Site-Specific Profiling of N-Glycans in Drosophila Melanogaster

Background: Drosophila melanogaster is a well-studied and highly tractable genetic model system for deciphering the molecular mechanisms underlying various biological processes. Although being one of the most critical post-translational modifications of proteins, the understanding of glycosylation in Drosophila is still lagging behind compared with that of other model organisms. Methods: In this study, we systematically investigated the site-specific N-glycan profile of Drosophila melanogaster using intact glycopeptide analysis technique. This approach identified the glycans, proteins, and their glycosites in Drosophila, as well as information on site-specific glycosylation, which allowed us to know which glycans are attached to which glycosylation sites. Results: The results showed that the majority of N-glycans in Drosophila were high-mannose type (69.3%), consistent with reports in other insects. Meanwhile, fucosylated N-glycans were also highly abundant (22.7%), and the majority of them were mono-fucosylated. In addition, 24 different sialylated glycans attached with 16 glycoproteins were identified, and these proteins were mainly associated with developmental processes. Gene ontology analysis showed that N-glycosylated proteins in Drosophila were involved in multiple biological processes, such as axon guidance, N-linked glycosylation, cell migration, cell spreading, and tissue development. Interestingly, we found that seven glycosyltransferases and four glycosidases were N-glycosylated, which suggested that N-glycans may play a regulatory role in the synthesis and degradation of N-glycans and glycoproteins. Conclusions: To our knowledge, this work represents the first comprehensive analysis of site-specific N-glycosylation in Drosophila, thereby providing new perspectives for the understanding of biological functions of glycosylation in insects.