Mutant glycosidases for labeling sialoglycans with high specificity and affinity

Shuyu Liang, Qi Tang, Xunzi Guo, Zi’an Li, Yilan Guo, Jinghan Chang, Bo Cheng, Qitao Song, Jiayu Sun, Peng Dai () and Xing Chen ()
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Shuyu Liang: Peking University
Qi Tang: Peking University
Xunzi Guo: Peking University
Zi’an Li: Peking University
Yilan Guo: Peking University
Jinghan Chang: Peking University
Bo Cheng: Peking University
Qitao Song: Peking University
Jiayu Sun: Peking University
Peng Dai: Peking University
Xing Chen: Peking University

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: Abstract Affinity labeling of biomacromolecules is vital for bioimaging and functional studies. However, affinity probes recognizing glycans with high specificity remain scarce. Here we report the development of glycan recombinant affinity binders (GRABs) based on mutant bacterial sialidases, which are enzymatically inactive but preserve stringent specificity for sialoglycan substrates. By mutating a key catalytic residue of Streptococcus pneumoniae neuraminidase A (SpNanA) and Ruminococcus gnavus neuraminidase H (RgNanH), we develop GRAB-Sia and GRAB-Sia3 recognizing total sialoglycans and α2,3-sialosides, respectively. The GRABs exhibit strict substrate and linkage specificity, and tetramerization with streptavidin substantially increases their avidity. The GRABs and tetrameric GRABs (tetra-GRABs) are effective tools for probing sialoglycans in immunoblotting, flow cytometry, immunoprecipitation, and fluorescence imaging. Furthermore, multiplex analysis with tetra-GRABs uncovers spatially distinct sialoglycans in the various mouse organs. This work provides a versatile toolkit for labeling and analyzing sialoglycans with high specificity, sensitivity, and convenience.

Date: 2025
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DOI: 10.1038/s41467-025-56629-9

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