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 heterodim eric proteins that share a common alpha-subunit and differ in their ho rmone-specific beta-subunit. The asparagine-linked (N-linked) oligosac charides on these hormones are important in signal transduction. The N -linked oligosaccharides on the alpha-subunit have no effect on hCG an d hFSH receptor binding, but are critical for their biological activit y. Here, we analyzed the role of alpha-subunit N-linked oligosaccharid es in human TSH (hTSH) bioactivity by site-directed mutagenesis and ge ne transfer. This was achieved by mutating the asparagine (Asn) residu e in the N-linked glycosylation consensus sequence (Asn-X-Thr/Ser) to aspartic acid. The wild-type hTSH and its variants were expressed in C hinese hamster ovary cells. Wild-type alpha-subunit and its mutants (a lpha(1), alpha(2), and alpha((1+2))) were efficiently combined with TS H beta-subunit and secreted as dimers. The bioactivity of TSH glycosyl ation variants was determined by measuring their abilities to stimulat e cAMP formation and T-3 secretion using a serum-free culture system o f human thyroid follicles. Using this system, wildtype hTSH was signif icantly effective in the stimulation of cAMP formation and T-3 secreti on. Deletion of the oligosaccharide units from either site 1 (alpha(1) ) or site 2 (alpha(2)) of the alpha-subunit increased the biological a ctivity of the dimer by about 30%. However, deletion of carbohydrate u nits from both Sites of hTSH alpha-subunit (alpha((1 divided by 2))) r esulted in a significant reduction in cAMP formation (by similar to 70 %) and T-3 secretion (by similar to 40%) compared to that with wild-ty pe hTSH. These findings emphasize the importance of the alpha-subunit N-linked oligosaccharide chains on hTSH bioactivity.