Characterization of an Iodothyronine 5'-Deiodinase in Gilthead Seabream (Sparus auratus) that Is Inhibited by Dithiothreitol
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《内分泌学杂志》
Department of Organismal Animal Physiology (P.H.M.K., R.H., J.R.M., L.M.C.N., G.F.), Institute for Neuroscience, Radboud University Nijmegen, NL-6525 ED Nijmegen, The Netherlands
Laboratory of Comparative Endocrinology (R.H., V.M.D., S.V.d.G.), Zoological Institute, K.U. Leuven, B-3000 Leuven, Belgium
Abstract
Iodothyronine deiodinases catalyze the conversion of the thyroid prohormone T4 to T3 by outer ring deiodination (ORD) of the iodothyronine molecule. The catalytic cycle of deiodinases is considered to be critically dependent on a reducing thiol cosubstrate that regenerates the selenoenzyme to its native state. The endogenous cosubstrate has still not been firmly identified; in studies in vitro the sulfhydryl reagent dithiothreitol (DTT) is commonly used to activate ORD. We now have characterized an ORD activity in the teleost gilthead seabream (Sparus auratus) that is inhibited by DTT. DTT inhibited reverse T3 (rT3) ORD by 70 and 100% in kidney homogenates (IC50 0.4 mmol/liter) and microsomes (IC50 0.1 mmol/liter), respectively. The omission of DTT from the incubation medium restored renal ORD Michaelis-Menten kinetics with a Michaelis constant value of 5 μmol/liter rT3 and unmasked the inhibition by 6-n-propyl-2-thiouracil. A putative seabream deiodinase type 1 (saD1), derived from kidney mRNA, showed high homology ( 41% amino acid identity) with vertebrate deiodinases type 1. Features of this putative saD1 include a selenocysteine encoded by an in-frame UGA codon, consensus sequences, and a predicted secondary structure for a selenocysteine insertion sequence and an amino acid composition of the catalytic center that is identical with reported consensus sequences for deiodinase type 1. Remarkably, three of six cysteines that are present in the deduced saD1 protein occur in the predicted amino terminal hydrophobic region. We suggest that the effects of DTT on rT3 ORD can be explained by interactions with the cysteines unique to the putative saD1 protein.
Introduction
THE CONVERSION, by outer ring deiodination (ORD), of the thyroid hormone T4 to the potent and biologically active T3 is an important activation pathway in the metabolism of thyroid hormones. ORD occurs mainly in peripheral tissues, of which liver and kidney are most important, and is catalyzed by the enzymatic action of iodothyronine deiodinases. Deiodinases constitute a family of at least three distinctly different selenoenzymes, designated D1, D2, and D3. In the catalytic site of the D1 protein, a selenocysteine residue functions as acceptor for the iodonium ion that is liberated from the iodothyronine substrate during catalysis. The catalytic cycle is completed by the reduction of the substituted enzyme intermediate by a thiol cosubstrate, which returns the selenocysteine to its native state and thus regenerates the enzyme molecule (1, 2, 3).
The reduction of the enzyme intermediate by the thiol cosubstrate is considered pivotal to deiodinase-catalyzed ORD. To date, however, the natural endogenous cosubstrate has still not been firmly recognized. In in vitro assays for ORD the sulfhydryl reagent dithiothreitol (DTT), in millimolar concentrations, is routinely and exclusively used as the reducing cosubstrate to activate deiodinase catalysis. DTT, or 2,3-hydroxy-1,4-dithiolbutane, is a dithiol with a low redox potential that forms a cyclic disulfide in the fully oxidized form (4). These properties make DTT a potent sulfhydryl reagent to keep monothiols in a reduced state, and its role in the ORD catalytic cycle is generally accepted to be the reduction of the selenenyl-iodide substituted enzyme complex (1).
The gilthead seabream (Sparus auratus) is a euryhaline teleost species that can adapt successfully to a wide range of ambient salinities, ranging from seawater (approximately 1000 mOsmol, or 3.5% salinity) to fresh water. Gills and kidney are important osmoregulatory organs that contain high activities of the sodium pump Na+,K+-ATPase and are targets for thyroid hormone action (5, 6, 7, 8). Fish liver and kidney express relatively high ORD activities (9, 10, 11) and are therefore important determinants of the thyroid status of the animal. In the course of our research on the involvement of the thyroid axis in fish osmoregulation, we measured ORD activities in preparations derived from the gills, liver, and kidney of gilthead seabream. When we assessed the optimum enzyme assay conditions, we obtained unexpected results with respect to the DTT requirement of reverse T3 (rT3) ORD in gilthead seabream kidney.
Materials and Methods
Animals
Juvenile gilthead seabream (Sp. auratus, hereafter called seabream) and adult Mozambique tilapia (Oreochromis mossambicus, hereafter called tilapia) were from laboratory stock. Seabream were kept in artificial seawater at 20 C and a photoperiod of 12-h light alternating with 12-h darkness. Artificial seawater was prepared by dissolving natural sea salt (Aqua Medic, Bissendorf, Germany) to a salinity of 3.4% in Nijmegen city tap water. Tilapia were kept in running Nijmegen city tap water at 25 C and a photoperiod of 16 h light alternating with 8 h darkness. Fish were fed a commercially available feed (Pro-Aqua, Trouvit, The Netherlands) once daily at a ration of 1% of the estimated body weight. Animals were anesthetized in 0.1% 2-phenoxyethanol and killed by spinal transection. All animal procedures were approved by the local ethical review committee.
Materials
rT3, T3, T4, and 6-n-propyl-2-thiouracil (PTU) were purchased from Sigma Chemical Co. (St. Louis, MO). 3-Monoiodothyronine from InoVar Chemicals (Gaithersburg, MD) was kindly made available to us by Professor T. J. Visser (Erasmus Medical Center, Rotterdam, The Netherlands). Sephadex LH-20 was from Amersham Pharmacia Biotech (Uppsala, Sweden). Radioactively labeled [125I]rT3 (24.4 TBq/mmol) was from NEN Life Science Products, Inc. (Boston, MA). [125I]T4 and [125I]3,3'-T2 were prepared by radioiodination of T3 and monoiodothyronine, respectively, with carrier-free Na125I using the chloramine-T method.
X-Gal was from AppliChem GmbH (Darmstadt, Germany). The ABI Prism Big Dye terminator cycle sequencing ready reaction kit was obtained from Applied Biosystems (Foster City, CA). DNase I (1 U/μl), 10x DNase I reaction buffer, DTT, EDTA, GeneRacer kit, deoxynucleotide triphosphates, oligo(dT)15 primer, TOPO TA cloning kit, and TRIzol reagent were from Invitrogen Co. (Carlsbad, CA). QIAEX II gel extraction kit was from QIAGEN Inc. (Valencia, CA). Agarose UP, avian myeloblastosis virus-reverse transcriptase (AMV-RTase, 25 U/μl), AMV-RT buffer, EcoRI (10 U/μl), expand long template PCR system, 10x H buffer high pure plasmid isolation kit, and Rnase inhibitor (RNasin, 40 U/μl) were obtained from Roche (Basel, Switzerland). The GeneAmp PCR 9700 system was from PerkinElmer (Shelton, CT). All other chemicals were of highest purity and obtained from commercial suppliers.
Tissue preparations
Liver and kidney from seabream and tilapia were homogenized in, respectively, 3 and 1 ml phosphate buffer [100 mmol/liter Na-phosphate (pH 7.2), 2 mmol/liter EDTA] in a glass dounce homogenizer equipped with a tightly fitting Teflon pestle. Homogenates were stored at –80 C until further analysis. To obtain a seabream renal microsomal fraction, kidney tissue was homogenized in 4 volumes (wt/vol) buffered solution [100 mmol/liter Tris/HCl (pH 7.4), 250 mmol/liter sucrose, 1 mmol/liter EDTA] and centrifuged at 20,000 x g for 20 min in a Sorvall SM24 rotor. The resulting supernatant was centrifuged at 100,000 x g for 60 min in a Beckman Sw40 rotor. The pellet thus obtained was resuspended in 100 mmol/liter Na-phosphate buffer (pH 7.2) and stored at –80 C until further analysis. Protein was measured with a commercial Coomassie Brilliant Blue reagent kit (Bio-Rad Laboratories, München, Germany) using BSA as a reference.
Iodothyronine 5'-deiodinase assays
5'-Deiodinase activities were assayed in duplicate by incubating 50 μg homogenate or microsomal protein for 15 min at 37 C in 200 μl 100 mmol/liter phosphate buffer (pH 7.2) to which were added: T4 or rT3 and DTT in concentrations as indicated in the legends to the figures, [125I]T4 or [125I]rT3 to a specific activity of 9 x 1012 to 8 x 1014 cpm/mol iodothyronine, and 2 mmol/liter EDTA. Nonenzymatic ORD (5'-deiodination) was determined in the absence of a preparation. Radiotracer was purified on a 10% (wt/vol) Sephadex LH-20 minicolumn shortly before use as described earlier (12). The incubation was quenched by adding 100 μl 5% (wt/vol) ice-cold BSA. Quenched incubates were deproteinized with 500 μl 10% (wt/vol) ice-cold trichloroacetic acid followed by precipitation of denatured proteins at 1400 x g (15 min, 4 C). To 0.5 ml of the supernatant thus obtained, an equal volume of 1.0 mol/liter HCl was added, and liberated iodide was separated from the native iodothyronine with the use of Sephadex LH-20 column chromatography as described earlier (12), collecting 125I– in the first three 1-ml 0.1 mol/liter HCl eluates. HPLC analyses showed that equal amounts of iodide and 3,3'-T2 were produced by renal homogenates and microsomal fractions from rT3 in the absence or presence of 10 mmol/liter DTT (results not shown). Interestingly, when we incubated seabream kidney homogenates and microsomes with [125I]3,3'-T2, an ORD activity became apparent, and this explained why 3,3'-T2 amounts in the incubates, although still detectable, not always exactly equaled the amounts of liberated iodide from rT3 (results not shown). 125I radioactivity was measured in an LKB-1272 Clinigamma -counter (Wallac Oy, Turku, Finland). The specific D1 activity was expressed as femtomoles rT3 deiodinated per minute per milligram protein. Our calculations included a correction factor of 2 to take into account the random labeling of the 3'- and 5'-positions of [125I]rT3 and [125I]T4.
Cloning and sequencing
Total RNA was isolated from seabream kidney using the TRIzol reagent and treated with DNase before reverse transcription. cDNA was obtained using oligo(dT)15 primers and AMV-RTase in the presence of RNasin. Based on homologies of conserved regions in the D1 nucleotide sequences of guppy (Poecilia reticulata), Nile tilapia (O. niloticus), house musk shrew (Suncus murinus), chicken, dog, rat, mouse, and man the following primers were designed: 5'-GGC AAC AGA CCG CTG CTG CTG-3' (forward), and 5'-ATC TTC TCC AAG AAG GAT CGC ACC TC-3' and 5'-ATC TTC TGC AGG ACC GAA CGC ACC TC-3' (reverse), and PCR was performed using Taq DNA polymerase with proofreading activity. PCR products were separated on 2% agarose, subcloned into a TOPO vector, and transferred to One Shot chemically competent Escherichia coli (DH5). Transformed cells were lysed and plasmids isolated; only those plasmids that upon EcoRI restriction yielded cleavage products of the predicted size (3.9 and 403 bp) were subjected to automatic sequencing using the dideoxy method of Sanger et al. (13). A 404-bp cDNA fragment was thus obtained, the sequence of which was used to design the following primers for the rapid amplification of 5'- and 3'- rapid amplification of cDNA ends (RACE): 5'-TGA AGT CCC TGA CGA GTC GCT TGA A-3' and 5'-CGC CAG ACT CGC CTG TGG TCA CCA T-3' for 5'-RACE and 3'-RACE, respectively. Total RNA from Sp. auratus kidney was isolated, dephosphorylated, decapped and ligated, and then reverse transcribed into cDNA. 5'- and 3'-RACE was performed using the primers described above and GeneRacer 5'- and 3'-primers.
Analysis and statistics
ORD kinetic data were analyzed using a weighted nonlinear regression data analysis program (14) in which Marquardt’s algorithm for least squares estimation of parameters (15) is employed. Data points were not transformed before analysis, and the SD was used as an explicit weighting value for each data point. Statistical significance was evaluated with Student’s t test and was accepted at P < 0.05.
Results
We first measured iodothyronine ORD in seabream and tilapia kidney homogenates in the presence of DTT at concentrations that we, based on observations by Mol et al. (10, 11), considered optimal for rT3 and T4 ORD in teleost tissues, i.e. 10 and 20 mmol/liter DTT, respectively. rT3 ORD by seabream and tilapia kidney homogenates, measured at 5 μmol/liter rT3 and 10 mmol/liter DTT, proceeded linearly up to 50 min incubation time (results not shown); a time point of 15 min was chosen to measure initial deiodination rates. Seabream renal ORD activities, measured at initial rate, toward 1.0 μmol/liter rT3 and T4 were 0.9 ± 0.3 and 0.04 ± 0.01 fmol/min per microgram protein (n = 5, mean ± SD), respectively. The apparent 20-fold higher ORD substrate preference for rT3 over T4 is consistent with that reported for mammalian and Nile tilapia (O. niloticus) iodothyronine D1 (16, 17) and could well be the expression of a specific seabream D1 activity.
Figure 1 shows that the initial rate of rT3 ORD activity in seabream kidney homogenates, measured in the presence of 10 mmol/liter DTT, did not saturate at high substrate concentrations. Contrastingly, renal homogenates from tilapia (O. mossambicus), assayed in parallel with the seabream preparations, displayed the rectangular hyperbola characteristic for single-site Michaelis-Menten kinetics. The calculated Michaelis constant (Km) value for renal ORD in tilapia was 0.8 μmol/liter rT3, which is in the same range as that reported for other teleost species (11). Tilapia data points, and the values for Km and limiting rate (Vmax) calculated from these, converged on a linear Eadie-Hofstee transformation of the Michaelis-Menten equation, indicative for a single catalytically active component in renal rT3 ORD. The nonsaturating behavior of rT3 ORD in seabream was not tissue-specific because it was also observed in liver homogenates (Fig. 2), and it thus appears to be a property of a seabream 5'-deiodinase proper.
Proceeding from these observations, we chose to investigate biochemical and pharmacological properties of seabream renal rT3 ORD. Figure 3 shows that the effects of DTT on rT3 ORD in seabream renal homogenates were inhibitory, not stimulatory. An IC50 value of 0.4 mmol/liter DTT was calculated, which is lower than the lowest DTT concentration tested in this series of experiments. Moreover, an ORD component insensitive to DTT, comprising 32% of the uninhibited rT3 ORD activity, could be resolved in renal homogenates.
When DTT was omitted from the incubation medium, rT3 ORD activity obeyed simple Michaelis-Menten kinetics (Fig. 4). The value for Km was calculated to be 5 μmol/liter rT3, which is in the same order of magnitude as that reported for tilapia (this study) and other teleost species (11). In the absence of DTT, data points and calculated values for Km and Vmax converged on a linear Eadie-Hofstee transformation, indicative for a single catalytically active component in rT3 ORD in seabream renal homogenates. The progress curve of rT3 ORD by seabream renal homogenates, measured in the absence of DTT, is described by a first-order exponential equation (Fig. 5), indicative for a single enzymatic component and in line with the observed single-site Michaelis-Menten kinetics (Fig. 4). The maximum rate, calculated from the slope of the tangent to the progress curve at time point 0, is 53 fmol/μg·min. The calculated first-order rate constant equals 0.006 min–1, and the rT3 ORD reaction rate falls by 0.6%/min to reach 30% of the maximum rate at the 200-min time point in our assay.
To exclude the possible effect of endogenous cytosolic compounds, the effect of DTT on rT3 ORD was assessed in seabream renal microsomal fractions. As in whole kidney homogenates, rT3 ORD was inhibited by DTT, but here inhibition was virtually complete at 1 mmol/liter DTT (Fig. 6). The progress curve of rT3 ORD by seabream renal microsomal fraction, measured in the absence of DTT, is also described by a first-order exponential equation (Fig. 7), again indicative for a single enzymatic component. The maximum rate is calculated to be 192 fmol/μg·min, and this value is 3.6 times higher than that for renal homogenates, indicating an enrichment of the microsomal fraction with respect to rT3 ORD and putative D1 activity, compared with the homogenate. The calculated first-order rate constant for the microsomal fraction equals 0.009 min–1 and is similar to that of homogenates. At 200 min, the ORD reaction rate has decreased to 17% of the maximum rate.
In the presence of 10 mmol/liter DTT, rT3 ORD activity in seabream kidney homogenates was insensitive to inhibition by PTU (Fig. 8A). However, when DTT was omitted from the incubation medium, rT3 ORD activity was inhibited by approximately 70% in the presence of 0.1 mmol/liter PTU (Fig. 8B). Interestingly, a rat liver homogenate preparation, which was included in our assay as a positive control and was also incubated in the absence of DTT, contained an rT3 ORD activity that was inhibited by 90% in the presence of 0.1 mmol/liter PTU (result not shown).
The results obtained so far indicate that seabream possesses an iodothyronine 5'-deiodinase, most likely a D1, with a peculiar behavior toward the reducing cosubstrate that is considered to be essential to the catalytic action of the enzyme. We therefore decided to characterize a D1 cDNA derived from seabream kidney. The full-length nucleotide and deduced amino acid sequence of putative seabream D1 (saD1) cDNA with a size of 1545 bp are shown in Fig. 9. A large 3'-untranslated region (UTR) contains sequences identical with mammalian selenocysteine insertion sequence (SECIS) consensus motifs (18, 19). A SECIS secondary mRNA structure could be resolved (Fig. 10) using a computer algorithm for the analysis of structural and thermodynamic features of SECIS elements (20), and it can therefore be assumed that UGA at codon position 126 is translated as selenocysteine. The non-Watson-Crick base pair quartet UGAC_AGAU in the saD1 SECIS core was preceded 5' by a guanosine, not adenosine (position 1073). This less common variant (19) is present in SECIS elements of Nile tilapia D1 and D3 (16, 21) and can be found in killifish (Fundulus heteroclitus) D1 3'UTR as well. The substitution of G for A 5' to the UGAN quartet appears to be a typical feature of teleost SECIS elements.
The open reading frame (nt 52–798) putatively encodes a 248-amino acid saD1 protein with an estimated molecular weight of 27.7 x 103. Figures 11 and 12 show alignments of the deduced amino acid sequence of saD1 with that of nonteleost vertebrates and other teleost species, respectively. Sequence identity with man, dog, rat, mouse, house musk shrew (Su. murinus), and chicken (Gallus gallus) ranges from 41 to 47%, whereas that with the teleost species Nile tilapia, killifish, and zebrafish (Danio rerio) is 71, 69, and 56%, respectively. A hydropathy analysis based on the augmented Wimley-White whole-residue hydrophobicity scale (22) (Fig. 13) indicates a hydrophobic domain formed by amino acids at positions 1–33, of which positions 15–33 are predicted to form a transmembrane segment. The amino acids at positions 115–133, which include the consensus sequence for the catalytic center (positions 115–129) in the saD1 protein, are also predicted to form a hydrophobic domain.
The deduced amino acid sequence of the catalytic center of the saD1 protein is identical with that of Nile tilapia, killifish, zebrafish, and the consensus sequence for D1 and has a high homology with the consensus sequences for D2 and D3 as reported by Bianco et al. (3). Callebaut et al. (23) list 13 amino acid positions in the D1 active center, nine of which are shared by saD1. Conserved cysteine residues (24) are at positions 124 and 194 in saD1, whereas Cys108 replaces a tryptophane residue that appears to be conserved in mammals. Other conserved amino acids that, in mammals, are critical for correct enzyme function are Phe65 (25, 26, 27) and Gly45 and Glu46 (27). Recently a conserved D1 dimerization domain was described (28), which is also present in the putative saD1 protein (consensus residues in bold, one-letter code): D 148FLVVYIAEAHSTDGW163, located 22 amino acid positions 3' to Sec.
Differences between the D1 protein of seabream and other vertebrates are concentrated in the amino-terminal hydrophobic domain. Most notably, three of six cysteines that are present in saD1 occur here (i.e. at positions 10, 17, and 33). Positively charged amino acid residues in the hydrophobic region that are conserved in mammals (29) are uncharged polar or nonpolar residues in seabream (i.e. Ser11, Leu12, Gln27, and Leu36).
Discussion
We show here that DTT inhibits rT3 ORD in Sp. auratus kidney homogenates and microsomal fractions. This is contrary to all reported mammalian ORD activities, which are stimulated by DTT. Moreover, the reactivity of rT3 ORD in Sparus toward the thyrostatic PTU is affected profoundly by the presence of DTT in the incubation medium.
Our observations on the nonclassical action of DTT on rT3 ORD in seabream kidney are not unique. ORD activities in turbot (Psetta maxima) ovaries were reduced by approximately 25–30% in the presence of 10 and 100 mmol/liter DTT, respectively (Mol, K. A., unpublished results)1, a result that corroborates our data. Moreover, 65–80% of the maximal rT3 ORD activities in kidney homogenates from rainbow trout (Oncorhynchus mykiss), Nile tilapia, and turbot were measured in the absence of DTT from the incubation medium (ibid.), which implies that DTT has only a relatively modest activating effect on rT3 ORD in these species. Reverse T3 ORD in kidney homogenates from blue tilapia (O. aureus) is insensitive to DTT, i.e. is neither activated nor inhibited by DTT in concentrations up to 50 mmol/liter (10). We recently measured a potent dose-dependent inhibition by DTT of rT3 ORD in renal homogenates from Senegalese sole (Solea senegalensis), in which 1 mmol/liter DTT inhibited ORD by 78% (Klaren, P. H. M., and F. Arjona Madueo, 2005, unpublished results). All in all, teleost fish appear to represent a group of vertebrates in which ORD of iodothyronines is less critically dependent on the presence of DTT than in mammals. Teleosts could provide interesting natural models to study the interactions between deiodinases and their thiol cosubstrates.
Our results prompted us to critically consider the use of DTT in in vitro assays. DTT is an exogenous substrate and is applied in vitro because the natural, endogenous thiol cosubstrate has not been unequivocally recognized. Still, ORD activities of tissue preparations and expressed D1 transcripts have been shown to be stimulated by the endogenous intracellular peptidyl thiols glutathione (30, 31, 32, 33, 34, 35) and thioredoxin (35, 36, 37). Goswami and Rosenberg (34) demonstrated that the activation of ORD in a rat kidney microsomal preparation by glutathione, but not that by DTT, was lost upon preincubation by DTT. This indicates that these thiols have different sites of action on the deiodinase protein to activate ORD. Most of the ORD activities stimulated by glutathione or thioredoxin were measured at low, i.e. nanomolar substrate (rT3, T4), concentrations, and the stimulatory effects of the endogenous thiols were not detectable at micromolar substrate concentrations. However, considering the plasma total T3 and T4 concentrations in vertebrates that are in the nanomolar range, it is likely that cytosolic thyroid hormone concentrations are in the (sub)nanomolar rather than micromolar range. The reported stimulatory actions in vitro of glutathione and thioredoxin, albeit less pronounced than those of DTT, could therefore very well reflect an in vivo physiologically relevant ORD activation in mammals. It remains to be established whether the inhibition of rT3 ORD by DTT reflects inhibitory actions of endogenous thiols in Sp. auratus kidney in vivo. Indeed, the inhibition of rT3 ORD by DTT can be considered an artifact of the substitution for the unknown endogenous reducing cosubstrate. The argument could be equally valid for the activation of ORD by DTT in mammalian preparations.
Dihydrolipoamide is the only endogenous thiol compound tested that has been shown to be at least as potent as DTT in the activation of rT3 ORD (31, 38, 39, 40). Both DTT and dihydrolipoamide are potent nonpeptidyl dithiol reductants of which the redox couples have low redox potentials and that form a cyclic disulfide in the fully oxidized form in which properties are considered to favor a high reducing potential (4, 41). Although DTT and dihydrolipoamide have equivalent potencies in the activation of ORD, these compounds differ markedly in their reducing potential of disulfides, which has been estimated to be approximately 20-fold less for dihydrolipoamide, compared with DTT (41). The reactivities of thiols toward disulfides apparently do not predict the potency to reduce selenenyl-iodide enzyme intermediates. Interestingly, the reduction of a synthetic selenenyl iodide compound was found to be catalyzed by mammalian and bacterial thioredoxin reductases (of which only the mammalian enzyme contains a selenocysteine), whose reactions were only slightly enhanced by the addition of thioredoxin (42). There appear to be several thiol and nonthiol candidates that can fulfill the role of reducing cosubstrate in the catalytic cycle of iodothyronine ORD. We are currently evaluating the effects of these on rT3 ORD in seabream.
The reaction rates of rT3 ORD by seabream renal homogenates and microsomes, measured in the absence of DTT, fall exponentially by 0.6 and 0.9%/min, respectively, and this could be due to substrate depletion, product inhibition, or endogenous cosubstrate depletion. The latter, however, is less likely in view of the rT3 ORD activities that we observed in purified microsomes and that were enhanced, compared with the homogenates from which the microsomal fractions were derived. We are currently investigating the catalytic mechanism of rT3 ORD by seabream renal microsomes.
The omission of DTT from the incubation medium revealed a hidden sensitivity to inhibition by PTU of rT3 ORD in Sp. auratus kidney homogenates, albeit that the inhibition by the thyrostatic, measured at a concentration of 0.1 mmol/liter PTU, was less pronounced than in mammalian preparations. The inhibition of mammalian D1 by PTU is well established, and the insensitivity of teleost D1 activity for inhibition by PTU defines a marked difference between these vertebrate orthologs (16, 43, 44, 45). PTU is suggested to compete with thiol cosubstrates for binding to the selenenyl iodide enzyme intermediate in which binding of PTU results in a dead-end complex (46, 47). However, to date it is still unknown what structural feature confers the insensitivity for inhibition by PTU to the teleost D1 protein. Mammalian D1 proteins, i.e. human, rat, mouse, and dog, which are PTU sensitive, contain a serine two positions 3' from selenocysteine (25, 48, 49, 50). The first PTU-insensitive D1 cloned, that of Nile tilapia, was found to contain a proline at this position (16), as does Sp. auratus putative D1 (this study), here Pro128. This suggests that position 128 plays a pivotal role in the interactions of the selenenyl iodide intermediate with PTU. However, rT3 ORD by a Pro128Ser mutant of Nile tilapia D1 was inhibited by only about 15% in the presence of 1 mmol/liter PTU, and it was concluded that the PTU insensitivity of Nile tilapia D1 was not due to the presence of Pro for Ser at position 128 (16). Remarkably, the reverse substitution, i.e. Ser128Pro in human D1, resulted in a mutant that was resistant to PTU (23), the result of which leaves the role of position 128 in the interactions between selenoenzyme and PTU equivocal. The amino acid homology of the catalytic centers of tilapia D1 (16) and Sp. auratus putative D1 with those of mammalian D1s is very high, which suggests that the reactivity of the selenenyl iodide intermediate with PTU is determined by amino acid residues outside the catalytic center. ORD activities in a number of teleost preparations are well measurable in the absence of DTT, and it would be interesting to measure the effects of PTU under these in vitro conditions.
It is tempting to correlate the actions of the classical sulfhydryl reagent DTT with the presence of cysteine residues unique to the deduced putative saD1 protein, i.e. Cys108 near the catalytic center, and the three cysteine residues at positions 10, 17, and 33 in the hydrophobic amino terminal region. Cys108 is predicted to reside in the cytosol, and its thiol group can therefore presumed to be in the reduced state. In many mammals a conserved Cys105 is present in D1. Cys108 and Cys105 could be analogous, but it is not known whether these residues can interact with the active site of the enzyme. It has been shown that overexpressed mammalian D1 forms homodimers in vivo (51), possibly through specific dimerization sites (28) but possibly also through disulfide bridges. It can be hypothesized that the cysteine residues in the amino terminal region are involved in the formation of disulfide bridges in saD1 dimers, which are reduced by DTT, yielding catalytically inactive monomers. We are currently using a site-directed mutagenesis strategy to investigate the biochemical properties of wild-type saD1 and saD1 mutants in which selected cysteines are replaced.
The inhibition of rT3 ORD by DTT was complete in a 100,000 x g renal microsomal fraction, but in whole kidney homogenates, a DTT-insensitive component comprising 30% of the uninhibited activity was resolved. Our kinetic analyses clearly indicated the presence of only one catalytically active ORD component in whole kidney homogenates. Both the DTT-sensitive and DTT-insensitive components in seabream rT3 ORD are therefore most probably expressions of the same deiodinase protein. It could be that some cytosolic factor, present in whole organ homogenates but absent in isolated microsomes, is responsible for the observed behavior of rT3 ORD in Sp. auratus.
In conclusion, our data show that the substrate concentration dependence as well as the PTU-sensitivity of rT3 ORD in Sp. auratus kidney is profoundly affected by DTT in vitro. The presence of a relatively large number of cysteine residues in the deduced putative saD1 offers possible targets for interaction with the sulfhydryl reagent.
Acknowledgments
The authors are grateful to Professor Theo Visser (Erasmus Medical Center, Rotterdam, The Netherlands) for the generous donation of 3'-T1; Fransisco Arjona Madueo and Dr. Juan-Miguel Mancera (University of Cádiz, Cádiz, Spain) for making Solea senegalensis kidney available to us; and Tom Spanings for excellent fish husbandry.
Footnotes
This work was supported by the European Socrates program (grant to R.H.). S.V.d.G. was supported by the Fund for Scientific Research–Flanders.
First Published Online September 15, 2005
Abbreviations: AMV-RTase, Avian myeloblastosis virus-reverse transcriptase; D1, type 1 iodothyronine deiodinase; DTT, dithiothreitol; Km, Michaelis constant; ORD, outer ring deiodination; PTU, 6-n-propyl-2-thiouracil; RACE, rapid amplification of cDNA ends; rT3, reverse T3; saD1, Sparus auratus D1; SECIS, selenocysteine insertion sequence; UTR, untranslated region; Vmax, limiting rate.
1 Mol KA 1996 A study on peripheral deiodination of thyroid hormones in fish. Ph.D. Dissertation, K.U. Leuven, Belgium.
Accepted for publication September 7, 2005.
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Laboratory of Comparative Endocrinology (R.H., V.M.D., S.V.d.G.), Zoological Institute, K.U. Leuven, B-3000 Leuven, Belgium
Abstract
Iodothyronine deiodinases catalyze the conversion of the thyroid prohormone T4 to T3 by outer ring deiodination (ORD) of the iodothyronine molecule. The catalytic cycle of deiodinases is considered to be critically dependent on a reducing thiol cosubstrate that regenerates the selenoenzyme to its native state. The endogenous cosubstrate has still not been firmly identified; in studies in vitro the sulfhydryl reagent dithiothreitol (DTT) is commonly used to activate ORD. We now have characterized an ORD activity in the teleost gilthead seabream (Sparus auratus) that is inhibited by DTT. DTT inhibited reverse T3 (rT3) ORD by 70 and 100% in kidney homogenates (IC50 0.4 mmol/liter) and microsomes (IC50 0.1 mmol/liter), respectively. The omission of DTT from the incubation medium restored renal ORD Michaelis-Menten kinetics with a Michaelis constant value of 5 μmol/liter rT3 and unmasked the inhibition by 6-n-propyl-2-thiouracil. A putative seabream deiodinase type 1 (saD1), derived from kidney mRNA, showed high homology ( 41% amino acid identity) with vertebrate deiodinases type 1. Features of this putative saD1 include a selenocysteine encoded by an in-frame UGA codon, consensus sequences, and a predicted secondary structure for a selenocysteine insertion sequence and an amino acid composition of the catalytic center that is identical with reported consensus sequences for deiodinase type 1. Remarkably, three of six cysteines that are present in the deduced saD1 protein occur in the predicted amino terminal hydrophobic region. We suggest that the effects of DTT on rT3 ORD can be explained by interactions with the cysteines unique to the putative saD1 protein.
Introduction
THE CONVERSION, by outer ring deiodination (ORD), of the thyroid hormone T4 to the potent and biologically active T3 is an important activation pathway in the metabolism of thyroid hormones. ORD occurs mainly in peripheral tissues, of which liver and kidney are most important, and is catalyzed by the enzymatic action of iodothyronine deiodinases. Deiodinases constitute a family of at least three distinctly different selenoenzymes, designated D1, D2, and D3. In the catalytic site of the D1 protein, a selenocysteine residue functions as acceptor for the iodonium ion that is liberated from the iodothyronine substrate during catalysis. The catalytic cycle is completed by the reduction of the substituted enzyme intermediate by a thiol cosubstrate, which returns the selenocysteine to its native state and thus regenerates the enzyme molecule (1, 2, 3).
The reduction of the enzyme intermediate by the thiol cosubstrate is considered pivotal to deiodinase-catalyzed ORD. To date, however, the natural endogenous cosubstrate has still not been firmly recognized. In in vitro assays for ORD the sulfhydryl reagent dithiothreitol (DTT), in millimolar concentrations, is routinely and exclusively used as the reducing cosubstrate to activate deiodinase catalysis. DTT, or 2,3-hydroxy-1,4-dithiolbutane, is a dithiol with a low redox potential that forms a cyclic disulfide in the fully oxidized form (4). These properties make DTT a potent sulfhydryl reagent to keep monothiols in a reduced state, and its role in the ORD catalytic cycle is generally accepted to be the reduction of the selenenyl-iodide substituted enzyme complex (1).
The gilthead seabream (Sparus auratus) is a euryhaline teleost species that can adapt successfully to a wide range of ambient salinities, ranging from seawater (approximately 1000 mOsmol, or 3.5% salinity) to fresh water. Gills and kidney are important osmoregulatory organs that contain high activities of the sodium pump Na+,K+-ATPase and are targets for thyroid hormone action (5, 6, 7, 8). Fish liver and kidney express relatively high ORD activities (9, 10, 11) and are therefore important determinants of the thyroid status of the animal. In the course of our research on the involvement of the thyroid axis in fish osmoregulation, we measured ORD activities in preparations derived from the gills, liver, and kidney of gilthead seabream. When we assessed the optimum enzyme assay conditions, we obtained unexpected results with respect to the DTT requirement of reverse T3 (rT3) ORD in gilthead seabream kidney.
Materials and Methods
Animals
Juvenile gilthead seabream (Sp. auratus, hereafter called seabream) and adult Mozambique tilapia (Oreochromis mossambicus, hereafter called tilapia) were from laboratory stock. Seabream were kept in artificial seawater at 20 C and a photoperiod of 12-h light alternating with 12-h darkness. Artificial seawater was prepared by dissolving natural sea salt (Aqua Medic, Bissendorf, Germany) to a salinity of 3.4% in Nijmegen city tap water. Tilapia were kept in running Nijmegen city tap water at 25 C and a photoperiod of 16 h light alternating with 8 h darkness. Fish were fed a commercially available feed (Pro-Aqua, Trouvit, The Netherlands) once daily at a ration of 1% of the estimated body weight. Animals were anesthetized in 0.1% 2-phenoxyethanol and killed by spinal transection. All animal procedures were approved by the local ethical review committee.
Materials
rT3, T3, T4, and 6-n-propyl-2-thiouracil (PTU) were purchased from Sigma Chemical Co. (St. Louis, MO). 3-Monoiodothyronine from InoVar Chemicals (Gaithersburg, MD) was kindly made available to us by Professor T. J. Visser (Erasmus Medical Center, Rotterdam, The Netherlands). Sephadex LH-20 was from Amersham Pharmacia Biotech (Uppsala, Sweden). Radioactively labeled [125I]rT3 (24.4 TBq/mmol) was from NEN Life Science Products, Inc. (Boston, MA). [125I]T4 and [125I]3,3'-T2 were prepared by radioiodination of T3 and monoiodothyronine, respectively, with carrier-free Na125I using the chloramine-T method.
X-Gal was from AppliChem GmbH (Darmstadt, Germany). The ABI Prism Big Dye terminator cycle sequencing ready reaction kit was obtained from Applied Biosystems (Foster City, CA). DNase I (1 U/μl), 10x DNase I reaction buffer, DTT, EDTA, GeneRacer kit, deoxynucleotide triphosphates, oligo(dT)15 primer, TOPO TA cloning kit, and TRIzol reagent were from Invitrogen Co. (Carlsbad, CA). QIAEX II gel extraction kit was from QIAGEN Inc. (Valencia, CA). Agarose UP, avian myeloblastosis virus-reverse transcriptase (AMV-RTase, 25 U/μl), AMV-RT buffer, EcoRI (10 U/μl), expand long template PCR system, 10x H buffer high pure plasmid isolation kit, and Rnase inhibitor (RNasin, 40 U/μl) were obtained from Roche (Basel, Switzerland). The GeneAmp PCR 9700 system was from PerkinElmer (Shelton, CT). All other chemicals were of highest purity and obtained from commercial suppliers.
Tissue preparations
Liver and kidney from seabream and tilapia were homogenized in, respectively, 3 and 1 ml phosphate buffer [100 mmol/liter Na-phosphate (pH 7.2), 2 mmol/liter EDTA] in a glass dounce homogenizer equipped with a tightly fitting Teflon pestle. Homogenates were stored at –80 C until further analysis. To obtain a seabream renal microsomal fraction, kidney tissue was homogenized in 4 volumes (wt/vol) buffered solution [100 mmol/liter Tris/HCl (pH 7.4), 250 mmol/liter sucrose, 1 mmol/liter EDTA] and centrifuged at 20,000 x g for 20 min in a Sorvall SM24 rotor. The resulting supernatant was centrifuged at 100,000 x g for 60 min in a Beckman Sw40 rotor. The pellet thus obtained was resuspended in 100 mmol/liter Na-phosphate buffer (pH 7.2) and stored at –80 C until further analysis. Protein was measured with a commercial Coomassie Brilliant Blue reagent kit (Bio-Rad Laboratories, München, Germany) using BSA as a reference.
Iodothyronine 5'-deiodinase assays
5'-Deiodinase activities were assayed in duplicate by incubating 50 μg homogenate or microsomal protein for 15 min at 37 C in 200 μl 100 mmol/liter phosphate buffer (pH 7.2) to which were added: T4 or rT3 and DTT in concentrations as indicated in the legends to the figures, [125I]T4 or [125I]rT3 to a specific activity of 9 x 1012 to 8 x 1014 cpm/mol iodothyronine, and 2 mmol/liter EDTA. Nonenzymatic ORD (5'-deiodination) was determined in the absence of a preparation. Radiotracer was purified on a 10% (wt/vol) Sephadex LH-20 minicolumn shortly before use as described earlier (12). The incubation was quenched by adding 100 μl 5% (wt/vol) ice-cold BSA. Quenched incubates were deproteinized with 500 μl 10% (wt/vol) ice-cold trichloroacetic acid followed by precipitation of denatured proteins at 1400 x g (15 min, 4 C). To 0.5 ml of the supernatant thus obtained, an equal volume of 1.0 mol/liter HCl was added, and liberated iodide was separated from the native iodothyronine with the use of Sephadex LH-20 column chromatography as described earlier (12), collecting 125I– in the first three 1-ml 0.1 mol/liter HCl eluates. HPLC analyses showed that equal amounts of iodide and 3,3'-T2 were produced by renal homogenates and microsomal fractions from rT3 in the absence or presence of 10 mmol/liter DTT (results not shown). Interestingly, when we incubated seabream kidney homogenates and microsomes with [125I]3,3'-T2, an ORD activity became apparent, and this explained why 3,3'-T2 amounts in the incubates, although still detectable, not always exactly equaled the amounts of liberated iodide from rT3 (results not shown). 125I radioactivity was measured in an LKB-1272 Clinigamma -counter (Wallac Oy, Turku, Finland). The specific D1 activity was expressed as femtomoles rT3 deiodinated per minute per milligram protein. Our calculations included a correction factor of 2 to take into account the random labeling of the 3'- and 5'-positions of [125I]rT3 and [125I]T4.
Cloning and sequencing
Total RNA was isolated from seabream kidney using the TRIzol reagent and treated with DNase before reverse transcription. cDNA was obtained using oligo(dT)15 primers and AMV-RTase in the presence of RNasin. Based on homologies of conserved regions in the D1 nucleotide sequences of guppy (Poecilia reticulata), Nile tilapia (O. niloticus), house musk shrew (Suncus murinus), chicken, dog, rat, mouse, and man the following primers were designed: 5'-GGC AAC AGA CCG CTG CTG CTG-3' (forward), and 5'-ATC TTC TCC AAG AAG GAT CGC ACC TC-3' and 5'-ATC TTC TGC AGG ACC GAA CGC ACC TC-3' (reverse), and PCR was performed using Taq DNA polymerase with proofreading activity. PCR products were separated on 2% agarose, subcloned into a TOPO vector, and transferred to One Shot chemically competent Escherichia coli (DH5). Transformed cells were lysed and plasmids isolated; only those plasmids that upon EcoRI restriction yielded cleavage products of the predicted size (3.9 and 403 bp) were subjected to automatic sequencing using the dideoxy method of Sanger et al. (13). A 404-bp cDNA fragment was thus obtained, the sequence of which was used to design the following primers for the rapid amplification of 5'- and 3'- rapid amplification of cDNA ends (RACE): 5'-TGA AGT CCC TGA CGA GTC GCT TGA A-3' and 5'-CGC CAG ACT CGC CTG TGG TCA CCA T-3' for 5'-RACE and 3'-RACE, respectively. Total RNA from Sp. auratus kidney was isolated, dephosphorylated, decapped and ligated, and then reverse transcribed into cDNA. 5'- and 3'-RACE was performed using the primers described above and GeneRacer 5'- and 3'-primers.
Analysis and statistics
ORD kinetic data were analyzed using a weighted nonlinear regression data analysis program (14) in which Marquardt’s algorithm for least squares estimation of parameters (15) is employed. Data points were not transformed before analysis, and the SD was used as an explicit weighting value for each data point. Statistical significance was evaluated with Student’s t test and was accepted at P < 0.05.
Results
We first measured iodothyronine ORD in seabream and tilapia kidney homogenates in the presence of DTT at concentrations that we, based on observations by Mol et al. (10, 11), considered optimal for rT3 and T4 ORD in teleost tissues, i.e. 10 and 20 mmol/liter DTT, respectively. rT3 ORD by seabream and tilapia kidney homogenates, measured at 5 μmol/liter rT3 and 10 mmol/liter DTT, proceeded linearly up to 50 min incubation time (results not shown); a time point of 15 min was chosen to measure initial deiodination rates. Seabream renal ORD activities, measured at initial rate, toward 1.0 μmol/liter rT3 and T4 were 0.9 ± 0.3 and 0.04 ± 0.01 fmol/min per microgram protein (n = 5, mean ± SD), respectively. The apparent 20-fold higher ORD substrate preference for rT3 over T4 is consistent with that reported for mammalian and Nile tilapia (O. niloticus) iodothyronine D1 (16, 17) and could well be the expression of a specific seabream D1 activity.
Figure 1 shows that the initial rate of rT3 ORD activity in seabream kidney homogenates, measured in the presence of 10 mmol/liter DTT, did not saturate at high substrate concentrations. Contrastingly, renal homogenates from tilapia (O. mossambicus), assayed in parallel with the seabream preparations, displayed the rectangular hyperbola characteristic for single-site Michaelis-Menten kinetics. The calculated Michaelis constant (Km) value for renal ORD in tilapia was 0.8 μmol/liter rT3, which is in the same range as that reported for other teleost species (11). Tilapia data points, and the values for Km and limiting rate (Vmax) calculated from these, converged on a linear Eadie-Hofstee transformation of the Michaelis-Menten equation, indicative for a single catalytically active component in renal rT3 ORD. The nonsaturating behavior of rT3 ORD in seabream was not tissue-specific because it was also observed in liver homogenates (Fig. 2), and it thus appears to be a property of a seabream 5'-deiodinase proper.
Proceeding from these observations, we chose to investigate biochemical and pharmacological properties of seabream renal rT3 ORD. Figure 3 shows that the effects of DTT on rT3 ORD in seabream renal homogenates were inhibitory, not stimulatory. An IC50 value of 0.4 mmol/liter DTT was calculated, which is lower than the lowest DTT concentration tested in this series of experiments. Moreover, an ORD component insensitive to DTT, comprising 32% of the uninhibited rT3 ORD activity, could be resolved in renal homogenates.
When DTT was omitted from the incubation medium, rT3 ORD activity obeyed simple Michaelis-Menten kinetics (Fig. 4). The value for Km was calculated to be 5 μmol/liter rT3, which is in the same order of magnitude as that reported for tilapia (this study) and other teleost species (11). In the absence of DTT, data points and calculated values for Km and Vmax converged on a linear Eadie-Hofstee transformation, indicative for a single catalytically active component in rT3 ORD in seabream renal homogenates. The progress curve of rT3 ORD by seabream renal homogenates, measured in the absence of DTT, is described by a first-order exponential equation (Fig. 5), indicative for a single enzymatic component and in line with the observed single-site Michaelis-Menten kinetics (Fig. 4). The maximum rate, calculated from the slope of the tangent to the progress curve at time point 0, is 53 fmol/μg·min. The calculated first-order rate constant equals 0.006 min–1, and the rT3 ORD reaction rate falls by 0.6%/min to reach 30% of the maximum rate at the 200-min time point in our assay.
To exclude the possible effect of endogenous cytosolic compounds, the effect of DTT on rT3 ORD was assessed in seabream renal microsomal fractions. As in whole kidney homogenates, rT3 ORD was inhibited by DTT, but here inhibition was virtually complete at 1 mmol/liter DTT (Fig. 6). The progress curve of rT3 ORD by seabream renal microsomal fraction, measured in the absence of DTT, is also described by a first-order exponential equation (Fig. 7), again indicative for a single enzymatic component. The maximum rate is calculated to be 192 fmol/μg·min, and this value is 3.6 times higher than that for renal homogenates, indicating an enrichment of the microsomal fraction with respect to rT3 ORD and putative D1 activity, compared with the homogenate. The calculated first-order rate constant for the microsomal fraction equals 0.009 min–1 and is similar to that of homogenates. At 200 min, the ORD reaction rate has decreased to 17% of the maximum rate.
In the presence of 10 mmol/liter DTT, rT3 ORD activity in seabream kidney homogenates was insensitive to inhibition by PTU (Fig. 8A). However, when DTT was omitted from the incubation medium, rT3 ORD activity was inhibited by approximately 70% in the presence of 0.1 mmol/liter PTU (Fig. 8B). Interestingly, a rat liver homogenate preparation, which was included in our assay as a positive control and was also incubated in the absence of DTT, contained an rT3 ORD activity that was inhibited by 90% in the presence of 0.1 mmol/liter PTU (result not shown).
The results obtained so far indicate that seabream possesses an iodothyronine 5'-deiodinase, most likely a D1, with a peculiar behavior toward the reducing cosubstrate that is considered to be essential to the catalytic action of the enzyme. We therefore decided to characterize a D1 cDNA derived from seabream kidney. The full-length nucleotide and deduced amino acid sequence of putative seabream D1 (saD1) cDNA with a size of 1545 bp are shown in Fig. 9. A large 3'-untranslated region (UTR) contains sequences identical with mammalian selenocysteine insertion sequence (SECIS) consensus motifs (18, 19). A SECIS secondary mRNA structure could be resolved (Fig. 10) using a computer algorithm for the analysis of structural and thermodynamic features of SECIS elements (20), and it can therefore be assumed that UGA at codon position 126 is translated as selenocysteine. The non-Watson-Crick base pair quartet UGAC_AGAU in the saD1 SECIS core was preceded 5' by a guanosine, not adenosine (position 1073). This less common variant (19) is present in SECIS elements of Nile tilapia D1 and D3 (16, 21) and can be found in killifish (Fundulus heteroclitus) D1 3'UTR as well. The substitution of G for A 5' to the UGAN quartet appears to be a typical feature of teleost SECIS elements.
The open reading frame (nt 52–798) putatively encodes a 248-amino acid saD1 protein with an estimated molecular weight of 27.7 x 103. Figures 11 and 12 show alignments of the deduced amino acid sequence of saD1 with that of nonteleost vertebrates and other teleost species, respectively. Sequence identity with man, dog, rat, mouse, house musk shrew (Su. murinus), and chicken (Gallus gallus) ranges from 41 to 47%, whereas that with the teleost species Nile tilapia, killifish, and zebrafish (Danio rerio) is 71, 69, and 56%, respectively. A hydropathy analysis based on the augmented Wimley-White whole-residue hydrophobicity scale (22) (Fig. 13) indicates a hydrophobic domain formed by amino acids at positions 1–33, of which positions 15–33 are predicted to form a transmembrane segment. The amino acids at positions 115–133, which include the consensus sequence for the catalytic center (positions 115–129) in the saD1 protein, are also predicted to form a hydrophobic domain.
The deduced amino acid sequence of the catalytic center of the saD1 protein is identical with that of Nile tilapia, killifish, zebrafish, and the consensus sequence for D1 and has a high homology with the consensus sequences for D2 and D3 as reported by Bianco et al. (3). Callebaut et al. (23) list 13 amino acid positions in the D1 active center, nine of which are shared by saD1. Conserved cysteine residues (24) are at positions 124 and 194 in saD1, whereas Cys108 replaces a tryptophane residue that appears to be conserved in mammals. Other conserved amino acids that, in mammals, are critical for correct enzyme function are Phe65 (25, 26, 27) and Gly45 and Glu46 (27). Recently a conserved D1 dimerization domain was described (28), which is also present in the putative saD1 protein (consensus residues in bold, one-letter code): D 148FLVVYIAEAHSTDGW163, located 22 amino acid positions 3' to Sec.
Differences between the D1 protein of seabream and other vertebrates are concentrated in the amino-terminal hydrophobic domain. Most notably, three of six cysteines that are present in saD1 occur here (i.e. at positions 10, 17, and 33). Positively charged amino acid residues in the hydrophobic region that are conserved in mammals (29) are uncharged polar or nonpolar residues in seabream (i.e. Ser11, Leu12, Gln27, and Leu36).
Discussion
We show here that DTT inhibits rT3 ORD in Sp. auratus kidney homogenates and microsomal fractions. This is contrary to all reported mammalian ORD activities, which are stimulated by DTT. Moreover, the reactivity of rT3 ORD in Sparus toward the thyrostatic PTU is affected profoundly by the presence of DTT in the incubation medium.
Our observations on the nonclassical action of DTT on rT3 ORD in seabream kidney are not unique. ORD activities in turbot (Psetta maxima) ovaries were reduced by approximately 25–30% in the presence of 10 and 100 mmol/liter DTT, respectively (Mol, K. A., unpublished results)1, a result that corroborates our data. Moreover, 65–80% of the maximal rT3 ORD activities in kidney homogenates from rainbow trout (Oncorhynchus mykiss), Nile tilapia, and turbot were measured in the absence of DTT from the incubation medium (ibid.), which implies that DTT has only a relatively modest activating effect on rT3 ORD in these species. Reverse T3 ORD in kidney homogenates from blue tilapia (O. aureus) is insensitive to DTT, i.e. is neither activated nor inhibited by DTT in concentrations up to 50 mmol/liter (10). We recently measured a potent dose-dependent inhibition by DTT of rT3 ORD in renal homogenates from Senegalese sole (Solea senegalensis), in which 1 mmol/liter DTT inhibited ORD by 78% (Klaren, P. H. M., and F. Arjona Madueo, 2005, unpublished results). All in all, teleost fish appear to represent a group of vertebrates in which ORD of iodothyronines is less critically dependent on the presence of DTT than in mammals. Teleosts could provide interesting natural models to study the interactions between deiodinases and their thiol cosubstrates.
Our results prompted us to critically consider the use of DTT in in vitro assays. DTT is an exogenous substrate and is applied in vitro because the natural, endogenous thiol cosubstrate has not been unequivocally recognized. Still, ORD activities of tissue preparations and expressed D1 transcripts have been shown to be stimulated by the endogenous intracellular peptidyl thiols glutathione (30, 31, 32, 33, 34, 35) and thioredoxin (35, 36, 37). Goswami and Rosenberg (34) demonstrated that the activation of ORD in a rat kidney microsomal preparation by glutathione, but not that by DTT, was lost upon preincubation by DTT. This indicates that these thiols have different sites of action on the deiodinase protein to activate ORD. Most of the ORD activities stimulated by glutathione or thioredoxin were measured at low, i.e. nanomolar substrate (rT3, T4), concentrations, and the stimulatory effects of the endogenous thiols were not detectable at micromolar substrate concentrations. However, considering the plasma total T3 and T4 concentrations in vertebrates that are in the nanomolar range, it is likely that cytosolic thyroid hormone concentrations are in the (sub)nanomolar rather than micromolar range. The reported stimulatory actions in vitro of glutathione and thioredoxin, albeit less pronounced than those of DTT, could therefore very well reflect an in vivo physiologically relevant ORD activation in mammals. It remains to be established whether the inhibition of rT3 ORD by DTT reflects inhibitory actions of endogenous thiols in Sp. auratus kidney in vivo. Indeed, the inhibition of rT3 ORD by DTT can be considered an artifact of the substitution for the unknown endogenous reducing cosubstrate. The argument could be equally valid for the activation of ORD by DTT in mammalian preparations.
Dihydrolipoamide is the only endogenous thiol compound tested that has been shown to be at least as potent as DTT in the activation of rT3 ORD (31, 38, 39, 40). Both DTT and dihydrolipoamide are potent nonpeptidyl dithiol reductants of which the redox couples have low redox potentials and that form a cyclic disulfide in the fully oxidized form in which properties are considered to favor a high reducing potential (4, 41). Although DTT and dihydrolipoamide have equivalent potencies in the activation of ORD, these compounds differ markedly in their reducing potential of disulfides, which has been estimated to be approximately 20-fold less for dihydrolipoamide, compared with DTT (41). The reactivities of thiols toward disulfides apparently do not predict the potency to reduce selenenyl-iodide enzyme intermediates. Interestingly, the reduction of a synthetic selenenyl iodide compound was found to be catalyzed by mammalian and bacterial thioredoxin reductases (of which only the mammalian enzyme contains a selenocysteine), whose reactions were only slightly enhanced by the addition of thioredoxin (42). There appear to be several thiol and nonthiol candidates that can fulfill the role of reducing cosubstrate in the catalytic cycle of iodothyronine ORD. We are currently evaluating the effects of these on rT3 ORD in seabream.
The reaction rates of rT3 ORD by seabream renal homogenates and microsomes, measured in the absence of DTT, fall exponentially by 0.6 and 0.9%/min, respectively, and this could be due to substrate depletion, product inhibition, or endogenous cosubstrate depletion. The latter, however, is less likely in view of the rT3 ORD activities that we observed in purified microsomes and that were enhanced, compared with the homogenates from which the microsomal fractions were derived. We are currently investigating the catalytic mechanism of rT3 ORD by seabream renal microsomes.
The omission of DTT from the incubation medium revealed a hidden sensitivity to inhibition by PTU of rT3 ORD in Sp. auratus kidney homogenates, albeit that the inhibition by the thyrostatic, measured at a concentration of 0.1 mmol/liter PTU, was less pronounced than in mammalian preparations. The inhibition of mammalian D1 by PTU is well established, and the insensitivity of teleost D1 activity for inhibition by PTU defines a marked difference between these vertebrate orthologs (16, 43, 44, 45). PTU is suggested to compete with thiol cosubstrates for binding to the selenenyl iodide enzyme intermediate in which binding of PTU results in a dead-end complex (46, 47). However, to date it is still unknown what structural feature confers the insensitivity for inhibition by PTU to the teleost D1 protein. Mammalian D1 proteins, i.e. human, rat, mouse, and dog, which are PTU sensitive, contain a serine two positions 3' from selenocysteine (25, 48, 49, 50). The first PTU-insensitive D1 cloned, that of Nile tilapia, was found to contain a proline at this position (16), as does Sp. auratus putative D1 (this study), here Pro128. This suggests that position 128 plays a pivotal role in the interactions of the selenenyl iodide intermediate with PTU. However, rT3 ORD by a Pro128Ser mutant of Nile tilapia D1 was inhibited by only about 15% in the presence of 1 mmol/liter PTU, and it was concluded that the PTU insensitivity of Nile tilapia D1 was not due to the presence of Pro for Ser at position 128 (16). Remarkably, the reverse substitution, i.e. Ser128Pro in human D1, resulted in a mutant that was resistant to PTU (23), the result of which leaves the role of position 128 in the interactions between selenoenzyme and PTU equivocal. The amino acid homology of the catalytic centers of tilapia D1 (16) and Sp. auratus putative D1 with those of mammalian D1s is very high, which suggests that the reactivity of the selenenyl iodide intermediate with PTU is determined by amino acid residues outside the catalytic center. ORD activities in a number of teleost preparations are well measurable in the absence of DTT, and it would be interesting to measure the effects of PTU under these in vitro conditions.
It is tempting to correlate the actions of the classical sulfhydryl reagent DTT with the presence of cysteine residues unique to the deduced putative saD1 protein, i.e. Cys108 near the catalytic center, and the three cysteine residues at positions 10, 17, and 33 in the hydrophobic amino terminal region. Cys108 is predicted to reside in the cytosol, and its thiol group can therefore presumed to be in the reduced state. In many mammals a conserved Cys105 is present in D1. Cys108 and Cys105 could be analogous, but it is not known whether these residues can interact with the active site of the enzyme. It has been shown that overexpressed mammalian D1 forms homodimers in vivo (51), possibly through specific dimerization sites (28) but possibly also through disulfide bridges. It can be hypothesized that the cysteine residues in the amino terminal region are involved in the formation of disulfide bridges in saD1 dimers, which are reduced by DTT, yielding catalytically inactive monomers. We are currently using a site-directed mutagenesis strategy to investigate the biochemical properties of wild-type saD1 and saD1 mutants in which selected cysteines are replaced.
The inhibition of rT3 ORD by DTT was complete in a 100,000 x g renal microsomal fraction, but in whole kidney homogenates, a DTT-insensitive component comprising 30% of the uninhibited activity was resolved. Our kinetic analyses clearly indicated the presence of only one catalytically active ORD component in whole kidney homogenates. Both the DTT-sensitive and DTT-insensitive components in seabream rT3 ORD are therefore most probably expressions of the same deiodinase protein. It could be that some cytosolic factor, present in whole organ homogenates but absent in isolated microsomes, is responsible for the observed behavior of rT3 ORD in Sp. auratus.
In conclusion, our data show that the substrate concentration dependence as well as the PTU-sensitivity of rT3 ORD in Sp. auratus kidney is profoundly affected by DTT in vitro. The presence of a relatively large number of cysteine residues in the deduced putative saD1 offers possible targets for interaction with the sulfhydryl reagent.
Acknowledgments
The authors are grateful to Professor Theo Visser (Erasmus Medical Center, Rotterdam, The Netherlands) for the generous donation of 3'-T1; Fransisco Arjona Madueo and Dr. Juan-Miguel Mancera (University of Cádiz, Cádiz, Spain) for making Solea senegalensis kidney available to us; and Tom Spanings for excellent fish husbandry.
Footnotes
This work was supported by the European Socrates program (grant to R.H.). S.V.d.G. was supported by the Fund for Scientific Research–Flanders.
First Published Online September 15, 2005
Abbreviations: AMV-RTase, Avian myeloblastosis virus-reverse transcriptase; D1, type 1 iodothyronine deiodinase; DTT, dithiothreitol; Km, Michaelis constant; ORD, outer ring deiodination; PTU, 6-n-propyl-2-thiouracil; RACE, rapid amplification of cDNA ends; rT3, reverse T3; saD1, Sparus auratus D1; SECIS, selenocysteine insertion sequence; UTR, untranslated region; Vmax, limiting rate.
1 Mol KA 1996 A study on peripheral deiodination of thyroid hormones in fish. Ph.D. Dissertation, K.U. Leuven, Belgium.
Accepted for publication September 7, 2005.
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