Kim Jung-Min, Munkhuu Orgilkhatan, Byambaragchaa Munkhzaya, Lee Bae-Ik, Kim Shin-Kwon, Kang Myung-Hwa, Kim Dae-Jung, Min Kwan-Sik
Animal Biotechnology, Graduate School of Future Convergence Technology, Department of Animal Life Science, Institute of Genetic Engineering, Hankyong National University, Ansung 17579, Republic of Korea.
Aquaculture Research Division, National Institute of Fisher Science (NIFS), Busan 46083, Republic of Korea.
Gen Comp Endocrinol. 2019 May 15;276:37-44. doi: 10.1016/j.ygcen.2019.03.003. Epub 2019 Mar 2.
Eel follicle-stimulating hormone (eelFSH) is composed of a common α-subunit and a hormone specific β-subunit, both of which contain two N-linked carbohydrate residues. We characterized the biologically active single chains by fusing the α-subunit to the carboxyl terminal region of the eelFSH β-subunit. Expression vectors were constructed and the biological activity of the recombinant hormones (rec-hormones) was characterized using Chinese hamster ovary (CHO) K1 cells expressing the eelFSH receptor gene. Mutagenesis of the individual and double glycosylated sites was performed to determine the functions of the oligosaccharide chains on signal transduction. The absence of the Asn (eelFSHβΔ22/α) and Asn (eelFSHβΔ5.22/α) N-linked oligosaccharide chain in the eelFSH β-subunit completely reduced the secretion level in the medium and cell lysate of CHO-K1 cells. The expression levels of eelFSHβ/α wild-type in CHO suspension (CHO-S) cells was approximately 4-fold higher in CHO-k1 cells. The molecular weight of rec-eelFSHβ/α wild-type by western blotting analysis was found to be 34 kDa. Mutants (β/αΔ56, β/αΔ79, and βΔ5/α) lacking single oligosaccharide sites showed molecular weights that were reduced by approximately 10%. The digestion of N-linked oligosaccharides using PNGaseF treatment showed that the molecular weights of all mutants were reduced to 27-kDa. The oligosaccharide chains in rec-eelFSHβ/α wild-type were modified to a molecular weight of approximately 7-10 kDa in CHO-K1 and CHO-S cells. Oligosaccharide site deletions at positions Asn and Asn on the α-subunit and Asn on the β-subunit were found to play an essential role in cAMP signal transduction through the eelFSH receptor. The EC values of Asn and Asn resulted in a significant decrease in potency to 64% and 53% of the wild type, respectively. Specifically, the removal of the carbohydrates at Asn of the α-subunit (β/αΔ79) was drastically reduced to 53.8% of the wild-type levels in maximum response. These results have allowed for the identification of the site-specific roles of carbohydrate residues in eel FSH. Our data suggest that N-linked oligosaccharide chains play a pivotal role in biological activity through the eelFSH receptor as suggested in similar studies of other mammalian FSH hormones.
鳗鲡促卵泡激素（eelFSH）由一个共同的α亚基和一个激素特异性β亚基组成，二者均含有两个N-连接的碳水化合物残基。我们通过将α亚基融合到eelFSH β亚基的羧基末端区域来表征具有生物活性的单链。构建了表达载体，并使用表达鳗鲡促卵泡激素受体基因的中国仓鼠卵巢（CHO）K1细胞来表征重组激素（重组激素）的生物活性。对单个和双糖基化位点进行诱变，以确定寡糖链在信号转导中的功能。鳗鲡促卵泡激素β亚基中Asn（eelFSHβΔ22/α）和Asn（eelFSHβΔ5.22/α）N-连接寡糖链的缺失完全降低了CHO-K1细胞培养基和细胞裂解物中的分泌水平。鳗鲡促卵泡激素β/α野生型在CHO悬浮（CHO-S）细胞中的表达水平比在CHO-K1细胞中高约4倍。通过蛋白质印迹分析发现重组鳗鲡促卵泡激素β/α野生型的分子量为34 kDa。缺乏单个寡糖位点的突变体（β/αΔ56、β/αΔ79和βΔ5/α）的分子量降低了约10%。使用PNGaseF处理消化N-连接寡糖后发现，所有突变体的分子量均降至27 kDa。在CHO-K1和CHO-S细胞中，重组鳗鲡促卵泡激素β/α野生型中的寡糖链被修饰为分子量约为7-10 kDa。发现α亚基上Asn和Asn以及β亚基上Asn处的寡糖位点缺失在通过鳗鲡促卵泡激素受体的cAMP信号转导中起重要作用。Asn和Asn的EC值导致效力分别显著降低至野生型的64%和53%。具体而言，α亚基Asn处碳水化合物的去除（β/αΔ79）在最大反应中急剧降低至野生型水平的53.8%。这些结果使得能够确定鳗鲡促卵泡激素中碳水化合物残基的位点特异性作用。我们的数据表明，N-连接寡糖链在通过鳗鲡促卵泡激素受体的生物活性中起关键作用，这与其他哺乳动物促卵泡激素的类似研究结果一致。
