A New Method For Tissue-Specific Enrichment Of O-GlcNAcylated Proteins

Research purpose:O-GlcNAcylation plays a biological role by linking a single N-acetylglucosamine(GlcNAc)to the serine or threonine residues of target proteins.It is a common protein post-translational modification.In cells,the level of O-GlcNAcylation is determined by two mutually antagonistic enzymes:O-GlcNAc transferase(OGT)and O-GlcNAcase(OGA),both of which regulate the dynamic balance of O-GlcNAcylated level by catalyzing the addition and removal of GlcNAc on proteins.The homeostasis of O-GlcNAcylation plays an important role in the development of the brain and the maintenance of the function of the nervous system.Abnormal changes in the O-GlcNAcylated level are related to the occurrence and development of neurological diseases such as intellectual disability and neurodegenerative diseases.As a common model organism,Drosophila is often used to study related pathways in the nervous system.In Drosophila,O-GlcNAcylation and OGA have been reported to have conserved cognitive regulatory functions,but the underlying molecular mechanisms remain to be further explored.In view of the existing methods for analyzing the substrates of O-GlcNAcylation,they cannot be used for tissue-specific enrichment and detection of O-GlcNAcylated proteomes in different brain regions of Drosophila,which limits the in-depth study of the function of O-GlcNAcylation in different brain regions of Drosophila.Method:In this study,we designed and developed a novel tissue-specific O-GlcNAcylated proteins enrichment method(TurboID-CpOGA~M)based on the highly efficient biotin ligase TurboID and the CpOGA mutant developed by our collaborative research group.It provides a powerful means to study the specific molecular mechanism of O-GlcNAcylation regulating brain development and function.Results:First,we verified the protein-specific enrichment of TurboID-CpOGA~Mfor O-GlcNAcylated proteins in HEK293T cells,and successfully analyzed and improved the O-GlcNAcylated proteome of HEK293T cells;secondly,based on UAS/Gal4 system,we applied TurboID-CpOGA~Mto the Drosophila model and identified the O-GlcNAcylated proteome of Drosophila adults;then,we demonstrated through Drosophila behavioral experiments that the decreased O-GlcNAcylated levels in Drosophila mushroom body lead to defects in olfactory learning.At the same time,we successfully achieved tissue-specific enrichment and identification of O-GlcNAcylated proteins in Drosophila mushroom body using TurboID-CpOGA~M,and analyzed the potential molecular network that may be involved in the regulation of cognitive function.Conclusion:This study developed a novel method for the enrichment and detection of O-GlcNAcylated proteins(TurboID-CpOGA~M)and applied it to the specific O-GlcNAcylated proteins in Drosophila mushroom body Analysis of groups.It will not only contribute to the in-depth understanding of the regulation of protein O-GlcNAcylation on brain development and function,but also provide more ideas for the study of O-GlcNAcylation-associated neurological diseases.