Fig. 1: Development of GRAB-Sia.
From: Mutant glycosidases for labeling sialoglycans with high specificity and affinity
a Schematic showing the catalytic mechanism of sialidases in the GH33 family. b Close-up view of the catalytic pocket from the previously determined crystal structure of SpNanA in complex with Neu5Ac (PDB: 3H72)60. Three catalytic residues of SpNanA are labeled and shown as sticks; the Neu5Ac molecule is shown in yellow. c Schematic showing the domain organization of SpNanA and the recombinant SpNanA catalytic domain. d SDS-PAGE analysis of fetuin incubated with WT SpNanA, SpNanA variants, or vehicle. CBB, Coomassie brilliant blue. Representative results are shown from three replicates. e HeLa cells were incubated with vehicle, His-tagged WT SpNanA, or His-tagged SpNanA 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. f WT Hela cells with or without pretreatment of SpNanA and CMAS−/− HeLa cells were incubated with vehicle or His-tagged SpNanAD372N, followed by staining with APC-anti-His tag antibody and flow cytometry analysis. In (e, f), MFI median fluorescence intensity, a.u. arbitrary units. Error bars represent the standard deviation (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.
