Effect of Ganglioside GM3 Synthase Gene Knockout on the Glycoprotein N-Glycan Profile of Mouse Embryonic Fibroblast

Authors

  • Noriko Nagahori,

    1. Graduate School of Advanced Life Science, and Frontier Research Center for the Post-Genome Science and Technology, Hokkaido University, N21, W11, Sapporo 001-0021 (Japan)
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  • Tadashi Yamashita,

    1. Graduate School of Advanced Life Science, and Frontier Research Center for the Post-Genome Science and Technology, Hokkaido University, N21, W11, Sapporo 001-0021 (Japan)
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  • Maho Amano,

    1. Graduate School of Advanced Life Science, and Frontier Research Center for the Post-Genome Science and Technology, Hokkaido University, N21, W11, Sapporo 001-0021 (Japan)
    2. Medicinal Chemistry Pharmaceuticals, Co. Ltd. N21, W12, Sapporo 001-0021 (Japan)
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  • Shin-Ichiro Nishimura

    Corresponding author
    1. Graduate School of Advanced Life Science, and Frontier Research Center for the Post-Genome Science and Technology, Hokkaido University, N21, W11, Sapporo 001-0021 (Japan)
    2. Medicinal Chemistry Pharmaceuticals, Co. Ltd. N21, W12, Sapporo 001-0021 (Japan)
    • Graduate School of Advanced Life Science, and Frontier Research Center for the Post-Genome Science and Technology, Hokkaido University, N21, W11, Sapporo 001-0021 (Japan)
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Abstract

The structural and clinical significance of cellular glycoproteins and glycosphingolipids (GSLs) are often separately discussed. Considering the biosynthetic pathway of glycoconjugates, glycans of cell-surface glycoproteins and GSLs might partially share functions in maintaining cellular homeostatis. The purpose of this study is to establish a general and comprehensive glycomics protocol for cellular GSLs and N-glycans of glycoproteins. To test the feasibility of a glycoblotting-based protocol, whole glycans released both from GSLs and glycoproteins were profiled concurrently by using GM3 synthase-deficient mouse embryonic fibroblast GM3(−/−). GM3(−/−) cells did not synthesize GM3 or any downstream product of GM3 synthase. Instead, expression levels of o-series gangliosides involving GM1-b and GD1-α increased dramatically, whereas a-/b-series gangliosides were predominantly detected in wild-type (WT) cells. We also discovered that glycoprotein N-glycan profiles of GM3(−/−) cells are significantly altered as compared to WT cells, although GM3 synthase is responsible only for GSLs synthesis and is not associated with glycoprotein N-glycan biosynthesis. The present approach allows for high-throughput profiling of cellular glycomes enriched by different classes of glycoconjugates, and our results demonstrated that gene knockout of the enzymes responsible for GSL biosynthesis significantly influences the N-glycans of glycoproteins.

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