Characterization of a novel polypeptide N-acetylgalactosaminyltransferase (dGalNAc-T3) from Drosophila.
Polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases) catalyze the initial reaction of mucin-type O-glycosylation. Here, we report the first biochemical characterization of one of the Drosophila GalNAc-transferases, dGalNAc-T3. This enzyme retains conserved motifs essential for the catalytic activity, but is a novel isozyme in that it has several inserted sequences in its lectin-like domain. Northern hybridization analysis of this isozyme identified a 2.5-kb mRNA in Drosophila larva. Biochemical characterization was carried out using the recombinant soluble dGalNAc-T3 expressed in COS7 cells. dGalNAc-T3, which required Mn2+ for the activity, had a pH optimum ranging from pH 7.5 to 8.5, and glycosylated most effectively at 29-33 degrees C. Its Km for UDP-GalNAc was 10.7 microM, which is as low as that of mammalian isozymes. dGalNAc-T3 glycosylated the peptides containing a sequence of XTPXP or TTAAP most efficiently. The enzyme was irreversibly inhibited by p-chloromercuriphenylsulphonic acid, indicating the presence of essential Cys residues for the activity.
Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Japan.
Naosuke
N
Nakamura
Kenji
K
Katano
Shinya
S
Toba
Akira
A
Kurosaka
D002731:4-Chloromercuribenzenesulfonate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004330:Drosophila; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry*; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature
D007527:Isoenzymes; D009077:Mucins; D010455:Peptides; D011994:Recombinant Proteins; D002731:4-Chloromercuribenzenesulfonate; D017350:N-Acetylgalactosaminyltransferases