©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Expression of a cDNA for a Mammalian Type III Iodothyronine Deiodinase (*)

Walburga Croteau , Susan L. Whittemore (§) , Mark J. Schneider , Donald L. St. Germain (¶)

From the (1)Departments of Medicine and Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The type III iodothyronine deiodinase metabolizes the active thyroid hormones thyroxine and 3,5,3`-triiodothyronine to inactive compounds. Recently, we have characterized a Xenopus laevis cDNA (XL-15) that encodes a selenoprotein with type III deiodinase activity (St. Germain, D. L., Schwartzman, R., Croteau, W., Kanamori, A., Wang, Z., Brown, D. D., and Galton, V. A.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7767-7771). Using the XL-15 as a probe, we screened a rat neonatal skin cDNA library. Among the clones isolated was one (rNS43-1) which contained a 2.1-kilobase pair cDNA insert that manifested significant homology to both the XL-15 and the G21 rat type I deiodinase cDNAs, including the presence of an in-frame TGA codon. Expression studies demonstrated that the rNS43-1 cDNA encodes a protein with 5-, but not 5`-, deiodinase activity that is resistant to inhibition by propylthiouracil and aurothioglucose. Northern analysis demonstrated a pattern of tissue expression in the rat consistent with that of the type III deiodinase and site directed mutagenesis confirmed that the TGA triplet codes for selenocysteine. We conclude that the rNS43-1 cDNA encodes the rat type III deiodinase and that the types I and III deiodinases present in amphibians and mammals constitute a family of conserved selenoproteins important in the metabolism of thyroid hormones.


INTRODUCTION

The varying patterns of thyroid hormone metabolism occurring in different tissues and during different stages of development are dictated in large part by the selective expression of one or more of several iodothyronine deiodinases. During fetal development in many species, including mammals, the predominant metabolic process involves the catalytic removal of iodine at the 5-, or the chemically equivalent 3-, position on the tyrosyl, or inner, ring of thyroxine (T)()and 3,5,3`-triiodothyronine (T). This 5-deiodinase (5-D) activity results in the formation of the essentially inactive metabolites 3,3`,5`-triiodothyronine (reverse T, rT) and 3,3`-diiodothyronine (T), respectively(1) . The type III deiodinase, which functions exclusively as a 5-D, is highly expressed in both mammalian placenta and several fetal tissues and thus likely contributes significantly to this metabolic process(2) . After birth, the expression of type III 5-D is more limited and in rats occurs primarily in the central nervous system and skin, with particularly high levels noted in the latter tissue during the neonatal period(3) . In contrast, the type I deiodinase is highly expressed in the liver, kidney, and thyroid gland of adult rats and can function as both a 5`-deiodinase (5`-D) and a 5-D. However, it efficiently catalyzes the 5-deiodination of sulfated iodothyronine substrates only (4) and its expression is low in most tissues during development(5) .

We have recently reported the characterization of the XL-15 cDNA(6) , which was isolated by Wang and Brown from a Xenopus laevis tadpole tail cDNA library (7) and encodes a type III 5-D. Of note, this amphibian cDNA, like the previously isolated mammalian type I deiodinase cDNAs(8, 9, 10) , contains an in-frame TGA codon which encodes selenocysteine at the enzyme's active site. The structural features of the mammalian type III deiodinase are uncertain, and several lines of evidence have suggested that this subclass of enzymes does not contain selenocysteine. Thus, the rat type III deiodinase is resistant to inhibition by propylthiouracil and gold compounds such as aurothioglucose(11) , properties which Berry et al.(12) have suggested are consistent with the presence of cysteine at the enzyme's catalytic site. In addition, type III activity is maintained at essentially normal levels in the brain(13) , placenta(14) , and skin (15) of selenium-deficient rats, whereas type I deiodinase activity in the liver and kidney is markedly decreased. And finally, protein labeling studies using Se have failed to identify candidate selenoproteins in rat tissues manifesting type III activity (11).

In the present report we describe the isolation from a rat neonatal skin cDNA library of a cDNA that manifests significant sequence homology to the mammalian type I and amphibian type III deiodinase cDNAs, including the presence of an in-frame TGA codon. X. laevis oocytes injected with RNA transcripts derived from this cDNA express abundant 5-D activity with characteristics of the type III enzyme.


MATERIALS AND METHODS

Animals and Tissue Preparation

For the preparation of RNA, skin samples from 7-14-day-old neonatal rats and their corresponding dams were harvested and homogenized as described previously(16) . Poly(A) RNA was isolated by two cycles of chromatography on oligo(dT)-cellulose and used for injection into X. laevis oocytes and cDNA library construction. RNA from other tissues used for Northern analysis was prepared by the same methods. In one experiment, rats were rendered hyperthyroid by the injection of 50 µg T subcutaneously/100 g body weight for 4 days prior to sacrifice.

cDNA Library Construction and Screening

Poly(A) RNA from neonatal rat skin was used with oligo(dT) primers to prepare double-stranded cDNA using reagents from the Choice kit (Life Technologies, Inc.). Following the addition of EcoRI linkers, this material was used to construct a cDNA library in the Lambda-Zap II vector according to the manufacturer's instructions (Stratagene, La Jolla, CA). Screening of the library was performed using plaque hybridization under low stringency conditions according to the methods of Lees et al.(17) . The first 732 nucleotides of the XL-15 cDNA, which includes most of the coding region, was used as a probe. Positive plaques were detected by autoradiography and purified by additional rounds of screening using the same hybridization conditions. cDNA inserts were sequenced on both strands using gene-specific primers and an automated sequencing system with fluorescent dye terminators (Applied Biosystems, Foster City, CA).

Expression Studies in X. laevis Oocytes

Stage 5-6 X. laevis oocytes were isolated and each microinjected as described previously (16) with 5-50 ng of either poly(A) RNA or 50 ng of in vitro synthesized capped RNA transcripts prepared using the MEGAscript kit (Ambion, Austin, TX). After injection, oocytes were incubated for 4 days in Barth's medium (for determination of 5-D activity) or L-15 medium (for determination of 5`-D activity), then harvested, membrane fractions prepared as described previously (18) and deiodinase activity determined according to published methods(18, 19) . In kinetic studies, 5-D activity was determined using 1-20 nM [I]T and 50 mM dithiothreitol as cofactor. Kinetic constants were determined from double reciprocal plots. I-Labeled iodothyronines used as substrates were obtained from E. I. Du Pont de Nemours & Co. and purified by chromatography using Sephadex LH-20 (Sigma) prior to use.

In other experiments, the deiodinase activities in oocyte membrane preparations were determined in the absence or presence of propylthiouracil (10-1000 µM) or aurothioglucose (0.01-10 µM). 5`-D activity was measured using 67 nM [I]rT as substrate and 20 mM dithiothreitol as cofactor. The 5-D assay utilized 1 nM [I]T and 20 or 50 mM dithiothreitol. Protein concentrations were determined by the method of Bradford (20) with reagents obtained from Bio-Rad. The G21 cDNA was kindly provided by Drs. M. Berry and P. R. Larsen (Boston, MA).

Reverse Transcriptase-Polymerase Chain Reaction

The reverse transcriptase-polymerase chain reaction (PCR) was performed as described previously(21) . In brief, 0.5 µg of poly(A) RNA from rat neonatal skin or placenta was digested with 0.1 unit of RNase-free DNase I (Life Technologies, Inc.), then reverse-transcribed using Superscript II reverse transcriptase (Life Technologies, Inc.) and the rNS43-1-derived antisense oligonucleotide designated 1148AS (5`-GTCACTTGTCCCTTGGTTTT-3`) as primer. Following treatment with RNase H (Life Technologies, Inc.), 5 µl of the reaction mixture (total volume, 30 µl) was used directly in PCR. PCR was performed using the hot start method with five pairs of rNS43-1-derived oligonucleotides as primers used in different reactions. In all cases, 668AS (5`-GATGCGCTGGCTCTGGAA-3`) was used as the antisense primer. Sense primers included Bluescript/M13 reverse, 133S (5`-GGGCGGCTGTGTGAGT-3`), 226S (5`-CAGGGAGACCAGAAAGCAGAG-3`), 262S (5`-GGTCGGAGAAGGTGAAGGG-3`), and 319S (5`-CTCCCTGCTGCTTCACTCT-3`). Reaction conditions included 32 cycles of 94 °C 1 min, 55 °C 45 s, and 72 °C 1 min and a final 10-min extension period. Reaction products were separated on a 3% agarose gel and stained with ethidium bromide.

Southern Blotting

The PCR reaction products separated by agarose gel electrophoresis were transferred by capillary blotting to nylon membranes (MagnaCharge, MSI, Inc., Westboro, MA) and probed using the P-radiolabeled rNS43-1-derived 430AS oligonucleotide (5`-GGAAATGCTTGCGGATG-3`). Following washing, membranes were autoradiographed for 10-60 min.

Northern Analysis

Northern analysis was performed using 5-10 µg of poly(A) RNA/sample as described previously (22), except that hybridization was performed at 42 °C and the blots were subsequently washed twice at room temperature in 2 SSPE (1 SSPE: 0.15 M NaCl, 10 mM NaHPO, 1 nM EDTA, pH 7.4), 0.1% SDS 10 min; once at room temperature in 0.1 x SSPE, 0.1% SDS 10 min; then once at 42 °C in 0.1 x SSPE, 0.1% SDS 60 min prior to autoradiography. In some experiments, blots were also washed an additional time at 60 °C in 0.1 x SSPE, 0.1% SDS.

Site-directed Mutagenesis

Site-directed mutagenesis was performed using the Altered Sites mutagenesis system according to the manufacturer's instructions (Promega). All mutations were confirmed by DNA sequence analysis using the methods described above. For each construct, oocytes were injected with 50 ng of capped RNA transcripts synthesized in vitro. Four days later 5-D activity was determined as described above. Activity was compared with that obtained in uninjected oocytes and those injected with an equivalent amount of RNA synthesized from the wild type cDNA in the pAlter vector (Promega).


RESULTS

The choice of tissue for constructing a cDNA library was based on expression studies in X. laevis oocytes (Fig. 1). Injection into oocytes of 5 ng of poly(A) RNA isolated from neonatal rat skin or rat placenta induced significant amounts of 5-D activity which manifested a K (using T as substrate) of 3 nM, consistent with the properties of a type III deiodinase(1, 3) . In contrast, injection of 50 ng/oocyte of poly(A) RNA isolated from the skin of lactating dams induced little or no 5-D activity, a finding in agreement with the low levels of activity previously noted in homogenates prepared from this tissue(3) .


Figure 1: Expression of 5-D activity in X. laevis oocytes following the injection of poly(A) RNA derived from rat placenta or rat skin of 7-day-old neonatal pups or a lactating dam. Control oocytes were not injected with RNA. Activity was determined in oocyte homogenates. Values represent the percentage of [I]T substrate (1 nM) converted to [I]T during the 2-h incubation period and are the mean of closely agreeing duplicate determinations.



Screening of a neonatal skin library by plaque hybridization under low stringency conditions with the coding region of the XL-15 amphibian type III deiodinase cDNA as a probe resulted in the identification and isolation of seven different clones. Partial DNA sequencing of several of the cDNA inserts from these clones revealed areas that had significant homology to the XL-15 cDNA. Two clones designated rNS27-1 and rNS43-1, containing 1.5- and 2.1-kb inserts, respectively, were selected for detailed expression studies and complete sequencing.

Functional characterization of the protein products of the isolated cDNA was carried out using the X. laevis oocyte translational assay system. Oocytes injected with RNA synthesized in vitro using clone rNS43-1 as template expressed high levels of 5-D activity as determined by the conversion of [I]T to [I]T. Such activity was dependent on the presence of relatively high concentrations of dithiothreitol (Fig. 2), requiring 50 mM dithiothreitol for maximal activity. In contrast, type I 5`-D activity induced by the injection of RNA derived from the G21 cDNA was near-maximal with only 0.1 mM dithiothreitol in the reaction mixture. Unlike the 5`-D activity of the type I deiodinase(18) , glutathione at concentrations of 5 and 50 mM did not support 5-D activity in oocyte membranes injected with rNS43-1-derived RNA (data not shown). Using 50 mM dithiothreitol as cofactor, kinetic analysis of the rNS43-1-induced 5-D activity revealed a K value for T of 1 nM. Using rT as substrate and 20 mM dithiothreitol as cofactor, no 5`-D activity was detected in rNS43-1-injected oocyte membrane fractions. Injection into oocytes of RNA derived from clone rNS27-1, which lacks sequences in the 5` region of the open reading frame of rNS43-1 (see below), did not induce 5-D activity (data not shown).


Figure 2: The effect of dithiothreitol concentration on the activity of the G21 and the rNS43-1 deiodinases. 5`-D or 5-D activity was determined in membranes isolated from X. laevis oocytes previously injected with RNA transcripts synthesized in vitro using the two cDNA clones as templates.



A comparison was made of the sensitivity of the rNS43-1 5-D and the G21 type I 5`-D to inhibition by propylthiouracil and aurothioglucose. As expected(8) , the G21-induced 5`-D activity was highly sensitive to inhibition by propylthiouracil with a concentration of 100 µM inhibiting activity by essentially 100% (Fig. 3A). In contrast, the induced rNS43-1 5-D activity was resistant to high concentrations of propylthiouracil. Similarly, the G21 deiodinase was at least 100-fold more sensitive than the rNS43-1 deiodinase to the inhibitory effects of aurothioglucose; 50% inhibition of G21-induced type I activity occurred at approximately 0.01 µM aurothioglucose, whereas 1 µM was required to obtain the same level of inhibition of the rNS43-1-induced activity (Fig. 3B). The studies depicted in Fig. 3were performed using different concentrations of dithiothreitol in the 5`-D and 5-D assays. Because the sensitivity of these processes to propylthiouracil can be influenced by the concentration of cofactor(23, 24) , we determined in other experiments that at equivalent concentrations of dithiothreitol (20 mM), the marked disparity in the inhibitory effects of propylthiouracil on the G21 and rNS43-1 deiodinases persist (data not shown).


Figure 3: Sensitivity of the G21 and rNS43-1 deiodinase activity to the inhibitory effects of propylthiouracil (A) and aurothioglucose (B). Membrane preparations isolated from oocytes previously injected with RNA synthesized in vitro using the G21 type I deiodinase or rNS43-1 cDNAs as templates were assayed for 5`-D or 5-D activity in the presence of the concentrations of the inhibitors noted. Control incubations were performed in aliquots of the same membrane preparations in the absence of inhibitors. Dithiothreitol concentrations were 20 and 50 mM in the 5`-D and 5-D assays, respectively.



A diagram of the rNS27-1 and rNS43-1 cDNAs is shown in Fig. 4. The sequence of the rNS27-1 cDNA is identical to nucleotides 549-2081 of the rNS43-1 cDNA. The latter cDNA contains an open reading frame of 834 nucleotides encoding a protein of 278 amino acids with a predicted molecular mass of 31.6 kDa. Of note is an in-frame TGA codon, presumably coding for selenocysteine, beginning at nucleotide 740 and located in a region of high homology to the XL-15 protein and the mammalian type I deiodinases (see below). A search of GenBank for homologous nucleotide sequences identified the XL-15 cDNA, previously isolated mammalian type I deiodinase cDNAs (from rat, human, dog), and a 283-nucleotide partial cDNA isolated from a human skeletal muscle library (accession number Z19511) which shows 80% sequence identity to nucleotides 1-196 of the rNS43-1 cDNA. The identity of this human cDNA has not been established.


Figure 4: Structural features of the rNS27-1 and rNS43-1 cDNAs. The nucleotide sequence of cDNA 27-1 is identical to nucleotides 549-2081 of cDNA rNS43-1 and, in addition, contains a longer poly(A) tail. Nucleotides 1-196 of rNS43-1 (open bar) show 80% identity to a partial cDNA isolated from a human skeletal muscle library (GenBank accession number Z19511). Nucleotides 311-1144 of rNS43-1 constitute an 834-nucleotide open reading frame and show significant homology to previously isolated cDNAs for the mammalian type I and amphibian type III deiodinases. Included in this reading frame is a TGA codon which presumably codes for selenocysteine.



Because of the unexpected homology of a portion of the rNS43-1 cDNA to a human skeletal muscle cDNA, and the finding that previously isolated cDNAs for other deiodinases have relatively short 5`-untranslated regions (6-24 nucleotides), reverse transcriptase-PCR was conducted to determine the extent to which sequences in the 5` region of the rNS43-1 cDNA were derived from the corresponding rat deiodinase mRNA. The experimental approach is depicted in Fig. 5A. Reverse transcription was carried out using poly(A) RNA from rat neonatal skin or rat placenta and the rNS43-1-derived antisense oligonucleotide 1148AS as a primer. The resulting cDNA was then used in the PCR with a number of different primer pairs derived from the rNS43-1 cDNA sequence (see ``Materials and Methods'' and Fig. 5A). In addition, to detect any contamination with the rNS43-1 plasmid during the procedures, a primer pair which included the Bluescript/M13 reverse sequencing primer was also used. PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining followed by transfer of the products to nylon membranes and Southern blotting using the nested oligonucleotide 430AS as a probe. Expected amplicon sizes for each primer pair, based on the rNS43-1 sequence, were 882 (rev/668AS), 553 (133S/668AS), 460 (226S/668AS), 424 (262S/668AS), and 367 (319S/668AS) nucleotides.


Figure 5: Reverse transcriptase-PCR studies conducted to define the 5` nucleotide sequences of the rNS43-1 cDNA that are derived from the type III 5-D mRNA. A, location of the oligonucleotides used as primers and the sizes of the expected amplicons in the reverse transcriptase-PCR experiments. Oligonucleotide 1148AS was used in the reverse transcriptase step, 668AS was used as the antisense primer in all PCR, and 430AS was utilized as a probe in Southern analysis. B, Southern blot of PCR products. Templates and PCR primer pairs used in the reactions are designated above each lane. After hybridization and washing, the membrane was autoradiographed for 10 min.



As shown in Fig. 5B, control PCR reactions using the rNS43-1 plasmid as template (lanes 7-11) demonstrated the expected size products for all primer combinations, thus confirming the usefulness of these primer pairs for amplifying the desired rNS43-1-related sequences. In contrast, only primer pairs 262S/668AS and 319S/668AS gave demonstrable products of the correct size by ethidium bromide staining (data not shown) and Southern blotting when cDNA derived from rat neonatal skin (lanes 1-5) or rat placenta (lanes 12-16) were used as templates in the PCR. No amplification was noted with the primer pair rev/668AS using either cDNA preparation (lanes 1 and 12, respectively). Nor was amplification noted when (a) reverse transcriptase (lanes 6, 17, and 18) or (b) RNA (lanes 19-21) were omitted from the initial cDNA synthesis step or when (c) PCR reaction tubes contained water in place of an aliquot of the cDNA synthesis mixture (data not shown). These results strongly suggest that sequences in the rNS43-1 cDNA insert located 5` to oligonucleotide 262S are not present in the type III deiodinase mRNA from neonatal skin and placenta. At present, the identity of these sequences is uncertain; their presence in the rNS43-1 cDNA may represent a cloning artifact secondary to the inclusion of two unrelated cDNA inserts in this clone.

The nucleotide and deduced amino acid sequence of the rNS43-1 cDNA are shown in Fig. 6. Uppercase letters represent the sequences proven for the type III deiodinase and start at the point corresponding to oligonucleotide 262S. The 261 nucleotides of unknown origin in the region 5` to this are shown in lowercase letters.


Figure 6: Nucleotide and deduced amino acid sequence of the rNS43-1 cDNA. Uppercase letter nucleotides refer to those demonstrated by reverse transcriptase-PCR (Fig. 5) to be derived from the 5-D mRNA. Lowercase letter nucleotides are of uncertain origin. Underlined sequences correspond to the four sense oligonucleotides used as primers in the reverse transcriptase-PCR reactions depicted in Fig. 5. SC, selenocysteine.



Northern analysis of rat tissues was performed using the rNS27-1 cDNA, which contains only deiodinase cDNA sequences, as a probe (Fig. 7). Using poly(A) RNA from neonatal skin and placenta, a single RNA species of 2.2 kb in size was noted. No hybridization was detected to RNA derived from liver, kidney, or the skin of postpartum (lactating) rats, all tissues which contain little or no type III deiodinase activity. In the cerebral cortex of euthyroid adult rats, a more complex hybridization pattern was noted with several faint bands visualized. Of note, the two larger RNA species of approximately 3.3 and 3.6 kb appeared more abundant after T administration to induce hyperthyroidism. Type III activity in this tissue was also increased with T administration (data not shown). In both skeletal muscle and cerebral cortex, a smaller band at 1.6 kb was faintly visible. These hybridization patterns did not significantly change when blots were washed at 60 °C.


Figure 7: Northern analysis of poly(A) RNA derived from several rat tissues using the rNS27-1 cDNA as probe. Nonadjacent lanes from a single blot are shown. RNA samples of cerebral cortex were prepared from a euthyroid rat as well as from one which had been injected with T for 4 days prior to sacrifice to render it hyperthyroid.



Previous studies of the rat type I deiodinase and the XL-15 type III deiodinase have demonstrated that the presence of selenocysteine is critical for the catalytic activity of the protein(6, 8) . Thus, substitution of cysteine for selenocysteine in these proteins reduces expressed catalytic activity by approximately 76-90%, in spite of increased translational efficiency of the cysteine mutant(25) . To investigate the importance of selenocysteine to the functional activity of the rNS43-1-encoded deiodinase, site-directed mutagenesis of the TGA codon was performed. Conversion of this triplet to a TAA stop codon, or a TTA leucine codon, abolished expression in oocytes. The TGT cysteine mutant was also essentially inactive, manifesting less than 5% of wild type 5-D activity in four experiments. To ensure that this lack of activity of the cysteine mutant did not result from some other unplanned alteration in structure, the mutant cDNA was sequenced in its entirety and found to be otherwise identical to the rNS43-1. In addition, RNA derived from a second isolated cysteine mutant clone also exhibited a comparably low level of activity when expressed in X. laevis oocytes.

A comparison of the deduced amino acid sequence of the rNS43-1 protein with that of other deiodinases (6, 8) revealed 58% sequence identity and 73% similarity with the XL-15 type III deiodinase, but only 39 identity and 57% similarity to the rat G21 type I deiodinase. On alignment of the deiodinase protein sequences published to date(6, 8, 9, 10) , several regions of conservation in the mid- and carboxyl-terminal portions of the molecules are evident (Fig. 8). High homology is noted in the regions surrounding the selenocysteine (designated ``X'' at amino acid 144 in r5DIII [rNS43-1]) and surrounding a histidine residue (amino acid 176) demonstrated by Berry et al.(26) to be essential for type I deiodinase activity. Amino acid identity in these two regions is 83 and 75%, respectively.


Figure 8: Comparison of the deduced amino acid sequences of the rat type III deiodinase (rDIII; rNS43-1 cDNA) with the X. laevis type III deiodinase (xDIII, XL-15 cDNA), the rat type I deiodinase (rDI; G21 cDNA), and the human and dog type I deiodinases (hDI and dDI, respectively). Amino acid residues conserved in all five proteins are shown with a black background, whereas residues in boxes are common to four of the proteins or found only in the two type III deiodinases or found only in the three type I deiodinases. X, selenocysteine.




DISCUSSION

The structural and functional data presented herein, as well as the patterns of tissue expression as determined by Northern analysis, indicate that the rNS43-1 cDNA encodes the rat type III 5-D. Of paramount importance is the finding that this cDNA contains an in-frame TGA codon. Although we have not directly demonstrated that the rNS43-1-encoded enzyme is a selenoprotein, the mutagenesis studies, the strong amino acid conservation present in all the deiodinases in the region of this codon, and preliminary studies identifying a selenocysteine insertion element in the 3`-untranslated region of the cDNA()provide compelling evidence that the TGA encodes selenocysteine rather than a termination signal. Thus, the type I and type III deiodinases constitute a family of structurally related selenoproteins. To date, the type II 5`-D has not yet been purified, nor have cDNAs been isolated. The structural characteristics of the type II enzyme therefore remain uncertain.

The finding that the rNS43-1 cDNA encodes a selenoprotein has important implications for the study of selenoproteins in general and deiodinases in particular. First, previous studies by Berry et al.(12) have demonstrated that mutagenesis of the selenocysteine in the type I deiodinase to cysteine renders that enzyme insensitive to propylthiouracil and gold compounds. This finding suggested that the unique biochemical properties of selenocysteine confer sensitivity to these inhibitors. However, the present studies, as well as our previous demonstration that the XL-15 deiodinase is resistant to these agents (6), indicates that factors other than the presence of selenocysteine, such as those related to protein structure or kinetic mechanisms, must dictate insensitivity to propylthiouracil and aurothioglucose in the type III deiodinases.

Second, it has previously been demonstrated that type III deiodinase activity in rat brain, placenta, and skin are largely preserved in the face of nutritional selenium deprivation severe enough to markedly lower type I protein and activity levels in the liver and kidney (13-15). Our demonstration that the type III deiodinase in this species is a selenoprotein indicates that selenium stores in the former tissues are preserved in the face of moderate selenium deficiency. Although prior studies of selenium turnover have demonstrated that the brain, like the thyroid gland, testes, and other endocrine organs (27-29), has a marked propensity to conserve selenium, the data presented herein are the first to suggest that placenta and skin share this property. This is of particular interest given that placental selenium stores must be accumulated de novo during pregnancy, a circumstance which presumably should predisposes this organ to selenium deficiency in the setting of nutritional selenium deprivation.

Third, the inability of others using Se to label a candidate type III deiodinase protein of 32 kDa in appropriate tissues (e.g. brain, placenta) points out the insensitivity of these methods to identify selenoproteins(11, 27) . This is not surprising given that little is known about the rates of entry of selenium into various tissues, selenium pool sizes, or the abundance and turnover rates of most selenoproteins.

The importance of selenocysteine to the catalytic activity of the deiodinases is reaffirmed by the present studies. Indeed, unlike the type I enzymes and the XL-15 deiodinase which retain a significant, albeit substantially reduced (10-24%), level of activity when cysteine is substituted for selenocysteine(6, 8) , the cysteine mutant of the rNS43-1 cDNA is essentially inactive. Given that activity was determined using an in vivo expression system, it is uncertain whether this lack of activity reflects an intrinsically inactive protein or whether expression of the protein was impaired due to a decreased half-life of the injected RNA or the cysteine mutant protein. However, the latter two possibilities appear unlikely given that the cysteine mutants of other deiodinases induce significant levels of activity in X. laevis oocytes(6, 8) .

Northern analysis revealed hybridization of the type III deiodinase probe to multiple species in different tissues. In neonatal skin and placenta only a single transcript was identified that was somewhat larger in size than the 1.8-kb size of the rNS43-1 type III deiodinase sequences. In the cerebral cortex, however, larger hybridizing species were observed, and a low intensity 1.6-kb band was noted in both cerebral cortex and skeletal muscle. Whether such species represent alternatively processed transcripts or products of a related gene remains uncertain. Of note, relatively high levels of 5-D activity have been measured in fetal rat skeletal muscle(30) . In the adult rat, however, levels in this tissue are only 2% of that found in cerebral cortex or skin(30) .

A comparison of the deduced amino acid sequences of the five mammalian and amphibian deiodinase proteins reported to date (6, 8, 9, 10) indicates that the rNS43-1 protein is more closely related to the amphibian XL-15 enzyme than to the mammalian type I deiodinases. This finding is consistent with the similar functional characteristics of the 5-D activity displayed by these two proteins. In contrast, the activity of the rNS43-1 deiodinase differs markedly from that of the mammalian type I enzymes in that it manifests no 5`-D activity and is able to catalyze efficiently the 5-deiodination of non-sulfated iodothyronine substrates.

Although little primary sequence homology is present in the amino-terminal portions of the deiodinases (Fig. 8), all contain a region of marked hydrophobicity, which in the rNS43-1 protein encompasses the initial 42 amino acid residues. Toyoda et al.(31) have provided evidence that the analogous hydrophobic region in the rat type I deiodinase serves to anchor the protein in the endoplasmic reticulum. Given that the type III deiodinases are also membrane proteins, such regions in the rNS43-1 and XL-15 proteins likely serve a similar function. In other studies, these investigators have recently demonstrated that mutation of a phenylalanine at position 65 in the human and rat type I deiodinases results in marked inefficiency of these enzymes in utilizing rT as a substrate for 5`-deiodination(10) , but does not affect the inner ring deiodination of appropriate sulfated substrates(4) . The observation that the rNS43-1 and XL-15 deiodinases lack a phenylalanine at the corresponding locations provides further evidence that such a residue is not critical for 5-deiodination of T or T.

The ability of the mammalian type III deiodinase to convert T and T to metabolically inactive metabolites, as well as its patterns of expression and regulation in the placenta during fetal development, and in the adult brain, suggest that it plays a protective role in preventing exposure of tissues to excessive levels of active thyroid hormones(1) . This concept is strengthened by the observations of Silva and Matthews (32) that in the cerebral cortex of neonatal rats the residency time of T, a parameter likely dependent in large part on the activity of the type III deiodinase, is critical to thyroid hormone homeostasis in this organ. The isolation of the rNS43-1 cDNA thus provides an important tool for the further study of iodothyronine metabolism. In particular, the development of animal models with targeted deletion or overexpression of the type III deiodinase gene will allow a direct assessment of its physiologic role in thyroid hormone metabolism and a better understanding of thyroid hormone action during development and in selected tissues such as the brain.


FOOTNOTES

*
This work was supported by the National Institutes of Health in the form of Grants DK-42271 (to D. L. S.), DK-08671 (to S. L. W.), and HD-27706 (to M. J. S.) and by Norris Cotton Cancer Center Core Grant CA 23108. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U24282.

§
Current address: Dept. of Biology, Keene State College, Keene, NH 03431.

To whom all correspondence should be addressed: Dartmouth Medical School, One Medical Center Dr., Lebanon, NH 03756. Tel.: 603-650-7910; Fax: 603-650-6130.

The abbreviations used are: T, thyroxine; T, 3,5,3`-triiodothyronine; T, 3,3`-diiodothyronine; rT, 3,3`,5`-triiodothyronine; 5-D, 5-deiodinase; 5`-D, 5`-deiodinase; PCR, polymerase chain reaction; kb, kilobase pair(s).

B. Moyer and D. L. St. Germain, unpublished observations.


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