Characterization of the Oligosaccharides Associated with the Human Ovarian Tumor Marker CA125*

Nyet Kui Wong {ddagger} §, Richard L. Easton {ddagger}, Maria Panico {ddagger}, Mark Sutton-Smith {ddagger}, Jamie C. Morrison ¶, Frank A. Lattanzio ¶, Howard R. Morris {ddagger} ||, Gary F. Clark ¶ **, Anne Dell {ddagger} {ddagger}{ddagger} and Manish S. Patankar ¶ §§

From the {ddagger}Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AY, United Kingdom, the Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23501-1980, and the ||M-SCAN Mass Spectrometry Research and Training Centre, Silwood Park, Ascot SL5 7PZ, United Kingdom

Received for publication, March 18, 2003 , and in revised form, May 5, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CA125 is a mucin commonly employed as a diagnostic marker for epithelial ovarian cancer. Induction of humoral responses to CA125 leads to increased survival times in patients with this form of cancer, suggesting a potential role for this mucin in tumor progression. In this study, oligosaccharides linked to CA125 derived from the human ovarian tumor cell line OVCAR-3 were subjected to rigorous biophysical analysis. Sequencing of the O-glycans indicates the presence of both core type 1 and type 2 glycans. An unusual feature is the expression of branched core 1 antennae in the core type 2 glycans. CA125 is also N-glycosylated, expressing primarily high mannose and complex bisecting type N-linked glycans. High mannose type glycans include Man5-Man9GlcNAc2. The predominant N-glycans are the biantennary, triantennary, and tetraantennary bisecting type oligosaccharides. Remarkably, the N-glycosylation profiles of CA125 and the envelope glycoprotein gp120 (derived from H9 lymphoblastoid cells chronically infected with HIV-1) are very similar. The CA125-associated N-glycans have also recently been implicated in crucial recognition events involved in both the innate and adaptive arms of the cell-mediated immune response. CA125 may therefore induce specific immunomodulatory effects by employing its carbohydrate sequences as functional groups, thereby promoting tumor progression. Immunotherapy directed against CA125 may attenuate these immunosuppressive effects, leading to the prolonged survival of patients with this extremely serious form of cancer.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CA125 is a mucin first detected by Bast et al. using the monoclonal antibody OC125 (1, 2). CA125 is also a significant tumor marker associated with many human cancers but is most widely utilized for the diagnosis of epithelial ovarian cancer (3). A recent study indicates that the induction of anti-CA125 responses in ovarian cancer patients leads to their prolonged survival compared with untreated controls (4). Therefore, CA125 could act as a targeting antigen to elicit antibody-dependent, cell-mediated cytotoxicity against ovarian tumor cells. Another possibility is that CA125 plays some key physiological role that promotes tumor development in individuals with ovarian cancer. Its complete biochemical analysis is therefore crucial for defining its potential functional roles.

CA125 is an extremely complex molecule from both the proteomic and glycomic perspectives (5). Recent sequencing of its gene has yielded substantial information about the peptide backbone of this mucin (6, 7). CA125 is composed of an N-terminal domain, a tandem repeat region, and a short cytoplasmic tail. The N-terminal domain of CA125 consists of 1637 amino acids (8). The tandem repeat domain is made up of 40–60 repeats of 156 amino acids (7). The cytoplasmic tail of CA125 consists of 256 amino acids. Recently, an additional 10,431-amino acid extension of the N-terminal domain was reported (8). Thus, the core protein sequence of CA125 could have a mass approaching 2.5 million Da (8).

CA125 is also highly enriched in serine and threonine residues, consistent with its recent designation as the MUC16 mucin (6). The carbohydrate content of CA125 based on its mass is estimated to be ~24–28% (5, 9), with the majority being O-linked glycans (7). This very high degree of glycosylation suggests that the average molecular mass of CA125 may be 3.5 million Da (8).

Complete structural analysis of the CA125 molecule also requires sequencing of its oligosaccharides and localization of its glycosylation sites. Previously, Lloyd et al. identified several O-linked glycans expressed on CA125 (9). However, the results obtained were based solely on indirect structural analysis. Moreover, this approach did not yield complete characterization of all the oligosaccharides linked to CA125.

Unambiguous sequencing of CA125-associated glycans requires the use of precise biophysical methods. We have characterized the glycans associated with CA125 using this very rigorous approach. The analysis detailed in this report reveals several notable structural features of the O-glycans associated with CA125. Surprisingly, the data also confirm robust N-glycosylation of CA125, supporting previous studies suggesting this possibility (5, 10). This sequencing data, other more recent immunological findings, and the observed beneficial effects of vaccination strategies directed against CA125 suggest the distinct possibility that the carbohydrate sequences linked to this mucin may play a crucial role in promoting modulation of the immune response in patients with epithelial ovarian cancer.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The epithelial ovarian tumor cell line, OVCAR-3, was purchased from the American Type Culture Collection. The anti-CA125 monoclonal antibody, OC125, was the generous gift from Dr. Robert Bast of the University of Texas M. D. Anderson Cancer Center, Houston, TX. Reagents for gel electrophoresis and Western blot analysis were purchased from Bio-Rad. Fetal calf serum used for tissue culture was obtained from Atlanta Biologicals, and RPMI 1640 media was from Invitrogen. All other chemicals and reagents used in this study were purchased from Sigma unless otherwise stated.

Isolation of CA125 from OVCAR-3 Cells—Isolation of CA125 was performed as described previously (11). Briefly, OVCAR-3 cells were cultured in 225-cm2 tissue culture flasks using RPMI 1640 medium containing 20% fetal calf serum until the cells were confluent. The culture medium was removed, and the cells were washed twice with serum-free RPMI 1640 medium that was devoid of phenol red. The cells were then cultured for 5 days in this medium, after which the culture medium was removed and saved for extraction of CA125. Fresh, serum-free, non-phenol red RPMI 1640 medium was added to the tissue culture flasks, and the process was repeated two more times.

The spent medium was pooled and dialyzed through a 3,500 molecular weight cutoff membrane against water containing 0.02% sodium azide. Material retained in the dialysis bag was lyophilized and resuspended in a small volume of 10 mM ammonium bicarbonate. Total protein content of this material was determined by BCA assay (Pierce). 10 mg of total protein in 500 µl of 10 mM ammonium bicarbonate buffer was loaded on a 1x 45-cm Sephacryl S500 HR size exclusion column. The column was eluted with 10 mM ammonium bicarbonate buffer, and 0.8-ml fractions were collected. Fractions were monitored for absorbance at 280 nm. The two peaks obtained were pooled, lyophilized, and monitored for CA125 using Western blot analysis. CA125 isolated from different column runs was pooled, and the amount of this tumor marker was determined by using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Glycotech).

Electrophoretic and Western Blot Analysis of CA125—Electrophoresis of CA125 under denaturing conditions (SDS-PAGE) was routinely conducted on 5 or 7.5% separating and 3% or 5% stacking gels. Silver staining of the gels was conducted according to the supplier's (Bio-Rad) specifications. Gels were stained with PAS1 reagent to detect glycoproteins. For Western blot analysis, CA125 separated on denaturing gels was transferred to nylon membranes using Bio-Rad mini-gel apparatus. The nylon membranes were preincubated with bovine serum albumin and then layered with OC125 for 1 h. A goat anti-mouse horseradish peroxidase-labeled secondary antibody (Amersham Biosciences) was used for detection of the CA125 bands with chemiluminescence reagents (Amersham Biosciences).

To detect binding of CA125 to ConA, the mucin was transferred to nylon membranes as described above. After preincubation with bovine serum albumin, the membranes were layered with 1 µg/ml horseradish peroxidase-labeled ConA (E-Y Laboratories) in 0.1% bovine serum albumin solution. After incubation for 1 h, the membrane was washed thoroughly, and lectin binding to CA125 was detected by chemiluminescence.

Digestion of CA125 with PNGase F for Western Blotting Experiments—Lyophilized CA125 (50 µg) was dissolved in 50 mM ammonium bicarbonate (pH 8.4) containing 5 mM mercaptoethanol. 10 milliunits of PNGase F (EC 3.5.1.52 [EC] ; Roche Molecular Biochemicals) was added, and the mixture was incubated overnight at 37 °C. A parallel experiment was conducted under the same conditions, except that PNGase F was omitted.

Reduction and Carboxymethylation—Reduction and carboxymethylation was carried out as described (12). CA125 was reduced in 50 mM Tris-HCl buffer, pH 8.5, containing dithiothreitol in a 4-fold molar excess over the number of disulfide bridges. Reduction was performed under nitrogen atmosphere at 37 °C for 1 h. Carboxymethylation was carried out in iodoacetic acid (5-fold molar excess over dithiothreitol), and the reaction was allowed to proceed under a nitrogen atmosphere at 37 °C for 1 h. Carboxymethylation was terminated by dialysis against 4 x 2.5 liters of 50 mM ammonium bicarbonate, pH 8.5, at 4 °C for 48 h. After dialysis, the CA125 was lyophilized.

Tryptic Digestion—CA125 was incubated with trypsin (EC 3.4.21.4 [EC] ; Sigma) at a 50:1 ratio (w/w) in 50 mM ammonium bicarbonate, pH 8.5, for 5 h at 37 °C. The digestion was terminated by placement in boiling water for 3 min, followed by lyophilization.

PNGase F Digestion of Tryptic Glycopeptides—PNGaseF digestion was carried out in 50 mM ammonium bicarbonate, pH 8.5, for 16 h at 37 °C with 3 units of enzyme. The reaction was terminated by lyophilization, and the released N-glycans were separated from peptides and O-glycopeptides by Sep-Pak C18 (Waters Corp.) as described (12).

Reductive Elimination—O-glycans were released by reductive elimination, which was performed in 400 µl of sodium borohydride (1 mg/ml in 0.05 M sodium hydroxide) at 45 °C for 16 h. The reaction was terminated by dropwise addition of glacial acetic acid followed by Dowex chromatography and borate removal (12).

Sequential Exoglycosidase Digestion—The released glycans were incubated with the following enzymes and conditions: neuraminidase (Vibro cholerae, EC 3.2.1.18 [EC] ; Roche Molecular Biochemicals), 50 milliunits in 100 µl of 50 mM ammonium acetate buffer, pH 5.5; {alpha}-mannosidase (jack bean, EC 3.2.1.24 [EC] ; Glyko), 0.5 units in 100 µl of 50 mM ammonium acetate buffer, pH 4.6; {beta}-galactosidase (bovine testes, EC 3.2.1.23 [EC] ; Calbiochem), 10 milliunits in 100 µl of 50 mM ammonium formate buffer, pH 4.6; and {beta}-N-acetylhexosaminidase (jack bean, EC 3.2.1.30 [EC] ; Calbiochem), 0.2 units in 100 µl of 50 mM ammonium formate buffer, pH 4.6. All of the enzyme digestions were carried out at 37 °C for 48 h with a fresh aliquot of enzyme added after 24 h. Each digestion was terminated by boiling for 3 min before lyophilization.

Endo-{beta}-galactosidase Digestion—Endo-{beta}-galactosidase (Escherichia freundii, EC.3.2.1.103; Calbiochem) digestion was carried out in 100 µl of 100 mM ammonium acetate, pH 5.5, 10 milliunits at 37 °C for 48 h with a fresh aliquot of enzyme added after 24 h.

Periodate Cleavage—A solution of 40 mM sodium periodate in 100 mM ammonium acetate, pH 6.5, was prepared. Released and dried O-glycans were dissolved in 100 µl of this reagent. The reaction was allowed to proceed in the dark at 0 °C for 48 h. The reaction was terminated by the addition of 2–3 µl of ethylene glycol and incubation in the dark at room temperature for 30–60 min. The products of the periodate cleavage were lyophilized and reduced with 200 µl of sodium borohydride (10 mg/ml) in 2 M ammonium hydroxide. The reaction was carried out at room temperature for 2 h and terminated by the dropwise addition of glacial acetic acid. This sample was subjected to Dowex chromatography, borate removal, permethylation, and Sep-Pak cleanup using an acetonitrile gradient as described (12).

Methanolysis—The reagent was prepared by bubbling dry hydrochloric acid gas into methanol as described in a previous study (13). After cooling, 20 µl of this reagent was added to the permethylated sample. The reaction was performed at room temperature for 15 min and terminated by drying under a nitrogen stream. The sample was then resuspended in methanol, and an aliquot of 1 µl was removed for MALDI-TOF analysis. The free hydroxyl groups were then deuteromethylated and subjected to linkage analysis.

Chemical Derivatization for FAB-MS, GC-MS, MALDI-TOF, and CAD-MS/MS Analysis—Permethylation was performed using the sodium hydroxide procedure as described previously (12). Briefly, sodium hydroxide pellets were crushed with dimethyl sulfoxide to form a slurry. An aliquot of this slurry was added to dried glycans along with 1 ml of methyl iodide. The reaction was terminated by the addition of water, and permethylated glycans were recovered by chloroform extraction. The chloroform layer was washed several times with water to remove any impurities. Partially methylated alditol acetates were prepared from permethylated samples for GC-MS linkage analysis as described (14). Briefly, the permethylated glycans were hydrolyzed with 2 M trifluoroacetic acid for 2 h at 121 °C, reduced with 10 mg/ml sodium borodeuteride in 2 M aqueous ammonium hydroxide at room temperature for 2 h, and then acetylated with acetic anhydride at 100 °C for 1h.

FAB-MS Analysis—FAB mass spectra were acquired using a Fisons Instruments VG ZAB-2SE-2FPD mass spectrometer fitted with a cesium ion gun operated at 30 kV. The matrix used was monothioglycerol, and all samples were dissolved in methanol prior to loading. Data acquisition and processing were performed using VG Analytical Opus software.

MALDI-TOF Analysis—MALDI data were acquired using a Perseptive Biosystems Voyager-DETM STR mass spectrometer in the reflectron mode with delayed extraction. A permethylated sample was dissolved in 10 µl of methanol, and 1 µl of dissolved sample was premixed with 1 µl of matrix (2,5-dihydrobenzoic acid) before loading onto a metal plate.

GC-MS Analysis—Linkage analysis of partially methylated alditol acetates was carried out on a Fisons Instruments MD 800 apparatus fitted with a RTX-5 fused silica capillary column (30 m x 0.32 mm internal diameter; Restek Corp.). The sample was dissolved in hexanes and injected onto the column at 65 °C. The column was maintained at this temperature for 1 min and then heated to 290 °C at a rate of 8 °C per min.

CAD-ES-MS/MS Analysis—CAD-ES-MS/MS spectra were acquired using Q-TOF (Micromass, Manchester, United Kingdom) and Q-STAR (Applied Biosystems) instruments. The permethylated glycans were dissolved in methanol before loading into a spray capillary coated with a thin layer of gold/palladium, inner diameter 2 µl (Proxeon, Odense, Denmark). A potential of 1.5 kV was applied to a nanoflow tip to produce a flow rate of 10–30 nl/min. The drying gas used was N2 and the collision gas was argon, with the collision gas pressure maintained at 104 millibar. Collision energies varied depending on the size of the carbohydrate, typically between 30 and 90 eV.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of CA125 from OVCAR-3 Cells—An established two-step isolation procedure was utilized to purify CA125 from the spent media of OVCAR-3 cells (11). In the first step, the media were dialyzed against a 3.5-kDa molecular mass cutoff membrane to remove low molecular weight impurities. The dialyzed material was lyophilized and separated on a Sephacryl S500 HR size exclusion column equilibrated in 10 mM ammonium bicarbonate. Fractions obtained from this column were monitored for absorbance at 260 and 280 nm. In a typical run, two peaks were obtained (Fig. 1). CA125 was detected exclusively in the first peak when the fractions were analyzed by Western blot (data not shown). This peak was pooled, lyophilized, and used for further analysis. Total protein content in this sample was determined. Approximately 2.5–3 million units of CA125 were detected per milligram of protein by enzyme-linked immunosorbent assay. Similar values have been reported for highly enriched CA125 preparations isolated in other studies (6).



View larger version (17K):
[in this window]
[in a new window]
 
FIG. 1.
Gel filtration separation of CA125. Dialyzed OVCAR-3 media was loaded on a column (1 x 45 cm) of Sephacryl S500 HR and eluted with 10 mM ammonium bicarbonate buffer (pH 8.0). Fractions were monitored for absorbance at 280 nm (closed circles) and 260 nm (closed squares). CA125-containing fractions were identified by Western blot analysis. The peak containing CA125 is indicated.

 

Electrophoretic and Western blot analyses were initially employed to determine the purity of the isolated CA125 sample. Silver stain analysis (Fig. 2A) indicated a broad band above the 200-kDa molecular mass marker. Some staining was also observed in the stacking gel as reported previously (9). These high molecular mass bands were easily detected by Western blot analysis using the anti-CA125 monoclonal antibody OC125 (Fig. 2B). Similar high molecular mass bands were also detected by PAS staining (Fig. 2C).



View larger version (34K):
[in this window]
[in a new window]
 
FIG. 2.
Electrophoretic and Western blot analysis of CA125. Purified CA125 (5–10 µg of total protein) was separated by electrophoresis. The gels were stained with silver reagent (A) or with PAS reagent (B). Western blotting was performed using OC125 as the primary antibody (C). Molecular masses of markers are indicated for each panel. A single-headed arrow indicates the interface between stacking and separating gel. The double-headed arrow indicates migration of the dye front.

 

In addition to the band corresponding to CA125, some low molecular mass bands were also detected in the silver stained gel (Fig. 2A). Such lower molecular mass bands are always present in CA125 preparations examined in other studies (6). However, these lower molecular mass bands did not bind OC125 and were also not stained by PAS, confirming that they are not glycosylated (Fig. 2, B and C) and, therefore, would not interfere with oligosaccharide sequencing.

Confirmation that the high molecular mass band was CA125 was provided by proteomics analysis. ES-MS/MS analysis of an in-gel tryptic digest of this band gave data that corresponded to the peptide LTLLRPEK, which is a tandem repeat sequence that occurs 27 times in the CA125 sequence (7). In a complementary experiment, MALDI-TOF analysis of a tryptic digest of a sample that had not been subjected to SDS-PAGE gave molecular ions mapping to residues 2451–2479, 4637–4665, 5105–5133, 6651–6661, 6819–6847, 7287–7315, 8223–8251, 9938–9966, 10094–10122, 10862–10872, and 10406–10434 that have been shown to be present in CA125 (6). Peptides from proteins or glycoproteins other than CA125 were not detected in this analysis. The restricted PAS staining of the high molecular mass band within this preparation in combination with this proteomics data provides very strong evidence that the glycans analyzed in this study are covalently linked to CA125.

Structural Analysis Strategy—The overall structural strategy employed to characterize CA125 glycosylation is summarized in Scheme 1. Oligosaccharides were derivatized and recovered in 35 and 50% acetonitrile fractions from the C18 Sep-Pak cartridge prior to analysis by FAB-MS, MALDI-TOF, CAD-MS/MS, and linkage analysis. Data from these experiments were complemented by sequential exoglycosidase digestions, periodate oxidation, and methanolysis experiments.



View larger version (22K):
[in this window]
[in a new window]
 
SCHEME 1.
Summary of overall experimental strategy employed to characterize CA125.

 

Analyses of PNGase F Digests Reveals Abundant N-Glycosylation of CA125—N-glycans were released from tryptic digests of reduced carboxymethylated CA125 by digestion with PNGase F. Fig. 3 shows the MALDI data of the 50% acetonitrile fraction with the assignments given in Table I. The MALDI data obtained from the 35% acetonitrile fraction (data not shown) are similar to the 50% fraction, except that the signals are of lower abundance. The data indicate that CA125 is rich in N-glycans, having compositions consistent with high mannose structures (Hex5–9HexNAc2), and complex type structures of compositions NeuAc0–1Fuc0–2Hex5–7HexNAc4–7. After {alpha}-mannosidase digestion, signals corresponding to high mannose structures disappeared, and the putative complex type structures were unaffected. Notable features of the data include the following. (i) Complex type structures contribute to over 80% of the N-glycans with the remainder being high mannose. (ii) The most abundant complex type N-glycans have compositions consistent with mono-fucosylated bisected biantennary structures (m/z 2243, 2285, 2489, 2605, and 2850). (iii) The higher molecular mass components have compositions that are consistent with tri and tetraantennary structures; this result was confirmed by observing mass shifts after {beta}-galactosidase digestion (data not shown). (iv) Two minor components have compositions consistent with fucose being present both on the core and on one of the antennae (m/z 2664 and 2780). (v) The presence of a minor A-type fragment ion in the FAB spectrum (data not shown) at m/z 638 (FucHexHexNAc+) provides supportive evidence for fucosylated antennae. (vi) The level of sialylation is relatively low, with no components carrying more than a single sialic acid.



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 3.
MALDI-TOF mass spectrum of CA125 N-glycans. Glycans were released by PNGase F, permethylated, and subjected to Sep-Pak cleanup. Data from the 50% acetonitrile fraction are shown. The signals are assigned in Table I.

 

View this table:
[in this window]
[in a new window]
 
TABLE I
Assignments of molecular ([M + Na]+) ions observed in the MALDI spectrum of permethylated N-glycans derived from the 50% acetonitrile fraction of CA125

 

CAD-ES-MS/MS Defines Sites of Fucosylation—Collisionally activated decomposition electrospray tandem mass spectrometry of the major components observed in the MALDI experiment yielded fragment ions that allowed the assignment of the positions of fucose substitution. Data derived from Fuc1Hex5HexNAc4, NeuAc1Fuc1Hex5HexNAc4, and Fuc2Hex5HexNAc5 are shown in Fig. 4, A, B, and Fig. 4C, respectively. Assignments of key signals are given in the insets. The diagnostic signal for fucose being attached to the terminal GlcNAc of the core is m/z 474 (whereas antennae substituted with Fuc give a major fragment ion at m/z 660). The difucosylated molecule exhibits major signals at both of these m/z values, indicating that the majority of the glycans of this composition carry one Fuc on the core and one on the antennae. In contrast, the monofucosylated glycans show a major signal at m/z 474 and a minor signal at m/z 660, indicating that these glycans are predominantly fucosylated on the core.



View larger version (24K):
[in this window]
[in a new window]
 
FIG. 4A.
CAD-MS/MS spectra of the [M + 2Na]2+ molecular ions of Fuc1Hex5HexNAc4 (A), NeuAc1Fuc1Hex5HexNAc4 (B), and Fuc2Hex5HexNAc5 (C). The major component in each case is shown in the schematic. Additionally, panels A and B contain a minor component that lacks the fucose on the core and instead carries a fucose on a LacNAc antenna, giving the fragment ion at m/z 660.

 


View larger version (17K):
[in this window]
[in a new window]
 
FIG. 4C.
CAD-MS/MS spectra of the [M + 2Na]2+ molecular ions of Fuc1Hex5HexNAc4 (A), NeuAc1Fuc1Hex5HexNAc4 (B), and Fuc2Hex5HexNAc5 (C). The major component in each case is shown in the schematic. Additionally, panels A and B contain a minor component that lacks the fucose on the core and instead carries a fucose on a LacNAc antenna, giving the fragment ion at m/z 660.

 

Linkage Analysis of N-Glycans Released by PNGase F—Linkage analysis data for the PNGase F-released glycans and their desialylated counterparts are shown in Table II. These results are fully in agreement with the idea that high mannose and complex type structures are major constituents. Key features of these data are as follows. (i) The 3,4,6-linked Man confirms the presence of bisected glycans. (ii) The high abundance of 2-Man is consistent with the MALDI data, which showed that biantennary structures are the most dominant N-glycans. (iii) The 3,4- and 4,6-linked GlcNAcs are in accord with the CAD-ES-MS/MS data. (iv) The presence of 3- and 6-linked Gal, which were both diminished after sialidase treatment, indicates that the sialic acids are attached at either the 3- or the 6-position of Gal.


View this table:
[in this window]
[in a new window]
 
TABLE II
GC-MS analysis of partially methylated alditol acetates obtained from the PNGase F-released N-glycans of CA125

The 50% acetonitrile fraction after Sep-Pak purification of permethylated glycans was hydrolyzed, reduced, acetylated, and analyzed by GC-MS.

 

Polylactosamine-type Antennae Revealed by Endo-{beta}-galactosidase Digestion—Digestion with endo-{beta}-galactosidase was carried out to establish whether any of the N-glycans contain polylactosamine chains whose existence should be revealed by new molecular ions in the low mass region corresponding to antennae fragments released by the enzyme. The permethylated products gave the data shown in Fig. 5. The molecular ions at m/z 722, 896, and 1083 are consistent with the sequences Gal-GlcNAc-Gal, Gal-(Fuc)GlcNAc-Gal, and NeuAc-Gal-GlcNAc-Gal, respectively, which are the predicted digestions of uncapped, fucosylated, and sialylated polylactosamine chains, respectively. Disaccharides released from within the polylactosamine antennae give the signal at m/z 518 (GlcNAc-Gal). These data confirm the existence of polylactosamine chains. Evidence for the m/z 896 component being terminated by the Lewisx epitope (Gal{beta}1–4[Fuc{alpha}1–3]GlcNAc{beta}1-), was obtained by mild methanolysis under conditions that are known to rapidly liberate fucose residues that are 3-linked to GlcNAc (15). This experiment afforded a new signal at m/z 708 concomitant with loss of m/z 896, which is consistent with methanolytic removal of Fuc (data not shown). Additional evidence for the Lewisx structure was provided by CAD-ES-MS/MS of m/z 896 (Fig. 6), which yielded a major fragment ion at m/z 690 corresponding to the removal of fucose accompanied by loss of water, a fragmentation pathway that is diagnostic of 3-linked fucose (16). Corroborative evidence for this assignment was provided by the major signal at m/z 660 that corresponds to A-type cleavage to give [Gal(Fuc)GlcNAc+Na]+.



View larger version (32K):
[in this window]
[in a new window]
 
FIG. 5.
Partial FAB mass spectrum of products of endo-{beta}-galactosidase digestion. Glycans were released from CA125 by PNGase F, digested with endo-{beta}-galactosidase, permethylated, and subjected to Sep-Pak cleanup. Data from the 35% acetonitrile fraction are shown. The schematics represent products of digestion. Non-annotated signals were present in samples that had not been treated with endo-{beta}-galactosidase.

 


View larger version (14K):
[in this window]
[in a new window]
 
FIG. 6.
CAD-MS/MS spectrum of the [M + Na]+ molecular ion of the m/z 896 product of endo-{beta}-galactosidase digestion. Assignments are given on the schematic structure.

 

Sequential Enzyme Digestions Define the Cores of the Polylactosaminyl Glycans—To define the core structures that carry polylactosaminyl chains, N-glycans were sequentially digested with {beta}-galactosidase, {beta}-N-acetylhexosaminidase, and endo-{beta}-galactosidase. Under these conditions, short sialylated or fucosylated antennae are unaffected, whereas their polylactosaminyl counterparts will be degraded by the endo-{beta}-galactosidase. In addition, uncapped antennae with a single LacNAc backbone will be completely removed, and uncapped antennae containing two LacNAc repeats will be shortened by a single LacNAc moiety. Aliquots were taken after each digestion, permethylated, and examined by MALDI-TOF after Sep-Pak purification. Partial MALDI data from the products of {beta}-galactosidase plus {beta}-N-acetylhexosaminidase and the products of sequential treatment with all three enzymes are shown in Fig. 7, A and B, respectively. Signals that show notable increases in abundance before and after endo-{beta}-galactosidase are observed at m/z 2215 (Fuc2Hex4HexNAc4), m/z 2401 (NeuAcFucHex4HexNAc4), m/z 2460 (Fuc2Hex4HexNAc5), and m/z 2646 (NeuAcFucHex4HexNAc5). As indicated by the structural annotations in Fig. 7, these data show that the glycans that carry the polylactosaminyl chains are core fucosylated bi- and/or triantennary glycans with a single short sialylated or Lewisx antenna.



View larger version (45K):
[in this window]
[in a new window]
 
FIG. 7.
MALDI-TOF mass spectra of glycosidase digests of CA125 N-glycans. Glycans were released by PNGase F, digested sequentially with {beta}-galactosidase, {beta}-N-acetylhexosaminidase and endo-{beta}-galactosidase, permethylated, and subjected to Sep-Pak cleanup. Data from the 50% acetonitrile fractions of {beta}-galactosidase plus {beta}-N-acetylhexosaminidase (A) and {beta}-galactosidase plus {beta}-N-acetylhexosaminidase plus endo-{beta}-galactosidase (B) are shown. Signals not present prior to digestion are annotated with schematics.

 

Structural Conclusions—Taking into consideration the MALDI, FAB, linkage, CAD-ES-MS/MS, methanolysis, and exoglycosidase data, we conclude that the major N-glycans associated with CA125 have the structures shown in Fig. 8.



View larger version (20K):
[in this window]
[in a new window]
 
FIG. 8.
Proposed structures of the major N-glycans present in CA125. As noted in the text (under "Results") and the legend to Fig. 3, a minor portion of the mono-fucosylated glycans carry fucose on an antenna rather than the core. The polylactosamine-containing structures shown in the final schematic were not rigorously characterized with respect to chain length. In addition, the sialic acid and fucose residues are mutually exclusive.

 

Additional Evidence for Expression of N-Linked Glycans on CA125—Our sequencing data indicated that a substantial subset of the glycans present in CA125 include the ConA-reactive high mannose and complex type biantennary N-glycans (17). To provide further evidence for the presence of such glycans on CA125, a Western blot was performed using horseradish peroxidase-labeled ConA lectin. Strong binding of this lectin was observed to the >200-kDa band corresponding to CA125 (data not shown). ConA-horseradish peroxidase did not bind to any of the low molecular mass proteins present in CA125, suggesting that the N-linked glycans detected in the FAB-MS analysis were likely not derived from any of these impurities.

FAB and MALDI Screening of CA125 O-Glycans—Permethylated O-glycans were analyzed by FAB and MALDI after Sep-Pak purification. The annotated MALDI spectrum of the 50% acetonitrile fraction is shown in Fig. 9. The structures shown by the annotations are based on the combined data of the analyses described below. All assignments were corroborated by observing mass shifts after deuteromethylation (data not shown). From these data we conclude the following. (i) O-glycans containing up to at least fourteen sugar residues are present. (ii) The majority of O-glycans are sialylated and/or fucosylated and carry a maximum of two fucoses or two sialic acids or two fucoses and two sialic acids. (iii) Two major families are present, with the first family being mono- and di-sialylated core type 1 (Gal{beta}1–3GalNAc), whereas the members of the second family have compositions corresponding to core type 2 (Gal{beta}1–3(GlcNAc{beta}1–6)GalNAc) with non, mono, and difucosylation as well as sialylation. (iv) An A-type fragment ion at m/z 638 (FucHexHexNAc+) in the FAB mass spectrum (data not shown) indicated the presence of the Lewisx epitope in the O-glycans as well as the N-glycans (see above).



View larger version (35K):
[in this window]
[in a new window]
 
FIG. 9.
MALDI-TOF mass spectrum of CA125 O-glycans. Glycans were released by reductive elimination, permethylated and subjected to Sep-Pak cleanup. Data from the 35% acetonitrile fraction are shown in the top two panels, and high mass data from the 50% acetonitrile fraction are shown in the bottom panel. The structural assignments are derived from the combined data described in the text (under "Results"). For clarity, the schematics show major components. The CAD-MS/MS and periodate experiments indicate that a minor portion of the components that are annotated with branched core 1 glycans are unbranched and have linear oligolactosamine core 1 antennae. Compositions are given for ions of masses greater than m/z 2800, because the sequences of these glycans have not been determined. {blacksquare} with a diagonal connecter pointing downward from the left, 3-GalNAcitol; {blacksquare} with diagonal connectors pointed upward and downward from the left, 3,6-GalNAcitol; •, terminal-Gal; • with a horizontal connector pointing to the left; 3-Gal; • with a horizontal connector pointing to the left and a vertical connector pointing downward; 3,6-Gal; {square} with a horizontal connector pointing to the left, 4-GlcNAc; {diamond}, NeuAc; and {triangleup}, Fuc. NeuAc is attached to either 3- or 6-positions of Gal.

 

Linkage Analysis of the O-Glycans—The data for the linkage analysis are given in Table III. Notable features include the following. (i) Both 3- and 3,6-linked GalNAcitol are observed consistent with the presence of core type 1 and 2 structures, respectively. (ii) The detection of 3-Gal and 6-Gal suggests that sialic acids are attached at both the 3-linked and 6-linked positions of galactose. (iii) 3,4-GlcNAc and 2-Gal suggest the probable presence of Lewisx and blood group H and/or Lewisy, respectively.


View this table:
[in this window]
[in a new window]
 
TABLE III
GC-MS analysis of partially methylated alditol acetates obtained from the reductively eliminated O-glycans of CA125

The 50% acetonitrile fraction after Sep-Pak purification of permethylated glycans was hydrolyzed, reduced, acetylated, and analyzed by GC-MS.

 

Characterization of O-Glycan Structures by CAD-ES-MS/MS Analyses—Each of the components observed in the MALDI experiment were subjected to CAD-ES-MS/MS to assign sequences. Good quality fragment ion data were obtained on components up to 2000 Da, allowing the sequence assignments shown in Fig. 9. Corroborative data were obtained from similar analysis of a sample that was reductively eliminated using sodium borodeuteride instead of sodium borohydride, which shifted the mass of each molecular ion by one mass unit. Representative data from three components of special interest are discussed below.

CAD-MS/MS analysis of deutero-reduced Fuc1Hex3HexNAc2HexNAcitol (m/z 8152+, Fig. 10A) unambiguously defines the presence of the Lewisx epitope on the core 1 antenna of a core 2 structure (see assignments on Fig. 10A inset). In contrast, the difucosylated counterpart of this component (which gives the signal at m/z 1780 in the MALDI spectrum shown in Fig. 9) yielded a complex set of fragment ions consistent with the presence of variously fucosylated antennae (data not shown), suggesting that the difucosylated component is a mixture of glycans carrying Lewisy and blood group H antennae as well as Lewisx antennae. The low abundance of m/z 1780 precluded further characterization. Finally, the CAD-MS/MS spectrum of reduced Hex4HexNAc3HexNAcitol (m/z 9532+, Fig. 10B) confirms the fact that extended core 1 antennae are a feature of CA125 O-glycans (see assignments on Fig. 10B inset). Furthermore, the data also suggest that the core 1 arm is branched, because the major fragment ion for terminal LacNAc (m/z 486) is not accompanied by an ion at m/z 935, which would be predicted to occur if the two LacNAc moieties were in a tandem repeat. To further investigate this unexpected finding, the O-glycan sample was subjected to periodate degradation (see below).



View larger version (27K):
[in this window]
[in a new window]
 
FIG. 10.
CAD-MS/MS spectra of the [M + 2Na]2+ molecular ions of Fuc1Hex3HexNAc2HexNAcitol (A) and Hex4HexNAc3HexNAcitol (B). Sequence-informative fragment ions are shown on the structures.

 

Periodate Cleavage Confirms Core Types and Antennae Backbones of O-Glycans—Reductively eliminated O-glycans were subjected to periodate oxidation, followed by reduction, permethylation, Sep-Pak cleanup, and MALDI-TOF analysis. Under the relatively rigorous conditions used, a majority of the linear structures carrying vicinal hydroxyls (such as GalNAcitol) are cleaved. In addition, partial cleavage and subsequent hydrolytic loss of more resistant structures, such as terminal Fuc and Gal, also occurred. The partial MALDI spectrum of the 35% acetonitrile fraction (Fig. 11) showed molecular ions corresponding to the two sets of products expected from cleavage of the GalNAcitol, i.e. one containing the C-1 to C-4 carbons (denoted C-4) attached to the 3-linked antenna, and the other containing the C-5 and C-6 carbons (denoted C-2) attached to the 6-linked antenna. Thus sequences containing the C-2 moiety represent the 6-arms of core type 2 components, whereas sequences containing the C-4 moiety correspond to the 3-arms of core types 1 and 2. The assignments are given in Table IV. Corroborative evidence for these assignments was provided by CAD-ES-MS/MS experiments. Representative data from m/z 807 (HexHexNAc2-C2), m/z 835 (NeuAcHexHexNAc-C2), and m/z 936 (HexHexNAc2-C4) are given in Fig. 12,Fig. 12 (see Fig. 12,Fig. 12 insets for assignments). From these spectra it is evident that core type 2 structures are abundant in CA125 but, as indicated by the experiments described earlier, many of these are extended on the 3-arm rather than the 6-arm. Importantly the data for the m/z 936 component (Fig. 12C) confirm that the core 1 arm is branched in a significant portion of the larger O-glycans.



View larger version (15K):
[in this window]
[in a new window]
 
FIG. 11.
Partial MALDI-TOF mass spectrum of periodate oxidized CA125 O-glycans. Glycans were released by reductive elimination, treated with periodate, permethylated, and subjected to Sep-Pak cleanup. Data from the 35% acetonitrile fraction are shown. See Table IV for assignments.

 

View this table:
[in this window]
[in a new window]
 
TABLE IV
Assignments of molecular ([M + Na]+) ions observed in the MALDI spectrum for the products of O-glycans after periodate oxidation, reduction, and permethylation

The denoted C4 contains C1 to C4 carbons attached to the 3-linked antenna, whereas the denoted C2 contains C5 and C6 carbons attached to the 6-linked antenna. NeuAc residues lack C8 and C9 as a result of periodate cleavage.

 


View larger version (17K):
[in this window]
[in a new window]
 
FIG. 12.
CAD-MS/MS spectra of the [M + Na]+ molecular ions at m/z 807 (A), m/z 835 (B), and m/z 936 (C). Sequence-informative fragment ions are shown on the structures. The non-reducing HexNAc residues are a result of periodate oxidation and subsequent hydrolytic loss of terminal Gal and Fuc residues in the original glycans.

 


View larger version (19K):
[in this window]
[in a new window]
 
FIG. 12.
CAD-MS/MS spectra of the [M + Na]+ molecular ions at m/z 807 (A), m/z 835 (B), and m/z 936 (C). Sequence-informative fragment ions are shown on the structures. The non-reducing HexNAc residues are a result of periodate oxidation and subsequent hydrolytic loss of terminal Gal and Fuc residues in the original glycans.

 

Structural Conclusions—Taking into consideration the MALDI, FAB, linkage, CAD-ES-MS/MS, and periodate data, we conclude that the major O-glycans found in CA125 have the structures shown in the annotations of Fig. 9.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Analysis of the structure of CA125 is essential for determining the physiological role of this very significant tumor antigen. However, its very high molecular weight and mucinous nature pose major obstacles for performing structural characterization studies. Here we report the sequence of the glycans linked to CA125. To the best of our knowledge, this study outlines the first exhaustive analysis of CA125 glycans derived from epithelial ovarian tumor cells.

Previously, Lloyd et al. employed indirect approaches for characterizing the oligosaccharides associated with CA125 derived from OVCAR-3 cells (9). Because of the relatively insensitive methods of detection that were employed, only a limited number of oligosaccharides were analyzed. We have circumvented these problems by using very sensitive and precise biophysical methods of carbohydrate structural analysis. This approach has provided unambiguous sequencing of the oligosaccharides linked to CA125.

Core 1 and core 2 type glycans are the major O-linked glycans expressed on CA125 based on the present results. Minor amounts of core type 2 glycans with up to two polylactosamine units were also detected. A small subset of the O-linked glycans are also decorated with Lewisx antigens on their terminal ends. O-linked glycans carrying two fucose residues were also detected. However, because these difucosylated glycans were present in very low amounts, it was not possible to determine whether these O-linked glycans carried two Lewisx epitopes or one Lewisy antigen or two blood group H epitopes. Lloyd et al. also reported the presence of both Lewisx and Lewisy antigens on CA125 but suggested that they were major components (9). This discrepancy cannot be readily explained, because CA125 analyzed in both studies was isolated from the OVCAR-3 cells using identical tissue culture conditions. However, more definitive methods of structural analysis were employed in the current study.

An unusual feature associated with CA125 O-linked glycans is the expression of branched core 1 structures. Only uromodulin, a pregnancy-associated isoform of the Tamm-Horsfall glycoprotein, expresses this sequence (18). Like uromodulin, these branched core 1 structures linked to CA125 carry sialic acid and fucose. However, the branches of CA125 O-glycans appear to be either sialylated or fucosylated in contrast to uromodulin, which carries abundant sialyl Lewisx moieties. The functional roles of these unusually branched core 1 antennae in the core type 2 O-linked glycans have not been clearly established.

Conclusive evidence for the N-glycosylation of CA125 was also presented in the current study. These results are consistent with the existing literature. Previous compositional analyses indicate that CA125 from either OVCAR-3 cells or amniotic fluid contain mannose, albeit in much lower amounts than does Gal, GlcNAc, or GalNAc (9, 19). However, significantly higher amounts of mannose have been reported to be associated with CA125 isolated from the ovarian tumor cell line OVCA 433 (5).

Clinical CA125 samples exhibit reduced binding to OC125 after treatment with PNGase F (10), an enzyme that specifically clips N-glycans. Treatment of CA125 with PNGase F resulted in a significant reduction in the molecular mass of this mucin and also a lower affinity for OC125 as detected by Western blot analysis (data not shown). The reduction in binding of OC125 to CA125 cannot be readily explained, because this antibody recognizes a peptide epitope within the CA125 molecule (7). However, these observations clearly provide supportive evidence for the presence of N-linked glycans on CA125.

CA125 isolated from tumor tissues and the sera of ovarian cancer patients is also specifically retained during affinity chromatography on ConA-agarose in a carbohydrate-dependent manner, consistent with the association of high mannose and biantennary complex N-linked glycans with this mucin (10). A prior study by Nagata et al. confirms that CA125 isolated from serum and ascites fluid of ovarian cancer patients also binds to erythroagglutinating phytohemagglutinin (EPHA) (10), consistent with the presence of biantennary and triantennary bisecting type glycans. Therefore both biophysical and lectin binding studies are consistent with these earlier findings.

A recent study (4) indicates that vaccination with the murine monoclonal anti-idiotypic antibody ACA125 elicits an immune response to CA125 in a subset of patients. Ovarian cancer patients that mediate potent anti-CA125 response show an increase in their survival time from 5.3 + 4.3 months to 19.9 + 13.1 months (4, 20). This result suggests that CA125 could play a crucial role as a surface tumor antigen that facilitates antibody-dependent, cell-mediated cytotoxicity.

Another distinct possibility is that CA125 could promote tumor progression by modulating the human immune response, perhaps by using its carbohydrate sequences as functional groups. The N-linked glycans associated with CA125 fall into two major classes, i.e. bisecting complex type and high mannose type oligosaccharides.

Recent data indicate that the specific cell surface lectin DC-SIGN interacts with ICAM-3 to enable screening of the MHC-peptide complexes required for subsequent formation of the immunological synapse (reviewed in Refs. 21 and 22). DCSIGN also acts as a novel HIV-1 attachment receptor that facilitates infection of T lymphocytes (23, 24). More recent data indicate that DC-SIGN specifically recognizes both high mannose type N-glycans and Lewisx/Lewisy-terminated glycans (25, 26). It is possible that CA125 could interfere with immunological synapse formation by binding to DC-SIGN via its high mannose type N-glycans. This possibility is under investigation.

Tumor survival and expansion likely rely upon overcoming potential cell-mediated immune responses. It is well established that tumor cells often down-regulate MHC class I molecules during the later stages of tumor development (27 and 28) (reviewed in Ref. 29). This loss is thought to enable tumor cells to escape antigen-driven lytic responses by class I-restricted cytotoxic T lymphocytes, thereby allowing them to evade this adaptive arm of the immune cell-mediated response. Epithelial ovarian tumor cells often lose their MHC class I molecules (30, 31). However, this evasion should then make the tumor cells more susceptible to NK cells, which detect and kill MHC class I negative tumor cells (reviewed in Ref. 32).

Bisecting type N-glycans are found on a great variety of different glycoproteins in the human. They are major sequences associated with human immunoglobulins, MHC class I molecules, and glycophorin, a major erythrocyte membrane glycoprotein (3335). Bisecting type glycans are also prominently expressed on human gametes (36) and neurons (37).

Bisecting type N-glycans may play a role in inducing suppression of NK cell-mediated cytolytic responses when presented on cell surfaces. Electroinsertion of glycophorin, a glycoprotein expressing primarily bisecting type glycans, into the plasma membrane of K562 cells decreases their cytolysis by human NK cells (38). Similar insertion of glycophorin lacking its N-glycans does not mediate this protective effect (38). In addition, soluble forms of glycophorin also do not inhibit NK cell-mediated responses in vitro.

K562 cells overexpressing N-acetylglucosaminyltransferase III, the enzyme that synthesizes the bisecting GlcNAc sequence, also exhibit greatly decreased sensitivity to NK cell-mediated lysis (39). Increased expression of N-acetylglucosaminyltransferase III is also positively correlated with the increased metastatic potential of tumor cells (40, 41).

An attractive hypothesis is that epithelial ovarian tumor cells down-regulate their MHC class I molecules and up-regulate CA125 expression on their cell surfaces in the late stages of ovarian cancer to escape responses mediated both by cytotoxic T lymphocytes and NK cells. CA125 or other glycoproteins secreted by ovarian tumors may not suppress NK cell-mediated responses. However, transient expression of CA125 on the surfaces of ovarian tumor cells may enable them to evade NK cell-mediated responses. This reasoning could explain why CA125 expression increases during tumor progression. This linkage is currently under investigation.

This type of subterfuge could also be linked to the reproductive imperative. NK cells constitute 70–80% of the maternal immune effector cells in the uterus during pregnancy (42). Localization studies indicate that CA125 is expressed in significant amounts by the human decidua and the amnion (4345). Serum CA125 levels of women in their first trimester of pregnancy are elevated (43, 45). It is therefore possible that, similar to its potential tumor promoting activities, CA125 may also play a role in protecting the human embryo from the maternal immune response.

Human germ cells down-regulate class I expression during their development, yielding gametes that lack MHC class I molecules (46, 47). Human gametes also express substantial amounts of bisecting type glycans on their surfaces based on lectin binding studies (36). Therefore, tumor cells may be employing the same strategy that gametes use to avoid potential immune responses.

In addition, several glycoconjugates expressed in the human male and female reproductive system or in other organs under the influence of pregnancy-related hormones have been shown to suppress specific immune responses in vitro (reviewed in Ref. 48). Many of the carbohydrate sequences linked to these glycoconjugates are also prevalently expressed on persistent human pathogens such as HIV-1, helminthic parasites, Helico-bacter pylori, and other human tumor cells (48). A hypothetical model linking pathological subterfuge to the suppression of immune responses essential to fulfill the reproductive imperative has been proposed (48, 49).

The expression of many of the same carbohydrate sequences (Lewisx sequences, bisecting type N-glycans, and high mannose type N-glycans) previously implicated in immune suppression and reproduction on CA125 is entirely consistent with this model. Another rather remarkable correlation in this respect is that the N-glycans associated with CA125 closely resemble the oligosaccharides attached to the viral coat glycoprotein gp120 synthesized in the HIV-infected human T lymphoblastoid cell line H9 (50). The fact that both CA125 and gp120 carry similar N-glycans may not be purely coincidental. Each could employ their oligosaccharides as functional groups to induce suppression of both innate and adaptive arms of the human immune response. By using this type of subterfuge, tumor cells and HIV-1 could, in effect, couple their survival to the human reproductive imperative.

In conclusion, we have characterized the oligosaccharides associated with CA125 derived from OVCAR-3 tumor cells. The results of lectin and antibody binding data suggest that similar carbohydrate sequences are expressed on CA125 from ovarian cancer patients. This data, together with the genomic and proteomic analysis reported earlier, will provide a solid basis for understanding the physiological role of CA125, defining new methods for detecting this tumor marker, and developing novel strategies that effectively combat epithelial ovarian cancer.


    FOOTNOTES
 
* This work was supported by Jeffress Research Grant J-584 and the Elsa U. Pardee Foundation (to M. S. P.), the Biotechnology and Biological Sciences Research Council and the Wellcome Trust (to A. D. and H. R. M.), and National Institutes of Health Grant R01 HD35652 (to G. F. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a Malaysian Government Scholarship. Back

** Member of the Consortium for Functional Glycomics supported by NIGMS, National Institutes of Health and to whom correspondence may be addressed. Tel.: 757-446-5653; Fax: 757-624-2269; E-mail: clarkgf{at}evms.edu.

{ddagger}{ddagger} Member of the Consortium for Functional Glycomics supported by NIGMS, National Institutes of Health and to whom correspondence may be addressed. Tel.: 44-207-225-5219; Fax: 44-207-225-0458; E-mail: a.dell{at}ic.ac.uk.

§§ To whom correspondence may be addressed. Tel.: 757-446-5755; Fax: 757-624-2269; E-mail: pantankms{at}evmsmail.evms.edu.

1 The abbreviations used are: PAS, periodic acid-Schiff; CAD, collisionally activated decomposition; ConA, concanavalin A; DC-SIGN, dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin; ES, electrospray; FAB, fast atom bombardment; GC, gas chromatography; Hex, hexose; HexNAc, N-acetylhexosamine; LacNAc, N-acetyllactosamine; MALDI-TOF, matrix assisted laser desorption ionization time of flight; MHC, major histocompatibility complex; MS, mass spectrometry; NK, natural killer; PNGase F, peptide N-glycosidase F. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bast, R. C., Jr., Feeney, M., Lazarus, H., Nadler, L. M., Colvin, R. B., and Knapp, R. C. (1981) J. Clin. Invest. 68, 1331–1337[Medline] [Order article via Infotrieve]
  2. Bast, R. C., Jr., Klug, T. L., St. John, E., Jenison, E., Niloff, J. M., Lazarus, H., Berkowitz, R. S., Leavitt, T., Griffiths, C. T., Parker, L., Zurawski, V. R., Jr., and Knapp, R. C. (1983) N. Engl. J. Med. 309, 883–887[Abstract]
  3. Maggino, T., and Gadducci, A. (2000) Eur. J. Gynaecol. Oncol. 21, 64–69[Medline] [Order article via Infotrieve]
  4. Wagner, U., Kohler, S., Reinartz, S., Giffels, P., Huober, J., Renke, K., Schlebusch, H., Biersack, H. J., Mobus, V., Kreienberg, R., Bauknecht, T., Krebs, D., and Wallwiener, D. (2001) Clin. Cancer Res. 7, 1154–1162[Abstract/Free Full Text]
  5. Davis, H. M., Zurawski, V. R., Jr., Bast, R. C., Jr., and Klug, T. L. (1986) Cancer Res. 46, 6143–6148[Abstract]
  6. Yin, B. W., and Lloyd, K. O. (2001) J. Biol. Chem. 276, 27371–27375[Abstract/Free Full Text]
  7. O'Brien, T. J., Beard, J. B., Underwood, L. J., Dennis, R. A., Santin, A. D., and York, L. (2001) Tumour Biol. 22, 348–366[CrossRef][Medline] [Order article via Infotrieve]
  8. O'Brien, T. J., Beard, J. B., Underwood, L. J., and Shigemasa, K. (2002) Tumour Biol. 23, 154–169[CrossRef][Medline] [Order article via Infotrieve]
  9. Lloyd, K. O., Yin, B. W., and Kudryashov, V. (1997) Int. J. Cancer 71, 842–850[CrossRef][Medline] [Order article via Infotrieve]
  10. Nagata, A., Hirota, N., Sakai, T., Fujimoto, M., and Komoda, T. (1991) Tumour Biol. 12, 279–286[Medline] [Order article via Infotrieve]
  11. Schultes, B. C., Baum, R. P., Niesen, A., Noujaim, A. A., and Madiyalakan, R. (1998) Cancer Immunol. Immunother. 46, 201–212[CrossRef][Medline] [Order article via Infotrieve]
  12. Dell, A., Khoo, K.-H., Panico, M., McDowell, R. A., Etienne, A. T., Reason, A. J., and Morris, H. R. (1993) in Glycobiology: A Practical Approach (Fukuda, M., and Kobata, A., eds), pp. 187–222, Oxford University Press, Oxford
  13. Dell, A., Reason, A. J., Khoo, K. H., Panico, M., McDowell, R. A., and Morris, H. R. (1994) Methods Enzymol. 230, 108–132[Medline] [Order article via Infotrieve]
  14. Albersheim, P., Nevins, D. J., English, P. D., and Karr, A. (1967) Carbohydr. Res. 5, 340–345[CrossRef]
  15. Haslam, S. M., Coles, G. C., Munn, E. A., Smith, T. S., Smith, H. F., Morris, H. R., and Dell, A. (1996) J. Biol. Chem. 271, 30561–30570[Abstract/Free Full Text]
  16. Chalabi, S., Easton, R. L., Patankar, M. S., Lattanzio, F. A., Morrison, J. C., Panico, M., Morris, H. R., Dell, A., and Clark, G. F. (2002) J. Biol. Chem. 12, 12
  17. Merkle, R. K., and Cummings, R. D. (1987) Methods Enzymol. 138, 232–259[Medline] [Order article via Infotrieve]
  18. Easton, R. L., Patankar, M. S., Clark, G. F., Morris, H. R., and Dell, A. (2000) J. Biol. Chem. 275, 21928–21938[Abstract/Free Full Text]
  19. Hanisch, F. G., Uhlenbruck, G., Peter-Katalinic, J., and Egge, H. (1988) Carbohydr. Res. 178, 29–47[CrossRef][Medline] [Order article via Infotrieve]
  20. Wagner, U., Schlebusch, H., Kohler, S., Schmolling, J., Grunn, U., and Krebs, D. (1997) Hybridoma 16, 33–40[Medline] [Order article via Infotrieve]
  21. van Kooyk, Y., and Geijtenbeek, T. B. (2002) Immunol. Rev. 186, 47–56[CrossRef][Medline] [Order article via Infotrieve]
  22. Geijtenbeek, T. B., Torensma, R., van Vliet, S. J., van Duijnhoven, G. C., Adema, G. J., van Kooyk, Y., and Figdor, C. G. (2000) Cell 100, 575–585[Medline] [Order article via Infotrieve]
  23. Curtis, B. M., Scharnowske, S., and Watson, A. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8356–8360[Abstract]
  24. Geijtenbeek, T. B., Kwon, D. S., Torensma, R., van Vliet, S. J., van Duijnhoven, G. C., Middel, J., Cornelissen, I. L., Nottet, H. S., KewalRamani, V. N., Littman, D. R., Figdor, C. G., and van Kooyk, Y. (2000) Cell 100, 587–597[Medline] [Order article via Infotrieve]
  25. Mitchell, D. A., Fadden, A. J., and Drickamer, K. (2001) J. Biol. Chem. 276, 28939–28945[Abstract/Free Full Text]
  26. Van Die, I., Van Vliet, S. J., Schiphorst, W. E., Bank, C. M. C., Appelmelk, B. J., Nyame, A. K., Cummings, R. D., Geijtenbeek, T. B., and van Kooyk, Y. (2002) Glycobiology 12, 641–642 (abstr.)
  27. Garrido, F., Festenstein, H., and Schirrmacher, V. (1976) Nature 261, 705–707[Medline] [Order article via Infotrieve]
  28. Garrido, F., Cabrera, T., Concha, A., Glew, S., Ruiz-Cabello, F., and Stern, P. L. (1993) Immunol. Today 14, 491–499[CrossRef][Medline] [Order article via Infotrieve]
  29. Garrido, F., and Algarra, I. (2001) Adv. Cancer Res. 83, 117–158[Medline] [Order article via Infotrieve]
  30. Ferguson, A., Moore, M., and Fox, H. (1985) Br. J. Cancer 52, 551–563[Medline] [Order article via Infotrieve]
  31. Le, Y. S., Kim, T. E., Kim, B. K., Park, Y. G., Kim, G. M., Jee, S. B., Ryu, K. S., Kim, I. K., and Kim, J. W. (2002) Exp. Mol. Med. 34, 18–26[Medline] [Order article via Infotrieve]
  32. Karre, K. (2002) Scand J. Immunol. 55, 221–228[CrossRef][Medline] [Order article via Infotrieve]
  33. Kornfeld, R., Keller, J., Baenziger, J., and Kornfeld, S. (1971) J. Biol. Chem. 246, 3259–3268[Abstract/Free Full Text]
  34. Barber, L. D., Patel, T. P., Percival, L., Gumperz, J. E., Lanier, L. L., Phillips, J. H., Bigge, J. C., Wormwald, M. R., Parekh, R. B., and Parham, P. (1996) J. Immunol. 156, 3275–3284[Abstract]
  35. Irimura, T., Tsuji, T., Tagami, S., Yamamoto, K., and Osawa, T. (1981) Biochemistry 20, 560–566[Medline] [Order article via Infotrieve]
  36. Patankar, M. S., Ozgur, K., Oehninger, S., Dell, A., Morris, H., Seppala, M., and Clark, G. F. (1997) Mol. Hum. Reprod. 3, 501–505[Abstract]
  37. Warner, T. G., deKremer, R. D., Sjoberg, E. R., and Mock, A. K. (1985) J. Biol. Chem. 260, 6194–6199[Abstract/Free Full Text]
  38. El Ouagari, K., Teissie, J., and Benoist, H. (1995) J. Biol. Chem. 270, 26970–26975[Abstract/Free Full Text]
  39. Nishikawa, A., Ihara, Y., Hatakeyama, M., Kangawa, K., and Taniguchi, N. (1992) J. Biol. Chem. 267, 18199–18204[Abstract/Free Full Text]
  40. Yang, X., Bhaumik, M., Bhattacharyya, R., Gong, S., Rogler, C. E., and Stanley, P. (2000) Cancer Res. 60, 3313–3319[Abstract/Free Full Text]
  41. Taniguchi, N., Miyoshi, E., Ko, J. H., Ikeda, Y., and Ihara, Y. (1999) Biochim. Biophys. Acta 1455, 287–300[Medline] [Order article via Infotrieve]
  42. Christmas, S. E., Bulmer, J. N., Meager, A., and Johnson, P. M. (1990) Immunology 71, 182–189[Medline] [Order article via Infotrieve]
  43. Jacobs, I. J., Fay, T. N., Stabile, I., Bridges, J. E., Oram, D. H., and Grudzinskas, J. G. (1988) Br. J. Obstet. Gynaecol. 95, 1190–1194[Medline] [Order article via Infotrieve]
  44. Bischof, P. (1993) Eur. J. Obstet. Gynecol. Reprod. Biol. 49, 93–98[Medline] [Order article via Infotrieve]
  45. Niloff, J. M., Knapp, R. C., Schaetzl, E., Reynolds, C., and Bast, R. C., Jr. (1984) Obstet Gynecol. 64, 703–707[Abstract]
  46. Anderson, D. J., Bach, D. L., Yunis, E. J., and DeWolf, W. C. (1982) J. Immunol. 129, 452–454[Free Full Text]
  47. Dohr, G. A., Motter, W., Leitinger, S., Desoye, G., Urdl, W., Winter, R., Wilders-Truschnig, M. M., Uchanska-Ziegler, B., and Ziegler, A. (1987) J. Immunol. 138, 3766–3770[Abstract/Free Full Text]
  48. Clark, G. F., and Patankar, M. S. (1997) Mol. Hum. Reprod. 3, 985–987[Free Full Text]
  49. Clark, G. F., Dell, A., Morris, H. R., Patankar, M. S., and Easton, R. L. (2001) Cells Tissues Organs 168, 113–121[CrossRef][Medline] [Order article via Infotrieve]
  50. Mizuochi, T., Matthews, T. J., Kato, M., Hamako, J., Titani, K., Solomon, J., and Feizi, T. (1990) J. Biol. Chem. 265, 8519–8524[Abstract/Free Full Text]