title: Subunit-specific functions of N-linked oligosaccharides in human thyrotropin: role of terminal residues of alpha- and beta-subunit oligosaccharides in metabolic clearance and bioactivity.
abstract: The recombinant human thyroid stimulating hormone (rhTSH) containing oligosaccharides terminated with NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc beta 1 showed higher in vivo activity and lower metabolic clearance rate (MCR) than pituitary human TSH (phTSH), which contains oligosaccharides terminating predominantly in SO(4)4GalNAc(beta 1-4)GlcNAc beta 1. To elucidate the relative contribution of the sulfated and sialylated carbohydrate chains of each subunit in the MCR and bioactivity of the hormone, the alpha and beta subunits of phTSH, rhTSH, and enzymatically desialylated rhTSH (asialo-rhTSH; asrhTSH) were isolated, their oligosaccharides were analyzed, and the respective subunits were dimerized in various combinations. The hybrids containing alpha subunit from phTSH or asrhTSH showed higher in vitro activity than those with alpha subunit from rhTSH, indicating that sialylation of alpha but not beta subunit attenuates the intrinsic activity of TSH. In contrast, hybrids with beta subunit from rhTSH displayed lower MCR compared to those with beta subunit from phTSH. The phTSH alpha-rhTSH beta hybrid had the highest in vivo bioactivity followed by rhTSH alpha-rhTSH beta, rhTSH alpha-phTSH beta, phTSH alpha-phTSH beta, and asrhTSH dimers. These differences indicated that hybrids with beta subunit from rhTSH displayed the highest in vivo activity and relatively low MCR, probably due to higher sialylation, more multiantennary structure, and/or the unique location of the beta-subunit oligosaccharide chain in the molecule. Thus, the N-linked oligosaccharides of the beta subunit of glycoprotein hormones have a more pronounced role than those from the alpha subunit in the metabolic clearance and thereby in the in vivo bioactivity. In contrast, the terminal residues of alpha-subunit oligosaccharides have a major impact on TSH intrinsic potency.
authors.affiliation: Molecular and Cellular Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: M W
authors.initials: MW
authors.lastname: Szkudlinski
authors.forename: N R
authors.initials: NR
authors.lastname: Thotakura
authors.forename: B D
authors.initials: BD
authors.lastname: Weintraub
mesh_terms: D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism*; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D006801:Humans; D046911:Macromolecular Substances; D008297:Male; D008657:Metabolic Clearance Rate; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D055503:Protein Multimerization; D051381:Rats; D017207:Rats, Sprague-Dawley; D011989:Receptors, Thyrotropin / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013972:Thyrotropin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D014162:Transfection; D014284:Triiodothyronine / Q000097:blood / Q000493:pharmacokinetics
chemical_list: D002241:Carbohydrates; D015292:Glycoprotein Hormones, alpha Subunit; D046911:Macromolecular Substances; D009844:Oligosaccharides; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D014284:Triiodothyronine; D013972:Thyrotropin; D000242:Cyclic AMP