(Received for publication, August 3, 1995; and in revised form, August 23, 1995)
From the
Three types of iodothyronine deiodinase have been identified in vertebrate tissues. cDNAs for the types I and III have been cloned and shown to contain an in-frame TGA that codes for selenocysteine at the active site of the enzyme. We now report the cloning of a cDNA for a type II deiodinase using a reverse transcription/polymerase chain reaction strategy and RNA obtained from Rana catesbeiana tissues. This cDNA (RC5`DII) manifests limited but significant homology with other deiodinase cDNAs and contains a conserved in-frame TGA codon. Injection of capped in vitro synthesized transcripts of the cDNA into Xenopuslaevis oocytes results in the induction of deiodinase activity with characteristics typical of a type II deiodinase. The levels of RC5`DII transcripts in R. catesbeiana tadpole tail and liver mRNA at stages XII and XXIII correspond well with that of type II deiodinase activity but not that of the type III activity in these tissues. These findings indicate that the amphibian type II 5`-deiodinase is a structurally unique member of the family of selenocysteine-containing deiodinases.
Intracellular concentrations of the thyroid hormones, T(
)and T
, are profoundly influenced by the
activity of three iodothyronine deiodinases, classified as types I, II,
and III (1) . In mammals, the type I enzyme (5DI) catalyzes
5`-deiodination (5`D), the removal of iodine from the 5` (or 3`)
positions of T
and its derivatives. The enzyme can also
catalyze 5-deiodination (5D), the removal of an iodine located at
either the 5 (or 3) positions of iodothyronines, but does so
efficiently only with sulfated iodothyronine substrates(2) .
The type II enzyme (5DII) also catalyzes 5`D, but it is readily
distinguished from the 5DI by its kinetics, substrate specificity,
sensitivity to propylthiouracil (PTU) and aurothioglucose
(AThG)(1, 3) , and response to thyroid
status(1) . The type III enzyme (5DIII) catalyzes primarily 5D
activity(1) , a process that results in derivatives with little
or no thyromimetic activity(1) .
The primary function of the
types I and II deiodinases is to convert T to its
metabolically more active derivative, T
. However, the
tissue distribution and physiological roles of the two enzymes are very
different. The principal role of the 5DI in mammals is to provide a
source of plasma T
by deiodination of T
in
peripheral tissues such as liver and kidney. In contrast, the 5DII is
responsible for the majority of the intracellular T
in
tissues such as the pituitary, brain, and brown fat by mediating local
deiodination of T
and is considered to be of major
importance in regulating thyroid hormone action in these
tissues(1, 3) . The 5DII also plays a major role
during development. 5DII is the principal 5`-deiodinase expressed in
the mammalian fetus, and it is notable that 5DII activity in brain
peaks in the neonatal period, the time that is critical for thyroid
hormone-dependent development in this tissue(4) . Moreover,
5DII is the only 5`-deiodinase present in the developing frog in which
the orderly progression of developmental processes is dependent on the
ability to attain appropriate intracellular levels of
T
(5) . Thus, the 5DII appears to play an essential
role in intracellular T3 production in those circumstances where
thyroid hormone-dependent processes take on critical significance.
cDNAs for the type I enzyme of rat(6) , dog(7) , and human (8) have been cloned. These cDNAs contain an in-frame TGA coding for selenocysteine, which is necessary for maximal enzyme activity(6) . Three cDNAs for the type III enzyme have also been cloned; we have shown that XL-15, a cDNA isolated by Wang and Brown (9) from a Xenopuslaevis tadpole tail cDNA library, encodes a 5DIII (10) and, using XL-15 as a probe, we have isolated 5DIII cDNAs for Ranacatesbeiana(11) and rat (12) . These cDNAs exhibit significant sequence homology to the mammalian 5DI cDNAs including the in-frame TGA codon, which codes for selenocysteine.
Isolation of a cDNA for a 5DII has yet to be reported. To this end we predicted that this enzyme would share significant sequence homology with other deiodinases. Close examination of the sequences of the known 5DI and 5DIII cDNAs revealed that, although the overall similarity between the two types is relatively low, there are three limited regions that are highly conserved. One is near the TGA codon that codes for selenocysteine, and the other two are approximately 60 and 230 bp 3` of this codon. We hypothesized that these regions would also be conserved in the 5DII gene.
This hypothesis proved to be correct. In the present report we describe the cloning of a cDNA for the 5DII of R. catesbeiana using a reverse transcription/polymerase chain reaction (RT/PCR) strategy, oligonucleotide primers based on the sequences of these conserved regions, and RNA from R. catesbeiana tissues that contain relatively high levels of type II 5`D activity. Once a portion of the putative coding region of the 5DII cDNA was obtained, gene-specific primers were used to synthesize the 3`- and 5`-ends of the cDNA using rapid amplification of cDNA ends (RACE) procedures(13) . The resulting cDNA (RC5`DII) contains the conserved TGA codon and codes for a protein with characteristics typical of a 5DII.
Figure 1: Schematic representation of the RT/PCR-based technique used to synthesize and clone RC5`DII. A, a ``generic'' deiodinase cDNA coding region showing the approximate locations, relative to each other, of all the oligonucleotide primers employed. The sequences of primers A-D are based on the ``conserved'' regions of the three 5DI and three 5DIII deiodinase cDNAs; the sequences of primers 1-6 are specific to RC5`DII. UAP* is a modified version of the universal amplification primers in the RACE kits (Life Technologies, Inc.) (see ``Materials and Methods'' for details). Sense primers are placed above, and antisense primers below, the line. B, PCR-based approach used to synthesize the full-length RC5`DII cDNA (see ``Materials and Methods'' for details).
The nucleotide and deduced amino acid sequences of RC5`DII are shown in Fig. 2A. RC5`DII is a 1459-bp cDNA with an open reading frame extending from bp 11 to 802 and an in-frame TGA codon at bp 380-382 that, by analogy with the cDNAs for the 5DI and 5DIII, is likely to code for selenocysteine. A schematic comparison of the RC5`DII protein with those of R. catesbeiana type III and the rat type I is shown in Fig. 2B. The areas of homology are indicated, and it is also noted that the proteins exhibit a hydrophobic region at the amino-terminal end and two histidine residues 3` to the TGA. In the rat 5DI, these histidines have been shown to be critical for 5`D activity (20) .
Figure 2: A, nucleotide and deduced amino acid sequences of RC5`DII. SC, selenocysteine. The sequence for RC5`DII has been deposited in GenBank under accession number L42815. B, comparison of the structural features of three types of deiodinase (the types II and III of R. catesbeiana and the type I of rat) as predicted by the cDNA sequences of RC5`DII, RC5D, and G21. Regions of homology and other shared features are noted.
5`D activity
was induced in X. laevis oocytes after injection of capped RNA
transcripts obtained by in vitro transcription of RC5`DII (Fig. 3A). No 5D activity was detected (data not
shown). The 5`D activity was not inhibited by PTU (0.1 mM),
but the percent deiodination of the [I]rT
was greatly reduced in the presence of 3 nM non-radioactive rT
, indicating that the enzyme
manifests a low K
for this substrate. We have
shown previously that the 5`D activity in tadpole tissues exhibits a
low K
and is resistant to inhibition by
PTU(5, 21) , characteristics that are typical of the
type II 5`D activity described in mammals(1) . In contrast,
capped transcripts of the 5DI cDNA, G21, induced activity that was
highly sensitive to PTU and appeared to have a relatively high K
. In addition, the 5`D activity induced by the
RC5`DII transcripts was relatively insensitive to inhibition by AThG (Fig. 3B). The 5`D activity induced in oocytes by
RC5`DII transcripts was considerably lower than that induced by
transcripts of the G21 cDNA. The reason for this difference is not
known. However, 5`D activity is only minimally induced in X. laevis oocytes by rat brown adipose tissue mRNA, a tissue containing
considerable type II 5`D activity, (
)and it is possible that
other factors important for type II 5`D activity are not optimal in
this oocyte system.
Figure 3:
A, induction of 5`D activity in X.
laevis oocytes following injection of capped RNA transcripts (50
ng/oocyte) synthesized in vitro from the RC5`DII and the 5`DI
(G21) cDNAs. Control oocytes received no injection. Reaction mixtures
contained 45, 49.5, and 54 µg of membrane protein from control,
RC5`DII, and G21 mRNA-injected oocytes, respectively. B,
sensitivity to aurothioglucose of the 5`D activity induced in X.
laevis oocytes by RC5`DII and G21 transcripts. Values are
corrected for the 5`D activity (2%) observed in uninjected
oocytes. Reaction mixtures contained 48 and 21 µg of membrane
protein from the RC5`DII and G21 mRNA-injected oocytes,
respectively.
In view of the indirect evidence suggesting that the mammalian 5DII is not a selenoprotein(3) , including the fact that it catalyzes 5`D activity that is relatively insensitive to PTU and AThG, the possibility that the protein coded by RC5`DII is not a selenoprotein or that selenocysteine is not involved in the activity of the enzyme was investigated. It was found that 5`D activity in oocytes injected with capped transcripts derived from mutant RC5`DII cDNAs, where the TGA codon had been changed to TAA (stop) or TGT (cysteine), was essentially the same as levels obtained in uninjected oocytes. Furthermore, no induction of activity was observed after injection of transcripts derived from RC5`DII truncated at bp 906, suggesting that the deleted 3`-untranslated region contains a selenocysteine insertion sequence. These findings provide strong evidence that RC5`DII codes for a protein with selenocysteine at its catalytic site. Previous studies by Berry and Larsen (3) had led to the suggestion that sensitivity of a deiodinase to PTU and AThG could serve as a marker for the presence of selenocysteine at the enzyme's active site. That this is not a valid criterion is demonstrated by the presence of the TGA coding for selenocysteine in the RC5`DII cDNA described herein and in three recently isolated type III deiodinase cDNAs, all of which encode enzymes resistant to PTU and AThG(10, 11, 12) . Thus other structural properties of these enzymes or differences in kinetic mechanisms may dictate PTU and AThG sensitivity.
The ontogenic profiles of 5`D and
5D activities in R. catesbeiana tadpole tail and liver are
very different; 5`D activity is minimal in tadpole tail until the onset
of metamorphic climax when it increases markedly reaching a maximum by
stage XXIII, while liver is devoid of 5`D activity at all stages of
development (21) . In contrast, 5D activity is present in both
tail and liver during premetamorphosis, and in liver it is greatly
reduced when the tadpoles reach metamorphic climax(22) . To
obtain additional evidence concerning the identity of RC5`DII, the size
and relative abundance of RC5`DII-related transcripts in tail and liver
poly(A) RNA obtained from premetamorphic (stage
X-XII) and metamorphosing (stage XXIII) tadpoles were determined
by Northern analysis (Fig. 4). No RC5`DII transcripts were
detected in liver mRNA at either stage of development. This finding is
consistent with the absence of 5`D activity in this tissue at all
stages of the life cycle(5, 21) . However, a major RNA
species of approximately 1.5 kb was detected in tail RNA; two minor
species were just discernible at approximately 1.8 and 2.2 kb. The
2.2-kb species may represent cross-hybridization with RC5D transcripts
since comparable blots probed with RC5D exhibit a signal at 2.2 kb
(data not shown). In other blots a minor species was also detected at
approximately 7.4 kb. The level of the 1.5-kb species was much higher
in RNA from metamorphosing tail than in that from premetamorphic tail.
Thus the profile of RC5`DII transcripts in liver and tail corresponds
closely to that of 5`D but not 5D activity in these tissues. The
increase in RC5`DII mRNA species in tail during metamorphic climax was
quantified using slot blot analysis. Densitometric analysis indicated
that the hybridization signals in tadpole tail at stages X-XII
and stages XXIII-XXIV were, respectively, 280 ± 50 (S.E.)
and 5151 ± 345 units (p < 0.001). As with the
Northern blot, no signal was observed in liver RNA. Reprobing of the
blot with RC5D revealed that levels of RC5D transcripts, which were
clearly evident in both liver and tail at both stages of development,
were not increased during metamorphic climax (data not shown). Thus,
the observed increase in the level of RC5`DII-related transcripts on
the blot cannot be attributed even in part to cross-hybridization of
RC5`DII with RC5D transcripts.
Figure 4:
Northern analysis of RC5`DII-related
transcripts in poly(A) RNA from tadpole tail and
liver. P, poly(A)
RNA from premetamorphic
(stage XII) tadpoles; M, poly(A)
RNA from
metamorphosing (stage XXIII) tadpoles.
Additional evidence that RC5`DII is
an amphibian type II deiodinase is provided by our recent
identification of rat and human homologues of this cDNA. Both are
highly homologous to RC5`DII within the coding region (rat, 71%; human,
73%). Furthermore, the tissue distribution of their related mRNA
transcripts, as determined by Northern analysis, is characteristic of
the mammalian type II enzyme. ()
We thus conclude that RC5`DII is the cDNA for the 5DII in R. catesbeiana. The characteristics of the deiodinase for which it codes are comparable with those of the 5`D activity in R. catesbeiana tadpole tissues (5) and in mammalian brain, pituitary, and brown fat(1) . The fact that type I 5`D activity has not been detected in tissues of this amphibian species (5, 21) makes it highly unlikely that RC5`DII codes for a form of the type I deiodinase. In addition, the data strongly suggest that the type II deiodinase coded by RC5`DII is a selenoprotein. We have compared the protein sequences deduced from the seven cloned deiodinase cDNAs, and it is evident that the RC5`DII protein has limited but significant homology with both the type I and the type III enzymes. Thus the amphibian type II deiodinase represents a structurally unique member of this family of selenocysteine-containing enzymes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L42815[GenBank].