Mutant glycosidases for labeling sialoglycans with high specificity and affinity

Area of Science:

• Biochemistry
• Glycobiology
• Molecular Biology

Background:

• Affinity labeling is crucial for studying biomacromolecules, but specific glycan-binding probes are limited.
• Developing tools for precise glycan detection is essential for understanding biological processes.

Purpose of the Study:

• To engineer novel affinity probes for specific sialoglycan detection.
• To create a versatile toolkit for glycan analysis in various biological applications.

Main Methods:

• Mutating key catalytic residues of bacterial sialidases (SpNanA and RgNanH) to create inactive but specific glycan recombinant affinity binders (GRABs).
• Developing GRAB-Sia for total sialoglycans and GRAB-Sia3 for α2,3-sialosides.
• Enhancing avidity through tetramerization with streptavidin (tetra-GRABs).

Main Results:

• Engineered GRABs demonstrated strict substrate and linkage specificity for sialoglycans.
• Tetramerization significantly increased the avidity of GRABs.
• GRABs and tetra-GRABs proved effective in immunoblotting, flow cytometry, immunoprecipitation, and fluorescence imaging.
• Multiplex analysis with tetra-GRABs revealed distinct spatial distributions of sialoglycans in mouse organs.

Conclusions:

• The developed GRABs offer a highly specific and sensitive method for labeling and analyzing sialoglycans.
• This toolkit provides a convenient approach for glycan research, advancing bioimaging and functional studies.
• The findings enable deeper insights into the biological roles of sialoglycans.