DmSAS Is Required for Sialic Acid Biosynthesis in Cultured DrosophilaThird Instar Larvae CNS neurons

Sialylation is an important carbohydrate modification of glycoconjugates that has been shown to modulate many cellular/molecular interactions in vertebrates. In Drosophila melanogaster(Dm), using sequence homology, several enzymes of the sialylation pathway have been cloned and their function tested in expression systems. Here we investigated whether sialic acid incorporation in cultured Dmcentral nervous system (CNS) neurons required endogenously expressed Dmsialic acid synthase (DmSAS). We compared neurons derived from wild type Dmlarvae with those containing a DmSAS mutation (148 bp deletion). The ability of these cells to produce Sia5NAz (sialic acid form) from Ac4ManNAz (azide-derivatized N-acetylmannosamine) and incorporate it into their glycoconjugates was measured by tagging the azide group of Sia5NAz with fluorescent agents viaClick-iT chemistry. We found that most of the wild type DmCNS neurons incorporated Sia5NAz into their glycoconjugates. Sialic acid incorporation was higher at the soma than at the neurite and could also be detected at perinuclear regions and the plasma membrane. In contrast, neurons from the DmSAS mutant did not incorporate Sia5NAz unless DmSAS was reintroduced (rescue mutant). Most of the neurons expressed α2,6-sialyltransferase. These results confirm that the mutation was a null mutation and that no redundant sialic acid biosynthetic activity exists in Dmcells, i.e., there is only one DmSAS. They also provide the strongest proof to date that DmSAS is a key enzyme in the biosynthesis of sialic acids in DmCNS neurons, and the observed subcellular distribution of the newly synthesized sialic acids offers insights into their biological function.