Mapping ST3GAL Transferase Specificities in the Glycosylation Landscape of N/TERT-1 Keratinocytes Using Glycogenomics and Mass Spectrometry

Glycan sialylation is vital for proper cellular function and signaling. The six-membered ST3GAL family of sialyltransferases catalyzes the transfer of sialic acid in an α2,3-linkage to galactose residues on the outermost glycan epitopes. Dysregulation of sialyltransferase activity has been linked to diverse pathological processes. To expand our understanding of the substrate specificity and cooperative function of the ST3GAL family enzymes in protein and lipid glycosylation, we systematically analyzed the function of individual ST3GAL enzymes in glycan biosynthesis using a panel of CRISPR/Cas9-engineered human keratinocyte (N/TERT-1) cell lines with single or combined ST3GAL gene knockouts (KO). For protein glycosylation, KO of ST3GAL1 reduced sialylation of type 3 epitopes (Galβ1,3-GalNAc-) on both core 1 and 2 O-glycans, while complete ablation of sialylation was observed for the combined ST3GAL1 and ST3GAL2 KO. ST3GAL2 KO alone had limited effect, but reduced sialylation of specifically core 1 O-glycans. KO of ST3GAL4 and the combined KO of ST3GAL4 and ST3GAL6 reduced sialylation of type 1 and 2 epitopes (Galβ1,3/4-GlcNAc-) on both N- and O-glycans, while no effect was observed for the single KO of ST3GAL6. In GSL glycan biosynthesis, ST3GAL5 regulated lactosylceramide sialylation, as anticipated. ST3GAL2 and ST3GAL6 mediate sialylation of type 3 motifs, whereas ST3GAL3 and ST3GAL6 target type 2 epitopes, with ST3GAL3 exhibiting no discernible preference between type 1 and type 2 substrates. Our findings reveal that glycosyltransferase specificities are shaped by substrate availability, epitope distribution across glycan classes, and enzyme competition, which can only be captured by investigating this within the cellular context.