Fig. 2: GRAB-Sia3 recognizing α2,3-sialosides.

a Close-up view of the catalytic pocket from the previously determined crystal structure of the RgNanH in complex with 2-deoxy 2,3-didehydro Neu5Ac (Neu5Ac2en) (PDB: 4X47)³⁶. Three catalytic residues of RgNanH are labeled and shown as sticks; the Neu5Ac2en molecule is shown in yellow. b Schematic showing the domain organization of RgNanH and the recombinant RgNanH catalytic domain. c SDS-PAGE analysis of fetuin incubated with WT RgNanH, RgNanH variants, or vehicle. Representative results are shown from three replicates. d HeLa cells were incubated with vehicle, His-tagged WT RgNanH, or His-tagged RgNanH variants, followed by staining with APC-anti-His tag antibody and flow cytometry analysis. Representative flow cytometry histograms and the bar graph of statistical analysis are shown in the left and right panels, respectively. e WT Hela cells with or without pretreatment of SpNanA and CMAS^−/− HeLa cells were incubated with vehicle or His-tagged RgNanH^D282A, followed by staining with APC-anti-His tag antibody and flow cytometry analysis. f Fetuin was incubated with vehicle, RgNanH, SpNanA, SpNanC, or AuNan, followed by blotting with monobiotinylated RgNanH^D282A. HRP-conjugated streptavidin was used for visualization. Representative results are shown from three replicates. g WT and CMAS^−/− HEK293T cells were incubated with vehicle, biotinylated GRABs (100 μg/mL), SNA (0.2 μg/mL), MAL-II (1 μg/mL), pan-specific SiaFind^TM Lectenz (version 1.0 or 2.0, 5 μg/mL), or α2,3-specific SiaFind^TM Lectenz (25 μg/mL), followed by staining with streptavidin-AF647 and flow cytometry analysis. Fold changes of MFI are shown above each pair of bars. In (d, e, g), MFI median fluorescence intensity, a.u. arbitrary units. Error bars represent the s.d. from three biological replicates with cells cultured in different wells/dishes in a single experiment. P values were calculated by one-way ANOVA. Source data are provided as a Source Data file.