N-Glycosylation profiling of recombinant mouse extracellular superoxide dismutase produced in Chinese hamster ovary cells
N-Glycosylation profiling of recombinant mouse extracellular superoxide dismutase produced in Chinese hamster ovary cells
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中国仓鼠卵巢细胞产生的重组小鼠胞外超氧化物歧化酶的 N-糖基化分析
DOI：
10.1007/s10719-011-9333-6
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Korekane H
中科院分区：
文献类型：
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作者：
Okamoto I;Arao T;Miyazaki M;Satoh T;Okamoto K;Tsunoda T;Nishio K;Nakagawa K.;Korekane H
细胞外超氧化物歧化酶（EC-SOD）是生物体液中主要的SOD同工酶，与大多数糖蛋白一样具有N-糖基化和异质性。然而，在中国仓鼠卵巢（CHO）细胞中产生的重组人EC-SOD中仅报道了一个N-聚糖结构。因此，重组EC-SOD的精确N-聚糖谱不可用。在这项研究中，我们报告的N-聚糖重组小鼠EC-SOD在CHO细胞中产生的高分辨率技术，包括释放N-聚糖的处理与PNGase F，荧光标记的吡啶胺化，表征的阴离子交换，正相和反相HPLC分离，和质谱分析。我们成功地鉴定了重组酶中26种不同类型的N-聚糖。EC-SODN-聚糖基本上是核心岩藻糖基化的（占总N-聚糖含量的98.3%），并且是高甘露糖糖链，以及表现出不同程度唾液酸化的单-、双-、三-和四-触角复合糖链。其中4个N-聚糖被硫酸基团、Lewisx结构或α-Gal表位独特修饰。这些发现将为EC-SODN-聚糖的结构-功能关系提供新的线索。
Extracellular superoxide dismutase (EC-SOD), the major SOD isoenzyme in biological fluids, is known to beN-glycosylated and heterogeneous as was detected in most glycoproteins. However, only oneN-glycan structure has been reported in recombinant human EC-SOD produced in Chinese hamster ovary (CHO) cells. Thus, a preciseN-glycan profile of the recombinant EC-SOD is not available. In this study, we report profiling of theN-glycan in the recombinant mouse EC-SOD produced in CHO cells using high-resolution techniques, including the liberation ofN-glycans by treatment with PNGase F, fluorescence labeling by pyridylamination, characterization by anion-exchange, normal and reversed phase-HPLC separation, and mass spectrometry. We succeeded in identifying 26 different types ofN-glycans in the recombinant enzyme. The EC-SODN-glycans were basically core-fucosylated (98.3% of the totalN-glycan content), and were high mannose sugar chain, and mono-, bi-, tri-, and tetra-antennary complex sugar chains exhibiting varying degrees of sialylation. Four of the identifiedN-glycans were uniquely modified with a sulfate group, a Lewisxstructure, or an α-Gal epitope. The findings will shed new light on the structure-function relationships of EC-SODN-glycans.