Glycoproteins from Human Colonic Adenocarcinoma ISOLATION AND CHARACTERIZATION OF CELL SURFACE CARCINOEMBRYONIC ANTIGEN FROM A CULTURED TUMOR CELL LINE*

Alterations in cell surface glycoproteins have been implicated in malignancy. We examined surface membrane proteins of a cultured cell line, SKCO-1, which had been derived from a human colonic adenocarcinoma. Cell surface labeling of SKCO-1 cells with galactose oxidase, followed by reduction with sodium borotritide, revealed five major labeled glycoproteins upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. At least three additional labeled glycoproteins could be detected if galactose oxidase treatment was preceded by neuraminidase treatment. Some, but not all, of the glycoproteins could be iodinated by lactoperoxidase. The predominantly labeled glycoprotein (GPI) had a molecular weight of 200,000 and co-migrated in SDS gel with carcinoembryonic antigen (CEA). GPI was not removed from the cell surface by EDTA, hypertonic saline, or sonication but was released from the membrane by detergents. This glycoprotein was subsequently purified using lectin-agarose columns and gel filtration. GPI was judged homogenous by protein- and carbohydrate-stained SDS-polyacrylamide gels and had an amino acid composition similar to that of CEA. The carbohydrate composition of GPI was qualitatively similar to CEA but quantitatively distinct. GPI had a greater proportion of sialic acid and galactosamine and less fucose and glucosamine than CEA. Immunological studies, however, demonstrated identity between GPI and CEA. A study of the turnover rate of GPI showed it to have a half-life of 5 days.


Alterations
in cell surface glycoproteins have been implicated in malignancy.
We examined surface membrane proteins of a cultured cell line, SKCO-1, which had been derived from a human colonic adenocarcinoma.
Cell surface labeling of SKCO-1 cells with galactose oxidase, followed by reduction with sodium borotritide, revealed five major labeled glycoproteins upon sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. At least three additional labeled glycoproteins could be detected if galactose oxidase treatment was preceded by neuraminidase treatment. Some, but not all, of the glycoproteins could be iodinated by lactoperoxidase.
The predominantly labeled glycoprotein (GPI) had a molecular weight of 200,000 and co-migrated in SDS gel with carcinoembryonic antigen (CEA). GPI was not removed from the cell surface by EDTA, hypertonic saline, or sonication but was released from the membrane by detergents. This glycoprotein was subsequently purified using lectin-agarose columns and gel filtration. GPI was judged homogenous by protein-and carbohydrate-stained SDS-polyacrylamide gels and had an amino acid composition similar to that of CEA. The carbohydrate composition of GPI was qualitatively similar to CEA but quantitatively distinct. GPI had a greater proportion of sialic acid and galactosamine and less fucose and glucosamine than CEA. Immunological studies, however, demonstrated identity between GPI and CEA. A study of the turnover rate of GPI showed it to have a half-life of 5 days. Since  (12). The gels containing tritium were sliced at 2-mm intervals, added to toluene scintillation mixture containing 5% NCS solubilizer (AmershamiSearle) and 0.4% Omnifluor (New England Nuclear) and counted after incubation at 50" overnight. Gels containing '*51 were sliced similarly but were counted directly in a y-spectrometer.
Iodination of CEA -Immobilization of lactoperoxidase on Affi-Gel 10 was performed according to the instructions provided by Bio-Rad. It was found that 0.346 mg of enzyme (0.7 mg of total protein) was coupled to 1 ml of agarose gel using A::jM = 114 (13) for lactoperoxidase. Agarose gel was suspended in an equal volume of NaCl/P,, pH 7.4. Iodination of CEA with lzsI was carried out as follows. To 100 ~1 of CEA solution (1 mg/ml in NaCl/P,) were added 5 ~1 of carrier-free lrsI (0.5 mCi), 20 ~1 of the lactoperoxidase-agarose suspension (3.5 pg of immobilized enzyme), and H,O, at a final concentration of 1 PLM. The reaction was carried out at 4" for 30 min with gentle shaking and was stopped by gel filtration of the reaction mixture on a Sephadex G-25 column after removal of the lactoperoxidase-agarose gel by centrifugation.
Radioactively labeled CEA was collected at the void volume of the column and was stored at -20" until used. A specific activity of 5 x 10" cpmlwg was obtained for CEA after enzymatic radioiodination. -Two methods were utilized to study the turnover of GPI in SKCO-1 cells. Cells at 50% confluency in 25.cm" tissue culture flasks were labeled with galactose oxidase and NaB:'H, as described above. Labeled cells were washed and incubated at 10 ml of growth medium (Dulbecco's modified Eagle's medium with 10% fetal calf serum, penicillin, and streptomycin) overnight. Less than 10% of the cells were found dead or detached.
The culture medium was replaced with 10 ml of fresh medium. Labeled cells were harvested at this point as a zero-time control and at l-day intervals up to 6 days with one change of medium at Day 3. Two to four flasks of cells were used for each time point.
An alternate approach was to label confluent cells in 75.cm' tissue culture flasks. Labeled cells were detached from plates by 15-min incubation at 37" with 27 ITIM citrate/isotonic phosphate buffer, pH 7.4, washed once with growth medium, resuspended in the same medium, and replated to a 50 to 60% confluency in 25cm' tissue culture flasks. About 70% of the cells were found to attach to the plates after overnight incubation. Again, the culture medium was replaced with 10 ml of fresh medium.
Cells were harvested at this point as a zero-time control and at l-day intervals up to 6 days. The membrane fractions were obtained from harvested cells, solubilized in 2% NPX, 2% NP-40 in NaCl/P,, diluted 5-fold with NaCl/P,, centrifuged, and applied to a Con A-Sepharose column. GPI was eluted from the column with 0.5 M ol-methyl-n-mannoside in detergent buffer, concentrated, and assayed for radioactivity. The growth media from each time period were dialyzed against NaCl/P,, pH 7.4. Determination of recovery of labeled GPI in the media was achieved by applying dialyzed media to a Con A-Sepharose column equilibrated in NaCl/P,, pH 7.4, concentrated, and assayed for radioactivity. by guest on March 24, 2020 http://www.jbc.org/

Cell Surface Proteins
-For these studies, monolayer SKCO-1 cells in culture flasks at confluency were washed and incubated with enzymes (neuraminidase, galactose oxidase) in Hanks' balanced salt solution. Properties of cell surface proteins were examined with enzymes which modify the surface proteins of intact cells and could be monitored by the incorporation of isotopes into the membrane proteins or by the change of protein patterns after gel electrophoresis.
The radioactively labeled membranes obtained by ultracentrifugation of sonicated cells were solubilized in SDS, and the membrane proteins were analyzed by SDS-polyacrylamide gel electrophoresis.
A scintillant (2,5-diphenyloxazole) was introduced into the gel to speed the visualization of tritium-labeled protein in polyacrylamide gels by autoradiography. Membranes from cells labeled with galactose oxidase treatment showed the presence of a large number of labeled polypeptides of molecular weights 200,000 (GPI), 115,000 (GPII), 100,000 (GPIII), 78,000 (GPIV), 49,000 (GPV), and 42,000 (GPVI). GPI was the major labeled component, accounting for more than 50% of the total radioactivity incorporated into cell membrane (Fig. 1). When galactose oxidase treatment was preceded by neuraminidase treatment, labeling of bands corresponding to GPI, GPIV, and GPVI were enhanced. Also, additional bands of molecular weights > 500,000 (GPNl), 280,000 (GPNB), and 62,000 (GPN3) were detected. Trypsin treatment of labeled cells produced a diminution of GPII, GPIII, and GPNl. None of the other bands showed any significant change after trypsin treatment. GPNl was found to be radioiodinated by lactoper- FIG. 1. Autoradiographic pattern of 3H-and '2SI-labeled SKCO-I membrane proteins separated in SDS-polyacrylamide slab gel. Membranes of monolayer SKCO-I cells were reduced with LIHlborohydride after the conditions described (A to F) or iodinated with lzsI (G to I) under the conditions described. A, no treatment; B, galactose oxidase treatment; C, galactose oxidase followed by trypsin treatment; D, treatment with neuraminidase; E, treatment with neuraminidase followed by galactose oxidase; F, treatment with neuraminidase followed by galactose oxidase with subsequent trypsin treatment; G, lactoperoxidase alone; H, lactoperoxidase and H202; I, lactoperoxidase and H,O, with subsequent trypsin treatment. Affinity chromatography of membrane galactoproteins on Con A-Sepharose. Membrane galactoproteins eiuted &om RCA-Sepharose were dialyzed, concentrated, and applied to a column (1.5 x 5 cm) containing Con A-Sepharose. After washing, membrane galactoproteins were eluted in z-ml fractions with Cl.5 M a-methyl-Dmannoside in 0.1% NPX, 0.1% NP-40 in NaCl/P,, pH 7.4. The arrow denotes the point of application of the cr-methyl-D-mannoside solution. The flow rate was 20 ml/h and chromatography was carried out at room temperature. Aliquots of 50 ~1 were assayed for radioactivity.
oxidase, and the label could be removed from the cells by treatment with 0.05% trypsin (Fig. 1). GPI, GPV, and GPVI were also iodinated. Additional '251-labeled membrane proteins of 180,000, 150,000. and 20,000 daltons were detected. Zsolation and Purification of GPZ -Preliminary data showed that GPI remained associated with the membrane fraction after sonication, extraction with 0.1 M EDTA, or treatment with 3 M KCl. However, it could be solubilized readily with detergents. Results of affinity chromatography of solubilized tritium-labeled membrane glycoproteins on a column of RCA-Sepharose is shown in Fig. 2. A small amount of labeled material emerged with the effluent (Peak I), while most of the radioactivity bound to the column and could be eluted with lactose (Peak II). Peak I contained most of the membrane proteins judging from the large quantity of nondialyzable, lyophilized material (protein determinations of this fraction were greatly affected by NP-40 and NPX, even after extensive dialysis Peak II to a column of Con A-Sepharose are shown in Fig. 3. About 36% of the radioactivity was not bound. Elution of the column with 0.5 M cu-methyl-n-mannoside produced a peak which contained over 60% ,of the labeled material applied to the column. This Con A-bound galactoprotein fraction consisted mainly of GPI. Sephadex G-200 chromatography appeared to separate the major component GPI from the minor bands as shown in Fig. 4A. This major labeled protein was shown to be homogeneous by Coomassie stained SDS-polyacrylamide gel shown in Fig. 4B. SDS-polyacrylamide gels stained for glycoproteins with periodic acid-Schiff reagent also showed only a single band corresponding to labeled GPI. gel. Y-labeled CEA from hepatic metastases of colonic carcinoma (10,800 cpm) was mixed with "H-labeled GPI (22,000 cpm) and dissolved in 2% SDS, 5% fl-mercaptoethanol, 0.1 M phosphate sample buffer, pH 7.0. The mixture was incubated at 100" for 3 min, and glycerol was added to a final concentration of 10%. The sample was subjected to electrophoresis on a 7.5% polyacrylamide disc gel. After electrophoresis the gel was sliced at 2-mm intervals and counted in a y spectrometer for lz51 (O---0). Gel slices were then transferred to scintillation vials, incubated overnight at 45" in a toluene mixture containing 5% NCS and 0.4% Omnifluor and counted for "H (0-O).
the medium of the cells examined." Over 90% of the CEA of SKCO-1 cells was found to be associated with cellular membranes. Similarity in molecular weights between GPI and CEA prompted us to compare their chemical properties. An identical migration of 3H-labeled GPI and '251-labeled CEA was found using SDS-polyacrylamide gel electrophoresis as shown in. Anti-CEA antiserum, 1 to 25 ~1, was allowed to react with purified GPI (O---O) or galactoproteins which did not bind to Con A-Sepharose (A-A) for 30 min at 45" in a volume of 50 yl. The tubes were centrifuged at 1100 x g for 15 min, and the radioactivity was measured in the supernatant fraction. Control incubations of GPI or galactoproteins which did not bind to Con A-Sepharose with normal goat serum were also carried out. Radioactivity was determined by adding 6 ml of toluene mixture containing 5% NCS and 0.4% Omnifluor to 25 ~1 of supernatant which was carefully removed from the top of each tube. The radioactivity remaining in the pellet was calculated and divided by the total radioactivity added to the tube to obtain the per cent of total radioactivity precipitated.  Fig. 7. GPI could be completely precipitated by CEA The cell density was 1.8 x lo5 cells/ml, and the cells were 85% viable by trypan blue exclusion. After overnight incubation at 37", the medium was replaced with 10 ml of fresh growth medium.
About 70% of the cells were attached to the culture flask. Labeled cells were harvested at this point and at l-day intervals up to 6 days with one change of medium at Day 3. Membrane fractions were obtained from harvested cells, solubilized with detergent, diluted B-fold with NaCl/ Pi and applied to a column of Con A-Sepharose.
GPI was eluted from the column with 0.5 M o-methyl-n-mannoside in NaCl/P,, concentrated, electrophoresed in SDS-polyacrylamide gel, and assayed for radioactivity.
Each point represented an average of three flasks.
antiserum. None of the other glycoproteins which bound to RCA-Sepharose contained appreciable CEA activity. As shown in Table I, the amino acid composition of GPI is similar to those reported previously for CEA by Banjo et al. (171,Kupchik et al. (18), Coligan et al. (191,Terry et al. (20), and Westwood et al. (21). However, GPI was slightly higher in the basic amino acids lysine and histidine than was CEA. The carbohydrate compositions of GPI and other CEA preparations are compared in Table II. GPI is much lower in fucose and Nacetylglucosamine and higher in sialic acid and N-acetylgalactosamine than the CEA preparations isolated from liver. PIG. 9. Profile of radioactivity in SDS-polyacrylamide disc gel of labeled GPI isolated from membranes and media. SKCO-1 cells at 50% confluency in 25cmL tissue culture flasks were labeled with galactose oxidase and tritiated sodium borohydride. After culturing the cells for 3 days, media was removed from the flasks and dialyzed. Labeled glycoproteins were prepared by centrifuging sonicated cells at 105,000 x g for 60 min and solubilizing membranes (0-O) and from dialyzed media (O---0) was dissolved in 2% SDS, 5% 2mercaptoethanol, 0.1 M phosphate, pH 7.0. The mixture was incubated at 100" for 3 min, glycerol was added (to lo%), and the sample was subjected to electrophoresis on a 7.5% polyacrylamide disc gel containing 0.1% SDS and 0.1 M phosphate, pH 7.0. After electrophoresis, gels were sliced into 2-mm slices which were incubated overnight at 45" in a toluene mixture containing 5% NCS and 0.4% Omnifluor and counted in a Tri-Carb scintillation counter.
isolated from cells immediately after and 5 days after galactose oxidase labeling. No difference was found in the mobility of the labeled proteins using SDS-polyacrylamide gel electrophoresis. After growing labeled SKCO-1 cells in medium for 3 days, the labeled GPI present in the medium was isolated and examined by SDS-polyacrylamide gel electrophoresis. The results in Fig. 9 show that GPI recovered from the medium had the same mobility as GPI recovered from cell membranes.

DISCUSSION
Cell surface labeling methods utilizing galactose oxidase or lactoperoxidase have been previously used to probe externally oriented proteins of mammalian cell membranes (24). We have used these techniques to study membrane proteins of SKCO-1 cells, a well defined colonic adenocarcinoma line cultured in vitro. The experiments were performed either with monolayer cell cultures or with cells detached from culture flasks and suspended in Hanks' balanced salt solution, but a much higher background of nonspecific incorporation of label (experiments performed in the absence of galactose oxidase) was found when using cell suspensions. This may be due to differences in viability of cells which is crucial in ensuring specific labeling of cell surface proteins. Cells of low viability tend to show some undesirable labeling of internal components (8). For these reasons monolayer cell cultures of SKCO-1 cells were used throughout the study.
Labeling of glycoproteins of intact SKCO-1 cells with tritiated sodium borohydride after oxidation of terminal galactosyl-and N-acetylgalactosaminyl residues with galactose oxidase consistently produced five major and several minor labeled membrane glycoproteins. A membrane glycoprotein of approximately 200,000 daltons was the most intensely labeled. Lactoperoxidase-catalyzed iodination of SKCO-1 cells was characterized by the labeling of at least eight membrane polypeptides with apparent molecular weights > 500, 000, 200,000, 150,000, 65,000, 49,000, 45,000, 40,000, and 20,000. Immunogenicity of cells has been reported to be modified by various methods, such as treating the cells with enzymes, attaching foreign proteins to their surface, or treating them with diazotized sulfanilic acid (25)(26)(27)(28). A number of observations have been made that suggest that exposure in VCN in vitro increases the immunogenicity of cells, e.g.. (a) VCNtreated fetal tissue evokes a greater degree of sensitization than untreated tissues when injected into allogenic recipients; (b) a variety of tumors grow less well in normal susceptible recipients if the tumor cells have been incubated in VCN; and (c), VCN-treated cells cause immunospecific regression in leukemia or in firmly established solid syngeneic transplantable methyl cholanthrene-induced fibrosarcoma (26). In an attempt to expose other tumor antigenic sites on the cell surface, SKCO-1 cells were treated with neuraminidase prior to galactose oxidase treatment.
Most of the components showed an increase in labeling. Also, three additional membrane surface glycoproteins were found to be labeled following neuraminidase treatment. This indicates that in GPI, GPIV, and GPVI and in the newly labeled glycoproteins (GPNl, GPNB, GPNS), sialic acid affects the exposure of galactosyl and N-acetylgalactosaminyl residues at the cell surface. These effects could be the result of direct blocking of galactose and N-acetylgalactosamine residues by sialic acid, or sialic acid residues on other components may provide a charge barrier to normally reactive galactosyl groups.
Some of the glycoproteins, GPNl, GPI, GPV, and GPVI, could be labeled both by galactose oxidase and lactoperoxidase, while the others were not susceptible to lactoperoxidase iodination. This is interpreted to mean that some of the surface membrane glycoproteins do not have exposed tyrosine groups in their amino acid sequences.
Singer has classified membrane proteins into two groups, peripheral and integral, based on the requirements of their dissociation from membranes (29). The former are weakly bound, while the later are intercollated into the lipid bilayer. GPI could only be solubilized with detergents, suggesting that GPI is tightly bound to the membrane and apparently should be considered an integral membrane protein. GPI is apparently exposed at the cell surface and oriented toward the extracellular environment since: (a) GPI was labeled by galactose oxidase; (b) GPI was also labeled by lactoperoxidase and; (c) labeling was enhanced by prior treatment with neuraminidase.
Nonionic detergents, NPX and NP-40, were used to extract labeled cell surface glycoproteins and after centrifugation at 105,000 x g for 60 min, more than 80% of the radioactivity was found in the supernatant.
An identical labeling pattern after SDS-polyacrylamide gel electrophoresis was found in the supernatant as that of the original membrane solubilized directly in SDS. Analysis by SDS-polyacrylamide gel electrophoresis of radioactive components in fractions obtained after lectin affinity chromatography indicated that Peak I from RCA-Sepharose column contained only the radioactive components which migrated in the dye-front region while all other high molecular weight labeled glycoproteins were bound to the column. No radioactivity which corresponded to GPI was found in the Peak I fraction from the Con A-Sepharose