Site-specific N-glycosylation of ovine lutropin. Structural analysis by one- and two-dimensional 1H-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 1H-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. 1H-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.
Department of Biochemistry, University of Auckland, New Zealand.
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Weisshaar
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Hiyama
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Renwick
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D007986:Luteinizing Hormone / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D012756:Sheep; D014357:Trypsin
D002241:Carbohydrates; D006023:Glycoproteins; D009844:Oligosaccharides; D007986:Luteinizing Hormone; D014357:Trypsin