Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity.
Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity.
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DOI：
10.1074/jbc.m109.068353
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发表时间：
2010-02-19
期刊：
影响因子：
--
通讯作者：
Haslam SM
中科院分区：
文献类型：
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作者：
North SJ;Huang HH;Sundaram S;Jang-Lee J;Etienne AT;Trollope A;Chalabi S;Dell A;Stanley P;Haslam SM
通过对培养的细胞系和模式生物的糖基化突变体的研究，极大地方便了确定哺乳动物多糖的生物学作用和合成途径。根据中国仓鼠卵巢(CHO)对凝集素的抗性而获得的糖基化突变体在重组糖蛋白的糖基化工程中具有重要的应用价值。为了进一步加强这些突变体的应用，并深入了解改变一个特定的糖基转移酶或糖基化活性对细胞多糖整体表达的影响，利用基质辅助激光解吸电离飞行时间/飞行时间质谱学分析了一组CHO突变体的N-糖和主要O-糖。我们在这里报告了9个不同的CHO糖基化突变体中存在的主要N-糖链和O-糖链的互补。单层培养和悬浮培养的亲本CHO细胞具有相似的N-GalNAc和O-GalNAc糖链，而糖基化突变体Lec1、Lec2、Lec3.2.8.1、Lec4、Lec10、LEC11、LEC12、Lec13和LEC30的图谱与已有的遗传和生化数据一致。然而，观察到的N-糖链范围的复杂性是意想不到的。一些复杂的N-聚糖谱包含m/z∼13,000的结构，代表复杂的N-聚糖链，总共26个N-乙酰乳糖胺(Galβ1-4GlcNAc)n个单元。重要的是，LEC11、LEC12和LEC30 CHO突变体显示了岩藻糖化复合体N-糖链的独特补体，其末端是Lewis-X和sialyl-Lewis-X决定簇。这一分析揭示了CHO细胞突变体中N-葡聚糖的复杂性比预期的要大，这些突变可能用于广泛的功能糖组学研究和制造重组糖蛋白。
Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z ∼13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Galβ1–4GlcNAc)n units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewisx and sialyl-Lewisx determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.