NMR INVESTIGATIONS OF THE N-LINKED OLIGOSACCHARIDES AT INDIVIDUAL GLYCOSYLATION SITES OF HUMAN LUTROPIN

Human lutropin or luteinizing hormone (hLH) is a heterodimeric glycoprotein, composed of two subunits, hLH-alpha (N-glycosylated at Asn52 and Asn78) and hLH-beta (N-glycosylated at Asn30). The sugar chains were liberated by hydrazinolysis from intact hLH-beta and from glycopeptides obtained after tryptic digestion of hLH-alpha, subsequently reduced and fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC and identified mainly by one-dimensional (1D) and two-dimensional (2D) H-1-NMR spectroscopy. The results indicate predominantly diantennary, N-acetyllactosamine-type structures at all three glycosylation sites. The oligosaccharides attached to Asn52 (hLH-alpha) and Asn30 (hLH-beta) show a remarkably similar pattern, with mainly chain-terminating 4-sulphated 2-deoxy-2-N-acetylamino-D-galactose (GalNAc) and a sulphated/sialylated structure as the major single component. However, virtually all N-glycans on the beta subunit bear a fucose residue alpha-1-6-linked to the proximal GlcNAc, whereas those at Asn52 (and Asn78) of the alpha subunit are predominantly non-fucosylated. The oligosaccharides at Asn78 (hLH-alpha) are sialylated rather than sulphated and contain the unique sequence NeuAc-alpha-2-6GalNAc-beta-1-4GlcNAc-beta-1-2Man-alpha-1-3 as part of the majority of mono- and disialylated compounds. The major single constituent at Asn78 has the following structure: [GRAPHICS]