Structural Analysis of N-Linked Oligosaccharides of Equine Chorionic Gonadotropin and Lutropin .beta.-Subunits

Abstract

Equine chorionic gonadotropin (eCG) and lutropin (eLH) are composed of alpha- and beta-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 beta-subunit was determined. N-linked sugar chains, liberated as tritum-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 beta-subunit contained a single N-glycosylation site, but a high degree of microheterogeneity was observed in the structure of 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 GlcNAc and about 20% of poly-N-acetyllactosamine structures. Elongation of N-acetyllactosamine units showed a preference to the Man alpha 1-->6 side rather than the Man alpha 1-->3 side. Triantennary chains had only a C-2, 4-branched structure. eLH beta contained only mono- and biantennary complex-type and hybrid-type oligosaccharides in a ratio of approximately 18:67:10. eLH beta also contained bisected structures in about 18%. Oligosaccharides derived from the sulfated fraction of eLH beta 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 trimannosyl core. These results clearly indicate that eCG beta and eLH beta 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.

Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

“…These data are in agreement with our previous reports showing that carbohydrate side chains play a primary role in the stability of heterodimers at 37 8C (Galet et al 2004) in which the interchange of disulfide bonds is involved (Belghazi et al 2006). The carbohydrate moieties of eCG are much bulkier and more acidic than those in pituitary eLH and in recombinant eLH/CGs (Smith et al 1993, Matsui et al 1994, Bousfield & Butnev 2001. The present report reinforces the view that the carbohydrate chains in eCG play a stabilizing role on the heterodimeric structure of the hormone.…”

“…As an exception in equids, eCG and LH (eLH) are encoded by the same a and b genes (Sherman et al 1992) and thus exhibit the same peptidic moiety. However, they strongly differ in their N and O-linked carbohydrate side chains (Smith et al 1993, Matsui et al 1994, Bousfield & Butnev 2001, conferring different in vivo biological potencies due to a prolonged eCG halflife as compared with eLH (Klett et al 2003), as well as different thermal stabilities (Galet et al 2004).…”

Biological activities of recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) expressed in Sf9 and Mimic insect cell lines

“…In addition to complex sialylated biantennary N-glycans (Smith et al 1993, Matsui et al 1994, eCG possesses 12 uncommon disialylated poly-N-acetyllactosaminyl O-glycans located at the C-terminus of the -subunit (Hokke et al 1994, Bousfield & Butnev 2001. The N-glycans of eLH occupy the same three sites as those in eCG (Fig.…”

“…In addition to N-glycans, both eLH and eCG -subunits possess a carboxy-terminal peptide (CTP) of 28 amino acids ( 122-149), which is O-glycosylated at the same twelve serine or threonine residues (Bousfield & Butnev 2001). Placental and pituitary hormones also strongly differ by their N-glycan termination with sialic acids (Sia 2,3 Gal) on eCG and sulphated Nacetylgalactosamines (SO4-4-GalNAc) on eLH (Smith et al 1993, Matsui et al 1994). The remarkable difference in their molecular weight is essentially due to the presence of longer disialylated poly-N-acetyllactosaminyl O-glycans on eCG, with a greater percentage of O-glycans attached at serine and threonine sites (Hokke et al 1994, Bousfield & Butnev 2001 (Fig.…”

Involvement of equine chorionic gonadotropin (eCG) carbohydrate side chains in its bioactivity; lessons from recombinant hormone expressed in insect cells

“…In addition to N-glycans, both eLH and eCG β-subunits possess a carboxy-terminal peptide (CTP) of 29 amino acids (β121-149), which is O-glycosylated at the same twelve serine or threonine residues [6]. Placental and pituitary hormones also strongly differ by their N-glycan termination with sialic acids (Siaα2, 3Gal) on eCG and sulfated N-acetylgalactosamines (SO 4 -4-GalNac) in eLH [7,8]. The remarkable difference in their molecular weight is essentially due to the presence of longer disialylated poly-N-acetyllactosaminyl O-glycans on eCG with a greater percentage of O-glycans attached at serine and threonine sites [6,9] (Fig.…”