Hormone formation in the isolated fragment 1–171 of human thyroglobulin involves the couple tyrosine 5 and tyrosine 130

Abstract

The 22 kDa fragment (Asn 1—Met 171) purified from iodine-poor human thyroglobulin (hTg) is capable by itself to synthesize thyroxine at Tyr 5, the preferential hormonogenic acceptor site of the protein, after iodination in vitro. To identify the corresponding donor site in this model we studied the fate of the six Tyr residues present in the 22 kDa peptide after in vitro hormone synthesis. Structural studies of the tyrosyl peptides showed that Tyr 5 was the only thyroxine-forming site, the other tyrosines (29, 89, 97 and 107) were noniodinated and Tyr 130 was recovered in alanine form after CNBH 4 treatment of the Tyr 130-containing peptide. Taking into account that alanine could arise from aminoreduced pyruvate species, these results showed that in the 22 kDa fragment (1) hormone formation involves the couple Tyr 5 (acceptor)-Tyr 130 (donor), and (2) dehydroalanine, the resultant product of donor tyrosine after hormone synthesis, has evolved in pyruvoyl form. To test whether Tyr 130 could also act as donor in hTg hormone synthesis, the 22 kDa peptide was isolated from hTg iodinated under conditions leading to iodotyrosine formation followed or not by hormone formation and the tyrosyl peptides were analyzed. After hTg iodination and before coupling (i.e. hormone synthesis) only Tyr 5 and Tyr 130 were recovered in iodotyrosine form; after coupling thyroxine was found at Tyr 5 whereas Tyr 130 disappeared. Taken together these results, correlated with the previously reported cleavage of hTg chain at Tyr 130 occurring during in vivo hormone synthesis, support the theory that the couple Tyr 5 (acceptor)-Tyr 130 (donor) would be the preferential hormonogenic site in human Tg.