Phosphoproteomic analysis of Drosophila embryos deficient in neural-specific glycosylation. Varshika Kotu^1,2, Peng Zhao^1,3, Toshihiko Katoh¹, Lance Wells^1,2,3, Michael Tiemeyer^1,2. 1) The Complex Carbohydrate Research Center, University of Georgia, Athens, GA; 2) The Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA; 3) The Department of Chemistry, University of Georgia, Athens, GA.

   Appropriate glycan expression is essential for development and normal tissue function. However, a complete mechanistic understanding of the pathways regulating glycoprotein glycosylation is lacking. A family of structurally related N-linked glycans known as HRP epitopes are specifically expressed in Drosophila neural tissue, providing a platform to understand the regulatory mechanisms controlling tissue-specific glycosylation. We previously generated and characterized a Drosophila mutant called sugar-free frosting (sff) which affects HRP-epitope expression in the embryonic nervous system. The sff mutation mapped to the Drosophila homologue of a serine/threonine kinase known as SAD-1 in other invertebrate and vertebrate species. In mid-stage Drosophila embryos, confocal analysis demonstrated that the sff mutation alters Golgi compartmental distributions such that glycoprotein glycosylation is shifted in favor of greater glycan complexity and decreased HRP-epitope expression. In order to further characterize the molecular mechanisms underlying altered neural glycan expression, we have undertaken differential phosphoproteomic analysis of OreR and sff mutant embryos. By LC-MS/MS, we identified phosphoprotein serine/threonine phosphorylation sites that were utilized in wild-type but not detected in the sff mutant and undertook the validation of these proteins as Sff/SAD kinase substrates by orthogonal approaches. Detection of genetic interaction with our mutant sff allele nominated three phosphoproteins (Bifocal, Rasputin and Liprin-alpha) as candidate substrates for Sff/SAD kinase in relation to glycoprotein glycosylation. Our on-going efforts are directed towards understanding the functional significance of these identified phosphoproteins and of Sff/SAD kinase signaling in the context of neural specific glycosylation.