The mammalian homolog of the frog type II selenodeiodinase does not encode a functional enzyme in the rat
Molecular Endocrinology Laboratories; Department of Physiology; Department of Medicine, Division of Endocrinology & Metabolism; Program in Molecular Medicine
Adipose Tissue, Brown; Animals; Astrocytes; Astrocytoma; Blotting, Western; Bucladesine; Cloning, Molecular; DNA, Complementary; Gene Expression; Immunohistochemistry; Immunosorbent Techniques; Iodide Peroxidase; Protein Biosynthesis; RNA, Messenger; Rats; Transfection; Tumor Cells, Cultured; Xenopus laevis
Life Sciences | Medicine and Health Sciences
Type II iodothyronine deiodinase is a short-lived, membrane-bound enzyme found in rat brain, brown adipose tissue, and cAMP-stimulated astrocytes. Recently, a full-length complementary DNA (cDNA) encoding a 30-kDa, type II-like selenodeiodinase was cloned from frog, and a homologous partial cDNA (rBAT 1.1), containing two in-frame selenocysteine codons (UGA), was isolated from rat brown adipose tissue. Importantly, the rBAT 1.1 cDNA was derived from a 7.5-kb messenger RNA (mRNA) and did not encode a functional selenoenzyne unless an enabling selenocysteine insertion sequence was appended to the presumed coding region and this cDNA. In this study we determined whether the native 7.5-kb SeD2 mRNA in rat tissues programmed the synthesis of the native type II deiodinase using specific antibodies that were raised against the C-terminus of full-length, 30-kDa SeD2 protein and against the catalytic core of SeD2. Direct analysis of the translation products programmed by the native SeD2 mRNA in cAMP-stimulated astrocytes was performed using antisense deoxynucleotides and hybrid selection strategies. (Bu)2cAMP-stimulated rat astrocytes expressed both type II deiodinase activity (approximately 2500 U/mg protein) and contained abundant levels of the 7.5-kb SeD2 mRNA. However, no immunoreactive 30-kDa SeD2 protein was identified by Western analysis, immunoprecipitation, or immunocytochemistry, and the specific C-terminus antiserum failed to immunoprecipitate deiodinase activity from (Bu)2cAMP-stimulated astrocytes, brown adipose tissue or brain. Instead, the native 7.5-kb SeD2 mRNA encoded a 15-kDa protein that terminated at the first UGA codon and contained the catalytically inactive, N-terminal 129 amino acids of SeD2. These data show that the native 7.5-kb SeD2 mRNA in stimulated astrocytes does not encode D2.
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Citation: Endocrinology. 1999 May;140(5):2206-15.