Cloning of a cDNA for the type II iodothyronine deiodinase.
ABSTRACT 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 inframe 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 Xenopus laevis 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.
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ABSTRACT: In embryonic chicken liver (ECL) two types of iodothyronine deiodinases are expressed: D1 and D3. D1 catalyzes the activation as well as the inactivation of thyroid hormone by outer and inner ring deiodination, respectively. D3 only catalyzes inner ring deiodination. D1 and D3 have been cloned from mammals and amphibians and shown to contain a selenocysteine (Sec) residue. We characterized chicken D1 and D3 complementary DNAs (cDNAs) and studied the expression of hepatic D1 and D3 messenger RNAs (mRNAs) during embryonic development. Oligonucleotides based on two amino acid sequences strongly conserved in the different deiodinases (NFGSCTSecP and YIEEAH) were used for reverse transcription-PCR of poly(A+) RNA isolated from embryonic day 17 (E17) chicken liver, resulting in the amplification of two 117-bp DNA fragments. Screening of an E17 chicken liver cDNA library with these probes led to the isolation of two cDNA clones, ECL1711 and ECL1715. The ECL1711 clone was 1360 bp long and lacked a translation start site. Sequence alignment showed that it shared highest sequence identity with D1s from other vertebrates and that the coding sequence probably lacked the first five nucleotides. An ATG start codon was engineered by site-directed mutagenesis, generating a mutant (ECL1711M) with four additional codons (coding for MGTR). The open reading frame of ECL1711M coded for a 249-amino acid protein showing 58-62% identity with mammalian D1s. An in-frame TGA codon was located at position 127, which is translated as Sec in the presence ofa Sec insertion sequence (SECIS) identified in the 3'-untranslated region. Enzyme activity expressed in COS-1 cells by transfection with ECL1711M showed the same catalytic, substrate, and inhibitor specificities as native chicken D1. The ECL1715 clone was 1366 bp long and also lacked a translation start site. Sequence alignment showed that it was most homologous with D3 from other species and that the coding sequence lacked approximately the first 46 nucleotides. The deduced amino acid sequence showed 62-72% identity with the D3 sequences from other species, including a putative Sec residue at a corresponding position. The 3'-untranslated region of ECL1715 also contained a SECIS element. These results indicate that ECL1711 and ECL1715 are near-full-length cDNA clones for chicken D1 and D3 selenoproteins, respectively. The ontogeny of D1 and D3 expression in chicken liver was studied between E14 and 1 day after hatching (C1). D1 activity showed a gradual increase from E14 until C1, whereas D1 mRNA level remained relatively constant. D3 activity and mRNA level were highly significantly correlated, showing an increase from E14 to E17 and a strong decrease thereafter. These results suggest that the regulation of chicken hepatic D3 expression during embryonic development occurs predominantly at the pretranslational level.Endocrinology 01/1998; 138(12):5144-52. · 4.72 Impact Factor
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ABSTRACT: Mammalian type I iodothyronine deiodinase (D1) activates and inactivates thyroid hormone by outer ring deiodination (ORD) and inner ring deiodination (IRD), respectively, and is potently inhibited by propylthiouracil (PTU). Here we describe the cloning and characterization of a complementary DNA encoding a PTU-insensitive D1 from teleost fish (Oreochromis niloticus, tilapia). This complementary DNA codes for a protein of 248 amino acids, including a putative selenocysteine (Sec) residue, encoded by a TGA triplet, at position 126. The 3' untranslated region contains two putative Sec insertion sequence (SECIS) elements. Recombinant enzyme expressed in COS-1 cells catalyzes both ORD of T4 and rT3 and IRD of T3 and T3 sulfate with the same substrate specificity as native tilapia D1 (tD1), i.e. rT3 > T4 > T3 sulfate > T3. Native and recombinant tD1 show equally low sensitivities to inhibition by PTU, iodoacetate, and gold thioglucose compared with the potent inhibitions observed with mammalian D1s. Because the residue 2 positions downstream from Sec is Pro in tD1 and in all (PTU-insensitive) type II and type III iodothyronine deiodinases but Ser in all PTU-sensitive D1s, we prepared the Pro128Ser mutant of tD1. The mutant enzyme showed strongly decreased ORD and somewhat increased IRD activity, but was still insensitive to PTU. These results provide new information about the structure-activity relationship of D1 concerning two characteristic properties, i.e. catalysis of both ORD and IRD, and inhibition by PTU.Endocrinology 01/1998; 138(12):5153-60. · 4.72 Impact Factor
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ABSTRACT: Recent molecular cloning studies in mammals and amphibians have demonstrated that the types I, II, and III deiodinases constitute a family of selenoproteins of critical importance in metabolizing T4 to active (i.e. T3) and inactive (i.e. rT3) metabolites. In several tissues of teleost fish, various deiodinase processes have been described, but the structural and functional characteristics of these enzymes and their relationship to the deiodinases present in higher vertebrates remains uncertain. Using a complementary DNA library derived from the liver of the teleost Fundulus heteroclitus, we have identified a complementary DNA that codes for a deiodinase with functional characteristics virtually identical to those of the mammalian and amphibian type II deiodinase. Sequence analysis demonstrates a high degree of homology at both the nucleotide and predicted amino acid levels between the Fundulus clone and these previously characterized type II enzymes, including the presence of an in-frame TGA codon that codes for selenocysteine. These findings demonstrate that the deiodinase family of selenoproteins has been highly conserved during vertebrate evolution and underscores their importance in the regulation of thyroid hormone action.Endocrinology 03/1997; 138(2):642-8. · 4.72 Impact Factor