Chitlaru T, Kronman C, Zeevi M, Kam M, Harel A, Ordentlich A, Velan B, Shafferman A
Department of Biochemistry and Molecular Biology, Israel Institute for Biological Research, P.O.B. 19 Ness-Ziona, 70450, Israel.
Biochem J. 1998 Dec 15;336 ( Pt 3)(Pt 3):647-58. doi: 10.1042/bj3360647.
Sialylation of N-glycans associated with recombinant human acetylcholinesterase (rHuAChE) has a central role in determining its circulatory clearance rate. Human embryonal kidney 293 (HEK-293) cells, which are widely used for the expression of recombinant proteins, seem to be limited in their ability to sialylate overexpressed rHuAChE. High-resolution N-glycan structural analysis, by gel permeation, HPLC anion-exchange chromatography and high-pH anion-exchange chromatography (HPAEC), revealed that the N-glycans associated with rHuAChE produced in HEK-293 cells belong mainly to the complex-biantennary class and are only partly sialylated, with approx. 60% of the glycans being monosialylated. This partial sialylation characterizes rHuAChE produced by cells selected for high-level expression of the recombinant protein. In low-level producer lines, the enzyme exhibits a higher sialic acid content, suggesting that undersialylation of rHuAChE in high-level producer lines stems from a limited endogenous glycosyltransferase activity. To improve sialylation in HEK-293 cells, rat liver beta-galactoside alpha-2,6-sialyltransferase cDNA was stably transfected into cells expressing high levels of rHuAChE. rHuAChE produced by the modified cells displayed a significantly higher proportion of fully sialylated glycans as shown by sialic acid incorporation assays, direct measurement of sialic acid, and HPAEC glycan profiling. Genetically modified sialylated rHuAChE exhibited increased circulatory retention (the slow-phase half-life, t12beta, was 130 min, compared with 80 min for the undersialylated enzyme). Interestingly, the same increase in circulatory residence was observed when rHuAChE was subjected to extensive sialylation in vitro. The engineered HEK-293 cells in which the glycosylation machinery was modified might represent a valuable tool for the high level of expression of recombinant glycoproteins whose sialic acid content is important for their function or for pharmacokinetic behaviour.
与重组人乙酰胆碱酯酶（rHuAChE）相关的N-聚糖的唾液酸化在决定其循环清除率方面起着核心作用。广泛用于重组蛋白表达的人胚肾293（HEK-293）细胞，其对过表达的rHuAChE进行唾液酸化的能力似乎有限。通过凝胶渗透、HPLC阴离子交换色谱和高pH值阴离子交换色谱（HPAEC）进行的高分辨率N-聚糖结构分析表明，HEK-293细胞中产生的与rHuAChE相关的N-聚糖主要属于复杂双天线类，并且只是部分被唾液酸化，约60%的聚糖为单唾液酸化。这种部分唾液酸化是为重组蛋白高水平表达而选择的细胞所产生的rHuAChE的特征。在低水平生产细胞系中，该酶表现出较高的唾液酸含量，这表明高水平生产细胞系中rHuAChE的唾液酸化不足源于有限的内源性糖基转移酶活性。为了改善HEK-293细胞中的唾液酸化，将大鼠肝脏β-半乳糖苷α-2,6-唾液酸转移酶cDNA稳定转染到高水平表达rHuAChE的细胞中。如唾液酸掺入试验、唾液酸直接测量和HPAEC聚糖谱分析所示由修饰细胞产生的rHuAChE显示出完全唾液酸化聚糖的比例显著更高。基因修饰的唾液酸化rHuAChE表现出循环保留增加（慢相半衰期t12β为130分钟，而唾液酸化不足的酶为80分钟）。有趣的是，当rHuAChE在体外进行广泛唾液酸化时，也观察到了相同的循环停留时间增加。其中糖基化机制被修饰的工程化HEK-293细胞可能代表了一种有价值的工具，用于高水平表达其唾液酸含量对其功能或药代动力学行为很重要的重组糖蛋白。
