Relationship between the dimerization of thyroglobulin and its ability to form triiodothyronine.

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.

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