RESUMO
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 glycosylation-deficient 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 (500microM) 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.
Assuntos
Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Manose/farmacologia , Manosefosfatos/metabolismo , Animais , Desoxirribonuclease I/metabolismo , Glicosilação/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Lipopolissacarídeos/metabolismo , Manose-6-Fosfato Isomerase/deficiência , Manose-6-Fosfato Isomerase/metabolismo , CamundongosRESUMO
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 approximately 13 microm 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 microm 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.