Engineering Glyco-Enzymes for Substrate Identification and Targeting

Glycoconjugates are assembled by the coordinate actions of glycosyltransferases, which add sugars, and glycosidases, which remove sugars. These glyco-enzymes comprise families of enzymes that catalyze the same reaction, making it difficult to identify the direct substrates of each isozyme. To solve this challenge, mutagenesis of glyco-enzymes has been used to enable incorporation of unnatural sugar analogs that can be employed to tag and isolate the protein substrates of an individual glycosyltransferase. A second challenge arises in efforts to determine which substrates mediate biological effects. Engineering a glycosyltransferase to target its activity toward select acceptor substrates allows deconvolution of the roles of specific glycosylation events. Similarly, glycosidases can be engineered to target specific substrates, with basic science and translational applications. This review describes recent efforts at engineering glyco-enzymes to identify and target their distinct substrates.