Role of Sulfated Tyrosines of Thyroglobulin in Thyroid Hormonosynthesis

Abstract

Our previous studies showed that sulfated tyrosines (Tyr-S) are involved in thyroid hormone synthesis and that Tyr5, the main hormonogenic site of thyroglobulin (Tg), is sulfated. In the present paper, we studied the role of Tyr-S in the formation and activity of the peroxidase-Tg complex. Results show that noniodinated 35SO3-Tg specifically binds (Kd = 1.758 μm) to immobilized lactoperoxidase (LPO) via Tyr-S linkage by using saturation binding and competition experiments. We found that NIFEY-S, a 15-amino acid peptide corresponding to the NH2-end sequence of Tg and containing the hormonogenic acceptor Tyr5-S, was a better competitor than cholecystokinin and Tyr-S. 35SO3-Tg, iodinated without peroxidase, bound to LPO with a Kd (1.668 μm) similar to that of noniodinated Tg, suggesting that 1) its binding occurs via Tyr-S linkage and 2) Tyr-S requires peroxidase to be iodinated, whereas nonsulfated Tyr does not. Iodination of NIFEY-S with [125I]iodide showed that Tyr5-S iodination increased with LPO concentration, whereas iodination of a nonsulfated peptide containing the donor Tyr130 was barely dependent on LPO concentration. Enzymatic hydrolysis of iodinated Tg or NIFEY-S showed that the amounts of sulfated iodotyrosines also depended on LPO amount. Sulfated iodotyrosines were detectable in the enzyme-substrate complex, suggesting they have a short life before the coupling reaction occurs. Our data suggest that after Tyr-S binding to peroxidase where it is iodinated, the sulfate group is removed, releasing an iodophenoxy anion available for coupling with an iodotyrosine donor.