Mannose efflux from the cells: a potential source of mannose in blood

Abstract

All mammals have 50-100 μM mannose in their blood. However, the source of the dynamic pool of mannose in blood is unknown. Most of it is thought to be derived from glucose in the cells. We studied mannose uptake and release by various cell types. Interestingly, our results show that mannose taken up by the cells through transporters is handled differently from the mannose released within the cells due to glycan processing of protein-bound oligosaccharides. Although more than 95% of incoming mannose is catabolized, most of the mannose released by intracellular processing is expelled from the cells as free mannose predominantly via a nocodazole-sensitive sugar transporter. Under physiological conditions, incoming mannose is more accessible to hexokinase, whereas mannose released within the cells is protected from HK and therefore has a different fate. Our data also suggest that generation of free mannose due to the processing of glycoconjugates composed of glucose-derived mannose and its efflux from the cells can account for most of the mannose found in blood and its steady state maintenance.

SCHEME 1.

Mannose metabolic pathway.

FIGURE 1.

Steady state levels of precursors during labeling. C3a cells were labeled with [2-³H]mannose in DMEM containing 0.5 mm glucose. At the indicated times, the medium was collected, and the cells were extracted as mentioned under “Experimental Procedures.” Glycosylation intermediates were measured using standard protocols.

FIGURE 2.

a, pulse-chase depicting turnover of various glycosylation intermediates. C3a cells were labeled in DMEM containing 0.5 mm glucose and 50 μCi of [2-³H]mannose for 1 h. The cells were washed and chased in fresh DMEM with 5 mm glucose. At the indicated times, the medium was collected, and the cells were extracted as mentioned under “Experimental Procedures.” Glycosylation intermediates were measured using standard protocols. b, total radiolabel associated with cells and medium at each time point was determined during chase. c, composition of the medium during pulse-chase. At different time points, free [³H]mannose, ³H₂O, and ³H-labeled protein were estimated as described under “Experimental Procedures.” The results shown are representative of two such experiments.

FIGURE 3.

Glycan processing contributes the majority of mannose secreted in the medium. C3a cells were labeled for 1 h with 50 μCi of [2-³H]mannose in DMEM containing 0.5 mm glucose and then chased in 5 mm glucose DMEM with a mixture of mannosidase inhibitors (MI) (50 μg/ml kifunensine, 20 μg/ml swainsonine, 20 μg/ml deoxymannojiramycin) and/or lysosomotropic agents (1 mm chloroquine (CHlQ) or 10 mm ammonium chloride (NH₄Cl)) for 1 h. Radiolabel was measured in the medium. The data are an average of triplicate determinations.

FIGURE 4.

a, nocodazole inhibits mannose efflux. C3a cells were labeled for 1 h. After 30 min, during labeling, the indicated amount of nocodazole was added for preincubation. This was followed by a chase in fresh DMEM containing the indicated amounts of nocodazole. At different times, an aliquot of medium was collected, and [³H]mannose was estimated using hexokinase and MPI (as mentioned under “Experimental Procedures”). b, inhibition of mannose release by nocodazole is independent of microtubule depolymerization. The procedure is the same as mentioned above except that chase was done for 1 h with medium containing nocodazole either in the absence or presence of taxol. Free [³H]mannose was estimated in the medium using hexokinase and MPI (as mentioned under “Experimental Procedures”). c–f, microtubule stabilization by paclitaxel under experimental conditions. C3a cells were grown on glass coverslips and treated with nocodazole in the absence (e) and presence (f) of paclitaxel as described under “Experimental Procedures” and compared with no treatment (c) and paclitaxel alone (d). The cells were fixed, permeabilized, and stained for α-tubulin. Error bars, S.E.

FIGURE 5.

Effect of phloretin, sugars, and sugar analogs. C3a cells were labeled with [2-³H]mannose for 1 h. This was followed by a chase in fresh DMEM containing the indicated amounts of the additives. a, phloretin-[³H]mannose and ³H₂O were determined in the medium. Sugars (b) and sugar analogs (c) ³H₂O were estimated in the medium. The data represent the average of triplicate determinations. Error bars, S.E.

FIGURE 6.

Endogenously generated mannose from glycan processing escapes catabolism due to inaccessibility to hexokinase. CHO cells and HK-deficient CHO and (MI-54) cells were labeled for 1 h. This was followed by chase in fresh DMEM containing the indicated amounts of glucose. ³H₂O formation was estimated by counting medium with and without evaporation. The data represent an average of triplicate determinations. Error bars, S.E.