BIOSYNTHESIS AND GLYCOSYLATION OF THYROTROPIN

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The oligosaccharide structure of secreted TSH in perinatal and mature rats with hypothyroidism was compared to euthyroid controls using newly developed methods of anion exchange HPLC. Compared to control TSH, hypothyroid TSH showed relatively more sialic acid and less terminal sulfate, as well as more complicated structure with 3 or more negative charges, probably representing multiantennary structures. TSH derived from chronically hypothyroid animals was similar to recombinant TSH produced after transfection of alpha and TSH-beta genes into Chinese hamster ovary cells. Such recombinant TSH was completely sialylated and showed no evidence of Ga1Nac or terminal sulfate moieties. The recombinant hormone was 3- to 8-fold less potent in various in vitro bioassays, but showed a considerably longer half-life when injected into euthyroid rats. Hypothyroid as well as recombinant TSH will be useful in determining the physiologic roles of sialic acid vs. sulfate in the action of human TSH. In addition, the recombinant hormone will be of clinical utility in the diagnosis and treatment of patients with thyroid cancer. Using site-directed mutagenesis, we are currently determining the structure-function relationships of human TSH. We have already mutated various amino acids as well as glycosylation sites and determined important functional roles of various codons in the beta subunit. We are also generating several stable cell lines producing these TSH mutants so that detailed studies of biosynthesis, degradation and secretion can be determined. Finally, we have used various inhibitors of oligosaccharide processing to determine the role of carbohydrate structure on the secretion of TSH from dispersed rodent pituitary cells. Inhibitors of both glucosidases as well as mannosidases impair the assembly and secretion of TSH and the high mannose forms containing glucose are particularly susceptible to intracellular degradation. Several stable mutant cell lines producing TSH molecules with altered carbohydrate chains are being established to extend these studies.