STRUCTURAL-ANALYSIS OF N-LINKED OLIGOSACCHARIDES OF EQUINE CHORIONIC-GONADOTROPIN AND LUTROPIN BETA-SUBUNITS

Equine chorionic gonadotropin (eCG) and lutropin (eLH) are composed of alpha- and beta-subunits with an identical amino acid sequence but sh ow different biological activities. To elucidate the molecular differe nce between these gonadotropins, the structure of the N-linked oligosa ccharides of each beta-subunit was determined. N-Linked sugar chains, liberated as tritium-labeled oligosaccharides by hydrazinolysis follow ed by N-acetylation and reduction with (NaBH4)-H-3, were neutralized b y sialidase digestion and/or methanolytic desulfation. Neutralized oli gosaccharides were fractionated by sequential chromatography on serial lectin affinity columns and on a Bio-Gel P-4 column. Each oligosaccha ride structure was determined by sequential exoglycosidase digestion i n conjunction with elution profiles on lectin columns and methylation analysis. Each beta-subunit contained a single N-glycosylation site, b ut a high degree of microheterogeneity was observed in the structure o f its N-linked oligosaccharides. eCG beta contained mono-, bi-, tri-, and tetraantennary complex-type oligosaccharides in a ratio of 3:63:13 :1. eCG beta oligosaccharides contained about 16% of the bisecting Glc NAc and about 20% of poly-N-acetyllactosamine structures. Elongation o f N-acetyllactosamine units showed a preference to the Man alpha 1-->6 side rather than the Man alpha 1-->3 side. Triantennary chains had on ly a C-2, 4-branched structure. eLH beta contained only mono- and bian tennary complex-type and hybrid-type oligosaccharides in a ratio of ap proximately 18:67:10. eLH beta also contained bisected structures in a bout 18%. Oligosaccharides derived from the sulfated fraction of eLH b eta contained GalNAc residues at nonreducing termini. Oligosaccharides from the sialylated/sulfated fraction of eLH beta contained both Gal and GalNAc residues at nonreducing termini, and those GalNAc residues were preferentially distributed to the Man alpha 1-->3 side of the tri mannosyl core. These results clearly indicate that eCG beta and eLH be ta possess structurally distinct N-linked oligosaccharides in addition to different charge groups even though they have a protein moiety ide ntical to each other. Our results suggest that the biological activity of these hormones might be modulated by its terminal charge groups an d stem structures of carbohydrate moiety synthesized in different orga ns.