From the
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.
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
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
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.
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 [
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)
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 [
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.
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.
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
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
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
The ability of the mammalian type III deiodinase to
convert T
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
)
(
)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) .
Se have failed to identify candidate
selenoproteins in rat tissues manifesting type III activity (11).
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.
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 NaH
PO
, 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).
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.
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.
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.
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.
(
)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.
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.
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
.
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.
/EMBL Data Bank with accession number(s)
U24282.
, 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).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.