| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Articles |
Universidad National Autonoma de Mexico (C.V.-R., A.O.), Mexico 04510 D. F.; Departments of Medicine and Physiology (W.C., D.L.S.), Dartmouth Medical School, Lebanon, New Hampshire 03756; and The Whitney Laboratory (G.J.L.), University of Florida, St. Augustine, Florida 32086
Address all correspondence and requests for reprints to: Donald L. St. Germain, M.D., Dartmouth Medical School, One Medical Center Drive, Lebanon, New Hampshire 03756. E-mail: stgermain{at}dartmouth.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The deiodination of iodothyronines is catalyzed by a family of selenoenzymes that have differing catalytic properties and are expressed in both tissue-specific and developmentally-specific fashions (8). Information concerning the biochemical properties of these enzyme are derived primarily from studies in mammals and amphibians. Two deiodinases, the types I (DI) and II (DII), serve an activating role by converting T4 to T3 by 5'-deiodination, whereas the type III deiodinase (DIII) facilitates 5-deiodination, which converts T4 and T3 to inactive metabolites (rT3 and 3, 3'-diiodothyronine (T2), respectively). Similar processes of deiodination have also been reported in fish (5, 9). In particular, both DI- and DII-like activity have recently been reported in certain teleost species [Orozco, A., J. Silva, and C. Valverde-R, submitted for publication and (10)]. For example, our recent studies in Fundulus heteroclitus, a small estuarine teleost native to North America (11), have shown that the liver of this species contains high levels of DII-like activity (10).
Complementary DNAs (cDNAs) for the DI, DII, and DIII from several mammalian and amphibian species have recently been identified (12, 13, 14, 15, 16, 17, 18, 19, 20). To date, however, little is known of the structural and molecular features of the enzymes catalyzing deiodination in fish. Given the apparent primacy in fish of extrathyroidal 5'-deiodination in the generation of circulating and tissue T3, we sought to identify a cDNA that codes for a fish 5'-deiodinase. We report herein the successful identification and expression of such a cDNA from a F. heteroclitus liver cDNA library.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
gt10 cDNA library was prepared using poly(A)+
RNA derived from a pool of five livers from male F.
heteroclitus treated with estradiol-17ß. The preparation of this
library has previously been described (21) and used tissue from
estrogen-treated animals so as to enhance the representation of
vitellogenin cDNAs. The library was screened by plaque hybridization
under low stringency conditions according to the methods of Lees
et al. (22). The first 305 nucleotides of the coding region
of an amphibian DII cDNA (RC5'DII) and a 714-nucleotide rat DI cDNA
(6b-short) that encompasses 92% of the coding region of that enzyme
were used together as probes in the initial screening protocol. Details
concerning the isolation and structures of these cDNAs have previously
been published (19, 23). Positive plaques were detected by
autoradiography and purified by additional rounds of screening using
the hybridization conditions described above and either the amphibian
DII or rat DI cDNAs separately as probes. cDNA inserts from the
positive clones were amplified by PCR using vector-based primers. (PCR
amplification was necessitated by the loss of the EcoRI
restriction enzyme sites in the gt10 vector during the library
construction process.) The PCR reaction mixture of a selected clone
(designated FhDII) was then subjected to agarose gel electrophoresis
and the reaction product purified using the QIAquick Gel Extraction Kit
(Qiagen, Chatsworth, CA) and subcloned into pBluescript using the
pCR-Script Cloning Kit (Stratagene, La Jolla, CA). The cDNA insert was
sequenced on both strands using vector-based and gene-specific primers
and an automated sequencing system with fluorescent dye terminators
(Applied Biosystems, Foster City, CA). Radiolabeled cDNA probes for library screening and Northern analysis were prepared as previously described (20).
The hydrophobicity profile of the FhDII protein was performed using the Kyte-Doolittle method with the MacDNASIS Pro computer program (Hitachi Software Engineering, San Bruno, CA), whereas prediction of the transmembrane domain was made using the TopPred II program (24).
Preparation of a chimeric FhDII cDNA
A chimeric cDNA was constructed by splicing part of the
3'-untranslated region of the full length rat DIII cDNA (rNS43-1),
which contains an active selenocysteine insertion sequence (SECIS)
element, to the 3' end of the coding region of the FhDII cDNA using
overlap PCR methods analogous to those previously described (20). The
entire coding region of the FhDII cDNA remained intact in this
construct. The splice region of the chimeric cDNA was sequenced to
ensure the accuracy of the construction method.
Expression studies in COS-7 cells
For expression in COS-7 cells, the FhDII chimera cDNA was
subcloned into the pcDNA3 mammalian expression vector (Invitrogen, San
Diego, CA). cDNAs for the rat DI (G21, kindly provided by Drs. M. Berry
and P. R. Larsen, Boston MA), rat DII (rBAT1-1 chimera), and amphibian
DIII (XL-15) were subcloned into the same vector as previously
described (20). COS-7 cells were cultured and transfected as previously
described (20), then maintained in culture medium for 48 h before
harvesting. After aspiration of the medium, cell monolayers were washed
twice with PBS, and the cells then scraped from the dish, pelleted, and
sonicated in 0.25 M sucrose, 0.02 M Tris/HCl,
pH 7.4.
5'D and 5D activities were determined in COS-7 cell sonicates according to published methods (25, 26). For the 5'D assay, the reaction buffer contained 1 mM EDTA. In kinetic studies using rT3 and T4, 5'D activity was determined during a 1-h incubation in a 50 µl reaction mixture volume containing 2 µg of sonicate protein and either 0.5–6 nM 125I-rT3 or 125I-T4 as substrate. In these reactions, dithiothreitol at a concentration of 20 mM was used as cofactor. The extent of substrate utilization was less than 52%. To correct for this degree of substrate utilization in kinetic studies, average substrate concentrations during the incubation mixture were used in the analysis as detailed by Lee and Wilson (27). Kinetic constants were determined from double reciprocal or Eadie-Hofstee plots (28).
In other experiments, the 5'-deiodinase activity in sonicates from COS-7 cells transfected with the G21 rat DI cDNA (13), the BAT1-1 rat DII cDNA (20) or the FhDII cDNA were determined at different assay incubation temperatures, or in the absence or presence of PTU (10–100 µM) or aurothioglucose (0.01–10 µM). In these assays, deiodinase activity was measured during a 1 h (for temperature studies) or 2 h (for PTU and aurothioglucose studies) incubation using 1.5 nM 125I-rT3 as substrate and 20 mM dithiothreitol as cofactor. Substrate utilization was less than 35% in these studies.
Initial studies employing T3 as substrate used 1 nM 125I-T3, 50 mM dithiothreitol as cofactor, and a 2-h incubation period. For kinetic studies with T3, a 1-h incubation period, 25 µl reaction volume containing 20 µg of sonicate protein, 20 mM DTT, and 0.5–15 nM or 15–1000 nM 125I-T3 were used. Substrate utilization was less than 25%.
125I-labeled iodothyronines were obtained from du Pont de Nemours (Boston, MA) and purified by chromatography using Sephadex LH-20 (Sigma, St. Louis, MO) before use. Protein concentrations were determined by the method of Bradford (29) with reagents obtained from Bio-Rad (Richmond, CA).
RNA Preparation and northern analysis
RNA was prepared as previously described (21) from livers of
male F. heteroclitus. Poly(A)+ RNA was isolated
by two cycles of chromatography over oligo(dT)-cellulose (Collaborative
Biomedical Products, Bedford, MA). RNA gel electrophoresis, transfer to
nylon membranes, hybridization and washing of Northern blots were
performed as previously described for rat tissues (30) with the final
wash performed at 60 C. The FhDII cDNA was used as the probe, and the
blots were exposed to x-ray film for 1 week.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 kilobase pair (kb)]. The
nucleotide sequence of one of these cDNAs (designated FhDII) was
determined and is shown in Fig. 1A
. An open reading
frame, 798 bp in length, extends from nucleotides #132-929 and codes
for a protein with a predicted molecular mass of 29.6 kDa. Included in
the open reading frame is an in-frame TGA triplet at codon #134
(nucleotide nos. 531–533) that is predicted, like the other deiodinase
cDNAs isolated to date (13, 14, 15, 16, 17, 18, 19, 20), to code for selenocysteine. A
comparison of the FhDII with DII cDNAs isolated from R.
catesbeiana (19), rat, and human (20) reveals 65–68% nucleotide
homology within the coding region and the same percentage of amino acid
identity (Fig. 1B). In contrast, homology to DI and DIII cDNAs from
several mammalian and amphibian species is considerably lower (less
than 50% nucleotide conservation and only 30% amino acid identity).
Nonetheless, several structural features of the FhDII protein are
common to all types of deiodinases including, the highly conserved
region surrounding the selenocysteine residue, two histidine residues
at amino acid nos. 166 and 186 (31), and a highly hydrophobic region in
the amino terminus that is predicted to form a membrane spanning domain
encompassing residue nos. 8–28 (Fig. 1C).
|
Initial studies in COS-7 cell sonicates demonstrated that transfection with the FhDII chimeric cDNA induced significant levels of 5'-deiodination (52% deiodination of 1 nM radiolabeled rT3 during a 2-h incubation at 37 C; velocity = 29 fmol/min·mg protein). In comparison, expression and assay of the G21 rat DI cDNA in the same experiment induced a considerably higher level of activity (velocity = 283 fmol/min·mg protein), a finding consistent with previous activity comparisons between other DII cDNAs and the G21 clone (20). [We have shown previously that COS-7 cells transfected with the empty pcDNA3 vector contain undetectable levels of 5'-deiodinase activity (20).]
Kinetic analysis of the FhDII-induced 5'-deiodinase activity using
T4 and rT3 as substrates demonstrated low
Km values (0.5 and 1.0 nM, respectively)
typical of a DII (Fig. 2). The Vmax value
using rT3 as substrate was approximately twice the value
observed with T4, resulting in
Vmax/Km ratios that were equivalent for both
substrates. Thus, the FhDII enzyme appears to catalyze the
5'-deiodination of T4 and rT3 with equal
efficiency.
|
). Such results are again
consistent with the properties of a DII. As a positive control,
abundant T2 formation (90% conversion of T3
to T2) was obtained in COS-7 cell sonicates expressing the
XL-15 amphibian DIII cDNA. An unexpected finding in these studies was
that considerable iodide (representing 18% of the radioactive
T3 substrate added) was formed in the FhDII assay mixture
indicating that outer ring, or 5'-, deiodination of T3 was
occurring (Fig. 3). In contrast, no iodide formation occurred in the
XL-15 sonicate mixture.
|
, 40% of the T3 added as substrate was deiodinated at the
5'-position by the Fundulus enzyme, whereas minimal or no
iodide formation was noted in sonicates from cells transfected with
cDNAs for the rat DI (G21), the rat DII (rBAT1-1 chimera), or an
amphibian DIII (XL-15). (As noted above, and previously (20), these
sonicates demonstrated very high levels of 5'-deiodinase activity (rat
DI and rat DII) when rT3 was used as substrate, or
5-deiodinase activity (XL-15) when assayed using T3,
indicating that the relevant deiodinases were expressed in these
sonicates.)
|
and compared with the kinetic data for
T4 cited above. The Km for T3 was
considerably higher (340 nM) than for T4 or
rT3, and although a higher Vmax value was
observed, the Vmax/Km ratio was markedly lower
than for the other two substrates (0.005 for T3vs. 0.4 for T4 and rT3).
|
. Analogous to amphibian and mammalian
DIIs (19, 20), the Fundulus enzyme, when compared with the
expressed rat DI (G21), was markedly resistant to inhibition by PTU and
10-fold less sensitive to the effects of AThG.
|
, maximal 5'-deiodinase activity was
observed at 37 C, similar to the pattern observed with the expressed
rat DI and DII enzymes.
|
). In the lane containing total RNA, hybridization
occurred predominantly to a 1.3-kb RNA species, with a fainter band at
6 kb noted. In the poly(A)+ lane, hybridization occurred
only to the larger RNA species.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The F. heteroclitus is an excellent model for conducting these studies in that it posses many features typical of teleosts (21). In addition, thyroid function has been studied in this species and a marked seasonal variation in T4 secretion has been noted (34), as is true of many fish species (35, 36). In addition, we have recently characterized the deiodinase activity in the F. heteroclitus liver and have demonstrated the presence of both DI and DII-like activity (10). Studies of 5'-deiodination in liver homogenates of other fish species by different investigators have yielded somewhat conflicting data on the characteristics of the deiodinase activities present in this tissue. However, Eales and colleagues (37, 38) have consistently demonstrated a low Km, DII-like activity in the liver of rainbow trout, and we have recently confirmed this findings and also demonstrated the presence of DI-like activity in this tissue (Orozco, A., J. Silva, and C. Valverde-R, submitted for publication). It is notable that the functional characteristics of the FhDII deiodinase match closely with the DII-like activities defined in Fundulus and trout liver and other tissues, suggesting that this cDNA, and its homologues, which are presumably present in other teleost species, indeed code for these enzymes.
The functional activity of the FhDII as expressed in COS-7 cells is typical of a DII (39). Thus, this enzyme demonstrates a Km value for T4 and rT3 in the nanomolar range when using DTT as the thiol cofactor, and it catalyzes the 5'-deiodination of both these substrates with approximately equal efficiency. Furthermore, the FhDII deiodinase is resistant to inhibition by high concentrations of PTU and demonstrates diminished sensitivity to AThG as compared with the mammalian DI. The structural features of the FhDII correlate well with these functional characteristics in that the cDNA and predicted amino acid sequences are most homologous to the DII subfamily of deiodinases. In particular, the FhDII cDNA contains an alanine at amino acid no. 132, which is two residues toward the amino terminus from the selenocysteine. The presence of an alanine at this position has been a feature of all of the DII proteins characterized to date (19, 20). In contrast, the DI and DIII enzymes contain a cysteine in this location. The functional significance of this difference in structure is as yet uncertain.
An unexpected structural feature of the rat and human DII cDNAs is the presence of a second in-frame TGA codon located just 5' to an unambiguous TAG (rat) or TAA (human) stop codon (20). Whether this TGA codes for a second selenocysteine residue in these proteins is uncertain, and could depend on the selenium status of the cell. This downstream TGA codon is not present in the FhDII cDNA, however. In this respect, the FhDII more closely resembles the R. catesbeiana DII, which also contains only a single TGA codon (19). This suggests that incorporation of a second selenocysteine is not essential for DII activity, a finding that we have recently confirmed by demonstrating that mutagenesis of the second TGA codon to TAA in the rat DII cDNA does not alter the functional activity of this enzyme (Croteau and St. Germain, unpublished data). However, the essential nature of the first selenocysteine to the catalytic activity of this family of enzymes has clearly been demonstrated by us (15, 16, 19) and other investigators (13, 40); mutagenesis of this residue to cysteine either renders the enzyme inactive or reduces the catalytic efficiency by 1000-fold. The presence of selenocysteine in the FhDII enzyme demonstrates its conservation through vertebrate evolution, and thus further emphasizes the requirement of this rare amino acid for efficient catalysis of the reductive deiodination reaction.
Incorporation of selenocysteine into proteins occurs during translation and requires the presence of a specific stem loop structure (the SECIS element) in the 3'-untranslated region of the mRNA (41). The 1-kb FhDII cDNA is shorter than the corresponding 1.3- and 6-kb RNA species identified in Fundulus liver by Northern analysis and appears to have a truncated 3'-untranslated region that lacks such a SECIS element. Thus the relative efficiency of translation of the FhDII when utilizing its native SECIS element is unknown and may differ from that of the chimeric cDNA construct used in these studies.
The hybridizing 1.3-kb RNA seen in the total RNA sample was not present when poly(A)+ RNA was used indicating that this short RNA lacks a poly(A)+ tail. Its presence suggests that alternative mRNA processing may be involved in regulating the expression of DII in this tissue. The 6-kb species, however, is similar in size to the predominant DII RNA species identified in samples of poly(A)+ RNA prepared from several mammalian tissues (20) but is larger than the predominant 1.5-kb mRNA present in R. catesbeiana (19). In contrast, the mRNAs coding for the DI and DIII enzymes are approximately 2 kb in size (12, 16).
The FhDII deiodinase demonstrates a unique functional activity: namely, its ability to catalyze, albeit with relatively low efficiency, the 5'-deiodination of T3. Such activity has not previously been noted in any of the deiodinases characterized to date, including the amphibian RCDII cDNA (V. A. Galton, personal communication). Thus, comparative studies with deiodinases from other species may provide interesting insights into the structure-function correlates of this unique catalytic activity. Notably, 5'-deiodination of T3 has been observed in teleost tissue homogenates; Pimlott and Eales (37) have described "weak" 5'-deiodination of T3 in rainbow trout liver homogenates incubated at 20 C, the highest temperature at which they performed their assays. The physiological significance of this catalytic activity, however, may be relatively minor given the marked preference of the FhDII to utilize T4 and rT3 as substrates.
Early studies conducted by Leatherland (42) in trout liver homogenates suggested that the optimal temperature for 5'-deiodination in this tissue was 20 C. In these studies, no thiol co-factors were included in the trout liver reaction mixtures and the substrate concentration was so high (1.4 µM T4) as to preclude distinguishing between DI and DII activity. Thus, a comparison of these data with ours is problematic. However, more recent data from our laboratory have demonstrated that DII activity as determined in vitro in trout liver homogenates is maximal at 25 C (Orozco, A., J. Silva, and C. Valverde-R, submitted for publication). It was, therefore, somewhat surprising to find that the thermal activity profile of the FhDII deiodinase was the same as that of the mammalian DI and DII enzymes with maximal activity noted at 37 C. Such differences between the temperature sensitivities of the trout and Fundulus enzymes could reflect their different habitats in that the latter species tends to reside in warmer waters. Similar observations showing maximal activity of an amphibian DII at 37 C have been made in tissue homogenates from R. catesbeiana (V. A. Galton, personal communication).
In summary, although previous studies have documented that there are important species differences in the tissue and developmental patterns of expression of the DII, the present study demonstrates that the structural and functional characteristics of the DII protein have been highly conserved during evolution. This provides additional evidence that this enzyme serves an important physiological role. The availability of the FhDII cDNA should facilitate further investigations into the role of thyroid hormones and their metabolic fate in fish.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received August 15, 1996.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  S Van der Geyten, N Byamungu, G E Reyns, E R Kuhn, and V M Darras Iodothyronine deiodinases and the control of plasma and tissue thyroid hormone levels in hyperthyroid tilapia (Oreochromis niloticus) J. Endocrinol., March 1, 2005; 184(3): 467 - 479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Shepherdley, W. Klootwijk, K. W. Makabe, T. J. Visser, and G. G. J. M. Kuiper An Ascidian Homolog of Vertebrate Iodothyronine Deiodinases Endocrinology, March 1, 2004; 145(3): 1255 - 1268. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. G. J. M. Kuiper, W. Klootwijk, and T. J. Visser Substitution of Cysteine for a Conserved Alanine Residue in the Catalytic Center of Type II Iodothyronine Deiodinase Alters Interaction with Reducing Cofactor Endocrinology, April 1, 2002; 143(4): 1190 - 1198. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Bianco, D. Salvatore, B. Gereben, M. J. Berry, and P. R. Larsen Biochemistry, Cellular and Molecular Biology, and Physiological Roles of the Iodothyronine Selenodeiodinases Endocr. Rev., February 1, 2002; 23(1): 38 - 89. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Gereben, D. Salvatore, J. W. Harney, H. M. Tu, and P. R. Larsen The Human, but Not Rat, dio2 Gene Is Stimulated by Thyroid Transcription Factor-1 (TTF-1) Mol. Endocrinol., January 1, 2001; 15(1): 112 - 124. [Abstract] [Full Text]
|
|
|
|
|
|
J. P. Sanders, S. Van der Geyten, E. Kaptein, V. M. Darras, E. R. Kühn, J. L. Leonard, and T. J. Visser Cloning and Characterization of Type III Iodothyronine Deiodinase from the Fish Oreochromis niloticus Endocrinology, August 1, 1999; 140(8): 3666 - 3673. [Abstract] [Full Text]
|
|
|
|
 |
|
 N. Montuori, F. Müller, S. De Riu, G. Fenzi, M. E. Sobel, G. Rossi, and M. Vitale Laminin Receptors in Differentiated Thyroid Tumors: Restricted Expression of the 67-Kilodalton Laminin Receptor in Follicular Carcinoma Cells J. Clin. Endocrinol. Metab., June 1, 1999; 84(6): 2086 - 2092. [Abstract] [Full Text]
|
|
|
|
 |
|
 B. Gereben, T. Bartha, H. M. Tu, J. W. Harney, P. Rudas, and P. R. Larsen Cloning and Expression of the Chicken Type 2 Iodothyronine 5'-Deiodinase J. Biol. Chem., May 14, 1999; 274(20): 13768 - 13776. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Van der Geyten, J. P. Sanders, E. Kaptein, V. M. Darras, E. R. Kuhn, J. L. Leonard, and T. J. Visser Expression of Chicken Hepatic Type I and Type III Iodothyronine Deiodinases during Embryonic Development Endocrinology, December 1, 1997; 138(12): 5144 - 5152. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Sanders, S. Van der Geyten, E. Kaptein, V. M. Darras, E. R. Kuhn, J. L. Leonard, and T. J. Visser Characterization of a Propylthiouracil-Insensitive Type I Iodothyronine Deiodinase Endocrinology, December 1, 1997; 138(12): 5153 - 5160. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Pallud, A.-M. Lennon, M. Ramauge, J.-M. Gavaret, W. Croteau, M. Pierre, F. Courtin, and D. L. St. Germain Expression of the Type II Iodothyronine Deiodinase in Cultured Rat Astrocytes Is Selenium-dependent J. Biol. Chem., July 18, 1997; 272(29): 18104 - 18110. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
