Journal: Nature Communications

Article Title: Engineering glycosyltransferases into glycan binding proteins using a mammalian surface display platform

doi: 10.1038/s41467-025-62018-z

Figure Lengend Snippet: a Fc-pST3Gal1 (PS1) has four structural motifs. Amino acids shown in cyan (Q108A, Y233A, and Y269F) and magenta (H302A) were mutated in Dead and H302A mutants, respectively. b pST3Gal1 co-crystalized with CMP and Galβ1,3GalNAcα- p NP (PDB: 2WNB). Protein is in green, with selected residues and ligands shown as sticks. CMP is orange, Galβ1,3GalNAcα- p NP is yellow, amino acids mutated in Dead mutant are cyan, and H302A is magenta. c Western blots of Fc-fusion proteins expressed using HEK293T cells under reducing and non-reducing conditions. Detection was performed using anti-human Fc specific IgG. d LC-MS/MS analysis of ST3Gal1 enzymatic activity. Recombinant hST3Gal1 served as positive control. H302A and dead mutants did not yield product (N = 1). e Lectin binding to HEK293T (gray bars), COLO357-FG (green bars) or Calu-3 (blue bars) cells, with or without sialidase treatment, measured using flow cytometry. Cells were incubated with 5 μg/mL fluorescent MALII, PNA, or 1 μg/mL purified Fc-fusion proteins pre-complexed with AF647-conjugated anti-human Fc specific IgG. Data are mean ± STD (N = 3 biological replicates). * p < 0.05 with respect to all other samples; ‡ p < 0.05 with respect to all other samples except samples marked by ‡ are not different from each other. P values calculated using one-way ANOVA followed by Tukey post-test. f Dose dependent binding of PS1 and H302A pre-complexed with AF488-conjugated anti-human Fc specific IgG. HEK293T cells treated with or without sialidase were used. Data are mean (N = 2 biological replicates). Source data including exact P values. Created in BioRender. Neelamegham, S. ( https://BioRender.com/tigz92o ).

Article Snippet: Recombinant human ST3Gal1 (rhST3Gal1, Product code: 6905-GT-020), Recombinant human P-selectin Fc chimera (P-selectin-Fc, Product code: 137-PS-050), sheep anti-hGCNT1 IgG (Product code: AF7248-SP) and HRP donkey anti-sheep H + L IgG (Product code: HAF016) were from R&D Systems (Minneapolis, MN).

Techniques: Mutagenesis, Western Blot, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Recombinant, Positive Control, Binding Assay, Flow Cytometry, Incubation, Purification

Journal: Nature Communications

Article Title: Engineering glycosyltransferases into glycan binding proteins using a mammalian surface display platform

doi: 10.1038/s41467-025-62018-z

Figure Lengend Snippet: a Isogenic HEK293T KO cells lacking selected glycan-processing enzymes or transporters were produced. b Next-generation sequencing (NGS) results for isogenic clones lacking GCNT1 , SLC35A1 , and ST3Gal1 . Wildtype DNA and amino acid sequence corresponding to sense strand are shown above, with gene edited sequence below. Exons and introns appear in uppercase and lowercase, respectively. As two sgRNA were added simultaneously to create KOs, red and green represent the first and second editing regions, and blue presents the protospacer adjacent motif (PAM) on sense ( GCNT1 and ST3Gal1 KO) or anti-sense strands ( SLC35A1 KO). The entire sequence between the sgRNA cut sites was excised in the GCNT1 and SLC35A1 KO. This resulted in a few different closely-resembling read sequences. The frequency of occurrence of each of these edit patterns is shown on the right side of the DNA sequence (underlined). c Flow cytometry binding assay using 5 μg/mL fluorescent lectins (MALII and PNA for O -glycans, PHA-L and ECL for N -glycans). HEK293T WT cells and panel of isogenic HEK293T KO cells were used. d Flow cytometry binding assay using 1 μg/mL purified Fc-fusion proteins pre-complexed with AF488-conjugated anti-human Fc specific IgG. Data are mean ± STD (N = 6 for ( c , d ), except N = 5 for PHA-L and ECL, biological replicates). P values calculated using one-way ANOVA followed by the Tukey post-test. Data show that H302A binds sialylated core-2 O -linked glycans. * p < 0.05 with respect to all other samples; ‡ p < 0.05 with respect to all other samples except that samples marked by ‡ are not different from each other; † p < 0.05 with respect to WT cells. Source data are provided in file. Created in BioRender. Neelamegham, S. ( https://BioRender.com/tigz92o .

Article Snippet: Recombinant human ST3Gal1 (rhST3Gal1, Product code: 6905-GT-020), Recombinant human P-selectin Fc chimera (P-selectin-Fc, Product code: 137-PS-050), sheep anti-hGCNT1 IgG (Product code: AF7248-SP) and HRP donkey anti-sheep H + L IgG (Product code: HAF016) were from R&D Systems (Minneapolis, MN).

Techniques: Glycoproteomics, Produced, Next-Generation Sequencing, Clone Assay, Sequencing, Flow Cytometry, Binding Assay, Purification