Identification of O-GalNAc-modified proteins interacting with GALNT3 using proximity labeling method

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Protein glycosylation, specifically O-N-acetylgalactosamine (O-GalNAc) modification, is a critical post-translational modification.
• O-GalNAc is catalyzed by N-acetylgalactosaminyltransferases (GALNTs) and is implicated in various diseases.
• Traditionally, O-GalNAc was thought to occur only in the secretory pathway, but recent findings suggest nuclear occurrence.

Purpose of the Study:

• To comprehensively characterize O-GalNAc modifications within the cell nucleus.
• To identify novel nuclear O-GalNAc-modified proteins and sites.

Main Methods:

• Utilized TurboID, an efficient proximity labeling enzyme, fused with GALNT3.
• Performed in vivo biotinylation of proteins interacting with GALNT3 in living cells.
• Employed mass spectrometry to identify biotinylated peptides and O-GalNAc sites.

Main Results:

• Identified 25 high-confidence and 10 potential O-GalNAc-modified sites across 30 proteins.
• Discovered 52 putative O-GalNAc-modified peptides from 52 distinct proteins.
• The majority of identified modifications and proteins were nuclear-localized and previously uncharacterized for O-GalNAc modification.

Conclusions:

• This study provides the first comprehensive glycoproteomic characterization of nuclear O-GalNAc modifications.
• The findings expand the known landscape of O-GalNAc modification, revealing its presence and significance in the nucleus.
• This work opens new avenues for understanding the role of nuclear O-GalNAc in cellular functions and disease pathogenesis.