SITE-SPECIFIC N-GLYCOSYLATION OF OVINE LUTROPIN - STRUCTURAL-ANALYSIS BY ONE-DIMENSIONAL AND 2-DIMENSIONAL H-1-NMR SPECTROSCOPY

The Asn-linked carbohydrate structures of the heterodimeric glycoprotein hormone lutropin from ovine pituitary glands have been investigated at each of its three glycosylation sites using one- and two-dimensional 400-MHz H-1-NMR spectroscopy. Highly purified, biologically active ovine lutropin (oLH) was dissociated and separated into its alpha and beta subunits (oLH-alpha, glycosylated at Asn56 and Asn82; oLH-beta glycosylated at Asn13). Oligosaccharides from intact oLH-beta and from glycopeptides obtained after tryptic digestion of oLH-alpha were released by hydrazinolysis and subsequently fractionated according to charge and size by anion-exchange and ion-suppression amine-adsorption HPLC, respectively. H-1-NMR analysis revealed, that monosulphated, mostly hybrid-type, oligosaccharides predominate at both glycosylation sites of oLH-alpha, whereas a disulphated, diantennary N-acetyllactosamine-type structure accounts for more than 60% of total oligosaccharides in the beta subunit. Furthermore, the saccharides attached to the beta subunit are almost completely fucosylated (Fuc-alpha-1-6) at the reducing terminal GlcNAc, whereas the sugar chains in oLH-alpha are either approximately 50% fucosylated (Asn82) or contain fucose only to a minor extent (Asn56). The results clearly indicate a distinct subunit- and site-specific synthesis of oligosaccharides in ovine lutropin and suggest that biosynthesis is effectively influenced by the surrounding polypeptide chain(s) at a given site.