N-Glycosylation profiling of recombinant mouse extracellular superoxide dismutase produced in Chinese hamster ovary cells

Abstract

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.

N‐Glycosylation is essential for the secretion of extracellular superoxide dismutase

“…Since N-glycans of human and mouse EC-SOD were both reported to possess a core-fucose structure [16,33], we also checked whether deletion of core-fucose affects EC-SOD secretion, by using HEK293T cells lacking the core-fucose-synthesizing enzyme FUT8 [35]. The presence of core-fucose on wild-type EC-SOD was confirmed by its reactivity with PhoSL lectin (Fig.…”

“…Notably, a naturally occurring mutation in the C-terminal region (R213G) that dramatically reduces the affinity to heparin was found in humans [28]. Previous glycan analyses of recombinant mouse and human EC-SODs showed that they are mainly modified with complextype N-glycans with core-fucose and sialic acids [16,33], but the physiological functions of the EC-SOD glycan are poorly understood. Positions of the N-glycosylation site and cysteine residues are indicated in the overview diagram.…”

“…Extracellular superoxide dismutase is the only glycosylated SOD with one N-glycosylation site, which is present at asparagine 89 [16,32]. Previous glycan analyses of recombinant mouse and human EC-SODs showed that they are mainly modified with complextype N-glycans with core-fucose and sialic acids [16,33], but the physiological functions of the EC-SOD glycan are poorly understood. The only detailed report on the glycosylation of EC-SOD by Edlund et al [32] suggested that the glycan on EC-SOD regulates its heparin-binding ability and solubility.…”

Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms

“…Purified N-glycans were quantitatively determined based on the peak areas by weak anion-exchange chromatography on a DEAE-5PW column (27). The neutral fraction (flow-through from the anion exchange column) was further separated by reversed-phase HPLC to quantify high mannose and asialo complex type N-glycans separately (28).…”

“…Total N-glycans derived from both cell lines were separated by anion exchange chromatography according to their sialic acid content (27), and neutral N-glycans were further analyzed by reversed-phase mode (supplemental Fig. S8) (28). As shown in Fig.…”