Structural analysis of N-linked oligosaccharides of equine chorionic gonadotropin and lutropin beta-subunits

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Issue Date
1994-11
Authors
Matsui, Taei
Mizuochi, Tsuguo
Titani, Koiti
Okinaga, Tatsuyuki
Hoshi, Motonori
Bousfield, George R.
Sugino, Hiromu
Ward, Darrell N.
Advisor
Citation

Taei Matsui, Tsuguo Mizuochi, Koiti Titani, Tatsuyuki Okinaga, Motonori Hoshi, George R. Bousfield, Hiromu Sugino, Darrell N. Ward; Structural Analysis of N-Linked Oligosaccharides of Equine Chorionic Gonadotropin and Lutropin .beta.-Subunits; Biochemistry 1994 33 (47), 14039-14048; DOI: 10.1021/bi00251a012

Abstract

Equine chorionic gonadotropin (eCG) and lutropin (eLH) are composed of a- and P-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 P-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 andor 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 P-subunit contained a single N-glycosylation site, but a high degree of microheterogeneity was observed in the structure of its N-linked oligosaccharides. eCGP contained mono-, bi-, tri-, and tetraantennary complex-type oligosaccharides in a ratio of 3:63: 13: 1. eCGP 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 Manal-6 side rather than the Manal-3 side. Triantennary chains had only a C-2,4-branched structure. eLHP contained only mono- and biantennary complex-type and hybrid-type oligosaccharides in a ratio of approximately 18:67: 10. eLHP also contained bisected structures in about 18%. Oligosaccharides derived from the sulfated fraction of eLHP contained GalNAc residues at nonreducing termini. Oligosaccharides from the sialylatedsulfated fraction of eLHP contained both Gal and GalNAc residues at nonreducing termini, and those GalNAc residues were preferentially distributed to the Manal-3 side of the trimannosyl core. These results clearly indicate that eCGp and eLHP 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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