Abstract
Thyroglobulin (TG) is the most abundant thyroid gland protein, a dimeric iodoglycoprotein (660 kDa). TG serves as the protein precursor in the synthesis of thyroid hormones tetraiodothyronine (T4) and triiodothyronine (T3). The primary site for T3 synthesis in TG involves an iodotyrosine acceptor at the antepenultimate Tyr residue (at the extreme carboxyl terminus of the protein). The carboxyl-terminal region of TG comprises a cholinesterase-like (ChEL) domain followed by a short unique tail sequence. Despite many studies, the monoiodotyrosine donor residue needed for the coupling reaction to create T3 at this evolutionarily conserved site remains unidentified. In this report, we have utilized a novel, convenient immunoblotting assay to detect T3 formation after protein iodination in vitro, enabling the study of T3 formation in recombinant TG secreted from thyrocytes or heterologous cells. With this assay, we confirm the antepenultimate residue of TG as a major T3-forming site, but also demonstrate that the side chain of this residue intimately interacts with the same residue in the apposed monomer of the TG dimer. T3 formation in TG, or the isolated carboxyl-terminal region, is inhibited by mutation of this antepenultimate residue, but we describe the first substitution mutation that actually increases T3 hormonogenesis by engineering a novel cysteine, 10 residues upstream of the antepenultimate residue, allowing for covalent association of the unique tail sequences, and that helps to bring residues Tyr2744 from apposed monomers into closer proximity.
Highlights
Thyroglobulin (TG) is the most abundant thyroid gland protein, a dimeric iodoglycoprotein (660 kDa)
Thyrocyte cell lines growing as monolayer cultures cannot iodinate and form thyroid hormone within their own secreted TG; upon in vitro iodination of TG secreted into cell culture medium, de novo T3 formation can be measured by immunoblotting with a mAb that recognizes T3, in parallel with immunoblotting using a polyclonal anti-TG antibody [5]
T3 formation in TG did not require TSH stimulation of thyrocytes, but we confirmed that T3 formation upon iodination was even greater when the recombinant TG was secreted from thyrocytes that had been stimulated by TSH (Fig. 1, B–D), consistent with what has been observed for endogenously expressed TG [5]
Summary
T4, thyroxine; TG, thyroglobulin; ChEL, cholinesterase-like; T3, tri-iodothyronine; TSH, thyroid-stimulating hormone; DIT, di-iodotyrosine; MIT, mono-iodotyrosine; DMEM, Dulbecco’s modified Eagle’s medium; HRP, horseradish peroxidase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; mTG, mouse TG. The Tgn gene encodes a single polypeptide synthesized in the thyrocyte endoplasmic reticulum as a ϳ330 kDa glycoprotein comprised of repeat domains within region I (bearing type 1 repeats plus linker and hinge segments), region II ϩ III (bearing type 2 and type 3 repeats), the carboxyl-terminal cholinesterase-like (ChEL) domain (containing both of the potential donor MIT residues noted above), and a unique (ϳ50 residue) tail sequence bearing the antepenultimate Tyr residue that can. This approach has allowed us to explore the relationship between TG dimerization and its efficiency in T3 formation at the antepenultimate Tyr residue that has been conserved in evolution for 500 million years
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