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Abstract：
Human chorionic gonadotropin (hCG) is a glycoprotein hormone that exists as a heterodimer comprised of an α subunit and β subunit linked with disulfide bridges. The β subunit contains four O-glycosylation sites. Previous studies have found that the translation of mRNA to polypeptides of the β subunit was a severely limiting step for the expression of recombinant hCG protein in Chinese hamster ovary (CHO) cells. The effects of O-glycosylation on recombinant hCG protein expression were assessed by adding O-glycan precursors and overexpressing and knocking down key regulatory genes of O-glycan precursor synthesis and O-glycan sugar chain synthesis or hydrolases. The results indicated that O-glycosylation was indeed limiting in the expression of recombinant hCG protein, and N-acetylgalactosamine (GalNAc) was the major limiting precursor. Glutamine-fructose-6-phosphate transaminase 2 (Gfat2) and Uridine diphosphate-glucose pyrophosphorylase 2 (Ugp2), key regulatory genes of O-glycan precursor synthesis, were overexpressed. Ugp2 overexpression significantly increased the recombinant hCG protein level by 1.92 times compared to that of the control. The LC-MS/MS analysis and Phaseolus vulgaris leucoagglutinin (PHA-L) lectin blot analysis showed that Ugp2 overexpression significantly increased the total galactosylation levels of intracellular proteins and the O-glycosylation of recombinant hCG protein. The stability of the hCG protein to trypsin digestion was also enhanced. Ugp2 is the major limiting enzyme of the O-glycan precursor synthesis in recombinant hCG protein production. Furthermore, the effects and mechanisms of the key genes of O-glycan sugar chain synthesis and hydrolases such as polypeptide N-acetylgalactosaminyltransferase1 (Galnt1), Core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase (C1galt1), O-linked N-acetylglucosamine transferase (Ogt) and Hexosaminidase (Hex), were evaluated. The results indicated that Galnt1 overexpression increased the recombinant hCG protein level by 1.57 times and improved the total galactosylation of intracellular proteins, O-glycosylation and the stability of recombinant hCG protein. Galnt1 is the major limiting enzyme of O-glycan sugar chain synthesis. Overexpression of Ugp2 and Galnt1 simultaneously improved the recombinant hCG protein level by 2.44 times, and both had synergistic effects. Based on the results of overexpression of Galnt1, the major limiting gene of O-Glycan chain synthesis, the precursors GalNAc and Gal were added and increased the recombinant hCG protein level by 3.68 times. This study revealed the major limiting factors of O-glycosylation of recombinant hCG protein in CHO cells and proposed an effective expression regulation strategy.
摘要：
人绒毛膜促性腺激素（hCG）是一种糖蛋白激素，以异源二聚体的形式存在，由与二硫键连接的α亚基和β亚基组成。β亚基含有四个O-糖基化位点。先前的研究发现，mRNA翻译为β亚基多肽是重组hCG蛋白在中国仓鼠卵巢（CHO）细胞中表达的严重限制步骤。通过添加O-聚糖前体并过表达和敲低O-聚糖前体合成和O-聚糖糖链合成或水解酶的关键调节基因来评估O-糖基化对重组hCG蛋白表达的影响。结果表明，O-糖基化确实限制了重组hCG蛋白的表达，N-乙酰半乳糖胺（GalNAc）是主要的限制性前体。谷氨酰胺-果糖-6-磷酸转氨酶2（Gfat2）和尿苷二磷酸-葡萄糖焦磷酸化酶2（Ugp2），O-聚糖前体合成的关键调控基因，过度表达。与对照相比,Ugp2过表达使重组hCG蛋白水平显著增加1.92倍。LC-MS/MS分析和菜豆白细胞凝集素（PHA-L）凝集素印迹分析表明，Ugp2过表达显着增加了细胞内蛋白的总半乳糖基化水平和重组hCG蛋白的O糖基化。hCG蛋白对胰蛋白酶消化的稳定性也得到增强。Ugp2是重组hCG蛋白生产中O-聚糖前体合成的主要限制性酶。此外，O-聚糖糖链合成和水解酶的关键基因如多肽N-乙酰半乳糖胺基转移酶1（Galnt1）的作用和机制，核心1合成酶，糖蛋白-N-乙酰半乳糖胺3-β-半乳糖基转移酶（C1galt1），O-连接的N-乙酰葡糖胺转移酶（Ogt）和氨基己糖苷酶（Hex），进行了评估。结果表明，Galnt1过表达使重组hCG蛋白水平提高了1.57倍，并提高了细胞内蛋白的总半乳糖基化。重组hCG蛋白的O-糖基化和稳定性。Galnt1是O-聚糖糖链合成的主要限制酶。同时过表达Ugp2和Galnt1使重组hCG蛋白水平提高了2.44倍,两者都有协同作用。基于O-聚糖链合成的主要限制性基因Galnt1的过表达结果,添加前体GalNAc和Gal并使重组hCG蛋白水平增加3.68倍。本研究揭示了重组hCG蛋白在CHO细胞中O-糖基化的主要限制因素，并提出了有效的表达调控策略。
