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Abstract
Appropriate glycosylation on biological macromolecules such as proteins and lipids is vital for the normal growth and development of organisms. However, the elaborate set of biological events ensuring proper glycosylation has not been completely elucidated. Drosophila melanogaster embryonic nervous system synthesizes a family of related glycan species that are structurally similar and recognized by antibodies against the plant glycoprotein, Horseradish peroxidase (HRP). The specific presentation of HRP epitopes in the developing insect nervous system provides a platform for examining the genetic and molecular pathways controlling tissue-specific glycan expression. Drosophila mutations such as neurally altered carbohydrate (nac), sugar free frosting (sff) and tollo have been shown to specifically affect the neural expression of HRP epitopes. Transgenic overexpression of a Golgi GDP-fucose transporter or of a Drosophila homolog of the vertebrate SAD kinases rescues HRP-epitope expression in nac1 and sff mutants, respectively. By in-situ hybridization, sff mRNA is detected at reduced levels in tollo mutants, indicating that sff expression is regulated through Tollo signaling. To further characterize the signaling pathways regulating neural specific glycan complexity in Drosophila embryos, we undertook differential phosphoproteomic analysis to identify molecular targets of Sff/SAD kinase. Bifocal (Bif), Rasputin (Rin) and Liprin-alpha (Lip-) were detected among a set of proteins phosphorylated in wild-type but not in sff mutant embryos. Rin and lip- genetically interact with sff to affect glycoprotein glycosylation. Confocal analysis of Stage 14 embryos reveals an increase in the colocalization of Rin with Golgi compartments in sff mutants compared to wild-type. Western analysis detected a decrease in the steady state concentration of Rin protein in sff mutants, consistent with the genetic interaction detected between these two genes. In lip-/lip- mutants, compartmentation of the Golgi apparatus is altered in a manner similar to disruptions previously detected in sff mutants, consistent with a role for phosphorylation of this multi-domain protein in modulating glycoprotein glycan processing. Taken together, our results provide mechanistic insights into the signaling pathways through which Sff/SAD kinase influences cellular glycosylation in response to intra and extracellular cues.