Tissue-specific <i>O-</i>GlcNAcylation profiling identifies substrates in translational machinery in <i>Drosophila</i> mushroom body contributing to olfactory learning.

Abstract

<i>O-</i>GlcNAcylation is a dynamic post-translational modification that diversifies the proteome. Its dysregulation is associated with neurological disorders that impair cognitive function, and yet identification of phenotype-relevant candidate substrates in a brain-region specific manner remains unfeasible. By combining an <i>O-</i>GlcNAc binding activity derived from <i>Clostridium perfringens</i> OGA (<i>Cp</i>OGA) with TurboID proximity labeling in <i>Drosophila</i>, we developed an <i>O-</i>GlcNAcylation profiling tool that translates <i>O-</i>GlcNAc modification into biotin conjugation for tissue-specific candidate substrates enrichment. We mapped the <i>O-</i>GlcNAc interactome in major brain regions of <i>Drosophila</i> and found that components of the translational machinery, particularly ribosomal subunits, were abundantly <i>O-</i>GlcNAcylated in the mushroom body of <i>Drosophila</i> brain. Hypo-<i>O-</i>GlcNAcylation induced by ectopic expression of active <i>Cp</i>OGA in the mushroom body decreased local translational activity, leading to olfactory learning deficits that could be rescued by dMyc overexpression-induced increase of protein synthesis. Our study provides a useful tool for future dissection of tissue-specific functions of <i>O-</i>GlcNAcylation in <i>Drosophila</i>, and suggests a possibility that <i>O-</i>GlcNAcylation impacts cognitive function via regulating regional translational activity in the brain.