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• W2087339901 abstract "The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T3, exhibits unique functions. Specific acceptor substrates are used by GalNAc-T3 and not by other GalNAc-transferases. The expression pattern of GalNAc-T3 is restricted, and loss of expression is a characteristic feature of poorly differentiated pancreatic tumors. In the present study, a sixth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T6, with high similarity to GalNAc-T3, was characterized. GalNAc-T6 exhibited high sequence similarity to GalNAc-T3 throughout the coding region, in contrast to the limited similarity that exists between homologous glycosyltransferase genes, which is usually restricted to the putative catalytic domain. The genomic organizations of GALNT3 and GALNT6 are identical with the coding regions placed in 10 exons, but the genes are localized differently at 2q31 and 12q13, respectively. Acceptor substrate specificities of GalNAc-T3 and -T6 were similar and different from other GalNAc-transferases. Northern analysis revealed distinct expression patterns, which were confirmed by immunocytology using monoclonal antibodies. In contrast to GalNAc-T3, GalNAc-T6 was expressed in WI38 fibroblast cells, indicating that GalNAc-T6 represents a candidate for synthesis of oncofetal fibronectin. The results demonstrate the existence of genetic redundancy of a polypeptide GalNAc-transferase that does not provide full functional redundancy. The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T3, exhibits unique functions. Specific acceptor substrates are used by GalNAc-T3 and not by other GalNAc-transferases. The expression pattern of GalNAc-T3 is restricted, and loss of expression is a characteristic feature of poorly differentiated pancreatic tumors. In the present study, a sixth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T6, with high similarity to GalNAc-T3, was characterized. GalNAc-T6 exhibited high sequence similarity to GalNAc-T3 throughout the coding region, in contrast to the limited similarity that exists between homologous glycosyltransferase genes, which is usually restricted to the putative catalytic domain. The genomic organizations of GALNT3 and GALNT6 are identical with the coding regions placed in 10 exons, but the genes are localized differently at 2q31 and 12q13, respectively. Acceptor substrate specificities of GalNAc-T3 and -T6 were similar and different from other GalNAc-transferases. Northern analysis revealed distinct expression patterns, which were confirmed by immunocytology using monoclonal antibodies. In contrast to GalNAc-T3, GalNAc-T6 was expressed in WI38 fibroblast cells, indicating that GalNAc-T6 represents a candidate for synthesis of oncofetal fibronectin. The results demonstrate the existence of genetic redundancy of a polypeptide GalNAc-transferase that does not provide full functional redundancy. UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase -T2, -T3, and -T4 represents human GalNAc-transferases cloned and expressed by Meurer et al. (4Meurer J.A. Naylor J.M. Baker C.A. Thomsen D.R. Homa F.L. Elhammer A.P. J. Biochem. (Tokyo). 1995; 118: 568-574Crossref PubMed Scopus (20) Google Scholar), White et al. (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 (184) Google Scholar), Bennett et al. (5Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), and Bennett et al. (6Bennett E.P. Hassan H. Mandel U. Mirgorodskaya E. Roepstorff P. Burchell J. Taylor-Papadamitriou J. Hollingsworth M.A. Merkx G. Geurts van Kessel A. Eiberg H. Steffensen R. Clausen H. J. Biol. Chem. 1998; 273: 30472-30481Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) (GenBank™ accession numbers are U41514, X85018, X85019, X92689, and Y08564) EST, expressed sequence tag human immunodeficiency virus base pairs kilobase pairs polymerase chain reaction fluorescein isothiocyanate matrix-assisted laser desorption/ionization mass spectrometry time of flight monoclonal antibody The initiation of mucin-type O-linked protein glycosylation is controlled by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases)1 (EC2.4.1.41) (1Clausen H. Bennett E.P. Glycobiology. 1996; 6: 635-646Crossref PubMed Scopus (226) Google Scholar). To date five distinct members of the animal GalNAc-transferase family have been reported (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 (184) Google Scholar, 4Meurer J.A. Naylor J.M. Baker C.A. Thomsen D.R. Homa F.L. Elhammer A.P. J. Biochem. (Tokyo). 1995; 118: 568-574Crossref PubMed Scopus (20) Google Scholar, 5Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 6Bennett E.P. Hassan H. Mandel U. Mirgorodskaya E. Roepstorff P. Burchell J. Taylor-Papadamitriou J. Hollingsworth M.A. Merkx G. Geurts van Kessel A. Eiberg H. Steffensen R. Clausen H. J. Biol. Chem. 1998; 273: 30472-30481Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 7Ten Hagen K.G. Hagen F.K. Balys M.M. Beres T.M. Van Wuyckhuyse B. Tabak L.A. J. Biol. Chem. 1998; 273: 27749-27754Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The GalNAc-transferase family appears to be highly conserved as nine distinct genes have been identified in Caenorhabditis elegans (8Hagen F.K. Nehrke K. J. Biol. Chem. 1998; 273: 8268-8277Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Four human GalNAc-transferases have been characterized, and several characteristics of the human family of GalNAc-transferases are now apparent. (i) There is overall sequence similarities of approximately 40–45%, with regions of high similarity (80%) in GalNAc-transferase motifs but little similarity among the N-terminal regions encoding the cytoplasmic tail, the putative signal anchor sequence, and the stem regions (1Clausen H. Bennett E.P. Glycobiology. 1996; 6: 635-646Crossref PubMed Scopus (226) Google Scholar); (ii) the chromosomal localizations and genomic organizations are different (1Clausen H. Bennett E.P. Glycobiology. 1996; 6: 635-646Crossref PubMed Scopus (226) Google Scholar,9Bennett 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); (iii) the substrate specificities as determined by in vitro assays are different, but there is overlap among some substrates, especially those derived from mucin tandem repeats (5Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 6Bennett E.P. Hassan H. Mandel U. Mirgorodskaya E. Roepstorff P. Burchell J. Taylor-Papadamitriou J. Hollingsworth M.A. Merkx G. Geurts van Kessel A. Eiberg H. Steffensen R. Clausen H. J. Biol. Chem. 1998; 273: 30472-30481Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar,10Wandall H.H. Hassan H. Mirgorodskaya E. Kristensen A.K. Roepstorff P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. Taylor-Papadamitriou J. Clausen H. J. Biol. Chem. 1997; 272: 23503-23514Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar); and (iv) the patterns of expression in human cells and organs are different (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 (184) Google Scholar, 5Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 6Bennett E.P. Hassan H. Mandel U. Mirgorodskaya E. Roepstorff P. Burchell J. Taylor-Papadamitriou J. Hollingsworth M.A. Merkx G. Geurts van Kessel A. Eiberg H. Steffensen R. Clausen H. J. Biol. Chem. 1998; 273: 30472-30481Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Furthermore, the catalytic action of the different GalNAc-transferases can be cooperative, since glycosylation of certain acceptor sites in the MUC1 tandem repeat by one GalNAc-transferase is required before other sites can be glycosylated by another GalNActransferase (6Bennett E.P. Hassan H. Mandel U. Mirgorodskaya E. Roepstorff P. Burchell J. Taylor-Papadamitriou J. Hollingsworth M.A. Merkx G. Geurts van Kessel A. Eiberg H. Steffensen R. Clausen H. J. Biol. Chem. 1998; 273: 30472-30481Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). These data suggest that each GalNAc-transferase has distinct biological functions that are mainly determined by the kinetic properties and expression patterns of the enzymes. Many properties of the enzyme function are still not fully understood: the importance of sub-Golgi localization (11Rottger S. White R. Wandall H.H. Bennett E.P. Stark A. Olivo J.-C. Whitehouse C. Berger E.G. Clausen H. Nilsson T. J. Cell Sci. 1998; 111: 45-60Crossref PubMed Google Scholar), the importance of large variation in the length of stem regions (1Clausen H. Bennett E.P. Glycobiology. 1996; 6: 635-646Crossref PubMed Scopus (226) Google Scholar, 7Ten Hagen K.G. Hagen F.K. Balys M.M. Beres T.M. Van Wuyckhuyse B. Tabak L.A. J. Biol. Chem. 1998; 273: 27749-27754Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), and the significance of a putative C-terminal lectin-like domain of approximately 100 amino acids, which does not appear to be essential for catalytic activity (12Imberty A. Piller V. Piller F. Breton C. Protein Eng. 1997; 10: 1353-1356Crossref PubMed Scopus (45) Google Scholar, 13Hazes B. Protein Sci. 1996; 5: 1490-1501Crossref PubMed Scopus (177) Google Scholar). GalNAc-T3 exhibits acceptor substrate specificities not seen with other enzymes, including glycosylation of a single in vivo defined O-glycosylation site in fibronectin, which forms the oncofetal fibronectin isoform (14Matsuura H. Takio K. Titani K. Greene T. Levery S.B. Salyan M.E. Hakomori S. J. Biol. Chem. 1988; 263: 3314-3322Abstract Full Text PDF PubMed Google Scholar) and a single site in the V3 loop of HIV gp120 (5Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). The unique specificity of GalNAc-T3 for the oncofetal fibronectin peptide was reproduced with plasma fibronectin, clearly indicating the importance of the primary sequence context as a major factor in determining O-glycosylation (10Wandall H.H. Hassan H. Mirgorodskaya E. Kristensen A.K. Roepstorff P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. Taylor-Papadamitriou J. Clausen H. J. Biol. Chem. 1997; 272: 23503-23514Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Furthermore, the specificity of GalNAc-T3 for the HIV sequence has been confirmed in vivo. A reporter construct containing the acceptor sequence was O-glycosylated only if GalNAc-T3 was co-expressed in the host cell (15Nehrke K. Hagen T.K.G. Hagen F.K. Tabak L.A. Glycobiology. 1997; 7: 1053-1060Crossref PubMed Scopus (25) Google Scholar). This demonstrated that specificity in vitro reflects in vivo specificity. Recent studies with a panel of monoclonal antibodies to human GalNAc-transferases demonstrated that GalNAc-T3 is not expressed in connective tissue cells in normal or tumor tissues or in a fibroblast cell line synthesizing oncofetal fibronectin (16Mandel U. Hassan H. Therkildsen M.H. Rygaard J. Jacobsen M. Juhl B.R. Dabelsteen E. Clausen H. Glycobiology. 1999; 9: 43-52Crossref PubMed Scopus (105) Google Scholar). It is therefore unlikely that GalNAc-T3 represents the native fibronectin GalNAc-transferase activity found in tumor tissues and fibroblast cell lines, as originally described by Matsuura et al. (17Matsuura H. Greene T. Hakomori S. J. Biol. Chem. 1989; 264: 10472-10476Abstract Full Text PDF PubMed Google Scholar). This suggests the existence of an additional GalNAc-transferase with similar properties as GalNAc-T3 but with a different expression pattern. Here, we report the cloning and expression of such a novel human GalNAc-transferase, which appears to represent a high similarity duplicate gene of GalNAc-T3. The novel GalNAc-transferase, designated GalNAc-T6, displayed an acceptor substrate specificity similar to GalNAc-T3, although GalNAc-T6 showed better kinetic properties with the fibronectin substrate. The two GalNAc-transferases exhibit different expression patterns as analyzed by Northern analysis and immunocytology. GalNAc-T6 was expressed in a fibroblast cell line synthesizing oncofetal fibronectin. The existence of high similarity pairs of genes within the GalNAc-transferase gene family is significant to the biological function of this large gene family and of practical significance for studies of transgenic knock-out models. The dbEST data base at The National Center for Biotechnology Information (NCBI), was searched for sequences similar to the coding region of the human GalNAc-T3 gene (5Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar) using the tBLASTn and BLASTn algorithms. The 5′ region of GalNAc-T3, with no known similarity to other human GalNAc-transferases (bp 1–600), was used to identify a rat EST (GenBank™ accession number H32001) predicted to encode a protein sequence with 63% similarity to residues 99–181 of GalNAc-T3. The putative human counterpart of this rat sequence was isolated by PCR with a sense primer, EBHC500 (5′-AGCGGATCCACTCCTGCCTTCCGGGGTTC-3′), derived from the rat EST sequence, and an antisense primer, EBHC106N (5′-AGCGGATCCGTATTCGTCCATCCAIACITCTG-3′), derived from the conserved GalNAc-transferase motif (5Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar) (Fig. 1, panel A). Four cDNA libraries from MKN45 (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 (184) Google Scholar), Colo205 (Stratagene), salivary glands (5Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), and spleen were screened by PCR with 0.5 μmprimers and library lysates containing 1 × 106plaque-forming units. A spleen λ ZAP (Stratagene) random-primed cDNA library was prepared from 2.0 μg of human spleen mRNA (CLONTECH) using a time saver cDNA kit (Amersham Pharmacia Biotech) and constructed as recommended by the manufacturer. PCR was performed by using 35 cycles of 95 °C for 20 s, 55 °C for 5 s, 72 °C for 2 min under standard conditions. Two cDNA libraries derived from salivary glands and spleen yielded a single PCR product, whereas two libraries derived from the cell lines Colo205 and MKN45 gave no product. The PCR products from the salivary gland and spleen libraries were cloned and sequenced, and the sequences were found to be identical. The identified novel human sequence (clone #1) covering the central part of a putative GalNAc-transferase gene was further studied by cloning 5′ and 3′ sequences using rapid cDNA library screening (5Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar) (Fig. 1, panel B). Briefly, the spleen library was aliquoted into 20 sublibraries, and these were screened by PCR using primer pairs based on the identified novel sequence and the λZAP vector primers T3 and T7, and the products were confirmed by hybridization with either EBHC500 or EBHC505 (5′-AGCGGATCCACTCTGCCCCTCTGGACGGGC-3′). EBHC503 (5′-AGCGGATCCGACAAGACAGTGGTGGTGAGC-3′) was used for the 3′ PCR, and EBHC504 (5′-AGCGGATCCGGGTCTCCAGGGGGGTCCAC-3′) was used for the 5′ PCR (Fig. 1, panel B). PCR with EBHC503/T7 yielded a single product of 2 kbp for the 3′ sequence, and PCR with EBHC504/T3 gave a single product of 250 bp. Both products were blunt end-cloned and sequenced. The 3′ sequence contained an in-frame stop codon. The 5′ sequence had a potential open reading frame but was shorter than the coding region of GalNAc-T3 and had no translation initiation codon and hydrophobic sequences, suggesting the existence of further 5′ sequence. Attempts to obtain additional 5′ sequence information by use of various 5′ rapid amplification of cDNA ends (RACE) techniques failed. A P1 human foreskin genomic library (DuPont Merck Pharmaceutical Co. Human Foreskin Fibroblast P1 Library) was screened using primer pairs EBHC500/EBHC504. Three clones were obtained from Genome Systems DMPC-HFF#1-235-B10 (P1-12423), DMPC-HFF#1-826-D3 (P1-12424), DMPC-HFF#1-994-B6 (P1-12425). DNA from P1 phage was prepared as recommended by Genome Systems Inc., and P1-12423 was selected for partial sequence analysis. The entire sequence of the open reading frame compiled from the PCR cDNA cloning strategy was confirmed with minor sequence corrections. The most 5′-coding sequence of the putative GalNAc-transferase gene was obtained by 5′ sequencing of the P1 DNA (Fig. 1, panel A). This sequence included a translation initiation codon, a putative cytoplasmic tail, and a putative hydrophobic transmembrane-spanning domain. The intron/exon organization of the gene was determined by comparing cDNA and P1 sequences. Fluorescence in situ hybridization (FISH) was performed on normal human lymphocyte metaphase chromosomes using procedures described previously (6Bennett E.P. Hassan H. Mandel U. Mirgorodskaya E. Roepstorff P. Burchell J. Taylor-Papadamitriou J. Hollingsworth M.A. Merkx G. Geurts van Kessel A. Eiberg H. Steffensen R. Clausen H. J. Biol. Chem. 1998; 273: 30472-30481Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 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. Genet. 1991; 48: 269-273PubMed Google Scholar). Briefly, P1 DNA (P1-12423) was labeled with biotin-14-dATP, precipitated with human Cot 1 DNA, dissolved in hybridization solution (2× SSC (1× SSC = 0.15 m NaCl and 0.015m sodium citrate), 10% dextran sulfate, 1% Tween 20, and 50% formamide, pH 7.0), and heat-denatured. Slides were incubated with the probe for 45 h, followed by immunochemical detection using avidin fluorescein isothiocyanate (FITC) and successive steps with rabbit-anti-FITC and mouse-rabbit FITC-conjugated antibodies. Slides were evaluated in a Zeiss epifluorescence microscope, and hybridization signals and chromosomes counter-stained with 4′,6-diamidino-2-phenylindole·2HCl (DAPI) were analyzed using the BDS-image™ software package (Oncor). An expression construct of a secreted form of the putative GalNAc-transferase gene, pAcGP67-GalNAc-T6-sol, was prepared by reverse transcription-PCR using primers EBHC514 (5′-AGCGGATCCTGGACCTCATGCTGGAGGCCATG-3′) and EBHC511 (5′-AGCGGATCCTGGGGATGATCTGGGTCCTAGAC-3′) with Bam HI overhangs (Fig. 1, panel A), and the product was cloned into the vector pAcGP67 (Pharmingen) and fully sequenced. Salivary gland mRNA (CLONTECH) was used as template for reverse transcription-PCR. Control constructs included pAcGP67-GalNAc-T1-sol, pAcGP67-GalNAc-T2-sol, pAcGP67-GalNAc-T3-sol, pAcGP67-GalNAc-T4-sol, and pAcGP67-O2-sol, which 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 (184) Google Scholar, 5Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 6Bennett E.P. Hassan H. Mandel U. Mirgorodskaya E. Roepstorff P. Burchell J. Taylor-Papadamitriou J. Hollingsworth M.A. Merkx G. Geurts van Kessel A. Eiberg H. Steffensen R. Clausen H. J. Biol. Chem. 1998; 273: 30472-30481Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Co-transfection of Sf9 cells with pAcGP67 constructs and BaculoGoldTMDNA was performed according to the manufacturer's specifications. Briefly, 0.4 μg of construct was mixed with 0.1 μg of BaculoGoldTM DNA and co-transfected in Sf9 cells in 24-well plates. Ninety-six h post-transfection recombinant virus was amplified in 6-well plates at dilutions of 1:10 and 1:50. The titer of amplified virus was estimated by titration in 24-well plates with monitoring of GalNAc-transferase activities (5Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Standard assays were performed in 50 μl of total reaction mixtures containing 25 mm Tris (pH 7.4), 10 mm MnCl2, 0.25% Triton X-100, 50 μm UDP[14C]GalNAc (2,000 cpm/nmol) (Amersham Pharmacia Biotech), 200–500 μm acceptor peptide (see Tables I and II for structures), and 5–10 μl of culture supernatants. In some experiments, as indicated, purified recombinant GalNAc-transferase preparations were used. GalNAc-T6 was purified as described previously (10Wandall H.H. Hassan H. Mirgorodskaya E. Kristensen A.K. Roepstorff P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. Taylor-Papadamitriou J. Clausen H. J. Biol. Chem. 1997; 272: 23503-23514Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) using sequential ion-exchange chromatographies on Amberlite (IRA95, Sigma) and DEAE-Sephacel, S-Sepharose fast flow, and Mono-S (PC1.6/5, Smart-System) (Amersham Pharmacia Biotech) columns. Final purification to apparent homogeneity was performed on S12 gel filtration (Superose 12 pc3.2/30, Smart-System, Amersham Pharmacia Biotech). Purity and protein concentration of the final fractions were assessed by S12 gel filtration and SDS-polyacrylamide gel electrophoresis using bovine serum albumin as a standard. The specific activity of the purified GalNAc-T6 was estimated to be 2.35 units/mg using 250 μmMuc1a (Table I) as the acceptor peptide. Previously, soluble forms of human GalNAc-T1, -T2, and -T3 were expressed in Sf9 cells and purified to near homogeneity with specific activities of 0.6 unit/mg for GalNAc-T1, 0.5 unit/mg for GalNAc-T2, 0.5 unit/mg for GalNAc-T3, and 0.05 unit/mg for GalNAc-T4, measured using peptides derived from MUC2, MUC1, and MUC7 tandem repeats (6Bennett E.P. Hassan H. Mandel U. Mirgorodskaya E. Roepstorff P. Burchell J. Taylor-Papadamitriou J. Hollingsworth M.A. Merkx G. Geurts van Kessel A. Eiberg H. Steffensen R. Clausen H. J. Biol. Chem. 1998; 273: 30472-30481Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 10Wandall H.H. Hassan H. Mirgorodskaya E. Kristensen A.K. Roepstorff P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. Taylor-Papadamitriou J. Clausen H. J. Biol. Chem. 1997; 272: 23503-23514Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar).Table IExpression of human recombinant GalNAc-transferases in Sf9 cellsPeptideSequenceGalNAc-T11-aThe assay was performed using culture supernatants of Sf9 cells expressing soluble recombinant human GalNAc-transferases as described under “Experimental Procedures.”GalNAc-T2GalNAc-T3GalNAc-T4GalNAc-T6milliunits/ml1-bOne unit is defined as the amount of enzyme that transfers 1 μmol of GalNAc in 1 min using the standard reaction mixture.Muc1aAHGVTSAPDTR2.71 ± 0.281-cBackground values obtained with irrelevant expression constructs were subtracted. Assays with Muc2 and EA2 substrates yielded background levels.1.47 ± 0.197.14 ± 0.120 ± 04.69 ± 0.16Muc1bRPAPGSTAPPA0 ± 06.36 ± 0.020.17 ± 0.060 ± 00.15 ± 0.02Muc2PTTTPISTTTMVTPTPTPTC3.84 ± 0.065.62 ± 0.414.94 ± 0.270.79 ± 0.093.56 ± 0.43Muc7Ac-CPPTPSATTPAPPSSSAPPETTAA9.30 ± 0.1414.34 ± 0.151.42 ± 0.151.21 ± 0.120.69 ± 0.16OSM fragmentLSESTTQLPGGGPGCA2.21 ± 0.380 ± 00.29 ± 0.060 ± 00.50 ± 0.16EA2PTTDSTTPAPTTK11.4 ± 0.2521 ± 0.344.12 ± 0.280.92 ± 0.031.59 ± 0.34hCG-βPRFQDSSSSKAPPPSLPSPSRLPG0 ± 01.34 ± 0.030 ± 00 ± 00 ± 0HIVHIBgp120Ac-CIRIQRGPGRAFVTIGKIGNMR0 ± 00 ± 02.42 ± 0.010 ± 00.88 ± 0.01FibronectinAc-PFVTHPGYD0 ± 00 ± 00.82 ± 0.060 ± 01.59 ± 0.011-a The assay was performed using culture supernatants of Sf9 cells expressing soluble recombinant human GalNAc-transferases as described under “Experimental Procedures.”1-b One unit is defined as the amount of enzyme that transfers 1 μmol of GalNAc in 1 min using the standard reaction mixture.1-c Background values obtained with irrelevant expression constructs were subtracted. Assays with Muc2 and EA2 substrates yielded background levels. Open table in a new tab Table IIKinetic constants of purified recombinant GalNAc-transferasesPeptideSequenceGalNAc-T1GalNAc-T2GalNAc-T3GalNAc-T6milliunits/mlKm, app.Vmaxmilliunits/mlKm, app.Vmaxmilliunits/mlKm, app.Vmaxmilliunits/mlKm, app.Vmax(1 mm)2-aAcceptor substrate concentration used in standard reaction mixture.mmnmol/min/μg(1 mm)mmnmol/min/μg(1 mm)mmnmol/min/μg(1 mm)mmnmol/min/μgHIVHIBgp120Ac-CIRIQRGPGRAFVTIGKIGNMRNA2-bNA, not applicable.NANANANANA938 ± 590.611.43421 ± 40.580.66FibronectinAc-PFVTHPGYDNANANANANANA580 ± 103.232.37970 ± 202.463.64Prion-aKQHTVTTTTKGEN6 ± 0.4ND2-cND, not determined.NDNANANA337 ± 20.580.53230 ± 20.630.40Prion-bKGENFTETDIKINANANANANANANANANANANANACD59NFNDVTTRLRENEL4.5 ± 0.2NDNDNANANA13.6 ± 0.1NDND132 ± 4NDNDZonadhesinPTERTTTPTKRTTTPTIR52 ± 10.140.0537 ± 70.120.05NM2-dNM, not measured due to substrate inhibition at concentrations greater than 0.5 mm.0.24NM362 ± 40.800.302-a Acceptor substrate concentration used in standard reaction mixture.2-b NA, not applicable.2-c ND, not determined.2-d NM, not measured due to substrate inhibition at concentrations greater than 0.5 mm. Open table in a new tab Peptides were synthesized by ourselves, by Carlbiotech (Copenhagen), or Neosystems (Strasbourg), and quality was ascertained by amino acid analysis and mass spectrometry. For analysis of the donor substrate specificity, assays were performed with 100 μmUDP[14C]Gal or UDP[14C]GlcNAc (4,000 cpm/nmol). Products were routinely quantified by scintillation counting after Dowex-1 formic acid cycle chromatography. At least once for all combinations of enzyme sources and peptides, the products were evaluated by C-18 reverse phase chromatography (PC3.2/3 or mRPC C2/C18 SC2.1/10 Amersham Pharmacia Biotech, Smart System) with scintillation counting of peptide peak fractions. Peptides and products produced by in vitro glycosylation were confirmed by mass spectrometry, and reaction kinetics were monitored by capillary electrophoresis. Reaction mixtures for preparative glycosylation included 2 mm cold UDP-GalNAc and 25 μg of acceptor peptide in a total volume of 100 μl. Reactions were incubated in the sample carousel of an Applied Biosystems model HT270 (Perkin-Elmer) at 30 °C, and injections were performed at 60-min intervals. Capillary zone electrophoresis was performed on coated fused silica capillaries, 72 cm × 50 μm, with 49 cm between sample injection and optical cell. Electrophoresis were performed at 30 °C using 50 mm phosphate buffer (pH 2.5). Voltage across the capillary was 20 kV in positive mode with the anode at the injection side, and the runs were monitored at 210 nm. At the beginning of each cycle the capillary was flushed with 0.1 m NaOH for 2 min followed by flushing with 50 mm phosphate buffer (pH 2.5) for 4 min. After 8 h of reaction the glycopeptides were purified by C-18 high performance liquid chromatography and analyzed by matrix-assisted laser desorption/ionization mass spectrometry time of flight (MALDI-TOF). Spectra were acquired on either Voyager-DE mass spectrometer (Perseptive Biosystem Inc.) equipped with delay extraction. The matrix used was 2,5-dihydroxybenzoic acid (25 mg/ml) 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 80 fmol–2 pmol/μl 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. Cell line blots were prepared with total RNA isolated from human colon and pancreatic adenocarcinoma cell lines SUIT2 and sublines (S2-007, S2-013, S2-020, S2-028, S2-CP9, and S2-VP10), PANC-1, HPAF, Capan-2, BxPC3, ASPC1, COLO 357, SW979, MiaPaca, HCG25, HT-29(+Gal), HT-29(+Glu), and the fibroblast line WI38, essentially as described (19Sutherlin M.E. Nishimori I. Caffrey T. Bennett E.P. Hassan H. Mandel U. Mack D. Iwamura T. Clausen H. Hollingsworth M.A. Cancer Res. 1997; 57: 4744-4748PubMed Google Scholar). Twenty-five μg of total RNA was subjected to electrophoresis on a 1% denaturing agarose gel and transferred to nitrocellulose. The soluble expression construct (containing bp 158 to 1869) was used as the GalNAc-T6 probe. The probe was random prime-labeled using [α-P32]dCTP (Amersham Pharmacia Biotech) and oligo labeling kit (Amersham Pharmacia Biotech). Blots were probed as described previously (5Bennett E.P. Hassan H. Clausen H. J. Biol. Chem. 1996; 271: 17006-17012Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar) and washed 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 m" @default.
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• W2087339901 date "1999-09-01" @default.
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• W2087339901 title "Cloning and Characterization of a Close Homologue of Human UDP-N-acetyl-α-d-galactosamine:Polypeptide N-Acetylgalactosaminyltransferase-T3, Designated GalNAc-T6" @default.
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