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• W2000588306 abstract "Many tumor-associated epitopes possess carbohydrate as a key component, and thus changes in the activity of glycosyltransferases could play a role in generating these epitopes. In this report we describe the stable transfection of a human pancreatic adenocarcinoma cell line, Panc1-MUC1, with the cDNA for mucin core 2 GlcNAc-transferase (C2GnT), which creates the core 2 β-1,6 branch in mucin-type glycans. These cells lack endogenous C2GnT activity but express a recombinant human MUC1 cDNA. C2GnT-transfected clones expressing different levels of C2GnT were characterized using monoclonal antibodies CC49, CSLEX-1, and SM-3, which recognize tumor-associated epitopes. Increased C2GnT expression led to greatly diminished expression of the CC49 epitope, which we identified as NeuAcα2,6(Galβ1,3)GalNAcα-Ser/Thr in the Panc1-MUC1 cells. This was accompanied by the emergence of the CSLEX-1 epitope, sialyl Lewis x (NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAc-R), an important selectin ligand. Despite this, however, the C2GnT transfectants could not bind to selectins. Increased C2GnT expression also led to masking of the SM-3 peptide epitope, which persisted after the removal of sialic acid, further suggesting greater complexity of the core 2-associated O- glycans on MUC1. The results of this study suggest that C2GnT could play a regulatory role in the expression of certain tumor-associated epitopes. Many tumor-associated epitopes possess carbohydrate as a key component, and thus changes in the activity of glycosyltransferases could play a role in generating these epitopes. In this report we describe the stable transfection of a human pancreatic adenocarcinoma cell line, Panc1-MUC1, with the cDNA for mucin core 2 GlcNAc-transferase (C2GnT), which creates the core 2 β-1,6 branch in mucin-type glycans. These cells lack endogenous C2GnT activity but express a recombinant human MUC1 cDNA. C2GnT-transfected clones expressing different levels of C2GnT were characterized using monoclonal antibodies CC49, CSLEX-1, and SM-3, which recognize tumor-associated epitopes. Increased C2GnT expression led to greatly diminished expression of the CC49 epitope, which we identified as NeuAcα2,6(Galβ1,3)GalNAcα-Ser/Thr in the Panc1-MUC1 cells. This was accompanied by the emergence of the CSLEX-1 epitope, sialyl Lewis x (NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAc-R), an important selectin ligand. Despite this, however, the C2GnT transfectants could not bind to selectins. Increased C2GnT expression also led to masking of the SM-3 peptide epitope, which persisted after the removal of sialic acid, further suggesting greater complexity of the core 2-associated O- glycans on MUC1. The results of this study suggest that C2GnT could play a regulatory role in the expression of certain tumor-associated epitopes. UDP-GlcNAc:Galβ1–3GalNAc (GlcNAc to GalNAc) β1–6GlcNAc-transferase UDP-GalNAc:Polypeptide GalNAc transferase UDP-Gal:GalNAc β1–3Gal transferase CMP-NeuAc:GalNAc/Galβ1–3GalNAc (NeuAc to GalNAc) α2–6NeuAc transferase CMP-NeuAc:Galβ1–3GalNAc (NeuAc to GalNAc) α2–6NeuAc transferase CMP-NeuAc:Galβ1–3GalNAc (NeuAc to Gal) α2–3-NeuAc transferase Dolichos biflorus agglutinin Arachis hypogaea agglutinin galactose Panc1 cells transfected with FLAG epitope-tagged MUC1 cDNA sialyl Lewis benzyl phosphate-buffered saline Tris-buffered saline bovine serum albumin transferase freezing point-depressing glycoprotein Many cancer cells are distinguished from their normal counterparts by the presence of certain cell surface epitopes. These tumor-associated epitopes are potential targets for diagnosis, imaging, and therapeutic treatment (reviewed in Refs. 1Farah R.A. Clinchy B. Herrera L. Vitetta E.S. Crit. Rev. Eukaryotic Gene Expression. 1998; 8: 321-356Crossref PubMed Scopus (69) Google Scholar and 2Rowlinson-Busza G. Epenetos A.A. Curr. Opin. Oncol. 1992; 4: 1142-1148Crossref PubMed Scopus (20) Google Scholar). The mechanism by which these epitopes arise is not well understood but may involve changes in the activity of glycosyltransferases, because carbohydrate is an essential component of many tumor-associated epitopes. Mucin-type glycan structures are influenced by both the level of expression and the Golgi localization of glycosyltransferases, which compete with one another for common acceptor structures (3Brockhausen I. Montreuil J. Vliegenthart J.F.G. Schachter H. New Comprehensive Biochemistry. 29a. Elsevier Publishing Co., Inc., New York1996: 201-259Google Scholar). When competing glycosyltransferases share the same Golgi localization, the final O- glycan structure is likely to be governed primarily by the relative activities of the enzymes. However, if the enzymes reside in different Golgi compartments, the earlier Golgi enzyme will have an advantage in dictating the oligosaccharide structure. Glycosyltransferase competition can occur following the creation of the core 1 acceptor structure Galβ1–3GalNAcαSer/Thr. UDP-GlcNAc:Galβ1–3GalNAc (GlcNAc to GalNAc) β-1,6GlcNAc-transferase (C2GnT;1 EC 2.4.1.102) attaches GlcNAc in β-1,6 linkage to GalNAc of the core 1 acceptor creating the core 2 β-1,6 branch in the O- glycan chain.UDP-GlcNAc+Galβ1-3GalNAcα-Ser/Thr→C2GnTGlcNAcβ1-6Galβ1-3GalNAcα-Ser/Thr+UDPREACTION I Creation of the core 2 branch enables the O- glycan chain to be extended into complex structures such as polylactosamine chains (reviewed in Ref. 4Dennis J.W. Glycobiology. 1993; 3: 91-96Crossref PubMed Scopus (11) Google Scholar). The sialyl transferases ST6GalNAc I and II compete with C2GnT for the core 1 acceptor substrate (5Kurosawa N. Kojima N. Inoue M. Hamamoto T. Tsuji S. J. Biol. Chem. 1994; 269: 19048-19053Abstract Full Text PDF PubMed Google Scholar, 6Kurosawa N. Hamamoto T. Lee Y.-C. Nakaoka T. Kojima N. Tsuji S. J. Biol. Chem. 1994; 269: 1402-1409Abstract Full Text PDF PubMed Google Scholar) and direct the glycosylation pathway toward simpler structures lacking the core 2 branch, such as NeuAcα2,6(Galβ1,3)GalNAc and NeuAcα2,6GalNAc. The latter structures are recognized by the monoclonal antibody CC49 (7Hanisch F.-G. Uhlenbruck G. Egge H. Peter-Katalinic J. Biol. Chem. Hoppe-Seyler. 1989; 370: 21-26Crossref PubMed Scopus (40) Google Scholar, 8O'Boyle K.P. Markowitz A.L. Khorshidi M. Lalezari P. Longenecker B.M. Lloyd K.O. Welt S. Wright S.E. Hybridoma. 1996; 15: 401-408Crossref PubMed Scopus (27) Google Scholar), and therefore creation of the CC49 epitope should be influenced by the relative activities as well as the specific Golgi localization of both C2GnT and these sialyl transferases. C2GnT activity is regulated under certain growth conditions, including maturation of granulocytes (9Fukuda M. Carlsson S.R. Klock J.C. Dell A. J. Biol. Chem. 1986; 261: 12796-12806Abstract Full Text PDF PubMed Google Scholar) and T cells (10Baum L.G. Pang M. Perillo N.L. Wu T. Delegeane A. Uittenbogaart C.H. Fukuda M. Seilhamer J.J. J. Exp. Med. 1995; 181: 877-887Crossref PubMed Scopus (258) Google Scholar) as well as T cell activation (11Piller F. Piller V. Fox R.I. Fukuda M. J. Biol. Chem. 1988; 263: 15146-15150Abstract Full Text PDF PubMed Google Scholar). Transgenic mice in which C2GnT was overexpressed showed normal T cell development but an impaired T cell immune response (12Tsuboi S. Fukuda M. EMBO J. 1997; 16: 6364-6373Crossref PubMed Scopus (83) Google Scholar). The core 2 branched structure has been associated with the sialyl Lewis x (sLex) determinant (13Maemura K. Fukuda M. J. Biol. Chem. 1992; 267: 24379-24386Abstract Full Text PDF PubMed Google Scholar, 14Heffernan M. Lotan R. Amos B. Palcic M. Takano Y. Dennis J.W. J. Biol. Chem. 1991; 268: 1242-1251Abstract Full Text PDF Google Scholar, 15Wilkins P.P. McEver R.P. Cummings R.D. J. Biol. Chem. 1996; 271: 18732-18742Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 16Ohmori K. Takada A. Yoneda T. Buma Y. Hirashima K. Tsuyoka K. Kannagi R. Blood. 1993; 81: 101-111Crossref PubMed Google Scholar), NeuAcα2–3Galβ1–4(Fucα1–3)GlcNAc-R, recognized by the monoclonal antibody CSLEX-1 (17Fukushima K. Hirota M. Terasaki P.I. Wakisaka A. Togashi H. Chia D. Suyama N. Fukushi Y. Nudelman E. Hakomori S.-I. Cancer Res. 1984; 44: 5279-5285PubMed Google Scholar). sLex acts as a ligand for binding of tumor cells (18Walz G. Aruffo A. Kolanus W. Bevilacqua M. Seed B. Science. 1990; 250: 1132-1135Crossref PubMed Scopus (887) Google Scholar, 19Majuri M.-L. Mattila P. Renkonen R. Biochem. Biophys. Res. Commun. 1992; 182: 1376-1382Crossref PubMed Scopus (99) Google Scholar, 20Takada A. Ohmori K. Yoneda T. Tsuyuoka K. Hasegawa A. Kiso M. Kannagi R. Cancer Res. 1993; 53: 354-361PubMed Google Scholar) and leukocytes (reviewed in Refs. 21Varki A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7390-7397Crossref PubMed Scopus (954) Google Scholarand 22McEver R.P. Moore K.L. Cummings R.D. J. Biol. Chem. 1995; 270: 11025-11028Abstract Full Text Full Text PDF PubMed Scopus (593) Google Scholar) to selectins on the surface of endothelial cells. The importance of C2GnT in vivo was recently shown in a study of mice in which the C2GnT gene was deleted from the germ line (23Ellies L.G. Tsuboi S. Petryniak B. Lowe J.B. Fukuda M. Marth J.D. Immunity. 1998; 9: 881-890Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Leukocyte interactions with selectins and endothelial cells were impaired, leading to a weakened inflammatory response. The mucin MUC1 is a type I membrane-bound glycoprotein, which is aberrantly glycosylated in many cancer tissues (reviewed in Ref. 24Ho S.B. Kim Y.S. Semin. Cancer Biol. 1991; 2: 389-400PubMed Google Scholar), displaying several tumor-associated epitopes. For example, monoclonal antibodies SM-3 and HMFG-2 recognize peptide epitopes within the MUC1 tandem repeat region and react preferentially with MUC1 in cancer tissues (25Burchell J. Gendler S.J. Taylor-Papadimitriou J. Girling A. Lewis A. Millis R. Lamport D. Cancer Res. 1987; 47: 5476-5482PubMed Google Scholar, 26Girling A. Bartkova J. Burchell J. Gendler S. Gillett C. Taylor-Papadimitriou J. Int. J. Cancer. 1989; 43: 1072-1076Crossref PubMed Scopus (307) Google Scholar, 27Arklie J. Taylor-Papadimitriou J. Bodmer W. Egan M. Millis R. Int. J. Cancer. 1981; 28: 23-29Crossref PubMed Scopus (265) Google Scholar) where aberrant glycosylation results in epitope exposure. The purpose of this study was to examine the effects of C2GnT on the expression of certain MUC1 tumor-associated epitopes. We stably transfected the human pancreatic adenocarcinoma cell line Panc1-MUC1, which expresses a recombinant human MUC1 cDNA, with a bovine C2GnT cDNA. We found that increased expression of C2GnT resulted in de novo expression of the sLex epitope. However, this did not render the cells capable of binding selectins. C2GnT expression also led to the elimination of the CC49 epitope. Furthermore, the SM-3 and HMFG-2 tumor-associated MUC1 peptide epitopes were masked by high levels of C2GnT expression. In summary, introduction of C2GnT into a cancer cell line significantly altered the expression of MUC1 tumor-associated epitopes by shifting the mucin-type glycosylation pathway toward more complex O- glycans. This suggests a potential regulatory role for C2GnT in the generation of tumor-associated epitopes. The Panc1 cell line was purchased from the ATCC (Manassas, VA). S2–013 is a subline of a human pancreatic tumor cell line derived from a liver metastasis (28Burdick M.D. Harris A. Reid C.J. Iwamura T. Hollinsworth M.A. J. Biol. Chem. 1997; 272: 24198-24202Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). UDP-[14C]GlcNAc, UDP-[3H]Gal, and CMP-[3H]NeuAc were from American Radiolabeled Chemicals (St. Louis, MO). Asialo ovine submaxillary mucin (aOSM) was prepared as described (29Cheng P.-W. Bona S.J. J. Biol. Chem. 1982; 257: 6251-6258Abstract Full Text PDF PubMed Google Scholar). FPDG was isolated as described (30Lin Y. DeVries A. Biochem. Biophys. Res. Commun. 1974; 59: 1192-1196Crossref PubMed Scopus (3) Google Scholar) from the serum of the antarctic fish Dissostichus mawsoni, provided by Dr. Arthur DeVries at the University of Illinois at Champaign-Urbana. E-, P- and L-selectin/IgM chimeras were provided by Dr. John Lowe at the University of Michigan, Ann Arbor. Transferrin (iron-saturated) was from Collaborative Biomedical Products (Bedford, MA). Immobilon P polyvinylidene difluoride membrane was from Millipore (Bedford, MA). Bond Elut C18 cartridges were from Varian (Sunny Vale, CA). M2 anti-FLAG monoclonal antibody was from Kodak IBI. CC49 monoclonal antibody was a gift from Dr. David Colcher at the University of Nebraska Medical Center. CSLEX-1 monoclonal antibody was obtained from the ATCC. HMFG-2 and SM-3 monoclonal antibodies were gifts of Dr. Sandra Gendler at the Mayo Clinic, Scottsdale, AZ. C2GnT monoclonal antibody B5–1 was obtained as described (31Li C.-M. Adler K.B. Cheng P.-W. Am. J. Respir. Cell Mol. Biol. 1998; 18: 343-352Crossref PubMed Scopus (15) Google Scholar). Biotin-conjugated lectins DBA and PNA were obtained from EY Laboratories (San Mateo, CA). Other chemicals were from Sigma unless otherwise noted. Panc1 and Panc1-MUC1 cells were grown in minimal essential medium supplemented with 5% fetal bovine serum and antibiotics (50 units/ml penicillin and 50 μg/ml streptomycin). Panc1-MUC1 cells stably transfected with C2GnT cDNA were grown in minimal essential medium supplemented with 5% fetal bovine serum and 300 μg/ml Zeocin. A 1.6-kilobase fragment of bovine C2GnT cDNA (containing the complete open reading frame of C2GnT) (31Li C.-M. Adler K.B. Cheng P.-W. Am. J. Respir. Cell Mol. Biol. 1998; 18: 343-352Crossref PubMed Scopus (15) Google Scholar) was subcloned into the mammalian expression vector pcDNA 3.1/Zeo+ (Invitrogen) using Kpn I and Not I. Transfection of pcDNA 3.1-C2GnT into Panc1-MUC1 cells was performed using a transferrin-assisted lipofection protocol as described previously (32Cheng P.-W. Hum. Gene Ther. 1996; 7: 275-282Crossref PubMed Scopus (170) Google Scholar), and clones, which stably express C2GnT, were selected based on resistance to the antibiotic Zeocin. Enzyme assays were carried out on total cell homogenates prepared by washing confluent monolayers of the cells twice with cold PBS, scraping the cells off the flask in 0.25 m sucrose, and disrupting the cells by successive passage of the sucrose suspension through 18-, 20-, and 25-gauge syringe needles. Protein concentration was measured by the Bio-Rad assay (Bio-Rad) using BSA as standard. All assays were conducted at least in duplicate under conditions in which product formation was linear with respect to time and enzyme amount. An additional reaction without exogenous acceptor was performed to measure endogenous enzyme activity. Enzyme activity was calculated by subtracting endogenous activity from total activity and was expressed as nmol of sugar donor transferred/hour/mg protein. GalNAc TF, which catalyzes the attachment of GalNAc in α-linkage to serine or threonine of the mucin peptide, was assayed as described (33Nishimori I. Johnson N.R. Sanderson S.D. Perini F. Mountjoy K. Cerny R.L. Gross M.L. Hollingsworth M.A. J. Biol. Chem. 1994; 269: 16123-16130Abstract Full Text PDF PubMed Google Scholar), using a synthetic 29-amino acid MUC2 peptide as acceptor having the sequence PTTTPITTTTTVTPTPTPTGTQTPTTTPI. Core 1 GalTF, which catalyzes attachment of galactose in β-1,3 linkage to GalNAcα-Ser/Thr, was assayed as described (29Cheng P.-W. Bona S.J. J. Biol. Chem. 1982; 257: 6251-6258Abstract Full Text PDF PubMed Google Scholar) using aOSM as acceptor. C2GnT activity was assayed as described (34Ropp P.A. Little M.R. Cheng P.-W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar) using Galβ1–3GalNAcα-Bzl as acceptor. ST6GalNAc I, which catalyzes attachment of neuraminic acid in α-2,6 linkage to GalNAc in GalNAcα-Ser/Thr and Galβ1–3GalNAcα-Ser/Thr (6Kurosawa N. Hamamoto T. Lee Y.-C. Nakaoka T. Kojima N. Tsuji S. J. Biol. Chem. 1994; 269: 1402-1409Abstract Full Text PDF PubMed Google Scholar), was assayed as described (35Cheng P.-W. Moeller S.L. Boat T.F. Fed. Proc. 1980; 39: 2002Google Scholar) using aOSM as acceptor. ST6GalNAc II, which catalyzes attachment of neuraminic acid in α-2,6 linkage to GalNAc in Galβ1–3GalNAcα-Ser/Thr (but not in GalNAcα-Ser/Thr) (5Kurosawa N. Kojima N. Inoue M. Hamamoto T. Tsuji S. J. Biol. Chem. 1994; 269: 19048-19053Abstract Full Text PDF PubMed Google Scholar), was assayed as described (35Cheng P.-W. Moeller S.L. Boat T.F. Fed. Proc. 1980; 39: 2002Google Scholar) using FPDG as acceptor. FPDG also acts as an acceptor for ST6GalNAc I and ST3Gal I, and therefore care must be taken in interpreting assay results using this acceptor. ST3Gal I, which catalyzes attachment of neuraminic acid in α-2,3 linkage to Gal of Galβ1–3GalNAcα-Ser/Thr (36Rearick J.I. Sadler J.E. Paulson J.C. Hill R.L. J. Biol. Chem. 1979; 254: 4444-4451Abstract Full Text PDF PubMed Google Scholar), was measured in the same manner as ST6GalNAc I and II, except Galβ1–3GalNAcα-Bzl was used as acceptor. ST6GalNAc I and II cannot utilize Galβ1–3GalNAcα-Bzl as acceptor (5Kurosawa N. Kojima N. Inoue M. Hamamoto T. Tsuji S. J. Biol. Chem. 1994; 269: 19048-19053Abstract Full Text PDF PubMed Google Scholar, 6Kurosawa N. Hamamoto T. Lee Y.-C. Nakaoka T. Kojima N. Tsuji S. J. Biol. Chem. 1994; 269: 1402-1409Abstract Full Text PDF PubMed Google Scholar), thus preventing their interference with the measurement of ST3Gal I activity. Confluent cells were washed twice with cold PBS and scraped from culture flasks in lysis buffer (500 μl for a T25 flask) containing 10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, and 1% Triton X-100, using a rubber cell scraper. Following a 40-min incubation on ice, lysates were centrifuged at 2000 rpm for 2 min in a microcentrifuge to pellet cell debris. Supernatant was then transferred to a new tube and stored at −20 °C. Cell lysates (300 μg of total protein) prepared as described above were treated with 100 milliunits of Clostridium perfringens neuraminidase in a total volume of 200 μl for 3.5 h at 37 °C in 0.05 m sodium acetate, pH 5.5. One-third of the neuraminidase-treated lysates was exposed to 1 unit of peptide N-glycosidase F in 200 mm sodium phosphate, 10 mm EDTA, pH 7.2, at 37 °C for 18 h. Another third of the neuraminidase-treated lysates was treated with 10 milliunits of Diplococcus pneumoniae β-galactosidase in 200 mm sodium cacodylate, pH 6.0, at 37 °C for 44 h. Samples were stored at −20 °C after treatment prior to SDS-polyacrylamide gel electrophoresis analysis and Western blotting or lectin blotting. For Western blot analysis of MUC1 and C2GnT, proteins in cell lysates were resolved by 6% SDS-polyacrylamide gel electrophoresis (with 3% polyacrylamide stacking gels) and 10% SDS-polyacrylamide gel electrophoresis (with 6% polyacrylamide stacking gels), respectively. Protein was electroblotted to Immobilon P polyvinylidene difluoride membrane overnight at 300 mA, then blocked in 5% nonfat milk in TBS (0.9% NaCl, 10 mm Tris, pH 7.5) at room temperature for 1 h. The MUC1 blots were then probed for 1 h at room temperature with various primary antibodies in 5% nonfat milk in TBS, whereas the C2GnT blot was probed with the C2GnT antibody B5–1 diluted 1:2500 in TBS, 1% BSA. Membranes were then washed 15 min in 5% nonfat milk in TBS (for MUC1 blots) or TBS, 1% BSA (for C2GnT blots) followed by two additional 5-min washes with fresh wash mixtures. The membranes were then exposed for 1 h at room temperature to peroxidase-conjugated goat anti-mouse IgG/IgM secondary antibody diluted 1:2000 in 5% nonfat milk in TBS (for MUC1 blots) or TBS, 1% BSA (for C2GnT blots). Washes were repeated as described above, and then ECL reagents (Pierce) were applied per the manufacturer's instructions; the blots were then exposed to ECL-sensitive film (Amersham Pharmacia Biotech). Proteins in whole cell lysates were resolved by 6% SDS-polyacrylamide gel electrophoresis (with 3% polyacrylamide stacking gels). Protein was electroblotted to Immobilon P polyvinylidene difluoride membrane overnight at 300 mA and blocked in 2% BSA (fraction V) in PBS at room temperature for 1 h. The blots were probed for 1.5 h at room temperature with biotin conjugates of either DBA or PNA 1:500 in TBT (TBS + 0.1% BSA + 0.025% Tween 20). Membranes were washed three times, 5 min each, in TBT. Next, the membranes were exposed for 1 h at room temperature to peroxidase-conjugated streptavidin diluted 1:1000 in TBT. Washes were repeated as described above. ECL reagents were applied as per the manufacturer's instructions, and the blots were exposed to ECL-sensitive film. 500 μl of Panc1-MUC1 C2#5 total cell lysate was incubated with 200 μl of antibody HMFG-2 at 4 °C with mild agitation for 3 h. Protein G-Sepharose (150 μl) was then added, and the mixture was incubated for 16 h at 4 °C with mild agitation. The immunoprecipitate was washed three times with 1 ml of cold PBS. SDS-polyacrylamide gel electrophoresis sample buffer was added, and the mixture was boiled for 5 min; the supernatant was resolved on 6% SDS-polyacrylamide gel electrophoresis. S2–013 cells and Panc1-MUC1 C2#7 cells were grown to approximately 90% confluence and released from the tissue culture flask by incubation for 30–60 min in PBS containing 0.5 mm EDTA and 0.1% BSA. Aliquots containing 5 × 105 cells were pelleted in wells of a 96-well plate and washed with staining medium (Dulbecco's modified Eagle's medium containing 0.1% BSA and 0.1% sodium azide). The cells were then stained for 1 h on ice with IgM-conjugated P-, E-, or L-selectin in staining medium with or without 5 mm EDTA added. Cells were washed twice with staining medium (with or without 5 mm EDTA, as appropriate) and stained 1 h on ice in the dark with 10 μg/ml fluorescein isothiocyanate-conjugated goat anti-human IgM in staining medium (with or without 5 mmEDTA, as appropriate). Cells were washed twice with staining medium and fixed for 10 min in 2% formaldehyde. The cells were resuspended in PBS containing 0.1% BSA and 0.1% sodium azide and analyzed on a Becton Dickinson FACScan or FACStarPlus. MUC1-transfected Panc1 (Panc1-MUC1) cells were chosen as the host for stable transfection of C2GnT because this cell line lacks endogenous C2GnT expression but expresses core 1 Gal TF, which creates the acceptor substrate utilized by C2GnT. These cells also express a recombinant human MUC1 bearing a FLAG epitope (Fig. 1), which provides for ease of detection and purification of the MUC1. Furthermore, MUC1 in the Panc1-MUC1 cells has been previously characterized with a panel of antibodies recognizing specific carbohydrate antigens and was found to express the tumor-associated epitope recognized by CC49 (28Burdick M.D. Harris A. Reid C.J. Iwamura T. Hollinsworth M.A. J. Biol. Chem. 1997; 272: 24198-24202Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), which does not possess a core 2 branch (7Hanisch F.-G. Uhlenbruck G. Egge H. Peter-Katalinic J. Biol. Chem. Hoppe-Seyler. 1989; 370: 21-26Crossref PubMed Scopus (40) Google Scholar, 8O'Boyle K.P. Markowitz A.L. Khorshidi M. Lalezari P. Longenecker B.M. Lloyd K.O. Welt S. Wright S.E. Hybridoma. 1996; 15: 401-408Crossref PubMed Scopus (27) Google Scholar). Following transfection of the C2GnT cDNA into Panc1-MUC1 cells, stable clones were isolated and screened for expression of C2GnT. Three clones representing a wide range of C2GnT expression (Fig.2) were chosen for characterization of the effects of C2GnT on MUC1. The three clones displayed an approximate 40-fold difference in C2GnT activity between the high expressing (C2#7) and low expressing (C2#5) clones (Fig. 2 A). C2GnT expression levels in the clones assayed via Western blotting gave results consistent with those of the C2GnT enzyme assays (Fig.2 B). The presence of the core 2 branch in mucin-type glycans has been implicated in the generation of the sLex epitope (13Maemura K. Fukuda M. J. Biol. Chem. 1992; 267: 24379-24386Abstract Full Text PDF PubMed Google Scholar, 14Heffernan M. Lotan R. Amos B. Palcic M. Takano Y. Dennis J.W. J. Biol. Chem. 1991; 268: 1242-1251Abstract Full Text PDF Google Scholar, 15Wilkins P.P. McEver R.P. Cummings R.D. J. Biol. Chem. 1996; 271: 18732-18742Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 16Ohmori K. Takada A. Yoneda T. Buma Y. Hirashima K. Tsuyoka K. Kannagi R. Blood. 1993; 81: 101-111Crossref PubMed Google Scholar), and transfection of C2GnT cDNA into a cell line, which expresses the P-selectin ligand P-selectin glycoprotein ligand-1, rendered the cells capable of binding to P-selectin (37Li F. Wilkins P.P. Crawley S. Weinstein J. Cummings R.D. McEver R.P. J. Biol. Chem. 1996; 271: 3255-3264Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 38Kumar R. Camphausen R.T. Sullivan F.X. Cumming D. Blood. 1996; 88: 3872-3879Crossref PubMed Google Scholar). Therefore, we examined whether expression of C2GnT in Panc1-MUC1 cells could generate the sLex epitope. Immunoblotting with CSLEX-1, which recognizes sLex(NeuAcα2–3Galβ1–4(Fucα1–3)GlcNAc-R) (17Fukushima K. Hirota M. Terasaki P.I. Wakisaka A. Togashi H. Chia D. Suyama N. Fukushi Y. Nudelman E. Hakomori S.-I. Cancer Res. 1984; 44: 5279-5285PubMed Google Scholar), revealed that the sLex epitope was not present in Panc1-MUC1 parental cells or in the low C2GnT-expressing clone C2#5. However, the epitope appeared in clones C2#14 and C2#7, which have higher C2GnT expression (Fig. 3 A). MUC1 expression levels in the clones varied little (Fig. 3 B), showing that the changes in sLex detection were not because of different amounts of MUC1 in the samples. Because we detected sLex on MUC1 in the high C2GnT-expressing transfectants, we tested these clones for their ability to bind to P-, E-, and L-selectins using IgM-selectin fusion proteins followed by staining with a fluorescein isothiocyanate-conjugated anti-IgM secondary antibody and subsequent flow cytometry analysis. Despite the presence of the sLexepitope, no binding of the C2GnT transfectants to the selectins was detectable under conditions in which the positive control cell line, S2–013, was found to bind to the selectins (data not shown). Because CC49 can recognize both NeuAcα2,6(Galβ1,3)GalNAc and NeuAcα2,6GalNAc (7Hanisch F.-G. Uhlenbruck G. Egge H. Peter-Katalinic J. Biol. Chem. Hoppe-Seyler. 1989; 370: 21-26Crossref PubMed Scopus (40) Google Scholar,8O'Boyle K.P. Markowitz A.L. Khorshidi M. Lalezari P. Longenecker B.M. Lloyd K.O. Welt S. Wright S.E. Hybridoma. 1996; 15: 401-408Crossref PubMed Scopus (27) Google Scholar), we sought to identify which of these was present on MUC1 in the Panc1-MUC1 parental cells, as detected previously by Burdick et al. (28Burdick M.D. Harris A. Reid C.J. Iwamura T. Hollinsworth M.A. J. Biol. Chem. 1997; 272: 24198-24202Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). We performed lectin blotting using DBA and PNA on Panc1-MUC1 cell lysates treated or untreated with neuraminidase. As shown in Fig. 4, A and B, lanes 1 and 2, PNA reacted intensely with MUC1 in neuraminidase-treated lysate, whereas DBA showed no reaction. Because DBA is specific for α-linked GalNAc, whereas PNA recognizes Galβ 1–3 GalNAc, these results strongly suggest the presence of Galβ 1–3 (NeuAc α 2–6) GalNAc, rather than sialyl Tn (NeuAcα 2–6GalNAc), on MUC1 in the Panc1-MUC1 parental cells. To rule out the possibility that the structure recognized by PNA is located on asparagine-linked glycans on MUC1 rather than on mucin-type glycans, we treated desialylated lysate with peptide N-glycosidase F to remove N-linked chains. No change was seen in the intensity of PNA recognition (Fig. 4, A and B, lane 3), confirming that the recognized structure is present on O- glycans. Finally, because PNA can react with terminal β1,4-linked galactose in addition to Galβ 1–3 GalNAc, we treated desialylated lysate with D. pneumoniae β-galactosidase, which cleaves β-1,4-linked galactose (but not β-1,3-linked galactose). Once again, no change was seen in PNA recognition (Fig. 4, A and B, lane 4), further supporting Gal β-1–3GalNAc as the structure recognized by PNA. From these results, we conclude that the structure detected by CC49 in the Panc1-MUC1 parental cells consists mainly of the trisaccharide Gal β-1–3 (NeuAcα 2–6) GalNAc. This conclusion is supported by our detection of substantial core 1 GalTF enzyme activity in Panc1 cells (Table I), which synthesizes Galβ-1–3GalNAc.Table IGlycosyltransferase activities in Panc1 and Panc1-MUC1 C2#7 cellsEnzymeAcceptor usedLinkage catalyzedSpecific activityPanc1Panc1-MUC1 C2#7nmol/h/mg proteinGalNAc TFMUC2 peptide1-aSee “Experimental Procedures” for the sequence of MUC2 acceptor peptide.GalNAcα ⑧ Ser/Thr25.026.6Core 1 Gal TFaOSMGalβ1,3 ⑧ GalNAc-Ser/Thr14.113.8ST3Gal IGalβ1,3GalNAc-BzlNeuAcα2,3 ⑧ Galβ1,3GalNAc-Bzl1.11.2ST6GalNAc IaOSMNeuAcα2,6 ⑧ GalNAc-Ser/Thr0.70.7ST6GalNAc IIFPDGNeuAcα2,61.71.7↘Galβ1,3GalNAc-Ser/Thr1-bThe linkage shown is that catalyzed by ST6GalNAc II using FPDG. However, FPDG is also utilized by ST6GalNAc I and ST3Gal I. The fact that ST6GalNAc I and ST3Gal I activities were the same in Panc1 and Panc1-MUC1 C2#7, as was the total activity measured using FPDG, suggests that ST6GalNAc II activity was also unchanged.C2GnTGalβ1,3GalNAc-BzlGlcNAcβ1,6ND1-cND, not detectable (<0.5 nmol/h/mg for C2GnT assay).40.1↘Galβ1,3GalNAc-BzlEnzyme activities are shown as the values of donor substrates incorporated into the exogenous acceptor. Assays were performed at least in duplicate as described under “Experimental Procedures.”1-a See “Experimental Procedures” for the sequence of MUC2 acceptor peptide.1-b The linkage shown is that catalyzed by ST6GalNAc II using FPDG. However, FPDG is also utilized by ST6GalNAc I and ST3Gal I. The fact that ST6GalNAc I and ST3Gal I act" @default.
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• W2000588306 title "Expression of Core 2 β-1,6-N-Acetylglucosaminyltransferase in a Human Pancreatic Cancer Cell Line Results in Altered Expression of MUC1 Tumor-associated Epitopes" @default.
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• W2000588306 cites W1511676968 @default.
• W2000588306 cites W1516183342 @default.
• W2000588306 cites W1522003637 @default.
• W2000588306 cites W1529741804 @default.
• W2000588306 cites W1578452610 @default.
• W2000588306 cites W1587612947 @default.
• W2000588306 cites W1591284352 @default.
• W2000588306 cites W1598071319 @default.
• W2000588306 cites W1895563716 @default.
• W2000588306 cites W1967729931 @default.
• W2000588306 cites W1970697917 @default.
• W2000588306 cites W1970751638 @default.
• W2000588306 cites W1973341387 @default.
• W2000588306 cites W1974281619 @default.
• W2000588306 cites W1982680133 @default.
• W2000588306 cites W1983177609 @default.
• W2000588306 cites W1988723846 @default.
• W2000588306 cites W1994018335 @default.
• W2000588306 cites W1994241126 @default.
• W2000588306 cites W1999624209 @default.
• W2000588306 cites W2000543336 @default.
• W2000588306 cites W2005648650 @default.
• W2000588306 cites W2010427283 @default.
• W2000588306 cites W2017150849 @default.
• W2000588306 cites W2041310039 @default.
• W2000588306 cites W2053559087 @default.
• W2000588306 cites W2055090102 @default.
• W2000588306 cites W2076603801 @default.
• W2000588306 cites W2087359011 @default.
• W2000588306 cites W2124594675 @default.
• W2000588306 cites W2129405766 @default.
• W2000588306 cites W2130561621 @default.
• W2000588306 cites W2155058151 @default.
• W2000588306 cites W2171872740 @default.
• W2000588306 cites W2262294238 @default.
• W2000588306 cites W2316698910 @default.
• W2000588306 cites W2576742884 @default.
• W2000588306 cites W53883719 @default.
• W2000588306 doi "https://doi.org/10.1074/jbc.274.35.24641" @default.
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