Equine chorionic gonadotropin (eCG) and lutropin (eLH) are composed of α- and β-subunits with an identical amino acid sequence but show different biological activities. To elucidate the molecular difference between these gonadotropins, the structure of the N-linked oligosaccharides of each β-subunit was determined. N-Linked sugar chains, liberated as tritium-labeled oligosaccharides by hydrazinolysis followed by N-acetylation and reduction with NaB3H4, were neutralized by sialidase digestion and/or methanolytic desulfation. Neutralized oligosaccharides were fractionated by sequential chromatography on serial lectin affinity columns and on a Bio-Gel P-4 column. Each oligosaccharide structure was determined by sequential exoglycosidase digestion in conjunction with elution profiles on lectin columns and methylation analysis. Each β-subunit contained a single N-glycosylation site, but a high degree of microheterogeneity was observed in the structure of its N-linked oligosaccharides. eCGβ contained mono-, bi-, tri-, and tetraantennary complex-type oligosaccharides in a ratio of 3:63:13:1. eCGβ oligosaccharides contained about 16% of the bisecting GlcNAc and about 20% of poly-N-acetyllactosamine structures. Elongation of N-acetyllactosamine units showed a preference to the Manα1→6 side rather than the Manα1→3 side. Triantennary chains had only a C-2, 4-branched structure. eLHβ contained only mono- and biantennary complex-type and hybrid-type oligosaccharides in a ratio of approximately 18:67:10. eLHβ also contained bisected structures in about 18%. Oligosaccharides derived from the sulfated fraction of eLHβ contained GalNAc residues at nonreducing termini. Oligosaccharides from the sialylated/sulfated fraction of eLHβ contained both Gal and GalNAc residues at nonreducing termini, and those GalNAc residues were preferentially distributed to the Manα1→3 side of the trimannosyl core. These results clearly indicate that eCGβ and eLHβ possess structurally distinct N-linked oligosaccharides in addition to different charge groups even though they have a protein moiety identical to each other. Our results suggest that the biological activity of these hormones might be modulated by its terminal charge groups and stem structures of carbohydrate moiety synthesized in different organs.
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