©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Mannose Enters Mammalian Cells Using a Specific Transporter That Is Insensitive to Glucose (*)

(Received for publication, December 12, 1995; and in revised form, February 15, 1996)

K. Panneerselvam Hudson H. Freeze (§)

From the La Jolla Cancer Research Foundation, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The concentration of D-mannose in serum is 20-50 µM, but its physiological significance for glycoprotein synthesis is unknown. Here, we show that the uptake of D-mannose by different mammalian cell lines involves a mannose-specific transporter(s) with a K of about 30-70 µM and a V(max) which is probably sufficient to account for the bulk of mannose needed for glycoprotein synthesis. Mannose uptake appears to be through a facilitated transport process since it is not inhibited by cyanide. Phloretin completely inhibits mannose uptake, but phloridzin inhibits only 25-30%. Both of these inhibitors can block 2-deoxyglucose uptake in fibroblasts which occurs through the typical glucose transporters. None of 9 other sugars tested inhibited mannose transport. Most importantly, 5 mMD-glucose only inhibits mannose uptake by 50% showing that it is not an efficient competitor. These results suggest that this transporter(s) may use serum mannose for glycoprotein synthesis.


INTRODUCTION

In mammalian cells, mannose for glycoprotein synthesis is assumed to originate mostly, if not entirely, from intracellular glucose through the pathway Glc Glc-6-P Fru-6-P Man-6-P(1) . Extracellular mannose is seldom considered as a significant source for glycoprotein synthesis. This is probably due to the fact that even though mannose can be transported by the well-known glucose transporters(2, 3, 4) , its concentration of 30-50 µM in plasma is at least 100 times less than glucose(5, 6) . Thus, any mannose transport through this pathway is likely to be very small and nearly totally competed by glucose(7) . Almost all mammalian cells appear to have plasma membrane glucose transporters. Two different types are known. The first type is a facilitated transport system that allows the movement of glucose across the plasma membrane down its chemical gradient and is sensitive to phloretin (8, 9, 10) . Six different isoforms of facilitative type glucose transporters have been identified (GLUT 1-6) with GLUT 1 being the most widely distributed isoform(8) . The second type is an Na-dependent active transport that is sensitive to phloridzin(11, 12) . Three different Na-dependent glucose transporter isoforms have been reported (SGLT 1-3). SGLT 2 is in kidney cortex, but the other two isoforms are expressed in kidney, small intestine, lung, and liver (11) .

In previous studies, we showed that fibroblasts from patients with carbohydrate-deficient glycoprotein syndrome (CDGS) (^1)are deficient in [^3H]mannose incorporation into glycoproteins, and that some of this reduction appears to result from impaired entry of mannose into the cells(40) . However, entry of 2-[^3H]deoxyglucose, which is assumed to measure glucose uptake, appeared to be nearly normal. The difference in the entry of these closely related hexoses prompted us to explore the possibility that mannose may enter the cell via a transporter distinct from the well-studied glucose transporters. Here we present evidence to support the existence of such a mannose-specific transporter in several types of cells. The K is near the plasma concentration of mannose, and its substantial activity at normal plasma glucose concentration raises the possibility that plasma mannose may be used for glycoprotein synthesis.


EXPERIMENTAL PROCEDURES

Materials

D-Glucose, D-mannose, L-mannose, D-galactose, D-fructose, D-ribose, L-fucose, myo-inositol, L-rhamnose, phloretin, and phloridzin were obtained from Sigma. alpha-Minimal essential medium (alpha-MEM) and Dulbecco's modified essential medium (DMEM) were from Life Technologies, Inc. Fetal bovine serum was from Hyclone Laboratories (Logan, Utah).

Radiolabels

[2-^3H]Mannose (15 Ci/mmol) and 2-deoxy-[1,2-^3H]glucose (40 Ci/mmol) were obtained from American Radiolabeled Chemicals, Inc., St. Louis, MO.

Cell Lines and Culture

Normal skin fibroblasts (922sk; CRL 1828), normal rat kidney fibroblasts (NRK-49F; CRL 1570), Madin Darby canine kidney (MDCK NBL-2; CCL 34), and macrophages (RAW 264) were obtained form American Type Culture Collection. Rat glial-like cells (B28) were provided by Dr. William Stallcup(13) . Mast cells (ABFTL-3) were from Dr. Greg Henkel(14) .

Fibroblasts (922sk, NRK-49F) were grown in alpha-MEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, penicillin, and streptomycin. MDCK and glial-like cells (B28) were grown in DMEM containing high glucose, 10% fetal bovine serum, and antibiotics. Macrophage (RAW 264) and mast cells (ABFTL-3) were grown in RPMI medium supplemented with 10% fetal bovine serum and antibiotics.

Measurement of [^3H]Mannose Uptake

Nearly confluent cells in 35-mm multiwell plates were rinsed with DMEM containing no glucose. Uptake was initiated by the addition of labeling medium containing 20 µCi/ml [^3H]mannose, 2 mM glutamine in DMEM in the absence or presence (0.5 mM) of glucose and incubated at 37 °C for the required time. To study the effect of hexose inhibitors (phloretin and phloridzin), they were included in the labeling medium at the concentrations indicated in Fig. 2. To determine the energy dependence of mannose uptake, cells were preincubated with 1 mM potassium cyanide for 10 min and then labeled with [^3H]mannose (20 µCi/ml) for 10 min at 37 °C. After removal of the radiolabeled medium, cells were washed three times in ice-cold phosphate-buffered saline, harvested by trypsinization, and solubilized in 0.1% SDS. An aliquot of the cell lysate was counted for radioactivity and normalized to protein content.


Figure 2: Effect of glucose transporter inhibitors on mannose uptake. Fibroblasts (922sk) were labeled with DMEM containing either 10 µCi/ml 2-[^3H]deoxyglucose (box) or 20 µCi/ml [^3H]mannose (circle) in the presence of increasing amounts of phloretin or phloridzin for 10 min at 37 °C. The cell-associated radiolabel was calculated as described under ``Experimental Procedures.''



Incorporation of [^3H]mannose into protein was determined by adding 10% trichloroacetic acid to an aliquot of the cell lysate using 100 µg of bovine serum albumin as carrier. After vortexing and standing 10 min on ice, the precipitated protein was collected by centrifugation. The pellet was washed with 10% trichloroacetic acid, neutralized with 0.1 N NaOH, transferred to a scintillation vial, and counted.

Fractionation of [^3H]Mannose-labeled Products

Fibroblasts grown in glass plates were labeled for 45 min at 37 °C with 20 µCi/ml [^3H]mannose in DMEM containing 0.5 mM glucose and 2 mM glutamine. After removing the medium, cells were washed a few times with ice-cold phosphate-buffered saline to remove free label, scraped, and extracted three times with 5 ml of CHCl(3)/MeOH (2:1, v/v). This extract contains mostly Dol-P-Man. The residual pellet was dried, sonicated, centrifuged, and washed three times with water to isolate the free oligosaccharides and low molecular weight lipid-linked oligosaccharide (LLO) precursors. The pellet after water wash was extracted three times with 5 ml of CHCl(3)/MeOH/H(2)O (10:10:3, v/v) to isolate LLO. The final pellet was solubilized in 0.1% SDS and digested with peptide-N^4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F) to release the protein-bound N-linked oligosaccharides.


RESULTS

[^3H]Mannose Uptake by Human Fibroblasts

-Human skin fibroblasts (922sk) were incubated for short times with serum-free medium and 20 µCi/ml [^3H]mannose (1 µM) in the absence or presence of 500 µM glucose. As shown in Fig. 1, glucose made little difference in the rate of [^3H]mannose uptake, indicating that mannose entry is insensitive to a large excess of glucose. This result is unexpected since both mannose and glucose are assumed to enter the cells by the same transporter and a 500-fold excess of glucose should inhibit [^3H]mannose uptake. Phloretin, at all concentrations tested, similarly inhibits the entry of both [^3H]mannose and 2-[^3H]deoxyglucose into the cells (Fig. 2A). Phloridzin, a well-known inhibitor of Na-dependent active hexose transport (K(i) <10 µM)(15, 16) , showed about a 25% inhibition of [^3H]mannose and 2-[^3H]deoxyglucose at a concentration of 50 µM. At higher concentrations, it variably blocked 2-[^3H]deoxyglucose uptake but had little further effect on [^3H]mannose uptake (Fig. 2B). Mannose uptake was not significantly inhibited either by including 1 mM potassium cyanide in the labeling medium or by preincubating the cells in 1 mM potassium cyanide for 10 min prior to labeling. So, it is unlikely that mannose enters the cells using Na-dependent active transport. Our results are consistent with mannose entering the cells using facilitative type transporter(s), a portion of which is sensitive to phloridzin.


Figure 1: Glucose has very little effect on mannose entry into the cells. Fibroblasts (922sk) were labeled with 20 µCi/ml [^3H]mannose in the presence (circle) and absence (box) of 0.5 mM glucose at 37 °C for the time periods indicated. The cell-associated ^3H was calculated as described under ``Experimental Procedures.''



Determination of K

Similar short-term labeling with increasing amounts of mannose was done in the presence of 0.5 mM glucose to determine the K of mannose. The amount of cell-associated mannose was calculated based on the known specific activity of mannose in the medium. A biphasic saturation curve was obtained using a wide range of mannose concentrations, suggesting that mannose enters the cell using two different transporters. In three different labelings, K of mannose was calculated at 30-70 µM and a V(max) of 3.0-9.0 nmol/mg/h. A representative experiment is shown in Fig. 3A (K 35 µM; V(max) of 3.2 nmol/mg/h). This is significant because the plasma mannose concentration has been measured at 20-50 µM(5, 6) , suggesting that such a transporter could be effective at physiological levels of mannose. Our calculated K is considerably lower than those usually seen for facilitative type or Na-dependent type glucose transporters which are normally in the millimolar range(8, 11) . At higher concentrations of mannose, a second saturation curve can be plotted which gives a K of 850 µM (Fig. 3B) and a V(max) of 78 nmol/mg/h. This K is comparable to the range (0.8-8.6 mM) reported for 2-deoxyglucose entry using the GLUT1 transporter that is normally found in all cultured cells(17, 18) . Since GLUT 1 can also transport mannose(2) , it is likely to account for the uptake of mannose at high concentrations as seen in Fig. 3B. When the results obtained using all mannose concentrations are plotted as V(0)versus V(0)/[Man] (Fig. 3C), two kinetically distinct transporters are evident.


Figure 3: Measurement of K in fibroblasts. Cells (922sk) were labeled with 20 µCi/ml [^3H]mannose for 5 min at 37 °C in the presence of varying amounts of unlabeled D-mannose. The cell-associated radiolabel was measured to calculate the K using Lineweaver-Burke (A and B) and Eadie-Hofstee (C) plots.



Incorporation of [^3H]Mannose into Glycoproteins

To determine whether the high affinity transporter provides mannose for glycoprotein synthesis, fibroblasts were labeled for 45 min with a constant amount of [^3H]mannose and variable amounts of unlabeled mannose. We calculated the amount of mannose in the various fractions based on the specific activity of [^3H]mannose in the medium, assuming that the endogenous pools rapidly equilibrate with the label in the medium. This is probably valid since the GDP-Man pool in mammalian cells is quite small (2-25 pmol/10^6 cells) and would completely turn over within few minutes. (^2)As shown in Fig. 4, the curves for total cell-associated mannose and that in glycoproteins are similar, with about 25-30% incorporated into glycoproteins. About 85% of the trichloroacetic acid-precipitable ^3H label in protein was released with PNGase F and consisted of mostly high mannose oligosaccharides (data not shown). The PNGase F-insensitive label is probably in glycophospholipid anchors. The remaining cell-associated label is found in lipid-linked (20-25%) and free oligosaccharides (15%) and in low molecular weight precursors (15%). Although [2-^3H]mannose can be metabolized by the glycolytic pathway and lost in the medium as ^3H(2)O, this accounts for only about 15-20% of the total, which is comparable to our previous results(19) .


Figure 4: Measurement of [^3H]mannose incorporation into proteins. Fibroblasts (922sk) were labeled with 20 µCi/ml [^3H]mannose for 45 min at 37 °C in the presence of varying amounts of unlabeled D-mannose. The cell-associated radiolabel (circle) and that incorporated into protein (box) was measured as described under ``Experimental Procedures.''



The calculated V(max) of 5.1 nmol/mg/h and K of 80 µM are comparable with those obtained in the short term labeling experiments. These results suggest that much of the mannose predicted to be taken up by the high affinity transporter is used for oligosaccharide synthesis. Similar kinetic values were also obtained for macrophages, NRK, MDCK, glial-like, and mast cells (see Table 1). The occurrence of similar saturation curves and kinetic values in several different cell types means that mannose transport for glycoprotein synthesis may be a common feature of mammalian cells.



Specificity of Mannose Transporter

To determine whether the mannose uptake was specific and that the transported mannose contributed to glycoprotein synthesis, we incubated cells with 1 µM [^3H]mannose and increasing amounts of nonlabeled mannose or with other sugars for 45 min. The longer incubation times were needed to allow significant incorporation into glycoprotein. Irrespective of the sugar present, 25% of the radiolabel present in the cell lysate was trichloroacetic acid-precipitable (data not shown). Table 2shows the cell-associated radiolabel. Mannose itself was the only effective competitor. Glucose had a much less pronounced effect; even at a 5000-fold excess over mannose, glucose inhibited [^3H]mannose uptake by only 50%. 2-Deoxyglucose which is a known competitor of glucose uptake, had minimal effects on [^3H]mannose transport and suggests that it is not recognized by the putative mannose transporter. Thus, efficient mannose uptake and incorporation into glycoproteins is quite tolerant of physiological concentrations of glucose. Galactose and other sugars such as xylose, rhamnose, fucose, fructose, N-acetylglucosamine, L-mannose, and myo-inositol produced negligible inhibition at 5000-fold excess (Table 2). Only talose which is the C-4 epimer of mannose inhibited the mannose uptake by 50%. Conversely, 1 mM mannose produces significant inhibition of [^3H]deoxyglucose uptake (not shown), as expected, since at high concentrations, mannose can be carried by the more common hexose transporters(2) .




DISCUSSION

We present evidence that human fibroblasts efficiently transport mannose and use it for glycoprotein synthesis. The three essential findings are: 1) transport is mannose-specific, 2) it is only slightly inhibited by glucose at normal plasma concentrations, and 3) the calculated K for mannose is in the physiological range of plasma mannose concentration. This raises the possibility that exogenous mannose could supply a significant proportion of mannose for glycoprotein synthesis. Assuming that 10% of cellular proteins are glycosylated and that mannose accounts for 3% of the weight of an average glycoprotein, cells growing with a doubling time of 24 h would need to synthesize at least 35 nmol of LLO/mg of protein/24 h. If transfer from LLO to protein were complete, this would require the synthesis of an average of 1.5 nmol of mannose/h in LLO. This calculated value is comparable to the amounts experimentally determined in different cell lines(20) . Mannose uptake by the high affinity transporter in the presence of normal plasma glucose (5.0 mM) is about 1.75 nmol/h which would be roughly sufficient to supply all of the mannose required for glycoprotein synthesis. Since we see similar results in macrophages, NRK, MDCK, glial-like, and mast cells, it suggests that mannose-specific transport may be a common feature of mammalian cells.

At the cellular level, mannose is used for biosynthesis of oligosaccharides and glycophospholipid anchors(21) , and, under normal circumstances, it does not appear to make a large contribution to general energy metabolism(22, 23) . Several studies have shown that mannose can be converted into glycogen (22, 24) and, in general, at high concentrations (>5 mM) it is metabolized like glucose at both the cellular and organismic levels(25, 26, 27, 28, 29, 30) . We could find no studies that used the normal physiological concentration of mannose for any experiments. This lack of attention probably results from the assumption that glucose normally provides sufficient mannose for glycoprotein synthesis. Also, it has been assumed that plasma mannose entry should not be significant because the overwhelming excess of glucose would compete out mannose transport through the typical hexose transporter. Our results bring these assumptions into question. However, at this point we do not know the relative contributions of glucose and mannose to glycoprotein synthesis in any system. In some cases, exogenous mannose may be a significant or preferential precursor for glycoprotein synthesis.

The plasma mannose presumably comes from a combination of dietary sources(23, 24) , normal oligosaccharide processing(31) , and from turnover of endogenous glycoproteins and free oligosaccharides (32, 33, 34) . Since mammalian cells generally contain a higher proportion of complex oligosaccharides compared to unprocessed high mannose-type chains, it is clear that the majority of nine mannose residues initially incorporated into the lipid-linked oligosaccharide (LLO) precursor will be lost as free mannose. When this amount is added to that generated by catabolism of LLO in cultured cells(40) , perhaps as much as 75-80% of the mannose initially incorporated into LLO does not become incorporated into stable protein products. It would be reasonable to have a salvage system that recycles the large fraction of rapidly turning over mannose. Mammalian cells are equipped with a diverse collection of broad specificity and highly specific alpha-mannosidases (35) that degrade oligosaccharides within the cell, but the fate of the released mannose has not been studied. Some of it would probably reach the plasma where it could be reabsorbed or excreted. Studies in rabbit kidney shows that mannose reabsorption is blocked by phloretin but not by phloridzin whereas glucose reabsorption is blocked by both the inhibitors(29) . These results were interpreted to mean that there were different binding sites on the same transporter, but they could also be interpreted as mannose and glucose using two different transporters.

Clearly, mannose can be derived from glucose. The assumption that glucose is the primary source of mannose seems to be based on the ubiquitous distribution of phosphomannose isomerase (PMI) and the fact that PMI deficiency is lethal in yeast(36) . This comparison may not be appropriate since the demand for extended high mannose-type oligosaccharide synthesis in yeast is much greater and the specific activity of their PMI is 20-100 times higher than in mammalian cells (37) . There are no comparable mutations in mammalian cells or specific inhibitors of PMI to directly determine its contribution to glycoprotein synthesis.

Our previous findings that CDGS cells are deficient in mannose uptake and underglycosylate their proteins might be partially explained by an altered mannose transport system, but this will require identification of the mannose transporter(s) itself.


FOOTNOTES

*
This work was supported by NIGMS Grant RO1 49096 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: La Jolla Cancer Research Foundation, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-455-6480; Fax: 619-450-2101; hudson{at}ljcrf.edu.

(^1)
The abbreviations used are: CDGS, carbohydrate-deficient glycoprotein syndrome; LLO, lipid-linked oligosaccharide; PNGase F, peptide-N^4-(N-acetyl-beta-glucosaminyl)asparagine amidase; alpha-MEM, alpha-minimal essential medium; DMEM, Dulbecco's modified essential medium; PMI, phosphomannose isomerase.

(^2)
Several laboratories have estimated the GDP-Man level as 2-25 pmol/10^6 cells (Refs. 20, 38, and 39). Since different cell lines have been found to have 300 µg of protein in 10^6 cells, we normalized the GDP-Man content to 6-80 pmol/mg. We have calculated (see ``Discussion'') that cells require approximately 1.5 nmol of mannose per h for glycoprotein synthesis. That means the GDP-Man pool should turn over 20-250 times per h.


ACKNOWLEDGEMENTS

We thank Drs. William Stallcup and Greg Henkel of La Jolla Cancer Research Foundation for providing rat glial-like cells and mast cells, respectively. We also thank Susan Greaney for secretarial assistance and David Davies for technical assistance.


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