Cloning and characterization of type III iodothyronine deiodinase from the fish Oreochromis niloticus.
Type III iodothyronine deiodinase (D3) catalyzes the inner ring deiodination (IRD) of T4 and T3 to the inactive metabolites rT3 and 3,3'-diiodothyronine (3,3'-T2), respectively. Here we describe the cloning and characterization of complementary DNA (cDNA) coding for D3 in fish (Oreochromis niloticus, tilapia). This cDNA contains 1478 nucleotides and codes for a protein of 267 amino acids, including a putative selenocysteine (Sec) residue, encoded by a TGA triplet, at position 131. The deduced amino acid sequence shows 57-67% identity with frog, chicken, and mammalian D3, 33-39% identity with frog, fish (Fundulus heteroclitus) and mammalian D2, and 30-35% identity with fish (tilapia), chicken, and mammalian D1. The 3' UTR contains a putative Sec insertion sequence (SECIS) element. Recombinant tilapia D3 (tD3) expressed in COS-1 cells and native tD3 in tilapia brain microsomes show identical catalytic activities, with a strong preference for IRD of T3 (Km approximately 20 nM). IRD of [3,5-125I]T3 by native and recombinant tD3 are equally sensitive to inhibition by substrate analogs (T3 > T4 >> rT3) and inhibitors (gold thioglucose >> iodoacetate > propylthiouracil). Northern analysis using a tD3 riboprobe shows high expression of a 1.6-kb messenger RNA in gill and brain, although D3 activity is much higher in brain than in gill. The characterization of tD3 cDNA provides new information about the structure-activity relationship of iodothyronine deiodinases and an important tool to study the regulation of thyroid hormone bioactivity in fish.
Sanders
J P
JP
Department of Internal Medicine III, Erasmus University Medical School, Rotterdam, The Netherlands.
Van der Geyten
S
S
Kaptein
E
E
Darras
V M
VM
Kühn
E R
ER
Leonard
J L
JL
Visser
T J
TJ
D020413:3' Untranslated Regions / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D007453:Iodide Peroxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009928:Organ Specificity; D011892:Rana catesbeiana; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D017210:Tilapia / Q000235:genetics*; D014982:Xenopus laevis
D020413:3' Untranslated Regions; D018076:DNA, Complementary; D011994:Recombinant Proteins; C117274:iodothyronine deiodinase type III; D007453:Iodide Peroxidase