Cloning and Expression of the Chicken Type 2 Iodothyronine 5'-Deiodinase*

Balazs GerebenDagger §, Tibor Bartha§, Helen M. TuDagger , John W. HarneyDagger , Peter Rudas§, and P. Reed LarsenDagger parallel

From the Dagger  Thyroid Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115 and the § Department of Physiology and Biochemistry, University of Veterinary Science, P.O. Box 2, Budapest H-1400, Hungary

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The type 2 iodothyronine deiodinase (D2) is critical for the intracellular production of 3,5,3'-triiodothyronine from thyroxine. The D2 mRNA of higher vertebrates is over 6 kilobases (kb), and no complete cDNA clones have been reported. Using 5'- and 3'-rapid amplification of cDNA ends and two cDNA libraries, we have cloned the 6094-base pair full-length chicken D2 cDNA. The deduced protein is ~31 kDa and contains two in-frame UGA codons presumably encoding selenocysteine. One of these is in the highly conserved active catalytic center; the other is near the carboxyl terminus. Unusual features of the cDNA include a selenocysteine insertion sequence element ~4.8 kb 3' to the UGA codon in the active center and three short open reading frames in the 5'-untranslated region. The Km of D2 is ~1.0 nM for thyroxine, and the reaction is insensitive to inhibition by 6-n-propylthiouracil. Chicken D2 is expressed as a single transcript of ~6 kb in different brain regions and in the thyroid and lung. Hypothyroidism increases D2 mRNA in the telencephalon. Unlike in mammals, D2 mRNA and activity are expressed in the liver of the chicken, suggesting a role for D2 in the generation of plasma 3,5,3'-triiodothyronine in this species.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

3,5,3'-Triiodothyronine (T3),1 which accounts for the biological effects of the thyroid prohormone thyroxine (T4), is generated by the removal of a single iodine from its phenolic ring. This deiodination process is catalyzed by two different enzymes, types 1 and 2 deiodinase. Type 1 deiodinase (D1) is a high Vmax, high Km (~10 µM for T4) enzyme, which is highly sensitive to inhibition by 6-n-propylthiouracil (PTU), contains selenocysteine in its active center and can also catalyze the removal of iodine from the tyrosyl ring of T4 or T3 or their SO4 derivatives. The presence of a PTU-insensitive 5'-deiodinase termed D2 was first suspected from in vivo studies showing that T3 generation from T4 in the hypothyroid rat pituitary was not inhibited by PTU (1). In contrast to D1, D2 has an exclusive 5'-activity. It has a low Vmax with high affinity for T4 and rT3 with the Km values in the nanomolar range. D2 plays a particularly important role in the thyroid status of the central nervous system, since it produces more than 75% of the nuclear T3 in the cerebral cortex (2). To date, D2 activity has been reported in the chicken brain, and the low Km and the increased D2 activity during hypothyroidism are similar to those characteristics of the mammalian D2 (3). Limited information regarding the presence of D2 activity in other avian tissues is available, although PTU-insensitive 5'-deiodinase activity has been found in the quail liver (4).

In the avian species, as in other vertebrates, thyroid hormone is required for many critical steps in development and maturation (5). Triiodothyronine specifically increases at the time of pipping when the animal begins active respiration (6, 7). This is analogous to the marked increase in circulating T3 in human neonates, which is thought to occur through a combination of the marked increase in thyrotropin at delivery and increased fractional T4 to T3 conversion perhaps involving activation of D2 in neonatal brown fat (8-10). In the chicken, the increase in T3 is at least partly due to a decrease in hepatic type 3 iodothyronine deiodinase (D3) activity, but hepatic D1 activity changes very little during that period (11). An increase in D2 in a tissue such as liver could be an important aspect of the perihatching changes in birds.

The sequences of the D2 coding regions of several species have been reported (Rana catesbeiana (GenBankTM accession number L42815) (12), human (GenBankTM Z44085 and U53506) (13, 14), rat (GenBankTM U53505) (14), and a fish (GenBankTM S83468) (Fundulus heteroclitus) (15)). However, the only full-length D2 reported is the ~1.4-kb cDNA of Rana. This corresponds to the most abundant D2 transcript found in this species by Northern blot, although several longer (up to 7 kb), less abundant transcripts were also detected (12). This may be important, since the D2 mRNA in the higher vertebrates is 6 kb or more. The deduced protein sequences of D2 show that the active centers of the rat (rD2) and human D2 (hD2) proteins are highly similar to those of D1 and D3. In this region, the mRNAs contain an in-frame UGA codon that encodes selenocysteine instead of serving as a stop codon (16). In order for recoding of UGAs to occur in eukaryotic mRNAs, a stem-loop structure with specific conserved nucleotides, the SECIS element, must be present in the 3'-untranslated region (17, 18). We have recently observed that the hD2 mRNA contains a SECIS element in the extreme 3' terminus of the mRNA, almost 5 kb 3' to the UGA codon (19). It is not clear whether this distance is typical for the D2 mRNA of higher vertebrates in general or is unique to the human species.

The goal of the present studies was to clone the full-length cDNA of the chicken D2, determine its kinetic characteristics, compare its tissue distribution with those of other species, identify the SECIS element, and analyze its function. The identification of this cDNA will then permit ontological studies of the roles of D1, D2, and D3 in the ontogeny of the vertebrate in a readily controlled paradigm.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Library Screening-- A chicken embryonic lambda  gt11 cDNA library was kindly provided by Dr. B. Vennström (Karolinska Institute, Stockholm, Sweden) (20), and a lambda  ZAP II chicken pituitary cDNA library was provided by Dr. Y. M. Sun (University of Cape Town, South Africa) (21). For competent cells, Y1090- and XL-Blue bacteria were used, respectively, and the plating and screening were performed using standard techniques. The chicken embryonic library was screened using a full-length human D2 coding probe (Genethon clone Z44085, kindly provided by Dr. D. L. St. Germain and V. A. Galton, which they and we have previously analyzed; Refs. 13 and 14). Five positive clones were identified, and the longest fragment (fragment B in Fig. 1) was subcloned by PCR into pGEM-T vector using lambda  phage-specific primers. Probes prepared from 5' or 3' portions of the subcloned fragment did not hybridize with other phage clones. The chicken pituitary library was screened using a 508-bp PCR product (nucleotides 5525-6032) obtained from the most 3' portion of the 3'-rapid amplification of cDNA ends (RACE) product (see below). Five clones (see below) were isolated and converted to phagemid using in vivo excision.

5'-RACE-- Three antisense nested oligonucleotides (Tp17, Tp18, and Tp22) were designed for the chicken D2 coding region on the basis of the sequence of the 5' portion of fragment B. (All cD2 oligonucleotides are shown in Table I). The 5'-RACE system of Life Technologies. was used according to the instructions of the manufacturer. Briefly, total RNA was isolated from the telencephalon of 17-day-old New Hampshire chicken embryo using the guanidium thiocyanate/phenol/chloroform extraction method (22) by Trizol (Life Technologies). Reverse transcription was started with the gene-specific primer Tp22. First strand product was digested by RNase H/T1, and poly-C tailing was performed by terminal deoxynucleotidyl transferase. Tailed cDNA was amplified by the abridged anchor primer of the kit (5'-GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3') and the Tp17 nested gene-specific antisense primer using the following PCR conditions: 94 °C for 2 min; 94 °C for 50 s, 63 °C for 40 s, 72 °C for 2 min; 35 cycles of 72 °C for 5 min. After electrophoresis, a ~1-kb band was reamplified by a second PCR reaction, using the abridged universal amplification primer of the kit (5'-GGC CAC GCG TCG ACT AGT AC-3') and the nested antisense Tp18 primer. This product, fragment A in Fig. 1, was purified, ligated into pGEM-T plasmid (Promega), and sequenced. The sequence contained a 109-bp overlap with fragment B (Fig. 1).

3'-Long Expand RACE PCR-- Total RNA was isolated from the brain of a 2-week-old broiler chicken, using Trizol (Life Technologies). The reverse transcription was started with the Tp27 primer, which includes a poly-T sequence and a NotI site. Two nested antisense primers were prepared for the 3' extension of Tp27, the 5' Tp41 (outer) and 3' Tp42 (inner). Two cD2-specific sense primers were also designed, the 5' Tp29 (outer) and 3' Tp30 (inner) (see Table I). After the RT reaction, a PCR using Tp29 and Tp41 was performed. The reaction mixture was prepared according to the instructions of the Expand Long Template PCR System (Roche Molecular Biochemicals) using system number 3. In the first cycle the annealing temperature was 50 °C, and in the following cycles this was increased to 56 °C. PCR cycles were as follows: 94.5 °C for 2 min; 10 cycles of 94 °C for 20 s, 56 °C for 30 s, and 68 °C for 6 min; 15 cycles of 94 °C for 20 s, 56 °C for 30 s, and 68 °C for 2 min with 20 s extension/cycle; and 68 °C for 7 min. In the second PCR, the diluted product of the first PCR was amplified as template using the Tp30 and Tp42 nested primers. Other conditions were the same, except the 50 °C annealing cycle was omitted. The ~3-kb RACE fragment (fragment C in Fig. 1) was subcloned into a pGEM-T vector. The fragment C had a 182-bp overlap with the 3' portion of fragment B (Fig. 1).

Assembling of cD2 by Long Expand RT-PCR-- Total telencephalic RNA of an adult broiler chicken was reverse transcribed using Tp58. The cDNA was amplified by the proofreading polymerase enzyme mix of the Expand Long Template PCR System (Roche Molecular Biochemicals) system number 3 according to the instructions of the manufacturer. PCR cycles were as follows: 94 °C for 2 min; 10 cycles of 94 °C for 10 s, 65 °C for 30 s, and 68 °C for 5 min; 25 cycles of 94 °C for 10 s, 65 °C for 30 s, and 68 °C for 5 min with 20 s extension/cycle; and 68 °C for 7 min. A sense oligonucleotide starting at position 33 of the full sequence and incorporating a SacII-containing tail (Bp15) was designed on the basis of the sequence of the 5'-RACE product. Bp12, located 3' to a unique BsaBI site (position 3249), was used as antisense primer (Fig. 1). The PCR product was ligated into the pGEM-T vector and confirmed by sequencing. A clone with an insert in the proper orientation was digested by BsaBI and at the 3' NotI site from the vector. The 3'-RACE product was cut out from pGEM-T by the same enzymes, and the fragment was ligated into the former clone. The BsaBI junction was checked by restriction digestion, PCR, and sequencing. The assembled native cD2 cDNA contains nucleotides 33-6094. The GenBankTM accession number for the full-length cD2 sequence is AF125575.


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Fig. 1.   Schematic diagram of the cloning strategy used to obtain the full-length chicken D2 cDNA. Fragment B was identified by probing a chicken embryonic cDNA library with coding sequences from the human D2. The 5' and 3' sequences of this clone were used to design oligonucleotides for the 5'- and 3'-RACE reactions, which generated the missing fragments A and C. The terminal 508 bp of fragment C were also used to probe a cDNA library prepared from mRNA from adult chicken pituitary glands. This resulted in the isolation of several fragments of identical sequence or with minor polymorphisms (see "Results"). The diagram is not proportional.

Eukaryotic Expression Vectors and Constructs-- A pUHD10-3 based, modified vector was the parent vector in this study to express cD2 in a transient system in HEK-293 cells (23). The parent vector was prepared by mutating the HindIII site and inserting a truncated, but fully active rat D1 (rD1) coding region 3' to a SacII and EcoRI site. A truncation of rD1 was performed at the HindIII site close to the 3'-end of the coding region, and a stop codon was inserted (24). A minimal rD1 SECIS element was then inserted between the stop codon and a NotI site (25). For kinetic studies of the transiently expressed cD2, the RT-PCR fragment containing the cD2 coding region and the translation initiation sequence (560-1491) was generated using the Bp3 and Bp5 oligonucleotides (Table I). The reaction was performed using the following program: 94.5 °C for 1 min; 33 cycles of 94 °C for 50 s, 60 °C for 40 s, and 72 °C for 90 s; 72 °C for 8 min. The fragment was confirmed by sequencing and then inserted between the EcoRI and HindIII sites of the parent vector, thus removing the rD1 coding region. The nearly full-length assembled cD2 sequence was also subcloned into the same parent vector between the SacII and NotI sites, removing both the rD1 coding and SECIS element sequences.

                              
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Table I
cD2 oligonucleotide sequences (5'-3')
Chicken D2 sequences are indicated by capitals, and non-cD2 sequences are indicated by lowercase letters.

SECIS Activity Test Plasmids-- For testing the SECIS activity of cDNA fragments using rD1 as a reporter gene, we prepared a further modification of the pUHD10-3 vector. The parent vector was cleaved by BamHI, and a polylinker region was inserted in two orientations (Fig. 2). To remove the rD1 SECIS element and reinsert a stop codon (TAG) at the end of the truncated rD1 coding region, the resulting plasmids were digested with HindIII and XbaI, and the ends were filled in by Klenow treatment and ligated. This results in the reformation of an XbaI (TCTAGA) site that places a TAG codon conveniently in frame 3' to the rD1 coding region. The plasmid pHT1427 is identical to pHT1429 except that the polylinker is in the reverse orientation. The 3' cD2 cDNA fragments identified in the pituitary library were cut from pBluescript (Stratagene) using NotI and ApaI sites located in the multiple cloning site and inserted into pHT1427. The 911-X fragment was also inserted into pHT1429 to check the influence of the inverted orientation on the SECIS activity of the fragment. The 3-1 fragment in pHT1427 was truncated by BamHI partial digestion using the endogenous BamHI site (position 5705 in cD2) and the BamHI sites, located 5' (from pBluescript) and 3' (from pHT1427) from the cD2 part of the sequence. In this way, the 389-bp cD2 fragment between the poly(A) tail and position 5705 (pHT1450) and the more 5' ~710-bp fragment (~4995-5705, pHT1452) were tested separately for SECIS activity. For testing the SECIS activity of the 3'-RACE fragment and the longest clone (2-1) from the pituitary library, the ~2.8-kb XbaI (position 3291) and NotI (in Tp27) digest of the 3'-RACE PCR product and the same region from the pituitary library clone (XbaI-ApaI) was inserted into pHT1427 (p#16 and p#19, respectively). To compare the activity of the D1 and D2 SECIS elements, the wild-type minimal rD1 SECIS element (1533-1585 in the rD1 cDNA GenBankTM sequence accession number X57999 (25)) in pHT1427 (pBG12) was compared with pHT1450.


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Fig. 2.   Vectors used to test the SECIS activity of the various 3' cDNA fragments. These are modified from earlier versions of pUHD10-3 (23) as described under "Experimental Procedures." Rat type 1 deiodinase (D1) is the reporter gene, which requires a functional SECIS element for expression. pHT1427 and pHT1429 are identical except that the polylinker is in the reverse orientation.

DNA Transfection-- Transient expression was performed by introducing the cD2 cDNA containing expression vectors into human embryonic kidney cells (HEK-293) by calcium phosphate precipitation (26). HEK-293 cells do not express D1 or D2 after transfection with empty vector. For each transfection, 10 µg of pUHD10-3-based vector was used, cotransfected with 4 µg of pUHD-15, which is required for the transcriptional activation of the pUHD10-3 promoter (23). For the SECIS test constructs, transfection efficiency was monitored by the cotransfection of 3 µg of TKGH, and human growth hormone was assayed in the media (24). Each SECIS test construct was expressed in duplicate in at least three separate transfections.

Deiodinase Assays-- Cell homogenates were assayed in duplicate for either D2 or D1 activity. D2 assays contained 30-150 µg of cell sonicate protein, 125I-labeled T4, 2 nM T4, and 20 mM DTT in 0.1 M potassium phosphate, 1 mM EDTA, pH 6.9 (PE buffer) in a final volume of 300 µl. For D1 assays, 125I-labeled rT3, 1 µM rT3, and 10 mM DTT was used. Incubation was carried out for 60 min at 37 °C, and 125I- was separated by trichloroacetic acid precipitation (27). Under the conditions used, deiodination was linear both with time and protein, and protein was adjusted to consume <30% of the substrate. For the determination of the Km for T4 or rT3 of the transiently expressed cD2, varying concentration of unlabeled T4 (1, 1.5, 3, and 10 nM) or rT3 (1, 1.5, 2.5, and 7.5 nM) was used in PE buffer containing 20 mM DTT, 1 mM PTU, and 125I-labeled T4 or rT3, respectively. Activity was expressed as pmol/min/mg of protein. For the SECIS test constructs, activity was expressed as D1 activity/µl of cell sonicate (~11 µg of protein/µl) × 1000 corrected for human growth hormone (cpm/100 µl of media). A liver sample from an adult chicken was homogenized in PE buffer containing 0.25 M sucrose and 10 mM DTT. 300 µg of protein was assayed in duplicate for 5'-deiodinase activity in a final volume of 300 µl of PE buffer containing 20 mM DTT, 125I-labeled T4, and 2 or 100 nM T4 or 10 µM reverse T3. Incubation was for 120 min at 37 °C. Activity was expressed as fmol/h/mg of protein. For the Km (T4) determination, 1, 1.5, 3, and 10 nM T4 was used in the presence of 125I-labeled T4, 20 mM DTT, and 1 mM PTU.

Northern Blot-- For the multitissue blots, the organs of an adult chicken were used. To induce hypothyroidism, a 1-week-old broiler chicken was surgically thyroidectomized, and one week after the surgery the telencephalon of hypothyroid and euthyroid control animals were quickly removed and frozen. Serum T3 and T4 were monitored by radioimmunoassay (28). Total RNA was isolated by the single-step method of Chomczynski (22), and 30 µg was run in 1% formaldehyde gel. Equal loading and transfer efficiency was monitored by ethidium bromide staining. Animal experiments were approved by the Animal Experiments Board of the University of Veterinary Science, Budapest. For D2, a 450-bp-long cDNA probe was used, generated by PCR from the coding region using Tp21 and Tp22 (positions 844-1293). The fragment was either labeled by [alpha -32P]deoxycytidine triphosphate by the random hexamer method (Prime-It II; Stratagene) used for multitissue blots or digoxigeninized by linear PCR using Tp22 oligonucleotide and DIG-dUTP (used for the hypothyroid blot). For beta -actin, a 741-bp-long chicken beta -actin coding region fragment (312-1053 in the GenBankTM sequence accession number L08165) was obtained by RT-PCR (using the primers; sense: 5'-ATG GAG AAG ATC TGG CAC CA-3'; antisense: 5'-TCT TGA TCT TCA TGG TGC T-3'), cloned and confirmed by sequencing. It was labeled by [alpha -32P]deoxycytidine. All blots were hybridized under high stringency conditions. Prehybridization was carried out at 42 °C for 1-3 h and hybridization for 16-20 h at the same temperature. Composition of the solution for the radioactive probe was 50% formamide, 5× SSPE, 5× Denhardt's solution, 10% dextran sulfate, sodium salt (Mr 500,000), 50 µg/ml salmon sperm, 1% SDS; composition of the nonradioactive probe was 50% formamide, 5× SSPE, pH 7.4, 5× Denhardt's solution, 100 µg/ml salmon sperm, 0.1% SDS. All blots were washed as follows: 2× SSC, 0.1% SDS for 20 min at room temperature three times; 2× SSC, 0.1% SDS for 20 min at 42 °C; 0.5× SSC, 0.1% SDS 20 min at 42 °C. Radioactive blots were autoradiographed for 46 h (for D2) or 3 h for beta -actin with an intensifying screen at -80 °C. Nonradioactive signals were detected overnight by the Dig Nucleic Acid Detection Kit (Roche Molecular Biochemicals). Densitometry was carried out by the NIH Image Software version 1.61. As denominator, the density of the 28 S band was used.

Sequencing-- The fragments were sequenced by automated sequencing in their entirety and partly by manual sequencing (fmol DNA Sequencing System (Promega) and Sequenase (U. S. Biochemical Corp./Amersham Pharmacia Biotech).

Reagents-- 125I-Labeled iodothyronines and [alpha -32P]deoxycytidine were purchased from DuPont Life Science Products and Isotope Institute Ltd. (Budapest, Hungary). Other chemicals were of molecular biology or reagent grade. All primers were synthesized by Life Technologies.

Sequence Analyses-- The Sequence Analysis Software Package of the Genetics Computer Group (University Research Park, Madison, WI) was used to analyze nucleotide and protein sequences.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of the Chicken D2-- Screening of the chicken embryonic library with hD2 provided the 2250-bp fragment B (corresponding to 826-3076 in the full sequence), which contained a large part of the coding region and a portion of the 3'-UTR (Fig. 1). The fragment could be aligned to the Z44085 cDNA showing a 77% identity to hD2 (13, 14). This library fragment provided the basis for the 5'-RACE, which produced fragment A, a 934-bp cDNA encoding the 595-bp 5'-UTR and the remaining portion of the coding region (fragment A in Fig. 1). Fragment C, containing the SECIS element, was obtained by two parallel approaches, i.e. long expand 3'-RACE PCR followed by a screening of chicken pituitary cDNA library with the 3'-RACE fragment. The latter yielded clones of ~3550 (2-1), ~2290 (6-2), ~2060 (911-X), ~1100 (3-1), and ~710 bp (4-1), all containing a 3' poly(A) tail. Although they were virtually identical in the overlapping sequences, two fragments (3-1 and 6-2 and in addition that from the 3'-RACE reaction) contained a G at position 5706 creating a BamHI site, while in three others (2-1, 911-X, 4-1) this restriction site was absent. In the 2-1 fragment, G was replaced by A at this position. T was present at position 5627 in the BamHI-positive 6-2 clone and the 3'-RACE product, while the 2-1 BamHI negative clone contained an A at this position. The complete cD2 cDNA is 6094 nucleotides exclusive of the poly(A) tail and has the GenBankTM accession number AF125575.

Sequence of the cD2 cDNA-- In the 595-bp-long 5'-UTR, three short open reading frames (sORFs) were present (positions 241-249, 408-434, and 496-537). Two of these end with the universal stop codons TAG and TAA, but the most 3' ends with TGA (Fig. 3). The translation initiation sequence of the cD2 contains the preferred A at position -3 and G at +4 (29). The 840-bp D2 ORF is located between nucleotides 596 and 1435 and contains two in-frame TGA codons (positions 989-991 and 1388-1390) and an unambiguous TAA stop codon 15 codons 3' to this. A 279-amino acid protein (~31 kDa) is predicted on the basis of the deduced amino acid sequence including the last 15 amino acids (Fig. 3). The deduced cD2 protein sequence is only 41% identical with the cD1 and 40% with the cD3 protein partial sequence (258 amino acids of the cD3 (11, 30). A Kyte-Doolittle hydropathy analysis of the deduced protein shows that the N-terminal portion of the protein is highly hydrophobic (Fig. 4). This region is followed by a highly hydrophilic region (residues 40-60) containing five basic amino acids. The remainder of the protein is hydrophilic with the exception of the region surrounding the active catalytic site.


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Fig. 3.   Nucleotide sequence of the cD2 5'-untranslated and coding region. Several short open reading frames in the 5'-UTR are shaded. The predicted amino acid sequence of the D2 coding region is indicated. Two in-frame TGA codons presumably encode selenocysteine (SeC). The TAA stop codon is marked by an asterisk.


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Fig. 4.   Hydropathy analysis of the chicken D2 protein. The Kyte-Doolittle algorithm with a window of 21 amino acids was generated by GCG PEPPLOT.

A putative SECIS element (stem-loop sequence) is located between positions 5788 and 5836 on the basis of computer-predicted RNA folding analysis (Fig. 5) (31). At position 5836, the 3'-RACE product and the 6-2 library fragment contained a G; however, it was replaced by an A in the 2-1 library fragment. Nine AUUUA RNA instability motifs were detected in the 3'-UTR (starting at positions 2266, 2826, 2844, 3424, 3665, 3998, 4442, 4717, and 5362). The motif starting at 3665 was a UU AUUUA AU nonamer (32). The sequences surrounding the motifs were very A/U-rich; e.g. the 184-bp 3' of the first motif (2266-2450) contained 20.6% A and 53.2% T; the 174-bp region containing two AUUUA motifs (2826-3000) contained 37.3% A and 29.9% T; the 100-bp fragment 3' of the fourth motif (3424-3523) was 34% A and 37% T. 


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Fig. 5.   Structure of the cD2 SECIS element (nucleotides 5788-5836) predicted by GCG FOLDRNA (31). The predicted structure conforms to that of a form II SECIS element (46). The G in the most 3' position was replaced by A in the 2-1 clone, but this had no major effect on its function.

Kinetic Studies of the Transiently Expressed cD2-- The apparent Km was 1.1 nm for thyroxine (T4), and the maximum velocity (Vmax) was 0.34 pmol/min/mg sonicate protein with a Vmax/Km ratio of 0.3 (Table II). Reverse T3 was also a good, but less favored, substrate, with a Vmax/Km ratio ~10-fold less than for T4. The 5'-deiodination by cD2 was relatively insensitive to GTG, with Ki (GTG) ~0.3 µM (Fig. 6, A and B). PTU (1 mM) did not significantly inhibit the 5'-deiodinase activity of the overexpressed cD2 at 2 or 100 nM T4 (Fig. 6C).

                              
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Table II
Kinetic parameters of transiently expressed cD2
The coding region of cD2 was expressed in HEK-293 cells to generate the enzyme. Cells were sonicated to provide a source of active enzyme for two separate kinetic studies (experiment (Expt.) 1 and 2).


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Fig. 6.   Kinetic studies of the transiently expressed cD2. A, Lineweaver-Burk plot of the 5'-deiodination of T4 in the absence and presence of GTG. A plot of the 5'-deiodination of rT3 in the absence of GTG is shown. Reactions were performed in 300 µl of PE buffer containing 20 mM DTT, 1 mM PTU, and 125I-labeled T4 or rT3 as described under "Experimental Procedures." Each data point is the average of closely agreeing duplicate determinations. B, slope replot of the T4 deiodination rates in the presence and absence of GTG. C, effect of PTU on the 5'-deiodination of T4 at two different concentrations. The study was performed using transiently expressed cD2 protein assayed in triplicate. Data are mean ± S.D.

Demonstration of a Functional SECIS Element in the 3'-UTR of cD2-- On the basis of the computer predictions, different cD2 3'-UTR fragments were tested for SECIS activity in pHT1427 and pHT1429 and compared with the SECIS element of rat D1 (25) (Fig. 7). Both the 3'-RACE PCR product and the fragments from the pituitary library (with or without the BamHI site) have SECIS activity. The element is found in the most 3' 400 bp of the cDNA in the location predicted by the FOLDRNA program. Activity was eliminated by inverting the orientation of the fragment. The potency of the D2 element was equivalent to that of the rD1 minimal SECIS element when these were placed in the same position 3' to rD1 in the SECIS test plasmid (Fig. 7). The native assembled cD2 cDNA with the SECIS element in its wild-type location encoded an active D2 protein after transient expression. Its activity was 0.011 ± 0.0002% (mean ± SD) deiodination at 2 nM T4/µl of cell sonicate (~11 µg of protein) × 1000 corrected for human growth hormone (cpm/100 µl of media). This is about 10% of that when the rat D1 SECIS element was placed immediately 3' to the cD2 coding sequence (0.15 ± 0.06; mean ± SD). Thus, the native SECIS element is active in its wild-type position.


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Fig. 7.   SECIS activity assays of various fragments of the cD2 cDNA using the pHT constructs (Fig. 2) in HEK-293 cells using D1 as the reporter gene. Results are the mean ± S.D. of duplicate D1/human growth hormone ratios of three different transient expression assays, each assayed in duplicate. The D1 activity expressed by the rat D1 SECIS element is shown for comparison.

Northern Blotting-- A single cD2 mRNA of ~6 kb was present in telencephalon, hippocampus, cerebellum, and brainstem and was also highly expressed in the liver (Fig. 8A). Faint bands of the correct size were also detected in thyroid, lung, and small intestine but not in skeletal muscle (leg), heart atria or ventricle, gizzard, or kidney. The D2/28 S density ratios were 4 for telencephalon, 1.7 for hippocampus, 2 for cerebellum, 1.4 for bainstem, 1 for thyroid, and on the second blot 4 for liver and 0.6 for lung. There were no transcripts smaller than ~6 kb present. There was a 4-fold increase in the mRNA level in the telencephalon from a hypothyroid chicken, indicating pretranslational regulation of D2 mRNA in this species (Fig. 8B).


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Fig. 8.   Northern blots of cD2 mRNA. A, expression of cD2 in tissues of the euthyroid adult chicken. Northern blots of 30 µg of total RNA were probed with the coding sequences of cD2 (nucleotides 844-1293) followed by hybridization with a chicken beta -actin probe. Exposure was 46 h for the cD2 blots and 3 h for the actin blots. B, effect of hypothyroidism on cD2 expression in the telencephalon. Northern blot of 30 µg of total RNA from the telencephalon of a control and a chicken thyroidectomized 7 days earlier. The same cD2 sequence was used for probing as for Fig. 8A. The ethidium bromide-stained 28 S band was used as the denominator for densitometry.

D2 Activity Is Expressed in Chicken Liver-- There are no previous studies evaluating adult chicken liver for D2 activity. To establish that the D2 mRNA is translated in the liver of the chicken, we prepared liver homogenate of tissue from an adult broiler. Using 125I-labeled T4 (2 nM) as substrate in the absence of PTU, we found low levels of total T4 5'-deiodinase activity, 205 fmol/h/mg of protein. Release of 125I- was reduced 55% by increasing the T4 concentration to 100 nM, which does not inhibit the fractional conversion of T4 to T3 by in vitro expressed mammalian or chicken D1 (11, 33). The deiodination of 125I-labeled T4 was almost completely blocked by 10 µM reverse T3, which competes with T4 for both D1 and D2 pathways. Thus, both D2 and D1 activities are expressed in liver of the adult chicken. Kinetic assays of D2 were performed by including 1 mM PTU and gave a mean Km of 4 nM and Vmax of 156 fmol/h/mg of protein, indicating the presence of D2 activity in the liver of this species.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The data presented establish that we have cloned the chicken D2, the first full-length cDNA for this protein from a higher vertebrate to be reported. This conclusion is supported by a number of observations. The 6-kb size of the cDNA corresponds to that of the cD2 mRNA obtained by Northern blot. The assembled cDNA encodes a protein with the classical D2 kinetics (low Km, preference for T4 over rT3, PTU, and GTG insensitivity). It contains a functional form II SECIS element. The deduced sequence of the cD2 protein is highly homologous to that of the rat, human, Rana, and Fundulus D2 (~80%; see below). However, it is only ~40% similar to the recently reported cD1 and partial cD3 deduced protein sequences (11, 30). The presence of D2 activity in chicken brain and liver correlates with Northern blots showing significant levels of cD2 message in these tissues.

Comparison of the cD2 Protein with Other Deiodinases-- The deduced cD2 protein sequence is highly similar to those of rat (83% identity), human (82%), Rana (80%), and Fundulus (67%) (Fig. 9). Comparing the protein sequences by the unweighted pair-group method with arithmetic averages, chicken and Rana are more similar (Fig. 10). However, the cD2 is more closely related to the mammalian enzymes on the basis of its two in-frame UGA codons and the large size of the mRNA. The hydropathy analysis of the cD2 protein shows that it contains a hydrophobic amino terminus, similar to other D2 proteins (Fig. 4) (13). This sequence is highly conserved, e.g. the first 30 residues are 93% identical to the corresponding portion of the hD2 (Fig. 9). Types 1 and 3 deiodinases also have an extremely hydrophobic NH2 terminus, also followed, as in cD2, by several charged residues (34). Between residues 42 and 61 in the D2 proteins there are eight charged amino acids (Fig. 9). Topological analysis of rD1 has shown that it is a type 1 integral membrane protein with a single ~21-residue transmembrane domain, which is again highly conserved between species (34). The hydrophilic positively charged region in the rD1 and presumably in the D2 proteins may function as a stop-transfer sequence. Thus, D2, like D1, would be predicted to have its catalytic portion in the cytosolic compartment.


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Fig. 9.   Comparison of the deduced amino acid sequence of D2 proteins. D2 sequences from chicken, rat, human, frog (R. catesbeiana), and fish (F. heteroclitus) were compared by GCG PILEUP, and the consensus sequence was prepared by LINEUP. Selenocysteines encoded by in-frame UGA codons and stop codons terminating the open reading frame are indicated by asterisks.


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Fig. 10.   Dendrogram of D2 proteins from various species. The unweighted pair-group method with arithmetic averages was used to analyze the relationships using GCG PILEUP.

The high conservation in the active center of the D2 enzymes is remarkable, with 15 of 15 identical amino acids in the five different species (Fig. 9). This portion of the protein also shows high similarity with that of D1 and D3 (16, 35). However, all of the D2 proteins including cD2 have an alanine two residues amino-terminal to the selenocysteine, while all D1 and D3 proteins contain a conserved cysteine in this position. The replacement of alanine by cysteine in the active center of D1 causes a 2-fold reduction in PTU sensitivity and a marked increase in the limiting Km for DTT in in vitro systems (16, 35). This alanine substitution may thus have an influence on the turnover number of the D2 enzyme, although it appears that this change may not be rate-limiting when transiently expressed enzymes are examined in in whole cells (35). There are other highly conserved regions in the D2 proteins, e.g. in the segment between 151 and 170 in Fig. 9. Interestingly, the amino acids carboxyl-terminal to the second UGA are not conserved, suggesting that this portion of the protein is not critical to its catalytic function. Previous studies with the wild-type hD2 showed two 75Se-labeled bands were transiently expressed, which corresponded to hD2 proteins terminating at the second UGA or at the canonical stop codon 8 codons carboxyl-terminal to it (13). In recent studies, we found no difference in the kinetic properties of an hD2 that terminated at a UAA residue at 266 and one containing cysteine at 266 that terminated at residue 273 (36). The nonconservation of the carboxyl-terminal 14-residue peptide further confirms this.

In the cD2 mRNA, as in all other D2 mRNAs, the base following the UGA in the catalytic center is a C, while that following the 3' UGA is A. It has been previously shown that the base following a stop codon can influence the efficiency of translational termination in that pyrimidine nucleotides are less effective than are purines (37). Thus, it would be predicted that D2 mRNA translation would be more likely to terminate at the 3' than at the 5' UGA.

The Km for T4 and rT3 of the transiently expressed cD2 is very similar to those reported for the human and rat D2 enzymes (13, 14). Catalysis by chicken D2 is also relatively resistant to GTG, as was first shown with the rat D2 (38). Its sensitivity to inhibition by GTG is ~10 times lower than that of either the transiently expressed or the native cD1 (11).

Comparison of the cD2 mRNA with Those of Other Species-- The cD2 mRNA was expressed in all of the brain regions studied, telencephalon, hippocampus, cerebellum, and brainstem as in the case of other vertebrates and was especially abundant in the telencephalon (14). Its presence in the brain correlates with the well established crucial role of D2 in maintaining intracellular T3 concentration in the brain and the previous demonstration of D2 activity mentioned earlier (2, 3). The transcript is also present in chicken pituitary, since the D2 cDNA was found in the pituitary cDNA library (21).

Unlike other higher vertebrates, cD2 mRNA is expressed in liver at levels comparable with that in brain. The D2 message is absent in human and rat liver (13, 14). No D2 message or 5'-deiodinase activity was detected in this organ in Rana tadpoles or in frogs (39, 40). However, D2 mRNA is present in liver of F. heteroclitus and D2 activity in that of the fish Oreochromis niloticus (15, 41). Kinetic studies have already suggested the presence of a PTU-insensitive 5'-deiodinase pathway in quail liver (42). A PTU-insensitive D1 has also been identified in tilapia kidney, but the Km of this enzyme is 2 µM for rT3, a range typical for D1 (43).

Chicken liver expresses both D1 and D2, making demonstration of low levels of D2 activity potentially difficult. The same problem occurs in human thyroid, in which D1 activity is much higher relative to D2 than is the case in the chicken liver (33). With hD2, the problem is further complicated in that PTU can inhibit about 20% of D2-catalyzed 5'-deiodination of nanomolar concentrations of T4 (33). Fortunately, this is not the case in the chicken, in which D2 activity is inhibited only 10% by 1 mM PTU (Fig. 6C), a concentration that completely inhibits cD1 (11, 28). The presence of D2 activity in chicken liver is shown in two ways. First, increasing the T4 concentration from 2 to 100 nM reduces the fractional deiodination by 55%. This cannot be due to saturation of D1 activity, since the fractional deiodination of T4 by chicken D1 is not inhibited by 100 nM T4 (11). However, there is T4 deiodination by D1 as shown by the reduction in the fractional deiodination of 125I-labeled T4 almost to background in the presence of 10 µM rT3. In addition, Lineweaver-Burk plots of T4 deiodination in the presence of 1 mM PTU show a Km for T4 of 4 nM, quite similar to that of the transiently expressed cD2 and that of the native D2 measured in chicken brain (2 nM), while the Vmax is ~10 times higher than the D2 Vmax measured in the euthyroid chicken brain (3). Thus, the chicken liver D2 activity is authentic. A functional role of liver D2 in the production of plasma T3 in the chicken seems likely but remains to be demonstrated.

As in humans, but not adult rats, D2 mRNA is expressed in chicken thyroid. Our recent studies of human thyroid have shown that the D2 mRNA in human thyroid, brain, pituitary, and heart is actually a doublet corresponding to transcripts of ~6.5 and 7.5 kb.2 This is due to alternate transcriptional start sites in the human D2 gene. This is not true in chicken. D2 is also expressed weakly in chicken lung and intestine, which does not occur in mammals but does in Rana (13, 14, 39).

The increased expression of D2 mRNA during hypothyroidism is typical of what has been observed in rats (14, 44, 45). In addition, the D2 Vmax increases ~9-fold in chicken brain during hypothyroidism (3). We have shown that the elevated D2 mRNA level is due to transcriptional changes in rat pituitary cells, since T3 does not reduce the half-life of D2 mRNA (45). We also found that the half-life of rat D2 mRNA is quite short, about 2 h. We found a number of AUUUA instability elements; a UU AUUUA AU nonamer and U-rich fragments in the cD2 mRNA. A similar A/U-rich 3'-UTR is found in hD2 mRNA (19). These have been associated with rapidly degraded mRNAs, suggesting that cD2 mRNA may also show this characteristic (32).

The SECIS Element of cD2 mRNA-- A SECIS element is required for translation of in-frame UGAs as selenocysteine (17). Presumably because of the large size of the D2 mRNA of most vertebrates as well as the high A/U content, no full-length D2 cDNA clones have been isolated. The 3'-RACE reaction used here was performed to generate the large missing 3' portion of this cDNA. While this was shown to contain sequences with SECIS activity, we confirmed that it was a valid sequence by probing a chicken pituitary library and identifying several other clones of identical sequence, as well as cDNAs with minor polymorphisms (Fig. 7). Computer analyses of the sequences predicted the stem-loop structure shown in Fig. 5, which indeed was equipotent with the SECIS element of rat D1 when assayed using a D1 SECIS assay plasmid (Figs. 2 and 7). SECIS elements can be grouped into two different classes or forms based on the structure of the terminal loop. In form I elements, the conserved adenines are located in this terminal loop, while in form II structures these are found in a bulge on the ascending limb of the stem (46). In fact, the form II structure results from predicted intraloop base pairing (nucleotides 20-23 pair with nucleotides 30-33; Fig. 5). The G/A polymorphism at the 3' terminus of the predicted structure is not in a critical region of the element and does not have a major effect on its function (46).

In the wild-type cD2 mRNA, the SECIS element is separated from the UGA codon in the active site by ~5 kb of A/U-rich sequences. The efficiency of the transient expression of D2 falls 10-fold as a consequence. We noted a similar result with the SECIS element of the hD2, which is located in an identical position in the hD2 mRNA (19). The fact that an element located at this distance is functional supports the looping model for the eukaryotic SECIS element proposed by Berry et al. (18). While steric factors may be important in limiting the efficiency of SECIS element placed at this distance, one must also know the ratio of D2 activity to D2 mRNA to establish the precise cause of the lower transient expression of the full-length, as opposed to the abbreviated, cD2. Given the A/U-rich nature of the 3'-UTR that intervenes between the coding region and the SECIS element, it seems possible that faster decay of the full-length mRNA will also be found to play a role.

The presence of putative SECIS elements in cD1 (1217-1303 in the GenBankTM sequence accession number Y11110) and cD3 (1121-1228 in the GenBankTM sequence accession number Y11273) has been suggested (11). Our analysis by the Genetics Computer Group (GCG) FOLDRNA program predicts a form I structure for the cD1 and a form II for the cD3 (31). The same form difference is predicted for hD1 and hD3 SECIS elements (47, 48), but all of these are within 2 kb of the UGA codon. Given the overall similarities in the SECIS locations between the human and chicken D2 mRNA 3'-UTRs and the large size of the rat D2 mRNA, it seems likely that this feature will be typical of D2 mRNAs. This speculation is further confirmed by a report of the the 3'-UTR of the mouse D2 mRNA, which appeared after submission of this paper. This showed that in this species the SECIS element is also located near the end of the 3'-UTR (49).

The 5'-Untranslated Region of the Chicken D2 mRNA-- The cD2 5' UTR also has a novel feature in that it contains three nonoverlapping sORFs. Interestingly, the UGA at the end of the third and longest ORF in the 5'-UTR is also followed by a G, which speaks in favor of that UGA codon serving here as a stop codon as opposed to a selenocysteine. In this case, the frame will be reset, since the coding region is not in frame with this ORF. The available portion of the rD2 5'-UTR contains four nonoverlapping sORFs (14). However, the deduced protein sequence of the chicken and rD2 ORFs is not conserved. Short ORFs in the 5'-UTR of certain mRNAs of Saccharomyces cerevisiae and the human cytomegalovirus can negatively regulate the translation of the main reading frame in an ORF sequence-dependent or -independent manner (50). Further analyses are required to investigate the effect of the sORFs that are also present in the rat D2 mRNA (14). These sORFs, together with the instability elements of the 3'-UTR and the extreme distance of the SECIS element from the UGA, would contribute to the typical low expression level of this enzyme.

In summary, the cloning of the full-length cD2 mRNA has confirmed that the coding regions of this protein are highly conserved among avian, fish, amphibian, and mammalian species. The availability of chicken cDNA will allow the exploration of the potential role of D2 in ontogeny in an embryonic system that can be easily manipulated and observed to illuminate what appear to be carefully regulated pathways for controlling the availability of T3 to the embryo and newborn of all vertebrate species.

    ACKNOWLEDGEMENTS

We thank Drs. I. Kacskovics, M. Berry, and G. Martin for helpful discussions. We also thank Drs. B. Vennström and Y. M. Sun for providing cDNA libraries prepared from chicken embryo and adult pituitaries, respectively. We thank Drs. D. L. St. Germain and V. A. Galton for the human D2 cDNA Genethon clone.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK-36256 and DK-07529 (to P. R. L.), by FKFP-0672 Hungary, and National Research Fund of Hungary Grants OTKA-16545 (to P. R.) and 26600 (to T. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These two authors contributed equally to this work.

parallel To whom correspondence should be addressed: Thyroid Division, Harvard Institutes of Medicine, Rm. 560, Boston, MA 02115. Tel.: 617-525-5155; Fax: 617-731-4718; E-mail: larsen{at}rascal.med.harvard.edu.

2 T. Bartha, S. W. Kim, D. Salvatore, B. Gereben, H. M. Tu, J. W. Harney, P. Rudas, and P. R. Larsen, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: T3, 3,5,3'-triiodothyronine; T4, thyroxine; reverse T3 or rT3, 3,3',5'-triiodothyronine; D1, D2, and D3, type 1, 2, and 3 iodothyronine deiodinase, respectively; rD1, rat type 1 iodothyronine deiodinase; cD2, hD2, rD2, chicken, human, and rat type 2 iodothyronine deiodinase, respectively; DTT, dithiothreitol; GTG, gold thioglucose; PTU, 6-n-propylthiouracil; RACE, rapid amplification of cDNA ends; RT, reverse transcription; PCR, polymerase chain reaction; SECIS, selenocysteine insertion sequence; ORF, open reading frame; sORF, short ORF; UTR, untranslated region; bp, base pair(s); kb, kilobase(s).

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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