Glycosylation failure extends to glycoproteins in gestational diabetes mellitus: evidence from reduced α2-6 sialylation and impaired immunomodulatory activities of pregnancy-related glycodelin-A.

Abstract

Gestational diabetes mellitus (GDM) is a common metabolic disorder of pregnancy. Patients with GDM are at risk for high fetal mortality and gestational complications associated with reduced immune tolerance and abnormal carbohydrate metabolism. Glycodelin-A (GdA) is an abundant decidual glycoprotein with glycosylation-dependent immunomodulatory activities. We hypothesized that aberrant carbohydrate metabolism in GDM was associated with changes in glycosylation of GdA, leading to defective immunomodulatory activities.
GdA in the amniotic fluid from women with normal (NGdA) and GDM (DGdA) pregnancies was purified by affinity chromatography. Structural analysis of protein glycosylation was preformed by lectin-binding assay and mass spectrometry. Cytotoxicity, cell death, cytokine secretion, and GdA binding of the GdA-treated lymphocytes and natural killer (NK) cells were determined. The sialidase activity in the placental tissue from normal and GDM patients was measured.
GDM affected the glycosylation but not the protein core of GdA. Specifically, DGdA had a lower abundance of α2-6-sialylated and high-mannose glycans and a higher abundance of glycans with Sda (NeuAcα2-3[GalNAcβ1-4]Gal) epitopes compared with NGdA. DGdA had reduced immuosuppressive activities in terms of cytotoxicity on lymphocytes, inhibitory activities on interleukin (IL)-2 secretion by lymphocytes, stimulatory activities on IL-6 secretion by NK cells, and binding to these cells. Desialylation abolished the immunomodulation and binding of NGdA. Placental sialidase activity was increased in GDM patients, which may account for the reduced sialic acid content of DGdA.
Taken together, this study provides the first direct evidence for altered enzymatic glycosylation and impaired bioactivity of GdA in GDM patients.

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Glycosylation Failure Extends to Glycoproteins in
Gestational Diabetes Mellitus
Evidence From Reduced a2-6 Sialylation and Impaired
Immunomodulatory Activities of Pregnancy-Related
Glycodelin-A
Cheuk-Lun Lee,1,2,3Philip C.N. Chiu,1,3Poh-Choo Pang,4Ivan K. Chu,2Kai-Fai Lee,1,3
Riitta Koistinen,5Hannu Koistinen,5Markku Seppälä,5Howard R. Morris,4Bérangère Tissot,4
Maria Panico,4Anne Dell,4and William S.B. Yeung1,3
OBJECTIVE—Gestational diabetes mellitus (GDM) is a common
metabolic disorder of pregnancy. Patients with GDM are at risk
for high fetal mortality and gestational complications associated
with reduced immune tolerance and abnormal carbohydrate
metabolism. Glycodelin-A (GdA) is an abundant decidual glyco-
protein with glycosylation-dependent immunomodulatory activi-
ties. We hypothesized that aberrant carbohydrate metabolism in
GDM was associated with changes in glycosylation of GdA,
leading to defective immunomodulatory activities.
RESEARCH DESIGN AND METHODS—GdA in the amniotic
fluid from women with normal (NGdA) and GDM (DGdA)
pregnancies was purified by affinity chromatography. Structural
analysis of protein glycosylation was preformed by lectin-binding
assay and mass spectrometry. Cytotoxicity, cell death, cytokine
secretion, and GdA binding of the GdA-treated lymphocytes
and natural killer (NK) cells were determined. The sialidase ac-
tivity in the placental tissue from normal and GDM patients was
measured.
RESULTS—GDM affected the glycosylation but not the protein
core of GdA. Specifically, DGdA had a lower abundance of a2-6–
sialylated and high-mannose glycans and a higher abundance of
glycans with Sda (NeuAca2-3[GalNAcb1-4]Gal) epitopes com-
pared with NGdA. DGdA had reduced immuosuppressive activi-
ties in terms of cytotoxicity on lymphocytes, inhibitory activities
on interleukin (IL)-2 secretion by lymphocytes, stimulatory activ-
ities on IL-6 secretion by NK cells, and binding to these cells.
Desialylation abolished the immunomodulation and binding of
NGdA. Placental sialidase activity was increased in GDM patients,
which may account for the reduced sialic acid content of DGdA.
CONCLUSIONS—Taken together, this study provides the first
direct evidence for altered enzymatic glycosylation and impaired
bioactivity of GdA in GDM patients. Diabetes 60:909–917, 2011
G
of pregnancies in Hong Kong (3). Compared with normal
pregnancy, GDM is associated with a higher risk of preg-
nancy complications such as macrosomia, preeclampsia,
fetal mortality, placental alterations, and increased risk of
diabetes of the mother and the offspring later in life (2,4,5).
The underlying pathophysiology of GDM has been associ-
ated with dysregulated immune responses, as demonstrated
by changes in the immune cell subpopulations and the cy-
tokine profile in women with GDM (6,7).
Glycodelin-A (GdA) is an abundant secretory glycopro-
tein of the pregnancy decidua (8). It is proposed to be
involved in fetomaternal defense (9,10) through its im-
munomodulatory activities. These include induction of
apoptosis of T-cells (11), skewing of T-cell response to-
ward the Th-2 phenotype (12), and modulation of the ac-
tivities of natural killer (NK) cells (13), B-cells (14), and
dendritic cells (15). Decreased secretion of glycodelin is
associated with recurrent spontaneous abortion and un-
explained infertility (8,10).
Glycosylation is crucial to the biological activities of
glycodelins (8,10). Its importance is shown by the lack
of immunomodulatory activity in two other glycodelin
isoforms with different glycosylation, glycodelin-S and
glycodelin-C (11). Although there is no difference in the
glycodelin concentration of the maternal serum in the first
trimester (16) and of the cord serum (17) between GDM
and normal pregnancy, the glycosylation of glycodelin in
GDM pregnancies is unknown. Hyperglycemia in diabetes
causes abnormal carbohydrate metabolism and the pro-
duction of advanced glycation end products, leading to
alteration of gene expression and activities of the cellular
glycosyltransferases and glycosidases (18). Diabetic preg-
nancy has been associated with changes in the glycosyla-
tion and subsequently the biological activities of human
chorionic gonadotrophin (19) and the placental transferrin
receptor (20). However, because of the lack of advanced
methodological procedures, these studies did not provide
any detailed information of the glycan structures and the
resultant changes in biological activities.
estational diabetes mellitus (GDM) is a meta-
bolic disorder manifested as glucose intolerance
with an onset during pregnancy (1,2). It occurs
in ~7% of pregnancies in the U.S. (1) and in 14%
From the1Department of Obstetrics and Gynecology, University of Hong
Kong, Hong Kong, China; the2Department of Chemistry, University of Hong
Kong, Hong Kong, China; the3Centre for Reproduction, Development, and
Growth, University of Hong Kong, Hong Kong, China; the4Division of Mo-
lecular Biosciences, Faculty of Natural Sciences, Imperial College London,
London, U.K.; and the5Department of Clinical Chemistry, University of
Helsinki and Helsinki University Central Hospital, Helsinki, Finland.
Corresponding author: Philip C.N. Chiu, pchiucn@hkucc.hku.hk.
Received 19 August 2010 and accepted 7 December 2010.
DOI: 10.2337/db10-1186
This article contains Supplementary Data online at http://diabetes.
diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1186/-/DC1.
C.-L.L. and P.C.N.C. contributed equally to this work.
? 2011 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
diabetes.diabetesjournals.orgDIABETES, VOL. 60, MARCH 2011909
ORIGINAL ARTICLE

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We hypothesized that the aberrant carbohydrate metab-
olism in GDM was associated with alteration in glycosylation
of GdA, thereby leading to defective immunomodulatory
activities of the molecule during pregnancy. To test this
hypothesis, we compared the immunomodulatory activi-
ties of GdA from normal (NGdA) and GDM (DGdA)
pregnancy and determined the changes in their N-glycan
structures by mass spectrometric analysis. The results
showed differences in glycosylation between NGdA and
DGdA. In particular, DGdA had reduced sialylation, leading
to reduced binding to lympohocytes and therefore de-
creased immunomodulatory activity of the molecule. The re-
sults support the hypothesis that GDM-associated changes
in glycosylation alter the biological activities of GdA.
RESEARCH DESIGN AND METHODS
Normal and diabetic amniotic fluid samples. The study protocol was ap-
proved by the institutional review board of the University of Hong Kong/
Hospital Authority Hong Kong West Cluster. A total of 35 amniotic fluid
samples (20 normal and 15 GDM) were collected from women at term preg-
nancy during cesarean delivery at the Queen Mary Hospital, Hong Kong. The
diagnosis of GDM was according to the World Health Organization criteria
using a 2-h 75-g oral glucose tolerance test (OGTT), as described (3). Blood
glucose levels .7.8 mmol/L were defined as having GDM. Two women with
GDM were treated by insulin and 13 women with GDM were treated through
diet control. Maternal and infant demographic information of normal and GDM
participants are shown in Table 1. The two groups of women were similar in
gravidity, parity, age, BMI, gestational age at birth, fasting plasma glucose, and
placental weight and fetal birth weight and differed only in 2-h plasma glucose
during the OGTT. The amount of the GdA isolated from each amniotic fluid
sample was limited. Therefore, NGdA and DGdA were randomly pooled into
five groups for experimentation.
Cell culture. Human female peripheral blood was obtained from the Hong
KongRed Cross. Peripheral bloodmononuclearcells (PBMCs) were isolated by
the Ficoll-Paque PLUS method (GE Healthcare, Uppsala, Sweden). Peripheral
blood NK (pbNK) cells with a purity of .95% were isolated from PBMCs using
a negative isolation kit (Dynal Biotech, Oslo, Norway). These cells were cul-
tured in 10% FBS supplemented with RPMI 1640 medium containing 500 IU/mL
recombinant interleukin (IL)-2 (Sigma-Aldrich, St. Louis, MO).
PMBCs, Jurkat (T-lymphoma cells), and OE-E6/E7 (oviductal cell line) were
cultured in RPMI-1640 medium (Sigma-Aldrich). TEV-1 (trophoblast cell line)
was cultured in DMEM medium (Sigma-Aldrich). All the culture media were
supplemented with 10% FBS.
Determination of glycodelin concentration in amniotic fluid. The glyco-
delin content from the amniotic fluid of normal and GDM pregnancies was
assayed by enzyme-linked immunosorbent assay (ELISA). Polyclonal goat anti-
human glycodelin antibody (1 mg in 100 mL; R&D Systems, Minneapolis, MN)-
coated assay wells were incubated successively with the amniotic fluid sample
or GdA standard (0–10 mg/mL), murine monoclonal antiglycodelin antibody
(clone F43-7F9), and horseradish peroxidase–conjugated goat anti-mouse IgG
(1:5000 vol/vol; Sigma-Aldrich). The ELISA signal was determined by absorp-
tion at 450 nm using a microplate reader (MR5000; Dynatech Laboratories,
Chantilly, VA). GdA content was expressed as a ratio to the total protein
content of the amniotic fluid.
Purification of GdA. NGdA and DGdA were purified from amniotic fluid as
described (21). Desialylation of NGdA was performed using sialidase-coated
agarose beads (Sigma-Aldrich) at 37°C for 18 h. The success of desialylation
was verified by the decreased binding to wheat germ agglutinin lectin (11).
The concentration of purified GdA was estimated by a protein assay kit
(Bio-Rad, Hercules, CA). The purity of the isolated GdA was checked by 12%
SDS-PAGE, whereas the identity of the isolated GdA was confirmed by peptide
mass fingerprinting of the GdA band in SDS-PAGE using matrix-assisted laser
desorption/ionization–tandem time-of-flight mass spectrometer (4800 MALDI-
TOF/TOF; Applied Biosystems, Warrington, U.K.).
Lectin binding assay. Glycosylation of glycodelins was studied by a lectin-
binding assay as described (11). Briefly, assay wells coated with various lectins
(7.5 pmol per well) were blocked by 5% casein before successive incubation
with GdA (250 ng per well), monoclonal antiglycodelin antibody (1 mg per
well), and horseradish peroxidase–conjugated anti-mouse IgG (1:5000 vol/vol;
Sigma-Aldrich). The signal was developed with o-phenylenediamine (Sigma-
Aldrich) and determined as described above.
Glycan sequencing by MALDI-TOF and MALDI-TOF/TOF. Purified NGdA
and DGdA (~15 mg each) were digested using trypsin (Sigma-Aldrich) and
purified by a reverse-phase Sep-Pak C18 cartridge (Waters Corporation,
Manchester, U.K.) as described (22). The N-glycans were then released by
N-glycosidase F (Roche Applied Science, West Sussex, U.K.) and purified on
a Sep-Pak C18 cartridge. The purified native N-glycans were permethylated as
described (23), purified using a Sep-Pak C18 cartridge, dissolved in methanol,
and mixed with 20 mg/mL 2,5-dihydrobenzoic acid in 70% methanol at a 1:1
ratio (vol/vol). The glycan-matrix mixture (1 mL) was spotted on a stainless-
steel target plate and dried in a vacuum. MALDI-TOF and -TOF/TOF data were
obtained using a 4800 MALDI-TOF/TOF mass spectrometer (AB Sciex UK
Limited, Warrington, U.K.). Argon with a collision energy of 1 kV was used.
The mass spectrometry (MS) and tandem MS (MS/MS) data obtained were
analyzed using Data Explorer 4.9. The assignment of glycan sequence was
done by manual annotation informed by knowledge of human biosynthetic
pathways and aided by the glycobioinformatics tool, GlycoWorkBench (24).
Relative quantification of the glycan abundance in a single spectrum was
calculated relative to the total ion counts from all of the observed glycans
(percentage of total abundance = [ion count of the glycan/total ion count of all
glycans] 3 100%).
Linkage analysis by gas chromatography–mass spectrometry. The
N-glycans were analyzed by gas chromatography–mass spectrometry (GC-MS)
as described (23). The samples were dissolved in hexane before injection into
the gas chromatographer–mass spectrometer (Clarus 500; PerkinElmer, Wal-
tham, MA) fitted with an RTX-5 column (30 m 3 0.32 mm internal diameter;
Restek, Bellefonte, PA). The oven temperature was held at 90°C for 1 min and
subsequently ramped to 290°C at a rate of 8°C per min. The acquired data were
analyzed by TurboMass version 4.5.0.007 (Perkin Elmer Instruments, Shelton, CT).
TABLE 1
Maternal and infant demographic information of normal and GDM participants
Demographic informationNormal (n = 20)GDM (n = 15)
Gravidity
Parity
Maternal age (years)
Maternal BMI (kg/m2)
Gestational age at birth (weeks)
Fasting plasma glucose (mmol/L)
2-h plasma glucose (mmol/L)
Placental weight (g)
Fetal birth weight (g)
Indications for caesarean section
2.8 (1–5) 6 1.25
1.8 (1–3) 6 0.62
35.0 (28–43) 6 3.40
27.1 (22.2–31.8) 6 2.55
38.8 (37.6–41.1) 6 1.06
4.4 (3.5–5.3) 6 0.50
6.1 (5.2–7.6) 6 0.57
559.5 (430–740) 6 88.9
3,178.5 (2,165–4,295) 6 460.7
Normal (n = 20)
Pervious caesarean section scar 3 16
Fetal malpresentation 3 2
High head 3 2
2.4 (1–5) 6 1.33
1.7 (1–4) 6 0.95
35.6 (29–40) 6 3.36
27.5 (20.9–38.6) 6 5.22
38.3 (37.6–40.9) 6 0.90
4.8 (3.8–7.4) 6 0.96
9.4 (7.8–11.6) 6 1.18*
553.6 (460–700) 6 87.9
3,108.1 (2,570–3,755) 6 396.7
GDM (n = 15)
Pervious caesarean section scar 3 8
Fetal malpresentation 3 1
High head 3 1
Cervical incompetence 3 2
Cephalopelvic disproportion 3 3
Data are means (range) 6 SD. *P , 0.001 vs. normal participants.
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XTT cell viability assay. Cell viability was determined by the XTT assay
(Roche Diagnostics, Basel, Switzerland), according to the manufacturer’s
protocol. The absorbance of the resulting color product was measured at
450 nm with a l correction at 595 nm. The changes in cell viability were
expressed as the suppression index (SI) using the following equation: sup-
pression index (%) = (Abs GdA 2 Abs blank) 3 100%.
Cell death analysis by flow cytometry. Apoptotic and necrotic cell death
were determined by flow cytometry using Yo-Pro-1 and propidium iodide dye
(Invitrogen, Carlsbad, CA) and analyzed with a flow cytometer (Beckman
Coulter, Fullerton, CA) equipped with a 488-nm argon laser. The fluorescence
signal was measured using the 525-nm and 610-nm band pass filters and was
analyzed by the Winlist software (Verity Software House, Topsham, ME).
Determination of cytokine production by ELISA. The levels of IL-2 andIL-6
in the conditioned media were measured by ELISA (IL-2, BD Biosciences,
FranklinLakes,NJ;IL-6,Invitrogen,Carlsbad,CA),accordingtothemanufacturer’s
procedure. The absorbance derived from 3.39,5.59-tetramethylbenzidine was
measured at 450 nm with a l correction of 595 nm as above.
Glycodelin binding assay. The binding of GdA was visualized by flow cyto-
metric analysis. In brief, GdA was fluorescence labeled using the Alexa Fluor
488 microscale fluorescence labeling kit (Invitrogen) as described (25). The
cells (5 3 105) were incubated with 1 mg of Alexa Fluor 488–labeled GdA for
2 h, washed with PBS twice, and analyzed by flow cytometry as above.
Determination of placental sialidase activity. Human placentae were
obtained from 20 singleton pregnancies (10 normal pregnancies and 10 GDM
pregnancies) after elective cesarean section at term before the onset of labor at
the Queen Mary Hospital, Hong Kong. The tissues were dissected, washed with
PBS, and homogenized in the presence of protease inhibitors and phosphatase
inhibitor. The sialidase activity in the total cell lysates (50 mg) was determined
by a fluorimetric assay using an artificial substrate, 4-methylumbelliferyl
N-acetylneuraminic acid (Sigma-Aldrich) as described (11). Fluorescence
emission was measured by a fluorometer with excitation at 340 nm and
emission at 505 nm (Infinite F200; Tecan, Männedorf, Switzerland).
Data analysis. All values were expressed as means 6 SEM. The data were
compared by ANOVA to discern differences between groups. Parametric
Student t test or a nonparametric Mann-Whitney U test were used where ap-
propriate as the posttest. A P value ,0.05 was considered significant.
RESULTS
Purification and identification of NGdA and DGdA.
There was no significant difference in the amount of GdA
in the amniotic fluid from normal (0.90 6 0.32 mg/mg total
protein; n = 20) and GDM (0.71 6 0.56 mg/mg; n = 15)
pregnancies. Purified NGdA and DGdA had a similar mo-
lecular size in SDS-PAGE (~30 kDa, Supplementary Fig. 1)
and peptide mass fingerprinting in MS/MS (Supplementary
Fig. 1). The peptide mass fingerprints of both NGdA and
DGdA were significantly matched to the product of the
progesterone-associated endometrial protein gene (protein
score = 162 for NGdA, P , 0.001; protein score = 120 for
DGdA, P , 0.001), a gene-encoding glycodelin.
NGdA and DGdA have different lectin-binding affinities.
DGdA reacted weakly to concanavalin A (ConA), suggest-
ing a low abundance of mannose/glucose in its glycans.
DGdA also had reduced affinity to sialic acid (N-acetyl-5-
neuraminic acid) and N-acetylglucosamine (GlcNAc)-
binding lectin and wheat germ agglutinin (WGA) (Table 2).
Because NGdA and DGdA had similar affinity to succinylated
wheat germ agglutinin (S-WGA), a lectin that binds to N-
acetylglucosamine, the reduced binding affinity of DGdA to
WGA reflected a lower amount of sialylated glycans in the
molecule.
Differential glycomics between NGdA and DGdA.
Glycomics analysis was performed using strategies pre-
viously optimized for GdA characterization (11). The per-
methylated N-glycans were subjected to MALDI-MS
profiling and MALDI-MS/MS sequencing. Linkage analysis
using GC-MS was subsequently carried out on the re-
maining samples. The complete MALDI spectra of NGdA
and DGdA glycans are shown in single panels in Fig. 1 to
facilitate visual comparison. For clarity, only the most
informative molecular ions are annotated with glycan
structures in this figure. Comprehensive annotations are
shown on the magnified spectra, which are reproduced in
Supplementary Fig. 2. Because the amounts of purified
NGdA and DGdA were limited, only strong signals in the
spectra could be sequenced by MALDI-TOF/TOF MS/MS.
These components are flagged in Supplementary Fig. 2.
Combining information on the glycan compositions, struc-
ture, and linkage, the structures were assigned manually,
based on knowledge of human N-glycan biosynthetic path-
ways.
The N-glycans were highly complex, and most of the
abundant glycans were biantennary and triantennary gly-
cans. Some mass peaks of the same m/z value contained
mixtures of glycans (e.g., glycans of m/z of 1,662, 1,836,
1,866, 2,070, 2,244, 2,285, 2,489, 2,693, 2,717, 2,850, and
3,253). The glycans of both NGdA and DGdA comprised
high-mannose, hybrid, and abundant complex structures.
NGdA from term pregnancy shared the same glycosylation
pattern as that reported for GdA from midtrimester preg-
nancy (11); both had heavily sialylated glycans, Lewis
X glycans, the Sda (NeuAca2-3[GalNAcb1-4]Gal) epitope
in the high–molecular weight glycans and bisecting N-
acetylglucosamine on some of the biantennary glycans.
Some common characteristics of N-glycans in many glyco-
proteins were observed, such as lacNAc and lacdiNAc as
antenna backbones, sialylated lacNAc or lacdiNAc anten-
nae, fucosylated lacNAc or lacdiNAc, and fucosylated core
GlcNAc. Linkage analysis by GC-MS (Supplementary Table 1)
showed that DGdA contained terminal fucose, mannose,
galactose, N-acetylglucosamine and N-acetylgalactosamine,
2-linked mannose, 6-linked galactose, 3,4-linked galactose,
2,4-linked mannose, 3,6-linked mannose, 3,4,6-linked man-
nose, 4-linked GlcNAc, and 4,6-linked GlcNAc. In addi-
tion, linkage analysis gave evidence for the specific glycan
structures of GdA, such as Sda (3,4-linked galactose),
bisecting GlcNAc (3,4,6-linked mannose), core fucose (4,6-
linked GlcNAc), and terminal sialic acid (6-linked galactose).
Comparison of these DGdA linkage data with published
linkage data for NGdA (11) reveals substantially lower levels
of 6-linked galactose and 6-linked N-acetylgalactosamine in
the former compared with the latter. This is indicative of
lower levels of a2-6 sialylation in DGdA compared with
NGdA.
The relative abundances of the glycans of NGdA and
DGdA were compared (Fig. 1). Some important differences
were found: 1) Of 147 glycoforms identified, .55% were
TABLE 2
Binding of lectins with NGdA and DGdA
Lectin (specificity)
Lectin immunoassay
at OD450
NGdADGdA
Wisteria floribunda agglutinin
(GalNAc)
Sambucus nigra bark agglutinin
(2NeuNAc[2-6]Gal/GalNAc)
Concanavalin A (ConA)
(2Man, 2Glc)
WGA ([GlcNAc]2, NeuNAc)
S-WGA (GlcNAc or its
oligomer)
0.55 6 0.090.53 6 0.02
0.63 6 0.030.58 6 0.11
0.59 6 0.04
0.42 6 0.05
0.42 6 0.05*
0.04 6 0.02*
0.11 6 0.05 0.17 6 0.04
Data are means 6 SEM, n = 5. *P , 0.05 vs. NGdA at the same
concentration.
C.-L. LEE AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 60, MARCH 2011911

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found in both NGdA and DGdA (Supplementary Fig. 2). 2)
The most notable difference between the two samples was
that DGdA contained much lower levels of sialylated gly-
cans (DGdA: 24.7%; N.GdA: 53.4%). From the MALDI-MS
spectra, many of the strong peaks of NGdA shifted toward
low molecular weight by 361 mass units in DGdA, corre-
sponding to the molecular weight of permethylated sialic
acid, indicating that these glycans lacked sialic acid in
DGdA (see the orange, red, and blue panels in Fig. 1).
3) DGdA contained a lower abundance (1.9 vs. 10.3% of
NGdA) of high-mannose glycans (m/z 1,580, 1,784, and
1,988). This is consistent with the reduced ConA binding
affinity of DGdA in the lectin-binding assay. The low
abundance of high-mannose glycans suggested that they
were unlikely to occupy a glycosylation site fully, as pre-
viously described for the Asn28 site of GdS, which is ex-
clusively occupied by high-mannose glycans (10). 4) DGdA
contained more glycans capped with the Sda epitope
FIG. 1. MALDI-TOF mass spectra of N-glycans of NGdA and DGdA (m/z 1500–4000). The N-glycans from purified glycodelin preparations were
released by PNGase F and permethylated (RESEARCH DESIGN AND METHODS) prior to MALDI-TOF profiling. For ease of semiquantitative comparison,
each spectrum is shown in a single panel, and all data are normalized to the most abundant component, which is designated as 100%. For clarity,
not all molecular ions are annotated with their m/z values. The annotated signals exemplify the major differences between the NGdA and DGdA
glycomes. Color coding has been used to distinguish families of glycans. The a2-6 sialylated glycans and their desialylated counterparts are flagged
as orange, red, and blue peaks in the spectra, and their annotations are shown in the respective orange, red, and blue panels. Thus, orange
indicates glycans that are bisected and fucosylated, those in red are bisected but not fucosylated, and those in blue are neither bisected nor
fucosylated. The upper structures in each of these panels are fully sialylated, and the arrows depict loss of sialic acid. The green and magenta
inserts show high-mannose glycans and the Sda family of complex glycans, respectively. Fully annotated spectra, which have been expanded on the
m/z axis to enable all components to be visualized, are presented in Supplementary Fig. 2. (A high-quality color representation of this figure is
available in the online issue.)
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(e.g., m/z 2,717, 2,850, 2,891, 3,253, 3,300, 3,341, 3,545, and
4,267; see the magenta panel in Fig. 1), which was a re-
cently identified characteristic of the female glycodelins
(11). The smaller amount of sialylated glycans in DGdA
compared with NGdA was further confirmed by the de-
creased WGA-binding of DGdA in a second population of
normal and GDM patients (Supplementary Table 2).
Reduced cytotoxicity of DGdA and desialylated NGdA
on human lymphocytes. Treatment with both NGdA and
DGdA at concentrations of $0.01 mg/mL for 36 h signifi-
cantly (P , 0.05) decreased the viability of PBMCs, the
cytotoxicity of the latter was significantly (P , 0.05) lower
than that of the former at concentrations of 0.01 and 0.1
mg/mL (Table 3). NGdA, but not DGdA, at a concentration
of 0.1 mg/mL, significantly (P , 0.05) decreased the viability
of Jurkat cells. At 1 mg/mL, the cytotoxic effect of NGdA on
Jurkat cells was also significantly (P , 0.05) higher than
that of DGdA (viability: NGdA, 41.7 6 4.9%; DGdA, 79.4 6
8.2%). At the tested concentrations, neither NGdA nor
DGdA affected the viability of TEV-1 and OE-E6/E7 cells.
Compared with NGdA, the cytotoxic effect of desialylated
NGdA could only be observed at higher concentrations
(PBMCs: $0.1 mg/mL; Jurkat cells: $1 mg/mL). In ad-
dition, the suppression index of desialylated NGdA was
higher than that of NGdA when tested at the same con-
centrations.
DGdA and desialylated NGdA have impaired ability to
induce cell death of lymphocytes. Treatment with 0.1
mg/mL NGdA significantly (P , 0.01) decreased the viable
population of PBMCs from 87.0 6 5.7% to 26.9 6 2.9% in
the YoPro-PI assay (Table 4 and Supplementary Fig. 3).
The corresponding decrease by DGdA was significantly
(P , 0.05) smaller (from 87.0 6 5.7% to 42.4 6 15.5%),
indicating a lower cytotoxic activity of DGdA on PBMCs.
Treatment with 0.1 mg/mL NGdA for 48 h significantly
(P , 0.05) decreased the percentage of viable Jurkat cells
from 86.6 6 2.4% to 77.2 6 3.5% (Table 4 and Supplementary
Fig. 3). By contrast, DGdA at the same concentration had no
significant effect on the viability of these cells (82.9 6 1.8%).
A differential response (P , 0.05) of Jurkat cells to NGdA
and DGdA was also observed at the concentration of
1 mg/mL.
After desialylation, the ability of NGdA to induce cell
death of PBMCs and Jurkat cells was abolished (Table 4
and Supplementary Fig. 3). No significant difference was
observed on the viable population after treatment with
desialylated NGdA when compared with the control.
DGdA and desialylated NGdA have reduced ability in
modulating the cytokine secretion by lymphocytes
and NK cells. NGdA dose-dependently inhibited IL-2 se-
cretion by PBMCs and Jurkat cells (Table 5). DGdA and
desialylated NGdA had a significantly (P , 0.05) lower
suppressive effect on IL-2 secretion than that of NGdA at
the same concentration in either cell types. Neither NGdA
nor DGdA affected cell viability within the treatment pe-
riod (data not shown). For IL-6, DGdA and desialylated
NGdA had a significantly (P , 0.05) smaller stimulatory
effect on pbNKs when compared with NGdA at the con-
centration of 1 mg/mL (Table 5).
DGdA and desialylated NGdA have reduced binding
affinity to the lymphocytes. The binding affinity of
DGdA on Jurkat and pbNK cells was significantly lower
(P , 0.05) than that of NGdA (Fig. 2). The binding of
DGdA on PBMCs was also somewhat lower, though the
difference did not reach statistical significance (NGdA:
35.7 6 5.3%; DGdA: 27.6 6 6.0%). Upon desialylation, the
binding of desialylated NGdA became significantly re-
duced in all the cells tested.
Placental tissue of GDM has a higher sialidase activity.
The sialidase activity of the GDM placental tissue increased
the production of the 4-methylumbelliferone in a time-
dependent manner and was significantly (P , 0.05) higher
than that of the normal placental tissue (Fig. 3).
DISCUSSION
Changes in glycosylation of glycoproteins occur in the
normal menstrual cycle and during pregnancy (26,27).
TABLE 3
Effect of NGdA, DGdA, and desialylated NGdA on viability of PBMCs, Jurkat cells, TEV-1, and OE-E6/E7 by XTT assay
Suppression index (SI6 SEM)
Jurkat cellsPBMCsTEV-1OE-E6/E7
NGdA
GdA (mg/mL)
0.001
0.01
0.1
1
DGdA
GdA (mg/mL)
0.001
0.01
0.1
1
Desialylated NGdA
GdA (mg/mL)
0.001
0.01
0.1
1
95.0 6 1.7
67.9 6 1.4*
40.1 6 0.8*
37.1 6 0.5*
104.7 6 1.7
93.0 6 8.3
68.6 6 2.8*
41.7 6 4.9*
103.8 6 3.5
103.5 6 3.0
101.0 6 2.2
98.8 6 6.7
100.4 6 1.6
102.4 6 1.2
101.1 6 1.5
97.9 6 1.5
97.6 6 8.7
83.7 6 3.8*†
74.5 6 3.8*†
42.6 6 2.0*
100.5 6 1.3
96.6 6 3.8
102.7 6 2.4†
79.4 6 8.2*†
105.8 6 2.7
105.5 6 1.6
103.1 6 1.6
94.2 6 10.8
101.3 6 1.1
102.1 6 1.4
100.4 6 1.6
97.3 6 1.9
100.1 6 4.1
100.3 6 4.0†
81.6 6 11.6*†
53.1 6 10.0*
100.5 6 1.4
100.8 6 1.4
98.7 6 1.9†
89.8 6 3.7*†
—
—
—
—
—
—
—
—
Data are mean 6 SEM, n = 5. Cells (3 3 104) were incubated with 0.001, 0.01, 0.1, and 1 mg/mL glycodelin for 36 h, and XTT-labeling mixture
was added 12 h before measurement. Suppression index (%) = (Abs GdA 2 Abs blank) / (Abs control 2 Abs blank) 3 100%. *P , 0.05 vs.
control without treatment. †P , 0.05 vs. NGdA at the same concentration.
C.-L. LEE AND ASSOCIATES
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Altered glycosylation of glycoproteins and glycolipids oc-
cur in diabetes, cancer, AIDS, Alzheimer’s disease, and
inflammatory diseases (28,29). Two observations in this
study demonstrate for the first time changes in glycosyla-
tion of GdA in GDM. First, the binding affinities of DGdA to
ConA and WGA were lower than that of NGdA. Second,
glycomics analyses of the N-glycans revealed substantive
quantitative and qualitative differences in the glycan struc-
tures between NGdA and DGdA. The main quantitative dif-
ference is the smaller amount of a2-6 sialylated glycans
in DGdA. An interesting qualitative difference is that most
of the major sialylated glycans in NGdA appear as non-
sialylated in DGdA.
Sialic acid levels on glycoproteins are regulated by
sialyltransferases during their cellular biosynthesis and,
in some instances, by sialidase(s) after secretion from
cells. Decreases in sialyltransferase (30) and increases in
sialidase activities (30,31) as well as changes in other gly-
cosidase activities (32) have been reported in humans and
animals suffering from diabetes. These findings are in ac-
cordance with the increased free sialic acid level in the
serum of type 2 diabetes (33). Human endometrial tissues
expresses both sialidase and sialyltransferase (27,34). In
this study, placental sialidase activity is higher in GDM than
in normal pregnancy, consistent with the reported abnormal
carbohydrate metabolism in the placental-decidual unit of
GDM pregnancy (35). Therefore, it is not surprising that the
altered carbohydrate metabolism in GDM leads to the pro-
duction of GdA with reduced sialic acid content.
Sialic acid is usually the terminal monosaccharide in
human N-glycans, and it affects the conformation, binding,
and biological activities of glycoproteins (29). The relative
amount of some sialic acid–containing glycoproteins in
amniotic fluid (36) and maternal plasma (37) is elevated
during pregnancy and increases with advancing gestation.
The results of this study emphasize the role of sialylation
in pregnancy; a decrease in sialic acid content reduces the
immunomodulatory activities of DGdA. Indeed, the abun-
dance of sialic acid in different glycodelin isoforms corre-
lates with their apoptosis-inducing activity on lymphocytes
(11). Consistently, DGdA with less sialylated glycans has
reduced apoptosis-inducing activity on lymphocytes, sup-
porting the importance of sialic acid in mediating the im-
munomodulatory function of GdA.
In a normal pregnancy, selective deletion of T-cells
occurs at the fetomaternal interface throughout gestation
(38). Suppression of the response of maternal lymphocytes
to fetal alloantigen is necessary for fetal survival (39). GdA
modulates the T-cell population by inducing apoptosis
of T-cells (11) and expression of Fas in Th-1 lymphocytes
(40). The reduced ability of DGdA to induce T-cell apo-
ptosis could be, at least in part, responsible for the ob-
served increase of lymphocytes in the GDM patients (7,41).
The involvement of carbohydrate metabolism in alteration
of the T-cell population in GDM is reflected by the re-
duction of T-cells after insulin treatment of these women
(7,41).
Changes in GdA glycosylation may also lead to in-
appropriate cytokine profiles in GDM. A shift in cytokine
profile in women with GDM has been documented (6,7).
Whether this is related to an increased risk of complica-
tions in GDM remains to be investigated. Significantly,
T-cells treated with DGdA produce more IL-2 than those
treated with NGdA. Excessive production of Th-1 cyto-
kines including IL-2 would mediate rejection of the fetal
semiallograft (42). On the other hand, DGdA has impaired
TABLE 4
Effect of NGdA, DGdA, and desialylated NGdA on the cell death of PBMCs and Jurkat cells
Control
NGdA (mg/mL)
DGdA (mg/mL)
Desialylated NGdA (mg/mL)
0.01
0.1
1
0.01
0.1
1
0.01
0.1
1
PBMCs (%)
Viable
87.0 6 5.7
44.3 6 15.4*
26.9 6 2.9*
20.8 6 3.2*
57.1 6 1.3*†
42.4 6 15.5*†
23.7 6 5.4*
76.7 6 7.7
75.7 6 8.4†
71.3 6 8.3†
Necrosis
8.0 6 4.0
42.5 6 13.7*
59.9 6 1.8*
64.7 6 3.4*
25.7 6 1.4*
41.2 6 16.2*
62.0 6 5.5*
15.0 6 4.7†
15.8 6 5.6†
19.6 6 5.7†
Apoptosis
4.8 6 1.3
12.8 6 1.8*
12.9 6 1.3*
14.1 6 0.6*
16.5 6 0.3*†
15.8 6 1.6*
13.8 6 1.2*
7.4 6 2.7†
7.6 6 2.7†
8.1 6 2.5†
Jurkat cells (%)
Viable
86.6 6 2.4
77.0 6 3.3*
77.2 6 3.5*
61.9 6 11.8*
82.0 6 2.6
82.9 6 1.8†
77.0 6 4.1*†
83.9 6 1.5
83.0 6 1.4†
83.9 6 1.3†
Necrosis
6.6 6 1.8
13.2 6 2.2*
16.7 6 2.8*
34.9 6 11.5*
10.2 6 0.9*
9.2 6 0.7
15.8 6 2.4*†
9.7 6 1.2*
10.1 6 0.5*
9.0 6 1.2†
Apoptosis
4.2 6 1.5
8.7 6 1.2*
5.5 6 0.8
2.7 6 0.7
7.1 6 1.8
7.2 6 1.2*
6.6 6 1.9†
6.3 6 0.4
6.8 6 1.1
7.0 6 1.0*†
Data are means 6 SEM, n = 5. PBMC and Jurkat cells (5 3 105) were incubated with 0–1 mg/mL glycodelins for 48 h. Viable, necrotic, and apoptotic cells were identified and quantified by
bivariate Yo-Pro-1/PI flow cytometry. Cells without stain were counted as viable cells. Cells labeled with Yo-Pro-1 only were counted as apoptotic cells. Cells labeled with both Yo-Pro-1 and
PI was counted as necrotic cells. *P , 0.05 vs. control without treatment. †P , 0.05 vs. NGdA at the same concentration.
GLYCODELIN-A GLYCOSYLATION IN GDM
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Page 7

stimulatory effect on IL-6 secretion by pbNK cells. IL-6 has
a wide range of biological activities, including stimulation
of trophoblast invasion (43) and hCG production (44). In-
adequate IL-6 concentration in the placenta and endome-
trium has been associated with fetal growth restriction and
recurrent miscarriage (45,46).
Both poorly sialylated DGdA and desialylated NGdA
have impaired binding affinities to lymphocytes and pbNK
cells. Sialic acid receptors, such as sialic acid–binding
immunoglobulin-like lectin receptor, on leukocytes (29)
have been proposed to mediate the action of glycodelin on
B-cells (47). Consistently, the reported receptors of glyco-
delin isoforms on spermatozoa (10) and lymphocytes (48)
are known to bind sialic acid–containing epitopes. The
identity of the receptor(s) mediating the action of GdA on
lymphocytes and pbNK cells remains unknown.
DGdA has proportionally more sialylated glycans with
the Sda epitope (NeuAca2–3[GalNAcb1-4]Gal) and less
high-mannose glycans compared with NGdA. Two obser-
vations suggest that these changes may not be related to
the change in immunomodulatory activities of DGdA. First,
two other glycodelin isoforms, namely glycodelin-F and
FIG. 2. The binding of NGdA, DGdA, and desialylated NGdA to PBMCs, Jurkat cells, and pbNK. PBMCs, Jurkat cells, and pbNK (1 3 106) were
incubated with 1 mg/mL fluorescence-labeled mouse IgG (black), NGdA (red), DGdA (green), and desialylated NGdA (blue) for 2 h. GdA-bound
cells were quantified by flow cytometry. Data are means 6 SEM, n = 5. †P < 0.05 when compared with the NGdA at the same concentration. The
results shown are representative of five replicate experiments.
TABLE 5
Effect of NGdA, DGdA, and desialylated NGdA on IL-2 secretion of PBMCs and Jurkat cells and IL-6 secretion of pbNKs
IL-2 (pg/mL)
IL-6 (pg/mL)
pbNK PBMCsJurkat cells
Control
GdA (mg/mL)
0
NGdA
GdA (mg/mL)
0.01
0.1
1
DGdA
GdA (mg/mL)
0.01
0.1
1
Desialylated NGdA
GdA (mg/mL)
0.01
0.1
1
1,045.2 6 53.5 1,117.5 6 138.330.1 6 0.3
549.3 6 42.5*
343.5 6 113.9*
267.9 6 87.8*
389.3 6 130.6*
390.1 6 59.6*
351.8 6 99.8*
27.0 6 0.8*
224.6 6 130.7*
999.8 6 294.7*
1,012.7 6 35.5†
777.3 6 108.6*†
500.0 6 160.3*†
727.6 6 78.3*†
567.2 6 62.1*†
435.7 6 93.5*
52.3 6 19.2
262.9 6 104.1*
287.1 6 113.0*†
1,037.6 6 119.8†
933.8 6 88.9†
875.0 6 110.5*†
1,123.8 6 120.3†
949.1 6 74.9†
981.4 6 78.3†
76.6 6 41.7
264.8 6 142.4
596.9 6 139.0*†
Data are means 6 SEM, n = 5. PBMCs (1 3 106) primed by PHA (5 mg/mL) and Jurkat cells were incubated with 0–1 mg/mL glycodelins for
16 h. pbNK cells (1 3 106) were incubated with 0–1 mg/mL glycodelins for 14 h. IL-2 and IL-6 secretions were quantified by ELISA. *P , 0.05
vs. the control without treatment. †P , 0.05 vs. NGdA at the same concentration.
C.-L. LEE AND ASSOCIATES
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Page 8

glycodelin-C, also carry the Sda epitopes, but only the
former has immunomodulatory activity (11). Second, an-
other glycodelin isoform, glycodelin-S, contains more high-
mannose glycans than GdA (10), but glycodelin-S is not
immunosuppressive (11). Additional investigation is re-
quired to understand the biological implication of these
glycosylation changes.
In conclusion, this study provides the first direct evi-
dence that changes in the glycosylation of decidual gly-
coprotein GdA is associated with defective binding and
immunomodulatory activities of this molecule. These dis-
coveries may give a new lead to the study of protein gly-
cosylation in the pathophysiology of GDM. It is possible
that the changes in GdA glycosylation in GDM are related
to increased placental sialidase activity and, therefore, are
not applicable to all types of diabetes. It also remains to be
seen whether the changes described herein have any
connections with fetal complications or have clinical
consequences of altered immune cell reactivity. In type 2
diabetes, an increase in serum sialic acid levels is an in-
dication of the loss of sialylation from circulatory and
membrane glycoproteins (33). Approaches aimed at fixing
the glycosylation changes may help to alleviate some of
the complications associated with GDM. In this connec-
tion, the glycosidase inhibitor miglitol used to treat type 2
diabetes (49) has been shown to modify the N-linked gly-
cosylation of secretory glycoproteins (50). The application
of the MS-based glycomics strategies described herein will
also open a new avenue for understanding the association
of structural and protein-specific glycosylation in diabetes
and its associated pathological conditions.
ACKNOWLEDGMENTS
P.-C.P., H.R.M., B.T., M.P., and A.D. were supported by the
Biotechnology and Biological Sciences Research Council
(BBF0083091), and R.K., H.K., and M.S. were supported by
grants from the Helsinki University Central Hospital Re-
search Fund and the Academy of Finland.
No potential conflicts of interest relevant to this article
were reported.
C.-L.L., P.C.N.C., and P.-C.P. researched data; contrib-
uted to discussion; and wrote, reviewed, and edited the
manuscript. I.K.C. reviewed and edited the manuscript.
K.-F.L. contributed to discussion and reviewed and edited
the manuscript. R.K. and H.K. reviewed and edited the
manuscript. M.S. researched data, contributed to discus-
sion, and reviewed and edited the manuscript. H.R.M.
contributed to discussion and reviewed and edited the
manuscript. B.T. researched data. M.P. researched data,
contributed to discussion, and reviewed and edited the
manuscript. A.D. researched data; contributed to discussion;
and wrote, reviewed, and edited the manuscript. W.S.B.Y.
contributed to discussion and reviewed and edited the
manuscript.
The authors thank Prof. Terence Lao, Dr. Maggie Cheng,
and Dr. Noel Shek and colleagues of the obstetrics team
of the Department of Obstetrics and Gynaecology, The
University of Hong Kong, for the collection of the amniotic
fluid samples.
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