From the Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California 94143-0556
Received for publication, November 20, 2000, and in revised form, February 6, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human glycodelin is synthesized by
endometrial cells in the late secretory phase and early pregnancy under
hormonal regulation. Whereas the precise physiological functions of
glycodelin are unknown, its expression during embryonic nidation and
its inhibition of T cell proliferation suggest an immunomodulatory
role. We purified human glycodelin from first trimester human decidual
cytosol by using a rapid two-step high-performance liquid
chromatography method and investigated its effects on human monocyte
migration. Human U937 cells were used as a model of monocyte chemotaxis
in Boyden chamber migration assays. N-Formyl-Met-Leu-Phe
and the The primary function of the human endometrium is to support
successful blastocyst implantation. In our species and in other higher
primates, placentation is hemochorial, i.e. the endometrial stroma is penetrated by invasive cytotrophoblast cells that establish direct contact with the maternal blood (1). The intermixing of
semiallogeneic cells at the maternal-embryonic interface creates a
fundamental immunological paradox. What prevents embryonic rejection within the immunocompetent mother? This question was first posed formally by Sir Peter Medawar (2), who postulated that cytotrophoblasts evaded recognition by cytotoxic maternal immune cells by lacking HLA
class I surface proteins. This idea has since been modified following
the discovery of monomorphic nonclassical HLA class I molecules
expressed on the surface of human (HLA-G) (3) and rhesus (Mamu-I) (4)
trophoblasts. It is now postulated that the immunotolerizing effect of
human trophoblast HLA-G may be further enhanced by extensive
polylactosamine units added co-translationally to the nascent protein
(5).
Other heavily glycosylated proteins at the human maternal-embryonic
interface, the expression of which temporally coincides with blastocyst
implantation, also are of interest: Glycodelin, a member of the lipocalin family of proteins, is
essentially undetectable in proliferative phase endometrium and is
dramatically up-regulated during the secretory phase of the ovulatory
cycle and in early pregnancy (14). Progesterone (15, 16), relaxin (17,
18), and chorionic gonadotropin (19) have been proposed as the major
physiological stimuli of glycodelin synthesis and secretion by the
primate endometrium. Low glycodelin concentrations were observed in
secretory phase uterine flushings from women with a history of
recurrent miscarriages (20). Although functional studies are limited,
preliminary data indicate that glycodelin has immunosuppressive effects
that are postulated to facilitate the embryonic evasion of the maternal
immune response (21). The objective of the current study was to
investigate the hypothesis that glycodelin could inhibit immune cell
migration and to assess the importance of its sugar moieties in this process.
Tissue Collection--
First trimester human maternal decidual
tissues were separated from fetal placenta tissues by "floating" in
cold saline, washed several times, and processed immediately. The
tissues were minced into small pieces, resuspended in cold 10 mM Tris, pH 7.2, and homogenized with a Potter apparatus
(Thomas Scientific, Swedesboro, NJ) at a concentration of 500 mg of
tissue, wet weight/ml of buffer. The resulting homogenate was
centrifuged at 2,000 × g for 20 min, and the
supernatant was centrifuged at 80,000 × g at 4 °C
for 60 min in a 60-Ti rotor of a Beckman ultracentrifuge
(Beckman Instruments). The final high speed supernatant (protein
concentration, 10 mg/ml) was used to conduct the purification, as noted
below. Protein concentration was determined according to the BCA method (Pierce).
Cells--
U937 and Ishikawa cell lines were obtained from the
Cell Culture Facility at the University of California, San Francisco. U937 cells were treated with 1 mM 8-bromo-cAMP for 48 h to induce a monocyte phenotype and up-regulate oligopeptide
chemoattractant receptors (22).
Protein Purification--
Glycodelin was purified from human
decidual tissue homogenates by using a modification of the procedures
described by Westwood et al. (23) and Vigne et
al. (24). A Protein-Pak Q 8HR chromatography column (10 × 100 mm) (Waters, Milford, MA) was preequilibrated with 20 mM Tris, pH 7.2, and 10 ml of high speed supernatant were loaded onto the column at a flow rate of 0.5 ml/min. After loading, the
column was washed with 10 bed volumes of equilibration buffer, and the
retained proteins were eluted by linearly increasing the salt
concentration to 0.5 M NaCl.
Pooled fractions containing the proteins of interest were acidified
with diluted trifluoroacetic acid, centrifuged in a Micro16 apparatus (Fisher, Pittsburgh, PA) to remove denatured material, and
then loaded onto a Vydac C-18 reverse phase column (4.6 × 250 mm)
(Vydac, Hesperia, CA) equilibrated with 2% acetonitrile and 0.1%
trifluoroacetic acid in water (Solvent A). Retained proteins were
eluted from the column with a linear gradient of Solvent A and Solvent
B (90% acetonitrile and 0.1% trifluoroacetic acid in water) to a
final concentration of 80% acetonitrile.
Gel Electrophoresis and NH2-terminal Sequence
Analysis--
Purified proteins were analyzed by SDS-polyacrylamide
gel electrophoresis. In some experiments, gels were stained using
Coomassie Brilliant Blue R-250. In other experiments, proteins were
transferred to polyvinylidene difluoride membrane for protein
sequencing, 0.2 µm (Bio-Rad) in the presence of 10 mM CAPS1
buffer, pH 11, and 10% methanol.
Blotted proteins were stained with 0.1% Coomassie Brilliant Blue R-250
in 50% methanol and 1% acetic acid and then destained in 50%
methanol. Peptide bands were excised from the polyvinylidene difluoride
membrane and directly microsequenced on a vapor phase Beckman-Porton
PI2090 Sequencer (Beckman Instruments) by using the Edman degradation
procedure. Sequence homologies were searched in the PIR/Swiss
Protein Database.
Deglycosylation of Glycodelin--
N-Linked
oligosaccharides were released from the purified glycodelin by
digestion with PNGase (1 unit/250 µg of glycodelin) in ammonium
bicarbonate buffer (50 mM, pH 8.4) containing 2% Triton X-100 for 16 h at 37 °C. The reaction was terminated by
lyophilization, and the products were purified by reverse phase
chromatography on a C-18 Vydac column.
Preparation of Cell Membranes--
U937 cells were washed twice
with ice-cold phosphate-buffered saline (PBS) and then centrifuged and
resuspended in Buffer A (200 mM glycine, 150 mM
NaCl, 50 mM EGTA, 50 mM EDTA, and 300 mM sucrose, pH 9.0, containing protease inhibitors) at the
concentration of 150 × 106 cells/ml. The cell
suspension was sonicated twice with 8-s bursts, and the cell lysate was
centrifuged for 10 min at 4 °C for 10 min. The resulting supernatant
was centrifuged at 100,000 × g for 2 h at
4 °C. The pellet was resuspended in 25 mM Tris and 10 mM MgCl2, pH 7.4, containing protease
inhibitors by using a Dounce homogenizer. Five-hundred-µl aliquots
were frozen immediately at Protein Labeling and Binding Studies--
Pure glycodelin (4 µg) was iodinated by using the chloramine-T method to a specific
activity ranging between 100 and 200 µCi/µg. 125I-Glycodelin was separated from free iodine by gel
permeation chromatography using a Sephadex G-75 column equilibrated in
phosphate-buffered saline containing 0.1% bovine serum albumin
(PBS/BSA). Binding assays were performed using U937 cells in 12-well
plates. Each well contained 500,000 cells/600 µl of PBS/BSA. Cells
were incubated for 2 h at 37 °C with
125I-glycodelin in the presence of increasing
concentrations of unlabeled glycodelin. Cells were then transferred to
prebaked glass tubes and pelleted by centrifugation to separate the
bound from the unbound radioactive glycodelin. Cell pellets were washed
twice with 3 ml of PBS/BSA, and bound radioligand was measured using a
Cross-linking Experiments--
Two hundred µg of U937 cell
membranes were suspended in 300 µl of buffer (100 mM
Tris, MgCl2, pH 8.3), and labeled glycodelin was added at a
final concentration of 300 nM in the presence or absence of
30 µM purified glycodelin and incubated for 5 h at room temperature. After incubation, 3 ml of phosphate-buffered saline
were added, and membranes were pelleted by centrifugation and
resuspended in 300 µl of PBS. The cross-linking reagent,
bis(sulfosuccinimidyl)suberate (Pierce), was added at the final
concentration of 2 mM. 125I-Glycodelin and
membranes were incubated on ice for 2 h, and the reaction was
stopped by the addition of 1 M Tris, pH 7.5, to achieve a
final concentration of 20 mM. The reaction was continued for another 30 min. After cross-linking, the membranes were diluted with cold buffer PBS/BSA and sedimented by centrifugation at 4000 × g for 30 min. Membranes were washed one more time with
PBS/BSA and dissolved in Triton X-100/SDS sample buffer before they
were subjected to SDS-polyacrylamide gel electrophoresis. Radiolabeled proteins were identified by direct autoradiography with Kodak X-Omat film (Eastman Kodak Co.).
Monocyte Chemotaxis Assay--
Glycodelin bioactivity was
assessed by a chemotaxis assay using a human histiocytic cell line
(U937). To induce monocytic differentiation, U937 cells were first
activated by cAMP. For the measurements, 24-well plates assembled with
Boyden chambers (0.4-µm pore size polycarbonate membranes) were used.
Samples (600 µl) containing the chemotactic peptide
N-formyl-methionyl-leucyl-phenylalanine (0.1 µM fMLP) (Sigma) or RANTES (30-60 nM)
(R & D, Minneapolis, MN) in PBS/BSA were placed in the
lower chamber as positive controls for cell migration. Previous studies
have shown these concentrations to provide optimal monocyte chemotaxis
(25). Purified glycodelin was added to the lower chamber to test its
activity. After assembly of the Boyden chambers, 200-µl suspensions
of U937 cells (106 cells) in PBS/BSA were placed in the
upper chamber. The chambers were then incubated in a humidified
CO2 incubator at 37 °C for 90 min. Nonmigrating cells
were removed by washes with PBS, and cells migrating across the
membrane were fixed, stained, and counted directly or estimated by
photometric absorption at 450 nm. In some experiments, purified
glycodelin samples were preincubated with high affinity, anti-human
glycodelin rabbit IgG (provided by Dr. Markkü
Seppälä) or nonspecific rabbit IgG, and the chemotactic activity was measured as described above. The results were expressed as
the percentage of chemotaxis obtained in response to a maximal stimulation with the fMLP chemoattractant alone.
Statistical Analyses--
Results are presented as means ± S.E. of independent experiments.
Purification of Glycodelin in a Two-step Procedure--
Several
different methods have been described for the purification of
glycodelin (23, 26). However, each of these is long and complex. We
designed a two-step protocol that can be achieved in a day with a yield
of hundreds of micrograms of purified glycodelin from 1.25 g of
human decidual tissue. In a typical experiment, 10 ml of decidual
homogenate were loaded onto a Protein-Pak Q-8HR column equilibrated in
20 mM Tris, pH 7.2. Nonretained proteins were eluted by
washing the column with the same equilibration buffer. Retained
proteins were eluted by a linear gradient of NaCl. Fig.
1 shows the profile of total proteins
eluted from the anion exchange column and detected by absorption at 280 nm. Glycodelin concentrations in each fraction were monitored by
SDS-polyacrylamide gel electrophoresis (inset). Verification
of the 27-kDa glycodelin band was confirmed by NH2-terminal
sequencing (Table I) and Western blotting
analysis (15). About 50% of the total glycodelin eluted from the anion
exchange column in fraction 18, which corresponded to a concentration
of 125 mM NaCl. In this fraction, albumin was not detected
and neither was the putative dimeric form of glycodelin previously
reported by Westwood et al. (23). However, in the subsequent
fractions, we detected the presence of albumin as a major 68-kDa
protein co-eluting with glycodelin. The identification of albumin was
evidenced by amino acid sequencing (Table I).
Fig. 2 shows the elution profile obtained
when fraction 18 from the anion exchange column was further separated
by reverse phase chromatography. An SDS-polyacrylamide gel of the
eluted fractions is also shown (inset). Fraction 55 off the
latter column yielded a single homogeneous band migrating at about 27 kDa, which has been identified by NH2-terminal sequencing
analysis as bona fide glycodelin.
Production of Deglycosylated Glycodelin--
To assess the
function of the complex glycan decoration of the glycodelin protein, we
undertook a series of experiments to remove these sugar moieties.
Digests of glycodelin by PNGase were analyzed by SDS-polyacrylamide gel
electrophoresis. Fig. 3A shows the products of digestion. After glycosidase digestion, we observed a
shift in the molecular mass of glycodelin from 27 kDa (lane 1) to 18 kDa (lane 3), predicted by the glycodelin
cDNA sequence (27). Gentler conditions yielded a major band at 18 kDa and two minor bands at 27 and 22 kDa (lane 2). The
higher molecular mass band represents the undigested form of glycodelin
with two N-glycosylated sites, one at Asn-28 and another at
Asn-63. The lowest molecular mass band represents the fully
deglycosylated form. The 22-kDa band represents glycodelin with only a
single glycosylated site. The precise structure of the intermediate
species has not yet been established. Immediately after digestion, the deglycosylated form was separated from the glycosylated forms by
reverse phase chromatography on a C-18 Vydac column (Fig.
3B). The purified deglycosylated product eluted in fraction
36 and is shown by SDS-polyacrylamide gel electrophoresis in the
inset of Fig. 3B.
Effect of Purified Glycosylated and Deglycosylated Glycodelin on
the Migration of U937 Cells--
The biological activity of glycodelin
was evaluated by measuring its effect on the migration of activated
U937 cells exposed to a chemotactic stimulus of fMLP, which previous
studies demonstrated attracts human monocytes (25). As shown in Fig.
4, purified glycodelin inhibits the
migration of U937 in a dose-responsive manner with a calculated
IC50 of 550 nM. This may be an overestimate because it is based upon the assumption that all the purified glycodelin retains its bioactivity. That the chemorepellent activity is
specific was demonstrated by the elimination of this effect when the
purified protein was heat-inactivated or preincubated with specific
anti-glycodelin IgG. When glycodelin was preincubated with nonspecific
IgG, it retained almost all of its inhibitory activity. Furthermore,
this effect is specific because other proteins tested (ovalbumin, human
placenta lactogen, and bovine serum albumin) had no inhibitory activity
on monocyte migration at a concentration of 25 µg/ml. By contrast,
the deglycosylated form of glycodelin expressed the same biopotency as
the intact glycoprotein.
Binding of 125I-Glycodelin to U937 Cells
and Membranes--
To substantiate this activity, we conducted binding
experiments to verify if radiolabeled glycodelin was able to bind to
the surface of U937 cells in a specific manner. Fig.
5 shows the results of a representative
competitive binding experiment using 125I-glycodelin and
unlabeled purified glycodelin bound to the surface of U937 cells. We
observed that the binding is saturable, and Scatchard analyses
estimated a Kd of 48 ± 21 nM from three independent experiments. We were unable to
demonstrate the binding of labeled glycodelin to a human endometrial
cell line (Ishikawa cells), demonstrating the specificity of glycodelin receptor expression on the surface of monocytes. Scatchard analyses of
125I-glycodelin binding to cell-free U937 plasma membranes
yielded a Kd of 121 ± 65 nM (data
not shown).
Cross-linking Experiments--
U937 cell membranes were incubated
with 125I-glycodelin in the presence or absence of
unlabeled glycodelin. The labeled glycodelin was cross-linked to its
receptor by treatment with bis(sulfosuccinimidyl)suberate, and the
cross-linked complexes were solubilized in SDS sample buffer and
analyzed by electrophoresis on a 7.5% acrylamide gel in the presence
of 0.1% SDS. Fig. 6 shows high molecular
weight cross-linked complexes measured by SDS-polyacrylamide
electrophoresis. The incubation of 125I-glycodelin in the
presence of a 100-fold excess of unlabeled glycodelin reduced
accumulation of the labeled complexes, indicating ligand-dependent specificity. We have calculated the
size of the complexes to be ~250 kDa. Ishikawa cell membranes failed
to display specific 125I-glycodelin cross-linking.
Metabolic labeling studies from our group (15) and others (23, 26,
28) established that glycodelin is the predominant uterine protein
produced by secretory phase endometrial epithelial cells and expressed
in early pregnancy endometrial glands. The evidence supports a role for
progestational regulation of glycodelin mRNA and protein expression
(15, 16) in isolated endometrial cells; however, this observation has
not been universally confirmed (29). Furthermore, the role of relaxin
in the secretion and synthesis of glycodelin also appears to be
controversial (16, 17, 18).
Equally unclear is the functional role of this abundant
secretory product. Based upon its expression at the maternal-embryonic interface and preliminary evidence of immunomodulatory activity in vitro (13, 21, 30), glycodelin has been postulated to serve as an inhibitor of the maternal immune response to a
semiallogeneic embryo. Because glycodelin has been observed to
interfere with sperm-oocyte binding, some authors have attributed the
immunosuppressive activity of glycodelin to its complex
GalNAc In the present study, we report a modified method for the purification
of glycodelin from human decidua. This two-step method is simple and
rapid with a purification factor of 100 and a yield of about 15%. The
highly purified material exhibits a unique band of 27 kDa following
SDS-polyacrylamide gel electrophoresis analysis, which was identified
throughout all the purification steps as a monomeric form of
glycodelin. This result differs from that published by Westwood
et al. (23) in which a putative dimeric ~60-kDa form of
glycodelin was observed. Our experiments failed to reveal such a
dimeric form; however, serum albumin (68 kDa) was noted to be a
prominent contaminant in the first step of the purification.
Our studies indicate that glycodelin purified from human
decidual tissue can inhibit the chemotaxis of U937 cells in a Boyden chamber model. The data demonstrate that the ability of glycodelin to
inhibit monocyte migration is not dependent upon its glycan moieties.
Deglycosylated glycodelin was equally efficacious in the inhibition of
U937 chemotaxis. This finding is consistent with the observations of
Rachmilewitz et al. (21), who showed that recombinant
glycodelin synthesized in Escherichia coli (and hence, not
expressing eukaryotic posttranslational modifications) possessed
inhibitory effects on T lymphocyte proliferation in response to lectin
stimulation. Whereas synthetic sugars might be useful in the mapping
and ultimate inhibition of sperm-oocyte interactions (31), this
strategy is not likely to be successful for the interference of
glycodelin actions on immune cell function.
We detected a saturable, high affinity (Kd
~50-120 nM) receptor for 125I-glycodelin on
U937 cells and purified plasma membranes. Miller et al. (32)
identified a glycodelin receptor of similar affinity (Kd ~7-28 nM) on circulating human
monocytes. Our preliminary analysis of the molecular mass of the
cross-linked glycodelin-receptor complex suggests that it is ~250
kDa, but this may reflect the sum mass of multiple proteins involved in
the complex.
The apparent IC50 of the monocyte
chemorepellent effect of glycodelin in our experiments was 550 nM. According to the Cheng-Prusoff equation
(Ki = IC50/(1 + [free
ligand]/Kd)), the inhibition constant
(Ki) for glycodelin is calculated to be 220 nM. This concentration is 6-fold higher than the peak circulating level of glycodelin in maternal blood during the first trimester of pregnancy (14) but is very likely to be attainable locally
within the secretory endometrium and early pregnancy decidua. A similar
IC50 of 200 nM was observed by Rachmilewitz
et al. (21) for glycodelin inhibition of T cell
proliferation. The physiological implication of these calculations is
that the inhibition of the maternal monocyte function in the peripheral
circulation would not be anticipated, even during peak glycodelin
biosynthesis. However, the local inhibition of monocyte migration into
the endometrium would be predicted during the critical period of
embryonic implantation. Teleologically such a mechanism preserves the
normal systemic immunocompetence of the mother while providing local
endometrial immunosuppression and tolerance of the semiallogeneic conceptus.
It is of interest that glycodelin inhibits U937 migration in response
to fMLP, recombinant human RANTES, or human peritoneal fluid.2 Hence, it appears
that this endometrial protein can modulate monocyte chemotaxis
independent of the specific chemotactic stimulus. It is conceivable
that its site of action is in a pathway shared by each of these
stimuli. Alternatively, glycodelin may activate an independent
chemorepellent pathway unrelated to the chemotactic pathways that
mediate monocyte migration. Further characterization of the glycodelin
receptor and postreceptor pathways are the foci of ongoing
investigations in our laboratory.
-chemokine RANTES (regulated on activation normal T cell
expressed and secreted) were used as monocyte chemoattractants.
Purified glycodelin inhibited monocyte migration in a
dose-dependent fashion (IC50 = 550 nM). Glycodelin activity was totally reversed by heat inactivation (95 °C × 15 min) and neutralized by pretreatment with specific anti-glycodelin antibodies. Deglycosylated glycodelin was
equipotent to intact glycodelin in the monocyte migration assay.
125I-Glycodelin binding to whole U937 cells revealed
a single, saturable site with a Kd = 48 ± 21 nM by Scatchard analysis. Cross-linking studies indicated
that glycodelin binds to a high molecular mass (~250 kDa)
protein complex at the monocyte cell surface. Our findings support the
hypothesis that glycodelin reduces the local maternal inflammatory
response toward the implantation of a semiallogeneic conceptus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
integrin (6), mucin-1 (7), leukemia inhibitory factor (8), and glycodelin (9). Recent studies suggest that glycosylation plays an
important role in the activities of these classes of molecules (10-13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C and stored for later use.
counter (Packard Instrument Co.). Competition studies were performed similarly using Ishikawa cells as a negative control. Data
were analyzed and Scatchard plots were generated using the PRISM for
Windows program.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (113K):
[in a new window]
Fig. 1.
Anion exchange chromatography of decidual
cytosol. Ten mg of total protein were injected onto a Protein-Pak
Q 8HR column from Waters Corporation equilibrated with 20 mM Tris, pH 7.2. The retained proteins were eluted with a
linear gradient of NaCl. Three-ml fractions were collected, and
aliquots of the fractions were immediately subjected to SDS gel
electrophoresis (inset). The asterisk indicates
the fraction used for further purification. The major protein bands
represent glycodelin (27 kDa) and albumin (68 kDa).
Amino acid sequences of the major endometrial proteins eluted during
chromatographic purification and their database matches
View larger version (111K):
[in a new window]
Fig. 2.
HPLC C-18 reverse phase chromatography
of protein Pak Q 8HR fraction 18. One mg
of total protein from fraction 18 was injected into a Waters HPLC
system using a reverse phase Vydac C-18 column equilibrated with 2%
acetonitrile and 0.1% trifluoroacetic acid in water. Retained proteins
were eluted from the column by increasing the acetonitrile
concentration. The solvent was immediately removed by vacuum, and
proteins were resuspended in phosphate-buffered saline. Aliquots from
peak fractions were subjected to SDS gel electrophoresis
(inset). The asterisk indicates fraction 55 containing the purified glycodelin (27 kDa).
View larger version (74K):
[in a new window]
Fig. 3.
Deglycosylation of purified glycodelin.
A, SDS-polyacrylamide gel electrophoresis analysis.
N-Linked oligosaccharides were released by the digestion of
purified glycodelin (lane 1) with polypeptide glycosidase
(PNGase, 1 unit) in ammonium bicarbonate buffer (pH 8.4) containing 2%
Triton X-100 for 16 h at 37 °C (lane 2). Purified
deglycosylated glycodelin is shown after 24 h of PNGase digestion
(lane 3). B, HPLC C-18 reverse phase
chromatography of digestion products obtained from purified glycodelin
treated with PNGase. Peak fractions (34-37) were subjected
to SDS-polyacrylamide gel electrophoresis analysis (inset).
Asterisk indicates fraction 36 containing purified
deglycosylated glycodelin.
View larger version (37K):
[in a new window]
Fig. 4.
Inhibition of monocyte chemotaxis by
glycodelin. In these experiments, data were normalized to U937
chemotaxis in response to 0.1 µM fMLP in PBS/BSA, which
was used to stimulate cell migration (100%). When partially purified
glycodelin (Protein Pak Q 8HR fraction 18) was added to the lower
chamber at three different concentrations (6.25, 12.5, and 25 µg/ml),
cell migration was inhibited in a dose-dependent manner.
Purified glycodelin (25 µg/ml) and purified deglycosylated glycodelin
(25 µg/ml) caused an almost complete inhibition of cell migration.
Heat inactivation (95 °C × 15 min) or preincubation with
specific, anti-glycodelin IgG neutralized the inhibitory activity of
glycodelin in the migration assay, whereas nonspecific IgG failed to
neutralize the inhibitory effect. Ovalbumin, human placental lactogen,
and bovine serum albumin (25 µg/ml) had no inhibitory effects on
monocyte chemotaxis (data not shown).
View larger version (12K):
[in a new window]
Fig. 5.
Saturation ligand binding analysis. U937
cells were incubated in the presence of 125I-glycodelin and
increasing amounts of unlabeled glycodelin. Binding of
125I-glycodelin to whole cells demonstrated specific,
saturable receptors on the surface of U937 cells with an estimated
Kd of 38 nM. Specific binding is
indicated in counts/min.
View larger version (82K):
[in a new window]
Fig. 6.
Receptor cross-linking experiments.
125I-Glycodelin (lane 3) was incubated with
membranes prepared from activated U937 cells in the presence
(lane 2) or absence (lane 1) of unlabeled
glycodelin. The glycodelin was cross-linked to its receptor by
treatment with bis(sulfosuccinimidyl)suberate, which was added at a
final concentration of 2 mM. The cross-linked complexes
were solubilized in SDS sample buffer and analyzed by electrophoresis
on a 7.5% polyacrylamide gel containing 0.1% SDS.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-4GlcNAc glycosylation pattern (13).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Professor Markku Seppälä (University of Helsinki) for providing anti-human glycodelin antibodies and Cynthia Voytek for assistance with the illustrations.
![]() |
FOOTNOTES |
---|
* This work was supported by the Philippe Foundation (Paris, France) and a National Institutes of Health grant from the NICHD, through cooperative agreement U54-HD37321, as part of the Specialized Cooperative Centers Program in Reproduction Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Director, Center for
Reproductive Sciences, HSE 1689, Dept. of Obstetrics, Gynecology and
Reproductive Sciences, University of California, San Francisco, CA
94143-0556. Tel.: 415-476-9214; Fax: 415-753-3271; E-mail: rtaylor@socrates.ucsf.edu.
Published, JBC Papers in Press, March 5, 2001, DOI 10.1074/jbc.M010451200
2 J.-L. Vigne, D. Hornung, M. D. Mueller, and R. N. Taylor, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CAPS, 3-[cyclohexylamine]-1-propane sulfonic acid; PBS, phosphate-buffered saline; BSA, bovine serum albumin; fMLP, N-formyl-methionyl-leucyl-phenylalanine; HPLC, high-performance liquid chromatography; PNGase, polypeptide glycosidase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Damsky, C. H., and Fisher, S. J. (1998) Curr. Opin. Cell Biol. 5, 660-666[CrossRef] |
2. | Medawar, P. (1953) Society for Experimental Biology, Evolution Symposium , pp. 320-338, Academic Press, New York |
3. | Kovats, S., Main, E. K., Librach, C., Stubblebine, M., Fisher, S. J., and DeMars, R. (1990) Science 248, 220-223[Medline] [Order article via Infotrieve] |
4. |
Urvater, J.,
Otting, N.,
Loehrke, J.,
Rudersdorf, R.,
Slukvin, I.,
Piekarczyk, M.,
Golos, T.,
Hughes, A.,
Bontrop, R.,
and Watkins, D.
(2000)
J. Immunol.
164,
1386-1398 |
5. | McMaster, M., Lim, K.-H., and Taylor, R. (1998) Curr. Probl. Obstet. Gynecol. Fertil. 21, 1-24 |
6. | Lessey, B. A., Castelbaum, A. J., Buck, C. A., Lei, Y., Yowell, C. W., and Sun, J. (1994) Fertil. Steril. 62, 497-506[Medline] [Order article via Infotrieve] |
7. | Hey, N. A., Graham, R. A., Seif, M. W., and Aplin, J. D. (1994) J. Clin. Endocrinol. Metab. 78, 337-342[Abstract] |
8. |
Cullinan, E. B.,
Abbondanzo, S. J.,
Anderson, P. S.,
Pollard, J. W.,
Lessey, B. A.,
and Stewart, C. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
3115-3120 |
9. | Seppälä, M., and Tiitinen, A. (1995) Hum. Reprod. 10, 67-76 |
10. | Lehmann, M., El Battari, A., Abadie, B., Martin, J., and Marvaldi, J. (1996) J. Cell. Biochem. 61, 266-277[CrossRef][Medline] [Order article via Infotrieve] |
11. |
DeLoia, J.,
Krasnow, J.,
Brekosky, J.,
Babaknia, A.,
Julian, J.,
and Carson, D.
(1998)
Hum. Reprod.
13,
2902-2909 |
12. |
Blanchard, F.,
Raher, S.,
Duplomb, L.,
Vusio, P.,
Pitard, V.,
Taupin, J.,
Moreau, J.,
Hoflack, B.,
Minvielle, S.,
and Jacques, Y.
(1998)
J. Biol. Chem.
273,
20886-20893 |
13. |
Dell, A.,
Morris, H. R.,
Easton, R. L.,
Panico, M.,
Patankar, M.,
Oehniger, S.,
Koistinen, R.,
Koistinen, H.,
Seppala, M.,
and Clark, G. F.
(1995)
J. Biol. Chem.
270,
24116-24126 |
14. | Mueller, M., Vigne, J.-L., Vaisse, C., and Taylor, R. (2001) Semin. Reprod. Med., in press |
15. |
Taylor, R. N.,
Savouret, J.-F.,
Vaisse, C.,
Vigne, J.-L.,
Ryan, I.,
Hornung, D.,
Seppälä, M.,
and Milgrom, E.
(1998)
J. Clin. Endocrinol. Metab.
83,
4006-4012 |
16. | Taylor, R. N., Vigne, J. L., Zhang, P., Hoang, P., Lebovic, D. I., and Mueller, M. D. (2000) Am. J. Obstet. Gynecol. 182, 841-847[Medline] [Order article via Infotrieve] |
17. |
Stewart, D. R.,
Erikson, M. S.,
Erikson, M. E.,
Nakajima, S. T.,
Overstreet, J. W.,
Lasley, B. L.,
Amento, E. P.,
and Seppälä, M.
(1997)
J. Clin. Endocrinol. Metab.
82,
839-846 |
18. |
Tseng, L.,
Zhu, H. H.,
Mazella, J.,
Koistinen, H.,
and Seppälä, M.
(1999)
Mol. Hum. Reprod.
5,
372-375 |
19. |
Fazleabas, A. T.,
Donnelly, K. M.,
Hild-Petito, S.,
Hausermann, H. M.,
and Verhage, H. G.
(1997)
Hum. Reprod. Update
3,
553-559 |
20. | Dalton, C. F., Laird, S. M., Estdale, S. E., Saravelos, H. G., and Li, T. C. (1998) Hum. Reprod. (Oxf) 13, 3197-3202[Abstract] |
21. | Rachmilewitz, J., Riely, G. J., and Tykocinski, M. L. (1999) Cell. Immunol. 191, 26-33[CrossRef][Medline] [Order article via Infotrieve] |
22. | Kay, G., Lane, B. C., and Snyderman, R. (1983) Infect. Immun. 41, 1166-1174[Medline] [Order article via Infotrieve] |
23. | Westwood, O. M., Chapman, M. G., Totty, N., Philp, R., Bolton, A. E., and Lazarus, N. R. (1988) J. Reprod. Fertil. 82, 493-500[Abstract] |
24. | Vigne, J.-L., Mueller, M., Vaisse, C., and Taylor, R. (2001) Référ. Gynécol. Obstét., in press |
25. |
Hornung, D.,
Bentzien, F.,
Kiesel, L.,
Wallwiener, D.,
and Taylor, R. N.
(2001)
Mol. Hum. Reprod.
7,
163-168 |
26. | Bell, S. C., and Drife, J. O. (1989) Baillières Clin. Obstet. Gynaecol. 3, 271-291[Medline] [Order article via Infotrieve] |
27. | Julkunen, M., Seppälä, M., and Jänne, O. A. (1991) Ann. N. Y. Acad. Sci. 626, 284-294[Abstract] |
28. | Bolton, A., Pockley, A., Clough, K., Mowles, E., Stoker, R., Westwood, O., and Chapman, M. (1987) Lancet 1, 593-595[CrossRef][Medline] [Order article via Infotrieve] |
29. | Ren, S., and Braunstein, G. (1990) J. Clin. Endocrinol. Metab. 70, 983-989[Abstract] |
30. | Morrow, D. M., Xiong, N., Getty, R. R., Ratajczak, M. Z., Morgan, D., Seppala, M., Riittinen, L., Gewirtz, A. M., and Tykocinski, M. L. (1994) Am. J. Pathol. 145, 1485-1495[Abstract] |
31. | Seppälä, M. (1999) Curr. Opin. Obstet. Gynecol. 3, 261-263[CrossRef] |
32. | Miller, R., Fayen, J., Chakraborty, S., Weber, M., and Tykocinski, M. (1998) FEBS Lett. 436, 455-460[CrossRef][Medline] [Order article via Infotrieve] |