title: Functional characterization and expression analysis of members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family from Drosophila melanogaster.
abstract: Here we report the cloning and functional characterization of eight members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase gene family from Drosophila melanogaster (polypeptide GalNAc transferase = pgant1-8). Full-length cDNAs were isolated from a Drosophila embryonic library based on homology to known ppGaNTases. Alignments with characterized mammalian isoforms revealed strong sequence similarities between certain fly and mammalian isoforms, highlighting putative orthologues between the species. In vitro activity assays demonstrated biochemical transferase activity for each gene, with three isoforms requiring glycosylated substrates. Comparison of the activities of Drosophila and mammalian orthologues revealed conservation of substrate preferences against a panel of peptide and glycopeptide substrates. Furthermore, Edman degradation analysis demonstrated that preferred sites of GalNac addition were also conserved between certain fly and mammalian orthologues. Semi-quantitative PCR amplification of Drosophila cDNA revealed expression of most isoforms at each developmental stage, with some isoforms being less abundant at certain stages relative to others. In situ hybridization to Drosophila embryos revealed specific staining of pgant5 and pgant6 in the salivary glands and pgant5 in the developing hindgut. Additionally, pgant5 and pgant6 expression within the egg chamber was restricted to the follicle cells, cells known to be involved in egg formation and subsequent embryonic patterning. The characterization reported here provides additional insight into the use of this model system to dissect the biological role of this enzyme family in vivo during both fly and mammalian development.
authors.affiliation: Section of Biological Chemistry, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. Kelly.Tenhagen@nih.gov
authors.forename: Kelly G
authors.initials: KG
authors.lastname: Ten Hagen
authors.forename: Duy T
authors.initials: DT
authors.lastname: Tran
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Gerken
authors.forename: David S
authors.initials: DS
authors.lastname: Stein
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004331:Drosophila melanogaster / Q000196:embryology / Q000201:enzymology* / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000201:enzymology; D015698:Genomic Library; D017403:In Situ Hybridization; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008322:Mammals; D008969:Molecular Sequence Data; D005810:Multigene Family; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics* / Q000378:metabolism*; D009865:Oocytes / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D017350:N-Acetylgalactosaminyltransferases; D011994:Recombinant Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase