Expanding the Glycoscope: New chemical tools to understand protein glycosylation

Glycosylation is the most abundant and most complex posttranslational protein modification. The vast majority of all proteins trafficking through the secretory pathway are glycosylated. To individual proteins, glycans add function and alter both physical and biochemical properties. While extremely abundant, particular structural changes in protein glycosylation can profoundly impact the physiological properties as well as interaction with other biomolecules.
Glycans are not directly encoded in the genome but biosynthesised by the combinatorial interplay of more than 250 glycosyltransferases. This interplay generates a vast complexity of glycans that is only beginning to be understood. At the same time, individual glycosyltransferases are of profound disease relevance, especially in cancer. To delineate how glycan biosynthesis is coordinated, methods are needed to inform on the activities of individual glycosyltransferases.
We have recently developed a tactic to directly profile the substrates of individual glycosyltransferase enzymes on cells and in the living animal. Key to this innovation was a process termed bump-and-hole engineering in which a glycosyltransferase is engineered to specifically accept a chemically modified nucleotide-sugar substrate. Transfer of the modified sugar to substrate proteins enables profiling by methods of click chemistry and subsequent imaging or mass spectrometry. We have optimised methods for both biosynthesis of nucleotide-sugars as well as mass spectrometry. Here, we will develop chemical tools as traceable reporters for the activities of individual glycosyltransferases. These tools will allow direct insight into the enzymes that introduce glycans as one of the most abundant and complex protein modifications. Cutting-edge methods of imaging, mass spectrometry glycoproteomics and genome engineering will collectively reveal the substrate proteins and glycosylation sites modified by disease-relevant individual glycosyltransferase enzymes.