From the
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.
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ABSTRACT |
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INTRODUCTION |
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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 4060 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 2428%
(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.
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EXPERIMENTAL PROCEDURES |
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Isolation of CA125 from OVCAR-3 CellsIsolation 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 CA125Electrophoresis 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 ExperimentsLyophilized 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 CarboxymethylationReduction 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 DigestionCA125 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 GlycopeptidesPNGaseF 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 EliminationO-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 DigestionThe 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;
-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;
-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
-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--galactosidase
DigestionEndo-
-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 CleavageA 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 23 µl of ethylene glycol and incubation in the dark at room temperature for 3060 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).
MethanolysisThe 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 AnalysisPermethylation 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 AnalysisFAB 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 AnalysisMALDI 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 AnalysisLinkage 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 AnalysisCAD-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 1030 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.
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RESULTS |
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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).
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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 24512479, 46374665, 51055133, 66516661, 68196847, 72877315, 82238251, 99389966, 1009410122, 1086210872, and 1040610434 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 StrategyThe 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.
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Analyses of PNGase F Digests Reveals Abundant N-Glycosylation of
CA125N-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
(Hex59HexNAc2), and complex type structures of
compositions
NeuAc01Fuc02Hex57HexNAc47.
After -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
-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.
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CAD-ES-MS/MS Defines Sites of FucosylationCollisionally 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.
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Linkage Analysis of N-Glycans Released by PNGase FLinkage 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.
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Polylactosamine-type Antennae Revealed by
Endo--galactosidase DigestionDigestion with
endo-
-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
14[Fuc
13]GlcNAc
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]+.
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Sequential Enzyme Digestions Define the Cores of the Polylactosaminyl
GlycansTo define the core structures that carry polylactosaminyl
chains, N-glycans were sequentially digested with
-galactosidase,
-N-acetylhexosaminidase, and
endo-
-galactosidase. Under these conditions, short sialylated or
fucosylated antennae are unaffected, whereas their polylactosaminyl
counterparts will be degraded by the endo-
-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
-galactosidase plus
-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-
-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.
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Structural ConclusionsTaking 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.
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Additional Evidence for Expression of N-Linked Glycans on CA125Our 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-GlycansPermethylated
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
(Gal13GalNAc), whereas the members of the second family have
compositions corresponding to core type 2
(Gal
13(GlcNAc
16)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).
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Linkage Analysis of the O-GlycansThe 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.
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Characterization of O-Glycan Structures by CAD-ES-MS/MS AnalysesEach 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).
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Periodate Cleavage Confirms Core Types and Antennae Backbones of O-GlycansReductively 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.
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Structural ConclusionsTaking 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.
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DISCUSSION |
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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 7080% 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.
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FOOTNOTES |
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Supported by a Malaysian Government Scholarship.
** 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.
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.
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