The relative contribution of mannose salvage pathways to glycosylation in PMI-deficient mouse embryonic fibroblast cells

Naonobu Fujita, Ayako Tamura, Aya Higashidani, Takashi Tonozuka, Hudson H. Freeze, Atsushi Nishikawa

    Research output: Contribution to journalArticle

    15 Citations (Scopus)

    Abstract

    Mannose for mammalian glycan biosynthesis can be imported directly from the medium, derived from glucose or salvaged from endogenous or external glycans. All pathways must generate mannose 6-phosphate, the activated form of mannose. Imported or salvaged mannose is directly phosphorylated by hexokinase, whereas fructose 6-phosphate from glucose is converted to mannose 6-phosphate by phosphomannose isomerase (PMI). Normally, PMI provides the majority of mannose for glycan synthesis. To assess the contribution of PMI-independent pathways, we used PMI-null fibroblasts to study N-glycosylation of DNase I, a highly sensitive indicator protein. In PMI-null cells, imported mannose and salvaged mannose make a significant contribution to N-glycosylation. When these cells were grown in mannose-free medium along with the mannosidase inhibitor, swainsonine, to block the salvage pathways, N-glycosylation of DNase I was almost completely eliminated. Adding ∼13 μm mannose to the medium completely restored normal glycosylation. Treatment with bafilomycin A 1, an inhibitor of lysosomal acidification, also markedly reduced N-glycosylation of DNase I, but in this case only 8 μm mannose was required to restore full glycosylation, indicating that a nonlysosomal source of mannose made a significant contribution. Glycosylation levels were greatly also reduced in glycoconjugate-free medium, when endosomal membrane trafficking was blocked by expression of a mutant SKD1. From these data, we conclude that PMI-null cells can salvage mannose from both endogenous and external glycoconjugates via lysosomal and nonlysosomal degradation pathways.

    Original languageEnglish
    Pages (from-to)788-798
    Number of pages11
    JournalFEBS Journal
    Volume275
    Issue number4
    DOIs
    Publication statusPublished - 2008 Feb 1

    Keywords

    • Congenital disorders of glycosylation
    • Lipid-linked oligosaccharide
    • Mannose
    • N-linked oligosaccharide
    • Phosphomannose isomerase

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Fingerprint Dive into the research topics of 'The relative contribution of mannose salvage pathways to glycosylation in PMI-deficient mouse embryonic fibroblast cells'. Together they form a unique fingerprint.

  • Cite this