Researchers have developed mutant glycosidases capable of labeling sialoglycans with unprecedented specificity and affinity, offering significant advancements for bioimaging and cellular analysis.
Affinity labeling is pivotal for our comprehension of biomolecular interactions, particularly when it involves the structurally complex glycans present on cell surfaces. These glycans play pivotal roles across various physiological processes, from cellular communication to pathogen recognition. Until now, the tools available for such specificity were limited. The newly created glycan recombinant affinity binders (GRABs), engineered from mutant bacterial sialidases, promise to fill this gap.
By muting specific catalytic residues of the sialidases produced by Streptococcus pneumoniae and Ruminococcus gnavus, researchers created GRAB-Sia and GRAB-Sia3, which are sensitive to total sialoglycans and α2,3-sialosides, respectively. Employment of these novel tools allows scientists to explore the diverse roles of glycans without the interference or bias commonly encountered with traditional labeling methods.
According to the study authors, the GRABs exhibit "high specificity, sensitivity, and convenience" for labeling sialoglycans, which are terminal 9-carbon monosaccharides important for numerous cellular interactions. The work reported shows how modifications to the enzymes allowed them to recognize target glycans without enzymatic activity, making them ideal for experimental applications.
One of the key innovations was the tetramerization of GRABs with streptavidin, which afforded increased binding affinity. This step significantly enhances the utility of GRABs for various techniques such as flow cytometry and fluorescence imaging. Researchers observed notable improvements, with tetramerized GRABs yielding fluorescence intensity more than two hundred times greater than the monomeric versions.
The impact of this study on the science community is immense. It not only introduces versatile tools for glycan analysis but also paves the way for new diagnostic and therapeutic applications, particularly within the realms of cancer research, where sialoglycan recognition by immune cells influences treatment outcomes.
The findings derived from multiplex analysis using tetra-GRABs revealed spatial distinctions of sialoglycan distributions across different mouse organs, showcasing their effectiveness. Researchers stated, "The GRABs neither bound non-sialylated glycans nor non-specifically interacted with other biomolecules... highlighting their superb specificity."
Insights gained from this novel approach could enrich our knowledge about the pivotal roles glycans play across various biological systems and contribute toward the design of high-fidelity glycomic tools. Brown and his team view this as just the beginning—a versatile platform indicative of broader applications.