Iterative Bump-and-Hole Engineering Creates a Bioorthogonal Reporter for N-Acetylglucosaminyltransferase I

Area of Science:

• Biochemistry
• Glycobiology
• Chemical Biology

Background:

• Asparagine-linked protein glycosylation is a common protein modification in the secretory pathway.
• N-acetylglucosamine (GlcNAc) incorporation, initiated by MGAT1, modulates glycan complexity.
• Current methods for incorporating tagged GlcNAc analogs lack specificity for individual glycosyltransferases.

Purpose of the Study:

• To engineer a bioorthogonal tool for precise study of MGAT1 activity in mammalian cells.
• To develop a method for selective chemical tagging of MGAT1 substrate proteins.
• To overcome the promiscuity of GlcNAc transferases for targeted glycan analysis.

Main Methods:

• Iterative bump-and-hole protein engineering of MGAT1.
• Structure-informed design to abrogate wild-type substrate acceptance and enable acceptance of azide-modified analogs.
• Kinetic and computational analyses, including neural networks, to guide synthesis of tailored UDP-GlcNAc analogs.
• Selective incorporation of chemical tags in living mammalian cells.

Main Results:

• Engineered MGAT1 variant showed abrogated activity toward UDP-GlcNAc but retained activity toward azide-modified analogs.
• A tailored UDP-GlcNAc analog was synthesized with preferential acceptance by the engineered enzyme.
• Selective chemical tagging of MGAT1 substrate proteins was achieved in mammalian cells with minimal background labeling.
• The developed tool demonstrated high specificity for MGAT1 substrates.

Conclusions:

• The study presents a novel, structure-informed bioorthogonal tool for precise investigation of MGAT1 activity.
• This approach enables specific labeling of MGAT1 substrates, overcoming limitations of existing methods.
• The developed strategy expands the toolbox for glycan-based reporter compounds and chemical biology studies.