Subunit-Specific Functions of N-Linked Oligosaccharides in Human Thyrotropin: Role of Terminal Residues of α- and β-Subunit Oligosaccharides in Metabolic Clearance and Bioactivity

• Published in: Proceedings of the National Academy of Sciences - PNAS 1995-09, Vol.92 (20), p.9062-9066
• Main Authors: Szkudlinski, Mariusz W., Thotakura, N. Rao, Weintraub, Bruce D.
• Format: Article
• Language: English
• Subjects: Animals; Bioassay; Carbohydrate Sequence; Carbohydrates; Carbohydrates - analysis; CHO Cells; Cricetinae; Cyclic AMP - metabolism; Dimers; Endocrinology; Glycoprotein Hormones, alpha Subunit - chemistry; Glycoprotein Hormones, alpha Subunit - metabolism; Glycoprotein Hormones, alpha Subunit - pharmacology; Glycoproteins; Hormones; Humans; Macromolecular Substances; Male; Medical research; Metabolic Clearance Rate; Metabolism; Molecular Sequence Data; Oligosaccharides; Oligosaccharides - chemistry; Oligosaccharides - metabolism; Oligosaccharides - pharmacology; Protein Multimerization; Rats; Rats, Sprague-Dawley; Receptors; Receptors, Thyrotropin - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Structure-Activity Relationship; Sulfates; Thyrotropin - chemistry; Thyrotropin - metabolism; Thyrotropin - pharmacology; Transfection; Triiodothyronine - blood; Triiodothyronine - pharmacokinetics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.92.20.9062

The recombinant human thyroid stimulating hormone (rhTSH) containing oligosaccharides terminated with NeuAc(α2-3)Gal(β1-4)GlcNAcβ1 showed higher in vivo activity and lower metabolic clearance rate (MCR) than pituitary human TSH (phTSH), which contains oligosaccharides terminating predominantly in SO44GalNAc(β1-4)GlcNAcβ1. To elucidate the relative contribution of the sulfated and sialylated carbohydrate chains of each subunit in the MCR and bioactivity of the hormone, the α and β 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 α subunit from phTSH or asrhTSH showed higher in vitro activity than those with α subunit from rhTSH, indicating that sialylation of α but not β subunit attenuates the intrinsic activity of TSH. In contrast, hybrids with β subunit from rhTSH displayed lower MCR compared to those with β subunit from phTSH. The phTSHα-rhTSHβ hybrid had the highest in vivo bioactivity followed by rhTSHα-rhTSHβ, rhTSHα-phTSHβ, phTSHα-phTSHβ, and asrhTSH dimers. These differences indicated that hybrids with β 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 β-subunit oligosaccharide chain in the molecule. Thus, the N-linked oligosaccharides of the β subunit of glycoprotein hormones have a more pronounced role than those from the α subunit in the metabolic clearance and thereby in the in vivo bioactivity. In contrast, the terminal residues of α-subunit oligosaccharides have a major impact on TSH intrinsic potency.