PDF(Pubmed)
Abstract：
Biological experiments are often conducted in vitro using immortalized cells due to their accessibility and ease of propagation compared to primary cells and live animals. However, immortalized cells may present different proteomic and glycoproteomic characteristics from the primary cell source due to the introduction of genes that enhance proliferation (e.g. CDK4) or enable telomere lengthening. To demonstrate the changes in phenotype upon CDK4-transformation, we performed LC-MS/MS glycomic and proteomic characterizations of a human lung cancer primary cell line (DTW75) and a CDK4-transformed cell line (GL01) derived from DTW75. We observed that the primary and CDK4-transformed cells expressed significantly different levels of sialylated, fucosylated, and sialofucosylated N-glycans. Specifically, the primary cells expressed higher levels of hybrid- and complex-type sialylated N-glycans, while CDK4-transformed cells expressed higher levels of complex-type fucosylated and sialofucosylated N-glycans. Further, we compared the proteomic differences between the cell lines and found that CDK4-transformed cells expressed higher levels of RNA-binding and adhesion proteins. Further, we observed that the CDK4-transformed cells changed N-glycosylation after 31 days in cell culture, with a decrease in high-mannose and increase in fucosylated, sialylated, and sialofucosylated N-glycans. Identifying these changes between primary and CDK4-transformed cells will provide useful insight when adapting cell lines that more closely resemble in vivo physiological conditions.
摘要：
由于与原代细胞和活体动物相比，它们的可及性和易于繁殖，因此通常使用永生化细胞在体外进行生物学实验。然而,由于引入了增强增殖(例如CDK4)或使端粒延长的基因,永生化细胞可呈现与原代细胞来源不同的蛋白质组和糖蛋白质组特征。为了证明CDK4转化后表型的变化，我们对人类肺癌原代细胞系（DTW75）和衍生自DTW75的CDK4转化细胞系（GL01）进行了LC-MS/MS糖组学和蛋白质组学表征。我们观察到，原代和CDK4转化的细胞表达显著不同水平的唾液酸化，岩藻糖基化,和唾液酸岩藻糖基化的N-聚糖。具体来说,原代细胞表达更高水平的杂交型和复合型唾液酸化N-聚糖，而CDK4转化的细胞表达更高水平的复合型岩藻糖基化和唾液酸岩藻糖基化N-聚糖。Further,我们比较了细胞系之间的蛋白质组差异,发现CDK4转化的细胞表达更高水平的RNA结合和粘附蛋白.Further,我们观察到CDK4转化的细胞在细胞培养31天后改变N-糖基化，随着高甘露糖的减少和岩藻糖基化的增加，唾液酸化,和唾液酸岩藻糖基化的N-聚糖。鉴定原代和CDK4转化细胞之间的这些变化将在适应更接近体内生理条件的细胞系时提供有用的见解。
