Abstract
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.
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