NMR investigations of the N‐linked oligosaccharides at individual glycosylation sites of human lutropin

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Abstract
Human lutropin or luteinizing hormone (hLH) is a heterodimeric glycoprotein, composed of two subunits, hLHα (N-glycosylated at Asn52 and Asn78) and hLHβ (N-glycosylated at Asn30). The sugar chains were liberated by hydrazinolysis from intact hLHβ and from glycopeptides obtained after tryptic digestion of hLHα, 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) 1H-NMR spectroscopy. The results indicate predominantly diantennary, N-acetyllactosamine-type structures at all three glycosylation sites. The oligosaccharides attached to Asn52 (hLHα) and Asn30 (hLHβ) 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 β subunit bear a fucose residue α1-6-linked to the proximal GlcNAc, whereas those at Asn52 (and Asn78) of the α subunit are predominantly non-fucosylated. The oligosaccharides at Asn78 (hLHα) are sialylated rather than sulphated and contain the unique sequence NeuAcα2-6GalNAcβ1-4GlcNAcβ1-2Manα1-3 as part of the majority of mono- and disialylated compounds. The major single constituent at Asn78 has the following structure: