A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase is essential for viability in Drosophila melanogaster

Abstract

We report the first demonstration that the activity of a member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase gene family is necessary for viability in Drosophila melanogaster. Expression of the wild-type recombinant pgant35A gene in COS7 cells resulted in in vitro activity against peptide and glycopeptide substrates, demonstrating that this gene encodes a biochemically active transferase. Previous mutagenesis studies identified recessive lethal mutations that were rescued by a genomic fragment containing the pgant35A gene; however, the presence of additional open reading frames within this fragment left open the possibility that another gene was responsible for rescue of the observed lethality. Here, we have determined the molecular nature of the mutations in three independent mutant alleles. Two of the mutant alleles contain premature stop codons within the coding region of pgant35A. The third mutant contains an arginine to tryptophan amino acid change, which, when expressed in COS7 cells, resulted in a dramatic reduction of transferase activity in vitro. PCR amplification of this gene from Drosophila cDNA panels and Northern analysis revealed that it is expressed throughout embryonic, larval, and pupal stages as well as in adult males and females. This study provides the first direct evidence for the involvement of a member of this conserved multigene family in eukaryotic development and viability.