An efficient system for bioconjugation based on a widely applicable engineered O-glycosylation tag

Area of Science:

• Biotechnology
• Glycobiology
• Protein Engineering

Background:

• O-glycan-based metabolic glycoengineering is crucial for therapeutic molecules but limited by complex endogenous pathways and lack of consensus sequences.
• Existing methods struggle with precise control over O-glycosylation, hindering the development of sophisticated bioconjugates.

Purpose of the Study:

• To develop a versatile, on-demand O-glycosylation system for precise control over glycan structures.
• To create a novel O-glycosylation tag and a metabolically engineered cell line for producing uniformly modified proteins.
• To demonstrate the utility of this system for generating site-specific bioconjugates for research and drug discovery.

Main Methods:

• Development of a novel 5 amino acid O-glycosylation tag.
• Metabolic engineering of a UDP-galactose-4-eperimase (GALE) knock-out cell line.
• Optimization of tag sequence for single or multiple O-glycans, controlled by media supplementation.
• Utilizing N-azido-acetylgalactosamine for CLICK chemistry-based bioconjugation.

Main Results:

• Production of Fc-based proteins with controlled single or multiple O-glycans.
• Generation of uniformly labeled proteins with exclusively N-azido-acetylgalactosamine.
• Successful creation of site-specifically fluorochrome-labeled antibodies, dual-payload molecules, and Fc-peptides.
• Demonstration of the system's applicability in basic research and drug discovery.

Conclusions:

• The developed system provides unprecedented control over O-glycosylation.
• This novel approach enables site-specific bioconjugation, advancing therapeutic molecule development.
• This is the first reported system combining an O-glycosylation tag and a metabolically engineered cell line for site-specific O-glycosylation.