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• W2120521563 abstract "A fourth human UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferase, designated GalNAc-T4, was cloned and expressed. The genomic organization of GalNAc-T4 is distinct from GalNAc-T1, -T2, and -T3, which contain multiple coding exons, in that the coding region is contained in a single exon. GalNAc-T4 was placed at human chromosome 12q21.3-q22 by in situ hybridization and linkage analysis. GalNAc-T4 expressed in Sf9 cells or in a stably transfected Chinese hamster ovary cell line exhibited a unique acceptor substrate specificity. GalNAc-T4 transferred GalNAc to two sites in the MUC1 tandem repeat sequence (Ser in GVTSA and Thr in PDTR) using a 24-mer glycopeptide with GalNAc residues attached at sites utilized by GalNAc-T1, -T2, and -T3 (TAPPAHGVTSAPDTRPAPGSTAPPA, GalNAc attachment sites underlined). Furthermore, GalNAc-T4 showed the best kinetic properties with an O-glycosylation site in the P-selectin glycoprotein ligand-1 molecule. Northern analysis of human organs revealed a wide expression pattern. Immunohistology with a monoclonal antibody showed the expected Golgi-like localization in salivary glands. A single base polymorphism, G1516A (Val to Ile), was identified (allele frequency 34%). The function of GalNAc-T4 complements other GalNAc-transferases in O-glycosylation of MUC1 showing that glycosylation of MUC1 is a highly ordered process and changes in the repertoire or topology of GalNAc-transferases will result in altered pattern of O-glycan attachments. A fourth human UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferase, designated GalNAc-T4, was cloned and expressed. The genomic organization of GalNAc-T4 is distinct from GalNAc-T1, -T2, and -T3, which contain multiple coding exons, in that the coding region is contained in a single exon. GalNAc-T4 was placed at human chromosome 12q21.3-q22 by in situ hybridization and linkage analysis. GalNAc-T4 expressed in Sf9 cells or in a stably transfected Chinese hamster ovary cell line exhibited a unique acceptor substrate specificity. GalNAc-T4 transferred GalNAc to two sites in the MUC1 tandem repeat sequence (Ser in GVTSA and Thr in PDTR) using a 24-mer glycopeptide with GalNAc residues attached at sites utilized by GalNAc-T1, -T2, and -T3 (TAPPAHGVTSAPDTRPAPGSTAPPA, GalNAc attachment sites underlined). Furthermore, GalNAc-T4 showed the best kinetic properties with an O-glycosylation site in the P-selectin glycoprotein ligand-1 molecule. Northern analysis of human organs revealed a wide expression pattern. Immunohistology with a monoclonal antibody showed the expected Golgi-like localization in salivary glands. A single base polymorphism, G1516A (Val to Ile), was identified (allele frequency 34%). The function of GalNAc-T4 complements other GalNAc-transferases in O-glycosylation of MUC1 showing that glycosylation of MUC1 is a highly ordered process and changes in the repertoire or topology of GalNAc-transferases will result in altered pattern of O-glycan attachments. UDP-N-acetyl-α-d-galactosamine:polypeptideN-acetylgalactosaminyltransferase -T2, and -T3 represents human GalNAc-transferases, GenBank accession numbers X85018,X85019, X92689, respectively (3White T. Bennett E.P. Takio K. Sorensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 4Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) expressed sequence tags rapid amplification of cDNA ends polymerase chain reaction base pair(s) high performance liquid chromatography Chinese hamster ovary matrix-assisted laser desorption/ionization polyacrylamide gel electrophoresis. A family of UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferases (GalNAc-transferases)1(EC 2.4.1.41) control the initiation of mucin-type O-linked protein glycosylation, in which N-acetylgalactosamine is transferred to serine and threonine amino acid residues (1Clausen H. Bennett E.P. Glycobiology. 1996; 6: 635-646Crossref PubMed Scopus (228) Google Scholar). Four members of the animal GalNAc-transferase family have been reported (1Clausen H. Bennett E.P. Glycobiology. 1996; 6: 635-646Crossref PubMed Scopus (228) Google Scholar, 2Homa F.L. Hollander T. Lehman D.J. Thomsen D.R. Elhammer A.P. J. Biol. Chem. 1993; 268: 12609-12616Abstract Full Text PDF PubMed Google Scholar, 3White T. Bennett E.P. Takio K. Sorensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 4Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 5Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The GalNAc-transferase gene family in animals contain several additional members 2E. P. Bennett and H. Clausen, unpublished observation. and recently it was reported that a number of GalNAc-transferase homologues exist in Caenorhabditis elegans (6Hagen F.K. Nehrke K. J. Biol. Chem. 1998; 273: 8268-8277Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The GalNAc-transferases characterized so far have distinct acceptor substrate specificities (4Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar,5Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 7Sorensen T. White T. Wandall H.H. Kristensen A.K. Roepstorff P. Clausen H. J. Biol. Chem. 1995; 270: 24166-24173Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 8Wandall H.H. Hassan H. Mirgorodskaya E. Kristensen A.K. Roepstorff P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. Taylor-Papadimitriou J. Clausen H. J. Biol. Chem. 1997; 272: 23503-23514Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar), and show different patterns of expression in human cells and organs (4Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 5Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 9Mandel, U., Hassan, H., Therkildsen, M. H., Nielsen, P. A., Rygaard, J., Jacobsen, M., Juhl, B. R., Dabelsteen, E., and Clausen, H. (1998) Glycobiology, in pressGoogle Scholar). The chromosomal localization and genomic organization of human GalNAc-T1, -T2, and -T3 (GALNT1, GALNT2, and GALNT3) are different; however, a number of conserved intron/exon boundaries confirm their evolutionary relationships (1Clausen H. Bennett E.P. Glycobiology. 1996; 6: 635-646Crossref PubMed Scopus (228) Google Scholar, 10Meurer J.A. Drong R.F. Homa F.L. Slightom J.L. Elhammer A.P. Glycobiology. 1996; 6: 231-241Crossref PubMed Scopus (11) Google Scholar,11Bennett E.P. Weghuis D.O. Merkx G. Geurts van Kessel A. Eiberg H. Clausen H. Glycobiology. 1998; 8: 547-555Crossref PubMed Scopus (34) Google Scholar). Taken together, these features strongly suggest that each GalNAc-transferase has distinct functions, and that the large number of members of this gene family has evolved as a consequence of the need for O-glycosylation of different sequences. The fine substrate specificities of GalNAc-transferases may represent a major determining factor for sites of O-glycan attachments. O-Glycosylation of MUC1 has attracted attention because it is altered in cancer cells with smaller and fewer O-glycans (12Taylor-Papadimitriou J. Stewart L. Burchell J. Beverley P. Ann. N. Y. Acad. Sci. 1993; 690: 69-79Crossref PubMed Scopus (43) Google Scholar, 13Brockhausen I. Yang J.M. Burchell J. Whitehouse C. Taylor-Papadimitriou J. Eur. J. Biochem. 1995; 233: 607-617Crossref PubMed Scopus (311) Google Scholar, 14Lloyd K.O. Burchell J. Kudryashov V. Yin B.W.T. Taylor-Papadimitriou J. J. Biol. Chem. 1996; 271: 33325-33334Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). The change in O-glycosylation leads to the exposure of cancer-associated peptide epitopes within the tandem repeat region of MUC1, although the exact molecular nature of this phenomenon is still unknown (15Girling A. Bartkova J. Burchell J. Gendler S. Gillett C. Taylor-Papadimitriou J. Int. J. Cancer. 1989; 43: 1072-1076Crossref PubMed Scopus (307) Google Scholar, 16Taylor-Papadimitriou J. Finn O.J. Immunol. Today. 1997; 18: 105-107Abstract Full Text PDF PubMed Scopus (35) Google Scholar). Analysis of the in vitro O-glycosylation properties of various GalNAc-transferase preparations including purified recombinant GalNAc-T1, -T2, and -T3 suggests that only three of the potential five sites in the repeat were glycosylated (AHGVTSAPDTRPAPGSTAPPA, in vitro glycosylation sites underlined). However, recently Mulleret al. (17Muller S. Goletz S. Packer N. Gooley A. Lawson A.M. Hanisch F.G. J. Biol. Chem. 1997; 272: 24780-24793Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) elegantly established that all five sites can be occupied in purified milk MUC1. Two explanations for this discrepancy are: 1) that in vitro O-glycosylation assays fail to reflect the in vivo function of GalNAc-transferases, or 2) that additional GalNAc-transferases have substrate specificity for the two last sites in the MUC1 repeat. The present study presents evidence supporting the latter explanation. Previously, a sequence motif shared between GalNAc-T1 and -T2 was used to identify and clone GalNAc-T3 (4Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Two candidate cDNA sequences containing the GalNAc-transferase motif were identified: one was the GalNAc-T3 gene. Here, we report the cloning and expression of the second identified cDNA, which was designated GalNAc-T4 (1Clausen H. Bennett E.P. Glycobiology. 1996; 6: 635-646Crossref PubMed Scopus (228) Google Scholar). A putative murine orthologue also designated GalNAc-T4 was recently reported by Hagen et al. (5Hagen F.K. Ten Hagen K.G. Beres T.M. Balys M.M. VanWuyckhuyse B.C. Tabak L.A. J. Biol. Chem. 1997; 272: 13843-13848Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The recombinant human GalNAc-T4 had a highly restricted acceptor substrate specificity, which is distinct from that of previously reported GalNAc-transferases. Thus, GalNAc-T4 was found to complement GalNAc-T1, -T2, and -T3, in O-glycosylation of the MUC1 tandem repeat by transferring GalNAc to two sites in the MUC1 repeat not utilized by other known GalNAc-transferases. Importantly, GalNAc-T4 showed preference for the MUC1 glycopeptide, GalNAc4TAP24, which was previously glycosylated with GalNAc-T2. A cDNA sequence (TE4) with extensive similarity to the GalNAc-transferase motif was previously identified following reverse transcriptase-PCR of MKN45 mRNA using degenerate primers (4Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). The PCR product was cleaved by BstNI to remove GalNAc-T1 and -T2 derived products. Uncleaved product was isolated using the prep-A-gene kit (Bio-Rad) and cloned into the pT7T3U19 vector (Pharmacia). A rapid cDNA library screening strategy was performed as described previously (4Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). A human salivary gland λgt11 library (CLONTECH) was amplified and aliquoted into 40 sublibraries, which were screened to identify phage clones containing TE4. Fourteen sublibraries found to contain TE4 λgt11 clones were further assayed by PCR using EBHC201 (5′-GCGGATCCGCGGGCACCATATGCTCG) or EBHC203 (5′-GAGCAGTETTCTGTAGGAAATTGG) primers combined with primers based on flanking λgt11 vector sequence to estimate lengths of cDNA inserts for selection of sublibraries with the largest 3′ or 5′ sequences. Amplifications were: 35 cycles of 95 °C for 45 s; 53 °C for 1 s; 72 °C for 2 min. One sublibrary (number 22) generated a 3′ PCR product (EBHC201/λgt11 REV) of approximately 750 bp, and one sublibrary (number 34) generated a 5′ PCR product (EBHC203/λgt11 REV) of approximately 800 bp. Additional 5′ sequence was obtained by 5′ RACE using a 5′ Ready-RACE Lung cDNA Kit (CLONTECH) in combination with antisense primer EBHC309 (5′-TCAAAAGAACTGCAGGAGAAG): 35 cycles of 95 °C for 45 s; 60 °C for 15 s; 72 °C for 3 min. The RACE products were blunt-end cloned into pT7T3U19 and multiple clones were sequenced. The longest insert was approximately 550 bp and did not contain the full 5′ coding sequence. Further 5′ RACE failed and the remaining 5′ sequence was obtained by sequencing P1 genomic clones. A P1 human foreskin genomic library (DuPont Merck Pharmaceutical Co., Human Foreskin Fibroblast P1 Library) was screened using primer pairs EBHC302 (5′-GCCACTGAAATGTTAAACGCC)/EBHC304 (5′-ACGAAGCCTGGTCGACTTTGC). Three clones, DPMC-HFF 1-0160-B2 (P1–6211), DPMC-HFF 1-0972-B9 (P1–6212), and DPMC-HFF 1-1275-A10 (P1–6213), were obtained from Genome Systems. DNA from P1 phage was prepared as recommended by Genome Systems. The coding sequence of GalNAc-T4 was sequenced from all P1 clones using automated sequencing (ABI377, Perkin-Elmer) with dye terminator chemistry. A single base substitution was identified in the coding region between P1–6213 (nucleotides 1516 G) and P1–6212 (nucleotide 1516 A) (Figs. 1 and 2). The substitution removes a uniqueKpnI restriction site for the 1516 G allele. A PCR genotyping assay using EBHC300 (5′-AGCGGATCCTGACAACAACCCCACAGG)/EBHC307 (5′-AGCGGATCCGACGAAAGTGCTGTTGTGCTC) (conditions: 35 cycles of 95 °C, 20 s; 55 °C, 1 s; 72 °C, 30 s) followed by KpnI restriction analysis was performed on DNA samples from 206 healthy blood donors, in order to evaluate the allele frequencies.Figure 2Fluorescence in situhybridization of GalNAc-T4 to metaphase chromosomes localized the gene to 12q21.3–22.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fluorescencein situ hybridization was performed on normal human lymphocyte metaphase chromosomes, using procedures described previously (18Suijkerbuijk R.F. van de Veen A.Y. van Echten J. Buys C.H. de Jong B. Oosterhuis J.W. Warburton D.A. Cassiman J.J. Schonk D. Geurts van Kessel A. Am. J. Hum. Gen. 1991; 48: 269-273PubMed Google Scholar). Briefly, P1 DNA was labeled with digoxigenin-14-dUTP (Boehringer Mannheim) using the BioNICK labeling system (Life Technologies). The labeled DNA was precipitated with ethanol in the presence of herring sperm DNA. A total of 200 ng of P1 DNA was precipitated with 50 × human Cot 1 DNA (Life Technologies, Inc.) and dissolved in 12 ml of hybridization solution (2 × SSC, 10% dextran sulfate, 1% Tween 20, and 50% formamide, pH 7.0). Prior to hybridization the probe was heat-denatured at 80 °C for 10 min, chilled on ice, and incubated at 37 °C to allow re-annealing of highly repetitive sequences. After denaturation of the slides, probe incubations were carried out under a 18 × 18-mm coverslip in a moist chamber for 45 h. Immunochemical detection of the probe was achieved using sheep anti-digoxigenin (fluorescein isothiocyanate) (Boehringer) and donkey anti-sheep (fluorescein isothiocyanate) (Jackson Laboratories) antibodies. For evaluation of the chromosomal slides, a Zeiss epifluorescence microscope equipped with appropriate filters for visualization of fluorescein isothiocyanate was used. Hybridization signals and 4,6-diamidino-2-phenylindole-counterstained chromosomes were transformed into pseudo-colored images using image analysis software. For precise localization and chromosome identification 4,6-diamidino-2-phenylindole-converted banding patterns were generated using the BDS-imageTM software package (ONCOR). Seven normal families with an average of 10 children from the Copenhagen Family Bank (19Eiberg H. Nielsen L.S. Klausen J. Dahlen M. Kristensen M. Bisgaard M.L. Moller N. Mohr J. Clin. Genet. 1989; 35: 313-321Crossref PubMed Scopus (46) Google Scholar) were analyzed for the G/A polymorphism at bp position 1516 using the PCR assay with EBHC300/EBHC307. To confirm in situ localization on 12q21.3-q22, a set of DNA markers with known physical localization were used. The order and distances for these were drawn from the Généthon Linkage map (20Dib C. Faure S. Fizames C. Samson D. Drouot N. Vignal A. Millasseau P. Marc S. Hazan J. Seboun E. Lathrop M. Gyapay G. Morissette J. Weissenbach J. Nature. 1996; 380: 152-154Crossref PubMed Scopus (2730) Google Scholar): D12S90-(18.8)-D12S80-(10.8)-D12S81-(7.5)-D12S101. The physical map of these markers are: D12S80, 12p13.2-q21.1; D12S81, 12q21; and D12S101, 12q22. Lod scores were calculated with the LIPED software (21Ott J. Am. J. Hum. Gen. 1976; 28: 528-529PubMed Google Scholar). Expression constructs designed to contain amino acid residues 32–578 of the coding sequence of the putative GalNAc-T4 gene were prepared by genomic PCR on the two different P1 clones using the primer pair EBHC318 (5′-AGCGGATCCTTTTCATGCCTCCGCAGGAGCCG) and EBHC307 with BamHI restriction sites (Fig. 1). These PCR products were cloned into aBamHI site of the expression vector pAcGP67 (Pharmingen), and the expression construct was sequenced. The constructs, pAcGP67-GalNAc-T4506Vsol and pAcGP67-GalNAc-T4506Isol, were designed to yield a putative soluble form of the GalNAc-T4 protein with an N-terminal end positioned immediately C-terminal to the potential transmembrane domain and including the entire sequence expected to contain the catalytic domain. Control constructs pAcGP67-GalNAc-T1-sol, pAcGP67-GalNAc-T2-sol, pAcGP67-GalNAc-T3-sol, and pAcGP67-O2-sol were prepared as described previously (3White T. Bennett E.P. Takio K. Sorensen T. Bonding N. Clausen H. J. Biol. Chem. 1995; 270: 24156-24165Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 4Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 22Bennett E.P. Steffensen R. Clausen H. Weghuis D.O. Geurts van Kessel A. Biochem. Biophys. Res. Commun. 1995; 211: 347Crossref PubMed Scopus (16) Google Scholar). A full coding expression construct was prepared by PCR of P1 clone P1–6213 (GalNAc-T4506V) using primer pair EBHC320 (5′-AGCGGATCCCACCATGGCGGTGAGGTGGACTTGG)/EBHC307. The product was cloned into BamHI sites of the expression vector pVL1392 (Pharmingen). Co-transfection of Sf9 cells with pAcGP67-constructs or pVL-constructs and Baculo-GoldTM DNA was performed according to the manufacturer's specifications. Briefly, 0.4 μg of construct was mixed with 0.1 μg of Baculo-Gold DNA and co-transfected in Sf9 cells in 24-well plates. Ninety-six hours post-transfection recombinant virus was amplified in 6-well plates at dilutions of 1:10 and 1:50. Titer of amplified virus was estimated by titration in 24-well plates with monitoring of GalNAc-transferase activities. Initial transferase assays were performed on supernatants of Sf9 cells in 6-well plates infected with first or second amplified virus. Titers representing end point dilutions giving optimal enzyme activities were used. Transferase assays of the full coding expression construct was performed by extracting washed cells in 1% Triton X-100 as described previously (7Sorensen T. White T. Wandall H.H. Kristensen A.K. Roepstorff P. Clausen H. J. Biol. Chem. 1995; 270: 24166-24173Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). pAcGP67-GalNAc-T4506Vsol was expressed in High Five™ cells grown in serum-free medium in upright roller bottles shaking at 140 rpm in 27 °C waterbaths. GalNAc-T4506Vwas purified essentially as described previously (8Wandall H.H. Hassan H. Mirgorodskaya E. Kristensen A.K. Roepstorff P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. Taylor-Papadimitriou J. Clausen H. J. Biol. Chem. 1997; 272: 23503-23514Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar), using successive chromatography on Amberlite (IRA95, Sigma), DEAE-Sephacel (Pharmacia), and S-Sepharose Fast Flow (Pharmacia). Final purification was performed on Mini-S™ (PC 3.2/3, Pharmacia) using the Smart System (Pharmacia), and peak fractions used for immunizing BALB/c mice as described previously (5–10 μg/subcutaneous injection three times with one intravenous boost) (9Mandel, U., Hassan, H., Therkildsen, M. H., Nielsen, P. A., Rygaard, J., Jacobsen, M., Juhl, B. R., Dabelsteen, E., and Clausen, H. (1998) Glycobiology, in pressGoogle Scholar). Monoclonal antibodies were selected and characterized by immunocytology on Sf9 cells infected with various GalNAc-transferase expression constructs and selective immunoprecipitation of active GalNAc-T4 activity. Immunofluorescence staining of frozen sections of submandibular glands was performed as described previously (9Mandel, U., Hassan, H., Therkildsen, M. H., Nielsen, P. A., Rygaard, J., Jacobsen, M., Juhl, B. R., Dabelsteen, E., and Clausen, H. (1998) Glycobiology, in pressGoogle Scholar), and monoclonal antibody PANH2reacting with MUC5B (23Nielsen P.A. Mandel U. Therkildsen M.H. Clausen H. J. Dent. Res. 1996; 75: 1820-1826Crossref PubMed Scopus (65) Google Scholar), was used as marker of mucous cells. The insert of pAcGP67-GalNAc-T4506Vsol was excised and cloned into a modified pCDNA3 vector (Invitrogen), which includes 19 amino acids of the γ-interferon secretion signal sequence (24Gray P.W. Leung D.W. Pennica D. Najarian R. Simonsen C.C. Derynck R. Sherwood P.J. Wallace D.M. Berger S.L. Levinson A.D. Goeddel D.V. Nature. 1982; 295: 503-508Crossref PubMed Scopus (526) Google Scholar). CHO-K1 (ATCC) was transfected using 0.2 μg of DNA and 5 μl of LipofectAMINE (Invitrogen) in subconfluent 6-well plates according to the manufacturer's protocol. After 48 h the medium was changed and 400 μg/ml G418 was added. At 72 h 10–20% of the wells were trypsinized and the percentage of cells expressing GalNAc-T4 was evaluated by immunocytology as described previously (9Mandel, U., Hassan, H., Therkildsen, M. H., Nielsen, P. A., Rygaard, J., Jacobsen, M., Juhl, B. R., Dabelsteen, E., and Clausen, H. (1998) Glycobiology, in pressGoogle Scholar), using a novel anti-GalNAc-T4 monoclonal antibody, UH6. Based on the frequency of positive cells the residual transfectant cells were trypsinized and plated in 96-well plates. Two rounds of screening and cloning by limiting dilution using immunoreactivity with UH6 were performed and clones reaching over 50% positive cells were selected, and tested for level of secreted enzyme in supernatant of confluent cultures. Standard assays were performed in 50-μl total reaction mixtures containing 25 mm cacodylate (pH 7.5), 10 mmMnCl2, 0.1–0.25% Triton X-100, 150–200 μmUDP-[14C]GalNAc (4,000 cpm/nmol) (Amersham), and 0.06–1 mm acceptor peptides. Peptides were synthesized by ourselves, by Carlbiotech (Copenhagen), or Neosystems (Strasbourg), and quality was ascertained by amino acid analysis and mass spectrometry. Peptides Muc1a, Muc1b, and TAP24, were derived from the tandem repeat of human MUC1 (25Gendler S.J. Lancaster C.A. Taylor-Papadimitriou J. Duhig T. Peat N. Burchell J. Pemberton L. Lalani E.N. Wilson D. J. Biol. Chem. 1990; 265: 15286-15293Abstract Full Text PDF PubMed Google Scholar), and Muc2, Muc5AC, and Muc7 from the tandem repeats of MUC2, MUC5AC, and MUC7, respectively (26Gum J.R. Byrd J.C. Hicks J.W. Toribara N.W. Lamport D.T. Kim Y.S. J. Biol. Chem. 1989; 264: 6480-6487Abstract Full Text PDF PubMed Google Scholar, 27Porchet N. Pigny P. Buisine M.P. Debailleul V. Degand P. Laine A. Aubert J.P. Biochem. Soc. Trans. 1995; 23: 800-805Crossref PubMed Scopus (49) Google Scholar, 28Bobek L.A. Tsai H. Biesbrock A.R. Levine M.J. J. Biol. Chem. 1993; 268: 20563-20569Abstract Full Text PDF PubMed Google Scholar). GalNAc4TAP24 was produced by in vitroglycosylation using GalNAc-T2 as described previously (8Wandall H.H. Hassan H. Mirgorodskaya E. Kristensen A.K. Roepstorff P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. Taylor-Papadimitriou J. Clausen H. J. Biol. Chem. 1997; 272: 23503-23514Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Products were quantified by scintillation counting after chromatography on Dowex-1, octadecyl silica cartridges (Bakerbond), or HPLC (PC3.2/3 or mRPC C2/C18 SC2.1/10 Pharmacia, Smart System). Products produced byin vitro glycosylation were in most cases also confirmed by mass spectrometry. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) the mass spectra were acquired on either Voyager-DE or Voyager-Elite MALDI time of flight mass spectrometers (Perseptive Biosystem Inc.), equipped with delay extraction. The MALDI matrix was a 9:1 mixture of 2,5-dihydroxybenzoic acid (2,5-DHB) 25 g/liter and 2-hydroxy-5-methoxybenzoic acid 25 g/liter (Aldrich) dissolved in a 2:1 mixture of 0.1% trifluoroacetic acid in water and acetonitrile. Samples dissolved in 0.1% trifluoroacetic acid to a concentration of approximately 2 pmol/ml were prepared for analysis by placing 1 μl of sample solution on a probe tip followed by 1 μl of matrix. Multiple tissue Northern (MTN) blots were obtained from CLONTECH. The soluble expression construct (contained nucleotides 92–1757) was used as the GalNAc-T4 probe. The probe was random prime labeled using [α-32P]dCTP (Amersham) and Oligo labeling kit (Pharmacia). Blots were probed as described previously (4Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), and washed at 5 × at 42 °C with 2 × SSC, 0.1% SDS, once with 0.5 × SSC, 0.1% SDS, and once at 55 °C with 0.1 × SSC, 0.1% SDS, in a mini-hybridization oven (HYBAID). Previously, reverse transcriptase-PCR was performed on mRNA from a variety of human organs and cell lines, using a pair of degenerate primers (EBHC100/EBHC106) corresponding to sequences flanking a putative GalNAc-transferase motif. Two novel sequences were identified, TE3 and TE4, with approximately 80% similarity in sequence to GalNAc-T1 and -T2 (4Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). The reverse transcriptase-PCR product obtained from the human gastric carcinoma cell line MKN45 was cleaved with BstNI (known to cleave GalNAc-T1 and -T2 sequences at non-conserved restriction sites), and the remaining uncleaved product was subcloned and sequenced. Eight out of 40 independent clones contained sequences similar but not identical to GalNAc-T1 and -T2. Six of these clones were derived from GalNAc-T3 (TE3) (4Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), and two clones contained a novel sequence designated TE4. Cloning and sequencing of the complete coding sequence of GalNAc-T4 was achieved by a combination of PCR screening of 40 sublibraries from a human salivary gland λgt11 library, 5′ RACE, and genomic P1 cloning. The combined sequences contained an open reading frame of 1734 bp (GenBank accession number Y08564) (Fig. 1). The entire coding sequence was confirmed by sequencing of P1 clones in both directions. The deduced sequence of GalNAc-T4 is predicted to be a type II transmembrane protein with a hydrophobic retention signal in residues 11–31. A BLAST search of the EST data base (GenBank/NCBI) with the coding region of GalNAc-T4 only detected one human GalNAc-T4 EST, which is in contrast to several other GalNAc-transferases that are highly represented by ESTs. GalNAc-T4 is highly similar to the other GalNAc-transferases in the COOH-terminal region (amino acid residues 85–559, GalNAc-T1; 105–571, GalNAc-T2; 151–633, GalNAc-T3; and 103- 578, GalNAc-T4), and there is conservation of 12 cysteine residues (Fig. 1) (1Clausen H. Bennett E.P. Glycobiology. 1996; 6: 635-646Crossref PubMed Scopus (228) Google Scholar). The NH2-terminal regions vary considerably in length and have no significant similarity. Three P1 clones each covering the entire coding sequence of GalNAc-T4 were isolated. Sequencing of all three P1 clones showed that the entire coding region of GalNAc-T4 was contained in a single exon. Fluorescence in situ hybridization revealed that the GalNAc-T4 gene resides at human chromosome 12q21.3-q22 (Fig. 2). No specific hybridization signals were observed at other chromosomal sites. A total of 20 cells in metaphase were analyzed. Further confirmation of the chromosomal location was achieved by linkage analysis using the PCR assay for the polymorphism at position 1516 combined with chromosome 12 microsatellite markers (Table I). Analysis of 10 families yielded significant Lod score (Z > 3) between D12S80, D12S81, D12S101, and the GalNAc-T4 polymorphism. One recombination to the marker D12S81 was detected in an intercross mating (Z = 10.50 at θ (M=F) = 0.02). A marker D12S7 (12q14-q24.1) showed no recombination with GalNAc-T4, but the marker is not on the Généthon map. The suggested order according to the Généthon map was as follows: D12S90-D12S80-D12S81-(“GalNAc-T4,” D12S7)-D12S101.Table ILinkage analysis of GalNAc-T4 with chromosome 12 markersMarker(θM = θF)0.010.050.100.200.300.40D12S90−18.88−7.1−2.760.371.030.56D12S80−3.262.153.723.992.911.21D12S8110.4210.219.337.094.521.79D1" @default.
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• W2120521563 date "1998-11-01" @default.
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• W2120521563 title "Cloning of a Human UDP-N-Acetyl-α-d-Galactosamine:PolypeptideN-Acetylgalactosaminyltransferase That Complements Other GalNAc-Transferases in Complete O-Glycosylation of the MUC1 Tandem Repeat" @default.
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• W2120521563 doi "https://doi.org/10.1074/jbc.273.46.30472" @default.
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