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Endocrinology Vol. 147, No. 7 3519-3529
Copyright © 2006 by The Endocrine Society

Characterization of Recombinant Xenopus laevis Type I Iodothyronine Deiodinase: Substitution of a Proline Residue in the Catalytic Center by Serine (Pro132Ser) Restores Sensitivity to 6-Propyl-2-Thiouracil

George G. J. M. Kuiper, Willem Klootwijk, Ghislaine Morvan Dubois, Olivier Destree, Veerle M. Darras, Serge Van der Geyten, Barbara Demeneix and Theo J. Visser

Department of Internal Medicine (G.G.J.M.K., W.K., T.J.V.), Erasmus Medical Center, 3015 GE Rotterdam, The Netherlands; Laboratory of Comparative Endocrinology (S.V.d.G., V.M.D.), Catholic University Leuven, B3000 Leuven, Belgium; Hubrecht Laboratory (O.D.), Netherlands Institute for Developmental Biology, 3584 CT Utrecht, The Netherlands; and Department Regulations, Development, and Molecular Diversity (G.M.D., B.D.), Museum National d’Histoire Naturelle, Cedex 05, F75231 Paris, France

Address all correspondence and requests for reprints to: Theo J. Visser, Ph.D., Department of Internal Medicine, Room Ee 502, Erasmus Medical Center, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: t.j.visser{at}erasmusmc.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In frogs such as Rana and Xenopus, metamorphosis does not occur in the absence of a functional thyroid gland. Previous studies indicated that coordinated development in frogs requires tissue and stage-dependent type II and type III iodothyronine deiodinase expression patterns to obtain requisite levels of intracellular T3 in tissues at the appropriate stages of metamorphosis. No type I iodothyronine deiodinase (D1), defined as T4 or reverse T3 (rT3) outer-ring deiodinase (ORD) activity with Michaelis constant (Km) values in the micromolar range and sensitivity to 6-propyl-2-thiouracil (6-PTU), could be detected in tadpoles so far. We obtained a X. laevis D1 cDNA clone from brain tissue. The complete sequence of this clone (1.1 kb, including poly A tail) encodes an ORF of 252 amino acid residues with high homology to other vertebrate D1 enzymes. The core catalytic center includes a UGA-encoded selenocysteine residue, and the 3' untranslated region (about 300 nt) contains a selenocysteine insertion sequence element. Transfection of cells with an expression vector containing the full-length cDNA resulted in generation of significant deiodinase activity in the homogenates. The enzyme displayed ORD activity with T4 (Km 0.5 µM) and rT3 (Km 0.5 µM) and inner-ring deiodinase activity with T4 (Km 0.4 µM). Recombinant Xenopus D1 was essentially insensitive to inhibition by 6-PTU (IC50 > 1 mM) but was sensitive to gold thioglucose (IC50 0.1 µM) and iodoacetate (IC50 10 µM). Because the residue 2 positions downstream from the selenocysteine is Pro in Xenopus D1 but Ser in all cloned PTU-sensitive D1 enzymes, we prepared the Pro132Ser mutant of Xenopus D1. The mutant enzyme showed strongly increased ORD activity with T4 and rT3 (Km about 4 µM) and was highly sensitive to 6-PTU (IC50 2 µM). Little native D1 activity could be detected in Xenopus liver, kidney, brain, and gut, but significant D1 mRNA expression was observed in juvenile brain and adult liver and kidney. These results indicate the existence of a 6-PTU-insensitive D1 enzyme in X. laevis tissues, but its role during tadpole metamorphosis remains to be defined.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE IS produced by the thyroid predominantly in the form of the biologically inactive precursor T4. The principal bioactive form of the hormone is T3, and most T3 is derived from outer-ring deiodination (ORD) of T4 in peripheral tissues. Both T4 and T3 undergo inner-ring deiodination (IRD) to metabolites, which do not interact with T3 nuclear receptors, reverse T3 (rT3) and 3,3'-diiodothyronine, respectively. Thus, ORD is regarded as an activating pathway and IRD as an inactivating pathway. Three iodothyronine deiodinases are involved in the deiodination of iodothyronines, i.e. D1, D2, and D3 (1, 2, 3, 4, 5).

In mammals D1 is located primarily in liver, kidney, and thyroid. It has both ORD and IRD activities and appears to be particularly important for the generation of plasma T3 and clearance of plasma rT3. Michaelis constant (Km) values for substrates of D1 are in the micromolar range, and D1 is potently inhibited by the thyrostatic drug 6-propyl-2-thiouracil (6-PTU). D2 activity is found in brain, pituitary, placenta, thyroid, and skeletal muscle; it has only ORD activity, preferring T4 over rT3 with Km values in the nanomolar range, and it is insensitive to PTU. D3 is located in brain, placenta, uterus, and fetal tissues; it has only IRD activity and is thus important for the inactivation of T4 and T3.

Local metabolism of thyroid hormone is a major factor in controlling the timing of tissue responsiveness to thyroid hormone during development, i.e. in the human fetal brain (6). Another well-known example of thyroid hormone action on development is frog metamorphosis (7, 8, 9). Different tissues undergo thyroid hormone-dependent metamorphic changes at different times and rates in the presence of highly elevated circulating levels of thyroid hormone. As in the human fetal brain, local metabolism of thyroid hormone is a major factor in controlling the timing of tissue responsiveness (9, 10, 11, 12, 13). Various studies have indicated that coordinated development in amphibia as Rana catesbeiana and Xenopus laevis requires tissue and stage-dependent D2 and D3 expression patterns to obtain requisite levels of intracellular T3 in tissues at the appropriate stages of metamorphosis (12, 13). During metamorphic climax T4 ORD activity is present in skin, gut, limbs, and tail. This T4 ORD activity has the typical characteristics of a mammalian D2 enzyme; Km values for rT3 and T4 in the nanomolar range and insensitive to inhibition by 6-PTU (10, 11). A frog homolog of the mammalian D1 enzyme, defined as T4 and rT3 ORD activity with Km values in the micromolar range and sensitive to 6-PTU, does not seem to play a role during the onset and climax stages of metamorphosis (10, 11, 13). Only in the gut of prometamorphic tadpoles, rT3 ORD activity with Km values in the micromolar range (0.2–0.3 µM) and insensitive to 6-PTU (up to 1 mM tested) was described (11). This ORD activity could have represented the frog homolog of mammalian D1 despite the fact that it was insensitive to 6-PTU. We know now that 6-PTU sensitivity is not a reliable criterion to differentiate D1 from D2 ORD activity. Whereas mammalian D1 enzymes are highly sensitive to 6-PTU (IC50 1–10 µM), in contrast to D2 enzymes (5, 14, 15, 16, 17, 18), the D1 enzymes from various teleost fish species (19, 20, 21, 22, 23) are relatively insensitive to 6-PTU (IC50 > 1000 µM). Only on the basis of additional criteria such as substrate preference (including sulfated iodothyronines), substrate Km values and ultimately amino acid sequence homology, it is possible to differentiate between D1 and D2 ORD activity.

We obtained a X. laevis cDNA clone that showed significant homology with the mammalian D1 sequences, including the highly conserved core catalytic center with the selenocysteine (SeC) residue. In this paper we examine the characteristics of this X. laevis deiodinase homolog with emphasis on 6-PTU sensitivity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Nonradioactive iodothyronines were obtained from Henning (Berlin, Germany) or Calbiochem (San Diego, CA). [3'-125I]T3, [3',5'-125I]T4, and [3',5'-125I]rT3 (1500–2000 mCi/µmol) were either obtained from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK) or in the case of radioactive rT3 prepared by radioiodination of 3,3'-diiodothyronine by the chloramine-T method (24). Gold-thioglucose (GTG), iodoacetate (IAc), 6-PTU, 5-propyl-2-thiouracil (5-PTU), 6-methyl-2-thiouracil (MTU), 2-thiouracil (TU), and 5-iodouracil (IU) were obtained from Sigma or Aldrich (St. Louis, MO). 5-iodo-2-thiouracil (ITU), also known as Itrumil, was obtained from Ciba Geigy. DTT (dithiothreitol) was obtained from ICN Biochemicals (Costa Mesa, CA). Pyrococcus furiosus thermostable DNA polymerase and DpnI restriction endonuclease were obtained from Promega Corp. (Madison, WI). XL-10 ultracompetent Escherichia coli cells were obtained from Stratagene (La Jolla, CA). Synthetic oligonucleotides were ordered from Invitrogen (Paisley, UK). FuGENE 6 transfection reagent was obtained from Roche (Almelo, The Netherlands).

X. laevis deiodinase (xlD1) cDNA
One expressed sequence tag clone (EST, no. IMAGE 4740778, GenBank accession no. BX844453) from the Washington University Xenopus EST project (brain library) was obtained from the RZPD (Deutsches Ressourcenzentrum fur Genomforschung, RZPD clone ID IMAGp998B1110584Q2). The EST clone was obtained in the pCMV-SPORT6 vector and was sequenced. The insert (1050 nt including poly A tail) contained an open reading frame of 252 amino acid residues (Fig. 1AGo) and a 3'-untranslated region (UTR) of 300 nt.


Figure 1
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  		FIG. 1. SECIS element and deduced amino acid sequence of xlD1 protein. A, Comparison of the deduced amino acid sequence of xlD1 with the sequences of human (hs) D1–3, chicken (gg) D1–3, xlD2–3, X. tropicalis (xt) D1, and zebrafish (dr) D1–3. The deduced amino acid sequence of the putative xtD1 enzyme is based on data from the Xenopus genome project (42 ). B, SECIS element structure present in the xlD1 cDNA 3'UTR between nucleotides 897 and 974, as predicted by SECISearch analysis (25 ).

 
Construction of xlD1 expression vector
Using the SECISearch 2.0 program (25), we identified a putative SeC insertion sequence (SECIS) element in the 3'UTR (nt 897–974) of xlD1 cDNA (Fig. 1BGo). The xlD1 cDNA was PCR amplified with primers 5'-CGCCACCATGGAAAGCTTGCTCCAAACG (Kozak start consensus underlined) and 5'-GGGCTTAAAAATGCTTTTATT (nt 984-1004) and cloned in pTargeT expression vector (Promega). The integrity and orientation of the insert was confirmed by plasmid DNA sequencing.

Site-directed mutagenesis of catalytic center
The expression vector containing the xlD1 cDNA was used as a template for site-directed mutagenesis via the circular mutagenesis procedure, followed by selection for mutants by DpnI digestion (26). The xlD1 wild-type sequence was mutated using overlapping sense and antisense primers containing the nucleotide changes needed to produce the xlD1 Pro132Ser and xlD1 Ser124Asn mutants and the xlD1 Ser124Asn/Pro132Ser double mutant: 5'-CCTTTGGTATTGAATTTTGGGAGTTGTACATGACCCT-CATTCCTCTTCAGAC-3' (52 nucleotides with mutations underlined). Mutagenesis reactions were performed as previously described (27). Plasmid DNA isolated from 10 clones was sequenced to verify that both single mutants and the double-mutant xlD1 clones were obtained. Plasmids were maintained in E. coli DH5{alpha} cells and purified for transfection with QIAfilter cartridges (QIAGEN, Hilden, Germany).

Expression of xlD1 protein
The wild-type and mutant xlD1 enzymes were expressed in COS-1 or HEK-293 cells after FuGENE-mediated plasmid DNA transfection (28). COS-1 or HEK-293 cells (65 cm2 dishes) were grown in DMEM-Ham’s F-12 medium containing 9% fetal calf serum and 40 nM sodium selenite. FuGENE transfection reagent (6 µl) and plasmid DNA (2 µg) were incubated for 20 min at room temperature in serum-free medium (total volume, 200 µl). The mixture was then added to cell cultures in medium with 9% fetal calf serum. After 24–36 h incubation (37 C/5% CO2 incubator), the cells were rinsed with PBS and collected in 0.5 ml 0.1 M phosphate buffer (pH 7.2) and 2 mM EDTA (P100E2), to which was added either no DTT or 1 mM DTT. Harvested cells were sonicated, aliquoted, and stored at –80 C. Protein concentrations were determined with the Bradford method using the Bio-Rad (Munchen, Germany) protein assay reagent and BSA as a standard.

Assay of ORD activity in cell homogenates by LH-20 chromatography
Deiodinase activities were analyzed by quantitation of radioiodide released by ORD of [3',5'-125I]rT3 or [3',5'-125I]T4. Incubations contained about 100,000 cpm-labeled rT3 or T4 with varying amounts of unlabeled substrate and cell homogenate in a final volume of 0.1 ml P100E2 buffer (pH 7.2) and varying amounts of DTT (0.1–10 mM). Mixtures were incubated in duplicate for 60 min at 37 C. In some experiments a temperature profile was obtained by simultaneous incubations at 10, 20, 30, or 37 C. Protein was adjusted to consume less than 30% of substrate, and in control experiments it was determined that the deiodination rate was linear up to 60 min of incubation. Blank incubations were carried out with homogenates of nontransfected cells (protein blank). PTU and other thiouracil derivatives were freshly dissolved in dimethylsulfoxide (100 mM stock solution), and the final dimethylsulfoxide concentration was always 1%. Reactions were stopped by addition of 0.1 ml 5% (wt/vol) BSA in water followed by addition of 0.5 ml 10% (wt/vol) trichloroacetic acid in water. After pelleting of the precipitated [125I]iodothyronines, [125I]iodide was further isolated from the supernatant by chromatography on LH-20 minicolumns (29). Deiodinase activities were corrected for nonenzymatic deiodination observed in the protein blanks.

Assay of ORD and IRD activity in cell homogenates by HPLC analysis
This assay is based on the determination of product formation (125I-rT3, 125I-T3, and/or 125I-T2) by reverse-phase HPLC analysis of reaction mixtures containing outer ring-labeled T4 ([3',5'-125I]T4) or rT3 ([3',5'-125I]rT3). Incubations contained about 200,000 cpm-labeled T4 or rT3 with varying amounts of unlabeled substrate and cell homogenate in a final volume of 0.1 ml P100E2 buffer (pH 7.2) with 1 mM DTT (P100E2D1). Mixtures were incubated for 60 min at 37 C, whereafter the reaction was stopped by addition of 0.1 ml ice-cold methanol. After centrifugation, 0.1 ml (equivalent to 25 µl reaction volume) of the mixture was applied to a 250 x 4.6 mm Symmetry C18 column connected to an Alliance HPLC system (Waters Chromatography Division, Millipore Corp., Milford, MA) and eluted with a 15-min linear gradient of acetonitrile (25–42%) in 0.02 M ammonium acetate (pH 4) at a flow rate of 1.2 ml/min. Radioactivity in the eluate was monitored online using a Radiomatic A-500 flow scintillation detector (Packard, Meriden, CT).

Deiodinase activity in adult Xenopus organ homogenates
Adult (X. laevis and X. tropicalis) frogs were used. Experiments were performed in accordance with the animal care regulations of Utrecht University and with prior approval of the Utrecht University Animal Care Committee. Frogs were killed by pithing and organs including liver, kidney, gut, and brain were rapidly dissected, put in capsules, frozen in liquid nitrogen and stored at –80 C. Organs were homogenized in ice-cold P100E2D1 buffer using several rounds of homogenization with a ultraturrax and a glass-Teflon Potter-Elvehjem homogenizer. Homogenates were centrifuged (15 min, 2500 x g) and the supernatants were stored at –80 C. ORD activity was measured with 2 or 100 nM T4 or rT3 (100,000 cpm-labeled substrate) in P100E2 buffer (pH 7.2) with 20 mM DTT. The homogenates were diluted 10-fold in the assay (final protein concentration 0.3–1 mg/ml) and were incubated for 60 min at 37 C. In parallel experiments 100 µM PTU were included. The amount of 125I– released was determined by LH-20 chromatography as described. In some experiments reaction mixtures were analyzed by HPLC as described.

In additional experiments, liver and kidney tissue was homogenized, and microsomal fractions were prepared by differential centrifugation as described (30). Microsomal fractions were incubated with T4 (1, 100, or 1,000 nM; with 200,000 cpm-labeled substrate) for 60 min at 37 C, and the reaction mixture was analyzed by HPLC as described.

Expression of xlD1 mRNA in X. laevis tissues
Animals.
Adult (8 months), juvenile (4 months), and stage NF 63 X. laevis animals were raised in the Paris laboratory. Frogs and tadpoles were killed by pithing and decapitation. The laboratory has the official approval of the Institutional Animal Care and Use Committee of the Animal Protection and Health, Veterinary Services Direction, Paris (France), for breeding and raising Xenopus. All aspects of animal care and experimentation performed in this study were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Real-time PCR quantification.
Organs including liver, kidney, gut, and brain were rapidly dissected and frozen in liquid nitrogen and stored at –80 C. RNA extraction was performed using RNAble reagent (Eurobio, les Ulis, France) according to the manufacturer’s guidelines. RNA was treated with 10 U/ml DNase (Promega) and then precipitated in ethanol. The amount and purity of the total RNA was determined by spectrophotometer. Five micrograms total RNA were used in each 50-µl reverse transcription reaction, which was carried out at 42 C with 100 U Superscript II (Invitrogen) per microgram RNA and 250 ng oligo-dT primers. Real-time PCR was performed on a Stratagene MX3000, with Brilliant SYBR Green reagent (Stratagene) using forward primer 5'-CTTGCTCCAAA-CGATTAAGCTGA-3' and reverse primer 5'-GTATGTAAAGAAAGTAGGACCCCAA-3' (6 ng each). Using MX3000P software (Stratagene), the amount of xlD1 mRNA in each tissue sample was normalized against the amount of RPL8 mRNA (accession code BC043823; forward primer 5'-TGGCAATGTACTCCCTGTTG-3'and reverse primer 5'-TTGCTGGAGGTGGTCGTATT-3'; 6 ng each).

In situ hybridization.
D1 sense and antisense probes were prepared by performing RT-PCR on a pool of RNA extracted from stage NF 20 embryos to stage NF 58 tadpoles using forward primer 5'-TTGCTCCAAACGATTAAGCTG-3' and reverse primer 5'-AGCCGATCCTGGAGACTTC-3'. The PCR product was cloned using the dual-promoter TOPO TA cloning kit (Invitrogen) and sequenced to check the orientation. Probes were prepared using the digoxigenin (DIG) labeling kit (Roche). Brains were rapidly dissected and fixed overnight in 4% paraformaldehyde in PBST (PBS, 0.1% Tween 20) at 4 C. They were rinsed in PBST and progressively dehydrated through methanol series. They were stored for at least 2 h in 100% methanol at –20 C. After rehydration in PBST, in situ hybridization was performed on whole brains. They were then treated for 20 (stage NF 63 tadpoles) or 30 (juveniles) min at room temperature with 15 mg/ml proteinase K (Fluka, Buchs, Switzerland), washed in PBST, and postfixed for 15 min in 4% PFA in PSBT. Brains were then prehybridized for 2 h at 65 C in 50% formamide, 5x saline sodium citrate, 1% sodium dodecyl sulfate, 50 µg/ml heparin, and 50 µg/ml yeast RNA. DIG-labeled RNA probe was added (1.5 µg/ml) to the prehybridization buffer, and hybridization was carried out overnight at 65 C. Brains were washed twice for 30 min at 65 C in 50% formamide/2x SSCT [150 mM NaCl, 15 mM sodium citrate (pH 7.0), 0.05% Tween], for 20 min in 2x SSCT, and twice for 30 min in 0.2x SSCT. Brains were preblocked for 60–120 min with 10% decomplemented goat serum (Sigma) and incubated overnight at 4 C with alkaline phosphatase-conjugated anti-DIG antibody diluted 1:5000 (Roche). The enzyme activity was revealed by addition of 5-bromo-4-chloro-3-indoyl-phosphate/nitro blue tetrazolium chloride (Roche). After hybridization, brains were postfixed and included for cryostat sectioning. Twenty-micrometer slices were mounted in glycerol and photographed with an AX70 microscope (Olympus, Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The X. laevis D1 (xlD1) homolog
The deduced amino acid sequence of the xlD1 clone is presented in Fig. 1AGo, aligned with the sequences of X. tropicalis (xt) D1, xlD2 and xlD3, human (Homo sapiens, hs) D1–3, chicken (Gallus gallus, gg) D1–3, and zebrafish (Danio rerio, dr) D1–3. The amino acid sequence of xlD1 shows 51% identity with human D1, 56% with ggD1, 92% with xtD1, and 47% with drD1; 35% with hsD2, 34% with ggD2, 34% with xlD2, and 32% with drD2; and 34% with hsD3, 38% with ggD3, 38% with xlD3, and 34% with drD3. These findings establish that xlD1 represents the X. laevis homolog of the D1 enzymes in other species. The core catalytic center around the essential SeC residue is highly conserved (Arg-Pro-Leu-Val-Leu-Ser124-Phe-Gly-Ser-Cys-Thr-SeC130-Pro-Pro132-Phe in xlD1), except Ser124 and Pro132. According to the HMMTOP transmembrane topology prediction server (31), the xlD1 protein contains a single transmembrane domain between amino acid residues 19 and 38.

SeC is cotranslationally incorporated into nascent polypeptides in response to UGA codons when a specific stem-loop structure, designated the SECIS element, is present in the 3'UTR (25, 32, 33). The SECISearch program (25) identified a SECIS element (nt 897–974) in the 3'UTR of the xlD1 clone (Fig. 1BGo). Almost all eukaryotic SECIS elements contain an adenosine preceding the quartet of non-Watson-Crick base pairs, a UGA_GA motif in the quartet, and two adenosines in the loop (the AUGA_AA_GA motif) (25, 33). The xlD1 SECIS element seems to be one of the few exceptions; the TR2 thioredoxin reductase SECIS element is another example (25), in which there is no adenosine preceding the quartet but in this case a guanosine.

Characterization of xlD1 enzyme
Homogenates of transfected cells were incubated with 100 nM 125I-T4 or 125I-rT3 at four temperatures (10, 20, 30, and 37 C), and thereafter the reaction mixtures were analyzed by HPLC. Already at 10 C, the xlD1 enzyme has significant activity, and especially for T4 ORD, the largest increase is between 20 and 30 C (Fig. 2AGo). Because at 37 C both ORD and IRD activity is even higher, all further experiments were performed at 37 C. In the absence of the reducing cofactor DTT, deiodinase activity was negligible, and the optimum concentration was 1 mM (data not shown).


Figure 2
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  		FIG. 2. Kinetic characterization of recombinant xlD1 enzyme. A, Effect of incubation temperature on the deiodinase (ORD and IRD) activity of xlD1 enzyme in the presence of 100 nM T4 or 100 nM rT3 and 10 mM DTT. B, Double-reciprocal plot of T4 (ORD and IRD), rT3 (ORD), and T3 (IRD) deiodination by xlD1 enzyme. All incubations in the presence of 10 mM DTT. C, Double-reciprocal plots of T4 ORD by xlD1 enzyme in the presence of increasing concentrations of DTT. The Vmax and Km values at increasing DTT concentrations (0.1, 1, and 10 mM) are 2.4, 4.2, and 4.5 pmol/min·mg and 0.3, 0.5, and 0.5 µM, respectively. D, Inhibition of rT3 ORD (100 nM) by 6-PTU, GTG, or IAc.

 
Incubations in the presence of increasing concentrations (0.1–10 µM) of rT3, T3, and T4 resulted in the inhibition of the ORD of 10 nM 125I-rT3 in the order T4 = rT3 > T3. The IC50 values were 0.3 µM T4, 0.3 µM rT3, and 2–3 µM T3 (data not shown). More detailed kinetic characterization (Fig. 2BGo and Table 1Go) confirmed that xlD1 is a typical D1 enzyme, showing both ORD and IRD activity with Km values in the micromolar range, i.e. about 0.3 µM for T4 as well as rT3 and slightly higher for T3. In contrast to mammalian D1 enzymes, rT3 is not the preferred substrate; rather rT3 and T4 are equally good substrates [see maximum velocity (Vmax) to Km ratios in Table 1Go]. Another difference with mammalian D1 enzymes is the fact that the IRD of T3 and the ORD of rT3 were not stimulated by substrate sulfation (data not shown).


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  		TABLE 1. Kinetic characteristics of recombinant xlD1 enzyme

 
Rat and human D1 enzymes show ping-pong-type enzyme kinetics (1, 2, 34), which is indicative for the alternate reaction of substrate and cofactor (DTT in vitro) with different enzyme forms. These are the native enzyme and the selenenyl iodide (SeI) intermediate generated during the catalytic cycle. Also the xlD1 enzyme displayed ping-pong-type kinetics, as evidenced by the parallel lines in the Lineweaver-Burk plots of deiodination rate vs. substrate concentration at different fixed DTT concentrations (Fig. 2CGo).

Similar to rat, porcine, and human D1, the xlD1 enzyme is potently inhibited (Fig. 2DGo) by GTG and IAc, although judging from the IC50 values (GTG around 0.1 µM, IAc around 10 µM), it appears that the xlD1 enzyme is approximately 10-fold less sensitive to GTG and IAc than rat, porcine, and human D1 (5, 17, 35). Remarkably, in contrast to mammalian D1 enzymes, the xlD1 enzyme is insensitive to 6-PTU.

Site-directed mutagenesis within the catalytic center of xlD1 and inhibition by 6-PTU
The mechanism of D1 inhibition by 6-PTU has been investigated in some detail for rat and human D1 (14, 15, 16, 17, 30, 34, 35). The SeC residue in the catalytic center of D1 is thought to function as the iodonium acceptor, thus leading to the formation of the SeI intermediate form. In the presence of 6-PTU, the SeI intermediate reacts with either the reducing cofactor (DTT in vitro) or 6-PTU, leading to the formation of a SeC-PTU adduct. Kinetic studies have shown that 6-PTU inhibition of D1 is uncompetitive with the iodothyronine substrate (14, 16). The 6-PTU insensitivity of xlD1 could be explained by a reduced rate of formation of the SeI intermediate because this determines the susceptibility to 6-PTU. An alternative explanation could be that due to structural constraints the SeI intermediate in xlD1 is less likely to interact with 6-PTU and instead favors the reduced cofactor. In this regard it is interesting to note that in the 6-PTU-sensitive D1 enzymes (human, pig, cat, dog, mouse, rat, chicken), the residue two positions downstream of SeC is Ser, but Pro in all so far characterized teleost and amphibian 6-PTU-insensitive D1 enzymes (X. laevis, Oreochromis niloticus, Fundulus heteroclitus, Oncorhynchus mykiss) (1, 17, 21, 22, 23, 36). Another difference concerns the residue six positions upstream of the SeC residue, which is Asn in all 6-PTU-sensitive D1 enzymes but Ser in the 6-PTU-insensitive D1 enzymes (see Fig. 1AGo).

Therefore, we produced the Pro132Ser and Ser124Asn mutants as well as the double mutant of xlD1 by site-directed mutagenesis and expressed these mutant proteins in cells (see Materials and Methods). It was found that especially the Pro132Ser mutation strongly increased the 6-PTU sensitivity of xlD1 when ORD activity was measured with 100 nM T4 as substrate (Fig. 3AGo). The Pro132Ser mutation caused a 10-fold increase in the Km and Vmax values for ORD of T4 as well as rT3 (Fig. 3BGo and Table 2Go). This indicated that the capacity and probably also the substrate turnover rate of the Pro132Ser D1 mutant are increased, compared with the wild-type (wt) enzyme, assuming equal D1 protein concentrations. Therefore, the test conditions used in the experiment of Fig. 3AGo might have underestimated the 6-PTU sensitivity. When this experiment was repeated with 2 µM T4 or rT3 as substrate (about half the Km value), the IC50 value for 6-PTU decreased to about 2 µM both for the xlD1-Pro132Ser and the xlD1-Pro132Ser/Ser124Asn enzyme (Fig. 3CGo). This IC50 value is in the same range as the IC50 values (1–5 µM) of the 6-PTU-sensitive mammalian D1 enzymes (5, 15, 16, 17, 35, 37). Under similar conditions (0.1 µM T4 for xlD1 wt enzyme, 2 µM T4 for xlD1-Pro132Ser), various thiouracil derivatives were tested for their inhibitory activity (Table 3Go). The xlD1 enzyme is inhibited only by the iodine-containing compounds (ITU and IU), whereas the xlD1-Pro132Ser enzyme is also inhibited by TU, MTU, 6-PTU, and 5-PTU (6-PTU = 5-PTU > MTU > TU). Uracil does not affect ORD activity, pointing to the essential nature of the 2-mercapto group. In comparison with rat and human D1 (Table 3Go), the alkyl substituents of TU more strongly increase the inhibitory potency for the xlD1-Pro132Ser mutant.


Figure 3
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  		FIG. 3. Characterization of xlD1 mutant enzymes. A, Inhibition of T4 ORD (100 nM T4, 10 mM DTT) by 6-PTU of xlD1-wt, xlD1-Pro132Ser, xlD1-Ser124Asn, and xlD1-Pro132Ser/Ser124Asn enzymes. B, Double-reciprocal plot of T4 ORD by xlD1-wt and mutant enzymes in the presence of 1 mM DTT. For Km and Vmax values, see Table 2Go. C, Inhibition of T4 ORD (2 µM T4, 1 mM DTT) by 6-PTU of xlD1-Pro132Ser and xlD1-Pro132Ser/Ser124Asn enzymes. D, Double-reciprocal plots of T4 ORD by xlD1-Pro132Ser enzyme in the presence of increasing concentrations of DTT. The Vmax and Km values at the different DTT concentrations (0.1, 1, and 10 mM) are 8.7, 21, and 26 pmol/min·mg and 1.3, 3.3, and 4.3 µM, respectively.

 

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  		TABLE 2. Kinetic characteristics (ORD activity) of mutant xlD1 enzymes

 

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  		TABLE 3. Inhibitory activity of thiouracil derivatives

 
Double-reciprocal plots of product formation with T4 as the variable substrate at several DTT concentrations (0.1–10 mM) yielded patterns of parallel lines for the xlD1-Pro132Ser enzyme (Fig. 3DGo), indicating ping-pong kinetics as for the wt enzyme. The same is true for the xlD1-Ser124Asn and the xlD1-Pro132Ser/Ser124Asn enzymes (not shown).

Mechanism of inhibition of the xlD1-Pro132Ser mutant enzyme by 6-PTU
Thiouracil and derivatives, such as 5-PTU and 6-PTU, are uncompetitive inhibitors with respect to the iodothyronine substrate of rat and human D1 (17, 38). The mechanism of inhibition of the xlD1-Pro132Ser enzyme by 6-PTU was investigated by measurement of the amount of T3 produced at different initial T4 concentrations in the absence or presence of different mounts of 6-PTU. The data were analyzed by plotting a function of the rate (1/V in the so-called Dixon plot) against the inhibitor concentration at different substrate levels (Fig. 4AGo). In the case of pure uncompetitive inhibition, this yields a set of parallel lines, and the intercept of an individual line on the abscissa provides the value of –IC50 for the particular substrate value of the line plotted (39). The IC50 value decreased when the iodothyronine substrate concentration increased, in other words the degree of inhibition by 6-PTU increases with the substrate concentration.


Figure 4
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  		FIG. 4. Mechanism of xlD1-Pro132Ser inhibition by 6-PTU and xlD1 inhibition by ITU. A, Dixon plots of T4 ORD by xlD1-Pro132Ser enzyme at 1 mM DTT and different 6-PTU concentrations. The IC50 values at the different T4 concentrations (1, 2, and 8 µM) are 3.5, 2.0, and 1.3 µM 6-PTU, respectively. B, Dixon plots of T4 ORD by xlD1-wt enzyme at 1 mM DTT and different ITU concentrations. The IC50 values at the different T4 concentrations (0.1, 0.2, 0.4, and 1.0 µM) are 13, 16, 21, and 29 µM ITU, respectively.

 
The inhibition by ITU of xlD1 wt enzyme was competitive with substrate (Fig. 4BGo), as indicated by the intersecting lines in the Dixon plots (39). The competitive inhibition by this iodinated thiouracil derivative is in line with the competitive inhibition of rat and human D1 by iodinated compounds such as certain radiographic contrast agents (iopanoic acid, ipodic acid) and antiarrhythmic drugs such as amiodarone (40, 41).

Deiodinase activity in adult Xenopus organs
Using T4 as substrate, ORD activity (measured as 125I– production by LH20-chromatography) was detected in X. laevis and X. tropicalis liver, kidney, gut, and brain homogenates (Fig. 5Go). The homogenates were incubated for 60 min at 37 C with either 2 or 100 nM T4. Comparing the ORD activities at 100 and 2 nM T4, it appeared that liver, kidney, and brain might contain a high Km (>100 nM) deiodinase component because the ORD activities were about 50-fold higher during incubations with 100 nM T4 than at 2 nM T4. In contrast, gut might contain a low Km deiodinase component because in this case the increase was only 5- to 10-fold. Addition of 100 µM 6-PTU did not inhibit ORD activity, and by using rT3 as substrate similar ORD activities were obtained (not shown).


Figure 5
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  		FIG. 5. ORD activity in Xenopus organ homogenates. Liver, kidney, brain, and gut homogenates derived from adult X. laevis (XL) or X. tropicalis (XT) were incubated for 60 min at 37 C with 2 or 100 nM T4 in P100E2 buffer with 20 mM DTT. The quantity of 125I– produced was measured by LH-20 chromatography (see Materials and Methods).

 
In parallel experiments reaction mixtures were analyzed by reverse-phase HPLC after incubation with 1 or 100 nM 125I-T4 or 125I-T3. Using gut homogenates, T3 production from T4 (ORD) and T2 production from T3 (IRD) could be detected at low (1 nM) concentrations of substrate, whereas at high (100 nM) concentrations of substrate, these products could not be detected anymore (not shown). This indicated the presence in gut homogenates of low Km (10 nM or less) ORD and IRD activity, probably D2- and D3-like enzymes as has also been described for gut tissue from R. catesbeiana tadpoles (12). Using kidney and brain homogenates, no 125I-T3 (ORD) production from 125I-T4 could be detected but only 125I– and 125I-rT3 (IRD, especially at 1 nM T4). With T3 as substrate, T2 production (IRD) was detected (not shown). These results indicated the presence of D3 activity in kidney and brain, as was also described for R. catesbeiana tadpoles (12). Regarding liver homogenates no 125I-T3 (ORD) production from 125I-T4 could be detected, but minute amounts of rT3 (IRD) and significant amounts of 125I– were produced. Because we also detected T2 formation (IRD) from T3, we concluded that liver contains D3 activity.

Microsomal fractions were prepared from liver and kidney tissue. Upon incubation with 125I-T4 (100 nM) and HPLC analysis also in this case, no 125I-T3 production (ORD) but only small amounts of 125I-rT3 and significant amounts of 125I– could be detected (not shown).

All in all, although significant amounts of 125I– were produced using 125I-T4 and 125I-rT3 as substrates, we found no convincing evidence for a high Km (about 0.25 µM) D1-like enzyme in adult Xenopus tissues because either no T3 was produced from T4 or the product formation was reduced or absent at the higher (100 nM) substrate concentration. In liver, kidney, and brain homogenates and liver microsomal fractions, there was significant nondeiodinase-mediated 125I– production, which was not saturable at higher (up to 1000 nM) T4 concentrations.

D1 mRNA expression in juvenile and adult tissues
We investigated the expression of D1 mRNA relative to that of the ribosomal protein RPL8 in juvenile and adult tissues by quantitative RT-PCR using total RNA extracted from whole kidney, liver, gut, and brain, including pituitary. The results are presented in Fig. 6Go. There is no major difference between males and females at the same stage. In postmetamorphic juvenile Xenopus, D1 is mostly expressed in the brain, whereas in young adult Xenopus D1 is expressed mainly in kidney but also the liver.


Figure 6
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  		FIG. 6. Relative xlD1 mRNA expression in different tissues of juvenile (stage NF 66) and adult X. laevis. D1 mRNA was normalized against RPL8 mRNA in each tissue. Data are expressed as 2{Delta}Ct ({Delta}Ct = CtRPL8 – CtD1) and presented as means ± SE (based on the {Delta}Ct data; n = 3). In juveniles, D1 is mostly expressed in the brain, whereas in adults highest expression of D1 is in kidney.

 
Because the brain is a highly heterogenous organ, we investigated the localization of D1 mRNA expression in juvenile Xenopus brain (Fig. 7Go, A–E) and compared it with metamorphic Xenopus brain (Fig. 7Go, F–I). D1 is expressed in the ventricular zone in both stages (Fig. 7Go, B, C, and G), although more strongly in metamorphic tadpoles than juveniles. At a higher magnification (Fig. 7Go, E and I), one notes that some cells lining the ventricle express higher levels of D1 mRNA. These cells present astrocytic morphologies. D1 is also expressed in a more diffuse pattern in isolated, relatively large cells (open arrows in Fig. 7Go, C and D).


Figure 7
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  		FIG. 7. xlD1 mRNA expression in the subventricular zone of the brains of metamorphic (stage NF 63, A–E) and juvenile (4 months, F–I) X. laevis as studied by in situ hybridization. Dissected brains were hybridized with either D1 sense (A, F, and H) or antisense (B–E, G, and I) DIG-labeled probes. After staining with 5-bromo-4-chloro-3-indoyl-phosphate/nitro blue tetrazolium chloride (Roche) (see Materials and Methods), cryostat sections (20 µm) were cut and mounted for photography in glycerol. Cytoplasmic localization of D1 mRNA is observed in subventricular cells in metamorphic X. laevis (open arrows in C and D) and cells lining the ventricles (black arrows in C, E, G, and I) in both metamorphic and young adult X. laevis. The boxed areas in B, F, and G correspond to the higher magnifications shown in C, H, and I, respectively. D and E represent further magnifications of C. Bar, 50 µm (A, B, F, and G); 5 µm, (C–E, H, and I).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of the present study was to investigate the characteristics of a iodothyronine deiodinase homolog identified in the X. laevis EST project (42). The Xenopus EST clone primary sequence was clearly different from the R. catesbeina D2 and D3 primary sequences (43, 44), and therefore we hypothesized that this EST clone might represent the Xenopus D1 homolog.

The evidence that the X. laevis deiodinase homolog (xlD1) characterized in this study represents a typical D1 enzyme can be summarized as follows: 1) the amino acid sequence of xlD1 is much more homologous with reported D1 sequences from other species than with the D2 and D3 sequences from X. laevis and other species and in the phylogenetic tree (1, 27) of the deiodinases xlD1 clusters in the D1 group; 2) it displays ORD (T4, rT3) as well as IRD (T3, T4) activities, which are typical D1 properties; 3) the apparent Km values are in the micromolar range (0.2–0.4 µM), which is another D1 property; 4) it displays ping-pong-type kinetics in contrast to the sequential reaction kinetics of D2 and D3 enzymes; and 5) the relatively high sensitivity to GTG and IAc is another characteristic shared with D1 enzymes.

Another question is whether this recombinant xlD1 enzyme has similar characteristics as the putative native X. laevis D1 enzyme. So far, both in R. catesbeina and X. laevis tadpoles, the expression of D2 and D3 has been described by enzyme activity assays, Northern blots, and in situ hybridization (12, 13). In premetamorphic and prometamorphic X. laevis tadpoles, constitutive D2 expression was found by in situ hybridization in the limb buds and the cells that line the ventricles in the brain as well as the spinal cord. During metamorphic climax D2 is expressed in the anterior pituitary, the tail, and the intestine, whereas the functional significance of D2 expression is underlined by the fact that it precedes major thyroid hormone-induced developmental changes (13, 45). On the basis of the described characteristics of T4 ORD activity (PTU-insensitive, Km ~10 nM), it was concluded that a typical D1 enzyme is not present in frogs during metamorphosis (10, 11, 12). We have not been able to demonstrate the presence of a native D1 enzyme with similar characteristics as the recombinant xlD1 enzyme (high Km ORD/IRD of T4, PTU insensitive) in tissue homogenates prepared from adult frogs (X. laevis and X. tropicalis). However, D1 mRNA expression has been demonstrated, in particular in liver and kidney of adult X. laevis and brain of juvenile animals. The physiological role of D1 during these developmental stages is unclear; it could be involved in either T4 activation (T3 production by ORD) or T4 inactivation (rT3 production by IRD). The fact that in R. catesbeiana tadpoles, no 6-PTU sensitive ORD activity was detected during any stage of metamorphosis (10, 11, 12) is in agreement with the finding that the xlD1 enzyme is insensitive to 6-PTU.

The Pro132Ser mutation in the catalytic center converts the 6-PTU-insensitive xlD1 enzyme to a 6-PTU-sensitive enzyme, whereas the Ser124Asn mutation, neither on its own nor in combination with the Pro132Ser mutation, did not influence the 6-PTU-sensitivity. Both xlD1-wt and xlD1-Pro132Ser displayed ping-pong-type kinetics, and the inhibition of xlD1-Pro132Ser by 6-PTU was uncompetitive with substrate. These properties are similar to those of the 6-PTU-sensitive D1 proteins (14, 16, 34, 38). However, the Pro132Ser mutation caused a 10-fold increase in the Km values for T4 and rT3 ORD; in other words more substrate is needed to saturate the enzyme. In comparison with the PTU-sensitive mammalian D1 enzymes, the Km value for rT3 ORD is even more increased (20- to 30-fold). Unfortunately, we could not determine the substrate turnover number of the xlD1 enzyme by saturation analysis of bromoacetyl-[125I]T3 affinity labeling (37, 46) because it was not possible to affinity label the protein. Another approach, quantitative immunoblotting, was also not possible because the xlD1 protein did not cross-react with the available D1 antisera (5, 37). As a consequence, it is not possible to answer the question whether the 6-PTU-sensitivity of the xlD1-Pro132Ser enzyme is due to an increased turnover number, i.e. the formation of more SeI intermediates per time unit.

Another explanation for the PTU insensitivity of xlD1 could be that due to the presence of Pro at amino acid position 131 and 132, the SeI intermediate is not accessible for 6-PTU but is accessible for the reducing cofactor. It remains to be determined whether it is the presence of a Pro as such at position 132 or the absence of the hydroxyl group of Ser, which makes xlD1-wt insensitive to 6-PTU, and vice versa, which makes xlD1-Pro132Ser sensitive to 6-PTU. In this regard it is remarkable that the reverse mutation in rat D1, i.e. rat D1-Ser128Pro, made the enzyme resistant to 6-PTU (47). And the same mutation, i.e. human D2-Pro135Ser, made human D2 sensitive to 6-PTU (47). Undoubtedly the amino acid two positions downstream from the SeC residue is important, but there are probably additional factor(s) that determine 6-PTU sensitivity of deiodinases. This is evident from the fact that the same Pro to Ser mutation in tilapia D1, i.e. tilapia D1-Pro128Ser, did not make tilapia D1 sensitive to 6-PTU (21). These additional factor(s) could be investigated by analysis of the primary amino acid sequences and 6-PTU-sensitivity of more amphibian (salamander), reptile (crocodile, snake, turtle), and teleost fish (zebrafish, killifish, trout, Sparus aurata, Oryzias latipes, Cyprinus carpio) D1 enzymes, which have either been (partially) characterized (22, 23, 48, 49) and/or for which EST clones are available.

It is likely that all vertebrate deiodinases, and maybe also the invertebrate deiodinases (27), stem from a single ancestral SeC-containing protein. Despite their general structural homology, the deiodinases have undergone considerable evolutionary divergence with regard to function. Of course, during evolution the D1 enzymes have not been selected for their sensitivity to inhibition by 6-PTU, IAc, or GTG. Rather, the sensitivity of the mammalian D1 enzymes and the insensitivity of amphibian as well as teleost fish D1 enzymes for 6-PTU probably reflect a difference in other properties and thereby also in function. In mammals and chicken, D1 is usually highly expressed in the liver, and the liver plays an important role in the regulation of plasma T3 levels (1, 2, 3, 4, 5, 50). In teleost fish a role for liver D1 in regulation of plasma T3 levels is less clear (22). This might also apply to frogs because D1 is not expressed in the liver. Another indication for a different function might be that whereas sulfated iodothyronines (rT3S, T3S, T4S) are very good substrates for mammalian D1 enzymes, teleost fish and frog D1 enzymes do not readily deiodinate sulfated iodothyronines (21, 22, 51).

Whereas this study has described the characteristics of a recombinant 6-PTU-insensitive X. laevis D1 enzyme, its physiological role during tadpole metamorphosis remains to be investigated.


   Acknowledgments
 
We thank Dr. Vadim Gladyshev (Department of Biochemistry, University of Nebraska, Lincoln, NE) for advice in identifying the SECIS element, the RZPD (Deutsches Ressourcenzentrum für Genomforschung, Berlin, Germany) for the X. laevis EST clone, and Ronald van der Wal (DNA-sequencing core facility, Department of Internal Medicine, Erasmus Medical Center) for sequencing of plasmids.


   Footnotes
 
This work was presented in part at the 76th Annual Meeting of the American Thyroid Association, Vancouver, Canada, 2004 (Short Call Abstract 9), p 876.

The nucleotide sequence reported in this paper has been submitted to the GenBank database with accession no. DQ098656.

First Published Online April 6, 2006

Abbreviations: D1, Type I iodothyronine deiodinase; D2, type II iodothyronine deiodinase; D3, type III iodothyronine deiodinase; DIG, digoxigenin; DTT, dithiothreitol; EST, expressed sequence tag; GTG, gold-thioglucose; IAc, iodoacetate; ITU, 5-iodo-2-thiouracil; IU, 5-iodouracil; Km, Michaelis constant; MTU, 6-methyl-2-thiouracil; ORD, outer-ring deiodinase; PBST, PBS and Tween 20; 5-PTU, 5-propyl-2-thiouracil; 6-PTU, 6-propyl-2-thiouracil; rT3, reverse T3; SeC, selenocysteine; SECIS, SeC insertion sequence; SeI, selenenyl iodide; TU, 2-thiouracil; UTR, untranslated region; Vmax, maximum velocity; wt, wild type; xlD1, Xenopus laevis deiodinase.

Received June 14, 2005.

Accepted for publication March 23, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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G. Morvan Dubois, A. Sebillot, G. G. J. M. Kuiper, C. H. J. Verhoelst, V. M. Darras, T. J. Visser, and B. A. Demeneix
Deiodinase Activity Is Present in Xenopus laevis during Early Embryogenesis
Endocrinology, October 1, 2006; 147(10): 4941 - 4949.
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