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Department of Internal Medicine III (J.P.S., E.K., S.V.d.G., T.J.V.), Erasmus University Medical School, 3000 DR Rotterdam, The Netherlands; Laboratory of Comparative Endocrinology (S.V.d.G., V.M.D., E.R.K.), K. U. Leuven, 3000 Leuven, Belgium; and Department of Nuclear Medicine (J.L.L.), University of Massachusetts, Medical Center, Worcester, Massachusetts 01655
Address all correspondence and requests for reprints to: Theo J. Visser, Department of Internal Medicine III, Erasmus University Medical School, Room Bd 234, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: visser{at}inw3.azr.nl
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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30
kDa proteins, featuring in corresponding positions a selenocysteine
(Sec) residue that is important for catalysis (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). The catalytic
cycle of D1 appears to consist of two half reactions: first, transfer
of an I+ from the substrate to the selenolate
(Se-) anion of Sec, and second, reduction of the selenenyl
iodide (SeI) generated by a thiol cofactor. In vitro,
dithiotreitol (DTT) substitutes for the endogenous cofactor (1, 2, 3).
6-n-Propyl-2-thiouracil (PTU) is a thyroid
peroxidase-blocking drug that is used for treatment of hyperthyroidism.
PTU is also a potent inhibitor of mammalian D1 but has no effect on D2
and D3 (1, 2, 3). PTU is an uncompetitive D1 inhibitor that is thought to
react with the SeI intermediate. The nucleophile-directed reagents
iodoacetate (IAc) and gold thioglucose (GTG) also inhibit D1 more
effectively than D2 and D3 (16, 17, 18). Both IAc and GTG are competitive
D1 inhibitors that probably interact with the Se- anion
(1, 2, 3). Before the amino acid sequences of D2 and D3 were known, the findings that PTU and GTG are less potent inhibitors of these enzymes and of the Sec126Cys mutant of D1 than of the wild-type D1 led to the proposal that Sec was absent in the catalytic centers of D2 and D3 (17, 18). This was supported by the relatively minor decreases in D2 and D3 activities compared with the strong decreases in hepatic and renal D1 activities in selenium-deficient rats (19, 20). A D1 variant has recently been identified in the kidney of the teleost fish Oreochromis niloticus (tilapia), showing characteristic catalytic and substrate specificities but low sensitivities to inhibition by PTU, IAc, and GTG (21). Similar to D2 and D3, it was proposed that Sec was not present in the active site of this tilapia D1 (tD1). To determine the molecular basis for the differences in catalytic mechanisms and inhibitor sensitivities between the deiodinase isoenzymes, we cloned and characterized complementary DNA (cDNA) coding for tD1. This involved RT-PCR of tilapia kidney messenger RNA (mRNA) using primers based on conserved amino acid sequences (NFGSCTSecP and YIEEAH) present in rat, human, and dog D1s and Xenopus laevis D3 sequences (4, 5, 7, 8). The resultant RT-PCR products were sequenced to confirm that they were related to known deiodinases and used as probes for cDNA library screening. Expression of the full-length cDNA clone obtained resulted in production of a functional deiodinase, the catalytic properties of which were examined.
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Materials and Methods |
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-32P]deoxyATP were purchased from
Amersham (Buckinghamshire, UK); polyethyleneglycol (PEG6000) was from
Merck (Hohenbrunn, Germany); diethylaminoethyl (DEAE)-dextran and
Sephadex LH-20 were from Pharmacia. Nonradioactive iodothyronines were
obtained from Henning Berlin R & D (Berlin, Germany),
[3',5'-125I]T4 (1200 Ci/mmol) from
Amersham, and [3,5-125I]T3 (35 Ci/mmol)
from Mr. R. Thoma (Formula GmbH, Berlin, Germany) courtesy of Dr. G.
Decker (Hennig Berlin GmbH, Berlin, Germany).
[3',5'-125I]rT3 (2000 Ci/mmol) and
[3,5-125I]T3 sulfate (T3S) were
prepared in our laboratory as described previously (22, 23). PTU, IAc,
GTG, DTT, and chloroquine were obtained from Sigma (St. Louis, MO). All
other reagents were of the highest purity commercially available.
Cloning
Total RNA was isolated from tilapia kidney using TRIzol reagent,
and Poly(A+) RNA was isolated on oligo(dT) cellulose. cDNA
was obtained by oligo(dT)-primed reverse transcription using AMV
reverse transcriptase. PCR was performed using the primers
5'-AATTTTGGCAGTTGTACCTGACC-3' and 5'-RTGIGCTTCCTCIATGTA-3' and SuperTaq
DNA polymerase. The PCR products were TA-cloned into pCR-II and
sequenced. The tilapia kidney cDNA library was constructed in Lambda
ZAP-Express (Stratagene, La Jolla, CA). The library was blotted on
Hybond-N+ and screened with the RT-PCR product labeled by
primer extension using Klenow DNA polymerase in the presence of
[-32P]deoxyATP. The phagemids carried in selected
positive bacteriophages were excised, generating cDNA clones in
pBK-CMV. The inserts were sequenced manually and by automatic
sequencing in both directions using the dideoxy method of Sanger
et al. (23a). Site-directed mutagenesis was carried out
using the MORPH kit (5prime
3prime, Boulder, CO) and the
oligonucleotide 5'-CACCTGACCCTGGTTTATGTATAA-3'; the mutation was
confirmed by sequencing. RNA secondary structure prediction was done
using the MFOLD program provided by Dr. M. Zuker (Institute for
Biomedical Computing, Washington University, St. Louis, MO) on the
Internet (http://www.ibc.wustl.edu/
zuker) (24).
Expression
cDNA was subcloned into pCI-Neo and expressed in COS-1 cells
grown in DMEM/F12 containing 10% FCS (Gibco BRL) and 40 nM
Na2SeO3 (25). One day before transfection,
COS-1 cells were seeded at 50% confluency in 55-cm2 cell
culture dishes. Expression constructs (7 µg), isolated by alkaline
lysis and polyethyleneglycol precipitation (26), were added to
serum-free DMEM/F12 medium containing 100 µg/ml DEAE-dextran. After
2 h, the medium was replaced by serum-free DMEM/F12 medium
containing 100 µM chloroquine. Again 2 h later the
medium was replaced by DMEM/F12 containing 10% FCS and 40
nM Na2SO3. After 3 days, the cells
were rinsed with PBS; collected in 0.3 ml 0.1 M phosphate
(pH 6.9), 1 mM EDTA, and 10 mM DTT; sonicated;
snap-frozen on dry-ice/ethanol; and stored at -80 C.
Northern analysis
Tilapia tissues and microsomal fractions thereof were obtained
as described previously (21). Northern blots were prepared using 20
µg total tissue RNA and Hybond-N membranes, which were hybridized for
16 h at 60 C with random hexamer-labeled full-length cDNA in
6 x SSC, 0.5% SDS, 5 x Denhardts’, and salmon sperm DNA
(100 µg/ml). Blots were washed twice for 15 min at 55 C in 3 x
SSC, 0.5% SDS at 55 C, and twice for 15 min at 60 C in 1 x SSC,
0.5% SDS. Autoradiographs were scanned using a Hewlett Packard Scanjet
IIcx (Amstelveen, The Netherlands), and signals were quantified using
software developed by Dr. R. Docter (Department of Internal Medicine
III, Erasmus University Medical School, Rotterdam, The Netherlands).
Results were standardized relative to the total amount of RNA applied,
which was determined similarly by photography and quantitation of the
fluorescence of the ethidium bromide-stained gel.
Enzyme assays
Deiodinase activities of native and recombinant enzyme
preparations were analyzed by quantitation of radioiodide released by
ORD of outer ring 125I-labeled T4 or
rT3 or by IRD of inner ring 125I-labeled
T3 or T3S (22, 23). In short, appropriate amounts of enzyme
protein were incubated in triplicate for 30–60 min at 37 C with 10
nM 125I-labeled substrate in 0.2 ml 0.1
M phosphate (pH 7.2), 2 mM EDTA, and 10
mM DTT. Reactions were stopped and
[125I]iodothyronines were precipitated by successive
addition of 0.1 ml 5% BSA and 0.5 ml 10% TCA.
125I- was further isolated from the
supernatant on Sephadex LH-20 minicolumns (22, 23). Unless indicated
otherwise, the results presented are taken from representative
experiments and were reproduced in at least two other experiments.
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Results |
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). Like the mammalian D1s, TN12 contains
a large 3' untranslated region (UTR). RNA secondary structure
prediction (24) and comparison with published consensus sequences (27, 28) revealed two almost identical, putative Sec insertion sequence
(SECIS) elements (Fig. 2), which are
essential for Sec incorporation at the UGA opal stop codon.
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), including the Sec residue (4, 5, 7).
The N-terminal region of TN12 showed a large divergence from the
hitherto described mammalian D1s. We have, therefore, sequenced the 5'
region of another, independent cDNA clone that proved to be identical.
The TN12 protein appeared to be much more acidic (pI 7.0) than other
D1s (pI 8.7–9.8), the biological significance of which is unknown. The
amino acid sequence of TN12 showed only 31%, 33%, and 28% identity
with human (13, 14), rat (13, 14), and Fundulus heteroclitus
(teleost fish) (15) D2, respectively, and 38% identity with rat (10)
and human (11) D3.
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).
Shorter exposure of the Northern blot shown in Fig. 4 revealed only one
prominent band of approximately 2.5 kb in kidney.
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).
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). The apparent
Michaelis-Menten constant (Km) value of rT3 for
both native and recombinant enzyme amounted to 2 µM,
which is 5- to 10-fold higher compared with rat and human D1 (22, 23).
Native tD1 and the TN12-encoded enzyme showed identical patterns of
inhibition by (in decreasing order of potency) GTG, IAc, and PTU (Fig. 7). These results indicated that both
native and recombinant enzyme activities are approximately
103-fold less sensitive to PTU inhibition, and
approximately 10-fold less sensitive to GTG and IAc inhibition than rat
and human D1 (5, 21).
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). IRD of
T3 by both wild-type tD1 and the mutant was too slow to
allow accurate measurements. Addition of 1 mM PTU had
little effect on the ORD of rT3 as well as on the IRD of
T3S by the Pro128Ser mutant (Fig. 9). T4 ORD by the mutant was
too slow to exactly determine the effect of PTU. These results suggest
that the PTU insensitivity of tD1 is not due to the presence of Pro
instead of Ser at position 128.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although we have not directly shown that the enzyme expressed after transfection of COS-1 cells with tD1 cDNA is a selenoprotein, indirect evidence strongly suggests that this is the case. First, mRNA transcribed from tD1 cDNA contains the opal UGA stop codon at a position identical to that in other D1 mRNAs shown to be translated as Sec. Furthermore, like the cDNAs for the other D1s cloned to date, tD1 cDNA contains a large 3' UTR. Comparison with reported consensus sequences for SECIS elements, which are essential for Sec incorporation at the UGA codon (27, 28), and RNA secondary structure prediction (24) reveal that two almost identical, putative SECIS elements are present in the 3' UTR of tD1. Although multiple SECIS elements have been described in selenoprotein P mRNA (27, 30), tD1 is the first deiodinase mRNA having more than one putative SECIS element. The function of multiple SECIS elements is unknown, but it may increase the efficiency of UGA codon read-through. Mutational analyses of mammalian D1s have indicated that the Sec residue is essential for enzyme activity. Replacement of Sec by Cys reduces catalytic activity approximately 100-fold, whereas replacement with Leu results in a complete loss of enzyme activity (4). Also, the protein truncated at the site of the Sec residue, which takes place in the absence of a SECIS element and, hence, if the UGA codon functions as a translation termination codon, is enzymatically inactive (4). Therefore, it is impossible that expression of TN12 cDNA yields a functional deiodinase if the UGA codon were not translated as Sec.
The most remarkable property of both native and recombinant tD1 is their insensitivity to PTU inhibition. Only weak inhibition is observed with as high as 1 mM PTU, whereas 1 µM of this drug strongly inhibits rat and human D1 (5, 21). In addition, tD1 is also approximately 10-fold less sensitive to inhibition by GTG and IAc than rat and human D1 (5, 21). We have demonstrated that this low sensitivity of tD1 to PTU, IAc and GTG is not caused by the absence of Sec, as previously hypothesized (21). Therefore, other structural determinants should be responsible for this anomality, one of which could be the Pro residue two positions downstream from Sec. All PTU-sensitive deiodinases, i.e. human, rat, mouse, and dog D1 (4, 5, 6, 7), have Ser, and all PTU-insensitive iodothyronine deiodinases, i.e. all D2s (12, 13, 14, 15), all D3s (8, 9, 10, 11), and tD1, have Pro at this position. If PTU inhibition is dependent on the nature of this residue, this could be explained by differences in effects of Pro and Ser on protein structure and, thus, on enzyme-inhibitor interaction, or by the requirement of the OH group of Ser for reaction of the inhibitors with Sec. Therefore, we studied the effect of the substitution of Pro128 by Ser on the catalytic properties of tD1, expecting that this mutation would increase the PTU sensitivity of the enzyme. One mM PTU was found to have as little effect on the Pro128Ser mutant as on the wild-type enzyme, suggesting that the PTU insensitivity of the latter is not solely due to the presence of Pro instead of Ser at position 128. However, in particular the low rT3 ORD activity of the Pro128Ser mutant hampers the interpretation of the lack of its inhibition by PTU (see below). It would be interesting to determine the effect of the reverse Ser128Pro mutation of other D1s on their inhibition by PTU.
The lower sensitivities of D2, D3, and tD1 to PTU, IAc, and GTG compared with mammalian D1s may be caused by the lower reactivity of the Sec residue. It is unknown whether other amino acid residues enhance Sec reactivity, much like Cys and Ser are activated by His and Asp residues in cysteine and serine proteases (31, 32). Essential His residues have indeed been identified in D1 (33), and these residues (His158 and His174) are also conserved in tD1. Because PTU supposedly reacts with an enzyme SeI group, the rate of formation of this intermediate also determines susceptibility to PTU inhibition. Our findings suggest that tD1 has a lower kcat than mammalian D1s, because the deiodinase activity expressed by transfection of COS-1 cells with tD1 is consistently approximately 10-fold lower than that produced by transfection with rat D1. This could be explained by a lower reactivity of Sec in tD1 than in mammalian D1, although the amount of tD1 protein expressed is unknown. In this regard it should be mentioned that the basic Arg and Lys residues at positions 11 and 12 of mammalian D1, which are important for membrane insertion (34), are lacking in tD1. Interestingly, a Phe residue that has been shown to be involved in rT3 binding to mammalian D1 (7, 22) is also conserved in tD1 (Phe65).
The Pro128Ser mutation in tD1 results in a selective and strong reduction of its ORD activity, whereas its IRD activity is not affected or even increased. These findings suggest that protein structural changes induced by the Pro128Ser mutation interfere in the interaction with substrates undergoing ORD, whereas they do not affect the interaction with substrates undergoing IRD. This is remarkable, because all other D1s that have Ser at this position catalyze ORD very effectively. Therefore, the catalytic specificities of the different deiodinases must be determined by additional structural elements in these proteins. Further mutational analyses should reveal the molecular basis for D1 having both ORD and IRD activity as opposed to D2 and D3, which have only ORD or only IRD activity, respectively, as well as for the different inhibitor susceptibilities of the different deiodinase isoenzymes. This may lead to the rational design of new and potent deiodinase inhibitors for research and clinical application.
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Acknowledgments |
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Footnotes |
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Received June 9, 1997.
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