The metabolic origins of mannose in glycoproteins

Abstract

Mannose in N-glycans is derived from glucose through phosphomannose isomerase (MPI, Fru-6-P ↔ Man-6-P) whose deficiency causes a congenital disorder of glycosylation (CDG)-Ib (MPI-CDG). Mannose supplements improve patients' symptoms because exogenous mannose can also directly contribute to N-glycan synthesis through Man-6-P. However, the quantitative contributions of these and other potential pathways to glycosylation are still unknown. We developed a sensitive GC-MS-based method using [1,2-(13)C]glucose and [4-(13)C]mannose to measure their contribution to N-glycans synthesized under physiological conditions (5 mm glucose and 50 μm mannose). Mannose directly provides ∼10-45% of the mannose found in N-glycans, showing up to a 100-fold preference for mannose over exogenous glucose based on their exogenous concentrations. Normal human fibroblasts normally derive 25-30% of their mannose directly from exogenous mannose, whereas MPI-deficient CDG fibroblasts with reduced glucose flux secure 80% of their mannose directly. Thus, both MPI activity and exogenous mannose concentration determine the metabolic flux into the N-glycosylation pathway. Using various stable isotopes, we found that gluconeogenesis, glycogen, and mannose salvaged from glycoprotein degradation do not contribute mannose to N-glycans in fibroblasts under physiological conditions. This quantitative assessment of mannose contribution and its metabolic fate provides information that can help bolster therapeutic strategies for treating glycosylation disorders with exogenous mannose.

Keywords: GC-MS; Genetic Diseases; Glucose Metabolism; Glycoconjugate; Metabolic Tracers.

FIGURE 1.

Example of mass distribution calculation. A fragment of C1-5 gives a molecular mass of 314, which is an isotopomer without stable isotope and defined as m0. If mannose is originated from glucose, two additional mass units will be observed (m2) because glucose contains two ¹³C at C-1 and 2. The percentage of contribution from glucose was calculated as m2/(m0 + m2). In a similar manner, the percentage of contribution from mannose was calculated as m1/(m0 + m1). Natural abundance heavy isotope was corrected using matrix based probabilistic methods.

SCHEME. 1

Mannose and glucose metabolic pathway.

FIGURE 2.

GC and MS chromatogram of mannose. A, GC chromatogram of before (black line) and after N-glycosidase F (PNGase F) digestion (pink line). (Glucose is a routine contaminant.) B, location of heavy isotopic labels in glucose and mannose. C, MS fragments of mannose. Upper panel, MS fragment without heavy isotope; lower panel, MS fragment with heavy isotope.

FIGURE 3.

Labeling and turnover of labeled N-glycans. A, incorporation of mannose into N-glycans is shown as Man^G, indicating contribution from glucose to mannose, and as Man^M, indicating contribution of mannose to mannose. Control fibroblasts were labeled with 5 mm [1,2-¹³C]Glc and 50 μm [4-¹³C]Man for 1, 2 or 4 h and followed by glycan analysis. B, turnover rates of mannose in N-glycans. Control fibroblasts were labeled with 5 mm [1,2-¹³C]Glc and 50 μm [4-¹³C]Man for 72 h and chased in unlabeled medium. Total ¹³C-labeled from glucose and mannose (Man^G+M) was defined as 100% at the beginning of the chase. Contribution from Glc (Man^G) and Man (Man^M) was calculated as Man^G/Man^G+M and Man^M/Man^G+M, respectively.

FIGURE 4.

Fate of mannose, including incorporation into glycogen, glycolysis, and other monosaccharides. A, glycogen was analyzed by labeling control human fibroblasts with 5 mm [1,2-¹³C]Glc and 50 μm or 1 mm [4-¹³C]Man for 24 h, respectively. B, glycolytic metabolite analysis was carried out by labeling control human fibroblasts with 5 mm [1,2-¹³C]Glc and 50 μm Man or 5 mm Glc and 1 mm [1,2-¹³C]Man for 24 h, and culture medium was processed as described under “Experimental Procedures.” C–E, origin of mannose (C), galactose (D), or GlcNAc (E) in N-glycans was analyzed with increasing concentrations of exogenous [4-¹³C]Man with 5 mm [1,2-¹³C]Glc. Contributions from ¹³C-Glc (G) and ¹³C-Man (M) origin were calculated as G/(G+M), M/(G+M), respectively, except for glycolysis (B), where the contribution of ¹³C-Glc or ¹³C-Man was adjusted to twice the percentage of incorporation into these metabolites because one glucose/mannose unit produces two pyruvate/lactate/alanine molecules.

FIGURE 5.

Metabolic detour by MPI. A, mechanism for incorporation of deuterium into [1,2-¹³C]Man-6-P through Fru-6-P (Man^F) catalyzed by MPI. B, control human fibroblasts were labeled with 100 or 200 μm [1,2-¹³C]Man and 5 mm Glc in the presence of D₂O for 12 h prior to glycan analysis to quantify the contribution of Man^M and Man^F into N-glycans. C, the same experiments were done with Mpi WT MEF and Mpi KO MEF.

SCHEME. 2

Deuterium incorporation in mannose of N-glycans from glucose via MPI-dependent pathway.

FIGURE 6.

GC and MS chromatogram of sugar phosphates. A, GC chromatogram corresponding to Fru-6-P, Man-6-P, and Glc-6-P as TMS derivatives (E- and Z-form). Peaks corresponding to E-forms were used for characterization. B, MS fragments of TMS-derivatized sugar phosphates. Characteristic fragments are highlighted as C4-6 (m/z 459) for Fru and C3-6 (m/z 471) for Glc and Man.

FIGURE 7.

Sugar phosphate analysis. Contributions from Man and Glc to sugar phosphates (Man-6-P, Glc-6-P, and Fru-6-P) were assessed by labeling wild type MEF with 5 mm [UL-¹³C]Glc and 50 μm Man or 5 mm Glc and 50 or 500 μm [UL-¹³C]Man for 6 h prior to GC-MS analysis described under “Experimental Procedures.” For comparison, N-glycans were also analyzed under the same labeling conditions except that incubation was done for 24 h.

FIGURE 8.

Other sources of mannose for N-glycans. A, contribution of glycogen to N-glycans. Control fibroblasts were labeled for 48 h with 5 mm [1,2-¹³C]Glc and chased in D₂O for 24 h with 0.5–5.0 mm Glc except for 12 h with 0 mm as indicated by (*) in the presence or absence of 1 mm DTT, and glycogen-derived mannose of N-glycans (Man^GL) was analyzed. White bar shows glucose contribution to mannose in N-glycans (Man^G) without chase in D₂O for 24 h. B, contribution of gluconeogenesis to N-glycans was assessed by labeling control fibroblasts with 1 mm [2-¹³C]glycerol or [3-¹³C]pyruvate and 5 mm [6,6-²H]glucose for 24 h. Contribution from [2-D]Glc (Man^G) and gluconeogenesis was calculated as the percentage of total incorporation of stable isotope into N-glycans. C, reutilization of mannose from glycan salvage was assessed by labeling control fibroblasts with 500 μm [1,2-¹³C]Man and 5 mm [¹²C]Glc for 72 h and chasing in D₂O for 12 h with or without 5 mm Glc and 50 μm Man. White bar shows percentage of labeled N-glycans from ¹³C-Man (Man^M) before D₂O chase. Black bar represents salvaged mannose from N-glycans (Man^S) during D₂O chase.

FIGURE 9.

Direct mannose contribution to glycans in various cell lines. A, various cell lines were labeled with 5 mm [1,2-¹³C]Glc and 50–200 μm [4-¹³C]Man for 24 h, and the proportion of ¹³C-Man contributing to total mannose of N-glycans was plotted. B, correlation between Man contribution to N-glycans and ratio of PMM2/MPI enzyme specific activity of cell lines tested in A was displayed.