Quantitative and site-specific chemoproteomic profiling of O-GlcNAcylation in Drosophila

Cong Lei; Zihan Chen; Yi Hao; Wanping Huang; Tianyu Chu; Kangming Xiao; Che Zhang; Wen Zhou; Chenjian Li; Xing Chen

doi:10.1016/j.bmc.2025.118191

Quantitative and site-specific chemoproteomic profiling of O-GlcNAcylation in Drosophila

Bioorg Med Chem. 2025 Jul 1:124:118191. doi: 10.1016/j.bmc.2025.118191. Epub 2025 Apr 10.

Authors

Cong Lei¹, Zihan Chen², Yi Hao³, Wanping Huang⁴, Tianyu Chu², Kangming Xiao², Che Zhang², Wen Zhou³, Chenjian Li⁴, Xing Chen⁵

Affiliations

¹ College of Chemistry and Molecular Engineering, Peking University, Beijing, China; Beijing National Laboratory for Molecular Sciences, Peking University, Beijing, China. Electronic address: conglei@pku.edu.cn.
² College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
³ College of Chemistry and Molecular Engineering, Peking University, Beijing, China; Beijing National Laboratory for Molecular Sciences, Peking University, Beijing, China.
⁴ The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China.
⁵ College of Chemistry and Molecular Engineering, Peking University, Beijing, China; Beijing National Laboratory for Molecular Sciences, Peking University, Beijing, China; Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China; Synthetic and Functional Biomolecules Center, Peking University, Beijing, China; Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, China. Electronic address: xingchen@pku.edu.cn.

PMID: 40245499
DOI: 10.1016/j.bmc.2025.118191

Abstract

Protein O-GlcNAcylation plays a crucial role in Drosophila melanogaster development. Dysregulation of O-GlcNAc transferase (sxc/Ogt) and O-GlcNAcase (Oga) disrupts early embryogenesis and locomotor behavior. It is therefore of great interest to identify and quantitatively analyze O-GlcNAcylation sites in Drosophila. Here, we perform quantitative and site-specific profiling of O-GlcNAcylation in Drosophila by employing a chemoenzymatic labeling strategy. A total of 2196 unambiguous O-GlcNAcylation sites and 1308 O-GlcNAcylated proteins are identified. Quantitative analysis of O-GlcNAcylation in the head of Drosophila with sxc/Ogt knockdown in GABAergic neurons reveals a reduction in O-GlcNAcylation of several proteins involved in muscle development, consistent with the phenotypic defects observed in sxc/Ogt RNAi Drosophila. Furthermore, quantitative analysis of O-GlcNAcylation under a high-sugar diet reveals altered O-GlcNAcylation of several proteins associated with obesity and neurological diseases, such as Hex-A and Ankyrin 2. Our study not only establishes an effective method for large-scale identification of O-GlcNAcylation sites, but also provides a valuable resource for studying O-GlcNAc biology in Drosophila.

Keywords: Chemoproteomic; Drosophila melanogaster; High-sugar diet; Muscle development; O-GlcNAcylation.

MeSH terms

Acetylglucosamine* / metabolism
Animals
Drosophila Proteins* / genetics
Drosophila Proteins* / metabolism
Drosophila melanogaster* / metabolism
Glycosylation
N-Acetylglucosaminyltransferases* / genetics
N-Acetylglucosaminyltransferases* / metabolism
Proteomics*
beta-N-Acetylhexosaminidases / metabolism

Substances

N-Acetylglucosaminyltransferases
O-GlcNAc transferase
Drosophila Proteins
Acetylglucosamine
beta-N-Acetylhexosaminidases