Cloning and characterization of type III iodothyronine deiodinase from thefish Oreochromis niloticus

Type III iodothyronine deiodinase (D3) catalyzes the inner ring deiodinatio n (IRD) of T-4 and T-3 to the inactive metabolites rT(3) and 3,3'-diiodothy ronine (3,3'-T2), respectively. Here we describe the cloning and characteri zation of complementary DNA (cDNA) coding for D3 in fish (Oreochromis nilot icus, tilapia). This cDNA contains 1478 nucleotides and codes for a protein of 267 amino acids, including a putative selenocysteine (Sec) residue, enc oded by a TGA triplet, at position 131. The deduced amino acid sequence sho ws 57-67% identity with frog, chicken, and mammalian D3, 33-39% identity wi th frog, fish (Fundulus heteroclitus) and mammalian D2, and 30-35% identity with fish (tilapia), chicken, and mammalian D1. The 3' UTR contains a puta tive Sec insertion sequence (SECIS) element. Recombinant tilapia D3 (tD3) e xpressed in COS-1 cells and native tD3 in tilapia brain microsomes show ide ntical catalytic activities, with a strong preference for IRD of T-3 (K-m s imilar to 20 nM), IRD of [3,5-I-125]T-3 by native and recombinant tD3 are e qually sensitive to inhibition by substrate analogs (T-3 > T-4 much greater than rT(3)) and inhibitors (gold thioglucose >> iodoacetate > propylthiour acil). 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 inform ation about the structure-activity relationship of iodothyronine deiodinase s and an important tool to study the regulation of thyroid hormone bioactiv ity in fish.