TY - JOUR
T1 - The relative contribution of mannose salvage pathways to glycosylation in PMI-deficient mouse embryonic fibroblast cells
AU - Fujita, Naonobu
AU - Tamura, Ayako
AU - Higashidani, Aya
AU - Tonozuka, Takashi
AU - Freeze, Hudson H.
AU - Nishikawa, Atsushi
PY - 2008/2
Y1 - 2008/2
N2 - 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.
AB - 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.
KW - Congenital disorders of glycosylation
KW - Lipid-linked oligosaccharide
KW - Mannose
KW - N-linked oligosaccharide
KW - Phosphomannose isomerase
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U2 - 10.1111/j.1742-4658.2008.06246.x
DO - 10.1111/j.1742-4658.2008.06246.x
M3 - Article
C2 - 18215164
AN - SCOPUS:38549150688
VL - 275
SP - 788
EP - 798
JO - FEBS Journal
JF - FEBS Journal
SN - 1742-464X
IS - 4
ER -