From the
Department of Hematology, Oncology and Tumorimmunology and the ||Department of Glycoconjugates, Max-Delbrück-Center for Molecular Medicine, Berlin 13125, the
Universitätsklinikum Charité, Robert-Rössle-Klinik, Department of Hematology, Oncology and Tumorimmunology, Humboldt University, Berlin 13125, and the ¶Forschungsinstitut für Molekulare Pharmakologie, Berlin 13125, Germany
Received for publication, February 7, 2003 , and in revised form, April 1, 2003.
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ABSTRACT |
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INTRODUCTION |
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Recently, a novel tumor-associated antigen that was attributed an important role in tumor immune interactions, RCAS1, was described (4, 5). Initially, RCAS1 was defined by the 22.1.1 monoclonal antibody (mAb), which was raised by immunization of mice with the human uterine cervical adenocarcinoma cell line SiSo (4). Cell surface staining with mAb 22.1.1 was shown immunohistochemically in a large number of different tumor tissues, and in some tumor entities 22.1.1 staining correlated well with poor clinical prognosis (68). A soluble form of the 22.1.1 antigen was purified from the supernatant of cultured SiSo cells (4).
Expression cloning led to the identification of a cDNA apparently encoding the 22.1.1 antigen. The gene product was termed "receptor binding cancer antigen expressed on SiSo cells" (RCAS1) and is identical with the estrogen-responsive protein EBAG9 (5, 9, 10).
Recombinantly expressed RCAS1 was shown to bind to a yet unidentified receptor, which was predominantly expressed on activated immune cells. Cell culture supernatant from SiSo cells inhibited proliferation of activated T cells and induced apoptotic cell death in receptor bearing cells. Therefore, RCAS1 was introduced as a new death receptor ligand involved in tumor immune escape (5). Moreover, it has been suggested that RCAS1 is involved in down-regulation of the maternal immune response during pregnancy (11). Matsushima et al. (12) demonstrated that RCAS1 expression by bone marrow macrophages is important in the regulation of erythropoiesis by induction of apoptosis in erythroid progenitor cells, thereby extending the role of RCAS1 to a general cell death-inducing system according to the Fas/Fas ligand system (13).
Although RCAS1 seems to be highly conserved in phylogeny (14) and was demonstrated to have important physiological functions, little is known about the gene product itself. Data bank screens revealed that there are no homologies to any known genes or proteins. According to sequence predictions, RCAS1 has been postulated to be a type II transmembrane (TM) protein with an N-terminal TM region (amino acids 827) and an extracellular C-terminal coiled-coil region (amino acids 179206). Data available on the molecular mass are confusing. Calculations attribute RCAS1 a molecular mass of about 24 kDa, whereas in SDS-PAGE the gene product migrates around 32 kDa (14). According to Sonoda et al. (4, 5), mAb 22.1.1 immunoprecipitates generated a 78-kDa signal in SDS-PAGE gels under reducing and non-reducing conditions. A homodimerization via the C-terminal coiled-coil region was suggested (4, 5).
Recombinantly expressed RCAS1-GST fusion protein was shown to bind a putative receptor, whereas a truncated version of RCAS1-GST lacking the postulated TM region did not show biological activity. However, full-length RCAS1 is poorly soluble in aqueous solutions because of its hydrophobic TM domain. This raises the question how RCAS1 acquires solubility and functional effects in an aqueous solution.
Here we studied the biochemical properties of RCAS1 and its subcellular localization in two human cell lines, HEK293 and MCF7. We show that RCAS1 is a Golgi resident protein with a cytoplasmic orientation and, unexpectedly, is not recognized by the 22.1.1 mAb. Instead, the 22.1.1 mAb recognizes the Tn glycan antigen, which appears upon RCAS1 overexpression on the cell surface of HEK293 cells.
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EXPERIMENTAL PROCEDURES |
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Expression Plasmid ConstructsRCAS1 cDNA was a kind gift of T. Watanabe, Kyushu University, Fukuoka, Japan. All restriction enzymes were purchased from New England BioLabs (Beverly, MA), and T4 DNA ligase and Pwo polymerase for PCR amplifications were from Roche Applied Science, Mannheim, Germany. For transfection studies, the RCAS1 cDNA was cloned into pcDNA3.1(+) (Invitrogen, Karlsruhe, Germany) via XhoI and EcoRI sites. For the construction of RCAS1 deletion variants fused to GFP, the following oligonucleotide pairs were used. All sense primers contained a KpnI site and the ATG start codon; antisense primers contained a BamHI site. Primers were as follows: full-length RCAS1, 5'-GGGGTACCCACCATGGCCATCACCCAGTTT-3' and 5'-CGGGATCCTGAAAGTTTCACACCAAT-3'; RCAS1 127, 5'-GGGGTACCCACCATGAGATCTGGCAGAGGACGG-3' and 5'-CGGGATCCTGAAAGTTTCACACCAAT-3'; RCAS1
30213, 5'-GGGGTACCCACCATGGCCATCACCCAGTTT-3' and 5'-CGGGATCCAGATCTGCATATTAATCT-3'; RCAS1
179213, 5'-GGGGTACCCACCATGGCCATCACCCAGTTT-3' and 5'-CGGGATCCTAGTTTCTGCTGTCTCAG-3'; RCAS1
1163, 5'-GGGGTACCCACCATGGAAGATGCAGCCTGGCAA-3'. PCR-products were cloned into pEGFP-N3 (Clontech, Palo Alto, CA), and sequences were confirmed by DNA sequencing.
Cell Culture and TransfectionsHuman embryonal kidney HEK293 and mammary carcinoma MCF7 cells were maintained in RPMI medium (PAA, Cölbe, Germany), supplemented with 5% fetal calf serum (Biochrom, Berlin, Germany), 2 mM L-glutamine (Invitrogen, Karlsruhe, Germany), and 100 units/ml penicillin-streptomycin (Invitrogen). For transfections, 2 x 106 cells were resuspended in 500 µl of serum-free medium and, after addition of the plasmids indicated, electroporated at 170 V and 960 microfarads in 4-mm gap electroporation cuvettes (Bio-Rad, Munich, Germany) using a Bio-Rad Gene Pulser.
Inhibition of O-linked glycosylation was carried out by addition of 2.5 mM benzyl-2-acetamido-2-deoxy--D-galactopyranoside (benzyl-
-GalNAc, Calbiochem, San Diego, CA) in 1% (v/v) ethanol to cell cultures. Addition of ethanol was used in control experiments.
Laser Scanning Microscopy and Fluorescence MicroscopyFor visualization of GFP-tagged RCAS1 variants in living HEK293 and MCF7 cells, cells were cotransfected with the indicated RCAS1-GFP variants and 1,4-galactosyl transferase-YFP (1,4-GT-YFP, Clontech). Cells were grown on coated coverslips for 48 h and washed once with phosphate-buffered saline (PBS), and coverslips were transferred into a self-made humidified chamber. Cells were covered with 1 ml of PBS containing 0.05% trypan blue for plasma membrane staining (17) and immediately subjected to microscopic analysis on a Zeiss LSM510 inverted laser scanning microscope. Fluorescence signals were detected using the following configurations: GFP:
exc = 488 nm, BP
em = 496517 nm; YFP:
exc = 514 nm, BP
em = 517.7646 nm. Trypan blue fluorescence was detected on a separate channel (
exc = 514 nm,
em ≥ 690 nm). For optimal visualization of overlapping signals, YFP-fluorescence color was changed to red, and trypan blue fluorescence color was changed to light blue using the Zeiss LSM image browser software.
For the detection of the 22.1.1 antigen, RCAS1-GFP-transfected cells were grown on coverslips for 48 h, then fixed with PBS containing 5% paraformaldehyde for 15 min, followed by permeabilization with PBS containing 0.1% Triton X-100 for 10 min. Cells were stained for 2 h with 22.1.1 mAb or IgM isotype control diluted 1:200 in PBS with 1% bovine serum albumin and 10% normal goat serum then rinsed with PBS and incubated with a phycoerythrin (PE)-conjugated goat anti-mouse antibody (Dako, Hamburg, Germany). Nuclei were stained with DAPI. PE and DAPI signals were recorded with exc = 543 nm, BP
em = 560646 nm and
exc = 364 nm, BP
em = 385470 nm wavelengths, respectively.
For the detection of endogenous RCAS1 in MCF7 cells, cells were grown on coverslips, washed with PBS, and fixed with methanol. RCAS1 antiserum, control serum, or mAb AM-75 were diluted 1:100 in PBS containing 10% normal goat serum and incubated overnight at 4 °C. Slides were rinsed, and bound antibodies were detected with a biotinylated goat anti-rabbit or goat anti-mouse antibody (Dako) and streptavidin-conjugated Alexa FluorTM 488 or Alexa FluorTM 568 (Molecular Probes, Eugene, OR). Signals were visualized on a Leica DM IRBE microscope equipped with a Zeiss Axiocam digital camera. All images were processed in Corel Photo Paint.
Periodate OxidationSodium periodate oxidation of carbohydrates was carried out essentially as described previously (18). Cells were grown on coverslips and fixed with 5% (w/v) paraformaldehyde in PBS for 15 min. After washing with PBS and acetate buffer (50 mM sodium acetate, pH 4.5), coverslips were incubated with 20 mM sodium periodate in acetate buffer for 1 h in the dark at RT. Coverslips were rinsed with acetate buffer and treated with 50 mM sodium borohydride in acetate buffer for 30 min at RT and processed for immunostaining with the indicated antibodies. Bound antibodies were visualized with a PE-conjugated goat anti-mouse antibody. Nuclei were stained with DAPI, and cells were analyzed using a Leica DM IRBE microscope and an Axiocam digital camera.
Subcellular Fractionation and High Salt Treatment2 x 107 cells were washed with PBS, resuspended in 10 mM Tris, pH 7.4, 250 mM sucrose, 1 mM PMSF, 1.5 µg/ml aprotinin, and homogenized in a Dounce homogenizer by 20 strokes with a tight fitting pestle. After nuclei were pelleted by centrifugation at 250 x g for 10 min, a postnuclear supernatant was ultracentrifuged at 100,000 x g in a Beckman TLA100.4 rotor for 1 h. Crude membrane pellets were washed with either 10 mM Tris, pH 7.4, 1 M KCl, 10 mM EDTA, 100 mM Na2CO3, 4 M urea, or 1% Triton X-100 (TX-100) for 1 h at 4 °C under agitation. Membranes were repelleted at 100,000 x g for 30 min, and membrane proteins were solubilized in reducing Laemmli sample buffer (rSB). Proteins from the supernatants were precipitated with acetone and solubilized in rSB.
Protease Protection Assays from Cell LinesProtease protection assays at intracellular compartments were performed essentially as described previously (19). Briefly, 2 x 107 cells were resuspended in 50 mM Tris-Cl, pH 7.4, 150 mM KCl, 250 mM sucrose, 1 mM EDTA and gently homogenized by five strokes in a Dounce homogenizer with a tight fitting pestle. Nuclei were pelleted at 250 x g for 10 min and supernatant was mock treated or treated with 60 µg/ml proteinase K for 1, 5, and 15 min. The reaction was stopped by the addition of 5 mM PMSF for 5 min. 2x rSB was added, and samples were boiled for 10 min and subjected to SDS-PAGE.
Cell Surface Biotinylation2 x 107 cells were washed twice in PBS before cell surface biotinylation was performed for 30 min at room temperature with 1 mg of sulfo-N-hydroxysulfosuccinimide-LC-biotin (Pierce, Rockford, IL) in 2 ml of PBS, pH 7.4. Cells were washed three times in PBS containing 10 mM lysine and lysed in 1 ml of 50 mM Tris, pH 7.4, 5 mM MgCl2, 0.5% Nonidet P-40, 1 mM PMSF, 1.5 µg/ml aprotinin for 30 min at 4 °C. Nuclei were pelleted for 10 min at 13,000 rpm, and streptavidin-Sepharose (Amersham Biosciences) was added to the supernatant for 1 h under agitation at 4 °C. Streptavidin-Sepharose beads were pelleted, 500 µl of the supernatant was carefully removed, and the unbound proteins were precipitated with 20% (v/v) trichloroacetic acid and solubilized in 100 µl of rSB. Streptavidin-Sepharose beads were washed five times with ice-cold PBS, before biotinylated surface proteins were recovered from the beads by boiling in 100 µl of rSB for 5 min. Proteins were subjected to SDS-PAGE.
In Vitro Transcription, Translation, and Protease Protection AssayIn vitro transcriptions and translations were performed in the presence of [35S]methionine according to the manufacturer's instructions using the Promega Riboprobe kit (Promega, Mannheim, Germany) and the Promega Flexi rabbit reticulocyte system. Where indicated, canine pancreatic microsomal membranes (Promega) were added to the reaction. Reaction products were analyzed directly by SDS-PAGE or further processed in a protease protection assay essentially as described (20). Briefly, microsomes were resuspended in ice-cold PBS and treated or mock treated with 4 µg/ml proteinase K (Merck, Darmstadt, Germany) for 15 min on ice. Microsomes were pelleted immediately for 10 min at 13,000 rpm at 4 °C in a microcentrifuge, followed by one washing step with ice-cold PBS containing 1 mM PMSF and 1.5 µg/ml aprotinin. Pellets were dissolved in rSB and boiled for 10 min. Endoglycosidase H (Endo H, New England BioLabs) digestions were performed as described by the manufacturer.
Gel Electrophoresis and ImmunoblottingCell lysates were separated on 12.5% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (0.45-µm pore size, Schleicher & Schuell, Dassel, Germany) using a Bio-Rad tank blot chamber. Membranes were incubated with the first antibodies for 60 min in PBS containing 0.05% Tween 20 (PBS-T) and 1% nonfat dry milk, followed by horseradish peroxidase-conjugated secondary antibody (Southern Biotechnology, Birmingham, AL) diluted 1:5000 in PBS-T. Bound antibody was visualized by chemoluminescence (ECL Western Blot Detection kit, Amersham Biosciences, Braunschweig, Germany).
Flow Cytometric AnalysisAfter blocking in FACS buffer containing 10% normal goat serum, primary antibodies were added to the cells at a dilution of 1:200 for1hon ice. Cells were washed twice and incubated for 15 min with PE-conjugated goat anti-mouse secondary antibody. For the detection of 22.1.1, Tn, or TF surface staining of GFP fusion protein-expressing cells, a biotinylated goat anti-mouse IgM secondary antibody (Dianova, Hamburg, Germany) was used. Bound antibody was detected with Cy5-conjugated streptavidin (Dianova), and flow cytometric analysis was carried out on a FACSCaliburTM cytometer (BD Biosciences, Heidelberg, Germany).
ELISAMaxisorb ELISA plates (Nunc, Wiesbaden, Germany) were coated with the indicated polyacrylamide-conjugated glycans (Synthesome, Munich, Germany) overnight in 50 mM carbonate buffer, pH 9.6, at RT. After washing with PBS, plates were incubated with the 22.1.1 antibody or IgM isotype control, diluted 1:200 in PBS containing 1% bovine serum albumin for 2 h at 4 °C. Plates were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Southern Biotechnology). Bound antibodies were visualized by incubation with 0.05% 2,2'-azino-di-[3-ethylbenzthiazone sulfonate]diammonium salt (6) (Roche Applied Science), 0.05% H2O2 in 50 mM citrate buffer, pH 4.0, and analyzed with a Bio-Rad Microplate Reader at 405 nm.
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RESULTS |
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To explore the membrane association, signal peptide cleavage, and membrane orientation of RCAS1, we employed a cell-free translation system in the presence of canine pancreatic microsomes that provides a functional environment for membrane insertion. RCAS1 was found to be associated with microsomal membranes without an apparent cleavage of a signal peptide (Fig. 1A). In SDS-PAGE the full-length form migration is nearly identical to that of RCAS1 as obtained from translation without microsomes. The shift in molecular weight shown in Fig. 1A is unlikely to account for a signal peptide cleavage. On average, signal peptides in eukaryotes are 1550 amino acids long and are usually located at the N terminus (25). Therefore, the shift expected for signal peptide cleavage would be at least 2 kDa. The signal obtained in the presence of microsomal membranes reflects a doublet that could also be observed in immunoblots from cell extracts using the polyclonal anti-RCAS1 serum (Fig. 1E). The doublet might indicate modifications or conformational changes that occur at the endoplasmic reticulum (ER) membrane. Because an N-linked glycosylation motif was absent from the RCAS1 polypeptide sequence and RCAS1 was not Endo H-sensitive (Fig. 1B), we could exclude that the occurrence of a doublet was due to glycosylation modifications.
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RCAS1 was suggested to be a type II TM protein with a large C-terminal luminal/extracellular domain and a short N-terminal cytoplasmic part (5). The orientation of the RCAS1 gene product in microsomal membranes was probed by protease protection assays. After translation, addition of proteinase K to the microsomes led to proteolytic cleavage of cytoplasmic protein domains, whereas luminal regions were protected (Fig. 1C). As a control for a protein with a large luminal part and a short cytoplasmic tail, we made use of the human MHC class I heavy chain molecule HLA-A3. Proteinase K treatment effectuated the complete digestion of RCAS1, whereas the large luminal part of HLA-A3 remained protected from proteolytic cleavage (Fig. 1C).
Results from in vitro experiments were confirmed in cell lines. We performed protease protection assays in the mAb 22.1.1-negative human embryonal kidney cell line HEK293 and in the mAb 22.1.1-positive mammary carcinoma cell line MCF7. Whole cells were homogenized under conditions that leave intracellular compartments intact, and homogenates were exposed to proteinase K. This treatment leads to the degradation of the cytoplasmic parts of organelle-associated proteins (19). Whereas the ER-resident intraluminal chaperone calreticulin and the large luminal part of the MHC class I heavy chain remained unaffected, RCAS1 was subject to complete degradation in both cell lines (Fig. 1D). A potential intraluminal/TM N terminus of about 30 amino acids was not resolved by the gel system employed.
Two alternatives might account for the membrane orientation of RCAS1. First, the non-polar region at the N terminus of RCAS1 constitutes a TM segment indicative of a type III TM protein with a reverse signal anchor, according to the classification of Spiess (26). Type III TM proteins possess a large cytoplasmically oriented C terminus and a single TM-spanning region at the N terminus that serves as non-cleaved membrane targeting signal. Second, a peripheral membrane association could be considered. To explore if RCAS1 might be an integral membrane protein, crude cell membranes from MCF7 and HEK293 cells were prepared and washed with 1 M KCl, 10 mM EDTA, 100 mM Na2CO3, or 4 M urea. In general, high salt treatment releases soluble and peripheral membrane proteins, whereas integral membrane proteins remain inserted in the lipid bilayer. RCAS1 could not be detected in the cytosolic fractions but was recovered from untreated and high salt treated postnuclear membranes (Fig. 1E). Detergent treatment with Triton X-100 allowed the solubilization of membrane-bound RCAS1 (Fig. 1E). Taken together, we conclude that RCAS1 is a type III TM protein with its large C terminus localized in the cytoplasm.
Subcellular Localization of RCAS1Applying the mAb 22.1.1 to immunohistochemistry, RCAS1 was reported to be localized to intracellular compartments and to the plasma membrane. However, the subcellular localization was reported to vary among tissues and degree of malignancy of the tumors explored (27, 28).
When we employed our polyclonal serum for the detection of RCAS1 in acetone-fixed MCF7 cells, a perinuclear reticular staining pattern characteristic of the Golgi complex in these cells was seen (Fig. 2, A and B). To further resolve the subcellular localization of the RCAS1-encoded gene product, confocal laser scanning microscopy on living cells was carried out. The 22.1.1-negative cell line HEK293 and the 22.1.1-positive cell line MCF7 were chosen for direct comparison. Both cells lines were transiently cotransfected with RCAS1-GFP and the Golgi marker 1,4-GT-YFP. After 48 h, plasma membranes were stained with trypan blue immediately followed by confocal microscopic analysis. Comparison of RCAS1-GFP with 1,4-GT-YFP revealed a considerable overlap of the fluorescence signals. RCAS1-GFP signals could not be detected at the plasma membranes or at any other subcellular localization in either of the two cell lines analyzed (shown for MCF7, Fig. 3, AD). RCAS1-GFP and endogenous RCAS1, as detected by our polyclonal serum (Fig. 2, C and D) and by the commercially available mAb AM-75 directed against the RCAS1 gene product (Fig. 2, FH), shared the same Golgi predominant expression pattern.
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The N-terminal Transmembrane Domain and the C-terminal Coiled-coil Region Determine Organelle AssociationTo elucidate the polypeptide domains responsible for the Golgi localization of RCAS1, four deletion variants of RCAS1 were constructed as GFP fusion proteins (Fig. 3). Subcellular distribution patterns of these mutants were analyzed in HEK293 and MCF7 cells using confocal microscopy. Deletion of the C-terminal coiled-coil region did not alter the subcellular localization, compared with wild-type RCAS1 (Fig. 3, AD and EH), whereas deletion of the N-terminal 27 amino acids led to a cytosolic staining pattern, as would be expected if a functional membrane anchor was deleted (Fig. 3, MP). To confirm that the N terminus was responsible for the Golgi membrane association, this region was directly fused to GFP. Confocal microscopy revealed a fluorescence pattern that was almost indistinguishable from that of full-length RCAS1 (Fig. 3, IL). Thus, the N-terminal 30 amino acids appear to be responsible for membrane association and Golgi targeting of RCAS1. Analysis of the 127 variant revealed a predominant cytosolic protein distribution. In addition, a GFP-fluorescence signal enhancement was observed at regions that were labeled with the Golgi marker 1,4-GT-YFP (Fig. 3, MP). An identical subcellular expression pattern was observed when the C-terminal coiled-coil domain was fused directly to GFP, indicating that the coiled-coil region also participates in organelle attachment (Fig. 3, QT).
The 22.1.1 Antibody Fails to Recognize the RCAS1 Gene ProductIt has been suggested that the 22.1.1-defined tumor antigen is encoded by the cDNA for RCAS1/EBAG9. This conclusion has been facilitated by expression cloning and subsequent screening of transfected HEK293 cells with mAb 22.1.1.
To our surprise, when we employed confocal microscopy, we failed to detect full-length RCAS1-GFP or any of the deletion variants at the plasma membrane of mAb 22.1.1 surface positive MCF7 cells (Fig. 3, AD). Lack of RCAS1-encoded antigen expression on the cell surfaces of HEK293 and MCF7 cells could also be confirmed biochemically in non-transfected cells. HEK293 and MCF7 cells were biotinylated, and surface proteins were recovered using streptavidin beads. Intracellular, non-biotinylated proteins were precipitated with trichloroacetic acid. Both pools, surface and intracellular proteins, were analyzed by immunoblot with the anti-RCAS1 serum (Fig. 4A). The anti MHC class I heavy chain mAb HC10 served as control for a plasma membrane glycoprotein. In our hands, the IgM mAb 22.1.1 was only applicable to immunocytochemistry and ELISA. Therefore, 22.1.1 surface expression was assessed by flow cytometric analysis in parallel. MCF7 cells were strongly stained with mAb 22.1.1, and HEK293 cells were essentially negative (Fig. 4B). In contrast to this observation, but in agreement with the topology and subcellular distribution of the RCAS1 gene product as demonstrated before (Figs. 1, 2, 3), neither cell line exhibited RCAS1 surface expression after surface biotinylation (Fig. 4A).
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The biochemical properties of RCAS1, as specified by its membrane orientation and subcellular distribution, were found to be identical in HEK293 and MCF7 cells. In conclusion, our failure to detect surface expression of RCAS1 in cell lines that were stained with mAb 22.1.1 on their surfaces raised the concern of whether the 22.1.1 mAb recognizes the RCAS1 gene product, as defined by the published cDNA sequence. To further resolve this discrepancy, MCF7 cells were transfected with RCAS1-GFP fusion protein, grown on coverslips for 48 h, fixed, permeabilized, and stained with 22.1.1 mAb or IgM isotype control. Confocal microscopy images suggested that RCAS1 was not recognized by the 22.1.1 mAb (Fig. 5, AF). We failed to observe a significant overlap between RCAS1-GFP and the 22.1.1 mAb-defined antigen. Instead, 22.1.1 mAb showed reactivity for vesicle-like structures mainly localized in the vicinity of the plasma membrane. Results from flow cytometry, antibody feeding experiments (data not shown), and the observation that the 22.1.1 antigen is shed into the cell culture supernatant (29) are indicative of a close relationship between the 22.1.1-positive vesicles and the cell surface. More specifically, our observation from confocal microscopy suggests that the 22.1.1-defined antigen might be stored in secretory vesicles prior to plasma membrane fusion and surface release.
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Overexpression of RCAS1 Renders HEK293 Cells Positive for 22.1.1 Surface StainingBased on its membrane topology and predominant Golgi localization, it appears unlikely that RCAS1 serves as soluble or plasma membrane-bound receptor ligand. We therefore revisited the data on RCAS1 expression cloning and transfected HEK293 cells with different amounts of RCAS1 expression plasmid, followed by flow cytometry using mAb 22.1.1. Depending on the amount of RCAS1 cDNA transfected, permeabilized and non-permeabilized HEK293 cells stained positively for 22.1.1 mAb (Fig. 6A). To analyze the localization of the 22.1.1 mAb-defined antigen in RCAS1-overexpressing HEK293 cells, cells were transfected with RCAS1-GFP, grown on coverslips, stained with 22.1.1 or isotype controls, and analyzed by confocal microscopy. The subcellular localization of the 22.1.1 antigen was indistinguishable from that in MCF7 cells (Fig. 5, GI). In agreement with our data from MCF7 cells, there was essentially no overlap between fluorescence signals from RCAS1-GFP and the 22.1.1-defined antigen. Transfection of the GFP mock control failed to induce 22.1.1 expression (Fig. 5, MO).
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The relevance of the major domains of RCAS1 for 22.1.1 staining was analyzed by flow cytometry after transfection of RCAS1-GFP deletion variants. The percentage of 22.1.1-positive cells was calculated as the ratio of 22.1.1 and GFP-positive cells to total GFP-positive cells. Identical region settings were used in all experiments. Fig. 6B shows mean values of five independent experiments. An average of 20% of transfected cells were stained with the 22.1.1 mAb when full-length RCAS1 or the coiled-coil variant (
179213-GFP) were transfected. Transfection of other variants did not lead to a significant 22.1.1 epitope up-regulation. We conclude that the coiled-coil domain did not contribute to the generation of the 22.1.1 antigen.
The 22.1.1 Antibody Recognizes the Tn AntigenThe mAb 22.1.1 exhibits a broad tumor-specific reactivity, suggesting that a common tumor-associated antigen might be recognized. It has been appreciated that cancer cells of diverse histological origin often exhibit abnormal O-linked glycoprotein structures that might elicit humoral or cellular immune responses. In addition, such humoral immune responses, as obtained from cancer patients or from mice immunized with tumor cell lines, are predominantly of the IgM type.
Based on our observation that the IgM antibody 22.1.1 failed to react with the RCAS1-encoded gene product, we wanted to test if the 22.1.1 mAb might recognize a tumor-specific O-linked carbohydrate instead. We employed benzyl--GalNAc as an inhibitor of O-linked glycosylation. Benzyl-
-GalNAc treatment facilitates incomplete O-linked glycosylation due to inhibition of sialyltransferases and galactosyltransferases, resulting in premature O-linked glycan structures like terminal GalNAc (Tn antigen) or Gal
13GalNAc (TF antigen) that are often found on tumor cells (3032). Although benzyl-
-GalNAc treatment of MCF7 cells had no influence on 22.1.1 staining, HEK293 and HeLa cells were rendered 22.1.1-positive (shown for HEK293, Fig. 7). Simultaneous overexpression of RCAS1-GFP led to an additional effect on mAb 22.1.1 surface staining, as determined by flow cytometry.
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The possible reactivity of 22.1.1 with one of the known tumor-associated carbohydrate antigens or closely related structures was investigated by ELISA (Fig. 8A). It was evident that the mAb 22.1.1 showed strong binding toward Tn. Similar structures, like sialylated Tn or TF, were not recognized. Carbohydrate specificity was also confirmed by periodate oxidation of fixed MCF7 cells. Treatment with periodate destroys terminal saccharide rings with vicinal OH groups, thereby abolishing immunological recognition (18). Fig. 8B shows that the 22.1.1 signal is extinguished after periodate treatment. An established Tn-specific mAb, 5F4, was used as a control. For additional proof of antibody specificity, we transiently transfected HEK293 cells with RCAS1-GFP or GFP alone and performed flow cytometry with the 22.1.1 mAb and the 5F4 mAb. Staining with both antibodies exhibited an identical pattern in dot blot diagrams (Fig. 8C). From these data we conclude that overexpression of the RCAS1-cDNA contributes indirectly to the generation of the Tn antigen. Interestingly, overexpression of RCAS1 also led to the enhanced surface expression of the closely related TF antigen as analyzed by flow cytometry using the mAb G/A7 (Fig. 8C). We note that an RCAS1 mutant with a deletion of a potential O-linked glycan acceptor site at the N terminus (Thr4 Ala) had identical effects on the generation of the 22.1.1 epitope, compared with wild-type RCAS1 (data not shown).
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DISCUSSION |
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The subcellular localization and membrane topology of the RCAS1-encoded gene product were the subject of this study. Employing RCAS1 transfectants and GFP-tagged truncation variants derived thereof, we found the RCAS1-encoded gene product to be localized to the Golgi complex. Protease protection assays revealed the overall membrane topology of RCAS1 with a predominant C terminus facing toward the cytoplasm. Such type III TM protein orientation would leave the RCAS1-encoded gene product with an extremely short span of seven amino acids encompassing the N terminus. This structural characteristic imposes the problem of how the 22.1.1 mAb can recognize such extracellular domain in surface staining. Second, the short N terminus at the outer site of a cell obviously would limit the capacity of the RCAS1-encoded molecule to interact with the postulated death receptor on immune cells (5).
To resolve these discrepancies, we compared 22.1.1 mAb staining with the subcellular localization of the RCAS1-GFP-tagged constructs. In addition, we made use of our polyclonal RCAS1-antiserum raised against recombinant RCAS1 fusion protein. Confocal microscopic analysis revealed that 22.1.1 failed to recognize the RCAS1-GFP constructs in diverse cell lines. Although the mAb exhibited a plasma membrane predominant staining pattern in MCF7 cells, RCAS1-GFP signals were obtained from the Golgi region, exclusively. Although the addition of a GFP tag might alter some properties of a native molecule, we consider it unlikely that in our case the endogenous RCAS1 behaves differently from its tagged derivatives. This could be concluded from the staining experiments of MCF7 cells with the polyclonal anti-RCAS1 serum (Fig. 2). Furthermore, GFP-tagged constructs of RCAS1 as well as the wild-type sequence-encoded antigen behaved identically in the generation of the 22.1.1 epitope. From our deletion mutant studies we conclude that the transmembrane domain of RCAS1 is responsible for its localization and sorting in the Golgi complex.
RCAS1 has attracted particular attention because of its suggested role as a tumor-associated antigen. In contrast to the tumor specificity as proposed, mRNA expression was found in all tissues and cell lines examined (5). The human genomic upstream region of RCAS1 contains CpG-rich islands (9, 10). In general, CpG-rich promoter regions are characteristic features of housekeeping genes and promote constitutive basal expression levels (33). The initial observation that RCAS1 antigen expression was missing from normal human tissues is best explained by the method employed, because the mAb 22.1.1 was used for immunostaining. In contrast, using a polyclonal serum for RCAS1 detection in murine tissues, we2 and others (14) confirmed its expression in essentially all murine tissues examined. Likewise, we found RCAS1 expression in all human and murine cell lines examined at the protein level (data not shown), suggesting that RCAS1 is a ubiquitously expressed protein but not tumor-specific. Based on the extensive homology at the protein level where only five amino acids are different, it seems reasonable to suggest that the RCAS1 gene product is expressed in most normal human tissues as well.
When we revisited the data on RCAS1 expression cloning, we could show that RCAS1 overexpression leads to the generation of the antigen recognized by the 22.1.1 mAb, which is identical to the Tn glycan antigen. These results were indicative of an indirect, modulatory role of RCAS1 in the process of Tn antigen generation. Further functional analysis of RCAS1-deletion constructs support our notion that a suggested multimerization of RCAS1, mediated by a coiled-coil domain (5), is not critical for the generation of Tn after RCAS1 overexpression.
To reconcile our cell biological characterization of RCAS1 with the functional results originally reported by Nakashima et al. (5), we would like to point out that the induction of apoptosis in activated T cells was seen upon exposure to SiSo culture supernatant. In our hands, recombinant RCAS1-GST fusion protein is not soluble in aqueous solutions and cannot be purified in the absence of detergent. Moreover, recombinant RCAS1-GST lacking the transmembrane domain failed to bind a putative receptor (5), nor has it any effect on activated T cells.2 We conclude that full-length RCAS1 is not applicable to cell culture experiments, and effects seen on cell viability and integrity are most likely due to residual detergent. RCAS1-induced alterations were not limited to the generation of Tn, a concomitant generation of the TF antigen points to a more general mechanism of interference with the O-glycan extension processes in the Golgi apparatus.
In general, TF and Tn antigens are thought to be linked with cell adhesion, invasion, and metastasis of cancer cells (34). Such glycan epitopes are normally cryptic in healthy and benign tissues, except in early embryonic stages (3).
With respect to its generalized expression pattern, a potential difference in expression and regulation of RCAS1 in benign and tumor tissues warrants further analysis. This could add to the problem if aberrant glycosylation is a result or a prerequisite of initial oncogenic transformation (1, 35).
Several mechanisms for the generation of Tn and TF facilitated by RCAS1-overexpression can be envisaged. Golgi-resident glycan-nucleotide transporters might be affected. There is evidence that transport of nucleotide-activated sugars into the Golgi lumen participates in the regulation of polypeptide glycosylation (36). Massive import of GalNAc-UDP into the Golgi lumen could lead to an excessive basal glycosylation of serine or threonine residues and of glycosphingolipids that cannot further be extended into more complex core structures. A stimulatory effect on such nucleotide-glycan transporters could mimic the observed appearance of the precursor glycans Tn and TF on the cell surface.
Glycosyltransferases are arranged in the Golgi apparatus in a spatial and functional hierarchy. Overexpression of RCAS1 might induce a disturbance of the Golgi architecture, and consequently a dislocation of glycosyltransferases. Interestingly, the flow cytometric analysis of RCAS1-transfected HEK293 (Fig. 8C) points to a non-linear correlation between RCAS1 overexpression and 22.1.1 antigen generation. Instead, an apparent threshold of RCAS1 expression is required for the appearance of Tn and TF.
An alternative explanation for the modulatory function of RCAS1 on Tn and TF antigen expression would include a role in vesicle trafficking from the Golgi apparatus to the cell surface (32). It could be hypothesized that RCAS1 facilitates the release of premature polypeptides or glycolipids carrying O-linked glycans.
In view of its supposed role as tumor-associated antigen, a more detailed analysis of the modulatory role of RCAS1 in the generation of the tumor-associated Tn and TF glycan structures is required. In particular, it remains unresolved at what level RCAS1 interferes with the secretory pathway for O-linked glycoproteins and if this ubiquitously expressed gene product is related to the malignant transformation of cells.
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FOOTNOTES |
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** To whom correspondence should be addressed. Tel.: 49-30-9406-2695; Fax: 49-30-9406-2698; E-mail: arehm{at}mdc-berlin.de.
1 The abbreviations used are: TF, Thomsen-Friedenreich antigen; 1,4-GT, 1,4-galactosyltransferase; benzyl-
-GalNAc, benzyl-2-acetamido-2-deoxy-
-D-galactopyranoside;DAPI,4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; GST, glutathione S-transferase; MHC, major histocompatibility complex; PE, phycoerythrin; rSB, reducing sample buffer; RT, room temperature; TM, transmembrane; YFP, yellow fluorescent protein; Tn, N-acetyl-D-galactosamine, GalNAc; RCAS1, receptor binding cancer antigen expressed on SiSo cells; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PMSF, phenyl-methylsulfonyl fluoride; TX-100, Triton X-100; Endo H, endoglycosidase H; FACS, fluorescence-activate cell sorting; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; BP, bandpass.
2 A. Engelsberg, R. Hermosilla, U. Karsten, R. Schülein, B. Dörken, and A. Rehm, unpublished observations.
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ACKNOWLEDGMENTS |
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