title: Cloning, expression and properties of porcine trachea UDP-galnac: polypeptide N-acetylgalactosaminyl transferase.
abstract: A UDP-GalNAc:polypeptide N-acetyl-galactosaminyl transferase which catalyses the transfer of GalNAc from UDP-GalNAc to serine and threonine residues in mucin polypeptide chains was purified to homogeneity from swine trachea epithelium (Mendicino J, Sangadala S: Mol Cell Biochem 185: 135-145, 1998). Peptides obtained by proteolysis of the purified enzyme were isolated, sequenced and used to prepare degenerate oligonucleotide primers. Amplified segments of a gene encoding GalNAc transferase were synthesised using the primers and a swine trachea epithelial cDNA library. Selected cDNA fragments were then used to screen the cDNA library, and a clone containing an open reading frame encoding 559 amino acids was isolated. The predicted amino acid sequence contains type II transmembrane region, three potential N-glycosylation sites as well as all of the isolated peptide sequences. The nucleotide sequence and predicted primary protein structure of the transferase were very similar to those of type T-1 GalNAc transferases. The isolated clone was transiently expressed in COS 7 cells and the recombinant enzyme, which contained an N-terminal hexa-histidine tag, was purified to homogeneity and its enzymatic properties were examined. The Vmax of the recombinant enzyme, 2.08 micromol/(min mg), was nearly the same as the native enzyme, 2.12 micromol/(min mg), when assayed with partially deglycosylated mucins as glycosyl acceptors. Both enzymes showed much higher activities when assayed with peptides prepared by limited acid hydrolysis of incompletely deglycosylated Cowper's gland, swine, and human respiratory mucins and tryptic peptides isolated from deglycosylated mucin polypeptide chains. However, as noted earlier (Mendicino J, Sangadala S: Mol Cell Biochem 185: 135-145, 1998), these enzymes showed very little activity with completely deglycosylated mucin polypeptide chains. When completely deglycosylated polypeptide chains were partially glycosylated by incubation with microsome preparations they were again good glycosyl acceptors for the T1-GalNAc transferases isolated from swine trachea. These results show for the first time that multiple isoforms of GalNAc transferases acting in sequence may be required for the complete O-glycosylation of mucin polypeptide chains, and those acting on the nacent polypeptide chain synthesize intermediates which can serve as glycosyl acceptors for other isoforms of the enzyme.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
authors.forename: Sreedhara
authors.initials: S
authors.lastname: Sangadala
authors.forename: Ja Baris
authors.initials: JB
authors.lastname: Swain
authors.forename: Adrian
authors.initials: A
authors.lastname: McNear
authors.forename: Joseph
authors.initials: J
authors.lastname: Mendicino
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D015870:Gene Expression; D015723:Gene Library; D006031:Glycosylation; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry*; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013552:Swine; D014132:Trachea / Q000201:enzymology*; D014530:Uridine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D017931:DNA Primers; D007527:Isoenzymes; D009077:Mucins; D017350:N-Acetylgalactosaminyltransferases; D014530:Uridine Diphosphate; D000097763:Polypeptide N-acetylgalactosaminyltransferase