Installation of O-glycan sulfation capacities in human HEK293 cells

The human genome contains at least 35 genes that encode Golgi sulfotransferases functioning in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Our insight into the sulfotransferases that serve glycoproteins and in particular GalNAc-type O-glycoproteins is limited, yet a number of important interactions with sulfated glycans by e.g. Selectins, Galectins, and Siglecs are thought to mainly rely on sulfated O-glycans. Moreover, sulfated mucins appear to be accumulated in respiratory diseases, arthritis and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, we have started to expand a cell-based glycan array in the human HEK293 cell line with sulfation capacities. Here, we stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knock-in of sulfotransferase genes, in combination with knockout of endogenous core2 and/or sialylation capacities, to enable production of mucin reporters with sulfated O-glycans. Expression of GAL3ST2 in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of GAL3ST4 resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan