title: Biological activity and metabolic clearance of a recombinant human thyrotropin produced in Chinese hamster ovary cells.
abstract: The presence and specific structures of the oligosaccharides on TSH have been shown to be important for its production and bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the biological activity and metabolic clearance of a preparation of purified recombinant human (rh) TSH derived from a stable transfectant of Chinese hamster ovary cells. Carbohydrate compositional analysis of this rTSH showed it to be more highly sialylated than a nonrecombinant, cadaver-derived pituitary hTSH. In addition, no N-acetyl galactosamine was detectable in rhTSH, which implies the absence of terminal sulfate moieties, both of which are present in pituitary-derived TSH. The immunologic activity and porcine TSH receptor-binding activity of the preparation of rhTSH were 3- to 4-fold lower than those of a standard pituitary hTSH. The rhTSH showed a maximum stimulatory activity similar to that of pituitary hTSH in two different in vitro bioassays. However, rhTSH elicited about 3-fold and 5-fold less cAMP than pituitary TSH after stimulation of adenylyl cyclase in bovine thyroid membranes and the rat FRTL-5 cell line, respectively. Removal of sialic acid did not alter the immunologic activity of rhTSH. However, the potencies of rhTSH in receptor-binding, adenylyl cyclase, and FRTL-5 assays were increased 2.4-, 2.6- and 26.7-fold, respectively after sialic acid removal. These data suggest that the in vitro biological activity of rhTSH is influenced by its highly sialylated oligosaccharide chains. The rhTSH had a 2-fold lower metabolic clearance rate than pituitary TSH, resulting in a greater than 10-fold higher serum concentration of rhTSH at 3 h as compared to pituitary hTSH. After sialic acid removal, the rhTSH was cleared faster (7.5-fold) than pituitary hTSH, showing that its longer plasma half-life was due to its higher sialylation. Biologically active rhTSH should be of clinical value in the diagnosis and treatment of patients with thyroid cancer and as a pure hTSH reference preparation.
authors.affiliation: Molecular, Cellular and Nutritional Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: N R
authors.initials: NR
authors.lastname: Thotakura
authors.forename: R K
authors.initials: RK
authors.lastname: Desai
authors.forename: L G
authors.initials: LG
authors.lastname: Bates
authors.forename: E S
authors.initials: ES
authors.lastname: Cole
authors.forename: B M
authors.initials: BM
authors.lastname: Pratt
authors.forename: B D
authors.initials: BD
authors.lastname: Weintraub
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D005260:Female; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D008657:Metabolic Clearance Rate; D010053:Ovary; D010902:Pituitary Gland / Q000502:physiology; D051381:Rats; D011989:Receptors, Thyrotropin / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology; D013961:Thyroid Gland; D013972:Thyrotropin / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology; D014162:Transfection
chemical_list: D002241:Carbohydrates; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013972:Thyrotropin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases