Matsui T, Mizuochi T, Titani K, Okinaga T, Hoshi M, Bousfield G R, Sugino H, Ward D N
Division of Biomedical Polymer Science, Fujita Health University, Aichi, Japan.
Biochemistry. 1994 Nov 29;33(47):14039-48. doi: 10.1021/bi00251a012.
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.
马绒毛膜促性腺激素（eCG）和促黄体素（eLH）由α-和β-亚基组成，它们的氨基酸序列相同，但具有不同的生物学活性。为了阐明这些促性腺激素之间的分子差异，测定了每个β-亚基的N-连接寡糖的结构。通过肼解将N-连接糖链释放为氚标记的寡糖，然后进行N-乙酰化并用NaB3H4还原，通过唾液酸酶消化和/或甲醇解脱硫使其中和。中和后的寡糖通过在系列凝集素亲和柱和Bio-Gel P-4柱上的顺序色谱法进行分级分离。通过外切糖苷酶顺序消化结合凝集素柱上的洗脱图谱和甲基化分析来确定每个寡糖结构。每个β-亚基包含一个单一的N-糖基化位点，但其N-连接寡糖的结构存在高度的微不均一性。eCGβ包含单天线、双天线、三天线和四天线复合型寡糖，比例为3:63:13:1。eCGβ寡糖包含约16%的平分型GlcNAc和约20%的多聚N-乙酰乳糖胺结构。N-乙酰乳糖胺单元的延伸显示出对Manα1→6侧而非Manα1→3侧的偏好。三天线链仅具有C-2,4-分支结构。eLHβ仅包含单天线和双天线复合型及杂合型寡糖，比例约为18:67:10。eLHβ也包含约18%的平分结构。来自eLHβ硫酸化部分的寡糖在非还原末端含有GalNAc残基。来自eLHβ唾液酸化/硫酸化部分的寡糖在非还原末端同时含有Gal和GalNAc残基，并且那些GalNAc残基优先分布在三甘露糖核心的Manα1→3侧。这些结果清楚地表明，即使eCGβ和eLHβ具有彼此相同的蛋白质部分，但除了不同的电荷基团外，它们还具有结构上不同的N-连接寡糖。我们的结果表明，这些激素的生物学活性可能受其末端电荷基团和在不同器官中合成的碳水化合物部分的茎结构的调节。
