Publications

• Isolation and expression of a cDNA clone encoding a bovine UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase.

  Homa F.L., Hollander T., Lehman D.J., Thomsen D.R., Elhammer A.P.

  NH2-terminal amino acid sequence obtained from a UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase (GalNAc-transferase) isolated from bovine colostrum was used for the construction of synthetic oligonucleotide primers. Subsequent polymerase chain reaction and library screenings of a bovine ... >> More

  NH2-terminal amino acid sequence obtained from a UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase (GalNAc-transferase) isolated from bovine colostrum was used for the construction of synthetic oligonucleotide primers. Subsequent polymerase chain reaction and library screenings of a bovine intestine cDNA library produced seven positive clones. The largest clone had a 2294-base pair insert that contained an open reading frame coding for a protein composed of 559 amino acids with a predicted polypeptide molecular mass of 64,173 Da. The cloned molecule has no significant sequence homology to previously reported cloned glycosyltransferases, but appears to have a similar domain structure. It is a type II membrane protein with a 23-amino acid putative transmembrane region starting 8 amino acids from the NH2 terminus. The transmembrane segment of the molecule is immediately followed by a sequence rich in proline residues. The molecule contains three consensus sequences for N-linked glycosylation and five predicted sites for O-glycosylation. Northern blot analysis of poly(A+) mRNA isolated from Madin-Darby bovine kidney cells, bovine mammary tissue, and eight human tissues demonstrated the expression of two transcripts differing in size by approximately 1 kilobase. The cloned DNA was expressed in insect cells using a baculovirus vector. This resulted in an almost 100-fold increase in GalNAc-transferase activity in lysates prepared from cells infected with virus containing the GalNAc-transferase gene compared to cells infected with virus containing DNA coding for an unrelated molecule or uninfected cells. Immunoprecipitation from lysates prepared from infected cells labeled in vivo with [35S] methionine showed a large increase in the recovery of an approximately 67-kDa protein. << Less

  J. Biol. Chem. 268:12609-12616(1993) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and cDNA cloning of a human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase.

  White T., Bennett E.P., Takio K., Soerensen T., Bonding N., Clausen H.

  A UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase (GalNAc-transferase) from human placenta was purified to apparent homogeneity using a synthetic acceptor peptide as affinity ligand. The purified GalNAc-transferase migrated as a single band with an approximate molecular weight of 52,000 ... >> More

  A UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase (GalNAc-transferase) from human placenta was purified to apparent homogeneity using a synthetic acceptor peptide as affinity ligand. The purified GalNAc-transferase migrated as a single band with an approximate molecular weight of 52,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on a partial amino acid sequence, the cDNA encoding the transferase was cloned and sequenced from a cDNA library of a human cancer cell line. The cDNA sequence has a 571-amino acid coding region indicating a protein of 64.7 kDa with a type II domain structure. The deduced protein sequence showed significant similarity to a recently cloned bovine polypeptide GalNAc-transferase (Homa, F.L., Hollanders, T., Lehman, D.J., Thomsen, D.R., and Elhammer, A.P. (1993) J. Biol. Chem. 268, 12609-12616). A polymerase chain reaction construct was expressed in insect cells using a baculovirus vector. Northern analysis of eight human tissues differed clearly from that of the bovine GalNAc-transferase. Polymerase chain reaction cloning and sequencing of the human version of the bovine transferase are presented, and 98% similarity at the amino acid level was found. The data suggest that the purified human GalNAc-transferase is a novel member of a family of polypeptide GalNAc-transferases, and a nomenclature GalNAc-T1 and GalNAc-T2 is introduced to distinguish the members. << Less

  J. Biol. Chem. 270:24156-24165(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• cDNA cloning and expression of a novel UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase.

  Hagen F.K., Ten Hagen K.G., Beres T.M., Balys M.M., VanWuyckhuyse B.C., Tabak L.A.

  The cDNA for a fourth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, termed ppGaNTase-T4, has been cloned from a murine spleen cDNA library and expressed transiently in COS7 cells as a secreted functional enzyme. Degenerate primers, based upon regions that ... >> More

  The cDNA for a fourth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, termed ppGaNTase-T4, has been cloned from a murine spleen cDNA library and expressed transiently in COS7 cells as a secreted functional enzyme. Degenerate primers, based upon regions that are conserved among the known mammalian members of the enzyme family (ppGaNTase-T1, -T2, and -T3) and three Caenorhabditis elegans homologues (ppGaNTase-TA, -TB, and -TC), were used in polymerase chain reactions to identify and clone this new isoform. Substrate preferences for recombinant murine ppGaNTase-T1 and ppGaNTase-T4 isozymes were readily distinguished. ppGaNTase-T1 glycosylated a broader range of synthetic peptide substrates; in contrast, the ppGaNTase-T4 preferentially glycosylated a single substrate among the panel of 11 peptides tested. Using Northern blot analysis, a ppGaNTase-T4 message of 5.5 kilobases was detectable in murine embryonic tissues, as well as the adult sublingual gland, stomach, colon, small intestine, lung, cervix, and uterus with lower levels detected in kidney, liver, heart, brain, spleen, and ovary. Thus, the pattern of expression for ppGaNTase-T4 is more restricted than for the three previously reported isoforms of the enzyme. The variation in expression patterns and substrate specificities of the ppGaNTase enzyme family suggests that differential expression of these isoenzymes may be responsible for the cell-specific repertoire of mucin-type oligosaccharides on cell-surface and secreted O-linked glycoproteins. << Less

  J. Biol. Chem. 272:13843-13848(1997) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Dynamic association between the catalytic and lectin domains of human UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase-2.

  Fritz T.A., Raman J., Tabak L.A.

  The family of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAcTs) is unique among glycosyltransferases, containing both catalytic and lectin domains that we have previously shown to be closely associated. Here we describe the x-ray crystal structures of human ppGalNAcT-2 ( ... >> More

  The family of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAcTs) is unique among glycosyltransferases, containing both catalytic and lectin domains that we have previously shown to be closely associated. Here we describe the x-ray crystal structures of human ppGalNAcT-2 (hT2) bound to the product UDP at 2.75 A resolution and to UDP and an acceptor peptide substrate EA2 (PTTDSTTPAPTTK) at 1.64 A resolution. The conformations of both UDP and residues Arg362-Ser372 vary greatly between the two structures. In the hT2-UDP-EA2 complex, residues Arg362-Ser373 comprise a loop that forms a lid over UDP, sealing it in the active site, whereas in the hT2-UDP complex this loop is folded back, exposing UDP to bulk solvent. EA2 binds in a shallow groove with threonine 7 positioned consistent with in vitro data showing it to be the preferred site of glycosylation. The relative orientations of the hT2 catalytic and lectin domains differ dramatically from that of murine ppGalNAcT-1 and also vary considerably between the two hT2 complexes. Indeed, in the hT2-UDP-EA2 complex essentially no contact is made between the catalytic and lectin domains except for the peptide bridge between them. Thus, the hT2 structures reveal an unexpected flexibility between the catalytic and lectin domains and suggest a new mechanism used by hT2 to capture glycosylated substrates. Kinetic analysis of hT2 lacking the lectin domain confirmed the importance of this domain in acting on glycopeptide but not peptide substrates. The structure of the hT2-UDP-EA2 complex also resolves long standing questions regarding ppGalNAcT acceptor substrate specificity. << Less

  J. Biol. Chem. 281:8613-8619(2006) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and characterization of UDP-GalNAc:polypeptide N-acetylgalactosamine transferase from an ascites hepatoma, AH 66.

  Sugiura M., Kawasaki T., Yamashina I.

  The membrane-bound UDP-GalNAc:polypeptide N-acetylgalactosamine transferase from an ascites hepatoma, AH 66, has been purified 48,100-fold, mainly by affinity chromatography in aqueous Triton X-100 on apomucin (deglycosylated bovine submaxillary mucin) coupled to Sepharose. The purified preparatio ... >> More

  The membrane-bound UDP-GalNAc:polypeptide N-acetylgalactosamine transferase from an ascites hepatoma, AH 66, has been purified 48,100-fold, mainly by affinity chromatography in aqueous Triton X-100 on apomucin (deglycosylated bovine submaxillary mucin) coupled to Sepharose. The purified preparation behaved homogeneously on gel filtration on Sephadex G-150 in aqueous Triton X-100 and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of about 55,000. The enzyme requires Mn2+, and only UDP-GalNAc served as a sugar donor. Apomucin, A1 protein, kappa-casein, apofetuin, and apoantifreeze glycoproteins served as acceptors, but the rate and amount of the transfer varied considerably from one acceptor to another. The transfer reaction terminated at the level of glycosylation of from only a few to at most about 40% of the serine plus threonine residues from which mucin-type oligosaccharides had been removed. This indicates that the transferase requires a certain conformation surrounding the acceptor site, but suggests also that a special mechanism may be functioning in vivo for frequent glycosylation of the abundant serine plus threonine residues of mucins. Lacto-N-fucopentaose I, ceramide di- and trihexosides, and globoside were not acceptors. << Less

  J Biol Chem 257:9501-9507(1982) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.