Title: Molecular Cloning and Enzymatic Characterization of a UDP-GalNAc:GlcNAcβ-R β1,4-N-Acetylgalactosaminyltransferase fromCaenorhabditis elegans
Abstract: A common terminal structure in glycans from animal glycoproteins and glycolipids is the lactosamine sequence Galβ4GlcNAc-R (LacNAc or LN). An alternative sequence that occurs in vertebrate as well as in invertebrate glycoconjugates is GalNAcβ4GlcNAc-R (LacdiNAc or LDN). Whereas genes encoding β4GalTs responsible for LN synthesis have been reported, the β4GalNAcT(s) responsible for LDN synthesis has not been identified. Here we report the identification of a gene fromCaenorhabditis elegans encoding a UDP-GalNAc:GlcNAcβ-R β1,4-N-acetylgalactosaminyltransferase (Ceβ4GalNAcT) that synthesizes the LDN structure. Ceβ4GalNAcT is a member of the β4GalT family, and its cDNA is predicted to encode a 383-amino acid type 2 membrane glycoprotein. A soluble, epitope-tagged recombinant form of Ceβ4GalNAcT expressed in CHO-Lec8 cells was active using UDP-GalNAc, but not UDP-Gal, as a donor toward a variety of acceptor substrates containing terminal β-linked GlcNAc in both N- and O-glycan type structures. The LDN structure of the product was verified by co-chromatography with authentic standards and 1H NMR spectroscopy. Moreover, Chinese hamster ovary CHO-Lec8 and CHO-Lec2 cells expressing Ceβ4GalNAcT acquired LDN determinants on endogenous glycoprotein N-glycans, demonstrating that the enzyme is active in mammalian cells as an authentic β4GalNAcT. The identification and availability of this novel enzyme should enhance our understanding of the structure and function of LDN-containing glycoconjugates. A common terminal structure in glycans from animal glycoproteins and glycolipids is the lactosamine sequence Galβ4GlcNAc-R (LacNAc or LN). An alternative sequence that occurs in vertebrate as well as in invertebrate glycoconjugates is GalNAcβ4GlcNAc-R (LacdiNAc or LDN). Whereas genes encoding β4GalTs responsible for LN synthesis have been reported, the β4GalNAcT(s) responsible for LDN synthesis has not been identified. Here we report the identification of a gene fromCaenorhabditis elegans encoding a UDP-GalNAc:GlcNAcβ-R β1,4-N-acetylgalactosaminyltransferase (Ceβ4GalNAcT) that synthesizes the LDN structure. Ceβ4GalNAcT is a member of the β4GalT family, and its cDNA is predicted to encode a 383-amino acid type 2 membrane glycoprotein. A soluble, epitope-tagged recombinant form of Ceβ4GalNAcT expressed in CHO-Lec8 cells was active using UDP-GalNAc, but not UDP-Gal, as a donor toward a variety of acceptor substrates containing terminal β-linked GlcNAc in both N- and O-glycan type structures. The LDN structure of the product was verified by co-chromatography with authentic standards and 1H NMR spectroscopy. Moreover, Chinese hamster ovary CHO-Lec8 and CHO-Lec2 cells expressing Ceβ4GalNAcT acquired LDN determinants on endogenous glycoprotein N-glycans, demonstrating that the enzyme is active in mammalian cells as an authentic β4GalNAcT. The identification and availability of this novel enzyme should enhance our understanding of the structure and function of LDN-containing glycoconjugates. Many of the functional moieties of complex glycoconjugates are in the terminal sequences of N- and O-glycans of glycoproteins and in glycolipids, which are recognized by a growing number of carbohydrate binding proteins (1Figdor C.G. van Kooyk Y. Adema G.J. Nat. Rev. Immunol. 2002; 2: 77-84Crossref PubMed Scopus (708) Google Scholar, 2Dodd R.B. Drickamer K. Glycobiology. 2001; 11: 71R-79RCrossref PubMed Scopus (325) Google Scholar, 3Leffler H. Results Probl. Cell Differ. 2001; 33: 57-83Crossref PubMed Scopus (74) Google Scholar, 4Angata T. Kerr S.C. Greaves D.R. Varki N.M. Crocker P.R. Varki A. J. Biol. Chem. 2002; 277: 24466-24474Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). A common terminal motif that is modified in a variety of ways by the additions of other sugars and sulfate groups is the lactosamine sequence Galβ4GlcNAc-R (LacNAc or LN), 1The abbreviations used are: LN or LacNAc, Galβ4GlcNAc; β4GalT, UDP-Gal:GlcNAcβ-R β1,4galactosyltransferase; LDN or LacdiNAc, GalNAcβ4GlcNAc; ORF, open reading frame; β4GalNAcT, UDP- GalNAc:GlcNAcβ-R β1,4-N-acetylgalactosaminyltransferase; pNP, 4-nitrophenyl; CHO, Chinese hamster ovary; HPAEC-PAD, high pH anion exchange chromatography with pulsed amperometric detection. which is generated by a large family of UDP-Gal:GlcNAcβ-R β1,4-galactosyltransferases (β4GalTs) acting on terminal GlcNAc residues (5Amado M. Almeida R. Schwientek T. Clausen H. Biochim. Biophys. Acta. 1999; 1473: 35-53Crossref PubMed Scopus (263) Google Scholar). 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Levery S.B. Mandel U. Kresse H. Schwientek T. Bennett E.P. Clausen H. J. Biol. Chem. 1999; 274: 26165-26171Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 34Guo S. Sato T. Shirane K. Furukawa K. Glycobiology. 2001; 11: 813-820Crossref PubMed Scopus (70) Google Scholar, 35Lee J. Sundaram S. Shaper N.L. Raju T.S. Stanley P. J. Biol. Chem. 2001; 276: 13924-13934Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 36Nakamura N. Yamakawa N. Sato T. Tojo H. Tachi C. Furukawa K. J. Neurochem. 2001; 76: 29-38Crossref PubMed Scopus (42) Google Scholar). Interestingly, these regions of homology are also found within the amino acid sequence of a snail UDP-GlcNAc:GlcNAcβ-R β1,4-N-acetylglucosaminyltransferase (37Bakker H. Van Tetering A. Agterberg M. Smit A.B. Van den Eijnden D.H. Van Die I. J. Biol. Chem. 1997; 272: 18580-18585Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 38Bakker H. Agterberg M. Van Tetering A. Koeleman C.A. 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The C. elegans genome contains three open reading frames that encode proteins with sequence homology to the β4GalT family. One of these open reading frames (ORF R10E11.4; sqv-3) is predicted to encode a protein involved in vulval invagination (47Herman T. Horvitz H.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 974-979Crossref PubMed Scopus (120) Google Scholar) and is likely to be a UDP-Gal:xylose β-R β1,4-galactosyltransferase (33Almeida R. Levery S.B. Mandel U. Kresse H. Schwientek T. Bennett E.P. Clausen H. J. Biol. Chem. 1999; 274: 26165-26171Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 48Okajima T. Yoshida K. Kondo T. Furukawa K. J. Biol. Chem. 1999; 274: 22915-22918Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Another of these open reading frames (ORF W02B12.11) encodes a protein for which no enzymatic activity has yet been reported. The third open reading frame (ORF Y73E7A.7) was identified more recently than the two mentioned above and therefore had not been reported in previous studies (27Van Die I. Bakker H. Van den Eijnden D.H. Glycobiology. 1997; 7: v-viiiCrossref PubMed Google Scholar, 31Lo N.W. Shaper J.H. Pevsner J. Shaper N.L. Glycobiology. 1998; 8: 517-526Crossref PubMed Scopus (172) Google Scholar). In this study, we have cloned a cDNA corresponding to the latter open reading frame and demonstrate that it encodes a β4GalNAcT, which we have termed Ceβ4GalNAcT. Ceβ4GalNAcT is active when expressed in mammalian cells in generating LDN determinants on N-glycans of glycoproteins. All chemicals and reagents used in this study, unless otherwise indicated, were from Sigma. The C. eleganscDNA library was a gift from Dr. Robert Barstead (Oklahoma Medical Research Foundation, Oklahoma City, OK). The QIA Quick gel extraction kit was from Qiagen (Valencia, CA). Restriction enzymes were from New England Biolabs (Beverly, MA). The pCR 2.1 vector was from Invitrogen. The pcDNA3.1(+)-TH was a gift from Dr. Alireza R. Rezaie (Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, MO). FuGENE 6 and Complete protease inhibitor mixture were from Roche Molecular Biochemicals.N-Glycanase was from Glyko (Novato, CA). HighSignal West Pico Chemiluminescent Substrate was from Pierce. GlcNAcβ1–3GalNAcα1-O-pNP (core 3-O-pNP) and GlcNAcβ1–6GalNAcα1-O-pNP (core 6-O-pNP) were obtained from Toronto Research Chemicals (Toronto, Canada). Acceptor compounds (see Table II) 1–3, 5, 9, and 12 were purchased from Sigma, 4 was from Koch-Light Laboratories, and 6–8 were from Toronto Research Chemicals. Compounds 10 and 11 were a kind gift from Dr. L. Anderson (University of Wisconsin, Madison, WI), and14–17 were from Dr. J. Lönngren (University of Stockholm). Compounds 13 (39Bakker H. Schoenmakers P.S. Koeleman C.A. Joziasse D.H. van Die I. van den Eijnden D.H. Glycobiology. 1997; 7: 539-548Crossref PubMed Scopus (23) Google Scholar) and 18–21 (32van Die I. van Tetering A. Schiphorst W.E. Sato T. Furukawa K. van den Eijnden D.H. FEBS Lett. 1999; 450: 52-56Crossref PubMed Scopus (49) Google Scholar) were synthesized as described previously. Radiolabeled nucleotide sugars were obtained from PerkinElmer Life Sciences and were diluted with unlabeled nucleotide sugars (Sigma) to give the desired specific radioactivity.Table IIAcceptor Specificity of Ceβ4GalNAcT and Comparison to Other Members of the β4GalT FamilyAcceptorRelative activityaAssays were carried out in duplicate as described under "Experimental Procedures" using SH-Ceβ4GalNAcT attached to HPC4 beads with a donor concentration of 0.5 mm and an acceptor concentration of 1 mmterminal GlcNAc. For comparison, 100% activity (using free GlcNAc as acceptor) corresponds to 2.1 nmol/min/ml bead suspension.Ceβ4-GalNAcTHuman β4GalT 1bAlso for comparison, relative activities with the same acceptors for human β4GalT I (32) andL. stagnalis β4GlcNAcT (39) are taken from previous publications.Lymnaea stagnalis β4GlcNAcTbAlso for comparison, relative activities with the same acceptors for human β4GalT I (32) andL. stagnalis β4GlcNAcT (39) are taken from previous publications.%%%1. GlcNAcβ-S-pNP28523253802. GlcNAcα1-pNP1439953. Galβ-pNP14. Glcβ1-methyl-umbelliferone0.55. GalNAcβ-pNP0.5<106. SO4-6-GlcNAcβ1-pNP6257. GlcNAcβ1–3GalNAcα-pNP1451972508. GlcNAcβ1–6(Galβ1–3)GalNAcα-pNP15919555709. GlcNAc10010010010. GlcNAcβ1–3Gal12117611. GlcNAcβ1–6Gal328159012. GlcNAcβ1–4GlcNAcβ1–4GlcNAc1152413. GlcNAcβ1–6GlcNAc10946714. GlcNAcβ1–2Man1323415. GlcNAcβ1–6Man15642516. 11517617. 1125818. 7136019. 12238120. 11137221. 48365a Assays were carried out in duplicate as described under "Experimental Procedures" using SH-Ceβ4GalNAcT attached to HPC4 beads with a donor concentration of 0.5 mm and an acceptor concentration of 1 mmterminal GlcNAc. For comparison, 100% activity (using free GlcNAc as acceptor) corresponds to 2.1 nmol/min/ml bead suspension.b Also for comparison, relative activities with the same acceptors for human β4GalT I (32van Die I. van Tetering A. Schiphorst W.E. Sato T. Furukawa K. van den Eijnden D.H. FEBS Lett. 1999; 450: 52-56Crossref PubMed Scopus (49) Google Scholar) andL. stagnalis β4GlcNAcT (39Bakker H. Schoenmakers P.S. Koeleman C.A. Joziasse D.H. van Die I. van den Eijnden D.H. Glycobiology. 1997; 7: 539-548Crossref PubMed Scopus (23) Google Scholar) are taken from previous publications. Open table in a new tab Table ISugar nucleotide specificity of the Ceβ4GalNAcTAcceptorUDP donorRelative activityaAssays were carried out in duplicate as described under "Experimental Procedures" using SH-Ceβ4GalNAcT attached to HPC4 beads with a donor concentration of 0.5 mm and an acceptor concentration of 1 mm. For comparison, 100% activity corresponds to 5.9 nmol/min/ml of bead suspension.%GlcNAcβ-S-pNPUDP-GalNAc100GlcNAcβ-S-pNPUDP-GlcNAc0.7GlcNAcβ-S-pNPUDP-Glc0.2GlcNAcβ-S-pNPUDP-Gal1a Assays were carried out in duplicate as described under "Experimental Procedures" using SH-Ceβ4GalNAcT attached to HPC4 beads with a donor concentration of 0.5 mm and an acceptor concentration of 1 mm. For comparison, 100% activity corresponds to 5.9 nmol/min/ml of bead suspension. Open table in a new tab A BlastP search of the NCBI nonredundant protein data base for homologues of the human β4GalT I (accession number CAA39074) identified a hypothetical protein encoded by an open reading frame in the C. elegans genome designated Y73E7A.7. A cDNA was amplified by PCR from a mixed stage C. elegans cDNA library using primers corresponding to the 5′- and 3′-ends of this open reading frame (5′-GCCACCATGGCTTTTCGTCATTTGGC-3′; 5′-CTAAAAACACGTTGGAAAGTCC-3′). Amplification was carried out at 95 °C for 2:30 min followed by 35 cycles at 95 °C for 50 s, 53 °C for 50 s, and 72 °C for 1:50 min and then at 72 °C for 10 min. The PCR product was purified from an agarose gel slice using a QIA Quick gel extraction kit, cloned into the pCR 2.1 vector, and sequenced on both strands at the Sequencing Facility of the Oklahoma Medical Research Foundation (Oklahoma City, OK). A PsiI (partial)/PvuII DNA fragment starting at bp 87 of the Ceβ4GalNAcT open reading frame and extending beyond the stop codon was subcloned into the EcoRV site of the pcDNA 3.1(+)-TH vector. The resulting vector (pCMV-SH-Ceβ4GalNAcT) encodes a fusion protein, designated SH-Ceβ4GalNAcT, which consists of a signal peptide at the N terminus followed by an HPC4 epitope and then the catalytic domain of the Ceβ4GalNAcT (beginning at Lys30, the first amino acid after the transmembrane domain). The HPC4 epitope is recognized by the Ca2+-dependent monoclonal antibody HPC4 (49Stearns D.J. Kurosawa S. Sims P.J. Esmon N.L. Esmon C.T. J. Biol. Chem. 1988; 263: 826-832Abstract Full Text PDF PubMed Google Scholar,50Rezaie A.R. Fiore M.M. Neuenschwander P.F. Esmon C.T. Morrissey J.H. Protein Expression Purif. 1992; 3: 453-460Crossref PubMed Scopus (78) Google Scholar). SH-Ceβ4GalNAcT is under the transcriptional control of the cytomegalovirus promoter, which is present in the vector. CHO-Lec8 and CHO-Lec2 cells were transfected with pCMV-SH-Ceβ4GalNAcT using FuGENE 6, according to the manufacturer's instructions, and cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 600 μg/ml Geneticin to select for stably transformed cells. After 4 weeks of culturing in medium containing Geneticin, the cells were cultured in the same medium without Geneticin, and the culture medium was harvested every 3 days and used to purify SH-Ceβ4GalNAcT. To assay intracellular β4GalNAcT activity and for Western blots, cells were washed with 75 mm sodium cacodylate, pH 7.0, and lysed in a buffer of 50 mm sodium cacodylate, pH 7.0, 20 mm MnCl2, 1% Triton X-100, 1× Complete protease inhibitor mixture (EDTA-free). The lysates were centrifuged at 12,000 × g for 3 min, and the supernatants were used for further analyses. Medium containing SH-Ceβ4GalNAcT was centrifuged at 1,500 ×g for 5 min to remove cellular debris and then incubated with HPC4-UltraLink beads (5 mg of HPC4 antibody/ml of beads; 0.1 μl of beads/ml of medium) for 1 h at room temperature on a rotating platform. The beads were collected by centrifugation at 600 ×g for 3 min and washed three times with 10 ml of 100 mm sodium cacodylate, pH 7.0, 2 mmCaCl2. The beads were then resuspended in the same buffer with the addition of 20 mm MnCl2 and used as the enzyme source. For Western blot analysis, the bound material was released by incubating the beads in a buffer of 50 mmsodium cacodylate, pH 7.0, 20 mm EDTA for 10 min at room temperature and then collecting the supernatant. Cell lysates were treated with N-glycanase in a buffer of 20 mmsodium phosphate, pH 7.5, 50 mm β-mercaptoethanol, 0.1% SDS, 0.75% Nonidet P-40 for 3 h at 37 °C. Control treatments were carried out in the same way but without addingN-glycanase. The lysates were then mixed with loading buffer, resolved by SDS-PAGE (4–20% gradient), and transferred to a nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin in a buffer of 20 mm Tris-HCl, pH 7.2, 150 mm NaCl, 2 mm CaCl2, 0.05% Tween 20 for 5 h at 4 °C. It was then incubated with the primary antibody (mouse monoclonal anti-LDN IgM SMLDN1.1 (16Nyame A.K. Leppanen A.M. DeBose-Boyd R. Cummings R.D. Glycobiology. 1999; 9: 1029-1035Crossref PubMed Scopus (61) Google Scholar) or HPC4 IgG) in the same buffer (without bovine serum albumin) for 1 h at room temperature, washed in the same buffer, and incubated with the secondary antibody (horseradish peroxidase-conjugated, goat anti-mouse IgM or IgG) as before. The membrane was then washed again, incubated in HighSignal West Pico Chemiluminescent Substrate for 2 min at room temperature, and exposed to a BioMax film (Eastman Kodak Co.) for 1 min. The film was then developed using a processing machine (Konica SRX-101). Standard assays were performed essentially as described previously (45van Die I. van Tetering A. Bakker H. van den Eijnden D.H. Joziasse D.H. Glycobiology. 1996; 6: 157-164Crossref PubMed Scopus (80) Google Scholar) in a 25-μl reaction mixture containing 2.5 μmol of sodium cacodylate, pH 7.2, 12.5 nmol of UDP-[3H]GalNAc (2.5 Ci/mol), 1 μmol of MnCl2, 0.1 μmol of ATP, 0.1 μl of Triton X-100, 2 μl of beads, and acceptor substrate, containing 25 nmol of terminal GlcNAc at the nonreducing end unless otherwise indicated. Control assays lacking the acceptor substrate were carried out to correct for incorporation into endogenous acceptors, and all assays were carried out in duplicate. All assays were linear with time for up to 180 min. After incubation at 37 °C for 180 min, the reaction was stopped. When oligosaccharides or glycopeptides were the acceptor, the labeled product was separated from unincorporated label by chromatography on a 1-ml column of Dowex 1-X8 (Cl− form) according to Eastonet al. (51Easton E.W. Blokland I. Geldof A.A. Rao B.R. van den Eijnden D.H. FEBS Lett. 1992; 308: 46-49Crossref PubMed Scopus (16) Google Scholar). When oligosaccharide acceptors with hydrophobic aglycon (pNP) were used as the acceptor, the product was isolated using Sep-Pak C-18 cartridges (Waters) as described (52Palcic M.M. Heerze L.D. Pierce M. Hindsgaul O. Glycoconj. J. 1988; 5: 49-63Crossref Scopus (279) Google Scholar). The isolated products were assayed for incorporation of radioactivity by liquid scintillation. The product catalyzed by SH-Ceβ4GalNAcT using GlcNAcβ1-O-pNP as acceptor was isolated using a Sep-Pak C-18 cartridge (1 cm3) and lyophilized. Three nmol of the product (dissolved in water) were analyzed by a Dionex HPAEC-PAD system, using a PA-1 column with a 100 mm NaOH solution at a flow rate of 1 ml/min. The standard containing the authentic LDN structure GalNAcβ1–4GlcNAcβ1-O-pNP was synthesized using bovine β4GalT I and GlcNAcβ1-O-pNP as the acceptor for UDP-GalNAc in the standard assay described above. Commercially acquired GlcNAcβ1–3GalNAcα1-O-pNP (core 3-O-pNP) and GlcNAcβ1–6GalNAcα1-O-pNP (core 6-O-pNP) were also used as standards. Synthesis was carried out overnight at 37 °C in a 1-ml reaction mixture containing 50 μmol of sodium cacodylate, pH 7.0, 300 nmol of GlcNAcβ1-S-pNP, 1 μmol of UDP-GalNAc, 20 μmol of MnCl2, 5 μmol of ATP, 3 μmol of NaN3, and 100 μl of beads. The product was then isolated using a Sep-Pak C-18 cartridge (1 cm3) and lyophilized. 150 nmol of the product catalyzed by SH-Ceβ4GalNAcT using GlcNAcβ1-S-pNP as acceptor were treated with D2O (99.75 atom %; Merck) three times with intermediate lyophilization. Finally, the sample was redissolved in 400 μl of D2O (99.96 atom %; Sigma-Aldrich). 1H NMR spectroscopy was performed on a Bruker MSL 400 spectrometer operating at 400 MHz at a probe temperature of 300 K. Resolution enhancement was achieved by Lorentzian to Gaussian transformation. Chemical shifts are expressed in ppm downfield from internal sodium 4,4-dimethyl-4-silapentane-1-sulfonate but were actually measured by reference to internal acetone (δ = 2.225 ppm in D2O). A potential C. elegans open reading frame designated Y73E7A.7 was identified by a BlastP search as encoding a homologue of the human β4GalT I. An identical cDNA (GenBankTM accession number AY130767) was amplified by PCR from a mixed stage C. elegans cDNA library using primers corresponding to the 5′- and 3′-ends of this open reading frame, establishing that the gene is expressed in vivo. The cDNA of Y73E7A.7 encodes a predicted 383-amino acid protein with a single transmembrane domain in a type 2 topology, which is a common topological motif in glycosyltransferases. The protein encoded by Y73E7A.7 is predicted to contain six potentialN-glycosylation sites and two DVD motifs, which are thought to participate in metal ion binding (53Wiggins C.A. Munro S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7945-7950Crossref PubMed Scopus (321) Google Scholar) (Fig.1). Curiously, the last four potentialN-glycosylation sites share an identical sequon (NQT), the significance of which is not clear at this time. The protein sequence encoded by Y73E7A.7 is 35.5% identical to human β4GalT I (Fig.2 A) and is more closely related to the first four members of the β4GalT family (I, II, III, and IV) than to the other three (Fig. 2 B).Figure 2Protein sequence comparisons between the protein encoded by Y73E7A.7 (Ceβ4GalNAcT) and members of the β4GalT family. A, alignment of Y73E7A.7 (Ceβ4GalNAcT) with human β4GalT I using the Align and Boxshade programs. Black boxes, identical residues; gray boxes, similar residues.B, phylogenic analysis of Ceβ4GalNAcT and other β4GalT family members using the ClustalW and Drawgram programs.View Large Image Figure ViewerDownload (PPT) To assess whether Y73E7A.7 encodes an active β4-galactosyltransferase or possibly a β4-N-acetylgalactosaminyltransferase, a soluble, recombinant form of the protein was generated lacking the cytoplasmic N terminus and transmembrane domain and containing the HPC4 peptide epitope at the new N terminus. This construct was stably expressed in Chinese hamster ovary CHO-Lec8 cells. These cells are impaired in the transport of UDP-Gal into the Golgi (54Deutscher S.L. Hirschberg C.B. J. Biol. Chem. 1986; 261: 96-100Abstract Full Text PDF PubMed Google Scholar) and consequently generate hybrid- and complex-type N-glycans containing terminal GlcNAc and O-glycans containing the simple Tn antigen GalNAcα1-Ser/Thr (55Stanley P. Siminovitch L. Somatic Cell Genet. 1977; 3: 391-405Crossref PubMed Scopus (124) Google Scholar, 56Do S.I. Cummings R.D. J. Biochem. Biophys. Methods. 1992; 24: 153-165Crossref PubMed Scopus (9) Google Scholar, 57Nagayama Y. Namba H. Yokoyama N. Yamashita S. Niwa M. J. Biol. Chem. 1998; 273: 33423-33428Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The transfected cells expressing Y73E7A.7, but not the control mock-transfected cells, acquired a novel intracellular GalNAcT activity in the cell extracts capable of utilizing UDP-GalNAc as the donor and GlcNAcβ1-S-pNP as the acceptor (Fig. 3 A). The recombinant protein containing the HPC4 epitope from extracellular medium was bound by HPC4-conjugated beads, confirming the β4GalNAcT activity of the enzyme encoded by the Y73E7A.7 (Fig. 3 A). A Western blot of the material bound to the HPC4-conjugated beads (Fig. 3 B) confirmed that it corresponded to the predicted size of the HPC4 epitope-tagged protein (43.1-kDa peptide plus N-glycans) as discussed below. These data demonstrate that Y73E7A.7 encodes an active β4GalNAcT and the enzyme was designated the C. elegansUDP-GalN