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

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 A1, 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.

Identification of Intercellular Cell Adhesion Molecule 1 (ICAM-1) as a Hypoglycosylation Marker in Congenital Disorders of Glycosylation Cells

“…However, the published data have focused mainly on the alteration of glycan structure rather than glycosylation site occupancy (25)(26)(27). Some CDG patient cell lines show underglyco- sylation of adenovirus-transfected DNase I (28,29), but those results are inconsistent and not easily applied to routine examination.…”

Metabolically programmed quality control system for dolichol-linked oligosaccharides

“…1B). Because MPI −/− MEFs are devoid of this interconversion pathway, GDP-Man biosynthesis in these cells is exclusively dependent upon the supply of mannose (28,30), providing an ideal system to modulate the GDP-Man biosynthetic pathway without altering the glucose supply. Mannose can be taken up from culture medium and/or salvaged from the degradation of mannose-containing glycoconjugates by class II α-mannosidases (28,30).…”

“…Because MPI −/− MEFs are devoid of this interconversion pathway, GDP-Man biosynthesis in these cells is exclusively dependent upon the supply of mannose (28,30), providing an ideal system to modulate the GDP-Man biosynthetic pathway without altering the glucose supply. Mannose can be taken up from culture medium and/or salvaged from the degradation of mannose-containing glycoconjugates by class II α-mannosidases (28,30). Accordingly, to shut down the mannose utilization pathway in MPI −/− MEFs, the cells were incubated in medium supplemented with 5 mM glucose, 10% (vol/vol) dialyzed FBS, and 10 μM class II α-mannosidase inhibitor swainsonine (SW) (28,30).…”

“…Mannose can be taken up from culture medium and/or salvaged from the degradation of mannose-containing glycoconjugates by class II α-mannosidases (28,30). Accordingly, to shut down the mannose utilization pathway in MPI −/− MEFs, the cells were incubated in medium supplemented with 5 mM glucose, 10% (vol/vol) dialyzed FBS, and 10 μM class II α-mannosidase inhibitor swainsonine (SW) (28,30). After only 6 h of mannose deprivation, the level of GDP-Man was significantly reduced, and it was further decreased up to 18 h (Fig.…”

MPI depletion enhances O-GlcNAcylation of p53 and suppresses the Warburg effect

“…MPI has primarily been studied for its role in converting Fru6P to Man6P for protein N-glycosylation (de la Fuente et al, 1986, Fujita et al, 2008; Sharma et al, 2011; Cline et al, 2012). We therefore predicted that MPI depletion would result in increased Fru6P levels.…”