Title : The
asparagine-linked
oligosaccharides at individual glycosylation
sites in human
thyrotrophin
Abstract :
- The asparagine-linked carbohydrate structures asparagine-linked carbohydrate structures at each of the three glycosylation sites of human thyrotrophin were investigated by 400 MHz 1H-NMR spectroscopy
- Highly purified, biologically active human thyrotrophin ( hTSH ) was dissociated into its subunits hTSH alpha (glycosylated at Asn 52 and Asn 78 ) and hTSH beta (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 hTSH alpha glycopeptides and from intact hTSH beta 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 Man alpha 1-3 branch that terminated with 4-O-sulphated GalNAc.
- The Man alpha 1-6 branch displayed structural heterogeneity in the terminal sequence , with chiefly alpha 2-3-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 (Fuc alpha 1-3GlcNAc) in the Man alpha 1-6 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