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.

Increasing intracellular mannose-6-phosphate (Man-6-P) was previously reported to reduce the amount of the major lipid linked oligosaccharide (LLO) precursor of N-glycans; a loss that might decrease cellular N-glycosylation. If so, providing dietary mannose supplements to glycosylationdeficient patients might further impair their glycosylation. To address this question, we studied the effects of exogenous mannose on intracellular levels of Man-6-P, LLO, and N-glycosylation in human and mouse fibroblasts. Mannose (500μM) did not increase Man-6-P pools in human fibroblasts from controls or from patients with Congenital Disorders of Glycosylation (CDG), who have 90-95% deficiencies in either phosphomannomutase (CDG-Ia) or phosphomannose isomerase (MPI) (CDG-Ib), enzymes that both use Man-6-P as a substrate. In the extreme case of fibroblasts derived from Mpi null mice (<0.001% MPI activity), intracellular Man-6-P levels greatly increased in response to exogenous mannose, and this produced a dose-dependent decrease in the steady state level of the major LLO precursor. However, LLO loss did not decrease total protein N-glycosylation or that of a hypoglycosylation indicator protein, DNaseI. These results make it very unlikely that exogenous mannose could impair N-glycosylation in glycosylation-deficient CDG patients.