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 primaril y from glucose through phosphomannose isomerase (PMI), The discovery o f mammalian mannose-specific transporters that function at physio logi cal 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 tric hloroacetic 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. Bot h sugars labeled a similar array of oligosaccharides, and acid hydroly sis of these chains showed that [2-H-3]mannose contributed 65-75% of t he [H-3]mannose in cells labeled for 1 h, despite the 100-fold higher concentration of exogenous glucose, Mannose consumption and [2-H-3]man nose utilization were within the range of rates expected for mannose t ransport via the mannose-specific transporter. About 7-14% of the [2-H -3]mannose is used for glycosylation, while the rest (86-93%) is catab olized to (H2O)-H-3 via PMI, Increasing the exogenous mannose concentr ation 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 gl ycoproteins reached 77% of the specific activity of [2-H-3]mannose add ed to the medium, These results show that when fibroblasts are provide d with physiological concentrations of mannose, they use the mannose-s pecific transporter to supply the majority of mannose needed for glyco protein synthesis, PMI may normally be used to catabolize excess manno se rather than to primarily supply Man-6-P for glycoprotein synthesis.