Thyroglobulin (Tg) is the substrate for thyroid hormone biosynthesis, which requires tyrosine iodination and iodotyrosine coupling and occurs at the apical membrane of the thyrocytes. Tg glycoconjugates have been shown to play a major role in Tg routing through cellular compartments and recycling after endocytosis. Here we show that glycoconjugates also play a direct role in hormonosynthesis. The N-terminal domain (NTD; Asn 1 -Met 171 ) of human Tg, which bears the preferential hormonogenic

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... , brings two N-glycans (Asn 57 and Asn 91 ). NTD preparations were purified from Tg with low and mild iodine content in vivo and from poorly iodinated Tg after in vitro iodination and coupling. NTD separated from poorly iodinated Tg was also submitted to iodination and coupling after desialylation and deglycosylation. The various NTD isoforms were analyzed for their N-glycan structures and hormone contents. Our results show that 1) in vivo as well as in vitro unglycosylated isoforms did not synthesize hormones, whereas fully or partially (at Asn 91 ) glycosylated isoforms did; 2) high mannose type structures enhanced the hormone content; and 3) desialylation did not affect in vitro hormone synthesis. Evidence of a direct involvement in hormonosynthesis adds to the role of N-glycans in Tg function and opens the way to new mechanisms for regulation (e.g. TSH modulation of N-glycan) or alteration (e.g. Asn 91 mutation) of thyroid hormone synthesis.