PMC

LLO profiles in Normal and Mpi^−/− fibroblasts in response to mannose. Cells were incubated in the presence of the indicted amount of mannose in medium containing 5.5 mM glucose and 20% FBS for 24 hours and LLO glycans prepared and analyzed in the Experimental Procedures. Identity of the glycans was determined by comparison with authentic standards. Representative experiments are shown Panels: (a) control mouse fibroblasts, 0μM mannose; (b), control mouse fibroblasts, 500μM mannose; (c), Mpi^−/− mouse fibroblasts 0μM mannose; (d), Mpi^−/− mouse fibroblasts 500μM mannose; (e), Mpi^−/− mouse fibroblasts 0μM mannose; (f), Mpi^−/− mouse fibroblasts 20μM mannose; (g), Mpi^−/−mouse fibroblasts 100μM mannose; (h), Mpi^−/− mouse fibroblasts 500μM mannose.

Effects of exogenous mannose on intracellular Man-6-P. Human control, CDG-Ia (PMM2 deficient), CDG-Ib (MPI-deficient), CDG-Ic (ALG6-deficient) and mouse fibroblasts from Mpi^+/+ and Mpi^−/− embryos were incubated with medium containing 10% serum and either 0 or 500μM mannose for 8 hr. Samples were extracted and analyzed by HPLC as previously described to determine Man-6-P (). Controls of known amount of Man-6-P and Glc-6-P were analyzed in parallel and recoveries normalized to an established standard curve to determine nmol Man-6-P/100μg cellular protein.

Effects of mannose on N-glycosylation of DNaseI and total secreted proteins. Mpi^−/− fibroblasts were incubated in medium containing the indicated concentration of mannose and for 4 h and labeled with ³⁵S-amino acids. Panel A. DNaseI was precipitated from the medium and glycosylated (upper band) and non-glycosylated (lower band) forms were separated by SDS-PAGE and visualized via fluorography. Numbers indicate the percentage of fully glycosylated molecules. Panel B. Medium from the same experiment using 10 and 500μM mannose was analyzed by binding to Concanavalin A. Figure shows 1× and 5× amounts of total, bound, and unbound proteins from medium containing 10μM and 500μM mannose separated by SDS-PAGE and visualized via fluorography. Both Panels show that increased mannose in Mpi^−/− cells does not alter the efficiency of N-glycosylation.

Mannose-6-P (Man-6-P) is a precursor for N-glycan synthesis. Man-6-P is converted to Man-1-P, then to GDP-Man and dolichol-P-Man for synthesis of the universal lipid linked oligosaccharide (LLO) donors of N-glycans. (See ) (). Two pathways can generate Man-6-P: phosphorylation of mannose via hexokinase or conversion of fructose-6-P via phosphomannose isomerase (MPI) (). Both pathways contribute mannose to N-glycosylation. Patients with congenital disorder of glycosylation (CDG) Type Ib have an 85–95% deficiency in MPI activity (, ), resulting in unoccupied N-glycosylation sites in many proteins. Dietary supplements of mannose correct the patients’ abnormal glycosylation and pathological features (, ). In mice, ablation of Mpi is lethal by embryonic day 10.5 due to intracellular accumulation of toxic levels of Man-6-P that can reach 25mM ().