THE ASPARAGINE-LINKED OLIGOSACCHARIDES AT INDIVIDUAL GLYCOSYLATION SITES IN HUMAN THYROTROPIN

The asparagine-linked carbohydrate structures at each of the three glycosylation sites of human thyrotrophin were investigated by 400 MHz H-1-NMR spectroscopy. Highly purified, biologically active human thyrotrophin (hTSH) was dissociated into its subunits hTSHalpha (glycosylated at Asn 52 and Asn 78) and hTSHbeta (glycosylated at Asn 23). The alpha-subunit was further treated with trypsin which gave two glycopeptides that were subsequently separated by reverse-phase HPLC and identified by amino acid sequence analysis. The oligosaccharides were liberated from hTSHalpha glycopeptides and from intact hTSHbeta by hydrazinolysis, and were fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC preparatory to structural analysis. The N-glycans present on hTSH were mainly diantennary complex-type structures with a common Manalpha1-3 branch that terminated with 4-O-sulphated GalNAc. The Manalpha1-6 branch displayed structural hetero-geneity in the terminal sequence, with chiefly alpha2-3-sialylated Gal and/or 4-O-sulphated GalNAc. The relative amounts of the two major complete diantennary oligosaccharides and their core fucosylation differed according to glycosylation site; the sulphated/sialylated diantennary oligosaccharide was most abundant at the two sites on the alpha-subunit, whereas the disulphated, core-fucosylated oligosaccharide was more plentiful on the beta-subunit. Some interesting structural features, not previously reported for the N-glycans of hTSH, included 3-O-sulphated galactose (SO4-3Gal) and peripheral fucose (Fucalpha1-3GlcNAc) in the Manalpha1-6 branch of some diantennary structures; the former suggests the presence of a hitherto uncharacterized galactose-3-O-sulphotransferase in thyrotroph cells of the human anterior pituitary gland.