Structure determination of the major N- and O-linked carbohydrate chains of the β subunit from equine chorionic gonadotropin

Abstract

The carbohydrate moieties of equine chorionic gonadotropin α and β subunits were released from the protein backbones by successive treatments with peptide-(N-acetyl-β-glucosaminyl)asparagine amidase F and alkaline borohydride and then fractionated by FPLC and HPLC. The major N- and O-linked glycans of the β subunit were characterized by 500-MHz 1H-NMR spectroscopy, showing a remarkable structural heterogeneity for the N-glycosidically linked chains, comprising mono-,di-, tri- and tri'-antennary N-acetyllactosamine type of glycans, being partly α1-6 fucosylated at the Asn-bound GlcNAc residue and having α2-6 and α2-3 linked N-acetyl- and N-acetyl-4-O-acetylneuraminic acid residues as sialic acid constituents. Significant differences in this respect were detected for the partially characterized glycans of the α subunit. The major part of the O-linked carbohydrate chains, occurring solely in the β subunit, is formed by tri-, tetra-, penta- and hexa-saccharides. There are indications for the presence of oligo(N-acetyllactosamine) units in both the N- and O-linked glycans of the β subunit.