Department of Disease Glycomics (Seikagaku Corporation), The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047, Japan.
Glycoconj J. 2011 May;28(3-4):183-96. doi: 10.1007/s10719-011-9333-6. Epub 2011 May 15.
Extracellular superoxide dismutase (EC-SOD), the major SOD isoenzyme in biological fluids, is known to be N-glycosylated and heterogeneous as was detected in most glycoproteins. However, only one N-glycan structure has been reported in recombinant human EC-SOD produced in Chinese hamster ovary (CHO) cells. Thus, a precise N-glycan profile of the recombinant EC-SOD is not available. In this study, we report profiling of the N-glycan in the recombinant mouse EC-SOD produced in CHO cells using high-resolution techniques, including the liberation of N-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 of N-glycans in the recombinant enzyme. The EC-SOD N-glycans were basically core-fucosylated (98.3% of the total N-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 identified N-glycans were uniquely modified with a sulfate group, a Lewis(x) structure, or an α-Gal epitope. The findings will shed new light on the structure-function relationships of EC-SOD N-glycans.
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，已知其为 N-糖基化且具有不均一性，就像大多数糖蛋白中检测到的那样。然而，在重组人 EC-SOD 中只报道了一种 N-聚糖结构在中国仓鼠卵巢（CHO）细胞中产生。因此，重组 EC-SOD 的精确 N-聚糖图谱尚不可用。在这项研究中，我们使用高分辨率技术报告了重组小鼠 EC-SOD 在 CHO 细胞中产生的 N-聚糖的图谱，包括用 PNGase F 处理释放 N-聚糖、通过吡啶氨化进行荧光标记、通过阴离子交换、正相和反相-HPLC 分离以及质谱进行表征。我们成功地鉴定了重组酶中 26 种不同类型的 N-聚糖。EC-SOD N-聚糖基本上是核心岩藻糖基化的（总 N-聚糖含量的 98.3%），并且是高甘露糖糖链，单、二、三、四触角复杂糖链表现出不同程度的唾液酸化。鉴定出的 4 种 N-聚糖具有独特的硫酸基团、Lewis(x)结构或α-Gal 表位修饰。这些发现将为 EC-SOD N-聚糖的结构-功能关系提供新的线索。
