An Investigation of Intracellular Glycosylation Activities in CHO Cells: Effects of Nucleotide Sugar Precursor Feeding
An Investigation of Intracellular Glycosylation Activities in CHO Cells: Effects of Nucleotide Sugar Precursor Feeding
复制标题
DOI：
10.1002/bit.22812
复制
发表时间：
2010-10-01
影响因子：
3.8
通讯作者：
Yap, Miranda G. S.
中科院分区：
文献类型：
--
作者：
Wong, Niki S. C.;Wati, Lydia;Yap, Miranda G. S.
控制由CHO细胞产生的重组蛋白的糖基化是高度期望的，因为它可以针对维持或提高产品质量。为了进一步了解影响糖基化的不同因素，使用79个基因的糖基化子阵列和同时分析12个核苷酸和7个核苷酸糖的毛细管电泳方法来产生细胞内N-糖基化谱。具体而言，在产生重组人干扰素-γ（IFN-γ）的CHO细胞中分析核苷酸糖前体补料对细胞内糖基化活性的影响。与未处理的对照培养物相比，半乳糖（+/-尿苷）、葡糖胺（+/-尿苷）和N-乙酰甘露糖胺（ManNAc）（+/-胞苷）补料导致IFN-γ唾液酸化增加12%、28%和32%。这可能直接归因于核苷酸糖底物UDP-Hex（相似于20倍）、UDP-HexNAc（6- 15倍）和CMP-唾液酸（30- 120倍）的增加。B4 gal和St 3gal的上调也可以增强聚糖添加到蛋白质上，导致更完全的糖基化（唾液酸化）。与单独饲喂糖前体相比，葡萄糖胺+尿苷和ManNAc +胞苷的组合饲喂使UDP-Hex-NAc和CMP-唾液酸增加了另外2至4倍。然而，它没有导致IFN-γ唾液酸化的协同增加。其他因素，如糖基转移酶或聚糖底物水平可能成为限制。此外，尿苷喂养增加水平的尿苷和胞苷激活的核苷酸糖同时，这可能意味着尿苷是在研究中的核苷酸糖合成的限制性底物之一。因此，细胞内糖基化活性的表征增加了我们对核苷酸糖前体补料如何影响CHO细胞中产生的重组蛋白的糖基化的理解。它还导致优化更有效的策略来操纵聚糖质量。Biotechnol. Bioeng. 2010;107：321-336. (c)2010 Wiley Periodicals，Inc.
Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub-array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N-glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon-gamma (IFN-gamma). Galactose (+/- uridine), glucosamine (+/- uridine), and N-acetylmannosamine (ManNAc) (+/- cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN-gamma sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP-Hex (similar to 20-fold), UDP-HexNAc (6- to 15-fold) and CMP-sialic acid (30- to 120-fold), respectively. Upregulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP-Hex-NAc and CMP-sialic acid by another two- to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN-gamma sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine- and cytidine-activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality. Biotechnol. Bioeng. 2010;107: 321-336. (c) 2010 Wiley Periodicals, Inc.