal binding residue, we predicted that the protein would be catalytically inactive. Indeed, the H95A mutant did not show any detectable cysteamine dioxygenase activity when incubated with 8 mM cysteamine (limit of assay detection was ~18 nmol·mg–1·min–1 or ~0.8% of WT maximal activity). The absence of activity was consistent with the metal analysis of the mutant protein, which showed it to be devoid of metal (see below).

Characterization of Metal Binding and Its Contribution to Catalytic Activity—Many cupin proteins are capable of binding a transition metal and use this metal as a cofactor to assist in their intrinsic biological activity (16). CDO, for instance, requires ferrous iron for activity (6–8). Moreover, because of issues with very low iron occupancy (<10–25%) after aerobic purification of the enzyme, ferrous iron must be added to assays of CDO to achieve full activity (7, 13). Because ADO is also a cupin protein, we wanted to test whether it binds a transition metal and, if so, whether it is required for catalytic activity. To answer this question metal analysis was conducted on WT ADO protein via both plasma emission spectroscopy and graphite furnace atomic absorption spectrometry. Plasma emission spectroscopy analysis revealed the presence of iron at an occupancy of 96.1%, zinc at an occupancy of 1.5%, nickel at an occupancy of 1%, and trace amounts of copper. Graphite furnace atomic absorption spectrometry also confirmed that the occupancy of iron in wild-type ADO protein was greater than 90%.

To test the specificity of iron binding as well as its requirement for cysteamine dioxygenase activity, we conducted a metal analysis on the H95A mutant. Mutation of histidine 95 in cupin motif 1 to an alanine, which ablates a highly conserved residue known to be involved in metal coordination in other cupin proteins, reduced bound iron levels to trace amounts in the recombinant purified protein. Concomitant with the loss of iron, the H95A mutant displayed no detectable catalytic activity (see above). These two pieces of evidence suggested that iron is indeed necessary for cysteamine dioxygenase activity.


Figure 5
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  		FIGURE 5.
Ectopic expression of 3x FLAG-tagged ADO in HepG2/C3A cells. A, a representative Western blot verifying the expression of 3x FLAG-tagged wild-type or H95A ADO proteins using anti-FLAG antibody. Lanes designated as Vector Control were loaded with lysate from cells transfected with 4 µg of the empty backbone vector. Transfections with the wild-type construct were conducted with 2, 3, or 4 µg of plasmid (empty vector was used to bring the final amount of transfected DNA to 4µg). Transfections with the H95A construct were conducted with 4 µg of expression plasmid. Cystamine was included in the culture medium as indicated for the final 30 h of culture. B, the effect of 3x FLAG-tagged ADO expression on intracellular hypotaurine levels. Results are the mean ± S.D. for 3–5 independent experiments. Bars not designated with the same letter (a–d) represent significantly different values (p ≤ 0.05).

 
Hypotaurine Production from Cysteamine Is Increased in HepG2/C3A Hepatoma Cells Transfected with ADO—Having established that ADO was capable of catalyzing cysteamine dioxygenase activity in vitro, we proceeded to test if it could also perform the same activity in a living system. To accomplish this objective, we transiently transfected 3x FLAG-tagged forms of catalytically active WT or catalytically incompetent H95A ADO into the human hepatoma HepG2/C3A cell line and then assayed for changes in the capacity of hypotaurine production from cysteamine. Western blots probing for the 3x FLAG tag confirmed that cells were able to ectopically express both the WT and mutant isoforms of the protein and to do so in a manner that was dependent upon the amount of expression construct that was transfected into the cells (Fig. 5A).

When cells were incubated in the presence of cystamine, a disulfide form of cysteamine that is efficiently taken up by cells and reduced to cysteamine intracellularly (23, 24), transfection with wild-type ADO significantly increased hypotaurine content in a dose-dependent fashion relative to control cells transfected with the empty vector alone (Fig. 5B). Transfection with the catalytically inactive mutant, on the other hand, had no significant effect on intracellular hypotaurine production.


Figure 6
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  		FIGURE 6.
siRNA knockdown of C10orf22, the ADO homolog endogenously expressed in HepG2/C3A cells. A, a Western blot evaluating the effect of siRNA transfection on C10orf22 (using anti-ADO antibody) and actin (using anti-actin antibody) expression in HepG2/C3A cells. Control siRNA is a negative control siRNA with no significant sequence similarity to any human gene sequences. siRNAs #1, #2, and #3 were designed to specifically target C10orf22. B, effect of siRNA transfection on intracellular hypotaurine levels. Medium was replaced with medium containing 0.5 mM cystamine for the last 24 h of culture. Each value represents the mean ± S.D. for three independent experiments. Bars not designated by the same letter (a–d) represent values that are significantly different (p ≤ 0.05).

 
We also observed in this series of experiments that control cells treated with cystamine contained significantly higher levels of hypotaurine than control cells treated without cystamine. One possible source of this hypotaurine production was a cysteamine dioxygenase that was being endogenously expressed by the HepG2/C3A cells. Further experiments were, therefore, designed to test this hypothesis.

Knockdown of ADO Homolog Expression in HepG2/C3A Cells Reduced Hypotaurine Synthesis from Cysteamine—HepG2/C3A cells were originally derived from a human hepatoblastoma (26). In the human genome, there is an ADO homolog known as C10orf22 that encodes a protein that shares ~85% identity with murine ADO (Fig. 2B). Initial results from reverse transcription-PCR studies confirmed that C10orf22 protein was expressed in HepG2/C3A cells, at least at the level of mRNA (data not shown). Expression of mature protein (~30 kDa) was subsequently confirmed by Western blot analysis of cell lysate using the polyclonal antibody generated against recombinant murine ADO (Fig. 6A).

To examine the contribution of C10orf22 to endogenous hypotaurine production, expression of the protein was reduced by employing RNA interference. Transfection with siRNAs specifically designed to target exon 1 of C10orf22 resulted in varying levels of reduction in protein expression (Fig. 6A). siRNA constructs #1 and #3 produced >70% reductions in C10orf22 protein expression, whereas siRNA construct #2 had no discernible effect. The negative control siRNA also had no effect. Blots for actin expression provided supporting evidence that the siRNA effects on C10orf22 expression were specific to C10orf22 (Fig. 6A).


Figure 7
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  		FIGURE 7.
The comparative tissue distributions of ADO and CDO protein expression in mouse. A, Western blot probing for ADO protein expression in various mouse tissues. B, Western blot of the same tissues using anti-CDO antibody. A total of 70 µg of protein was loaded into each lane. Epididymal fat was used for Fat, and gastrocnemius was used for Skeletal Muscle.

 
Reduction of C10orf22 expression was associated with a reduction in intracellular hypotaurine levels (Fig. 6B). siRNA construct #1 significantly (p ≤ 0.05) reduced hypotaurine levels by ~30% relative to mock-transfected cells, and siRNA construct #3 significantly (p ≤ 0.01) reduced levels by ~40% relative to mock-transfected cells. No significant perturbations in hypotaurine were seen with the negative control siRNA or siRNA construct #2. The association between C10orf22 protein expression and hypotaurine levels after incubation with cystamine is certainly consistent with the idea that C10orf22 protein acts as a cysteamine dioxygenase in intact cells.

Tissue Expression in Mice—Although EST data have confirmed that ADO mRNA is synthesized in murine tissues, ADO is nonetheless considered to be a hypothetical protein in this species. We, therefore, attempted to verify its expression. Using a polyclonal antibody generated from recombinant protein, several major tissues of the mouse were probed for ADO expression (Fig. 7). Most tissues showed a double band migrating at the appropriate molecular mass for ADO (~28 kDa). Brain, heart, and gastrocnemius also showed a third band that migrated at a slightly higher molecular weight than the 28-kDa doublet. These signals were specific to the primary antibody; incubation with preimmune serum and secondary antibody did not generate a detectable signal. The double band phenomenon is not unique to murine ADO because the human ortholog C10orf22 also migrated as a double band (Fig. 6A). Precisely why endogenous ADO migrates as a double band upon SDS-PAGE is not known. The result is particularly interesting, however, in light of the fact that endogenously expressed CDO also runs as a doublet on SDS-PAGE (Fig. 7) (11, 12, 27). The identity of the third higher molecular weight band seen in blots for ADO in brain, heart, and skeletal muscle is not known, although it could represent a post-translational modification of the protein. CDO, for instance, is post-translationally modified by ubiquitination (11, 12).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Identification of the protein responsible for cysteamine dioxygenase activity in mammalian tissues has been an elusive issue in sulfur amino acid metabolism for the past 40 years. As one of only two known thiol dioxygenases in mammals, this protein is of chemical interest for the unusual nature of the reaction it catalyzes. It is also of metabolic interest for its unique role in coupling the pathway of coenzyme A degradation with a pathway for taurine biosynthesis. We have discovered that ADO, a cupin protein of previously unknown function that shares some sequence similarity (14.2% identity) to cysteine dioxygenase, possesses highly specific cysteamine dioxygenase activity and, thus, may be the endogenous cysteamine dioxygenase.

Early attempts to isolate cysteamine dioxygenase from horse and porcine tissues identified proteins with conflicting molecular weights, a requirement for exotic electron-donating cofactors, and a broad range of substrate specificities. ADO, in contrast, shares homology with the known thiol dioxygenase CDO, binds a common iron cofactor, and catalyzes the dioxygenation of cysteamine with a high degree of specificity in vitro. We have also demonstrated that titrating the expression of the enzyme either through transient overexpression of ectopic protein or knockdown of endogenous protein expression produces a direct effect on the hypotaurine content of cells incubated with a source of cysteamine. For these reasons we propose that the intrinsic biological function of ADO is cysteamine dioxygenase activity.

This work represents the first biological activity ascribed to ADO and the larger protein family, known as Domain of Unknown Function 1637 (DUF1637; protein family accession number PF07847), to which it belongs. The DUF1637 family encompasses eukaryotic proteins from three different kingdoms of life (see Fig. 2B for representative species), all of which contain cupin protein motifs and are, thus, cupin superfamily members. Like many cupin superfamily members, ADO binds a transition metal (i.e. iron) that appears to be essential for activity. Elimination of iron binding by mutation of His-95, a strictly conserved putative metal coordinating residue, produced a catalytically inactive protein. With the exception of residues His-97 and His-179, which are also predicted to be involved in iron coordination, it is difficult to predict the function of the other strictly conserved residues found in this protein family. We speculate, however, that the vicinal proline motif (PP) conserved across all DUF1637 family members probably plays a structural role in the protein. Mammalian CDOs contain a similar vicinal proline motif that appears to permit the formation of a loop between two beta-strands containing important active site residues (13–15). Future structural analysis of the protein via either x-ray crystallography or nuclear magnetic resonance studies may shed light on the function of the conserved residues in ADO as well as reveal additional insights into the catalytic mechanism of this protein and the related thiol dioxygenase CDO. This is an important issue as there is currently much uncertainty in the literature over the catalytic mechanism of CDO (13–15, 29).

ADO appears to be a mammalian paralog of CDO. Both are cupin proteins that possess thiol dioxygenase activity. Despite catalyzing the oxidation of two structurally similar thiol compounds, however, the two proteins show no efficient cross-utilization of substrates. CDO will not use cysteamine as a substrate (6, 29), and ADO will not use cysteine as a substrate. This interesting divergence in substrate specificity may reflect an underlying homeostatic need by mammals to separately regulate the sulfoxidation of cysteine and cysteamine. This would be consistent with the largely disparate biological roles of the two compounds. Cysteine is used for many metabolic pathways including the synthesis of proteins, glutathione, inorganic sulfate, coenzyme A, and taurine. The availability of cysteine can fluctuate dramatically as a function of both dietary protein composition and the frequency of eating. CDO expression, in turn, is directly regulated by cysteine availability and plays an important role in disposing of excess cysteine. Cysteamine, on the other hand, has only one known function, and that is as a precursor for the formation of hypotaurine, which is subsequently oxidized to taurine. The rate of cysteamine production as a result of coenzyme A breakdown is not well understood (33), but it is clear that cysteamine levels are not as dramatically affected by dietary habits as are cysteine levels (12, 19). Coenzyme A degradation is suspected to be a constitutive process that occurs in all tissues, although some tissues such as brain and heart may have exceptionally high coenzyme A turnover rates (33–36). The fact that ADO shows a much broader range of tissue expression than CDO, with higher levels in brain and muscle, is certainly consistent with this hypothesis.

The broad tissue expression of ADO also requires a reassessment of the contribution of cysteamine dioxygenase to the synthesis of hypotaurine and taurine in vivo. The importance of the cysteamine ->hypotaurine -> taurine pathway has been largely regarded as minor relative to the cysteine -> cysteine sulfinic acid -> hypotaurine -> taurine pathway. Indeed, it is the reaction catalyzed by CDO that has been implicated as the major rate-determining step in the synthesis of hypotaurine and taurine (9, 28). Evidence for this assertion, however, has been biased by the relative paucity of information about cysteamine dioxygenase. More importantly the assertion inadequately explains certain tissue-specific discrepancies between taurine biosynthesis and CDO expression. The brain, for instance, is capable of synthesizing taurine and yet expresses relatively little CDO (25). Indeed, we have now shown that the brain expresses the highest level of ADO of any mouse organ tested, and it is, thus, possible that the cysteamine dioxygenase-mediated pathway is largely responsible for hypotaurine/taurine synthesis in the brain. Further work is definitely required to quantify how much hypotaurine and taurine is synthesized via the cysteamine -> hypotaurine -> taurine route versus the cysteine -> cysteine sulfinic acid -> hypotaurine -> taurine route.

Taken together, our results present strong evidence for the identification of Gm237 and its orthologs within the DUF1637 protein family as the elusive cysteamine dioxygenase. The identification of the gene encoding cysteamine dioxygenase opens a new avenue of research for expanding our understanding of the basic chemistry of thiol dioxygenases as well as our understanding of cysteamine metabolism and taurine biosynthesis in mammals.


   FOOTNOTES
 
* This research was supported in part by NIDDK, National Institutes of Health Public Health Service Grant DK056649 (to M. H. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a National Science Foundation graduate student fellowship award. Back

2 To whom correspondence should be addressed: Division of Nutritional Sciences, 227 Savage Hall, Cornell University, Ithaca, NY 14853. Tel.: 607-255-2638; Fax: 607-255-1033; E-mail: mhs6{at}cornell.edu.

3 The abbreviations used are: CDO, cysteine dioxygenase; ADO, 2-aminoethanethiol dioxygenase; SSA, sulfosalicylic acid; siRNA, small interfering RNA duplexes; MES, 2-(N-morpholino)ethanesulfonic acid; ORF, open reading frame; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank the staff of the Cornell University United States Plant, Soil, and Nutrition Laboratory and the staff of Cornell University Metabolic Mass Spectrometry Laboratory for assistance in conducting metal analyses of recombinant protein.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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