The role of the asparagine-linked oligosaccharides of the alpha-subunit in human thyrotropin bioactivity

TSH and the gonadotropins (FSH, LH, and hCG) are a family of heterodimeric proteins that share a common alpha-subunit and differ in their hormone-specific beta-subunit. The asparagine-linked (N-linked) oligosaccharides on these hormones are important in signal transduction. The N-linked oligosaccharides on the alpha-subunit have no effect on hCG and hFSH receptor binding, but are critical for their biological activity. Here, we analyzed the role of alpha-subunit N-linked oligosaccharides in human TSH (hTSH) bioactivity by site-directed mutagenesis and gene transfer. This was achieved by mutating the asparagine (Asn) residue in the N-linked glycosylation consensus sequence (Asn-X-Thr/Ser) to aspartic acid. The wild-type hTSH and its variants were expressed in Chinese hamster ovary cells. Wild-type alpha-subunit and its mutants (alpha(1), alpha(2), and alpha((1+2))) were efficiently combined with TSH beta-subunit and secreted as dimers. The bioactivity of TSH glycosylation variants was determined by measuring their abilities to stimulate cAMP formation and T-3 secretion using a serum-free culture system of human thyroid follicles. Using this system, wildtype hTSH was significantly effective in the stimulation of cAMP formation and T-3 secretion. Deletion of the oligosaccharide units from either site 1 (alpha(1)) or site 2 (alpha(2)) of the alpha-subunit increased the biological activity of the dimer by about 30%. However, deletion of carbohydrate units from both Sites of hTSH alpha-subunit (alpha((1 divided by 2))) resulted in a significant reduction in cAMP formation (by similar to 70%) and T-3 secretion (by similar to 40%) compared to that with wild-type hTSH. These findings emphasize the importance of the alpha-subunit N-linked oligosaccharide chains on hTSH bioactivity.