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• W2087359011 abstract "Mucin type O-glycans with core 2 branches are distinct from nonbranched O-glycans, and the amount of core 2 branched O-glycans changes dramatically during T cell differentiation. This oligosaccharide is synthesized only when core 2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) is present, and the expression of this glycosyltransferase is highly regulated. To understand how O-glycan synthesis is regulated by the orderly appearance of glycosyltransferases that form core 2 branched O-glycans, the subcellular localization of C2GnT was determined by using antibodies generated that are specific to C2GnT. The studies using confocal light microscopy demonstrated that C2GnT was localized mainly in cis tomedial-cisternae of the Golgi. We then converted C2GnT to atrans-Golgi enzyme by replacing its Golgi retention signal with that of α-2,6-sialyltransferase, which resides intrans-Golgi. Chinese hamster ovary cells expressing wild type C2GnT and the chimeric C2GnT were then subjected to oligosaccharide analysis. The results obtained clearly indicate that the conversion of C2GnT into a trans-Golgi enzyme resulted in a substantial decrease of core 2 branched oligosaccharides.These results, taken together, strongly suggest that the predominance of core 2 branched oligosaccharides in those cells expressing C2GnT is due to the fact that C2GnT is located earlier in the Golgi than α-2,3-sialyltransferase that competes with C2GnT for the common substrate. Furthermore, alteration of Golgi localization renders the chimeric C2GnT much less efficient in synthesizing core 2 branched oligosaccharides, indicating the critical role of orderly subcellular localization of glycosyltransferases. Mucin type O-glycans with core 2 branches are distinct from nonbranched O-glycans, and the amount of core 2 branched O-glycans changes dramatically during T cell differentiation. This oligosaccharide is synthesized only when core 2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) is present, and the expression of this glycosyltransferase is highly regulated. To understand how O-glycan synthesis is regulated by the orderly appearance of glycosyltransferases that form core 2 branched O-glycans, the subcellular localization of C2GnT was determined by using antibodies generated that are specific to C2GnT. The studies using confocal light microscopy demonstrated that C2GnT was localized mainly in cis tomedial-cisternae of the Golgi. We then converted C2GnT to atrans-Golgi enzyme by replacing its Golgi retention signal with that of α-2,6-sialyltransferase, which resides intrans-Golgi. Chinese hamster ovary cells expressing wild type C2GnT and the chimeric C2GnT were then subjected to oligosaccharide analysis. The results obtained clearly indicate that the conversion of C2GnT into a trans-Golgi enzyme resulted in a substantial decrease of core 2 branched oligosaccharides. These results, taken together, strongly suggest that the predominance of core 2 branched oligosaccharides in those cells expressing C2GnT is due to the fact that C2GnT is located earlier in the Golgi than α-2,3-sialyltransferase that competes with C2GnT for the common substrate. Furthermore, alteration of Golgi localization renders the chimeric C2GnT much less efficient in synthesizing core 2 branched oligosaccharides, indicating the critical role of orderly subcellular localization of glycosyltransferases. Leukosialin (CD43) is a major sialoglycoprotein present in leukocytes and heavily glycosylated by mucin-type O-glycans (1Fukuda M. Glycobiology. 1991; 1: 347-356Crossref PubMed Scopus (105) Google Scholar, 2Andersson L.C. Gahmberg C.D. Blood. 1978; 52: 57-67Crossref PubMed Google Scholar, 3Brown W.R. Barclay A.N. Sunderland C.A. Williams A.F. Nature. 1981; 289: 456-460Crossref PubMed Scopus (157) Google Scholar, 4Carlsson S.R. Fukuda M. J Biol. Chem. 1986; 261: 12779-12786Abstract Full Text PDF PubMed Google Scholar, 5Remold-O'Donnell E. Kenney D.M. Parkman R. Cairns L. Savage B. Rosen F.S. J. Exp. Med. 1984; 159: 1705-1723Crossref PubMed Scopus (156) Google Scholar). This glycoprotein of human origin contains approximately 80O-linked oligosaccharides in its extracellular domain consisting of 234 amino acids (1Fukuda M. Glycobiology. 1991; 1: 347-356Crossref PubMed Scopus (105) Google Scholar, 6Remold-O'Donnell E. Rosen F.S. Immunodefic. Rev. 1990; 2: 151-174PubMed Google Scholar). These O-linked oligosaccharides are highly sialylated and have been shown to exhibit an antiadhesive property (7Ardman B. Sikorski M.A. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5001-5005Crossref PubMed Scopus (149) Google Scholar). It has been also shown that the structure of oligosaccharides attached to leukosialin changes significantly during development of T cells. While resting human T lymphocytes express tetrasaccharides, NeuNAcα2→3Galβ1→3(NeuNAcα2→6)GalNAc, activated T lymphocytes almost exclusively express branched hexasaccharides, NeuNAcα2→3Galβ1→3(NeuNAcα2→3Galβ1→4GlcNAc β1→6) GalNAc (8Piller F. Piller V. Fox R.I. Fukuda M. J Biol. Chem. 1988; 263: 15146-15150Abstract Full Text PDF PubMed Google Scholar). Moreover, such change is associated with T cell development in thymus; while immature thymocytes in cortical thymus express the hexasaccharides, relatively mature medullary thymocytes express the tetrasaccharides (9Baum 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 (255) Google Scholar). The conversion of O-glycan biosynthesis is due to the turning on or off of core 2 β-1,6-N-acetylglucosaminyltransferase (C2GnT). 1The abbreviations used are: C2GnT, core 2 β-1,6-N-acetylglucosaminyltransferase; GST, glutathioneS-transferase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; CHO, Chinese hamster ovary; GalT, β-1,4-galactosyltransferase; ST6Gal I, Galβ1→ 4GlcNAc α-2,6-sialyltransferase; α-ManII, α-mannosidase II.1The abbreviations used are: C2GnT, core 2 β-1,6-N-acetylglucosaminyltransferase; GST, glutathioneS-transferase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; CHO, Chinese hamster ovary; GalT, β-1,4-galactosyltransferase; ST6Gal I, Galβ1→ 4GlcNAc α-2,6-sialyltransferase; α-ManII, α-mannosidase II. It has been demonstrated that activated T lymphocytes express a substantial amount of C2GnT activity, while resting T lymphocytes express negligible C2GnT activity (8Piller F. Piller V. Fox R.I. Fukuda M. J Biol. Chem. 1988; 263: 15146-15150Abstract Full Text PDF PubMed Google Scholar). By in situ hybridization of the transcript, it has been shown that immature cortical thymocytes express a substantial amount of C2GnT mRNA, while it was not detected in medullary thymocytes (9Baum 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 (255) Google Scholar). The conversion of O-glycan structures during thymocyte development may be critical for the apoptotic process in thymus, since such a process is modulated by the presence ofO-glycans on thymocytes (10Perillo N.L. Pace K.E. Seilhamer J.J. Baum L.G. Nature. 1995; 378: 736-739Crossref PubMed Scopus (931) Google Scholar). Expression of the branched hexasaccharide in peripheral blood T lymphocytes has been also observed in patients with immunodeficient syndromes such as Wiskott-Aldrich syndrome (11Piller F. Le Deist F. Weinberg K. Parkman R. Fukuda M. J. Exp. Med. 1991; 173: 1501-1510Crossref PubMed Scopus (125) Google Scholar, 12Higgins E.A. Siminovitch K.A. Zhuang D. Brockhausen I. Dennis J.W. J. Biol. Chem. 1991; 266: 6280-6290Abstract Full Text PDF PubMed Google Scholar). It has also been shown that AIDS patients express substantially increased amounts of the hexasaccharide or its monosialylated forms (13Saitoh O. Piller F. Fox R.I. Fukuda M. Blood. 1991; 77: 1491-1499Crossref PubMed Google Scholar, 14Lefebvre J.-C. Giordanengo V. Limouse M. Doglio A. Cucchiarini M. Monpoux F. Mariani R. Peyron J.-F. J. Exp. Med. 1994; 180: 1609-1617Crossref PubMed Scopus (59) Google Scholar). AIDS patients produce antibodies against leukosialin expressing those oligosaccharides, and such antibodies are implicated in causing T lymphocyte depletion, which may be a cause of pathological conditions in these diseases (15Ardman B. Sikorski M.A. Settles M. Staunton D.E. J. Exp. Med. 1990; 172: 1151-1158Crossref PubMed Scopus (66) Google Scholar). These combined results indicate that it is critical to understand how core 2 branchings are synthesized. The biosynthesis of oligosaccharides is also controlled by specific localization of glycosyltransferases that add a specific monosaccharide in each reaction (16Sadler J.E. Ginsburg V. Robbins P.W. Biology of Carbohydrates. John Wiley & Sons, Inc., New York1984: 199-228Google Scholar). If a glycosyltransferase is misplaced, sequential reactions would not take place, since a given glycosyltransferase adds a monosaccharide to a particular acceptor that was formed by another glycosyltransferase that resides in an earlier compartment(s). Although subcellular localization of glycosyltransferases that form N-glycans is relatively well studied (17Roth J. Biochim. Biophys. Acta. 1987; 906: 405-436Crossref PubMed Scopus (215) Google Scholar), very little is known about subcellular distribution of glycosyltransferases that form O-glycans (see Ref.18Roth J. Wang Y. Eckhardt A.E. Hill R.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8935-8939Crossref PubMed Scopus (115) Google Scholar). In the present study, we have first determined that C2GnT is localized in cis to medial-Golgi using antibodies specific for C2GnT. We then converted C2GnT into a trans-Golgi enzyme by replacing its domain responsible for Golgi retention with that of Galβ1→4GlcNAc α-2,6-sialyltransferase, ST6Gal I (19Weinstein J. Lee E.U. McEntee K. Lai P.-H. Paulson J.C. J. Biol. Chem. 1987; 262: 17735-17743Abstract Full Text PDF PubMed Google Scholar). Such altered localization of C2GnT was found to result in altered synthesis of oligosaccharides, demonstrating the importance of the orderly presence of glycosyltransferases. To prepare antibodies specific to C2GnT, a cDNA encoding the catalytic domain of C2GnT was amplified by PCR using C2GnT cDNA (20Bierhuizen M.F. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Crossref PubMed Scopus (279) Google Scholar) as a template and fused with GST protein. The 5′-primer for PCR is 5′-aaacgtggatccCATCATCATCATCATCAT ccc ggg TCTTCTTTCATC, (BamHI site and 6-His linker are singly and doubly underlined, respectively, while the italic type corresponds to residues 101–104 of C2GnT). The 3′-primer is 5′-aaaacggaattccccgggTCAGTGTTTTAATGT-3′ (the last 15 nucleotides correspond to residue 425 to the stop codon). PCR was carried out as described (21Ogata S. Fukuda M. J Biol. Chem. 1994; 269: 5210-5217Abstract Full Text PDF PubMed Google Scholar), and the amplified DNA was digested withBamHI and EcoRI and cloned into the same sites of pGEX-KG expression vector (Pharmacia). The resultant cDNA encodes a fusion protein composed of GST and a thrombin cleavage site, six histidines, and the catalytic domain (residues 101–428) of C2GnT.Escherichia coli HB101 was transformed with this plasmid vector, and a GST fusion protein was produced after isopropyl-1-thio-β-d-galactopyranoside induction. HB101 cells were recovered by centrifugation and frozen at −80 °C. After thawing on ice, the pellet was digested with 5 mg/ml lysozyme in 25 mmTris-HCl, pH 8.0, containing 10 mm EDTA and 1% Triton X-100 (buffer A). After the addition of DNase I (Amersham Corp.), the sample was then sonicated and centrifuged. The resulting pellet was resuspended in 25 mm Tris-HCl, pH 8.0, containing 10 mm EDTA and 1.5% N-lauroylsarcosine (Sigma) (buffer B). The suspended residue was then centrifuged, and the sarcosyl extract was obtained as described (22Frankel S. Sohn R. Leinwand L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1192-1196Crossref PubMed Scopus (97) Google Scholar). Glutathione-Sepharose beads were equilibrated with buffer B and added to the sarcosyl extract. The suspension was mixed gently at 4 °C for 90 min using a rotary mixer and then briefly centrifuged to recover the beads. After washing the beads with buffer C (50 mm Tris-HCl, pH 8.0, containing 150 mm NaCl and 2.5 mmCaCl2), the beads were suspended in 2 ml of buffer C containing 20 units of thrombin and mixed overnight at room temperature. Thrombin-released material was recovered in the supernatant after centrifugation of the above mixture. The proteins that remained on beads were then released by SDS-polyacrylamide gel electrophoresis sample buffer, which contained no reducing reagent, and the C2GnT protein fragment was recovered in this extraction and separated from other contaminating proteins by SDS-polyacrylamide gel electrophoresis. The purified protein sample, extracted from polyacrylamide gels by electroelution, was immunized in rabbits. The antiserum was applied to a protein A-Sepharose column, bound antibodies were eluted with glycine-HCl, pH 2.5, and the eluent was immediately neutralized by the addition of 1.0 m Tris-HCl buffer, pH 8.0. The partially purified antibodies were further applied to a column of Sepharose 4B conjugated to E. coli proteins, and the unbound fraction was used as a purified antibody sample. A cDNA encoding ST6Gal I was cloned by PCR using a human HL-60 cDNA library (20Bierhuizen M.F. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Crossref PubMed Scopus (279) Google Scholar) as the template. The 5′-primer for this PCR corresponds to nucleotides −15 to 15 with respect to the translation initiation site (23Wang X. Vertino A. Eddy R.L. Byers M.G. Jani-Sait S.N. Shows T.B. Lau J.T.Y. J. Biol. Chem. 1993; 268: 4355-4361Abstract Full Text PDF PubMed Google Scholar) plus SmaI site. The 3′-primer is 5′-aaacccggctcgag TGCTTAGCAGTGAATGGTCC-3′. TheXhoI site is underlined, while the italic type corresponds to nucleotides 1201–1221 (nucleotides 1216–1218 encode the stop codon). The PCR product was digested with SmaI andXhoI and cloned into the same sites in the pMSG vector (Pharmacia). A cDNA encoding the cytoplasmic, transmembrane, and stem regions of ST6Gal I was amplified by PCR using the above plasmid vector as a template. The 5′-primer, DS23, corresponds to nucleotides −9 to 11 in relation to the translation initiation site of ST6Gal I, with the BamHI site at the 5′-end. The 3′-primer sequence was 5′-ATCACTACTAGGGTCCTGGGTGCTGCTT-3′. The first 12 nucleotides of this primer (shown by italics) correspond in antisense to residues 53–56 of C2GnT, and the last 16 nucleotides correspond in antisense to nucleotides 195–210 of ST6Gal I (nucleotides 196–210 encode codons 66–70). This PCR product encodes the first 70 amino acid residues of ST6Gal I plus 4 amino acids in the stem region of C2GnT. A cDNA encoding the catalytic domain of C2GnT was amplified by PCR. The 5′-primer sequence was 5′-AGCACCCAGGACCCTAGTAGTGATATTAATTG-3′. In this sequence, the first 12 nucleotides encode residues 67–70 of ST6Gal I, and the following 20 nucleotides encode residues 53–58 plus the portion of residue 59 of C2GnT. The 3′-primer, DS26, encodes the stop codon plus the following fifteen 3′-untranslated nucleotides of C2GnT sequence with the addition of the XhoI site. The PCR products of the C2GnT catalytic domain and ST6Gal I sequence overlap at sequences corresponding toSer-Thr-Gln-Asp-Pro-Ser-Ser-Asp, in whichSer-Thr-Gln-Asp comes from ST6Gal I andPro-Ser-Ser-Asp comes from C2GnT. To make a chimera of the NH2-terminal region of ST6Gal I and the catalytic domain of C2GnT, PCR was carried out using DS23 and DS26 (shown above) as primers and a mixture of the above two PCR products as templates (21Ogata S. Fukuda M. J Biol. Chem. 1994; 269: 5210-5217Abstract Full Text PDF PubMed Google Scholar). After amplification under the same conditions as described, the PCR product was digested with BamHI and XhoI and then ligated into the same sites of pcDNAI, yielding pcDNAI-ST6Gal I/C2GnT. CHO DG44 cells were transfected with pZIPNEO-leu alone, with pZIPNEO-leu and pcDNAI-C2GnT, or with pZIPNEO-leu and pcDNAI-ST6Gal I/C2GnT using LipofectAMINE and were subsequently selected for G418 resistance. Clonal cell lines expressing a substantial amount of either leukosialin (CHO-leu) or both leukosialin and core 2 branched oligosaccharides (CHO-leu·C2GnT, CHO-leu·ST6Gal I/C2GnT) were selected as described (24Bierhuizen M.F.A. Maemura K. Fukuda M. J. Biol. Chem. 1994; 269: 4473-4479Abstract Full Text PDF PubMed Google Scholar). CHO-leu·C2GnT and CHO-leu·ST6Gal I/C2GnT cells were grown on coverslips and fixed in 4% paraformaldehyde in PBS and immersed in 0.05% saponin, 0.1% bovine serum albumin solution in PBS for 10 min at room temperature. They were then incubated with rabbit anti-C2GnT antibodies followed by rhodamine-conjugated goat anti-rabbit IgG as described previously (25Williams M.A. Fukuda M. J. Cell Biol. 1990; 111: 955-966Crossref PubMed Scopus (192) Google Scholar). After washing with PBS containing 0.1% bovine serum albumin, they were sequentially washed with PBS containing 1% normal goat serum, 10 μg/ml unconjugated secondary antibody (goat anti-rabbit IgG), and then 100 μg of unconjugated protein A/ml of PBS for 10 min each. The cells were then incubated with rabbit anti-α-mannosidase II antibodies (26Moremen K.W. Robbins P.W. J. Cell Biol. 1991; 115: 1521-1534Crossref PubMed Scopus (117) Google Scholar) followed by fluorescein isothiocyanate-conjugated goat F(ab′)2 fragment of IgG that is specific to the Fc portion of rabbit IgG (Axell). After washing with PBS containing 0.1% bovine serum albumin followed by PBS, the samples were visualized with a Zeiss Axioplan microscope (25Williams M.A. Fukuda M. J. Cell Biol. 1990; 111: 955-966Crossref PubMed Scopus (192) Google Scholar) or Zeiss CSM410 confocal laser scanning microscope (27Fukuda M.N. Sato T. Nakayama J. Klier G. Mikami M. Aoki D. Nozawa S. Genes & Dev. 1995; 9: 1199-1210Crossref PubMed Scopus (167) Google Scholar) as described. To detect C2GnT and β-1,4-galactosyltransferase in the same sample, pcDNAI-GalT (28Aoki D. Lee N. Yamaguchi N. Dubois C. Fukuda M.N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4319-4323Crossref PubMed Scopus (124) Google Scholar) was transiently transfected in the above CHO cells. Simple double immunofluorescent staining was then carried out as described (25Williams M.A. Fukuda M. J. Cell Biol. 1990; 111: 955-966Crossref PubMed Scopus (192) Google Scholar), since a mouse monoclonal antibody specific to human β-1,4-galactosyltransferase (29Uemura M. Sakaguchi T. Uejima T. Nozawa S. Narimatsu H. Cancer Res. 1992; 52: 6153-6157PubMed Google Scholar) was available. Controls were performed by omitting the primary antibodies. Rabbit antibodies specific to mouse (and hamster) α-mannosidase II (26Moremen K.W. Robbins P.W. J. Cell Biol. 1991; 115: 1521-1534Crossref PubMed Scopus (117) Google Scholar) were kindly provided by Dr. Kelley Moremen (University of Georgia). Mouse monoclonal antibody specific to human β-1,4-galactosyltransferase (29Uemura M. Sakaguchi T. Uejima T. Nozawa S. Narimatsu H. Cancer Res. 1992; 52: 6153-6157PubMed Google Scholar) was kindly provided by Dr. Hisashi Narimatsu (Soka University). Staining of specimens was performed as described previously (30Kerjaschki D. Schulze M. Binder S. Kain R. Ojha P.P. Susani M. Horvat R. Baker P.J. Couser W.G. J Immunol. 1989; 143: 546-552PubMed Google Scholar, 31Kain R. Matsui K. Exner M. Binder S. Schaffner G. Sommer E.M. Kerjaschki D. J. Exp. Med. 1995; 181: 585-597Crossref PubMed Scopus (137) Google Scholar). Briefly, frozen sections of human kidney specimens, CHO-leu·C2GnT and CHO-leu·ST6GalI/C2GnT cells, were fixed with paraformaldehyde-lysine-periodate, and incubation with primary antibody (rabbit anti-human C2GnT IgG, absorbed againstE. coli proteins) was performed overnight at 4 °C, followed by three washes in PBS containing 1% egg albumin and 0.075% saponin and incubation with secondary antibodies (goat horseradish peroxidase-conjugated anti-rabbit IgG, Amersham) for 1 h at room temperature. After three washes, bound antibodies were visualized with 0.05% diaminobenzidine (Sigma) and 3% H2O2 in 20 mm Tris-HCl, pH 7.4. After fixation in 2.5% glutaraldehyde in 20 mm phosphate buffer, pH 7.4, and embedding in epon, ultra thin sections were cut and examined in a Jeol 1200 microscope. Controls were performed by either omitting the primary antibody or by replacing it with rabbit preimmune serum. C2GnT was assayed by using the acceptor Galβ-1→3GalNAc-α-p-nitrophenol (Toronto Chemicals) as described (20Bierhuizen M.F. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Crossref PubMed Scopus (279) Google Scholar). The CHO cells (∼1 × 107 cells) were metabolically labeled with [3H]glucosamine (10 μCi/ml), and the cell residues were subjected to Pronase digestion as described (24Bierhuizen M.F.A. Maemura K. Fukuda M. J. Biol. Chem. 1994; 269: 4473-4479Abstract Full Text PDF PubMed Google Scholar). The glycopeptides obtained were applied to a Sephadex G-50 (superfine) column (1.0 × 110 cm) equilibrated with 0.1 mNH4HCO3. Higher molecular weight glycopeptides were subjected to β-elimination as described (24Bierhuizen M.F.A. Maemura K. Fukuda M. J. Biol. Chem. 1994; 269: 4473-4479Abstract Full Text PDF PubMed Google Scholar), and releasedO-glycans were separated from remaining glycopeptides using the same Sephadex G-50 gel filtration. The obtainedO-glycans were analyzed by Bio-Gel P-4 gel filtration using the same conditions as described (24Bierhuizen M.F.A. Maemura K. Fukuda M. J. Biol. Chem. 1994; 269: 4473-4479Abstract Full Text PDF PubMed Google Scholar). Oligosaccharide peaks were desialyzed by clostridial neuraminidase, and the digest was again analyzed by Bio-Gel P-4 gel filtration. The standard oligosaccharides used were obtained as described previously (24Bierhuizen M.F.A. Maemura K. Fukuda M. J. Biol. Chem. 1994; 269: 4473-4479Abstract Full Text PDF PubMed Google Scholar). To obtain the ratio of the oligosaccharides synthesized, the amount of the radioactivity was determined after converting all of these oligosaccharides to Galβ1→3GalNAcOH by various exoglycosidase treatments as described (24Bierhuizen M.F.A. Maemura K. Fukuda M. J. Biol. Chem. 1994; 269: 4473-4479Abstract Full Text PDF PubMed Google Scholar). After correcting the yield during chromatography, the relative amount of each oligosaccharide could be calculated (32Piller V. Piller F. Fukuda M. J. Biol. Chem. 1990; 265: 9264-9271Abstract Full Text PDF PubMed Google Scholar). The ratio of the specific radioactivity of GlcNAc and GalNAcOH was found to be 1:0.7, as seen before (32Piller V. Piller F. Fukuda M. J. Biol. Chem. 1990; 265: 9264-9271Abstract Full Text PDF PubMed Google Scholar). Membrane proteins were extracted in 200 mmNa2CO3, pH 10.5, and Triton X-114 phase partition as described previously (31Kain R. Matsui K. Exner M. Binder S. Schaffner G. Sommer E.M. Kerjaschki D. J. Exp. Med. 1995; 181: 585-597Crossref PubMed Scopus (137) Google Scholar). Twenty μg of the membrane proteins were separated on SDS-polyacrylamide (10%) gel electrophoresis, transferred onto nitrocellulose, and probed with anti-C2GnT antibodies. Alkaline phosphatase-conjugated anti-rabbit IgG (Promega) was used as a secondary antibody and detected by nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate chromogenic substrate (Kierkergard and Perry, Gaithersberg, MD). As a control, the duplicate blot was probed with IgG purified from rabbit preimmune sera. Cells were metabolically labeled with Tran35S-label and immunoprecipitated as described (21Ogata S. Fukuda M. J Biol. Chem. 1994; 269: 5210-5217Abstract Full Text PDF PubMed Google Scholar). To determine the subcellular distribution of C2GnT, it was essential to produce antibodies specific to C2GnT. First, the catalytic domain of C2GnT was fused with GST protein and expressed in E. coli. The produced protein was then immunized in rabbits. After two additional boost immunizations, the titer of the antibodies was increased enough to detect C2GnT in CHO cells expressing C2GnT (Fig.1 A). The same antibodies also reacted with COS-1 cells, which transfected with C2GnT cDNA (Fig.2 B) but not with untransfected COS-1 cells (data not shown). Moreover, the antibodies did not react with CHO cells expressing I-branching β-1,6-N-acetylglucosaminyltransferase, which shares homology with C2GnT (33Bierhuizen M.F. Mattei M.G. Fukuda M. Genes & Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar) (Fig. 1 C). To confirm that the antibodies reacted with C2GnT, Western blot analysis was performed on the protein products used for immunization. Fig. 2 A shows that the antibodies reacted with a fusion protein of ∼68 kDa before thrombin digestion (lanes 1 and 2) and reacted with ∼36-kDa protein after the digestion (lanes 3 and4). The results are consistent with the calculated molecular mass for the GST-C2GnT fusion protein (∼68 kDa) and C2GnT catalytic domain (36 kDa).Figure 2Western blot analysis and immunoprecipitation of C2GnT. A, GST-C2GnT fusion protein (lanes 1and 2) and its proteolytic digest (lanes 3 and4) were subjected to Western blot analysis using anti-C2GnT antibodies. Lanes 1 and 3 contained 10 times more sample than lanes 2 and 4. The bands migrating at ∼75 kDa are most likely the dimers. B, Western blot analysis of human kidney membrane proteins (20 μg) using anti-C2GnT antibodies. Lane 5 is a control experiment omitting the primary antibody. C, immunoprecipitation of [35S]methionine-labeled CHO cells stably expressing C2GnT (lane 7) and wild-type CHO cells (lane 8).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our preliminary studies on rat tissues showed that kidney had the highest activity of C2GnT. Western blot analysis of human kidney membrane proteins demonstrated that a ∼57-kDa protein strongly reacted with the antibodies, while the control experiment gave negative results (Fig. 2 B, lanes 5 and 6). Finally, immunoprecipitation of [35S]methionine-labeled CHO cells stably expressing C2GnT produced a specific band at ∼60 kDa, which was absent in wild type CHO cells (Fig. 2 C,lanes 7 and 8). These results combined clearly indicate that the antibodies generated are specific to C2GnT. To determine the subcellular distribution of C2GnT, CHO cells were transfected with pcDNAI-C2GnT and pZipNeo-leu, and those cells stably expressing C2GnT and leukosialin (CHO-leu·C2GnT) were established (24Bierhuizen M.F.A. Maemura K. Fukuda M. J. Biol. Chem. 1994; 269: 4473-4479Abstract Full Text PDF PubMed Google Scholar). As shown previously, C2GnT is absent in CHO cells (24Bierhuizen M.F.A. Maemura K. Fukuda M. J. Biol. Chem. 1994; 269: 4473-4479Abstract Full Text PDF PubMed Google Scholar, 34Sasaki H. Bothner B. Dell A. Fukuda M. J. Biol. Chem. 1987; 262: 12059-12076Abstract Full Text PDF PubMed Google Scholar); thus, only introduced C2GnT can be detected in CHO cells. When C2GnT is localized differently by the replacement of the Golgi retention signal, such change should be clearly detected. Leukosialin was co-transfected, since those cells expressing core 2 oligosaccharides on leukosialin can be detected by T305 antibody (24Bierhuizen M.F.A. Maemura K. Fukuda M. J. Biol. Chem. 1994; 269: 4473-4479Abstract Full Text PDF PubMed Google Scholar). CHO-leu·C2GnT cells (clone 1) were stained by rabbit antibodies specific to C2GnT followed by rhodamine-conjugated goat anti-rabbit IgG. After chasing the remaining antibodies by protein A, as detailed under “Experimental Procedures,” the same specimens were incubated with rabbit antibodies specific to α-ManII, a glycosidase normally found in cis to medial-Golgi (26Moremen K.W. Robbins P.W. J. Cell Biol. 1991; 115: 1521-1534Crossref PubMed Scopus (117) Google Scholar), followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Preliminary experiments showed that the antibodies raised against mouse α-ManII cross-reacted with CHO α-ManII. As shown in Fig.3 (top left), the majority of C2GnT and α-ManII are overlapping in their distributions, showing strong yellow staining. In the second set of experiments, pcDNAI-human GalT cDNA (28Aoki D. Lee N. Yamaguchi N. Dubois C. Fukuda M.N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4319-4323Crossref PubMed Scopus (124) Google Scholar) was transiently introduced into CHO-leu·C2GnT cells, and the expressed GalT, trans-Golgi enzyme, was similarly visualized by immunofluorescent staining. Since a mouse monoclonal antibody specific to human β-galactosyltransferase was available (29Uemura M. Sakaguchi T. Uejima T. Nozawa S. Narimatsu H. Cancer Res. 1992; 52: 6153-6157PubMed Google Scholar), the transfected cells were stained with rabbit anti-C2GnT antibodies and rhodamine-conjugated goat anti-rabbit IgG, followed by mouse monoclonal anti-GalT antibodies and goat FITC-conjugated anti-mouse IgG. The results, as shown in Fig. 3 (bottom left) demonstrated that there was almost no overlap in the distribution of C2GnT and GalT. These results, combined, established that C2GnT is present in thecis to medial-Golgi. The results also demonstrated that two-step immunostaining was specific, since staining for only C2GnT or α-ManII can be seen in certain cells (Fig. 3). These results were confirmed by immunoelectron microscopy using specimens of human kidney and stably transfected CHO cells (data not shown). The next question we asked was whether or not we could shift the Golgi localization of C2GnT by replacing the Golgi retention signal in C2GnT with that of a glycosyltransferase present in" @default.
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• W2087359011 date "1997-09-01" @default.
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• W2087359011 title "Altered Golgi Localization of Core 2 β-1,6-N-Acetylglucosaminyltransferase Leads to Decreased Synthesis of Branched O-Glycans" @default.
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