SUBUNIT-SPECIFIC FUNCTIONS OF N-LINKED OLIGOSACCHARIDES IN HUMAN THYROTROPIN - ROLE OF TERMINAL RESIDUES OF ALPHA-SUBUNIT AND BETA-SUBUNIT OLIGOSACCHARIDES IN METABOLIC-CLEARANCE AND BIOACTIVITY

The recombinant human thyroid stimulating hormone (rhTSH) containing o ligosaccharides terminated with NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc be ta 1 showed higher in vivo activity and lower metabolic clearance rate (MCR) than pituitary human TSH (phTSH), which contains oligosaccharid es terminating predominantly in SO(4)GalNAc(beta 1-4)GlcNAc beta 1. To elucidate the relative contribution of the sulfated and sialylated ca rbohydrate chains of each subunit in the MCR and bioactivity of the ho rmone, the alpha and beta subunits of phTSH, rhTSH, and enzymatically desialylated rhTSH (asialo-rhTSH; asrhTSH) were isolated, their oligos accharides were analyzed, and the respective subunits were dimerized i n various combinations, The hybrids containing alpha subunit from phTS H or asrhTSH showed higher in vitro activity than those with alpha sub unit from rhTSH, indicating that sialylation of alpha but not beta sub unit attenuates the intrinsic activity of TSH. In contrast, hybrids wi th beta subunit from rhTSH displayed lower MCR compared to those with beta subunit from phTSH. The phTSH alpha-rhTSH beta hybrid had the hig hest in vivo bioactivity followed by rhTSH alpha-rhTSH beta, rhTSH alp ha-phTSH beta, phTSH alpha-phTSR beta, and asrhTSH dimers, These diffe rences indicated that hybrids with beta subunit from rhTSH displayed t he highest in vivo activity and relatively low MCR, probably due to hi gher sialylation, more multiantennary structure, and/or the unique loc ation of the beta-subunit oligosacharide chain in the molecule, Thus, the N-linked oligosaccharides of the beta subunit of glycoprotein horm ones have a more pronounced role than those from the alpha subunit in the metabolic clearance and thereby in the in vivo bioactivity, In con trast, the terminal residues of alpha-subunit oligosaccharides have a major impact on TSH intrinsic potency.