New Strategy for Affinity Labeling of Sialyl Glycans
Affinity labeling proteins are important detection and analysis tools in the field of life sciences. For example, high-affinity monoclonal antibodies against proteins can achieve accurate and sensitive detection of proteins in different experimental scenarios.
Glycans, as an important class of biomacromolecules, carry a wealth of life information. Effective affinity labeling of glycans is an important technical prerequisite for exploring the functions of glycans in different biological systems. However, due to the low immunogenicity of glycans, high-affinity antibodies against glycans are very rare.
Lectins, as a class of naturally occurring glycan-binding proteins, have been widely used for glycan affinity labeling, but their low substrate specificity limits their application prospects in glycan function research.
Sialic Acids are a class of negatively charged nine-carbon sugars. As a capping sugar, sialic acid is often connected to the end of the sugar chain in different bonding forms (such as α2,3, α2,6 or α2,8), and plays an important role in biological processes such as cell migration, pathogen infection and Tumor immunoregulation. However, it is still very difficult to accurately label sialylated glycomotifs with specific bonding methods.
In order to solve the above problems, the team of Peng Dai and Xing Chen from Peking University published an article titled "Mutant glycosidases for labeling sialoglycans with high specificity and affinity" online in Nature Communications on February 7, 2025. This work reports a class of sialylated sugar affinity labeling tools glycan recombinant affinity binders (GRABs) based on the Glycosidases mutation strategy.
The authors used sialidases, a class of proteins that have evolved over a long period of time in nature and can efficiently recognize sialylated glycomotifs. Starting from the catalytic domains of Streptococcus pneumoniae neuraminidase A (SpNanA) and Ruminococcus gnavus neuraminidase H (RgNanH), based on their conservative catalytic mechanisms, they used a rational design approach to conduct small-scale mutation screening, and obtained proteins that recognize various connection modes and specifically recognize α2,3-linked sialic acid binding proteins, respectively, and named them GRAB-Sia and GRAB-Sia3.
Fig. 1 Development of GRAB-Sia. (Liang, et al., 2025)
Compared with the "gold standard" SNA and MAL-II lectins currently used for sialic acid affinity labeling, which have significant nonspecific binding, cell labeling experiments verified that GRABs have strict sialic acid binding specificity. GRAB monomers were assembled into tetramers (tetra-GRABs) through a biotin/streptavidin system, further improving the apparent affinity.
Using tetra-GRABs, the authors analyzed the distribution of sialylated glycans in various organ tissues of mouse and revealed the distribution characteristics of sialylated glycans with different connection modes in the mouse intestine.
GRABs and tetra-GRABs have good effects in various glycan labeling and analysis fields, such as immunoblotting, flow cytometry, immunoprecipitation and fluorescence imaging, providing a class of labeling tools with high specificity, high affinity and high convenience for sialylated glycan research.
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