We used a novel approach to study the role of the Asn-linked oligosaccharides for human thyrotropin (hTSH) activity. Mutagenesis of Asn(N) within individual glycosylation recognition sequences to Gln (Q) was combined with expression of wild type and mutant hTSH in cell lines with different glycosylation patterns. The in vitro activity of hTSH lacking the Asn oligosaccharide (Q52/TSH) expressed in CHO-K1 cells (sialylated oligosaccharides) was increased 6-fold compared with wild type, whereas the activities of Q78/TSH and /TSHQ23 were increased 2-3-fold. Deletion of the Asn oligosaccharide also increased the thyrotropic activity of human chorionic gonadotropin, in contrast to previous findings at its native receptor. The in vitro activity of wild type hTSH expressed in CHO-LEC2 cells (sialic acid-deficient oligosaccharides), CHO-LEC1 cells (ManGlcNAc intermediates), and 293 cells (sulfated oligosaccharides) was 5-8-fold higher than of wild type from CHO-K1 cells. In contrast to CHO-K1 cells, there was no difference in the activity between wild type and selectively deglycosylated mutants expressed in these cell lines. Thus, in hTSH, the oligosaccharide at Asn and, specifically, its terminal sialic acid residues attenuate in vitro activity, in contrast to the previously reported stimulatory role of this chain for human chorionic gonadotropin and human follitropin activity. The increased thyrotropic activity of Q52/CG suggests that receptor-related mechanisms may be responsible for these differences among the glycoprotein hormones. Despite their increased in vitro activity, Q52/TSH, and Q78/TSH from CHO-K1 cells had a faster serum disappearance rate and decreased effect on T production in mice. These findings highlight the importance of individual oligosaccharides in maintaining circulatory half-life and hence in vivo activity of hTSH.

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