Purification and characterization of an immunomodulatory endometrial protein, glycodelin.

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 beta-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 degrees C x 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 (approximately 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.

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 ␤-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 (IC 50 ‫؍‬ 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. 125 I-Glycodelin binding to whole U937 cells revealed a single, saturable site with a K d ‫؍‬ 48 ؎ 21 nM by Scatchard analysis. Cross-linking studies indicated that glycodelin binds to a high molecular mass (ϳ250 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.
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 cotranslationally to the nascent protein (5).
Other heavily glycosylated proteins at the human maternalembryonic interface, the expression of which temporally coincides with blastocyst implantation, also are of interest: ␣ ␤ 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).
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.

EXPERIMENTAL PROCEDURES
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 ϫ g for 20 min, and the supernatant was centrifuged at 80,000 ϫ 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 ϫ 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 in-creasing 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 ϫ 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 NH 2 -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 m (Bio-Rad) in the presence of 10 mM CAPS 1 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 g 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 ϫ 10 6 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 ϫ g for 2 h at 4°C. The pellet was resuspended in 25 mM Tris and 10 mM MgCl 2 , pH 7.4, containing protease inhibitors by using a Dounce homogenizer. Five-hundred-l aliquots were frozen immediately at Ϫ80°C and stored for later use.
Protein Labeling and Binding Studies-Pure glycodelin (4 g) was iodinated by using the chloramine-T method to a specific activity ranging between 100 and 200 Ci/g. 125 I-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 l of PBS/BSA. Cells were incubated for 2 h at 37°C with 125 I-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 ␥ 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.
Cross-linking Experiments-Two hundred g of U937 cell membranes were suspended in 300 l of buffer (100 mM Tris, MgCl 2 , pH 8.3), and labeled glycodelin was added at a final concentration of 300 nM in the presence or absence of 30 M 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 l of PBS. The cross-linking reagent, bis(sulfosuccinimidyl)suberate (Pierce), was added at the final concentration of 2 mM. 125 I-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 ϫ 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-m pore size polycarbonate membranes) were used. Samples (600 l) containing the chemotactic peptide N-formyl-methionyl-leucyl-phe- 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-l suspensions of U937 cells (10 6 cells) in PBS/BSA were placed in the upper chamber. The chambers were then incubated in a humidified CO 2 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 Ϯ S.E. of independent experiments.

RESULTS
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 NH 2 -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 1 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.

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). 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 NH 2 -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 IC 50 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 g/ml. By contrast, the deglycosylated form of glycodelin expressed the same biopotency as the intact glycoprotein.
Binding of 125 I-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 125 Iglycodelin and unlabeled purified glycodelin bound to the surface of U937 cells. We observed that the binding is saturable, and Scatchard analyses estimated a K d of 48 Ϯ 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 125 I-glycodelin binding to cell-free U937 plasma membranes yielded a K d of 121 Ϯ 65 nM (data not shown).
Cross-linking Experiments-U937 cell membranes were incubated with 125 I-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 125 I-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 ϳ250 kDa. Ishikawa cell membranes failed to display specific 125 I-glycodelin cross-linking. 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 phosphatebuffered saline. Aliquots from peak fractions were subjected to SDS gel electrophoresis (inset). The asterisk indicates fraction 55 containing the purified glycodelin (27 kDa).

DISCUSSION
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 epi-thelial 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 In these experiments, data were normalized to U937 chemotaxis in response to 0.1 M 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 g/ml), cell migration was inhibited in a dose-dependent manner. Purified glycodelin (25 g/ml) and purified deglycosylated glycodelin (25 g/ml) caused an almost complete inhibition of cell migration. Heat inactivation (95°C ϫ 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 g/ml) had no inhibitory effects on monocyte chemotaxis (data not shown). 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 maternalembryonic 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␤1-4GlcNAc glycosylation pattern (13).
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 ϳ60-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 (K d ϳ50 -120 nM) receptor for 125 I-glycodelin on U937 cells and purified plasma membranes. Miller et al. (32) identified a glycodelin receptor of similar affinity (K d ϳ7-28 nM) on circulating human monocytes. Our preliminary analysis of the molecular mass of the cross-linked glycodelin-receptor complex suggests that it is ϳ250 kDa, but this may reflect the sum mass of multiple proteins involved in the complex.
The apparent IC 50 of the monocyte chemorepellent effect of glycodelin in our experiments was 550 nM. According to the Cheng-Prusoff equation (K i ϭ IC 50 /(1 ϩ [free ligand]/K d )), the inhibition constant (K i ) 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 IC 50 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.