Expanding the repertoire of GalNAc analogues for cell-specific bioorthogonal tagging of glycoproteins

Glycosylation is a ubiquitous modification of proteins, necessitating approaches for its visualization and characterization. Bioorthogonally tagged monosaccharides have been instrumental to this end, offering a chemical view into the cell biology of glycans. Understanding the use of such monosaccharides by cellular biosynthetic pathways has expanded their applicability in cell biology, for instance through the strategy Bioorthogonal Cell-specific Tagging of Glycoproteins (BOCTAG). Here, we show that the cellular use of two azide-tagged analogues of the monosaccharide N-acetylgalactosamine can be promoted through expression of two biosynthetic enzymes. Cellular expression of the kinase NahK and the engineered pyrophosphorylase AGX1F383A led to biosynthesis of the corresponding activated nucleotide-sugars and subsequent bioorthogonal tagging of the cellular glycoproteome. We explore the use of both sugars for BOCTAG, demonstrating the visualization of cell surface glycosylation in a specific cell line in a co-culture system. Our work adds to the toolbox of glycoprotein analysis in biomedicine.