Mannose corrects altered N-glycosylation in carbohydrate-deficient glycoprotein syndrome fibroblasts

Abstract

Type I carbohydrate-deficient glycoprotein syndrome (CDGS) patients fail to add entire N-linked oligosaccharide chains to some serum glycoproteins. Here we show that four CDGS fibroblast cell lines have two related glycosylation abnormalities. First, they incorporate 3-10-fold less [3H] mannose into proteins, and, second, the size of the lipid-linked oligosaccharide precursor (LLO) is much smaller than in controls. Addition of exogenous mannose, but not glucose, to these CDGS cells corrects both the lowered [3H] mannose incorporation and the size of LLO. These corrections are not permanent, and the defects immediately reappear when mannose is removed. To explore further the basis of mannose correction, we analyzed the amount of 3H-labeled LLO intermediates. Except for dolichol-P-mannose, other precursors, including mannose, mannose-6-phosphate, mannose-1-phosphate, and GDP-mannose, all showed a 3-10-fold decrease in CDGS cells. Thus, there are no obvious lesions in the intracellular conversion of mannose into LLO, and, once inside the cell, [3H]mannose appeared to be metabolized normally. Initial velocities of [3H]mannose uptake were two- to threefold less in CDGS cells compared with controls, and this slower transport may partially explain the reduced [3H]mannose incorporation in CDGS cells. Since we previously showed that the enzymes converting glucose to mannose-6-phosphate appear to be normal, our results suggest that cells may acquire or generate mannose in other ways. Although we have not identified the primary defect in CDGS, these studies show that intracellular mannose is limited and that some patients might benefit from including mannose in their regular diets.

Full text

1478
K. Panneerselvam and H.H. Freeze
J. Clin. Invest.
© The American Society for Clinical Investigation, Inc.
0021-9738/96/03/1478/10 $2.00
Volume 97, Number 6, March 1996, 1478–1487
Mannose Corrects Altered N-Glycosylation in Carbohydrate-deficient Glycoprotein
Syndrome Fibroblasts
K. Panneerselvam and Hudson H. Freeze
La Jolla Cancer Research Foundation, La Jolla, California 92037
Abstract
Type I carbohydrate-deficient glycoprotein syndrome (CDGS)
patients fail to add entire N-linked oligosaccharide chains
to some serum glycoproteins. Here we show that four CDGS
fibroblast cell lines have two related glycosylation abnor-
malities. First, they incorporate 3–10-fold less [
3
H] mannose
into proteins, and, second, the size of the lipid-linked oli-
gosaccharide precursor (LLO) is much smaller than in con-
trols. Addition of exogenous mannose, but not glucose, to
these CDGS cells corrects both the lowered [
3
H]mannose in-
corporation and the size of LLO. These corrections are not
permanent, and the defects immediately reappear when
mannose is removed. To explore further the basis of man-
nose correction, we analyzed the amount of
3
H-labeled LLO
intermediates. Except for dolichol-P-mannose, other pre-
cursors, including mannose, mannose-6-phosphate, man-
nose-1-phosphate, and GDP-mannose, all showed a 3–10-
fold decrease in CDGS cells. Thus, there are no obvious
lesions in the intracellular conversion of mannose into LLO,
and, once inside the cell, [
3
H]mannose appeared to be me-
tabolized normally. Initial velocities of [
3
H] mannose up-
take were two- to threefold less in CDGS cells compared
with controls, and this slower transport may partially ex-
plain the reduced [
3
H]mannose incorporation in CDGS
cells. Since we previously showed that the enzymes convert-
ing glucose to mannose-6-phosphate appear to be normal,
our results suggest that cells may acquire or generate man-
nose in other ways. Although we have not identified the pri-
mary defect in CDGS, these studies show that intracellular
mannose is limited and that some patients might benefit
from including mannose in their regular diets. (
J. Clin. In-
vest.
1996. 97:1478–1487.) Key words: glycosylation
•
lipid-
linked oligosaccharide
•
glycoprotein
•
hexose transporter
•
CDG syndrome
Introduction
Carbohydrate-deficient glycoprotein syndromes (CDGS)
1
are
autosomal recessive disorders that affect multiple organ sys-
tems (1, 2). All patients show altered isoelectric focusing pat-
terns of multiple serum glycoproteins that result from under-
sialylation of their N-linked oligosaccharides (3, 4). The
reduced sialylation in one patient with the rarer type II CDGS
can be fully explained by the absence of Golgi enzyme
GlcNAc transferase II (1, 5). This enzyme is required to make
sialylated biantennary N-linked oligosaccharides typical of
many serum glycoproteins (6). The undersialylation in the
more common type I CDGS clearly results from the complete
absence of one or more N-linked oligosaccharide chains on the
proteins (7–10). The basis of this defect has been proposed to
be in the biosynthesis of the lipid-linked oligosaccharide
(LLO) precursor or its transfer to proteins (1, 7, 8). However,
CDGS fibroblasts appear to be normal in oligosaccharyl trans-
ferase and in UDP-GlcNAc:dolichol phosphate GlcNAc-1-P
transferase, the first enzyme in LLO biosynthesis (11, 12).
We recently reported that the carbohydrate content of to-
tal serum glycoproteins from two CDGS patients was reduced
by
z
30%, but the monosaccharide composition and types of
N-linked oligosaccharides were normal (13). Metabolic label-
ing of two fibroblasts with [
3
H]mannose showed a 2.5–5-fold
decrease in the incorporation into both glycoproteins and
LLO of CDGS cells compared with controls. We have now ex-
panded this study to four CDGS patients and found that re-
duced protein glycosylation probably results from an inade-
quate supply of mannose needed for N-linked oligosaccharide
biosynthesis, and not from a clear lesion in any step leading to
protein glycosylation. Adding exogenous mannose to cells in
culture corrects the biochemical lesions.
Methods
Materials.
Most of the materials were obtained from Sigma Chemical
Co. (St. Louis, MO), except for the following: concanavalin A (Con
A)–Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ),
a
-MEM
(GIBCO BRL, Baltimore, MD), FBS (Hyclone Laboratories, Logan,
UT), Micropak AX-5 HPLC column (Varian Instruments, Walnut
Creek, CA), and microspin filters (Lida Manufacturing Corp.,
Kenosha, WI).
Radiolabels.
2-[
3
H]Mannose (15 Ci/mmol), 2-deoxy[1,2-
3
H(N)]-
glucose (40 Ci/mmol), and [
35
S]methionine (1,217 Ci/mmol) were ob-
tained from American Radiolabeled Chemicals, Inc. (St. Louis, MO).
Cell lines.
The CDGS and control cell lines were all obtained
from Dr. Neil Buist (Department of Pediatrics, Oregon Health Sci-
ences University, Portland, OR). CDGS 1 and 2, our designation, are
patients from the same family (samples F27467 and F27468) de-
scribed in our previous study (13). CDGS cell lines 3 and 4 are pa-
tients from another family (samples F20660 and F02661, coded by Dr.
N. Buist). All these patients are diagnosed as type I CDGS based on
the clinical and biochemical (transferrin pattern) analyses. Disease
Address correspondence to Hudson H. Freeze, La Jolla Cancer Re-
search Foundation, 10901 N. Torrey Pines Rd., La Jolla, CA 92037.
Phone: 619-455-6480; FAX: 619-450-2101.
Received for publication 10 November 1995 and accepted in re-
vised form 5 January 1996.
1.
Abbreviations used in this paper:
a
-MeMan,
a
-methyl mannoside;
CDGS, carbohydrate-deficient glycoprotein syndrome; Con A, con-
canavalin A; dol-P-Man, dolichol-P-mannose; Endo H, endoglycosi-
dase H; GDP-Fuc, GDP-fucose; GDP-Man, GDP-mannose; LLO,
lipid-linked oligosaccharide; Man, mannose; Man-1-P, mannose-1-
phosphate; Man-6-P, mannose-6-phosphate; PMI, phosphomannose
isomerase.

Carbohydrate-deficient Glycoprotein Syndrome
1479
control fibroblasts are from pediatric patients with lactic acidosis,
fatty acid/glutaric disorders, and movement disorders, expressing nor-
mal transferrin patterns. Normal adult fibroblasts (CRL 1826) were
obtained from American Type Culture Collection (Rockville, MD).
Cell lines were grown in
a
-MEM containing 10% heat-inactivated
FBS and 2 mM glutamine. When the long-term effect of mannose was
studied, both normal and CDGS cells were grown for 10 d in the reg-
ular growth medium containing 1 mM
d
-mannose, and the medium
was changed every day.
Determination of incorporation of [
3
H]mannose.
Fibroblasts in 35-
mm multiwell plates were labeled with [
3
H]mannose (20
m
Ci/ml) and
[
35
S]methionine (2
m
Ci/ml) for 60 min in DMEM containing 0.5 mM
glucose and 2 mM glutamine. After removal of the radioactive me-
dium, cells were quickly washed three times with ice-cold PBS and
harvested by trypsinization, counted, sonicated, and solubilized in
0.1% SDS. The lysate was divided into four aliquots and used for pro-
tein determination and TCA precipitation before and after peptide:
N
-glycosidase F (PNGase F) or endoglycosidase H (Endo H) diges-
tion. TCA-precipitated radiolabel was counted and normalized to
protein content. PNGase F and Endo H digestions were done as de-
scribed previously (14).
Isolation and characterization of [
3
H]mannose-labeled products
from cellular fraction.
Fibroblasts grown in glass plates were labeled
for 30 min with [
3
H]mannose (100
m
Ci/ml) in DMEM containing 0.5
mM glucose and 2 mM glutamine (15). After removing the labeling
medium, cells were quickly washed three times with ice-cold PBS to
remove free label, and the plate was immediately flooded directly
with 5 ml of chloroform/methanol (CHCl
3
/MeOH, 2:1 vol/vol) at 0
8
C
(15). Cells were scraped and stored at
2
20
8
C until processed. Cells
were vortexed to disperse cell clumps and centrifuged at 3,000 rpm
for 5 min. Pellets were extracted twice with 5 ml of the same solvent,
and this combined extract (fraction I) was used to measure dolichol-
P-mannose (dol-P-Man) content. The residual pellet after CHCl
3
/
MeOH extraction was dried under a stream of nitrogen, suspended in
1 ml of water, sonicated, centrifuged, and washed three times with
5 ml of water. This combined water wash (fraction II) was used to
measure labeled free oligosaccharides, mannose phosphates, and
sugar nucleotides. The remaining cell pellet was extracted three times
with 5 ml of CHCl
3
/MeOH/H
2
O (10:10:3 vol/vol) (fraction III) to iso-
late LLO. Oligosaccharides from the lipid were released by mild acid
hydrolysis (15). The final cell pellet was solubilized in 0.1% SDS
(fraction IV) and digested with PNGase F to release the protein-
bound N-linked oligosaccharides (14), which were reduced with so-
dium borohydride in sodium borate buffer, pH 9.8, desalted, and ana-
lyzed by HPLC on an amine adsorption column with a linear gradient
of acetonitrile (65–35%) in water as described previously (16).
Measurement of dol-P-Man.
CHCl
3
/MeOH extract (fraction I)
was partitioned into CHCl
3/
MeOH/H
2
O (3:2:1 vol/vol). The organic
phase was dried, hydrolyzed in 0.01 N HCl for 15 min at 100
8
C, and
again partitioned into CHCl
3/
MeOH/H
2
O (3:2:1 vol/vol). Radioactiv-
ity partitioning into the aqueous phase was taken as dol-P-Man con-
tent (17). Authentic dol-P-Man was completely hydrolyzed under
these conditions and partitioned into the aqueous phase. The quanti-
tation of labeled dol-P-Man by this method was confirmed by thin-
layer chromatography and by counting individual 0.5-cm sections.
Separation of water soluble components on Con A–Sepharose.
The water-soluble material in fraction II was fractionated on Con
A–Sepharose in microfuge spin filter baskets as described below. Ma-
terial that did not bind to Con A was used for estimating the amount
of mannose phosphates and sugar nucleotides. Con A–bound mate-
rial was eluted with 100 mM
a
-methyl mannoside (
a
-MeMan) and
applied to a G-10 Sephadex column to remove
a
-MeMan. The single
radioactive peak near the void volume (Vo) was pooled, concen-
trated, and loaded on to QAE-Sephadex (in spin columns). The un-
bound neutral oligosaccharides were reduced and analyzed by HPLC
as described above. Anionic oligosaccharides bound to QAE were
eluted with 70 mM NaCl, desalted on Sephadex G-10, and concen-
trated. They were subsequently digested with alkaline phosphatase or
treated with 0.1 N HCl for 10 min at 100
8
C to remove the negative
charge before reduction and HPLC analysis.
Spin columns.
Samples in a volume of 50
m
l were applied to 200
m
l of Con A–Sepharose or QAE-Sephadex packed in spin columns
and spun at 1,500 rpm in a microfuge tube for 1–2 min. Columns were
washed four times with 200
m
l of water, and the bound material was
eluted with 5
3
200
m
l of preheated (60
8
C) 100 mM
a
-MeMan (for
Con A–Sepharose) or 70 mM NaCl (for QAE).
Determination of [
3
H]mannose phosphates and sugar nucle-
otides.
The Con A–Sepharose unbound material is expected to con-
tain
3
H-labeled mannose (Man), mannose-6-phosphate (Man-6-P),
mannose-1-phosphate (Man-1-P), GDP-mannose (GDP-Man), and
GDP-fucose (GDP-Fuc). We measured the amount of each of these
components using a series of enzymatic reactions. All of the determi-
nations are based on the ability of phosphomannose isomerase (PMI)
to convert [
3
H]Man-6-P into fructose-6-P with the release of
3
H
2
O
(18, 19), which can be removed by evaporation. The amount of Man-
6-P is measured by direct conversion to
3
H
2
O using PMI. Mannose is
measured by converting it to Man-6-P with hexokinase. Man-1-P is
converted to Man by alkaline phosphatase digestion, and then to
Man-6-P with hexokinase. The remaining labeled material represents
GDP-Man and GDP-Fuc. All reactions were carried out at 37
8
C for
1 h in a volume of 50
m
l containing the sample, 50 mM Tris-HCl, pH
8.0, 6 mM MgCl
2
, and 1
mU of required enzymes, PMI, HK, or alka-
line phosphatase (20, 21). Measurements were done on a single sam-
ple in the order described above, or by separate treatments of the
sample divided into three aliquots. The results of both methods were
the same. Conditions for these determinations were first worked out
on individual standards or in mixtures containing various proportions
of each component. In all cases, the conversions were
.
95%, and as
little as 10% of any one product in the mixture could be accurately
measured. A detailed description of the above method will be pre-
sented elsewhere (Panneerselvam, K., and H.H. Freeze, manuscript
in preparation).
Labeling of fibroblasts with [
3
H]2-deoxyglucose.
[
3
H]2-deoxyglu-
cose uptake by fibroblasts was performed according to Asano et al.
(22) with some modifications. The preincubation in the absence of se-
rum was omitted, and the labeling was done in DMEM. Nearly con-
fluent monolayers of fibroblasts in 35-mm multiwell plates were incu-
bated with [
3
H]2-deoxyglucose (10
m
Ci/ml) in DMEM containing
2 mM glutamine for 15 min at 37
8
C. Labeling was terminated by the
addition of 2 ml of ice-cold PBS containing 10 mM glucose and 0.3
mM phloretin, and the cells were rapidly washed three times with ice-
cold PBS containing glucose and subsequently solubilized with 0.1%
SDS. Aliquots of these were assayed for radioactivity and protein.
Results
Lipid and protein-bound oligosaccharides in CDGS cells.
In previous studies, we found that two CDGS cell lines incor-
porated 2.5–5-fold less [
3
H]mannose into LLO and protein
compared with normal cells (13). To extend these results, we
analyzed the size of labeled oligosaccharides released from
LLO or protein of controls and four CDGS cell lines. Normal
cells make predominantly large LLO (Glc
1–3
Man
9
GlcNAc
2
)
(Fig. 1). Protein linked oligosaccharides released by PNGase F
digestion had a size distribution similar to LLO, suggesting
that little mannose processing occurs during labeling. This is
supported by the finding that these oligosaccharides bind
strongly to Con A–Sepharose and are not sialylated (not
shown). In contrast, CDGS cells usually synthesize and trans-
fer much smaller (Man
4–6
GlcNAc
2
) oligosaccharides to pro-
teins (Fig. 1). The LLOs do not appear to be glucosylated since
a
-mannosidase digestion degrades them to free [
3
H]mannose
and Man
b
GlcNAc
b
GlcNAc (HPLC data not shown). How-
ever, our results did not rule out the possibility that CDGS

1480
K. Panneerselvam and H.H. Freeze
cells synthesize a smaller proportion of fully glucosylated
LLO (Glc
1–3
Man
5
GlcNAc
2
), which is preferentially trans-
ferred to protein. This kind of preferential transfer has been
reported earlier in other cells that make truncated LLO (23).
In
z
20% of the experiments under apparently identical label-
ing conditions, the CDGS cells synthesize and transfer mostly
normal-sized LLOs (HPLC data not shown). Even in these
cases, CDGS cells showed at least a threefold lower incorpora-
tion of [
3
H]mannose into LLO and protein compared with
normal. The size variations were never seen in normal cells.
These results show that underglycosylation of proteins is not
due to a permanent inability to make the normal-sized LLO
structure or to transfer it to protein.
Analysis of protein glycosylation using PNGase F and Endo
H.
Since isolation and analysis of LLO and protein bound
N-linked chains is time consuming, we used PNGase F to mea-
sure the amount of N-linked glycosylation and Endo H to
determine the size of newly transferred oligosaccharides.
PNGase F cleaves nearly all known N-linked oligosaccharides
from proteins, while Endo H cleaves high-mannose type oli-
gosaccharides larger than Man
5
GlcNAc
2
(14). Therefore, mea-
suring the proportion of Endo H–sensitive [
3
H]mannose-
labeled chains measures the proportion of oligosaccharides
.
Man
5
GlcNAc
2
.
Duplicate cultures of normal, disease control, and four
CDGS cell lines were labeled with [
3
H]mannose and [
35
S]me-
thionine, and a portion of the lysates was analyzed by TCA
precipitation before and after Endo H or PNGase F digestions.
The results of one representative labeling are shown in Table
I. In all CDGS cells, [
3
H]mannose incorporation into protein
was 3–20-fold less than controls when normalized either to
protein content or to [
35
S]methionine incorporation. PNGase
F digestion released 83–87% of the [
3
H]mannose from all cell
lysates, and Endo H digestion released 60–70% of [
3
H]man-
nose from protein of normal and disease control cells. In con-
trast, Endo H released only 16–29% from CDGS cell proteins.
The smaller size of the Endo H–resistant oligosaccharides was
confirmed by HPLC (data not shown).
Since cell passage number and density can influence the
size of the LLO (24) and the amount of [
3
H]mannose incorpo-
Figure 1. CDGS cells synthesize truncated LLO and transfer them to
protein. Fibroblasts were labeled with 100 mCi/ml [
3
H]mannose for 30
min, and the neutral oligosaccharides from fractions II (water wash),
III (LLO), and IV (protein) were isolated and processed as described
in Methods. Sizes of the oligosaccharides were analyzed using an
amine adsorption HPLC column that separates oligosaccharides ac-
cording to size, with larger eluting later. 1-ml fractions were collected
and counted for radioactivity. The shaded area in the water wash of
control indicates the elution pattern of Con A–bound anionic oli-
gosaccharides derived from fraction II after dephosphorylation
with alkaline phosphatase. The elution position of the standards
are indicated by arrows labeled with 5 (Man
5
GlcNAc
2
) and 9
(Glc
1-3
Man
9
GlcNAc
2
).
Table I. Labeling of Control and CDGS Fibroblasts with
[
3
H]Mannose and [
35
S]Methionine*
Cell line
3
H/
35
S
‡
% PNGase F
sensitive
§
% Endo H
sensitive
i
Normal 159 (100) 86 61
Disease control 164 (103) 84 63
CDGS 1 53 (33) 85 16
2 15 (9) 83 10
3 36 (22) 87 26
4 52 (32) 83 24
*Labeling for 60 min. and incorporation into protein was determined as
described in Methods.
‡
Numbers in parentheses indicate the percentage
of normal which is defined as 100, and other samples are presented as a
percentage of this value.
§
Defined as the percentage of total TCA-pre-
cipitable
3
H released by PNGase F.
i
Defined as the percentage of total
TCA-precipitable
3
H released by Endo H.
Figure 2. Potential sources of mannose and its metabolism in relation
to protein glycosylation. Mannose for N-linked glycosylation may (a)
come from glucose through a pathway involving PMI; (b) plasma
mannose may be transported (Transporter) inside the cell; or (c) it
may also be salvaged from LLO degradation and typical oligosaccha-
ride processing (Turnover). The relative contribution of mannose
from each of these sources is unknown. Mannose is converted into
Man-6-P and then to Man-1-P, which is used to synthesize GDP-Man
and dol-P-Man for LLO. INSIDE and OUTSIDE refer to the cell.

Carbohydrate-deficient Glycoprotein Syndrome
1481
ration into protein, we compared these parameters in normal,
disease control, and CDGS cells at different passages. Cells of
the same passage, from widely different passages or at differ-
ent densities, were labeled on the same day with [
3
H]mannose
and digested with PNGase F and Endo H. Regardless of the
passage number and cell density, normal and disease control
cells always make primarily (80%) Endo H–sensitive oligosac-
charides. In contrast, the proportion of Endo H–sensitive oli-
gosaccharides varied from 10–80% in CDGS cells, and had a
3–10-fold decrease in [
3
H]mannose incorporation. It appears
that N-linked oligosaccharide synthesis in CDGS cells under
these conditions is inherently variable. The control and CDGS
cells grew at the same rate (with 60 h doubling time), and there
were no obvious differences in the cell size, morphology, or
protein content compared with the controls. Since fibroblasts
from normal and each of three different disease control cells
behaved the same in these experiments, we sometimes used
only the disease control cells or normal cells in subsequent ex-
periments.
Glycosylation in CDGS cells resembles glucose starvation.
The combination of underglycosylation and truncated LLO
size has been seen in cells during glucose starvation (24–32).
These cells mostly synthesize Man
5
GlcNAc
2
and in some cases
fully glucosylated LLO (Glc
3
Man
5
GlcNAc
2
) (31, 32). There is
a considerable cell-type variation in the degree of underglyco-
sylation and time required to see the effects, but, in all cases,
only the addition of either glucose or mannose corrects both of
the glycosylation defects. Although the basis of the glucose
starvation effect is unknown, the similar efficacy of glucose
and mannose can be easily explained by the assumption that
the bulk of Man-6-P used for glycoprotein synthesis is derived
from glucose through a pathway involving PMI (Fig. 2).
Based on this rationale, we considered the possibility that
CDGS cells have insufficient intracellular glucose, perhaps in-
duced by replacing the normal glucose (5.0 mM) medium with
labeling medium that contains 0.5 mM glucose. This medium is
commonly used to improve [
3
H]Man labeling efficiency by re-
ducing the mutual competition of mannose and glucose for the
hexose transporters (18, 33). To test this possibility, the glu-
cose concentration was increased up to 5 mM during or 1 h be-
fore the labeling, but this did not have any effect on either un-
derglycosylation or oligosaccharide size (Fig. 3) in CDGS cells.
These results show altered glycosylation in CDGS cells is not
identical to that seen in glucose starvation.
Addition of
d
-mannose corrects oligosaccharide size and
[
3
H]mannose incorporation into proteins. In contrast, when CDGS
cell lines were labeled in the presence of increasing amounts of
exogenous d-mannose, both the decreased incorporation of
[
3
H]mannose and Endo H sensitivity are corrected in parallel
(Fig. 4). Addition of mannose to CDGS cells corrected the
LLO size to Glc
1–3
Man
9
GlcNAc
2
(Fig. 5), and these normal-
sized chains were transferred to protein (data not shown). In-
Figure 3. d-Glucose has no effect on the underglycosylation and oli-
gosaccharide size of CDGS cells. Fibroblasts were labeled with 20
mCi/ml [
3
H]mannose for 1 h with increasing amounts of d-glucose.
The incorporation into glycoprotein was measured by TCA precipita-
tion, and the oligosaccharide size was measured by Endo H sensitivity
as described in Methods. TCA precipitable counts were normalized
to protein content and taken as the total incorporation into glycopro-
teins (glycosylation). The difference in the TCA precipitable counts
before and after Endo H digestion represents the oligosaccharides
with structure . Man
5
GlcNAc
2
(Endo H sensitive). Glycosylation
(A) and Endo H sensitivity (B) in normal cells are taken as 100%,
and those in CDGS patients are shown as a percentage of this value.
Figure 4. Correction of underglycosylation and oligosaccharide size
by d-mannose in CDGS cells. Fibroblast cultures were incubated with
20 mCi/ml of [
3
H]mannose and increasing amounts of nonlabeled
d-mannose for 1 h, and the
3
H-label incorporation into glycoprotein
(h) and Endo H sensitivity (s) were measured by TCA precipitation
as described in the legend to Fig. 3.

1482 K. Panneerselvam and H.H. Freeze
creased mannose does not alter the size of the oligosaccharides
made by normal cells. The effect of mannose on CDGS cells
was seen even in the presence of 5 mM glucose (Fig. 6), show-
ing that the effect is exclusive for mannose under these condi-
tions. l-Mannose is also ineffective, presumably since it cannot
be transported and/or phosphorylated by hexokinase.
Effects of long-term incubation of mannose on cells. To de-
termine whether prolonged incubation with mannose corrects
the altered glycosylation, CDGS fibroblasts were grown in
1 mM d-mannose for up to 10 d before labeling. The exoge-
nous mannose was removed and cells were labeled with only
[
3
H]mannose (1 mM) in the presence of 0.5 mM glucose. Un-
der these conditions, the amount of incorporation and Endo H
sensitivity of the labeled oligosaccharides were the same as
those of cells that had never been grown in mannose (data not
shown). This finding supports the idea that mannose must be
continually present to correct the underglycosylation and the
truncated size of the oligosaccharides in CDGS fibroblasts.
Fractionation and analysis of [
3
H]mannose-labeled prod-
ucts in control and CDGS lines. The correction of abnormal
glycosylation in CDGS cells by mannose suggested that there
might be a specific deficiency in one or more of the intermedi-
ates leading to LLO. To search for a specific deficiency, we an-
alyzed a broad range of [
3
H]mannose-labeled products from
control and four CDGS cells.
Passage- and density-matched control and CDGS cells
were labeled for 30 min with [
3
H]mannose as described in
Methods, and four fractions (I–IV) were prepared in the fol-
lowing order: The first (I) is a CHCl
3
/MeOH (2:1 vol/vol) ex-
tract that contains mostly small lipid-linked molecules such as
Figure 5. d-Mannose cor-
rects LLO size. Normal fi-
broblasts or CDGS 2 and
CDGS 3 fibroblasts were
labeled with 150 mCi/ml
[
3
H]mannose in the pres-
ence and absence of 1 mM
unlabeled d-mannose for
30 min. The LLOs were iso-
lated as described in Meth-
ods. Oligosaccharides
(1,500–3,000-cpm aliquots)
were applied to an amine
adsorption HPLC column
connected to a radioactivity
flow detector. Profiles show
%
3
H vs elution time for
LLO from control or
CDGS fibroblasts. The elu-
tion positions of standards
are indicated by arrows
labeled with 5
(Man
5
GlcNAc
2
) and 9
(Glc
1–3
Man
9
GlcNAc
2
).

Carbohydrate-deficient Glycoprotein Syndrome 1483
dol-P-Man. The second (II) is a water wash containing low–
molecular weight intermediates, and perhaps free oligosaccha-
rides. The third (III) is a CHCl
3
/MeOH/H
2
O (10:10:3 vol/vol)
extraction that contains the LLO, and, finally, an insoluble pel-
let, fraction IV, contains protein-bound N-linked oligosaccha-
rides. As shown in Table II, CDGS cells incorporate 3–20-fold
less [
3
H]mannose into protein (fraction IV) and 3–40-fold less
in LLO (fraction III) compared with control. The size of the
oligosaccharides from lipid or protein in CDGS cells is
Man
4–6
GlcNAc
2
(data not shown). The
3
H protein/LLO ratio
in CDGS cells is equal to or greater than control, showing that
the CDGS cells use the reduced amount of LLO as efficiently
as control cells.
Fraction I. The majority of labeled material in fraction I is
dol-P-Man, and the amount is similar in CDGS cells and the
control. This was surprising in view of the reduction in the
amount of label in all of the other fractions analyzed.
Fraction II, Con A–bound material. The amount of label in
fraction II is also 3–10-fold lower in CDGS cells than in the
control. This fraction should contain
3
H-labeled Man and met-
abolic intermediates Man-6-P, Man-1-P, GDP-Man, and GDP-
Fuc, but z 35–60% of the label in fraction II from each cell
line bound to Con A–Sepharose. This material is composed of
free oligosaccharides rather than glycopeptides, since it did not
bind to cation exchange resin (Dowex-50). The size of these
oligosaccharides also closely resembles that of those derived
from protein-bound oligosaccharides (shown in Fig. 1), and
they are most likely cleaved from protein or LLO as reported
by others (34–40).
About 15–40% of the Con A–Sepharose–bound material
also binds to QAE-Sephadex and is eluted by 70 mM NaCl,
suggesting that this material contains two negative charges
(41). Alkaline phosphatase digestion, or mild acid hydrolysis
(0.1 N HCl, 10 min, 1008C) eliminates binding to the resin.
These molecules probably have a phosphomonoester on the
reducing GlcNAc and were derived from LLO by pyrophos-
phatase-type cleavage. The alkaline phosphatase–neutralized
oligosaccharides from the control are small (Man
5
GlcNAc
2
-
size; Fig. 1, top right panel, shaded area), and not typical of the
entire LLO pattern. This shows that selected LLOs are prefer-
entially hydrolyzed in control cells. In CDGS, the size of the
anionic oligosaccharides is also small, and, in this case, they re-
flect the truncated LLO pattern (data not shown).
The amount of Con A–Sepharose–bound material differs
in each cell line (Table III), but it is nearly the same propor-
tion of the total
3
H label in all cells. This shows that decrease
of
3
H in glycoproteins seen in CDGS cells is not due to acceler-
ated degradation of either LLO or newly synthesized glyco-
proteins.
Fraction II, not bound to Con A. The amounts of
3
H-labeled
Man, Man-6-P, Man-1-P, and GDP-Man 1 GDP-Fuc in frac-
tion II were determined as described in Methods (Table III).
The amount of each and the total amount of cell-associated
3
H
(total counts per minute in fractions I–IV; Table II) are much
lower in all CDGS cells. Since these decreases correspond
quite well to the decrease in protein glycosylation and LLO, it
appears that CDGS cells are not deficient in any of the biosyn-
thetic steps leading to the synthesis of LLO or its transfer to
protein. Once [
3
H]mannose is inside the cell, it appears to be
metabolized normally. Thus, it appears that [
3
H]mannose itself
may be limited.
Mannose entry in CDGS cells is impaired. As shown in Fig.
7, CDGS cells are two- to threefold slower in [
3
H]mannose up-
take than the normal cells. Mannose uptake is completely
blocked by phloretin, showing that it is transporter mediated
(42). Since the plasma membrane glucose transporters are able
Figure 6. Effect of d-mannose on the underglycosylation of CDGS
cells at a higher concentration of glucose. Fibroblasts were labeled
with 20 mCi/ml [
3
H]mannose for 1 h in DMEM containing 5 mM glu-
cose and increasing amount of unlabeled d-mannose. Cell lysates
were precipitated with TCA, counted for radioactivity, and normal-
ized to protein content. [
3
H]Mannose incorporation into glycoprotein
in normal is taken as 100% and that in CDGS patients is presented as
a percentage of this value.
Table II. Distribution of [
3
H]Mannose in Various Fractions of Control and CDGS Fibroblasts*
Cell line
2:1 CHCl
3
/MeOH
Fraction I
Water wash
Fraction II
LLO
Fraction III
Protein
Fraction IV
Total
3
H in cells
Fractions I–IV Protein/LLO % Endo H sensitive
‡
3
H cpm
3
10
2
4
/mg protein
Disease control 41.9 (37.7)
§
685.5 418.0 613.9 1,759.3 (100.0)
i
1.46 83
CDGS 1 36.7 (29.4)
§
158.5 15.7 105.3 316.2 (18.0)
i
6.60 31
2 41.0 (35.7)
§
80.5 10.9 34.4 166.8 (9.5)
i
3.10 16
3 35.3 (33.5)
§
272.0 78.7 202.5 588.5 (33.4)
i
2.50 29
4 51.5 (36.4)
§
160.6 17.9 93.3 323.3 (18.3)
i
5.20 25
* Duplicate cultures were labeled consecutively with [
3
H]Mannose for 30 min and fractionated as described in Methods.
§
Dol-P-Man content.
i
Numbers in parentheses indicate the percentage of control which is defined as 100, and CDGS samples are presented as a percentage of this value.
‡
Defined as the percentage of total TCA-precipitable
3
H released by Endo H.

1484 K. Panneerselvam and H.H. Freeze
to transport both mannose and glucose (43–45), one possible
explanation for decreased [
3
H]mannose uptake is a defect in
one of these transporters. To determine this, we used a stan-
dard [
3
H]2-deoxyglucose transport assay as a general measure
of hexose uptake. All the CDGS cells showed only a 10% de-
crease in [
3
H]2-deoxyglucose uptake except in one experi-
ment, where one CDGS cell line showed a 30% decrease (data
not shown) compared with normal cells. This makes it unlikely
that the transporters measured by [
3
H]2-deoxyglucose uptake
are responsible for the more pronounced decrease in [
3
H]man-
nose uptake by CDGS cells.
Addition of
d
-mannose corrects [
3
H]mannose incorpora-
tion into LLO precursors fractions. To determine whether in-
creased exogenous mannose normalized the incorporation
into various fractions, normal and two CDGS cell lines were
labeled in the absence and presence of 1 mM exogenous
d-mannose, and the products were analyzed as before (Table
IV). In this experiment the amount of dol-P-Man is 1.5–2-fold
less than in normal cells. This was in contrast to four previous
experiments where CDGS and normals had the same amount
of dol-P-Man. All the other fractions (II–IV) had 2–20-fold
less label than the normal, and addition of mannose produces
normal levels. These results support the idea that CDGS fibro-
blasts are limited in the availability of mannose inside the cell
for N-linked protein glycosylation.
Discussion
Type I CDGS patients show reduced N-linked glycosylation of
many proteins (1). In previous studies, fibroblasts from two
CDGS patients incorporated less [
3
H]mannose into both gly-
coproteins and LLO compared with controls (13). Based on
these results, we suggested that there was a deficiency in the
synthesis of LLO. Another possibility is that the LLO is rap-
idly degraded, but this is ruled out by the finding that the pro-
portion of free oligosaccharides is nearly the same in CDGS
and controls. We also found that cells from CDGS patients
usually make smaller LLO species (Man
4–6
GlcNAc
2
) and al-
ways incorporate less [
3
H]mannose into glycoproteins. Re-
cently, another group also showed that one CDGS cell line
synthesizes a truncated LLO and incorporates less [
3
H]man-
nose into protein (46). In some of our experiments, CDGS
cells made mostly normal-sized LLO, showing that the defect
in CDGS is not in one of the LLO mannosyl transferases. We
also found that CDGS cells usually synthesize a normal
amount of dol-P-Man, suggesting that the defect in CDGS is
not the same as in Thy E cells that invariably synthesize a spe-
cific LLO (Glc
3
Man
5
GlcNAc
2
) due to dol-P-Man synthase de-
ficiency (47).
Similar aberrant LLO synthesis and underglycosylation are
seen in cultured cells during glucose starvation (24–32). This
well-documented but unexplained phenomenon shows consid-
erable cell-type variation. Normal glycosylation is restored by
the addition of 50 mM of either glucose or mannose but not by
any other sugars or by pyruvate and glutamine (24–27). In
baby hamster kidney cells, galactose also partially reverses the
starvation effects (25). Glucose-starved Chinese hamster ovary
cells also show a drastic reduction in dol-P-Man, and in this
way they resemble dol-P-Man synthase deficiency seen in Thy
E cells (31, 47). More recently, similar effects of glucose limita-
tion have been seen in the underglycosylation of recombinant
proteins (48–51). Although the physiological basis of this phe-
nomenon remains unknown, the weight of the evidence seems
to point to a deficiency in obtaining or generating mannose for
glycoprotein synthesis.
Table III. Detailed Analysis of Fraction II from [
3
H]Mannose-labeled Control and CDGS Fibroblasts*
Con A bound Con A unbound
Cell line Total Anionic Total Man Man-6-P Man-1-P GDP-Man 1 GDP-Fuc
cpm
3
10
2
4
/mg protein
Disease control 376.4 (100.0)
‡
82.8 309.1 (100.0)
‡
70.7 60.9 49.8 127.7
CDGS 1 74.2 (20.0)
‡
29.7 84.3 (27.2)
‡
11.1 31.7 15.2 26.6
2 34.4 (9.1)
‡
13.7 46.1 (14.9)
‡
9.5 12.9 4.8 18.9
3 142.5 (37.8)
‡
54.2 129.5 (41.9)
‡
18.2 35.2 25.2 50.9
4 60.1 (15.9)
‡
24.0 100.5 (32.5)
‡
16.1 26.7 17.7 40.2
* Fraction II from the same experiment described in Table II was separated into Con A–Sepharose–bound and unbound fractions and analyzed as de-
scribed in Methods.
‡
Numbers in parentheses indicate the percentage of control which is defined as 100, and CDGS samples are presented as a per-
centage of this value.
Figure 7. Measurement of initial velocities of [
3
H]mannose uptake.
Cells in multiwell plates were labeled with 20 mCi/ml of [
3
H]mannose
for times shown in the figure. At each point, medium was removed,
and cells were washed three times with cold PBS. Cells were solubi-
lized in 0.5% SDS, and cell-associated [
3
H]mannose was normalized
to protein.

Carbohydrate-deficient Glycoprotein Syndrome 1485
Based on the similarity to the glucose starvation effect, it
was surprising that mannose, but not glucose, corrects the al-
tered glycosylation in CDGS cells, since the bulk of endoge-
nous mannose for glycoprotein synthesis is assumed to be de-
rived from glucose through PMI (Fig. 2). However, we have
already shown that this enzyme and others involved in this
pathway (hexokinase and phosphoglucose isomerase) appear
to be normal in CDGS cells (21). This may mean that glucose
is not actually the predominant source of mannose for glyco-
protein synthesis and that adding mannose to CDGS cells by-
passes the deficiency from the other pathway(s). In this regard,
it is important to note that the quantitative contribution of glu-
cose-derived mannose in mammalian glycoprotein synthesis
has not actually been established. Cells may acquire mannose
in other ways (Fig. 2). One possible source is from the plasma,
which contains z 30–50 mM mannose (52). Another is from
the degradation of pinocytosed serum glycoproteins or from
the turnover of endogenous glycoproteins and oligosaccha-
rides. Mannose released from LLO degradation and oligosac-
charide “trimming” alone could total 80% of the mannose ini-
tially incorporated into LLO. Mammalian cells are equipped
with an array of a-mannosidases to degrade many linkages and
generate free mannose (53). The absence of solid results on
the origin of mannose in higher organisms is further compli-
cated by untested assumptions and extrapolations from other
systems. For instance, although PMI is ubiquitous, the effects
of a gene deletion have not been tested in mammalian cells.
Loss of PMI in yeast is lethal (54), but the much larger meta-
bolic load and 100-fold higher specific activity in yeast than in
mammalian cells may make such a comparison unwarranted.
Since glucose and mannose can be carried by the same hex-
ose transporter, a possible explanation for decreased mannose
labeling in CDGS is a defective glucose transporter. This is un-
likely since CDGS fibroblasts were only slightly less efficient
in [
3
H]2-deoxyglucose transport compared with normal cells.
The decrease could be a secondary effect of transporter under-
glycosylation (55). The more dramatic two- to threefold de-
crease in [
3
H]mannose entry into the cells may partially ex-
plain the variable decrease in [
3
H]mannose incorporation in
CDGS cells. This observation could mean that mannose enters
the cell using a separate transporter that does not transport
[
3
H]2-deoxyglucose.
We found that CDGS cells also had considerably less
3
H-labeled intermediates, GDP-Man 1 GDP-Fuc, Man-1-P,
Man-6-P, and Man, compared with controls, and these de-
creases corresponded well to the lowered amount of LLO and
protein-bound oligosaccharides. The exception to this pattern
is dol-P-Man, which seems to be much closer to normal in CDGS
cells. The reasons for this are unknown, but dol-P-Man syn-
thase may preferentially use GDP-Man because of a lower K
m
,
or dol-P-Man may accumulate because of lack of utilization in
making larger LLOs. Alternatively, dol-P-Man might use a sep-
arate pool of GDP-Man from that used in the other reactions.
Could the glycosylation abnormalities as measured by
[
3
H]mannose labeling be explained simply by changes in the
size of the precursor pools or in the specific activities of la-
beled products? We think this is unlikely since [
3
H]mannose,
as a tracer, makes only a small quantitative contribution to oli-
gosaccharide synthesis, and this would not explain the pres-
ence of smaller size oligosaccharides in CDGS cells.
Since all of the
3
H-labeled intermediates except dol-P-Man
decreased in the same proportion and were similar to de-
creases in LLO and protein-linked oligosaccharides, it appears
that CDGS cells metabolize mannose normally. Because glu-
cose could not correct both defects, it is tempting to speculate
that CDGS cells may be defective in generating mannose from
both exogenous and endogenous sources (Fig. 2). The defect
in type I CDGS has been mapped to a region on chromosome
16p (56), but it is not clear how a single mutation can account
for underutilization of both endogenous and exogenous
sources of mannose. Regardless of the source of mannose for
glycoprotein synthesis, it is clear that mannose can correct the
defects in CDGS fibroblasts. This leads to the intriguing possi-
bility that patients may benefit from an ongoing dietary sup-
plement of mannose or mannose-containing foods. Although
very few studies have been done on mannose metabolism in
mammals, in moderate doses, mannose should be nontoxic
(57). More basic physiological analysis will be required before
we can assess the potential benefits of mannose for CDGS pa-
tients.
Table IV. Distribution of [
3
H]Mannose in Various Fractions of Control and CDGS Fibroblasts in the Presence and Absence of
Unlabeled
D-Mannose*
Cell line
2:1 CHCl
3
/MeOH
Fraction I
Water wash
Fraction II
LLO
Fraction III
Protein
Fraction IV
Total
3
H in cells
Fractions I–IV % Endo H sensitive
‡
3
H cpm 3 10
2
3
/mg protein
2 Man
Normal 170.7 (138.3)
§
1340.0 354.2 67.7 1932.6 (100.0)
i
80
CDGS 1 126.9 (100.2)
§
226.0 90.5 15.3 458.7 (23.7)
i
22
2 83.4 (61.7)
§
169.5 28.8 13.3 295.0 (15.3)
i
34
1 1 mM Man
Normal 48.2 (39.5)
§
368.8 14.8 9.8 441.6 (100.0)
i
82
CDGS 1 37.8 (32.2)
§
316.8 13.0 8.7 376.4 (85.2)
i
85
2 34.6 (30.1)
§
268.5 16.3 10.2 329.7 (74.6)
i
78
* Fibroblast cultures were labeled with 150 mCi/ml [
3
H]mannose in the presence and absence of unlabeled d-mannose for 30 min and fractionated as
described in Methods.
‡
Defined as the percentage of total TCA-precipitable
3
H released by Endo H.
§
Dol-P-Man content.
i
Numbers in parentheses
indicate the percentage of normal which is defined as 100, and CDGS samples are presented as a percentage of this value.

1486 K. Panneerselvam and H.H. Freeze
Note added in proof: A recent report (van Shaftingen, E., and
J. Jaeken, 1996. FEBS Lett. 377:318–320) showed that four
Type I CDGS patients were z 95% deficient in phosphoman-
nomutase. The authors concluded that this deficiency is proba-
bly the major cause of the disorder. However, their results do
not explain how mannose corrects the altered glycosylation in
the patients we studied. It remains to be seen whether there
are other causes of Type I CDGS.
Acknowledgments
The authors thank Dr. Ajit Varki for helpful discussions during this
work, Dr. Neil Buist for providing CDGS and control cell lines, Dr.
James Etchison for help in HPLC analysis, and Drs. Donna M. Kras-
newich and Gordon D. Holt for sharing their results before publica-
tion. We also thank Felicia Fong for technical assistance and Susan
Greaney for secretarial assistance.
This work was supported by a grant from the National Institute of
General Medical Sciences (R01 49096).
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