Distinct contributions of beta 4GalNAcTA and beta 4GalNAcTB to Drosophila glycosphingolipid biosynthesis

Drosophila melanogaster has two beta4-N-acetylgalactosaminyltransferases, beta4GalNAcTA and beta4GalNAcTB, that are able to catalyse the formation of lacdiNAc (GalNAcbeta,4GlcNAc). LacdiNAc is found as a structural element of Drosophila glycosphingolipids (GSLs) suggesting that beta4GalNAcTs contribute to the generation of GSL structures in vivo. Mutations in Egghead and Brainaic, enzymes that generate the beta4GalNAcT trisaccharide acceptor structure GlcNAcbeta,3Manbeta,4GlcbetaCer, are lethal. In contrast, flies doubly mutant for the beta4GalNAcTs are viable and fertile. Here, we describe the structural analysis of the GSLs in beta4GalNAcT mutants and find that in double mutant flies no lacdiNAc structure is generated and the trisaccharide GlcNAcbeta,3Manbeta,4GlcbetaCer accumulates. We also find that phosphoethanolamine transfer to GlcNAc in the trisaccharide does not occur, demonstrating that this step is dependent on prior or simultaneous transfer of GalNAc. By comparing GSL structures generated in the beta4GalNAcT single mutants we show that beta4GalNAcTB is the major enzyme for the overall GSL biosynthesis in adult flies. In beta4GalNAcTA mutants, composition of GSL structures is indistinguishable from wild-type animals. However, in beta4GalNAcTB mutants precursor structures are accumulating in different steps of GSL biosynthesis, without the complete loss of lacdiNAc, indicating that beta4GalNAcTA plays a minor role in generating GSL structures. Together our results demonstrate that both beta4GalNAcTs are able to generate lacdiNAc structures in Drosophila GSL, although with different contributions in vivo, and that the trisaccharide GlcNAcbeta,3Manbeta,4GlcbetaCer is sufficient to avoid the major phenotypic consequences associated with the GSL biosynthetic defects in Brainiac or Egghead.