(Received for publication, December 12, 1995; and in revised form, February 15, 1996)
From the
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
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.
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) ()are deficient in [
H]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-[
H]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.
Fibroblasts (922sk, NRK-49F) were grown in
-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.
Figure 2:
Effect of glucose transporter inhibitors
on mannose uptake. Fibroblasts (922sk) were labeled with DMEM
containing either 10 µCi/ml 2-[H]deoxyglucose
(
) or 20 µCi/ml [
H]mannose (
) 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 [H]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.
Figure 1:
Glucose has very little effect on
mannose entry into the cells. Fibroblasts (922sk) were labeled with 20
µCi/ml [H]mannose in the presence (
) and
absence (
) of 0.5 mM glucose at 37 °C for the time
periods indicated. The cell-associated
H was calculated as
described under ``Experimental
Procedures.''
Figure 3:
Measurement of K in
fibroblasts. Cells (922sk) were labeled with 20 µCi/ml
[
H]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.
Figure 4:
Measurement of
[H]mannose incorporation into proteins.
Fibroblasts (922sk) were labeled with 20 µCi/ml
[
H]mannose for 45 min at 37 °C in the
presence of varying amounts of unlabeled D-mannose. The
cell-associated radiolabel (
) and that incorporated into protein
(
) was measured as described under ``Experimental
Procedures.''
The calculated V 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.
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
-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.