Purification and Characterization of a Membrane-bound and a ...

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Secreted Mucin-type Glycoprotein Carrying the Carcinoma-associated. Sialyl-Le” Epitope ... drate epitopes present on gangliosides (4, 5): sialyl-Lewis a (Si-Le” ...
THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 266,No. 32,Issue of November 15,pp. 21537-21547,1991 Printed in U.S.A.

Purification and Characterizationof a Membrane-bound and a Secreted Mucin-type GlycoproteinCarrying theCarcinoma-associated Sialyl-Le” Epitopeon Distinct Core Proteins* (Received for publication, June 21, 1991)

Dan BaeckstromSOlI, Gunnar C. HanssonS, OlleNilssonf, Christina Johanssonfll, Sandra J. Gendler**, and Leif Lindholmf 1 I From the $Department of Medical Biochemistry, University of Goteborg, Box 33031, S-400 33 Goteborg, Sweden, 3Pharmacin CanAg, Box 12136, S-402 42 Goteborg, Sweden, the IlDepattment of Medical Microbiology and Immunology, Guldhedsgatan IO, S-413 46 Goteborg, Sweden, and the **Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom

Two mucin-type glycoproteins detected by the mono- polymorphic epithelial mucin (PEM) and DU-PAN-2, clonal antibody CSO, which reacts with the carcinoma-reacted withH-CanAg. After deglycosylation with triassociated sialyl-Lewis a and sialyl-lactotetraose epi- fluoromethanesulfonic acid, H-CanAg but not L-CanAg topes, were found in secreted and solubilized materials was recognized by the monoclonal antibodies SM-3 and from the colon carcinoma cell line COLO 205. The HMFG-2, directed to the tandem repeat of the PEM larger glycoprotein (H-CanAg; heavy cancer antigen) apoprotein. However, theseantibodies which react was predominantly found in extractsof cells grown in with PEM from mammary carcinomas without prior vitro or as nude mice xenografts whereas the smaller deglycosylation were unable to recognize intact Hspecies (L-CanAg;light cancer antigen) was the major CanAg, probably as a consequence of a more extensive component in spent culture medium and serum from glycosylation of this glycoprotein. Comparison of grafted mice. Using detergent in the extraction bufferamino acid compositions confirmed that H-CanAg apodoubled the yield of H-CanAg, suggesting that this protein isclosely related to the MUCl protein whereas glycoprotein is membrane bound whereas the yield of L-CanAg is not. These findings show that colon carciL-CanAg was relativelyunaffected. The twoglycopro- noma cells can express twomucins with several carciteins were purified from xenograft extracts and spent noma-associated carbohydrate epitopes in common but culture medium using perchloric acid precipitation, differing in core protein structure and cellularlocalmonoclonal antibodyaffinitypurification, ion ex- ization. change chromatography, and gel filtration. Both glycoproteins were unaffected by reduction and alkylation in guanidine HCl. Using sodium dodecyl sulfatepolyacrylamide gel electrophoresis, relativemolecular The monoclonal mouse antibody (350, whichwas raised 600-800 kDafor H- against the human colon adenocarcinoma-derived cell line masses wereestimatedtobe CanAg and 150-300 kDa forL-CanAg. COLO 205, has proved useful in the detection of carcinomaCarbohydrate analysis revealed that theCanAg gly- associated antigens in serological and immunohistochemical coproteins were highly glycosylated (81-89% carboassays (1-3). C50 has been shown to react with two carbohyhydrate by weight), carrying carbohydrate chains with average lengths of 13-18 sugars which were rich in drate epitopes present on gangliosides (4, 5): sialyl-Lewis fucose andsialic acid (2-3 residues/chain for each a(Si-Le”; NeuAccu2-+3Ga1/31+3[Fuccul-+4]GlcNAc/31+),* sugar). L-CanAg isolated from spentmedium was gly- identified previously as the colorectal carcinoma-associated sialyl-lactotetraosyl chain cosylated to a higher degreethan its counterpart from CA19-9 epitope (6); andthe These two structures xenograft extract. Immunochemical studies of the in- (NeuAccu2~3Gal~1~3G1cNAc/31-+). tact glycoproteins showed that both H-CanAg and L- have collectively been named CA50 (5). A CA5O-carrying CanAg expressed the monoclonal antibody-defined, glycoprotein, called CanAg (for cancer antigen), has been sialic acid-containing carbohydrate epitopes CA203 examined with immunochemical methods by Johansson et al. and CA242 as well as the Lewis a blood group antigen (7), demonstrating that several other carbohydrate-reactive whereas only H-CanAg appeared to carry the sialyl- monoclonal antibodies (mAbs) recognize distinct carcinomaLewis xepitope. associated epitopes on this antigen. Among these were the The amino acid compositionswere typicalof mucins, Lewis a (Le”) and thesialidase-sensitive, but otherwise struccontaining high amounts of serine, threonine (more turally unknown CA203 and CA242 epitopes (defined by than 25%together), and proline(11-18%).Significant mAbs C203 and C242, respectively). However, the antigenic differences in amino acid composition between H- material studied was neither purified nor fractionated, and it CanAg and L-CanAg were found. A rabbit antiserum was therefore not possible to conclude whether these CanAgagainst the cytoplasmic C-terminal part of the MUCl gene product, core proteinof the carcinoma-associated ’The abbreviations used are: Si-, sialyl-; Le,Lewisblood group antigen; PBS, phosphate-buffered saline; PEM, polymorphic epithelial mucin; BSA, bovine serum albumin; mAb, monoclonal antibody; the payment of page charges. This article must therefore be hereby SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophomarked “aduertisement” in accordance with 18 U.S.C. Section 1734 resis; IFMA, immunofluorometric assay; CanAg, cancer antigen; Hsolely to indicate this fact. CanAg and L-CanAg, heavy and light CanAg, respectively; Hx-CanAg llTo whom correspondence should be sent. Tel.: 46-31-853497; and Lx-CanAg, heavy and light CanAg, respectively, from xenograft Fax: 46-31-416108. extract; Lm-CanAg, L-CanAg from spent medium.

* The costs of publication of this article were defrayed in part by

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associated epitopes always were expressed on the same glycoprotein as Si-Le" or if they can appear separately. Si-Le" and othertumor-associated carbohydrate structures, suchas the TAG-72 and DU-PAN-:! epitopes, have been detected previously on molecules having the biochemical properties of mucins (8-11). This group of substances is characterized by high molecular masses and a large number of sugar chains (comprising 50% or more of the totalweight), 0-linked via N-acetylgalactosamine to serine or threonine residues in the polypeptide core. The major mucins of the normal large intestine areproduced by goblet cells and appear as extracellular, disulfide-stabilized, gel-forming glycoproteins (12) that are insoluble also in strongly chaotropic solvents such as 6 M guanidine HC1 (13). There is no general knowledge about the structural relationship between tumorassociated mucins and those of the goblet cell type. However, a core protein common to carcinoma-associated mucins described by several groups as episialin or polymorphic epithelial mucin (PEM), among numerous other names (14, 15), has been the subject of detailed studies revealing marked differences to goblet cell mucins (15-17). PEM was originally isolated from human skim milk and has also been detected in normal epithelium of the breast, lung, and pancreas(18).The core protein of PEM has also been shown to be identical with that of a pancreatic tumor-associated mucin carrying the DUPAN-2 epitope (19). mAbs have been raised which detect core protein epitopes on the PEMmucin. Some of these antibodies bind preferentially to carcinoma cells, probably because tumor-associated changes in the glycosylation of PEM expose otherwise hidden epitopes (20, 21). All such core protein-reactive mAbs whose epitopes have been localized bind to structures contained in a tandem repeated 20-amino acid sequence. The amino acid sequence deduced from cDNA cloning (15, 16, 17, 19) of the core protein gene, called MUCl (22), shows that the tandem repeats are flanked by unique N- and C-terminal sequences, the latter containing a transmembrane and a cytoplasmic region. The tandemrepeat domain is subject to variable number tandem repeat polymorphism and accounts for 65% of the total protein contentin the most common allele product. One colon carcinoma-associated mucin has been isolated and characterized on the criterion of its Si-Le" expression (8). The 210-kilodalton molecule identified by this study and named CA 19-9 containing glycoprotein (19-9CGP) was suggested to be the main Si-Le"-carrying structure in both tumor tissue and secreted materials. However, only antigen shed into spent tissue culture medium of the colorectal carcinoma cell line SW1116 and a patientserum were examined. The aim of this study was to purify cell-bound and secreted glycoproteins carrying the Si-Le" epitope, using a colon carcinoma cell line as antigensource. We have investigated their relationships to each other and to MUCl and other tumorassociated mucins with respect to physical properties, chemical composition, and immunochemical reactivities of their oligosaccharide side chains and proteincores. EXPERIMENTALPROCEDURES

Materials Phenylmethylsulfonyl fluoride, streptavidin, and bovine serum albumin (BSA) were obtained from Sigma. Sepharose CL-4B, protein A-Sepharose, Q-Sepharose, Superose 6, and dissociation-enhanced lanthanide fluoroimmunoassay enhancement solution were from Pharmacia LKB Biotechnology Inc. Amplify solution, sulfur labeling reagent, and N a T were from Amersham Corp. IODO-GEN was from Pierce Chemical Co.; Iscove'smodifiedDulbecco's medium, from

GIBCO; and Vibrio cholerae neuraminidase, from Behringwerke AG, Marburg, Germany. Guanidine HCl (Merck) was purified by repeated filtrations through charcoal. Antibodies The generation of hybridomas producing monoclonal antibodies C50, C151, C241 C203, and C242 has been described previously (1, 7). For the production of these mAbs, hybridoma cells were cultured in Iscove's medium, supplemented with gentamycin, amino acid supplement (23), and 5% fetal bovine serum. Cells were kept in 10layered Cell Factory flasks (Nunc A/S, Roskilde, Denmark) in a total volume of2,500 ml at aconcentration of 2 X lo4 cells/ml, in a humidified atmosphere containing 8% COZ at 37 "C. After 7-8 days in culture the cell culture supernatants were decanted and filtered through a glass fiber filter to remove suspended cells and debris and were concentrated by ultrafiltrationina Pellicon cell (Millipore Corp., Bedford, MA.). mAbs were purified from the concentrates by adsorption to protein A-Sepharose and elution at acid pH. mAbs SM-3 andHMFG-2 were produced as described (20,24), and the "CT-1" rabbit antiserum was obtained by immunization with a synthetic 17-amino acid peptide corresponding to the C-terminal portion of the polymorphic epithelial mucin? Hybridomas C0431 (anti-Leb) and B72.3 (against sialyl-Tn: NeuAca2+3GalNAcal-+) were supplied by American Type Culture Collection (ATCC), Rockville MD, and mAbs were produced in cell culture according to the supplier's recommendations. mAbs X001 and YOO1, directed to the Le" and LeYantigens, respectively, were obtained from BioCarb, Lund, Sweden. Peroxidase-conjugated rabbit antiserum to mouse imunoglobulins and o-phenylenediamine were from DAKO Immunoglobulins a/s, Glostrup, Denmark. Goat anti-mouse antiserum was from Jackson Immunoresearch Laboratories, West Grove, PA. Biotinylated goat anti-rabbit immunoglobulins were from Zymed Laboratories, South SanFrancisco, CA. Preparation of Sources of C a d g Glycoprotein Cell Culture, Serum, and Xenograft Preparation-COLO 205 cells (ATCC CCL 222) were cultured, harvested, and injected into nude mice as described (23). Tumors wereallowed to grow for about 4 weeks to anaverage diameter of 1cm before the mice were killed; the tumors were excised, frozen in liquid nitrogen, and subsequently stored at -70 "C. From some of the mice serum was collected, pooled, and frozen. The spent cell culture medium was filtered to remove residual cells and concentrated 10-fold by ultrafiltration on an Amicon Diaflo stirred cell (Amicon Corporation, Danvers, MA), using a YM-10 filter. Preparation of Extracts from Xenografts and Cells-The entire preparation was carried out at 4 "C. The frozen xenografts were ground with a cold (-20 "C) mortar and pestle and immediately afterward suspended in extraction buffer (10 mM phosphate, pH 7.1, 0.15 M NaCl (PBS) containing 0.5% Triton X-100,0.02% NaN3, and 1mM phenylmethylsulfonyl fluoride) using 10 ml of buffer/g of tissue. The suspension was homogenized with six strokes in a Teflon homogenizer on an ice bath. The homogenate was allowed to standfor 1h with magnetic stirring before centrifugation a t 35,000 X g for 3 h. The supernatantwas aliquoted and frozen at -70 "C. This extraction procedure was also applied directly to cells from tissue culture, although without mortar grinding. In one xenograft extract preparation, extraction buffer without Triton X-I00 was used for the firstthreestrokesin the Teflon homogenizer, and the homogenate was then divided into two equal volumes, to one of which was added Triton X-100 to a final concentration of 0.5%. Both samples were then homogenized with three additional strokes and subjected to the rest of the extraction procedure as described above. Gel Filtration Size distribution of antigenic activity was determined using gel filtrations on a 1.6 X 70-cm Sepharose CL-4B column, using 0.1 M Tris-HC1, pH 8.0, as eluent with a flow rate of 5 ml/h. The column was also used for separations with the addition of 0.1% SDS (sample boiled in 2% SDS) or 6 M guanidine HC1 (sample reduced and alkylated, seebelow) in the eluent. Fractions were analyzed for antigenic activity (see "Fluoroimmunoassays" below). L. Pemberton and S. J. Gender, unpublished results.

Carcinoma-associated Mucins Reduction and Alkylation Samples were reduced by incubation in 0.1 M Tris-HC1, pH 8.0, 2 mM EDTA, 6 M guanidine HCI, and 0.1 M dithiothreitol for 2 h at room temperature. Sulfhydryl groups were then alkylated by treatment with 0.3 M iodoacetamide for 2 h in the dark. Purification of CanAg Glycoproteins Perchloric Acid Prec+itation-COLO 205 xenograft extract was precipitated by adding 2 M perchloric acid to a final concentrationof 0.1 M. With spent COLO 205 medium, concentrated perchloric acid was added to a final concentration of 0.5 M. After centrifugation a t 2,000 X g for 5 min the supernatantwas collected, neutralized by the addition of 1 M Tris-HC1, pH 8.0, and dialyzed against PBS buffer. The dialysate was passed through a 0.22-pm filter before affinity purification. When spent mediumwas purified, the dialysate was concentrated further by ultrafiltration before the next step. Affinity Chromatography-mAb C241 was covalently coupled to protein A-Sepharose according to themethod by Schneider et al. (25). Before each purification, the affinity column was washed with 6 M urea, 0.1% SDS and with 0.2 M sodium phosphate, pH 11.2, each step followed by washing with 0.1 M Tris-HC1, pH 8.0. The gel was then mixed with the dialyzed perchloric acid-soluble material on a rotary mixer for 1 h, poured onto a column, washed, and eluted according to a modified version of the method by Schneider et al. (25). Briefly, after washing with the threedetergent-containing neutralbuffers, the column was washed with 0.1 M Tris-HC1, pH 8.0, and then eluted with 0.2 M sodium phosphate, pH 11.2. The eluate was collected in 1 M sodium borate, pH 7.5, to neutralize pH. After dialysis against water, the eluate was concentrated by ultrafiltration. Zon Exchange Chromatography-Q-Sepharose (typically 2-3 ml for IO6 units CA50) was preequilibrated with 50 mM Tris-HCI, pH 7.0, 8 M urea (starting buffer). To the affinity-purified material urea was added to a concentration of 8 M and Tris-HC1, pH 7.0, to a final concentration of 50 mM. After a 30-min incubation with the sample on a rotary mixer the gel was poured onto a column, washed with starting buffer, and eluted with a 0-0.5 M gradient of NaCl (constant pH and urea concentration). Fractionswere analyzed for CA50 activity (see "Immunoassays" below), and antigen-containing fractions were pooled, dialyzed against water, and concentrated by ultrafiltration. Size Fractionation-Saturated guanidine HCl solution was added to the ion exchange-purified glycoprotein to a final concentration of 4 M. The sample was then subjected to gel filtration using 50 mM Tris-HC1, pH 7.0,4 M guanidine HCl as eluent. The highest molecular mass antigenic material was first isolated on a 1.6 X 70-cm Sephacryl S-500 (Pharmacia) column and then further purified on a 2.6 X 100cm Superose 6 preparation grade (Pharmacia) column. To prepare the lower molecular weight materials, only the Superose 6 column was employed. Fractions were analyzed for CA50 activity, and peak activity fractions were extensively dialyzed against water and concentrated by ultrafiltration. Labeling Methods Radioiodination-In a reaction tube coated with 25 pg of IODOGEN, thepurified glycoprotein sample (5-10 pg ofprotein) was mixed with 20 pCi of Na'? in a totalvolume of 150 pl of sodium phosphate, pH 7.5, for 3 min before desalting on a PD-10 column (Pharmacia) using PBS with 0.05% Tween 20 as eluent. 35SLabeling-Purified glycoprotein samples (5-10 pg of protein) were labeled with 50 pCi of sulfur labeling reagent according to the manufacturer's description. After termination of the labeling reaction, samples were desalted on a PD-10 column, using PBS with 0.05% Tween 20 as eluent. Purity Analysis Total Protein Determination-Total protein concentrationof crude antigen preparations was determined with Bio-Rad protein assay using BSA as standard. After affinity chromatography and subsequent steps, amino acid analysis was employed to calculate protein content. SDS-PAGE, Autoradiography, and Diffusion Elution-SDS-PAGE was run on 1.5-mm-thick 2-15% gradient gels without stacking gels a t a constant currentof 35 mA/gelin adiscontinuous Laemmli buffer system (26). The gelswere fixed with 30% ethanol and 10% HAC for >3 h, soaked in Amplify solution (35S-labeledsamples) or in 7% HAC and

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5% glycerol ('251-labeled samples), dried, and exposed to Hyperfilm MP (Amersham) at -70 "C for 1-7 days. Lanes containing molecular mass marker proteins were fixed as above and visualized by the silver stain method of Heukeshoven and Dernick (27). In diffusion elution experiments, lanes were cut into 3-mm slices which then were crushed with a pipette tip and mixed with 0.5 ml of electrode buffer (26) each on a rotary mixer overnight. The resulting eluates were analyzed for CA50 activity. Amino Acid Analysis Samples were prepared and run as described (28) on an Alpha Plus amino acid analyzer (Pharmacia). Cysteine and tryptophan were not analyzed. Carbohydrate Analysis The sugar composition was determined by gas chromatography of the corresponding alditol acetates (29) after acid hydrolysis with 4 M trifluoroacetic acid for 4 h at 100 "C. Samples were separated on a 20-m X 0.22-mm column coated with silarylene, using hydrogen as carrier gas with a lineargas velocityof 60 cm/s. The temperature was increased linearly from 70 to 260 "C. Detector temperature was 300 "C. Peaks were evaluated with a Hewlett-Packard 3396 series I1 integrator. Sialic acids were analyzed after release with 0.1 M HCl for 1 h at 80 "C, the sample lyophilized and analyzed by high pressure liquid chromatography (Dionex AS-6 column/AG-6A precolumn), using a pulsed amperometric detector (Dionex, Sunnyvale, CA). The column was eluted with 0.5 mM NaOH, 15 mM NaAc, at a flow rate of 1 ml/ min. Deglycosylation Purified samples, each corresponding to 5 pgof protein, were lyophilized in Reacti-Vials (Pierce Chemical Co.), dried under vacuum overnight over P205at room temperature, and subjected to trifluoromethanesulfonic acid treatment according to Sojar and Bahl (30). Briefly, samples were incubated with 150 pl of trifluoromethanesulfonic acid for 5 h on ice with magnetic stirring, neutralized with 60% pyridine in water, extensively dialyzed against 50 mM pyridinium acetate, pH 5.4, lyophilized, and resuspended in 1 ml of PBS. Inhibition Enzyme-linked Immunosorbent Assay of intact and SiLe"-depleted COLO 205 Xenograft Extract One aliquot of xenograft extract was divided into two portions. In one portion Si-Le" activity was reduced 200-fold by repeated extraction with Sepharose-coupled Si-Le" reactive mAbC241. To match the depleted extract inSi-Le" activity, the other portion of the extract was diluted 1:200 in PBS containing 1%BSA. Aliquots of the two samples and PBS, 1%BSA as control were then incubated with mAbs C50, C203, C242, and C151 (mAb concentration 0.5 pg/ml) for 1 h. Free, uninhibited mAb was then immobilized by transferring the reaction mixtures to microtiter strips coated with intact extract (2 pg of protein/well (7)) and incubated for 2 h. After washing, the bound mAb was detected by a 2-h incubation with horseradish peroxidase-conjugated rabbit antiserum to mouse immunoglobulins and visualized with o-phenylenediamine according to the manufacturer's instructions.Inhibition was calculated as 1 (A450. sample/A450. An inhibition ratio, defined asthe inhibition value obtained with the depleted extract divided by the value for the diluted, intact extract, was then calculated for each mAb. Fluoroimmunoassays General Methods-All fluoroimmunoassays were performed in polystyrene mictrotiterstrips (Labsystems Oy, Helsinki, Finland) using the dissociation-enhanced lanthanide fluoroimmunoassay system (Pharmacia). Europium labelings were performed using the Euchelate of isothiocyanatobenzyl diethylenetriamine tetraacetic acid to a specific activity of 2-5 Eu/protein molecule (31). Conditions for coating of the strips have been described previously (7). Antibody and antigen dilutions were made in the assay buffer described by Hemmila et al. (31). After incubation on a shaking apparatus, strips were aspirated and washed with 5 mM Tris-HC1, pH 7.75,0.15 M NaCl, 0.005% Tween 20,0.05% NaN3. Strips were washedthree times after incubation with unlabeled reagents and six times after incubation with europium conjugates. Europium ions were released by the addition of dissociation-enhanced lanthanide fluoroimmunoassay en-

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hancement solution, and fluorescence was measured in an Arcus fluorometer (Pharmacia). Homologous Assay for the Detection of CanAg Glycoproteins-Homologous immunofluorometric assay (IFMA), using C50 mAb both as solid phase-bound catching antibody and as europium-labeled tracer, was performed as described by Johansson et al. (3). Dilutions of COLO 205 spent tissue culture medium were used as standards, and theantigenic activity was expressed in arbitraryunits/ml, defined in relation to a reference medium batch (3). Analysis of Carbohydrate Structures on Intact andNeuraminidasetreated CanAg Glycoproteins-Microtiter strips were coated with serial dilutions (1:3) of CanAg glycoprotein samples (starting concentration, 1,000 CA50 units /ml). Neuraminidase treatment was performed on the adsorbed glycoproteins using V. chokrae neuraminidase, 0.015 units/ml, in 50 mM sodium acetate, pH 5.5, 0.15 M NaC1, 9 mM CaC12, 1%BSA, 100 pl/well, for 3 h at 37 “C with shaking. Intact and enzyme-treated strips were incubated with mAbs against carbohydrate epitopes for 1 h. Bound antibodies were detected by a 1-h incubation with europium-labeled goat anti-mouse antiserum. Analysis of Apoprotein Structures in Intact and Deglycosylated CanAg Glycoproteins-Microtiter strips were coated with serial dilutions (1:3) of solutions of deglycosylated CanAg glycoproteins, prepared as described under “Deglycosylation” above. Intact glycoprotein, diluted to a concentration corresponding to 5 pg of protein/ml, was coated to strips in the same manner. The immunoassay was performed as described above, using mAbs SM-3 andHMFG-2. Assay for the Detection of C-terminal MUCl Structures-Polystyrene strips coated with mAb C50 were incubated for 2 h with serial dilutions (1:3) of intact CanAg glycoproteins (starting concentration 0.5 pglml). The strips were then incubated for 1h with the polyclonal rabbit antiserum CT-1,directed against the C-terminal, cytoplasmic part of the MUCl protein. Bound antiserum was detected by incubation for 1 h with biotinylated goat anti-rabbit immunoglobulins followed by a 30-min incubation with europium-labeled streptavidin. Normal mouse serum was added to a concentration of 1%in the rabbit and goat antiserum solutions to suppress anti-species-specific binding to the mouse IgM mAb C50.

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FIG. 1. Gel filtration chromatography of CanAg glycoproteins from COLO 205 cells grown as xenografts and in vitro. Samples were chromatographed on a 1.6 X 70-cm Sepharose CL-4B column, and fractions were analyzed for CA50 activity by homologous IFMA using mAb C50. A comparison between extract from nude mice RESULTS xenografts and serum from the grafted mice is shown (A) as well as Secreted and Cell-bound CanAg Glycoproteins from FOLO a comparison between extract from in uitro cultured cells and the 205-Gel filtration on Sepharose CL-4B wasemployed to spent culture medium ( B ) . Also shown are the void (VO)and total examine the size distribution of CA50 activity (as measured (V,) volumes of the column and the retention volumes of thyroglobby the immunofluorometric assay using C50 as both catching ulin (Thy, 669 kDa), aldolase (AZd, 158 kDa), and BSA (67 kDa).

and tracing antibody) of cell-bound and secreted glycoprotein. Extract of COLO 205 xenografts was compared with serum from the grafted mice (Fig. Ut), and extract from cells taken directly from tissue culture was compared with the spent culture medium (Fig. 1 B ) . In both of these comparisons, the CanAg glycoproteins could be roughly divided into two size groups: one that eluted close to or in the void volume of the Sepharose CL-4B column (the “heavy” fraction, referred to as H-CanAg below) and one that eluted considerably later (“light” fraction, or L-CanAg). The abundance of H-CanAg was low in the secreted antigen sources whereas it was the major component in the cell and xenograft extracts. To estimate the degree of membrane association of the CanAg glycoproteins, xenograft extracts made with and without TritonX-100 in the extraction buffer were compared. The use of detergent increased the total yield of CA50 activity by a factor of 1.8. As can be seen from the gel filtration profiles in Fig. 2, this gainwas mainly aresult of an increased extraction of H-CanAg, suggesting that hydrophobic interaction plays a part in the cell association of this molecule. The yield of L-CanAg, on theother hand,was relatively unaffected by the addition of detergent. To examine whether these substances might be aggregates susceptible to denaturing treatments, COLO 205 xenograft extract was boiledwith SDS with subsequent gel filtration in SDS-containing buffer. Reduction and alkylation in guanidine HC1, followed by gel filtration in guanidine HC1 buffer, were also performed. Neither of these treatmentschanged the shape of the CA50gel filtration profile (data not shown),

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FIG. 2. Gel filtration chromatography showing the influence of detergent in the extraction of CanAg glycoproteins from nude mice xenografts of COLO 205 cells. Extracts made without and with 0.5% Triton X-100 in the extraction buffer were chromatographed on a 1.6 X 70-cm Sepharose CL-4B column. Fractions were analyzed for CA50 activity by homologous IFMA using mAb C50.

indicating that neither H-CanAg nor L-CanAg is aggregated in the native state. Purification of CanAg Glycoprotein from COLO 205 Cells and Medium-To characterize both H-CanAg and L-CanAg, COLO 205 xenograft extract and spent tissue culture medium

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TABLEI Yield and purity datafrom the different steps in the Purification of CanAg glycoprotein from xenograftextract and spent tissue culture medium. Yield in antigen activity Sample

Xenografts This step

Specific activity

Medium

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units/pg protein

%

Starting materials" Perchloric acid supernatant" 87 Affinity chromatography eluate" Ion exchange chromatography eluateb Gel filtration fractions*,' H-CanAg

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Purification

100 81 49 48

81 40 19

17.2 62.5 14,300 17,000

2.14 3.10 1.4 2,380 16,400

This step

Overall

-fold

1.4 3.6 229 1.2

3.6 831 988

768 6.9

1,112 7,660

22,200 11,800 22,700 Protein concentrations determined using Bio-Rad protein assay. * Protein concentrations determinedby amino acid analysis. e In the last step, only peak fractions, ie. a small amount of the total recovered antigen, were selected, and therefore no yield data are presented. Also, this step includes fractionation into different antigen species with different apparentspecific activities, which makes fold purification calculations irrelevant.

were chosen as starting materials for purification. The amount of CA50 activity extracted from xenografts varied between 5 x lo6 and 2 x lo6 CA50 units/g of tissue, and the activity in spent media before concentration varied between 500 and 1,500 units/ml. Typical yields, specific activities, and fold purifications from the different purification steps aresummarized in Table I. Perchloric acid precipitation was employed to remove bulk proteins from the crude materials but gave modest fold purification values (1.4-5). Higher concentrations of perchloric acid, while removing more protein, drastically reduced the yields of antigen activity (data not shown). However, the perchloric acid precipitations under the relatively mild conditions used were sufficient to maintain the efficency of the subsequently used affinity column for more than 30 repeated purification cycles whereas it deteriorated rapidly if cruder samples were applied (data not shown). C241, the anti-Si-Le" IgG mAb used in the affinity purification step, was able to extract more than 99% of the CA50 actitvity from the perchloric acid supernatant.Thisstep accounted for the main increase in purity,giving a 200-1,000fold purification with yields varying from 35 to 65%, both parameters depending on the age of the affinity gel and the condition of the sample. After affinity chromatography the sample contained some copurifying contaminant proteins and mAb that had leaked from the affinity column. These were removed by ion exchange chromatography on Q-Sepharose in the presence of 8 M urea, which was usedto disrupt both specific mAb binding and nonspecific interactions between the antigen and contaminating proteins. Finally, the CanAg glycoproteins were purified by gel filtration chromatography in the presence of 4 M guanidine HCl. H-CanAg eluted partly in the void volume of a Superose 6 column, and therefore material purified from xenograft extract was first run on a Sephacryl S-500 column (Fig. 3). The final fractionation of all CanAg samples was then carried out on a Superose 6 column. Peak fractions containingH-CanAg and L-CanAg from xenograft extract (abbreviated Hx-CanAg and Lx-CanAg, respectively) as well as those containing LCanAgfrom spent medium (Lm-CanAg) were selected for further analyses (Fig. 4). Gel Electrophoresis-The purity of the antigen preparations after the affinity chromatography step was monitored with SDS-PAGE of radioiodinated samples. The autoradiograph

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160

ml FIG. 3. Preparation of Hx-CanAg by gel filtration chromatography on Sephacryl S-500. Ion exchange-purified CanAg glycoproteins from xenograft extractwere subjected to gel filtration on a 1.6 X 70-cm Sephacryl S-500 column, using buffer containing 4 M guanidine HCI as eluent. Fractions were analyzed for CA50 activity by homologous IFMA using mAb C50. Fractions indicatedby the bar were pooled and used for further purificationas shown in Fig. 4.

in Fig. 5 illustrates the final steps in the purification. The removal of low molecular mass contaminants as well as separation of CanAg species are evident for both Lx-CanAg and Lm-CanAg. Hx-CanAg, on the other hand, appeared to be contaminated with significant amounts of both Lx-CanAg and low molecular mass bands (lane 3). However, Hx-CanAg was radioiodinated to a much lower specific radioactivity than Lx-CanAg, exaggerating the apparent degree of contamination with Lx-CanAg present in the Hx-CanAg sample. This also gives the false impression that Lx-CanAg is the more abundant species in lanes 1 and 2 of Fig. 5 whereas Hx-CanAg is actually dominating, in termsof both antigenic activity and protein content. To circumvent this bias in labeling efficiency and todetermine whether the low molecular bands seen in the lane of Hx-CanAg might be artifacts from the oxidative IODO-GEN labeling method, 35Slabeling of primary amines with sulfur labeling reagent reagent followedby SDS-PAGE was employed to visualize the finally purified samples. The resulting autoradiograph is showl. Fig. 6 together with the results of diffusion elution of unlabeled samples run in the same gel. These results show that the purified antigen fractions were I

Mucins

21542

Carcinoma-associated

120000 .

A

A

H

-

100000

-. .

80000 -

1 ~

2

B 3

4

5

6

7 kDa 4- 870 4+ 670

40000 20000 60000

t-330

200

100

300

400

150

50000 B

40000

-E 5

30000

.-d

gm

20000

+43

0 v)

5

10000 0 100

200

300

I 30000

400 1

H

I

4- 20

4- 14

FIG. 5. SDS-polyacrylamide gel electrophoresis and autoradiography of radioiodinated samples from the finalstages in the purficationof CanAg glycoproteins. Samples taken after affinity chromatography, ion exchange chromatography, and gel fil10000 tration chromatography stepswere radioiodinated, separatedon a 215%gradient SDS-PAGEgel, and autoradiographed. Lanes in group A show materials from xenograft extract; group B shows materials from spent culture medium. Lanes I and 5, affinity chromtography 0 eluates. Lanes 2 and 6,ion exchange purified materials. Lanes 3, 4, 100 200 300 400 and 7, Hx-CanAg, Lx-CanAg,and Lm-CanAg, respectively, after final Superose 6 gel filtrations. Molecular mass markersindicatedare ml FIG. 4. Preparative gel filtration on Superose 6 of Hx- mouse IgM (870 kDa), thyroglobulin (670 kDa), thyroglobulin halfCanAg, Lx-CanAg, and Lm-CanAg glycoproteins. CanAg gly- unit (330 kDa), mouse IgG (150 kDa), all nonreduced; BSA (67 kDa), coproteins werechromatographed on a 2.6 X 100-cm Superose 6 ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor preparation grade column using buffer containing 4 M guanidine HCl (20 kDa), and lactalbumin(14 kDa), all reduced. as eluent. Fractions were analyzed for CA50 activity by homologous IFMA using mAb C50. Bars indicate fractions pooled as the finally purified glycoproteins. This procedure was applied to samples after differences between Hx-CanAg on one hand and Lx-CanAg gel filtration chromatography on Sepahcryl S-500 in the case of Hx- and Lm-CanAg on the other hand,especially with respect to the content of alanine, proline, and glutamic acid (including CanAg (panel A ) , as shown inFig. 3, or after the ionexchange chromatography step in the case of Lx-CanAg (panel B ) and Lm- glutamine) and alsofor several of the minor aminoacids. CanAg (panelC). Carbohydrate Composition-Results of the sugar analyses for the CanAg glycoproteinsare shown in Table111, calculated essentially homogeneous, although giving relatively broad as sugar residues/GalNAc. Gal and GlcNAc dominated the bands, and that the antigenic reactivity cochromatographed compositions, which were also rich in Fuc and NeuAc (2-3 with the labeled glycoproteins. The actual contamination of residueseach/GalNAc).NeuGcwasonly detectedinHxHx-CanAg with Lx-CanAg, as estimated from the diffusion CanAg, there constituting10%of the totalsialic acidcontent. elution results, was only about 5%. Mannose, typical of N-linked glycans, was found in amounts On the basis of the positions of molecular mass marker far below those of other sugars. proteins shown in Fig. 5, the apparent molecular masses of These data, taken together with amino acid compositions the antigens were estimated, giving a value of 600-800 kDa and immunometric results, permit the calculation of total for H-CanAg, 100-150 kDa for Lx-CanAg, and 150-300 kDa carbohydratecontent, CA50 activityrelatedto sialic acid for Lm-CanAg. content, average sugar chain length, and the degree of glycoAmino Acicl Composition-Table I1 shows the amino acid sylation of serines and threonines (Table IV). The calculacompositions for the CanAgglycoproteins. In all samples, tions show that theCanAg molecules are highly glycosylated. serine and threonine together accounted for more than25% Lm-CanAg had the highest carbohydrate content as well as of the total aminoacid content. Proline, glycine, and alanine the highest average oligosaccharide chain length, followed in were also major components. However, there were marked both respects by Hx-CanAg and Lx-CanAg. Table IV also 20000

21543

Carcinoma-associated Mucins CA50 activity, Ulml N

P

0

0

w 0

0 0

~ 0 0

m 0 0

m

0 0

0 I

mm

100

.

-

N

o

0

0

0

0

0

0

l

.

1

.

I

P .

0 0 I

.

i

FIG.6. SDS-polyacrylamide gel electrophoresis and autoradiography of ?3-1abeled purified HxCanAg (A), Lx-CanAg ( B ) ,and Lm-CanAg (C) glycoproteins shown together with CA50 activity in diffusion eluates from the same gel. Samples of purified CanAg glycoproteins were labeled with 3JSand run together with their unlabeled counterparts on a 2-15% gradient SDS-PAGE gel. The part of the gel containing labeled samples was autoradiographed whereas lanes containing unlabeled sampleswere cut in 3-mmslices which were eluted by diffusion. The eluateswere tested for CA50 activity by homologous IFMA using mAb C50.

shows estimates of the molecular masses of the apoproteins of the CanAg glycoproteins. Carbohydrate Epitopes on CanAg Glycoproteins-To examine the expression of carbohydrate epitopes on the purified CanAg glycoproteins, their binding by carbohydrate-reactive mAbs was analyzed in solid phase immunoassays. Hx-, Lx-, and Lm-CanAg were used as solid phase-bound antigens. The maximum fluorescence was related to that obtained with SiLe"-reactive mAb C50 (Table V). The mAbs C203 and C242, directed against carcinoma-associated, sialic acid-containing carbohydrate epitopes of unknown strucure, previously shown to be coexpressed with Si-Le" (7), bound strongly to all intact CanAg glycoproteins. Their binding was stronger toHxCanAg than to theL-CanAg samples, suggesting differences in therelative density of these carbohydrateepitopes between the CanAg glycoproteins. The mAb B72.3, reactive with the carcinoma-associated sialyl-Tn epitope (32, 33), bound very weakly to all intact CanAg samples. The same assay procedure was used to detect binding of antibodies against the Lewis blood group antigens to intact and neuraminidase-treated CanAg glycoproteins. The moderate binding of the Le"-reactive mAb C151 to all three intact glycoproteins increased strongly after neuraminidase treatment (Table V). The Lex-reactive mAb X001 did not bind to any of the intact samples, but Hx-CanAg was detected after neuraminidase treatment. In contrast, Lx-CanAg and Lm-

CanAg remained unreactive. No Leb or LeYactivity could be detected in any of the samples. Coexpression of Carbohydrate Epitopes-To determine whether the CA203, CA242, and Le" epitopes were expressed only on the same glycoproteins as Si-Le" in COLO 205 cells, COLO 205 xenograft extract was depleted of Si-Le" activity by affinity extraction. The depleted extract was then tested for its capacity to inhibit binding of mAbs C50, C203, C242, and C151 (anti-Le"). Intact extract, diluted to match the depleted extract in Si-Le" activity, was tested in the same manner. As shown in Table VI, the inhibition by depleted extract of mAbs C50, (2203, and C242 all matched the values obtained with the diluted, intact extract. In contrast, (2151 was inhibited 5.3 times more by the depleted extract. These results show that extraction of Si-Le" activity removed CA203 and CA242 to the same extent although there was a greater residual Le" reactivity. Polypeptide Core Structures of CanAg Glycoproteins-Intact and deglycosylated CanAg glycoproteins were adsorbed to microtiter strips and examined for binding by the mAbs HMFG-2 and SM-3, reactive with the MUCl tandem repeat sequence (14). Except for a very weak reactivity with LxCanAg, the antibodies did not bind to the intact samples (Fig. 7B). However, after deglycosylation with trifluoromethanesulfonic acid, both mAbs reacted strongly with Hx-CanAg (Fig. 7A). Lm-CanAg remained unreactive whereas Lx-CanAg still showed a weak reactivity.

.3

Mucins

21544

Carcinoma-associated

Intact CanAg glycoprotein samples immobilized to C50coated microtiter strips were also tested for binding by a rabbit antiserum (CT-1) against the cytoplasmic C-terminal portion of the MUCl protein. This assay showed that the antiserum reacted strongly with Hx-CanAg, weaker with LxCanAg, and not detectably with Lm-CanAg (Fig. 7C). However, the difference between Hx-CanAg and Lx-CanAg was

much less pronounced than with the other anti-apoprotein antibodies. DISCUSSION

Cells of the colorectal carcinoma cell line COLO 205contain and secrete Si-Le"-carrying glycoproteins of at least two molecular sizes. H-CanAg, with an apparent molecular mass of 600-800 kDa, appears almost exclusively to be a membraneassociated structure whereas L-CanAg (150-300 kDa) is seTABLEI1 Amino acid compositions of purifiedCanAg glycoproteins fromCOLO creted from the cells. Both H-CanAg and L-CanAg display 205 xenograft extract (Hx-CanAg, Lx-CanAgJ and spent tissue culture several features typical of mucins, including perchloric acid medium (Lm-CanAg) solubility, high carbohydratecontent,and an amino acid Also shown aredata for the MUCl protein (derived from sequence composition rich in serine, threonine, and proline. Glycoprodata (16) and deleting a 20-amino acid hydrophobic leadersequence) teins of the mucin type arenotoriously difficult to characterize and for the 19-9CGP mucin purified from the SW1116 colorectal with respect to molecular size because of their elongated carcinoma cell line (8). "bottle-brush" structure, large negative electrostatic charge, Aminoacidresidue Hx-CanAe MUCl Lx-CanAe Lm-CanAe 19-9CGP and tendency to display structural microheterogeneity. ThereAlanine 17.0 16.4 9.1 7.5 7.5 fore, the molecular masses presented above must be regarded Arginine 4.3 4.3 2.1 1.8 5.5 as highly tentative. Aspartic acid and 5.1 5.8 8.1 5.7 5.1 Mucin characteristics were also evident in the carbohydrate asparagine compositions of the CanAg glycoproteins. Mannose occurred Cysteine NA" 0.2 NA 0.0 NA 3.3 Glutamic acid and 2.2 7.5 8.6 9.8 in very low amounts, indicating that N-linked glycans are glutamine rare. Both fucose and sialic acid were abundantly expressed Glycine 9.6 8.6 8.4 9.0 7.9 (2-3 residues/chain for each sugar) on theoligosaccharides of Histidine 4.7 2.0 2.4 4.8 1.5 the CanAg glycoproteins, admitting the possibility of nonterIsoleucine 0.9 3.2 0.9 3.4 3.0 minally situated fucoses and sialic acids as well as concatenLeucine 2.3 5.6 4.9 2.2 5.3 ated cu243-linked sialic acids. High fucosyl and/or sialyl Lysine 1.2 4.1 2.3 0.6 2.5 Methionine 0.3 0.9 2.0 0.9 0.2 transferase activities have been discussed as a carcinomaPhenylalanine 1.1 1.1 2.1 1.2 2.7 associated phenomenon (34). Proline 18.6 11.87.3 10.6 19.6 N-Glycoloyl neuraminic acid was found in Hx-CanAg but Serine 12.4 12.1 16.2 17.0 19.7 not in Lx-CanAg, probably because of the small amounts Threonine 13.3 15.413.6 17.6 8.8 analyzed in the lattercase. This type of sialic acid is usually Tryptophan NA 0.0 NA NA NA 0.5 Tyrosine 1.0 1.3 1.3 2.5 not found in human cell lines (35) but has been detected Valine 5.5 6.0 4.7 5.4 5.1 before in gangliosides from a human tumorcell line grown in NA, not analyzed. nude mice (36). The mouse has enzymes for converting NeuAc to NeuGc and can probably supply NeuGc to the tumor cell sialyl transferases. However, Higashi et al. (37) proposed a TABLE111 tumor-associated expression of NeuGc in humancolon cancer. Total carbohydrateanalyses of purified CanAg glycoproteins from The carbohydrate chains released from human skim milk COLO 205 xenograft extract (Hx-CanAg, Lx-CanAg)and spent tissue mucins have been studied by Hanisch et al., who found expresculture medium (Lm-CanAgJ sion of the Si-Le"epitope (38) and anatypical polylactosamine ResidueslN-Acetylgalactosamine Sugar residue chain (( l-&Gal@1+4GlcNAc@),,n = 1-3) as amajor internal Hx-CanAg Lx-CanAg Lm-CanAg sequence (39). Polylactosamine has not been detected in rnol/mol normal mucins of the colon, but thehigh and nearly equimolar N-Acetylgalactosamine 1.0 1.0 1.0 amounts of GlcNAc and Gal in the CanAg glycoproteins N-Acetylglucosamine 3.8 4.3 5.3 indicate the possible presence of polylactosamine units in Galactose3.6 4.2 5.3 these carbohydrate chains. Fucose 2.5 2.4 H-CanAg and L-CanAg were similar to each other with N-acetylneuraminic acid 2.4 1.7 2.1 N-glycoloylneuraminic acid 0.26 respect to totalcarbohydrate composition. However, LxMannose 0.07 0.22 0.12 CanAgwas less glycosylated, probably because of shorter a Below limits of detection. oligosaccharide chains, than its counterpart from spent me~

~

~~~~

TABLEIV Glycosylatwn data derived from carbohydrate, amino acid, and immunometric analyses and molecular mass estimations Carbohydrate CA50 content % of total

weight

units/nmol Average sugar chain sialicmassb acidsmolecular residues" threonine length" unitslnmol

no. of

residues

Glycosylated serine/

Estimated apoprotein

kDa % of total

Hx-CanAg

85

3,300

14.6

70

90-120

Lx-CanAg

81

4,300

12.8

54

20-30

88 4.200 17.1 63 Lm-CanAp Assuming that N-acetylgalactosamine only appears next to the polypeptide core. Based on total molecular mass estimatesobtained by gel electrophoresis.

20-35

Carcinoma-associated Mucins

21545

TABLEV Binding of carbohydrate-reactive monoclonal antibodies to purified CanAg samples without and with neuraminidase treatment Antibody binding was detected with europium-labeled secondary antibody and measured as fluorescence of released europiumions. Antibody binding relativeto that of C50" Lx-CanAg Epitope

mAb

Hx-CanAg neuraminidaseneuraminidase neuraminidase

-

+

-

Lm-CanAg

+

-

NT NT

100

+

%

C50 C203 C242 C151

sialyllactotetraose Si-Le",

100

CA203' CA242' NT

98

Le"

108 152 20