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.