Molecular forms and microheterogeneity of the oligosaccharide chains of pregnancy-associated CA125 antigen

Miroslava M. Jankovic1 and Bojana S. Tapuskovic

Institute for the Application of Nuclear Energy – INEP, Belgrade, Banatska 31b, 11080 Zemun-Belgrade, Serbia and Montenegro

1 To whom correspondence should be addressed. Email: miraj{at}inep.co.yu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The cancer antigen CA125 has a very complex molecular architecture in terms of both protein backbone and oligosaccharide chains. In this study, we examined the molecular forms and microheterogeneity of oligosaccharide chains of pregnancy-associated CA125, as a first step towards gaining an insight into its possible involvement as a ligand in carbohydrate-dependent interactions. The glycobiochemical properties of CA125 may be of diagnostic and biomedical importance as specific markers of physiological and pathological conditions of early pregnancy, as well as targets in different therapeutic procedures. METHODS: Pregnancy-associated CA125 was characterized by gel filtration and ion-exchange chromatography, followed by lectin-affinity chromatography with a panel of plant lectins as ligands. RESULTS: CA125 antigen isolated from first trimester placental extract was found to be heterogeneous in respect to molecular mass and the existence of different glyco-isoforms. Thus, elution profiles from the lectin-affinity columns demonstrated molecular subpopulations bound with low, intermediate and high affinity. Under the applied experimental conditions, CA125 bound most strongly to Triticum vulgaris agglutinin (WGA) and Ricinus communis agglutinin (RCA), but low affinity interactions occurred with the other lectins tested. CONCLUSIONS: The assessment of the carbohydrate composition of N- and O-glycans of pregnancy-associated CA125 was in general agreement with available data on CA125 of cancer origin. The main difference was observed in reactivity to Canavalia ensiformis agglutinin (ConA) and Phaseolus vulgaris erythroagglutinin (PHA-E) binding.

Key words: CA125 antigen/glycosylation/lectin-binding pattern/placenta/pregnancy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
CA125, coelomic epithelium-related antigen, is defined by the monoclonal antibody, OC125 (Bast et al., 1981Go). This antibody was obtained by somatic hybridization of spleen cells from mice immunized with the ovarian carcinoma cell line, OVCA433 (Bast et al., 1981Go, 1983Go). Accumulated experimental evidence has shown that CA125 epitopes reside on a molecule of very complex architecture in terms of both protein backbone and oligosaccharide structures (Davis et al., 1986Go). Detailed structural characterization is still lacking and only part of this gene has been cloned so far (O'Brien et al., 1998Go, 2001; Yin and Lloyd, 2001Go).

The CA125 molecule is composed of an N-terminal domain, a tandem repeat region and a cytoplasmic domain and there is a typically high content of proline, serine and threonine (Yin and Lloyd, 2001Go). Although the initial data indicated that the N-terminal domain is made up of 1637 amino acids, it has recently been shown to be vastly larger, i.e. an additional 10 431 amino acids have been found (O'Brien et al., 2001Go, 2002Go). The tandem repeat domain consists of 40–60 repeats, each 156 amino acids long with a disulphide bridge loop of 19 amino acids (O'Brien et al., 2001Go). Although the overall structure is well conserved, only a few repeats have identical amino acid sequences. The cytoplasmic tail is relatively short, consisting of 256 amino acids, and it possesses a phosphorylation site (O'Brien et al., 2001Go). The CA125 protein backbone is phosphorylated on either or both serine and threonine before secretion (Nagata et al., 1991Go; O'Brien et al., 1998Go), after which the molecule is dephosphorylated. Its release is thought to be linked to the epidermal growth factor-receptor signal transduction pathway (Konishi et al., 1994Go; Fendrick et al., 1997Go). As for glycosylation, O-linked oligosaccharide chains forming a hairbrush structure at its N-terminal domain predominate (Davis et al., 1986Go; Wong et al., 2003Go). The molecule contains 28% of carbohydrates including N-linked sugar chains. The oligosaccharide units are rich in galactose, GalNAc, GlcNAC, mannose, sialic acid and fucose forming distinct carbohydrate epitopes (Wong et al., 2003Go).

The available structural data on CA125 mostly refer to isolates from ovarian carcinoma cell line(s) or malignant tissue. However, CA125 is also expressed in normal physiological conditions. During embryonic development, CA125-related antigen has been localized in the gastrointestinal tract, enteric duct, the mesonephric duct, skin, periderm, notocord, myocardium, remnant of the umbilical cord and the amnion (Zurawski et al., 1988Go; Hardardottir et al., 1990Go). It was also detected in normal adult tissues: endocervix, endometrium, tubes, pleura, pericard, peritoneum, active secreting mammary gland, apocrine sweat glands, and occasionally in the intestine, lung and kidney (Zurawski et al., 1988Go; Hardardottir et al., 1990Go). Moreover, CA125 has been found in sera, breast milk, cyst fluid, amniotic fluid, ascites, cervical and uterine secretion, seminal plasma and WISH cell culture medium (Halila, 1985Go; Hanisch et al., 1985Go; de Bruijn et al., 1986Go; O'Brien et al., 1986Go). CA125 concentrations change in conditions affecting the endometrium, such as pregnancy, menstruation and endometriosis (Kan et al., 1992Go; Montz, 1992Go).

In this study, first trimester human placental extract was used as a readily available source of pregnancy-associated CA125 antigen. It was characterized by gel filtration and ion-exchange chromatography followed by lectin affinity chromatography with a panel of plant lectins of different carbohydrate specificity. We examined the molecular forms and microheterogeneity of the oligosaccharide chains of CA125, as a first step towards gaining an insight into its possible involvement as a ligand in carbohydrate-dependent interactions. Carbohydrate recognition is an initial step in cell adhesion, migration and invasion and is very important in the regulation of growth, differentiation and development (Sinowatz et al., 1997Go; Zanetta, 1997Go; Perillo et al., 1998Go). An understanding of the signal and recognitive properties of CA125–oligosaccharide epitopes may have significant biomedical implications in creating a strategy to modify the carbohydrate-dependent interactions of CA125. The use of glycomimetics, carbohydrate-blocking or modifying drugs and carbohydrate-based vaccines is still in its infancy, but this field of medicinal glycoscience is rapidly growing and shows promise (Davis, 1999Go; Macmillan and Daines, 2003Go). Employing this approach, it should be possible to influence and control cell motility, adhesion and invasion during normal or neoplastic growth and differentiation. Elucidation of the molecular nature of CA125 may help to explore its potential as a diagnostic marker. This might be achieved by the development of diagnostic assays that take advantage of particular properties of those oligosaccharide chains which are different in normal and pathological conditions.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Materials
Mouse monoclonal anti-CA125 antibodies, clone X325 (M-11-like) and clone X306 (OC125-like), were from HyTest (PharmaCity, Turku, Finland). Radioiodine (125I) was from the Institute of Isotopes Co. Ltd (Budapest, Hungary). Bovine serum albumin (BSA), mannose (Man), fucose, N-acetylglucosamine and molecular mass markers were from Sigma (St Louis, USA). Lactose and galactose (Gal) were from ICN Biochemicals (Cleveland, Ohio, USA). Sephadex G-75, Sepharose 4B and diethylaminoethyl (DEAE) Sephadex A-50 were purchased from Pharmacia Fine Chemicals AB (Uppsala, Sweden). Affinity columns with immobilized plant lectins were from Vector Laboratories (Burlingame, USA). The concentration of CA125 was estimated immunoradiometrically using an ELSA CA125 II kit (Cis-bio international subsidiary of Shering S.A., Gif-sur-Yvette cedex, France). All other chemicals were reagent grade.

Preparation of placental extract
First trimester human placentas, from patients undergoing elective termination of pregnancy at 6–12 weeks of gestation as estimated from the date of the last menstrual period were used with local ethics committee approval. Tissue was collected in cold 0.1 mol/l phosphate-buffered saline (PBS), pH 7.2, brought to the laboratory within 60 min. It was washed free of blood and contaminating tissue, decidua and the placental bed. The tissue was homogenized in 0.1 mol/l PBS, pH 7.2, and the cytosol fraction was separated by ultracentrifugation at 36 000 r.p.m. for 60 min in a Beckman L8-60M ultracentrifuge (Beckman Instruments, Inc., Palo Alto, CA, USA). The placental extract was used for further examination immediately or stored at –20°C, until processed.

Gel filtration
Placental extract was separated on a Sepharose 4B column (bed volume 40 ml) equilibrated and eluted with 0.1 mol/l PBS, pH 7.2. The optical density at 280 nm of each fraction (0.5 ml) was measured using a double beam spectrophotometer (CE594 CECIL, Cambridge, UK). CA125-Immunoreactivity of each fraction (0.1 ml) was recorded by solid phase assay using monoclonal anti-CA125 (clone X-325) for capture and radiolabelled monoclonal anti-CA125 (clone X-306, 0.1 ml) as the tracer, after overnight incubation at room temperature. After rinsing the tubes with distilled water (3x1 ml), the bound radioactivity (c.p.m.) was counted on an Isomedix 4/6000 gamma counter (ICN biochemicals, Cleveland, OH, USA). The immunoreactive fractions (20–35) were pooled, concentrated by ultrafiltration, dialysed and used for further characterization. The column was calibrated with BSA (66 kDa) and internal reference standard thyroglobulin (660 kDa) as molecular mass standards.

Ion-exchange chromatography
Ion-exchange chromatography of placental extract was performed on a DEAE Sephadex A-50 column (20 ml bed volume) equilibrated with 50 mmol/l Tris–HCl buffer, pH 7.6. The column was washed free of protein with the same buffer and the bound material was eluted batchwise using: 50 mmol/l NaCl, 100 mmol/l NaCl, 200 mmol/l NaCl, 1 mol/l NaCl and 3 mol/l NaCl in 50 mmol/l Tris–HCl buffer, pH 7.6. Fractions (2 ml) were collected and CA125 immunoreactivity was determined as described above.

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE)
Pregnancy-associated CA125 was resolved on 6% separating gel with 3% stacking gel under denaturating and reducing conditions, according to Laemmli (1970)Go. The gel was calibrated with the following molecular mass standards: myosin 205 kDa, {beta}-galactosidase 116.7 kDa and phosphorylase b 97.4 kDa. The gel was stained with silver nitrate using a Roti black P kit (Carl Roth GambH+Co., Karlsruhe, Germany) according to the manufacturer's instructions.

Mass spectrometric analysis
The CA125 preparation was subjected to peptide mass fingerprinting analysis at the Proteome Factory AG (Berlin, Germany). Empirical data were matched to theoretical mass values with the FindPept and FindMode tools at www.expasy.org/tools, taking into account non-specific cleavages as well as post-translational modification of the discrete masses obtained. Peptide mass maps for human CA125 (entry code Q96RK2/Q8WX17) from the SWISS-Prot/TrEMBL databases were searched as templates.

Iodination
Isolated CA125 and anti-CA125 antibody were labelled with 125I, using the chloramine T method as described by Greenwood et al. (1963)Go. The labelled CA125 was separated from free iodine on a Sephadex G-75 column (10 ml bed volume), equilibrated with 0.1 mol/l PBS, pH 7.2, containing 0.05% bovine serum albumin.

Lectin affinity chromatography
Affinity chromatography of [125I]CA125 was performed on columns with the following immobilized plant lectins: RCA I (Ricinus communis agglutinin I), Con A (lectin from Canavalia ensiformis), WGA (wheat germ agglutinin), PHA-E (Phaseolus vulgaris erythroagglutinin), PHA-L (Phaseolus vulgaris leukoagglutinin), UEA (Ulex europaeus agglutinin), AAA (Aleuria aurantia agglutinin), SNA (Sambucus nigra agglutinin), MAA (Maackia amurensis agglutinin), PNA (Arachis hypogaea agglutinin), SBA (Glycine max agglutinin), WFA (Wisteria floribunda agglutinin). The bed volume was 5 ml for the Con-A, RCA, WGA, MAA, PNA and SBA columns and 2 ml for the AAA, UEA, PHA-L and PHA-E, SNA and WFA columns. A common chromatographic scheme was applied to all columns, according to the manufacturer's instructions. [125I]CA125 (0.5 ml; 300 000–400 000 c.p.m.) was loaded on each of the columns and after 3 h incubation at room temperature, eluted fractions (1 ml for 5 ml columns or 0.5 ml for 2 ml columns) were collected and the radioactivity of each was recorded. Unbound and retarded fractions were eluted with binding buffer (0.1 mol/l PBS pH 7.2), except for the Con-A column, which was washed with 0.1 mol/l acetate buffer pH 6.0, supplemented with 100 mmol/l CaCl2, MgCl2 and MnCl2. Specifically bound fractions were eluted by adding the corresponding competitive sugars: 0.1 mol/l mannose (for Con-A), 0.1 mol/l lactose (for SNA and RCA), 0.1 mol/l galactose (for PNA and SBA), 0.1 mol/l N-acetylglucosamine (for PHA-E, PHA-L and WGA) and 0.1 mol/l fucose (for UEA and AAA). Finally, tightly bound fractions were eluted with low pH buffers: 0.1 mol/l glycine–HCl, pH 3.0 (for RCA, PHA-E, PHA-L, RCA, AAA and UEA), 0.2 mol/l acetic acid (for WGA) or 0.3 mol/l lactose in 0.2 mol/l acetic acid (for SNA). The Con-A column was finally eluted with 0.1 mol/l borate buffer, pH 8.4. WGA was additionally eluted with 0.5 mol/l N-acetylglucosamine in 0.2 mol/l acetic acid and 1 mol/l KCl in 10% dioxan. Since there is no appropriate competitive sugar for MAA and WFA, these columns were eluted with 0.2 mol/l acetic acid.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Molecular forms of pregnancy-associated CA125
Molecular forms of CA125 in the cytosol extract of first trimester pregnancy placenta were initially characterized by gel filtration and ion-exchange chromatography. Gel filtration of soluble placental extract on Sepharose 4B revealed several protein peaks at 280 nm (Figure 1). When these fractions were probed in the CA125 assay, one CA125-immunoreactive peak was detected in the very high molecular mass region (column void volume), but immunoreactivity tailed all the way down to species of ~100 kDa.



View larger version (10K):
[in this window]
[in a new window]
 
Figure 1. Sepharose 4B gel filtration of human first trimester placental extract. The elution was monitored by measuring: optical density at 280 nm (a) and CA125 immunoreactivity (b) in each fraction. Fraction volume was 1 ml. 79x59 mm (72x72 DPI).

 
Ion-exchange chromatography of placental extract on Sephadex DEAE A50 with a discontinuous gradient of NaCl (50 mmol/l–3 mol/l NaCl) revealed one major CA125-immunoreactive peak eluted with 1 mol/l NaCl, in addition to minor peaks which appeared in the non-bound fraction and in fractions eluted with 200 mmol/l NaCl and 3 mol/l NaCl (Figure 2).



View larger version (12K):
[in this window]
[in a new window]
 
Figure 2. DEAE Sephadex A-50 ion-exchange chromatography of human first trimester placental extract. The elution was monitored by measuring CA125 immunoreactivity (c.p.m.) in each fraction. Fraction volume was 2 ml. Arrows indicate the start of elution with a discontinuous gradient of NaCl. 79x59 mm (72x72 DPI).

 
Electrophoretic and mass spectrometric analysis of the CA125-immunoreactive preparation
The CA125-immunoreactive peaks separated by Sepharose 4B gel filtration (Figure 1, volumes 20–35) during several individual runs were pooled and concentrated by ultrafiltration. When analysed by SDS–PAGE, one broad band just below the stacking gel was detected by silver staining (Figure 3). No lower molecular mass bands were observed. The isolated pregnancy-associated CA125 was subjected to tryptic digestion and subsequently analysed by mass spectrometry. When the estimated experimental monoisotopic masses were compared with virtually digested protein masses using FindMod and FindPep tools, molecular ions mapping to residues 541–555, 984–989, 1066–1076, 1109–1120, 1121–1135, 2194–2197, 2498–2509, 2986–2989, 3799–3803, 3805–3810, 3811–3821, 4182–4194, 4304–4308, 4461–4465, 4738–4742, 4761–4772, 4773–4780, 4900–4905, 4917–4928, 5362–5366, 5429–5440, 5518–5522, 5717–5722, 5765–5775, 5879–5890, 5998–6008, 6021–6027, 6035–6046, 6077–6087, 6154–6160, 6461–6465, 6471–6479, 6499–6513, 6514–6521, 6717–6721 and 6840–6850 were identified. Among the experimentally obtained peptides that matched over the entire CA125 sequence, there were molecular ions mapping to residues 6021–6027 and 6514–6521, which correspond to the well-conserved tandem repeat region typical of mucin primary structure. In addition, other peptide matches based on rules taking into account post-translational modifications, such as glycosylation, were also found.



View larger version (32K):
[in this window]
[in a new window]
 
Figure 3. SDS–PAGE of pregnancy-associated CA125. The gel was stained with silver. Positions of molecular mass standards (kDa) are marked with arrows. The concentration of isolated CA125 antigen was 6000 IU/ml. 32x59 mm (72x72 DPI).

 
Lectin-affinity chromatography of pregnancy-associated CA125 antigen
Isolated CA125 antigen was radiolabelled and subjected to lectin-affinity chromatography on columns with immobilized plant lectins in order to assess its carbohydrate composition. The representative elution profiles are shown in Figure 4.



View larger version (16K):
[in this window]
[in a new window]
 
Figure 4. Lectin affinity chromatography of [125I]CA125. Arrows indicate the start of elution with: (1) 0.1 mol/l mannose (for Con-A), 0.1 mol/l lactose (for SNA and RCA), 0.1 mol/l galactose (for PNA and SBA), 0.1 mol/l N-acetylglucosamine (for PHA-E, PHA-L and WGA) and 0.1 mol/l fucose (for UEA and AAA); (2) 0.1 mol/l glycine–HCl pH 3.0 (for RCA, PHA-E, PHA-L, RCA, AAA and UEA), 0.2 mol/l acetic acid (for WGA, WFA, MAA) or 0.3 mol/l lactose in 0.2 mol/l acetic acid (for SNA), 0.1 mol/l borate buffer, pH 8.4 (for Con-A); (3) 0.5 mol/l N-acetylglucosamine in 0.2 mol/l acetic acid; (4) 1 mol/l KCl in 10% dioxan. The elution was monitored by measuring radioactivity in each fraction. 84x113 mm (72x72 DPI).

 
Among the lectins used, WGA was found to interact most strongly with the examined antigen resulting in an inability to desorb the bound material from the affinity column. Recovery was only 33.8% of total antigen loaded and it consisted of a CA125 fraction not reactive with WGA. Thus, elution with a competitive sugar at 0.1 and 0.5 mol/l concentrations, followed by 1 mol/l KCl in 10% dioxan, released no radioactivity, i.e. the majority of the antigen remained on the affinity matrix.

RCA also showed high affinity binding to the examined CA125 preparation. However, the elution profile was different from that obtained with WGA. A significant sugar-elutable CA125 fraction appeared as one sharp peak, while the low pH buffer eluted no more radioactivity from the RCA column.

On the Con-A column, one broad peak of CA125 was released by 0.1 mol/l mannose but its abundance was lower than the WGA- and RCA-reactive fractions.

Pregnancy-associated CA125 was not retarded on the PHA-E column, whereas partial separation of at least two retarded CA125 subpopulations occurred on the PHA-L column.

Elution profiles from the SNA column were typical of similarly abundant sugar-elutable and tightly bound fractions, both of which appeared as broadened peaks. As for MAA reactivity, CA125 was moderately retarded during elution with buffer lacking sugar and a small low pH buffer-elutable peak, i.e. a tightly bound fraction was also separated.

The fucose-specific lectins, AAA and UEA, showed distinct binding patterns. Fucose eluted no radioactivity from the UEA column, whereas, in addition to retardation during elution, one sharp sugar-elutable peak of CA125 was separated on the AAA column.

A lactose-elutable fraction was obtained on the column with immobilized PNA as one broadened peak, but its abundance was lower than the non-bound and retarded CA125 fractions separated on the other columns with immobilized lectins.

Monotone elution fronts with buffer lacking sugar were typical for CA125 on the SBA column, and only a small amount of radioactivity was eluted with 0.1 mol/l galactose. On the WFA column, there was no sugar-eluted fraction, but one retarded CA125 fraction was partially separated.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the available literature, the molecular nature of CA125 antigen is variously claimed to be typical of a glycoprotein or a glycosylphosphoinositol-linked glycoprotein or a mucin-type molecule (Zurawski et al., 1988Go; Nagata et al., 1991Go). The data we report here for pregnancy-associated CA125 antigen support mucin-like properties. They were obtained using proteomic analysis and glycobiochemical characterization of the isolated antigen. The profiles obtained by gel filtration, ion-exchange chromatography and lectin affinity chromatography showed CA125 antigen from human first trimester placental extract to be heterogeneous in respect to molecular mass species and the existence of different glyco-isoforms. The immunoreactive epitopes, detected by a well-characterized pair of monoclonal antibodies (Nap et al., 1996Go; Nustad et al., 1996Go, 2002Go), were found to reside mainly on a high molecular mass fraction, which was further characterized. CA125 can exist in a number of molecular mass forms, among which the largest (1–4 MD) have been found in normal abdominal fluid and cervical mucus (de Bruijn et al., 1986Go; Nustad et al., 1998). Endogenous protease activity and continuous deglycosylation of oligosaccharide chains in body fluids are thought to be responsible for the heterogeneity in both size and charge (Konishi et al., 1994Go). Our results revealed that the majority of pregnancy-associated CA125 molecules bound strongly to anionic exchanger, whereas the elution profiles obtained from the lectin-affinity columns reflected its microheterogeneity, i.e. fractionation into molecular subpopulations bound with low, intermediate and high affinity. Strong binding to WGA was observed, confirming earlier data on its high affinity to various mucin-type molecules (Madiyalakan et al., 1996Go). RCA also strongly bound the examined antigen, but with lower affinity than WGA, since the bound fraction was completely eluted with the specific sugar solution. In contrast to this, ConA, SNA, MAA, AAA, SBA and PNA bound significantly smaller amounts of CA125. These and the other lectins tested here showed low affinity interactions with the examined preparation, i.e. CA125-retarded fractions were predominant. The carbohydrate specificity of the lectins used and the CA125 elution profiles pointed to the structural composition of the N- and O-oligosaccharide moietes. N-Glycan units can be divided into three groups: high mannose, complex and hybrid (Sharon and Lis, 1997Go). They share a common pentasaccharide core chain consisting of two N-acetylglucosamine (GlcNAc) and three mannose (Man) residues. Addition of GlcNAc, galactose (Gal), fucose and sialic acid (NeuNAc) leads to the formation of a diverse array of oligosaccharides that are termed complex. These latter units may have two to four branches arising from the core mannoses. Each branch, called antenna, generally consists of an GlcNAc/Gal unit, which can bear fucose or sialic acid. Structures that contain two, three and four branches are termed bi, tri, and tetraantennar, respectively. The hybrid type consists of a mannose branch and a complex branch.

Thus, pregnancy-associated CA125 showed moderate binding to Con A, retardation on PHA-L and no binding to PHA-E. The distinct binding specificities of these lectins were used to assess the type and branching of N-glycan side chains (Cummings and Kornfeld, 1982Go; Osawa and Tsuji, 1987Go; Kobata and Endo, 1992Go; Cummings, 1994Go). The results obtained suggest a low content of high mannose glycans, which are high affinity ligands for ConA, and a lack of GlcNAc linked {beta}1,4 to core mannose which is essential for PHA-E binding. In addition, retardation on PHA-L indicated the presence of multiantennary oligosaccharide chains. Previously published data on CA125 derived from OVCAR-3 cell line showed predominance of high mannose type and complex bisected type N-glycans (Won et al., 2003). The observed discrepancy can be related to the difference in CA125 source and physiological conditions.

The content and the position of fucose in pregnancy-associated CA125 oligosaccharide chains were deduced from the AAA- and UEA-binding patterns. Core fucosylated N-linked structures are high affinity ligands for AAA, whereas structures containing outer chain fucose residues do not bind but are retarded (Yamashita et al., 1985Go). The elution profile from the AAA column indicated the presence of fucose linked {alpha}1,6 to core mannose, as well as fucose linked {alpha}1,3 to side chains. However, {alpha}1,2-linked outer fucose residues cannot be claimed, since no binding to UEA was observed (Cummings, 1994Go, 1997).

The elution pattern from SNA and MAA suggested microheterogeneity in the extent and type of sialylation of pregnancy-associated CA125. SNA specifically recognizes sialic acid linked {alpha}2,6 to Gal, whereas MAA exclusively binds to sialic acid linked {alpha}2,3 to Gal (Goldstein and Poretz, 1986Go; Cummings, 1997Go). Our results indicate that both types of linkages as well as di- or monosialylated chains may have been present on the examined CA125 molecules. A relatively low level of sialylation of N-glycan was found on OVCAR-3 cell line derived CA125 and also sialic acid linked to Gal in the 3- and 6-positions in both N- and O-glycans (Wong et al., 2003Go).

The results of WGA and RCA affinity chromatography indicate the presence of polylactosamine structures on pregnancy-associated CA125-oligosaccharide chains (Gallagher et al., 1985). WGA binds strongly to GlcNAc and its {beta}1,4 oligomers and weakly to sialic acid (Wright, 1990). RCA exhibits high affinity towards terminal Gal linked {beta}1,4 to GlcNAc, and significantly lower affinity for terminal sugar linked {beta}1,3 (Osawa and Tsuji, 1987Go; Kobata and Endo, 1992Go; Cummings, 1994Go). However, WGA and RCA reactivity can also be due to the presence of O-glycans, as they have affinity for O-linked GlcNAc residues (Roquemore et al., 1994; Hayes et al., 1995Go). O-Glycans are very diverse and subclassified into at least four groups (Sharon and Lis, 1997Go; Brockhausen, 1999Go). Among them, the mucin type containing R-GlcNAc-Ser/Thr core and its distinct elongation by addition of specific oligosaccharides forming other core structures, as well as the O-linked GlcNAc-type containing only GlcNAc-Ser/Thr core, were addressed in this study. The lectins PNA and SBA preferentially bind O-glycans (Goldstein and Poretz, 1986Go; Osawa and Tsuji, 1987Go). PNA recognizes Gal{beta}1,3 GalNAc{alpha}1-Ser/Thr, whereas SBA shows higher affinity for GalNAc{alpha}1-Ser/Thr than for GalNAcGal{beta}1,3GalNAc{alpha}1-Ser/Thr. Taken together, the binding patterns of O-glycan-reactive lectins are in agreement with data on cancer-associated CA125 indicating Gal{beta}1–3GalNAc and (Gal{beta}1–3GlcNAc{beta}1–6) GalNAc bound to the protein backbone (Wong et al., 2003Go). The WFA-binding pattern (Cummings, 1997Go) pointed to the absence of oligosaccharides with terminal GlcNAc.

It is well known that pregnancy, similarly to malignant transformation, is accompanied by pronounced changes in the glycosylation pattern of related cells and tissues resulting in the expression and secretion of molecules with altered structural and physiological activity (Bohn, 1985Go; Hakomori, 1989Go; Brockhausen and Kuhns, 1997Go). The biological role of CA125 is still elusive. Regarding human reproduction, the possibility of its involvement in modulating the immune response, i.e. in protecting the human embryo from a maternal immune response, as well as an influence on cell adhesion have been speculated (Wong et al., 2003Go, Rump et al., 2004Go). However, the complexity of CA125 structure suggests that it can act as a multifunctional molecule, i.e. both protein and carbohydrate parts may be involved in different kinds of interactions. Recently, CA125 is classified as mucin-type molecule (Yin and Lloyd, 2001Go). Generally, mucins protruding from the surface of epithelial cells provide a physical barrier against chemical and microbial agents and inhibit sterically and electrostatically adhesive interactions between cell and external environment (Hilkens et al., 1992Go). O-Glycans of mucin-type maintain protein conformations, determine antigenicity and may influence the surface expression and function of cell receptors important for growth regulation (Varki, 1993Go; Brockhausen, 1999Go). Various mucins, including CA125, are reported to exibit anti-adhesive and gel-forming properties and they can significantly affect cell attachment, migration and invasion (Hardardottir et al., 1990Go; Fukuda, 2002Go). These processes based on carbohydrate recognition are hallmarks of placentation (Benirschke and Kaufmann, 1990Go; Pijnenborg, 1994Go; Vicovac and Aplin, 1996Go). In relation to this, the microheterogeneity of CA125 oligosaccharide chains may imply interactions with different carbohydrate recognition molecules as possible ligands. Thus, a role for CA125 in placentation, i.e. in the control of organization of the extracellular matrix and cell communication and motility, should be considered in the light of the dynamic network of many multifunctional molecules and its possible developmental changes in cell- and tissue-dependent expression, localization and ligand pattern.

Galectin-1 and mesothelin have been reported to bind to CA125 from ovarian cancer cell lines (Seelenmeyer et al., 2003Go; Rump et al., 2004Go). Interaction among galectins and complementary glycoconjugates is thought to be important for cell growth, immunomodulation, apoptosis, etc. (Zhou and Cummings, 1992; Leffler, 1997; Rabinovich et al., 2002Go). Specifically, galectin-1, a {beta}-galactoside-binding lectin, is abundantly expressed in pregnancy-related tissue with overlapping localization in relation to CA125 (Cuperlovic and Jankovic, 1989Go; Bevan et al., 1994Go; Vicovac et al., 1998Go). Although this may be important, possible interactions with other types of lectins or non-carbohydrate-binding ligands cannot be excluded. In order to obtain conclusive evidence about the role of the oligosaccharide chains of CA125, appropriate linkage to all putative endogenous ligands at different levels of cell and tissue organization should be studied further using various experimental approaches.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported by the Ministry of Science and Environmental Protection of the Republic of Serbia, project code 1504: Glycobiological aspects of physiological and pathophysiological processes.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bast RC Jr, Feeney M, Lazarus H, Nadler LM, Colvin RC and Knapp RC (1981) Reactivity of monoclonal antibody with human ovarian carcinoma. J Clin Invest 68, 1331–1337.[ISI][Medline]

Bast RC Jr, Klug TL, St John E, Jenison E, Niloff JM, Lazarus H, Berkowitz RS, Laevitt T, Griffiths CT, Parker L et al. (1983) A radioimmunoassay using monoclonal antibody to monitor the course of epihelial ovarian cancer. New Engl J Med 309, 883–887.[Abstract]

Benirschke K and Kaufmann P (1990) Pathology of the Human Placenta. Springer, New York, USA.

Bevan BH, Kilpatrick DC, Liston WA, Hirabayashi J and Kasai K (1994) Immunohistochemical localisation of a {beta}-d-galactoside-binding lectin at the human maternofetal interface. Histochem J 26, 582–586.[CrossRef][ISI][Medline]

Bohn H (1985) Biochemistry of placental proteins. In Bischof P and Klopper A (eds) Proteins of the Placenta. Karger, Basel, Switzerland, pp. 1–25.

Brockhausen I (1999) Pathways of O-glycan biosynthesis in cancer cells. Biochem Biophys Acta 1473, 67–95.[ISI][Medline]

Brockhausen I and Kuhns W (1997) Glycoproteins and Human Disease. Medical Intelligence Unit. Springer-Verlag, Heidelberg, Germany.

Cummings RD (1994) Use of lectins in analysis of glycoconjugates. Methods Enzymol 230, 66–86.[ISI][Medline]

Cummings RD (1997) Lectins as tools for glycoconjugate purification and characterization. In Gabius HJ and Gabius S (eds) Glycosciences Status and Perspectives. Chapman & Hall, Weinheim, Germany, pp. 191–198.

Cummings RD and Kornfeld S (1982) Characterization of the structural determinants required for the high affinity interaction of asparagine-linked oligosaccharides with immobilized Phaseolus vulgaris leukoagglutinating and Phaseolus vulgaris erythroagglutinating lectins. J Biol Chem 257, 11230–11234.[Abstract/Free Full Text]

Cuperlovic M and Jankovic M (1989) Endogenous beta-galactoside binding lectin of human placenta. In Genbacev O, Klopper A, and Beaconsfield R (eds) Placenta as a Model and Source. Plenum Press, New York, NY, pp. 145–153.

Davis B (1999) Recent developments in glycoconjugates. Chem Soc Perkin Trans 1, 3215–3237.

Davis HM, Zurawski VR Jr, Bast RC Jr and Klug TL (1986) Characterization of the CA 125 antigen associated with human epithelial ovarian carcinoms. Cancer Res 46, 6143–6148.[Abstract]

de Bruijn HW, van-Beeck-Calkoen C, Jager S, Duk JM, Aalders JG and Fleuren GJ (1986) The tumor marker CA125 is a common constituent of normal cervical mucus. Am J Obstet Gynecol 154, 1088–1091.[ISI][Medline]

Fendrick JL, Konishi I, Geary SM, Parmely TH, Quirk JG Jr and O'Brien TJ (1997) CA 125phosporylation is associated with its secretion from the WISH human amnion cell line. Tumor Biol 18, 278–289.[ISI]

Fukuda M (2002) Roles of mucin-type O-glycans in cell adhesion. Review. Biochim Biophys Acta 1573, 394–405.[ISI][Medline]

Gallagher JT, Morris A and Dexter TM (1985) Identification of two binding sites for WGA on polylactosamine-type oligosaccharides. Biochem J 231, 115–122.[ISI][Medline]

Goldstein IJ and Poretz RD (1986) Isolation, physicochemical characterization and carbohydrate-binding specificities of lectins. In Liener IE, Sharon N, and Goldstein IJ (eds) Lectins: Properties, Functions and Applications in Biology and Medicine. Academic Press, London, UK, pp. 33–248.

Greenwood PC, Hunter MW and Glover S (1963) The preparation of 131I labelled growth hormone of high specific radioactivity. Biochem J 89, 114–120.[ISI][Medline]

Hakomori SI (1989) Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens. Adv Cancer Res 52, 257–332.[ISI][Medline]

Halila H (1985) Detection of ovarian cancer marker CA 125 in human seminal plasma. Tumour Biol 6, 207–212.[Medline]

Hanisch F-G, Uhlenbruck G, Dienst C, Stottrop M and Hippauf E (1985) CA 125 and CA 19-9: two cancer-associated sialylsaccharide antigens on a mucus glycoprotein from human milk. Eur J Biochem 149, 323–330.[Abstract]

Hardardottir H, Parmley TH, Quirk JG Jr, Sanders MM, Miller FC and O'Brien TJ (1990) Distribution of CA 125 in embryonic tissues and adult derivatives of the fetal periderm. Am J Obstet Gynecol 163, 1925–1931.[ISI][Medline]

Hayes BK, Greis KD and Hart GW (1995) Specific isolation of O-linked N-acetylglucosamine glycopeptides from complex mixture. Anal Biochem 228, 115–122.[CrossRef][ISI][Medline]

Hilkens J, Lightenberg MJ, Vos HL and Litivinov SV (1992) Cell-membrane associated mucins and their adhesion-modulating property. Trends Biochem Sci 17, 359–363.[CrossRef][ISI][Medline]

Kan YY, Yeh SH, Ng HT and Lou CM (1992) Effect of menstruation on serum CA 125 levels. Asia-Oceania J Obstet Gynaecol 18, 339–343.

Kobata A and Endo E (1992) Immobilized lectin columns: useful tools for the fractionation and structural analysis of oligosacchraides. J Chromatogr 597, 111–122.[CrossRef][Medline]

Konishi I, Fendrick JL, Parmley TH, Quirk JG Jr and O'Brien TJ (1994) Epidermal growth factor enhances secretion of the ovarian tumor-asociated antigen CA 125 from the human amnion WISH cell line. J Soc Gynecol Invest 1, 89–96.[ISI][Medline]

Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 227, 680–685.[ISI][Medline]

Leffler H (1997) Introduction to galectins. Trends Glycosci Glycotechnol 9, 9–19.[ISI]

Macmillan D and Daines AM (2003) Recent developments in the synthesis and discovery of oligosaccharides and glycoconjugates for the treatment of disease. Curr Med Chem 10, 2733–2773.[CrossRef][ISI][Medline]

Madiyalakan R, Kuzma M, Noujaim AA and Suresh MR (1996) An antibody-lectin sandwich assay for the determination of CA 125 antigen in ovarian cancer patients. Glycoconj J 13, 513–517.[CrossRef][ISI][Medline]

Montz FJ (1992) Serological Cancer Markers. In Stewart S (ed.) Humana Press, Totowa, NJ, pp. 417–427.

Nagata A, Hirota N, Sakai T, Fujimoto M and Komoda T (1991) Molecular nature and possible presence of a membranous glycan-phosphatidylinositol anchor of CA 125 antigen. Tumor Biol 12, 279–286.[ISI]

Nap M, Vitali A, Nustad K, Bast RC Jr, O'Brien TJ, Nilsson O, Seguin P, Suresh MR, Bormer OP, Sage T et al. (1996) Immunohistochemical characterization of 22 monoclonal antibodies against the CA125 antigen: 2nd report from the ISOBM TD-1 Workshop. Tumour Biol 17, 325–331.[ISI][Medline]

Nustard K, Bast RC Jr, O'Brien TJ, Nilsson O, Seguin P, Suresh MR, Sage T, Nozawa S, Borwer OP, de Bruijn HWA et al. (1996) Specificity and affinity of 26 monoclonal antibodies against the CA 125 antigen: first report from the ISOBM TD-1 Workshop. Tumour Biol 17, 196–219.[ISI][Medline]

Nustard K, Onsurd M, Jansson B and Warren D (1998) CA125—epitopes and molecular size. Int J Biol Markers 13, 196–199.[ISI][Medline]

Nustard K, Lebedin Y, Lloyd KO, Shigemasa K, de Bruijn HWA, Jansson B, Nilsson O, Olsen KH and O'Brien TJ (2002) Epitopes on CA125 from cervical mucus and ascites fluid and characterization of six new antibodies. Tumor Biol 23, 303–314.[CrossRef][ISI]

O'Brien TJ, Hardin JW, Bannon GA, Noris JS and Quirk JG Jr (1986) CA 125 antigen in human amniotic fluid and fetal membranes. Am J Obstet Gynecol 155, 50–55.[ISI][Medline]

O'Brien TJ, Tanimoto H, Konishi I and Gee M (1998) More than 15 years of CA125: what is known about the antigen, its structure and its function. Int J Biol Markers 13, 188–195.[ISI][Medline]

O'Brien TJ, Beard JB, Underwood LJ, Dennis RA, Santin AD and York L (2001) The CA125 gene: an extracellular superstructure dominated by repeat sequences. Tumor Biol 22, 348–366.[CrossRef][ISI]

O'Brien TJ, Beard JB, Underwood LJ and Shigemasa K (2002) The CA125 gene: an extracellular superstructure dominated by repeat sequences. Tumor Biol 12, 279–286.[CrossRef]

Osawa T and Tsuji T (1987) Fractionation and structural assessment of oligosaccharides and glycopeptides by use of immobilized lectins. Annu Rev Biochem 56, 21–42.[CrossRef][ISI][Medline]

Perillo NL, Marcus ME and Baum LG (1998) Galectins: versatile modulators of cell adhesion, cell proliferation and cell death. J Mol Med 76, 402–412.[CrossRef][ISI][Medline]

Pijnenborg R (1994) Trophoblast invasion. Reprod Med Rev 3, 53–73.

Rabinovich GA, Rubinstein N and Fainboim L (2002) Unlock the secrets of galectins: a challenge at the frontier of the glyco-immunology. J Leukocyte Biol 71, 741–752.[Abstract/Free Full Text]

Roquemore E, Chou T-H and Hart GW (1994) Detection of O-linked N-acetylglucosamine (O-GlcNAc) on cytoplasmic and nuclear proteins. Methods Enzymol 230, 443–460.[ISI][Medline]

Rump A, Morikawa Y, Tanaka M, Minam S, Umesaki N, Takeuchi M and Miyajima A (2004) Binding of ovarian cancer antigen CA125/MUC16 to mesothelin mediates cell adhesion. J Biol Chem 279, 9190–9198.[Abstract/Free Full Text]

Seelenmeyer C, Wegehingel S, Lechner J and Nickel W (2003) The cancer antigen CA125 represents a novel counter receptor for galectin-1. J Cell Sci 116, 1305–1318.[Abstract/Free Full Text]

Sharon N and Lis H (1997) Glycoproteins: structure and function. In Gabius HJ and Gabius S (eds) Glycosciences Status and Perspectives. Chapman & Hall, Weinheim, Germany, pp. 133–162.

Sinowatz F, Petersen ET and Calvete J (1997) Glycobiology of fertilization. In Gabius HJ and Gabius S (eds) Glycosciences Status and Perspectives. Chapman & Hall, Weinheim, Germany, pp. 595–606.

Varki A (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3, 97–130.[Abstract]

Vicovac LJ and Aplin JD (1996) Epithelial-mesenchymal transition during trophoblast differentiation. Acta Anat 156, 202–216.[ISI][Medline]

Vicovac LJ, Jankovic M and Cuperlovic M (1998) Galectin-1 and -3 in cells of the first trimester placental bed. Hum Reprod 13, 730–735.[Abstract]

Wong NK, Easton RL, Pancio M, Sutton-Smith M, Morrison JC, Lattanzio FA, Moris HR, Clark GF, Dell A and Patankar MS (2003) Characterization of the oligosaccharides associated with human ovarian tumor marker CA125. J Biol Chem 278, 28619–28634.[Abstract/Free Full Text]

Wright CS (1990) 2.2 A resolution structure analysis of two refined N-acetylneuraminyl-lactose-wheat germ agglutinin isolectin complexes. J Mol Biol 215, 635–651.[CrossRef][ISI][Medline]

Yamashita K, Kochibe N, Ohkura T, Ueda I and Kobata A (1985) Fractionation of l-fucose-containing oligosaccharide on immobilized Aleuria aurantia lectin. J Biol Chem 8, 4688–4693.

Yin BWT and Lloyd KO (2001) Molecular cloning of the CA125 ovarian cancer antigen. J Biol Chem 276, 27371–27375.[Abstract/Free Full Text]

Zanetta JP (1997) Lectins and carbohydrates in animal cell adhesion and control of proliferation. In Gabius HJ and Gabius S (eds) Glycosciences Status and Perspectives. Chapman & Hall, Weinheim, Germany, pp. 439–452.

Zurawski VR Jr, Davis HM, Finkler NJ, Harrison CL, Bast RC Jr and Knapp RC (1988) Tissue distribution and characteristics of the CA 125 antigen. Cancer Rev 11-12, 102–118.

Submitted on February 1, 2005; resubmitted on April 6, 2005; accepted on April 25, 2005.





This Article
Abstract
Full Text (PDF )
All Versions of this Article:
20/9/2632    most recent
dei095v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Jankovic, M. M.
Articles by Tapuskovic, B. S.
PubMed
PubMed Citation
Articles by Jankovic, M. M.
Articles by Tapuskovic, B. S.