Animal Biotechnology, Graduate School of Future Convergence Technology, Institute of Genetic Engineering, Hankyong National University, Ansung, 17579, Korea.
Department of Animal Resource Science, Hankyong National University, Ansung, 17579, Korea.
BMC Biotechnol. 2019 Aug 13;19(1):60. doi: 10.1186/s12896-019-0550-6.
To directly assess the biological role of oligosaccharides in recombinant equine chorionic gonadotropin (rec-eCG) functioning, cDNA encoding the full-length eCGβ-subunit was fused with the mature protein part of the α-subunit, and we examined the expression levels of deglycosylated eCG mutants, the ovulation rate for deglycosylated mutants in C57BL/6 mice.
The characterizations of heterodimeric and tethered mutants were studied following their respective secretions in culture medium, molecular weight and ovulation in vivo. Rec-eCG variants containing mutations at glycosylation sites at Asn82 of the α-subunit (eCGβ/αΔ82) and Asn13 of the β-subunit (eCGβΔ13/α) were not efficiently secreted into the culture medium from transfected cells. Western blot analysis revealed that the rec-eCGβ/α proteins have an approximate broad range of molecular weights of 40-46 kDa. Three rec-eCG mutants-a deglycosylated site at Asn56 of the α-subunit (eCGβ/αΔ56), a deletion of the C-terminal region of the β-subunit (eCGβ-D/α), and the double mutant (eCGβ-D/αΔ56)-turned out to have clearly lower (approximately 4-23 kDa) molecular weights. Protein N-glycosydase F (PNGase F) treatment markedly decreased the molecular weight to approximately 2-10 kDa. Normal oocytes were significantly more abundant in the natural eCG-treated group than in mutant rec-eCG-treated groups. In particular, numbers of nonfuntional oocytes were remarkably lower in all rec-eCG groups.
Our results indicate that the ovulation rates of oocytes are not affected by the deglycosylated rec-eCGβ/α mutant proteins. There are around 20% non-functional oocytes with natural eCG and only 2% with the rec-eCGs tested. These results provide insight into the molecular mechanisms underlying the production of rec-eCG hormones with excellent bioactivity in vivo.
为了直接评估寡糖在重组马绒毛膜促性腺激素（rec-eCG）功能中的生物学作用，我们将 eCGβ-亚基的全长 cDNA 与α-亚基的成熟蛋白部分融合，并检测了去糖基化 eCG 突变体的表达水平以及去糖基化突变体在 C57BL/6 小鼠中的排卵率。
对各自在培养基中分泌的异源二聚体和连接突变体的特性、分子量以及体内排卵进行了研究。含有在 α-亚基的 Asn82 （eCGβ/αΔ82）和β-亚基的 Asn13 （eCGβΔ13/α）糖基化位点突变的 rec-eCG 变体不能有效地从转染细胞分泌到培养基中。Western blot 分析表明，rec-eCGβ/α 蛋白的分子量约为 40-46 kDa。三个 rec-eCG 突变体-α-亚基的一个去糖基化位点（eCGβ/αΔ56）、β-亚基的 C 端区域缺失（eCGβ-D/α）和双突变体（eCGβ-D/αΔ56）-分子量明显较低（约 4-23 kDa）。蛋白 N-糖苷酶 F（PNGase F）处理使分子量显著降低至约 2-10 kDa。天然 eCG 处理组中的正常卵母细胞明显多于突变 rec-eCG 处理组。特别是，所有 rec-eCG 组的非功能性卵母细胞数量明显较低。
我们的结果表明，卵母细胞的排卵率不受去糖基化 rec-eCGβ/α 突变蛋白的影响。天然 eCG 有 20%左右的非功能性卵母细胞，而测试的 rec-eCG 只有 2%。这些结果为体内具有优异生物活性的 rec-eCG 激素产生的分子机制提供了新的见解。
