Human fibroblasts prefer mannose over glucose as a source of mannose for N-glycosylation - Evidence for the functional importance of transported mannose

Mannose in N-linked oligosaccharides is assumed to be derived primarily from glucose through phosphomannose isomerase (PMI), The discovery of mammalian mannose-specific transporters that function at physio logical concentrations suggested that mannose might directly contribute to oligosaccharide synthesis, To determine the relative contribution of glucose and mannose, human fibroblasts were labeled with either [2-H-3]mannose or [1,5,6-H-3]glucose at the same specific activity, and the N-linked chains were released by PN-Gase F digestion, Most of the trichloroacetic acid-precipitable [H-3]mannose label was released by this digestion, but only about 10% of the trichloroacetic acid precipitable material was released from cells labeled with [1,5,6-H-3]glucose. Both sugars labeled a similar array of oligosaccharides, and acid hydrolysis of these chains showed that [2-H-3]mannose contributed 65-75% of the [H-3]mannose in cells labeled for 1 h, despite the 100-fold higher concentration of exogenous glucose, Mannose consumption and [2-H-3]mannose utilization were within the range of rates expected for mannose transport via the mannose-specific transporter. About 7-14% of the [2-H-3]mannose is used for glycosylation, while the rest (86-93%) is catabolized to (H2O)-H-3 via PMI, Increasing the exogenous mannose concentration beyond mannose transporter saturation results in the conversion of >99% of [2-H-3]mannose into (H2O)-H-3. Long term labeling of cells with [2-H-3]mannose showed that the specific activity of mannose in glycoproteins reached 77% of the specific activity of [2-H-3]mannose added to the medium, These results show that when fibroblasts are provided with physiological concentrations of mannose, they use the mannose-specific transporter to supply the majority of mannose needed for glycoprotein synthesis, PMI may normally be used to catabolize excess mannose rather than to primarily supply Man-6-P for glycoprotein synthesis.